甲型副伤寒杆菌ompX基因原核表达及表达产物免疫原性鉴定

Construction and prokaryotic expression of Salmonella paratyphi A ompX gene and immunological identification of the expressed product

目的构建甲型副伤寒杆菌外膜蛋白基因ompX的原核表达系统,确定其重组表达产物rOmpX免疫原性。方法采用PCR从甲型副伤寒杆菌标准株50001扩增目的基因并构建其原核表达系统,IPTG诱导表达目的蛋白,提取并纯化表达产物。采用免疫扩散法和Western blot鉴定其免疫原性。结果成功构建了ompX的原核表达系统,rOmpX表达量约为细菌总蛋白的28%。rOmpX免疫家兔可产生抗体并能与重组蛋白rOmpX产生阳性Western blot杂交信号。结论成功构建甲型副伤寒杆菌外膜蛋白基因ompX的原核表达系统,rOmpX具有良好的免疫原性,可作为甲型副伤寒杆菌基因工程疫苗候选抗原之一。

Objective To generate a prokaryotic expression system of Salmonella paratyphi A ompX gene that encoding an outer membrane protein （OMP）, and to determine immunologic identification of expressed product. Methods ompX gene was amplified by PCR and its prokaryotic expression system was then constructed. IPTG was applied to induce the expres- sion of the target recombinant protein （rOmpX）, and then rOmpX was extracted and purified. Results The expression output of rOmpX was approximately 28% of the total bacterial proteins. The extracted rOmpX showed a single protein band in gel after SDS-PAGE. The immunodiffusion titer of rabbit antiserum against rOmpX was 1：4. The rOmpX antiserum was able to recognize rOmpX. Conclusion The constructed prokaryotic expression system enable express rOmpX with high efficiency, and can be used as a candidate antigen for developing genetic engineering vaccine.