用蜂毒溶血肽和表皮生长因子构建膜毒性免疫毒素的研究

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。正电性抗菌肽有其独特的膜毒性细胞毒机制。本研究以小鼠表皮生长因子为导向部分，以蜂毒溶血肽为毒性部分，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的鳞状上皮癌A431细胞表现出显著杀伤力，其LD50值为52.6μg/mL。本研究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IT是可行的。

Studies on the Construction of Membrane-lytic Immunotoxin by Using Melittin and Epidermal Growth Factor

Epidermal Growth Factor Receptor is becoming a perfect target to kill carcinoma cells specially because of its overexpression on the surface of carcinoma cells. Cationic antimicrobial peptides (CAP) have its own special membrane-lytic cytotoxicity mechanism. In this study, the membrane-lytic immunotoxin (IT), named as chimeric protein MEGFMEL, which composed of mouse epidermal growth factor (MEGF) as targeting part and melittin (MEL) as cytotoxic part, was constructed to kill carcinoma cells with epidermal growth factor receptor (EGFR) overexpression. Using E.coli BL21 as expression strain and pET30a as expression vector, through low-temperature inducing expression and thawing & freezing purification without cytolysis procedure, the MEGFMEL obtained was of 63.45μg/mL concentration and 68% purity. In vitro activity measurement showed the interested MEGFMEL had significant killing effect to A431 cell of squamous epithelium carcinoma which overexpresses EGFR on its surface, with LD50 value 52.6μg/mL.