尘螨变应原Der f 2编码基因重组pCold TF体系构建及可溶性表达

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。

Construction and solvable expression of the recombinant plasmid pCold TF-Der f 2 of Dermatophagoides farinae

Objective To construct and express the recombinant plasmid pCold TF-Der f 2 of Dermatophagoides farinae.Methods Using the plasmid pMD19-T-Der f 2 as template,the target gene Der f 2 was amplified by polymerase chain reaction.The gene fragment coding for Der f 2 was inserted into the prokaryotic expression plasmid pCold TF DNA to construct pCold TF-Der f 2 and transformed into E.coli BL21 for protein expression induced by isopropyl β-D-1-thiogalactopyranosid(IPTG).The expression product was analyzed and identified by SDS-PAGE and Western blotting.Results The gene coding for Der f 2 was obtained by PCR and the expression plasmid pCold TF-Der f 2 was successfully constructed.SDS-PAGE showed that the plasmid pCold TF-Der f 2 was successfully expressed in E.coli BL21.Most of the products are soluble and can bind with mouse anti-penta-His antibody according to Western blotting.Conclusion The recombinant plasmid pCold TF-Der f 2 of D.farinae was successfully constructed and highly expressed in E.coli,and the expressed fusion protein is soluble.