鸡黑素瘤差异化相关基因5启动子的克隆及功能验证

发布时间：2018-01-10 10:24

本文关键词：鸡黑素瘤差异化相关基因5启动子的克隆及功能验证　出处：《广西大学》2016年博士论文　论文类型：学位论文

【摘要】：天然免疫是机体抵抗病原体入侵的第一道屏障,能够利用模式识别受体(Pattern recognition receptors,PRRs)识别病原体相关分子模式(Pathogen associated molecular pattern,PAMP)。模式识别受体可分为toll样受体(Toll-like receptors,TLRs)、核苷酸结合寡聚化结构域蛋白样受体(Nucleotide-binding oligomerization domain protein-like receptors,NLRs)和视黄酸诱导基因 Ⅰ 样受体(Retinoicacid-inducible gene Ⅰ(RIG-I)-like receptors,RLRs)等受体家族。RLR受体家族包括视黄酸诱导基因Ⅰ(RIG-I)、黑素瘤差异化相关基因 5(Melanoma differentiation-associated gene 5,MDA5)和实验室遗传与生理学蛋白2(Laboratory of genetics and physiology 2,LGP2)。这些家族成员在对胞内的RNA病毒识别过程中起着重要作用,并能够触发一系列抗病毒天然免疫反应。在哺乳动物中,RIG-I和MDA5能够识别不同但又有重叠的RNA病毒种类。RIG-I主要识别不同长度的、具有5'端三磷酸基团或者二磷酸基团的双链RNA病毒,或长度小于1 kb的短链人工合成病毒RNA类似物聚肌苷酸-聚胞苷酸(Polyinosinic-polycytidylic acid,poly(I:C))双链 RNA 病毒;MDA5则主要识别大于1 kb的长的、双链小核糖核酸病毒和长链poly(I:C)。然而,MDA5的配体尚未完全明确。RIG-I在鸡的基因组中是缺失的,这可能是相对于表达RIG-I基因的鸭和其他哺乳动物,鸡对某些特定的病原体更易感的原因。因此,在鸡上,MDA5对细胞质中的RNA病毒的识别及抗病毒天然免疫反应的触发显得尤为重要。但是,目前鸡MDA5基因的相关研究还较少,其表达的分子调控机制尚未明确。为此,在本研究中,我们克隆并分析MDA5全长启动子序列,研究其在病毒和非病毒物质刺激时的活性状态,进而为利用此启动子制备转基因抗病鸡种奠定基础。具体研究结果如下:1.本研究以白羽鸡基因组DNA为模板进行PCR扩增,成功克隆出长度约为2.5 kb的鸡MDA5全长启动子序列。经预测,这段启动子区域中含有多个转录因子结合位点,包括Sp1、GATA-1、TGGCA-结合蛋白、AP-1、AP-3、ER-alpha、T3R-alpha、C/EBP alpha、NF-1(-样蛋白)和NF-E4的结合位点;为了探讨鸡与其他物种在进化上的关系,本研究将此启动子和其他鸟类、鱼和哺乳动物的启动子作了进化树分析。结果显示,就MDA5启动子而言,鸡与其他鸟类的进化关系较近,与哺乳动物的进化关系较远。2.本研究构建了此启动子的报告载体质粒piggybac-MDA5-DsRed,并用此质粒对鸡DF-1细胞进行转染。经过药物筛选,本研究得到了稳定转染质粒 piggybac-MDA5-DsRed 的细胞系,命名为 Piggybac-MDA5-DsRed 细胞系。在此细胞系中,DsRed的表达情况代表着MDA5启动子的活性。通过荧光显微镜观察和流式细胞仪分析,本研究发现:在正常生理条件下,表达DsRed的细胞非常少,几乎为零,表明MDA5启动子的活性极低;然而,当细胞受到长链poly(I:C)(1 kb)和短链poly(I:C)(1 kb)、干扰素-β(Interferon β,IFN-β)和鸡传染性法氏囊病毒的刺激(Infectious bursal disease virus,IBDV)时,表达DsRed的细胞数量显著上升,分别达到了 18%和18.0%、9.86%、2.01%和1.01%,说明MDA5启动子的活性被极大地激活了。实时定量PCR结果显示,细胞在受到poly(I:C)、IFN-β和IBDV的刺激时,DsRed的mRNA水平均得到显著的提高,与内源性MDA5和IFN-β的mRNA水平变化趋势是一致的。由于DsRed的mRNA水平代表MDA5启动子的活性,故本研究中的启动子活性与内源性MDA5的表达水平是一致的,启动子活性可以反应内源性MDA5的表达情况。此外,本研究的结果显示,相对于短链poly(I:C),长链poly(I:C)刺激能诱导更多细胞表达DsRed,引起更大的mRNA水平变化,说明相对于短链poly(I:C),鸡MDA5能够更好地识别长链poly(I:C)。同时,长链poly(I:C)的刺激不仅能够促进细胞内IFN-β表达,还能够促进细胞内IFN-α表达,而短链poly(I:C)刺激未能引起IFN-α表达量的变化,故推测长链poly(I:C)和短链poly(I:C)引导的下游信号通路略有差异。3.为了验证鸡MDA5启动子在鸡转基因抗病育种中的应用价值,本研究以PB转座子为背景,构建了由鸡MDA5启动子启动表达鸭RIG-I基因的重组表达载体,命名为PB-chMDA5-duRIG-I-mkate。试验证明,在正常情况下,检测不到鸭RIG-I的表达;而在病毒模拟物poly(I:C)刺激后,以此质粒为依托的鸭RIG-I能够在鸡DF-1细胞中表达。本研究首次成功克隆出了鸡MDA5启动子并对其功能进行了初步研究。本研究的结果表明,此启动子与得到的Piggybac-MDA5-DsRed细胞系可以用来检测鸡MDA5的配体,验证其能否调节内源性MDA5基因的表达。此外,由于此启动子的活性在正常生理条件下极低,但又在外源刺激物的作用下极大地提高,因此可以被用来启动表达其他抗病毒蛋白等目的蛋白,具有较好的应用价值。本研究对鸡天然免疫系统中的重要模式识别受体MDA5的启动子进行初步探索,将为针对鸡MDA5基因表达调控机制的研究提供理论依据,有利于完善人们对鸡天然免疫系统的认知。同时,本研究构建的鸭RIG-I表达载体质粒PB-chMDA5-duRIG-I-mkate,为表达RIG-I的转基因鸡的生产奠定了基础,进而为生产抗病毒转基因鸡开辟新了的道路。
[Abstract]:Innate immunity is the first barrier against pathogen invasion in the organism, can use pattern recognition receptors (Pattern, recognition receptors, PRRs) recognition of pathogen associated molecular patterns (Pathogen associated molecular pattern, PAMP). Pattern recognition receptors can be divided into toll like receptors (Toll-like, receptors, TLRs) nucleotide binding oligomerization domain protein receptor (Nucleotide-binding oligomerization domain protein-like receptors, NLRs) and retinoic acid inducible