摘要
目的克隆表达猪链球菌谷氨酸脱氢酶(GDH),并测定其酶活性。方法设计引物采用PCR法自海安病人分离株Habb基因组扩增GDH的编码基因gdh,进行序列分析;构建重组表达质粒pGEX4T-2-gdh,转化E.coliBL21(DE3),IPTG诱导表达,PAGE检测;通过亲和层析纯化,获得融合蛋白GST-GDH,用凝血酶剪切去除GST,获得完整的GDH纯化蛋白后测定其酶活性。结果PCR法自猪链球菌菌株Habb基因组扩增出约1.3kb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;体外构建筛选原核表达质粒pGEX4T-2-gdh,诱导重组体,表达的蛋白经PAGE初步测定其相对分子质量(Mr)约为71×103;用凝血酶酶切去除融合蛋白中的GST标签后得到的蛋白相对分子质量为45×103;GDH活性测定显示猪链球菌GDH利用α-酮戊二酸做底物,用NADPH做辅酶催化氨的同化和谷氨酸的合成。结论克隆并表达出猪链球菌GDH;猪链球菌GDH属于GDH蛋白家族I,是NADP(H)-特异性GDH。
To clone and prokearyotically express the gene encoding the glutamate dehydrogenase of Streptococcus suis serotype 2 and to investigate activity of the gene product, the encoding gene gdh of Habb strain isolated from patients in Hal-an county of Jiangsu province was amplified by PCR with the designed primers, and then cloned into vector pMD18-T. Thereafter, the gene was cloned into prokaryotic expression plasmid pGEX4T-2, and the recombinant plasmid pGRX4T-2-gdh was transformed into E. coli BL21 （DE3）. After induced expression by IPTG, the GDH protein isolated was analyzed with SDS-PAGE and purified by chromatography. The fusion protein GST-GDH was then cleaved by thrombin to remove the GST tail of the fusion protein, thus obtaining the completely purified GDH protein, and its enzymatic activity was measured afterwards. The experimental results showed that a target gene fragment with size of 1.3 kb was amplified from the genomes of Habb strain of S. suis, and the characteristically conserved se- quence of GDH protein family-1 could be demonstrated in the GDH of S. suis as indicated in the sequence analysis. As shown by SDS-PAGE, the molecular weight of the fusion protein of GST was 71kDa and that of one of the cleaved protein of GDH was 45 kDa. The measurement of the enzymatic activity of GDH revealed that its activity was attributed to NADP-dependent GDH.
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2006年第1期22-25,共4页
Chinese Journal of Zoonoses
基金
"十五"国家科技攻关计划"新发传染病的防治技术研究与应用"项目资助(2003BA712A03-05)
