应用siRNA特异沉默EBV潜伏期基因EBNA2表达的初步研究

发布时间：2018-01-15 14:25

本文关键词：应用siRNA特异沉默EBV潜伏期基因EBNA2表达的初步研究　出处：《青岛大学》2007年硕士论文　论文类型：学位论文

【摘要】： EBNA2是EBV感染宿主细胞后最先表达的病毒基因产物之一，由BYRF1基因编码，与细胞内蛋白无任何明显的同源序列。EBNA2是一种病毒转录因子，不能直接与DNA结合，但可通过与细胞抑制重组信号结合蛋白RBP-Jκ及其它一些胞内DNA结合蛋白的结合间接作用于EBNA2反应启动子。EBNA2具有核定位序列、DNA结合部位及转录激活部位等功能区域，是首个被确定与病毒细胞转化功能有密切关系的病毒蛋白。EBNA2缺失的EBV毒株P3-HR1失去转化B细胞的能力，提示我们可以通过干扰EBNA2的表达影响其转化细胞的能力。同时作为EBV BYRF1基因特异性表达的产物，EBNA2可以反映EBV的存在情况以及在细胞内的拷贝数。 RNAi是由双链RNA介导的遗传干扰现象，能够特异、有效地降解mRNA，引起转录后水平的基因沉默；研究表明，21-23nt的小干扰RNA(small interferenceRNA，siRNA)可介导哺乳动物细胞特定基因的沉默。RNAi具有高效性和高度特异性，可能成为封闭基因的新技术而在基因功能研究和疾病基因治疗中发挥重要作用。大量研究结果证实，siRNA可以有效抑制所有内源性基因的mRNA从而敲除该基因的功能，目前siRNA已经成为敲除特定基因在哺乳动物细胞系中表达的强大工具。本研究中我们化学合成了靶向EBNA2编码基因的特异性siRNA，采用脂质体法转染EBVⅢ型潜伏的胃癌上皮细胞GT38，观察分析siRNA对EBNA2基因表达的抑制作用以及靶基因沉默对GT38细胞的影响。同时构建能转录产生靶向EBNA2编码基因的siRNA的pSUPER逆转录病毒载体，进而筛选出稳定产毒的细胞克隆，以观察比较两种干涉方式对目的基因表达的抑制效果以及对靶细胞的影响。第一部分化学合成siRNA对GT38细胞EBNA2基因表达的抑制作用目的采用化学合成法合成针对EBNA2编码基因的siRNA，分别转染EBV阳性肿瘤细胞GT38细胞，观察人工合成siRNA对EBNA2的特异沉默效果以及EBNA2特异性沉默对EBV阳性肿瘤细胞的影响。方法①化学合成3组靶向EBNA2编码基因的siRNA，以EBVⅢ型潜伏的胃癌上皮细胞GT38为靶细胞，采用脂质体法分别将3组siRNA转染GT38细胞，同时设脂质体对照和细胞对照。②采用RT-PCR检测靶基因EBNA2 mRNA的转录表达水平，并筛选出最佳转染浓度及抑制效果最为理想的siRNA链。③将抑制效果最好的siRNA链以最佳转染浓度转染靶细胞，采用Hoechst 33258、透射电镜和流式细胞术检测GT38细胞周期和凋亡的变化。结果①RT-PCR检测结果表明，与未转染siRNA的细胞对照相比，siRNA789、siRNA764和siRNA257对GT38细胞EBNA2的表达均具有明显的抑制作用(P＜0.01)，其中以siRNA789的抑制作用更强；②与未转染的细胞对照比较，转染siRNA789后48h和72h GT38细胞中EBNA2 mRNA表达水平明显下降，两两间比较均有显著性差异(P＜0.01)，而siRNA789转染后24h抑制效果不明显(P＞0.05)；③在0～50nM浓度范围内siRNA789转染72h后EBNA2 mRNA转录表达水平随浓度增加而逐渐降低，差别均有统计学意义(P＜0.05)；当siRNA作用浓度为50nM～100nM siRNA各组间差别不明显(P＞0.05)，转染非特异siRNA细胞组与细胞对照比较无显著性差异(P＞0.05)；④Hoechst 33258染色和透射电镜结果显示，与对照组比较，，实验组GT38细胞未见胞核浓缩，也未观察到凋亡小体；流式细胞分析结果表明，与对照组比较，实验组细胞未出现凋亡，但其S其细胞数量明显减少。结论化学合成siRNA能有效抑制GT38细胞中EBNA2编码基因的表达，其抑制作用具有时间依赖性，在一定浓度范围内呈明显的量效关系，且对靶基因的特定序列具有较强的选择偏向性。化学合成siRNA诱导EBNA2编码基因沉默后，不能引起靶细胞明显的凋亡，但可引起靶细胞S期数量减少。第二部分特异性沉默BV潜伏期基因EBNA2 pSUPER retro RNAi系统的构建目的构建能转录产生靶向EBV核抗原EBNA2编码基因siRNA的pSUPER逆转录病毒载体，并筛选出稳定产毒的细胞克隆。方法采用DNA重组技术将60nt能转录产生靶向EBNA2小发夹RNA(small hairpin RNA，shRNA)的寡核苷酸序列定向克隆入逆转录病毒载体pSUPER.retro.neo gfp，并用限制性内切酶酶切鉴定以及测序鉴定；脂质体法将重组逆转录病毒载体转染包装细胞系PA317，G418筛选获得稳定产生逆转录病毒的细胞克隆。结果酶切鉴定及测序鉴定结果表明插入片段的序列和方向完全正确；重组载体转染包装细胞后可表达绿色荧光蛋白，经G418筛选获得可稳定产生逆转录病毒的抗性细胞克隆；病毒滴度为2.5×10~5 CFU／ml。结论成功构建了能转录产生靶向EBV核抗原EBNA2编码基因siRNA的pSUPER逆转录病毒载体，经PA317细胞包装筛选出稳定产毒的细胞克隆，为进一步探讨EBNA2在EBV相关肿瘤发生发展的生物学意义提供了实验基础。
