【作者】 李薇；

【导师】 冉玉平；

【作者基本信息】 四川大学 ， 皮肤病与性病学， 2006， 博士

【摘要】 天疱疮(pemphigus)是自身抗体介导作用于表皮角质形成细胞桥粒芯糖蛋白的自身免疫性大疱性皮肤和粘膜疾病，寻常型天疱疮(pemphigus vulgaris，PV)是其中最为常见的亚型。尽管已经公认桥粒芯糖蛋白-1(desmoglein 1，Dsg1)和桥粒芯糖蛋白-3(desmoglein 3，Dsg3)及其自身抗体与天疱疮发病相关，但其确切的发病机制仍不清楚。新近有研究发现了人体组织桥粒芯糖蛋白-4(desmoglein4，Dsg4)是PV自身抗体的靶抗原，为该病的研究提供了新的线索。 本项研究目的是扩增人皮肤组织Dsg4胞外域(extracellular domains，ECD)EC1、EC2、EC3和EC4的核酸序列，进一步探讨其在PV发病中的作用。 首先根据Genebank中人Dsg4的cDNA序列资料，设计Dsg4胞外域EC1、EC2、EC3和EC4 4个DNA片段的特异性引物，通过逆转录聚合酶链反应(reverse-transcription polymerase chain reaction，RT-PCR)法，以提取的人正常皮肤组织总RNA为模板，扩增桥粒芯糖蛋白4胞外域EC1、EC2、EC3和EC4目的基因；应用基因重组技术将目的基因分别与原核表达质粒载体pET32a在T4DNA连接酶作用下相连接，转化大肠杆菌(Escherichia coli，E．coli)DH5α感受态细菌，用含氨苄青霉素的LB(Luria-bertani)培养基平板筛选转化菌，重组质粒DNA序列测定鉴定。结果显示RT-PCR扩增产物经凝胶电泳得到4条均约为350bp的条带；分别与载体pET32a相连接后获得4个重组质粒更多还原

【Abstract】 Pemphigus is an autoimmune disease that results in blistering of cutaneous and mucous membrane. It is caused by autoantibodies directed against desmoglein on keratinocytes, and pemphigus vulgaris (PV) is one of the subtypes which can be frequently seen. Although it has been proved that desmoglein l(Dsgl), desmoglein 3(Dsg3) and their autoantibodies are relevant to pemphigus onset, the precise pathogenesis is still dimness. Recent study showed that desmoglein 4 from human body is also the target antigen of autoantibodies in PV which provided a new clue to research the disease.The aim of the study is to amplify the nucleotide sequences of desmoglein 4(Dsg4) extracellular domains (ECs), as EC1, EC2, EC3 and EC4 (EC1-4) from human skin tissue, and then investigate their role in PV pathogenisis.Firstly, specificiticity primers were designed according to cDNA sequences of Dsg4 in Genebank data. RNA was obtained from normal human skin tissue and then cDNA was synthesized. Objective genes of desmoglein 4 extracellular domains (EC1-4) were amplified by polymerase chain reaction (PCR). With the technique of gene recombination, objective genes linkaged with pET32a plasmids respectively by T4DNA ligase, which translated into Escherichia coli (E.coli) DH5α competent germs. Screening of translated germs was done by Luria-bertani (LB) medium with Ampicillin. DNA sequences of positive recons were identified finally. The results showed that four bands with all the length of 350bp were obtained by RT-PCR. Consequently four expressed plasmids of desmoglein 4 extracellular domains更多还原