Construction And Identification Of Tropism Liver Gene Therapy Recombinant Adenoviral Vector

Objects: In this study, we based on high expression of recombinant adenovirus vector system pAdEasy System,to be constructed with a hepatotropic vetor of rAd by change wild fiber structure of pAdEasy-1, which lose the natural properties of adenovirus and with HBV hepatotropic,for targeted gene therapy of liver disease.Methods: We based on pAdEasy System.we Using in vivo homologous recombination,replaced the wild fiber DNA fragment of pAdEasy-1 with the Fiber DNA fragments which have the Encoding gene of HBV who target liver cells (PreS1 of 21-47 amino acids),and constructed the recombinant backbone plasmid pAdEasy-1-Fiber,use BJ5183/pAdEasy-1-Fiber competent cells and pAdTrack-CMV shuttle plasmid homologous re-composition of the virus plasmid pAd-Fiber. With PacI digested recombinant plasmid pAd-Fiber,Then the recombinant plasmid pAd-Fiber transfected HEK293 cells and be generate gene therapy vector rAd-Fiber.We infected variety cells with rAd-Fiber and test it on the targeting of liver cells. Results: we replaced the wild fiber DNA fragment of pAdEasy-1 with the Fiber DNA fragments which have the encoding gene of HBV who target liver cells (PreS1 of 21-47 amino acids) was absolutely correct by detecting the gene sequence; The pAd-Fiber transfected HEK293 cells, after 7 days, we found it expressed GFP;We obtained the Fiber by PCR based on rAd-Fiber as a template;We extracted the proteins of rAd-Fiber and confirmed the PreS1(21-47aa)by Western blot;The rAd-Fiber infected different cells , we found it relatively targeted of liver cancer cells .Conclusion: we replaced the wild fiber DNA fragment of pAdEasy-1 with the Fiber DNA fragments which have the encoding gene of HBV who target liver cells (PreS1 of 21-47 amino acids),we changed wild fiber of the pAd,the rAd-Fiber generated in HEK293 cells showed the feature of targeting liver cancer cells comparatively by cell-experiment in vitro.