两种不同来源新城疫病毒的感染对鸡免疫作用的分子机制
发布时间：2018-11-20 22:01
【摘要】：新城疫(Newcastle Disease,ND)是由新城疫病毒(Newcastle Disease Virus,NDV)引起的一种发病急、致死率高的禽传染病,在世界范围内广为传播,是危害养禽业最大的禽病之一。由于NDV有多个基因型,常规疫苗的免疫效果并不理想,新型疫苗的研制又总是落后于病原的变异。因此,深入了解家禽抗感染作用的天然免疫调控机制,提高禽机体的免疫能力,寻找抗NDV感染的新途径显得极为重要。禽β-防御素(Avian beta defensins,Av BDs)是富含阳离子的宿主防御肽,具有抗细菌、真菌以及病毒的活性,在禽先天性免疫及获得性免疫中发挥着重要作用。为探讨禽β-防御素体内对不同来源新城疫病毒的免疫调节作用机制,本试验选用30只20日龄SPF鸡,随机分为三组,即对照组和两组攻毒组。对照组不攻毒,攻毒组采用点眼滴鼻法分别接种鸡源NDV强毒(F48E9病毒株)和鸭源NDV强毒(Md/CH/LGD/1/2005)。在攻毒后24 h后和48 h,每组各取出5只,分别采集鸡脑、气管、肺脏、肾脏、肝脏、腺胃、骨髓、脾脏、盲肠扁桃体、哈德氏腺、法氏囊组织。采用实时荧光定量RT-PCR法检测攻毒前后各组织器官中的NDV病毒载量以及13种Av BDs、8种鸡Toll样受体(Toll-like receptors,TLRs)、3种信号转导分子、11种细胞因子基因的表达量。结果显示:对照组各组织均未检测到病毒。在感染两种NDV后24 h和48 h,两个攻毒组鸡各组织均检测到病毒,且在感染后48 h,各组织的病毒载量均高于感染后24 h各组织的病毒载量。在感染后48 h,鸭源Md在各组织的表达量均高于鸡源F48E9病毒株的表达量。与对照组相比,攻毒后鸡Av BD2、3、4、5、6、9、10在大部分组织内表达量均有不同程度的增加。其中,感染鸭源Md病毒株后48 h,Av BD2在肾脏中的表达量显著升高(P0.05),Av BD5在肺脏中的表达显著上调(P0.05)。同时发现,感染两种NDV后,TLR2、3、4、7在肺脏和肾脏的表达量均增加,感染鸭源Md病毒株后48 h,TLR2、3、4、7在肺脏中均显著表达(P0.05)。感染两种NDV后,My D88在11种组织内的表达均上调,而IRF-7和IFN-β均没有明显变化。感染鸭源Md病毒株后48 h,IL-8、IFN-γ在肺脏中的表达量显著增加(P0.05),在感染鸭源Md病毒株后24 h肾脏中IFN-γ的表达量以及在感染鸭源Md病毒株后24 h和48 h肾脏中MHC class II的表达量均显著下调(P0.05)。本研究结果显示,机体受到NDV的感染后,可能通过启动TLR2、4、7介导的My D88以及TLR3介导的TRIF途径,诱导Av BD2、5基因上调表达,同时刺激IL-8、IFN-γ以及MHC class II基因的表达量发生变化,从而参加机体的对新城疫病毒的免疫反应。为探讨鸡Av BDs是否具有直接抗NDV的作用,根据经NDV感染后,鸡组织中各Av BD表达变化,我们选择鸡Av BD2进一步研究鸡Av BD的抗NDV病毒作用。通过细胞接种和鸡胚接种试验测定重组鸡Av BD2重组蛋白的体外NDV的作用。结果发现:与对照组相比,经重组蛋白中和后的病毒液接种细胞和鸡胚,病毒含量均有降低趋势,但差异不显著,还需进一步的研究证明其体外抗NDV的作用。综上所述,NDV能够诱导鸡Av BD2、5基因的表达量显著上调。机体受到NDV的感染后,可能通过启动TLR2、4、7介导的My D88以及TLR3介导的TRIF途径,诱导Av BD2、5基因上调表达,同时刺激IL-8、IFN-γ以及MHC class II基因的表达量发生变化。
[Abstract]:Newcastle disease (ND) is an acute and high-incidence avian infectious disease caused by Newcastle disease virus (NDV), which is widely spread in the world and is one of the biggest poultry diseases which are harmful to the poultry industry. As the NDV has a plurality of genotypes, the immune effect of the conventional vaccine is not ideal, and the development of the novel vaccine is always behind the variation of the pathogen. Therefore, it is very important to understand the natural immune regulation mechanism of the anti-infection of the poultry, to improve the immunity of the bird body and to find a new way of anti-NDV infection. Avian influenza-defensins (Av BDs) is a cation-rich host defense peptide, and has the activity of resisting bacteria, fungi and viruses, and plays an important role in the immunity and the acquired immunity of the birds. In order to study the mechanism of the immunoregulation of Newcastle disease virus in avian influenza-defensin, 30-day-old SPF chickens were randomly divided into three groups: control group and two groups. The control group did not attack the poison, and the poison group was inoculated with NDV virulent virus (F48E9 virus strain) and duck source NDV virulent virus (Md/ CH/ LGD/ 1/ 2005). After 24 h and 48 h after the attack, 5 rats were taken out of each group to collect chicken's brain, trachea, lung, kidney, liver, glandular stomach, bone marrow, spleen, cecum tonsil, had's gland and bursa of Fabricius. The amount of NDV virus and the expression of 13 kinds of Av BDs, 8 chicken Toll-like receptors (TLRs), 3 signal transduction molecules and 11 cytokine genes were detected by real-time fluorescence quantitative RT-PCR. The results showed that no virus was detected in the control group. In 24 h and 48 h after the infection of two NDV, the virus was detected in the tissues of the two different groups, and the viral load of each tissue was higher than the viral load of the tissues after