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-SepharoseCL-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。
Objective To establish a rapid,sensitive and specific diagnostic test for detecting Neisse-ria gonorrhoea.Methods The major outer membrane proteins(P Ⅰ )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 P Ⅰ .Hybridoma cell lines producing McAbs against P Ⅰ were established with lymphocyte hybridoma techniques.Results The molecular weight of P Ⅰ A and P Ⅰ B were determined with SDS-PAGE as35.2kDa and36.7kDa,respectively.Five hybridoma cell lines producing McAbs continuouslly and stably against P Ⅰ A and P Ⅰ B were obtained,in-cluding two hybridoma cell lines producing McAbs against P Ⅰ A and three hybridoma cell lines producing McAbs against P Ⅰ B.The titers of McAbs in the supernatants in the cultures and in abdominal ascites of BALB/c were from1:64to1:256and from1:4096to1:16384,respectively;and the specificity of the McAbs against P Ⅰ A and P Ⅰ 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 P Ⅰ and the McAbs obtained in the study provide a basis to establish a rapid,sensitive and specific diagnostic test for detecting N.gonorrhoea.
出处
《中华皮肤科杂志》
CAS
CSCD
北大核心
2003年第2期91-93,共3页
Chinese Journal of Dermatology
基金
湖北省武汉市科委资助课题(986002056)
关键词
淋球菌
分离
纯化
制备
细菌外膜蛋白
单克隆抗体
Neisseria gonorrhoeae
Oter membrane proteins,bacterial
Antibodies,monoclonal
