人EYA3基因真核表达载体的构建及其活性检测

Construction of myc-Tagged Human EYA3 Eukaryotic Expression Vector and its Activity Detection

目的:构建带myc标签的人EYA3基因真核表达载体,获得myc-EYA3融合表达蛋白,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EYA3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后,Western印迹检测表达情况,并进行生长曲线实验。结果:双酶切和测序鉴定表明,myc-EYA3真核表达质粒构建成功,转染ZR75-1细胞后成功表达;生长曲线实验结果表明,EYA3可促进乳腺癌细胞的生长。结论:构建了带myc标签的人EYA3基因真核表达载体,myc-EYA3能在乳腺癌细胞ZR75-1中表达,且能促进该细胞的生长,本实验为进一步研究在乳腺癌中的功能奠定了基础。

Objective： To construct the eukaryotic expression vector of human EYA3 labeled with myc and de-tect its activity. Methods： Human EYA3 coding region was amplified from human mammary cDNA library by PCR and cloned into myc vector. Human ZR75-1 cells were transfected with the recombinant plasmid myc-EYA3 and the expression was detected by Western blot. The proliferation of ZR75-1 cells regulated by myc-EYA3 was iden-tified by growth curve. Results： EYA3 eukaryotic expression vector labeled with myc was successfully constructed by double digestion identification. The inserted fragment was confirmed to be correct by sequencing. Human EYA3 protein was expressed in human ZR75-1 cells as shown by Western blot and EYA3 protein could promote prolifer-ation in ZR75-1 cells. Conclusion： The eukaryotic expression vector of myc-EYA3 was successfully constructed. The myc-EYA3 could express and promote proliferation in ZR75-1 cells, which had laid foundation for further study on EYA3 in breast cancer.