infection at 48 h after infection. The expression of Md in each tissue was higher than that of F48E9 strain in chicken after 48 h after infection. The expression of Av BD2, 3, 4, 5, 6, 9, and 10 was increased in most tissues compared with the control group. The expression of Av BD2 in the kidney increased significantly (P0.05), and the expression of Av BD5 in the lung was significantly increased (P0.05). At the same time, the expression of TLR2, 3,4,7 in lung and kidney increased after infection of two NDV strains. The expression of TLR2, TLR2, 3, 4 and 7 in the lung was significantly increased (P0.05). After two NDV infections, the expression of My D88 was up-regulated in 11 tissues, while neither the IRF-7 nor the IFN-1 changes. The expression of IL-8 and IFN-2 in the lung was significantly increased after 48 h, IL-8, and IFN-1 in the lung of the infected ducks (P0.05). The expression of IFN-1 and the expression of MHC class II in the 24-h and 48-h kidneys of the infected duck-derived Md virus were significantly reduced (P0.05). The results showed that, after the body was infected with NDV, it was possible to induce the up-regulated expression of the Av BD2, 5 gene by starting TLR2, 4, 7-mediated My D88 and the TLR3-mediated TRIF pathway, while stimulating the expression of IL-8, IFN-1 and the MHC class II gene. so as to participate in the immune response of the body to the newcastle disease virus. In order to study whether the chicken Av BDs had a direct anti-NDV effect, according to the change of the expression of Av BD in the chicken tissue after the infection of NDV, we selected the chicken Av BD2 to further study the anti-NDV effect of the chicken Av BD. The effect of recombinant chicken Av BD2 recombinant protein in vitro was determined by cell inoculation and chick embryo inoculation test. The results showed that, compared with the control group, the virus content in both the cell and the chick embryo and the virus content in the recombinant protein was lower than that of the control group, but the difference was not significant, and further study was needed to prove the effect of the anti-NDV in vitro. In conclusion, NDV can induce a significant increase in the expression of Av BD2, 5 gene in chicken. After the body was infected with NDV, it was possible to induce the up-regulated expression of the Av BD2, 5 gene by starting TLR2, 4, 7-mediated My D88 and the TLR3-mediated TRIF pathway, while stimulating the expression of IL-8, IFN-1 and the MHC class II gene.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31
本文编号：2346173
[Abstract]:Newcastle disease (ND) is an acute and high-incidence avian infectious disease caused by Newcastle disease virus (NDV), which is widely spread in the world and is one of the biggest poultry diseases which are harmful to the poultry industry. As the NDV has a plurality of genotypes, the immune effect of the conventional vaccine is not ideal, and the development of the novel vaccine is always behind the variation of the pathogen. Therefore, it is very important to understand the natural immune regulation mechanism of the anti-infection of the poultry, to improve the immunity of the bird body and to find a new way of anti-NDV infection. Avian influenza-defensins (Av BDs) is a cation-rich host defense peptide, and has the activity of resisting bacteria, fungi and viruses, and plays an important role in the immunity and the acquired immunity of the birds. In order to study the mechanism of the immunoregulation of Newcastle disease virus in avian influenza-defensin, 30-day-old SPF chickens