本文關(guān)鍵詞： 牛病毒性腹瀉病毒 連翹酯苷A CD28-4-1BB DNA疫苗 Prime-boost　出處：《中國(guó)農(nóng)業(yè)大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：背景:牛病毒性腹瀉(Bovine viral diarrhea,BVD)是由牛病毒性腹瀉病毒(Bovine viral diarrhea virus,BVDV)引起的奶牛養(yǎng)殖業(yè)重大損失的疾病。牛病毒性腹瀉病原復(fù)雜,各型病毒交叉保護(hù)性差,目前缺乏有效的防治藥物,同時(shí)也缺乏有效的疫苗。連翹酯苷A(ForsythiasideA,FTA)能夠抑制BVDV復(fù)制,但具體的機(jī)制尚不清楚。目的:本研究的主要目的就是探究FTA抑制BVDV復(fù)制的具體機(jī)制,并制備BVDV DNA疫苗,評(píng)價(jià)免疫方式及其免疫效果。方法:從BVDV抗原和抗體均為陰性的牛外周血提取單個(gè)核細(xì)胞(Peripheral blood mononuclear cells,PBMCs)。以感染復(fù)數(shù)(Multiplicity of infection,MOI)為 0.1 的 BVDV C24V株感染PBMCs,FTA添加濃度為100μg/mL,使用時(shí)與病毒同一時(shí)間點(diǎn)加入PBMCs中。病毒感染細(xì)胞的方法為無(wú)血清條件下加入病毒與PBMCs孵育1 h,而后加入10%的胎牛血清或FTA繼續(xù)培養(yǎng)。擴(kuò)增BVDV臨床株E2基因,構(gòu)建DNA疫苗。6～8周齡BALB/c小鼠150只,分為5組,分別進(jìn)行三次免疫,分別免疫(100μLPBS;100μg空載體;100 μg pcDNA3.1-E2;40μgE2蛋白;pcDNA3.1-E2和E2蛋白)。第三次免疫后兩周腹腔注射6×106TCID50病毒。用活細(xì)胞計(jì)數(shù)和絕對(duì)定量PCR分析FTA對(duì)BVDV復(fù)制作用的影響;用Real-time PCR及Western blot方法定量分析共刺激分子相對(duì)表達(dá)水平;用ELISA方法定量分析細(xì)胞免疫相關(guān)因子的表達(dá)和抗體效價(jià)測(cè)定;利用流式細(xì)胞術(shù)評(píng)價(jià)FTA對(duì)PBMCs增殖、活化和凋亡的影響;用免疫熒光、HE染色等方法評(píng)價(jià)疫苗對(duì)小鼠攻毒的保護(hù)作用。結(jié)果:FTA促進(jìn)T細(xì)胞活化和增殖,抑制BVDV引起的PBMCs凋亡。FTA可以調(diào)節(jié)CD28/CTLA-4的表達(dá)水平,促進(jìn)4-1BB在BVDV侵染過(guò)程中的表達(dá),并在感染24 h后提高TRAF-2的表達(dá)。FTA可以提高BVDV侵染過(guò)程中的IgG2a和IL-2表達(dá),并調(diào)節(jié)IFN-γ的過(guò)度分泌。FTA能夠抑制BVDV在每個(gè)PBMC中的拷貝數(shù)目,且不影響病毒毒力。制備BVDV E2真核表達(dá)載體pcDNA3.1-E2,并進(jìn)行轉(zhuǎn)染和體外表達(dá)鑒定。同ISA61佐劑混合后制備的DNA疫苗和亞單位疫苗免疫小鼠能夠激發(fā)較高水平的抗體水平,促進(jìn)CD4+IFN-γ+和CD8+IFN-γ+T細(xì)胞的表達(dá),提高血清中IL-4的濃度,可以減輕BVDV引起的小鼠組織損傷,攻毒后疫苗組小鼠脾臟、肺臟和腎臟的病理變化明顯輕于攻毒對(duì)照組。DNA疫苗免疫可以促進(jìn)脾臟淋巴細(xì)胞增殖。制備的DNA疫苗和亞單位疫苗采用prime boost的策略免疫小鼠,能夠產(chǎn)生較高水平的中和抗體,主要的抗體亞型為IgG1、IgG2b和IgG2a。同時(shí)能夠激發(fā)有效的CTL以及Th1和Th2細(xì)胞免疫反應(yīng),促進(jìn)IL-4分泌,有效的保護(hù)小鼠抵抗BVDV感染。結(jié)論:FTA具有抗BVDV感染作用,部分通過(guò)抑制BVDV的復(fù)制和PBMC的凋亡以及促進(jìn)T細(xì)胞的激活。抗病毒機(jī)制可能與TRAF2依賴(lài)性CD28-4-1BB信號(hào)傳導(dǎo)有關(guān)。采用聯(lián)合免疫DNA疫苗和亞單位疫苗及采用prime boost免疫策略能夠有效提高機(jī)體免疫反應(yīng)的強(qiáng)度。
[Abstract]:Background: bovine viral diarrhea. BVDs are produced by bovine viral diarrhea virus. The disease caused by BVDV. the pathogen of bovine viral diarrhea is complex, the cross-protection of various viruses is poor, and there is a lack of effective drugs to prevent and cure the disease. Forsythias deoxynucleotidyl (FTAA) can inhibit the replication of BVDV. But the specific mechanism is not clear. Objective: the main purpose of this study is to explore the specific mechanism of FTA inhibiting BVDV replication and to prepare BVDV DNA vaccine. Methods: mononuclear cells (mononuclear cells) were extracted from bovine peripheral blood with negative BVDV antigen and antibody. Peripheral blood mononuclear cells. PBMCs was infected by BVDV C24V strain with the plural number of infection plural of infective moi (0.1). The concentration of FTA was 100 渭 g / mL and added to PBMCs at the same time as the virus. The virus infected cells were incubated with PBMCs for 1 h without serum. Then, 10% fetal bovine serum or FTA were added to further culture. The E2 gene of BVDV clinical strain was amplified and DNA vaccine was constructed. 