HCV基因组NS1区的分子克隆及序列测定

Molecular Cloning and Sequence of the HCV NS1 Genomic Region

对广东省一名慢性丙型肝炎病人血清中的ＨＣＶ基因组ＮＳ１区进行分子克隆及序列测定。采用微粒吸附法提取ＨＣＶＲＮＡ，随机引物逆转录后进行聚合酶链反应。所用引物位于ＮＳ１区，扩增产物７８０ｂｐ在低熔点琼脂糖中电泳，回收相应条带处凝胶，与ｐＵＣ１８的连接反应直接在低熔点琼脂糖中完成。重组体转化ＪＭ１０９，挑取菌落增殖后提取的质粒采用ＰＣＲ和酶切法鉴定阳性克隆。将其中３２０ｂｐ的片段亚克隆ｐＵＣ１８和ｐＵＣ１９，随后采用双脱氧链末端终止法测其核苷酸序列，与国内外多个株比较，同源性介乎６８．８６％—９０．８４％。

he NS1 genomic region of the hepatitis C virus(HCV),derived from the serum of a patient withchronic NANB hepetitis in Guangdong province,was cloned and sequenced.HCV RNA extracted bythe powder-adsorption procedure was converted to cDNA by everse transcription with random primer.Polymerase chain reaction was performed with the primers which span the NS1 region,The amplifiedproducts were separated by electrophoresis on a low melting-point agarose gel and a portion of gel,cor-respondlng to the expected size,was cut out.The PCR preduct was lisated to pUC18 vector in low melting-point agarose gel.After transfecting JM109 strain,the recombinants were screened and identifiedby PCR and digestion with restriction endonucleases.One fragment of 320bp,amplified from a reeombi-nant plasmid,was cloned into pUC18 and pUC19,and their nucleotide sequences were determined bythe dideoxy c lhain termination me thod.A com parlson of the sequ ence with several isolates reported pre-viously showed homologies of 68.86%to 90.84%.