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Thesis Advisor朱作言 ; 聂品
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline遗传学
Keyword免疫球蛋白 重链 轻链 基因克隆 基因表达
Abstract鳜(Siniperca chuatsi)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。在分离纯化鳜血清免疫球蛋白(IgM)的基础上,本研究试图对编码鳜免疫球蛋白重链(IgH)和轻链(IgL)的cDNA进行克隆,并尝试在大肠杆菌(Escherichia coli)中进行表达。主要研究内容及结果如下:在国内率先采用免疫亲和层析技术纯化了鳜血清免疫球蛋白。SDS-PAGE显示其IgH链和IgL链分子量分别为72和29kD。制备了兔抗鳜IgM的抗血清,在Western blotting分析中与鳜IgH链反应强度高于IgL链。另外,采用间接ELISA实验分析了鳜免疫血清的活性和反应特异性。采用RT-PCR和RACE等方法,在国内首次克隆了鳜IgH和IgL链的全长cDNA。比较了鳜与其它硬骨鱼类IgH链的氨基酸序列特征。以18种脊椎动物IgM CH4区氨基酸序列构建了系统发育树,并对CH4结构域的进化进行了探讨。根据脊椎动物IgM CH4区恒定的氨基酸取代率,推算了鳜与鲈形目其它几种鱼类的分歧时间。将鳜IgL链氨基酸序列同其它辐鳍鱼类相应预列进行比较,发现鳜和鲈形目另一种鱼类CL区都存在特有的丝氨酸三核苷酸密码子重复序列(AGC)_n。以20种脊椎动物的CL区氨基酸序列构建了系统发育树,表明鳜IgL链类似于斑点叉尾鲴G或虹鳟L1同种型,而不能划归为高等脊椎动物的κ或λ同种型。首次分别构建了鳜IgH链和IgL链的重组表达质粒,并在大肠杆菌中实现了高效表达。经Western blotting分析,目的基因表达产物都能被兔抗鳜IgM的抗血清特异性识别,进一步证实了上述cDNA克隆分别编码鳜IgH和IgL链。该工作的完成为鳜的基因工程抗体制备和抗体基因转移育种研究提供了分子基础。
Other AbstractThe mandarin fish, Siniperca chuatsi is an important species in the aquaculture industry of China. The understanding of its immune system is essential for such aquacultural purpose. On the basis of the serum immunoglobulin (IgM) purification from the fish, the present study aims to clone the cDNAs encoding the heavy (IgH) and light (IgL) chains of the IgM, and then express them respectively in Escherichia coli. The serum IgM from S. chuatsi was purified for the first time in China by using affinity chromatography techniques, with the molecular weight of the IgH and IgL being determined to the 72kD and 29kD, respectively. The rabbit antisera which were developed against the purified IgM, reacted more strongly with the IgH than with the IgL chain, as revealed by the Western blotting analyses. The full-length cDNAs encoding the IgH and IgL chains of the mandarin fish were for the first time cloned by RT-PCR and RACE. The amino acid (aa) sequence of the IgH was compared with those of other teleost fish, and the phylogeny tree was constructed according to the aa sequences of IgM-CH4 domains from 18 vertebrates. The evolution of CH4 domains was then discussed, and the divergence time between S. chuatsi and the other species estimated. When the aa sequences were compared between the IgL chain of the mandarin fish and those of other actinopterygian fishes, it is found that the unique trinucleotide sequence repeat (AGC)n encoding serine in the CL domains exists in S. chuatsi and another perciform species. The phylogeny tree constructed by aa sequences of the CL domains from 20 vertebrates suggested that the IgL of the mandarin fish was similar to the G isotype of Channel catfish, Ictalurus punctatus and to the L1 isotype of rainbow trout, Oncorhynchus mykiss, but not to the κ or λ isotype of higher vertebrates. The recombinant expression plasmids carrying the cDNA sequences of mandarin fish IgH and IgL chains were then constructed respectively, and subsequently expressed efficiently in E. coli. Using Western blotting analyses, the products expressed by the inserted genes were recognized by the rabbit anti- purified-mandarin-fish-IgM sera, confirming that the cloned cDNAs do encode the mandarin fish IgH and IgL chains.
Document Type学位论文
Recommended Citation
GB/T 7714
张永安. 鳜免疫球蛋白基因的克隆与表达研究[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,2001.
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