【摘要】 目的构建人可溶性肿瘤坏死因子受体Ⅱ(sTNFRⅡ)原核基因表达载体,并在大肠埃希菌中高效表达之。方法采用RT-PCR技术,从人外周血的单核细胞中扩增出肿瘤坏死因子受体Ⅱ(TNFRⅡ)基因的胞外区,将其克隆入pET28a(+)高效表达载体进行诱导表达,并对表达产物进行纯化及鉴定。结果在异丙基硫代-β-D-半乳糖苷(IPTG)诱导下,转入外源基因的大肠埃希菌BL21(DE3)可高效表达sTNFRⅡ蛋白,SDS-PAGE显示在32kD处有一特异表达条带,其表达量占菌体蛋白总量的30%左右。纯化的sTNFRⅡ经Western blot鉴定具有生物学活性;生物活性实验(MTT法)显示它可有效地封闭分泌性肿瘤坏死因子α(sTNF-α)对L929细胞的胞毒效应;直接免疫荧光显示它可以特异性抑制GFP-sTNF-α与靶细胞肿瘤坏死因子受体的结合。结论通过基因工程技术获得了人sTNFRⅡ的重组蛋白。更多还原

【Abstract】 Objective To construct and express the recombinant of human soluble tumor necrosis factor receptor Ⅱ(sTNFRⅡ) by E.coli.Methods The extracellular domain cDNA of human TNFRⅡ was obtained by RT-PCR from human monocytes.The gene fragment was inserted into pET28a(+) and expression was induced by IPTG in E.coli.The expression product was isolated and purified,furthermore its bioactivity was estimated.Results The highest expression was achieved after induction for 6 h with IPTG.SDS-PAGE showed an extra protein band which was around 32 kD in size,which occupied 30% of the total protein in E.coli.The bioactivity of sTNFRⅡ was identified by Western blot.Bioactivity assay revealed that sTNFRⅡ could effectively block the cytotoxicity mediated by sTNF-α on L929.And direct immune fluorescence implied that sTNFRⅡ could inhibit the binding of GFP-sTNF-α to TNFR on L929.Conclusion Using the genetic engineering technology,the recombinant human soluble TNFRⅡ was obtained.更多还原