新型重组毒素hIL15M/TNFαM的构建、表达与活性测定

Construction expression and activity evaluation of a novel fusion toxin hIL15M/TNFαM

目的 构建、表达hIL15M TNFαM融合毒素 ,为研制可消除hIL15受体高表达细胞的特异性导向药物奠定基础。方法 人白介素 15突变体 (hIL15M)对靶细胞不具有激动作用 ,将该突变体与TNFα突变体 (TNFαM)编码基因直接融合克隆至pET表达载体 ,并纯化出融合蛋白hIL15M TNFαM ,MTT法测定该蛋白的细胞毒活性。结果 SDS PAGE分析表明 ,重组菌株诱导后可以表达出 3 0× 10 3与预期大小一致的特异蛋白带。纯化的hIL15M TNFαM对PHA激活的T细胞的杀伤作用是静止性T细胞的 13倍。结论 hIL15M TNFαM融合蛋白对高表达hIL15受体的细胞具有较强的杀伤性作用 ,提示其对由活化的异常T细胞增多所致的疾病具有潜在的治疗作用。

Objective To construct and express fusion toxin human IL15 mutant/TNFα mutant (hIL15M/TNFαM) in order to develop a targeted toxin capable of eliminating abnormal cells expressing high levels of hIL15 receptors. Methods DNA fragments of truncated hIL15M and TNFαM coding gene were joined in frame and cloned into T7 expression vector pET16b. The expressed fusion protein was purified from E. coli . Cytotoxicity of hIL15M/TNFαM was measured by MTT assay. Results E. coli transformed with the recombinant plasmid expressed an extra band with the expected size of 30×10 3. MTT assay indicated that PHA activated T cells were 13 times more sensitive to the cytotoxic effect of hIL15M/TNFαM than to the resting T cells. Conclusion hIL15M/TNFαM has high toxic effect against cells expressing high levels of IL15 receptors, suggesting that hIL15M/TNFαM has potential therapeutic effect on T cell leukemia and other diseases related with improperly activated T cells.