中华皮肤科杂志 ›› 2012, Vol. 45 ›› Issue (1): 9-11.

• 论著 • 上一篇    下一篇

大疱性类天疱疮和妊娠疱疹患者血清抗BP180 NC16A抗体的纯化和鉴定

左亚刚1,LIU Zhi2,3   

  1. 1. 中国医学科学院北京协和医学院北京协和医院
    2.
    3. 美国北卡罗来纳大学
  • 收稿日期:2011-07-18 修回日期:2011-08-19 出版日期:2012-01-15 发布日期:2011-12-31
  • 通讯作者: 左亚刚 E-mail:zuoyagang@126.com
  • 基金资助:

    国家自然科学基金资助课题;人事部留学回国基金;教育部新世纪优秀人才项目

Purification and identification of anti-BP180 NC16A antibodies from the sera of patients with bullous pemphigoid or herpes gestationis

  • Received:2011-07-18 Revised:2011-08-19 Online:2012-01-15 Published:2011-12-31
  • Contact: Ya-gang ZUO E-mail:zuoyagang@126.com
  • Supported by:

    ;Scientific Research Foundation for the Returned overseas Chinese scholars，Ministry of Personnel of China;Program for New Century Excellent Talents in University of Ministry of Education of China

PDF

33

赞

摘要：

目的 探讨大疱性类天疱疮（BP）和妊娠疱疹（HG）患者血清抗BPl80 NC16A 抗体的纯化和鉴定方法。方法 原核表达系统pGEX-2TBP180NC16A表达GST/NC16A融合蛋白，将融合蛋白与谷胱甘肽琼脂糖凝聚微珠进行共价偶联。微珠亲和层析法纯化BP和HG患者血清中抗BP180 NC16A抗体，并用ELISA、免疫荧光、及Western印迹进行鉴定。结果 原核表达系统pGEX-2TBP180NC16A表达37 000 GST/NC16A融合蛋白，微珠亲和层析法纯化后得单一抗BP180 NC16A抗体。经ELISA方法定量后确定其含量为2.4 mg/ml；该抗体能与人皮肤基底膜带结合，证明抗体活性；免疫印迹可见单一片段，显示抗体纯度。结论 微珠亲和层析法纯化的BP和HG患者血清中抗BP180 NC16A自身抗体活性高、特异性强。

关键词: 鉴定

Abstract:

Objective To develop a method to purify and identify anti-BP180 NC16A antibodies from the sera of patients with bullous pemphigoid （BP） or herpes gestationis. Methods The GST/NC16A fusion protein was expressed in a prokaryotic expression vector pGEX-2TBP180NC16A, and then crosslinked to glutathione sepharose beads. Anti-BP180 NC16A antibodies were isolated from the sera of 3 patients with BP and 2 patients with herpes gestationis by affinity chromatography, and analyzed by immunofluorescence，Western blot and enzyme linked immunosorbent assay （ELISA）. Results The GST/NC16A fusion protein with a relative molecular mass of 37 000 was successfully expressed by the prokaryotic vector pGEX-2TBP180NC16A. Purified anti-BP180 NC16A antibodies were obtained from the sera of patients by the affinity chromatography, and ELISA revealed that the concentration of anti-BP180 NC16A was 2.4 mg/ml. The purified antibody could bind to the basement membrane zone of human skin, suggesting a strong biological activity of the antibodies. Western blot showed a single band corresponding to the expected molecular mass for anti-BP180 NC16A antibodies, indicating a high purity of the isolated antibodies. Conclusion The anti-BP180 NC16A antibodies purified by microbead-based affinity chromatography from the sera of patients with BP or herpes gestationis are highly active and specific.

Key words: identification