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

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.