与α-烯醇化酶相关的抗内皮细胞抗体ELISA的建立及临床初步应用

目的建立与α-烯醇化酶（ENOI）相关的抗内皮细胞抗体（AECA）的酶联免疫吸附试验（ELISA）,并探讨ENOI-AECA与自身免疫性疾病的关系。方法人工合成获得人ENOI基因编码序列并克隆该片段构建pUC57-ENOI重组质粒,再将重组质粒亚克隆入原核表达载体pET28（a）,回收酶切产物并经T4DNA连接酶连接,获得的重组表达质粒pET28（a）-ENOI,在大肠埃希菌BL21（DE3）中诱导表达蛋白,Ni NTA亲和层析纯化重组ENOI蛋白。以纯化的重组蛋白为包被抗原,并对ELISA反应条件进行优化,初步建立检测ENOI-AECAELISA并作分析性能评价。用建立的方法分别检测健康人及系统性红斑狼疮（SLE）、类风湿性关节炎（RA）患者血清中的ENOI-AECA。结果获得了相对分子质量为47 000的高纯度人ENOI重组蛋白。ELISA抗原最佳包被浓度为5μg/mL,血清最佳稀释倍数为1∶50。以免疫印迹法为参照,所建方法的敏感性和特异性分别为76.0%和90.2%。SLE和RA患者血清中ENOI-AECA的阳性率分别为23.1%和57.1%;RA组ENOI-AECA阳性率与健康对照组比较,差异有统计学意义（P=0.000）。结论以人ENOI重组蛋白为抗原建立的检测ENOI-AECA的ELISA有较高的敏感性和特异性,可用于ENOI-AECA的检测。ENOI-AECA阳性提示患者可能患有自身免疫性血管炎。

Establishment of a ELISA method for detecting anti-endothelial cell antibody related with human alphaenolase and its primary clinical application

Objective To establish a enzyme-linked immunosorbent assay（ELISA） method for detecting the anti-endothelial cell antibody（AECA） related with human alpha-enolase（ENOI）,and investigate the relationship between ENOI-AECA and autoimmune disease.Methods The artificially synthesized ENOI gene coding sequence was cloned,and the recombinant plasmid pUC57-ENOI was constructed with the fragment.The recombinant plasmid was subcloned to pET28（a） vector.Both pET28（a） vector DNA and plasmid DNA were cleaved by restriction enzymes and ligated with T4 ligase.The recombinant plasmid pET28（a）-ENOI was transformed into Escherichia coli BL21（DE3）.The cultures was induced and purified by the Ni NTA affinity chromatography.The method for detecting ENOI-AECA with ELISA was established with the purified recombinant protein as the coating antigen.The reaction conditions were optimized,and the analyzing efficiency was evaluated.The ENOI-AECAs in sera from patients with systemic lupus erythematosus（SLE） and rheumatoid arthritis（RA） and healthy controls were detected by the method.Results The high purity recombinant proteins of relative molecular mass 47 000 was acquired.The coating antigen concentration was 5 μg/mL,and best serum dilution was 1∶ 50.Compared with Western blot,the sensitivity and specificity of the established method were 76.0% and 90.2%.The ENOI-AECA positive rates in sera from SLE and RA patients were 23.1% and 57.1%,respectively.There was a statistical significance of ENOI-AECA positive rate between RA patients and healthy controls（P=0.000）.Conclusions The ELISA method established with the purified recombinant protein as the coating antigen has high sensitivity and specificity,and it could be applied into detecting ENOI-AECA.ENOI-AECA positivity suggests that patients probably suffer from autoimmune vasculitis.