人TNF-α点突变蛋白的制备及免疫原性分析  

Preparation and Research for Immunogenicity of Point Mutation Protein of Human Tumor Necrosis Factor alpha

作　　者：曾盼[1] 张丽[1] 邓立[1] 李荣秀[1] 

机构地区：[1]上海交通大学生命科学技术学院微生物代谢国家重点实验室,上海200240

出　　处：《基因组学与应用生物学》2016年第4期763-767,共5页Genomics and Applied Biology

基　　金：国家科技重大专项(2014ZX09101043-002)基金资助

摘　　要：肿瘤坏死因子-α(tumor necrosis factor alpha,TNF-α)在人体内的毒副作用可以通过定点突变的方式得以改善。本研究结合公开文献及空间结构展示软件(discovery studio 3.5)对人TNF-α进行了的点突变实验。运用重叠PCR方法成功引入S95F、Y115F、Y119W和S147Y四个单点突变。经原核表达及镍柱亲和层析纯化后得到人TNF-α及其点突变蛋白。通过测定人TNF-α及其点突变蛋白的生物活性发现三种点突变(S95F,Y115F,Y119W)都显著降低人TNF-α对L929细胞的杀伤率。小鼠免疫实验证明4个突变蛋白均可刺激机体产生针对人TNF-α的抗体效应。Tumor necrosis factor alpha（TNF-α）’s toxic and side effects on human body could be reduced by means of site-directed point mutation. In this study, we combined open literature and space structure display software（Discovery Studio 3.5） to implement the experiment of point mutation protein of TNF-α. We introduced four single point mutation（S95F, Y115 F, Y119 W, S147Y） by overlap PCR successfully. After prokaryotic expression and purification by Ni affinity chromatography human TNF-α and its mutant proteins were obtained. Then we detected bioactivity of human TNF-α and its mutant proteins, through which we found that three mutant proteins（S95F,Y115 F, Y119W） significantly reduced L929 cell killing rate that induced by human TNF-α. The mice immunity experiment showed that all the four mutant proteins could elicit the immune response against human TNF-α.

关 键 词：TNF-Α 点突变 L929细胞 免疫原性 

分 类 号：R392[医药卫生—免疫学]