Construction Of Tropism Liver Cells Recombinant Adenoviral Vector For Gene Therapy

Objects: The recombinant adenovirus (rAd) is commonly used in gene therapy ,but it lacks the feature of targeting the liver cells. we want to transform the structure of cilia in rAd to build a hepatotropic rAd vector for gene therapy of liver disease.Methods: We based on the high-expression vector system of rAd-AdEasy System and constructed the recombinant backbone plasmid pAdEasy-1-preS1 by cloning the preS1 encoding 21-47 amino acid sequence of PreS1 who is the key binding receptor of liver cells into the genes encoding HI Loop in the backbone plasmid pAdEasy-1 of AdEasy System. Then the homologous recombinant plasmid pAd-pres1 constructed by pAdEasy-1-preS1 and pShuttle- IRES-hrGFP-1 transfected HEK293 cells and be generated the rAd-PreS1.Results: The sequence of the recombinant plasmid cloned by preS1 was absolutely correct by detecting the gene sequence; The pAd-preS1 transfected HEK293 cells, after one week, we found it expressed fluorescent protein; We obtained the preS1 by PCR based on pAd-preS1 as a template;We extracted the proteins of rAd-PreS1 and confirmed the PreS1(21-47)by Western blotting;The rAd-PreS1 infected multiple cells ,we found it targeted the liver cells comparatively by calculating the titer of rAd-PreS1.Conclusion:The rAd-PreS1 generated in HEK293 cells whose genes containing preS1 showed the feature of targeting liver cells comparatively by cell-experiment in vitro.