【摘要】 构建结核分枝杆菌cfp10-esat6融合基因及其原核表达载体,在大肠杆菌中表达融合蛋白CFP10-ESAT6。用基因拼接(GeneSOEing)法扩增cfp10-esat6融合基因,并将其定向克隆至原核表达载体pGEX-4T-1,构建原核表达重组质粒pGcfp10-esat6。重组子经限制性内切酶分析、聚合酶链式反应及测序鉴定后转化宿主菌大肠杆菌BL21,IPTG(isopropy-β-D-thiogalactoside,异丙基硫代-β-D半乳糖苷)诱导表达约42kDa带谷胱苷肽硫转移酶(Glutathione-S-TransferasesGST)蛋白标签的rCFP10-ESAT6融合蛋白,经谷胱苷肽硫转移酶融合蛋白纯化试剂盒得到纯化的融合蛋白,产物进行SDS-PAGE电泳、Western-blot鉴定。重组质粒pGcfp10-esat6中目的基因测序结果与报道序列相同;在大肠杆菌中以可溶性非包涵体形式表达;表达量约占菌体总蛋白的40%,表达蛋白纯化后获得纯度为90%左右的重组蛋白;Western印迹结果证实重组蛋白与确诊的结核病患者血清发生特异免疫反应。本研究成功构建了原核表达载体pGcfp10-esat6,获得了rCFP10-ESAT6融合蛋白,为rCFP10-ESAT6融合蛋白在结核病诊断中的应用奠定了基础。更多还原

【Abstract】 To begin with,we constructed cfp10-esat6 fusion gene and its prokaryotic expression vector and had it express in E.coli.By GeneSOEing techniques,a fusion gene was constructed by splicing cfp10 gene and esat6 gene,and then was cloned into pGEX-4T-1 plasmid.Secondly,we constructed the prokaryotic expression recombinant plasmid pGcfp10-esat6.After identification with restriction enzyme analysis,PCR and nucleotide sequencing analysis,The E.coli BL21 containing the recombinant plasmid was induced by IPTG(Isopropy-β-D-thiogalatoside).The fusion protein CFP10-ESAT6 with GST-tag about 42 kDa was expressed and purified with GST-fusion protein purification kit,The expression of cfp10-esat6 fusion gene was subsequently detected by SDS-polyacrylamine gel electrophoresis and Western-blot analysis.The sequence of cfp10 and esat6 in recombinant plasmid was consistent with that of GenBank report.The fusion protein existed in cytoplasm in soluble form and represented about 40% total bacterial protein of E.coli.The fusion protein was purified and the purity reached 90%.Its antigenicity was confirmed by Western-blotting.The prokaryotic expression vector(pGcfp10-esat6)was constructed successfully,and the fusion protein CFP10-ESAT6 was obtained.This study provided an experimental basis for potential application of the recombinant CFP10-ESAT6 in the diagnosis of tuberculosis.更多还原