家蝇抗菌肽相关基因芯片的制备和应用及一个新抗菌肽基因的克隆分析
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摘要
抗菌肽(antibacterial peptides,简称ABP)是一类由结构基因编码产生的小分子肽类物质,广泛存在于生物体内,是构成其自身免疫体系的重要组分,能非特异性的抗细菌、真菌、病毒、寄生虫等病原体,且对肿瘤细胞、多重耐药菌也有明显的杀伤作用,且对正常生物体细胞无害,作用的机理非常独特,是一类具有巨大发展潜力的新型抗菌药,成为目前生命科学领域研究中的一大研究热点。
家蝇从幼虫到成虫常生活在垃圾堆、禽畜粪便等有多种病原菌孳生的环境中,携带多种有害病原微生物,能在人、畜中通过机械传播多种疾病,自身却不会因为感染病原菌而死亡,这可能缘于其独特的天然免疫活性物质抵抗各种微生物的浸染,抗菌肽在其中起着主要的作用。家蝇在我国分布广泛,容易饲养,且养殖的成本较低,在医药开发应用方面是一个很好的昆虫资源。但从家蝇体内提取天然抗菌活性物质的成本较高,人工合成也较昂贵,通过基因工程方法可能是规模制备其抗菌肽的理想途径之一,而该方法的前提是要获取其有关的抗菌肽基因。
基因芯片技术是近年来新兴的一种分子生物学技术,具有快速、高效、敏感、平行性等许多优点,克服了传统方法一次只能研究一条或少数几条基因的局限性,在基因差异表达的筛选、新基因的发现、突变基因的检测及基因多态性分析等方面显示出了很好的应用前景。用该技术用于家蝇抗菌肽相关基因的筛选研究还未见报道,本研究以GenBank中部分昆虫抗菌肽基因序列为目标设计寡核苷酸(oligonucleotide)探针,制备成寡核苷酸(oligo)芯片,以筛选家蝇抗菌肽相关基因,并对所筛选出的其中一个基因的cDNA序列进行克隆,运用生物信息学软件进行初步分析。
研究目的
运用基因芯片技术筛选家蝇抗菌肽相关基因,对其中筛选出的一个基因的cDNA序列进行克隆分析,为进一步研究家蝇抗菌肽结构和功能,揭示其在家蝇天然免疫体系中所起的作用,进而为用基因工程方法生产家蝇抗菌肽打下基础。
研究方法
1.家蝇抗菌肽基因芯片的制备与相关基因的筛选
从GenBank数据库中获取部分昆虫抗菌肽基因序列,运用NCBI中相关服务器对各基因的全长进行分析,找出其编码区,分析出各自的保守序列,以此作为设计oligo探针的备选序列,用生物学软件Array Designer 2.0对所选序列设计特异性高、Tm值接近、长度均一的oligo探针,用直接点样法将探针点印在特制玻片上制备成寡核苷酸(oligonucleotide)芯片;提取经大肠杆菌(E.coli)和金黄色葡萄球菌(Staphylococcus aureus)免疫诱导后24小时的家蝇三龄幼虫脂肪体总RNA,逆转录成cDNA并标记上荧光标记物Cy3,与制备的基因芯片杂交,经洗片、扫描处理后进行数据分析。
2.一个家蝇新抗菌肽基因cDNA的克隆与生物信息学分析对筛选出的其中一个有效杂交信号点所对应基因的氨基酸保守域设计简并引物,行RT-PCR扩增出一个同源片断;结合cDNA文库(本实验室已构建)载体上的下游引物,从所克隆到的同源片断中设计出一条特异性上游引物,以cDNA文库为模板,行PCR扩增出该基因的3′端;再结合文库载体上的上游引物,从所克隆到的3′端序列中设计出一条特异性下游引物,以cDNA文库为模板,行PCR扩增出该基因的5′端;将两次所克隆到的cDNA片断进行拼接,获得一条家蝇新抗菌肽基因的cDNA序列,并运用生物信息学相关软件对其进行初步分析。研究结果
1.家蝇抗菌肽基因芯片的制备与相关基因的筛选
据GenBank数据库中所获取的部分昆虫抗菌肽基因序列,用NCBI中相关生物学软件分析出各自的保守序列,根据oligo探针的设计原则,用ArrayDesigner 2.0生物学软件共设计出长度为50bp、Tm为72～77℃、GC含量为40～60%的oligo探针186条,将之点印在特制玻片上制备成oligo芯片;用逆转录PCR标记技术对诱导后家蝇三龄幼虫脂肪体总RNA反转录成cDNA并进行Cy3荧光素标记,与制备的芯片杂交,经洗片、扫描后进行数据分析,在两次重复杂交实验中均能检测到有效杂交信号的基因点共有15个,在其中一次杂交实验中检测到有效杂交信号的基因点共12个,其中所有阳性对照信号清晰,阴性对照和空白对照均为阴性。
2.一个新抗菌肽基因cDNA的克隆与生物信息学分析
对其中一个有效杂交信号的基因点所对应基因的氨基酸保守域设计简并引物,经RT-PCR扩增出一长为131bp的同源片断;结合cDNA文库载体上的上、下游引物,以cDNA文库为模板,分别行PCR扩增出长为366bp的3′端cDNA序列和457bp的5′端cDNA序列,将所得3′端与5′端cDNA序列进行拼接,得到一长为460bp的家蝇抗菌肽新基因的cDNA序列,生物信息学分析该基因序列所编码的蛋白质属于抗菌肽Attacin基因家族,其cDNA序列的3′端是完整的,存在有一个AATAAA的poly(A)加尾信号和一个poly(A)尾巴,但5′显示还不完整,Blast比较显示与双翅目刺舌蝇、新陆原伏蝇抗菌肽Diptericin基因的同源性较高,分别为80%、91%,推导出的所编码的氨基酸序列Blast比较显示与舌刺蝇Diptericin氨基酸序列同源性为77%、与果蝇Diptericin氨基酸序列同源性为59%、与新陆原伏蝇Diptericin氨基酸序列同源性为51%,初步判断该cDNA序列可能为家蝇一新抗菌肽基因。
结论
1.首次制备了家蝇抗菌肽相关基因芯片,并初步筛选到了27个可能与家蝇抗菌肽相关的基因,为进一步克隆其新抗菌肽基因奠定了良好的工作基础;
2.对其中一个有效杂交信号的基因点所对应的基因进行了克隆,成功克隆到了一长为460bp的家蝇新抗菌肽基因,并对其进行了相关的生物信息学分析,为下一步研究家蝇抗菌肽结构和功能,揭示其在家蝇天然免疫体系中所起的作用,进而为用基因工程方法生产家蝇抗菌肽打下了基础。
Antibacterial peptides(ABP) are small kinds of bioactive peptides in hameolymph encoded by some structural genes,and are widespread in the natural organisms, which are an important composition of its immune system, They have non-specific resistance on bacteria, fungi, viruses, parasites and other pathogens, at the same time, These peptides can inhibit the growth of tumor cell and multi-drug resistant bacteria without harmless on the normal cell organisms. Their mechanism is unique so that they have huge potential for the development of new antimicrobial agents. For that reasons, antibacterial peptides have increasingly become a hotspot in the area of life sciences research.
From larvae to adult, Musca domestica lives in the landfill, livestock manure ,where various pathogens reproduce, and can bring a variety of harmful pathogens to human and livestock without being infected and death strangely. This may have originated from its unique natural immune substances to combat various microorganisms, in which ABP play a important role. Musca domestica in China is a good insect resource in the development and application of the medicine because of their wide distributation, easy being raised and lower cost of breeding. Natural antibacterial peptides extracted from the Housefly body costs highly of material, and the way of synthesis are also very expensive. Through genetic engineering methods may be an ideal way to prepare ABP. But this method is on the basis of the obtaining of the related antimicrobial peptide genes.
As a new molecular biology technology ,the gene chip technology was developed in the field of life science in recent years,which has the fast, efficient, sensitive, parallel and many other advantages. Unlike the traditional method of studies by which only one or few genes has been studied, gene chip technology has a very good application prospect on screening in differential gene expression,discoverying the novel genes, detecting the gene of mutations and analyzing the gene polymorphism. Using this technology for the study of screening Musca domestica antibacterial peptide gene has not been reported. In this paper, The oligonucleotide hybrid probes were designed according to the part gene sequences in GenBank . Then, oligonucleotide (oligo) chips were constructed to screen the candidate genes associated with Musca domestica antibacterial peptides, and cloned and analyzed a novel antibacterial peptide gene of Musca domestica.
Objective
To screen the candidate genes associated with Musca domestica antibacterial peptides using gene chip technology, clone and analyze a novel antibacterial peptide gene of Musca domestica,which lay a solide foundation for the further research the Musca domestica antimicrobial peptide structure and functions, reveal their role in their innate immune system ,and then , prepare the Musca domestica antibacterial peptide through genetic engineering methods.
Methods
1. constructed a antibacterial peptide gene chip and screened the antibacterial peptide associated genes of Musca domestica Part insect antibacterial peptide gene orders were acquired from the GenBank, by analyzing mRNA sequence of those insect antibacterial peptide genes with associated free biology softwares in National Center for Biotechnology Informational ( NCBI ) , the encoded areas and the conservative domains of those antibacterial peptide genes were found, Then,the oligo software of Array Designer 2.0 was applied to select hybrid probes with high specificity, identical length and similar melting temperature(Tm) from the conservative sequences, and were synthesized by a chemical process, with the assistance of the automated Gen III Microarray Spotter , those oligo probes were printed on a special ready-made glass, and a cDNA microarray was constructed. The total RNA was extracted from the fat body of Musca domestic third-instar larve induced after 24 hours by E.coli and Staphylococcus aureus, the strands of cDNA were labled with fluoresceine Cy3 using the method of reverse transcription PCR, after prehybridization, hybridization and washing procedure, the results of hybridization were scanned using computer system, and the data were analysised using the software of MIDAS.
2.cloned and analyzed a novel antibacterial peptide gene of Musca domestica
A pair of degenerate primers were designed according the conservative domains in the amino acid orders of the insect antibacterial peptide gene Diptericin,which had been screened with gene chips in the first part, a homologous fragment was amplified by RT-PCR. A specific 5′primer was designed from the cloned homologous fragment ,combined the 3′primer in the plasmid ofλTripEx2 of the cDNA library of Musca domestic larve( which had been constructed by our laboratory),with the template of cDNA library, the 3′end cDNA sequence was amplified by PCR. With the same principle,a specific 3′primer was designed from the cloned 3′end cDNA sequence, combined the 5′primer in the plasmid ofλTripEx2 of the cDNA library, with the template of cDNA library, the 5′end cDNA sequence was amplified by PCR. Removed the overlapping sequences of the two cDNA sequence, a novel antibacterial peptide gene cDNA sequence of Musca domestica was spliced,and analyzed the cDNA sequence with the help of the free bioinformatics software in NCBI.
