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葛祥伟, 张德宇, 翟今朝, 卢迪, 叶棋浓, 汪进良. 人HPIP基因真核表达载体的构建及其对肺癌细胞的生物学影响[J]. 解放军医学院学报, 2021, 42(12): 1293-1297. DOI: 10.3969/j.issn.2095-5227.2021.12.012
引用本文: 葛祥伟, 张德宇, 翟今朝, 卢迪, 叶棋浓, 汪进良. 人HPIP基因真核表达载体的构建及其对肺癌细胞的生物学影响[J]. 解放军医学院学报, 2021, 42(12): 1293-1297. DOI: 10.3969/j.issn.2095-5227.2021.12.012
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GE Xiangwei, ZHANG Deyu, ZHAI Jinzhao, LU Di, YE Qinong, WANG Jinliang. Eukaryotic expression vector of human HPIP gene and its biological effect on lung cancer cells[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL, 2021, 42(12): 1293-1297. DOI: 10.3969/j.issn.2095-5227.2021.12.012
Citation: GE Xiangwei, ZHANG Deyu, ZHAI Jinzhao, LU Di, YE Qinong, WANG Jinliang. Eukaryotic expression vector of human HPIP gene and its biological effect on lung cancer cells[J]. ACADEMIC JOURNAL OF CHINESE PLA MEDICAL SCHOOL, 2021, 42(12): 1293-1297. DOI: 10.3969/j.issn.2095-5227.2021.12.012
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人HPIP基因真核表达载体的构建及其对肺癌细胞的生物学影响

Eukaryotic expression vector of human HPIP gene and its biological effect on lung cancer cells

    摘要
  • 摘要:
      背景   人造血前B细胞白血病转录因子相互作用蛋白(hematopoietic pre-B cell leukemia transcription factor interacting protein，HPIP)参与多种肿瘤的发生发展过程，其在肺癌中的机制仍有待研究。
      目的  构建带有Flag标签的HPIP真核表达载体，表达pcDNA3.0-Flag-HPIP融合蛋白，检测其对肺癌细胞增殖、迁移等生物学功能的影响。
      方法   利用人乳腺文库作为模板，PCR技术扩增HPIP基因的编码序列，将其酶切产物连接到pcDNA3.0-Flag载体并测序。将重组质粒转染至A549肺癌细胞系中，Western blot验证转染后HPIP的蛋白表达情况。采用克隆形成实验、CCK-8法及细胞划痕实验等明确其对肺癌细胞增殖、迁移等功能的影响。
      结果   PCR扩增得长度为2 109 bp的HPIP基因编码序列，并成功插入至pcDNA3.0-Flag载体中。测序和Western blot验证融合蛋白构建成功。CCK-8法和克隆形成实验表明，与对照组相比，转染Flag-HPIP后肺癌A549细胞增殖能力显著增强。划痕实验证实转染Flag-HPIP后可明显提高肺癌A549细胞的迁移能力。
      结论   本实验成功构建了带有Flag标签的HPIP基因真核表达载体，并证实其能够促进肺癌细胞的增殖和迁移，为后续HPIP在肺癌中的功能研究奠定了良好基础。

     

    Abstract:
      Background   Hematopoietic pre-B cell leukemia transcription factor (PBX)-interacting protein (HPIP) is involved in the development of many malignant tumors, but its mechanism in lung cancer remains to be explored.
      Objective  To construct the eukaryotic expression vector of HPIP labeled with Flag-tag, express the pcDNA3.0-Flag-HPIP fusion protein, and detect its effect on the proliferation, migration, and other biological functions of human lung cancer cells.
      Methods  Using human breast library as template, the coding sequence of HPIP gene was amplified by PCR technology, and inserted into pcDNA3.0-Flag vector. The recombinant plasmid was transfected into A549 lung cancer cell line, and the expression of HPIP after transfection was verified by western blotting. The effect of the protein on the cell proliferation and migration of lung cancer cell line were assessed by CCK-8, clone formation assay and wound healing assay, respectively.
      Results  The encoding sequence of HPIP gene with a length of about 2 109 bp was amplified by PCR and successfully inserted into pcDNA3.0-Flag vector. Sequencing and western blotting confirmed the successful construction of the fusion protein. CCK-8 assay and clone formation assay showed that the proliferation ability of lung cancer A549 cells transfected with Flag-HPIP was significantly enhanced compared with the control group. Wound healing assay confirmed that the transfection of Flag-HPIP could significantly improve the migration ability of lung cancer A549 cells.
      Conclusion  The eukaryotic expression vector of HPIP gene with Flag label is successfully constructed, and it is confirmed that the fusion protein can promote the proliferation and migration of lung cancer cells, which lays a good foundation for the subsequent functional studies of HPIP in lung cancer.

     

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