淋球菌外膜蛋白PI基因重组子的构建和表达

Construction and Expression of the Major Outer Membrane Protein PI of Neisseria gonorrhoeae in Escherichia coli

目的 扩增四株淋球菌外膜蛋白 PI基因 ,构建 p ET30 b- PI- NG重组子 ,在大肠杆菌中诱导表达外膜蛋白 PI。方法 采集临床淋球菌四株 ,提取细菌基因组 DNA,PCR扩增外膜蛋白 PI基因 ,与克隆载体 p BS- T连接 ,构建 p BS- T- PI- NG重组子 ,测序 ,目的基因插入表达质粒载体 p ET30 b中 ,构建 p ET30 b- PI NG重组子 ,转化表达宿主大肠杆菌 BL 2 1(DE3) ,IPTG诱导蛋白质表达 ,SDS- PAGE初步分析。结果 成功构建了四株淋球菌的p ET30 b- PI大肠杆菌表达重组子 ,经 IPTG诱导表达后 ,其中三株获得表达的目的蛋白 PI。结论 本研究为 PI蛋白免疫学特性的研究、抗体制备、以及预防淋病疫苗的研制奠定了基础。

Objective To construct Neisseria gonorrhoeae major outer membrane protein PI gene recombinants for expression of the target protein in E.coli. Methods Four clinic isolates of Neisseria gonorrhoeae were collected, and then the genome DNA of these strains was extracted. The gene encoding for PI of Neisseria gonorrhoeae was amplified by PCR, inserted into the cloning vector pBS-T; the recombinant plasmids pBS-T-PI-NG were constructed and sequence analysis was performed. Then PI gene fragments were inserted into expression vector pET30b to form pET30b-PI-NG recombinants. The PI protein expression was induced by adding IPTG in the inocula. The expressed proteins were analyzed by SDS-PAGE. Results The pET30b-PI-NG expression recombinants for four clinic isolates of Neisseria gonorrhoeae were constructed successfully; three of the four expression recombinants were expressed successfully. Conclusion The expressed PI protein will be applied in the further research for PI antigenicity and immunological activity. This will be very helpful for the further construction of preventve vaccines directed against Neisseria gonorrhoeae infection.