基础医学与临床 ›› 2012, Vol. 32 ›› Issue (11): 1337-1341.

• 研究论文 • 上一篇    下一篇

登革病毒4型E蛋白的表达与多克隆抗体的制备

李竹石1,林华1,杨健1,杨会强1,曾献武1,赵宇1,刘俐1,刘莉娜1,康庄1,俞永新2,李玉华2   

  1. 1. 成都生物制品研究所有限责任公司
    2. 中国食品药品检定研究院
  • 收稿日期:2012-06-28 修回日期:2012-09-15 出版日期:2012-11-05 发布日期:2012-10-19
  • 通讯作者: 李玉华 E-mail:liyuhua@hotmail.com
  • 基金资助:
    国家高技术研究发展计划

Expression of the envelope protein of Dengue virus type 4 and preparation of the polyclonal antibody

  • Received:2012-06-28 Revised:2012-09-15 Online:2012-11-05 Published:2012-10-19

摘要: 目的 在原核细胞中表达登革病毒4型E蛋白及E蛋白III区,分别以两种蛋白免疫家兔,获得可检测登革病毒的多克隆抗体。方法 将登革病毒4型E蛋白与E蛋白III区编码序列克隆到pET-32a(+)质粒中,分别构建两种蛋白的表达载体,以IPTG诱导其在Rosetta细胞中的大量表达,并进行SDS-PAGE检测。分别以两种蛋白免疫家兔,制备出针对E蛋白与E蛋白III区的多克隆抗体,并对其进行Western blot鉴定。结果 登革病毒4型E蛋白与E蛋白III区在Rosetta细胞中以包涵体的形式大量表达;利用所制备的多克隆抗体对登革病毒进行检测,出现了预期的条带。结论 本研究制备获得的多克隆抗体可用于登革病毒的检测,为登革病毒相关研究奠定了基础。

关键词: 登革病毒4型, E蛋白, 多克隆抗体

Abstract: Objective To express full length (DENV4E) and domain III (DENV4EIII) of the envelope protein of Dengue virus type 4 in procaryotic cells respectively, and to prepare the polyclonal antibody against dengue virus by immunizing rabbits with the expressed proteins. Methods The sequences encoding DENV4E and DENV4EIII proteins were cloned into pET-32a(+) plasmids respectively to construct expression vectors. Then DENV4E or DENV4EIII proteins were expressed in Rosetta cells by IPTG induction and detected by SDS-PAGE. Polyclonal antibody against DENV4E or DENV4EIII proteins was obtained by immunizing rabbits with the expressed proteins and was identified by Western blot. Results DENV4E and DENV4EIII proteins were both successfully expressed in Rosetta cells mainly in the form of inclusion body. The prepared polyclonal antibody could generate obvious bands as expected when reacting with the dengue virus. Conclusion The polyclonal antibody prepared is competent for the detection of dengue virus, laying a foundation for further research.

Key words: dengue virus type 4, envelope protein, polyclonal antibody

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