Construction of Recombinant Adenovirus Containing Apoptin VP3 Gene and Its inducing apoptosis effect on Human Colonic Carcinoma Cells alone or combination with Curcumin

Objective To construct recombinant adenovirus containing Apoptin VP3 gene by using the method of homologous recombination in bacteria,and observe its inducing apoptosis effect on human colonic carcinoma cells alone or combination with Curcumin in vitro. Methods The DNA sequence of VP3 was amplified from PET15b-VP3 and ligated into pShuttle-IRES-hrGFP The recombinant plasmid was named after pShuttle-VP3-hrGFP and was identified with PCR and EcoR Ⅴ digestion;pShuttle-VP3-hrGFP was linealized and transformed into ultracompletent BJ5183 containing pAdeasy-1,then recombinant adenovirus pAd-vp3 was constructed by homologous recombination in bacteria. The recombinant adenoviral plasmid pAd-VP3 was identified by PCR 、digestion and DNA sequencing. subsequently linealized recombinant advenovirus vector pAd-VP3 was transfected into AD293 cells by liposome to generate recombinant adenovirus particles which were purified and concentrated by CsCI density gradient centrifugation. Adenovirus particles 、Curcumin alone or combination were used to infect the human colonic carcinoma cells SW480,and apoptosis rate and cell phase were observed. Results Recombined adenovirus plasmid pShuttle-VP3-hrGFP was generated successfully. Linealized pShuttle-VP3-hrGFP transformed into ultracompletent colibacillus BJ5183,There were two bands 4. 5kb and larger than 23 kb when pAd-VP3 was digested with PacI. A 402 bp VP3 cDNA fragment was amplified by PCR. The target gene VP3-hrGFP was successfully cloned into Adenovirus,The sequence of VP3 segment was identical with that published in GenBank. we have successfully constructed recombinant advenovirus pAd-VP3. Adenovirus encoding Apoptin VP3 gene was generated with titers of about 1. 738 × 1012opu·L-1,pAd-VP3 Adenovirus and Curcumin all could arrest cell cycle in G0 /G1 phase. The effect above of Curcumin combination with pAd-VP3 was better than that of pAd-VP3 、Curcumin alone( P 0. 01). Conclusion Homologous recombination in bacteria can efficiently and conveniently construct high quality recombinant adenovirus containing VP3 gene,the adenovirus combination with Curcumin could induce SW480 cells apoptosis through arresting cell cycle in G0/G1 phase.