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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 and express the gene encoding Yersinia pseudotuberculosis in Escherichia coli.Methods Primers were designed according to the nucleotide sequences of Yersinia pseudotuberculosis registered in GenBank and the Yersinia pseudotuberculosis Then inserted into the cloning vector pMD18-T, and then sequenced and identified correctly, and then cloned into the prokaryotic expression vector pGEX-4T-1 to obtain the recombinant plasmid pGEX-4T-1-inv. The recombinant plasmid The recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells, induced by IPTG and detected by SDS-PAGE electrophoresis.Results A 591 bp fragment was obtained by PCR amplification of the genome of Yersinia pseudotuberculosis, which was consistent with the expected results. SDS -PAGE electrophoresis analysis, the molecular weight of the resulting recombinant protein was about 46 kDa, which was the same as the expected result. After the recombinant strain was lysed by SDS-PAGE and analyzed by 12% SDS-PAGE, the recombinant protein was inclusion body protein. The prime gene core fragment can be highly expressed in prokaryotic cells.