利用重组人α乳清蛋白和油酸大量快速制备高活性的HAMLET肿瘤杀伤复合物  

Fast and scalable production of cancer-killing complex from recombinant human alpha-lactalbumin and oleic acid

作　　者：张轶博[1,2] 曾瑞霞[3] 牛静[1] 郑少鹏[1] 贾弘禔[1] 丁卫[1] 

机构地区：[1]首都医科大学生物化学与分子生物学系,北京100069 [2]辽宁医学院免疫与病原生物学教研室,辽宁锦州121001 [3]辽宁医学院人体解剖教研室,辽宁锦州121001

出　　处：《首都医科大学学报》2012年第5期557-564,共8页Journal of Capital Medical University

基　　金：国家自然科学基金(30970161);北京市教委科技发展计划资助项目(KM201010025001)~~

摘　　要：目的可致肿瘤细胞死亡的人α乳清蛋白(human alpha-lactalbumin made lethal to tumor cells,HAMLET)是一种可导致多种癌细胞死亡的强效选择性肿瘤杀伤蛋白质-脂酸复合物。为了使HAMLET应用于肿瘤的治疗并对其杀伤机制进行探讨,我们需要建立和优化简便、快速、规模化制备与纯化HAMLET的方法。方法通过构建人α乳清蛋白分泌型细胞MCF-7的cDNA文库,PCR扩增出编码人α乳清蛋白成熟肽段的全长序列,然后克隆入pET30a(+)表达载体质粒,并转化BL21(DE3)宿主大肠杆菌。经过IPTG诱导,表达细菌的裂解,蛋白体外复性,Ni-NTA亲和层析柱纯化等步骤,获得了高纯度的重组人α乳清蛋白的C端His标签融合蛋白。纯化蛋白经EDTA脱钙后,与油酸(oleic acid,OA)在加热条件下制备HAMLET复合物。同时,对HAMLET的理化特性和生物学活性进行检测。结果通过与经典方法制备的牛α乳清蛋白和OA复合物(BAMLET)进行比较,发现我们制备的HAMLET在紫外吸收光谱、疏水性质特征及肿瘤杀伤活力方面与BAMLET活性相符,并且可引起HeLa肿瘤细胞的凋亡样程序性死亡。结论本研究自主制备的HAMLET成本经济、方法简便且具有较好的规模化放大前景,为日后临床应用奠定基础。Objective Human alpha-lactalbumin made lethal to tumor cells （HAMLET） is a novel potent tumoricidal agent that selectively induces apoptosis-like cell death in a variety of cancers. Methods As the sufficient amount and concentration of human alpha- lactalbumin （HLA） to form HAMLET is critical for its anti-tumor activity, mass production and rapid purification of recombinant HLA protein is highly desired. We first constructed a cDNA library of MCF-7 cell, known to secrete mature HLA peptides. Then amplified the full-length of HLA coding sequence by PCR, and cloned it into a pET30a （ ＋ ） vector with an C-terminus His-tag. The His-rHLA was highly expressed in BI221 E coli ceils with the induction of IPTG and purified by Ni-NTA affinity chromatography. The rHLA was refolded in an in vitro refolding buffer, de-calcinated with EDTA treatment, and then reacted with oleic acid （OA） water suspension under heated conditions to form the HAMLET complex. Results By comparing our HAMLET product with a reference preparation （BAMLET） from bovine alpha-lactalbumin and OA, we found that our preparations of HAMLET have a similar UV spectrum and a comparable tumoricidal activity as determined by cell viability assays and morphologic observations. Conclusion The cost-effectiveness and scalability of HAMLET production using recombinant HLA can be extremely useful for the further investigation of mechanistic studies of HAMLET functions, and may also be potentially considered as a practical anti-cancer agent for therapeutic trials.

关 键 词：可致肿瘤细胞死亡的人α乳清蛋白 α乳清蛋白 肿瘤细胞 程序性死亡 

分 类 号：R730.1[医药卫生—肿瘤]