The Inhibition Effect Of EFTUD2 On HBV Was Researched By NTCP-Huh7 Cell Model

Background and Aims: Hepatitis B has always been an important threat to global public health.In order to study HBV,a variety of cell models have been used at home and abroad,such as Hep G2.215 cells and Hep AD38 cells transfected with HBV plasmid,but lack of HBV invasion cells.Human primary hepatocytes and Hep RG cells can be infected by HBV in vitro,but the culture is complex and cannot be transmitted for infinite generations.Although hepatocellular carcinoma(HCC)can be transmitted for an infinite number of times,the expression of sodium taurocholate cotransporting polypeptide(NTCP)in the cell surface is low,and it is difficult to infect HBV virus.The above cells have certain limitations.In November 2012,professor Li announced the discovery of NTCP as a functional receptor for HBV,enabling HBV to infect HCC cell lines in vitro.Most of the existing technology using transient transfection carry NTCP gene into liver cells,thus NTCP expression is relatively short,and lead to low efficiency and unstable infection of HBV.In light of this disadvantage,this paper synthesized GV358-NTCP recombinant lentiviral vector by linking the sodium ionitrosulfolic acid co-transporter protein(NTCP)with gv-358 lentiviral vector.Then the GV358-NTCP was transfected into Huh7 cells,and the cells that could express NTCP protein steadily were established.The expression of various virus-related proteins after HBV infection was detected to verify whether NTCP-Huh7 cells had HBV susceptibility.By transfection of p EFTUD2 into cells,NTCP-Huh7 overexpressed EFTUD2.The expression of virus-related proteins and HBV DNA was determined again to verify the inhibitory effect of EFTUD2.Finally,p EFTUD2 and si RIG-1 were transfected into the cell,and the DNA content of HBV was determined on the cell,and it was confirmed whether EFTUD2 played an antiviral role through the RIG-1 dependence pathway.Methods: 1.GV358-NTCP recombinant plasmid was constructed,then GV358-NTCP was infected with Huh7 cells,and NTCP-Huh7 cells were successfully constructed.This cell can express NTCP proteins steadily.2.Supernate of Hep AD38 cells were collected and purified,and HBV was extracted.HBV was then incubated with NTCP-Huh7 cells for 500 qeg/ cell concentration.The cell culture plate was centrifuged for 30 minutes at 1000g/min,then cultured in the cell culture box for 18 hours,and then changed into the hepatocyte maintenance medium.Collect cell supernatant in 2,4,6,8,10,12 days,then use techniques such as immunofluorescence,ELISA and q PCR to detect virus protein expression and HBV DNA and HBV ccc DNA replication,so as to determine whether NTCP-Huh7 cells had HBV susceptibility.3,The NTCP cell lines were treated with p EFTUD2,and the levels of HBV DNA,HBe Ag and HBs Ag were observed in different groups of cells,further confirming the effect of EFTUD2 on HBV.4,p EFTUD2 and si RIG-1 were transfected into the cell,and the DNA content of HBV was determined on the control group,p EFTUD2 overexpression group,RIG-1 knockdown group and p EFTUD2 overexpression with RIG-1 knockdown group.And it was confirmed whether EFTUD2 played an antiviral role through the RIG-1 dependence pathway.Results: 1.Results of PCR and sequencing showed that the NTCP recombinant lentivirus was constructed,and the concentration of recombinant plasmid was 2 x 108TU/ml.Using GV358-NTCP to infect Huh7 cells,after a week of drug screening,the positive cells expressing green fluorescent protein increased from about 10% to more than 90%.Western blot showed that NTCP-Huh7 cells could express NTCP protein stably.2.After HBV infection of NTCP-Huh7,cell supernatant was collected for correlation detection.ELISA showed that the content of HBs Ag and HBe Ag in NTCP-Huh7 cells were higher than that in Huh7 group in the 4th,6th,8th and 10 th days(P<0.05).In the 8th day,the OD value of HBs Ag(0.866±0.040)and HBe Ag(0.603±0.053)in the NTCP-Huh7 group reached the highest,respectively.The expression of HBc Ag in NTCP-Huh7 was observed under confocal microscope after 8 days after HBV infection.Result of q PCR showed that the content of HBV DNA in NTCP-Huh7 cells were higher in the 4th,6th,8th and 10 th days,comparing with Huh7 group(P<0.05).In the 8th day,The secretion of HBV DNA reached the highest(9893.931±741.754 IU/ml).Real-Time PCR also showed a relatively high expression of HBV ccc DNA in NTCP-Huh7 cells in the 4th,6th,8th,10 th and 12 th days,than in Huh7 group(P<0.05).HBV ccc DNA expression was the highest in the 6th day.3.The Hep G2.2.15 and NTCP cell lines were treated with p EFTUD2.In Hep G2.2.15,the levels of HBs Ag in the control group and p EFTUD2 overexpression group were 3.180±0.132 and 1.779±0.096,respectively.The levels of HBe Ag in the two group were 3.613±0.021 and 2.101±0.065,respectively.When the plasmid used were 0.5ug,1ug and 2ug,the content of HBV DNA were 57738.333±1281.097?35607.971±1902.023?30296.495±687.261,respectively.In NTCP-Huh7,the levels of HBs Ag in the control group and p EFTUD2 overexpression group were 0.782±0.028 and 0.358±0.022,respectively.The levels of HBe Ag in the two group were 0.620±0.024 and 0.267±0.010,respectively.EFTUD2 can inhibit HBV replication.4.The content of HBV DNA in the control group,p EFTUD2 overexpression group,RIG-1 knockdown group and p EFTUD2 overexpression with RIG-1 knockdown group were 60983.076±1392.366?30889.608±1341.288?64242.599±1220.242?60469.652±1287.59,respectively.Comparative analysis found that EFTUD2 can play an antiviral role through the RIG-1 dependence pathway.Conclusions: NTCP-Huh7 cells obtained by transfection of GV358-NTCP recombinant plasmid into Huh7 can infected by HBV.EFTUD2 can play an antiviral role through the RIG-1 dependence pathway.