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.
Animals ; Capripoxvirus ; Epitopes, T-Lymphocyte/genetics* ; Peptides/genetics* ; Poxviridae Infections ; Sheep ; Sheep Diseases

The complete gene sequences of eight capripoxvirus strains in GenBank were aligned and analyzed with DNAStar software. We selected a size of 64 bp gene fragment that was located in gp064 region of goat pox virus (GPV) genome, and designed a pair of primers and a TaqMan-MGB probe against the gene fragment with Primer Express 2.0 software. Then, the fluorescence quantitative PCR (FQ-PCR) assay was developed and the standard curve of different dilution series was described. We extracted the DNA samples from clinical skin pox, scab and GPV infected materials of artificial challenge animals. The FQ-PCR assay has been performed for all kinds of DNA samples. The results showed that the FQ-PCR assay was sensitive, specific, stable and could be used for clinical diagnosis. This method provided an important tool for rapid diagnosis of goat pox clinically, and for study GPV pathogenesis in the course of disease occurrence, development and convalescence.
Animals ; Base Sequence ; Capripoxvirus ; genetics ; isolation & purification ; Goats ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; Poxviridae Infections ; diagnosis ; virology ; Sensitivity and Specificity

The full-length P32 gene and the truncated P32 gene (MP-32) were amplified from the recombinant plasmid pMD-P32 by polymerase chain reaction (PCR) and cloned into pcDNA3. 1(+) and pcDNA3.1-CpG respectively. The recombinant plasmids (pcDNA3.1-P32, pcDNA3.1-CpG-P32 and pcDNA3. 1-CpG-MP32) were transfected into BHK-21 cells by using lipofectin. The expressed P32 protein was confirmed by indirect immunofluorescence assay (IFA). The BALB/c mice were immunized with these recombinant plasmids by intramuscular injection. The specific antibodies aginst CPV were detected by ELISA kit weekly. The murine splenic T lymphocyte subgroups CD4+ and CD8+ were detected by flow cytometry. Results showed that the P32 protein was expressed successfully in vitro. After 2 weeks post im munization, the specific IgG antibodies against CPV were detected in the vaccinated mice. The percentage of CD4+ /CD8+ T cells was significantly higher than that of the control. In conclusion, these constructed eukaryotic vectors could induce humoral and celluar immune responses in mice.
Animals ; Antibodies, Viral ; blood ; Capripoxvirus ; genetics ; immunology ; Cell Line ; CpG Islands ; Cricetinae ; Female ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; immunology ; T-Lymphocyte Subsets ; immunology ; Vaccines, Synthetic ; immunology ; Viral Envelope Proteins ; immunology ; Viral Vaccines ; immunology

The purpose of the study is to construct recombinant goat pox virus (GPV) expressing Peste des petits ruminants virus (PPRV) H protein, and to evaluate the immunization effect. Recombinant GPV containing PPRV H gene (rGPV-PPRV-H) was selected and purified by gpt and eGFP utilizing plaque purification, and the final selected recombinant GPV was proved to be purified by PCR. Immunofluorescence and Western blotting showed that the recombinant virus could express H protein of PPRV while infecting lamb testis cells. Six goats were immunized with 2 x 10(6) PFU rGPV-PPRV-H through intradermal injection, and were immunized for the second time at 28 days with the same dose recombinant virus after first immunization. Serum was collected after immunization, and was analyzed for the neutralization antibodies. 21 days after first immunization, the neutralization antibodies of GPV were 40, 80, > or = 80, > or = 80, 40, > or = 80 in turn, and neutralization antibodies of PPRV were 80, 80, 80, 80, 40, 40, 10 in turn; 14 days after second immunization, the neutralization antibodies of GPV were all > or = 80, and the neutralization antibodies of PPRV were > 80, 80, > 80, 80, 80 and 40 in turn. This study established a foundation for the industrialization of the PPRV recombinant GPV vaccine.
Animals ; Capripoxvirus ; genetics ; immunology ; Goat Diseases ; immunology ; prevention & control ; virology ; Goats ; Hemagglutinins, Viral ; genetics ; immunology ; metabolism ; Peste-des-Petits-Ruminants ; immunology ; prevention & control ; Peste-des-petits-ruminants virus ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Vaccines, Combined ; immunology ; Vaccines, Synthetic ; immunology ; Viral Vaccines ; immunology