寻常型天疱疮自身抗原Dsg3片段的重组表达和免疫识别

RECOMBINANT EXPRESSION AND EPITOPE ANALYSIS OF AUTOANTIGEN Dsg3 IN PEMPHIGUS VULGARIS

探讨寻常型天疱疮(pemphigusvulgaris,PV)自身抗原桥粒芯蛋白 3(desmoglein 3,Dsg3)的抗原决定簇 ,为自身免疫性疾病机制的研究提供依据。根据GeneBank中的Dsg3序列分析 ,采用反转录 聚合酶链式反应 (RT PCR)法克隆自身抗原Dsg3E1、E2、E3、E4、E5多肽片段的cDNA ,定向插入表达载体 pGEX 2T ,并且导入大肠杆菌JM10 9中表达重组融合蛋白 ,经免疫印迹法与PV患者及疾病对照组、正常对照组血清进行反应。结果显示 ,Dsg3E1、E2和E4与PV患者血清反应 ,而不与疾病对照组、正常对照组血清反应。不同患者抗原优势表位有所不同。研究表明 ,Dsg3E1、E2和E4表位在PV发病中起重要作用。

The present study was to analyze the epitopes of autoantigen Dsg3 in pemphigus vulgaris (PV), and to elucidate the molecular mechanisms underlying autoimmune diseases. Based on the sequence analysis of autoantigen Dsg3 in Genbank five fragments of Dsg 3 cDNA (E1, E2, E3, E4, and E5) were cloned using RT PCR. Then PCR products were inserted into the expression vector pGEX 2T, and transformed into JM109 strain of E. coli. Dsg3 recombinant proteins were purified from the cell lysates. The epitopes of Dsg3 in PV patients were analyzed with five Dsg3 recombinant proteins by Western blot. The results showed that Dsg3 E1, E2 and E4 recombinant proteins reacted with PV patients′ sera, but not with control or normal sera. The dominant epitopes of Dsg3 in PV patients were different. The above data indicated that the epitopes of Dsg3 were located in E1, E2, and E4, which might play an important role in the pathogenesis of pemphigus vulgaris.