可溶性人TNFR75的表达、纯化及活性鉴定

将编码可溶性人TNFR75 (shTNFR75 )的cDNA与酵母整合载体pPICZαA重组 ,构建的重组质粒线性化后转染酵母细胞GS 115 ,获得了shTNFR75在酵母细胞中遗传性稳定表达酵母工程细胞 .甲醇诱导的pPICZαA shTNFR75 GS115重组酵母工程细胞培养上清 ,经CNBr活化的TNF Sepharose4B亲和层析柱纯化 ,纯化产物纯度为 92 % ;经Western印迹分析 ,可被TNFR75单克隆抗体特异性识别 ,分子量约为 31kD ;受体配体结合试验 ,shTNFR75纯化产物与rhTNFα和rhTNFβ的结合能力与其阳性对照基本相同 ;中和试验显示 ,该shTNFR75可完全阻断TNF对L92 9细胞的细胞毒活性 .表明酵母系统分泌表达的shTNFR75产物具有良好的结合TNF的能力 .

Expression,Purification and Activity Analysis of Soluble Human TNFR75

cDNA encoding soluble human tumor necrosis factor receptor 75(shTNFR75)was inserted into yeast integrative vector pPICZαA and transfected yeast cell GS115 by linear recombinant plasmid.The yeast engineering cells stably expressing shTNFR75 was obtained.The culture supernant of methanol induced pPICZαA| shTNFR 75/GS115 recombination yeast engineering cells was purified by CNBr|activated TNF Sepharose 4B affinity chromatography.The purity of the product was 92%,and Western|blot analysis showed that the product could be specially identified by TNFR75 monoclonal antibody,and its molecular weight was 31 kD.Receptor ligand combination test indicated that the combination ability of the purified product of TNFR75 with rhTNFα and rhTNFβ was rudimentarily similar to the combination ability of its standard product.Neutralization test showed that shTNFR75 could completely block the cytotoxicity of TNF to the L929.These results suggested that the shTNFR75 product of yeast system secretary expression had a good ability of combination with TNF.