中华皮肤科杂志 ›› 2003, Vol. 36 ›› Issue (2): 91-93.

• 论著 • 上一篇    下一篇

淋球菌主要外膜蛋白的分离纯化及其单克隆抗体的制备

周旺1, 郑卫1, 沈关心2, 朱慧芬2, 张悦2, 夏正喜1   

  1. 1. 武汉市皮肤病防治研究所 430030;
    2. 华中科技大学同济医学院 430030
  • 收稿日期:2001-12-25 出版日期:2003-02-15 发布日期:2003-02-15
  • 基金资助:
    湖北省武汉市科委资助课题(986002056)

Preparation of Monoclonal Antibodies against Major Outer Membrane Protein of Neisseria Gonor-rhoeae

ZHOU Wang1, ZHENG Wei1, SHEN Guan-xin2, ZHU Hui-fen2, ZHANG Yue2, XIA Zheng-xi1   

  1. Wuhan Institute of Dermatology and Venereology, Wuhan 430030, China
  • Received:2001-12-25 Online:2003-02-15 Published:2003-02-15

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摘要： 目的 建立快速、敏感、特异的淋球菌感染实验诊断方法。方法 采用溴化己二烯苯基三乙基胺-乙醇沉淀法提取淋球菌外膜蛋白复合物,以Z3,14、EDTA萃取外膜蛋白,经二乙氨基乙酸纤素-Sepharose CL-6B层析纯化获得主要外膜蛋白(PⅠ),并按免疫杂交瘤技术建立分泌抗PⅠ单克隆抗体(McAb)的杂交瘤细胞株。结果 获得了WⅠ群淋球菌的PⅠA和WⅡ/WⅢ群淋球菌的PⅠB,SDS鄄PAGE测得PⅠA的分子量为35200,PⅠB的分子量为36700。建立了可持续、稳定分泌抗PⅠAMcAb的杂交瘤细胞2株和抗PⅠBMcAb的杂交瘤细胞3株。5株杂交瘤细胞培养上清液的抗体滴度为1:64～1:256,其诱生的BALB/c小鼠腹水中的抗体滴度为1:4096～1:16384,与淋球菌呈高效价反应,与脑膜炎双球菌等其他抗原不发生反应。结论 获得的高纯度PⅠ及其McAb为建立快速、敏感、特异的淋球菌感染实验诊断方法奠定了基础。

关键词: 奈瑟氏球菌, 淋病, 细菌外膜蛋白质类, 抗体, 单克隆

Abstract: Objective To establish a rapid,sensitive and specific diagnostic test for detecting Neisseria gonorrhoea.Methods The major outer membrane proteins(PⅠ)in different gonococcal serogroups were obtained by isolation of outer membrane complex with CTB-ethanol precipitation,the outer membrane proteins were extracted with Z 3,14 and EDTA,and purified with DEAE-Sepharose CL-6B to obtain PⅠ.Hybridoma cell lines producing McAbs against PⅠ were established with lymphocyte hybridoma techniques.Results The molecular weight of PⅠA and PⅠB were determined with SDS-PAGE as 35.2k Da and 36.7k Da,respectively.Five hybridoma cell lines producing McAbs continuouslly and stably against PⅠA and PⅠB were obtained,in-cluding two hybridoma cell lines producing McAbs against PⅠ A and three hybridoma cell lines producing McAbs against PⅠ B.The titers of McAbs in the supernatants in the cultures and in abdominal ascites of BALB/c were from 1:64to1:256 and from 1:4096 to 1:16384,respectively;and the specificity of the McAbs against PⅠA and PⅠ B was so high that they easily reacted with N.gonorrhoeae but did not with other antigens such as N.meningitidis etc.Conclusion The purified PⅠ and the McAbs obtained in the study provide a basis to establish a rapid,sensitive and specific diagnostic test for detecting N.gonorrhoea.

Key words: Neisseria gonorrhoeae, Oter membrane proteins, bacterial, Antibodies, monoclonal