| Research background:Hepatitis B virus(HBV)infection is global and is one of the leading causes of acute/chronic hepatitis B,cirrhosis,and hepatocellular carcinoma(HCC).Chronic hepatitis B(CHB)has become a global health and safety problem due to the large number of people infected.Therefore,it is of great significance to study the pathogenesis of CHB and the relationship between HBV infection and host immunity for the treatment of CHB.Complement factor B(CFB)is an important molecule in the alternative pathway of the complement system.At present,the research on the relationship between HBV infection and immunity mainly focuses on adaptive immunity,while the relationship between innate immunity,especially the complement system,and HBV infection is still unclear.Therefore,the study of the role of CFB in HBV infection is of great significance to further improve the relationship between HBV infection and immune system.Previously,through a genome-wide association study,we found that a single nucleotide polymorphism(SNP)rs12614(R32W,CFB 32 codon from arginine to tryptophan)located on the CFB gene was associated with CHB risk of pathogenesis.but the specific mechanism of CFB R32W in HBV infection is still unclear.Purpose of the studyThis study is aimed to explore the mechanism of CFB gene and its functional polymorphism R32W in HBV infection,which will facilitate to further explain the relationship between CFB R32W and chronic hepatitis B.Research methods and contents(1)In HepG2 and Huh7 cells,HBV1.3 plasmids were transfected,and reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and enzyme-linked immunosorbent assay(ELISA)were used to analyze whether HBV had an inhibitory effect on CFB gene.(2)In HepG2 and Huh7 cells,HBV plasmids(HBp,HBc,HBx and HBs)of different components were transfected.RT-qPCR and ELISA were used to analyze whether proteins of different components of HBV had inhibitory effects on CFB gene.(3)In HepG2.2.15 cells,HBeAg and HBsAg in cell supernatant were detected by electroluminescence immunoassay(ECLIA)by plasmid transfection or lentivirus infection,overexpression or knockdown of CFB gene expression,and HBV DNA levels in cell supernatant were detected by qPCR Verify whether CFB inhibits HBV replication.(4)In HepG2 and 293T cells,the plasmids of CFB32R and CFB32W were transfected.RT-qPCR and ELISA were used to analyze whether the expressions of CFB32R and CFB32W in different cells were different.(5)In HepG2.2.15 cells,the plasmids of CFB32R and CFB32W were transfected,and the expression levels of CFB32R and CFB32W in HBV infection were analyzed by RT-qPCR and ELISA.ECLIA and qPCR were used to detect the levels of HBeAg,HBsAg and HBV DNA,and to analyze the differences between CFB32R and CFB32W in inhibiting HBV replication.The results of the study(1)HBV reduced the level of CFB protein by inhibiting the transcription of endogenous CFB.(2)HBs protein significantly inhibited the expression of endogenous CFB gene in cells.(3)The inhibition of HBV replication by CFB was mainly reflected in the reduction of the secretion levels of HBeAg and HBsAg and the inhibition of HBV DNA replication.(4)The expression level of CFB32W was higher than that of CFB32R.(5)The ability of CFB32W to inhibit HBV replication was weaker than that of CFB32R,which was mainly reflected in the fact that the ability of CFB32W to inhibit HBV DNA replication was weaker than that of CFB32R.ConclusionHBV can inhibit the expression of CFB,and at the same time,CFB can inhibit HBV replication.The expression of CFB32W was higher than that of CFB32R,but the ability of CFB32W to inhibit HBV replication was weaker than that of CFB32R. |
