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Alternative TitleCloning of Immunoglobulin Heavy Chain Genes for the Grass Carp, Ctenopharyngoden idellus
Thesis Advisor聂品
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword草鱼 免疫球蛋白 重链 Igm Igz 嵌合体ig 基因克隆 荧光定量 免疫印迹
Abstract草鱼是我国重要的水产养殖鱼类,对其免疫系统的研究在理论和应用方面都具有重要意义。本研究克隆了草鱼Ctenopharyngodon idellus免疫球蛋白IgM、IgZ和一种特殊的Ig嵌合体重链基因,并研究了它们在组织器官的表达。 采用RACE-PCR的方法扩增到草鱼IgM重链的cDNA全长,其开放阅读框包含1731核苷酸,编码576个氨基酸,编码区由前导链、可变区和恒定区组成。可变区又分为4个骨架区和3个高变区。在与其它鱼类可变区的氨基酸序列比对中,发现4个骨架区的序列相对保守。骨架区(framework region, FR)2和FR3存在GKGLEW和YYCAR的保守序列,FR4存在FDYWGKGT-VTV-S的保守序列。与其它鱼类IgM恒定区氨基酸序列比较的结果显示,4个外显子的半胱氨酸和色氨酸残基都比较保守。荧光定量PCR检测显示,草鱼IgM主要在头肾、中肾和脾脏这三个免疫器官中表达,肝脏、鳃、胸腺和肠中也有少量表达,而在心脏和脑中几乎没有表达。 采用RACE-PCR和简并引物扩增的方法克隆到草鱼IgZ和一种Ig嵌合体的cDNA部分序列,分别编码487个和402个氨基酸。草鱼的IgZ与斑马鱼Danio rerio和鲤Cyprinus carpio的IgZ同源越细撸徊萦?Ig嵌合体,其恒定区CH1和CH2与IgM的相应区域一样,CH3和CH4与IgZ的相应区域一致,即μ1 + μ2 + ζ3 + ζ4的结构。根据IgZ的开放阅读框设计含有酶切位点的表达引物,构建重组表达质粒,将重组质粒转入大肠杆菌Escherichia coli M15中,经IPTG诱导获得了31 kD的融合蛋白。用切胶回收的方法对表达的蛋白进行了纯化,制备了兔抗IgZ的抗血清。经免疫印迹分析,IgZ主要在胸腺、头肾、鳃和心脏中表达,在脾脏和中肾中也有少量表达,在脑和肝脏中几乎没有表达。
Other AbstractThe grass carp (Ctenopharyngoden idellus) is an important species in aquaculture industry in China. The understanding of its immune system is essential for its aquaculture. The present study aims to clone immunoglobulin heavy chain genes, such as IgM, IgZ and IgD, and express IgZ heavy chain in E.coli. The IgM cDNA full sequence of grass carp has been cloned using RACE-PCR and PCR. Its open reading frame has 1731 nucleotides which encode a 576 amino acid peptide. The coding region consists of leader segment, variable region and constant region. The variable region can be divided into four framework regions and three hypervariable regions. The deduced amino acid sequence of grass carp IgM was compared with those from other fish species, and the conserved regions, i.e. GKGLEW, YYCAR and FDYWGKGT-VTV-S were found in FR2, FR3 and FR4, respectively. The cysteines and tryptophans in constant regions were identified at conserved locations. The real-time PCR analysis revealed a higher level of IgM gene expression in head kidney, kidney and spleen and lower expression in liver, gill, thymus and intestines and almost none in heart and brain. An Ig cDNA sequence similar to the reported IgZ of Zebra fish (Danio rerio)or IgT of rainbow trout (Oncorhynchus mykiss) has been cloned in grass carp using degenerate primers and RACE-PCR, and a Ig chimera also has been cloned in this fish. They encode 487 and 402 amino acid peptides, respectively. The IgZ or IgT similar IgH chain has a high similarity with IgZ from zebrafish and common carp ( Mus musculus). The first two constant domains of the chimeric Ig heavy chain are similar to IgM, and the latter two to IgZ, being a structure of μ1 + μ2 + ζ3 + ζ4. The expression primers were coded for the open reading frame of IgZ and recombinant IgZ was expressed in Escherichia coli M15. The expressed product was 31 kD fusion protein, and was purified in the denatured condition. Rabbit polyclonal antisera were then developed against the purified protein. A higher level of IgZ expression was detected by using Western blotting analysis in head kidney, thymus, gills and heart, and lower level in spleen and kidney and almost none in liver and brain.
Document Type学位论文
Recommended Citation
GB/T 7714
王欣欣. 草鱼免疫球蛋白重链基因的克隆[D]. 水生生物研究所. 中国科学院水生生物研究所,2006.
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