| 大黄鱼β2-微球蛋白基因的克隆及其在大肠杆菌中的重组表达 |
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| 引用本文： | 余素红,王玉桥,陈新华.大黄鱼β2-微球蛋白基因的克隆及其在大肠杆菌中的重组表达[J].台湾海峡,2009,28(1):13-18. |
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| 作者姓名： | 余素红 王玉桥 陈新华 |
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| 作者单位： | 1. 厦门大学生命科学学院,福建,厦门,361005;国家海洋局第三海洋研究所,福建,厦门,361005;福建省农业科学院,福建,福州,350002
2. 国家海洋局第三海洋研究所,福建,厦门,361005 |
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| 摘 要： | 主要组织相容性复合体（major histocompatibility complex，MHC）是一类高度多态的基因群，它广泛分布于各种脊椎动物体内，在启动特异性免疫应答中起关键作用．MHC根据结构分为Ⅰ型和Ⅱ型两种．β2-微球蛋白（β2mcroglobulin，β2m）是MHCI型分子的轻链．本文根据大黄鱼β2m的表达序列标签（expressed sequence tags，EST）序列设计合成了特异性引物，利用RT—PCR技术从大黄鱼脾脏组织中克隆了β2m基因（Pscr-β2m）；构建了合β2m因的原核表达载体（pGEX4T-2-β2m，在0．1mmol／dm^3IPTG诱导下重组β2m得到了表达；采用Sepharose 4B亲合层析纯化目的蛋白并制备了抗体；Western—blotting分析证实了该抗体具有与天然大黄鱼脾脏组织中β2m蛋白反应的特性．这些结果为进一步研究鱼类β2m构与功能的关系，以及制备MHCI类分子四聚体奠定基础．
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| 关 键 词： | 大黄鱼 β2-微球蛋白 克隆 重组表达 Western-blotting |
Cloning of β2-microglobulin in large yellow croaker(Pseudosciaena croces) and efficient expression in Escherichia coli |
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| YU Su-hong,WANG Yu-qiao,CHEN Xin-hua.Cloning of β2-microglobulin in large yellow croaker(Pseudosciaena croces) and efficient expression in Escherichia coli[J].Journal of Oceanography In Taiwan Strait,2009,28(1):13-18. |
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| Authors: | YU Su-hong WANG Yu-qiao CHEN Xin-hua |
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| Institution: | YU Su-hong, WANG Yu-qiao, CHEN Xin-hua ( 1. Department of Biology, School of Life Science, Xiamen University, Xiamen 361005, China ;2. Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen 361005, China; 3. Fujian Academic of Agricultural Science, Fuzhou 350002, China) |
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| Abstract: | Major hiscompatibility complex of genes with extraordinary polymorphism （MHC） including MHC class Ⅰ and MHC class Ⅱ is an extended cluster spreading over nearly all vertebrates extensively and plays a critical role in the induction of immune responses. β2-mmicroglobulin .（β2m） is a light chain of MHC class Ⅰ molecule. In this experiment ,the β2m gene was cloned from spleen tissue of large yellow croaker by expressed sequence tags （EST） analysis and RT-PCR techniques. The expression vector pGEX-4T-2-β2m gene was presented,in which the mature peptide sequence of β2m gene was inserted. High-yield expression of β2m was achieved induced by 0. lmmol/dm^3 IPTG in E. coli transformed with the expression vector, and protein was completely purified using glutathione sepharose 4B affinity chromatography methods for preparing specific antibody. Western blotting assay antibody against pGEX-4T-2-β2m could react specifically with the native Pscr-β2m protein in high quality Pscr-β2m laid a good foundation for preparation of MHC Ⅰtetramer and the study between the configuration of fish MHC Ⅰ and its functions. indicated that the spleen tissue. The on the relationship |
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| Keywords: | Western-blotting |
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