连翘酯苷A对BVDV复制影响及BVDV DNA疫苗初步制备和小鼠免疫效果评价

发布时间：2018-01-20 20:09

本文关键词： 牛病毒性腹泻病毒连翘酯苷A CD28-4-1BB DNA疫苗 Prime-boost　出处：《中国农业大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:牛病毒性腹泻(Bovine viral diarrhea,BVD)是由牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)引起的奶牛养殖业重大损失的疾病。牛病毒性腹泻病原复杂,各型病毒交叉保护性差,目前缺乏有效的防治药物,同时也缺乏有效的疫苗。连翘酯苷A(ForsythiasideA,FTA)能够抑制BVDV复制,但具体的机制尚不清楚。目的:本研究的主要目的就是探究FTA抑制BVDV复制的具体机制,并制备BVDV DNA疫苗,评价免疫方式及其免疫效果。方法:从BVDV抗原和抗体均为阴性的牛外周血提取单个核细胞(Peripheral blood mononuclear cells,PBMCs)。以感染复数(Multiplicity of infection,MOI)为 0.1 的 BVDV C24V株感染PBMCs,FTA添加浓度为100μg/mL,使用时与病毒同一时间点加入PBMCs中。病毒感染细胞的方法为无血清条件下加入病毒与PBMCs孵育1 h,而后加入10%的胎牛血清或FTA继续培养。扩增BVDV临床株E2基因,构建DNA疫苗。6～8周龄BALB/c小鼠150只,分为5组,分别进行三次免疫,分别免疫(100μLPBS;100μg空载体;100 μg pcDNA3.1-E2;40μgE2蛋白;pcDNA3.1-E2和E2蛋白)。第三次免疫后两周腹腔注射6×106TCID50病毒。用活细胞计数和绝对定量PCR分析FTA对BVDV复制作用的影响;用Real-time PCR及Western blot方法定量分析共刺激分子相对表达水平;用ELISA方法定量分析细胞免疫相关因子的表达和抗体效价测定;利用流式细胞术评价FTA对PBMCs增殖、活化和凋亡的影响;用免疫荧光、HE染色等方法评价疫苗对小鼠攻毒的保护作用。结果:FTA促进T细胞活化和增殖,抑制BVDV引起的PBMCs凋亡。FTA可以调节CD28/CTLA-4的表达水平,促进4-1BB在BVDV侵染过程中的表达,并在感染24 h后提高TRAF-2的表达。FTA可以提高BVDV侵染过程中的IgG2a和IL-2表达,并调节IFN-γ的过度分泌。FTA能够抑制BVDV在每个PBMC中的拷贝数目,且不影响病毒毒力。制备BVDV E2真核表达载体pcDNA3.1-E2,并进行转染和体外表达鉴定。同ISA61佐剂混合后制备的DNA疫苗和亚单位疫苗免疫小鼠能够激发较高水平的抗体水平,促进CD4+IFN-γ+和CD8+IFN-γ+T细胞的表达,提高血清中IL-4的浓度,可以减轻BVDV引起的小鼠组织损伤,攻毒后疫苗组小鼠脾脏、肺脏和肾脏的病理变化明显轻于攻毒对照组。DNA疫苗免疫可以促进脾脏淋巴细胞增殖。制备的DNA疫苗和亚单位疫苗采用prime boost的策略免疫小鼠,能够产生较高水平的中和抗体,主要的抗体亚型为IgG1、IgG2b和IgG2a。同时能够激发有效的CTL以及Th1和Th2细胞免疫反应,促进IL-4分泌,有效的保护小鼠抵抗BVDV感染。结论:FTA具有抗BVDV感染作用,部分通过抑制BVDV的复制和PBMC的凋亡以及促进T细胞的激活。抗病毒机制可能与TRAF2依赖性CD28-4-1BB信号传导有关。采用联合免疫DNA疫苗和亚单位疫苗及采用prime boost免疫策略能够有效提高机体免疫反应的强度。
[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- 纬 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- 纬 and CD8 IFN- 纬 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.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.4

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