虾蟹类免疫相关基因的研究
|
摘要
中华绒螯蟹(Eriocheir sinensis)是我国重要的经济养殖蟹类之一,又俗称河蟹或大闸蟹,隶属于节肢动物门(Athropoda)、软甲纲(Malacostraca)、十足目(Decapoda)、方蟹科(Grapsidae)、绒螯蟹属(Eriocheir)、中华绒螯蟹种(Eriocheir sinensis),不仅肉味鲜美,营养丰富,而且是我国久负盛名的美食,因此具有极高的经济和养殖价值。红螯光壳螯虾(Cherax quadricarinatus),又俗称澳洲淡水龙虾或者红螯螯虾,隶属于甲壳纲(Crustacea)、十足目(Decapoda)、拟螯虾科(Parastacidae)、光壳虾属(Cherax),原产于澳大利亚,于1991年首次引入我国试养,并于次年开展人工繁殖和自然繁殖获得成功,因其具有肉质细嫩、生长速度快、养殖产量高、富含低胆固醇等特点而被广泛推广养殖。伴随着两种甲壳动物养殖规模的扩大以及集约化养殖的发展,各种养殖病害频发,严重制约了这两种甲壳动物养殖产业的发展。而这两种甲壳动物,均凭借其独特的先天性免疫系统,在外界不良生存环境中有效地抵御外界病原菌的入侵,因而其先天性免疫防御机制的研究备受关注。有鉴于此,本研究以这两种甲壳动物为研究对象,开展了对这两种甲壳动物免疫相关基因的功能研究,以期能够完善甲壳动物先天性免疫防御机制的理论研究,为制定有效地病害防御策略提供坚实的理论基础与支持。
本研究主要研究内容与结果如下：1)中华绒螯蟹Toll基因的克隆与表达分析
Toll样受体在诱导病原菌的先天性免疫应答反应中起着重要作用,因此在大部分昆虫中均已经有所报道,但是在中华绒螯蟹中却未见报道。在本文中,我们克隆了两个新的中华绒螯蟹Toll基因：EsTolll和EsToll2。EsTolll基因的cDNA全长3963bp,开放阅读框3042bp,编码1013个氨基酸。其胞外区主要由17个亮氨酸重复序列组成,胞内区主要由139个氨基酸残基的TIR结构域组成。EsToll2基因cDNA全长4419bp,开放阅读框2667bp,编码888个氨基酸。其胞外区主要由10个亮氨酸重复序列组成,胞内区主要由139个氨基酸残基的TIR结构域组成。聚类分析的结果显示,EsToll1和EsToll2首先与其它甲壳动物的Toll受体家族聚为一支,然后与其它节肢动物的Toll受体家族聚为一支。荧光定量PCR(qRT-PCR)分析结果表明：EsTolll和EsToll2在各组织中都能够检测到,经由脂多糖(LPS)、肽聚糖(PG)和葡聚糖(GLU)免疫刺激后,表达量具有不同程度的变化。比较重要的是,EsToll2mRNA的表达量几乎在所有免疫时间段内均有显著的增加。综上,EsTolll和EsToll2对于不同的病原相关分子的刺激具有不同的可诱导性,对中华绒螯蟹先天性免疫应答的研究具有重要意义。2)中华绒螯蟹Tube基因的克隆与表达分析
Tube作为Toll信号通路中的重要组分,在机体存活、发育以及先天性免疫等生理活动中具有重要的作用。目前已经在对虾中被成功识别,但是未见在中华绒螯蟹中有所报道。在本文中,我们成功克隆得到了中华绒螯蟹Tube基因,简称为EsTube。EsTube基因cDNA全长2247bp,开放阅读框1539bp,编码512个氨基酸。EsTube蛋白主要包括一个由116个氨基酸残基组成的死亡结构域(death domain, DD)和一个由272个氨基酸残基组成的丝组氨酸蛋白激酶结构域(S TKc)。聚类分析的结果显示,EsTube首先与其它无脊椎动物Tube和Tube样蛋白聚为一支,然后与脊椎动物的IRAK-4蛋白聚为一支,最后与其它无脊椎动物Pelle蛋白聚为一支。实时荧光定量PCR (qRT-PCR)的结果显示,EsTube高度表达在卵巢和精巢,中度表达在胸神经节和胃；在所有发育阶段均能检测到EsTube的表达,高度表达在精巢发育的精细胞阶段(十月份)和卵巢发育的Ⅲ-2阶段(十一月份)；在不同病原相关分子模式(LPS、β-1,3-glucan and PG)的免疫刺激下,具有不同的诱导表达水平。综上,本研究的结果表明,EsTube在中华绒螯蟹免疫和发育方面具有重要的作用,具有免疫和发育的双重功能。3)中华绒螯蟹Dorsal基因的克隆与表达分析
Dorsal作为Toll信号通路中的重要组分,在先天性免疫应答的诱导和识别中具有重要作用。在本文中,我们在中华绒螯蟹中克隆了一个NF-κB样核转录因子—Dorsal,简称为EsDorsal。EsDorsal基因cDNA全长2493bp,开放阅读框2022bp,编码673个氨基酸。EsDosal蛋白主要包括一个由171个氨基酸组成的RHD结构域和一个由102个氨基酸组成的IPT结构域。聚类分析的结果显示,EsDorsal首先与其它无脊椎动物的Dorsals或者NF-κBs聚为一支,然后与脊椎动物的NF-κBs聚为一支。实时荧光定量PCR(qRT-PCR)的结果显示,EsDorsal在免疫相关组织中表达量较高,经过脂多糖(LPS)、肽聚糖(PG)和葡聚糖(GLU)免疫刺激后,表达量具有不同程度的变化。重要的是,相对于对于肽聚糖(PG)和葡聚糖(GLU), EsDorsal对脂多糖(LPS)的免疫刺激更加敏感。综上,EsDorsal对于不同的病原相关分子的刺激具有不同的可诱导性,对中华绒螯蟹先天性免疫应答机制的研究具有重要意义。
4)红螯光壳螯虾Crustin基因的克隆与功能分析
抗菌肽作为一种重要的免疫效应因子,对于红螯光壳螯虾(Cherax quadricarinatus)先天性免疫应答防御反应具有重要作用。本研究综合同源克隆和SMART-RACE技术,从红螯光壳螯虾的血细胞中成功克隆得到了一个重要的抗菌肽Crustin,简称为CqCrustin。CqCrustin基因cDNA全长为608bp,开放式阅读框为324bp,编码107个氨基酸,在其推测的氨基酸序列的N端含有一个由16个氨基酸残基组成的信号肽,C端含有一个由48个氨基酸残基组成的乳清酸蛋白(WAP)结构域。基于其WAP结构域的序列信息,进行聚类分析的结果显示,CqCrustin首先与其它甲壳动物的Ⅰ型Crustin聚为一支,然后与其它甲壳动物Ⅱ型Crustin聚为一支,最后于Ⅲ型Crustin聚为一支。多序列比对的结果显示,CqCrustin与克氏原螯虾(Procambarus clarkii) PcCrustin2具有较高的序列相似性(69%)。实时荧光定量PCR (qRT-PCR)的结果显示,CqCrustin主要表达在免疫相关组织,且在红螯光壳螯虾血细胞中的表达量最高；利用LPS,GLU和PG三种病原相关分子模式免疫刺激后,CqCrustin的mRNA表达水平在红螯光壳螯虾血细胞中具有不同程度的显著上调。为了进一步研究其生物学活性,利用原核表达技术获得的重组蛋白rCqCrustin对于不同的病原菌具有不同程度的结合活性,而且对不同的病原菌的生长速度亦具有不同程度的抑制作用。综上可见,CqCrustin在红螯光壳螯虾先天性免疫识别和防御过程中具有重要作用。
5)红螯光壳螯虾Dscam基因的克隆与功能分析
细胞粘附分子(Dscam)属于免疫球蛋白超级家族中的重要一员,在无脊椎动物,特别是节肢动物的先天性免疫识别和防御中具有重要作用。本研究综合同源克隆和SMART-RACE技术,从红螯光壳螯虾(Cherax quadricarinatus)的血细胞中成功克隆得到了其Dscam基因,简称为CqDscam。GqDscam蛋白含有类似于其它无脊椎动物的Dscam蛋白同系物的典型结构域,主要包括一个信号肽、10个免疫球蛋白结构域(Ig)、6个Ⅲ型纤连蛋白结构域(FNⅢ)、一个跨膜区和一个细胞质尾结构域。聚类分析的结果显示,CqDscam蛋白首先与其它甲壳动物的Dscam同系物聚为一支,然后与其它无脊椎动物的Dscam同系物聚为一支,最后与脊椎动物的Dscam同系物聚为一支。胞外结构域的检测结果表明,CqDsccam蛋白的N端具有多个可变剪切亚型,其中Ig2结构域具有30个亚型,Ig3结构域具有33个亚型,Ig7具有19个亚型,跨膜区具有2个亚型,理论上,总共可产生多达37620个不同的亚型。qRT-PCR的检测结果表明,CqDscam组成性表达在红螯螯虾各组织,特别是神经和免疫相关的组织：利用脂多糖(LPS)、肽聚糖(PG)和β-1,3-葡聚糖(GLU)免疫刺激红螯螯虾后,CqDscam的mRNA表达水平具有不同程度的诱导上调。CqDscam重组蛋白的功能检测显示,rCqDscam能够不同程度地识别不同的细菌,并且能够不同程度地抑制革兰氏阳性菌(金黄色葡萄球菌)和革兰氏阴性菌(大肠杆菌)的生长。此外,免疫荧光的结果显示,CqDscam主要分布于红螯螯虾血细胞的表面。综上可见,CqDscam在红螯螯虾的先天性免疫识别和防御中具有重要作用。
The Chinese mitten crab(Eriocheir sinensis) as one of the commercially important crab, also was known as the fresh water crab、hairy crab and so on, belongs to Athropoda, Malacostraca, Decapoda, Grapsidae, Eriocheir, Eriocheir sinensis. The Chinese mitten crab not only is prestigious food, but also has better nutritive value and raising value because of its rich-nutrition and delicious taste. The Red claw crayfish (Cherax quadricarinatus), that also is known as the Australian freshwater lobster or red claw crayfish, belongs to Crustacea, Decapoda, Parastacidae. Cherax quadricarinatus, that is native to Australia and introduced into China in1991for the first time. It is breeding successfully and widely cultured in the next year because of its fast growth, delicious taste, high yield and adaptability. With the development of the intensive aquaculture and the enlargement of the cultivation scale, the frequent outbreaks of diseases have caused decreased production and catastrophic losses in the past decade. Crustacean can induce rapid and effective immune responses to clear the intruding pathogens relying largely on innate immunity, which attracts more and more attention. Upon this backdrop, studying the structure and transcriptional responses of potential immune-related genes may facilitate a better understanding of the crustacean immune defense and recognition mechanisms and support the sustainable development of better disease management strategies in the crustacean farming industry.
The main research contents and results are as follows:
1) Two novel Toll genes(EsTolll and EsToll1) from Eriocheir sinensis are differentially induced by lipopolysaccharide, peptidoglycan and zymosan
Tolls/Toll-like receptors (TLRs) play an essential role in initiating innate immune responses against pathogens and are found throughout the insect kingdom but have not yet been reported in the crustacean, Eriocheir sinensis. For this purpose, we cloned two novel Toll genes from E. sinensis, EsTolll and EsToll2. The full-length cDNA of EsTolll was3,963bp with a3,042-bp open reading frame (ORF) encoding a1,013-amino acid protein. The extracellular domain of this protein contains17leucine-rich repeats (LRRs) and a139-residue cytoplasmic Toll/interleukin-1receptor (TIR) domain. The cDNA full-length of EsToll2was4,419bp with a2,667-bp ORF encoding an888-amino acid protein with an extracellular domain containing10LRRs and a139-residue cytoplasmic TIR domain. By phylogenetic analysis, EsTolll and EsTolll clustered into one group together with Tolls from other crustaceans. Quantitative RT-PCR analysis demonstrated that a) both EsTolll and EsToll2were constitutively expressed in all tested crab tissues; b) EsTolll and EsToll2were differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsToll2expression was significantly upregulated at almost all time intervals post-challenge with LPS, PG and GLU. Our study indicated that EsTolll and EsToll2are differentially inducibility in response to various PAMPs, suggesting their involvement in a specific innate immune recognition mechanism in E. sinensis.
2) Molecular cloning and expression analysis of Tube from Eriocheir sinensis
As a key component of the Toll signaling pathway, Tube plays central roles in many biological activities, such as survival, development and innate immunity. Tube has been found in shrimps, but have not yet been reported in the crustacean, Eriocheir sinensis. In this study, we cloned the full-length cDNA of the adaptor Tube for the first time from E sinensis and designated the gene as EsTube. The full-length cDNA of EsTube was2,247-bp with a1,539-bp open reading frame (ORF) encoding a512-amino acid protein. The protein contained a116-residue death domain (DD) at its N-terminus and a272-residue serine/threonine-protein kinase domain (S_TKc) at its C-terminus. Phylogenetic analysis clustered EsTube initially in one group with other invertebrate Tube and Tube-like proteins, and then with the vertebrate IRAK-4proteins, finally with other invertebrate Pelle proteins. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that EsTube was highly expressed in ovary and testis, and moderately expressed in thoracic ganglia and stomach. EsTube was expressed at all selected stages and was highly expressed in the spermatid stage (October, testis) and the stage III-2(November, ovary). EsTube was differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (β-1,3-glucan). Our study indicated that EsTube might possess multiple functions in immunity and development in E. sinensis.
3) Molecular cloning and expression analysis of a dorsal homologue from Eriocheir sinensis
Dorsal as a crucial component of Toll signaling pathway, played important roles in induction and regulation of innate immune responses. In this study, we cloned a NF-κB-like transcription factor Dorsal from Eriocheir sinensis and designated it as EsDorsal. The full-length cDNA of EsDorsal was2,493bp with a2,022-bp open reading frame (ORF) encoding a673-amino acid protein. This protein contained a171-residue conserved Rel homology domain (RHD) and a102-residue Ig-like, plexins and transcription factors domain (IPT). By phylogenetic analysis, EsDorsal was clustered into one group together with other invertebrate Dorsals or NF-κBs, and then clustered with vertebrate NF-κBs. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that a) EsDorsal had higher expression level in immune organs; b) EsDorsal differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsDorsal was more responsive to LPS than GLU and PG. Collectively, EsDorsal was differentially inducibility in response to various PAMPs, suggesting its involvement in a specific innate immune regulation in E. sinensis.
4) A novel Crustin involves in antibacterial responses in the red claw crayfish, Cherax quadricarinatus
Antimicrobial peptides are important immune effectors and play important roles in mediating innate immune responses against intruding pathogens. Here, we successfully isolated and characterized a novel Crustin from Red claw crayfish Cherax quadricarinatus and named it as CqCrustin. The deduced amino acid sequence of CqCrustin exhibited the typical configuration similar to other crustacean Crustin orthologs, including one signal peptide region at N-terminus between1and16and a long whey acidic protein domain (WAP domain) at C-terminus between60and107along with a WAP-type "four-disulfide core" motif. Phylogenetic analysis showed that CqCrustin was clustered with other crustacean Type Ⅰ Crustins firstly, and then with other crustacean Type Ⅱ Crustins, finally with other crustacean Type Ⅲ Crustins. CqCrustin showed higher sequence similarity (69%) with Crustin2from Procambarus clarkii (Pc-Crustin2). Transcription of CqCrustin-Ⅰ were both1) detected in all tissues, especially in hemocytes and gill; b) differentially induced in hemocytes post β-1,3-glucans (GLU), lipopolysaccharides (LPS) and peptidoglycans (PG) injection at selected time points. To understand its biological activity, the recombinant CqCrustin protein was constructed and expressed in Escherichia coli BL21(DE3). Recombinant protein rCqCrustin exhibited distinct bacterial binding activities against different Gram-positive bacteria, Gram-negative bacteria and fungus. Furthermore, bacterial growth inhibition assays demonstrated that rCqCrustin responds positively to the growth inhibition of different Gram-positive bacteria, Gram-negative bacteria and fungus. These findings suggested CqCrustin may involve in a specific innate immune recognition and defense mechanism against bacteria and fungus in Cherax quadricarinatus.
5) Identification and characterization of Dscam isoforms isolated from the red claw crayfish Cherax quadricarinatus
The Down syndrome cell adhesion molecule (Dscam) belongs to the immunoglobulin superfamily (IgSF) member and has been identified and isolated from some vertebrates and invertebrates. Recently, many studies have confirmed the important role of Dscam in mediating innate immune response against intruding pathogens and wiring of the nerve system in invertebrates, especially in Arthropod. Here, we successfully isolated and characterized the Dscam from Red claw crayfish Cherax quadricarinatus and named it as CqDscam. The deduced amino acid sequence of CqDscam exhibited the typical configuration similar to other invertebrate Dscam orthologs, including one signal peptide,10immunoglobulin (Ig) domains,6fibronectin type III (FNIII) domains and one transmembrane (TM) domain and cytoplasmic tail domain. Phylogenetic analysis showed that CqDscam was clustered with other invertebrate Dscams firstly, and then with vertebrate Dscams. In the extracellular region, the variable regions of CqDscam were located in N-terminal half of Ig2and Ig3domains and the complete Ig7domain. The CqDscam extracellular variants and transmembrane domain variants were produced by mutually exclusive alternative splicing events and could generate more than37,620different unique isoforms. Transcription of CqDscam were both1) detected in all tissues, especially in immune tissues and nerve tissues; b) differentially induced in hemocytes post glucans (GLU), lipopolysaccharides (LPS) and peptidoglycans (PG) injection at selected time points. Importantly, we had detected membrane-bound and secreted Dscam isoforms in C. quadricarinatus and showed that secreted isoforms were extensively transcribed post different PAMPs challenge, respectively. Results from immuno-localization assay revealed that CqDscam evenly distributed in the cell surface of hemocytes. In addition, the PAMPs specific isoforms of CqDscam were shown to be associated with bacterial clearance and phagocytosis in Red claw crayfish. These findings suggested CqDscam may involve in a specific innate immune recognition and defense mechanism in Cherax quadricarinatus.
