铜绿假单胞菌重组质粒pGEX-PopB的构建、鉴定及其在大肠埃希菌中的表达效率

Construction,identification and expression of the recombinant plasmid pGEX-PopB derived from Pseudomonas aeruginosa in Escherichia coli

目的构建和鉴定铜绿假单胞菌重组质粒pGEX-PopB,并研究该质粒在大肠埃希菌BL21(DE3)中的表达。方法以铜绿假单胞菌PA01标准株的DNA为模板,通过PCR扩增PopB基因。经酶切后与载体pGEX-1λT连接,构建重组质粒pGEX-PopB,电穿孔将其转化至大肠埃希菌BL21(DE3)。异丙基硫代-β-D-半乳糖苷(IPTG)诱导重组菌BL21(pGEX-PopB)的表达,SDS-PAGE和Western blot对表达产物进行分析和鉴定。结果PCR扩增出1200 bp的PopB基因;双酶切和PCR证实PopB基因成功插入pGEX-1λT中;SDS-PAGE发现重组菌表达相对分子质量约66×103的融合蛋白,其蛋白表达量约占菌体总量的20%;Western blot证实铜绿假单胞菌感染的鼠血清能特异性识别该重组蛋白。结论成功构建了铜绿假单胞菌重组质粒pGEX-PopB,并在大肠埃希菌BL21(DE3)中高效表达具有抗原特异性的融合蛋白。

Objective To construct and identify the recombinant plasmid pGEX-PopB of Pseudomonas aeruginosa(P.aeruginosa)and study its expression in Escherichia coli(E.coli)BL21(DE3).Methods The PopB gene was amplified by PCR using the DNA of P.aeruginosa PA01 standard strain as template.After digestion with the vector pGEX-1λT,the recombinant plasmid pGEX-PopB was constructed and electroporated to transform E.coli BL21(DE3).The expression of recombinant BL21(pGEX-PopB)was induced by isopropylthio-β-D-galactoside(IPTG),and the expressed product was analyzed and identified by SDS-PAGE and Western blot.Results The PopB gene of 1200 bp was amplified by PCR,the PopB gene was successfully inserted into pGEX-1λT by double enzyme digestion and PCR,and the fusion protein with relative molecular weight of about 66×103 was expressed by SDS-PAGE.It accounted for 20%of the total bacterial cells.Western blot confirmed that the sera in mice infected with P.aeruginosa could specifically recognize the recombinant protein.Conclusion The recombinant plasmid pGEX-PopB of P.aeruginosa was successfully constructed and the antigen-specific fusion protein highly expressed in E.coli BL21(DE3).