鲈鱼头肾cDNA文库的构建与免疫相关基因的筛选及中肾NCC基因的研究
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摘要
本文构建了鲈鱼头肾cDNA文库,并筛选了免疫相关基因,同时对中肾中起离子调节作用的NCC基因进行了基因克隆和免疫组织化学定位等研究。
鲈鱼头肾cDNA文库的构建。鲈鱼是我国沿海一种重要的养殖鱼类,以其肉质细腻鲜美而深受欢迎,但是近年来随着鲈鱼养殖规模的不断扩大,病害也日趋严重。抗生素的使用不但会造成药物在鱼体内的富集、残留,也会危害人类的健康问题,造成环境污染和破坏生态平衡。因此从鱼体本身的抗病力出发,研究如何提高机体对疾病的抵抗力,增强免疫力,揭示机体的免疫抵御机制有助于减少病害的发生和对整体养殖环境的净化,进而提高生产,降低污染和药物残留,实现可持续健康养殖。
成年鱼类的主要免疫器官是头肾,其中无肾单位结构,而分布有大量的T、B淋巴细胞,吞噬细胞和粒细胞等,是特异性免疫和非特异性免疫赖以实现的物质基础。本文采用分子生物学方法构建了鲈鱼头肾λZAPExpress cDNA文库。提取鲈鱼头肾RNA,纯化得到了质量较高的mRNA。在Stratascriptase作用下将5μgmRNA反转录合成第一链,第二链用pfu Polymerase合成双链的cDNA,之后连接EcoRⅠ接头,经XhoⅠ酶切得到具有粘性末端的双链cDNA,经CHROMASPIN-400分级分离,将500bp以上的片段连接入λZAPExpress载体,体外包装成噬菌体粒子,测定cDNA文库初级库滴度为1.7×10~5pfu/mL,扩增后的文库滴度达到5×10~9pfu/mL,插入片段主要分布在500-2000bp,说明成功构建了鲈鱼头肾cDNA文库。采用小规模测序和设计特异引物PCR方法对构建的cDNA文库进行了筛选和分析。共得到76个EST序列,其中筛选到了8个免疫相关基因,占已知基因的15.1%,占筛选到的总基因的10.9%。与免疫相关的8个基因分别是,免疫球蛋白重连(Ig heavy chain),Mx,干扰素诱导的GiG2,TLR,MyD88和TNF-α,趋化因子,巨噬细胞集落刺激因子。对主要免疫相关基因分别进行了同源性比对和序列进化分析,结果表明,这些基因均与同属鲈形目的鱼类同源性最高,分子进化分析表明,这几类基因的进化地位与传统进化分析趋势一致。文库筛选结果表明,本次构建的鲈鱼头肾cDNA文库质量较高,并可以广泛用于免疫相关基因的筛选、表达和调控研究。
鲈鱼中肾NCC基因的研究。鲈鱼是一种广温、广盐性鱼类,对环境中盐度变化适应能力很强,能在海水、半咸水和淡水中生活,能迅速适应不同环境中盐度的变化。钠氯共转运子NCC(Na-Cl cotransporter)是分布于上皮细胞顶端的一类12次跨膜蛋白,向胞内同向转运等量的钠离子和氯离子,属于电中性离子转运体家族的成员。
本文根据Genbank中已有的NCC同源序列,从鲈鱼中肾中克隆到了NCC基因,利用PCR拼接和3'-RACE结合的方法,扩增到了3346bp的NCC片段,并对其序列特征进行了分析和同源比对。同时分别对在淡水适应0、3、7和14天的鳃组织、肠组织和中肾中NCC mRNA水平的变化进行了Real-time PCR检测。结果表明,这3种组织均在淡水适应第3天达到最高值,之后随着时间的增加,NCC的含量逐渐降低,在鳃组织和中肾中,在14天时仍高于初始值;肠组织中则随时间增加逐渐降低,在14天时降到最低水平,且低于初始值,上述结果表明,鳃组织、肠组织和中肾在鱼类适应淡水过程中起重要的Na~+、Cl~-调节作用,而NCC则表现为机体对外界环境盐度变化的应激反应和调节,待机体适应淡水环境后,又逐渐降低,表现为机体对淡水环境的适应。为研究NCC蛋白在鲈鱼中肾的分布情况,我们选择了NCC的羧基端氨基酸序列进行了大肠杆菌原核表达,经1mM IPTG诱导,结果表明表达的目的蛋白以包涵体的形式存在,选择表达量最高的16小时大量制备目的蛋白,经切胶免疫新西兰雄兔,得到了抗血清。对中肾进行石蜡切片,进行了中肾组织学和NCC蛋白的免疫组织化学定位研究。结果表明,鲈鱼中肾肾单位数量较少,分布有大量的肾小管,其中近端小管占比例较大,而远端小管相对较少;免疫组织化学定位表明,NCC蛋白特异分布在鲈鱼中肾的远端小管和集合管上皮细胞顶端。
This paper consists two parts,one is the construction and screening of immune-related genes of the head-kidney cDNA library from Japanese sea bass;the other is the research on the NCC of the mid-kiney.
