摘要
目的:从HL-60细胞中获得了sTrail基因片段,优化蛋白表达条件,并研究其抗肿瘤活性。方法:培养HL-60细胞,提取总RNA,通过RT-PCR扩增sTRAIL蛋白基因片段,并将目的基因克隆至原核表达载体p ET28a上,并电击转化E.coli BL21(DE3),IPTG诱导表达,优化蛋白表达条件,Ni-IDA柱纯化重组蛋白,SDS-PAGE蛋白电泳,胶内酶解质谱鉴定。纯化后的重组蛋白作用HUVEC,He La,Hep-3B,HCT-116,MDA-MB-231,H460细胞检测蛋白生物学作用。结果:DNA测序结果证实成功构建了重组质粒p ET28a-sTrail,SDS-PAGE蛋白电泳,胶内酶解质谱检测显示成功表达sTRAIL蛋白,MTT法和流式细胞术结果显示,sTRAIL蛋白对肿瘤细胞包括He La,HCT-116,MDA-MB-231,H460,Hep-3B细胞有良好的生物活性,对正常的HUVEC细胞无毒性。结论:成功构建可以高效表达sTRAIL蛋白的原核表达载体,优化蛋白的表达和纯化后所得sTRAIL蛋白具有良好的抗肿瘤生物活性,为研究和发展利用sTRAIL蛋白作为临床治疗抗肿瘤药物提供了重要基础。
Purpose： To study the induced expression condition and anti-tumor activity of protein sTRAIL from human HL-60 cell. Methods： Genomic RNA was extract from the human HL-60 cell. The sTrail gene segment was amplified with RT-PCR and cloned into the prokaryotic expression vector pET28a. The final expression vector pET28a-sTrail was transformed into E. coli BI21 （DE3） for sTRAIL expression. Through IPTG induced, SDS-PAGE and LC-MS were used to determine the correct expression of sTRAIL. After purification by nickel-affinity chromatography, the recombinant sTRAIL was used to HUVEC, HeLa, Hep-3B, HCT-116, MDA- MB-231 and H460 cells. Result： The recombinant plasmid pET28a-sTrail was successfully built by the analysis of DNA sequencing. The result of SDS-PAGE show that sTRAIL gene was successfully expressed in E. coli BL21 （DE3） and identified by LC-MS. The purified sTRAIL inhibited the growth of HeLa, HCT-116, MDA-MB-231, H460 and Hep-3B cell while not HUVEC. Flow cytometry showed the inducing apoptosis of Hep-3B cells by sTRAIL. Conclution： The prokaryotic expression vector of sTRAIL was successfully built, and the purified protein sTRAIL exhibited anti-tumor activity. This provided an important foundation for using sTRAIL protein as a antitumor drugs of clinical treatment.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2015年第12期1-7,共7页
China Biotechnology
基金
国家国际合作项目(2011DFA32610)
国家"973"计划专项(2012CB722301)
国家"863"计划(2011AA10A203)
湖南省协同创新中心项目(20134486)资助项目
关键词
肿瘤坏死因子相关凋亡配体
基因克隆
原核表达
蛋白纯化
抗肿瘤活性
TNF-related apoptosis inducing ligand purification Antineoplastic activity Gene cloning Prokaryotic expression Protein
