粉尘螨变应原第1组分原核表达质粒pCold-TF-Der f 1构建及鉴定

Construction and identification of prokaryotic expression plasmid pCold-TF-Der f 1 for the dust mite allergen Der f 1

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 construct prokaryotic expression plasmid pCold-TF-Der f 1 for the dust mite allergen Der f 1. Methods The Der f 1 gene in full length was amplified from pET-28a（＋）-Der f 1 plasmid and cloned to the vector pCold-TF-DNA, with the recombinants then transferred into Escherichia coli BL21. The genetically engineered bacteria with pCold-TF-Der f 1 plasmids were induced by IPTG and the expressed products were analyzed by SDS-PAGE and Western blotting. Results The Der f 1 gene was acquired by PCR, with pCold-TF-Der f 1 successfully constructed, and expressed in E. coli. which was indicated SDS-PAGE and western blotting. Conclusion The prokaryotic expression plasmid pCold-TF-Der f 1, which contain Der f 1 gene fragment of Dermatophagoides farinae, can be successfully constructed with its expression in E. coli BL21 accomplished.