Japanese sea bass(Lateolabrax japonicus) is a marine fish with great commercial value especially in Asia.In breeding farms,unfavorable conditions or poor management practices may put stress on fishes,which will cause reduction of growth rate,immune suppression and various infectious diseases.To date,genetic knowledge on sea bass immune relative genes responsible for resistance to pathogens is poor. Progress in these research fields will have significant value in the near future.
The head kidney of teleosts serves as main immune organ and plays an important role in specific and non-specific immune defences.
A cDNA expression library from head kidney of Japanese sea bass was constructed. The first-strand cDNA was synthesized with Moloney Murine Leukaemia virus reverse transcriptase with 5μg mRNA and the double-stranded cDNA was digested by XhoⅠenzyme.Size fractionation was performed on CHROMA SPIN-400 columns. cDNA fragments longer than 500 bp were ligated into theλZAPExpress vector.The recombinant DNA was packaged in vitro with GigapackⅢgold packaging extract. The titers of the primary and amplified library were 1.7×10~5 pfu/mL and 5.0×10~9 pfu/mL respectively.To characterize the constructed cDNA library,15 phage plaques were selected randomLy to test the inserted fragments.The results showed that the inserts were mostly longer than 500 bp.
Small-scale sequence and PCR with specific primers for immune-related genes were conducted for the screening of the cDNA library.76 EST sequences was obtained from the screening.8 immune-related genes(Ig heavy chain,TLR,MyD88, Mx,Gig2 and TNF-α,CC chemokine and CSF1-2) were obtained and showed 15.1% of knowned genes,10.9%of all the obtained EST sequences.The homology and phylogenetic analysis on the main genes showed that they all had the highest identity to the species of Perciformes,which was consistent with the systematic classification. The results indicated that the cDNA library was constructed successfully,and could be used for the screening of the immune-related genes.
Japanese sea bass is a euryhaline fish,is able to live in waters with a wide range of salinity and maintain nearly constant ion concentrations of body fluids irrespective of the external salinity,in patieular,displaying a remarkable plasticity when it comes to adjusting ion transport in response to changes in environment salinity.NCC(Na-Cl cotransporter) is a protein with 12 transmembrane-spanning segments,and apieally located in the epithelial cells,and is one of the members of the electroneutral, cation-chloride eotransporters family,which play a important role of ion regulation.
The NCC was cloned from the mid-kidney of the Japanese sea bass according to the specific primers designed by the known species.Based on the PCR combination and the 3'-RACE PCR,a cDNA,3346bp long,was obtained from the mid-kidney. The homology and phylogenetic analysis were conducted about the NCC sequence. On the other hand,time-course changes in the mRNA,following transfer of Japanese sea bass from seawater to freshwater for 0,3,7 and 14 days were examined in the tissue of gill,intestine and mid-kidney.The results showed that the NCC mRNA in all the 3 tissues displayed the similar trend.The mRNA in the 3 tissues all reached the maximum on 3 days acclimation and then decreased gradually.The abundance of the NCC on 14 days accilimation in gill and mid-kidney was higer than that of initial abundance;and the abundance in intestine was lower than that of initial abundance. The results implied that the NCC may participate in the cousrse of fast response of the organism to the environment.In order to uncover the location of the NCC in mid-kidey,the preparation of polyclonal antibody was conducted against the 645bp fragment(coding for 215 Aa fragment) located in the carboxyl termini.The pET-28a vector was chosen for the expression of the fusion protein.Induced by 1mM IPTG,the fusion protein was expressed in inclusion bodies.After purification,the protein was isolated by the SDS-PAGE electrophoresis.Then,the part of the protein was cut for immune New Zealand male rabbits for 1month.The anti serum was obtained and used for the immunohistochemistry of NCC.The reults showed that the NCC of Japanese sea bass was located in the distal ducting cells and collecting cells.