Results
1.Constructed a antibacterial peptide gene chip and screened the antibacterial peptide associated genes of Musca domestica
Part insect antibacterial peptide gene sequences were acquired from the GenBank, according the design principles of oligo probe, with the help of the biology software Array Designer 2.0, 186 oligo hybrid probes were designed from the conservative sequences , the length were 50bp, melting temperature(Tm)were 72 to 77 degree,and the percent of GC were 40～60, which will be deposited on chips as a microarray in screening the antibacterial peptide associated genes of Musca domestica. The total RNA was extracted from the fat body of Musca domestic third-instar larve induced after 24 hours by E.coli and Staphylococcus aureus, the strands of cDNA were labled with fluoresceine Cy3 using the method of reverse transcription PCR, after prehybridization, hybridization and washing procedure, the results of hybridization were scanned using computer system, and the data were analysised using the software of MIDAS,15 valid hybridization singals were detected through two times of hybridization and scanning, and the other 12 valid hybridization singals were also detected in the one hybridization and scanning, giving a clear positive control signal,whereas the negative and blank control were negative.
2.Cloned and analyzed a novel antibacterial peptide gene of Musca domestica
A pair of degenerate primers were designed according the conservative domains in the amino acid orders of the insect antibacterial peptide gene Diptericin,which had been screened with gene chips in the first part, a 131bp homologous fragment was amplified by RT-PCR.according to the 5′and 3′primers in the plasmid ofλTripEx2 of the cDNA library of Musca domestic larve, a 366bp cDNA sequences of 3′end and a 457bp 5′end were amplified by PCR, at last, a 460bp novel antibacterial peptide gene cDNA sequence of Musca domestica was spliced from the 3′end and 5′end cDNA sequence,and this sequence were analyzed with the help of the bioinformatics software in NCBI,the result indicate that the protein encoded by this cDNA sequence belongs to Attacin gene family, the sequence of the 3′end is complete, existing a putative poly (A) tail signal and a poly (A) tail,but the 5′end is incomplete. Blast comparison showed higher gene homology exists comparing to Diptericin of Glossina morsitans morsitans and Protophormia terraenovae antibacterial peptide gene, 80% and 91%,respectively,the coded amino acid sequence comparison showed homology is 77% , 59% and 51%,comparing to Glossina morsitans morsitans, Drosophila melanogaster and Protophormia terraenovae. from the result of comparison, we can judge the cDNA sequence may be a novel antibacterial peptide gene of Musca domestica.
Conclusion
1. A antibacterial peptide gene chip of Musca domestica was constructed for the first time,and used this gene chip, twenty seven genes possibly related to antibacterial peptide from Musca domestica were screened initially, which established satisfactory foundation for furtherly cloning new antibacterial peptide genes of it.
2. A 460bp antibacterial peptide gene of Musca domestica was successfully cloned, which had been screened with gene chips in the first part, and furtherly analyzed the cDNA sequence with the help of the free bioinformatics software in NCBI,which lay a solide foundation for the further research the Musca domestica antimicrobial peptide structure and functions, reveal their role in their innate immune system ,and then , prepare the Musca domestica antibacterial peptide through genetic engineering methods.
家蝇从幼虫到成虫常生活在垃圾堆、禽畜粪便等有多种病原菌孳生的环境中,携带多种有害病原微生物,能在人、畜中通过机械传播多种疾病,自身却不会因为感染病原菌而死亡,这可能缘于其独特的天然免疫活性物质抵抗各种微生物的浸染,抗菌肽在其中起着主要的作用。家蝇在我国分布广泛,容易饲养,且养殖的成本较低,在医药开发应用方面是一个很好的昆虫资源。但从家蝇体内提取天然抗菌活性物质的成本较高,人工合成也较昂贵,通过基因工程方法可能是规模制备其抗菌肽的理想途径之一,而该方法的前提是要获取其有关的抗菌肽基因。
基因芯片技术是近年来新兴的一种分子生物学技术,具有快速、高效、敏感、平行性等许多优点,克服了传统方法一次只能研究一条或少数几条基因的局限性,在基因差异表达的筛选、新基因的发现、突变基因的检测及基因多态性分析等方面显示出了很好的应用前景。用该技术用于家蝇抗菌肽相关基因的筛选研究还未见报道,本研究以GenBank中部分昆虫抗菌肽基因序列为目标设计寡核苷酸(oligonucleotide)探针,制备成寡核苷酸(oligo)芯片,以筛选家蝇抗菌肽相关基因,并对所筛选出的其中一个基因的cDNA序列进行克隆,运用生物信息学软件进行初步分析。
研究目的
运用基因芯片技术筛选家蝇抗菌肽相关基因,对其中筛选出的一个基因的cDNA序列进行克隆分析,为进一步研究家蝇抗菌肽结构和功能,揭示其在家蝇天然免疫体系中所起的作用,进而为用基因工程方法生产家蝇抗菌肽打下基础。
研究方法
1.家蝇抗菌肽基因芯片的制备与相关基因的筛选
从GenBank数据库中获取部分昆虫抗菌肽基因序列,运用NCBI中相关服务器对各基因的全长进行分析,找出其编码区,分析出各自的保守序列,以此作为设计oligo探针的备选序列,用生物学软件Array Designer 2.0对所选序列设计特异性高、Tm值接近、长度均一的oligo探针,用直接点样法将探针点印在特制玻片上制备成寡核苷酸(oligonucleotide)芯片;提取经大肠杆菌(E.coli)和金黄色葡萄球菌(Staphylococcus aureus)免疫诱导后24小时的家蝇三龄幼虫脂肪体总RNA,逆转录成cDNA并标记上荧光标记物Cy3,与制备的基因芯片杂交,经洗片、扫描处理后进行数据分析。
2.一个家蝇新抗菌肽基因cDNA的克隆与生物信息学分析对筛选出的其中一个有效杂交信号点所对应基因的氨基酸保守域设计简并引物,行RT-PCR扩增出一个同源片断;结合cDNA文库(本实验室已构建)载体上的下游引物,从所克隆到的同源片断中设计出一条特异性上游引物,以cDNA文库为模板,行PCR扩增出该基因的3′端;再结合文库载体上的上游引物,从所克隆到的3′端序列中设计出一条特异性下游引物,以cDNA文库为模板,行PCR扩增出该基因的5′端;将两次所克隆到的cDNA片断进行拼接,获得一条家蝇新抗菌肽基因的cDNA序列,并运用生物信息学相关软件对其进行初步分析。研究结果
1.家蝇抗菌肽基因芯片的制备与相关基因的筛选
据GenBank数据库中所获取的部分昆虫抗菌肽基因序列,用NCBI中相关生物学软件分析出各自的保守序列,根据oligo探针的设计原则,用ArrayDesigner 2.0生物学软件共设计出长度为50bp、Tm为72～77℃、GC含量为40～60%的oligo探针186条,将之点印在特制玻片上制备成oligo芯片;用逆转录PCR标记技术对诱导后家蝇三龄幼虫脂肪体总RNA反转录成cDNA并进行Cy3荧光素标记,与制备的芯片杂交,经洗片、扫描后进行数据分析,在两次重复杂交实验中均能检测到有效杂交信号的基因点共有15个,在其中一次杂交实验中检测到有效杂交信号的基因点共12个,其中所有阳性对照信号清晰,阴性对照和空白对照均为阴性。
2.一个新抗菌肽基因cDNA的克隆与生物信息学分析
对其中一个有效杂交信号的基因点所对应基因的氨基酸保守域设计简并引物,经RT-PCR扩增出一长为131bp的同源片断;结合cDNA文库载体上的上、下游引物,以cDNA文库为模板,分别行PCR扩增出长为366bp的3′端cDNA序列和457bp的5′端cDNA序列,将所得3′端与5′端cDNA序列进行拼接,得到一长为460bp的家蝇抗菌肽新基因的cDNA序列,生物信息学分析该基因序列所编码的蛋白质属于抗菌肽Attacin基因家族,其cDNA序列的3′端是完整的,存在有一个AATAAA的poly(A)加尾信号和一个poly(A)尾巴,但5′显示还不完整,Blast比较显示与双翅目刺舌蝇、新陆原伏蝇抗菌肽Diptericin基因的同源性较高,分别为80%、91%,推导出的所编码的氨基酸序列Blast比较显示与舌刺蝇Diptericin氨基酸序列同源性为77%、与果蝇Diptericin氨基酸序列同源性为59%、与新陆原伏蝇Diptericin氨基酸序列同源性为51%,初步判断该cDNA序列可能为家蝇一新抗菌肽基因。
结论
1.首次制备了家蝇抗菌肽相关基因芯片,并初步筛选到了27个可能与家蝇抗菌肽相关的基因,为进一步克隆其新抗菌肽基因奠定了良好的工作基础;
2.对其中一个有效杂交信号的基因点所对应的基因进行了克隆,成功克隆到了一长为460bp的家蝇新抗菌肽基因,并对其进行了相关的生物信息学分析,为下一步研究家蝇抗菌肽结构和功能,揭示其在家蝇天然免疫体系中所起的作用,进而为用基因工程方法生产家蝇抗菌肽打下了基础。
Antibacterial peptides(ABP) are small kinds of bioactive peptides in hameolymph encoded by some structural genes,and are widespread in the natural organisms, which are an important composition of its immune system, They have non-specific resistance on bacteria, fungi, viruses, parasites and other pathogens, at the same time, These peptides can inhibit the growth of tumor cell and multi-drug resistant bacteria without harmless on the normal cell organisms. Their mechanism is unique so that they have huge potential for the development of new antimicrobial agents. For that reasons, antibacterial peptides have increasingly become a hotspot in the area of life sciences research.
From larvae to adult, Musca domestica lives in the landfill, livestock manure ,where various pathogens reproduce, and can bring a variety of harmful pathogens to human and livestock without being infected and death strangely. This may have originated from its unique natural immune substances to combat various microorganisms, in which ABP play a important role. Musca domestica in China is a good insect resource in the development and application of the medicine because of their wide distributation, easy being raised and lower cost of breeding. Natural antibacterial peptides extracted from the Housefly body costs highly of material, and the way of synthesis are also very expensive. Through genetic engineering methods may be an ideal way to prepare ABP. But this method is on the basis of the obtaining of the related antimicrobial peptide genes.