本研究主要研究内容与结果如下：1)中华绒螯蟹Toll基因的克隆与表达分析
Toll样受体在诱导病原菌的先天性免疫应答反应中起着重要作用,因此在大部分昆虫中均已经有所报道,但是在中华绒螯蟹中却未见报道。在本文中,我们克隆了两个新的中华绒螯蟹Toll基因：EsTolll和EsToll2。EsTolll基因的cDNA全长3963bp,开放阅读框3042bp,编码1013个氨基酸。其胞外区主要由17个亮氨酸重复序列组成,胞内区主要由139个氨基酸残基的TIR结构域组成。EsToll2基因cDNA全长4419bp,开放阅读框2667bp,编码888个氨基酸。其胞外区主要由10个亮氨酸重复序列组成,胞内区主要由139个氨基酸残基的TIR结构域组成。聚类分析的结果显示,EsToll1和EsToll2首先与其它甲壳动物的Toll受体家族聚为一支,然后与其它节肢动物的Toll受体家族聚为一支。荧光定量PCR(qRT-PCR)分析结果表明：EsTolll和EsToll2在各组织中都能够检测到,经由脂多糖(LPS)、肽聚糖(PG)和葡聚糖(GLU)免疫刺激后,表达量具有不同程度的变化。比较重要的是,EsToll2mRNA的表达量几乎在所有免疫时间段内均有显著的增加。综上,EsTolll和EsToll2对于不同的病原相关分子的刺激具有不同的可诱导性,对中华绒螯蟹先天性免疫应答的研究具有重要意义。2)中华绒螯蟹Tube基因的克隆与表达分析
Tube作为Toll信号通路中的重要组分,在机体存活、发育以及先天性免疫等生理活动中具有重要的作用。目前已经在对虾中被成功识别,但是未见在中华绒螯蟹中有所报道。在本文中,我们成功克隆得到了中华绒螯蟹Tube基因,简称为EsTube。EsTube基因cDNA全长2247bp,开放阅读框1539bp,编码512个氨基酸。EsTube蛋白主要包括一个由116个氨基酸残基组成的死亡结构域(death domain, DD)和一个由272个氨基酸残基组成的丝组氨酸蛋白激酶结构域(S TKc)。聚类分析的结果显示,EsTube首先与其它无脊椎动物Tube和Tube样蛋白聚为一支,然后与脊椎动物的IRAK-4蛋白聚为一支,最后与其它无脊椎动物Pelle蛋白聚为一支。实时荧光定量PCR (qRT-PCR)的结果显示,EsTube高度表达在卵巢和精巢,中度表达在胸神经节和胃；在所有发育阶段均能检测到EsTube的表达,高度表达在精巢发育的精细胞阶段(十月份)和卵巢发育的Ⅲ-2阶段(十一月份)；在不同病原相关分子模式(LPS、β-1,3-glucan and PG)的免疫刺激下,具有不同的诱导表达水平。综上,本研究的结果表明,EsTube在中华绒螯蟹免疫和发育方面具有重要的作用,具有免疫和发育的双重功能。3)中华绒螯蟹Dorsal基因的克隆与表达分析
Dorsal作为Toll信号通路中的重要组分,在先天性免疫应答的诱导和识别中具有重要作用。在本文中,我们在中华绒螯蟹中克隆了一个NF-κB样核转录因子—Dorsal,简称为EsDorsal。EsDorsal基因cDNA全长2493bp,开放阅读框2022bp,编码673个氨基酸。EsDosal蛋白主要包括一个由171个氨基酸组成的RHD结构域和一个由102个氨基酸组成的IPT结构域。聚类分析的结果显示,EsDorsal首先与其它无脊椎动物的Dorsals或者NF-κBs聚为一支,然后与脊椎动物的NF-κBs聚为一支。实时荧光定量PCR(qRT-PCR)的结果显示,EsDorsal在免疫相关组织中表达量较高,经过脂多糖(LPS)、肽聚糖(PG)和葡聚糖(GLU)免疫刺激后,表达量具有不同程度的变化。重要的是,相对于对于肽聚糖(PG)和葡聚糖(GLU), EsDorsal对脂多糖(LPS)的免疫刺激更加敏感。综上,EsDorsal对于不同的病原相关分子的刺激具有不同的可诱导性,对中华绒螯蟹先天性免疫应答机制的研究具有重要意义。
4)红螯光壳螯虾Crustin基因的克隆与功能分析
抗菌肽作为一种重要的免疫效应因子,对于红螯光壳螯虾(Cherax quadricarinatus)先天性免疫应答防御反应具有重要作用。本研究综合同源克隆和SMART-RACE技术,从红螯光壳螯虾的血细胞中成功克隆得到了一个重要的抗菌肽Crustin,简称为CqCrustin。CqCrustin基因cDNA全长为608bp,开放式阅读框为324bp,编码107个氨基酸,在其推测的氨基酸序列的N端含有一个由16个氨基酸残基组成的信号肽,C端含有一个由48个氨基酸残基组成的乳清酸蛋白(WAP)结构域。基于其WAP结构域的序列信息,进行聚类分析的结果显示,CqCrustin首先与其它甲壳动物的Ⅰ型Crustin聚为一支,然后与其它甲壳动物Ⅱ型Crustin聚为一支,最后于Ⅲ型Crustin聚为一支。多序列比对的结果显示,CqCrustin与克氏原螯虾(Procambarus clarkii) PcCrustin2具有较高的序列相似性(69%)。实时荧光定量PCR (qRT-PCR)的结果显示,CqCrustin主要表达在免疫相关组织,且在红螯光壳螯虾血细胞中的表达量最高；利用LPS,GLU和PG三种病原相关分子模式免疫刺激后,CqCrustin的mRNA表达水平在红螯光壳螯虾血细胞中具有不同程度的显著上调。为了进一步研究其生物学活性,利用原核表达技术获得的重组蛋白rCqCrustin对于不同的病原菌具有不同程度的结合活性,而且对不同的病原菌的生长速度亦具有不同程度的抑制作用。综上可见,CqCrustin在红螯光壳螯虾先天性免疫识别和防御过程中具有重要作用。
5)红螯光壳螯虾Dscam基因的克隆与功能分析
细胞粘附分子(Dscam)属于免疫球蛋白超级家族中的重要一员,在无脊椎动物,特别是节肢动物的先天性免疫识别和防御中具有重要作用。本研究综合同源克隆和SMART-RACE技术,从红螯光壳螯虾(Cherax quadricarinatus)的血细胞中成功克隆得到了其Dscam基因,简称为CqDscam。GqDscam蛋白含有类似于其它无脊椎动物的Dscam蛋白同系物的典型结构域,主要包括一个信号肽、10个免疫球蛋白结构域(Ig)、6个Ⅲ型纤连蛋白结构域(FNⅢ)、一个跨膜区和一个细胞质尾结构域。聚类分析的结果显示,CqDscam蛋白首先与其它甲壳动物的Dscam同系物聚为一支,然后与其它无脊椎动物的Dscam同系物聚为一支,最后与脊椎动物的Dscam同系物聚为一支。胞外结构域的检测结果表明,CqDsccam蛋白的N端具有多个可变剪切亚型,其中Ig2结构域具有30个亚型,Ig3结构域具有33个亚型,Ig7具有19个亚型,跨膜区具有2个亚型,理论上,总共可产生多达37620个不同的亚型。qRT-PCR的检测结果表明,CqDscam组成性表达在红螯螯虾各组织,特别是神经和免疫相关的组织：利用脂多糖(LPS)、肽聚糖(PG)和β-1,3-葡聚糖(GLU)免疫刺激红螯螯虾后,CqDscam的mRNA表达水平具有不同程度的诱导上调。CqDscam重组蛋白的功能检测显示,rCqDscam能够不同程度地识别不同的细菌,并且能够不同程度地抑制革兰氏阳性菌(金黄色葡萄球菌)和革兰氏阴性菌(大肠杆菌)的生长。此外,免疫荧光的结果显示,CqDscam主要分布于红螯螯虾血细胞的表面。综上可见,CqDscam在红螯螯虾的先天性免疫识别和防御中具有重要作用。
The Chinese mitten crab(Eriocheir sinensis) as one of the commercially important crab, also was known as the fresh water crab、hairy crab and so on, belongs to Athropoda, Malacostraca, Decapoda, Grapsidae, Eriocheir, Eriocheir sinensis. The Chinese mitten crab not only is prestigious food, but also has better nutritive value and raising value because of its rich-nutrition and delicious taste. The Red claw crayfish (Cherax quadricarinatus), that also is known as the Australian freshwater lobster or red claw crayfish, belongs to Crustacea, Decapoda, Parastacidae. Cherax quadricarinatus, that is native to Australia and introduced into China in1991for the first time. It is breeding successfully and widely cultured in the next year because of its fast growth, delicious taste, high yield and adaptability. With the development of the intensive aquaculture and the enlargement of the cultivation scale, the frequent outbreaks of diseases have caused decreased production and catastrophic losses in the past decade. Crustacean can induce rapid and effective immune responses to clear the intruding pathogens relying largely on innate immunity, which attracts more and more attention. Upon this backdrop, studying the structure and transcriptional responses of potential immune-related genes may facilitate a better understanding of the crustacean immune defense and recognition mechanisms and support the sustainable development of better disease management strategies in the crustacean farming industry.
The main research contents and results are as follows:
1) Two novel Toll genes(EsTolll and EsToll1) from Eriocheir sinensis are differentially induced by lipopolysaccharide, peptidoglycan and zymosan
Tolls/Toll-like receptors (TLRs) play an essential role in initiating innate immune responses against pathogens and are found throughout the insect kingdom but have not yet been reported in the crustacean, Eriocheir sinensis. For this purpose, we cloned two novel Toll genes from E. sinensis, EsTolll and EsToll2. The full-length cDNA of EsTolll was3,963bp with a3,042-bp open reading frame (ORF) encoding a1,013-amino acid protein. The extracellular domain of this protein contains17leucine-rich repeats (LRRs) and a139-residue cytoplasmic Toll/interleukin-1receptor (TIR) domain. The cDNA full-length of EsToll2was4,419bp with a2,667-bp ORF encoding an888-amino acid protein with an extracellular domain containing10LRRs and a139-residue cytoplasmic TIR domain. By phylogenetic analysis, EsTolll and EsTolll clustered into one group together with Tolls from other crustaceans. Quantitative RT-PCR analysis demonstrated that a) both EsTolll and EsToll2were constitutively expressed in all tested crab tissues; b) EsTolll and EsToll2were differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsToll2expression was significantly upregulated at almost all time intervals post-challenge with LPS, PG and GLU. Our study indicated that EsTolll and EsToll2are differentially inducibility in response to various PAMPs, suggesting their involvement in a specific innate immune recognition mechanism in E. sinensis.
2) Molecular cloning and expression analysis of Tube from Eriocheir sinensis
As a key component of the Toll signaling pathway, Tube plays central roles in many biological activities, such as survival, development and innate immunity. Tube has been found in shrimps, but have not yet been reported in the crustacean, Eriocheir sinensis. In this study, we cloned the full-length cDNA of the adaptor Tube for the first time from E sinensis and designated the gene as EsTube. The full-length cDNA of EsTube was2,247-bp with a1,539-bp open reading frame (ORF) encoding a512-amino acid protein. The protein contained a116-residue death domain (DD) at its N-terminus and a272-residue serine/threonine-protein kinase domain (S_TKc) at its C-terminus. Phylogenetic analysis clustered EsTube initially in one group with other invertebrate Tube and Tube-like proteins, and then with the vertebrate IRAK-4proteins, finally with other invertebrate Pelle proteins. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that EsTube was highly expressed in ovary and testis, and moderately expressed in thoracic ganglia and stomach. EsTube was expressed at all selected stages and was highly expressed in the spermatid stage (October, testis) and the stage III-2(November, ovary). EsTube was differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (β-1,3-glucan). Our study indicated that EsTube might possess multiple functions in immunity and development in E. sinensis.
3) Molecular cloning and expression analysis of a dorsal homologue from Eriocheir sinensis
Dorsal as a crucial component of Toll signaling pathway, played important roles in induction and regulation of innate immune responses. In this study, we cloned a NF-κB-like transcription factor Dorsal from Eriocheir sinensis and designated it as EsDorsal. The full-length cDNA of EsDorsal was2,493bp with a2,022-bp open reading frame (ORF) encoding a673-amino acid protein. This protein contained a171-residue conserved Rel homology domain (RHD) and a102-residue Ig-like, plexins and transcription factors domain (IPT). By phylogenetic analysis, EsDorsal was clustered into one group together with other invertebrate Dorsals or NF-κBs, and then clustered with vertebrate NF-κBs. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that a) EsDorsal had higher expression level in immune organs; b) EsDorsal differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsDorsal was more responsive to LPS than GLU and PG. Collectively, EsDorsal was differentially inducibility in response to various PAMPs, suggesting its involvement in a specific innate immune regulation in E. sinensis.
4) A novel Crustin involves in antibacterial responses in the red claw crayfish, Cherax quadricarinatus
Antimicrobial peptides are important immune effectors and play important roles in mediating innate immune responses against intruding pathogens. Here, we successfully isolated and characterized a novel Crustin from Red claw crayfish Cherax quadricarinatus and named it as CqCrustin. The deduced amino acid sequence of CqCrustin exhibited the typical configuration similar to other crustacean Crustin orthologs, including one signal peptide region at N-terminus between1and16and a long whey acidic protein domain (WAP domain) at C-terminus between60and107along with a WAP-type "four-disulfide core" motif. Phylogenetic analysis showed that CqCrustin was clustered with other crustacean Type Ⅰ Crustins firstly, and then with other crustacean Type Ⅱ Crustins, finally with other crustacean Type Ⅲ Crustins. CqCrustin showed higher sequence similarity (69%) with Crustin2from Procambarus clarkii (Pc-Crustin2). Transcription of CqCrustin-Ⅰ were both1) detected in all tissues, especially in hemocytes and gill; b) differentially induced in hemocytes post β-1,3-glucans (GLU), lipopolysaccharides (LPS) and peptidoglycans (PG) injection at selected time points. To understand its biological activity, the recombinant CqCrustin protein was constructed and expressed in Escherichia coli BL21(DE3). Recombinant protein rCqCrustin exhibited distinct bacterial binding activities against different Gram-positive bacteria, Gram-negative bacteria and fungus. Furthermore, bacterial growth inhibition assays demonstrated that rCqCrustin responds positively to the growth inhibition of different Gram-positive bacteria, Gram-negative bacteria and fungus. These findings suggested CqCrustin may involve in a specific innate immune recognition and defense mechanism against bacteria and fungus in Cherax quadricarinatus.
5) Identification and characterization of Dscam isoforms isolated from the red claw crayfish Cherax quadricarinatus
The Down syndrome cell adhesion molecule (Dscam) belongs to the immunoglobulin superfamily (IgSF) member and has been identified and isolated from some vertebrates and invertebrates. Recently, many studies have confirmed the important role of Dscam in mediating innate immune response against intruding pathogens and wiring of the nerve system in invertebrates, especially in Arthropod. Here, we successfully isolated and characterized the Dscam from Red claw crayfish Cherax quadricarinatus and named it as CqDscam. The deduced amino acid sequence of CqDscam exhibited the typical configuration similar to other invertebrate Dscam orthologs, including one signal peptide,10immunoglobulin (Ig) domains,6fibronectin type III (FNIII) domains and one transmembrane (TM) domain and cytoplasmic tail domain. Phylogenetic analysis showed that CqDscam was clustered with other invertebrate Dscams firstly, and then with vertebrate Dscams. In the extracellular region, the variable regions of CqDscam were located in N-terminal half of Ig2and Ig3domains and the complete Ig7domain. The CqDscam extracellular variants and transmembrane domain variants were produced by mutually exclusive alternative splicing events and could generate more than37,620different unique isoforms. Transcription of CqDscam were both1) detected in all tissues, especially in immune tissues and nerve tissues; b) differentially induced in hemocytes post glucans (GLU), lipopolysaccharides (LPS) and peptidoglycans (PG) injection at selected time points. Importantly, we had detected membrane-bound and secreted Dscam isoforms in C. quadricarinatus and showed that secreted isoforms were extensively transcribed post different PAMPs challenge, respectively. Results from immuno-localization assay revealed that CqDscam evenly distributed in the cell surface of hemocytes. In addition, the PAMPs specific isoforms of CqDscam were shown to be associated with bacterial clearance and phagocytosis in Red claw crayfish. These findings suggested CqDscam may involve in a specific innate immune recognition and defense mechanism in Cherax quadricarinatus.
引文
[1]李伟微.基于中华绒螯蟹EST序列的深度发掘—免疫和营养相关基因的克隆与表达研究[D].上海：华东师范大学,2011.
[2]李风铃.鱼类适应性免疫系统的早期发生以及Ikaros基因的克隆和表达[D].青岛：中国海洋大学,2009.
[3]ROWLEY A F, POWELL A. Invertebrate immune systems-specific, quasi-specific, or nonspecific?[J]. The Journal of Immunology,2007,179(11):7209-7214.
[4]严芳.甲壳动物血蓝蛋白免疫学活性及其作用机制研究[D].汕头:汕头大学,2011.
[5]JOHANSSON M W, KEYSER P, SRITUNYALUCKSANA K, et al. Crustacean haemocytes and haematopoiesis[J]. Aquaculture,2000,191(1):45-52.
[6]郭青松.中华绒螯蟹血液指标研究[D].武汉：华中农业大学,2009.
[7]郭慧芝.中华绒鳌蟹cDNA文库的构建及免疫相关基因的克隆与表达[D].武汉：华中农业大学,2010.
[8]JIRAVANICHPAISAL P, LUEL L B, KENNETH S. Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization[J]. Immunobiology,2006, 211(4):213-236.
