Identification Of Containing PreS1Recombinant Adenovirus Gene Therapy Vector Of Liver Cell Tropism

Objective：Amplify and purify recombinant adenovirus gene therapyvector rAd-preS1, and a variety of cell assay in vitro to detect tropism ofliver cell.Methods：Fiber DNA gene fragment contains Pres1gene wasdesigned and amplified, and inserted into the pAdEasy-1which instead ofwild fiber DNA fragment， then produce rAd-PreS1by homogenousrecombination with shuttle plasmid，After being confirmed by restrictionenzyme digesion and DNA sequencing，the human embryonic kidney celllines was transfected to generate gene therapy vector rAd-preS1. Target genefragment preS1was detected by the polymerase chain reaction PCR. Afterinfected293cells repeatly, recombinant adenovirus was amplified and gethigher titer，using CSCL density gradient centrifugation to purify virus.weinfected variety cells as L02,A549, and Hep2with rAd-preS1, rAd wasnegative control group. Observe the infected cells carrier GFP and virustiters for statistical analysis.Results：Restriction endonuclease confirmed that the recombinantvirus were consistent and PCR results showed750bp gene fragment, provedPreS1fragment successful expression.Transfection results show GFP expression obviously significant and virus titers in cells have significantdifference between L02and A549，Hep2(P value are all<0.01)，but thegroup A549and Hep2had no statistically significant (P value are>0.05). thecontrol group all had no statistically significant.Conclusions：Carrying HBV PreS1recombinant adenoviral vectorrAd-preS1be verified its function of relatively strong tropism to liver cell invitro，while no significant effection on non-liver cells，Wild adenovirusinfected cells was no significant difference，rAd-preS1will be a goodfoundation for the research on the gene therapy for hepatic diseases.