重组金属蛋白酶蛋白的纯化及鉴定  

Purification and identification of Vibrio vulnificus vvp gene

作　　者：郑晶[1] 谢富生[1] 

机构地区：[1]海南医学院病理教研室,海南海口571199

出　　处：《中国热带医学》2013年第8期928-930,共3页China Tropical Medicine

基　　金：海南省自然科学基金资助项目(No.309036)

摘　　要：目的构建创伤弧菌(Vibrio vulnificus,Vv)金属蛋白酶(vvp)基因原核表达系统并鉴定其表达产物的免疫性。方法采用pET-32a质粒构建vvp基因原核表达载体,在E.coliBL21(DE3)宿主菌中用异丙基-β-D-硫代半乳糖苷(IPTG)诱导目的重组金属蛋白酶蛋白rvvp表达,采用His-Ni亲和层析法提纯rVvp,SDS-PAGE检测表达和提纯效果。采用兔抗Vv全菌抗体的Western Blot鉴定其特异性和免疫原性。结果在1mmol/L IPTG诱导下,rvvp产量较高。提纯的rvvp经SDS-PAGE后仅显示分子量为86KD的单一蛋白条带。重组蛋白rvvp能与兔抗Vv全菌抗体发生特异性结合。结论本研究成功地构建了创伤弧菌vvp基因高效原核表达系统,所表达的rvvp具有良好的免疫反应性,可作为Vv免疫检测试剂盒及疫苗的抗原。Objective To clone Vibrio vulnificus vvp gene and construct a pmkaryotic expression system of the gene and identify immunity of the recombinant protein rvvp. Methods Plasmid pET-32a was used to construct prokaryotic expression vectors of the vvp gene.rvvp was expressed in E.coli strain BL21 （DE3）. IPTG was used to induce the target recombinant protein rvvp and His-Ni affinity chromatography was applied to extract rvvp. By using SDS-PAGE plus BioRad Agarose Image Analyser. The effects of expression and purification of rvvp were determined. Western Blot was applied to determine immunoreactivity and immunogenicity of the rvvp and antibodies against whole cell of V.vulnificus and rabbit antiserum immunized with the rvvp. Results In comparison with the published results by GeneBank,the homology of nucleotide sequences of the cloned vvp gene was 99%. Under inducement of 1mmol/L IPTG,the output of rvvp could reach a high level. The purified rvvp only showed a single fragment in gel after SDS-PAGE. The rvvp 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 vvp gene with high efficiencies was successfully established in this study. The expressed rvvp showed well immunoreactivity and immunogenicity, which could be used as a antigen in development of V. vulnificus vaccine and detection kit.

关 键 词：金属蛋白酶 wp基因 克隆 原核表达 

分 类 号：R37[医药卫生—病原生物学]