山羊痘病毒CTL表位鉴定和GLAⅠ-肽结构与功能分析

CTL epitopes play an important role in the immune process in which poxviruses are involved. However, the study on CTL epitopes against goatpox virus is rarely reported. Based on the principle of antigen presentation of MHC I in vivo, α-chain, β2m and peptides were assembled to GLA I-peptide, which can effectively stimulate the proliferation of CD8+ T cells. In this study, the α-chain and β2m genes of GLA I were cloned from 4 varieties of goats and CTL epitopes against goatpox virus were predicted by three bioinformatic methods and identified by ELISPOT and cytokine detection assay. The complex proteins including α, β2m and peptides were refolded together after renaturation and the structural analyses were performed by the Swiss-Model and X-diffraction of protein crystallization technique. To analyze the function of GLA I-peptide, the tetramers were constructed, then identified by flow cytometry. This study will lay the foundation for the research of cell-mediated immune response mechanism against goatpox virus and provide a new insight for the development of epitopes vaccine.

CTL表位在痘病毒参与的免疫应答过程中具有重要作用，然而，对羊痘病毒CTL表位的研究目前仍然处于起步阶段。GLAⅠ-肽模仿生物体内MHCⅠ抗原提呈原理，在体外完成α链、β2m及其表位肽的装配过程，能够有效刺激CD8+T细胞的增殖反应，具有良好的免疫学功能。本研究针对四品系山羊克隆GLAⅠ的α和β2m基因，采用BIMAS，SYFPEITHI，NETMHCPAN三种生物信息学技术和ELISPOT及细胞因子检测方法完成山羊痘病毒CTL表位的预测及鉴定，然后通过蛋白共复性的方法完成GLAⅠ-肽复合蛋白的组装，使用Swiss-Model三维结构同源建模和蛋白结晶X衍射技术完成结构分析，最终构建GLAⅠ-肽四聚体，并进行流式检测以达到功能分析的目的。本研究将为阐明山羊痘病毒的细胞免疫机制研究奠定基础，并为其CTL表位疫苗的设计提供新思路。

CTL表位在痘病毒参与的免疫应答过程中具有重要作用，然而，对羊痘病毒CTL表位的研究目前仍然处于起步阶段。GLAⅠ-肽模仿生物体内MHCⅠ抗原提呈原理，在体外完成α链、β2m及其表位肽的装配过程，能够有效刺激CD8+T细胞的增殖反应，具有良好的免疫学功能。本研究针对四品系山羊克隆GLAⅠ的α和β2m基因，采用BIMAS，SYFPEITHI，NETMHCPAN三种生物信息学技术和ELISPOT及细胞因子检测方法完成山羊痘病毒CTL表位的预测及鉴定，然后通过蛋白共复性的方法完成GLAⅠ-肽复合蛋白的组装，使用Swiss-Model三维结构同源建模，最终构建体外构建OLAⅠ/肽复合体筛选绵羊痘病毒CTL表位。本研究将为阐明山羊痘病毒的细胞免疫机制研究奠定基础，并为其CTL表位疫苗的设计提供新思路。