MDV感染B19、B21单倍型SPF鸡外周血淋巴细胞中BLec、BNK和细胞因子转录水平的动态变化

发布时间：2018-03-02 16:41

本文选题：细胞因子　切入点：Blec　出处：《山西农业大学》2014年硕士论文　论文类型：学位论文

【摘要】：鸡主要组织相容性复合体(MHC)长期以来被认为是识别对疾病易感和耐受的免疫反应系统,尤其与马立克氏病(MD)密切相关。哈尔滨兽医研究所国家禽类实验动物种子中心根据MHC-B区域中的4个微卫星位点等位基因,筛选出B13、B15、B2、B5、B21和B19六种单倍型,这是我国唯一具有自主知识产权的无特定病原体鸡群。本试验用B15、B19和B21单倍型来作为实验动物进行试验。本研究对2月龄B15单倍型的胸腺组织提取RNA,经RT-PCR扩增ChBLec基因,并以pET-30a(+)为原核表达载体成功构建重组质粒pET-30a-ChBLec,并在BL21(DE3)中经IPTG (0.02mmol/L)诱导表达,获得大小约为17kDa以包涵体形式存在的ChBLec重组蛋白。把重组蛋白所在凝胶研磨后免疫新西兰兔,获得兔抗血清。用western blot检测后兔抗血清能检测到pET-30a-ChBLec外源蛋白。结果表明：ChBLec基因在大肠杆菌成功表达,且成功制备了兔抗ChBLec血清。为了了解鸡外周血淋巴细胞(PBL)中BNK, BLec基因以及IFN-γ、IL-4、IL-10、IL-18细胞因子的转录水平与遗传背景的联系。本试验通过肌肉注射的方法用MDV Md5细胞结合型病毒对1日龄鸡进行攻毒,每羽800PFU,对照组接种无菌的PBS,在4、7、10、13、19dpi时采集抗凝血,用鸡外周血淋巴细胞分离液分离PBL,然后用ConA(25uggmL)刺激,培养48h后收获细胞提取RNA并且反转录,然后用双重荧光定量PCR技术检测BNK、BLec和四种细胞因子转录水平的动态变化。BNK和BLec在PBL的动态变化表明：在4、7dpi(第一次溶细胞感染期)和19dpi BNK和BLec大多表现出不同程度的降低；在10dpi(潜伏期)B19单倍型鸡中BNK和BLec表现出不同程度的升高,B21单倍型鸡中BNK升高；在13dpi(第二次溶细胞感染期)B21单倍型鸡中BNK和BLec表现出不同程度的升高。四种细胞因子在PBL的动态变化表明：在4、7dpi(第一次溶细胞感染期)和19dpi四种细胞因子大多表现出不同程度的降低；在10dpi(潜伏期)B21单倍型鸡中IFN-γ(19%)、I-4(5%)和I-18(32%)表现出不同程度的升高,而B19单倍型鸡中IFN-γ(-16%)、IL-4(-29%)和IL,-18(-23%)表现出不同程度的降低；在13dpi(第二次溶细胞感染期)B21单倍型鸡中IL-18(47%)和I-10(69%)表现出不同程度的升高,而B19单倍型鸡中IL-18(-30%)和IL-10(-37%)表现出不同的降低。根据文献中对MD发病机理的阐述可知,在第一次溶细胞感染期淋巴器官会出现不同程度的萎缩,引起免疫抑制,这与本试验在4、7dpi时BNK、BLec和四种细胞因子转录水平下降结果一致；在感染后8-14d为第二次溶细胞感染期,耐受鸡一般不会经历,与之相反易感鸡会持续免疫抑制。以上结果说明这种变化与鸡的遗传背景是相关的,并且B21单倍型鸡比B19单倍型鸡对MD耐受。
[Abstract]:Chicken major histocompatibility complex (MHC) has long been considered as an immunoreactive system for identifying susceptibility and tolerance to disease. The National Laboratory Animal seed Center of Harbin Veterinary Research Institute screened six haplotypes B13, B15, B _ 2, B _ (5), B _ (21) and B _ (19) according to the alleles of four microsatellite loci in the MHC-B region. This is the only chicken flocks with independent intellectual property rights in China without specific pathogens. In this experiment, B15B19 and B21 haplotypes were used as experimental animals. In this study, ChBLec gene was amplified by RT-PCR from 2-month-old B15 haplotype thymus tissue. The recombinant plasmid pET-30a-ChBLecwas successfully constructed by using pET-30a () as prokaryotic expression vector, and was induced by IPTG 0.02mmolL / L in BL21DDE3. A recombinant ChBLec protein was obtained in the form of inclusion body of about 17kDa. The New Zealand rabbits were immunized by grinding the gel in which the recombinant protein was located. Rabbit antiserum was obtained and pET-30a-ChBLec exogenous protein was detected by western blot. The results showed that the pET-30a-ChBLec gene was successfully expressed in Escherichia coli and rabbit anti-#en3# serum was successfully prepared. In order to understand the relationship between genetic background and transcriptional level of BNK-, BLec gene and IL-10 IL-18 cytokines in peripheral blood lymphocytes of chicken, MDV Md5 cell-binding virus was injected intramuscularly to attack 1-day-old chicken. The control group was inoculated with aseptic PBSs. Anticoagulant blood was collected at 1319 dpi. PBLs were isolated from chicken peripheral blood lymphocytes, then stimulated with Cona 25uggm L. After 48 hours of culture, the RNA was extracted from the cells and reversed-transcripted. Then the dynamic changes of the transcription levels of BNK-BLec and four cytokines were detected by double fluorescence quantitative PCR. The dynamic changes of BNK and BLec in PBL showed that most of them were decreased in different degrees at 4dpiand 19dpi BNK and BLec. BNK and BLec increased in different degree in 10 d pii (incubation period B19 haplotype) and BNK increased in B21 haplotype. BNK and BLec increased in different degrees in 13dpi. the dynamic changes of four cytokines in PBL showed that the four cytokines were mostly expressed at 4dpi7dpiand 19dpi. Showing varying degrees of decline; IFN- 纬 ~ (19) and I-18 ~ (32) and IFN- 纬 ~ (19) and I-18 ~ (32) showed different degrees of elevation in 10dpi. while IFN- 纬 -16 + IL-4 ~ (-29) and IL-18-23 in B19 haplotypes showed different degrees of decrease, and increased at 13 d (IL-18 ~ (47) and I-10 ~ (69) in B21 haplotypes in the second stage of cytolytic infection). In B 19 haplotypes, IL-18-30) and IL-10-37) showed different decreases. According to the explanation of the pathogenesis of MD in the literature, the lymphoid organs of the first lysocytic infection stage will shrink in varying degrees, causing immunosuppression, which is consistent with the decrease of the transcription level of BNK-BLec and four cytokines at 4 ~ 7 dpi in this experiment. During the second cytolytic infection period from 8 to 14 days after infection, the tolerant chickens did not generally experience it, whereas the susceptible chickens continued to undergo immunosuppression. These results suggest that this change is related to the genetic background of the chickens. And B 21 haplotypes were more resistant to MD than B 19 haplotypes.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：S858.31

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