Construction of gene therapy vector rAd-preS1 and identification of liver cell targeting function

Abstract:

Objective：To prepare the recombinant adenoviruses carrying gene fragment PreS1 and to detect tropism of liver cell infect－ed-virus. Methods：Fiber DNA gene fragment containing PreS1 gene was designed and amplified and inserted into the pAdEasy-1. Fiber DNA gene fragment replaced wild fiber DNA fragment then produced rAd-preS1 by homogenous recombination. After being confirmed by restriction enzyme digesion，human embryonic kidney cell lines were transfected with pAd-preS1 to get rAd-preS1. Protein expression of preS1 was detected by Western blot. Human normal live cell L02，human lung cancer cell A549，human larngy－neal cancer cell Hep2，human overian cancer cell SKOV3 were infected with rAd-preS1 respectively and rAd was taken as negative control group. Green fluorescent protein（GFP） expression of each cell were observed and manifested by virus titers. Results：Restric－tion digestion confirmed the consistent of recombinant vertor. Western blot results showed that protein molecular weight was about 63 kD fragment stripe. Transfection results showed that GFP expression were higher in L02 group than in A549，Hep2，SKOV3 groups（P value were 0.000）. There was no staitical differnece in GFP expression among A549，Hep2 and SKOV3 groups（P >0.05）. There was no staitical differnece in GFP expression among control groups（P >0.05）. Conclusions：Recombinant adenovirus vector rAd-preS1 demonstrate its function of tropism to liver cell in vitro，which will be the foundation for the research of gene therapy of PreS1 for hepatic diseases.