Construction Of Recombinant Adenovirus Containing Apoptin VP3Gene And Its Expression In Colonic Carcinoma Cells

Objective To construct recombinant adenovirus plasmid containing Apoptin VP3gene by using the method of homologous recombination in bacteria, and provides abasis for VP3gene in the treatment of cancer research.Methods The VP3cDNA primers were designed, the DNA sequence of VP3wasamplified from recombinant vector PET15b-VP3and ligated intopShuttle-IRES-hrGFP-2, The recombinant plasmid was named afterpShuttle-VP3-hrGFP and was identified with PCR and EcoR Ⅴ digestion;pShuttle-VP3-hrGFP was linealized with PmeI and transformed into ultracompletentBJ5183containing pAdeasy-1, then recombinant adenovirus pAd-vp3-hrGFP wasconstructed by homologous recombination in bacteria. The recombinant adenoviralplasmid pAd-VP3-hrGFP was identified by PCR、PacI digestion and DNA sequencing.subsequently linealized recombinant advenovirus vector pAd-VP3-hrGFP wastransfected into AD293cells by liposome to generate recombinant adenovirus particleswhich were purified and concentrated by CsCI density gradient centrifugation.Adenovirus particles were used to infect the human colonic carcinoma cells SW480,andinfection rate and VP3expression were observed.Results Recombined adenovirus plasmid pShuttle-VP3-hrGFP was generatedsuccessfully. Linealized pShuttle-VP3-hrGFP transformed into ultracompletentcolibacillus BJ5183containing pAdeasy-1, There were two bands4.5kb and larger than 23kb when pAd-VP3-hrGFP was digested with PacI. A402bp VP3cDNA fragment wasamplified by PCR. The target gene VP3-hrGFP was successfully cloned intoAdenovirus,The sequence of VP3segment was identical with that published inGenBank.Our experiment verificated we have constructed recombinant advenoviruspAd-VP3-hrGFP successfully. Adenovirus encoding Apoptin VP3gene was generatedwith titers of about1.738×1012opu/ml，The Infection efficiency of adenovirus to SW480cells was more than98%. Apoptin VP3gene could be expressed in SW480cells.Conclusion Homologous recombination in bacteria can efficiently and convenientlyconstruct high quality recombinant adenovirus containing VP3gene, the adenoviruscould infect SW480cells efficiently and Apoptin VP3gene could express well, whichprovides a basis for for Apoptin VP3gene further research in cancer therapy.