中华皮肤科杂志 ›› 2007, Vol. 40 ›› Issue (3): 139-142.

• 论著 • 上一篇    下一篇

人酪氨酸酶抗原表位肽的表达、纯化及生物学活性分析

关翠萍, 李永伟, 尉晓冬, 张迪敏, 许爱娥   

  1. 杭州市第三人民医院皮肤科, 310009
  • 收稿日期:2006-04-05 出版日期:2007-03-15 发布日期:2007-03-15
  • 通讯作者: 许爱娥,email:xuaiehz@msn.com E-mail:xuaiehz@msn.com
  • 基金资助:
    浙江省科技厅科技计划般基金资助项目(2005C33017)

Expression, purification and biological activity analysis of the epitope peptide of human tyrosinase antigen

GUAN Cui-ping, LI Yong-wei, WEI Xiao-dong, ZHANG Di-min, XU Ai-e   

  1. Department of Dermatology, Third People's Hospital of Hangzhou, Hangzhou 310009, China
  • Received:2006-04-05 Online:2007-03-15 Published:2007-03-15

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摘要： 目的 探讨人酪氨酸酶抗原表位肽在白癜风患者自身抗体检测中的表达.方法 目的基因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SDS-PAGE分析和蛋白质印迹鉴定目的蛋白表达,ELISA检测其生物学活性.结果 成功构建重组表达载体,SDS-PAGE和蛋白质印迹结果表明,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90%.融合蛋白经过试剂盒纯化后,其蛋白纯度达95%以上.ELISA检测纯化蛋白的生物学活性,结果表明,纯化蛋白具有结合白癜风患者IgG的能力.结论 成功表达了人酪氨酸酶抗原表位肽,其表达蛋白具有生物学活性.

关键词: 肿瘤坏死因子α, 核因子- kappa B, p38丝裂原活化蛋白激酶, HB-13, HP-13, 白癫风, 一元酚单氧酶, 原核表达

Abstract: Objective To study the expression, purification and biological activity of the epitopc peptide of human tyrosinase antigen.Methods The target gene, encoding the 240-300 epitope regions of human tyrosinase, was cloned to the prokaryotic expression-veckor pGEX-4T-2, which was then transfected into E.coli BL21.The protein expression was induced by IPTG(isopropyl-(3-D-thiogalactoside)and examined with SDS-PAGE and Western blotting.The biological activity of the target protein was detected with ELISA.Results The recombinant expression vector was constructed successfully.SDS-PAGE and Western blotting verified the successful expression ofrecombinant protein Quantification by gel analysis system revealed that the recombinant protein amounted to 90% of the total protein content of the bacterial cells.With the application of glutathione S-transferase(GST)purification kit, the purity of the recombinant protein reached over 95%.The purified protein was confirmed by ELISA analysis to he able to bind to IgG from vitiligo patients.Conclusion Our results suggest that the epitope peptide of human tyrosinase antigen is expressed successfully, with biological activity confiimed.

Key words: Vitiligo, Monophenol monooxygenase, Prokaryotic expression