sTNFR1在昆虫细胞中的表达及鉴定  

Expression and identification of human soluble TNFR1 in baculovirus expression system

作　　者：高波[1] 张杰[1] 刘云[2] 王会信[1] 薛沿宁[1] 

机构地区：[1]军事医学科学院基础医学研究所,北京100850 [2]军事医学科学院放射医学研究所,北京100850

出　　处：《军事医学科学院院刊》2002年第4期250-253,共4页Bulletin of the Academy of Military Medical Sciences

基　　金：国家自然科学基金资助课题 (3 990 0 182 )

摘　　要：目的 :研究可溶性肿瘤坏死因子受体 (solubleTNFαreceptor1,sTNFR1)在昆虫细胞中的表达。方法 :应用BAC_TO_BAC杆状病毒表达系统 ,构建含有sTNFR1基因的杆状病毒穿梭载体Bacmid ,转染sf2 1昆虫细胞进行高效表达 ,利用TALON金属鏊合层析进行重组蛋白的纯化。免疫印迹和细胞测活方法鉴定重组sTNFR1的生物学活性。结果 :从无血清培养上清中可纯化得到约 3mg L的重组蛋白。免疫印迹反应和细胞活性实验表明 ,重组蛋白具有良好的抗原结合能力和抑制TNF对L92 9细胞的细胞毒作用。结论 :重组sTNFR1在昆虫细胞中表达稳定 ,易于纯化 。Objective: To study the expression of soluble TNF receptor1 (sTNFR1) in insect cells.Methods:Using BAC_TO_BAC baculovirus expression system, human soluble TNFR1 gene inserted into pF AST B ACS CD14 vector and the recombinant plasmids were transfered into DH10BAC competent cells for transposition into the Bacmid. The recombinant Bacmid DNAs were transfected into sf21 insect cells to attain effective expression and the recombinant protein could be purified by using TALON metal affinity resin. The biological activity of recombinant sTNFR1 was tested by Western blot and cellular assay.Results:The yield of purified recombinant proteins was about 3mg per liter cultural supernatant. Western blot showed that the purified proteins can bind to both anti 6×His McAb and anti_human sTNFR1 polyclonal antibody. The purified sTNFR1 can neutralize TNF cytotoxicity in a dose_dependent manner assayed on mouse L929 cells.Conclusions:sTNFR1 Protein, which would be easy to be purified, was capable of being expressed and appropriately processed in insect cells. The present results suggest that the recombinant sTNFR1 possesses good biological activity.

关 键 词：肿瘤坏死因子受体1 杆状病毒 昆虫细胞 炎症反应 生物学活性 

分 类 号：R346[医药卫生—基础医学]