应用pET32a(+)载体表达尘螨变应原Der f 3及其产物IgE反应性鉴定  

Expression of the group 3 allergen of Dermatophagoides farinaein pET32a(+) expression vector and its IgE-binding reactivity

作　　者：崔玉宝[1,2] 周鹰[2] 王楠[2] 俞黎黎[2] 卞勇华[2] 杨李[2] 张承伯 

机构地区：[1]上海浦南医院检验科,上海200125 [2]江苏盐城卫生职业技术学院,江苏盐城224006

出　　处：《中国病原生物学杂志》2014年第11期1012-1015,1019,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.NSFC31272369;NS-FC81001330;NSFC 30060166);江苏省卫生厅招标项目(No.Z200914);江苏省卫生职业技术教育研究立项课题(No.J200907);江苏省"六大人才高峰"第六批资助项目

摘　　要：目的获得粉尘螨变应原第3组分Der f 3原核表达产物并检测其与尘螨过敏性哮喘患儿血清抗体IgE反应性。方法酶切质粒pET28a(+)-Der f 3获得目的基因Der f 3,将其与pET32a载体连接成质粒pET32a(+)-Der f3,转化BL21细菌,用异丙基硫代半乳糖苷(IPTG)诱导表达,用Ni+离子亲和层析柱纯化表达产物,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)、Western blot和质谱鉴定纯化产物。以纯化产物为包被抗原,采用间接ELISA检测尘螨过敏性哮喘患儿血清抗体反应情况。结果成功构建了原核表达质粒pET32a(+)-Der f 3。将该质粒转化E.coli BL21诱导表达,亲和层析纯化后经SDS-PAGE鉴定获得目的蛋白,Western blot验证该蛋白的能够与载体的组氨酸标签结合,质谱鉴定其结构与天然Der f 3一致。以此产物为包被抗原采用间接ELISA检测尘螨过敏性哮喘患儿血清,阳性率为29.73%(11/37)。结论成功构建了原核表达质粒pET32a(+)-Der f 3,亲和纯化获得的目的蛋白具有良好的反应原性。Objectives To obtain a recombinant expressing agroup 3allergen of Dermatophagoides farinae（Der f 3）and to detect its IgE-binding reactivity. Methods The target gene Der f 3was obtained by enzyme digestion of pET28a（＋）-Der f 3with BamHⅠ plus XhoⅠ,and this gene was ligated into the vector pET32 ato construct the recombinant plasmid pET32a（＋）-Der f 3,which was then transformed into E.coli BL21 cells for expression via induction with IPTG.The expressed product was purified using nickel affinity chromatography and was then identified using SDSPAGE,Western blotting,and MALDI-TOF/TOF.Recombinant Der f 3was used as a coating antigen to detect reactivity to serum antibodies from children with asthma via indirect ELISA. Results The plasmid pET32a（＋）-Der f 3was successfully constructed and expressed in E.coli BL21.After purification with nickel affinity chromatography,the product was subjected to SDS-PAGE to identify the target protein.The recombinant protein was tagged with His-tag according to Western blotting,and its structure was consistent with native Der f 3.According to indirect ELISA testing,the recombinant fusion protein bound with sera from 29.73%（11/37）of serum samples from patients allergic to D.farinae. Conclusion The plasmid pET32a（＋）-Der f 3was successfully constructed and expressed in E.coli BL 21（DE3）.The recombinant fusion protein reacts strongly to sera from children with asthma.

关 键 词：尘螨 变应原 基因表达 蛋白质纯化 

分 类 号：R384.4[医药卫生—医学寄生虫学]