RESUMO
This study aims to construct a 3D structure of the avian major histocompatibility complex (MHC)-ß2M complex through homology modelling technology, perform molecular docking of the predicted infectious bronchitis virus (IBV) S1 protein potential epitope peptide Sp6 (NQFYIKLT) and the avian MHC-ß2M complex, and demonstrate the interactive mechanism between Sp6 and MHC using molecular dynamical simulations. The peptide Sp6 and the non-related peptide NP89-97 (PKKTGGPIY) were used to stimulate in vitro recombinant plasmid (pCAGGS-S1) avian splenic lymphocytes. Flow cytometric results show that CD8(+) T lymphocytes reproduce stimulated by the Sp6 and the nonrelated peptide proliferate by 34.8% and 2.6%, respectively. Meanwhile, fluorescent quantitative PCR results show that the secretion of IFN-γ in avian splenic lymphocytes increases after Sp6 stimulation. These data suggest that Sp6 can induce the activated avian lymphocytes in vitro to produce CTL, which is the CTL epitope in IBV S1.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epitopos de Linfócito T/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Vírus da Bronquite Infecciosa/imunologia , Simulação de Acoplamento Molecular , Glicoproteína da Espícula de Coronavírus/metabolismo , Proliferação de Células , Epitopos de Linfócito T/química , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/química , Interferon gama/biossíntese , Ligação Proteica , Reação em Cadeia da Polimerase em Tempo Real , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
Hemagglutinin gene of subtype H5 avian influenza virus was amplified by polymerase chain reaction to construct expression cassette containing FPV early, late promoter and SV40 polyA tail. Then delivery vector was constructed by subcloning hemagglutinin gene of subtype H5 and GFP gene into fowlpox virus recombinant arm. The delivery vector and Lipid were transfected into CEF cells preinfected with FPV 282E4 strain virus. Recombinant fowlpox virus expressing the green fluorescence protein and hemagglutinin gene was screened and plaques were purified in CEF cell. After a second cotransfection with Cre recombinase plasmid, a recombinant virus only including hemagglutinin gene was gained. The immunofluorescent assay and replication efficiency of virus proved the recombinant could replicate steadily and express subtype H5 hemagglutinin gene. Two groups of 8-day-old SPF chickens were vaccinated with rFPVH5 by the wing-web method at the dosage of 10(5) PFU and 2 x 10(5) PFU respectively. After 28 days,antibodies titer was tested by HI. The results showed that the recombinant fowlpox virus could activate high antibody response.