Preparation And Identification Of Monoclonal Antibody Against Nucleoprotion Of Infectious Bronchitis Virus

Infectious bronchitis(IB)is an acute,highly contagious disease caused by infectious bronchitis virus(IBV),which is one of the major infectious diseases endangering the chicken industry seriously.IBV is a highly variable avian coronavirus with rapid spread,numerous serotypes and lack of cross-protection between different serotypes.N protein is an important immunogen that induces immune responses.The gene of N protein is highly conserved.Preparation of monoclonal antibody against N protion of IBV,which has certain significance for the diagnosis,prevention and control of IBV.In this study,IBV H120 strain was expanded by inoculation of chicken embryos and total RNA was extracted from virus-containing allantoic fluid.Reverse transcribed of total RNA to cDNA.A pair of specific primers was designed to amplify the N gene.The fragment was 1230bp in length,which was linked with the cloning vector pMD19-T,transformed into E.coli DH5?competent cells.It is also linked with the expression vector pGEX-6p-1,transferred into the E.coli Transetta(DE3)competent cells.PGEX-6p-1-N-Transetta(DE3)was induced for expression.The recombinant N protein is 79kD which was identified by SDS-PAGE.The recombinant N protein was purified by GST protein purification column.The GST tag was excised by PreScission Protease.The antigenicity of N protein was detected by Western-blot.The result showes that N protein had good antigenicity.Purified N protein was used to immunize BALB/c mice.The B lymphocyte of mice with the best immune effect were selected by indirect ELISA for cell fusion.Positive hybridoma cells were screened by indirect ELISA.Thirteen hybridoma cell lines that stably produced anti-N protein antibodies were obtained after three rounds of subcloning.Eight hybridoma cell lines of them were able to bind to IBV.Clone1A12C5 displaying highest titer was used to generate ascites fluid,purified by using the caprylic/ammonium sulfate precipitation method.SDS-PAGE electrophoresis results showed 1A12C5 monoclonal antibody had higher purity.It has been identified that the 1A12C5 monoclonal antibody is immunoglobulinG2b,?-light chain isotype.The titer of purified 1A12C5 monoclonal antibody was 1:1.64×10~6.The affinity constant was 1.87×10~7 L/mol.The results of specificity test showed that 1A12C5monoclonal antibody was highly specific to IBV.1A12C5 monoclonal antibodies can react with different serotypes of IBV.The stability identification showed that it had good passaging stability.In this study,we successfully prepared 13 monoclonal antibodies against IBV N protein,of which 8 were able to bind to IBV.This research laid the foundation for the further development of diagnostic reagents.