HCV　NS_3区C_（33）c抗原基因cDNA的克隆及序列分析

Cloning and Sequencing of cDNA from C33c Protein Gene in NS3 Region of Hepatitis C Virus

自上海市及江苏省慢性丙型肝炎患者血清中分离丙型肝炎病毒（ＨＣＶ）ＲＮＡ，采用自行设计的引物，经逆转录－聚合酶链反应（ＲＴ－ＰＣＲ），获得长为６９４ｂｐ的Ｃ３３ｃ抗原基因ｃＤＮＡ，将其克隆于表达载体并作序列分析。结果表明，上海株ＨＣＶ－Ｓ１及江苏株ＨＣＶ－Ｓ２该区域核苷酸水平有很高的同源性（＞９９％），上海及江苏株ＨＣＶ与美国原型ＨＣＶ－１、日本株ＨＣＶ－Ｊ及河北株ＨＣＶ－ＨＢ在该区域核苷酸水平的差异分别为１８．６４％、５．５５％及８．０１％～８．１７％，氨基酸水平的差异在２．３１％～６．０２％之间。上海株同一份血清三个独立克隆间核苷酸水平有０．６％～０．９％的差异。本实验为进一步表达Ｃ３３ｃ蛋白作好了准备。

Sera from two chronic hepatitis C patients were used for isolation of HCV RNA, one from Shanghai and the other from Jiangsu Province.Using primers designed by ourself,a 694bp cDNA fragment of C33c protein gene located in NS3 region of HCV was generated by RT-PCR. After cloning into the expressing vector pBluescript Ⅱ, the fragments were sequenced by the dideoxynucleotide method.The cDNA fragment of Shanghai isolate HCV-S1 and Jiangsu isolate HCV-S2 showed a high identity(>99%) at the nucleotide(nt) level.When the two isolates were compared with the original isolate of US HCV-1、Japanese isolate HCV-J and Hebei isolate HCV-HB in the examined region,18.64%、5.55% and 8.01～8.17% diversity at the nucleotide level was found respectively and 2.31～6.02% diversity was found at the predicted amino acid (aa) level. In the study,0.6%～0.9% diversity at the nucleotide level was observed among three independent clones obtained from a single serum sample.The study here also served as a preparation for further expression of C33c protein.