重组肿瘤坏死因子-α衍生物的制备及聚乙二醇修饰  

Preparation and Pegylation of TNF-α Derivative

作　　者：毕研伟[1] 罗娜[1] 龙海亭[1] 杨增福[1] 杨旭[1] 李健锋[1] 徐维明[1] 

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所,昆明650118

出　　处：《中国生物工程杂志》2007年第12期17-21,共5页China Biotechnology

基　　金：云南省自然科学基金资助项目(2005C0062M)

摘　　要：应用搭桥PCR技术,将肿瘤坏死因子-α基因的前4位氨基酸的编码序列删除,对hTNF-α的第8/9/10/29/31/157位氨基酸的密码子进行定点突变,将突变后的cDNA插入到pBV220载体中构建重组质粒pBV220-tnf-αD4。将重组质粒转化大肠杆菌DH5α,筛选获得了高效表达TNF-αD4突变体的工程菌,表达的重组蛋白约占菌体蛋白总量的45%左右,经硫酸铵沉淀和阳离子交换层析纯化得到纯度达90%以上的重组目的蛋白,比活性达到8×107。用单甲氧基聚乙二醇-丁醛(mPEG-ButyrALD)对TNF-αD4进行修饰,经阳离子交换层析纯化得到mPEG-TNF-αD4,纯度达85%以上,比活性达到8.6×107,系统毒性也有了明显的降低。通过应用PEG修饰肿瘤坏死因子-α,为降低其毒性,增加其活性进行了有益的尝试,为其进一步研究与开发奠定了基础。The gene of mutated TNF-αD4 gene was amplified by overlap PCR and cloned into the prokaryotic expressive vector pBV220. TNF-αD4 contains two changes： substitutions of Pro8Arg, Ser9Lys, Asp10Arg,Ile157Phe,Leu29Ser,Arg31Val and a deletion of the N terminal four amino acids. The recombinant vector pBV220-TNF-αD4 was transformated into E. coli strain DH5α, and the high expression strain was obtained by screening monoclones. The level of expression was about 45 % of total cell protein. After purification, the purity of fusion protein was above 90% by HPLC and relative ability was 8 × 10^7. TNF-αD4 was modificated by mPEG -ButyrALDo After purification, the purity of mPEG-TNF-αD4 was above 85% and relative ability was 8.6 × 10^7. The in vivo systemic toxicity of mPEG-TNF-αD4, which is indicated by LD50, is lower than that of rhTNF-α. These results strongly supported for the further study and explOitation of TNF-antitumor drug.

关 键 词：重组肿瘤坏死因子-α突变体 表达纯化 单甲氧基聚乙二醇丁醛 生物活性 系统毒性 

分 类 号：Q819[生物学—生物工程]