高等学校化学学报

高等学校化学学报 ›› 2005, Vol. 26 ›› Issue (11): 2061.

• 研究论文 • 上一篇    下一篇

SARS病毒核衣壳蛋白的表达与鉴定

赵博2, 金宁一1, 王瑞琳1, 郑敏1, 张应玖2   

  1. 1. 军事医学科学院军事兽医研究所,长春130062;
    2. 吉林大学分子酶学工程教育部重点实验室,长春130023
  • 收稿日期:2004-11-30 出版日期:2005-11-10 发布日期:2005-11-10
  • 基金资助:

    吉林省科技厅重大项目(批准号:20030413)资助

Expression and Identification of SARS-CoV Nucleocapsid Protein

ZHAO Bo2, JIN Ning-Yi1, WANG Rui-Lin1, ZHENG Min1, ZHANG Ying-Jiu2   

  1. 1. Institute of Military Veterinary,Academy of Military Medical Sciences,Changchun 130062,China;
    2. College of Life Science,Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education,Jilin University,Changchun 130023,China
  • Received:2004-11-30 Online:2005-11-10 Published:2005-11-10

PDF (PC)

被引次数

6

摘要： 依据Genebank中SARS基因组序列和酵母菌对密码子的选择性,采用人工合成的方法,合成了优化的SARS病毒核衣壳蛋白(N)的全基因(1296bp),与CTL表位基因(195bp)重组后,将其克隆到酵母分泌型表达载体pPIC9K中,构建成重组表达载体pPIC9K-N.重组载体转化毕赤酵母GS115,并经MD平板和MM平板筛选及PCR鉴定,得到阳性重组酵母工程菌GS115-pPIC9K-N.用甲醇诱导其分泌表达目的蛋白并对表达产物进行分析、浓缩与鉴定.结果表明,SARS病毒核衣壳蛋白能实现在毕赤酵母中高效表达,表达量达到20%,初步纯化后的产物具有良好的抗原特异性.

关键词: 严重急性呼吸系统综合症(SARS), 核衣壳蛋白, 毕赤酵母

Abstract: According to the data in genebank and the characterization of the codon usage in yeast,the optimized gene encoding SARS nucleocapsid protein(1 296 bp) by mutating low-usage codon into synonymous ones was synthesized.After recombined with the CTL epitope gene(195 bp),the gene was cloned into secreted expression vector pPIC9K to pPIC9K-N.pPIC9K-N was transformed into the Pichia pastoris GS115,and then the positive recombinant GS115/pPIC9K-N was screened by MM and MD plates and identified by PCR further.Nucleocapsid protein was highly expressed with a yield of 20% as a secretion under methanol induction and the expressed nucleocapsid protein was isolated and purified by salting out.SDS-PAGE and Western blotting results showed that both the expressed and purified nucleocapsid protein were recognized specifically by SARS antiserum,showed good reactiongenicity and antigenic specificity.

Key words: Severe acute respiratory syndrome(SARS), Nucleocapsid protein, Pichia pastoris

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