RESUMO
Background@#Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) on the surface of Streptococcus dysgalactiae, coded with gapC, is a glycolytic enzyme that was reported to be a moonlighting protein and virulence factor. @*Objective@#This study assessed GAPDH as a potential immunization candidate protein to prevent streptococcus infections. @*Methods@#Mice were vaccinated subcutaneously with recombinant GAPDH and challenged with S. dysgalactiae in vivo. They were then evaluated using histological methods. rGAPDH of mouse bone marrow-derived dendritic cells (BMDCs) was evaluated using immunoblotting, reverse transcription quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay methods. @*Results@#Vaccination with rGAPDH improved the survival rates and decreased the bacterial burdens in the mammary glands compared to the control group. The mechanism by which rGAPDH vaccination protects against S. dysgalactiae was investigated. In vitro experiments showed that rGAPDH boosted the generation of interleukin-10 and tumor necrosis factor-α. Treatment of BMDCs with TAK-242, a toll-like receptor 4 inhibitor, or C29, a toll-like receptor 2 inhibitor, reduced cytokines substantially, suggesting that rGAPDH may be a potential ligand for both TLR2 and TLR4. Subsequent investigations showed that rGAPDH may activate the phosphorylation of MAPKs and nuclear factor-κB. @*Conclusions@#GAPDH is a promising immunization candidate protein for targeting virulence and enhancing immune-mediated protection. Further investigations are warranted to understand the mechanisms underlying the activation of BMDCs by rGAPDH in a TLR2- and TLR4-dependent manner and the regulation of inflammatory cytokines contributing to mastitis pathogenesis.
RESUMO
Immunoglobulin (Ig) is considered a part of the innate immune system and cooperates with the complementary system as the first line of defense. In this study, a monoclonal antibody (MAb) direct against the light chain of goose Ig (GoIgCL) was generated, characterized and identified in various immunoassays to detect goose Ig. An immunoaffinity chromatography column prepared with this MAb was used to separate the goose Ig from sera. After being conjugated with horseradish peroxidase (HRP), this MAb was used as the secondary antibody to evaluate the goose-specific antibody. In addition, this MAb distinguished and localized the SIg+ lymphocytes from peripheral blood lymphocytes. MAb against GoIgCL may be good candidate to detect or purify goose Ig under various conditions and as a powerful tool for humoral immunity research on goose.
RESUMO
After sequencing the Asia 1 foot-and-mouth disease virus (FMDV) (As01 strain), we amplified the two fragments covering the whole genome by overlapping PCR and long PCR. The 5' fragment was 1.8 kb in length including 15Cs, and the 3' fragment was 6.7 kb in length. The two fragments were cloned into the pBluescript SK vector to construct recombinant plasmid pBSAs carrying the full-length cDNA of FMDV As01 strain. The RNA transcript was synthesized in vitro using T7 polymerase and transfected into BHK-21 cells. We observed the typical CPE caused by rescued FMDV. The harvested virus was confirmed to be Asia 1 FMDV by RT-PCR, indirect immunofluorescence assay (IFA) and electron microscope observation. The rescued virus showed a similar pathogenicity in suckling mouse (LD50) compared to its wild-type virus. The infectious cDNA clone of the FMDV As01 strain laid a new ground for further investigation of FMDV virulence determinants and development of novel vaccines against FMD.