Preparation And Identification Of Monoclonal Antibodies Against Nucleoprotein Of Infectious Bronchitis Viruse

Avian infectious bronchitis (IB) is an acute and highly contagious disease in chickens which caused by infectious bronchitis virus (IBV). It can lead to a decline in the weight gain and feed conversion of chickens, decrease egg production and quality, and also induce respiratory or kidney infections chicks. IBV is a Coronaviruses (the family Coronaviridae, genera Coronavirus), representative member of subgroupγ. Because of the high mutation frequency and recombination of IBV and little cross-protection between serologically distinct viruses, although the vaccine has been used, IB is still ongoing outbreak and epidemic. It caused serious economic losses in poultry industry. Monoclonal antibody (McAbs) can aim directly at specified epitope structure, and have a strong specificity, it show great significance on diagnosis of the disease and the design of genetic engineering vaccine. N Protein is a amajor immunogenic protein which induce the immunoresponse, especially the cellular immunological response. IBV N protein is regard as a target protein in our study, we prepared a monoclonal antibody against the target protein, and it has a certain significance on the diagnosis, prevention and control of IBV.In this study, IBV genomic RNA was extracted from chicken embryo allantoic fluid. Using software to predict and analysis the epitope of N protein, A pair of specific primers was designed to amplify the fragments which showed good immunogenic, hydrophilicity and epitope possibility, amplified fragment was 818 bp. It ligated into the vector pET-32a(+) and then transformed into E. coli BL21. After induction of expression, the SDS-PAGE results showed that a protein about 50 kDa. Purified on a column packed with Ni-NTA His-Bind superflow, the recombinant protein then reaction with chicken anti-IBV serum by western blot to test their antigenicity, the results showed good antigenicity of recombinant proteins.BALB/c mice were immunized with recombinant N protein mixed with an equal volume of Freund's adjuvant. Detected by indirect ELISA, it found that after immunization, mice produced high titers of antibodies, and can be used for cell fusion. SP2/0 cells and immune mouse spleen cells were fused, and indirect ELISA method was used for detection of antibody titers 2 weeks after fusion. Two McAbs designated as 2B33 and 4E10 were prepared. The subclass of them were IgG1 and IgM respectively and their light strand wereκ. The antibody titers of ascites antibody preparaed by ascites induction method and cell culture supernatant were greater than 106 and 1:640 respectively. The two monoclonal antibodies had good specificity, and could specifically recognize the N protein. The indirect ELISA result indicated that they both have good stability passage.This successful development of the two McAbs against IBV N protein is important to further research on IBV N protein and the diagnosis of avian infectious bronchitis...