霍乱弧菌外膜蛋白W的原核表达及抗原性分析

摘要

目的 在大肠杆菌中表达霍乱弧菌种特异性外膜蛋白(Omp)W,纯化后制备检测用OmpW抗体.方法 采用PCR法以霍乱弧菌基因组为模板扩增ompW基因,插入表达载体pET-32a( + )的多克隆位点,构建重组表达质粒pET-32a-ompW;重组质粒转化至大肠杆菌BL21(DE3)中,筛选阳性重组菌株,经IPTG诱导获得目的 蛋白,纯化后免疫新西兰大耳白兔,制备OmpW的多克隆抗体并进行鉴定.结果 扩增得到ompW基因片段,并成功构建pET-32a-ompW原核表达系统,重组蛋白OmpW以包涵体形式高效表达;制备的OmpW抗体能与天然的O1群和O139群霍乱弧菌结合.结论 霍乱弧菌OmpW可由原核系统高效表达,免疫获得的抗体具有较好的效价,为进一步制备霍乱弧菌检测用抗体奠定基础.%Objective To obtain Vibrio cholerae outer membrane protein W expressed in E. Coli ( DE3 ) in order to produce its polyclonal antibody. Methods The ompW gene amplified from V. Cholerae by PCR was inserted into expression plasmid pET-32a( + ) to construct recombinant plasmid pET-32a( + )-ompW that was transformed into E. Coli BL21 ( DE3 ) with IPTG induction for the expression OmpW. The protein OmpW was purified with degeneration and renaturation and used to immunize rabbits to prepare anti-OmpW antibody,the property of which was identified. Results The ompW gene was obtained and the prokaryotic expression system constructed. The positive recombinant clone was identified by restriction enzyme digestion analysis and DNA sequencing. Induced with IPTG, OmpW was expressed in E. Coli BL2l( DE3 ). The purified recombinant protein was used to immunize rabbits. It could irritate the production of specific antibody. Conclusion The recombinant expression plasmid is constructed successfully and expressed in E. Coli. The anti-OmpW antibody has a high titer, which can be used to detect V. Cholerae in the future.