鳗弧菌溶血毒素基因vah4的克隆及表达

目的：对鳗弧菌溶血毒素基因vah4进行克隆与原核表达,为进一步深入研究其免疫原性及VAH4的功能奠定基础。方法：PCR扩增vah4,将扩增的产物连接于测序载体pMD18—T上,经测序反应确定无误后,再将PCR产物与原核表达载体pET-32a构建表达VAH4的重组质粒（pET-32a-VAH4）,经PCR鉴定后,再转入表达宿主大肠杆菌BL21菌株内,对转化菌株进行诱导表达,SDS-PAGE电泳检测。结果：含重组质粒的菌株有表达蛋白,其表达的蛋白质相对分子质量为40kDa,并经Western blot鉴定结果证实该条带即VAH4-His融合蛋白。结论：vah4基因成功克隆至pET-32a质粒内并成功表达,为进一步研究其免疫原性、VAH4的毒性作用效果及作用机制奠定基础。

Cloning of vah4 Gene of Hemolytic Toxin of Vibrio anguillarum and It's Expression in E. coli

Objective:Clone and express vah4 gene of hemolytic toxin of Vibrio anguillarum in order to establish the foundation for further study on it's immunogenicity and function of VAH4. Method: Amplified vah4 gene by PCR and the amplification products were connected with sequencing carrier pMD18-T.Then constructed recombinant plasmid vector (pET-32a- VAH4) expressing VAH4 by using the amplification products and prokaryotic expression vector pET- 32a after sequencing . And transfered it into E. coli BL21 after PCR identification. Induced the translated bacteria to express. Then identified the expression protein by SDS- PAGE electrophoresis. Result:The translated bacteria could express VAH4 with the molecular size of 40kDa.The results of Western blot showed that the fusion protein is VAH4- His. Conclusion: The success of cloning and expression of vahA gene provides basis for the further study on it's immunogenicity and toxicity and action mechanism of VAH4.