Construction Of IBV H120 Mutant Strain And Preparation Of Polyclonal Antibody For IBV Nucleocapsid Protein

Infectious bronchitis(IB)is a highly contagious,acute infectious disease caused by infectious bronchitis virus(IBV)and has caused significant economic loss to the poultry industry.It is classified as a class II animal disease in China.Vaccination is the most effective measure to control IBV.However,as IBV is prone to recombination and mutation,new serotypes appear from time to time,existing vaccines have no crossprotective effect against different serotypes,and virulence may return,which brings great difficulties to the prevention and control of this disease.Therefore,the development of polyvalent vaccines against different epidemic strains and serotypes has become a priority.The spike(S)protein encoded by IBV is responsible for recognizing cell surface receptors and entering cells.It has the main antigen epitopes of neutralizing antibodies and is associated with the tissue tropism and pathogenicity of the virus.There are two Furin protease cleavage sites in IBV-S protein.The first recognized motif is RXXR,and S protein is cleaved into S1 and S2 subunits.The second is located in S2 and identified as PXXR.The cleavage of these two sites plays an important role in the process of S protein mediated membrane fusion into cells.Our previous study found that mutating the second recognized motif PXXR of S protein of IBV-QX strain into the classical Furin recognized motif RXXR could enhance virulence and pathogenicity of the virus.Therefore,we speculate that if the second cleavage site of the vaccine strain H120 S protein mutated from PXXR to RXXR,it might promote the entry of the virus,resulting in the risk of the enhanced virulence of H120.To evaluate this risk,we used the established reverse genetics system of IBV to construct a full-length c DNA clone of H120 containing RRRR motif mutated from PRRR in the second Furin protease cleavage site of H120 S protein.The in vitro transcripts were transfected into BHK-21 cells by electroporation,and the chicken embryos were inoculated and passaged for 5 generations.Sequencing the RT-PCR products revealed the presence of genetic markers and introduced mutation sites on H120,indicating that the recombinant virus r H120-m S was successfully rescued.We will isolate the monoclonal recombinant strain and evaluate its growth characteristics,genetic stability and pathogenicity,in order to provide support for assessing the risk of H120 virulence enhancement.IBV-encoded 5a and 5b proteins are not required for viral replication but are associated with viral virulence.In order to study the biological functions of 5a and 5b of QX strain,we obtained 10 c DNA fragments covering the whole genome sequence of QX strain by RT-PCR and cloned them into vectors respectively.Full-length c DNA clones of r IBV-QX strain and r IBV-QX(Δ5a5b)deleting 5a and 5b regions were constructed by reverse genetics system.The establishment of this system will provide a powerful tool for studying the function of the genes from IBV-QX strain,and lay a foundation for the research and development of the attenuated vaccine for IBV-QX strain.The nucleocapsid(N)protein of IBV is highly conserved in various IBV serotypes and is one of the antigens for serological diagnosis.In order to study the function of N protein and develop diagnostic reagents,we constructed the prokaryotic expression vector of N gene of IBV Beaudette strain and prepared polyclonal antibodies against N protein using its prokaryotic expression product as antigen.Indirect immunofluorescence and Western blot identification showed that the antibody had good specificity.The successful preparation of antibodies against N protein provides a powerful tool for laboratory research and diagnosis on IBV.