[9]SMITH V J, RATCLIFFE N A. Cellular defense reactions of the shore crab, Carcinus maenas:in vivo hemocytic and histopathological responses to injected bacteria[J]. Journal of Invertebrate Pathology,1980,35(1):65-74.
[10]SMITH V J, RATCLIFFE N A. Host defence reactions of the shore crab, Carcinus maenas (L.):clearance and distribution of injected test particles[J]. Journal of the Marine Biological Association of the United Kingdom,1980,60(01):89-102.
[11]JOHANSSON M W, KENNETH S. Isolation and purification of a cell adhesion factor from crayfish blood cells[J]. The Journal of Cell Biology,1988,106(5):1795-1803.
[12]KOBAYASHIM, JOHANSSON M W, KENNETH S. The 76 kD cell-adhesion factor from crayfish haemocytes promotes encapsulation in vitro[J]. Cell and Tissue Research,1990, 260(1):13-18.
[13]KOBAYASHI M, KENNETH S. Comparison of concanavalin A reactive determinants on isolated haemocytes of parasite-infected and non-infected freshwater crayfish[J]. DIS Aquat ORG,1990,9(1):141-147.
[14]HANS-GILBERT H, ANNA A, SODERHALL K. Purification and characterization of a high-Mr proteinase inhibitor of pro-phenol oxidase activation from crayfish plasma[J]. Biochem. J,1987,248(1):223-228.
[15]SODERHALL K, SMITH V J. Separation of the haemocyte populations of Carcinusmaenas and other Marine decapods, and prophenoloxidase distribution[J]. Developmental & Comparative Immunology,1983,7(2):229-239.
[16]徐海圣,徐步进.甲壳动物细胞及体液免疫机理的研究进展[J].大连水产学院学报,2001,16(1)：49-56.
[17]邓欢,陈俅,刘卫东,等.中国对虾血细胞包掩作用的超微结构和组织化学观察[J].应用与环境生物学报,1999,5(3)：296-299.
[18]JOHANSSON M W, TORBJORN H, PER-OVE T, et al. A cell-surface superoxide dismutase is a binding protein for peroxinectin, a cell-adhesive peroxidase in crayfish[J]. Journal of Cell Science,1999,112(6):917-925.
[19]SRITUNYALUCKSANA K, WONGSUEBSANTATI K, JOHANSSON M W, et al. Peroxinectin, a cell adhesive protein associated with the proPO system from the black tiger shrimp, Penaeus monodon[J]. Developmental & Comparative Immunology,2001,25(5): 353-363.
[20]Liu C H, Cheng W, Kuo C M, et al. Molecular cloning and characterisation of a cell adhesion molecule, peroxinectin from the white shrimp Litopenaeus vannamei[J]. Fish & Shellfish Immunology,2004,17(1):13-26.
[21]Liu C H, Cheng W, Chen J C. The peroxinectin of white shrimp Litopenaeus vannamei is synthesised in the semi-granular and granular cells, and its transcription is up-regulated with Vibrio alginolyticus infection[J]. Fish & Shellfish Immunology,2005,18(5):431-444.
[22]HSU P, LIU C, TSENG D, et al. Molecular cloning and characterisation of peroxinectin, a cell adhesion molecule, from the giant freshwater prawn Macrobrachium rosenbergii[J]. Fish & Shellfish Immunology,2006,21(1):1-10.
[23]Liu C H, Yeh S P, Hsu P Y, et al. Peroxinectin gene transcription of the giant freshwater prawn Macrobrachium rosenbergii under intrinsic, immunostimulant, and chemotherapeutant influences[J]. Fish & Shellfish Immunology,2007,22(4):408-417.
[24]MARTIN G G, POOLE D, POOLE C, et al. Clearance of bacteria injected into the hemolymph of the penaeid shrimp, sicyonia ingentis[J]. Journal of Invertebrate Pathology, 1993,62(3):308-315.
[25]JOHANSSON M W, SODERHALL K. Cellular immunity in crustaceans and the proPO system[J]. Parasitology Today,1989,5(6):171-176.
[26]MARTIN G G, KAY J, POOLE D, et al. In vitro nodule formation in the ridgeback prawn, Sicyonia ingentis, and the American lobster, Homarus americanus[J]. Invertebrate Biology, 1998,117(2):155-168.
[27]MILLER J S, HOWARD R W, RANA R L, et al. Eicosanoids mediate nodulation reactions to bacterial infections in adults of the cricket, Gryllus assimilis[J]. Journal of Insect Physiology,1999,45(1):75-83.
[28]MILLER J S, HOWARD R W, NGUYEN T, et al. Eicosanoids mediate nodulation responses to bacterial infections in larvae of the tenebrionid Beetle, Zophobas atratus[J]. Journal of Insect Physiology,1996,42(1):3-12.
[29]董超华.克氏原螯虾两种模式识别受体基因的克隆、重组表达及功能分析[D].青岛：中国海洋大学,2009.
[30]杨军厚.克氏原螯虾C型凝集素的表达与功能研究[D].济南：山东大学,2011.
[31]LEONARD C, SODERHALL K, RATCLIFFE N A. Studies on prophenoloxidase and protease activity of Blaberus craniifer haemocytes[J]. Insect Biochemistry,1985,15(6): 803-810.
[32]王雷,李光友.中国对虾血淋巴中的抗菌,溶菌活力与酚氧化酶活力的测定及其特性研究[J].海洋与湖沼,1995,26(2)：179-185.
[33]KENNETH S, LENA H. Lipopolysaccharide-induced activation of prophenoloxidase activating system in crayfish haemocyte lysate[J]. Biochimica ET Biophysica ACTA (BBA)-General Subjects,1984,797(1):99-104.
[34]ASHIDA M, SODERHAL K. The prophenoloxidase activating system in crayfish[J]. Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1984,77(1): 21-26.
[35]孟凡伦,张玉臻.甲壳动物中的酚氧化酶原激活系统研究评价[J].海洋与湖沼,1999,30(1)：110-116.
[36]樊现远.中国卤虫(Artemia sinica)酚氧化酶的分离纯化及其生物化学性质和酶性质的研究[D].青岛：中国海洋大学,2011.
[37]PERAZZOLO L M, LORENZINI D M, DAFFRE S, et al. Purification and partial characterization of the plasma clotting protein from the pink shrimp Farfantepenaeus paulensis[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2005,142(3):302-307.
[38]杜欣军.中国明对虾先天免疫的模式识别与效应分子[D].济南：山东大学,2007.
[39]钱云霞,顾晓英.甲壳动物血液凝固的分子机制[J].生物技术通报,2011(06)：25-30.
[40]ZASLOFF M. Antimicrobial peptides of multicellular organisms[J]. Nature,2002,415(6870)：389-395.
[41]黄文树,王克坚,李少菁.甲壳动物抗菌肽研究进展[J].海洋科学,2005,29(2)：64-68.
[42]SCHNAPP D, KEMP G D, SMITH V J. Purification and characterization of a Proline-Rich antibacterial peptide, with sequence similarity to Bactenecin-7, from the haemocytes of the shore crab, carcinus maenas[J]. European Journal of Biochemistry,1996,240(3):532-539.
[43]CHISHOLM J R, SMITH V J. Comparison of antibacterial activity in the hemocytes of different crustacean species[J]. Comparative Biochemistry and Physiology Part a: Physiology,1995,110(1):39-45.
[44]NOGA E J, ARROLL T A, FAN Z. Specificity and some physicochemical characteristics of the antibacterial activity from blue crab Callinectes sapidus[J]. Fish & Shellfish Immunology,1996,6(6):403-412.
[45]EVANS E E, PAINTER B, EVANS M L, et al. An induced bactericidin in the spiny lobster, Panulirus Argus[C]//Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, NY),128,1968:394-398.
[46]EVANS E E, GUSHING J E, SAWYER S, et al. Induced bactericidal response in the California spiny lobster Panulirus interruptus[C]//Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (NewYork,NY),132,1969：111-114.
[47]STEWART J E, ZWICKER B M. Natural and induced bactericidal activities in the hemolymph of the lobster, Homarus americanus:products of hemocyte-plasma interaction[J]. Canadian Journal of Microbiology,1972,18(9):1499-1509.
[48]DESTOUMIEUX-GARZON D,SAULNIER D,GARNIER J.Crustacean immunityantifungal peptides are generated from the C terminus of shrimp hemocyanin in response to microbial challenge[J]. Journal of Biological Chemistry,2001,276(50):47070-47077.
[49]GROSS P, BARTLETT T, BROWDY C E. Immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopan creas in the Pacific White Shrimp, Litopenaeus vannamei, and the Atlantic White Shrimp, L. setiferus[J]. Developmental & Comparative Immunology,2001,25(7):565-577.
[50]ROJTINNAKORN J, HIRONO I, ITAMI T E. Gene expression in haemocytes of kuruma prawn, Penaeus japonicus, in response to infection with WSSV by EST approach[J]. Fish & Shellfish Immunology,2002,13(1):69-83.
[51]王秀英,邵庆均,黄磊.对虾抗菌肽Penaeidins的研究进展[J].华中农业大学学报,2003,22(06)：624-629.
[52]BARRACCO M A, LORGERIL J, GUEGUEN Y, et al. Molecular characterization of penaeidins from two Atlantic Brazilian shrimp species, Farfantepenaeus paulensis and Litopenaeus schmitti*[J]. FEMS Microbiology Letters,2005,250(1):117-120.
[53]BROCKTON V, SMITH V J. Crustin expression following bacterial injection and temperature change in the shore crab, Carcinus maenas[J]. Developmental & Comparative Immunology,2008,32(9):1027-1033.
[54]HAUTON C, BROCKTON V, SMITH V J. Cloning of a crustin-like, single whey-acidic-domain, antibacterial peptide from the haemocytes of the European lobster, Homarus gammarus, and its response to infection with bacteria[J]. Molecular Immunology, 2006,43(9):1490-1496.
[55]TAMURA H, TANAKA S, ODA T, et al. Purification and characterization of a (1→ 3)-β-d-glucan-binding protein from horseshoe crab (Tachypleus tridentatus) amoebocytes[J]. Carbohydrate Research,1996,295(1):103-116.
[56]Pan C Y, Chao T T, Chen J C, et al. Shrimp (penaeus monodon) anti-lipopolysaccharide factor reduces the lethality of pseudomonas aeruginosa sepsis in MICE[J]. International Immunopharmacology,2007,7(5):687-700.
[57]Li C H, Zhao J M, Song L S, et al. Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis[J]. Developmental & Comparative Immunology,2008,32(7):784-794.
[58]MIREILLE L, CAILLE A, LANDON C, et al. Solution structures of the antifungal heliomicin and a selected variant with both antibacterial and antifungal activities[J]. Biochemistry,2001,40(40):11995-12003.
[59]李红权,刘燕.虾类免疫机制的特点及其免疫增强剂的研究进展[J].畜牧兽医科技信息,2006(8)：87-89.
[60]HALL J L, ROWLANDS JR D T. Heterogeneity of lobster agglutinins. I. Purification and physicochemical characterization [J]. Biochemistry,1974,13(4):821-827.
[61]曹剑香,简纪常,吴灶和,等.凡纳滨对虾血清凝集素,溶血素的特性研究[J].海洋科学,2006,6(30)：1-5.
[62]PACE K E, BAUM L G. Insect galectins:roles in immunity and development[J]. Glycoconjugate Journal,2002,19(7-9):607-614.
[63]MOCK A, RENWRANTZ L. Isolation and characterization of a lectin from the cephalochordate Branchiostoma lanceolatum(pallas)[J]. Developmental & Comparative Immunology,1991,99(3):699-707.
[64]LOPEZ-CORTES L, CASTRO D, NAVAS J I, et al. Phagocytic and chemotactic responses of Manila and carpet shell clam haemocytes against Vibrio tapetis, the causative agent of brown ring disease[J]. Fish & Shellfish Immunology,1999,9(7):543-555.
[65]KAWABATA H, YANG R, HIRAMA T, et al. Molecular cloning of transferrin receptor 2 A new member of the transferrin receptor-like family[J]. Journal of Biological Chemistry, 1999,274(30):20826-20832.
[66]MARQUES M F, BARRACCO M A. Lectins, as non-self-recognition factors, in crustaceans[J]. Aquaculture,2000,191(1):23-44.
[67]RATANAPO S, CHULAVATNATOL M. Monodin-induced agglutination of Vibrio vulnificus, a major infective bacterium in black tiger prawn (Penaeus monodon)[J]. Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1992,102(4): 855-859.
[68]VARGAS-ALBORES F, MARIA-ANTONIA G, JOSE-LUIS O. An anticoagulant solution for haemolymph collection and prophenoloxidase studies of penaeid shrimp Penaeus californiensis[J]. Comparative Biochemistry and Physiology Part a:Physiology,1993, 106(2):299-303.
[69]LORENA V, FELIPE M, PATRICIA R, et al. Purification and characterization of a lectin from Macrobrachium rosenbergh(Crustacea, Decapoda) hemolymph[J]. Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1993,105(3):617-623.
[70]VAZQUEZ L, JARAMILLO L, LASCURAIN R, et al. Bacterial agglutination by the sialic acid specific serum lectin from Macrobrachium rosenbergii[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,1996,113(2):355-359.
[71]FRAGKIADAKIS G A, STRATAKIS E K. The lectin from the crustacean liocarcinus depurator recognizes O-acetylsialic acids[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,1997,117(4):545-552.
[72]MAHESWARI R, MULLAINADHAN P, ARUMUGAM M. Isolation and characterization of an acetyl group-recognizing agglutinin from the serum of the Indian white shrimp Fenneropenaeus indicus[J]. Archives of Biochemistry and Biophysics,2002,402(1):65-76.
[73]MAJUMDER M, CHATTOPADHYAY T, GUHA A K, et al. Inhibition of bacterial respiration by a low-molecular weight lectin, scyllin, from Scylla serrata crab hemolymph[J]. Indian Journal of Biochemistry & Biophysics,1996,34(1-2):87-89.
[74]LIN Y, VASEEHARAN B, CHEN J. Molecular cloning and phylogenetic analysis on a 2-macroglobulin (α2-M) of white shrimp Litopenaeus vannamei[J]. Developmental & Comparative Immunology,2008,32(4):317-329.
[75]RATTANACHAI A, HIRONO I, OHIRA T, et al. Molecular cloning and expression analysis of a2 macroglobulin in the kuruma shrimp, Marsupenaeus japonicus[J]. Fish& Shellfish Immunology,2004,16(5):599-611.
[76]HO P, CHENG C, CHENG W. Identification and cloning of the a2-macroglobulin of giant freshwater prawn Macrobrachium rosenbergii and its expression in relation with the molt stage and bacteria injection[J]. Fish & Shellfish Immunology,2009,26(3):459-466.
[77]Qin C J, Chen L Q, JIAN G Q, et al. Molecular cloning and characterization of a 2-macroglobulin (a2-M) from the haemocytes of Chinese mitten crab Eriocheir sinensis[J]. Fish & Shellfish Immunology,2010,29(2):195-203.
[78]牟海津,江晓路,刘树青,等.日本对虾溶血素的活性测定及性能研究[J].海洋与湖沼,1999,30(4)：362-367.
[79]赵大显,陈立侨.中华绒螯蟹免疫相关基因的研究进展[J].水产科学,2010,29(4)：243-248.
[80]LEE S Y, Wang R G, SODERHALL K. A Lipopolysaccharide-and beta-1, 3-Glucan-binding protein from hemocytes of the freshwater crayfish pacifastacus leniusculus purification, characterization, and CDNA cloning[J]. Journal of Biological Chemistry,2000,275(2):1337-1343.
[81]Cheng W, Liu C H, Tsai C H, et al. Molecular cloning and characterisation of a pattern recognition molecule, lipopolysaccharide-and β-1,3-glucan binding protein (LGBP) from the white shrimp Litopenaeus vannamei[J]. Fish & Shellfish Immunology,2005,18(4): 297-310.
[82]Zhao D X, Chen L Q, Qin C J, et al. Molecular cloning and characterization of the lipopolysaccharide and β-1,3-glucan binding protein in Chinese mitten crab(Eriocheir sinensis)[J]. COMP Biochem and Physiol B:Biochem Molbiol,2009,154(1):17-24.
[83]VARGAS-ALBORES F, JIMENEZ-VEGA F, YEPIZ-PLASCENCIA G M. Purification and comparison of β-1,3-Glucan binding protein from white shrimp (penaeus vannamei)[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology, 1997,116(4):453-458.
[84]Lai X F, Kong J, Wang Q Y, et al. Cloning and characterization of a (3-1,3-glucan-binding protein from shrimp Fenneropenaeus chinensis[J]. Molecular Biology Reports,2011,38(7): 4527-4535.
[85]曹慧,李宗芸,王秋香.果蝇先天性免疫研究进展[J].昆虫知识,2009,791(46)
[86]GOBERT V, GOTTAR M, MATSKEV1CH A A, et al. Dual activation of the Drosophila toll pathway by two pattern recognition receptors[J]. Science,2003,302(5653):2126-2130.
[87]CORBO J C, LEVINE M. Characterization of an immunodeficiency mutant in Drosophila[J]. Mechanisms of Development,1996,55(2):211-220.
[88]PERSSON M, CERENIUS L, SODERHALL K. The influence of haemocyte number on the resistance of the freshwater crayfish, Pacifastacus leniusculus Dana, to the parasitic fungus Aphanomyces astaci[J]. Journal of Fish Diseases,1987,10(6):471-477.
[89]SMITH V J, SODERHALL K. Induction of degranulation and lysis of haemocytes in the freshwater crayfish, Astacus astacus by components of the prophenoloxidase activating system in vitro[J]. Cell and Tissue Research,1983,233(2):295-303.
[90]WITTEVELDT J, VLAK J M, VAN HULTEN M C. Protection of penaeus monodon against white spot syndrome virus using a WSSV subunit vaccine[J]. Fish & Shellfish Immunology,2004,16(5):571-579.
