Construction of recombinant adenovirus of monocyte chemoattractant protein-1 by two-step homologous recombination in bacteria

Objective To construct replication defective recombinant adenovirus of human monocyte chemoattractant protein-1(MCP-1) by two-step homologous recombination in bacteria.Methods The adenoviral backbone plasmid pAdEasy-1 was transformed into E.coli BJ5183 cells.A single colony containing pAdEasy-1(BJ5183pAdEasy-1) was subsequently characterized by Hind Ⅲ restriction analysis of the plasmid,and underwent electrocompetent.The cDNA of MCP-1 gene was obtained by RT-PCR,and was sub-cloned into a transfer plasmid to make pAdTrack-MCP-1,which was then linearized by PmeⅠ and transformed into BJ5183pAdEasy-1.The recombinant adenovirus vector pAd-MCP-1 was characterized by PacⅠ restriction analysis.pAd-MCP-1 was linearized and then was transfected into HEK293 packing cells to produce virus particles.The detection of recombinant adenovirus was characterized by PCR.Results The simplified two-step homologous recombination in bacteria generated recombinant adenovirus plasmids at an efficacy of more than 50%.Recombinant adenovirus was obtained and identified by PCR.Conclusions The two-step protocol of homologous recombination in bacteria is an easier way to construct the recombinant adenovirus plasmid.The establishment of recombinant adenovirus of MCP-1 provides an important method for further study.