Expression and refolding of OLA Ⅰ protein with peptides derived from sheeppox virus / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 139-147, 2022.
Artigo
em Chinês
| WPRIM
| ID: wpr-927699
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 111 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 SuperdexTM 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.
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Peptídeos
/
Doenças dos Ovinos
/
Ovinos
/
Capripoxvirus
/
Infecções por Poxviridae
/
Epitopos de Linfócito T
Limite:
Animais
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2022
Tipo de documento:
Artigo
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