[91]STUART L M, EZEKOWITZ R A. Phagocytosis and comparative innate immunity: learning on the fly[J]. Nature Reviews Immunology,2008,8(2):131-141.
[92]董迎辉.泥蚶高通量转录组分析及生长相关基因的克隆与表达研究[D].青岛：中国海洋大学,2012.
[93]杨昌健.中国明对虾Toll样受体基因的克隆和表达分析[D].青岛：中国科学院研究生院(海洋研究所),2008.
[94]Wang P H, Liang J P, Gu Z H, . Molecular cloning, characterization and expression analysis of two novel Tolls (LvToll2 and LvToll3) and three putative Spatzle-like Toll ligands (LvSpzl-3) from Litopenaeus vannamei[J/OL]. Developmental & Comparative Immunology,2012,36(2):359-371.
[95]Yang L S, Yin Z X, Liao J X, et al. A toll receptor in shrimp[J]. Molecular Immunology, 2007,44(8):1999-2008.
[96]Lin Z Y, Jie Q, Zhang Y L, et al. Cloning and characterisation of the SpToll gene from green mud crab, Scylla paramamosain[J]. Developmental & Comparative Immunology,2012, 37(1):164-175.
[97]张樱.三疣梭子蟹LGBP基因的克隆与表达研究[D].宁波：宁波大学,2011.
[98]HOFFMANN A, FUNKNER A, NEUMANN P,等. Biophysical characterization of refolded Drosophila Spatzle, a cystine knot protein, reveals distinct properties of three isoforms[J]. Journal of Biological Chemistry,2008,283(47):32598-32609.
[99]Shi X Z, Zhang R R, Jia Y P,等. Identification and molecular characterization of a Spatzle-like protein from Chinese shrimp (Fenneropenaeus chinensis)[J]. Fish & Shellfish Immunology,2009,27(5):610-617.
[100]朱磊.拟穴青蟹(Scylla paramamosain) Toll样信号途径关键基因及抗菌肽SpALF的克隆、表达与功能分析[D].雅安市：四川农业大学,2012.
[101]BRUNO L, JULES H. The host defense of Drosophila melanogaster[J]. Annual Review of Immunology,2007,25(1):697-743.
[102]Wang P H, Gu Z H, Wan D H, et al. The shrimp NF-κB pathway is activated by white spot syndrome virus (WSSV) 449 to facilitate the expression of WSSV069 (iel), WSSV303 and WSSV371[J]. PLOS ONE,2011,6(9):e24773.
[103]Li X C, Zhu L, Li L G, et al. A novel myeloid differentiation factor 88 homolog, SpMyD88, exhibiting SpToll-binding activity in the mud crab Scylla paramamosain[J]. Developmental & Comparative Immunology,2013,39(4):313-322.
[104]Wang P H, Wan D H, Gu Z H, et al. Litopenaeus vannamei tumor necrosis factor receptor-associated factor 6 (TRAF6) responds to Vibrio alginolyticus and white spot syndrome virus (WSSV) infection and activates antimicrobial peptide genes[J]. Developmental & Comparative Immunology,2011,35(1):105-114.
[105]Li F H, Wang D D, Li S H, et al. A dorsal homolog (fcdorsal) in the Chinese shrimp fenneropenaeus chinensis is responsive to both bacteria and WSSV challenge[J]. Developmental & Comparative Immunology,2010,34(8):874-883.
[106]Huang X D, Yin Z X, Jia X T, et al. Identification and functional study of a shrimp Dorsal homologue[J]. Developmental & Comparative Immunology,2010,34(2):107-113.
[107]LEMAITRE B, KROMER-METZGER E, MICHAUT L, et al. A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense[J]. Proceedings of the National Academy of Sciences,1995,92(21):9465-9469.
[108]Wang P H, Gu Z H, Huang X D, et al. An immune deficiency homolog from the white shrimp, Litopenaeus vannamei, activates antimicrobial peptide genes[J]. Molecular Immunology,2009,46(8):1897-1904.
[109]冯宁宁.虾类IMD基因的功能分析及抗病指标的筛选[D].青岛：中国科学院研究生院(海洋研究所),2013.
[110]LI F, YAN H, WANG D, et al. Identification of a novel relish homolog in Chinese shrimp Fenneropenaeus chinensis and its function in regulating the transcription of antimicrobial peptides[J]. Developmenta l& Comparative Immunology,2009,33(10):1093-1101.
[111]Huang X D, Yin Z X, Liao J X, et al. Identification and functional study of a shrimp Relish homologue[J]. Fish & Shellfish Immunology,2009,27(2):230-238.
[112]刘文静.斑节对虾TLR22及Relish基因的克隆与表达分析[D].上海：上海海洋大学,2012.
[113]Sun C, Xu W T, Zhang H W, et al. An anti-lipopolysaccharide factor from red swamp crayfish, Procambarus clarkii, exhibited antimicrobial activities in vitro and in vivo[J]. Fish & Shellfish Immunology,2011,30(1):295-303.
[114]THARNTADA S, PONPRATEEP S, SOMBOONWIWAT K, et al. Role of anti-lipopolysaccharide factor from the black tiger shrimp, Penaeus monodon, in protection from white spot syndrome virus infection[J]. Journal of General Virology,2009,90(6): 1491-1498.
[115]SOMBOONWIWAT K, MARCOS M, TASSANAKAJON A, et al. Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor (ALF) from the black tiger shrimp Penaeus monodon[J]. Developmental & Comparative Immunology,2005, 29(10):841-851.
[116]YEDERY R D, REDDY K R. Identification, cloning, characterization and recombinant expression of an anti-lipopolysaccharide factor from the hemocytes of Indian mud crab, Scylla serrata[J]. Fish & Shellfish Immunology,2009,27(2):275-284.
[117]CUTHBERTSON B J, SHEPARD E F, CHAPMAN R W, et al. Diversity of the penaeidin antimicrobial peptides in two shrimp species[J]. Immunogenetics,2002,54(6):442-445.
[118]Kang C J, Wang J X, Zhao X F, et al. Molecular cloning and expression analysis of Ch-penaeidin, an antimicrobial peptide from Chinese shrimp, Fenneropenaeus chinensis[J]. Fish & Shellfish Immunology,2004,16(4):513-525.
[119]Li L, Wang J X, Zhao X F, et al. High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris[J]. Protein Expression and Purification,2005,39(2):144-151.
[120]Kang C J, Xue J F, Liu N, et al. Characterization and expression of a new subfamily member of penaeidin antimicrobial peptides (penaeidin 5) from Fenneropenaeus chinensis[J]. Molecular Immunology,2007,44(7):1535-1543.
[121]BLANCO-PORTALES R, LOPEZ-RAEZ J A, BELLIDO M L, et al. A strawberry fruit-specific and ripening-related gene codes for a HyPRP protein involved in polyphenol anchoring[J]. Plant Molecular Biology,2004,55(6):763-780.
[122]RELF J M, CHISHOLM J R, KEMP G D, et al. Purification and characterization of a cysteine-rich 11.5-kDa antibacterial protein from the granular haemocytes of the shore crab, Carcinus maenas[J]. European Journal of Biochemistry,1999,264(2):350-357.
[123]STOSS T D, NICKELL M D, HARDIN D, et al. Inducible transcript expressed by reactive epithelial cells at sites of olfactory sensory neuron proliferation[J]. Journal of Neurobiology, 2004,58(3):355-368.
[124]RATTANACHAI A, HIRONO I, OHIRA T, et al. Cloning of kuruma prawn Marsupenaeus japonicus crustin-like peptide cDNA and analysis of its expression[J]. Fisheries Science, 2004,70(5):765-771.
[125]SUPUNGUL P, TANG S, MANEERUTTANARUNGROJ C, et al. Cloning, expression and antimicrobial activity of crustin Pm 1, a major isoform of crustin, from the black tiger shrimp Penaeus monodon[J]. Developmental & Comparative Immunology,2008,32(1): 61-70.
[126]JIRAVANICHPAISAL P, LEE S Y, KIM Y, et al. Antibacterial peptides in hemocytes and hematopoietic tissue from freshwater crayfish Pacifastacus leniusculus:Characterization and expression pattern[J]. Developmental & Comparative Immunology,2007,31(5): 441-455.
[127]OKUMURA T. Effects of lipopolysaccharide on gene expression of antimicrobial peptides (penaeidins and crustin), serine proteinase and prophenoloxidase in haemocytes of the Pacific white shrimp, Litopenaeus vannamei[J]. Fish & Shellfish Immunology,2007,22(1): 68-76.
[128]BRITES D, MCTAGGART S, MORRIS K, et al. The dscam homologue of the crustacean daphnia is diversified by alternative splicing like in insects[J]. Molecular Biology and Evolution,2008,25(7):1429-1439.
[129]WATTHANASUROROT A, JIRAVANICHPAISAL P, LIU H, et al. Bacteria-induced Dscam isoforms of the crustacean, Pacifastacus leniusculus[J]. PLOS Pathogens,2011,7(6): e1002062.
[130]Chou P H, Chang H S, Chen I T, et al. Penaeus monodon Dscam (PmDscam) has a highly diverse cytoplasmic tail and is the first membrane-bound shrimp Dscam to be reported[J]. Fish & Shellfish Immunology,2011,30(4-5):1109-1123.
[131]Jin X K, Li W W, Wu M H, et al. Immunoglobulin superfamily protein Dscam exhibited molecular diversity by alternative splicing in hemocytes of crustacean,Eriocheir sinensis[J]. Fish & Shellfish Immunology,2013,35(3):900-909.
[132]Wang J J, WANG L L, Gao Y, et al. A tailless Dscam from Eriocheir sinensis diversified by alternative splicing[J]. Fish & Shellfish Immunology,2013,35(2):249-261.
[133]Liu Y C, Li F H, Dong B, et al. Molecular cloning, characterization and expression analysis of a putative C-type lectin (Fclectin) gene in Chinese shrimp Fenneropenaeus chinensis[J]. Molecular Immunology,2007,44(4):598-607.
[134]Sun Y D, Fu L D, Jia Y P, et al. A hepatopancreas-specific C-type lectin from the Chinese shrimp Fenneropenaeus chinensis exhibits antimicrobial activity [J]. Molecular Immunology, 2008,45(2):348-361.
[135]Zhang X W, Xu W T, Wang X W, et al. A novel C-type lectin with two CRD domains from Chinese shrimp Fenneropenaeus chinensis functions as a pattern recognition protein[J]. Molecular Immunology,2009,46(8):1626-1637.
[136]Wang X W, Xu W T, Zhang X W, et al. A C-type lectin is involved in the innate immune response of Chinese white shrimp[J]. Fish & Shellfish Immunology,2009,27(4):556-562.
[137]XU W, WANG X, ZHANG X, et al. A new C-type lectin (FcLec5) from the Chinese white shrimp Fenneropenaeus chinensis[J], Amino Acids,2010,39(5):1227-1239.
[138]Li F H, Xiang J H. Recent advances in researches on the innate immunity of shrimp in China[J]. Developmental & Comparative Immunology,2013,39(1):11-26.
[139]Jin X K, Li S, Guo X N, et al. Two antibacterial C-type lectins from crustacean, Eriocheir sinensis, stimulated cellular encapsulation in vitro[J]. Developmental & Comparative Immunology,2013,41(4):544-552.
[140]任乾.中国明对虾丝氨酸蛋白酶、活性氧的产生与消除及其半胱氨酸蛋白酶研究[D].济南：山东大学,2011.
[141]Ren Q, Xu Z L, Wang X W, et al. Clip domain serine protease and its homolog respond to Vibrio challenge in Chinese white shrimp, Fenneropenaeus chinensis[J]. Fish & Shellfish Immunology,2009,26(5):787-798.
[142]Liu Y C, Li F H, Wang B, et al. A serpin from Chinese shrimp Fenneropenaeus chinensis is responsive to bacteria and WSSV challenge[J]. Fish & Shellfish Immunology,2009,26(3): 345-351.
[143]刘树青,江晓路.免疫多糖对中国对虾血清溶菌酶,磷酸酶和过氧化物酶的作用[J].海洋与湖沼,1999,30(3)：278-283.
[144]魏克强,许梓荣.对虾的免疫机制及其疾病预防策略的研究[J].中国兽药杂志,2004,38(9)：25-29.
[145]杨志彪,赵云龙,周忠良,等.水体铜对中华绒螯蟹(Eriocheir sinensis)代谢酶活力的影响[J].海洋与湖沼,2006,37(2)：118-124.
[146]陈昌福,姚娟,陈超然,等.利用免疫刺激剂预防虾,蟹的传染性疾病[J].淡水渔业,2004,34(2)：55-57.
[147]管晓娟.甲壳动物体液免疫相关酶及免疫因子研究概况[J].生命科学仪器,2009(6)：3-7.
[148]宋林生,苏建国,崔朝霞,等.中华绒螯蟹(Eriocheir sinensis)幼蟹上岸病的免疫学研究[J].海洋与湖沼,2002,33(6)：657-662.
[149]王宜艳,孙虎山,李光友.复合免疫药物对中国对虾血淋巴氧化酶和抗氧化酶活力的影响[J].海洋科学进展,2002,20(3)：79-83.
[150]Wang D L, Zuo D, Wang L M, et al. Effects of white spot syndrome virus infection on immuno-enzyme activities and ultrastructure in gills of Cherax quadricarinatus[J]. Fish & Shellfish Immunology,2012,32(5):645-650.
[151]UEMATSU S, AKIRA S. Toll-Like receptors (tlrs) and their ligands[G]. Handbook of Experimental Pharmacology. [S.l.]:Springer Berlin Heidelberg,2008:1-20.
[152]CARTY M, BOWIE A G. Recent insights into the role of Toll-like receptors in viral infection[J]. Clinical and Experimental Immunology,2010,161(3):397-406.
[153]KAWAI T, AKIRA S. The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors[J]. Nature Immunology,2010,11(5):373-384.
[154]IMLER J, ZHENG L. Biology of toll receptors:lessons from insects and mammals[J]. Journal of Leukocyte Biology,2004,75(1):18-26.
[155]IMLER J, HOFFMANN J A. Toll receptors in innate immunity[J]. Trends in Cell Biology, 2001,11(7):304-311.
[156]BARTON G M, MEDZHITOV R. Toll-like receptors and their ligands,270[M]. [S.l.]: Berlin Heidelberg:Springer-Verlag,2002:81-92.
[157]BARTON G M, MEDZHITOV R. Toll-like receptor signalling[J]. Nature Reviews Immunology,2004,4(7):499-511.
[158]MULINARI S, HACKER U, CASTILLEJO-LOPEZ C. Expression and regulation of Spatzle-processing enzyme in Drosophila[J]. FEBS Letters,2006,580(22):5406-5410.
[159]TAUSZIG S, JOUANGUY E, HOFFMANN J A, et al. Toll-related receptors and the control of antimicrobial peptide expression in Drosophila[J]. Proceedings of the National Academy of Sciences,2000,97(19):10520-10525.
[160]IMLER J, HOFFMANN J A. Signaling mechanisms in the antimicrobial host defense of Drosophila[J]. Current Opinion in Microbiology,2000,3(1):16-22.
[161]Wang P H, Gu Z H, Huang X D, et al. An immune deficiency homolog from the white shrimp, Litopenaeus vannamei[J]. Molecular Immunology,2009,46(8-9):1897-1904.
[162]Ying X P, Yang W X, Zhang Y P. Comparative studies on fatty acid composition of the ovaries and hepatopancreas at different physiological stages of the Chinese mitten crab[J]. Aquaculture,2006,256(1-4):617-623.
[163]Gai Y C, Qiu L M, Wang L L, et al. A clip domain serine protease (cSP) from the Chinese mitten crab Eriocheir sinensis:cDNA characterization and mRNA expression[J]. Fish & Shellfish Immunology,2009,27(6):670-677.
[164]GAI, YUNCHAO, WANG, LINGLING, ZHAO, JIANMIN, et al. The construction of a cDNA library enriched for immune genes and the analysis of 7535 ESTs from Chinese mitten crab[J]. Fish & Shellfish Immunology,2009,27(6):684-694.
[165]ZHANG, HAO, CHEN, LIQ1AO, QIN, JIANGUANG, et al. Molecular cloning, characterization and expression of a C-type lectin cDNA in Chinese mitten crab,[J]. Fish & Shellfish Immunology,2011,31(2):358-363.
[166]JIN, XING-KUN, LI, WEI-WEI, HE, LIN, et al. Molecular cloning, characterization and expression analysis of two apoptosis genes, caspase and nm23, involved in the antibacterial response in Chinese mitten crab,[J]. Fish & Shellfish Immunology,2011,30(1):263-272.
[167]MU, CHANGKAO, ZHENG, PEILIN, ZHAO, JIANMIN, et al. Molecular characterization and expression of a crustin-like gene from Chinese mitten crab,[J]. Developmental & Comparative Immunology,2010,34(7):734-740.
[168]JIANG, HUI, CAI, YI-MEI, CHEN, LI-QIAO, et al. Functional annotation and analysis of expressed sequence TAGS from the hepatopancreas of mitten CRAB ([J]. Marine Biotechnology,2009,11(3):317-326.
[169]ZHAO D X, SONG S H, WANG Q, et al. Discovery of immune-related genes in Chinese mitten crab (Eriocheir sinensis) by expressed sequence tag analysis of haemocytes[J]. Aquaculture,2009,287(3-4):297-303.
[170]GUO, H. Z., ZOU, P. F., FU, J. P., et al. Characterization of two C-type lectin-like domain (CTLD)-containing proteins from the cDNA library of Chinese mitten crab[J]. Fish & Shellfish Immunology,2011,30(2):515-524.
[171]QIN, CHUANJIE, CHEN, LIQIAO, QIN, JIAN G., et al. Characterization of a serine proteinase homologous (SPH) in Chinese mitten crab[J]. Developmental & Comparative Immunology,2010,34(1):14-18.