As a new molecular biology technology ,the gene chip technology was developed in the field of life science in recent years,which has the fast, efficient, sensitive, parallel and many other advantages. Unlike the traditional method of studies by which only one or few genes has been studied, gene chip technology has a very good application prospect on screening in differential gene expression,discoverying the novel genes, detecting the gene of mutations and analyzing the gene polymorphism. Using this technology for the study of screening Musca domestica antibacterial peptide gene has not been reported. In this paper, The oligonucleotide hybrid probes were designed according to the part gene sequences in GenBank . Then, oligonucleotide (oligo) chips were constructed to screen the candidate genes associated with Musca domestica antibacterial peptides, and cloned and analyzed a novel antibacterial peptide gene of Musca domestica.
Objective
To screen the candidate genes associated with Musca domestica antibacterial peptides using gene chip technology, clone and analyze a novel antibacterial peptide gene of Musca domestica,which lay a solide foundation for the further research the Musca domestica antimicrobial peptide structure and functions, reveal their role in their innate immune system ,and then , prepare the Musca domestica antibacterial peptide through genetic engineering methods.
Methods
1. constructed a antibacterial peptide gene chip and screened the antibacterial peptide associated genes of Musca domestica Part insect antibacterial peptide gene orders were acquired from the GenBank, by analyzing mRNA sequence of those insect antibacterial peptide genes with associated free biology softwares in National Center for Biotechnology Informational ( NCBI ) , the encoded areas and the conservative domains of those antibacterial peptide genes were found, Then,the oligo software of Array Designer 2.0 was applied to select hybrid probes with high specificity, identical length and similar melting temperature(Tm) from the conservative sequences, and were synthesized by a chemical process, with the assistance of the automated Gen III Microarray Spotter , those oligo probes were printed on a special ready-made glass, and a cDNA microarray was constructed. The total RNA was extracted from the fat body of Musca domestic third-instar larve induced after 24 hours by E.coli and Staphylococcus aureus, the strands of cDNA were labled with fluoresceine Cy3 using the method of reverse transcription PCR, after prehybridization, hybridization and washing procedure, the results of hybridization were scanned using computer system, and the data were analysised using the software of MIDAS.
2.cloned and analyzed a novel antibacterial peptide gene of Musca domestica
A pair of degenerate primers were designed according the conservative domains in the amino acid orders of the insect antibacterial peptide gene Diptericin,which had been screened with gene chips in the first part, a homologous fragment was amplified by RT-PCR. A specific 5′primer was designed from the cloned homologous fragment ,combined the 3′primer in the plasmid ofλTripEx2 of the cDNA library of Musca domestic larve( which had been constructed by our laboratory),with the template of cDNA library, the 3′end cDNA sequence was amplified by PCR. With the same principle,a specific 3′primer was designed from the cloned 3′end cDNA sequence, combined the 5′primer in the plasmid ofλTripEx2 of the cDNA library, with the template of cDNA library, the 5′end cDNA sequence was amplified by PCR. Removed the overlapping sequences of the two cDNA sequence, a novel antibacterial peptide gene cDNA sequence of Musca domestica was spliced,and analyzed the cDNA sequence with the help of the free bioinformatics software in NCBI.
Results
1.Constructed a antibacterial peptide gene chip and screened the antibacterial peptide associated genes of Musca domestica
Part insect antibacterial peptide gene sequences were acquired from the GenBank, according the design principles of oligo probe, with the help of the biology software Array Designer 2.0, 186 oligo hybrid probes were designed from the conservative sequences , the length were 50bp, melting temperature(Tm)were 72 to 77 degree,and the percent of GC were 40～60, which will be deposited on chips as a microarray in screening the antibacterial peptide associated genes of Musca domestica. The total RNA was extracted from the fat body of Musca domestic third-instar larve induced after 24 hours by E.coli and Staphylococcus aureus, the strands of cDNA were labled with fluoresceine Cy3 using the method of reverse transcription PCR, after prehybridization, hybridization and washing procedure, the results of hybridization were scanned using computer system, and the data were analysised using the software of MIDAS,15 valid hybridization singals were detected through two times of hybridization and scanning, and the other 12 valid hybridization singals were also detected in the one hybridization and scanning, giving a clear positive control signal,whereas the negative and blank control were negative.
2.Cloned and analyzed a novel antibacterial peptide gene of Musca domestica
A pair of degenerate primers were designed according the conservative domains in the amino acid orders of the insect antibacterial peptide gene Diptericin,which had been screened with gene chips in the first part, a 131bp homologous fragment was amplified by RT-PCR.according to the 5′and 3′primers in the plasmid ofλTripEx2 of the cDNA library of Musca domestic larve, a 366bp cDNA sequences of 3′end and a 457bp 5′end were amplified by PCR, at last, a 460bp novel antibacterial peptide gene cDNA sequence of Musca domestica was spliced from the 3′end and 5′end cDNA sequence,and this sequence were analyzed with the help of the bioinformatics software in NCBI,the result indicate that the protein encoded by this cDNA sequence belongs to Attacin gene family, the sequence of the 3′end is complete, existing a putative poly (A) tail signal and a poly (A) tail,but the 5′end is incomplete. Blast comparison showed higher gene homology exists comparing to Diptericin of Glossina morsitans morsitans and Protophormia terraenovae antibacterial peptide gene, 80% and 91%,respectively,the coded amino acid sequence comparison showed homology is 77% , 59% and 51%,comparing to Glossina morsitans morsitans, Drosophila melanogaster and Protophormia terraenovae. from the result of comparison, we can judge the cDNA sequence may be a novel antibacterial peptide gene of Musca domestica.
Conclusion
1. A antibacterial peptide gene chip of Musca domestica was constructed for the first time,and used this gene chip, twenty seven genes possibly related to antibacterial peptide from Musca domestica were screened initially, which established satisfactory foundation for furtherly cloning new antibacterial peptide genes of it.
2. A 460bp antibacterial peptide gene of Musca domestica was successfully cloned, which had been screened with gene chips in the first part, and furtherly analyzed the cDNA sequence with the help of the free bioinformatics software in NCBI,which lay a solide foundation for the further research the Musca domestica antimicrobial peptide structure and functions, reveal their role in their innate immune system ,and then , prepare the Musca domestica antibacterial peptide through genetic engineering methods.
引文
[1] Newkirk, Marianna M. Antibacterial Antibodies and Arthritis: Is There a Link? [J].Journal of Clinical Rheumatology. 2006,12(1):1～2.
[2] 王荫长 主编.昆虫生物化学.北京:中国农业出版社,2001,240～312.
[3] Park S Y, Kim K M, Lee J H,et al. Extracellular Gelatinase of Enterococcus faecalis Destroys a Defense System in Insect Hemolymph and Human Serum. Infection and Immunity, 2007,75(4): 1861～1869.
[4] Pal S, Leger R J S, Wu L P.Fungal Peptide Destruxin A Plays a Specific Role in Suppressing the Innate Immune Response in Drosophila melanogaster. J. Biol. Chem., 2007,282(12):8969～8977.
[5] Nüsslein K, Arnt L, Rennie J, et al. Broad-spectrum antibacterial activity by a novel abiogenic peptide mimic.Microbiology,2006,152: 1913～1918.
[6] Kamysz W,Turecka K. Antimicrobial preservative effectiveness of natural peptide antibiotics. Acta Pol Pharm,2005,62(5):341～344.
[7] Yeaman M R, Yount N Y. Mechanisms of Antimicrobial Peptide Action and Resistance. pharmacological reviews, 2003,55(1):27～56.
[8] Franco O L, Murad A M, Leite J R,et al.Identification of a cowpea γ-thionin with bactericidal activity. FEBS Journal, 2006,273:3489～3497.
[9] Bommineni Y R, Dai H, Gong Y X,et al.Fowlicidin-3 is an -helical cationic host defense peptide with potent antibacterial and lipopolysaccharide-neutralizing activities. FEBS Journal, 2007, 274: 418～428.
[10] Silva P D, Jouvensal L, Lamberty M, et al.Solution structure of termicin, an antimicrobial peptide from the termite Pseudacanthotermes spiniger. Protein Science, 2003,12:438～446.
[11] 柏鸣,周立.家蝇抗菌蛋白的部分结构信息及生物学活性.中国生物化学与分子生物学报,2002,18(5):633～637.
[12] Gueguen Y, Herpin A, Aumelas A,et al.Characterization of a Defensinfrom the Oyster Crassostrea gigas: recombinant production, folding, solution structure, antimicrobial activities, and gene expression. J. Biol. Chem.,2006,281(1): 313～323.
[13] Fjell C D, Hancock R E.W, Cherkasov A. AMPer: a database and an automated discovery tool for antimicrobial peptides. Bioinformatics,2007 23(9):1148～1155.
[14] Xu X,Jin F,Yu X, et al. Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli. Protein Expr and Purif, 2007,53(2):293～301.
[15] Waniek P J, Stock P, Mayer C,et al. Sequence characterization and expression. patterns of defensin and lysozyme encoding genes from the gut of the reduviid bug Triatoma brasiliensis.Insect Biochemistry and Molecular Biology,2006,36(7): 547～560.
[16] Imler JL,Bulet P. Antimicrobial peptides in Drosophila: structures, activities and gene regulation. Chem Immunol Allergy, 2005,86:1～21.
[17] Hu Y, Aksoy S. An antimicrobial peptide with trypanocidal activity characterized from Glossina morsitans morsitans. Insect Biochemistry and Molecular Biology,2005,35(2):105～115.
[18] Rahuma N,Ghenghesh KS,Ben Aissa R,et al. Carriage by the housefly (Musca domestica) of multiple-antibiotic-resistant bacteria that are potentially pathogenic to humans, in hospital and other urban environments in Misurata, Libya. Ann trop Med Parasitol,2005,99(8):795～802.
[19] Moreira C K,Capurro M,Calvo E,et al. The Musca domestica larval hexamerin is composed of multiple, similar polypeptides.Insect Biochem Mol Biol,2003,33(4):389～395.
[20] Naitza S,Ligoxygakis P.Antimicrobial defences in Drosophila:the story so far.Mol Immunol.2004,40(12):887～896.
[21] Hultmark D.Drosophila immunity:paths and patterns.Curr Opin Immunol.2003,15(1):12～9.
[22] Xia Q Y, Zhou Z Y, Lu C, et al. A Draft Sequence for the Genome ofthe Domesticated Silkworm (Bombyx mori).Science,2004,306(10):1937～ 1940.