gene 1 receptor (Retinoicacid-inducible gene 1 (RIG-I) -like receptors, RLRs) and.RLR receptor family receptors including retinoic acid inducible gene 1 (RIG-I), melanoma differentiation associated gene 5 (Melanoma differentiation-associated 5 gene, MDA5) and genetic laboratory with the physiology of protein 2 (Laboratory of genetics and physiology 2, LGP2). The family members in the RNA plays an important role in the process of intracellular virus identification, and can trigger a series of antiviral innate immune responses. In mammals, RIG-I and MDA5 can identify different but have different length of RNA virus type.RIG-I mainly recognize overlapping, double stranded RNA virus with 5'terminal three phosphate groups or two phosphate groups short chain, synthetic analogues of RNA virus or less than 1 kb in length polyinosinic polycytidylic acid (Polyinosinic-polycytidylic, acid, poly (I:C)) double stranded RNA virus; MDA5 is mainly identified more than 1 KB long, small double stranded RNA viruses and long chain poly (I:C). However, MDA5 the ligand.RIG-I is not completely clear in the chicken genome is missing, which may be relative to the expression of RIG-I gene of duck and other mammals, chicken to certain pathogens are more susceptible. Therefore, in the chicken, MDA5 of RN in the cytoplasm Identification and antiviral innate immune response to A virus trigger is particularly important. However, the related research of chicken MDA5 gene is still less, the expression of the molecular regulation mechanism is not clear. Therefore, in this study, we cloned and analyzed the full-length MDA5 promoter sequence, to study the viral and non viral substances when stimulated the active state, and the promoter of preparation of disease resistant transgenic chicken to lay the foundation. The results are as follows: 1. in this study, white chicken genome DNA as template for PCR amplification, clone a length of about 2.5 KB of the chicken MDA5 full-length promoter sequence. After the forecast, contains a number of transcription factor binding sites this section of the promoter region including Sp1, GATA-1, TGGCA- binding protein, AP-1, AP-3, ER-alpha, T3R-alpha, C/EBP, alpha, NF-1 (- like protein) and NF-E4 binding sites; in order to investigate the chicken and other species in evolution. In this study the relationship between promoter and other birds, fish and mammalian promoter as phylogenetic analysis. The results showed that the MDA5 promoter, close evolutionary relationship of chicken and other birds and mammals, the evolutionary relationship is far.2. the establishment of this promoter reporter plasmid piggybac-MDA5-DsRed. And the chicken DF-1 cells were transfected with this plasmid. After drug screening, this study obtained the stable transfection of plasmid piggybac-MDA5-DsRed cell line, named Piggybac-MDA5-DsRed cells. This cell line, the expression of DsRed represents the situation of MDA5 promoter activity. By fluorescence microscopy and flow cytometric analysis, this study found: under normal physiological conditions, the expression of DsRed is very small, almost zero, showed that MDA5 promoter activity is very low; however, when cells were long chain poly (I:C) (1 