HCV5＇端非编码区cDNA的体外转录

In Vitro Transcription of HCV 5'Non-Coding Region cDNA

采用逆转录聚合酶链反应（ＲＴ－ＰＣＲ）从广东省一例慢性丙型肝炎病人血清中获得丙型肝炎病毒（ＨＣＶ）５＇端非编码区（５＇ＮＣＲ）３０２ｂｐ的ｃＤＮＡ片段，经补齐和提纯后插入ｐＵＣ１９质粒，获得的重组体ｐＵＮ进行序列测定。将ｐＵＮ的目的基因亚克隆进体外转录载体ｐＳＰＯＲＴＩ多克隆位点的ＥｏｏＲＩ和ＰｓｔＩ切点之间，所得重组体ｐＳＮ线性化后由Ｔ＿７ＲＮＡ多聚酶及ＳＰ６ＲＮＡ多聚酶引导体外转录反应，产物经凝胶电泳及特异引物ＲＴ－ＰＣＲ，证实ＳＰ６引导的是正义ＲＮＡ，Ｔ７合成的是反义ＲＮＡ，其大小分别力４２９ｂｐ和３６２ｂｐ。并证实所得ＲＮＡ力ＨＣＶ５＇ＮＣＲｃＤＮＡ转录而来。获得的ＨＣＶ５＇ＮＣＲｃＤＮＡ和ＲＮＡ在常规逆转录和ＰＣＲ步骤中用于设立有效的模板对照，对消除假用性及评估试剂有重要意义。同时，ＨＣＶ５＇ＮＣＲ体外转录载体的构建可用于制各ＲＮＡ探针和反义ＲＮＡ，改进后还可作为定量ＰＣＲ的竞争性模板。

epatitis C virus(HCV)5'non-coding region(5'NCR)cDNA with 302 bp obtained byreverse transcription polymerase chain reaction(RT-PCR)from the serum of a patient withchronic hepatitis C in Guangdong.Province was subsequently filled in recessed ends,purified andinserted into pUC19 plasmid vector.The recombinant plasmid pUN was sequenced.The targetgene in pUN was subcloned into EcoR I/Pst I sites of pSPORT I transcription vector.Alter therecombinant pSN was linearized,the transcription reaction in vitro was performed using SP6RNA polymerase or T_7 RNA polymerase。The sense RNA with 429 bp and anti-sense RNA with362 bp synthesized by SP6 RNA polymerase and T7 RNA polymerase respectively were identifiedby electrophoresis on agarose gel and by RT-PCR using HCV-specific primers.It was also ver-ified that the RNAs were transcripted from HCV5'NCR cDNA.The 5'NCR cDNA clone con-structed and RNA synthesized can be used as effective positive control templat for PCR and RTrespectively,which will be helpful in eliminating false-negative results and evaluating the qualityof reagents. In addition,transcription vector of HCV 5'NCR allows the production in vitro of RNA probes and anti-sense RNA. After being modified,the recombinant vector will also be uti-lized as a competitive template for quantitative assay of HCV RNA.