甲型副伤寒沙门菌鞭毛蛋白fliC-a基因的克隆、原核表达及鉴定

Cloning,constrction of prokaryotic expression system and identification of fliC-a gene from phase-1 flagellar antigens of S. Paratyphi A

目的:克隆甲型副伤寒沙门菌1相鞭毛蛋白抗原(H1抗原)fliC-a基因,构建原核表达系统并鉴定重组蛋白的抗原性。方法:PCR方法扩增fliC-a基因,克隆到质粒pET-42a构建原核表达载体pET-fliC-a,转化E.coli BL21(DE3),IPTG诱导目的重组蛋白rfliC-a表达,用SDS-PAGE鉴定rfliC-a,Western blot和双向免疫扩散法鉴定rfliC-a的抗原性和免疫原性。结果:成功表达并纯化获得相对分子量为14.4 kDa的重组蛋白rfliC-a,rfliC-a能与兔抗甲型副伤寒沙门菌全菌血清发生特异性结合,免疫家兔获得高效价抗体。结论:成功构建了fliC-a基因原核表达系统,所表达的rfliC-a具有良好的抗原性。

Objective：To clone flic-a gene from phase-1 flagellar（H1） antigens of S.Paratyphi A,construct a prokaryotic expression system of the gene and identify antigenicity and immunogenicity of rflic-a.Methods：The flic-a gene was amplified by PCR and cloned into plasmid Pet-42a to make pET-fliC-a for the prokaryotic expression of flic-a gene.The rflic-a was expressed in E.coli strain BL21（DE3）.IPTG was used to induce the target recombinant protein rflic-a.The purified protein was identified by SDS-PAGE,and the antigenicity and immunogenicity of the rflic-a were identified by Western Blot and double immunodiffusion.Results：The purified rflic-a protein was visualized on membrane at molecular mass 14.4kDa by SDS-PAGE.The rflic-a was able to combine with the commercial antibody against whole cell of S.Paratyphi A and could induce the immunized rabbits to produce antibodies with an double immunodiffusion titers of 1：8.Conclusion：A prokaryotic expression system of flic-a gene was established successfully.The expressed rflic-a showed well antigenicity.