| TRIM25 and IFN-β are important innate antiviral immune molecules in the body,which have been applied in the prevention and treatment of some diseases in humans and mammals,but their clinical applications in chicken production are rarely reported.In this study,the expression methods of chicken TRIM25 and chicken IFN-β in vitro were established,and the biological activities of the expressed products were analyzed in vivo and in vitro,so as to provide scientific basis for the development of new drugs.In this study,primers were designed according to chicken IFN-β gene sequence,and the target gene was amplified with chicken liver DNA gene as template.The prokaryotic expression vector pET-32a-IFN-β was induced by 1 mmol/L IPTG at 37℃ for 4 h,and the inclusion body protein was expressed.The inclusion body protein was broken and purified,and the target protein with high content was obtained.Secondly,the prokaryotic expression vector of chicken TRIM25 and the expression vector of Pichia pastoris were induced to obtain the corresponding expression products,which were purified and identified by Western blot.Third,SPF chickens were inoculated with chicken pET-32a-IFN-β protein and pET-32a-TRIM25 protein combined with chicken NDV attenuated vaccine and ALV-A protein antigen,respectively,and the titers of NDV vaccine antibody and ALV-A protein antibody and lymphocyte proliferation reaction were detected.SPF chickens were inoculated with pET-32a-TRIM25 and Ppic9k-TRIM25 protein combined with AIV inactivated vaccine and ALV-A protein antigen,respectively.The antibody titer and lymphocyte proliferation reaction of AIV vaccine and ALV-A protein were detected,and their immune enhancement effects were compared.Fourth,the effect of three proteins on ALV-A replication was analyzed by cell test in vitro.The results showed that:（1）the chicken IFN-β gene was amplified by PCR and the constructed pET-32a-IFN-βrecombinant expression plasmid could induce the expression of the target protein band of 37 k Da.The protein content was more than 95% after purification by nickel column chromatography.（2）the prokaryotic expression plasmid of chicken TRIM25 constructed in laboratory and the expression plasmid of Pichia pastoris were induced to obtain the target protein with the size of 75 k Da,the former being the inclusion body protein pET-32a-TRIM25 and the latter being the secretory protein Ppic9k-TRIM25.（3）pET-32a-TRIM25 protein and pET-32a-IFN-β protein could significantly enhance the antibody immune response and TRIM25/IFN-βlymphocyte proliferation response of NDV attenuated vaccine and ALV-A protein antigen,but the enhancement effect of pET-32a-TRIM25 protein was more significant.（4）both pET-32a-TRIM25 and Ppic9k-TRIM25 protein could significantly improve the antibody immune response and lymphocyte proliferation response of chicken AIV inactivated vaccine to ALV-A protein antigen,and the immune enhancement effect of Ppic9k-TRIM25 protein was significantly higher than that of pET-32a-TRIM25 protein.（5）2 μg pET-32a-IFN-βprotein could significantly inhibit ALV-A virus replication in DF-1 cells at different times,while pET-32a-TRIM25 and Ppic9k-TRIM25 proteins had no significant effect on ALV-A virus replication.Conclusion: in this study,a method for obtaining bioactive proteins of chicken IFN-β and chicken TRIM25 was established successfully.the protein products expressed in vitro increased the serum antibody reaction and lymphocyte proliferation of the vaccine in varying degrees,and chicken IFN-β could significantly inhibit ALV-A replication,which provided methodological and theoretical basis for the development and application of the two proteins. |
