The Expression And Purification Of The Truncated HCV Core Protein (HCV Core125) And Its Antibody Preparation

Hepatitis C virus (HCV) is the major cause of the acute and chronic viral Hepatitis, further lead to chronic progressive liver disease, Hepatocellular cirrhosis (HC) and Hepatocellular carcinoma (HCC). It was estimated that there are about170-200million people infected with HCV worldwide and more than40million people have been infected in China, which is one of the serious countries having been infected HCV. Up to date, there is no vaccine and effective drug of hepatitis C, the early accurate diagnose on this disease are particularly important for prevention of HCV spread and control of disease progression. At present, HCV detection technologies mainly include antibody detection, nucleic acid detection and core antigen detection. Generally, HCV antibody could be detected after7to10weeks of the window period. However, six genotypes and more than50subtype of HCV can cause false negative results. Although HCV nucleic acid exists immediately after HCV infection, the related detection cannot be widely used because of easily pollution and more expensive of PCR technique. HCV antigen is also a useful detection target, but its low quantity makes the detection more difficult. So, it is urgent to get high sensitive detection technique on core antigen. The purpose of this study is to express and purify HCV core protein, produce the high titer of specific antibody. Both of them are the necessary contents of a high sensitivity HCV core antigen diagnosis kit.In this study, the hydrophilicity profile of HCV core antigen was firstly analyzed and the1-120aa fragment was confirmed as the hydrophilicity region, which contains almost all of the conserved B cell epitopes of core antigen. But, hydrophobicity of C terminal can prohibit protein expression and purification. Therefore, we select the former125aa of HCV core protein (HCV core125) as a candidate protein. In order to get the high expressed core protein, a375bp (125aa) gene was amplified from HCV genome by PCR, subsequently cloned into PET28a expression plasmid. The recombinant PET28a-Core125plasmid which can express the partial core protein were successfully constructed, and than transferred into Escherichia coli host cell. The recombinant Prokaryote expression Escherichia coli is induced by the1.0mM IPTG, and contributes to the high expression of Core125protein (about21KD), which mainly exists as inclusion body. Core125protein was purified with Ni2+-NTA affinity chromatography column. Its molecular weight is determined to be about anticipanted21KD. However, some miscellaneous belts are still existed during the first purification step. So, Core125protein was further purified with Strep-Tactin system affinity chromatography, to get high purified target protein. Purification of the Core125protein was analyzed by Western blot, it was showed that the protein reacted with serum antibody from various HCV genotypes and subtypes (1a,1b,3a,3b,6a,6n,6u) infeted patients. It reveals the expression of the protein has highly antigenicity and versatility across different genotypes.The purified Corel25protein was used to immunize a rabbit, and polyclonal antibody titer in serum is determined as1:1024000with indirect ELISA. Polyclonal antibody was analyzed by Western blot, and the result shows that it has a very high specificity. Then, Core125protein was used to immune BALB/C mice, and the serum of antibody titer is1:51200with indirect ELISA. It means the core protein has better immune effect. It also meets the requirements for preparation of monoclonal antibodies. Unfortunetly, we haven't get monoclonal antibodies because of the low cell fusion rate.This study was designed to express truncated type of HCV core protein (Core125), finally got the high expressed and purified target protein. The gotted Core125protein could react with serum antibody of different HCV genotypes patient. It reveals the core protein has high antigenicity and versatility to different genotype. The sucessuful preparation of a polyclonal antibody lays a foundation on improving the sensitivity of HCV antigen detection. The preliminary result in preparation of monoclonal antibody is a useful try in the development of high sensitivity of HCV core antigen detection kit.