HCV Antigen Development And The Application In The Combined Detection Of HCV Antigen And Antibody

Hepatitis C virus(Hepatitis c-virus,HCV)is a kind of enveloped RNA viruses.It transmits mainly through blood,can cause acute and chronic viral hepatitis,protracted course of disease can lead to cirrhosis and liver cancer.3% of the world's population or about 170 million people are living with HCV virus,there are more in developing countries than in developed countries.About 40 million people in China are living with HCV virus;therefore,China is one of the highest HCV prevalence countries in the world.Since HCV vaccine has not been developed,thus controlling the spread of HCV has become a serious public health problem.The methods for clinical detection of HCV include nucleic acid test and,immunoassay.The current available HCV immunoassay products in the market are mainly the third generation HCV antibody detection kits.The fourth generation products have been developed recently in the developed countries.Since the HCV CORE antigen can be detected in the eraly stage of HCV infection,the fourth generation products can detect HCV infection with a significant shorter window period,through a combination detection of HCV antibodies and HCV CORE antigen.At the moment,the fourth generation products available in Chinese market are mainly produced by foreign companies.Despite this kind of products are being developed domestically,they are still at the early stage,and a good quality is still to be developed.Hence,there is a strong demand for the development of the fourth generation HCV detection kits with high quality in China.The achievements in the current study include: the successful construction of HCV C33C+CORE recombinant expression plasmid;an optimization of the fermentation conditions,and a stable expression strain was obtained;an optimization of the purification process,which is stable and easy to obtaine high-purity protein.This purification process is of great value.The purified recombinant antigen displayed high immunoactivity in immunoassay,suggesting that this antigen may be a promising biomaterial for the development of HCV Ag/Ab immunoassay diagnostic kits,the Successfully expressed proteins are used in the HCVAg/Ab joint detection kits,show good clinical performance in sensitivity and specificity,it has great clinical significance to shorten the detection window period for hepatitis c virus.The main results are as follows:1.C33 C and CORE gene fragments were obtained from the HCV positive serum.After a RNA extraction and RT-PCR,HCV cDNA was obtained.Finally they were cloned to pET-28 a and pET-30 a,two different plasmids,including pET-28a-C33C-CORE(C60)and pET-30a-C33C-CORE(C60),were generated.The two plasmids were transformed to BL21(DE3)strains,and proteins have been expressed.2.By optimizing process parameters,such as the concentration of IPTG and the duration of protein expression.The conditions of optimized protein expression with high density fermentation were determined.3.A large-scale protein purification method was established.This method is simple and efficient,and the scale of purification can be linear enlarged.Importantly,high purity protein can be obtained with this purification process,which includes several experimental sections,such as sinification of bacteria,centrifugations,affinity chromatography and ion-exchange chromatography,etc.4.By the improvement of coating and labelling parameters,finally we developed the combin detection kit for HCV Ag/Ab.