HER2基因的克隆及其在MCF-7细胞中的表达

目的 克隆人表皮生长因子受体(HER2)基因,建立共表达HER2基因和雌激素受体(ER)基因的MCF-7细胞模型。方法 从高表达HER2的人乳腺癌细胞株SKBR-3中,用RT-PCR技术克隆HER2基因全长cDNA,将其插入载体pcDNA3.1中构建重组真核表达载体pcDNA3.1-HER2。测序鉴定后,利用阳离子脂质体介导将其转染ER阳性的乳腺癌MCF-7细胞,经G418筛选阳性克隆。用免疫细胞化学、流式细胞术及Westernblotting等方法检测HER2基因在MCF-7细胞中的稳定表达情况。结果 DNA测序证明,获得的HER2基因序列与GenBank中报道序列完全一致。将其转染MCF-7细胞,经流式细胞术、Westernblotting及免疫细胞化学等方法检测证实,转染细胞内有HER2基因高表达。结论 成功克隆HER2基因并获得共表达HER2基因和ER基因的MCF-7细胞株。

Cloning and expression of the HER2 gene in MCF-7 cells

Objective To clone human epidermal growth factor receptor (HER2) gene and establish a MCF-7 cell line with stable co-expression of HER2 and estrogen receptor (ER). Methods The full-length HER2 gene fragment was amplified by RT-PCR from the total RNA extracted from human breast cancer cell line SKBR-3 which over-expressed HER2 protein. The gene fragment was then inserted into the vector pcDNA3.1 to construct the recombinant expression plasmid pcDNA3.1- HER2, which was identified by sequence analysis and transfected into ER-expressing MCF-7 cells via lipofectamine. The positive cell clones were obtained after G418 selection. The MCF-7 cells transfected with HER2-cDNA was assayed by immunocytochemistry, flow cytometry and Western blotting. Results DNA sequencing results showed that HER2 gene was exactly consistent with the sequence reported in GenBank. The MCF-7 cells tansfected with HER2 could highly express HER2 protein as shown by immunocytochemistry, flow cytometry and Western blotting. Conclusion HER2 gene has been cloned successfully and the MCF-7 cell line co-expressing HER2 and ER is obtained.