霍乱疫苗株溶血素hlyA基因敲除及绿色荧光蛋白构建

Construction of hemolysin hlyA gene knocked out of cholera vaccine candidate and inserted green fluorescent protein gene

目的:探讨霍乱疫苗株NFYY101溶血素hly A基因敲除及绿色荧光蛋白的构建。方法:将hlyA基因上游片段(hlyAup)、hlyA下游片段(hlyAdown),克隆入pUC18中,再在两者间插入绿色荧光蛋白(laczGFPuv)基因,获得重组质粒pUC18-hlyAup-laczGFPuv-hlyAdown,再构建重组自杀质粒pCVD442-hly Auplacz GFPuv-hly Adown,接合入霍乱疫苗株NFYY101中,进行同源重组。结果:正确构建重组自杀质粒pCVD442-hly Aup-lacz GFPuv-hly Adown表达绿色荧光蛋白,成功缺失了疫苗株候选株NFYY101的hlyA基因,在敲除位置同源重组入绿色荧光蛋白基因并表达。结论:成功构建表达绿色荧光蛋白的重组自杀质粒,可用于O1群及O139群霍乱疫苗株hlyA基因敲除及绿色荧光蛋白构建。

Objective To knock down hemolysin hlyA gene and insert green fluorescence protein gene of vibrio cholerae vaccine candidate NFYY101. Methods Amplified fragments of hlyA gene upstream （hlyAup） and downstream （hlyA down）, lacz-GFPuv, and hlyA up-laczGFPuv-hlyA down, and plasmids treated with specific enzymes were utilized to construct recombinant plasmids pUC18-hlyAup-laczGFPuv-hlyAdown and pCVD442- hlyAup-laczGFPuv-hlyA down. Following the construction of the recombinant suicide plasmids, a vaccine candidate was constructed by homologous recombination, while SM10hpir carrying pCVD442- hlyAup-laczGFPuv-hlyAdown was utilized as the donor strain to transfect NFYY101. Results Construction of recombinant suicide plasmids pCVD442- hlyAup-laczGFPuv-hlyAdown was satisfactory that hemolysin hlyA gene was knocked out and green fluorescence protein gene was successfully inserted of vibrlo cholerae vaccine candidate NFYY101. Conclusion Construction of the recombinant suicide plasmid pCVD442-hlyAup-laczGFPuv-hlyAdown successfully can be used for knocked out the hlyA gene and added green fluorescence protein gene as genetic marker into the chromosomal DNA of vibrio cholerae vaccine candidate.