Study Truncated Middle Surface Protein Of Hepatitis B Virus On Hepatocellular Carcinoma

OBJECTIVE:To study the types of MHBst in hepatocellular carcinoma patiens and MHBst biologic funcation in hepatocytes.METHODS:The S region of HBV was amplified by polymerase chain reaction (PCR) method from 80 cases of hepatocellular carcinoma, and the PCR products were cloned into pMD18-T vector and sequenced. Vector 10.0 software was emploied to analyze the sequences. Alu-polymerase chain reaction approach to detcet the HBV DNA integration into cellular genes. The PCR products were cloned into pMD18-T vector and sequenced. Apply NCBI Blast to analyze viral-host junctions. Artificial mutants used in this study were generated by GeneTailor Site-Directed Mutagenesis System. To express MHBst mutants in the mammalian cells, the MHBst fragment were cloned into the vector pcDNA3.1 by HindIII and BamH I sites. Positive clones were sequenced to confirm their compliance with the original results. Construction and expression of a recombinant adenovirus vector expressing MHBst. Construction the pGL3Basic-fos,myc-Luc reporter. SV40-Luc reporter from Promega reporter assay system. Apply CCK-8, soft agar assay, TUNEL methods to study MHBst biologic funcation. Immunoprecipitation to screen proteins interact with MHBst167.RESULTS:After sequencing, obtained 600 sequence and find 7 types MHBst:77, 90, 116, 124, 129, 227, 254. Most 254 occur in B genotype and other types occur in C genotype. 129,227,254 types show higher frequencies,15%(12/80),7.5%(6/80)and 20%(16/80). Alu-PCR approach obtained 9 kinds of viral-host junctions. Successfully mutate S start codons. Constructed pCDNA3.1, p3×flag-cmv-10 and adenovirus AD-77, 90, 116, 124, 129, 227, 254,167,281 expression vector. Constructed SV40, c-fos,c-myc luciferase repoter expression vector and screen SV40,c-myc cell strain. MHBst show diffrenrnt transactive and clone form.MHBst can through TRAIL to induce cell apoptosis. MHBst show different trans-activation, proliferation and colony formation. MHBst167 interact with Aldolase by Immunoprecipitation.CONCLUSION:There are 7 MHBst in HCC patients. Most MHBst belong to C genotyoe.HBV S gene show low integration rate in HCC and random integration,but the HBV S gene integration may involve in cell proliferation and apotoisis. This research indicates that HBV MHBst involve in trans-activation, cell proliferation and apoptosis. However, the roles of HBV envelope protein may be comprehensive and remain to be elucidated. MHBst167 interact with Aldolase offers an opportunity to probe into the role of MHBst in the pathogenesis of HBV-associated liver fibrosis and hepatic celluar carcinoma.