人FOXO3a基因真核表达载体的构建及其功能验证  

Construction of myc- Tagged Human FOXO3a Eukaryotic Expression Vector and Detection of its Biological Function

作　　者：郭芸菲[1] 王涛[1] 徐小洁[2] 周丽英[2] 梁迎春[2] 冯滢滢[3] 李玲[2] 郭靖[2] 叶棋浓[2] 江泽飞[1] 

机构地区：[1]军事医学科学院附属医院乳腺肿瘤科,北京100071 [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

出　　处：《生物技术通讯》2014年第5期625-627,648,共4页Letters in Biotechnology

基　　金：国家自然科学基金(31100604);北京市自然科学基金(7132155);北京市科技新星计划(xx2014B055);军事医学科学院创新基金转化医学项目(ZHYX003)

摘　　要：目的:构建带myc标签的人FOXO3a基因真核表达载体,并对其功能进行初步检测。方法:采用PCR技术,从乳腺文库中扩增人FOXO3a基因,并将其正确插入pXJ-40-myc载体;将重组质粒与空载体分别转染人乳腺癌细胞系ZR75-1、MCF-7后,通过Western印迹检测其表达情况,并用CCK8法测定细胞生长曲线。结果:双酶切和测序鉴定表明myc-FOXO3a真核表达质粒构建成功,转染乳腺癌ZR75-1、MCF-7细胞后目的基因成功表达;细胞生长曲线结果显示,转染myc-FOXO3a的乳腺癌细胞较空载体细胞生长较慢。结论:构建了带myc标签的人FOXO3a基因真核表达载体,为进一步研究FOXO3a在乳腺癌中的功能奠定了基础。Objective： To construct the eukaryotic expression vector of human FOXO3a labeled with myc tag and detect its biological function preliminarily. Methods： Human FOXO3a coding region was amplified from human mammary eDNA library by PCR and cloned into pXJ-40-myc vector. The recombinant plasmid myc-FOXO3a was identified by enzyme digestion and sequencing, transfeeted into breast cancer cell line and detected by Western blot. Then CCK8 assay was performed to investigate myc-FOXO3a transfected cell proliferation. The proliferation in ZR75-1 and MCF-7 cells which were supposed to be promoted by mye-FOXO3a was identified by its expression. Results： FOXO3a eukaryotic expression vector labeled with myc tag was successfully constructed by double digestion identification. The inserted fragment was confirmed to be correct by sequencing. Human FOXO3a pro- tein was expressed in human ZR75-1 and MCF-7 cells as shown by Western blot. Cell growth curve showed that cells transfectd with myc-FOXO3a grew significantly slower than the control cells. Conclusion： The eukaryotic expression vector of myc-FOXO3a was successfully constructed which lays a molecular foundation for the study of FOXO3a in breast cancer development and progression.

关 键 词：人FOXO3a基因 克隆 真核表达 

分 类 号：Q78[生物学—分子生物学]