Development Of Detection Method And Preparation Of Monoclonal Antibodies To σC Protein Of Chicken-derived Avian Orthoreovirus

Avian reovirus(ARV)belongs to the family Reovirdae Orthoreovirus,which mainly causes viral arthritis and tenosynovitis in broilers.In recent years,broiler farms and commercial broiler farms in Shandong,Jiangsu,Hebei,Fujian,Zhejiang provinces were infected with Reovirus and caused serious losses to the broiler breeding industry.In this study,a method of digoxin-labeled nucleic acid probes was established.At the same time,prokaryotic expression was achieved for its main antigenic protein σC,and an indirect ELISA method for ARV antibody was established.The monoclonal antibody were prepared,which provided technical support for the follow-up research and clinical detection of ARV.The specific research contents are as follows:According to the S1 gene sequence of the ARV strain LY383,a specific primer was designed,after that a 229 bp gene fragment was amplified by RT-PCR.The product was labeled with digoxin to prepare a nucleic acid probe.Specific test results indicate that except for ARV,H9 subtype avian influenza virus(H9-AIV),Infectious bronchitis virus(IBV),Infectious bursal disease virus(IBDV),Fowl adenovirus(FAV),Newcastle disease virus(NDV)could not hybridize with probe;the sensitivity test showed that the minimum detectable amount of the labeled probe was 50 pg/μL.The 13 suspected cases collected in the clinic were tested and the coincidence rate with the RT-PCR results was 100%.The prepared probe can be used for further detection after being stored in a refrigerator at-20 °C at least for 90 days.The results above indicate that the established ARV digoxigenin-labeled nucleic acid probe detection method has good specificity,sensitivity and stability,and can be used to detect disease samples in large quantities.Design a pair of specific primers to amplify the σC gene fragment of ARV strain LY383 by RT-PCR,and then clone into pMD18-T vector.The correct recombinant vector σC-pMD18-T and the expression vector pET-32a(+)were digested separately then the σC-pET-32 a recombinant plasmid was constructed and transferred into expressing bacteria E.coli BL21(DE3).By exploring the optimal conditions for protein-induced expression,a large amount of fusion protein was expressed and purified by urea gradient method.SDS-PAGE electrophoresis to confirm the expression form of the target protein as inclusion body.Western blot analysis showed that the protein has good immunogenicity.Indirect enzyme-linked immunosorbent assay detection for ARV was established using the expressed recombinant protein: the optimal dilution of the target protein was determined to be 1000 folds and the optimal dilution of the serum was determined to be 10 folds;the optimal blocking solution was 5% skim milk powder;HRP-labeled rabbit anti-chicken antibody dilution was determined to be 500 folds.Specificity tests showed that no positive reaction between ARV and other pathogens.The repeatability of this method was determined by the inter assay and intra assay tests with variability ranging from 4.85% to 7.93%.The above results indicate that the assay has strong specificity and good reproducibility which can be applied to large-scale serological investigation and monitoring of antibody levels in ARV infection.The purified σC protein was used as an immunogen to immunize BALB/c mice.After screening and subcloning,two hybridoma cell lines stably secreting σC protein monoclonal antibodies were obtained,named A1S9 and B2D9.The monoclonal antibody obtained by the subclass kit assay is an IgG1 subtype and the light chain is a kappa chain.The ELISA titer assay showed that the supernatant titers were 1:100,the mouse ascites titers were 1:128000 and 1:256000.The results of Western blot and IFA showed that the prepared monoclonal antibody reacted specifically with ARV.