[23] 宫霞,乐国伟,施用晖,付景春. 电泳制备家蝇幼虫抗菌肽及其性质. 无锡轻工大学学报,2003,22(6):25～30.
[24] 文彩虹,曲传智,李东英,等. 家蝇免疫血淋巴对 SMMC-7721 肝癌细胞超微结构和增殖周期的影响[J]. 河南肿瘤学杂志,2004,17(2):100～102.
[25] 刘晖,万启惠,贺莉芳,等. 家蝇幼虫抗菌蛋白的诱导及抗菌特性[J].中国公共卫生,2006,22(5):568 ～569.
[26] 万启惠,刘晖,贺莉芳,等. 家蝇幼虫抗菌蛋白的抗菌特性及其组成[J].中国媒介生物学及控制杂志,2006,17(3):191～193.
[27] 曹小红,陈一,张燕,等. 家蝇蛹凝集素的分离工艺和性质的初步研究[J]. 生物化学与生物物理进展,2003,30(3):442～445.
[28] 安春菊,耿华,郝友进,等. 不同提取工艺对家蝇幼虫蛋白粗提液抗菌活性的影响. 昆虫学报,2004,47(5):691～694.
[29] 安春菊,李德森,杜荣岑. 家蝇幼虫中与抗菌功能相关的蛋白和多肤的分析.卫生研究,2004,33(1):86～88.
[30] 安春菊,石明,盛长忠,等. 家蝇幼虫抗菌相关蛋白/多肤的诱导及抗菌活性分析. 昆虫学报,2003,46 ( 5) : 545～548.
[31] 齐静姣,曲传智. 不同诱导源诱导家蝇抗菌物质的动力学研究. 中国人兽共患病杂志 2004,20(6):554～555.
[32] Xanthopoulos KG,Lee JY,Gan RB,et al.The structure of the gene for cecropia B,an antibacterial immune protein from Hyalophora cecropia.Eur J Biochem,1988,172∶371-376.
[33] Lepage P, Bitsch F,van Dorsselaer A,et al. Determination of disulfide bridges in natural and recombinant insect defensin A.Eur. J. Biochem, 1991,196:735～742.
[34] Bonmatin J M, Genest M,Ptak M,et al.Progress in multidimensional NMR investigations of peptide and protein 3-D structures in solution. From structure to functional aspects.Biochimie,1992,74:825～836.
[35] Cornet B, Bonmatin J M,Vovelle F.Refined three-dimensional solution structure of insect defensin A.Structure,1995,3:435～448.
[36] 王来城,王金星,王来元,等. 家蝇防御素基因的cDNA克隆及序列分析. 动 物学报,2003,49(3):408～413.
[37] Liang Y,Wang JX,Zhao XF,et al.Molecular cloning and characterization of cecropin from the housefly (Musca domestica), and its expression in Escherichia coli. Dev Comp Immunol,2006,30(3):249～257.
[38] 耿华,安春菊,杜荣骞,等.家蝇攻击素(Attacin)基因的克隆与表达.遗传学报,2004,31(12):1344～1350.
[39] 许琴英,金小宝,朱家勇,等.家蝇幼虫防御素基因 cDNA 的克隆与序列分 析.热带医学杂志,2004;4(5):513～517.
[40] 金小宝,徐建华,朱家勇,等.家蝇幼虫天蚕素基因的克隆与序列分析.中国热带医学,2004,4(6):903～906.
[41] Nie D,Xiang Y. Molecular cloning and characterization of a novel human testis-specific gene by use of digital differential display.J Genet, 2006, 85(1):57～62.
[42] Tessitori M, Maria G, Capasso C,et al.Differential display analysis of gene expression in Etrog citron leaves infected by Citrus viroid III. Biochim Biophys Acta, In Press, Corrected Proof, Available online 24 March, 2007.
[43] Liu GY, Xiong YZ.Isolation, sequence analysis and expression profile of a novel porcine gene, NIP7, differentially expressed in the Longissimus dorsi muscle tissues from Meishan, Meishan x Large White cross and Large White pigs. Mol Biol Rep. Published online: December 23,2006.
[44] Hui XJ,Dean W, Yu L Ch,et al.Molecular Cloning and Localization of Human Ataxin2- like Gene. High Technology letters, 1998,4(2):100～104.
[45] 周国岭,杨光圣,傅廷栋. 基因克隆技术. 华中农业大学学报,2001,20(6):584～592.
[46] Lievens S, Goormachtig S,Holsters M. A critical evaluation of differential display as a tool to identify genes involved in legumenodulation: looking back and looking forward[J]. Nucleic Acids Research,2001,29(17):3459～3468.
[47] Mrazek J,Kreutmayer S B,Grasser F A, et al.Subtractive hybridization identifies novel differentially expressed ncRNA species in EBV-infected human B cells. Nucleic Acids Res. Published online: May 3,2007.
[48] Chen Z J, Shen H,Tew K D. Gene expression profiling using a novel method: amplified differential gene expression (ADGE). Nucleic Acids Res,2001,29, e46.
[49] Bertone P,Stolc V,Royce T E,et al. Global Identification of Human Transcribed Sequences with Genome Tiling Arrays[J]. Science,2004,306(24):2242～2246.
[50] Nagino K,Nomura O, Takii Y,et al.Ultrasensitive DNA Chip: Gene Expression Profile Analysis without RNA Amplification. Journal of Biochemistry,2006,139(4):697～703.
[51] 金小宝,朱家勇,王婷婷. 家蝇幼虫脂肪体cDNA文库的构建及初步鉴定. 中国人兽共患病杂志,2005,21(1):52～55.
[52] Nie D,Xiang Y. Molecular cloning and characterization of a novel human testis-specific gene by use of digital differential display. Genet, 2006,85(1):57～62.
[53] Frigessi A,Wiel M A,Holden M,et al. Genome-wide estimation of transcript concentrations from spotted cDNA microarray data. Nucleic Acids Res,2005,33(17): e143.
[54] Ramachandran N,Hainsworth E,Bhullar B. Self-Assembling Protein Microarrays. Science,2004,305(2):86～90.
[55] Wu C,Carta R,Zhang L. Sequence dependence of cross hybridization on short oligo microarrays. Nucleic Acids Research,2005,33(9):e84.
[56] He Zh L,Wu L Y,Li X Y,et al. Empirical Establishment of Oligonucleotide Probe Design Criteria. Appl Environ Microbiol, 2005,71(7): 3753～3760.
[57] 卢圣栋 主编.现代分子生物学实验技术(第二版).北京:中国协和医科大学出版社,1999,136～154.
[58] Lowenberger C,Maurice M,Vizioli J,et al.Antimicroial activity specerum,cDNA cloning,and mRNA expression of a newly isolated member of the cecropin family from the mosquito vetor.Aedes aegypti,J Biol Chem,1999,274:20092～20097.
[59] Knight J. When the chips are down. Nature,2001,410:860～861.
[60] Luscombe NM,Babu MM. GenCompass: a universal system for analysing gene expression for any genome. Trends Biotechnol, 2004, 22(11):552～555.
[61] He W,Ji Z Y,Huang C Y. Molecular cloning of a novel cDNA from Mus muscular BALB/c mice encoding glycosyl hydrolase family 1: a homolog of human lactase-phlorizin hydrolase. Biomed Environ Sci,2006,19(5): 340～345.
[62] Fang Ch J, Long R Zh, Ying K X,et al.Isolation Characterization and Expression Analysis of Male Sterility Gene Homology Sequence in Wheat. Actn Geneticn Sinica,2005,32(6):566～570.
[63] Grauvogel C,Petersen J.Isoprenoid biosynthesis authenticates the classification of the green alga Mesostigma viride as an ancient streptophyte. Gene, Published online: Mar,15,2007.
[64] Lee C C,Wu X,Gibbs R A,et al.Generation of cDNA probes directed by amina acid sequence: Cloning of urate oxidase[J].Science,1988,239: 1288～1291.
[65] Zhong J G, Mei Y B,Li F T, et al.Application of degenerate primers in cloning hsp70 gene families of Dunaliella salina. Journal of Zheng zhou Universitv(Medical Sciences),2003,38(6):875～877.
[66] 王洪振,周晓馥,宋朝霞,等.简并 PCR 技术及其在基因克隆中的应用.遗传,2003,25(2):201～204.
[67] 张新宇,高燕宁.PCR 引物设计及软件使用技巧. 生物信息学,2004,2(4):15～18.
[68] Wang N,Sun Y H, Liu J, et al. Molecular characterization of common carp (Cyprinus carpio)Sonic Hedgehog and discovery of its maternal expression.Dev Genes Evol,2007,217(4):299～305.
[69] Franco P F, Hedreyda C T. Amplification and sequence analysis of the full length toxR gene in Vibrio harveyi.J Gen Appl Microbiol,2006, 52(5):281～287.
[70] Kumar C G, Larson J H, Band M R,et al. Discovery and characterization of 91 novel transcripts expressed in cattle placenta. BMC Genomics. 2007 8(1):113～121.
[71] Clark JM.Novel non-templated nucleotide adittion reations catalyzed by procaryotic and eucaryotic DNA polymerase.Nucleic Acids Res.1988, 16:9677～9686.
[72] Marchuk D,Drumm M,Saulino A,et al.Construction of T-vectors,a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res.1991,19:1154～1157.
[73] 卢圣栋 主编.现代分子生物学实验技术(第二版).北京:中国协和医科大 学出版社,1999,287～290.
[74] Chen YB,Bergen P,Gadd C,etal. The Online Bioinformatics Resources Collection at the University of Pittsburgh Health Sciences Library System—a one-stop gateway to online bioinformatics databases and software tools. Nucleic Acids Research,2007,35(Database issue): D780～D785.
[75] Feng Zh Z,Qing S Ch, Cai F Y,et al.Isolation and Analysis of a Novel MYC Gene from Rice.Acta Genetica Sinica,2005,32(4):393～398.
[1] International Human Genome Sequence Consortium. Finishing the euchromatic sequence of the human genome. Nature,2004,431:931～945.
[2] Nygaard V,Hovig E. Options available for profiling small samples: a review of sample amplification technology when combined with microarray profiling. Nucleic Acids Research,2006,34(3):996～1014.
[3] Chaudhuri JD. Genes arrayed out for you: the amazing world of microarrays. Med Sci Monit, 2005, 11(2): RA52～62.
[4] Ramachandran N,Hainsworth E, Bhullar B ,et al. Self-Assembling Protein Microarrays. Science,2004,305(2):86～90.
