Construction of hemolysin hlyA gene knocked out of cholera vaccine candidate and inserted green fluorescent protein gene
10.3969/j.issn.1006-5725.2016.03.007
- VernacularTitle:霍乱疫苗株溶血素hlyA基因敲除及绿色荧光蛋白构建
- Author:
Yanyan ZHOU
;
Biao KAN
;
Yongyu RUI
- Publication Type:Journal Article
- Keywords:
Vibrio cholerae;
Live attenuated vaccine;
Genetic recombination;
Green fluorescence protein
- From:
The Journal of Practical Medicine
2016;32(3):362-366
- CountryChina
- Language:Chinese
-
Abstract:
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 (hlyAdown),lacz-GFPuv,and hlyAup-laczGFPuv-hlyAdown, and plasmids treated with specific enzymes were utilized to construct recombinant plasmids pUC18-hlyAup-laczGFPuv-hlyAdown and pCVD442-hlyAup-laczGFPuv-hlyAdown. Following the construction of the recombinant suicide plasmids ,a vaccine candidate was constructed by homologous recombination ,while SM10λpir 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 vibrio 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.
