HCV 广东株5’NCRc DNA的克隆及序列测定

MOLECULAR CLONING AND SEQUENCING OF HCV 5’NCR cDNA

从广东省１例慢性丙型肝炎病人血清中提取ＨＣＶＲＮＡ，随机引物逆转录为ｃＤＮＡ后用ＨＣＶ５’端非编码区（５’ＮＣＲ）特异引物进行聚合酶链反应（ＰＣＲ）。扩增产物３０２ｂｐ，经补齐和提纯后插入ｐＵＣ１９质粒，获得的重组质粒ｐＵＮ采用双脱氧链终止法测定核苷酸序列，与国内外多个株比较，核苷酸同源性介乎９２．６９％～１００％，其中与ＨＣＶⅡ（１ｂ）型的同源性最大。本文的测序结果可为引物设计提供依据。获得的ＨＣＶｃＤＮＡ在常规ＰＣＲ步骤中用于设立有效的模板对照，对消除假阴性及评估试剂有重要意义。

HCV RNA derived from the serum of a patient with chronic hepatitis C was extracted before it was converted to cDNA by reverse transcription using random primers. Polymerase chain reaction (PCR) was performed with primers specific for HCV 5'NCR. Amplified product with 302bp was subsequently filled in 3' recessed ends, isolated, purified and inserted into pUC19 plasmid vector. The recombinant plasmid pUN obtained was sequenced by dideoxy nucleotide chain termination method. A comparison of the nucleotide sequence of HCV 5'NCR with several previously reported strains showed the homology to be 92.69%￣100% and most identical to strains belong to genotype Ⅱ. The sequence of the HCV 5'NCR determined in this report was useful for the selection of primers. The cDNA clone constructed can be used as effective positive control template for PCR, which will be helpful in eliminating false negative results and evaluating the quality of reagents.