膜稳定型TNF突变体的构建、表达及活性研究

Construction of a human stable transmembrane mutant TNF-α recombinant and its eukaryotic expression

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

Objective: TNF α exists as both transmembrane form(M TNF)and secretory form(S TNF).A kind of M TNF recombinant with a stable,uncleavable mutant type(mTNFm)was constructed for its gene therapy.Methods: On the basis of cloning the M TNF cDNA encoding region,the authors constructed mTNFm,which could potentially express on the cell surface with a uncleavable type of TNF resulting in deletion of the sequence encoding enzymatic cleavage site of TNF α converting enzyme.The cell clones expressed mTNFm were selected and identified,and their cytotoxic effect analyzed subsequently. Results:mTNFm could express as a stable transmembrane type and act chiefly through cell to cell contact.In addition, in vivo experiments on mice demonstrated that both the gene mutant and its expression products were persistently present in the local injection sites by direct gene injection.Conclusion: This table M Tnf gene mutant clone can be suited for the research of bioactivity and gene therapy of M TNF.