【摘要】 目的克隆表达猪链球菌2型诊断性抗原谷氨酸脱氢酶GDH。方法根据基因库中的gdh基因序列设计PCR引物,自病人分离株2007SF0165基因组扩增GDH的编码基因gdh,克隆至pMD19-T载体后进行测序分析,并且构建重组表达质粒pGEX4T-2-gdh,转化E.coli BL21(DE3),IPTG诱导表达,SDS-PAGE检测。结果 PCR法自猪链球菌2型菌株2007SF0165基因组扩增出约1.3 kb的目的片段;序列分析显示猪链球菌的GDH具有GDH蛋白家族I的典型保守序列;构建筛选的原核重组表达质粒pGEX4T-2-gdh经诱导表达,获得蛋白相对分子质量为45 kDa目的蛋白。结论克隆并表达出猪链球菌2型诊断性抗原GDH,可为进一步研究奠定坚实的基础。更多还原

【Abstract】 Objective To clone and prokearyotically express the gene encoding the glutamate dehydrogenase(gdh)of Streptococcus suis(S.suis)serotype 2. Methods The encoding gene gdh of 2007SF0165 strain isolated from patients was amplified by PCR with the designed primers according to the Gene bank gdh sequence, and then was cloned into vector pMD19- T and sequenced. Thereafter, the gene was cloned into prokaryotic expression plasmid pGEX4T- 2, and the recombinant plasmid pGEX4T- 2- gdh was transformed into E.coli BL21(DE3). After the expression was induced by IPTG, the GDH protein isolated was detected by SDS- PAGE. Results A target gene fragment with the size of 1. 3 kb was amplified from the genomes of 2007SF0165 strain of S.suis serotype 2, and the characteristically conserved sequence of GDHprotein family- 1could be demonstrated in the GDH of S.suis as indicated in the sequence analysis. As shown by SDS- PAGE, the molecular weight of the expression protein of GDH was 45 kDa. Conclusions The cloning and expression of gene encoding the glutamate dehydrogenase of Streptococcus suis serotype 2 can provide a strong basis for further study on this issue.更多还原