Construction and identification of recombinant adenovirus expressing B7-1 genes

Objective To construct replication deficient recombinant adenovirus expressing b7-1 genes using AdEasy system, and detect the expression of b7-1 genes in Hep-2 after adenovirus infection. Methods A recombinant adenovirus shutter plasmid pAdTrackB7-1 containing human B7-1 gene was constructed by molecular cloning. The recombinant shutter plasmid was lined by Pme I and co-transformed with backbone vector pAdEasy-1 into bacteria BJ5183 for homologous recombination to obtain the recombinant adenoviral vector pAd-B7-1. The recombinant adenoviral vector was then transfected into HEK293 package cells to produce virus particles. The expression of B7-1 gene in cells were detected in human laryngeal epithelial carcinoma cells Hep- 2 by RT-PCR after adenovirus infection. Results The recombinant adenoviral vector was successfully established and confirmed by restriction endonuclease digestion. GFP expression was observed on the 3rd day after the adenovirus transfecting into HEK 293 cells. Recombinant adenovirus was identified by PCR. High level Expression of B7-1 mRNA was maintained for 6 days in Hep-2 cells after adenovirus infection. Conclusion The construction of recombinant adenovirus of Ad-B7-1 provided a basis for further studies of combined gene therapy for laryngeal cancers.