Construction,Identification And Biological Feature Study Of Human Hepatocellular Carcinoma Cells Stably Expressing HBeAg

ObjectiveAt present,researches on HBeAg are mostly focused on clinical analysis.There are few studies on the influence and mechanism of hepatoma cell and its microenvironment.We use lentivirus vector technology to construct the Hep G2 liver cancer cell line with HBe Ag gene stable expression,identify the expression of HBeAg in the transfected cells,and study the effect of HBeAg on the proliferation,migration and invasion of Hep G2 cells.METHODSAccording to the human HBe Ag gene sequence in GenBank,the target gene fragment was chemically synthesized,and the lentiviral vector carrying the HBe Ag gene sequence was constructed,and the HBe Ag gene was transfected into the Hep G2 cell genome by lentiviral transfection technology.Transfected cell lines were obtained by puromycin selection and monoclonal culture.RT-qPCR and Western blot techniques were used to detect the transcription and translation levels of HBeAg in transfected cells.The expression of HBeAg in the cell supernatant was detected by IFMA.CCK-8 experiment,clone formation experiment,Transwell cell experiments were used to detect the changes of cell proliferation,migration and invasion ability after transfection.After 3 months of passage,the HBe Ag mRNA and protein expression levels and cell biological characteristics of the transfected cell lines were detected.RESULTSThe lentiviral vector carrying the HBeAg gene sequence was successfully constructed.The homology of the sequencing result of the lentiviral product with the HBeAg sequence in the GenBank database was 100%.Hep G2 cells were transfected with lentivirus and screened by puromycin to obtain transfected Hep G2 cell line.And experiment results show,(1)RT-q PCR verification cells can express HBeAg mRNA.(2)Western blot experiments detected cells producing HBeAg protein.(3)Assay of HBeAg positive by cell supernatants by IFMA.(4)CCK-8 experiment and colony formation experiment proved that the cell proliferation ability decreased(P<0.01).(5)Transwell experiments showed that the cell migration and invasion ability decreased(P<0.01).After 3 months of cell culture,there was no significant change in HBeAg mRNA and protein expression level and cell biological characteristics.CONCLUSIONSIn this experiment,a recombinant lentiviral vector carrying the HBeAg gene was constructed.After transfection of Hep G2 cells with the lentivirus,Hep G2 cells can effectively express the HBeAg gene,translating and producing HBe Ag protein,and can secrete HBeAg out of the cell.So a hepatoma cell line that stably expressed HBeAg was successfully constructed.In vitro experiments confirmed that the proliferation,migration and invasion of Hep G2 hepatoma cells were inhibited after stable expression of HBeAg.