Preparation Of Monoclonal Antibody Against Non-structural Protein 3C Of Bovine Enterovirus HY12 Strain And Its Application For The Detection Of Viral Antigen

Enterovirus,a member of the genus Enterovirus in the family of Picornaviridae is the causative agents of the infectious diseases that poses a severe threat to human health and the development of the livstock industry.Currently,the genus of Enterovirus is divided into 12 Enterovirus species and 3 Rhinovirus species.Out of the 12 Entervirus species,the Enterovirus E(EV-E)and Enterovirus F(EV-F)mainly infects cattle with clinical signs characterized by severe digestive,respiratory,and reproductive disorders.HY12 strain is the first EV-E strain isolated by our laboratory from cattle herd with severe diarrhra and respiratory signs in Jilin Province in 2012.Although more progress has been made regarding this disease,there are few research on the non-structural protein encoded by the virus.In this study,specific primers for non-structural protein 3C were designed,synthesized and used to amplify the 3C gene fragment.The PCR-amplified fragment was cloned into the prokaryotic expression vector pGEX-4T-1 to construct the recombinant expression plasmid.After the expression,the recombinant protein was purified and used as immunogen to immunize BABL/c mice.Spleen cells collected was fused with SP2/0 myeloma cells.After screening and subcloning,a positive hybridoma cell line capable of stably expressing 3C monoclonal antibody was obtained.Further characterization showed that the monoclonal antibody has good reactivity and specificity to the HY12 virus antigen.Furthermore,an IPMA method for detection of viral antigens was established using the obtained monoclonal antibodies and the reaction conditions were optimized.Specificity and sensitivity results showed that the established IPMA method was specific,sensitive,rapid and simple.The established IPMA has high accuracy,and coincidence rate with immunofluresence assay and can be used for detection of bovine enterovirus antigen.The strains of EV-E,EV-F and EV-G in our laboratory were detected by the IPMA,and the results showed that the mathod can be used to detect the antigen of EV-E and EV-F.The IPMA detection method based on 3C monoclonal antibody provides a new and effective method for the detection,quarantine and prevention of bovine enterovirus infection.