scTRAIL在E.coli中的重组表达及抗肿瘤活性的研究  

Recombinant expression and anti-tumor effects of single-chain TRAIL in E.coli

作　　者：刘魏 卢红娟 许骥 王梁华[4] Liu Wei;Lu Hongjuan;Xu Ji;Wang Lianghua(Department of Laboratory Diagnostics;Department of Rheumatology,Changzheng Hospital,the Second Military Medical University,Shanghai 200003,China;Research Office of Diving Medicine,Research Institute of Naval Medical Sciences,the Second Military Medical University,Shanghai 200433,China;Teaching and Research Office of Biological Chemistry and Molecular Biology,Department of Basic Medical Sciences,the Second Military Medical University,Shanghai 200433,China)

机构地区：[1]海军军医大学附属长征医院实验诊断科,上海200003 [2]海军军医大学附属长征医院风湿免疫科,上海200003 [3]海军军医大学海军医学研究所潜水医学研究室,上海200433 [4]海军军医大学基础部生物化学与分子生物学教研室,上海200433

出　　处：《现代肿瘤医学》2018年第23期3708-3712,共5页Journal of Modern Oncology

基　　金：国家自然科学基金青年基金(编号:31500716)

摘　　要：目的:重组表达人单链TRAIL(single-chain TRAIL,sc TRAIL)及研究其在抗肿瘤中的作用。方法:通过重组PCR获得编码sc TRAIL的基因序列,构建含编码sc TRAIL基因的重组质粒,将重组表达质粒转化到大肠埃希菌(E. coli) BL21(DE3),通过IPTG诱导,破碎细菌,上清行Amylose Resin亲和层析初步纯化融合蛋白。通过MTT法检测融合蛋白对人胰腺胆管上皮细胞SW1990、人大细胞肺癌NCI-H460和人脐静脉内皮细胞(HUVECs)的增殖抑制作用。结果:成功构建并克隆编码sc TRAIL的基因序列,经IPTG诱导表达,表达产物的表达量约占菌体蛋白的20%左右,获得了相对分子质量约为110 k D的可溶性重组蛋白MBP-sc TRAIL。MBP-sc TRAIL分别作用于NCI-H460和SW1990,其增殖抑制作用IC50分别约为111 ng/ml和250 ng/ml,且MBP-sc TRAIL增殖抑制率明显高于TRAIL114-281(P <0. 01)。结论:sc TRAIL的抗肿瘤效果优于单体TRAIL114-281,是一种安全、有效的抗肿瘤生物制品。Objective：To investigate the expression of a single-chain TRAIL （scTRAIL） and study its anti-tumor effects.Methods：The genetic sequence of coded scTRAIL was obtained by means of PCR.One recombinant plasmid containing scTRAIL gene was structured,and it was transformed in E.coli BL21 and then expressed by isopropyl-β-d-thiogalactoside induction.Amylose resin affinity purification column was used to purify the obtained protein.The fusion protein inhibiting the growth of pancreatic cancer cells （SW1990）,lung cancer cells （NIC-H460） and human umbilical vein endothelial cells （HUVECs） were detected by means of MTT.Results：The genetic sequence of coded scTRAIL was successfully structured and cloned.The expression level of fusion protein was about 20% of total somatic protein by IPTG,while the relative molecular mass was about 110 kD.MBP-scTRAIL was obtained.The MBP-scTRAIL was capable of significantly inhibiting the proliferation of cancer cells （NCI-H460 and SW1990）.There was positive correlation between inhibitory effects and the density,and the inhibitory rate was obviously higher than that of TRAIL114-281（P〈0.01）.The levels of IC50 exerted on NCI-H460 and SW1990 are around 111 ng/ml and 250 ng/ml respectively.Conclusion：Proven to be a safe and effective anti-tumor biological medicine,scTRAIL has better effects than those of TRAIL114-281.

关 键 词：肿瘤坏死因子相关凋亡诱导配体 单链化 大肠埃希菌 重组表达 抗肿瘤活性 

分 类 号：R730.5[医药卫生—肿瘤]