鲈鱼头肾cDNA文库的构建。鲈鱼是我国沿海一种重要的养殖鱼类,以其肉质细腻鲜美而深受欢迎,但是近年来随着鲈鱼养殖规模的不断扩大,病害也日趋严重。抗生素的使用不但会造成药物在鱼体内的富集、残留,也会危害人类的健康问题,造成环境污染和破坏生态平衡。因此从鱼体本身的抗病力出发,研究如何提高机体对疾病的抵抗力,增强免疫力,揭示机体的免疫抵御机制有助于减少病害的发生和对整体养殖环境的净化,进而提高生产,降低污染和药物残留,实现可持续健康养殖。
成年鱼类的主要免疫器官是头肾,其中无肾单位结构,而分布有大量的T、B淋巴细胞,吞噬细胞和粒细胞等,是特异性免疫和非特异性免疫赖以实现的物质基础。本文采用分子生物学方法构建了鲈鱼头肾λZAPExpress cDNA文库。提取鲈鱼头肾RNA,纯化得到了质量较高的mRNA。在Stratascriptase作用下将5μgmRNA反转录合成第一链,第二链用pfu Polymerase合成双链的cDNA,之后连接EcoRⅠ接头,经XhoⅠ酶切得到具有粘性末端的双链cDNA,经CHROMASPIN-400分级分离,将500bp以上的片段连接入λZAPExpress载体,体外包装成噬菌体粒子,测定cDNA文库初级库滴度为1.7×10~5pfu/mL,扩增后的文库滴度达到5×10~9pfu/mL,插入片段主要分布在500-2000bp,说明成功构建了鲈鱼头肾cDNA文库。采用小规模测序和设计特异引物PCR方法对构建的cDNA文库进行了筛选和分析。共得到76个EST序列,其中筛选到了8个免疫相关基因,占已知基因的15.1%,占筛选到的总基因的10.9%。与免疫相关的8个基因分别是,免疫球蛋白重连(Ig heavy chain),Mx,干扰素诱导的GiG2,TLR,MyD88和TNF-α,趋化因子,巨噬细胞集落刺激因子。对主要免疫相关基因分别进行了同源性比对和序列进化分析,结果表明,这些基因均与同属鲈形目的鱼类同源性最高,分子进化分析表明,这几类基因的进化地位与传统进化分析趋势一致。文库筛选结果表明,本次构建的鲈鱼头肾cDNA文库质量较高,并可以广泛用于免疫相关基因的筛选、表达和调控研究。
鲈鱼中肾NCC基因的研究。鲈鱼是一种广温、广盐性鱼类,对环境中盐度变化适应能力很强,能在海水、半咸水和淡水中生活,能迅速适应不同环境中盐度的变化。钠氯共转运子NCC(Na-Cl cotransporter)是分布于上皮细胞顶端的一类12次跨膜蛋白,向胞内同向转运等量的钠离子和氯离子,属于电中性离子转运体家族的成员。
本文根据Genbank中已有的NCC同源序列,从鲈鱼中肾中克隆到了NCC基因,利用PCR拼接和3'-RACE结合的方法,扩增到了3346bp的NCC片段,并对其序列特征进行了分析和同源比对。同时分别对在淡水适应0、3、7和14天的鳃组织、肠组织和中肾中NCC mRNA水平的变化进行了Real-time PCR检测。结果表明,这3种组织均在淡水适应第3天达到最高值,之后随着时间的增加,NCC的含量逐渐降低,在鳃组织和中肾中,在14天时仍高于初始值;肠组织中则随时间增加逐渐降低,在14天时降到最低水平,且低于初始值,上述结果表明,鳃组织、肠组织和中肾在鱼类适应淡水过程中起重要的Na~+、Cl~-调节作用,而NCC则表现为机体对外界环境盐度变化的应激反应和调节,待机体适应淡水环境后,又逐渐降低,表现为机体对淡水环境的适应。为研究NCC蛋白在鲈鱼中肾的分布情况,我们选择了NCC的羧基端氨基酸序列进行了大肠杆菌原核表达,经1mM IPTG诱导,结果表明表达的目的蛋白以包涵体的形式存在,选择表达量最高的16小时大量制备目的蛋白,经切胶免疫新西兰雄兔,得到了抗血清。对中肾进行石蜡切片,进行了中肾组织学和NCC蛋白的免疫组织化学定位研究。结果表明,鲈鱼中肾肾单位数量较少,分布有大量的肾小管,其中近端小管占比例较大,而远端小管相对较少;免疫组织化学定位表明,NCC蛋白特异分布在鲈鱼中肾的远端小管和集合管上皮细胞顶端。
This paper consists two parts,one is the construction and screening of immune-related genes of the head-kidney cDNA library from Japanese sea bass;the other is the research on the NCC of the mid-kiney.
