Establishment And Preliminary Application Of ELISA Method Based On Infectious Bronchitis Virus 5b Protein

Infectious bronchitis virus(IBV)can caused acute respiratory disease in chickens.IBV has the property of being easily mutated,and the attenuated vaccine may have the risk of being scattered and poisoned.These pose great challenges for the purification of IBV diseases in the future,so attenuated vaccines will surely be eliminated with the development of technology.Therefore,the establishment of an IBV-based non-structural protein ELISA antibody detection method is useful for understanding the prevalence of Infectious Bronchitis(IB)in chickens,timely adjustment of immunization procedures,effective prevention of IB,and future treatment of IBV disease.Detection method.5b protein is a non-structural protein of IBV,and its structure and function are conserved.It is not involved in the composition of virus particles.In this study,chicken polyclonal antibodies against 5b protein were prepared and an indirect ELISA method for the detection of IBV was established.Non-structural protein 5b was also established.As a detection antigen,it has the potential to distinguish between inactivated vaccine immunity and live virus infection.In this study,pET-32a(+)was used as a vector to construct a recombinant expression plasmid expressing the 5b protein fragment,transform competent cells and induce expression,and purification.The purified 5b protein was used as an immunogen to immunize 4-week-old Specific Pathogen Free(SPF)chickens.The serum was collected after four free passages and stored.Further Western Blot(WB)identification showed that the obtained polyclonal antibody can specifically bind to 5b protein..The optimal reaction conditions were determined by IBV 5b-ELISA: the antigen coating concentration was 5 ?g/mL,the dilution ratio of the tested serum was 1:400,the dilution ratio of the enzyme-labeled antibody was 1:10 000,and the 0.05 M pH 9.6carbonic acid was used.Salt buffer was the coating liquid;5% skim milk powder was the blocking solution;5% skim milk was the sample diluent;the reaction time of the serum and enzyme-labeled secondary antibody to be detected was 60 min and 45 min,and the substrate reaction time was 10 min.The specific experiments showed that the established IBV 5b-ELISA antibody detection method had no cross reaction with avian influenza virus(AIV),Newcastle disease virus(ND),and K subtype avian leukemia(ALV-K).Reproducibility experiments show that the coefficient of variation of this method is less than 5%.Non-structural protein 5b is used as a coating antigen,which has a low cost.The method has the advantages of convenience,high sensitivity,good specificity,etc.It provides a new and effective detection method for IBV surveillance,epidemiological investigation and purification of IBV diseases.