HER2基因在MCF-7细胞中的克隆与表达

Rong Li, Hang Zheng, Rong-cheng Luo
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引用次数: 0

摘要

目的:克隆人表皮生长因子受体(HER2)基因,建立HER2与雌激素受体(ER)稳定共表达的MCF-7细胞系。方法:从过表达HER2蛋白的人乳腺癌细胞系SKBR-3中提取总RNA,采用RT-PCR扩增HER2基因全长片段。将该基因片段插入载体pcDNA3.1,构建重组表达质粒pcDNA3.1- her2,经序列分析鉴定后,通过脂质体转染表达er的MCF-7细胞。G418筛选后获得阳性细胞克隆。用免疫细胞化学、流式细胞术和Western blotting检测转染HER2-cDNA的MCF-7细胞。结果:DNA测序结果显示HER2基因与GenBank中报道的序列完全一致。免疫细胞化学、流式细胞术和Western blotting结果显示,转染HER2的MCF-7细胞能够高表达HER2蛋白。结论:成功克隆了HER2基因,获得了HER2和ER共表达的MCF-7细胞系。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
[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 transfected 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.

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