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
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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BL21/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639bp, 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, 为进一步研究该基因的功能打下基础。Abstract:Objective To amplify a new encoding gene of GST from adult C.sinensis(CsGST gene), determine whetherithad intron or not, express it in Escherichia coli(E.coli)and purify the recombinant protein.Methods The gene encoding Cs GST 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 p ET-30a(+)and expressed in E.coli BL21/DE3.The recombinant protein was purified according to the protocol of Ni-NTA agarose(QIA GEN, Germany).It was analyzed by SDS-PAGE and TLC.Results The full length of the gene encoding CsGST was 639 bp.Ithad no intron.The recombinant plasmid p ET-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.
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