【作者】 刘光亮；

【导师】 吴东来；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2007， 博士

【摘要】 MHCⅠ类分子/肽四聚体技术是1996年建立的一种检测特异性细胞毒性T淋巴细胞（cytotoxicT lymphocytes，CTLs）的方法，该方法已在人类及模式动物的相关领域得到较为广泛的研究和应用，然而在兽医学相关研究中只有近年来往猪和马上有了初步的研究，目前该项技术在禽类的研究尚属空白。为了构建鸡的MHC/肽四聚体，本实验从SPF莱航鸡全血克隆了鸡MHCⅠ重链α（BF2）和β2微球蛋白（Chβ2m）全基因，经系统进化分析表明本实验所获得的BF2基因为B15单倍型。然后利用软件分析了其各自的信号肽序列及BF2的跨膜区，利用引物分别缺失了这两段基因的信号肽序列以及BF2的跨膜区，并在BF2基因的3’端融合了BirA底物肽（BSP）序列。将经改造的两段基因分别克隆至融合蛋白重组表达载体中，分别命名为pET-BF₂-BSP和pET-Chβ2m，并将其分别转化至大肠杆菌感受态BL21（DE3）中进行诱导表达，获得了包涵体表达的重组蛋白，表达产物溶解于8M脲中，经SDS-PAGE分析，结果表明所得融合目的蛋白分别约为38kD和15kD，Western-blot结果显示该重组蛋白均成功融合了6×His标签。为了获得大量的目的蛋白，优化了诱导剂浓度、起始诱导菌体浓度及诱导时间等表达条件。此外，建立了重组蛋白BF2-BSP和Chβ2m在变性状态下通过镍柱亲和层析纯化包涵体的方法，获得了高纯度的重组蛋白BF2-BSP和Chβ2m。根据文献所报道，合成了鸡传染性支气管炎病毒（IBV）核蛋白N_71-78T细胞表位肽，将其与纯化后的重组蛋白BF2-BSP和Chβ2m在重折叠缓冲液中复性。复性后在快速蛋白液相色谱仪（FPLC）上分离出复性组装成功的BF2/肽单体复合物。将该单体复合物在体外进行生物素化，其产物再次采用FPLC通过分子筛分离出生物素化后的BF2/肽单体复合物；通过链霉亲和素迁移实验检测体外生物素化效率，然后将生物素化后的BF2/肽单体复合物与藻红蛋白（PE）标记的链霉亲和素按一定比例反应生成BF2/肽四聚体。为了检测所制备BF2/肽四聚体的功能，采用10^5.5EID₅₀的IBV H52株接种一周龄SPF鸡，10d后采抗凝血用淋巴细胞分离液分离PBMC，调整细胞浓度后取1×10⁶个细胞进行抗鸡CD8单抗和BF2/肽四聚体双色荧光染色，采用流式细胞术检测并分析染色结果，结果表明所制备的BF2/肽四聚体可用于检测IBV N蛋白特异性T淋巴细胞，检测一周龄SPF鸡接种H52后10d时针对N蛋白的特异性T细胞比率为3.65％。为了探讨SPF鸡在感染IBV后体内抗体水平及针对N蛋白特异性的T淋巴细胞的动态变化，将125羽SPF鸡随机分为三组分别作为接种免疫组、攻毒对照组和空白对照组。采用IBV H52标准毒株对其通过滴鼻方式进行免疫，之后每3天剖杀5羽观察各脏器的病理学变化、检测血清抗体效价并采用BF2/肽四聚体评价其N蛋白特异性T细胞的比例；免疫15d时采用IBV M41对免疫组和攻毒对照组进行攻毒，同样检测上述指标。最后综合评价IBV在免疫及攻毒后血清抗体和N蛋白特异性T细胞的动态变化趋势及两者之间的相互关系。检测结果表明H52滴鼻免疫后特异性T细胞免疫的产生早于血清抗体IgG的出现，攻毒结果显示在SPF鸡未产生坚强的体液免疫时仍能抵抗同型强毒的攻击，提示特异性T细胞在抵抗IBV早期感染免疫反应过程中可能起着重要作用。更多还原

【Abstract】 The major histocompatibility complex classⅠ（MHC classⅠ） peptide tetramer is a sensitive tool to evaluate antigen-specific cytotoxic T lymphocytes（CTLs）. However, no chicken MHC classⅠpeptide tetramer has been reported by now.In order to construct the chicken MHC classⅠpeptide tetramer, genes of the chicken MHCⅠαchain（BF2*15） andβ2 microglobulin（Chβ2m） were amplified from the total RNA of peripheral blood monouclear cells（PBMCs） by RT-PCR and cloned into pMD18-T. Then the PCR products deleted the signal sequence at the 5’ end of both the BF2 and Chβ2m genes and included the BirA substrate peptide（BSP） sequence at the 3’ end of the BF2 gene were sub-cloned into the expression vector pET-28a（+） and named as pET-BF2-BSP and pET-Chβ2m. High levels of BF2-BSP and Chβ2m proteins were successfully expressed in E. coli strain BL21（DE3） transformed with the recombinant vectors pET-BF2-BSP and pET-Chβ2m. The recombinant target proteins were purified with a Ni²⁺NTA column including His-Bind Resin, and then high-quality recombinant proteins were recovered.BF2-BSP and Chβ2m were refolded with synthetic peptide originated from infectious bronchitis virus nucleoprotein(IBV N_71-78) in refolding buffer to generate the monomer of BF2/peptide complex. The folded product was then subjected to enzymatic biotinylation by BirA enzyme. After biotinylation the sample was run over a gel filtration column to purify the BF2/peptide fraction. The tetrameric complexes of biotinylated BF2/peptide were produced by mixing purified, biotinylated monomer with PE-labeled streptavidin at a molar ratio of 4:1.In order to test the function of the prepared BF2/peptide tetramer, one-week SPF chicks were immunized with 10^5.5EID₅₀ IBV H52 strain through intranasal inoculation. The PBMCs were isolated at 10 days post infection（d.p.i.） and used for flow cytometry staining. The CTLs response of IBV-infected chicks was evaluated with the prepared BF2/peptide tetramer and 3.65% CTLs frequency specific to IBV N was detected.To discuss the kinetics of the antibody titer and the IBV N-specific T cell when the SPF chicks were infected with the IBV, 125 SPF chicks were divided into three groups at random, namely vaccinated group, challenge control group and negative control group. The SPF chicks from the vaccinated group were immunized with 10^5.5EID₅₀ IBV H52 strain through intranasal inoculation. Five chicks were sacrificed every three days for pathogenesis observation, assessment of antibody titer and IBV N-specific T cell measured by prepared BF2/peptide tetramer. The chicks from vaccinated group and challenge control group were challenged with IBV M41 at the day 15 after vaccination. Also, the same assessments were taken on as described above. All the results were pooled together to investigate the interaction between the antibody titer and nucleoprotein-specific T cell after vaccination and challenge. The results indicated that the CTL activity was shown to be effective during the initial elimination of virus in the chicks, later control of infection probably depends on antibody, especially the IgG response, which likely plays a important role in preventing recrudescence of infection.更多还原