论文部分内容阅读
AIM To study the epitope distribution of hepatitis G virus(HGV) and to seek for the potential recombinant antigensfor the development of HGV diagnositic reagents.METHODS Fourteen clones encompassing HGV genefragments from core to NS3 and NS5 were constructedusing prokaryotic expression vector pRSET and (or)pGEX,and expressed in E.coli.Western blotting andELISA were used to detect the immunoreactivity of theserecombinant proteins.RESULTS One clone with HGV fragment from core to E1(G1),one from E2 (G31),three from NS3 (G6,G61,G7),one from NS5B (G821) and one chimeric fragment fromNS3and NS5B (G61-821) could be expressed well andshowed obvious immunoreactivity by Western blotting.One clone with HGV framment from NSSB (G82) was alsowell expressed,but could not show immunoreactivity byWestern blotting.No obvious expression was found in theother six clones.All the expressed recombinant proteinswere in inclusion body form,except the protein G61 whichcould be expressed in soluble form.Further purifiedrecombinant proteins G1,G31,G61,G821 and G61-821were detected in indirected ELISA as coating antigenrespectively.Only recombinant G1 could still showimmunoreactivity,and the other four recombinantproteins failed to react to the HGV antibody positive sera.Western blotting results indicated that the immunoactivityof these four recombinant proteins were lost duringpurification.CONCLUSION Core to E1,E2,NS3 and NS5 fragment ofHGV contain antigenic epitopes,which could be producedin prokaryotically expressed recombinant proteins.Ahigh-yield recombinant protein (G1) located in HGV coreto E1 could remain its epitope after purification,whichshowed the potential that G1 could be used as a coatingantigen to develop an ELISA kit for HGV specific antibodydiagnosis.
AIM To study the epitope distribution of hepatitis G virus (HGV) and to seek for the potential recombinant antigens for the development of HGV diagnositic reagents. METHODS Fourteen clones encompassing HGV genefragments from core to NS3 and NS5 were constructedusing prokaryotic expression vectors pRSET and (or) pGEX, and expressed in E. coli. Western blotting and ELISA were used to detect the immunoreactivity of these recombinant plasmids .RESULTS One clone with HGV fragment from core to El (G1), one from E2 (G31), three from NS3 (G6, G61 , G7), one from NS5B (G821) and one chimeric fragment from NS3 and NS5B (G61-821) could be expressed well and shy obvious immunoreactivity by Western blotting. One clone with HGV framment from NSSB (G82) was alsowell expressed but could not show immunoreactivity by Western blotting. No obvious expression was found in theother six clones. All the recombinant proteinwere in inclusion body form, except the protein G61 whichcouldbebe expressed in soluble form.Further purifiedrecombinant proteins G1, G31, G61, G821 and G61-821were detected in indirected ELISA as coating antigenrespectively. Ofly recombinant G1 could still showimmunoreactivity, and the other four recombinant proteins failed to react to the HGV antibody positive sera. Western blotting results indicated that the immunoactivityof These four recombinant proteins were lost during purification. CONCLUSION Core to E1, E2, NS3 and NS5 fragment of HGV contain antigenic epitopes, which could be produced in prokaryotically expressed recombinant proteins. High-yield recombinant protein (G1) located in HGV core to E1 remained its epitope after purification, which showed the potential that G1 could be used as a coating antigen to develop an ELISA kit for HGV specific antibodydiagnosis.