RESUMO
The immune response in the fox (Vulpes vulpes), despite the success of the oral rabies vaccine is not well characterised, and specific immunological tools are needed. A quantitative RT-PCR using SyBR Green to investigate fox cytokine expression after antigen PBMC in vitro re-stimulation is presented here. First, we cloned by homology with dog cytokine sequences the fox IL2, IL6, IL10, IFNgamma and a partial 18S sequence. Fox specific primers were then defined and used to set up a species-specific quantitative RT-PCR assay using SyBR Green and 18S housekeeping gene as internal standard. The technique was validated using total RNA from fox PBMC stimulated with a polyclonal activator, Concanavaline A.
Assuntos
Concanavalina A/farmacologia , Raposas/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon gama/genética , Interleucina-10/genética , Interleucina-2/genética , Interleucina-6/genética , Sequência de Aminoácidos , Animais , Gatos , Clonagem Molecular , Cães , Perfilação da Expressão Gênica , Interferon gama/química , Interleucina-10/química , Interleucina-2/química , Interleucina-6/química , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
Identification of fox (Vulpes vulpes) sperm antigens was carried out to assess their interest as a potential target for a contraceptive vaccine. We report here the cloning and sequencing of fSP8, a fox sperm protein of 14.7 kd. fSP8 was isoantigenic in foxes, as it was recognized by sera of both male and female foxes immunized with fox sperm proteins. No glycosylation was detected, on fSP8, as shown both by deglycosylation assay and lectin labeling. To determine the fSP8 sequence, the NH2-terminal sequence was first obtained, and a piece of cDNA was amplified from testicular RNA by Rapid Amplification of cDNA extremities polymerase chain reaction. This piece was used to screen a cDNA library from fox testis by Southern blot. A sequence of 879 base pairs was obtained, which includes a major open reading frame coding for 128 amino acids. Mass spectrometric analyses have confirmed the position of the open reading frame. Analysis of the predicted amino acids sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated a homology with the Zinc binding site of the subunit Vb of the cytochrome c oxidase. On the C-terminal extremity, fSP8 presents a high homology to the Vb polypeptide of the cytochrome c oxidase from bovine, mouse, and human; however the 34 amino acids on the NH2-extremity were specific to fSP8. Moreover, it was demonstrated that this sequence was testis-specific. This could contribute to the antigenicity of this protein. fSP8 is one of the first fox sperm antigens to be cloned and sequenced.
Assuntos
DNA Complementar/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Raposas/genética , Espermatozoides/química , Testículo/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Complexo IV da Cadeia de Transporte de Elétrons/imunologia , Feminino , Biblioteca Gênica , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Subunidades Proteicas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermatozoides/imunologia , Testículo/imunologia , Vacinas Anticoncepcionais/imunologiaRESUMO
Fox (Vulpes vulpes) sperm antigens were identified to assess them as a potential target for a contraceptive vaccine. We report here the cloning and sequencing of fSP13, a fox sperm protein of 97 kDa. The fSP13 protein was both auto- and iso-antigenic in foxes; it was recognized by sera of foxes immunized with fox sperm proteins and vasectomized foxes. The NH2-terminal sequence of fSP13 was determined, and a piece of cDNA was amplified from testicular RNA by reverse transcription polymerase chain reaction. This piece was used to screen a cDNA library from fox testis by Southern blot. A sequence of 1662 base pairs was obtained, including a major open reading frame coding for 498 amino acid. Mass spectrometry analysis confirmed the position of the open reading frame and the presence of posttranscriptional modifications. Analysis of the predicted amino acid sequence revealed no apparent transmembrane regions. Comparison of the protein sequence with the Prosite database demonstrated the presence of four potential N-linked glycosylation sites. The fSP13 bears the closest amino acid similarity to two human sperm proteins: fibrousheathin 2 and testis-specific calcium binding protein 86-VII. The deduced 80 N-terminal amino acid sequence also presents similarity with the RIIalpha domain. By using a serum against fSP13, this antigen was localized on the principal piece of the fox spermatozoa. Northern blot analysis showed that fSP13 is specifically expressed in testis. The fSP13 is one of the first fox sperm antigens to be cloned and sequenced.
Assuntos
Antígenos/química , Raposas/genética , Cauda do Espermatozoide/química , Testículo/química , Sequência de Aminoácidos , Animais , Northern Blotting , DNA Complementar/biossíntese , DNA Complementar/genética , Eletroforese , Imunofluorescência , Focalização Isoelétrica , Masculino , Dados de Sequência Molecular , Peso Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vacinas AnticoncepcionaisRESUMO
The immune response in the fox (Vulpes vulpes), despite the success of the oral rabies vaccine is not well characterized, and specific immunological tools are needed. To investigate both the humoral and cellular immune response, we used ovalbumin (OVA) and cholera toxin B (CTB) as an antigenic model to set-up ELISA and ELISPOT antibodies secreting cells (ASC) assays in the fox model. Identification of antibodies that cross-react with fox immunoglobulin was performed by Western blot, and their use was adapted for both the ELISA and ELISPOT ASC assay. The humoral and cellular specific immune responses were assessed after intra-muscular or intra-nasal immunization. Intra-muscular immunization resulted in the development of both cellular and humoral anti-OVA and anti-CTB responses in peripheral blood mononuclear cells (PBMCs). Immunization via the intra-nasal route resulted in the development of a cellular and humoral response against CTB in PBMCs. This immune response was confirmed using splenocytes from immunized animals by ELISPOT assay at euthanasia. Females immunized via the intra-nasal route developed specific anti-CTB IgM, IgA and IgG in vaginal fluids after the initial boost (day 26) showing that mucosal immunization produces a vaginal immune response in foxes. These immunological tools developed here are now available to be adapted to other antigenic models to facilitate further immune studies in foxes.
