铜绿假单胞菌OprH基因在大肠埃希菌BL21(DE3)中的表达

Expression of the recombinant plasmid pGEX-OprH of Pseudomonas aeruginosa in E.coli BL21(DE3)

目的构建铜绿假单胞菌重组质粒pGEX-OprH,研究其在大肠埃希菌BL21(DE3)中的表达。方法以铜绿假单胞菌PAO1标准株双链DNA为模板,通过PCR扩增OprH基因。经酶切后与载体pGEX-1λT进行连接,构建重组质粒pGEX-OprH,转化E.coli BL21(DE3)感受态细胞,异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果 PCR扩增出660 bp的OprH编码基因;重组质粒经双酶切和PCR证实OprH基因成功插入pGEX-1λT中;SDS-PAGE电泳发现表达产物相对分子质量约为47 000 Mr,与前期预测结果一致,大肠埃希菌BL21(DE3)表达蛋白为菌体量的22%;免疫印渍表明Pa抗原免疫的小鼠血清能特异识别47 000 Mr重组蛋白。结论铜绿假单胞菌重组质粒pGEX-OprH在E.coli BL21(DE3)中高效融合表达,表达的融合蛋白具有抗原特异性。

Objective To construct and express the recombinant plasmid pGEX-OprH of Pseudomonas aeruginosa in Escherichia coli BL21（DE3）. Methods The ds DNA, extracted from Pseudomonas aeruginosa（PAO1）, was used as a template to amplify the OprH coding gene with PCR; the resulted PCR products was digested and ligated to vector pGEX-1λT, therefore the recombinant plasmid pGEX-OprH was constructed. The plasmid was then electroporated into E.coli BL21（DE3）. E.coli BL21（DE3） harbouring the recombinant plasmid pGEX-OprH was induced by IPTG; the expressed products were analyzed and identified with SDS-PAGE and Western blot. Results The PCR amplfied OprH gene was estimated as660 bp and cloned into pGEX-1λT successfully; the recombinant pGEX-OprH was successfully constructed. SDS-PAGE analysis demonstrated that the relative molecular weight of expressed recombinant protein was approximately 47 000 Mr. The expressing efficiency of the OprH-GST fusion protein was 22%. Immunoblotting result showed that the sera of mice immunized against crude outer membrane recognized the fusion protein. Conclusion The recombinant plasmid pGEX-OprH was successfully constructed and highly expressed in E.coli as a GST fused protein, and the expressed fusion protein showed specific antigenicity.