人酪氨酸酶抗原表位肽的表达、纯化及在白癜风患者中的抗原性检测

Expression and purification of epitope peptide of human tyrosinase and its antigenicity in vitiligo patients

目的表达人酪氨酸酶抗原表位肽,探讨其在白癜风患者自身抗体检测中的应用。方法目的基因合成后克隆至原核表达载体 pGEX-4T-2,转化大肠杆菌 BL21菌株,异丙基-β-D-硫代半乳糖苷(IPTG)诱导蛋白表达,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析和 Western 印迹鉴定目的蛋白表达,以此蛋白为抗原,检测100例进展期白癜风患者和30名正常人血清 IgG 抗体情况。结果成功构建重组表达载体,重组蛋白成功表达。应用凝胶分析系统分析蛋白表达量发现,目的蛋白的表达量占菌体总蛋白的70%。融合蛋白经过谷胱苷肽 S-转移酶试剂盒纯化后,其蛋白纯度达90%以上。100例白癜风患者中64例血清与目的蛋白结合反应呈阳性,30名正常人血清结合反应均呈阴性。结论成功表达了人酪氨酸酶抗原表位肽,其在白癜风患者血清中具有抗原性。

Objective To express the epitope peptide of human tyrosinase （TYR）, and discuss the application of the peptide in detecting autoantibody of the vitiligo patients. Methods The epitope areas 240-255,289-294,295-300,435-447, and 461-479 of human TYR were synthesized and connected to the vector pGEM-T. The target gene was cloned to the prokaryotic expression vector pGEX-4T-2, which was then transferred to Escherichia coli BL21 host cells. Isopropy-β-D-thiogalactoside （IPTG） was used to induce the protein expression that was examined with SDS-PAGE and Western blotting. Indirect ELISA was conducted to detect the antigenicity of the peptide in 100 blood specimens of active vitiligo patients and 30 healthy controls. Results The recombinant expression vector was constructed successfully. The SDS-PAGE and Western blotting results showed expression of the recombinant protein in E. coli. The amount of the recombinant protein reached about 70% of the total mass of bacterial protein with PAGE analysis system. With the glutathione S-transferase （ GST ） purification kit, the purity of recombinant protein reached over 90%. Indirect ELISA showed that reaction with the target protein was negative in all the 30 healthy controls and was positive in 64 of the 100 active vitiligo patients. Conclusion The epitope peptide of human TRY is expressed successfully, and it has antigenicity in the serum of vitiligo patients.