重组HER2真核表达载体的构建及其稳定转染EMT6细胞株的筛选

Construction of an eukaryotic plasmid encoding HER2 and screening of a cell line stably expressing clones

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。

AIM：To construct an eukaryotic vector encoding extracellular domain of human epidermal growth factor receptors（HER2）,pcDNA6/v5-his-HER2,and to screen HER2 positive clones from mouse breast cancer cell line EMT6.METHODS：The extracellular domain of HER2 was amplified from pcDNA3.1-HER2 by PCR.pcDNA6/v5-his-HER2 was prepared by inserting the fragment into the plasmid pcDNA6/v5-his.Then the recombinant vector was identified by restriction enzyme and sequencing.Next,pcDNA6/v5-his-HER2 was transfected into the EMT6 cell line and the positive clones（EMT6/HER2）were screened with blasticidin.Finally,the expression of HER2 in EMT6/HER2 was detected by RT-PCR and immunohistochemistry.RESULTS：The fragment of HER2 was amplified and pcDNA6/v5-his-HER2 was prepared successfully.No errors were found both in the sequence and ORF of the acquired fragment.The expected fragment of HER2（1896 bp）was amplified from EMT6/HER2 by RT-PCR and positive signals of HER2 were detected in EMT6/HER2 by immunohistochemistry.CONCLUSION：An eukaryotic plasmid encoding HER2（pcDNA6/v5-his-HER2）has been constructed and a cell line expressing HER2 stably has been prepared successfully.