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

目的 扩增人皮肤组织桥粒芯糖蛋白4(desmoglein 4,Dsg4)胞外区域EC1、EC2、EC3和EC4的核酸序列,为寻常型天疱疮(pemphigus vulgaris, PV)发病机制的研究提供依据.方法 根据GenBank中的Dsg4序列(登录号为AY177664)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cDNA,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pET32a在T4 DNA连接酶作用下相连接,转化E.coli DH5α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ELISA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pET32a连接的目的基因经序列测定后与在GenBank登录的Dsg4胞外区域EC1、EC2、EC3和EC4核酸序列完全一致,4个Dsg4胞外区域cDNA读码框架正确;Dsg4 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 Dsg4胞外区域多肽片断在PV发病中可能有作用.

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

OBJECTIVE: To amplify the nucleotide sequences of desmoglein 4 (Dsg4) extracellular domains (EC1-4) 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 cDNA was synthesized by RT-PCR. The target gene fragments of desmoglein 4 extracellular 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 DH5alpha competent germs. Screening of transformant germs was done by LB medium with Ampicillin. The DNA sequences of positive recombinants 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.