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丙型肝炎病毒 (HCV)依赖于RNA的RNA聚合酶 (RdRp)是参与病毒基因组RNA复制的一个关键蛋白因子 ,是研究抗HCV新型药物的重要靶标 ,获得纯化的RdRp产物是建立靶向RdRp药物高通量筛选体系和抗病毒药物研究的关键步骤。以HCV病毒基因组全长cDNA质粒 (p90 HCVFL longpU)为模板 ,设计了特异性扩增RdRp的引物 ,通过CPR扩增获得编码RdRp的基因。将该基因克隆到T载体中 ,构建了重组质粒 ,通过克隆快速筛选和酶切分析筛选到含RdRp基因的阳性克隆(克隆号为 4 4 ) ,DNA序列分析证实 4 4号阳性克隆RdRp基因序列与cDNA质粒中的相应序列完全一致。将含RdRp基因的重组质粒 (44号 )用NheⅠ XhoⅠ双酶切 ,获得RdRp基因 ,将该基因连接到用NheⅠ XhoⅠ双酶切的原核表达载体pET2 8(a)中 ,构建了重组质粒pET2 8(a) NS5B ,用该重组质粒转化大肠杆菌BL2 1(DE3) ,经 1mmol LIPTG诱导 ,SDS PAGE分析 ,发现一条特异性蛋白带 ,相对分子质量约 6 6× 10 3 ,与预期的完全一致。HCVRdRp基因的克隆与表达为靶向RdRp的新型抗HCV药物研究 ,特别是靶向RdRp药物高通量筛选技术的建立奠定了实验基础
Hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) is a key protein involved in the replication of viral genomic RNA and is an important target for the study of novel anti-HCV drugs. The purified RdRp product was established to target RdRp drugs High-throughput screening systems and key steps in antiviral drug research. The full length cDNA of HCV genome (p90 HCVFL longpU) was used as a template to design a specific primer for amplifying RdRp. The gene encoding RdRp was amplified by CPR. The recombinant plasmid was cloned into T vector. The positive clones containing RdRp gene (clone number 4 4) were screened by rapid cloning and restriction enzyme digestion. DNA sequence analysis confirmed the RdRp gene sequence The exact sequence in the cDNA plasmid is exactly the same. The RdRp gene was digested with Nhe I Xho I to obtain the RdRp gene. The gene was ligated into the prokaryotic expression vector pET28 (a) digested with Nhe I XhoI to construct the recombinant plasmid pET28 (a) NS5B. The recombinant plasmid was transformed into E. coli BL21 (DE3). After induced by 1 mmol LIPTG and analyzed by SDS PAGE, a specific protein band was found with the molecular weight of about 6 6 × 10 3, which was exactly as expected. Cloning and Expression of HCVRdRp Genes Lay a Foundation for New Anti-HCV Drug Targeting RdRp, in particular, the Establishment of High-Throughput Screening Technologies for Drugs Targeted RdRp