人可溶性TNF受体Ⅱ克隆、原核表达与活性鉴定

Cloning and Expression of Human Soluble Tumor Necrosis Factor Receptor Ⅱ by E.coli and Appraisement of Its Activity

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。

Objective To construct and express the recombinant of human soluble tumor necrosis factor receptor Ⅱ （sTNFRⅡ ） by E. coli. Methods The extraeellular domain cDNA of human TNFRⅡ was obtained by RT-PCR from human mono-ytes. 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 sTNFR Ⅱ could effectively block the cytotoxicity mediated by sTNF-α on 1.929. And direct immune fluorescence implied that sTNFR Ⅱ could inhibit the binding of GFP sTNF-α to TNFR on 1.929. Conclusion Using the genetic engineering technology, the recombinant human soluble TNFRⅡ was obtained.