摘要
目的 制备U1RNP70KD ,Sm D ,SSB及 β2GP1重组自身抗原 ,探讨自身免疫性疾病的机理及提高临床自身抗体检测的敏感性和特异性。方法 应用逆转录PCR方法克隆 β2GP1及其第 5功能区 (D5 ) ,U1RNP70KD及其U1RNA结合功能区 (BD) ,Sm D和SSB的编码基因 ,经原核表达系统 (PGEX 2T)进行表达、鉴定及纯化 ,并分析 β2GP1及U1RNP70KD抗原表位的定位 ,进而以重组抗原为基质建立新型免疫印迹及酶联免疫吸附检测法。结果 成功表达纯化了上述重组抗原 ;定位研究显示 β2GP1 D5及U1RNA BD分别为 β2GP1及U1RNP70KD的主要抗原表位区域 ;新型重组检测法的测定结果与常规检测系统比较基本相吻合。结论 重组蛋白具有良好的特异性 ,可进一步作为抗原表位研究的手段 ,并可用于临床自身抗体检测。
Objective Studies on the expression of recombinant autoantigens U1RNP70KD, Sm D, SSB and β2GP1 and evaluation of their serodiagnostic value. Methods By adapting reverse transcription PCR technology, we obtained the cDNA clones encoding β2GP1, β2GP1 D5, U1RNP70KD, U1BD, Sm D and SSB autoantigens. The recombinant proteins were expressed by the bacterial expression plasmid pGEX 2T and then idnetified and purified. The locations of epitopes of β2GP1 and U1RNP70KD were analysed by competitive inhibition ELISA and IBT respectively. CIE or IBT defined positive autoimmune sera were tested by ELISA or IBT of the recombinant autoantigens. Results The β2GP1 D5 and U1BD were confirmed to be the major epitopes of β2GP1 and U1RNP70KD respectively. There was a high correlation between binding values of IgG anti β2GP1 and IgG aCL antibodies ( r =0.667),and there was also relevance between binding values of IgM anti β2GP1, and IgM aCL antibodies ( r =0.553). The analysis by IBT demonstrated that 96%(48/50) of anti U1RNP70KD positive sera and 67.4%(29/43) of anti SSB positive sera could recognize the recombinant proteins. And 9 sera samples which showed 13.5kD positive band in IBT method also had positive reaction with recombinant Sm D by ELISA method. Conclusion The recombinant autoantigens have good specificity and can be used in epitopes mapping further and in clinical use for serodiagnosis.(Shanghai Med J, 2000,23∶263 266)
出处
《上海医学》
CAS
CSCD
北大核心
2000年第5期263-266,共4页
Shanghai Medical Journal
