热带剥爪螨变应原Blot 5的克隆表达、纯化及免疫学鉴定  

Blomia tropicalis allergen Blo t 5:cloning,expression,purification and identification

作　　者：蔡俊龙[1] 杨彬[1] 邓宜红[1] 陈实[1] 罗慕侠[1] 李勤[1] 付永锋[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》2012年第8期690-694,共5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:克隆热带剥爪螨主要变应原Blot 5基因,表达纯化该蛋白,并检测其免疫活性。方法:提取热带剥爪螨总RNA,采用RT-PCR的方法扩增Blot 5编码基因,将其连入原核表达载体pET-19b,将重组质粒转化入大肠杆菌BL21 Star(DE3)pLysS,IPTG诱导表达后,通过Ni2+亲和层析纯化重组变应原Blot 5。用Dot blot、Western blot和ELISA等方法检测重组变应原Blot 5免疫活性。结果:Dot blot和Western blot结果表明重组变应原Blot 5和热带剥爪螨粗提液都能和Blot 5鼠单克隆抗体结合。重组变应原Blot 5检测69份热带剥爪螨过敏患者血清和21份户尘螨过敏患者血清中的特异性IgE,阳性率分别为29.0%和33.3%。结论:表达和纯化了具有与天然蛋白相似的免疫活性的重组热带剥爪螨变应原Blot 5,可用于热带剥爪螨变应原的标准化,为标准化抗原的临床特异性诊断与治疗奠定基础。Objective：To clone,express and purify the gene of the major allergen Blot 5 from Blomia tropicalis and to investigate its immunological activity.Methods：The total RNA was acquired from B.tropicalis,then the Blot 5 gene fragments amplified by RT-PCR were cloned into vector pET-19b and then transformed to E.coli BL21 Star（DE3） pLysS.After inducted by IPTG,the recombinant Blot5 was purified by Ni2＋ chelating affinity chromatography.The immunological activity was identified by Dot blot,Western blot and ELISA.Results：The results of Dot blot and Western bolt showed both recombinant Blot 5 and B.tropicalis extract can specially react with monoclonal antibodies against Blot 5.The IgE antibodies from serum of 69 B.tropicalis allergic patients and 21 Dermatophagoides pteronyssinus allergic patients were tested.The result showed the positive rates were 29.0% and 33.3%,respectively.Conclusion：We have successfully obtained the recombinant allergen Blot 5 with immunological activity similar to its native counterpart,which could not only resolve the standardization of natural allergen extracts but also contribute to the design of better strategies for the diagnosis and treatment of allergic respiratory diseases.

关 键 词：热带剥爪螨 重组变应原 BLOT 5 蛋白表达 IgE 

分 类 号：R392.8[医药卫生—免疫学]