沙门菌外膜蛋白D的原核表达及多克隆抗体制备

目的:在大肠杆菌中表达沙门菌外膜蛋白(OMP)D,纯化后制备兔抗OMPD抗体。方法:用PCR方法从鼠伤寒沙门菌中扩增出ompD基因,并插入融合表达载体pET-28a(+)的多克隆位点,构建重组表达质粒pET28a(+)-ompD;以重组质粒转化大肠杆菌BL21(DE3),筛选阳性重组菌株,经IPTG诱导目的蛋白表达,在变性条件下对目的蛋白进行亲和层析纯化;以表达的OMPD蛋白免疫家兔,制备抗OMPD的多克隆抗体并进行鉴定。结果:扩增了ompD基因,测序证实正确后亚克隆于表达载体pET-28a(+)中,经PCR筛选和酶切鉴定获得阳性克隆,经诱导在大肠杆菌中表达出相对分子质量为40×103的目的蛋白并进行纯化;纯化的OMPD免疫家兔后,能有效地刺激特异性抗体的产生,抗血清的效价达到1∶10000以上,且具有良好的特异性。结论:构建ompD基因的原核表达载体,并在大肠杆菌中获得高效表达;制备出兔抗OMPD抗体,效价及特异性均良好,为进一步制备肠黏膜高亲和力疫苗奠定了基础。

Prokaryotic Expression of Salmonella Outer Membrane Protein D and Development of its Polyclonal Antibody

Objective： To obtain Salmonella outer membrane protein（OMP） D expressed in E.coli BL21（DE3） for producing its polyclonal antibody. Methods： The omp D gene amplified from S.typhimurium by PCR was inserted into expression plasmid pET-28a（＋） to construct recombinant plasmid pET-28a（＋）-omp D. The recombinant plasmid was transformed into E.coli BL21（DE3） for the expression OMP D under IPTG induction. The protein OMP D was purified with Ni＋-NTA system and used to immunize rabbits to prepare anti-OMP D antibody, and the antibody＇s properties were identified. Results： The Salmonella omp D gene was confirmed by DNA sequencing, and positive recombinant clones were identified by restriction enzyme digestion analysis and DNA sequencing. After induction with IPTG, OMP D with Mr being 40 kDa was expressed in E.coli BL21（DE3） and was purified. Rabbit polyclonal antibody with a good specificity was obtained, and the titer was above of 1：10 000. Conclusion： The recombinant expression plasmid of OMP D was constructed successfully and expressed in E.coli. The prepared rabbit anti-OMP D antibody had a high titer and specificity, which laid a foundation for the preparation of intestinal mucosa immunological vaccine in future.