假结核耶尔森菌侵袭素蛋白基因的克隆及原核表达

Gene Cloning and Prokaryotic Expression of Invasin Protein of Yersinia pseudotuberculosis

目的克隆假结核耶尔森菌侵袭素蛋白基因并在原核表达系统中表达.方法根据GenBank上登录的假结核耶尔森菌侵袭素蛋白核酸序列设计引物,用PCR方法扩增假结核耶尔森菌侵袭素蛋白基因,并将其插入克隆载体pMD18-T中,测序鉴定正确后再将其克隆到原核表达载体pGEX-4T-1上,得到重组质粒pGEX-4T-1-inv.将重组质粒转化大肠杆菌BL21(DE3)感受态细胞,经IPTG诱导,SDS-PAGE电泳检测.结果假结核耶尔森菌提取的基因组,经PCR扩增,得到一段591 bp的片段,与预期结果一致;SDS-PAGE电泳分析,所得重组蛋白的分子量约46 kDa,与预期结果相同;重组菌体超声裂解后,经12%的SDS-PAGE电泳分析,结果显示该重组蛋白为包涵体蛋白.结论克隆的侵袭素基因核心片段可以在原核细胞中高效表达.

Objective To clone invasin gene from Yersinia pseudotuberculosi and express it in prokaryotic system. Methods According to the nucleic acid sequence of Yersinia pseudotuberculosi invasin gene deposited in GenBank, a pair of primers were designed and synthesized. The invasin gene was amplified by PCR and inserted into the pMD18-T vector, after confirmed with sequencing, the gene was subcloned into the prokaryotic expression vector pGEX-4T-1 and the recombinant plasmid pGEX-4T-inv was constructed. For expression of recombinant invasin protein, the recombinant plasmid was transformed into competent cell of E.coli BL21 （DE3） after IPTG induction. The expressed fusion protein was confirmed by SDS-PAGE electrophoresis. Results An expected fragment 591 bp of invasin gene was amplified from the genomic DNA extracted from Yersinia pseudotuberculosis by PCR. The recombinant invasin gene was expressed after the induction with isopropyl-^-d- thiogalactoside （IPTG）. The expressed GST-fused invasin core protein was stained on SDS-PAGE gel which showed the expected molecular weight of 46 kDa compared with the standard protein marker. After ultrasonic degradation, the recombinant was analyzed by 12% SDS-PAGE electrophoresis and the result showed that the recombinant protein was inclusion body. Conclusions Core fragment of the clonal invasin gene can highly express in prokaryocyte.