[5] Ali TR,Li MS,Langford PR. Monitoring gene expression using DNA arrays. Meths Mol Med,2003,71:119～134.
[6] Nie D,Xiang Y. Molecular cloning and characterization of a novel human testis-specific gene by use of digital differential display.J Genet, 2006, 85(1):57～62.
[7] Lievens S,Goormachtig S,Holsters M. A critical evaluation of differential display as a tool to identify genes involved in legume nodulation: looking back and looking forward[J]. Nucleic Acids Research,2001,29(17):3459～3468.
[8] Bertone P,Stolc V,Royce T E,et al. Global Identification of Human Transcribed Sequences with Genome Tiling Arrays. Science,2004,306(24):2242～2246.
[9] Ciro M,Bracken AP,Helin K. Profiling cancer. Curr Opin Cell Biol,2003,15(2):213～220.
[10] Yao J,Weremowicz S,Feng B,et al. Combined cDNA Array Comparative Genomic Hybridization and Serial Analysis of Gene Expression Analysis of Breast Tumor Progression. Cancer Research,2006,66,4065-4078.
[11] Slater EP,Diehl SM,Langer P,et al. Analysis by cDNA microarrays of gene expression patterns of human adrenocortical tumors. European Journal of Endocrinology,2006,154(4):587～598.
[12] 彭桂福,马文丽,郑文岭,等. DNA 芯片技术筛选 K562 肿瘤细胞特异性基因. 第一军医大学学报,2004,24(5):525～528.
[13] Baelde HJ,Eikmans M,Doran PP,et al. Gene expression profiling in glomeruli from human kidneys with diabetic nephropathy. Am J Kidney Dis,2004,43(4):636～650.
[14] 郑敬民,刘志红,张鑫,等. 一个糖尿病肾病相关新基因的筛选、克隆和序列分析. 生物化学与生物物理进展,2004,31(1):47～53.
[15] 易著文,马祖祥,吴小川,等. 原发性肾病综合征相关基因的筛选. 中华儿科杂志,2002,40(12):724～727.
[16] Basile DP,Fredrich K,Alausa M,et al. Identification of persistently altered gene expression in the kidney after functional recovery from ischemic acute renal failure. Renal Physiol,2005,288:F953～F963.
[17] Zhang HG,Hyde K,Page GP,et al. Novel tumor necrosis factor alpha- regulated genes in rheumatoid arthritis. Arthritis Rheum,2004,50(2):420～431.
[18] Ishii T,Onda H,Tanigawa A, et al. Isolation and Expression Profiling of Genes Upregulated in the Peripheral Blood Cells of Systemic Lupus Erythematosus Patients. DNA Research,2005,12(6):429～439.
[19] Chowers I, Gunatilaka TL,Farkas RH,et al. Identification of Novel Genes Preferentially Expressed in the Retina Using a Custom Human Retina cDNA Microarray. Investigative Ophthalmology and Visual Science,2003,44:3732～3741.
[20] Nishimura DY,Swiderski RE,Searby CC,et al. Comparative genomics and gene expression analysis identifies BBS9,a new Bardet-Biedl syndrome gene[J]. Am J Hum Genet,2005,77(6):1021～1033.
[21] 陈海,郑军华,闵志廉,等. 基因表达谱芯片在免疫高敏相关基因筛选中的应用. 第二军医大学学报,2005,26(1):75～79.
[22] Roussoulieres ALS,Raisky O,Chalabreysse L,et al. Identification and Characterization of Two Genes (MIP-1?, VE-CADHERIN) Implicated in Acute Rejection in Human Heart Transplantation Use of Murine Models in Tandem With cDNA Arrays. Circulation,2005,111:2636～2644.
[23] Wang W,Cassidy J,O'Brien V,et al. Mechanistic and predictive profiling of 5-Fluorouracil resistance in human cancer cells. Cancer Res,2004,64(22):8167～8176.
[24] Aragon LM,Navarro F,Heiser V,et al. Rapid detection of specific gene mutations associated with isoniazid or rifampicin resistance in Mycobacterium tuberculosis clinical isolates using non-fluorescent low-density DNA microarrays. Journal of Antimicrobial Chemotherapy,2006,57(5):825～831.
[25] Kang JX,Liu J,Wang J D,et al. The extract of huanglian,a medicinal herb, induces cell growth arrest and apoptosis by up regulation of interferon-? and TNF- in human breast cancer cells. Carcinogenesis,2005,26(11):1934～1939.
[26] Chen JW,Sun CM,Sheng WL,et al. Expression Analysis of Up-RegulatedGenes Responding to Plumbagin in Escherichia coli. Journal of Bacteriology,2006,188(2):456～463.
[27] Vandepoele K,Vlieghe K,Florquin K,et al. Genome-Wide Identification of Potential Plant E2F Target Genes. Plant Physiology,2005,139,pp:316～328.
[28] Misson J,Raghothama KG,Jain A,et al. A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation. PLANT BIOLOGY,2005,102(33):11934～11939.
[29] Madsen M L,Nettleton D,Thacker E L,et al. Transcriptional Profiling of Mycoplasma hyopneumoniae during Heat Shock Using Microarrays. Infection and Immunity,2006,74(1):160～166.
[30] Cui L,Lian JQ,Neoh HM,et al. DNA Microarray-Based Identification of Genes Associated with Glycopeptide Resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy,2005,49(8):3404～3413.
[31] David JP , Strode C , Vontas J , et al. The Anopheles gambiae detoxification chip: A highly specific microarray to study metabolic-based insecticide resistance in malaria vectors. GENETICS,2005,102(11):4080～4084.
[32] Gregorio ED,Spellman PT, Rubin GM, et al. Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays. Proc Natl Acad Sci U S A : Genetics, 2001,98(22):12590～12595.
[33] Ooi SL,Shoemaker DD,Boeke JD. A DNA microarray-based genetic screen for nonhomologous end-joining mutants in Saccharomyces cerevisiae. Science,2001,294(5551):2552～2556.
[34] Sarropoulou1 E,Kotoulas1 G,Power D M, et al. Gene expression profiling of gilthead sea bream during early development and detection of stress-related genes by the application of cDNA microarray technology. Physiol Genomics,2005,23: 182～191.
[35] Knight J. When the chips are down. Nature,2001,410:860～861.
[36] Luscombe NM,Babu MM. GenCompass: a universal system for analysing gene expression for any genome. Trends Biotechnol, 2004, 22(11):552～555.
[1]Kamysz W,Turecka K. Antimicrobial preservative effectiveness of natural peptide antibiotics[J]. Acta Pol Pharm,2005,62(5):341～344.
[2] Rahuma N,Ghenghesh KS,Ben Aissa R,et al. Carriage by the housefly (Musca domestica) of multiple-antibiotic-resistant bacteria that are potentially pathogenic to humans, in hospital and other urban environments in Misurata, Libya. Ann trop Med Parasitol,2005,99(8):795～802.
[3] Moreira C K,Capurro M,Calvo E,et al. The Musca domestica larval hexamerin is composed of multiple, similar polypeptides[J].Insect Biochem Mol Biol,2003,33(4):389～395.
[4] 宫霞,乐国伟,施用晖,等. 电泳制备家蝇幼虫抗菌肽及其性质[J]. 无锡轻工大学学报,2003,22(6):25～30.
[5] 安春菊,李德森,杜荣骞. 家蝇幼虫中与抗菌功能相关的蛋白和多肽的分析[J].卫生研究,2004,33(1):868～8.
[6] 盛长忠,安春菊,耿 华,等. 一种家蝇幼虫热稳定抗菌肽的分离纯化[J]. 南开大学学报(自然科学).2002,35(4):6～10.
[7] 刘晖,万启惠,贺莉芳,等. 家蝇幼虫抗菌蛋白的诱导及抗菌特性[J].中国公共卫生,2006,22(5):568 ～569.
[8] 万启惠,刘晖,贺莉芳,等. 家蝇幼虫抗菌蛋白的抗菌特性及其组成[J].中国媒介生物学及控制杂志,2006,17(3):191～193.
[9] 陈留存,王金星,刘瑶,等. 家蝇抗菌肽的分离纯化及性质研究. 山东大学学报(自然科学版),2001,36(3):351～357.
[10] 柏鸣,周立.家蝇抗菌蛋白的部分结构信息及生物学活性[J].中国生物化学与分子生物学报,2002,18(5):633～637.
[11] 耿华,安春菊,杜荣骞,等.家蝇攻击素(Attacin)基因的克隆与表达[J].遗传学报,2004,31(12):1344～1350.
[12] Liang Y , Wang JX , Zhao XF , et al. Molecular cloning and characterization of cecropin from the housefly (Musca domestica), and its expression in Escherichia coli[J]. Dev Comp Immunol , 2006 ,30(3):249～257.
[13] 王来城,王金星,王来元,等. 家蝇防御素基因的cDNA克隆及序列分析[J]. 动物学报,2003,49(3):408～413.
[14] 安春菊,耿华,郝友进,等. 不同提取工艺对家蝇幼虫蛋白粗提液抗菌活性的影响[J]. 昆虫学报,2004,47(5):691～694.
[15] 周义文,尹一兵,涂植光,等. 家蝇抗菌肽抗菌活性及抗菌机制的初步研究[J].中国抗生素杂志,2004,29(5):272～274.
[16] 宫霞,施用晖,乐国伟. 家蝇幼虫抗菌肽MDL-1的分离纯化及其对大肠杆菌超微结构的影响[J]. 昆虫学报,2004,47(1):8～13.
[17] 宫霞,施用晖,乐国伟.抗菌活性肽与细菌染色体DNA的相互作用机理[J].自然科学进展,2004,14(5):509～513.
[18] 徐建华,朱家勇,金小宝,等. 抗菌肽Cecropin-His-6基因的真核表达及其生物活性研究[J].中国热带医学,2006,6(7):1119～1121.
[19] 金小宝,朱家勇、刘雷山,等. 家蝇抗菌肽Defensin基因在COS-7细胞中的瞬时表达[J].生物技术,2006,16(4):10～11.
[20] Gong X,Le GW,Li YF. Antibacterial spectrum of antibacterial peptides from Musca domestica larvae and synergic interaction between the peptides and antibiotics[J]. Wei Sheng Wu Xue Bao,2005,45(4):516～520.
[21] 赵东红,张双全,戴祝英,等. 重组家蚕抗菌肽 CM4 对癌细胞骨架及核骨架破坏作用的观察[J].高技术通讯,2000,10(1):23～27.
