人TNFβ缺失体融合表达及其产物一步纯化法研究  

Study on Construction of His6-Human TNFβ Fussion Expression Plasmid and Single-Step Purification of Its Product

作　　者：徐建芬[1] 周晴[1] 马志章[1] 余建法[2] 华明[1] 丁仁瑞 

机构地区：[1]浙江大学生命科学学院免疫工程实验室,杭州310012 [2]浙江中医学院,杭州310013

出　　处：《药物生物技术》2000年第1期1-5,共5页Pharmaceutical Biotechnology

基　　金：国家自然科学基金!79670 815;浙江省自然科学基金!3 970 5 0

摘　　要：本文将hTNFβN端缺失 2 3aa的缺失体基因克隆于 pET 2 8 C(+ ) 表达载体 ,构建成T7lac启动子控制下His6 TNFβ融合表达质粒 ,转化到E coliBL2 1(DE3) ,经IPTG诱导表达 ,His6 TNFβ表达量约占总菌体蛋白的 2 5 % ,Mr2 0 5 0 0。表达产物大部分以包涵体形式存在。包涵体经过洗涤 ,70mol/L脲变性溶解 ,用Ni2 + 亲和层析一步快速纯化 ,所得His6 TNFβ的纯度达 90 %以上 ,回收率在 90 %左右。纯化产物稀释复性后 ,其细胞毒活性为 (0 5～ 1 0 )× 10 6U/mg蛋白左右。这些研究结果将为制备重组人TNFβ缺失体以及进一步研究其生物学功能奠定了基础。In this research, a deletion of human TNFβ which lacking 23 N termial amino acid residues was cloned into over expression vector pET 28 C (+) , a T 7lac promoter based fusion plasmid was constructed. Then the recombinant plasmid was transformed into E.coli BL21(DE3).The protein(His6 TNFβ)expressed by induction of IPTG has a molecular weight 20 500KD by SDS PAGE. The expressed protein band constructed 25% of total bacteria protein. The product induced was mainly as in clusion bodies(IBs). The His6 TNFβ purified by Ni 2+ Sepharose 6B column under the denaturation after IBs were washed with 2mol/L urea buffer and dissolved in 7mol/L urea solution. The result of purification showed that the product purity was more than 90% and the recovery rate was about 90%. The purified His6 TNFβ fusion protein was slowing diluted with the rnaturation buffer and dialyzed against 0.02mol/L PB buffer. The cytotoxic activity of the His6 TNFβ was about(0.5～1.0)×10 6U/mg.p. These results lay the foundation of production and further research of the hTNFβ deletion.

关 键 词：肿瘤环死因子 一步 经法 缺失体 TNFΒ 

分 类 号：R977.6[医药卫生—药品]