目的 :TNF α可以分泌型 (S TNF)及其前体膜型 (M TNF)两种活性形式同时存在于体内。为膜型TNF的转基因应用 ,构建一种可表达于胞膜而不被酶解的膜稳定型TNF重组体 (mTNFm)。方法 :在克隆M TNFcDNA的基础上 ,删除两型TNF转换酶酶解部位的编码序列 ,构建出mTNFm并测序验证 ;建立只表达膜型TNF的真核细胞株。结果 :Westernblot分析及活性测定结果表明 ,mTNFm不再发生酶切转换且只通过效 靶细胞间直接接触发挥胞毒效应。通过基因直接注射方式 ,使mTNFm稳定表达于小鼠体内。结论 :所构建的mTNFm及其表达细胞株可用于膜型TNF生物学效应的研究及基因治疗 OBJECTIVE: Two active forms of TNFα secreted (S TNF) and its precursor membrane type (M TNF) exist in vivo simultaneously. For transgeneic use of membrane-bound TNF, a membrane-stable TNF recombinant (mTNFm), which can be expressed on the membrane but not enzymatically, was constructed. Methods: Based on the cloned M TNF cDNA, the coding sequence of the two types of TNF converting enzyme was deleted and mTNFm was constructed and verified by sequencing. The eukaryotic cell line expressing only membrane type TNF was established. Results: The results of Western blot analysis and activity assay showed that mTNFm did not undergo enzymatic cleavage conversion and exerted cytotoxic effect only through direct contact between target cells. Through direct gene injection, mTNFm was stably expressed in mice. Conclusion: The constructed mTNFm and its cell lines can be used for the study of membrane biological effects of TNF and gene therapy