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  

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

作　　者：朱伟[1] 黎桂仙 陈洪浪[2] 尹金宝[1] 姜恩平[1] 

机构地区：[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

出　　处：《吉林大学学报（医学版）》2016年第3期424-429,I0001,共7页Journal of Jilin University：Medicine Edition

基　　金：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)

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。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 CSF2 Afusion protein on the proliferation and apoptosis of EBV positive（EBV＋）tumor cells.Methods：According to the principle of overlap extension,the LMP2 Aand GM-CSF gene fragments were restructured by connecting the coding（Gly4Ser）3polypeptide 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-CSF2 A plasmid was established as experimental group,while the pIRES2-LMP2 Aplasmid 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 CSF2 A 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 CSF2 Afusion gene was inserted into the pIRES2-eGFP plasmid,and the recombinant eukaryotic expression vector named pIRES2-CSF2 A was successfully obtained.The CSF2 Aprotein expression was found in DC after transfected with pIRES2-CSF2 A.The MTT results prompted that the inhibitory rate of proliferation of the cells in pIRES2-CSF2 Aplasmid 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-CSF2 Agroup was increased significantly at 48 hafter treatment.Conclusion：The recombinant eukaryotic expression vect

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 

分 类 号：Q75[生物学—分子生物学] Q78