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

摘要/Abstract

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:

朱伟, 黎桂仙, 陈洪浪, 尹金宝, 姜恩平. 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[J]. 吉林大学学报(医学版), 2016, 42(03): 424-429.

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.

参考文献

[1] Zhang NN,Chen JN,Xiao L,et al.Accumulation mechanisms of CD4(+)CD25(+)FOXP3(+) regulatory T cells in EBV-associated gastric carcinoma [J].Sci Rep,2015,5:18057.

[2] Strong MJ,Xu G,Coco J,et al.Differences in gastric carcinoma microenvironment stratify according to EBV infection intensity:implications for possible immune adjuvant therapy [J].PLoS Pathog,2013,9(5):e1003341.

[3] Xue QJ,Dai J,Li XZ,et al.Construction of a recombinant-BCG containing the LMP2A and BZLF1 genes and its significance in the Epstein-Barr virus positive gastric carcinoma[J].J Med Virol,2014,86(10):1780-1787.

[4] Chen Y,Sun H,Liu G,et al.EBV LMP2A-specific T cell immune responses elicited by dendritic cells loaded with LMP2A protein [J].Cell Mol Immunol,2009,6(4):269-276.

[5] Huang CC,Kuo KK,Cheng TC,et al.Development of membrane-bound GM-CSF and IL-18 as an effective tumor vaccine [J].PLoS One,2015,10(7):e0133470.

[6] Xu H,Wang Q,Lin C,et al.Synergism between cryoablation and GM-CSF:enhanced immune function of splenic dendritic cells in mice with glioma [J].Neuroreport,2015,26(6):346-353.

[7] Baer R,Bankier AT,Biggen MD,et al.DNA sequence and expression of the B95-8 Epstein-Barr virus[J].Nature,1984,310(5974):207-211.

[8] Wong G,Witek J,Temple P,et al.Human GM-CSF:molecular cloning of the complementary DNA and purification of the natural and recombinant proteins [J].Science,1985,228(4701):810-815.

[9] Böttcher R,Hollmann M,Merk K,et al.Efficient chromosomal gene modification with CRISPR/cas9 and PCR-based homologous recombination donors in cultured Drosophila cells [J].Nucleic Acids Res,2014,42(11):e89.

[10] 蔡学敏,朱向情,刘凌,等.人外周血树突状细胞的诱导、鉴定及活性测定[J].中华细胞与干细胞杂志:电子版,2012,2(4):231-236.

[11] Lichtor T,Glick RP.Immunogene therapy[J].Adv Exp Med Biol,2012,746:151-165.

[12] Evans CH,Ghivizzani SC,Robbins PD.Getting arthritis gene therapy into the clini [J].Nat Rev Rheumatol,2011,7(4):244-249.

[13] Brewin J,Mancao C,Straathof K,et al.Generation of EBV-specific cytotoxic T cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease [J].Blood,2009,114(23):4792-4803.

[14] Berthomé M,Gallot G,Vivien R,et al.Viral DNA contamination is responsible for Epstein-Barr virus detection in cytotoxic T lymphocytes stimulated in vitro with Epstein-Barr virus B-lymphoblastoid cell line [J].Cancer Immunol Immunother,2010,59(12):1867-1875.

[15] Lee CH,Yeh TH,Lai HC,et al.Epstein-Barr virus zta-Induced immunomodulators from nasopharyngeal carcinoma cells upregulate interleukin-10 production from monocytes [J].J Virol,2011,85(14):7333-7342.

[16] Glasgow JN,Everts M,Curiel DT.Transductional targeting of adenovirus vectors for gene therapy [J].Cancer Gene Ther,2006,13(9):830-844.

[17] 段秀梅,邹亚斌,马洪喜,等.结核杆菌热休克蛋白70刺激表位在融合基因疫苗中的佐剂作用[J].吉林大学学报:医学版,2012,38(3):506-511.

[18] Jin J,Wang B,Zhu Z,et al.Construction of a recombinant eukaryotic expression vector containing a leptin gene and its expression in HPMSCs[J].Cytotechnology,2014,66(3):471-479.