Cloning of LIGHT gene and construction of its recombinant adenovirus vector

Aim To clone LIGHT full length cDNA and construct its recombinant adenovirus vector so as to observe the effects of LIGHT overexpression and adenovirus infection on the growth of infected cells. Methods Human LIGHT cDNA was amplified by RT-PCR from peripheral blood mononuclear cells(PBMCs) and it was cloned into shuttle vector pAdTrack-CMV by standard procedures. The resultant plasmid was linearized by enzyme digestion and subsequently cotransformed into E.coli BJ5183 with an adenoviral backbone plasmid pAdEasy-1. Recombinants were selected for kanamycin resistance and confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmid was transfected into adenovirus packaging 293 cell lines. LIGHT recombinant adenovirus production was observed by fluorescent microscope and confirmed by PCR and Western blot analyses. ResultsA 723bp cDNA was amplified by RT-PCR from PBMCs, its sequence analysis was documented as was expected. Analyses of fluorescent microscope observation , PCR and Western blot showed that LIGHT recombinant adenovirus could infect 293 cell lines and replicate in the cells. ConclusionHuman LIGHT gene is successfully cloned and its recombinant adenovirus vector has been constructed . This provide the basis of further researching function of LIGHT gene.