创伤弧菌vvhA基因的克隆、原核表达系统的构建及鉴定

目的 克隆创伤弧菌（Vibrio vulnificus,Vv）溶细胞素基因（υυhA）,构建原核表达系统并鉴定其表达产物的免疫性.方法 采用PCR技术从Vv GTC333和WZ01株DNA中扩增全长υυhA基因,T-A克隆后测定其核苷酸序列.采用pET32a质粒构建vvhA基因原核表达载体,在E coli BL21（DE3）宿主菌中用不同浓度的IPTG诱导目的重组蛋白rVvhA表达,采用Ni-NTA亲和层析法提纯rVvhA,SDS-PAGE检测表达和提纯效果.采用兔抗Vv全菌抗体的Western Blot和兔抗rVvhA血清的免疫扩散试验鉴定其免疫反应性和免疫原性.结果 所克隆的vvhA基因核苷酸序列与GeneBank公布的同源性分别为96.09%和98.26%.在0.5 mmol/L IPTG诱导下,rVvhA产量可占细菌总蛋白的18%.提纯的rVvhA经SDS-PAGE后仅显示单一的蛋白条带.重组蛋白rVvhA能与兔抗Vv全菌抗体发生特异性结合,免疫家兔可获得高效价抗体.结论 该研究成功地构建了创伤弧菌υυhA基因高效原核表达系统,所表达的rVvhA具有良好的免疫原性和免疫反应性,可作为Vv免疫检测试剂盒及疫苗的抗原.

Cloning, construction of prokaryotic expression system and identification of Vibrio vulni ficus vvhA gene

Objective To clone Vibrio vulnificus vvhA gene and construct a prokaryotic expression system of the gene and identify immunity of rVvhA.Methods The vvhA gene from strain V.vulnificus GTC333 and WZ01 were amplified by high fidelity PCR.The nucleotide sequences of the target DNA amplification fragments were sequenced after T-A cloning.Plasmid pET32a was used to construct prokaryotic expression vectors of the vvhA gene.rVvhA was expressed in E.coli strain BL21(DE3).IPTG was used to induce the target recombinant protein rVvhA and Ni-NTA affinity chromatography was applied to extract rVvhA.By using SDS-PAGE plus BioRad Agarose Image Analysor,the effects of expression and purification of rVvhA were determined.Western Blot was applied to determine immunoreactivity and immunogenicity of the rVvhA and antibodies against whole cell of V.vulnificus and rabbit antiserum immunized with the rVvhA.Results In comparison with the published by GeneBank,the homology of nucleotide sequences of the cloned vvhA gene was 96.09% and 98.26%.Under inducement of 0.5 mmol/L IPTG,the output of rVvhA could reach 18% of the total bacterial proteins.The purified rVvhA only showed a single fragment in gel after SDS-PAGE.The rVvhA was able to combine with antibodies against whole cell of V.vulnificus and induce rabbit to produce specific antibodies with high titres.Conclusions A prokaryotic expression system of V.vulnifcus vvhA gene with high efficiencies were successfully established in this study.The expressed rVvhA showed well immunoreactivity and immunogenicity,which could be used as a antigen in V.vulnificus vaccine and detection kit.