Recombinase-mediated in vitro rapid construction of replication-competent human adenovirus type 14 vector encoding EGFP / 中华微生物学和免疫学杂志

ABSTRACT

Objective@#To rapidly and efficiently construct a replication-competent human recombinant adenovirus type 14 vector expressing enhanced green fluorescence protein (rAd14-EGFP) using in vitro homologous recombination.@*Methods@#The skeleton plasmid pBRAd14 was constructed using homologous recombination in Escherichia coli (E.coli) BJ5183 competent cells. The plamid was linearized and transfected into AD293 cells to rescue Ad14. Exnase, a recombinase, was used to construct the shuttle plasmid pSK14-EGFP in vitro using homologous recombination among four fragments. The overlapping sequence was 15-20 bp. Three exogenous fragments generated with PCR including Ad14 E3L fragment, EGFP gene and Ad14 E3R fragment were cloned into the plasmid pBluescript Ⅱ SK(-) simultaneously. Recombinant plasmid pBRAd14-EGFP was constructed by in vitro homologous recombination between 27 kb fragment of plasmid pBRAd14 obtained through double digestion and Ad14 E3L-EGFP-Ad14 E3R fragment amplified by PCR using the shuttle plasmid pSK14-EGFP as template. The plasmid pBRAd14-EGFP was linearized and transfected into cells to obtain the viral vector rAd14-EGFP, which was then used to immunize mice to detect the induced immune responses.@*Results@#A replication-competent E3-deleted adenovirus vector rAd14-EGFP expressing EGFP was successfully constructed. Intracellular proliferation properties and immunogenicity of the vector were no significantly differences compared with those of Ad14.@*Conclusions@#In vitro homologous recombination using the commercial recombinase Exnase can be a rapid, efficient and accurate method to construct adenoviral vector.