[Abstract]:EBNA2 is one of the viral gene product EBV infection of host cells after the first expression, encoding by BYRF1 gene, no obvious homology.EBNA2 is a viral transcription factor and intracellular protein, not directly combined with DNA, but the RBP-J kappa binding protein and other intracellular DNA binding protein with an indirect effect on the reaction of EBNA2.EBNA2 promoter with nuclear localization sequence and cell inhibition of recombinant signal, DNA binding site and transcription activation site and other functional areas, EBV is the first P3-HR1 strain of virus protein was identified and transformed virus cell function have close relationship to the deletion of the.EBNA2 lost the ability to transform B cells, suggesting that we can influence the ability of transformed cells by the expression of EBNA2 interference. At the same time as the product of EBV BYRF1 gene specific expression, EBNA2 can reflect the existence of EBV and the copy number within the cell.
RNAi is the phenomenon of genetic interference mediated by double stranded RNA can specifically and efficiently degrade mRNA, cause post transcriptional gene silencing; studies show that small interfering RNA 21-23nt (small interferenceRNA siRNA) can mediate specific gene silencing in mammalian cells.RNAi with high efficiency and high specificity, may be the new technology of closed gene and gene in the gene function study and treatment of diseases play an important role. A large number of research results confirmed that siRNA can effectively inhibit all endogenous gene mRNA to knockdown the gene function, the siRNA has become a powerful tool for knockout specific gene expression in mammalian cell lines.
In this study we chemically synthesized specific siRNA EBNA2 encoding gene, gastric epithelial cells GT38 transfected with EBV type III latent by liposome method, observation and analysis of the inhibition effect of siRNA on the expression of EBNA2 gene and the target gene silencing on GT38 cells. At the same time to construct encoded targeting pSUPER retroviral vector siRNA the EBNA2 encoding gene, and then screened stable cell clones producing toxin, to observe the inhibitory effect of two kinds of interference on the expression of target gene and effect on target cells.
