Using techniques of molecular biology and serology, we research on the establishment of HCV diagnotic quality control standard, HCV diagnostic materials, anti-HCV EIA diagnostic method, detection of serum HCV RNA,detection of HCV RNA in liver tissue by in situ PCR, and the establishment of monoclonal hybridoma cell line of HCV C33c and NS5a. Successfully prepared diagnostic kits have applied in screening of blood donor, clinical diagnosis,observation of treatment effect and research on pathogenesis of hepatitis C. After the use of these reagents, the transmission rate of HCV through blood product has been dramatically reduced.
从中国华北地区采集到的抗-丙型肝炎病毒(Hepatitis C virus,HCV)阳性血清中分离RNA,经逆转录及DNA聚合酶链反应(RT-PCR),扩增HCV cDNA核心蛋白基因及包膜蛋白基因片段,并对扩增到的DNA片段进行分子克隆及序列分析。结果显示,中国人丙型肝炎病毒基因组的核心基因区核苷酸序列,与日本人及美国人相比较,其同源性分别为96％和91％,包膜基因区分别为93％及73％。比较氨基酸序列,核心多肽同源性分别为97％和96％,包膜多肽分别为93％和76％。本研究结果可用于丙型肝炎免疫诊断试剂和疫苗的研制。
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.