[22] 文彩虹,曲传智,李东英,等. 家蝇免疫血淋巴对 SMMC-7721 肝癌细胞超微结构和增殖周期的影响[J]. 河南肿瘤学杂志,2004,17(2):100～102.
[23] 贺莉芳,万启惠,刘晖,等. 家蝇幼虫抗菌蛋白诱导黑色素瘤 A375 细胞凋亡的探讨[J]. 中国媒介生物学及控制杂志,2006,17(1):20～21.
[24] 曹小红,陈一,张燕,等. 家蝇蛹凝集素的纯化及其免疫调节和抑菌作用[J]. 中华微生物学和免疫学杂志,2003,23(10):297～300.
[25] Wang Z. Wang G. APD: the Antimicrobial Peptide Database.Nucleic Acides Res,2004,32:D590～D592.
[26] Gallo S A,Wang W,Rawat S S,et al.θ-Defensins Prevent HIV-1 Env-mediated Fusion by Binding gp41 and Blocking 6-Helix Bundle Formation[J]. J. Biol. Chem.,2006,281(27): 18787～18792.
[27] 王芙蓉,艾辉,雷朝亮,等. 家蝇幼虫组织匀浆液的抗病毒活性[J].昆虫知识,2006,43(1):82～85.
[28] 陈艳,李金富. 家蝇幼虫组织匀浆液抗病毒活性的研究[J]. 热带医学杂志,2004,4(2):130～132.
[29] 李金富,陈艳. 家蝇幼虫匀浆液抗病毒作用初探. 寄生虫病与感染性疾病,2006,4(2):63～64.
[30] 张杰,张双全.抗真菌肽对真菌作用机制研究进展[J].生物化学与生物物理进展,2005,32(1):13～17.
[31] Jiggins FM, Kim KW. The Evolution of Antifungal Peptides in Drosophila[J]. Genetics,2005, 171:1847～1859.
[32] 吴建伟,吴健桦,杨鹤萍,等. 家蝇幼虫血淋巴抗白念珠菌作用的初步研究[J]. 中国人兽共患病杂志,2002,18(3):57～59.
[33] 赵瑞君,刘成芳,董建臻,等. 家蝇抗菌肽对弓形虫的抑制作用研究[J]. 中国媒介生物学及控制杂志,2005,16(3):189～190.
[34] 国果,吴建伟.家蝇幼虫分泌物对猪蛔虫卵发育的影响[J]. 贵阳医学院学报,2002,27(2):134～135.
[35] 金小宝,朱家勇. 家蝇幼虫差异表达基因的克隆筛选与分析[J].生物技术,2005,15(2):5～7.
[36] Y.Lee,J.Tsai,S.Sunkara,et al. The TIGR Gene Indices: clustering and assembling EST and known genes and integration with eukaryotic genomes[J]. Nucleic Acids Research,2005,33:D71～D74 .
[37] 付劲蓉,刘文励,周剑锋.等. 多药耐药相关基因 HV126 的电子克隆及在化疗前后白血病细胞中的表达[J].中华医学遗传学杂志,2005,22(2):158～162.
[38] Bertone P,Stolc V,Royce T E,et al. Global Identification of Human Transcribed Sequences with Genome Tiling Arrays[J]. Science,2004,306(24):2242～2246
[2] 王荫长 主编.昆虫生物化学.北京:中国农业出版社,2001,240～312.
[3] Park S Y, Kim K M, Lee J H,et al. Extracellular Gelatinase of Enterococcus faecalis Destroys a Defense System in Insect Hemolymph and Human Serum. Infection and Immunity, 2007,75(4): 1861～1869.
[4] Pal S, Leger R J S, Wu L P.Fungal Peptide Destruxin A Plays a Specific Role in Suppressing the Innate Immune Response in Drosophila melanogaster. J. Biol. Chem., 2007,282(12):8969～8977.
[5] Nüsslein K, Arnt L, Rennie J, et al. Broad-spectrum antibacterial activity by a novel abiogenic peptide mimic.Microbiology,2006,152: 1913～1918.
[6] Kamysz W,Turecka K. Antimicrobial preservative effectiveness of natural peptide antibiotics. Acta Pol Pharm,2005,62(5):341～344.
[7] Yeaman M R, Yount N Y. Mechanisms of Antimicrobial Peptide Action and Resistance. pharmacological reviews, 2003,55(1):27～56.
[8] Franco O L, Murad A M, Leite J R,et al.Identification of a cowpea γ-thionin with bactericidal activity. FEBS Journal, 2006,273:3489～3497.
[9] Bommineni Y R, Dai H, Gong Y X,et al.Fowlicidin-3 is an -helical cationic host defense peptide with potent antibacterial and lipopolysaccharide-neutralizing activities. FEBS Journal, 2007, 274: 418～428.
[10] Silva P D, Jouvensal L, Lamberty M, et al.Solution structure of termicin, an antimicrobial peptide from the termite Pseudacanthotermes spiniger. Protein Science, 2003,12:438～446.
[11] 柏鸣,周立.家蝇抗菌蛋白的部分结构信息及生物学活性.中国生物化学与分子生物学报,2002,18(5):633～637.
[12] Gueguen Y, Herpin A, Aumelas A,et al.Characterization of a Defensinfrom the Oyster Crassostrea gigas: recombinant production, folding, solution structure, antimicrobial activities, and gene expression. J. Biol. Chem.,2006,281(1): 313～323.
[13] Fjell C D, Hancock R E.W, Cherkasov A. AMPer: a database and an automated discovery tool for antimicrobial peptides. Bioinformatics,2007 23(9):1148～1155.
[14] Xu X,Jin F,Yu X, et al. Expression and purification of a recombinant antibacterial peptide, cecropin, from Escherichia coli. Protein Expr and Purif, 2007,53(2):293～301.
[15] Waniek P J, Stock P, Mayer C,et al. Sequence characterization and expression. patterns of defensin and lysozyme encoding genes from the gut of the reduviid bug Triatoma brasiliensis.Insect Biochemistry and Molecular Biology,2006,36(7): 547～560.
[16] Imler JL,Bulet P. Antimicrobial peptides in Drosophila: structures, activities and gene regulation. Chem Immunol Allergy, 2005,86:1～21.
[17] Hu Y, Aksoy S. An antimicrobial peptide with trypanocidal activity characterized from Glossina morsitans morsitans. Insect Biochemistry and Molecular Biology,2005,35(2):105～115.
[18] Rahuma N,Ghenghesh KS,Ben Aissa R,et al. Carriage by the housefly (Musca domestica) of multiple-antibiotic-resistant bacteria that are potentially pathogenic to humans, in hospital and other urban environments in Misurata, Libya. Ann trop Med Parasitol,2005,99(8):795～802.
[19] Moreira C K,Capurro M,Calvo E,et al. The Musca domestica larval hexamerin is composed of multiple, similar polypeptides.Insect Biochem Mol Biol,2003,33(4):389～395.
[20] Naitza S,Ligoxygakis P.Antimicrobial defences in Drosophila:the story so far.Mol Immunol.2004,40(12):887～896.
[21] Hultmark D.Drosophila immunity:paths and patterns.Curr Opin Immunol.2003,15(1):12～9.
[22] Xia Q Y, Zhou Z Y, Lu C, et al. A Draft Sequence for the Genome ofthe Domesticated Silkworm (Bombyx mori).Science,2004,306(10):1937～ 1940.
[23] 宫霞,乐国伟,施用晖,付景春. 电泳制备家蝇幼虫抗菌肽及其性质. 无锡轻工大学学报,2003,22(6):25～30.
[24] 文彩虹,曲传智,李东英,等. 家蝇免疫血淋巴对 SMMC-7721 肝癌细胞超微结构和增殖周期的影响[J]. 河南肿瘤学杂志,2004,17(2):100～102.
[25] 刘晖,万启惠,贺莉芳,等. 家蝇幼虫抗菌蛋白的诱导及抗菌特性[J].中国公共卫生,2006,22(5):568 ～569.
[26] 万启惠,刘晖,贺莉芳,等. 家蝇幼虫抗菌蛋白的抗菌特性及其组成[J].中国媒介生物学及控制杂志,2006,17(3):191～193.
[27] 曹小红,陈一,张燕,等. 家蝇蛹凝集素的分离工艺和性质的初步研究[J]. 生物化学与生物物理进展,2003,30(3):442～445.
[28] 安春菊,耿华,郝友进,等. 不同提取工艺对家蝇幼虫蛋白粗提液抗菌活性的影响. 昆虫学报,2004,47(5):691～694.
[29] 安春菊,李德森,杜荣岑. 家蝇幼虫中与抗菌功能相关的蛋白和多肤的分析.卫生研究,2004,33(1):86～88.
[30] 安春菊,石明,盛长忠,等. 家蝇幼虫抗菌相关蛋白/多肤的诱导及抗菌活性分析. 昆虫学报,2003,46 ( 5) : 545～548.
[31] 齐静姣,曲传智. 不同诱导源诱导家蝇抗菌物质的动力学研究. 中国人兽共患病杂志 2004,20(6):554～555.
[32] Xanthopoulos KG,Lee JY,Gan RB,et al.The structure of the gene for cecropia B,an antibacterial immune protein from Hyalophora cecropia.Eur J Biochem,1988,172∶371-376.
[33] Lepage P, Bitsch F,van Dorsselaer A,et al. Determination of disulfide bridges in natural and recombinant insect defensin A.Eur. J. Biochem, 1991,196:735～742.
[34] Bonmatin J M, Genest M,Ptak M,et al.Progress in multidimensional NMR investigations of peptide and protein 3-D structures in solution. From structure to functional aspects.Biochimie,1992,74:825～836.
[35] Cornet B, Bonmatin J M,Vovelle F.Refined three-dimensional solution structure of insect defensin A.Structure,1995,3:435～448.
[36] 王来城,王金星,王来元,等. 家蝇防御素基因的cDNA克隆及序列分析. 动 物学报,2003,49(3):408～413.
[37] Liang Y,Wang JX,Zhao XF,et al.Molecular cloning and characterization of cecropin from the housefly (Musca domestica), and its expression in Escherichia coli. Dev Comp Immunol,2006,30(3):249～257.
[38] 耿华,安春菊,杜荣骞,等.家蝇攻击素(Attacin)基因的克隆与表达.遗传学报,2004,31(12):1344～1350.
[39] 许琴英,金小宝,朱家勇,等.家蝇幼虫防御素基因 cDNA 的克隆与序列分 析.热带医学杂志,2004;4(5):513～517.
