人可溶性肿瘤坏死因子受体1基因原核表达及活性鉴定

Cloning,prokaryotic expression and activity identification of human soluble tumor necrosis factor receptor 1 gene in Escherichia Coli JM109

目的:构建人可溶性肿瘤坏死因子受体1基因的原核表达质粒,诱导sTNFR1-MBP融合蛋白表达,观察表达产物的生物学活性.方法:以HeLa细胞的总RNA为模板.用RT- PCR方法扩增sTNFR1全编码区基因片段,构建含有目的片段的T载体克隆及原核表达载体pMAL-c2x重组质粒亚克隆,经异丙基-β-D半乳糖苷酶(IPTG)诱导重组质粒菌表达sTNFR1,以淀粉树脂亲和层析法纯化重组蛋白,并对重组蛋白进行序列分析和Western blot鉴定.MTT法测定重组蛋白的生物学活性的测定.结果:经DNA序列分析和Western blot鉴定,成功构建了人sTNFR1重组质粒基因工程菌;在IPTG的诱导下,重组质粒菌能表达sTNFR1- MBP融合蛋白,SDS-PAGE显示在66 kDa处有一特异性表达条带;纯化的sTNFR1-MBP融合蛋白经Western blot证实具有生物学活性;生物活性实验(MTT法)显示可有效地封闭TNF-α对OSG7701的细胞毒性作用,随着重组蛋白浓度的增高(0.2,2,20,40,80 mg/L),对TNF-α细胞毒效应的抑制作用增强,细胞死亡率依次为69.98%±1.52%,60.05%±2.18%,46.27%±2.48%,37.02%±3.17%,1.83%±0.59%,1.71%±0.61%.结论:成功获得了具有生物学活性的人sTNFR1-MBP融合蛋白,为进一步研究奠定了基础.

AIM： To construct a prokaryotic expression plasmid of human soluble tumor necrosis factor receptor 1 （sTNFR1） gene, induce the expression of sTNFRl-maltose binding protein （MBP） fusion protein and investigate the bioactivity of expression products. METHODS： The total RNA was extracted from HeLa cells and used as a template to amplify human sTNFR1 gene by reverse transcriptionpolymerase chain reaction （RT-PCR）. The PCR products were cloned into T vector and subcloned into plasmid pMAL-c2x, a prokaryotic expression plasmid. The recombinant plasmid was transferred into Escherichia Coli JM109 and induced by isopropyl-β-D-thiogalactopyranoside （IPTG） to express fusion protein sTNFR1-MBP. sTNFR1-MBP was purified by amylose resin affinity chromatography and identified by sequencing and Western blot analysis. The bioactivity of sTNFR1-MBP was estimated by MTT assay. RESULTS： A 558-bp fragment of human sTNFR1 gene had been amplified by RT-PCR and successfully cloned into vector pMAL-c2x as .recombinant vector pMAL-c2x-sTNFR1, which was confirmed by DNA sequencing, sTNFR1- MBP was produced in Escherichia Coli with pMAL-c2x-sTNFR1 after IPTG inducement, and SDS-PAGE showed an extra protein band which was 66 kDa in size. The bioactivity of sTNFR1 was identified by Western blot. MTT assay revealed that sTNFR1 effectively blocked the TNF- α-mediated cytotoxicity on QSG7701 cells. The cytotoxicity was strengthened with the increase of sTNFR1 recombinant protein concentration （0.2, 2, 20, 40, 80 mg/L）, and the rates of cell death were 69.98% ± 1.52%, 60.05% ± 2.18%, 46.27% ± 2.48%, 37.02% ± 3.17%, 1.83% ± 0.59% and 1.71% ± 0.61%. CONCLUSION： The fusion protein sTNFR1- MBP with high bioactivity is obtained successfully, which lays a foundation for further study.