中华皮肤科杂志 ›› 2005, Vol. 38 ›› Issue (5): 288-290.

• 论著 • 上一篇    下一篇

TOPO定向克隆和表达Ⅱ型单纯疱疹病毒gG基因的免疫优势表位片段

王书崎, 尹跃平, 余艳华, 赖伟红, 蒋明军, 陈强, 陈祥生   

  1. 中国医学科学院、中国协和医科大学皮肤病研究所, 南京210042
  • 收稿日期:2004-05-14 出版日期:2005-05-15 发布日期:2005-05-15
  • 通讯作者: 尹跃平,E-mail:ypyinc@vip.163.com E-mail:ypyinc@vip.163.com
  • 基金资助:
    国家自然科学基金资助项目(30170857)

Cloning and Expression of an Immunodominant Fragment of Glycoprotein G of HSV-2 Using Directional TOPO Vector in E.coli

WANG Shu-qi, YIN Yue-ping, YU Yan-hua, LAI Wei-hong, JIANG Ming-jun, CHEN Qiang, CHEN Xiang-sheng   

  1. Institute of Dermatology, Chinese Academy of Medical Sciences & Peking Union Medical College, Nanjing 210042, China
  • Received:2004-05-14 Online:2005-05-15 Published:2005-05-15

摘要: 目的 通过基因工程技术,克隆和表达Ⅱ型单纯疱疹病毒gG基因的免疫优势表位片段。方法 以PCR法扩增HSV-2的gG基因的免疫优势表位片段;将PCR产物与TOPO定向表达载体连接;然后鉴定含有目的基因的重组表达载体;并转化BL21StarTM菌株诱导表达目的蛋白;以免疫印迹法检测表达的目的蛋白。结果 PCR法扩增出616bp的DNA片段;通过PCR法及测序法证实重组质粒中含有616bp的DNA片段,其中99.5%序列与目的基因序列一致。SDS-PAGE结果显示重组蛋白在3h时的表达产量最高。免疫印迹法通过抗gG的单克隆抗体,证实重组蛋白在大肠杆菌中的表达。结论 Ⅱ型单纯疱疹病毒的gG免疫优势表位基因片段的成功表达,为制备国产的HSV-2血清诊断试剂提供资料。

关键词: 基因,病毒, 基因表达, 疱疹2型,人

Abstract: Objective To clone and express immunodominant fragment of glycoprotein G of HSV-2 (FgG-2). Methods The target gene was amplified by polymerase chain reaction (PCR). The PCR products were ligated into directional TOPO expression vector. After identification, the recombinant expression vector was transferred into BL21 StarTM cell for expression. Finally, recombinant protein of FgG-2 (rFgG-2) was detected by Western Blot (WB). Results A 616 bp DNA fragment was obtained with PCR and then confirmed in recombinant vector by PCR and sequencing, bearing 99.5% consistent sequence with target gene. Highest recombinant protein production was obtained at the time point of 3 hours. Expression of target protein was confirmed by WB with anti-gG monoclonal antibody. Conclusions The immunodominant fragment of gG-2 has been successfully cloned and expressed in E.coli, which might be used for the development of serum diagnostics assay kits for HSV-2 infection.

Key words: Herpesvirus 2, human, Genes, viral, Gene expression