[40] 金小宝,徐建华,朱家勇,等.家蝇幼虫天蚕素基因的克隆与序列分析.中国热带医学,2004,4(6):903～906.
[41] Nie D,Xiang Y. Molecular cloning and characterization of a novel human testis-specific gene by use of digital differential display.J Genet, 2006, 85(1):57～62.
[42] Tessitori M, Maria G, Capasso C,et al.Differential display analysis of gene expression in Etrog citron leaves infected by Citrus viroid III. Biochim Biophys Acta, In Press, Corrected Proof, Available online 24 March, 2007.
[43] Liu GY, Xiong YZ.Isolation, sequence analysis and expression profile of a novel porcine gene, NIP7, differentially expressed in the Longissimus dorsi muscle tissues from Meishan, Meishan x Large White cross and Large White pigs. Mol Biol Rep. Published online: December 23,2006.
[44] Hui XJ,Dean W, Yu L Ch,et al.Molecular Cloning and Localization of Human Ataxin2- like Gene. High Technology letters, 1998,4(2):100～104.
[45] 周国岭,杨光圣,傅廷栋. 基因克隆技术. 华中农业大学学报,2001,20(6):584～592.
[46] Lievens S, Goormachtig S,Holsters M. A critical evaluation of differential display as a tool to identify genes involved in legumenodulation: looking back and looking forward[J]. Nucleic Acids Research,2001,29(17):3459～3468.
[47] Mrazek J,Kreutmayer S B,Grasser F A, et al.Subtractive hybridization identifies novel differentially expressed ncRNA species in EBV-infected human B cells. Nucleic Acids Res. Published online: May 3,2007.
[48] Chen Z J, Shen H,Tew K D. Gene expression profiling using a novel method: amplified differential gene expression (ADGE). Nucleic Acids Res,2001,29, e46.
[49] Bertone P,Stolc V,Royce T E,et al. Global Identification of Human Transcribed Sequences with Genome Tiling Arrays[J]. Science,2004,306(24):2242～2246.
[50] Nagino K,Nomura O, Takii Y,et al.Ultrasensitive DNA Chip: Gene Expression Profile Analysis without RNA Amplification. Journal of Biochemistry,2006,139(4):697～703.
[51] 金小宝,朱家勇,王婷婷. 家蝇幼虫脂肪体cDNA文库的构建及初步鉴定. 中国人兽共患病杂志,2005,21(1):52～55.
[52] Nie D,Xiang Y. Molecular cloning and characterization of a novel human testis-specific gene by use of digital differential display. Genet, 2006,85(1):57～62.
[53] Frigessi A,Wiel M A,Holden M,et al. Genome-wide estimation of transcript concentrations from spotted cDNA microarray data. Nucleic Acids Res,2005,33(17): e143.
[54] Ramachandran N,Hainsworth E,Bhullar B. Self-Assembling Protein Microarrays. Science,2004,305(2):86～90.
[55] Wu C,Carta R,Zhang L. Sequence dependence of cross hybridization on short oligo microarrays. Nucleic Acids Research,2005,33(9):e84.
[56] He Zh L,Wu L Y,Li X Y,et al. Empirical Establishment of Oligonucleotide Probe Design Criteria. Appl Environ Microbiol, 2005,71(7): 3753～3760.
[57] 卢圣栋 主编.现代分子生物学实验技术(第二版).北京:中国协和医科大学出版社,1999,136～154.
[58] Lowenberger C,Maurice M,Vizioli J,et al.Antimicroial activity specerum,cDNA cloning,and mRNA expression of a newly isolated member of the cecropin family from the mosquito vetor.Aedes aegypti,J Biol Chem,1999,274:20092～20097.
[59] Knight J. When the chips are down. Nature,2001,410:860～861.
[60] Luscombe NM,Babu MM. GenCompass: a universal system for analysing gene expression for any genome. Trends Biotechnol, 2004, 22(11):552～555.
[61] He W,Ji Z Y,Huang C Y. Molecular cloning of a novel cDNA from Mus muscular BALB/c mice encoding glycosyl hydrolase family 1: a homolog of human lactase-phlorizin hydrolase. Biomed Environ Sci,2006,19(5): 340～345.
[62] Fang Ch J, Long R Zh, Ying K X,et al.Isolation Characterization and Expression Analysis of Male Sterility Gene Homology Sequence in Wheat. Actn Geneticn Sinica,2005,32(6):566～570.
[63] Grauvogel C,Petersen J.Isoprenoid biosynthesis authenticates the classification of the green alga Mesostigma viride as an ancient streptophyte. Gene, Published online: Mar,15,2007.
[64] Lee C C,Wu X,Gibbs R A,et al.Generation of cDNA probes directed by amina acid sequence: Cloning of urate oxidase[J].Science,1988,239: 1288～1291.
[65] Zhong J G, Mei Y B,Li F T, et al.Application of degenerate primers in cloning hsp70 gene families of Dunaliella salina. Journal of Zheng zhou Universitv(Medical Sciences),2003,38(6):875～877.
[66] 王洪振,周晓馥,宋朝霞,等.简并 PCR 技术及其在基因克隆中的应用.遗传,2003,25(2):201～204.
[67] 张新宇,高燕宁.PCR 引物设计及软件使用技巧. 生物信息学,2004,2(4):15～18.
[68] Wang N,Sun Y H, Liu J, et al. Molecular characterization of common carp (Cyprinus carpio)Sonic Hedgehog and discovery of its maternal expression.Dev Genes Evol,2007,217(4):299～305.
[69] Franco P F, Hedreyda C T. Amplification and sequence analysis of the full length toxR gene in Vibrio harveyi.J Gen Appl Microbiol,2006, 52(5):281～287.
[70] Kumar C G, Larson J H, Band M R,et al. Discovery and characterization of 91 novel transcripts expressed in cattle placenta. BMC Genomics. 2007 8(1):113～121.
[71] Clark JM.Novel non-templated nucleotide adittion reations catalyzed by procaryotic and eucaryotic DNA polymerase.Nucleic Acids Res.1988, 16:9677～9686.
[72] Marchuk D,Drumm M,Saulino A,et al.Construction of T-vectors,a rapid and general system for direct cloning of unmodified PCR products. Nucleic Acids Res.1991,19:1154～1157.
[73] 卢圣栋 主编.现代分子生物学实验技术(第二版).北京:中国协和医科大 学出版社,1999,287～290.
[74] Chen YB,Bergen P,Gadd C,etal. The Online Bioinformatics Resources Collection at the University of Pittsburgh Health Sciences Library System—a one-stop gateway to online bioinformatics databases and software tools. Nucleic Acids Research,2007,35(Database issue): D780～D785.
[75] Feng Zh Z,Qing S Ch, Cai F Y,et al.Isolation and Analysis of a Novel MYC Gene from Rice.Acta Genetica Sinica,2005,32(4):393～398.
[1] International Human Genome Sequence Consortium. Finishing the euchromatic sequence of the human genome. Nature,2004,431:931～945.
[2] Nygaard V,Hovig E. Options available for profiling small samples: a review of sample amplification technology when combined with microarray profiling. Nucleic Acids Research,2006,34(3):996～1014.
[3] Chaudhuri JD. Genes arrayed out for you: the amazing world of microarrays. Med Sci Monit, 2005, 11(2): RA52～62.
[4] Ramachandran N,Hainsworth E, Bhullar B ,et al. Self-Assembling Protein Microarrays. Science,2004,305(2):86～90.
[5] Ali TR,Li MS,Langford PR. Monitoring gene expression using DNA arrays. Meths Mol Med,2003,71:119～134.
[6] Nie D,Xiang Y. Molecular cloning and characterization of a novel human testis-specific gene by use of digital differential display.J Genet, 2006, 85(1):57～62.
[7] Lievens S,Goormachtig S,Holsters M. A critical evaluation of differential display as a tool to identify genes involved in legume nodulation: looking back and looking forward[J]. Nucleic Acids Research,2001,29(17):3459～3468.
[8] Bertone P,Stolc V,Royce T E,et al. Global Identification of Human Transcribed Sequences with Genome Tiling Arrays. Science,2004,306(24):2242～2246.
[9] Ciro M,Bracken AP,Helin K. Profiling cancer. Curr Opin Cell Biol,2003,15(2):213～220.
[10] Yao J,Weremowicz S,Feng B,et al. Combined cDNA Array Comparative Genomic Hybridization and Serial Analysis of Gene Expression Analysis of Breast Tumor Progression. Cancer Research,2006,66,4065-4078.
[11] Slater EP,Diehl SM,Langer P,et al. Analysis by cDNA microarrays of gene expression patterns of human adrenocortical tumors. European Journal of Endocrinology,2006,154(4):587～598.
[12] 彭桂福,马文丽,郑文岭,等. DNA 芯片技术筛选 K562 肿瘤细胞特异性基因. 第一军医大学学报,2004,24(5):525～528.
[13] Baelde HJ,Eikmans M,Doran PP,et al. Gene expression profiling in glomeruli from human kidneys with diabetic nephropathy. Am J Kidney Dis,2004,43(4):636～650.
[14] 郑敬民,刘志红,张鑫,等. 一个糖尿病肾病相关新基因的筛选、克隆和序列分析. 生物化学与生物物理进展,2004,31(1):47～53.
[15] 易著文,马祖祥,吴小川,等. 原发性肾病综合征相关基因的筛选. 中华儿科杂志,2002,40(12):724～727.
[16] Basile DP,Fredrich K,Alausa M,et al. Identification of persistently altered gene expression in the kidney after functional recovery from ischemic acute renal failure. Renal Physiol,2005,288:F953～F963.
[17] Zhang HG,Hyde K,Page GP,et al. Novel tumor necrosis factor alpha- regulated genes in rheumatoid arthritis. Arthritis Rheum,2004,50(2):420～431.
[18] Ishii T,Onda H,Tanigawa A, et al. Isolation and Expression Profiling of Genes Upregulated in the Peripheral Blood Cells of Systemic Lupus Erythematosus Patients. DNA Research,2005,12(6):429～439.
[19] Chowers I, Gunatilaka TL,Farkas RH,et al. Identification of Novel Genes Preferentially Expressed in the Retina Using a Custom Human Retina cDNA Microarray. Investigative Ophthalmology and Visual Science,2003,44:3732～3741.
[20] Nishimura DY,Swiderski RE,Searby CC,et al. Comparative genomics and gene expression analysis identifies BBS9,a new Bardet-Biedl syndrome gene[J]. Am J Hum Genet,2005,77(6):1021～1033.
[21] 陈海,郑军华,闵志廉,等. 基因表达谱芯片在免疫高敏相关基因筛选中的应用. 第二军医大学学报,2005,26(1):75～79.