were randomly divided into three groups: control group and two groups. The control group did not attack the poison, and the poison group was inoculated with NDV virulent virus (F48E9 virus strain) and duck source NDV virulent virus (Md/ CH/ LGD/ 1/ 2005). After 24 h and 48 h after the attack, 5 rats were taken out of each group to collect chicken's brain, trachea, lung, kidney, liver, glandular stomach, bone marrow, spleen, cecum tonsil, had's gland and bursa of Fabricius. The amount of NDV virus and the expression of 13 kinds of Av BDs, 8 chicken Toll-like receptors (TLRs), 3 signal transduction molecules and 11 cytokine genes were detected by real-time fluorescence quantitative RT-PCR. The results showed that no virus was detected in the control group. In 24 h and 48 h after the infection of two NDV, the virus was detected in the tissues of the two different groups, and the viral load of each tissue was higher than the viral load of the tissues after infection at 48 h after infection. The expression of Md in each tissue was higher than that of F48E9 strain in chicken after 48 h after infection. The expression of Av BD2, 3, 4, 5, 6, 9, and 10 was increased in most tissues compared with the control group. The expression of Av BD2 in the kidney increased significantly (P0.05), and the expression of Av BD5 in the lung was significantly increased (P0.05). At the same time, the expression of TLR2, 3,4,7 in lung and kidney increased after infection of two NDV strains. The expression of TLR2, TLR2, 3, 4 and 7 in the lung was significantly increased (P0.05). After two NDV infections, the expression of My D88 was up-regulated in 11 tissues, while neither the IRF-7 nor the IFN-1 changes. The expression of IL-8 and IFN-2 in the lung was significantly increased after 48 h, IL-8, and IFN-1 in the lung of the infected ducks (P0.05). The expression of IFN-1 and the expression of MHC class II in the 24-h and 48-h kidneys of the infected duck-derived Md virus were significantly reduced (P0.05). The results showed that, after the body was infected with NDV, it was possible to induce the up-regulated expression of the Av BD2, 5 gene by starting TLR2, 4, 7-mediated My D88 and the TLR3-mediated TRIF pathway, while stimulating the expression of IL-8, IFN-1 and the MHC class II gene. so as to participate in the immune response of the body to the newcastle disease virus. In order to study whether the chicken Av BDs had a direct anti-NDV effect, according to the change of the expression of Av BD in the chicken tissue after the infection of NDV, we selected the chicken Av BD2 to further study the anti-NDV effect of the chicken Av BD. The effect of recombinant chicken Av BD2 recombinant protein in vitro was determined by cell inoculation and chick embryo inoculation test. The results showed that, compared with the control group, the virus content in both the cell and the chick embryo and the virus content in the recombinant protein was lower than that of the control group, but the difference was not significant, and further study was needed to prove the effect of the anti-NDV in vitro. In conclusion, NDV can induce a significant increase in the expression of Av BD2, 5 gene in chicken. After the body was infected with NDV, it was possible to induce the up-regulated expression of the Av BD2, 5 gene by starting TLR2, 4, 7-mediated My D88 and the TLR3-mediated TRIF pathway, while stimulating the expression of IL-8, IFN-1 and the MHC class II gene.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.31
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