[172]Jiang H, Yin Y X, Zhang X W, et al. Chasing relationships between nutrition and reproduction:A comparative transcriptome analysis of hepatopancreas and testis from Eriocheir sinensis[J]. Comparative Biochemistry and Physiology Part D:Genomics and Proteomics,2009,4(3):227-234.
[173]ZHANG, WEI, WAN, HAOLEI, JIANG, HUI, et al. A transcriptome analysis of mitten crab testes (Eriocheir sinensis)[J]. Genetics and Molecular Biology,2011,34(1):136-141.
[174]He L, Wang Q, Jin X K, et al. Transcriptome profiling of testis during sexual maturation stages in Eriocheir sinensis using Illumina sequencing[J]. PLOS ONE,2012,7(3):e33735.
[175]MEKATA T, KONO T, YOSHIDA T, et al. Identification of cDNA encoding Toll receptor, MjToll gene from kuruma shrimp, Marsupenaeus japonicus[J]. Fish & Shellfish Immunology,2008,24(1):122-133.
[176]ASSAVALAPSAKUL, W, PANYIM, S. Molecular cloning and tissue distribution of the Toll receptor in the black tiger shrimp, Penaeus monodon[J]. Genetics and Molecular Research,2012,11(1):484-493.
[177]Yang C J, Zhang J Q, Li F H, et al. A toll receptor from Chinese shrimp fenneropenaeus chinensis is responsive to vibrio anguillarum infection[J]. Fish & Shellfish Immunology, 2008,24(5):564-574.
[178]GAY N J, GANGLOFF M. Structure and function of toll receptors and their ligands[J]. Annual Review of Biochemistry,2007,76(1):141-165.
[179]LEULIER, FRANCOIS, LEMAITRE, BRUNO. Toll-like receptors [mdash] taking an evolutionary approach[J]. Nature Reviews. Genetics,2008,9(3):165-178.
[180]SCHNEIDER, DAVID S, HUDSON, KATHY L, LIN, TING-YI, et al. Dominant and recessive mutations define functional domains of Toll, a transmembrane protein required for dorsal-ventral polarity in the Drosophila embryo[J]. Genes and Development,1991,5(5): 797-807.
[181]QURESHI S T, LARIVIERE L, LEVEQUE G, et al. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4)[J]. The Journal of Experimental Medicine,1999, 189(4):615-625.
[182]TAKEUCHI O, HOSHINO K, KAWAI T, et al. Differential roles of TLR2 and TLR4 in recognition of Gram-Negative and Gram-Positive bacterial cell wall components[J]. Immunity,1999,11(4):443-451.
[183]RAMOS, HUGO CRUZ, RUMBO, MARTIN, SIRARD, JEAN-CLAUDE. Bacterial flagellins:mediators of pathogenicity and host immune responses in mucosa[J]. Trends in Microbiology,2004,12(11):509-517.
[184]HIROAKI H, OSAMU T, KAWAI T, et al. A Toll-like receptor recognizes bacterial DNA[J]. Nature,2000,408(6813):740-845.
[185]Zhang L L, Li L, Zhang G F. A crassostrea gigas Toll-like receptor and comparative analysis of TLR pathway in invertebrates[J]. Fish & Shellfish Immunology,2011,30(2):653-660.
[186]SODERHALL K, CERENIUS L. Crustacean immunity[J]. Annual Review of Fish Diseases, 1992,2(0):3-23.
[187]QIU L, SONG L, XU W, et al. Molecular cloning and expression of a Toll receptor gene homologue from Zhikong Scallop, Chlamys farreri[J]. Fish & Shellfish Immunology,2007, 22(5):451-466.
[188]OOI, JAMES Y., YAGI, YOSHIMASA, HU, XIAODI, et al. The drosophila Toll-9 activates a constitutive antimicrobial defense[J]. EMBO Reports,2002,3(1):82-87.
[189]BISCHOFF V, VIGNAL C, BONECA I G, et al. Function of the drosophila pattern-recognition receptor PGRP-SD in the detection of Gram-positive bacteria[J]. Nature Immunology,2004,5(11):1175-1180.
[190]ROYET J. Infectious non-self recognition in invertebrates:lessons from Drosophila and other insect models[J]. Molecular Immunology,2004,41(11):1063-1075.
[191]Wang P H, Gu Z H, Huang X D, et al. An immune deficiency homolog from the white shrimp, Litopenaeus vannamei, activates antimicrobial peptide genes[J]. Molecular Immunology,2009,46(8-9):1897-1904.
[192]TOWB P, SUN H, WASSERMAN S A. Tube is an IRAK-4 homolog in a toll pathway adapted for development and immunity[J]. Journal of Innate Immunity,2009,1 (4):309-321.
[193]GOSU V, BASITH S, DURAIP, et al. Molecular evolution and structural features of IRAK family members[J]. PLOS ONE,2012,7(11):e49771.
[194]Li C Z, Chen Y X, Weng S P, et al. Presence of tube isoforms in litopenaeus vannamei suggests various regulatory patterns of signal transduction in invertebrate NF-κB pathway[J]. Developmental & Comparative Immunology,2014,42(2):174-185.
[195]BRUNO L, EMMANUELLE N, LYDIA M,等. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antirungal response in drosophila adults[J]. Cell, 1996,86(6):973-983.
[196]SWANTEK J L, TSEN M F, COBB M H, et al. IL-1 receptor-associated kinase modulates host responsiveness to endotoxin[J]. The Journal of Immunology,2000,164(8):4301-4306.
[197]TAKEDA K, AKIRA S. Toll-like receptors in innate immunity[J]. International Immunology,2005,17(1):1-14.
[198]SUZUKI N, SUZUKI S, DUNCAN G S, et al. Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4[J]. Nature,2002,416(6882):750-756.
[199]Li X C, Zhang X W, Zhou J F, et al. Identification, characterization, and functional analysis of tube and pelle homologs in the mud CRAB scylla paramamosain[J]. PLOS ONE,2013, 8(10):e76728.
[200]ANDERSON K V. Toll signaling pathways in the innate immune response[J]. Current Opinion in Immunology,2000,12(1):13-19.
[201]LI F, WANG L, ZHANG H, et al. Molecular cloning and expression of a Relish gene in Chinese mitten crab Eriocheir sinensis[J]. International Journal of Immunogenetics,2010, 37(6):499-508.
[202]Yu A Q, Jin X K, Shuang L, et al. Molecular cloning and expression analysis of a dorsal homologue from Eriocheir sinensis[J]. Developmental & Comparative Immunology,2013, 41(4):723-727.
[203]Yu A Q, Jin X K, Guo X N, et al. Two novel Toll genes (EsToll1 and EsToll2) from Eriocheir sinensis are differentially induced by lipopolysaccharide, peptidoglycan and zymosan[J]. Fish & Shellfish Immunology,2013,35(4):1282-1292.
[204]WATTHANASUROROT A, SODERHALL K, JIRAVANICHPAISAL P. A mammalian like interleukin-1 receptor-associated kinase 4 (IRAK-4), a TIR signaling mediator in intestinal innate immunity of black tiger shrimp (Penaeus monodon)[J]. Biochemical and Biophysical Research Communications,2012,417(1):623-629.
[205]MULLER-HOLTKAMP F, KNIPPLE D C, SEIFERT E, et al. An early role of maternal mRNA in establishing the dorsoventral pattern in pelle mutant Drosophila embryos[J]. Developmental Biology,1985,110(1):238-246.
[206]Ge H, Wang G D, ZHANG L L, et al. Molecular cloning and expression of interleukin-1 receptor-associated kinase 4, an important mediator of Toll-like receptor signal pathway, from small abalone Haliotis diversicolor[J]. Fish & Shellfish Immunology,2011,30(4): 1138-1146.
[207]PHELAN P E, MELLON M T, KIM C H. Functional characterization of full-length TLR3, IRAK-4, and TRAF6 in zebrafish (Danio rerio)[J]. Molecular Immunology,2005,42(9): 1057-1071.
[208]NISHIMURA M, NAITO S. Tissue-specific mRNA expression profiles of human toll-like receptors and related genes[J]. BIOL Pharm Bull,2005,28(5):886-892.
[209]Yu Y, Zhong Q W, Li C M, et al. Identification and characterization of IL-1 receptor-associated kinase-4 (IRAK-4) in half-smooth tongue sole Cynoglossus semilaevis[J]. Fish & Shellfish Immunology,2012,32(4):609-615.
[210]VOLLMER W, BLANOT D, DE PEDRO M A. Peptidoglycan structure and architecture[J]. FEMS Microbiology Reviews,2008,32(2):149-167.
[211]CHETTRI J K, RAIDA M K, HOLTEN-ANDERSEN L, et al. PAMP induced expression of immune relevant genes in head kidney leukocytes of rainbow trout (Oncorhynchus mykiss)[J]. Developmental & Comparative Immunology,2011,35(4):476-482.
[212]KRAVCHENKO V V, KAUFMANN G F. Bacterial inhibition of inflammatory responses via TLR-independent mechanisms[J]. Cellular Microbiology,2013,15(4):527-536.
[213]Yu H H, Xiao Z Y, Yong X C, et al. Effects of pH, temperature, and osmolarity on the morphology and survival rate of primary hemocyte cultures from the Mitten Crab, Eriocheir sinensis[J]. In Vitro Cellular and Developmental Biology-Animal,2013,49(9):716-727.
[214]Wu S H, Chen Y J, Huang S Y, et al. Demonstration of expression of a neuropeptide-encoding gene in crustacean hemocytes[J]. Comparative Biochemistry and Physiology Part a:Molecular and Integrative Physiology,2012,161(4):463-468.
[215]DE GREGORIO E, SPELLMAN P T, TZOU P, et al. The toll and IMD pathways are the major regulators of the immune response in drosophila[J]. The EMBO Journal,2002,21(11): 2568-2579.
[216]MATEO D R, GREENWOOD S J, ARAYA M T, et al. Differential gene expression of y-actin, Toll-like receptor 2 (TLR-2) and interleukin-1 receptor-associated kinase 4 (IRAK-4) in Mya arenaria haemocytes induced by in vivo infections with two Vibrio splendidus strains[J]. Developmental & Comparative Immunology,2010,34(7):710-714.
[217]DIXIT V, MAK T W. NF-κB signaling:many roads Lead to Madrid[J]. Cell,2002,111(5): 615-619.
[218]GHOSH S, MAY M J, KOPP E B. NF-κB and Rel proteins:evolutionary conserved mediators of immune responses[J]. Annual Review of Immunology,1998,16(1):225-260.
[219]HAYDEN M S, GHOSH S. Shared principles in NF-κB signaling[J]. Cell,2008,132(3): 344-362.
[220]BAEUERLE P A, HENKEL T. Function and activation of NF-kappaB in the immune system[J]. Annual Review of Immunology,1994,12(1):141-179.
[221]DUSHAY M S, ASLING B, HULTMARK D. Origins of immunity:Relish, a compound Rel-like gene in the antibacterial defense of Drosophila[J]. Proceedings of the National Academy of Sciences,1996,93(19):10343-10347.
[222]REICHHART J M, GEORGEL P, MEISTER M, et al. Expression and nuclear translocation of the rel/NF-kappa B-related morphogen dorsal during the immune response of Drosophila[J]. Comptes Rendus DE iacademie DES Sciences. Serie Iii, Sciences DE LA VIE,1993,316(10):1218-1224.
[223]STEWARD R. Dorsal, an embryonic polarity gene in Drosophila, is homologous to the vertebrate proto-oncogene, c-rel[J]. Science,1987,238(4827):692-694.
[224]STEWARD R, MCNALLY F J, SCHEDL P. Isolation of the dorsal locus of Drosophila[J]. Nature,1984,311(1):262-265.
[225]IMLER, JEAN-LUC, HOFFMANN, JULES A.. Signaling mechanisms in the antimicrobial host defense of Drosophila[J]. Current Opinion in Microbiology,2000,3(1):16-22.
[226]HOFFMANN J A, REICHHART J. Drosophila innate immunity:an evolutionary perspective[J]. Nature Immunology,2002,3(2):121-126.
[227]Zhang J Q, Li F H, Wang Z Z, et al. Cloning and recombinant expression of a crustin-like gene from Chinese shrimp, Fenneropenaeus chinensis[J]. Journal of Biotechnology,2007, 127(4):605-614.
[228]王丹丽,左迪,王兰梅,等.红螯光壳螯虾酚氧化酶原基因的克隆与表达[J].水产学报,2013,37(001)：43-54.
[229]ROSE T M, HENIKOFF J G, HENIKOFF S. CODEHOP (COnsensus-DEgenerate hybrid oligonucleotide primer) PCR primer design[J]. Nucleic Acids Research,2003,31(13): 3763-3766.
[230]申望,叶茂, 石戈,et al.三疣梭子蟹 (Portunus trituberculatus) I 型 Crustin[J].海洋 与湖沼,2010,41(3)：371-377.
[231]AMPARYUP P, KONDO H, HIRONO I, et al. Molecular cloning, genomic organization and recombinant expression of a crustin-like antimicrobial peptide from black tiger shrimp Penaeus monodon[J]. Molecular Immunology,2008,45(4):1085-1093.
[232]Chen D D, He N H, Xu X. Mj-DWD, a double WAP domain-containing protein with antiviral relevance in Marsupenaeus japonicus[J]. Fish & Shellfish Immunology,2008, 25(6):775-781.
[233]Sun C, Du X J, Xu W T, et al. Molecular cloning and characterization of three crustins from the Chinese white shrimp, Fenneropenaeus chinensis[J]. Fish & Shellfish Immunology, 2010,28(4):517-524.
[234]MU C, ZHENG P, ZHAO J, et al. Molecular characterization and expression of a crustin-like gene from Chinese mitten crab,Eriocheir sinensis[J]. Developmental & Comparative Immunology,2010,34(7):734-740.
[235]MU C, ZHENG P, ZHAO J, et al. A novel type Ⅲ crustin (CrusEs2) identified from Chinese mitten crab Eriocheir sinensis[J]. Fish & Shellfish Immunology,2011,31(1):142-147.
[236]AROCKIARAJ J, GNANAM A J, MUTHUKRISHNAN D, et al. Crustin, a WAP domain containing antimicrobial peptide from freshwater prawn Macrobrachium rosenbergii: Immune characterization[J]. Fish & Shellfish Immunology,2013,34(1):109-118.
[237]LITTLE T J, O'CONNOR B, COLEGRAVE N, et al. Maternal transfer of strain-specific immunity in an invertebrate [J]. Current Biology,2003,13(6):489-492.
[238]SADD B M, SCHMID-HEMPEL P. Insect immunity shows specificity in protection upon secondary pathogen exposure[J]. Current Biology,2006,16(12):1206-1210.
[239]PHAM L N, DIONNE M S, SHIRASU-HIZA M, et al. A specific primed immune response in Drosophila is dependent on phagocytes[J]. PLOS Pathogens,2007,3(3):e26.
[240]LEONARD P M, ADEMA C M, ZHANG S, et al. Structure of two FREP genes that combine IgSF and fibrinogen domains, with comments on diversity of the FREP gene family in the snail Biomphalaria glabrata[J]. Gene,2001,269(1):155-165.
[241]Yue M D, TAYLOR E H, DIMOPOULOS G. AgDscam, a hypervariable immunoglobulin domain-containing receptor of the Anopheles gambiae innate immune system[J]. PLOS Biology,2006,4(7):e229.
[242]TERWILLIGER D P, BUCKLEY K M, BROCKTON V, et al. Distinctive expression patterns of 185/333 genes in the purple sea urchin, Strongylocentrotus purpuratus:an unexpectedly diverse family of transcripts in response to LPS,β-1,3-glucan, and dsRNA[J]. BMC Molecular Biology,2007,8(1):16.
[243]YAMAKAWA K, HUO Y, HAENDEL M A, et al. DSCAM:a novel member of the immunoglobulin superfamily maps in a Down syndrome region and is involved in the development of the nervous system[J]. Human Molecular Genetics,1998,7(2):227-237.
[244]SCHMUCKER D, CLEMENS J C, SHU H, et al. Drosophila dscam is an AXON guidance receptor exhibiting extraordinary molecular diversity[J]. Cell,2000,101(6):671-684.
[245]WATSON F L, PUTTMANN-HOLGADO R, THOMAS F, et al. Extensive diversity of Ig-superfamily proteins in the immune system of insects[J]. Science,2005,309(5742): 1874-1878.
[246]Chou P H, Chang H S, Chen I T, et al. The putative invertebrate adaptive immune protein Litopenaeus vannamei Dscam (LvDscam) is the first reported Dscam to lack a transmembrane domain and cytoplasmic tail[J]. Developmental & Comparative Immunology,2009,33(12):1258-1267.
[247]WATSON F L, PUTTMANN-HOLGADO R, THOMAS F, et al. Extensive diversity of Ig-Superfamily proteins in the immune system of insects[J]. Science,2005,309(5742): 1874-1878.
[248]DONG Y, CIRIMOTICH C M, PIKE A, et al. Anopheles NF-κB-Regulated splicing factors direct Pathogen-Specific repertoires of the hypervariable pattern recognition receptor agdscam[J]. Cell Host & Microbe,2012,12(4):521-530.
[2]李风铃.鱼类适应性免疫系统的早期发生以及Ikaros基因的克隆和表达[D].青岛：中国海洋大学,2009.
[3]ROWLEY A F, POWELL A. Invertebrate immune systems-specific, quasi-specific, or nonspecific?[J]. The Journal of Immunology,2007,179(11):7209-7214.
[4]严芳.甲壳动物血蓝蛋白免疫学活性及其作用机制研究[D].汕头:汕头大学,2011.
[5]JOHANSSON M W, KEYSER P, SRITUNYALUCKSANA K, et al. Crustacean haemocytes and haematopoiesis[J]. Aquaculture,2000,191(1):45-52.
[6]郭青松.中华绒螯蟹血液指标研究[D].武汉：华中农业大学,2009.
[7]郭慧芝.中华绒鳌蟹cDNA文库的构建及免疫相关基因的克隆与表达[D].武汉：华中农业大学,2010.
