重组α-胞衬蛋白的表达及其在原发性干燥综合征临床研究中的意义

Expression and identification of α-Fodrin and its application in Sjgren’s syndrome

目的:利用pGEX-4T-2表达载体在大肠杆菌BL21中表达重组的GST-α-胞衬蛋白(α-Fodrin),以纯化获得的GST-α-胞衬蛋白为抗原,利用免疫印迹法对其抗体在原发性干燥综合征(pSS)中的诊断意义进行研究。方法:将表达质粒pGEX-4T-2-α-Fodrin转入大肠杆菌BL21,IPTG诱导培养,收集的细菌经超声破碎后离心,利用Glutathione-Sepharose4B亲和层析及Sephacryl-S100分子筛获取纯化的α-胞衬蛋白,通过SDS-PAGE电泳及免疫印迹法进行鉴定。最终以纯化得到的GST-α-胞衬蛋白为抗原,运用免疫印迹法测定干燥综合征患者及其他疾病患者血清中的抗α-胞衬蛋白抗体。结果:SDS-PAGE凝胶电泳显示表达产物分子量为97kD,与GST-α-胞衬蛋白分子量一致。用原发性干燥综合征病人血清对纯化出的GST-α-胞衬蛋白和GST分别进行免疫印迹分析,结果表明血清中的抗α-胞衬蛋白抗体是针对α-胞衬蛋白抗原的特异性抗体。原发性干燥综合征(pSS)患者血清中抗α-胞衬蛋白抗体的阳性率为63·75%(51/80),明显高于正常人的21·67%(8/37)(P<0·01),且各例正常人的阳性条带均明显浅于原发性干燥综合征患者。其他疾病包括类风湿关节炎(RA)、系统性红斑狼疮(SLE)及骨性关节炎(OA)患者血清抗α-胞衬蛋白抗体的阳性率分别为33·33%(13/39)、25·71%(9/35)及33·33%(4/12)。结论:通过GST融合蛋白表达系统获得了具有抗原性的α-胞衬蛋白,抗α-胞衬蛋白抗体测定可能对原发性干燥综合征的诊断有重要意义。

Objective：To express recombinant GST-α-Fodrin fusion proteins in E. coil by plasmid pGEX-4T-2 and investigate the prevalenee of anti-α-Fodrin antibody in primary Sjtoegren＇ s syndrome using purified GST-α-Fodrin fusion proteins. Methods：pGEX- 4T-2 veetor inserted with α-Fodrin eDNA was transformed into E. coli BL21. Suspended eells were sonieated and eentrifuged after indueement with IPTG. The fusion protein GST-α-Fodrin was purified by affinity ehromatography and gel filtration on Sephaeryl-S 100. The anfigenie features of purified protein were analyzed using SDS-PAGE and Western blot. The prevalenee of anti-α-Fodrln antibodies were examined in Sjoegren＇ s syndrome （SS） and other patients by using GST-α-Fodrin fusion protein as antigen. Results：SDS-PAGE showed the moleeular weight of the produet was 97 kD. The sera from primary SjSgren＇ s syndrome patient reacted with fusion protein, but not with GST protein, suggesting that the sera were speeifie to α-Fodrin. 51 of 80 serum samples from patients with Sjoegren＇ s syndrome reacted with the purified α-Fodrin fusion protein in Western blot analysis. In the eontrast, only 13 out of 39 patients with rheu- matoid arthritis（ RA）, 9 out of 35 patients with systemie lupus erythematnsus（SLE）, 4 out of 12 patients with osteoarthritis（OA） and 8 out of 37 normal individuals were anti-α-Fodrin positive with low reactivity. Conclusion： Alpha-Fodrin is able to be expressed in E. coil, and purified by GST expression system. The anti-α-Fodrin antibody is likely to be a diagnostie marker for Sjoegren＇ s syndrome.