【作者】 李卫；

【导师】 胡薛英；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2009， 硕士

【摘要】 本研究通过原核表达樱桃谷鸭CD3ε胞外区,制备兔抗鸭的CD3ε多克隆抗体,利用制备的抗体建立一种用于测定或分选出鸭的CD3~+T细胞亚群的方法,由于鸭瘟病毒主要侵害的是上皮细胞和淋巴细胞,运用此方法为阐明鸭瘟的致病机理提供依据,为以后进一步研究水禽疾病发生发展规律提供手段。根据GenBank中收录北京鸭CD3ε基因序列(AF378704)设计合成了特异性的引物,扩增了樱桃谷鸭和麻鸭CD3εORF基因,测定基因序列,将其登录到Genbank,其登录号分别为FJ524847和FJ524848。应用BLAST和DNASTAR等生物学软件比对分析表明:樱桃谷鸭、麻鸭、北京鸭间CD3ε基因ORF序列相互之间同源性都为99%。通过Clustalx(1.83)分析鸭的CD3ε基因ORF推导氨基酸序列差异,最保守的部位是ε链多肽的胞质区,而最不保守的是胞外区。根据樱桃谷鸭CD3ε序列,设计特异性的一对引物克隆胞外区。分别构建了DuCD3ε胞外区基因的原核表达质粒(pET-28a-DuCD3ε),并在E.coli BL21(DE3)中成功获得了表达,将纯化的重组蛋白作为免疫原,通过设计好的免疫程序,成功制备了兔抗重组蛋白的高免血清,通过间接ELISA测定兔抗rDuCD3ε抗体效价为1:51200。所得高免血清分别采用饱和硫酸铵粗提和Sephadex G-200过柱纯化,得到纯化的抗体。用鸭瘟病毒感染35日龄樱桃谷鸭,在感染后4d开始,感染鸭出现鸭瘟的特征性形态学变化,如病鸭头颈部肿大;剖检变化有肠道开始明显环状出血病理变化,同时在感染病毒后期的脾脏组织中,应用透射电镜观察到病毒粒子,证实鸭瘟的病理模型构建成功。在感染后1d、2d、3d、4d、5d、6d分别采取外周血液,制作血涂片检测。应用制备的兔抗樱桃谷鸭CD3ε多克隆抗体建立了检测鸭外周血中CD3阳性T淋巴细胞的免疫组织化学染色方法,具有较好的特异性。运用此方法对鸭瘟病毒感染后1d、2d、3d、4d、5d、6d鸭外周血液中CD3~+T淋巴细胞的检测表明,在感染病毒后3d内,外周血液中CD3~+T淋巴细胞数量逐渐增加;感染后3d,外周血液中CD3~+T淋巴细胞数量开始下降。另外在试验组中,通过光学显微镜观察到在感染后3d的脾脏、法氏囊和胸腺淋巴细胞都出现数量减少的现象,由此可以推测,在感染病毒后3d内外周血液中CD3~+T淋巴细胞数量增加可能是由于免疫器官中淋巴细胞迁移到外周血液中所致;3d后外周血液CD3~+T中淋巴细胞数量减少,可能是病毒导致总的淋巴细胞坏死所致。更多还原

【Abstract】 In this study, through prokaryotic expression of Cherry Valley Duck CD3εextracellular domain,preparation of polyclonal antibodies of Ant-rDuCD3εand establishment the way of measurement and selected subsets of duck CD3~+T cells were foundation of understanding changes of ducks and even waterfowl cellular immune function.Ultimately illuminatting regularity of development of duck multiple diseases nosogenesis .According to Beijing duck CD3εgene sequence (AF378704)in GenBank,designed, synthesized a specific primer, and then amplificated Cherry Valley Duck and Sheldrake duck CD3εORF genes. Determinations result of the two sequences were loged on to Genbank,Accession number of Cherry Valley Duck and Sheldrake duck were FJ524847 and FJ524848 respectively. The two sequences were analyzed by biological software, such as BLAST and DNASTAR software, results showed that DuCD3εORF the sequence homology among Cherry Valley Duck, Sheldrake Duck and Beijing Duck were 99%. Through Clustalx (1.83) Analysis of differences of duck CD3εgene ORF deduced amino acid sequence,the most conservative parts of theεchain is the cytoplasmic peptide, while the most unstable is the extracellular domain. So according to Cherry Valley Duck DuCD3εORF sequence, synthesized specific primers of a pair of cloned extracellular domain,and then constructed plasmid of the extracellular domain of DuCD3εProkaryotic expression (pET-28a-DuCD3ε).The plasmid of pET-28a-DuCD3εcan successfully expressed rDuCD3εprotein in E.coli BL21 (DE3).The expressed recombinant protein was immunogen which prepared immune serum by designing a good immune procedures.The successful preparation of a rabbit anti-recombinant protein(rDuCD3ε) of the high-free serum was measured by indirect ELISA, the titer of rabbit anti-antibody was 1:102400.High-income-free serum were crudely purified by saturation ammonium sulfate and purified by Sephadex G-200.The purified antibodies have been prepared.Duck infected duck plague virus showed the characteristic morphological changes, such as diseases of the head and neck swelling, intestinal ring bleeding of autopsy significantly change. At the same time, in the spleen tissue, observed virus particles by transmission electron microscopy. The morphological changes proved that pathology model of duck plague was constructed successfully.Through detection of the peripheral blood CD3~+T lymphocytes showed that prepared rabbit anti-rDuCD3εpolyclonal antibodies can be used to identify duck CD3 positive T lymphocyte in peripheral blood ,and the antibody has good specificity.The number of the peripheral blood CD3~+T lymphocytes gradually increased in infection 3 days; the number of peripheral blood CD3~+T lymphocytes began to decline at 3 days after infection. In the test group ,we observed that the number of the spleen, bursa and thymus lymphocytes reducing phenomena by optical microscope after infection.it can be speculated that at 3 days post-infectio the reason of number of blood CD3~+T lymphoid increase may be due to the migration from immune organs to the peripheral blood; 3 days after infection the reason of number of blood CD3~+T lymphoid decrease may be due to necrosis of lymphocytes by viruse.更多还原