白喉毒素-白细胞介素2重组嵌合毒素的克隆表达及特异性细胞毒作用的研究

应用PCR技术分别扩增出编码白喉毒素氨基端 389个氨基酸 (DT3 89)的基因片段及人IL 2全基因 ,将两基因串连插入 pET3a载体 ,构建成含有DT3 89 IL 2融合基因的表达载体 ,转化大肠杆菌BL2 1,经表达、纯化后 ,用3 H Leucine掺入法测定其对HUT 10 2细胞的蛋白合成抑制作用。SDS PAGE电泳分析表明 ,表达产物分子质量 (Mr)约为 5 8kD ;重组嵌合毒素能够特异性地抑制高表达IL 2受体的HUT 10 2细胞的蛋白生物合成 ,且有一定的剂量反应关系 ,其细胞半数抑制浓度 (IC50 )约为 3 3× 10 -11mol/L。为进一步研制特异性的抗IL 2受体高表达肿瘤和相关疾病的药物打下了基础。

Cloning and Expression of Recombinant Chimeric Toxin DT389-IL-2 and Testing of Its Specific Cytotoxicity Toward Cells Which Bearing High Affinity IL-2 Receptor

The fragments of truncated diphtheria toxin（containing 389 aminoacids of N-terminus，DT389）and full length human IL-2 gene were amplified by PCR technique respectively. The two fragments were linked together and then inserted into prokaryotic expression vector pET3a.The fusion gene encoding the recombinant chimeric toxin was transformed into E.Coli BL21 strain and the expressed fusion protein was purified to test the cytotoxicity to HUT 102 cells line by3H-Leucine incorporation. SDS-PAGE analysis showed that the molecular weight of the recombinant chimeric toxin is about 58 kD. The purified recombinant chimeric toxin was found to inhibitory the protein synthesis of HUT 102 cells which bearing high affinity IL-2 receptors effectively，and resulted in dose-responsive relationship with 50％ inhibitory concentration（IC50）of 3.3×10-11mol/L.These results lay a foundation for preparing the therapeutic agent toward tumors highly expressing IL-2 receptors and its related diseases.