【作者】 侯艳霞；

【导师】 王靖飞；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2011， 硕士

【摘要】 禽流感是由禽流感病毒(Avian influenza virus, AIV)引起的禽的一种高度传染病。AIV中节段5编码的核蛋白(Nucleoprotein, NP)上含有大量的细胞毒性淋巴细胞识别的抗原表位,其诱导的特异性的细胞毒性T淋巴细胞(Cytotoxic T lymphocyte, CTL)反应在不同亚型的AIV之间具有一定的交叉保护力。目前许多关于流感病毒的T细胞表位在人和鼠中已获得鉴定。然而,由于禽主要组织相容性复合体(Major histocompatibility complex, MHC)的结构和功能方面研究很少,已鉴定的禽流感病毒结构蛋白中鸡MHC限制性T细胞表位非常有限,限制了禽类抗流感病毒细胞免疫研究的开展。为鉴定H5亚型禽流感病毒NP中能被鸡MHC分子特异性识别的CTL表位,本研究首先模建了B4、B12、B15和B19单倍型鸡的MHC I类分子的α1和α2结构域的三维结构,并分析了各个单倍型结构的肽结合槽的表面性质。结果显示:结合槽的表面都高度疏水;结合槽内B口袋比F口袋的柔性大;B4单倍型的结合槽表面大部分区域带正电,B15和B19的结合槽表面以负电荷聚集为主,B12的表面相对呈中性。将AIV代表毒株A/Goose/Guangdong/1/1996 (H5N1)的NP蛋白截取重叠肽,选出符合B4、B12、B15和B19单倍型MHC I类分子结合基序的多肽,利用分子对接方法将多肽与相应的MHC I类分子结构对接,预测可以和各单倍型具有高亲和力的多肽。经以构象和打分函数相结合的方法筛选,最终获得25条(其中结合B4、B12、B15和B19的多肽数分别为10、6、2和7)与各个单倍型的MHC I类分子结合较好的潜在CTL表位肽,并选出结合能最高的9条短肽用于实验室免疫原性检测。为了诱导SPF鸡产生针对AIV NP的记忆性T细胞,本研究将优化成鸡偏嗜性的NP基因双酶切后亚克隆到含禽源启动子的真核表达载体pCAGGS中,构建重组质粒pCAGGS-NP,经筛选鉴定正确的阳性重组质粒转染293T细胞,间接免疫荧光和Western blot检测表明NP蛋白在真核细胞中能正确表达并具有良好的免疫原性。重组质粒pCAGGS-NP以100 ug/羽的剂量腿部肌肉免疫3周龄SPF鸡,3周后以同样的剂量加强免疫,间接酶联免疫实验检测结果显示pCAGGS-NP能诱导SPF鸡产生针对AIV NP的抗体,抗体水平在加强免疫后迅速升高。取加强免疫三周后的鸡脾脏制成淋巴单细胞悬液,调整细胞浓度为1×106 /mL,一部分细胞加入上述9条短肽和1条不相关肽进行体外刺激培养,24 h后收集细胞,采用ELISA试剂盒检测培养上清中IFN-γ分泌量;一部分细胞加入CFSE贮存液和10条合成肽体外培养,5 d后用流式细胞术对培养后细胞中CD8~+T淋巴细胞增殖情况进行检测。检测结果一致显示肽NP89-97(PKKTGGPIY)和NP198-206(KRGINDRNF)能刺激活化的鸡脾淋巴细胞IFN-γ分泌量明显增加,CD8~+T淋巴细胞分别有13.7 %、11.9 %的增值,表明NP89-97和NP198-206能诱导较强的细胞免疫反应,为AIV NP的T细胞表位。本研究所构建的鸡MHC I类分子的结构及鉴定的表位为进一步探讨AIV所引起的细胞免疫及T细胞表位的鉴定奠定了基础。更多还原

【Abstract】 Avian influenza virus(AIV) is a causative agent of highly contagious viral disease in chickens. Previous studies showed that T cell-mediated immune responses play a critical role in defense against influenza virus infection. The nucleoprotein (NP) encoded by the fifth fragment of AIV is a conserved inner protein, and it contains many T cell epitopes. Those epitopes can induce Cytotoxic T lymphocyte(CTL) response against the infection of different subtypes of influenza viruses. Some T cell epitopes from various proteins of influenza virus have been identified in human and mouse. However, little is known about which in chicken partially for lacking of the structure of the chicken major histocompatibility complex (MHC).In this study, we have developed three-dimensional structures ofα1 andα2 domains of chicken MHC classΙmolecules from B4, B12, B15 and B19 haplotypes, those models provide clear insight into the structural characters of their peptide binding grooves. The surface of peptide binding grooves in those four structures are high lipothilic; The B pockets of the four structures are more flexible than the F pockets; The electrostatic potential of the peptide binding groove of B4 haplotype is highly positive charged,B15 and B19 haplotypes are negatively charged, while that of B12 is neutral. Based on structural properties, we screened antigen peptides derived from an AIV NP according to peptide-binding motifs and molecular docking, all together 25 peptides (10 for B4, 6 for B12, 2 for B15 and 7 for B19 haplotype) were predicted with potentials to bind to those MHC class I molecules. The top 9 peptides with high binding energy were selected to verify their activity.In order to induce CD8~+ CTL specific to AIV NP in SPF chicken, NP gene, which was optimized with chicken biased codons, was sub-cloned into the eukaryotic expression vector pCAGGS. The recombinant plasmid pCAGGS-NP was constructed and the expression of NP was confirmed by IFA and western blot in transfected 293T cells. SPF chickens were immunized with 100μg pCAGG-NP and boosted with the same dose three weeks later. A dramatically increase NP antibody was detected by ELISA after being boosted. The spleens were isolated from vaccinated chickens for the preparation of lymphocytes. The peptide-stimulated lymphocytes were used to detect the secretions of chicken IFN-γand proliferation of CD8~+ T cell by ELISA kit and flow cytometry analysis respectively. The results showed that the increase of chicken IFN-γsecretions and proliferation of CD8~+ T lymphocytes by 13.8%, 11.9 % were detected in cells stimulated with peptides NP89-97 and NP198-206. The results indicate that peptides NP89-97 and NP198-206 are NP T cell epitopes in chicken. The homology modeling structures of chicken MHC class I molecules and the identified epitopes will extend our understanding of the mechanisms of immune response to AIV in chickens.更多还原