人胰岛素样生长因子1受体β结构域蛋白的原核表达及活性检测  

Prokaryotic expression,purification and activity detection of the extracellular and intracellular domains of human IGF1R beta subunit

作　　者：郭靖[1,2] 冯滢滢[3] 王涛[1] 张柯[4] 李玲[1] 冀全博[1] 徐小洁[1] 叶棋浓[1] 

机构地区：[1]大连医科大学生物工程研究所,辽宁大连116044 [2]军事医学科学院生物工程研究所,北京100850 [3]第二炮兵总医院,北京100088 [4]军事医学科学院科技部,北京100850

出　　处：《细胞与分子免疫学杂志》2015年第9期1211-1215,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81472589;31100604);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);北京市自然科学基金(7132155)

摘　　要：目的构建人胰岛素样生长因子1受体(IGF1R)β亚基胞外结构域(ED)和胞内激酶(PKD)结构域的原核表达载体,获得纯化的GST-IGF1Rβ-ED和GST-IGF1Rβ-PKD融合蛋白,并检测其活性。方法用PCR方法从乳腺cDNA文库中扩增IGF1Rβ-ED和GST-IGF1Rβ-PKD基因编码序列,将其正确插入pGEX-KG载体。重组质粒转化大肠杆菌Rossate表达后,用GST-Sepharose 4B珠子纯化融合蛋白,并用Western blot法检测融合蛋白表达,最后用GST pull-down技术检测纯化蛋白与已知蛋白表皮生长因子受体2驱动的细胞运动调节蛋白(MEMO)的相互作用。结果构建得到GST-IGF1Rβ-ED和GST-IGF1Rβ-PKD基因的原核表达载体,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,测序后与目的序列一致;在Rossate菌中诱导表达出与预期位置相符的目的蛋白,并经过Western blot检测,融合蛋白成功表达;纯化得到GST-IGF1Rβ-ED和GST-IGF1Rβ-PKD两个融合蛋白,GST pull-down证明蛋白GST-IGF1Rβ-PKD可以和MEMO相互作用。结论成功克隆GST-IGF1Rβ-ED和GST-IGF1Rβ-PKD基因,并获得活性良好的GST-IGF1Rβ-ED和GST-IGF1Rβ-PKD蛋白。Objective To construct the prokaryotic expression vectors of extracellular domain（β-ED） and intracellular protein kinase domain（ β-PKD） of insulin-like growth factor 1 receptor beta（ IGF1R-β） subunit,purify the fusion proteins GST-IGF1R β-ED and GST-IGF1R β-PKD,and detect their activities. Methods Human GST-IGF1R β-ED and GST-IGF1Rβ-PKD coding regions were amplified from human mammary c DNA library by PCR and cloned into the prokaryotic expression vector pGEX-KG. The fusion proteins GST-IGF1R β-ED and GST-IGF1R β-PKD were expressed in E. coli Rossate and purified by GST-Sepharose 4B beads. The expression of the fusion proteins were detected by Western blotting. The interactions of the proteins with mediator of epidermal growth factor receptor-2（ ERBB2）-driven cell motility（ MEMO） protein were identified by GST pull-down assay. Results GST-IGF1R β-ED and GST-IGF1R β-PKD recombinant plasmids were successfully cloned. Double enzyme digestion and sequencing confirmed that the inserted fragments were identical to the target ones. The fusion proteins were successfully induced in Rossate and Western blotting showed the expression as expected. GST pull-down assay revealed that GST-IGF1R β-PKD could interact with MEMO in vitro. Conclusion GST-IGF1R β-ED and GST-IGF1R β-PKD were successfully cloned and purified. In addition,GST-IGF1R β-PKD could interact with MEMO in vitro,which demonstrated the good activity of the purified proteins.

关 键 词：胰岛素样生长因子1受体(IGF1R) β亚基胞外结构域 β亚基胞内蛋白激酶结构域 原核表达 纯化 表皮生长因子受体2驱动的细胞运动调节蛋白(MEMO) 

分 类 号：R392.33[医药卫生—免疫学] Q816[医药卫生—基础医学]