人表皮生长因子受体结构域蛋白的原核表达、纯化及活性检测  

Prokaryotic Expression,Purification and Activity Detection of the Extracellular and Intracellular Domains of Human EGFR Subunit

作　　者：郭靖[1,2] 王涛[3] 李齐宏[3] 李玲[2] 梁迎春[2] 洪甜[2] 梅国徽 冀全博[2] 纪贝贝 徐小洁[2] 叶棋浓[1,2] 

机构地区：[1]大连医科大学肿瘤干细胞研究院,辽宁大连116044 [2]军事医学科学院生物工程研究所,北京100850 [3]军事医学科学院附属医院,北京100071

出　　处：《生物技术通讯》2015年第5期615-618,共4页Letters in Biotechnology

基　　金：国家自然科学基金(81472589;31100604);北京市自然科学基金(7132155);北京市科技新星计划(Z141102001814055);军事医学科学院创新基金转化医学项目(ZHYX003)

摘　　要：目的:构建人表皮生长因子受体(EGFR)胞外结构域(ED)和胞内激酶结构域(PKD)的原核表达载体,获得纯化的GST-EGFR-ED和GST-EGFR-PKD融合蛋白,并检测其活性。方法:用PCR方法从乳腺文库中扩增EGFR的ED和PKD基因编码序列,将其正确插入p GEX-KG载体,重组质粒转化大肠杆菌Rossate表达后,用GST-Sepharose4B珠子纯化融合蛋白。结果:构建得到GST-EGFR-ED和GST-EGFR-PKD的原核表达载体,双酶切鉴定得到与预期片段大小相符的外源基因插入片段,经测序与目的序列完全一致;在Rossate菌中诱导表达出与预期位置相符的目的蛋白,经Western印迹检测,融合蛋白成功表达;纯化得到融合蛋白GST-EGFR-ED和GST-EGFR-PKD。结论:克隆了GST-EGFR-ED和GST-EGFR-PKD基因,并获得了活性良好的GST-EGFR-ED和GST-EGF-PKD融合蛋白,为研究EGFR在肿瘤中的作用奠定了实验基础。Objective： To clone prokaryotic expression vectors of extracellular domain（ED） and protein kinase do- main（PKD） of human epithelial growth factor receptor（EGFR）, and express and purify the fusion proteins of GST- EGFR-ED and GST-EGFR-PKD, and identify their functions preliminarily. Methods： ED and PKD coding re- gions of EGFR were amplified from human mammary cDNA library by PCR and cloned into the prokaryotic expres- sion vector pGEX-KG. The fusion proteins of GST-EGFR-ED and GST-EGFR-PKD were expressed in E.coli Ros- sate and purified by GST-Sepharose 4B beads. The interactions of the proteins with memo were identified by GST pull-down assay. Results： GST-EGFR-ED and GST-EGFR-PKD recombinant plasmids were successfully obtained by double digestion identification. The inserted fragments were confirmed correct by sequencing. The fusion pro- teins were successfully induced and identified by Western blot analysis. Conclusion： GST-EGFR-ED and GST- EGFR-PKD were successfully cloned and purified.

关 键 词：人表皮生长因子受体 胞外结构域 胞内激酶结构域 原核表达 纯化 

分 类 号：Q78[生物学—分子生物学]