华支睾吸虫GST的新编码基因的克隆与纯化  

Cloning,expression and purification of new encoding gene of GST from clonorchis sinensis

作　　者：伍忠銮[1] 余新炳[1] 吴德[1] 徐劲[1] 吴忠道[1] 王海[1] 

机构地区：[1]中山大学基础医学院寄生虫学教研室,广州510089

出　　处：《中国公共卫生》2004年第6期668-669,共2页Chinese Journal of Public Health

基　　金：广东省自然科学基金首批科研团队项目;广东省科技厅社会发展攻关计划 (2 0 0 2B31 0 0 5);广州市科技攻关计划(2 0 0 2 2 3 -E40 2 2 )

摘　　要：目的　扩增华支睾吸虫 (Cs)一个编码谷胱甘肽转移酶 (GST)的新基因 ,确认是否存在内含子 ,进行原核表达 ,纯化。方法　用PCR方法从成虫cDNA文库和基因组中扩增CsGST基因 ,测序 ,定向克隆到原核表达载体pET- 30a(+) ,在大肠杆菌BL2 1/DE3表达 ,按照Ni-NTAagarose说明书纯化重组蛋白 ,用十二烷基硫酸钠 -聚丙烯酰胺凝胶电泳 (SDS -PAGE)和薄层色谱 (TLC)分析。结果　CsGST基因全长 6 39bp ,没有内含子 ,构建的原核表达质粒 pET - 30a(+) -CsGST在大肠杆菌中得到了有效表达 ,融合蛋白的分子量约为 30kDa。重组蛋白达总蛋白的33% ,纯化后的重组蛋白纯度为 97%。结论　CsGST基因没有内含子 ,在大肠杆菌中能有效表达 ,重组蛋白得到了纯化 。Objective To amplify a new encoding gene of GST from adult C.sinensis (CsGST gene),determine whether it had intron or not,express it in Escherichia coli (E.coli) and purify the recombinant protein.Methods The gene encoding CsGST was obtained from the cDNA (plasmid) library and genomic DNA of adult C.sinensis by PCR and sequenced.The coding gene was cloned into the prokaryotic expression vector pET-30a(+) and expressed in E.coli BL21/DE3.The recombinant protein was purified according to the protocol of Ni-NTA agarose (QIAGEN,Germany).It was analyzed by SDS-PAGE and TLC.Results The full length of the gene encoding CsGST was 639 bp.It had no intron.The recombinant plasmid pET-30a(+)-CsGST was efficiently expressed in E.coli,its molecular weight was about 30 kDa.The recombinant protein occupied 33% of total bacterial protein.The purity of the purified recombinant protein was 97%.Conclusion The CsGST gene of C.sinensis had no intron.Its efficient expression was achieved in E.coli.The recombinant protein was purified.These can lay a base for its function research.

关 键 词：华支睾吸虫 谷胱甘肽转移酶 克隆 基因表达 纯化 

分 类 号：R383.22[医药卫生—医学寄生虫学]