重组肿瘤坏死因子-α衍生物的制备及聚乙二醇修饰

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纯的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。此项工作通过应用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。

Preparation and pegylation of TNF-α derivative

The gene of mutaed 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,leu 29ser,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 relactive ability was 8 ×107 .TNF-αD4 was modificated by mPEG-ButyrALD。After purification， the purity of mPEG-TNF-αD4 was above 85% and relactive ability was 8.6 ×107. and the in vivo systemic toxicity of mPEG-TNF-αD4, which is indicated by LD50, is lower than that of rhTNF-a .These results strongly supported for the further study and exploitation of TNF-antitumor drug.