大疱性类天疱疮和妊娠疱疹患者血清抗BP180NC16A抗体的纯化和鉴定  

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

作　　者：左亚刚[1] LIUZhi 

机构地区：[1]中国医学科学院北京协和医学院北京协和医院皮肤科,北京100730 [2]美国北卡罗来纳大学教堂山分校皮肤科

出　　处：《中华皮肤科杂志》2012年第1期9-11,共3页Chinese Journal of Dermatology

基　　金：基金项目：国家自然科学基金（81071301）;教育部新世纪人才项目（NCET-10-0964）

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BP180 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 000GST/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.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.

关 键 词：类天疱疮 大疱性 妊娠期类天疱疮 BP180 NC16A 

分 类 号：R758.66[医药卫生—皮肤病学与性病学]