稳定表达CHD5蛋白的293细胞系的构建及CHD5的功能研究
首发时间:2012-12-28
摘要:目的:构建CHD5基因真核表达载体, 并利用该载体构建稳定表达CHD5蛋白的人胚肾细胞(HEK 293)系,并对CHD5在HEK 293细胞中的功能进行研究。方法:以 PCR 的方法扩增CHD5的开放阅读框,把CHD5开放阅读框正向或反向插入pcDNA3.1(+)载体中以构建pcDNA3.1-CHD5和pcDNA3.1-CHD5-AS表达载体。将构建的真核表达载体以脂质体介导的方法体外转染HEK 293细胞,利用G418进行克隆筛选,以建立稳定表达CHD5蛋白或稳定转录CHD5反义序列的HEK 293细胞系,并利用QPCR和Western Blot技术检测细胞克隆中CHD5的表达水平。然后对稳定表达CHD5蛋白和稳定转录CHD5反义序列的HEK 293细胞系进行细胞增殖和细胞周期分析。结果:成功构建了CHD5的真核表达载体;并筛选出了稳定表达CHD5蛋白或稳定转录CHD5反义序列的HEK 293细胞各三株。其中稳定表达CHD5蛋白的HEK 293细胞系中CHD5蛋白的表达水平比转录CHD5反义序列的HEK 293细胞系有明显提高;稳定表达CHD5蛋白导致HEK 293细胞系增殖被抑制,细胞周期被阻滞在了G2/M期。而稳定转录CHD5反义序列对HEK 293细胞系的细胞增殖、细胞周期无影响。结论:成功构建了稳定表达CHD5蛋白和稳定转录CHD5反义序列的HEK293细胞系,为进一步体外研究CHD5的功能奠定了基础。CHD5能抑制293细胞的增殖,并将细胞阻滞在G2/M期。
关键词: CHD5 真核表达载体 稳定表达的HEK293
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Construction of CHD5 stable transfected HEK293 cell line and the study of CHD5 functions
Abstract:AIM: To construct eukaryotic expression vector of CHD5 gene, and establishment of its stable transfected HEK293 cell line. METHODS: The open reading frame (ORF) of CHD5 was amplified by PCR. Then the fragment was forward or backward subcloned into pcDNA3.1 (+), a eukaryotic expression vector. The recombinant plasmids were then transferred into HEK293 cells by lipofectin mediation. After screening culture by G418, stable transfected HEK293 cell lines were established, and Q-PCR and Western-blot were performed to identify the expression of CHD5, cell proliferation and cell cycle were detected . RESULTS: successfully construct eukaryotic expression vector of CHD5 gene, the expression of CHD5 was decreased significantly at both mRNA and protein levels tansfected by antisense vector , but elevated in sense group.In HEK293 cell line,CHD5 inhibited cell proliferation and Cell cycle was arrested at the G2 / M phase .Conclusion: The eukaryotic expression vector of CHD5 was successfully constructed and successfully stablishment of its stable transfected HEK293 cell line, CHD5 inhibited cell proliferation and Cell cycle was arrested at the G2 / M phase in HEK293 cell line. providing a foundation for further studies on the function of CHD5 in vitro.
Keywords: CHD5 eukaryotic expression vector stable transfected HEK293 cell line
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