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

doi:10.13481/j.1671-587x.20160302

Abstract

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

CLC Number:

ZHU Wei, LI Guixian, CHENG Honglang, YIN Jinbao, JIANG Enping. Construction of CSF2A fusion gene recombinant eukaryotic expression vector and its effect on proliferation and apoptosis of EBV⁺ tumor cells[J].Journal of Jilin University Medicine Edition, 2016, 42(03): 424-429.

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

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

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