重组人内皮细胞抗原成分α烯醇化酶的表达及其在弥漫性结缔组织疾病中的意义  

Prokaryotic expression of recombinant human α enolase and the prevalence of anti-α enolase antibody in connective tissue diseases

作　　者：阳洪波[1] 郑文洁[1] 王汉萍[1] 姚志建[2] 

机构地区：[1]中国医学科学院中国协和医科大学北京协和医院风湿免疫科,100730 [2]国家人类基因组北方研究中心

出　　处：《中华风湿病学杂志》2008年第11期743-746,共4页Chinese Journal of Rheumatology

基　　金：国家自然科学基金资助项目（30400411）

摘　　要：目的对蛋白质组学分离和鉴定的抗内皮细胞抗体（AECA）相关抗原-α烯醇化酶进行基因重组和表达，检测其特异性抗体在血管炎和其他自身免疫性疾病患者血清中的阳性率。方法聚合酶链反应（PCR）扩增人enol基因CDS片段并与表达载体重组，在大肠杆菌中表达和纯化重组人α烯醇化酶。用质谱鉴定后的重组蛋白作为抗原，用点印迹法和酶联免疫吸附试验（ELISA）法分别检测健康人和不同结缔组织病患者血清中抗人α烯醇化酶抗体水平。结果克隆了人enol基因cDNA片段，将重组的人α烯醇化酶在大肠杆菌中进行原核表达和纯化。用重组蛋白作为抗原，点印迹法检测健康对照和不同结缔组织病患者血清中抗人α烯醇化酶抗体，证实：①系统性血管炎（SV）、系统性红斑狼疮（SLE）、干燥综合征（SS）和类风湿关节炎（RA）患者血清中抗α烯醇化酶抗体的阳性率分别为76．7％、78．3％、63．6％和78．9％，均明显高于多发性肌炎／皮肌炎（PM／DM）组和健康对照组（P〈0．01）。②系统性血管炎中自塞病（BD）、多发性大动脉炎（TA）、韦格纳肉芽肿病（WG）、显微镜下多动脉炎（MPA）和Churg-Strauss综合征（CSS）患者血清中抗α烯醇化酶抗体阳性率各为74．0％、81．5％、62．5％、92．3％和80．0％。③SV和SLE患者血清中抗α烯醇化酶抗体的阳性率与SS和RA患者血清中该抗体的阳性率差异无统计学意义（P〉0．05）。④不同患者血清中抗α烯醇化酶抗体强阳性者所占比例分别为：SV51．7％，SLE33．3％，SS42．9％，RA20．0％。RA组与SV组差异有统计学意义（P〈0．05）。结论SV和SLE、SS、RA患者血清中均存在不同水平的抗α烯醇化酶抗体，α烯醇化酶是AECA识别的自身抗体之一。Objective In our previous work, the prevalence of anti-endothelial cell antibodies （AECA） in patients with systemic vasculitis and other autoimmune diseases was analyzed. AECA against a 47 000 endothelial cell antigen was found in patients of a variety of systemic vasculitis and systemic lupus erythematosus （SLE）. It was suggested to be α-enolase by the combination of immunoblotting and proteomics methods. The aim of this work is to demonstrate that α-enolase is one of the targets of AECA, and to detect the prevalence of anti-α-enolase antibody in sera of patients with autoimmune disorders including systemic vasculitis. Methods The CDS of human Enol gene was amplified by polymerase chain reaction （PCR）, with template of human placenta λZAP express cDNA library. The product was then recombined with expressiotl vector. After expression and purification from E.coli, the recombinant protein was analyzed by mass spectrometry. The prevalence of anti-α-enolase antibody in patients with autoimmune disorders including systemic vasculitis was tested by Western blot and enzyme-linked immunosorbent assay （ELISA）. Results The CDS of human Enol gene was subcloned to the expression vector. Recombinant human α-enolase was expressed and purified in E.coli. The recombinant protein was demonstrated to be his-tagged human α-enolase by mass spectrometry. Results of Dot-Blot revealed that the prevalence of anti-α-enolase antibody was 76.7% in systemic vasculitis [ including 74.0% in Behcet＇s disease （BD）, 81.5% in Takayasu arteritis （TA）, 62.5% in Wegener＇s granulomatosus （WG）, 92.3% in microscopic polyangitis （MPA） and 80.0% in Churg-Strauss syndrome （CSS）]. 78.3% in SLE, 63.6% in Sjogren＇s syndrome （SS） and 78.9% in rheumatoid arthritis （RA）. No positive signals were detected in sera of normal controls or patients with polymyositis/ dermatomyositis （PM/DM）. There was no statistical significance among positive rates of anti-α-enolase antibody in systemic vasculitis, SLE,

关 键 词：结缔组织疾病 磷酸丙酮酸水合酶 抗内皮细胞抗体 

分 类 号：R686[医药卫生—骨科学]