[8]JIRAVANICHPAISAL P, LUEL L B, KENNETH S. Cell-mediated immunity in arthropods: hematopoiesis, coagulation, melanization and opsonization[J]. Immunobiology,2006, 211(4):213-236.
[9]SMITH V J, RATCLIFFE N A. Cellular defense reactions of the shore crab, Carcinus maenas:in vivo hemocytic and histopathological responses to injected bacteria[J]. Journal of Invertebrate Pathology,1980,35(1):65-74.
[10]SMITH V J, RATCLIFFE N A. Host defence reactions of the shore crab, Carcinus maenas (L.):clearance and distribution of injected test particles[J]. Journal of the Marine Biological Association of the United Kingdom,1980,60(01):89-102.
[11]JOHANSSON M W, KENNETH S. Isolation and purification of a cell adhesion factor from crayfish blood cells[J]. The Journal of Cell Biology,1988,106(5):1795-1803.
[12]KOBAYASHIM, JOHANSSON M W, KENNETH S. The 76 kD cell-adhesion factor from crayfish haemocytes promotes encapsulation in vitro[J]. Cell and Tissue Research,1990, 260(1):13-18.
[13]KOBAYASHI M, KENNETH S. Comparison of concanavalin A reactive determinants on isolated haemocytes of parasite-infected and non-infected freshwater crayfish[J]. DIS Aquat ORG,1990,9(1):141-147.
[14]HANS-GILBERT H, ANNA A, SODERHALL K. Purification and characterization of a high-Mr proteinase inhibitor of pro-phenol oxidase activation from crayfish plasma[J]. Biochem. J,1987,248(1):223-228.
[15]SODERHALL K, SMITH V J. Separation of the haemocyte populations of Carcinusmaenas and other Marine decapods, and prophenoloxidase distribution[J]. Developmental & Comparative Immunology,1983,7(2):229-239.
[16]徐海圣,徐步进.甲壳动物细胞及体液免疫机理的研究进展[J].大连水产学院学报,2001,16(1)：49-56.
[17]邓欢,陈俅,刘卫东,等.中国对虾血细胞包掩作用的超微结构和组织化学观察[J].应用与环境生物学报,1999,5(3)：296-299.
[18]JOHANSSON M W, TORBJORN H, PER-OVE T, et al. A cell-surface superoxide dismutase is a binding protein for peroxinectin, a cell-adhesive peroxidase in crayfish[J]. Journal of Cell Science,1999,112(6):917-925.
[19]SRITUNYALUCKSANA K, WONGSUEBSANTATI K, JOHANSSON M W, et al. Peroxinectin, a cell adhesive protein associated with the proPO system from the black tiger shrimp, Penaeus monodon[J]. Developmental & Comparative Immunology,2001,25(5): 353-363.
[20]Liu C H, Cheng W, Kuo C M, et al. Molecular cloning and characterisation of a cell adhesion molecule, peroxinectin from the white shrimp Litopenaeus vannamei[J]. Fish & Shellfish Immunology,2004,17(1):13-26.
[21]Liu C H, Cheng W, Chen J C. The peroxinectin of white shrimp Litopenaeus vannamei is synthesised in the semi-granular and granular cells, and its transcription is up-regulated with Vibrio alginolyticus infection[J]. Fish & Shellfish Immunology,2005,18(5):431-444.
[22]HSU P, LIU C, TSENG D, et al. Molecular cloning and characterisation of peroxinectin, a cell adhesion molecule, from the giant freshwater prawn Macrobrachium rosenbergii[J]. Fish & Shellfish Immunology,2006,21(1):1-10.
[23]Liu C H, Yeh S P, Hsu P Y, et al. Peroxinectin gene transcription of the giant freshwater prawn Macrobrachium rosenbergii under intrinsic, immunostimulant, and chemotherapeutant influences[J]. Fish & Shellfish Immunology,2007,22(4):408-417.
[24]MARTIN G G, POOLE D, POOLE C, et al. Clearance of bacteria injected into the hemolymph of the penaeid shrimp, sicyonia ingentis[J]. Journal of Invertebrate Pathology, 1993,62(3):308-315.
[25]JOHANSSON M W, SODERHALL K. Cellular immunity in crustaceans and the proPO system[J]. Parasitology Today,1989,5(6):171-176.
[26]MARTIN G G, KAY J, POOLE D, et al. In vitro nodule formation in the ridgeback prawn, Sicyonia ingentis, and the American lobster, Homarus americanus[J]. Invertebrate Biology, 1998,117(2):155-168.
[27]MILLER J S, HOWARD R W, RANA R L, et al. Eicosanoids mediate nodulation reactions to bacterial infections in adults of the cricket, Gryllus assimilis[J]. Journal of Insect Physiology,1999,45(1):75-83.
[28]MILLER J S, HOWARD R W, NGUYEN T, et al. Eicosanoids mediate nodulation responses to bacterial infections in larvae of the tenebrionid Beetle, Zophobas atratus[J]. Journal of Insect Physiology,1996,42(1):3-12.
[29]董超华.克氏原螯虾两种模式识别受体基因的克隆、重组表达及功能分析[D].青岛：中国海洋大学,2009.
[30]杨军厚.克氏原螯虾C型凝集素的表达与功能研究[D].济南：山东大学,2011.
[31]LEONARD C, SODERHALL K, RATCLIFFE N A. Studies on prophenoloxidase and protease activity of Blaberus craniifer haemocytes[J]. Insect Biochemistry,1985,15(6): 803-810.
[32]王雷,李光友.中国对虾血淋巴中的抗菌,溶菌活力与酚氧化酶活力的测定及其特性研究[J].海洋与湖沼,1995,26(2)：179-185.
[33]KENNETH S, LENA H. Lipopolysaccharide-induced activation of prophenoloxidase activating system in crayfish haemocyte lysate[J]. Biochimica ET Biophysica ACTA (BBA)-General Subjects,1984,797(1):99-104.
[34]ASHIDA M, SODERHAL K. The prophenoloxidase activating system in crayfish[J]. Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1984,77(1): 21-26.
[35]孟凡伦,张玉臻.甲壳动物中的酚氧化酶原激活系统研究评价[J].海洋与湖沼,1999,30(1)：110-116.
[36]樊现远.中国卤虫(Artemia sinica)酚氧化酶的分离纯化及其生物化学性质和酶性质的研究[D].青岛：中国海洋大学,2011.
[37]PERAZZOLO L M, LORENZINI D M, DAFFRE S, et al. Purification and partial characterization of the plasma clotting protein from the pink shrimp Farfantepenaeus paulensis[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,2005,142(3):302-307.
[38]杜欣军.中国明对虾先天免疫的模式识别与效应分子[D].济南：山东大学,2007.
[39]钱云霞,顾晓英.甲壳动物血液凝固的分子机制[J].生物技术通报,2011(06)：25-30.
[40]ZASLOFF M. Antimicrobial peptides of multicellular organisms[J]. Nature,2002,415(6870)：389-395.
[41]黄文树,王克坚,李少菁.甲壳动物抗菌肽研究进展[J].海洋科学,2005,29(2)：64-68.
[42]SCHNAPP D, KEMP G D, SMITH V J. Purification and characterization of a Proline-Rich antibacterial peptide, with sequence similarity to Bactenecin-7, from the haemocytes of the shore crab, carcinus maenas[J]. European Journal of Biochemistry,1996,240(3):532-539.
[43]CHISHOLM J R, SMITH V J. Comparison of antibacterial activity in the hemocytes of different crustacean species[J]. Comparative Biochemistry and Physiology Part a: Physiology,1995,110(1):39-45.
[44]NOGA E J, ARROLL T A, FAN Z. Specificity and some physicochemical characteristics of the antibacterial activity from blue crab Callinectes sapidus[J]. Fish & Shellfish Immunology,1996,6(6):403-412.
[45]EVANS E E, PAINTER B, EVANS M L, et al. An induced bactericidin in the spiny lobster, Panulirus Argus[C]//Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, NY),128,1968:394-398.
[46]EVANS E E, GUSHING J E, SAWYER S, et al. Induced bactericidal response in the California spiny lobster Panulirus interruptus[C]//Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (NewYork,NY),132,1969：111-114.
[47]STEWART J E, ZWICKER B M. Natural and induced bactericidal activities in the hemolymph of the lobster, Homarus americanus:products of hemocyte-plasma interaction[J]. Canadian Journal of Microbiology,1972,18(9):1499-1509.
[48]DESTOUMIEUX-GARZON D,SAULNIER D,GARNIER J.Crustacean immunityantifungal peptides are generated from the C terminus of shrimp hemocyanin in response to microbial challenge[J]. Journal of Biological Chemistry,2001,276(50):47070-47077.
[49]GROSS P, BARTLETT T, BROWDY C E. Immune gene discovery by expressed sequence tag analysis of hemocytes and hepatopan creas in the Pacific White Shrimp, Litopenaeus vannamei, and the Atlantic White Shrimp, L. setiferus[J]. Developmental & Comparative Immunology,2001,25(7):565-577.
[50]ROJTINNAKORN J, HIRONO I, ITAMI T E. Gene expression in haemocytes of kuruma prawn, Penaeus japonicus, in response to infection with WSSV by EST approach[J]. Fish & Shellfish Immunology,2002,13(1):69-83.
[51]王秀英,邵庆均,黄磊.对虾抗菌肽Penaeidins的研究进展[J].华中农业大学学报,2003,22(06)：624-629.
[52]BARRACCO M A, LORGERIL J, GUEGUEN Y, et al. Molecular characterization of penaeidins from two Atlantic Brazilian shrimp species, Farfantepenaeus paulensis and Litopenaeus schmitti*[J]. FEMS Microbiology Letters,2005,250(1):117-120.
[53]BROCKTON V, SMITH V J. Crustin expression following bacterial injection and temperature change in the shore crab, Carcinus maenas[J]. Developmental & Comparative Immunology,2008,32(9):1027-1033.
[54]HAUTON C, BROCKTON V, SMITH V J. Cloning of a crustin-like, single whey-acidic-domain, antibacterial peptide from the haemocytes of the European lobster, Homarus gammarus, and its response to infection with bacteria[J]. Molecular Immunology, 2006,43(9):1490-1496.
[55]TAMURA H, TANAKA S, ODA T, et al. Purification and characterization of a (1→ 3)-β-d-glucan-binding protein from horseshoe crab (Tachypleus tridentatus) amoebocytes[J]. Carbohydrate Research,1996,295(1):103-116.
[56]Pan C Y, Chao T T, Chen J C, et al. Shrimp (penaeus monodon) anti-lipopolysaccharide factor reduces the lethality of pseudomonas aeruginosa sepsis in MICE[J]. International Immunopharmacology,2007,7(5):687-700.
[57]Li C H, Zhao J M, Song L S, et al. Molecular cloning, genomic organization and functional analysis of an anti-lipopolysaccharide factor from Chinese mitten crab Eriocheir sinensis[J]. Developmental & Comparative Immunology,2008,32(7):784-794.
[58]MIREILLE L, CAILLE A, LANDON C, et al. Solution structures of the antifungal heliomicin and a selected variant with both antibacterial and antifungal activities[J]. Biochemistry,2001,40(40):11995-12003.
[59]李红权,刘燕.虾类免疫机制的特点及其免疫增强剂的研究进展[J].畜牧兽医科技信息,2006(8)：87-89.
[60]HALL J L, ROWLANDS JR D T. Heterogeneity of lobster agglutinins. I. Purification and physicochemical characterization [J]. Biochemistry,1974,13(4):821-827.
[61]曹剑香,简纪常,吴灶和,等.凡纳滨对虾血清凝集素,溶血素的特性研究[J].海洋科学,2006,6(30)：1-5.
[62]PACE K E, BAUM L G. Insect galectins:roles in immunity and development[J]. Glycoconjugate Journal,2002,19(7-9):607-614.
[63]MOCK A, RENWRANTZ L. Isolation and characterization of a lectin from the cephalochordate Branchiostoma lanceolatum(pallas)[J]. Developmental & Comparative Immunology,1991,99(3):699-707.
[64]LOPEZ-CORTES L, CASTRO D, NAVAS J I, et al. Phagocytic and chemotactic responses of Manila and carpet shell clam haemocytes against Vibrio tapetis, the causative agent of brown ring disease[J]. Fish & Shellfish Immunology,1999,9(7):543-555.
[65]KAWABATA H, YANG R, HIRAMA T, et al. Molecular cloning of transferrin receptor 2 A new member of the transferrin receptor-like family[J]. Journal of Biological Chemistry, 1999,274(30):20826-20832.
[66]MARQUES M F, BARRACCO M A. Lectins, as non-self-recognition factors, in crustaceans[J]. Aquaculture,2000,191(1):23-44.
[67]RATANAPO S, CHULAVATNATOL M. Monodin-induced agglutination of Vibrio vulnificus, a major infective bacterium in black tiger prawn (Penaeus monodon)[J]. Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1992,102(4): 855-859.
[68]VARGAS-ALBORES F, MARIA-ANTONIA G, JOSE-LUIS O. An anticoagulant solution for haemolymph collection and prophenoloxidase studies of penaeid shrimp Penaeus californiensis[J]. Comparative Biochemistry and Physiology Part a:Physiology,1993, 106(2):299-303.
[69]LORENA V, FELIPE M, PATRICIA R, et al. Purification and characterization of a lectin from Macrobrachium rosenbergh(Crustacea, Decapoda) hemolymph[J]. Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1993,105(3):617-623.
[70]VAZQUEZ L, JARAMILLO L, LASCURAIN R, et al. Bacterial agglutination by the sialic acid specific serum lectin from Macrobrachium rosenbergii[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,1996,113(2):355-359.
[71]FRAGKIADAKIS G A, STRATAKIS E K. The lectin from the crustacean liocarcinus depurator recognizes O-acetylsialic acids[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology,1997,117(4):545-552.
[72]MAHESWARI R, MULLAINADHAN P, ARUMUGAM M. Isolation and characterization of an acetyl group-recognizing agglutinin from the serum of the Indian white shrimp Fenneropenaeus indicus[J]. Archives of Biochemistry and Biophysics,2002,402(1):65-76.
[73]MAJUMDER M, CHATTOPADHYAY T, GUHA A K, et al. Inhibition of bacterial respiration by a low-molecular weight lectin, scyllin, from Scylla serrata crab hemolymph[J]. Indian Journal of Biochemistry & Biophysics,1996,34(1-2):87-89.
[74]LIN Y, VASEEHARAN B, CHEN J. Molecular cloning and phylogenetic analysis on a 2-macroglobulin (α2-M) of white shrimp Litopenaeus vannamei[J]. Developmental & Comparative Immunology,2008,32(4):317-329.
[75]RATTANACHAI A, HIRONO I, OHIRA T, et al. Molecular cloning and expression analysis of a2 macroglobulin in the kuruma shrimp, Marsupenaeus japonicus[J]. Fish& Shellfish Immunology,2004,16(5):599-611.
[76]HO P, CHENG C, CHENG W. Identification and cloning of the a2-macroglobulin of giant freshwater prawn Macrobrachium rosenbergii and its expression in relation with the molt stage and bacteria injection[J]. Fish & Shellfish Immunology,2009,26(3):459-466.
[77]Qin C J, Chen L Q, JIAN G Q, et al. Molecular cloning and characterization of a 2-macroglobulin (a2-M) from the haemocytes of Chinese mitten crab Eriocheir sinensis[J]. Fish & Shellfish Immunology,2010,29(2):195-203.
[78]牟海津,江晓路,刘树青,等.日本对虾溶血素的活性测定及性能研究[J].海洋与湖沼,1999,30(4)：362-367.
[79]赵大显,陈立侨.中华绒螯蟹免疫相关基因的研究进展[J].水产科学,2010,29(4)：243-248.
[80]LEE S Y, Wang R G, SODERHALL K. A Lipopolysaccharide-and beta-1, 3-Glucan-binding protein from hemocytes of the freshwater crayfish pacifastacus leniusculus purification, characterization, and CDNA cloning[J]. Journal of Biological Chemistry,2000,275(2):1337-1343.
[81]Cheng W, Liu C H, Tsai C H, et al. Molecular cloning and characterisation of a pattern recognition molecule, lipopolysaccharide-and β-1,3-glucan binding protein (LGBP) from the white shrimp Litopenaeus vannamei[J]. Fish & Shellfish Immunology,2005,18(4): 297-310.
[82]Zhao D X, Chen L Q, Qin C J, et al. Molecular cloning and characterization of the lipopolysaccharide and β-1,3-glucan binding protein in Chinese mitten crab(Eriocheir sinensis)[J]. COMP Biochem and Physiol B:Biochem Molbiol,2009,154(1):17-24.
[83]VARGAS-ALBORES F, JIMENEZ-VEGA F, YEPIZ-PLASCENCIA G M. Purification and comparison of β-1,3-Glucan binding protein from white shrimp (penaeus vannamei)[J]. Comparative Biochemistry and Physiology Part B:Biochemistry and Molecular Biology, 1997,116(4):453-458.
[84]Lai X F, Kong J, Wang Q Y, et al. Cloning and characterization of a (3-1,3-glucan-binding protein from shrimp Fenneropenaeus chinensis[J]. Molecular Biology Reports,2011,38(7): 4527-4535.
[85]曹慧,李宗芸,王秋香.果蝇先天性免疫研究进展[J].昆虫知识,2009,791(46)
[86]GOBERT V, GOTTAR M, MATSKEV1CH A A, et al. Dual activation of the Drosophila toll pathway by two pattern recognition receptors[J]. Science,2003,302(5653):2126-2130.
[87]CORBO J C, LEVINE M. Characterization of an immunodeficiency mutant in Drosophila[J]. Mechanisms of Development,1996,55(2):211-220.
[88]PERSSON M, CERENIUS L, SODERHALL K. The influence of haemocyte number on the resistance of the freshwater crayfish, Pacifastacus leniusculus Dana, to the parasitic fungus Aphanomyces astaci[J]. Journal of Fish Diseases,1987,10(6):471-477.
