用蜂毒溶血肽和表皮生长因子构建膜毒性免疫毒素  

Construction of Membrane-lytic Immunotoxin byMelittin and Epidermal Growth Factor

作　　者：牛冬[1] 阮晖[1] 潘冰青[1] 王金玲[1] 吴德 陈美龄[1] 张佳佳[1] 陈启和[1] 何国庆[1] 

机构地区：[1]浙江大学生物系统工程与食品科学学院 [2]浙江金华省级高新技术园区,金华321017

出　　处：《农业生物技术学报》2008年第3期390-394,共5页Journal of Agricultural Biotechnology

基　　金：浙江省自然科学基金资助项目(No.Y204348);杭州市科技计划资助项目(No.2002121B08)资助

摘　　要：表皮生长因子受体由于在许多种肿瘤细胞表面过度表达而成为特异杀伤肿瘤细胞的理想靶位;正电性抗菌肽有独特的膜毒性细胞毒机制。研究以小鼠(Mus musculus)表皮生长因子(mouse epidermal growth factor,MEGF)为导向部分,以蜂毒溶血肽(melittin,Mel)为毒性部分,构建特异杀伤肿瘤细胞的膜毒性免疫毒素MEGFMEL嵌合蛋白。该嵌合蛋白以大肠杆菌(Escherichia coli)BL21为表达宿主,以pET30a为表达载体,采用低温诱导表达和无破胞程序的冻融法进行纯化,最终浓度为63.45μg/mL、纯度为68%。体外活性检测表明,MEGFMEL嵌合蛋白对表面过度表达EGFR的肝癌A431细胞表现出显著杀伤力,其LD50为52.6μg/mL。结果显示以正电性抗菌肽为毒性部分构建针对表皮生长因子受体的新型膜毒性免疫毒素(im-munotoxin,IT)是可行的。Epidermal growth factor receptor is becoming a perfect target to kill carcinoma cells specially because of its overexpression on the surface of carcinoma cells and Cationic antimicrobial peptides （CAP） have special membrane-lytic cytotoxicity mechanism. In this study, the membrane-lytic immunotoxin （IT）, named as the chimeric protein MEGFMEL, composed of mouse （Mus musculas） epidermal growth factor （MEGF） as targeting part and melittin （MEL） as cytotoxic part, was constructed to kill carcinoma cells by epidermal growth factor receptor （EGFR） overexpression. Using Escherichia coil BL21 as expression strain and pET30a as expression vector, through low-temperature inducing expression and thawing and freezing purification without cytolysis procedure, the MEGFMEL obtained was of 63.45 μg/mL concentration and 68% purity. In vitro activity measurement showed that the interested MEGFMEL had significant killing effect to A431 cell of liver carcinoma which overexpresses EGFR on its surface, with LD50 value 52.6 μg/mL. The results demonstrated that using CAP as toxic part to construct the unique membrane-lytic IT aiming at EGFR is feasible.

关 键 词：膜毒性免疫毒素 蜂毒溶血肽 表皮生长因子受体 

分 类 号：S188[农业科学—农业基础科学]