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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 human eukaryotic expression vector of human HER2 gene and verify its effect on proliferation and targeting drug sensitivity of breast cancer cells. Methods The HER2 gene was amplified by PCR and inserted into pXJ-40-myc vector. After digestion and sequencing, the HER2 gene was transfected into ZR75-1 breast cancer cells. The expression of HER2 gene was detected by Western blot. The cell growth curve was determined by CCK8 method. After the target trastuzumab monoclonal antibody was added, the response of the transfected cells to the drug was observed. Results The recombinant plasmid was successfully constructed by restriction enzyme digestion and sequencing. The results of Western blot showed that myc-HER2 protein was successfully expressed in transfected cells. The breast cancer cells transfected with myc-HER2 grew faster than empty vector cells. Bead monoclonal antibody transfected myc-HER2 cell growth was significantly inhibited. Conclusion The myc-tagged HER2 gene eukaryotic expression vector was successfully constructed, which laid the foundation for the further study of trastuzumab resistance.