Generation Of Monoclonal Antibody And Biopanning A Mimotope For Infectious Bronchitis Virus S1 Protein

Infectious bronchitis virus?IBV?is a highly contagious pathogen in the poultry industry.It easily infects the respiratory tract and causes kidney and reproductive tract diseases,causing severe economic damage in the poultry industry.IBV has a high mutation rate and multiple serotypes,and there is almost no cross protection between different serotypes,which brings great challenge to the diagnosis and treatment of IBV.As the main structural protein,S1 protein is responsible for the binding of the virus to the host cell membrane and can cause the orgnism to produce neutralizing antibodies.Therefore,the preparation of monoclonal antibody?m Ab?and the biopanning of mimotope of IBV M41 S1 protein can lay the foundation for the detection of IBV,and provide the reference for the analysis of S1 protein epitopes.In present study,the IBV M41 strain was expanded by inoculating of chicken embryos to obtain allantoic fluid containing the virus.The total RNA of the virus was extracted and the selected S1 gene was amplified by RT-PCR.Then,the recombinant vector p ET-28a-S1 was constructed and transformed into E.coli BL21?DE3?.The induced expression products were analyzed by SDS-PAGE.The recombinant S1protein was mainly expressed in inclusion bodies with the molecular weight of approximately 20 k Da.The results of Western-blot showed that the renatured S1protein could react with chicken positive serum,indicating that the recombinant S1protein showed reactionogenicity.The recombinant S1 protein was used as an immunogen to immunize Balb/c mice,a cell line 3D9 capable of stably secreting monoclonal antibodies against the IBV M41 S1 protein was obtained by cell fusion technology.The titer of ascites antibody was 1:2.56×10⁵and the affinity constant was 2.01×10⁹L/mol by indirect ELISA.The results of immunoperoxidase monolayer assay?IPMA?showed that the monoclonal antibody 3D9 could react with the IBV M41 strain.The monoclonal antibody 3D9 was used as the target in biopanning of Ph.D.-12^TMpeptide library.After 3 rounds of biopanning,the phages that specifically binds to monoclonal antibody 3D9 were sequenced and identified,and the mimotope of IBV M41 S1 protein was finally obtained as SFYDFEMQGFFI.In the study,a monoclonal antibody against IBV M41 S1 protein was successfully prepared,and could be combined with IBV M41 strain.Finally,the mimotope of IBV M41 S1 protein was finally biopanned by phage display technology as SFYDFEMQGFFI.