Construction and expression of the recombinant plasmid pGEX-OprF of Pseudomonas aeruginosa

Objective To construct and express the recombinant plasmid pGEX-OprF of Pseudomonas aeruginosa in Escherichia coli BL21(DE3). Methods Total RNA was extracted from Pseudomonas aeruginosa(PAO1) and used as a template.The OprF coding gene was amplified by RT-PCR,digested and then cloned into the E.coli-Bifidobacteria shuttle plasmid pGEX-1λT to construct the recombinant plasmid pGEX-OprF.The recombinant plasmid was transformed into E.coliBL21(DE3) and screened.The BL21(pGEX-OprF) was induced with isopropyl-β-D-thiogalactopyranoside(IPTG),and the expressed products were analyzed and identified with SDS-PAGE and Western blot. Results The 1 016 bp OprF coding gene was successfully amplified by RT-PCR and cloned into pGEX-1λT.The recombinant plasmid pGEX-OprF was successfully constructed,with a relative molecular weight of expressed recombinant protein of approximately 61×103 determined by SDS-PAGE.The amount of the expressed protein accounted for 16% of the total bacterial proteins.The fusion protein was recognized by the sera of mice immunized against crude outer membrane proteins.Conclusion The recombinant plasmid pGEX-OprF was successfully constructed and highly expressed in E.coli in a fused form with GST.The expressed fusion protein has specific antigenicity.