重组可溶性TRAIL的表达及其诱导A549和H460^(wt)细胞凋亡  

Expression of rsTRAIL and its apoptoic effect on A549 and H460^(wt) cells

作　　者：刘磊[1] 方艳秋[1] 许淑芬[1] 谭岩[1] 

机构地区：[1]吉林大学第一医院中心实验室,吉林长春130021

出　　处：《吉林大学学报（医学版）》2008年第2期270-273,共4页Journal of Jilin University：Medicine Edition

基　　金：吉林省杰出青年基金资助课题(20050113);吉林省科技厅国际合作项目资助课题(20060722)

摘　　要：目的:获得人重组可溶性肿瘤坏死因子相关凋亡诱导配体(rsTRAIL)蛋白,并探讨其应用于非小细胞肺癌(NSCLC)治疗的前景。方法:RT-PCR从外周血单核细胞(PBMCs)中获取目的基因片段,构建重组质粒PQE30-TRAIL并转化入大肠杆菌(E.coli)M15,IPTG诱导目的蛋白表达并经镍固定金属亲和层析纯化,MTT法检测细胞增殖情况,荧光显微镜观察H460wt细胞的形态学改变,流式细胞仪检测其凋亡率。结果:获得与GenBank中报道一致的TRAIL基因片段。SDS-PAGE电泳和免疫印迹分析显示,获得具有TRAIL免疫原性、相对分子质量约为21000目的蛋白。Ni2+-NTAagarose纯化得到单一条带蛋白。rsTRAIL处理H460wt细胞24h后,细胞凋亡率为43.2%,顺铂可以增强rsTRAIL对A549细胞的杀伤作用。结论:获得了与天然TRAIL蛋白具有相同生物学活性的目的蛋白,该目的蛋白具有诱导NSCLC细胞凋亡的作用,联合顺铂能够增强rsTRAIL对NSCLC的杀伤作用。Objective To construct prokaryotic expression plasmid of human recombinant soluble TNF-related apoptosis inducing ligand （rsTRAIL）, then to investigate the effects of rsTRAIL on apoptosis in non-small cell lung carcinoma （NSCLC）. Methods The encoding sequence for rsTRAIL was amplified with RT-PCR and cloned into PQE30 vector to establish the prokaryotic expression system. The competent ceils of host strain of M15 were transformed by the recombinant plasmid. The expression of the target protein was induced with IPTG and purified by Ni^2＋-NTA agarose column, rsTRAII, was added in the media of A549 and H460^wt ceils, then the viability was examined by MTT assay. Apoptosis of H460^wt cells was observed under fluorescence microscopy. The apoptotic rate of tumor ceils was examined by FACS. Results The cloned fragment of rsTRAIL was 100% consistent with that reported in GenBank. The expressed protein with molecular weight of 21 000 in SDS-PAGE as expected was obtained and recognized by a commercial McAb. The apoptotic rate of H460^wt cells after treated with rsTRAIL for 24 h was 43.2%. Cislatin enhanced the effect of rsTRAIL on A549 cells. Conclusion The rsTRAIL is obtained after Ni affinity chromatograph, rsTRAIL has a strong cytotoxic activity against NSCLC and cisplatin may enhance the antitumor effect of rsTRAIL .

关 键 词：肿瘤坏死因子相关凋亡诱导配体 细胞凋亡 癌 非小细胞肺 顺铂 

分 类 号：Q78[生物学—分子生物学] R734.2[医药卫生—肿瘤]