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

Zhanhong Wang; Zhixun Zhao; Guohua WU; Yang Deng; Guoqiang Zhu; Fangyan Zhao; Zengjun LU; Qiang Zhang

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

Author: Zhanhong WANG ¹ ; Zhixun ZHAO ¹ ; Guohua WU ¹ ; Yang DENG ¹ ; Guoqiang ZHU ¹ ; Fangyan ZHAO ¹ ; Zengjun LU ¹ ; Qiang ZHANG ¹
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1. State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, Gansu, China.
Publication Type:Journal Article
Keywords: CTL epitope; OLA Ⅰ; molecular exclusion chromatography; sheeppox virus
MeSH: Animals; Capripoxvirus; Epitopes, T-Lymphocyte/genetics*; Peptides/genetics*; Poxviridae Infections; Sheep; Sheep Diseases
From: Chinese Journal of Biotechnology 2022;38(1):139-147
CountryChina
Language:Chinese
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.