一组绵羊OLA Ⅰ蛋白与绵羊痘病毒多肽的复性组装

Expression and refolding of OLA Ⅰ protein with peptides derived from sheeppox virus

摘要:

本研究旨在体外组装小尾寒羊OLA Ⅰ蛋白与绵羊痘病毒多肽复合物，筛选绵羊痘病毒CTL表位肽。首先克隆小尾寒羊OLA Ⅰ重链基因胞外区OLA Ⅰ α-BSP和轻链基因OLA Ⅰ-β₂m，将获得的轻、重链基因分别插入原核表达载体pET-28a(+)并转化至BL21(DE3) 细胞进行诱导表达，收集菌体超声破碎，过镍柱纯化获得一定纯度的OLA Ⅰ重链和轻链β₂m包涵体蛋白，稀释复性法将获得的轻、重链包涵体蛋白与绵羊痘病毒多肽PV4按摩尔比为1︰1︰1的比例进行共复性，分子筛层析验证复性情况，ELISPOT试验评价OLA Ⅰ分子限制性多肽引起的T细胞免疫应答。结果表明，克隆获得的重、轻链基因正确表达，大小分别为36.3 kDa和16.7 kDa，分子筛和SDS-PAGE结果表明，绵羊痘病毒多肽PV4与OLA Ⅰ分子稳定结合，ELISPOT试验确定该多肽可刺激引起T细胞免疫应答。本研究建立了小尾寒羊OLA Ⅰ分子轻、重链的原核表达体系，实现该轻、重链与绵羊痘病毒多肽PV4的复性，确定了1个绵羊痘病毒CTL表位肽，为下一步解析该复合物的结构及对羊痘CTL表位筛选提供思路。

关键词:OLA Ⅰ;绵羊痘病毒;分子筛;CTL表位

Abstract:

The aim of this study was to refold the OvisAries leukocyte antigen (OLA) class Ⅰ protein with peptides derived from sheeppox virus (SPPV) to identify SPPV T cell epitopes. Two pairs of primers were designed based on the published sequence of a sheep major histocompatibility complex Ⅰ to amplify the heavy chain gene of OLA Ⅰ α-BSP and the light chain gene of OLA Ⅰ-β₂m. Both genes were cloned into a pET-28a(+) expression vector, respectively, and induced with ITPG for protein expression. After purification, the heavy chain and light chain proteins as well as peptides derived from SPPV were refolded at a ratio of 1:1:1 using a gradual dilution method. Molecular exclusion chromatography was used to test whether these peptides bind to the OLA Ⅰ complex. T-cell responses were assessed using freshly isolated PBMCs from immunized sheep through IFN-γ ELISPOT with peptides derived from SPPV protein. The results showed that the cloned heavy chain and light chain expressed sufficiently, with a molecular weight of 36.3 kDa and 16.7 kDa, respectively. The protein separated via a Superdex^TM 200 increase 10/300 GL column was collected and verified by SDS-PAGE after refolding. One SPPV CTL epitope was identified after combined refolding and functional studies based on T-cell epitopes derived from SPPV. An OLA Ⅰ/peptide complex was refolded correctly, which is necessary for the structural characterization. This study may contribute to the development of sheep vaccine based on peptides.

Key words:OLA Ⅰ;sheeppox virus;molecular exclusion chromatography;CTL epitope