The inhibitory effect of chemical synthesis of siRNA on the expression of EBNA2 gene in GT38 cells
Objective to synthesize siRNA targeting EBNA2 coding gene by chemical synthesis, and transfect EBV positive tumor cells into GT38 cells. Observe the effect of synthetic siRNA on EBNA2 and the effect of EBNA2 specific silence on EBV positive tumor cells.
The siRNA method of chemical synthesis of 3 groups of target gene encoding EBNA2, gastric epithelial cells GT38 with EBV type III latent target cells by liposome method. The 3 groups of siRNA were transfected into GT38 cells, and liposome control and control cells. The expression level of the transcription of RT-PCR target gene EBNA2 and mRNA. Select the most ideal siRNA chain for the best transfection concentration and inhibition effect. The best inhibitory effect of siRNA chain with the best transfection concentration of transfected target cells by Hoechst 33258, the change of GT38 cell cycle and apoptosis was detected by electron microscopy and flow cytometry.
Results the results of RT-PCR showed that siRNA789 and siRNA transfected cells compared to the control, the expression of siRNA764 and siRNA257 on GT38 EBNA2 cells were significantly inhibited (P < 0.01), the stronger the inhibition of siRNA789; and the non transfected cells compared with EBNA2 48h and 72h GT38 cells the expression level of mRNA was significantly decreased after transfection of siRNA789, there were significant differences between the 22 (P < 0.01), and siRNA789 24h after transfection had no obvious inhibition effects (P > 0.05); in 0 ~ 50nM in the concentration range of siRNA789 after transfection of 72h EBNA2 mRNA mRNA expression level decreased gradually with the increase of concentration difference statistical significance (P < 0.05); when the concentration of siRNA 50nM ~ 100nM siRNA no significant differences between groups (P > 0.05), non transfection of specific siRNA cells and cell control group showed no significant difference (P > 0.05); Hoechst 33258 The results of staining and transmission electron microscopy showed that compared with the control group, the GT38 cells in the experimental group did not concentrate on the nucleus and no apoptotic bodies were observed. Flow cytometry analysis showed that compared with the control group, the cells in the experimental group did not show apoptosis, but the number of S cells in the experimental group was significantly reduced.
Conclusion the chemical synthesis of siRNA can effectively inhibit the expression of EBNA2 encoding gene in GT38 cells, the inhibition is time dependent, was in a dose-dependent manner within a certain concentration range, and the specific sequence of target gene with strong selection bias. The chemical synthesis of siRNA induced by EBNA2 encoding gene silencing, can induce apoptosis of target cells, but can cause the number of target cells in S phase decreased.
Construction of the second part specific silent BV latency gene EBNA2 pSUPER retro RNAi system
Objective to construct a pSUPER retrovirus vector capable of transcriptional production of the target EBV nuclear antigen EBNA2 encoding gene siRNA, and to screen out the stable and toxic cell clones.
Methods the 60nt encoded targeting EBNA2 small hairpin RNA using recombinant DNA Technology (small hairpin RNA, shRNA) oligonucleotide sequences cloned into the retroviral vector pSUPER.retro.neo GFP and restriction enzyme digestion and sequencing; Lipofectamine recombinant retroviral vector was transfected into packaging cell line PA317 cell clone G418 stably produce retrovirus.
The results of enzyme digestion and sequencing showed that the sequence and direction of insert completely correct; green fluorescent protein expression recombinant vector was transfected into packaging cell, can obtain clones stably produce retrovirus by G418 screening; virus titer was 2.5 * 10~5 / ml. CFU
Conclusion we have successfully constructed the encoded targeting pSUPER retroviral vector EBNA2 encoding gene siRNA EBV nuclear antigen, PA317 cells were screened by packaging cell clones stably produce poison, it provides the experimental basis for further study of the biological significance of EBNA2 in EBV is related to the occurrence and development of tumor.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R373

【引证文献】