YANG Zhen, JIANG Jiang—dong, QI Zi—bai, CHEN Hong—shan, Zhang Hua—yuan and LI He—ming. Cloning,Expression and Analysis of HCV Ns5b Polymerase gene from Chinese serum[J]. Virologica Sinica, 2003, 18(6): 530-533.
Citation: YANG Zhen, JIANG Jiang—dong, QI Zi—bai, CHEN Hong—shan, Zhang Hua—yuan, LI He—ming. Cloning,Expression and Analysis of HCV Ns5b Polymerase gene from Chinese serum .VIROLOGICA SINICA, 2003, 18(6) : 530-533.

中国人血清中丙型肝炎病毒聚合酶全长基因的克隆表达及鉴定

  • 摘要:构建中国人丙型肝炎病毒(HCV)复制的RNA聚合酶原核表达载体pET30aNS5b,并在大肠杆菌中获得NS5B 聚合酶蛋白的高效表达,为建立HCV NS5b聚合酶细胞外分子复制模型的方法创造条件。使用高保真Pfu DNA 聚合酶进行反转录及套式PCR扩增,从我国HCV RNA阳性血清中扩增出HCV NS5b RNA多聚酶全基因序列, 经BamHI和Sa/l酶切。将其克隆至同样酶切的pET 30a载体中;转化大肠杆菌BL21,IFrG诱导表达。用抗HCV NS5b单克隆抗体做Western Blot进行鉴定。结果表明构建了原核表达载体,pET30aNS5bpET30aNS5b明显表达出 12 His NS5b聚合酶蛋白。测序结果表明,与已发表的相关HCV NS5b RNA聚合酶序列比较,其核苷酸和氨基酸 的同源性分别在69%~92.7%及88.8%~96.8%之间。在最佳表达条件下,可高效诱导表达融合蛋白(65kDa),最 高表达量占菌体蛋白18.9%。Western Blot结果显示表达蛋白为HCV NS5b酶。HCV聚合酶蛋白全长基因可以成 功地克隆在pET-30a载体上并有效表达出目的蛋白,为研究建立HCV NS5b聚合酶细胞外分子复制模型奠定了基 础。 Abstract:To ampUfy Hepatitis C virus(HCV)polymerase sequence and express HCV NS5b protein inorder to,in the next,establish a molecular model of HCV replication.RT—PCR method was used toamplify HCV NS5b polymerase gene from a Chinese HCV(+1 patient’S serum. e polymerasefragment Was constructed in the plasmid pET一30a an d expressed in E coli.HCV NS5b protein Wasidentified by W lestern blot with a monoclonal an tibody against HCV NS5b.Sequence analysis showedtllat the isolate had 69% 一95% an d 89%一97% homologies to the reported HCV sequences in respect tonucleotide an d amino acid respectively.HCV NS5b gene was expressed at a high level an d themolecular weight ofthe expressed prod uct was 65kDa.which was within the ran ge of expectation.Thisprotein Was confirmed to be HCV NS5b by Western blot.HCV NS5b from a Chinese patient Can beSuccess—fully cloned an d expressed with pET一30a plasmid an d E coli. is protein could be used toestablish a molecular mod el of HCV replication.

Cloning,Expression and Analysis of HCV Ns5b Polymerase gene from Chinese serum

  • Abstract:To ampUfy Hepatitis C virus(HCV)polymerase sequence and express HCV NS5b protein in order to,in the next,establish a molecular model of HCV replication.RT—PCR method was used to amplify HCV NS5b polymerase gene from a Chinese HCV(+1 patient’S serum. e polymerase fragment Was constructed in the plasmid pET一30a an d expressed in E coli.HCV NS5b protein Was identified by W lestern blot with a monoclonal an tibody against HCV NS5b.Sequence analysis showed tllat the isolate had 69% 一95% an d 89%一97% homologies to the reported HCV sequences in respect to nucleotide an d amino acid respectively.HCV NS5b gene was expressed at a high level an d the molecular weight ofthe expressed prod uct was 65kDa.which was within the ran ge of expectation.This protein Was confirmed to be HCV NS5b by Western blot.HCV NS5b from a Chinese patient Can be Success—fully cloned an d expressed with pET一30a plasmid an d E coli. is protein could be used to establish a molecular mod el of HCV replication.

  • 加载中
  • 加载中

Article Metrics

Article views(4153) PDF downloads(688) Cited by(0)

Related
Proportional views
    通讯作者: 陈斌, bchen63@163.com
    • 1. 

      沈阳化工大学材料科学与工程学院 沈阳 110142

    1. 本站搜索
    2. 百度学术搜索
    3. 万方数据库搜索
    4. CNKI搜索

    Cloning,Expression and Analysis of HCV Ns5b Polymerase gene from Chinese serum

    • 1. National lnsititute for the Control of Pharmaceutical and Biology Products.Beijing 100050,China

    Abstract: Abstract:To ampUfy Hepatitis C virus(HCV)polymerase sequence and express HCV NS5b protein in order to,in the next,establish a molecular model of HCV replication.RT—PCR method was used to amplify HCV NS5b polymerase gene from a Chinese HCV(+1 patient’S serum. e polymerase fragment Was constructed in the plasmid pET一30a an d expressed in E coli.HCV NS5b protein Was identified by W lestern blot with a monoclonal an tibody against HCV NS5b.Sequence analysis showed tllat the isolate had 69% 一95% an d 89%一97% homologies to the reported HCV sequences in respect to nucleotide an d amino acid respectively.HCV NS5b gene was expressed at a high level an d the molecular weight ofthe expressed prod uct was 65kDa.which was within the ran ge of expectation.This protein Was confirmed to be HCV NS5b by Western blot.HCV NS5b from a Chinese patient Can be Success—fully cloned an d expressed with pET一30a plasmid an d E coli. is protein could be used to establish a molecular mod el of HCV replication.

    Relative (20)

    目录

    /

    DownLoad:  Full-Size Img  PowerPoint
    Return
    Return