[22] Roussoulieres ALS,Raisky O,Chalabreysse L,et al. Identification and Characterization of Two Genes (MIP-1?, VE-CADHERIN) Implicated in Acute Rejection in Human Heart Transplantation Use of Murine Models in Tandem With cDNA Arrays. Circulation,2005,111:2636～2644.
[23] Wang W,Cassidy J,O'Brien V,et al. Mechanistic and predictive profiling of 5-Fluorouracil resistance in human cancer cells. Cancer Res,2004,64(22):8167～8176.
[24] Aragon LM,Navarro F,Heiser V,et al. Rapid detection of specific gene mutations associated with isoniazid or rifampicin resistance in Mycobacterium tuberculosis clinical isolates using non-fluorescent low-density DNA microarrays. Journal of Antimicrobial Chemotherapy,2006,57(5):825～831.
[25] Kang JX,Liu J,Wang J D,et al. The extract of huanglian,a medicinal herb, induces cell growth arrest and apoptosis by up regulation of interferon-? and TNF- in human breast cancer cells. Carcinogenesis,2005,26(11):1934～1939.
[26] Chen JW,Sun CM,Sheng WL,et al. Expression Analysis of Up-RegulatedGenes Responding to Plumbagin in Escherichia coli. Journal of Bacteriology,2006,188(2):456～463.
[27] Vandepoele K,Vlieghe K,Florquin K,et al. Genome-Wide Identification of Potential Plant E2F Target Genes. Plant Physiology,2005,139,pp:316～328.
[28] Misson J,Raghothama KG,Jain A,et al. A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation. PLANT BIOLOGY,2005,102(33):11934～11939.
[29] Madsen M L,Nettleton D,Thacker E L,et al. Transcriptional Profiling of Mycoplasma hyopneumoniae during Heat Shock Using Microarrays. Infection and Immunity,2006,74(1):160～166.
[30] Cui L,Lian JQ,Neoh HM,et al. DNA Microarray-Based Identification of Genes Associated with Glycopeptide Resistance in Staphylococcus aureus. Antimicrobial Agents and Chemotherapy,2005,49(8):3404～3413.
[31] David JP , Strode C , Vontas J , et al. The Anopheles gambiae detoxification chip: A highly specific microarray to study metabolic-based insecticide resistance in malaria vectors. GENETICS,2005,102(11):4080～4084.
[32] Gregorio ED,Spellman PT, Rubin GM, et al. Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays. Proc Natl Acad Sci U S A : Genetics, 2001,98(22):12590～12595.
[33] Ooi SL,Shoemaker DD,Boeke JD. A DNA microarray-based genetic screen for nonhomologous end-joining mutants in Saccharomyces cerevisiae. Science,2001,294(5551):2552～2556.
[34] Sarropoulou1 E,Kotoulas1 G,Power D M, et al. Gene expression profiling of gilthead sea bream during early development and detection of stress-related genes by the application of cDNA microarray technology. Physiol Genomics,2005,23: 182～191.
[35] Knight J. When the chips are down. Nature,2001,410:860～861.
[36] Luscombe NM,Babu MM. GenCompass: a universal system for analysing gene expression for any genome. Trends Biotechnol, 2004, 22(11):552～555.
[1]Kamysz W,Turecka K. Antimicrobial preservative effectiveness of natural peptide antibiotics[J]. Acta Pol Pharm,2005,62(5):341～344.
[2] Rahuma N,Ghenghesh KS,Ben Aissa R,et al. Carriage by the housefly (Musca domestica) of multiple-antibiotic-resistant bacteria that are potentially pathogenic to humans, in hospital and other urban environments in Misurata, Libya. Ann trop Med Parasitol,2005,99(8):795～802.
[3] Moreira C K,Capurro M,Calvo E,et al. The Musca domestica larval hexamerin is composed of multiple, similar polypeptides[J].Insect Biochem Mol Biol,2003,33(4):389～395.
[4] 宫霞,乐国伟,施用晖,等. 电泳制备家蝇幼虫抗菌肽及其性质[J]. 无锡轻工大学学报,2003,22(6):25～30.
[5] 安春菊,李德森,杜荣骞. 家蝇幼虫中与抗菌功能相关的蛋白和多肽的分析[J].卫生研究,2004,33(1):868～8.
[6] 盛长忠,安春菊,耿 华,等. 一种家蝇幼虫热稳定抗菌肽的分离纯化[J]. 南开大学学报(自然科学).2002,35(4):6～10.
[7] 刘晖,万启惠,贺莉芳,等. 家蝇幼虫抗菌蛋白的诱导及抗菌特性[J].中国公共卫生,2006,22(5):568 ～569.
[8] 万启惠,刘晖,贺莉芳,等. 家蝇幼虫抗菌蛋白的抗菌特性及其组成[J].中国媒介生物学及控制杂志,2006,17(3):191～193.
[9] 陈留存,王金星,刘瑶,等. 家蝇抗菌肽的分离纯化及性质研究. 山东大学学报(自然科学版),2001,36(3):351～357.
[10] 柏鸣,周立.家蝇抗菌蛋白的部分结构信息及生物学活性[J].中国生物化学与分子生物学报,2002,18(5):633～637.
[11] 耿华,安春菊,杜荣骞,等.家蝇攻击素(Attacin)基因的克隆与表达[J].遗传学报,2004,31(12):1344～1350.
[12] Liang Y , Wang JX , Zhao XF , et al. Molecular cloning and characterization of cecropin from the housefly (Musca domestica), and its expression in Escherichia coli[J]. Dev Comp Immunol , 2006 ,30(3):249～257.
[13] 王来城,王金星,王来元,等. 家蝇防御素基因的cDNA克隆及序列分析[J]. 动物学报,2003,49(3):408～413.
[14] 安春菊,耿华,郝友进,等. 不同提取工艺对家蝇幼虫蛋白粗提液抗菌活性的影响[J]. 昆虫学报,2004,47(5):691～694.
[15] 周义文,尹一兵,涂植光,等. 家蝇抗菌肽抗菌活性及抗菌机制的初步研究[J].中国抗生素杂志,2004,29(5):272～274.
[16] 宫霞,施用晖,乐国伟. 家蝇幼虫抗菌肽MDL-1的分离纯化及其对大肠杆菌超微结构的影响[J]. 昆虫学报,2004,47(1):8～13.
[17] 宫霞,施用晖,乐国伟.抗菌活性肽与细菌染色体DNA的相互作用机理[J].自然科学进展,2004,14(5):509～513.
[18] 徐建华,朱家勇,金小宝,等. 抗菌肽Cecropin-His-6基因的真核表达及其生物活性研究[J].中国热带医学,2006,6(7):1119～1121.
[19] 金小宝,朱家勇、刘雷山,等. 家蝇抗菌肽Defensin基因在COS-7细胞中的瞬时表达[J].生物技术,2006,16(4):10～11.
[20] Gong X,Le GW,Li YF. Antibacterial spectrum of antibacterial peptides from Musca domestica larvae and synergic interaction between the peptides and antibiotics[J]. Wei Sheng Wu Xue Bao,2005,45(4):516～520.
[21] 赵东红,张双全,戴祝英,等. 重组家蚕抗菌肽 CM4 对癌细胞骨架及核骨架破坏作用的观察[J].高技术通讯,2000,10(1):23～27.
[22] 文彩虹,曲传智,李东英,等. 家蝇免疫血淋巴对 SMMC-7721 肝癌细胞超微结构和增殖周期的影响[J]. 河南肿瘤学杂志,2004,17(2):100～102.
[23] 贺莉芳,万启惠,刘晖,等. 家蝇幼虫抗菌蛋白诱导黑色素瘤 A375 细胞凋亡的探讨[J]. 中国媒介生物学及控制杂志,2006,17(1):20～21.
[24] 曹小红,陈一,张燕,等. 家蝇蛹凝集素的纯化及其免疫调节和抑菌作用[J]. 中华微生物学和免疫学杂志,2003,23(10):297～300.
[25] Wang Z. Wang G. APD: the Antimicrobial Peptide Database.Nucleic Acides Res,2004,32:D590～D592.
[26] Gallo S A,Wang W,Rawat S S,et al.θ-Defensins Prevent HIV-1 Env-mediated Fusion by Binding gp41 and Blocking 6-Helix Bundle Formation[J]. J. Biol. Chem.,2006,281(27): 18787～18792.
[27] 王芙蓉,艾辉,雷朝亮,等. 家蝇幼虫组织匀浆液的抗病毒活性[J].昆虫知识,2006,43(1):82～85.
[28] 陈艳,李金富. 家蝇幼虫组织匀浆液抗病毒活性的研究[J]. 热带医学杂志,2004,4(2):130～132.
[29] 李金富,陈艳. 家蝇幼虫匀浆液抗病毒作用初探. 寄生虫病与感染性疾病,2006,4(2):63～64.
[30] 张杰,张双全.抗真菌肽对真菌作用机制研究进展[J].生物化学与生物物理进展,2005,32(1):13～17.
[31] Jiggins FM, Kim KW. The Evolution of Antifungal Peptides in Drosophila[J]. Genetics,2005, 171:1847～1859.
[32] 吴建伟,吴健桦,杨鹤萍,等. 家蝇幼虫血淋巴抗白念珠菌作用的初步研究[J]. 中国人兽共患病杂志,2002,18(3):57～59.
[33] 赵瑞君,刘成芳,董建臻,等. 家蝇抗菌肽对弓形虫的抑制作用研究[J]. 中国媒介生物学及控制杂志,2005,16(3):189～190.
[34] 国果,吴建伟.家蝇幼虫分泌物对猪蛔虫卵发育的影响[J]. 贵阳医学院学报,2002,27(2):134～135.
[35] 金小宝,朱家勇. 家蝇幼虫差异表达基因的克隆筛选与分析[J].生物技术,2005,15(2):5～7.
[36] Y.Lee,J.Tsai,S.Sunkara,et al. The TIGR Gene Indices: clustering and assembling EST and known genes and integration with eukaryotic genomes[J]. Nucleic Acids Research,2005,33:D71～D74 .
[37] 付劲蓉,刘文励,周剑锋.等. 多药耐药相关基因 HV126 的电子克隆及在化疗前后白血病细胞中的表达[J].中华医学遗传学杂志,2005,22(2):158～162.
[38] Bertone P,Stolc V,Royce T E,et al. Global Identification of Human Transcribed Sequences with Genome Tiling Arrays[J]. Science,2004,306(24):2242～2246