Construction Of Recombinant Adenovirus Vector Co-expressing HBcl-2 And HVEGF₁₆₅ Genes

Objective:To construct recombinant adenovirus vector co-expressing hBcl-2 and hVEGF165 genes , so as to lay a foundation for the subsequent gene therapy and cell transplantation of animal experiments.Methods:The recombinant adenovirus vector co-expressing hBcl-2 and hVEGF165 genes connectted by IRES element was constructed based on the AdEasy system and the mechanism of homologous recombination in bacteria . Firstly , the PCR primers were designed . The total RNA was extracted from A549 cells . hBcl-2 and hVEGF165 gene fragments were obtained from the total RNA by RT-PCR respectively and inserted into the pMD19T vector . Both the hBcl-2 gene and hVEGF165 gene were verified by gene sequencing . hBcl-2 gene , IRES element and hVEGF165 gene were cloned into the shuttle vector plasmid pAd-Track-CMV successively . After being verified by enzemy digestion , the recombinant shuttle vector plasmid was linearizated and electroporated into the BJ5183 bactetia which had carried the adenovirus skeleton plasmid pAd-Easy-1 for homologous recombination . After being verified by enzemy digestion , the recombinant adenovirus vector plasmid was linearizated and transfected into HEK293 cells by lipofectamine for packaging . The recombinant adenovirus were further amplificated and purified after PCR identification . The final virus production were titrated by counting GFP . Results:The hBcl-2 and hVEGF165 genes were verified by gene sequencing . Both the recombinant shuttle vector plasmid and the recombinant adenovirus vector plasmid were verified by enzemy digestion . The expression of GFP could be firstly observed in HEK293 cells on 3rd day after transfection . The CPE was observed at the 7th day of second round of amplificaion . PCR identification showed that the adenovirus carried the hBcl-2 and hVEGF165 genes . After four rounds of amplificaion and final purification , the determination of titer was 5×109 pfu /mL .Conclusions:1. We had cloned the hBcl-2 and hVEGF165 genes sucessfully .2. We had constructed and prepared the high-titer of co-expressing recombinant adenovirus vector carrying hBcl-2 and hVEGF165 genes sucessfully , which lay the experimental foundation for the treatment of myocardial infarction using united gene therapy .