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目的建立尘螨变应原Der f 2编码基因重组pCold TF体系并诱导表达。方法以质粒pMD19-T-Der f 2为模板扩增目的基因Der f 2,克隆至pCold TF DNA载体,转化BL21细菌,用IPTG诱导表达,用SDS-PAGE和West-ern blot鉴定表达产物。结果 PCR扩增获得Der f 2编码基因,成功构建表达质粒pCold TF-Der f 2。SDS-PAGE检测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,表达产物分子质量单位为69ku,Western blot检测该蛋白能被鼠抗Penta-His抗体识别。结论成功建立尘螨变应原Der f 2编码基因重组pCold TF体系,并实现其可溶性原核表达。
OBJECTIVE: To establish and induce the expression of Der f 2 gene encoding recombinant plasmid pCold TF. Methods The target gene Der f 2 was amplified by using the plasmid pMD19-T-Der f 2 as a template and cloned into the pCold TF DNA vector. The recombinant plasmid was transformed into BL21 and induced with IPTG. The expressed products were identified by SDS-PAGE and Western-blot. Results Der f 2 gene was amplified by PCR and the expression plasmid pCold TF-Der f 2 was successfully constructed. SDS-PAGE analysis showed that the plasmid was expressed normally in Escherichia coli and was soluble in E.coli. The molecular weight of the expressed product was 69ku. Western blot showed that the protein was recognized by mouse anti-Penta-His antibody. Conclusion The recombinant plasmid Der f 2 encoding pCold TF allergen has been successfully constructed and its soluble prokaryotic expression has been achieved.