ABSTRACT
BACKGROUND: Interferon-gamma (IFN-γ) is an important mediator of type I immune response and has antiviral, immunoregulatory and anti-tumor properties, plays a wide range of roles in inflammation and autoimmune diseases. The aim of this study was to obtain monoclonal antibody (mAb) against caprine IFN-γ by immunizing of BALB/c mice with the purified rIFN-γ. RESULTS: Recombinant caprine IFN-γ was expressed in Escherichia coli strain BL21 (DE3) and monoclonal antibodies against caprine IFN-γ were produced by immunizing of BALB/c mice with rIFN-γ. One hybridoma secreting mAb was screened by enzyme-linked immunosorbent assay (ELISA) which was designated as 2C. MAb secreted by this cell line were analyzed through ELISA, western blot and application of the mAb was evaluated by immunofluorescence analysis using goat lip tissues infected with Orf virus. ELISA analysis revealed that mAb 2C can specifically recognize rIFN-γ protein and culture supernatant of goat peripheral blood mononuclear cells (PBMCs) stimulated by concanavalin A (Con A) but cannot recognize the fusion tag protein of pET-32a. Western blot analysis showed that mAb 2C can specifically react with the purified 34.9 kDa rIFN-γ protein but does not react with the fusion tag protein of pET-32a. Immunofluorescence results demonstrated that mAb 2C can detect IFN-γ secreted in histopathological sites of goats infected with Orf virus. CONCLUSIONS: A caprine IFN-γ-specific mAb was successfully developed in this study. Further analyses showed that the mAb can be used to detect IFN-γ expression level during contagious ecthyma in goats.
Subject(s)
Antibodies, Monoclonal/analysis , Interferon-gamma/analysis , Interferon-gamma/immunology , Animals , Antibodies, Monoclonal/metabolism , Blotting, Western , Ecthyma, Contagious/blood , Ecthyma, Contagious/immunology , Ecthyma, Contagious/virology , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Goat Diseases/blood , Goat Diseases/immunology , Goat Diseases/virology , Goats , Hybridomas/metabolism , Interferon-gamma/blood , Interferon-gamma/genetics , Leukocytes, Mononuclear/immunology , Mice, Inbred BALB C , Orf virus/physiologyABSTRACT
The mammalian intestine is colonized by over a trillion microbes that comprise the "gut microbiota," a microbial community which has co-evolved with the host to form a mutually beneficial relationship. Accumulating evidence indicates that the gut microbiota participates in immune system maturation and also plays a central role in host defense against pathogens. Here we review some of the mechanisms employed by the gut microbiota to boost the innate immune response against pathogens present on epithelial mucosal surfaces. Antimicrobial peptide secretion, inflammasome activation and induction of host IL-22, IL-17, and IL-10 production are the most commonly observed strategies employed by the gut microbiota for host anti-pathogen defense. Taken together, the body of evidence suggests that the host gut microbiota can elicit innate immunity against pathogens.
