Recombinant HCV genotype 3a NS5 [His]

Based on B cell epitope predictions, a recombinant antigen with multiple epitopes from four Hepatitis C Virus fragments (C, NS3, NS4 and NS5) were engineered. The recombinant gene was then highly expressed in E. coli. The non-modified and C-terminal-modified recombinant proteins were used for coating and biotin labeling, respectively, to establish the double-antigen sandwich ELISA. Ten positive reference samples confirmed by the CHIRON RIBA HCV 3.0 SIA kit were detected positive, Forty one plasma samples were positive among samples from 441 volunteers, which indicated that the recombinant antigen could readily react well with plasma HCV antibody. As critical reagents of double-antigen sandwich ELISA, the recombinant multi-epitope antigen and the C-terminal-modified and biotin-conjugated antigen show good antigenicity. In this study, we provide a simple approach to produce multiple epitopes within one recombinant protein in order to avoid the costly expression of less-effective pools of multiple proteins, which is the conventional strategy of diagnostic antigen production for HCV antibody detection.

AIM: To study hepatic virus C (HCV) RNA and HCV protein expression in peripheral blood mononuclear cells (PBMCs) of patients with HCV infection, and explore the relationship between the HCV RNA in the PBMCs and response to interferon (IFN) therapy. METHODS: Type-specific primers were designed and RT- nested PCR was used to detect the plus- and minus- strands of HCV RNA in PBMCs of 54 patients with HCV infection; Indirect immunofluorescence assay was applied to identify HCVNS5 protein expression in PBMCs; 6 month-, 3 MU-IFN regiment was administrated to observe the responses to IFN in 35 chronic hepatitis C patients with different HCV RNA status in PBMCs. RESULTS: HCV plus strand RNA was found in 10 of 19 (52.6 %) acute hepatitis C patients and 22 of 35 (62.9 %) chronic hepatitis C patients. HCV minus strand RNA was detected in 14 of 35 (40.0 %) chronic hepatitis C patients, but only one patient (5.3 %) with acute HCV infection was found to be minus HCV RNA positive. Though no HCV NS5 protein expression was found in the examined 10 cases of acute HCV infection, it was positive in 17 of 20 (85.0 %) chronic hepatitis C patients by indirect immunofluoresence assay. There are significant differences of positive rate of the minus-strand and HCVNS5 protein between acute and chronic hepatitis C groups(u=2.07, P<0.05and u=4.43, P<0.01 respectively). The patients with minus-strand HCV RNA showed a significantly lower 6-month sustained response (SR6) to IFN compared to those without minus-strand HCVRNA in PBMCs (biologically 14.3 % vs 42.8 %, x(2) =4.12, P<0.05 and virologically 7.1 % vs 23.9 %, x(2) =4.24, P<0.05). CONCLUSION: HCV is capable of infecting and replicating in PBMCs, and HCVNS5 protein was expressed in PBMCs. The patients with minus strand HCV RNA in PBMCs showed a significantly lower 6-month sustained response to IFN, suggesting that minus-strand HCV RNA in PBMCs may be one of the factors influencing response to IFN therapy.