【摘要】 构建有利于人肿瘤坏死因子α(TNF-α)大量表达及有效纯化的原核表达载体,并在大肠杆菌中诱导表达。通过密码子优化构建表达质粒pET28-TNF,并将其转化入大肠杆菌,对表达条件进行优化后进行大量表达并通过两轮镍柱亲和纯化获得高纯度重组人TNF-α(rhTNF-α)蛋白。应用Western blot和细胞测活分别检测重组人TNF-α蛋白的抗原性和细胞毒活性。成功构建了重组人TNF-α原核表达载体,并通过表达纯化获得了纯度>95%,具有生物活性(LD50<10 ng/mL)的rhTNF-α蛋白。更多还原

【Abstract】 Tumor necrosis factor α(TNF-α) is a cytokine that plays a major role in several chronic inflammatory disorders.Anti-TNF-α therapy has been widely employed in clinic.However,the source of natural human TNF-α is so limited that it′s difficult to meet the research needs.The purpose of this project is to construct an effective prokaryotic expression system for recombinant human TNF α(rhTNF-α).The sequence of hTNF-α is retrieved from Entrez Gene at NCBI.The whole gene of hTNF-α is optimized,synthezied by a company,inserted into the expression vector pET-28a(+) and expressed in E.coli BL21(DE3).The N-terminus of hTNF-α contains a His-tag and a TEV protease site.The 10×His-tag is used to purify the protein by Ni+-NTA,and the TEV protease site is used to remove the His-tag.The soluble expression conditions of hTNF-α were optimized by orthogonal array design.The large-scale fermentation was carried out under optimized conditions.The expressed rhTNF-α protein was purified by Ni+ affinity chromatography.Then,the 10×His tag was removed by TEV protease treatment and followed though Ni+ column again.The purification effect was identified by SDS-PAGE and western blot.The biological activity of rhTNF-α was evaluated by MTT method with L929 cells as target cells.The recombinant cloning and expressing plasmid of rhTNF-α was successfully constructed.The outflow from the Ni column was the purified rhTNF-α,showing single polypeptide band by SDS-PAGE.The productivity of purified rhTNF-α is about 4.17 mg/L.L929 cell cytotoxicity assay showed IC50<10 ng/mL.An effective prokaryotic expression system for rhTNF α was successfully constructed in our study.The application of two-step Ni+ affinity chromatography purification method could produce soluble rhTNF-α protein with high purity and relative bioactivity.更多还原