带myc标签的人HER2基因真核表达载体的构建及其生物学功能

Construction and biological function of eukaryotic expression vector for myctagged HER2

目的构建人类HER2基因的真核表达载体,并对其促进乳腺癌细胞增殖效果及靶向药物敏感性进行验证。方法采用PCR技术扩增出HER2基因,将其插入到pXJ-40-myc载体中,酶切和测序验证后,将其转染到乳腺癌ZR75-1细胞中,Western blot法检测其表达情况;CCK8法测定细胞生长曲线;加入靶向药物曲妥珠单克隆抗体后,观察转染细胞对药物的反应。结果酶切和测序结果证实表达质粒构建成功;Western blot法结果显示,myc-HER2蛋白在转染细胞中成功表达;转染myc-HER2的乳腺癌细胞较空载体细胞生长较快;加入曲妥珠单克隆抗体后,转染myc-HER2的细胞生长明显受到抑制。结论成功构建了带myc标签的HER2基因真核表达载体,为进一步研究曲妥珠单抗的耐药奠定了实验基础。

Objective To construct eukaryotic expression vector of myc-tagged human HER2 and investigate its role in breast cancer cell proliferation and drug resistance. Methods Human HER2 gene was amplified by PCR and cloned into the pXJ-40-myc vector. The recombinant myc-HER2 was identified by enzyme digestion and sequencing, transfected into breast cancer cell line ZR75-1, and then was detected by Western blotting. Next, CCK8 assay was performed to investigate myc- Her2 transfected cell proliferation. Trastuzumab, anti-Her2 antibody, was added to determine the sensitivity of cells transfect- ed with myc-Her2. Results Enzyme digestion and sequencing confirmed the myc-HER2 vector was constructed successfully. Western blotting showed the expression of myc-HER2 in the ZR75-1 cells. The result of cell growth curve showed that cells transfected with myc-her2 grew significantly faster than the control cells. Moreover, trastuzumab obviously suppressed the growth of ZR75-1 cells transfected with myc-HER2. Conclusion The myc-HER2 vector was successfully constructed which lays an experimental foundation for the study of HER？. drug resistance to trastuzumab.