[89]SMITH V J, SODERHALL K. Induction of degranulation and lysis of haemocytes in the freshwater crayfish, Astacus astacus by components of the prophenoloxidase activating system in vitro[J]. Cell and Tissue Research,1983,233(2):295-303.
[90]WITTEVELDT J, VLAK J M, VAN HULTEN M C. Protection of penaeus monodon against white spot syndrome virus using a WSSV subunit vaccine[J]. Fish & Shellfish Immunology,2004,16(5):571-579.
[91]STUART L M, EZEKOWITZ R A. Phagocytosis and comparative innate immunity: learning on the fly[J]. Nature Reviews Immunology,2008,8(2):131-141.
[92]董迎辉.泥蚶高通量转录组分析及生长相关基因的克隆与表达研究[D].青岛：中国海洋大学,2012.
[93]杨昌健.中国明对虾Toll样受体基因的克隆和表达分析[D].青岛：中国科学院研究生院(海洋研究所),2008.
[94]Wang P H, Liang J P, Gu Z H, . Molecular cloning, characterization and expression analysis of two novel Tolls (LvToll2 and LvToll3) and three putative Spatzle-like Toll ligands (LvSpzl-3) from Litopenaeus vannamei[J/OL]. Developmental & Comparative Immunology,2012,36(2):359-371.
[95]Yang L S, Yin Z X, Liao J X, et al. A toll receptor in shrimp[J]. Molecular Immunology, 2007,44(8):1999-2008.
[96]Lin Z Y, Jie Q, Zhang Y L, et al. Cloning and characterisation of the SpToll gene from green mud crab, Scylla paramamosain[J]. Developmental & Comparative Immunology,2012, 37(1):164-175.
[97]张樱.三疣梭子蟹LGBP基因的克隆与表达研究[D].宁波：宁波大学,2011.
[98]HOFFMANN A, FUNKNER A, NEUMANN P,等. Biophysical characterization of refolded Drosophila Spatzle, a cystine knot protein, reveals distinct properties of three isoforms[J]. Journal of Biological Chemistry,2008,283(47):32598-32609.
[99]Shi X Z, Zhang R R, Jia Y P,等. Identification and molecular characterization of a Spatzle-like protein from Chinese shrimp (Fenneropenaeus chinensis)[J]. Fish & Shellfish Immunology,2009,27(5):610-617.
[100]朱磊.拟穴青蟹(Scylla paramamosain) Toll样信号途径关键基因及抗菌肽SpALF的克隆、表达与功能分析[D].雅安市：四川农业大学,2012.
[101]BRUNO L, JULES H. The host defense of Drosophila melanogaster[J]. Annual Review of Immunology,2007,25(1):697-743.
[102]Wang P H, Gu Z H, Wan D H, et al. The shrimp NF-κB pathway is activated by white spot syndrome virus (WSSV) 449 to facilitate the expression of WSSV069 (iel), WSSV303 and WSSV371[J]. PLOS ONE,2011,6(9):e24773.
[103]Li X C, Zhu L, Li L G, et al. A novel myeloid differentiation factor 88 homolog, SpMyD88, exhibiting SpToll-binding activity in the mud crab Scylla paramamosain[J]. Developmental & Comparative Immunology,2013,39(4):313-322.
[104]Wang P H, Wan D H, Gu Z H, et al. Litopenaeus vannamei tumor necrosis factor receptor-associated factor 6 (TRAF6) responds to Vibrio alginolyticus and white spot syndrome virus (WSSV) infection and activates antimicrobial peptide genes[J]. Developmental & Comparative Immunology,2011,35(1):105-114.
[105]Li F H, Wang D D, Li S H, et al. A dorsal homolog (fcdorsal) in the Chinese shrimp fenneropenaeus chinensis is responsive to both bacteria and WSSV challenge[J]. Developmental & Comparative Immunology,2010,34(8):874-883.
[106]Huang X D, Yin Z X, Jia X T, et al. Identification and functional study of a shrimp Dorsal homologue[J]. Developmental & Comparative Immunology,2010,34(2):107-113.
[107]LEMAITRE B, KROMER-METZGER E, MICHAUT L, et al. A recessive mutation, immune deficiency (imd), defines two distinct control pathways in the Drosophila host defense[J]. Proceedings of the National Academy of Sciences,1995,92(21):9465-9469.
[108]Wang P H, Gu Z H, Huang X D, et al. An immune deficiency homolog from the white shrimp, Litopenaeus vannamei, activates antimicrobial peptide genes[J]. Molecular Immunology,2009,46(8):1897-1904.
[109]冯宁宁.虾类IMD基因的功能分析及抗病指标的筛选[D].青岛：中国科学院研究生院(海洋研究所),2013.
[110]LI F, YAN H, WANG D, et al. Identification of a novel relish homolog in Chinese shrimp Fenneropenaeus chinensis and its function in regulating the transcription of antimicrobial peptides[J]. Developmenta l& Comparative Immunology,2009,33(10):1093-1101.
[111]Huang X D, Yin Z X, Liao J X, et al. Identification and functional study of a shrimp Relish homologue[J]. Fish & Shellfish Immunology,2009,27(2):230-238.
[112]刘文静.斑节对虾TLR22及Relish基因的克隆与表达分析[D].上海：上海海洋大学,2012.
[113]Sun C, Xu W T, Zhang H W, et al. An anti-lipopolysaccharide factor from red swamp crayfish, Procambarus clarkii, exhibited antimicrobial activities in vitro and in vivo[J]. Fish & Shellfish Immunology,2011,30(1):295-303.
[114]THARNTADA S, PONPRATEEP S, SOMBOONWIWAT K, et al. Role of anti-lipopolysaccharide factor from the black tiger shrimp, Penaeus monodon, in protection from white spot syndrome virus infection[J]. Journal of General Virology,2009,90(6): 1491-1498.
[115]SOMBOONWIWAT K, MARCOS M, TASSANAKAJON A, et al. Recombinant expression and anti-microbial activity of anti-lipopolysaccharide factor (ALF) from the black tiger shrimp Penaeus monodon[J]. Developmental & Comparative Immunology,2005, 29(10):841-851.
[116]YEDERY R D, REDDY K R. Identification, cloning, characterization and recombinant expression of an anti-lipopolysaccharide factor from the hemocytes of Indian mud crab, Scylla serrata[J]. Fish & Shellfish Immunology,2009,27(2):275-284.
[117]CUTHBERTSON B J, SHEPARD E F, CHAPMAN R W, et al. Diversity of the penaeidin antimicrobial peptides in two shrimp species[J]. Immunogenetics,2002,54(6):442-445.
[118]Kang C J, Wang J X, Zhao X F, et al. Molecular cloning and expression analysis of Ch-penaeidin, an antimicrobial peptide from Chinese shrimp, Fenneropenaeus chinensis[J]. Fish & Shellfish Immunology,2004,16(4):513-525.
[119]Li L, Wang J X, Zhao X F, et al. High level expression, purification, and characterization of the shrimp antimicrobial peptide, Ch-penaeidin, in Pichia pastoris[J]. Protein Expression and Purification,2005,39(2):144-151.
[120]Kang C J, Xue J F, Liu N, et al. Characterization and expression of a new subfamily member of penaeidin antimicrobial peptides (penaeidin 5) from Fenneropenaeus chinensis[J]. Molecular Immunology,2007,44(7):1535-1543.
[121]BLANCO-PORTALES R, LOPEZ-RAEZ J A, BELLIDO M L, et al. A strawberry fruit-specific and ripening-related gene codes for a HyPRP protein involved in polyphenol anchoring[J]. Plant Molecular Biology,2004,55(6):763-780.
[122]RELF J M, CHISHOLM J R, KEMP G D, et al. Purification and characterization of a cysteine-rich 11.5-kDa antibacterial protein from the granular haemocytes of the shore crab, Carcinus maenas[J]. European Journal of Biochemistry,1999,264(2):350-357.
[123]STOSS T D, NICKELL M D, HARDIN D, et al. Inducible transcript expressed by reactive epithelial cells at sites of olfactory sensory neuron proliferation[J]. Journal of Neurobiology, 2004,58(3):355-368.
[124]RATTANACHAI A, HIRONO I, OHIRA T, et al. Cloning of kuruma prawn Marsupenaeus japonicus crustin-like peptide cDNA and analysis of its expression[J]. Fisheries Science, 2004,70(5):765-771.
[125]SUPUNGUL P, TANG S, MANEERUTTANARUNGROJ C, et al. Cloning, expression and antimicrobial activity of crustin Pm 1, a major isoform of crustin, from the black tiger shrimp Penaeus monodon[J]. Developmental & Comparative Immunology,2008,32(1): 61-70.
[126]JIRAVANICHPAISAL P, LEE S Y, KIM Y, et al. Antibacterial peptides in hemocytes and hematopoietic tissue from freshwater crayfish Pacifastacus leniusculus:Characterization and expression pattern[J]. Developmental & Comparative Immunology,2007,31(5): 441-455.
[127]OKUMURA T. Effects of lipopolysaccharide on gene expression of antimicrobial peptides (penaeidins and crustin), serine proteinase and prophenoloxidase in haemocytes of the Pacific white shrimp, Litopenaeus vannamei[J]. Fish & Shellfish Immunology,2007,22(1): 68-76.
[128]BRITES D, MCTAGGART S, MORRIS K, et al. The dscam homologue of the crustacean daphnia is diversified by alternative splicing like in insects[J]. Molecular Biology and Evolution,2008,25(7):1429-1439.
[129]WATTHANASUROROT A, JIRAVANICHPAISAL P, LIU H, et al. Bacteria-induced Dscam isoforms of the crustacean, Pacifastacus leniusculus[J]. PLOS Pathogens,2011,7(6): e1002062.
[130]Chou P H, Chang H S, Chen I T, et al. Penaeus monodon Dscam (PmDscam) has a highly diverse cytoplasmic tail and is the first membrane-bound shrimp Dscam to be reported[J]. Fish & Shellfish Immunology,2011,30(4-5):1109-1123.
[131]Jin X K, Li W W, Wu M H, et al. Immunoglobulin superfamily protein Dscam exhibited molecular diversity by alternative splicing in hemocytes of crustacean,Eriocheir sinensis[J]. Fish & Shellfish Immunology,2013,35(3):900-909.
[132]Wang J J, WANG L L, Gao Y, et al. A tailless Dscam from Eriocheir sinensis diversified by alternative splicing[J]. Fish & Shellfish Immunology,2013,35(2):249-261.
[133]Liu Y C, Li F H, Dong B, et al. Molecular cloning, characterization and expression analysis of a putative C-type lectin (Fclectin) gene in Chinese shrimp Fenneropenaeus chinensis[J]. Molecular Immunology,2007,44(4):598-607.
[134]Sun Y D, Fu L D, Jia Y P, et al. A hepatopancreas-specific C-type lectin from the Chinese shrimp Fenneropenaeus chinensis exhibits antimicrobial activity [J]. Molecular Immunology, 2008,45(2):348-361.
[135]Zhang X W, Xu W T, Wang X W, et al. A novel C-type lectin with two CRD domains from Chinese shrimp Fenneropenaeus chinensis functions as a pattern recognition protein[J]. Molecular Immunology,2009,46(8):1626-1637.
[136]Wang X W, Xu W T, Zhang X W, et al. A C-type lectin is involved in the innate immune response of Chinese white shrimp[J]. Fish & Shellfish Immunology,2009,27(4):556-562.
[137]XU W, WANG X, ZHANG X, et al. A new C-type lectin (FcLec5) from the Chinese white shrimp Fenneropenaeus chinensis[J], Amino Acids,2010,39(5):1227-1239.
[138]Li F H, Xiang J H. Recent advances in researches on the innate immunity of shrimp in China[J]. Developmental & Comparative Immunology,2013,39(1):11-26.
[139]Jin X K, Li S, Guo X N, et al. Two antibacterial C-type lectins from crustacean, Eriocheir sinensis, stimulated cellular encapsulation in vitro[J]. Developmental & Comparative Immunology,2013,41(4):544-552.
[140]任乾.中国明对虾丝氨酸蛋白酶、活性氧的产生与消除及其半胱氨酸蛋白酶研究[D].济南：山东大学,2011.
[141]Ren Q, Xu Z L, Wang X W, et al. Clip domain serine protease and its homolog respond to Vibrio challenge in Chinese white shrimp, Fenneropenaeus chinensis[J]. Fish & Shellfish Immunology,2009,26(5):787-798.
[142]Liu Y C, Li F H, Wang B, et al. A serpin from Chinese shrimp Fenneropenaeus chinensis is responsive to bacteria and WSSV challenge[J]. Fish & Shellfish Immunology,2009,26(3): 345-351.
[143]刘树青,江晓路.免疫多糖对中国对虾血清溶菌酶,磷酸酶和过氧化物酶的作用[J].海洋与湖沼,1999,30(3)：278-283.
[144]魏克强,许梓荣.对虾的免疫机制及其疾病预防策略的研究[J].中国兽药杂志,2004,38(9)：25-29.
[145]杨志彪,赵云龙,周忠良,等.水体铜对中华绒螯蟹(Eriocheir sinensis)代谢酶活力的影响[J].海洋与湖沼,2006,37(2)：118-124.
[146]陈昌福,姚娟,陈超然,等.利用免疫刺激剂预防虾,蟹的传染性疾病[J].淡水渔业,2004,34(2)：55-57.
[147]管晓娟.甲壳动物体液免疫相关酶及免疫因子研究概况[J].生命科学仪器,2009(6)：3-7.
[148]宋林生,苏建国,崔朝霞,等.中华绒螯蟹(Eriocheir sinensis)幼蟹上岸病的免疫学研究[J].海洋与湖沼,2002,33(6)：657-662.
[149]王宜艳,孙虎山,李光友.复合免疫药物对中国对虾血淋巴氧化酶和抗氧化酶活力的影响[J].海洋科学进展,2002,20(3)：79-83.
[150]Wang D L, Zuo D, Wang L M, et al. Effects of white spot syndrome virus infection on immuno-enzyme activities and ultrastructure in gills of Cherax quadricarinatus[J]. Fish & Shellfish Immunology,2012,32(5):645-650.
[151]UEMATSU S, AKIRA S. Toll-Like receptors (tlrs) and their ligands[G]. Handbook of Experimental Pharmacology. [S.l.]:Springer Berlin Heidelberg,2008:1-20.
[152]CARTY M, BOWIE A G. Recent insights into the role of Toll-like receptors in viral infection[J]. Clinical and Experimental Immunology,2010,161(3):397-406.
[153]KAWAI T, AKIRA S. The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors[J]. Nature Immunology,2010,11(5):373-384.
[154]IMLER J, ZHENG L. Biology of toll receptors:lessons from insects and mammals[J]. Journal of Leukocyte Biology,2004,75(1):18-26.
[155]IMLER J, HOFFMANN J A. Toll receptors in innate immunity[J]. Trends in Cell Biology, 2001,11(7):304-311.
[156]BARTON G M, MEDZHITOV R. Toll-like receptors and their ligands,270[M]. [S.l.]: Berlin Heidelberg:Springer-Verlag,2002:81-92.
[157]BARTON G M, MEDZHITOV R. Toll-like receptor signalling[J]. Nature Reviews Immunology,2004,4(7):499-511.
[158]MULINARI S, HACKER U, CASTILLEJO-LOPEZ C. Expression and regulation of Spatzle-processing enzyme in Drosophila[J]. FEBS Letters,2006,580(22):5406-5410.
[159]TAUSZIG S, JOUANGUY E, HOFFMANN J A, et al. Toll-related receptors and the control of antimicrobial peptide expression in Drosophila[J]. Proceedings of the National Academy of Sciences,2000,97(19):10520-10525.
[160]IMLER J, HOFFMANN J A. Signaling mechanisms in the antimicrobial host defense of Drosophila[J]. Current Opinion in Microbiology,2000,3(1):16-22.
[161]Wang P H, Gu Z H, Huang X D, et al. An immune deficiency homolog from the white shrimp, Litopenaeus vannamei[J]. Molecular Immunology,2009,46(8-9):1897-1904.
[162]Ying X P, Yang W X, Zhang Y P. Comparative studies on fatty acid composition of the ovaries and hepatopancreas at different physiological stages of the Chinese mitten crab[J]. Aquaculture,2006,256(1-4):617-623.
[163]Gai Y C, Qiu L M, Wang L L, et al. A clip domain serine protease (cSP) from the Chinese mitten crab Eriocheir sinensis:cDNA characterization and mRNA expression[J]. Fish & Shellfish Immunology,2009,27(6):670-677.
[164]GAI, YUNCHAO, WANG, LINGLING, ZHAO, JIANMIN, et al. The construction of a cDNA library enriched for immune genes and the analysis of 7535 ESTs from Chinese mitten crab[J]. Fish & Shellfish Immunology,2009,27(6):684-694.
[165]ZHANG, HAO, CHEN, LIQ1AO, QIN, JIANGUANG, et al. Molecular cloning, characterization and expression of a C-type lectin cDNA in Chinese mitten crab,[J]. Fish & Shellfish Immunology,2011,31(2):358-363.
[166]JIN, XING-KUN, LI, WEI-WEI, HE, LIN, et al. Molecular cloning, characterization and expression analysis of two apoptosis genes, caspase and nm23, involved in the antibacterial response in Chinese mitten crab,[J]. Fish & Shellfish Immunology,2011,30(1):263-272.
[167]MU, CHANGKAO, ZHENG, PEILIN, ZHAO, JIANMIN, et al. Molecular characterization and expression of a crustin-like gene from Chinese mitten crab,[J]. Developmental & Comparative Immunology,2010,34(7):734-740.
[168]JIANG, HUI, CAI, YI-MEI, CHEN, LI-QIAO, et al. Functional annotation and analysis of expressed sequence TAGS from the hepatopancreas of mitten CRAB ([J]. Marine Biotechnology,2009,11(3):317-326.
