人Bcl2基因真核表达载体的构建及其抗凋亡功能研究
인Bcl2기인진핵표체재체적구건급기항조망공능연구
Construction of myc-Tagged Human Bcl2 Eukaryotic Expression Vector and its Anti-Apoptotic Activity Detection
  • 万方数据
    生物技术通讯 생물기술통신
    LETTERS IN BIOTECHNOLOGY
    2015年 2期 186-189 ,共4页

    史平安%徐小洁%闫志风%范忠义%王涛%冯滢滢%梁迎春%李玲%郭靖%叶棋浓%吕朝晖

    사평안%서소길%염지풍%범충의%왕도%풍형형%량영춘%리령%곽정%협기농%려조휘

    人Bcl2基因%克隆%细胞凋亡 人Bcl2基因%剋隆%細胞凋亡
    인Bcl2기인%극륭%세포조망
    Bcl2%cloning%apoptosis
    目的：构建带myc标签的Bcl2真核表达载体，获得myc-Bcl2融合蛋白，并对其生物学功能进行初步检测。方法：以本实验室保存的乳腺文库为模板，采用PCR技术扩增Bcl2编码序列，将其插入pCMV-myc载体，Western印迹检测表达情况；将重组质粒与空载体分别转染乳腺癌MCF-7细胞，通过流式细胞仪检测pCMV-myc-Bcl2重组质粒对细胞凋亡的影响。结果：双酶切和测序结果表明pCMV-myc-Bcl2真核表达质粒构建成功；转染293T细胞后myc-Bcl2蛋白成功表达；流式细胞仪检测结果显示，myc-Bcl2明显抑制乳腺癌细胞系的凋亡。结论：构建了带myc标签的人Bcl2真核表达载体，为进一步研究Bcl2在细胞凋亡中的功能奠定了基础。
    목적：구건대myc표첨적Bcl2진핵표체재체，획득myc-Bcl2융합단백，병대기생물학공능진행초보검측。방법：이본실험실보존적유선문고위모판，채용PCR기술확증Bcl2편마서렬，장기삽입pCMV-myc재체，Western인적검측표체정황；장중조질립여공재체분별전염유선암MCF-7세포，통과류식세포의검측pCMV-myc-Bcl2중조질립대세포조망적영향。결과：쌍매절화측서결과표명pCMV-myc-Bcl2진핵표체질립구건성공；전염293T세포후myc-Bcl2단백성공표체；류식세포의검측결과현시，myc-Bcl2명현억제유선암세포계적조망。결론：구건료대myc표첨적인Bcl2진핵표체재체，위진일보연구Bcl2재세포조망중적공능전정료기출。
    Objective: To construct the eukaryotic expression vector of human Bcl2 labeled with myc tag and de?tect its activity. Methods: Human Bcl2 gene was obtained from mammary gland library by PCR and cloned into pCMV-myc vector. Human 293T cells were transfected with the recombinant plasmid pCMV-myc-Bcl2 and the ex?pression of myc-Bcl2 was detected by Western blotting. Human breast cancer MCF-7 cells were transfected with pCMV-myc-Bcl2 and the cell apoptosis was examined by flow cytometry analysis. Results: The constructed hu?man Bcl2 eukaryotic expression vector was identified by double digestion, and the inserted fragment was confirmed by sequencing. The expression of human Bcl2 protein in human 293T cells was identified by Western blotting. In addition, flow cytometry analysis showed that human Bcl2 protein could restrain cell apoptosis significantly. Conclu?sion: The eukaryotic expression vector pCMV-myc-Bcl2 was constructed and myc-Bcl2 was expressed in human 293T cells, which laid foundation for the further study of the role of Bcl2 in cell apoptosis.
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