类风湿关节炎滑膜组织cDNA文库的构建及自身抗原的筛选和鉴定

Construction the cDNA library from synovium of rheumatoid arthritis and screening and testifying of self-antigens

目的 通过SEREX（serological analysis of recombinant cDNA expression library）技术对类风湿关节炎滑膜组织cDNA文库进行初步筛选,得到新的类风湿关节炎自身抗原.方法 利用SMART技术构建类风湿关节炎患者滑膜组织cDNA文库,并对该文库进行SEREX筛选,获得特异性结合阳性克隆,对阳性克隆同源性分析.然后进行酶联免疫吸附实验（ELISA）验证,分析该阳性克隆对于类风湿关节炎诊断临床敏感度和特异度.结果 构建cDNA文库滴度约为6.89×10^6 pfu/mL,筛选文库获得12个阳性克隆,同源性分析3个阳性克隆插入片段为已知蛋白,分别是：Filaggrin、SERPINE2和p68.进行血清学初步验证,抗-Filaggrin和抗-SERPINE2具有高敏感度和低特异度,而抗-p68具有高敏感度和高特异度.结论 抗-Filaggrin、抗-SERPINE2和抗-p68对于类风湿关节炎的诊断具有不同的敏感度和特异度.

Objective To obtain new RA antigen genes,we screened the cDNA library of RA Using SEREX technology.Methods We constructed the cDNA library using SMART technology.After evaluation,the cDNA library was screened with the RA sera.The positive clones were obtained and the nucleotide sequences of positive clones were identified and analyzed with BLAST software in GenBank.According to the results of ELISA,we analysed the specificity and sensitivity for the diagnosis and treatment of RA.Results The titer of cDNA library was 6.89 × 10^6 pfu/mL.After several rounds SEREX screening,12 positive clones were obtained.3 of 12 genes were homologous to the genes known in GenBank.They are Filaggrin,SERPINE2 and p68.The results of ELISA were as follows.Anti-Filaggrin and anti-SERPINE2 are of high sensitivity but low specificity; While,anti-p68 is of high sensitivity and specifity.Conclusion Anti-Filaggrin、anti-SERPINE2 and anti-p68 are of different sensitivity and specifity for diagnosis of RA.