ABSTRACT
Objective To evaluate the relationship between the serum free IGF-1 level and the severity and prognosis in patients with chronic severe hepatitis. Methods Serum free IGF-1 was assessed by ELISA in 44 patients with chronic severe hepatitis, 43 chronic viral hepatitis, 46 liver cirrhosis. At the same time the liver function, prothrombin activity and cholinesterase were also determined. Results Serum free IGF-1 in patients with chronic severe hepatitis, liver cirrhosis and chronic viral hepatitis was 0.24?0.15,0.33?0.17 and 1.06?0.70 (ng/ml), respectively. IGF-1 was significantly decreased in patients with chronic severe hepatitis and liver cirrhosis. IGF-1 level in patients with chronic severe hepatitis at early, middle and advanced stages was 0.28?0.07, 0.27?0.19 and 0.16?0.06 (ng/ml), respectively. The reduction in the value showed a positive correlation with different stages of chronic severe hepatitis. Patients with chronic severe hepatitis having a serum free IGF-1 below 0.2ng/ml had a higher mortality, and those with the value above 0.35ng/ml had a better chance to survive during the follow-up period. There was a significant positive correlation between serum free IGF-1 and prothrombin activity. Conclusion Serum free IGF-1 was decreased in the patients with chronic severe hepatitis. The clinical observation suggested that the serum free IGF-1 might be an important prognostic indicator in patients with chronic severs hepatitis.
ABSTRACT
HCV NS5B, acting as a RNA dependent replication enzyme,has emerged as an attractive protein used as a target for screenig of drugs against HCV NS5B, and plays an important role in HCV replication. In the report the gene expression of NS5B in E.coli was investigated. PCR was performed to gain the gene of HCV NS5B from plasmid pBRTM/HCV 1 which contains whole sequence of HCV, and the truncated NS5B gene containing no hydrophobic domain was cloned into pGEM Teasy vector. The gene of the truncated NS5B was cut from pGEM Teasy vector and cloned into E.coli expression plasmid pET 21b, then pET 21b NS5B was transfected into E.coli cells. The protein E.coli lysates were purified and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting assay. The RdRp activity of NS5B was examined by scintillation proximity assay (SPA). The truncated NS5B gene was successfully cloned into pET 21b. The results of SDS PAGE and Western blotting assay showed: ①the molecular weight of the expressed product was about 68 000 D, ②The truncated NS5B protein was existed in media of E.coli cells, ③The activity of NS5BDCT21 His from HCV 1b amounted to 6 900 cpm (total incorporation of approximately). These findings suggest that soluble NS5B can be successfully expressed in E.coli and could secret into media.