摘要
目的：克隆并表达寻常性天疱疮抗原（PVA，即桥粒芯糖蛋白-3）的EC1-2和EC3-4表位，用于通过血清学方法特异性诊断天疱疮和了解PVA的表位与抗PVA抗体间的联系。方法：从角朊细胞抽提RNA，逆转录合成cDNA,扩增目的基因EC1-2和EC3-4后与质粒载体PGEX－4T-1连接，电转导入大肠杆菌BI21，经IPTG诱导后表达EC1-2和EC3-4融合蛋白，SDS-PAGE电泳后转移至硝酸纤维素膜，免疫印迹法检测抗PVA抗体。结果：经限制性内切酶分析和DNA序列测定，克隆的EC1－2、EC3-4基因与PC／CGENE登录的序列一致；表达的重组蛋白仅与寻常性天疱疮血清反应，不与大疱性类天疱疮、系统性红斑狼疮以及正常人血清反应。结论：该重组蛋白具有高度抗原特异性，此项研究为通过血清学方法诊断寻常性天疱疮和鉴别诊断其它大疱性皮肤病提供了方法，为进一步研究粘附分子与天疱疮发病的时间关系奠定了基础。
objective: Cloning and expressing EC1-2 and EC3-4 epitopes of PVA(pemphigus vulgaris antigen, desmoglein-3) respectively in order to diagnose pemphigus specifically and study the relationship between epitopes of PVA and anti-PVA antibody. Methods: Extracting RNA from keratinacytes, synthesizing the cDNA of epitopes EC1-2 and EC3-4 by reverse transcription. Amplified genes of EC1-2, EC3-4 were inserted into expression plasmid PGEX-4T-1, which were transformed into E. coli BI21 by electric transduction .Recombinant fusion proteins of EC1-2, EC3-4 epitopes were expressed by IPTG induce. These proteins were separated on SDS-PAGE gel, electroblotted to nitrocellulose to de tect the anti-PVA antibody. Results: The sequence of cloned EC1-2,EC3-4 genes were identical to the sequence registered in PC/GENE. The expressed recombinant proteins reacted only to sera from patients with pemphigus vulgaris, not to sera from patients with bullous pemphigoid, patients with systemic lupus erythematosus and normal persons. Conclusion: These recombinant proteins are very specific in antigenicity. It provides a new method to make diagnosis of PV or make differential diagnosis of other bullous cutaneous diseases via patient's sera, and it is also help to realize the relationsip between adhesion molecules and pathogenic mechanism of PV.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2000年第7期349-352,共4页
Chinese Journal of Immunology
基金
中华医学会皮肤科专业委员会科研基金
上海市教育委员会科学研究基金!98B11
关键词
天疱疮
寻常性天疱疮抗原
表位
克隆
Pemphigus Pemphigus vulgaris antigen Desmoglein-3 Epitope
