人肿瘤坏死因子α组合突变体生物学特性的研究

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的结合能力的下和程度要大于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。

Studies on biological properties of hTNF

im To study the structure-function relationship of hTNFα . Methods We compared the cytotoxicity, 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 R32W-L157F-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-R32W-L157F-hTNFα )of hTNFα is obtained, which the mutant may possess potential application value in clinical therapy.