CDNA CLONING OF C33-C ANTIGEN GENE DERIVED FROM NS3 REGION OF CHINESEHCV GENOME, EXPRESSION IN ESCHERICHIA-COLI AND DEVELOPMENT OF HCV EIA2ND-GENERATION DIAGNOSTIC KIT

A cDNA fragment of about 860 bp corresponding to the c33-c gene in the non-structural region 3 (NS3) of HCV genome was obtained from one pla sma derived from a Chinese HCV carrier who came from Tai'an of Shandon g Province, China by the application of reverse transcription (RT) and polymerase chain reaction (PCR) techniques. After the sequence of the cDNA fragment was determined and compared with the equivalent region of. the HCV-I (HCV-US) and HCV-II (HCV-BK) genomes, the nucleotide/ am ino acid sequence homologies were found to be 79.2%/91.3% and 91.3%/93 .9%, respectively. The prokaryotic expression vector pBV220 was employ ed for the overproduction of c33-c native recombinant protein in E. co li cells. The expression products were detected by enzyme-linked immun osorbent assay (ELISA) and Western blotting with antisera of chronic h epatitis C patients, and a molecular weight 31 kD of c33-c viral prote in was shown to account for 14% of the total cellular soluble proteins . This product was extracted from the bacterial lysate by lysozyme, Tr iton X-100 and urea treatment, and purified through ion exchange chrom atography. The purified c33-c protein combined with a branch peptide M AP-C-19 representing immunodominant epitopes on the nucleocapsid regio n of HCV genome was used to develop a Chinese HCV EIA 2nd-generation d iagnostic kit for the detection of anti-HCV antibodies. Its specifity, sensitivity and reproducibility were all in keeping with the indexes of the national standard for the quality control of the HCV diagnostic kit. The agreement rate between our kit and Abbott company's HCV EIA second-generation diagnostic kit was 99.33%, and the identified rate o f positive anti-HCV of our kit was 2% more than that of the Abbott com pany's kit.