EGFR可溶性胞外段的表达、纯化及鉴定  

Expression,Purification and Identification of Soluble Extracellular Domain of EGFR

作　　者：赵志磊 段勤勇[1] 汪炬[1] 

机构地区：[1]广东省生物医学重点实验室暨南大学基因工程国家工程研究中心,广东广州510632

出　　处：《药物生物技术》2017年第5期377-380,共4页Pharmaceutical Biotechnology

基　　金：基金项目 抗肿瘤新药BAT-7205及tsFGFR2临床前研究及其规模制备技术(No.201300000041)

摘　　要：利用大肠杆菌表达表皮生长因子受体胞外段(ED-EGFR),将目的基因片段插入到p ET28a载体中,将插入目的基因载体转化入BL21表达。利用异丙基-β-D-硫代半乳糖苷(ITPG)诱导BL21表达ED-EGFR,破碎后80%的ED-EGFR为可溶性表达,其他则存在包涵体中,将菌液离心上清通过镍柱亲和层析纯化,纯化蛋白样品经过胶浓度为12%SDS-PAGE电泳,条带在相对分子质量58 k左右出现单一条带,并且纯度达到95%以上。通过BCA检测蛋白浓度,利用CCK8方法检测ED-EGFR具有较高活性,人肺非小细胞癌细胞(H1648细胞)增殖明显被抑制,并且在浓度320 ng/m L下抑制效果最好。The target gene fragment was inserted into the p ET28 a vector. The target gene vector was inserted into BL21 to express the extracellular fraction of epidermal growth factor receptor（ED-EGFR）. The expression of ED-EGFR in BL21 was induced with isopropyl-β-D-thiogalactoside（ITPG）. 80% of ED-EGFR was expressed as soluble after disintegration. Others were existing in inclusion bodies. The purity of the purified protein sample was 12% by SDS-PAGE. The band was a single band at about 58 ku,and the purity was over 95%. The proliferation of human lung non-small cell carcinoma cells（H1648 cells） was inhibited by CCK8 method,and the inhibitory effect was the best at the concentration of 320 ng/m L.

关 键 词：目的基因 EFGR胞外段蛋白(ED-EGFR) 包涵体 可溶性表达 纯化 检测 

分 类 号：Q786[生物学—分子生物学]