人肿瘤坏死因子α组合突变体生物学特性的研究  

Studies on biological properties of hTNFα multi-site mutants

作　　者：卢芳[1] 刘惠[1] 罗锐[1] 陈常庆[1] 

机构地区：[1]中国科学院上海生物工程研究中心,上海市漕宝路500号上海200233

出　　处：《细胞与分子免疫学杂志》2001年第1期5-7,15,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家"863"高技术计划资助!No.103-13-01-04

摘　　要：目的研究ｈＴＮＦα的结构与功能。方法比较了野生型 ｈＴＮＦα与其单突变体 Ｒ２Ｋ－、 Ｎ３０Ｓ－、 Ｒ３２Ｗ－和Ｌ１５７Ｆ－ｈＴＮＦα及两种组合突变体Ｒ３２Ｗ－Ｌ１５７Ｆ－ｈＴＮＦα和Ｒ２Ｋ－Ｎ３０Ｓ－Ｒ３２Ｗ－Ｌ１５７Ｆ－ｈＴＮＦα的细胞毒活性、受体结合能力，以及动物体内毒性。结果发现两种组合突变体基本保持了与野生型相当的对人肿瘤细胞的细胞毒活性，但对小鼠Ｌ９２９细胞的活性明显下降；两种组合突变体与ｈＴＲ７５的结合能力的下降程度要大于 ｈＴＲ５５ ；小鼠体内毒性测定实验表明，两种组合突变体的毒性显著降低，其中双突变体的ＬＤ５０为野生型的３００倍左右，而四突变体对小鼠的体内毒性比野生型下降了７００倍以上；四突变体对恒河猴的体内毒性也比野生型ｈＴＮＦα明显降低。结论获得具有潜在应用价值的ＴＮＦ－α四突变体。Aim To study the structure-function relationship of hTNFα.Methods We compared the cytototicity, receptor binding ability and toxicity in animal body of wild type(wt) hTNFα with its mutants including R2K-, N30S-, R32W-, L157F- hTNFα, and two multi-site mutants (R32W- L157F-hTNFα and R2K-N30S-R32W-L157F-hTNFα). Results We found that the two multi-site mutants remained similar cytotoxicity to several human tumor cell lines as wild type hTNFα. However, their cytotoxicity to L929 cells were decreased sharply as compared with those of wt hTNFα. The two multi-site hTNFα mutants had lower binding activity with hTR75 than hTR55. We also found that compared with the wild type, the LD50 of the mutant R32WL157F-hTNFα was decreased about 300 fold and the dose of mutant R2K-N30S-R32W-L157F-hTNFα resulted in 30 % death was 700 folds lower than LD50 of wt hTNFα. To certify the systematic toxicity of the mutant R2K-N30S-R32W-L157F-hTNFα, we assayed its toxicity to monkeys and found that its systematic toxicity was lower than that of wt hTNFα. Conclusion A 4-site mutants (R2k-N30S-R30W-L157F-hTNFα ) of hTNFα is obtained, which the mutant may possess potential application value in clinical therapy.

关 键 词：肿瘤坏死因子Α 组织突变体 生物学特性 细胞毒活性 

分 类 号：R392.12[医药卫生—免疫学]