人皮肤组织桥粒芯糖蛋白4片段的克隆和在寻常型天疱疮中免疫识别的初步研究  

Gene Fragments Cloned and Immune Recognition Studied Preliminarily for Desmoglein 4 in Pemphigus Vulgaris

作　　者：李薇[1] 冯云[2] 吕小岩[1] 黎静宜[1] 冉玉平[1] 

机构地区：[1]四川大学华西医院皮肤性病科,成都610041 [2]四川大学华西基础医学与法医学院感染免疫研究室

出　　处：《四川大学学报（医学版）》2007年第1期84-87,共4页Journal of Sichuan University(Medical Sciences)

摘　　要：目的扩增人皮肤组织桥粒芯糖蛋白4(desm og le in 4,D sg4)胞外区域EC1、EC2、EC3和EC4的核酸序列,为寻常型天疱疮(pem ph igus vu lgaris,PV)发病机制的研究提供依据。方法根据G enB ank中的D sg 4序列(登录号为AY 177664)分析,通过逆转录聚合酶链反应(RT-PCR)法从人正常皮肤组织中抽提RNA,逆转录合成cD-NA,聚合酶链反应(PCR)扩增皮肤组织桥粒芯糖蛋白4胞外区域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质粒pET 32a在T 4 DNA连接酶作用下相连接,转化E.coli DH 5α感受态细菌,用含氨苄青霉素的LB培养基平板筛选转化菌,阳性重组子DNA序列测定鉴定;重组融合蛋白经EL ISA法与PV患者、正常对照组血清进行反应。结果RT-PCR扩增产物经凝胶电泳得到均约为350 bp的4条条带;质粒载体pET 32a连接的目的基因经序列测定后与在G enB ank登录的D sg4胞外区域EC1、EC2、EC3和EC4核酸序列完全一致,4个D sg4胞外区域cDNA读码框架正确;D sg4 EC1、EC2、EC3和EC4与患者血清反应,而不与正常对照组血清反应。结论D sg4胞外区域多肽片断在PV发病中可能有作用。Objective To amplify the nueleotide sequences of desmoglein 4 （Dsg4） extracellular domains （ECI-4） from human skin tissue, and then to investigate their roles in pemphigus vulgans （PV） pathogenesis. Methods RNA was obtained from normal human skin tissue and then eDNA was synthesized by RT-PCR. The target gene fragments of desmoglein 4 extraeellular domains （EC1-4） were amplified by PCR. With the technique of gene recombination, these target gene fragments were inserted into pET32a plasmids respectively by T4 DNA ligase, which formed the recombinant plasmids used to transform the E. coli DHSa competent germs. Screening of transformant germs was done by LB medium with Ampieillin. The DNA sequences of positive reeombinants were then identified. The epitopes of four recombinant proteins of Dsg4 in PV patients were analyzed by ELISA. Results Four DNA bands with all the length of 350 bp were obtained by RT-PCR. Consequently four expression plasmids of desmoglein 4 extracellular domains were constructed, of which the nucleotide sequences and open reading frames were proved to be correct. It showed that the recombinant proteins of Dsg4 domains EC1, EC2, EC3 and EC4 reacted to PV patients＇ sera, but not to normal sera. Conclusion The above data indicate that the epitopes of Dsg4 may play a role in the pathogenesis of PV.

关 键 词：桥粒芯糖蛋白4 天疱疮 克隆 融合蛋白 免疫识别 

分 类 号：R758.66[医药卫生—皮肤病学与性病学]