寻常型天疱疮抗原Dsg3基因的真核表达

目的 探讨寻常型天疱疮自身抗原Dsg3EC1-EC5在哺乳动物细胞的克隆、表达及纯化,并以重组的Dsg抗原检测寻常型天疱疮患者及正常人血清中的天疱疮抗体.方法 根据基因库中的Dsg3基因序列分析,采用RT-PCR法扩增自身抗原Dsg3EC1-EC5多肽片段的cDNA,定向插入真核表达质粒pcDNA3.1TM/myc-His(-)B中,稳定转染人宫颈癌细胞系表达重组融合蛋白,并经Ni+亲和层析柱纯化.采用蛋白质印迹法鉴定蛋白表达产物.以重组Dsg3EC1-EC5蛋白为抗原用ELISA检测40份寻常型天疱疮患者及40份正常人对照组血清.结果 成功构建真核表达重组体pcDNA3.1TM/myc-His(-)B-Dsg3.纯化的Dsg3EC1-EC5蛋白能够与寻常型天疱疮患者血清中的天疱疮抗体发生反应.ELISA方法检测寻常型天疱疮患者血清,结果显示,天疱疮抗体的阳性率为95%.结论 真核表达的Dsg3EC1-EC5蛋白与寻常型天疱疮患者血清中的抗体发生特异性反应.

Eukaryotic expression of pemphigus vulgaris autoantigen desmoglein 3 and its clinical application

Objective To explore the cloning,expression and purification of pemphigus vulgaris (PV)autoantigen desmoglein(Dsg)3,and to utilize the recombinant Dsg 3 to detect pemphigus antibodies in sera of patients with PV.Methods Specific primers were designed aiming the EC1-EC5 fragment of Dsg 3 antigen.The specific gene fragment was amplified with RT-PCR,then inserted into the eukaryotic expression plasmid pcDNA3.1^TM/myc-His(-)B.The recombinant plasmid was then employed to transfect human cervical carcinoma cell line Hela,to express the fusion protein,which was purified with Ni⁺ affinity chromatography.Western blotting method was used to confirm the reactivity of the antigen.ELISA was utilized,with the recombinant protein as antigen,to detect PV antibodies in sera of 40 patients with PV and 40 normal human controls.Results The eukaryotic expression plasmid was successfully established,and the recombinant Dsg3 antigen was successfully expressed.The purified antigen could selectively react with sera from PV patients.ELISA revealed that the positivity rate of PV antibodies was 95% in sera from the PV patients.Conclusion The recombinant autoantigen Dsg3 could specifically react with serum antibodies from PV patients.