吉林大学学报(医学版) ›› 2016, Vol. 42 ›› Issue (03): 424-429.doi: 10.13481/j.1671-587x.20160302

• 基础研究 • 上一篇    下一篇

CSF2A融合基因真核表达载体的构建及其对EBV+肿瘤细胞增殖和凋亡的影响

朱伟1, 黎桂仙1, 陈洪浪2, 尹金宝1, 姜恩平1   

  1. 1. 广东医学院病理学教研室, 广东 东莞 523808;
    2. 广东医学院药理学教研室, 广东 东莞 523808
  • 收稿日期:2015-10-06 发布日期:2016-06-17
  • 通讯作者: 姜恩平,副教授,硕士研究生导师(Tel:0769-22896401,E-mail:jiangenping168@126.com) E-mail:jiangenping168@126.com
  • 作者简介:朱伟(1970-),男,山东省济宁市人,副教授,医学博士,主要从事EB病毒相关肿瘤免疫和结直肠癌发病机制方面的研究。
  • 基金资助:

    国家自然科学基金资助课题(81472275);广东省科技厅自然科学基金资助课题(2014A030313542);广东省卫生厅医学科研基金资助课题(A2015372);广东医学院博士启动基金资助课题(B2011004)

Construction of CSF2A fusion gene recombinant eukaryotic expression vector and its effect on proliferation and apoptosis of EBV+ tumor cells

ZHU Wei1, LI Guixian1, CHENG Honglang2, YIN Jinbao1, JIANG Enping1   

  1. 1. Department of Pathology, Guangdong Medical University, Dongguan 523808, China;
    2. Department of Phamacology, Guangdong Medical University, Dongguan 523808, China
  • Received:2015-10-06 Published:2016-06-17

摘要:

目的: 构建爱泼斯坦-巴尔(EB)病毒(EBV)潜伏膜蛋白2A(LMP2A)基因与粒细胞-巨噬细胞集落刺激因子(GM-CSF)融合基因(CSF2A)重组真核表达载体,探讨CSF2A融合蛋白对EBV阳性(EBV+)肿瘤细胞增殖和凋亡的影响。方法: 根据重叠延伸原理,将LMP2A与GM-CSF在编码(Gly4Ser)3多肽的DNA基因片段连接下进行重组。采用DNA重组技术将融合基因片段连接于pIRES2-eGFP真核表达载体上,行酶切电泳分析和DNA测序鉴定重组质粒。设pIRES2-CSF2A重组质粒转染组为实验组,pIRES2-LMP2A质粒转染组和pIRES2-eGFP空质粒转染组为对照组,分别转染树突状细胞(DC)。采用RT-PCR和Western blotting法检测细胞中CSF2A基因和蛋白表达;MTT和Hochest法检测各组EBV+肿瘤细胞增殖抑制率和细胞凋亡形态学。结果: 酶切实验和序列测定,CSF2A融合基因正确插入pIRES2-eGFP质粒,并成功获得重组真核表达载体pIRES2-CSF2A。转染pIRES2-CSF2A至DC后,检测到CSF2A蛋白的表达。MTT法检测,pIRES2-CSF2A质粒转染组细胞增殖抑制率高于对照组(P<0.05或P<0.01),且呈时间依赖性;Hochest检测,细胞出现凋亡形态学改变并产生凋亡小体。pIRES2-CSF2A质粒转染组作用于EBV+肿瘤细胞48h时凋亡细胞明显增多。结论: 重组真核表达载体pIRES2-CSF2A可有效转染DC,并明显抑制EBV+CNE2细胞增殖且促进其凋亡。

关键词: 潜伏膜蛋白2A, 粒细胞-巨噬细胞集落刺激因子, 融合基因, 真核表达载体

Abstract:

Objective: To construct the Epstein-Barr virus (EBV) latent membrane protein 2A(LMP2A) and gramulocyte-macrophase colony stimulating factor(GM-CSF) fusion gene (CSF2A) recombinant eukaryotic expression vector,and to discuss the effect of CSF2A fusion protein on the proliferation and apoptosis of EBV positive(EBV+) tumor cells. Methods: According to the principle of overlap extension,the LMP2A and GM-CSF gene fragments were restructured by connecting the coding (Gly4Ser) 3 polypeptide gene fragments of DNA.The fusion gene was connected with the pIRES2-eGFP eukaryotic expression vector by recombinant DNA technology,and the electrophoresis analysis of enzyme digestion and DNA sequencing methods were used to identify the recombinant plasmid.The pIRES2-CSF2A plasmid was established as experimental group,while the pIRES2-LMP2A plasmid and the pIRES2-eGFP plasmid were established as control groups.Then all the plasmids were transfected into the dendritic cells (DC),respectively.The expression levels of CSF2A gene and protein were tested by RT-PCR and Western blotting methods,separately,and the inhibitory rate of proliferation and apoptotic morphology of the EBV+ tumor cells in various groups were determined by MTT and Hochest methods. Results: The enzyme digestion experiment and sequence determination results showed that the CSF2A fusion gene was inserted into the pIRES2-eGFP plasmid,and the recombinant eukaryotic expression vector named pIRES2-CSF2A was successfully obtained.The CSF2A protein expression was found in DC after transfected with pIRES2-CSF2A.The MTT results prompted that the inhibitory rate of proliferation of the cells in pIRES2-CSF2A plasmid transfection group was obviously higher than those in control groups in a time-dependent manner.The Hochest test results showed the morphological changes of apoptosis and apoptotic body.The number of apoptotic cells in pIRES2-CSF2A group was increased significantly at 48 h after treatment. Conclusion: The recombinant eukaryotic expression vector pIRES2-CSF2A is effectively transfected into DC,and the fusion gene could significantly inhibit the proliferation of EBV+ CNE2 cells and also promote the apoptosis.

Key words: latent mumbrane protein 2A, gramulocyte-macrophase colony stimulating factor, fusion gene, eukaryotic expression vector

中图分类号: 

  • Q75