[169]ZHAO D X, SONG S H, WANG Q, et al. Discovery of immune-related genes in Chinese mitten crab (Eriocheir sinensis) by expressed sequence tag analysis of haemocytes[J]. Aquaculture,2009,287(3-4):297-303.
[170]GUO, H. Z., ZOU, P. F., FU, J. P., et al. Characterization of two C-type lectin-like domain (CTLD)-containing proteins from the cDNA library of Chinese mitten crab[J]. Fish & Shellfish Immunology,2011,30(2):515-524.
[171]QIN, CHUANJIE, CHEN, LIQIAO, QIN, JIAN G., et al. Characterization of a serine proteinase homologous (SPH) in Chinese mitten crab[J]. Developmental & Comparative Immunology,2010,34(1):14-18.
[172]Jiang H, Yin Y X, Zhang X W, et al. Chasing relationships between nutrition and reproduction:A comparative transcriptome analysis of hepatopancreas and testis from Eriocheir sinensis[J]. Comparative Biochemistry and Physiology Part D:Genomics and Proteomics,2009,4(3):227-234.
[173]ZHANG, WEI, WAN, HAOLEI, JIANG, HUI, et al. A transcriptome analysis of mitten crab testes (Eriocheir sinensis)[J]. Genetics and Molecular Biology,2011,34(1):136-141.
[174]He L, Wang Q, Jin X K, et al. Transcriptome profiling of testis during sexual maturation stages in Eriocheir sinensis using Illumina sequencing[J]. PLOS ONE,2012,7(3):e33735.
[175]MEKATA T, KONO T, YOSHIDA T, et al. Identification of cDNA encoding Toll receptor, MjToll gene from kuruma shrimp, Marsupenaeus japonicus[J]. Fish & Shellfish Immunology,2008,24(1):122-133.
[176]ASSAVALAPSAKUL, W, PANYIM, S. Molecular cloning and tissue distribution of the Toll receptor in the black tiger shrimp, Penaeus monodon[J]. Genetics and Molecular Research,2012,11(1):484-493.
[177]Yang C J, Zhang J Q, Li F H, et al. A toll receptor from Chinese shrimp fenneropenaeus chinensis is responsive to vibrio anguillarum infection[J]. Fish & Shellfish Immunology, 2008,24(5):564-574.
[178]GAY N J, GANGLOFF M. Structure and function of toll receptors and their ligands[J]. Annual Review of Biochemistry,2007,76(1):141-165.
[179]LEULIER, FRANCOIS, LEMAITRE, BRUNO. Toll-like receptors [mdash] taking an evolutionary approach[J]. Nature Reviews. Genetics,2008,9(3):165-178.
[180]SCHNEIDER, DAVID S, HUDSON, KATHY L, LIN, TING-YI, et al. Dominant and recessive mutations define functional domains of Toll, a transmembrane protein required for dorsal-ventral polarity in the Drosophila embryo[J]. Genes and Development,1991,5(5): 797-807.
[181]QURESHI S T, LARIVIERE L, LEVEQUE G, et al. Endotoxin-tolerant mice have mutations in Toll-like receptor 4 (Tlr4)[J]. The Journal of Experimental Medicine,1999, 189(4):615-625.
[182]TAKEUCHI O, HOSHINO K, KAWAI T, et al. Differential roles of TLR2 and TLR4 in recognition of Gram-Negative and Gram-Positive bacterial cell wall components[J]. Immunity,1999,11(4):443-451.
[183]RAMOS, HUGO CRUZ, RUMBO, MARTIN, SIRARD, JEAN-CLAUDE. Bacterial flagellins:mediators of pathogenicity and host immune responses in mucosa[J]. Trends in Microbiology,2004,12(11):509-517.
[184]HIROAKI H, OSAMU T, KAWAI T, et al. A Toll-like receptor recognizes bacterial DNA[J]. Nature,2000,408(6813):740-845.
[185]Zhang L L, Li L, Zhang G F. A crassostrea gigas Toll-like receptor and comparative analysis of TLR pathway in invertebrates[J]. Fish & Shellfish Immunology,2011,30(2):653-660.
[186]SODERHALL K, CERENIUS L. Crustacean immunity[J]. Annual Review of Fish Diseases, 1992,2(0):3-23.
[187]QIU L, SONG L, XU W, et al. Molecular cloning and expression of a Toll receptor gene homologue from Zhikong Scallop, Chlamys farreri[J]. Fish & Shellfish Immunology,2007, 22(5):451-466.
[188]OOI, JAMES Y., YAGI, YOSHIMASA, HU, XIAODI, et al. The drosophila Toll-9 activates a constitutive antimicrobial defense[J]. EMBO Reports,2002,3(1):82-87.
[189]BISCHOFF V, VIGNAL C, BONECA I G, et al. Function of the drosophila pattern-recognition receptor PGRP-SD in the detection of Gram-positive bacteria[J]. Nature Immunology,2004,5(11):1175-1180.
[190]ROYET J. Infectious non-self recognition in invertebrates:lessons from Drosophila and other insect models[J]. Molecular Immunology,2004,41(11):1063-1075.
[191]Wang P H, Gu Z H, Huang X D, et al. An immune deficiency homolog from the white shrimp, Litopenaeus vannamei, activates antimicrobial peptide genes[J]. Molecular Immunology,2009,46(8-9):1897-1904.
[192]TOWB P, SUN H, WASSERMAN S A. Tube is an IRAK-4 homolog in a toll pathway adapted for development and immunity[J]. Journal of Innate Immunity,2009,1 (4):309-321.
[193]GOSU V, BASITH S, DURAIP, et al. Molecular evolution and structural features of IRAK family members[J]. PLOS ONE,2012,7(11):e49771.
[194]Li C Z, Chen Y X, Weng S P, et al. Presence of tube isoforms in litopenaeus vannamei suggests various regulatory patterns of signal transduction in invertebrate NF-κB pathway[J]. Developmental & Comparative Immunology,2014,42(2):174-185.
[195]BRUNO L, EMMANUELLE N, LYDIA M,等. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antirungal response in drosophila adults[J]. Cell, 1996,86(6):973-983.
[196]SWANTEK J L, TSEN M F, COBB M H, et al. IL-1 receptor-associated kinase modulates host responsiveness to endotoxin[J]. The Journal of Immunology,2000,164(8):4301-4306.
[197]TAKEDA K, AKIRA S. Toll-like receptors in innate immunity[J]. International Immunology,2005,17(1):1-14.
[198]SUZUKI N, SUZUKI S, DUNCAN G S, et al. Severe impairment of interleukin-1 and Toll-like receptor signalling in mice lacking IRAK-4[J]. Nature,2002,416(6882):750-756.
[199]Li X C, Zhang X W, Zhou J F, et al. Identification, characterization, and functional analysis of tube and pelle homologs in the mud CRAB scylla paramamosain[J]. PLOS ONE,2013, 8(10):e76728.
[200]ANDERSON K V. Toll signaling pathways in the innate immune response[J]. Current Opinion in Immunology,2000,12(1):13-19.
[201]LI F, WANG L, ZHANG H, et al. Molecular cloning and expression of a Relish gene in Chinese mitten crab Eriocheir sinensis[J]. International Journal of Immunogenetics,2010, 37(6):499-508.
[202]Yu A Q, Jin X K, Shuang L, et al. Molecular cloning and expression analysis of a dorsal homologue from Eriocheir sinensis[J]. Developmental & Comparative Immunology,2013, 41(4):723-727.
[203]Yu A Q, Jin X K, Guo X N, et al. Two novel Toll genes (EsToll1 and EsToll2) from Eriocheir sinensis are differentially induced by lipopolysaccharide, peptidoglycan and zymosan[J]. Fish & Shellfish Immunology,2013,35(4):1282-1292.
[204]WATTHANASUROROT A, SODERHALL K, JIRAVANICHPAISAL P. A mammalian like interleukin-1 receptor-associated kinase 4 (IRAK-4), a TIR signaling mediator in intestinal innate immunity of black tiger shrimp (Penaeus monodon)[J]. Biochemical and Biophysical Research Communications,2012,417(1):623-629.
[205]MULLER-HOLTKAMP F, KNIPPLE D C, SEIFERT E, et al. An early role of maternal mRNA in establishing the dorsoventral pattern in pelle mutant Drosophila embryos[J]. Developmental Biology,1985,110(1):238-246.
[206]Ge H, Wang G D, ZHANG L L, et al. Molecular cloning and expression of interleukin-1 receptor-associated kinase 4, an important mediator of Toll-like receptor signal pathway, from small abalone Haliotis diversicolor[J]. Fish & Shellfish Immunology,2011,30(4): 1138-1146.
[207]PHELAN P E, MELLON M T, KIM C H. Functional characterization of full-length TLR3, IRAK-4, and TRAF6 in zebrafish (Danio rerio)[J]. Molecular Immunology,2005,42(9): 1057-1071.
[208]NISHIMURA M, NAITO S. Tissue-specific mRNA expression profiles of human toll-like receptors and related genes[J]. BIOL Pharm Bull,2005,28(5):886-892.
[209]Yu Y, Zhong Q W, Li C M, et al. Identification and characterization of IL-1 receptor-associated kinase-4 (IRAK-4) in half-smooth tongue sole Cynoglossus semilaevis[J]. Fish & Shellfish Immunology,2012,32(4):609-615.
[210]VOLLMER W, BLANOT D, DE PEDRO M A. Peptidoglycan structure and architecture[J]. FEMS Microbiology Reviews,2008,32(2):149-167.
[211]CHETTRI J K, RAIDA M K, HOLTEN-ANDERSEN L, et al. PAMP induced expression of immune relevant genes in head kidney leukocytes of rainbow trout (Oncorhynchus mykiss)[J]. Developmental & Comparative Immunology,2011,35(4):476-482.
[212]KRAVCHENKO V V, KAUFMANN G F. Bacterial inhibition of inflammatory responses via TLR-independent mechanisms[J]. Cellular Microbiology,2013,15(4):527-536.
[213]Yu H H, Xiao Z Y, Yong X C, et al. Effects of pH, temperature, and osmolarity on the morphology and survival rate of primary hemocyte cultures from the Mitten Crab, Eriocheir sinensis[J]. In Vitro Cellular and Developmental Biology-Animal,2013,49(9):716-727.
[214]Wu S H, Chen Y J, Huang S Y, et al. Demonstration of expression of a neuropeptide-encoding gene in crustacean hemocytes[J]. Comparative Biochemistry and Physiology Part a:Molecular and Integrative Physiology,2012,161(4):463-468.
[215]DE GREGORIO E, SPELLMAN P T, TZOU P, et al. The toll and IMD pathways are the major regulators of the immune response in drosophila[J]. The EMBO Journal,2002,21(11): 2568-2579.
[216]MATEO D R, GREENWOOD S J, ARAYA M T, et al. Differential gene expression of y-actin, Toll-like receptor 2 (TLR-2) and interleukin-1 receptor-associated kinase 4 (IRAK-4) in Mya arenaria haemocytes induced by in vivo infections with two Vibrio splendidus strains[J]. Developmental & Comparative Immunology,2010,34(7):710-714.
[217]DIXIT V, MAK T W. NF-κB signaling:many roads Lead to Madrid[J]. Cell,2002,111(5): 615-619.
[218]GHOSH S, MAY M J, KOPP E B. NF-κB and Rel proteins:evolutionary conserved mediators of immune responses[J]. Annual Review of Immunology,1998,16(1):225-260.
[219]HAYDEN M S, GHOSH S. Shared principles in NF-κB signaling[J]. Cell,2008,132(3): 344-362.
[220]BAEUERLE P A, HENKEL T. Function and activation of NF-kappaB in the immune system[J]. Annual Review of Immunology,1994,12(1):141-179.
[221]DUSHAY M S, ASLING B, HULTMARK D. Origins of immunity:Relish, a compound Rel-like gene in the antibacterial defense of Drosophila[J]. Proceedings of the National Academy of Sciences,1996,93(19):10343-10347.
[222]REICHHART J M, GEORGEL P, MEISTER M, et al. Expression and nuclear translocation of the rel/NF-kappa B-related morphogen dorsal during the immune response of Drosophila[J]. Comptes Rendus DE iacademie DES Sciences. Serie Iii, Sciences DE LA VIE,1993,316(10):1218-1224.
[223]STEWARD R. Dorsal, an embryonic polarity gene in Drosophila, is homologous to the vertebrate proto-oncogene, c-rel[J]. Science,1987,238(4827):692-694.
[224]STEWARD R, MCNALLY F J, SCHEDL P. Isolation of the dorsal locus of Drosophila[J]. Nature,1984,311(1):262-265.
[225]IMLER, JEAN-LUC, HOFFMANN, JULES A.. Signaling mechanisms in the antimicrobial host defense of Drosophila[J]. Current Opinion in Microbiology,2000,3(1):16-22.
[226]HOFFMANN J A, REICHHART J. Drosophila innate immunity:an evolutionary perspective[J]. Nature Immunology,2002,3(2):121-126.
[227]Zhang J Q, Li F H, Wang Z Z, et al. Cloning and recombinant expression of a crustin-like gene from Chinese shrimp, Fenneropenaeus chinensis[J]. Journal of Biotechnology,2007, 127(4):605-614.
[228]王丹丽,左迪,王兰梅,等.红螯光壳螯虾酚氧化酶原基因的克隆与表达[J].水产学报,2013,37(001)：43-54.
[229]ROSE T M, HENIKOFF J G, HENIKOFF S. CODEHOP (COnsensus-DEgenerate hybrid oligonucleotide primer) PCR primer design[J]. Nucleic Acids Research,2003,31(13): 3763-3766.
[230]申望,叶茂, 石戈,et al.三疣梭子蟹 (Portunus trituberculatus) I 型 Crustin[J].海洋 与湖沼,2010,41(3)：371-377.
[231]AMPARYUP P, KONDO H, HIRONO I, et al. Molecular cloning, genomic organization and recombinant expression of a crustin-like antimicrobial peptide from black tiger shrimp Penaeus monodon[J]. Molecular Immunology,2008,45(4):1085-1093.
[232]Chen D D, He N H, Xu X. Mj-DWD, a double WAP domain-containing protein with antiviral relevance in Marsupenaeus japonicus[J]. Fish & Shellfish Immunology,2008, 25(6):775-781.
[233]Sun C, Du X J, Xu W T, et al. Molecular cloning and characterization of three crustins from the Chinese white shrimp, Fenneropenaeus chinensis[J]. Fish & Shellfish Immunology, 2010,28(4):517-524.
[234]MU C, ZHENG P, ZHAO J, et al. Molecular characterization and expression of a crustin-like gene from Chinese mitten crab,Eriocheir sinensis[J]. Developmental & Comparative Immunology,2010,34(7):734-740.
[235]MU C, ZHENG P, ZHAO J, et al. A novel type Ⅲ crustin (CrusEs2) identified from Chinese mitten crab Eriocheir sinensis[J]. Fish & Shellfish Immunology,2011,31(1):142-147.
[236]AROCKIARAJ J, GNANAM A J, MUTHUKRISHNAN D, et al. Crustin, a WAP domain containing antimicrobial peptide from freshwater prawn Macrobrachium rosenbergii: Immune characterization[J]. Fish & Shellfish Immunology,2013,34(1):109-118.
[237]LITTLE T J, O'CONNOR B, COLEGRAVE N, et al. Maternal transfer of strain-specific immunity in an invertebrate [J]. Current Biology,2003,13(6):489-492.
[238]SADD B M, SCHMID-HEMPEL P. Insect immunity shows specificity in protection upon secondary pathogen exposure[J]. Current Biology,2006,16(12):1206-1210.
[239]PHAM L N, DIONNE M S, SHIRASU-HIZA M, et al. A specific primed immune response in Drosophila is dependent on phagocytes[J]. PLOS Pathogens,2007,3(3):e26.
[240]LEONARD P M, ADEMA C M, ZHANG S, et al. Structure of two FREP genes that combine IgSF and fibrinogen domains, with comments on diversity of the FREP gene family in the snail Biomphalaria glabrata[J]. Gene,2001,269(1):155-165.
[241]Yue M D, TAYLOR E H, DIMOPOULOS G. AgDscam, a hypervariable immunoglobulin domain-containing receptor of the Anopheles gambiae innate immune system[J]. PLOS Biology,2006,4(7):e229.
[242]TERWILLIGER D P, BUCKLEY K M, BROCKTON V, et al. Distinctive expression patterns of 185/333 genes in the purple sea urchin, Strongylocentrotus purpuratus:an unexpectedly diverse family of transcripts in response to LPS,β-1,3-glucan, and dsRNA[J]. BMC Molecular Biology,2007,8(1):16.
[243]YAMAKAWA K, HUO Y, HAENDEL M A, et al. DSCAM:a novel member of the immunoglobulin superfamily maps in a Down syndrome region and is involved in the development of the nervous system[J]. Human Molecular Genetics,1998,7(2):227-237.
[244]SCHMUCKER D, CLEMENS J C, SHU H, et al. Drosophila dscam is an AXON guidance receptor exhibiting extraordinary molecular diversity[J]. Cell,2000,101(6):671-684.
[245]WATSON F L, PUTTMANN-HOLGADO R, THOMAS F, et al. Extensive diversity of Ig-superfamily proteins in the immune system of insects[J]. Science,2005,309(5742): 1874-1878.
[246]Chou P H, Chang H S, Chen I T, et al. The putative invertebrate adaptive immune protein Litopenaeus vannamei Dscam (LvDscam) is the first reported Dscam to lack a transmembrane domain and cytoplasmic tail[J]. Developmental & Comparative Immunology,2009,33(12):1258-1267.
[247]WATSON F L, PUTTMANN-HOLGADO R, THOMAS F, et al. Extensive diversity of Ig-Superfamily proteins in the immune system of insects[J]. Science,2005,309(5742): 1874-1878.
[248]DONG Y, CIRIMOTICH C M, PIKE A, et al. Anopheles NF-κB-Regulated splicing factors direct Pathogen-Specific repertoires of the hypervariable pattern recognition receptor agdscam[J]. Cell Host & Microbe,2012,12(4):521-530.