摘要
目的研究类风湿关节炎(RA)特异性抗sa抗体识别的Sa抗原,探讨Sa抗原的相关蛋白质。方法从人胎盘组织阴离子交换柱层析法纯化Sa抗原,应用免疫印迹法检测155例各种风湿病患者血清的抗Sa抗体,155例患者中类风湿关节炎71例,强直性脊柱炎11例、银屑病关节炎7例、反应性关节炎7例、幼年特发性关节炎4例、骨性关节炎5例、风湿性多肌痛6例、痛风6例、系统性红斑狼疮7例、干燥综合征10例、炎性肌病8例、混合性结缔组织病4例、白塞病6例、成人Still病3例。切取与免疫印迹法阳性条带相对应的电泳胶内条带,胰蛋白酶胶内酶解,用高效液相色谱-电喷雾-离子阱质谱鉴定目标蛋白质。以大肠杆菌重组表达目标蛋白质作为抗原,以免疫印迹法检测相应抗体。瓜氨酸化修饰重组蛋白质作为抗原,以免疫印迹法检测相应抗体。结果抗Sa抗体表现为相对分子质量为50 000(和)55 000的阳性印迹条带,在155例风湿病中,对RA的诊断敏感性和特异性分别为47．9％和95．2％。质谱鉴定Sa抗原相关蛋白质为波形蛋白(Vimentin)。抗波形蛋白抗体在RA组与其他风湿病组的阳性率差异有统计学意义(P=0．005),对RA的诊断敏感性和特异性分别为36．6％和83．3％,但与抗Sa抗体的一致性差(Kappa=0．316)。抗瓜氨酸化波形蛋白抗体阳性率在RA组较其他风湿病组高(P<0．01),对RA的诊断敏感性和特异性分别为49．3％和86．9％,且与抗Sa抗体的一致性较高(Kappa=0．746)。结论运用质谱技术鉴定的Sa抗原相关蛋白质波形蛋白,瓜氡酸化修饰后的抗原性与Sa抗原相仿,提示瓜氨酸化波形蛋白与Sa抗原密切相关。
Objective To study the proteins associated with Sa antigen, a target of the anti-Sa antibodies specific for rheumatoid arthritis, and to elucidate the nature of these proteins. Methods Sa antigen was extracted from fresh human placental tissue by anion exchange chromatography and subjected to SDS-PAGE electrophoresis. Serum samples were collected from 155 patients with connective tissue diseases, including rheumatoid arthritis （71 cases） ,ankylosing spondylitis （11 cases）, psoriatic arthritis （7 cases）, reactive arthritis （ 7 cases ）, juvenile idiopathic arthritis （4 cases ）, osteoarthritis （ 5 cases ）, polymyalgia rheumatica （6 cases） , gout （6 cases） , systemic lupus erythematous （7 cases） , Sjogren＇s syndrome （ 10 cases）, adult onset still＇s disease （3 cases） and Sa antibodies were detected by immunoblotting. The gel bands corresponding to the stained bands were excised, trypsin-digested in gel, and analyzed by LC-ESI- MS/MS. Once identified, the protein was recombinated and expressed in Escherichia toll, and the antibodies were detected by immunoblotting. Then the protein was citrullinated to detect the antibodies again. Results Immunoblotting showed anti-Sa antibodies, band （s） with apparent molecular weight of 50 000（and） 55 000, in 34 of the 71 patients of rheumatoid arthritis and 4 of the 84 patients of other rheumatic diseases, with a sensitivity rate of 47.9% and a specificity rate of 95.2%. The target protein was identified as vimentin. The positive rate of anti-vimentin antibody was statistically different between the RA patients and the patients with other rheumatic diseases （P=0. 005） ,with a sensitivity rate of 36.6% and a specificity rate of 83.3%, respectively. But there was no obvious correlation between anti-vimentin antibody and anti-Sa antibodies （Kappa = 0. 316 ）. The positive rate of anti-citrullinated vimentin antibody was significantly higher in the RA patients than in the patients with other rheumatic diseases （ P 〈 0. 01 ）, with a sensitivity rate of 49. 3%. There was a high correlation between anti-citnAlinated vimentin antibody and anti-Sa antibodies （ Kappa = 0. 746）, albeit a low specificity rate （86. 9% ）. Conclusion Citrullinated vimentin is closely correlated with Sa antigen.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2006年第27期1896-1900,共5页
National Medical Journal of China
基金
"863"十五攻关项目(2002-BA711A11)
关键词
类风湿关节炎
抗体
抗原
波形蛋白
质谱
Rheumatoid arthritis
Antibodies
Antigen
Vimentin
Mass spectrometry
