| The hepatitis C virus (HCV) is associated with the vast majority of cases of post-transfusion hepatitis and sporadic non-A, non-B hepatitis worldwide. HCV has infected about 30,000,000-40,000,000 people in China. Most people infected with HCV give rise to an acute illness, 80% of which develop into chronic hepatitis and more serious consequences including cirrhosis and hepatocellular carcinoma. In current, there are still no developed vaccines and special drugs against HCV infection, so it is important to search and find the patient at early stage to control and prevent HCV infection. Detection of antibody to HCV is the principal method for the diagnosis of HCV infection in individuals with chronic hepatitis and for the screening of blood donors. The original assay based upon the c100-3 recombinant proteins derived from NS4 has proved to be not very specific and sensitive. The more recently developed assays that use recombinant proteins from the Core, NS3 and NS4 regions of the HCV genome (second-generation assays) and the the Core, NS3, NS4 and NS5 region of the HCV genome (third-generation assays) have proved to be more effective for the screening of blood donors. Although screening assays have been effective in reducing the risk of post-transfusion hepatitis C (HC), these assays do not detect anti-HCV activity in all patients with HCV infection. In addition, these assays are prone to false-positive results, especially noticeable in low-risk populations. The-third generation assay is slightly more sensitive than the-second one and HCV serologic diagnosis is yet not perfect. The HCV genome is very heterogeneity, which has led to the identification of distinct types that may differ from each other by as much as 33% over the entire genome. HCV can be classified into at least six major genotypes with each genotype containing multiple subtypes based upon the difference in sequence homology. Sequence heterogeneity among various HCV genotypes has been associated with variant antigenic and biological properties. This is especially significant as it relates to various serologic and virologic assays for HCV detection and their interpretation. The present commercial HCV tests based upon proteins or peptides derived solely from HCV genotype 1 may be less sensitive for the detection of antibody against HCV of other genotypes. Dow et al. found that sera from donors infected with different genotypes showed different patterns of reactivity by the third-generation confirmatory assay.In this study we constructed successfully four plasmids and expressed four recombinant antigens encoded by different gene fragments with different gene sequences so as to explore their antigenic reactivity and significance in anti-HCV detection.1. The construction, identification and expression of recombinant plasmids derived from HCV different gene fragmentsThe different gene fragments involved C, NS3 (1 type), NS3 (6 type) and NS5 were amplified by PCR and then inserted into the vector pET-28a-c(+), respectively. The four recombinant plasmids were all used to transfer the E.coli strain BL21; and to express the four recombinant proteins induced by the addition of IPTG at a final concentration of 1mM. The proteins were purified by ligand-affinity chromatography and then identified by polyacrylamide gel electrophoresis (PAGE). 2. The antigenic analysis of HCV recombinant proteins encoded by different genotypes and different fragmentsFive HCV recombinant proteins encoded respectively by C, NS3 (1 type), NS3 (6 type), NS4 (mosaic, kindly provided by Dr. Fields, CDC), and NS5 region were applied in anti-HCV ELISA detection. Ninety known anti-HCV-positive specimens were tested with each of the five antigens, respectively. One hundred and twenty-six specimens collected from normal undergraduates and 39 samples from an anti-HCV-negative reference panel were also tested with the same five antigens; It appeared that 83 out of 90 known anti-HCV-positive specimens were immunoreactive to one or more of the five HCV recombinant antigen...
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