Construction And Identification Of Recombinant Adenovirus Vector Carring Cytosine Deaminase Gene

Objective Recombinant adenoviruses vector carring cytosine deaminase(CD) gene was constructed by the homologous recombination method in bacteria.Methods The RNA of E.coli JM109 was extracted with the method of TRIzol. Targetgene of CD amplificated by RT-PCR was connected with high performance clone vectorpMD18-T, then was converted into E.coli JM109 competence bacteria. TA clone wereperformed to get full-length cDNA of CD gene. The gene was subcloned into thepAdTrack-CMV shuttle vector. By sequencing, the resultant plasmidpAdTrack-CMV-CD was cotransduced into E.coli BJ5183 competence bacteria withpAdEasy-1 plasmid and undergone homologous recombination. The recombinantspAd-CD was selected by kanamycin resistance and restriction endonuclease analysis,followed by CD gene sequencing. Results we completed the construction of adenovirusshuttle plasmid pAdTracCMV-CD, and further constructed recombined adenovirus vectorpAd-CD by homologous recombination in bacteria. By the restriction endonucleaseanalysis and PCR with pAd-CMV-CD as the follow board, a target fragment of 1293 basepair was obtained. By the restriction endonuclease analysis of pAd-CD, a long fragmentof 31kb and a marked strap of 3.0kb were obtained. Conclusion The homologousrecombination in bacteria is a conveniently efficient method to prepare recombinantadenovirus plasmid. It provided a condition for the experimental studies of usingadenovirus—mediated CD/5—FC suicide gene in treatment to pancreatic cancer.