Japanese sea bass(Lateolabrax japonicus) is a marine fish with great commercial value especially in Asia.In breeding farms,unfavorable conditions or poor management practices may put stress on fishes,which will cause reduction of growth rate,immune suppression and various infectious diseases.To date,genetic knowledge on sea bass immune relative genes responsible for resistance to pathogens is poor. Progress in these research fields will have significant value in the near future.
The head kidney of teleosts serves as main immune organ and plays an important role in specific and non-specific immune defences.
A cDNA expression library from head kidney of Japanese sea bass was constructed. The first-strand cDNA was synthesized with Moloney Murine Leukaemia virus reverse transcriptase with 5μg mRNA and the double-stranded cDNA was digested by XhoⅠenzyme.Size fractionation was performed on CHROMA SPIN-400 columns. cDNA fragments longer than 500 bp were ligated into theλZAPExpress vector.The recombinant DNA was packaged in vitro with GigapackⅢgold packaging extract. The titers of the primary and amplified library were 1.7×10~5 pfu/mL and 5.0×10~9 pfu/mL respectively.To characterize the constructed cDNA library,15 phage plaques were selected randomLy to test the inserted fragments.The results showed that the inserts were mostly longer than 500 bp.
Small-scale sequence and PCR with specific primers for immune-related genes were conducted for the screening of the cDNA library.76 EST sequences was obtained from the screening.8 immune-related genes(Ig heavy chain,TLR,MyD88, Mx,Gig2 and TNF-α,CC chemokine and CSF1-2) were obtained and showed 15.1% of knowned genes,10.9%of all the obtained EST sequences.The homology and phylogenetic analysis on the main genes showed that they all had the highest identity to the species of Perciformes,which was consistent with the systematic classification. The results indicated that the cDNA library was constructed successfully,and could be used for the screening of the immune-related genes.
Japanese sea bass is a euryhaline fish,is able to live in waters with a wide range of salinity and maintain nearly constant ion concentrations of body fluids irrespective of the external salinity,in patieular,displaying a remarkable plasticity when it comes to adjusting ion transport in response to changes in environment salinity.NCC(Na-Cl cotransporter) is a protein with 12 transmembrane-spanning segments,and apieally located in the epithelial cells,and is one of the members of the electroneutral, cation-chloride eotransporters family,which play a important role of ion regulation.
The NCC was cloned from the mid-kidney of the Japanese sea bass according to the specific primers designed by the known species.Based on the PCR combination and the 3'-RACE PCR,a cDNA,3346bp long,was obtained from the mid-kidney. The homology and phylogenetic analysis were conducted about the NCC sequence. On the other hand,time-course changes in the mRNA,following transfer of Japanese sea bass from seawater to freshwater for 0,3,7 and 14 days were examined in the tissue of gill,intestine and mid-kidney.The results showed that the NCC mRNA in all the 3 tissues displayed the similar trend.The mRNA in the 3 tissues all reached the maximum on 3 days acclimation and then decreased gradually.The abundance of the NCC on 14 days accilimation in gill and mid-kidney was higer than that of initial abundance;and the abundance in intestine was lower than that of initial abundance. The results implied that the NCC may participate in the cousrse of fast response of the organism to the environment.In order to uncover the location of the NCC in mid-kidey,the preparation of polyclonal antibody was conducted against the 645bp fragment(coding for 215 Aa fragment) located in the carboxyl termini.The pET-28a vector was chosen for the expression of the fusion protein.Induced by 1mM IPTG,the fusion protein was expressed in inclusion bodies.After purification,the protein was isolated by the SDS-PAGE electrophoresis.Then,the part of the protein was cut for immune New Zealand male rabbits for 1month.The anti serum was obtained and used for the immunohistochemistry of NCC.The reults showed that the NCC of Japanese sea bass was located in the distal ducting cells and collecting cells.
引文
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