猪SLA I分子超级型及猪源重要病毒CTL交叉表位的研究

Developing swine major histocompatibility complex (also named swine lymphocyte antigen, SLA) class I supertype system and indentifying CTL cross-reaction epitopes of important swine-origin viruses are essential for the design of porcine CTL epitope vaccine and control the swine-origin zoonoses. In this study, the SLA I molecules of domestic pigs will be massively cloned and expressed, and their 3D models will be constructed to analyze their antigen-binding domains. According to the amino acid compositions of the antigen-binding grooves, the SLA I molecules will be classified into different supertypes. The predominant SLA supertypes covering great percent of domestic pig will be identified. The CTL epitopes from important swine-origin virues (Classical swine fever virus，CSFV；Porcine reproductive and respiratory syndrome virus, PRRSV; Foot and mouth disease virus, FMDV; Swine-origin influenza virus, S-OIV) binding with major SLA I supertype molecules will be screened by using bioinformatics methods. The selected peptides will be synthesized, and their binding affinities with corresponding SLA I molecules will be checked by co-refolding and circular dichroism spectrum (CD) in order to test the SLA I supertype classification. At last, the functional CTL cross-reaction epitope peptides will be identified by SLA I tetramer， flow cytometry (FCM) and enzyme linked immunospot assay (ELISPOT). This study will benefit for expanding the applicable scope of CTL epitope vaccine in domestic pigs.

对猪主要组织相容性复合体（SLA）I分子进行超级型分类，鉴定猪源病毒的细胞毒性T淋巴细胞（CTL）交叉表位是开发猪用CTL表位疫苗的基础，对于控制猪源病毒性疾病有重要的价值。本项目拟大量克隆、表达我国猪群中存在的SLA I分子，对其进行精确的分子建模；在此基础上对不同SLA I分子的抗原结合区域进行分析，根据氨基酸组成的差异，对我国猪群中主要的SLA I分子进行超级型分类；利用生物信息学手段，筛选表型覆盖率高的SLA I超级型分子所能结合的猪源重要病毒（猪瘟、蓝耳病、口蹄疫、猪流感）的CTL表位；人工合成表位多肽，利用体外共复性、圆二色谱等技术鉴定多肽与相应SLA I分子的结合力，验证SLA I超级型分类的正确性；进一步利用四聚体、流失细胞术及酶联免疫斑点实验等技术，鉴定结合力强的多肽是否为功能性CTL交叉表位。研究结果将为解决CTL表位疫苗在猪群中适用范围窄的难题提供理论支持和数据储备。

本研究旨在对我国猪群SLA I 分子超级型及猪源重要病毒CTL交叉表位进行研究，因此我们克隆了94条我国主要养殖猪种的SLA I等位基因并进行了系统发育及变异频率的分析，将高变异位点定位到SLA I分子的结构中，发现这些位点集中分布在与抗原呈递相关的多肽结合槽中。对猪群中的广泛存在的SLA-1*0401及与其序列高度相似的等位基因进行了超级型分析，鉴定得到了3条流感病毒CTL交叉表位。构建了SLA-1*0401突变体并获得了其晶体结构，为阐释SLA-1*0401超级型呈递多肽的机制打下基础。解析了黑山猪SLA-3*0202的晶体结构，确定了其多肽结合基序，并对其能够呈递的猪流感病毒表位进行了筛选和鉴定。另外，本研究还解析了猪CD8αα的晶体结构，分析了其与不同SLA I分子结合的结构基础。本研究结果将推动对SLA I分子超级型的分类和猪流感病毒表位疫苗的开发的深入研究。