Expression And Purification Of HCV Core Protein And Preparation And Identification Of Core1-120aa Monoclonal Antibody

Hepatitis C Virus (HCV) is a single RNA virus, classified into Flaviviridae family and of Hepatovirus genus, transmitted mainly through blood or blood products. Viral hepatitis type C (HC) is an infectious disease caused by HCV, clinically characterized by chronicity,80%of HCV infected patients will go on to develop chronic hepatitis and a considerable percentage of patients to develop cirrhosis, or even worse, carcinoma. An estimated210million people worldwide are infected with HCV. Due to the lack of a suitable animal model and the high variability of HCV, it’s a hard job to develop HCV vaccine. Interferon and ribaviron-based therapy has been the mainstay of HCV treatment, but because of its side effects, patients tend to intolerate the above two drugs. Today, HCV immunodiagnosis tests mainly detected the presence of anti-HCV. To shorten the window stage, a diagnostic test kit which is able to detect the presence of HCV-cAg at an earlier stage (within two weeks of infection) is of crucial importance.We aim to develop stable HCV core proteins expressed by Escherichia coli, and screen several pairs of matched high purity and high specificity monoclonal antibodies by utilizing the expressed HCV core proteins. First, extracting HCV total RNA from HCV positive serum collected from patients. Amplify HCV corel-120and core1-60gene sequences through inverse transcription. Fuse the amplified genes with pET-28a and pET-41a expression vector which then is transformed to Escherichia coli BL21(DE3), followed by expression and purification induced by IPTG. Finally, core1-120, core1-60and GST-core1-60proteins are obtained. We utilized purified corel-120proteins as antigen to immunize BALB/C mice. Secondly, monoclonal antibodies were obtained through common used technique and indirect ELISA. Then we injected the selected monoclonal antibodies into peritoneal to induce ascetic fluid. Fourthly we prepared high purified monoclonal antibodies by using saturated ammonium sulfate and Protein A. Finally, we obtained seven prepared monoclonal antibody strains for corel-120protein, selected through ELISA, were named as W1.W2.W3, W4, W5, W6and W7. We determined the subtype of W1to W5and W7as IGg2b, while W6as IgG2a. In the respect of high relative affinity for corel-120protein, the affinity of W3, W4, W5and W6reached up to640000, W1and W7to320000, W2to160000and only W2didn’t react with corel-60protein. What’s more, there was no cross reaction between NS3protein and the7strains. We cultured the cell strains for6months and found that these cells endured stable titre except for a slight decrease of W6. SDS-PAGE determined the purity of the purified antibodies as100%. The affinity constants of W1to W7, calculated by utilizing antibody titre and affinity constant formula, were all greater than107L/mol, which demonstrated the stability of antigen-antibody complex. We determined the epitopes of W1to W7through antibody stacking experiment. The stacking percentages of W1, W3, W4, W6and W7are all less than50%. That is to say, for a particular epitope of corel-120protein, the stacking percentages of W2and W5with the other monoclonal antibodies are greater than50%. The result illustrated that the obtained7monoclonal antibodies are particularly for the3different epitopes of corel-120protein and12pairs of antibodies could react with the antigen, among which the fluorescent intensity of labeled W5reacting with W3and W7coating plate, labeled W3reacting with W5coating plate is the highest.The determined3pairs of matched antibodies can play a crucial role in providing valid raw materials for HCV antigen and antigen-antibody combo detection. However, due to a lack of HCV core positive serum, we are not sure whether these three pairs can react with natural core protein or natural HCV-cAg positive serum, which calls for our further efforts in researching.