Construction of the recombinant human adenovirus type 3 expressing Norovirus capsid protein gene
- VernacularTitle:诺如病毒衣壳蛋白重组人3型腺病毒的构建
- Author:
Xingui TIAN
;
Rong ZHOU
;
Haitao LI
;
Sitang GONG
;
Qiwei ZHANG
;
Bing ZHU
;
Huiying SHENG
;
Jiayu ZHONG
- Publication Type:Journal Article
- Keywords:
Norovirus capsid protein;
Human adenovirus type 3;
Homologous recombination
- From:
Chinese Journal of Microbiology and Immunology
2008;28(9):782-786
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare recombinant human adenovirus type 3 expressing Norovirus cap-sid protein gene(Noro-orf2). Methods The cDNA for Noro-orf2 was amplifed by RT-PCR from stool of in-fantile gastroenteritis and cloned into the adenovirus shuttle vector pBSE3CMV-egfp. The vector pBSE3CMV-Nor was linearized with EeoR Ⅴ and Not Ⅰ, and transformed into E. coil BJ5183 with lined edenovirus ge-nomic DNA pLasmid pBRAdv3 by Rsr Ⅱ. The identification of recombinant adenovirus plasmid pBRAdv3E3dNor was performed by PCR, enzyme digestion and DNA sequencing. Then pBRAdv3E3dNor was digested with AsiS Ⅰ and transfeeted into Hep-2 cells with LipofectAMINETM 2000 to package recombi-nant adenovirus particles. Results Noro-orf2 was successfully inserted into the shuttle vector. The recombi-nant adenoviral plasmid pBRAdv3E3dNor was generated by homologous recombination in E. coil BJ5183 and confirmed by PCR and enzyme digestion. The recombinant adenovirus was successfully packaged and puri-fied. Norovirus eapsid protein gene expression was confirmed in Hep-2 cells by immunecytochemistry assay. Conclusion The recombinant type 3 adenovirus expressing Norovirus eapsid protein gene was successfully constructed. This study laid a foundation for developing vaccine against Norovirus.
