多主枝孢霉重组变应原Cla h8的制备及其免疫活性鉴定  

Production of recombinant Cladosporium herbarum allergen Cla h8 and investigation on its immunological activities

作　　者：杨柳[1] 付永锋[1] 李雪萍[1] 冯萌[1] 程训佳[1] 

机构地区：[1]复旦大学上海医学院病原生物学系,上海200032

出　　处：《中国免疫学杂志》2011年第5期446-449,共4页Chinese Journal of Immunology

基　　金：国家高技术研究发展计划项目(2007AA02Z472);卫生行业科研专项项目(20082001)资助

摘　　要：目的:通过基因工程手段,获得重组多主枝孢霉变应原蛋白Cla h8,有利于进行变应原的标准化,为标准化抗原的临床特异性诊断与治疗奠定基础。方法:从多主枝孢霉菌体中提取总RNA,采用RT-PCR的方法扩增Cla h8编码基因,将其连入pET-19b载体。转入大肠杆菌BL21 Star(DE3)pLysS,经诱导表达后,进行提纯复性,用Western blot和Dot-blot检测其免疫活性。结果:重组多主枝孢霉变应原Cla h8蛋白可以与多主枝孢霉过敏患者的血清中IgE和IgG抗体特异性结合,与天然蛋白具有相似的免疫活性。结论:制备并获得了具有生物学活性的可溶性重组多主枝孢霉变应原Cla h8蛋白,可用于多主枝孢霉变应原的标准化,克服天然提取物的非单一性及标准化难的障碍。Objective：To gain large amount of recombinant allergen Cla h8 of Cladosporium herbarum through genetic engineering method,which not only facilitates the standardization of allergen vaccination,but also is more suitable for diagnosis and treatment.Methods：The total RNA was acquired from C.herbarum culture,then the cla h8 gene fragments amplified by RT-PCR was cloned into vector pET-19b and then transformed to E.coli BL21 Star（DE3）pLysS.After induction,Cla h8 was expressed as inclusion body in E.coli BL21 Star（DE3）pLysS.The soluble protein,following purification and renaturation,was obtained.The immunological activity was identified by Dot-blot and Western blot.Results：The IgE and IgG of the serum from C.herbarum allergic patients could specially react with the recombinant Cla h8（rCla h8）,and the immunological activity of rCla h8 was comparable with the native protein.Conclusion：We have successfully obtained the recombinant Cla h8 equipped with immunological activity,which leads to significant improvements in allergy diagnosis and treatment.The approach has also resolved the problems of natural allergen extracts associated with multi-component and difficult standardization.

关 键 词：多主枝孢霉 重组变应原Clah8 蛋白表达 变应原活性 

分 类 号：R392.8[医药卫生—免疫学]