摘要
【目的】扩增华支睾吸虫一个μ型谷胱甘肽移酶(mu-classglutathionetransferaseofClonorchissinen-sis,CsGSTM)基因,明确是否存在内含子,进行原核表达,纯化其表达产物,鉴定免疫反应性。【方法】用PCR方法从成虫cDNA文库和基因组中扩增出目的基因,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(sodiumdode-cylsulfate-polyacrylamidegelelectrophoresis,SDS-PAGE)、薄层色谱(thin-layerchromatography,TLC)和免疫印迹(Westernblotting)分析。【结果】CsGSTM基因全长657bp,没有内含子,构建的原核表达质粒pET-30a(+)-CsGSTM在大肠杆菌中得到了有效表达,Mr,pET-30a(+)-CsGSTM=31×103。重组蛋白达总蛋白的39%,纯化后的重组蛋白纯度为98%,重组蛋白在纯化前后均有免疫反应性。【结论】CsGSTM基因没有内含子,在大肠杆菌中能有效表达,纯化前后的重组蛋白都具有免疫反应性。
To amplify the CsGSTM gene of adult Clonorchis sinensis, determine whether it has intron or not, express it in Escherichia coli (E. coli), purify the recombinant protein and identify its immunoreactivity.The gene encoding CsGSTM was obtained from the cDNA (plasmid) library and genomic DNA of adult Clonorchis sinensis by PCR and was sequenced. The coding Seguence 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,TLC and Western blotting.The full length of the gene encoding CsGSTM was 657 bp. It has no intron. The recombinant plasmid pET 30a(+) CsGSTM was efficiently expressed in E. coli, Mr,pET 30a(+) CsGSTM=31×103 . The recombinant protein occupied 39%of total bacterial protein. The purity of the purified recombinant protein was 98% The recombinant protein has immunoreactivity before and after purification. [Conclusion]The CsGSTM gene of Clonorchis sinensis has no intron. Its high efficient expression has been achieved in E. coli. The recombinant CsGSTM has immunoreactivity before and after purification.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2004年第2期105-109,共5页
Journal of Sun Yat-Sen University:Medical Sciences
基金
广东省自然科学基金首批科研团队基金资助项目(2001年)
广东省科技厅社会发展攻关计划基金资助项目(2002B31005)
广州市科技攻关计划基金资助项目(200223-E4022)
