【摘要】：新城疫(Newcastle Disease,ND)是由新城疫病毒(Newcastle Disease Virus,NDV)引起的一種發(fā)病急、致死率高的禽傳染病,在世界范圍內(nèi)廣為傳播,是危害養(yǎng)禽業(yè)最大的禽病之一。由于NDV有多個(gè)基因型,常規(guī)疫苗的免疫效果并不理想,新型疫苗的研制又總是落后于病原的變異。因此,深入了解家禽抗感染作用的天然免疫調(diào)控機(jī)制,提高禽機(jī)體的免疫能力,尋找抗NDV感染的新途徑顯得極為重要。禽β-防御素(Avian beta defensins,Av BDs)是富含陽離子的宿主防御肽,具有抗細(xì)菌、真菌以及病毒的活性,在禽先天性免疫及獲得性免疫中發(fā)揮著重要作用。為探討禽β-防御素體內(nèi)對(duì)不同來源新城疫病毒的免疫調(diào)節(jié)作用機(jī)制,本試驗(yàn)選用30只20日齡SPF雞,隨機(jī)分為三組,即對(duì)照組和兩組攻毒組。對(duì)照組不攻毒,攻毒組采用點(diǎn)眼滴鼻法分別接種雞源NDV強(qiáng)毒(F48E9病毒株)和鴨源NDV強(qiáng)毒(Md/CH/LGD/1/2005)。在攻毒后24 h后和48 h,每組各取出5只,分別采集雞腦、氣管、肺臟、腎臟、肝臟、腺胃、骨髓、脾臟、盲腸扁桃體、哈德氏腺、法氏囊組織。采用實(shí)時(shí)熒光定量RT-PCR法檢測攻毒前后各組織器官中的NDV病毒載量以及13種Av BDs、8種雞Toll樣受體(Toll-like receptors,TLRs)、3種信號(hào)轉(zhuǎn)導(dǎo)分子、11種細(xì)胞因子基因的表達(dá)量。結(jié)果顯示:對(duì)照組各組織均未檢測到病毒。在感染兩種NDV后24 h和48 h,兩個(gè)攻毒組雞各組織均檢測到病毒,且在感染后48 h,各組織的病毒載量均高于感染后24 h各組織的病毒載量。在感染后48 h,鴨源Md在各組織的表達(dá)量均高于雞源F48E9病毒株的表達(dá)量。與對(duì)照組相比,攻毒后雞Av BD2、3、4、5、6、9、10在大部分組織內(nèi)表達(dá)量均有不同程度的增加。其中,感染鴨源Md病毒株后48 h,Av BD2在腎臟中的表達(dá)量顯著升高(P0.05),Av BD5在肺臟中的表達(dá)顯著上調(diào)(P0.05)。同時(shí)發(fā)現(xiàn),感染兩種NDV后,TLR2、3、4、7在肺臟和腎臟的表達(dá)量均增加,感染鴨源Md病毒株后48 h,TLR2、3、4、7在肺臟中均顯著表達(dá)(P0.05)。感染兩種NDV后,My D88在11種組織內(nèi)的表達(dá)均上調(diào),而IRF-7和IFN-β均沒有明顯變化。感染鴨源Md病毒株后48 h,IL-8、IFN-γ在肺臟中的表達(dá)量顯著增加(P0.05),在感染鴨源Md病毒株后24 h腎臟中IFN-γ的表達(dá)量以及在感染鴨源Md病毒株后24 h和48 h腎臟中MHC class II的表達(dá)量均顯著下調(diào)(P0.05)。本研究結(jié)果顯示,機(jī)體受到NDV的感染后,可能通過啟動(dòng)TLR2、4、7介導(dǎo)的My D88以及TLR3介導(dǎo)的TRIF途徑,誘導(dǎo)Av BD2、5基因上調(diào)表達(dá),同時(shí)刺激IL-8、IFN-γ以及MHC class II基因的表達(dá)量發(fā)生變化,從而參加機(jī)體的對(duì)新城疫病毒的免疫反應(yīng)。為探討雞Av BDs是否具有直接抗NDV的作用,根據(jù)經(jīng)NDV感染后,雞組織中各Av BD表達(dá)變化,我們選擇雞Av BD2進(jìn)一步研究雞Av BD的抗NDV病毒作用。通過細(xì)胞接種和雞胚接種試驗(yàn)測定重組雞Av BD2重組蛋白的體外NDV的作用。結(jié)果發(fā)現(xiàn):與對(duì)照組相比,經(jīng)重組蛋白中和后的病毒液接種細(xì)胞和雞胚,病毒含量均有降低趨勢,但差異不顯著,還需進(jìn)一步的研究證明其體外抗NDV的作用。綜上所述,NDV能夠誘導(dǎo)雞Av BD2、5基因的表達(dá)量顯著上調(diào)。機(jī)體受到NDV的感染后,可能通過啟動(dòng)TLR2、4、7介導(dǎo)的My D88以及TLR3介導(dǎo)的TRIF途徑,誘導(dǎo)Av BD2、5基因上調(diào)表達(dá),同時(shí)刺激IL-8、IFN-γ以及MHC class II基因的表達(dá)量發(fā)生變化。
[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.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.31

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本文編號(hào)：2346173