人 TNF-α 分泌型、膜型和膜稳定型重组体的构建、表达及杀瘤效应研究

Construction, Expression and in vitro Tumor killing Effect of Secretory, Transmembrane and Stable Transmembrane Mutant h TNF α Recombinants (received July 17, 1998)

通过用ＩＬ－２信号肽编码序列置换ＴＮＦ前导肽编码序列，构建出一种在真核细胞中仅产生分泌型ＴＮＦ的重组体；通过缺失二型ＴＮＦ转换的酶解部位而构建出跨膜表达的膜稳定型ＴＮＦ重组体，并建立分别只表达分泌型或膜型及可同时表达两型ＴＮＦ的真核细胞株，比较二型ＴＮＦ的杀瘤效应。结果显示，膜型ＴＮＦ主要经效－靶细胞间直接接触发挥胞毒效应，杀瘤谱比分泌型ＴＮＦ广；ＴＮＦ的分泌型和膜型具有协同效应。通过基因直接注射方式，使膜型及膜稳定型ＴＮＦ重组体稳定表达小于鼠体内，为膜型ＴＮＦ转基因应用奠定了基础。

TNF α is a kind of type II transmembrane proteins and exists as both transmembrane form (M TNF) and secretory form (S TNF). In order to study the bioactivities of these two TNF forms, two mutant forms of the wild type TNF cDNA are constructed. One was generated by substituting the nucleotide sequence of human IL 2 signal peptide for the leader sequence of TNF, which could encode an only secretory form of TNF. The other was constructed by deleting the enzymatic cleavage site of TNF, therefore, it could be present on the cell surface as an uncleavable type of TNF. These modified TNF genes expressed by cell genes were selected and identified, and their cytotoxic effects were analyzed and compared, successively. The results suggest that M TNF act chiefly through cell to cell contact, and has a broader oncolytic spectrum than S TNF. Another finding is that the two forms of TNF kill tumor cells synergistically in vitro. In addition, in the in vivo experiments on mice, the genes and expression products of M TNF and its uncleavable mutant form of recombinants were persistently present for 30 days in the local injection sites by direct gene injection.