Construction of the recombinant human adenovirus type 3 expressing Norovirus capsid protein gene / 中华微生物学和免疫学杂志

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.