Experimental Study Of Anti-angiogenic Treatment Of Gastric Cancer

Objective: To produce recombinant adenovirus via high efficient homologous recombination in bacteria with human endostatin as target gene. Methods: hEn gene was cut from plasmid pCA13-hEn and cloned into shuttle plasmid and formed transfer plasmid of pAdtrack-CMV-hEn. After linealized with PmeⅠ and co-transformed into BJ5183 bacterial cells with adenovirus genomic DNA plasmid of pAdeasy. The identified recombinant adenovirus plasmid DNA was digested with PacⅠand transfected to 293 cells for package of recombinant adenovirus particles. The PCR technique was used to detect target gene. The titer of the recombinant Ad was measured. RT-PCR proved target gene expression in infected tumor cells. Results: Positive recombinant bacterial clones have been identified after co-transformation of BJ5183 bacterial cells. Recombinant adenovirus was obtained from 293 cells transfected with Pac I-digested Ad genomic plasmid DNA. PCR test indicated that the recombinant Ad contained the hEn gene. The titer of purified Ad was 2.4×1011 pfu/ml. Meanwhile, mRNA product of hEn was detected in the infected tumor cells by RT-PCR. Conclusion: The method of homologous recombination in bacteria is a convenient and high efficient method to produce recombinant adenovirus. The recombinant adenoviruses can effectively mediate target gene expression in infected SGC-7901 cells.