融合蛋白DT389-hIL-13的克隆表达及其抗胶质瘤作用的初步研究

Construction and expression of the fusion protein DT389-hIL-13 and its cytotoxicity to glioma cell lines

目的 构建白喉毒素N端 389个氨基酸 (DT389)与人白细胞介素 13(hIL 13)的融合蛋白DT389 hIL 13,并检测其生物学活性。方法 通过聚合酶链反应 (PCR)扩增得到hIL 13的cDNA ,将序列正确的hIL 13及DT389的cDNA片段串联插入表达载体pET30a ,构建表达质粒pET30a/DT389 hIL 13,并对表达产物进行鉴定及初步纯化。该融合蛋白加入培养的U2 51胶质细胞中 ,应用MTS方法检测其对多型性胶质母细胞瘤细胞的杀伤作用。结果 得到序列正确的融合蛋白DT389 hIL 13的表达质粒pET30a/DT389 hIL 13,该质粒在大肠杆菌成功表达 ,经初步纯化后 ,十二烷基磺酸钠 聚丙烯酰胺 (SDS PAGE)凝胶电泳及Western印迹分析表明 ,该融合蛋白对白喉毒素多克隆抗体及hIL 13多克隆抗体都有很好的免疫反应性 ,相对分子质量约为 5 5 0 0 0 ;进一步活性检测发现 ,该融合蛋白对多型性胶质母细胞瘤细胞系U2 5 1的生长有较强的抑制作用 ,且作用存在剂量相关性 ,其半数抑制浓度(IC50 )约为 5× 10 -11mol/L。结论 成功构建了融合蛋白DT389 hIL 13的表达质粒 ,在细胞水平对表达产物进行了初步的活性检测 ,为进一步研制特异性的抗胶质瘤药物打下了基础。

ObjectiveTo construct a fusion protein toxin DT389-hIL-13 which comprises the N-terminal 389 amino acids of diphtheria toxin (DT389) and human interlukin 13 (hIL-13),and to explore its cytotoxicity on U251 glioma cells.MethodsThe cDNA of hIL-13 gene was amplified by PCR and linked with the 3′- terminus of the gene encoding the N-terminal 389 amino acids,which correspond to the enzyme domain and transmembrane domain of diphtheria toxin. The tandem constructed gene was then inserted into an E.coli expression vector pET30a. The resulted expression vector was transformed into E.coli BL21 and induced by IPTG. The expressed protein was analyzed by SDS-PAGE and Western blot analysis. U_ 251 glioma cells were cultured DT389-hIl-13 was added into the culture. The cytotoxicity was determined using colorimetric MTS proliferation assay. ResultsThe expression plasmid pET30a/DT389-hIL13 was constructed with correct sequence. The recombinant protein was successfully expressed in E.coli in manner of inclusion body and with a relative molecular weight of about 55 000,which reacted well with both anti-diphtheria toxin and anti-hIL-13 polyclonal antibody in Western blot assay. The purified recombinant chimeric toxin was found to effectively inhibit the prolifieration of glioblastoma multiforme cells bearing high affinity hIL-13 receptors,and resulted in dose-dependent relationship with 50% inhibition concentration (IC_ 50 ) of 5×10 -11 mol/L.ConclusionProkaryotic expression system can be recruited to produce recombinant chimeric toxin DT389-hIL-13. The results may lay a foundation for preparing specific the agent targets for tumors overexpressing IL-13 receptor.