丙型肝炎病毒Ns_3基因区部分序列的PCR克隆和鉴定

选取HCV－1、HCV－J、HCV－BK、台湾株HCV、中国河北株HCV序列的同源性部分，设计Ns3区部分序列的PCR引物．应用RT－PCR技术，从丙型肝炎患者血浆中扩增一长为448bp的cDNA片段作为目的基因，与经过限制性酶切的pUcI9载体连接，构建重组质粒pUHCV－Ns3．分别或混合应用HCV序列特异的引物和载作序列特异的通用引物，以PCR扩增重组质粒，结果扩增产物的分子量均与预期大小一致．限制性酶切位点分析结果也证实，克隆的基因是HCV的Ns3区部分序列的目的基因．克隆序列的特异性和插入方向同时得到了鉴定．

PCR Cloning and identification of a Partial Sequence of the Ns_3 Rogion of HCV Genome

Form homeologic region of HCV-J, HCV-BK, HCV-T3 and HCV-China, two conservative sequences were utilized to synthesize a pair of Primersin HCV-Ns3 region. By means of RT-PCR, a cDNA fragment of 448bp was amplified from plasma that was positive to HCV RNA. And by direct cloning, the fragment was ligated to PUC19 plasmid vector A recombinant plasmid pUHCV-Ns3 was generated. For identification of the insert, PCR was used employing a primer pair specific to HCV, a primer pair specific to pUC19, and 3 mixed primer pairs from the both HCV and pUC19. In each reaction, an anticipated product was produced to prove the insert to be the HCV Ns3 gene and its reverse direction. The result of the restrictive site analpsis also provided supportive evidence.