铜绿假单胞菌重组质粒pGEX.docVIP

铜绿假单胞菌重组质粒pGEX-OprF的构建及其表达 朱佑明 罗永艾 李文桂 【摘要】 目的 构建并鉴定铜绿假单胞菌重组质粒pGEX-OprF，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PAOl标准株提取总RNA为模板，自行设计引物，采用RT-PCR方法扩增获得OprF抗原编码基因，经酶切、连接定向克隆入大肠埃希菌-双歧杆菌穿梭表达载体pGEX-1λT，构建重组质粒pGEX-OprF。转化E.coliBL21(DE3)感受态细胞，经异丙基硫代-β-D-半乳糖苷（IPTG）诱导表达后用SDS和Western blot对表达产物进行分析和鉴定。结果 RT-PCR扩增出1016bp的OprF编码基因；重组质粒经双酶切和测序鉴定证实OprF基因成功插入pGEX-1λT中；SDS分析显示表达产物为相对分子量约为61×103的重组蛋白，与预期结果一致，表达的蛋白质占菌体总蛋白的16%；Western blot鉴定重组蛋白能被Pa外膜粗抗原免疫小鼠血清识别。结论 成功构建了铜绿假单胞菌重组质粒pGEX-OprF，该质粒在E.coliBL21(DE3)中高效融合表达，表达的融合蛋白具有特异性的抗原性。 【关键词】 铜绿假单胞菌；OprF；大肠埃希菌；表达 【中图分类号】R378.99+1 【文献标识码】A Construction and Expression of Recombinant Plasmid pGEX-OprF of Pseudomonas aeruginosa ZHU You-ming, LUO Yong-ai, LI Wen-gui （Department of Respiratory Medicine, the First Affiliated Hospital ,Chongqing medical University, Chongqing 400016,China） 【Abstract】 Objective To construct and express recombinant plasmid pGEX-OprF of Pseudomonas aeruginosa in Escherichia coli BL21(DE3). Methods Total RNA was extracted from Pseudomonas aeruginosa(PAO1), and used as template. The OprF coding gene was amplified by RT-PCR, digested and then cloned into E.coli-Bifidobacteria shuttle plasmid pGEX-1λT to construct 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-thiogalactopyranosid(IPTG), and the expressed products were analyzed and identified with SDSand Western blot. Results The 1016bp 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 at approximately 61×103 determined by SDS. The amount of the expressed protein comprised 16% of the total bacterial proteins. The fusion protein could be recognized by the sera of mice produced against the crude outer membrane protein. Conclusion The recombinant plasmid pGEX-OprF was successfully constructed, and