KB) and short Chain poly (I:C) (1 KB), interferon beta (Interferon beta, beta IFN-) and infectious bursal disease virus (Infectious bursal disease virus, stimulation, IBDV) expression significantly increased the number of DsRed cells, respectively 18% and 18%, 9.86%, 2.01% and 1.01%, said that MDA5 start the activity was greatly activated. Real time quantitative PCR results showed that cells in response to poly (I:C), IFN- beta and IBDV stimulation, DsRed mRNA levels were significantly improved, is consistent with the change of mRNA level of endogenous MDA5 and IFN- beta trend. Due to the DsRed level of mRNA on behalf of MDA5 promoter the expression level of activity, so the study of promoter activity and endogenous MDA5 is consistent with the expression of promoter activity can react with endogenous MDA5. In addition, the results of this study show that, compared with short chain poly (I:C), long chain poly (I:C) stimulation can induce more cells expressing Ds Red, mRNA level changes caused by the larger, relative to that of short chain poly (I:C), chicken MDA5 better able to identify long chain poly (I:C). At the same time, the long chain poly (I:C) stimulation can promote the expression of IFN- beta cells, but also can promote the expression of IFN- in cells, while the short chain poly (I:C) stimulation failed to induce changes in the expression of IFN- alpha, it is speculated that the long chain and short chain poly (I:C) poly (I:C) signaling pathways downstream of slight differences in the.3. guide in order to verify the application value of chicken MDA5 promoter in chicken transgenic breeding. In this study, PB transposon background, construct promoter expression vector expression of duck RIG-I gene by recombinant chicken MDA5 promoter, named as PB-chMDA5-duRIG-I-mkate. proof test, under normal circumstances, the expression of duck RIG-I was not detected; and the simulation of poly in virus (I:C) after stimulation with this plasmid based on duck RIG-I can be expressed in DF-1 cells in the chicken. The first study successfully cloned the chicken MDA5 promoter and its function were studied. The results of this study suggest that this ligand promoter and Piggybac-MDA5-DsRed cell lines can be used to detect the expression of chicken MDA5, verify whether the regulation of endogenous MDA5 gene. In addition, the promoter activity in normal physiological conditions under very low, but the exogenous stimulus greatly improved, therefore can be used to initiate the expression of other antiviral protein to protein, and has good application value. This study explores the promoter of important pattern recognition receptors of the innate immune system of chicken MDA5, provide a theoretical basis for the research the chicken MDA5 gene expression regulation mechanism, is conducive to improve the cognition of the chicken innate immune system. At the same time, this study constructed RIG-I expression vector plasmid PB-chMDA5-duRI duck G-I-mkate, which lays the foundation for the production of transgenic chicken with RIG-I, opens a new way for the production of antiviral transgenic chicken.

【学位授予单位】：广西大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S858.31

【相似文献】