150 BALB/c mice aged 68 weeks were divided into 5 groups. The mice were immunized with 100 渭 L PBSs for three times. 100 渭 g empty carrier; 100 渭 g pcDNA3.1-E2; 40 渭 gE2 protein; PcDNA3.1-E2 and E2 protein. Two weeks after the third immunization, 6 脳 106 TCID50 virus was injected intraperitoneally. The effect of FTA on BVDV replication was analyzed by living cell count and absolute quantitative PCR. The relative expression of costimulatory molecules was quantitatively analyzed by Real-time PCR and Western blot. The expression and antibody titer of cytokines were quantitatively analyzed by ELISA. The effects of FTA on proliferation, activation and apoptosis of PBMCs were evaluated by flow cytometry. Immunofluorescence and HE staining were used to evaluate the protective effect of the vaccine on mice. Results: FTA promoted the activation and proliferation of T cells. Inhibition of PBMCs apoptosis induced by BVDV. FTA could regulate the expression of CD28/CTLA-4 and promote the expression of 4-1BB in the process of BVDV infection. And after 24 hours of infection, the expression of TRAF-2 was increased. FTA could increase the expression of IgG2a and IL-2 in the process of BVDV infection. Regulation of IFN- 緯 over-secretion. FTA can inhibit the number of copies of BVDV in each PBMC. The BVDV E2 eukaryotic expression vector pcDNA3.1-E2 was prepared without affecting the virulence of the virus. After transfection and in vitro expression identification, the DNA vaccine and subunit vaccine mixed with ISA61 adjuvant could stimulate a high level of antibody level in mice. Promoting the expression of CD4 IFN- 緯 and CD8 IFN- 緯 T cells and increasing the concentration of IL-4 in serum can attenuate the tissue damage induced by BVDV in mice. The spleen of mice in the vaccine group after inoculation. The pathological changes of lung and kidney were significantly lighter than those of the control group. The immunized spleen lymphocytes were immunized with prime. The DNA vaccine and subunit vaccine were prepared with prime. Boost strategy immunized mice. It can produce a high level of neutralizing antibody, the main subtypes of antibody are IgG1G2b and IgG2a. it can also stimulate the effective CTL, Th1 and Th2 cellular immune reaction. Promote the secretion of IL-4 and protect mice against BVDV infection. Conclusion: FTAs have the effect of anti-#en2# infection. The antiviral mechanism may be related to the signal transduction of TRAF2 dependent CD28-4-1BB by inhibiting the replication of BVDV and the apoptosis of PBMC and promoting the activation of T cells. Epidemic DNA Vaccine and Subunit Vaccine and prime. Boost immune strategy can effectively improve the intensity of immune response.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S852.4

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