肿瘤坏死因子第59位酪氨酸对其细胞毒活性的意义

肿瘤坏死因子(Tumor Necrosis Factor,TNF)的基因克隆表达成功后,已进行了广泛的实验室研究,并进入Ⅰ期及Ⅱ期临床试验。但关于肿瘤坏死因子结构与功能关系人们尚所知甚少,阻碍了对肿瘤坏死因子各种生物活性的机制研究,也影响了肿瘤坏死因子的临床应用。本研究为肿瘤坏死因子结构与功能关系研究的一部分。肿瘤坏死因子不仅有广泛的生物学活性,也可引起严重的毒副作用。本文用寡核苷酸诱导的体外基因定点突变法,选择位于肿瘤坏死因子分子中央保守区第59位酪氨酸,将其改变为门冬氨酸。所选用的琥珀突变选择性突变系统,造成肿瘤坏死因子基因突变,获得较高的突变效率(约80％)。DNA序列分析证实第59位酪氨酸密码子TAC突变为门冬氨酸密码子GAT。将突变体肿瘤坏死因子基因于同一原核表达载体进行表达,结果表明,突变后不影响表达量及表达产物分子量,但其细胞毒比活性下降724倍,表明Tyr59对于维持肿瘤坏死因子细胞毒活性非常重要。

The Importance of Tyr59 to the Cytotoxicity of Tumor Necrosis Factor

Tumor necrosis factor, after being produced with bioengineering method, is now being widely studied and used for stage I and stage II clinical trial.The structure-function relations of TNF is not quite clear which limited the research on the mechanisms of various biological activities of TNF as well as its efficiency of clinical use.The present paper is one of the structure-function research series on TNF, in which a conserved amino acid residue Tyr59 was substituted by Asp59 using site-specific mutagenesis.The molecular weight and expression level of this mutant protein did not changed largely, but its specific activity decreased 724 times.This result showes that the Tyr59 residue or this region where Tyr59 located is critical for the cytotoxicity of tumor necrosis factor.