从噬菌体随机肽库筛选人肝再生增强因子-第三军医大学学报.docVIP

编号 201308195 刊用形式 论著 利用噬菌体随机肽库筛选人肝再生增强因子特异性结合肽及其抗肝癌作用的研究 冯 倩，刘燕倩，孙 航，刘 杞（400010重庆，重庆医科大学附属第二医院病毒性肝炎研究所，国家教育部感染性疾病分子生物学重点实验室） 【摘要】 目的 利用噬菌体随机肽库筛选人肝再生增强因子（hALR）特异性结合肽并研究其抗肝癌作用。方法 以hALR为靶分子，利用Ph.D.TM噬菌体肽展示系统筛选出与hALR特异性结合的单克隆噬菌体；ELISA法鉴定筛选出的单克隆噬菌体与hALR的结合性；对特异性结合噬菌体DNA中插入片段进行PCR扩增、测序及短肽合成；MTS法检测特异性结合肽对人HepG2肝癌细胞增殖的影响；观察特异性结合肽对裸鼠人HepG2肝癌移植瘤生长的影响。结果 经过3轮生物淘筛，特异性结合噬菌体得到富集；筛选到的单克隆噬菌体与hALR结合具有特异性；特异性结合肽在体外能抑制人HepG2肝癌细胞增殖，在体内能抑制裸鼠人HepG2肝癌移植瘤生长；该特异性结合肽免疫小鼠后机体内无相应抗体检出，且对裸鼠的重要脏器组织结构及功能无明显损害。结论 利用噬菌体肽库筛选到hALR特异性结合肽，该短肽通过阻断hALR促肝癌细胞增殖作用，能抑制人HepG2肝癌细胞在体内外增殖，达到抗肝癌的作用，同时产生较低的免疫原性及毒性损害，为肝癌的靶向治疗提供了新方向。 【关键词】 人肝再生增强因子，特异性结合肽，噬菌体肽库，肝癌，靶向治疗 [中图法分类号] [文献标志码] A [基金项目]国家自然科学基金 [通讯作者]刘杞；E-mail：liuqiz@ Screening of hALR-specific binding peptide from phage display peptide library and its biological activities of anti-liver cancer Feng Qian, Liu Yanqian, Sun Hang, Liu Qi (Key Laboratory of Molecular Biology for Infectious Disease of Ministry of Education, Institute for Viral Hepatitis, Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, China） 【Abstract】Objective To screen the human augmenter of liver regeneration (hALR)-specific binding peptide from phage display random peptide library and investigate its biological activities on anti-liver cancer. Methods The phage clones that specifically binded with target protein hALR were selected from phage display random peptide library. ELISA was used to identify the specificity of binding between selected phage and target protein hALR. The acquired DNA inserts were amplified using PCR, and the specific binding peptides were synthesised after sequencing. Methyl tolyl sulfide (MTS) assay was employed to measure the biological activities of the specific binding peptides in the proliferation of human HepG2 liver cancer cells. The biological activities of the specific binding peptides in the growth of human HepG2 cell xenograft tumor was studied in the tumor size in nu