Construction Of The Recombinant Adenovirus Of Inhibitor Of Differentiation 1 And Its Related Function Research

Objective: To construct the recombinant adenovirus(Rad)with expressing inhibitor 1 of differentiation(Id1),detect the expression level of RAd-Id1 in cytology and mice and its corresponding changes and study the effect of Id1 on hepatitis B virus(HBV)replication in HCC cell.Methods: Firstly,Id1 gene has been cloned to pAdtrack-TO4,which then was restructured in the skeleton adenovirus vector AdEasy-1.Secondly,p AdEasy-Id1,the recombinant adenovirus plasmid,was packaged in HEK293 cells by the methods of Prof.Tong-Chuan He to obtain recombinant adenovirus Id1(RAd-Id1).Finally,the titer of virus was determined by green fluorescent protein(GFP)labeling method.The over-expression efficiency of Id1 was measured by q RT-PCR and Western blot in HepG2 cells infected with RAd-Id1,and the effect of Id1 on proliferation of HepG2 cells was detected by MTS assay.Animal model of Id1 expressing in liver tissue was established through the way of tail vein injection of RAd-Id1.In order to determine the suitable load of RAd-Id1 virus for infecting the mice,viral load gradient experiment was divided into PBS control group,RAd-GFP control group and RAd-Id1 experimental group(n=5/group).Time gradient experiment examined once every 5 days as a group,a total of 7 group(n=5/group),to explore the appropriate time.The expression of Id1 as well as the changes of alpha fetal protein(AFP)and carcinoembryonic antigen(CEA)in liver tissues were determined by Western blot.The level of Id1 antibody and the concentrations of AFP and CEA in serum were detected by ELISA.The change of HBV replication level was observed in HepG2.2.15,one of liver cancer cell lines with HBV,after infected with RAd-Id1.Results: Recombinant adenovirus plasmid containing Id1 gene was successfully established,then the corresponding adenovirus were acquired and the measured titer was 1.5×1011 GFU/mL.Western blot and qRT-PCR analysis showed that Id1 mRNA and protein levels significantly increased in HepG2 infected with RAd-Id1 for 48 h and 72 h respectively(P<0.01).MTS assay reported that the values in RAd-Id1 group were higher than those in NC group and RAd-GFP group after 96 h;Western blot results showed: when compared with PBS group and RAd-GFP group,the RAd-Id1 group’s levels of Id1 as well as AFP and CEA in liver was higher.ELISA results showed: the concentrations of AFP and CEA in serum were positively correlated with the load of RAd-Id1.There was no significant difference among these concentrations of Id1 antibody in serum of mice infected with different load of RAd-Id1(P>0.05).HBV replication(HBV DNA)and expression levels(HBV pgRNA and HBc protein)decreased in Hep G2.2.15 after infected with RAd-Id1.Conclusion: The RAd-Id1 is successfully constructed,which serves as the vector tool to study the mechanism of action of Id1 on hepatic neoplasm and HBV at the cellular level.Mouse animal model of RAd-Id1 accompanied with the elevation of both AFP and CEA in liver also has been successfully established by tail vein injection,which suggests that the ectopic expression of Id1 may be related to the expression of AFP and CEA.Furthermore Id1 may participate in the process of inhibiting the HBV replication and expression.