Complementary DNA of partial HCV NS2 NS3 protein gene which encodes viral Zn 2+ dependent metalloprotease and serine protease was amplified by RT nested PCR from serum of a Chinese patient with chronic hepatitis C.The product was digested with Eco R Ⅰ/ Xba Ⅰ and cloned into pcDNA3.The nucleotide sequence was determined.Comparison of NS2 NS3 with the reported typical isolates HCV 1,HCV J,HC C2,HCV J6,HCV J8 showed as 73.72%,90.20%,91.02%,64.56%,63.37% identity in nucleotide sequence and 83.24%,92.09%,93.13%, 70.88% ,67.86% identity in amino acid sequence respectively, so the isolate could be classified as HCV genotype Ⅱ. This gene fragment was cloned into pBV220 to construct recombinant expression vector pBV220 NS2 3.It was expressed efficiently in E.coli . The cloning and expression of HCV NS2 NS3 may contribute to the investigation of the relation between structure and function of viral encoded protease.
HCV NS2-3 cDNA that encodes the Zn 2+ -dependent metalloprotease was amplified by RT-PCR from serum of a Chinese patient infected with HCV. By using a baculovirus expression system, the cloned NS2-3 cDNA was expressed in Sf9 insect cells. SDS-PAGE and Western blot analyses showed the NS2-3 protein product as well as the product corresponding to HCV protein cleaved from NS2-3 precursor. The results indicated that the expressed NS2-3 protein is likely of protease activity and carries out autocleavage at the NS2-NS3 junction. The expression of NS2-3 protease in Sf9 insect cells allows the study on the biological function of NS2-3 protease and constitutes the basis for testing influencing agents on NS2-3 protease activity.
