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

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.

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

Objective To rapidly and efficiently construct a replication-competent human recombi-nant 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 re-combination 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 trans-fected 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.