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目的建立粉尘螨变应原第1组分全长基因原核表达质粒pColdTFDer f 1。方法以质粒pET-28a(+)Der f 1为模板扩增目的基因Der f 1,克隆至pColdTF DNA载体,转化大肠埃希菌BL21,IPTG诱导表达并用SDSPAGE(十二烷基磺酸钠-聚丙烯酰胺凝胶电泳)和蛋白印迹法(Western blotting,WB)验证产物。结果 PCR扩增获得Der f 1编码全长基因,成功构建表达质粒pColdTFDer f 1,SDSPAGE和WB验证表明该质粒在大肠埃希菌中正常表达,且基本为可溶性表达。结论成功建立尘螨变应原原核表达质粒pColdTFDer f 1,并成功实现其原核表达,为进一步生产基因工程变应原提供基础依据。
Objective To establish the prokaryotic expression plasmid pColdTFDer f 1 of the first full-length gene of dust mite allergen. Methods The target gene Der f 1 was amplified by using the plasmid pET-28a (+) Der f 1 as a template and cloned into the pColdTF DNA vector. The recombinant plasmid was transformed into E. coli BL21 and induced with IPTG. SDFAGE (sodium dodecyl sulfate- Acrylamide gel electrophoresis) and Western blotting (WB). Results The full-length gene encoding Der f 1 was obtained by PCR. The expression plasmid pColdTFDer f 1 was constructed successfully. SDSPAGE and WB showed that the plasmid was expressed normally in Escherichia coli and was almost soluble. Conclusion The prokaryotic expression plasmid pColdTFDer f 1 of dust mite allergen has been successfully established and its prokaryotic expression has been successfully achieved, providing the basis for the further production of genetically engineered allergens.