RESUMO
The recombinant LaSota strain expressing a chimeric IBV S1 gene (rLaSota-S1) was constructed with the S1 gene of the LX4 type IBV ck/CH/LDL/091022. The expression of the S1 protein was detected by an indirect immunofluorescence assay and Western blotting. The rLaSota-S1 strain was slightly attenuated, and its growth dynamics were similar to that of the parental LaSota strain. Vaccination of specific pathogen-free chickens with the rLaSota-S1 strain induced NDV hemagglutination inhibition antibodies, and it protected chickens from challenge with virulent NDV. In addition, vaccination with the rLaSota-S1 strain induced IBV-specific IgG antibodies and cellular immunity; however, a single vaccination provided partial protection with reduced virus shedding. Better protection efficiency was observed after a booster vaccination, which resulted in higher antibody titers, significantly fewer disease symptoms, and reduced virus replication and shedding. Our results suggest that the rLaSota-S1 strain is a bivalent vaccine candidate against both NDV and IBV.
Assuntos
Infecções por Coronavirus/veterinária , Vírus da Bronquite Infecciosa/imunologia , Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle/imunologia , Doenças das Aves Domésticas/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Galinhas , Infecções por Coronavirus/prevenção & controle , Testes de Inibição da Hemaglutinação , Imunoglobulina G/sangue , Vírus da Bronquite Infecciosa/genética , Leucócitos Mononucleares/imunologia , Vírus da Doença de Newcastle/genética , Glicoproteína da Espícula de Coronavírus/genética , Resultado do Tratamento , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/isolamento & purificação , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/isolamento & purificaçãoRESUMO
OBJECTIVE: To study the structural characteristics of the mouse DNA-dependent activator of interferon-regulatory factors (DAI) and its related molecular mechanism in anti-viral innate immune responses and signal transduction. METHODS: The coding sequence of mouse DAI gene was amplified from splenic mononuclear cells by reverse transcription-PCR, and the genetic evolution and molecular structure of the mouse DAI gene were analyzed by bioinformatics softwares. After mouse DAI was stimulated by poly(dA-dT) and poly(dG-dC), the nuclear factor κB (NF-κB) and interferon-beta (IFN-ß) promoter-driven luciferase activity were detected by dual-luciferase reporter assay system. RESULTS: The open reading frame (ORF) of the cloned mouse DAI sequence was 1236 bp, encoding 411 amino acids, which exhibited identity with the corresponding sequences of cattle, pig, rat and other mammals ranging from 60%, 63.1%, 84%, and it contained two Z-DNA domains (Zα and Zß), DNA binding region (D3) and signaling domain (SD). The stimulation of poly (dA-dT) increased the expressions of mouse DAI activated transcription factors NF-κB and IFN-ß promoter. However, the stimulation of poly(dG-dC) only induced the activation of NF-κB but not IFN-ß promoter. CONCLUSION: Mouse DAI as an important cytosolic DNA sensor, is responsible for the recognition of A/T or G/C-rich DNA derived from DNA virus. It may play an important role in anti-viral innate immune responses.
Assuntos
DNA/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Camundongos/genética , Animais , Bovinos , Feminino , Glicoproteínas/química , Humanos , Masculino , Mamíferos/classificação , Mamíferos/genética , Camundongos/metabolismo , Camundongos Endogâmicos C57BL , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Ratos , SuínosRESUMO
Entire 3ABC sequence of FMDV containing a 6 x his tag coding sequence at the N-terminal was obtained through PCR amplification using a pair of specific primers, subcloned into shuttle plasmid of pMelBac-B with a melittin secretion signal sequence and finally constructed recombinant plasmid of pMel-3ABC. After co-transfected the recombinant plasmid and linearized Bac-N-Blue DNA into Sf9 insect cell under intermediary agent of the Cellfectin, the result showed that we have already acquired recombinant baculovirus by screen of plaque assay and identification of PCR. Though the recombinant baculovirus infecting the Sf9 cells again, experiments indicated that 3ABC gene could express in insect cells and the expressed protein was secreted in the supernatant of Sf9 cell culture possessing favourable biological activities detected by adopting two methods of SDS-PAGE and Western blot. The result verified that the protein could respond with sera derived from FMDV infected animals, but have no responsibility with sera derived from health animals and vaccinated animals detected by indirect ELISA using antigen of expressed protein after purification with Ni-NTA his bind resin. Therefore, this study has established a solid foundation for establishing an effective diagnosis method to discriminating the FMDV infected animals from vaccinated animals.