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目的:构建人表皮生长因子受体(HER2)胞外区(1896bp)基因的真核表达质粒(pcDNA6/v5-his-HER2),转染小鼠乳腺癌细胞(EMT6),获得其稳定表达细胞株(EMT6/HER2)。方法:用PCR方法从含HER2全长基因的pcDNA3.1-HER2质粒上扩增HER2胞外区基因序列;经酶切、连接构建pcDNA6/v5-his-HER2;转化大肠杆菌DH5α,筛选阳性克隆,对其进行酶切及测序鉴定;以PEI法将pcDNA6/v5-his-HER2导入EMT6小鼠乳腺癌细胞,经杀稻瘟菌素(Blasticidin)筛选1～2周,获得抗性克隆EMT6/HER2;用RT-PCR检测EMT6/HER2中HER2mRNA,免疫组化法检测其HER2蛋白的表达。结果:PCR产物与预期片段大小一致;pcDNA6/v5-his-HER2经酶切、琼脂糖凝胶电泳后,可见与PCR产物大小相同的片段;DNA测序结果显示,pcDNA6/v5-his-HER2中HER2基因序列无误,读码框正确;用RT-PCR可在EMT6/HER2中检测到HER2mRNA,免疫组化法证实,EMT6/HER2中有HER2的阳性信号。结论:成功地构建了HER2胞外区真核表达载体,获得稳定表达HER2基因的小鼠乳腺癌EMT6细胞株,为进一步研究HER2基因过表达与乳腺癌发生的关系及其基因治疗奠定基础。
OBJECTIVE: To construct the eukaryotic expression plasmid (pcDNA6 / v5-his-HER2) of human extracellular domain (1896bp) of human epidermal growth factor receptor (HER2) and to transfect mouse breast cancer cells (EMT6) Strain (EMT6 / HER2). Methods: The HER2 extracellular region was amplified by PCR from pcDNA3.1-HER2 plasmid containing HER2 gene. The recombinant plasmid pcDNA6 / v5-his-HER2 was constructed by digestion and cloning. The recombinant plasmid was transformed into E. coli DH5α and screened for positive clones , Which was digested with restriction endonucleases and sequenced. The pcDNA6 / v5-his-HER2 was transfected into EMT6 mouse breast cancer cells by PEI method and screened by Blasticidin for 1 to 2 weeks to obtain the resistant clone EMT6 / HER2. The HER2 mRNA in EMT6 / HER2 was detected by RT-PCR and the HER2 protein was detected by immunohistochemistry. Results: The PCR product was consistent with the expected fragment size. After digested with pcDNA6 / v5-his-HER2 and the agarose gel electrophoresis, the same size of the PCR product was observed. The DNA sequencing results showed that in the pcDNA6 / v5-his-HER2 The HER2 gene was correct with the correct reading frame. HER2 mRNA was detected by RT-PCR in EMT6 / HER2. Immunohistochemistry confirmed the positive signal of HER2 in EMT6 / HER2. CONCLUSION: The eukaryotic expression vector of HER2 extracellular domain was successfully constructed and the mouse breast cancer EMT6 cell line stably expressing HER2 gene was obtained. This study lays the foundation for further study on the relationship between HER2 gene overexpression and breast cancer occurrence and gene therapy.