Preparation And Application Of Monoclonal Antibodys Against VP2 Protein Of Duck-origin Novel Goose Parvovirus

Short beak and dwarfism syndrome(BADS)is a contagious disease that began to break out on a large scale in 2015 in Hebei,Shandong and Jiangsu,China.The disease is characterized by stunting,shortened beaks and swollen tongues,causing a significant reduction in the slaughter rate and huge economic losses in the duck industry in China.The pathogen of the disease was identified as Novel goose parvovirus(NGPV),also known as duck-derived goose parvovirus and novel duck parvovirus.In recent years,although many scholars in China have conducted studies on duck short beak and dwarf syndrome,there is little research base on this disease and NGPV.In this study,a pair of primers was designed to specifically amplify the VP2 sequence based on the NGPV VP2 gene sequence.the VP2 gene fragment was amplified in vitro using PCR,purified and cloned into the prokaryotic expression vector pET-32a,and the recombinant plasmid was transformed into E.coli receptor cells BL21(DE3)for induction.The protein expression,protein solubility and purification effect were identified by SDS-PAGE.The experimental results showed that the molecular weight of His-VP2 protein was about 83 kDa and was mainly expressed in the form of inclusion bodies.The purified protein had no obvious heterobands,indicating that the protein was successfully purified and used as the base material for the subsequent preparation of monoclonal antibodies.The VP2 protein was injected subcutaneously into BALB/c mice via the back of the neck according to standard immunization procedures.The mouse spleen was removed to make a cell suspension for cell fusion with SP2/0.The grown hybridoma cell line was identified using indirect ELISA.The reactivity of monoclonal antibodies with VP2 protein and virus was further characterized using Western blotting and IFA methods,respectively.Monoclonal antibodies were produced in large quantities by in vitro and in vivo methods,and the potency of monoclonal antibodies was determined by indirect ELISA.In this study,six positive hybridoma cell lines were successfully obtained and named as 2C4,4B12,4G6,6H1,7C8 and 7C10,respectively.4B12,4G6 and 7C8 had higher antibody potency,and all three monoclonal antibodies belonged to IgG1 type with kappa chain as light chain.Western blot and IFA results showed that these three monoclonal antibodies showed good binding reactivity with both protein and virus.It indicates that the monoclonal antibody was prepared successfully and provides an important tool for subsequent studies.Design of truncated peptide for VP2 protein,using monoclonal antibodies 4B12,4G6 and 7C8 as primary antibodies,and the binding of truncated protein to monoclonal antibodies was identified by Western blot,and then primers were designed according to the identification results.The further truncated proteins were prepared and identified in the same way as above until the minimal antigenic epitopes were identified.The results showed that three antigenic epitopes against VP2 were identified:"38DGGATAE44","546WNPEIQF552" and "461RDIYLQGPIWAK472".These three epitopes are more conserved in waterfowl micros,"38 DGGATAE44" and "546 WNPEIQF552" are located on the surface of VP2 protein and have good hydrophilicity,while "461RDIYLQGPIWAK472" has most amino acids located inside the protein and has the least hydrophilicity.It shows that the three antigenic epitopes were successfully identified,which can provide a reference for further research on the structure and function of NGPV molecules and pathogenesis.After optimization of the conditions,the procedure was determined as follows:the antigen was coated with 4μg/mL overnight at 4℃,5%FBS was used as the blocking solution for 1h at 37℃ the serum to be tested was diluted at 1:2 and incubated at 37℃ for 1.5h,the monoclonal antibody was diluted at 1:25600 and incubated at 37℃ for 0.5h,the enzyme secondary antibody was diluted at 1:8000 and incubated at 37℃ for 15min.The sample was judged as positive when the blocking rate was greater than 33.53%,negative when the blocking rate was less than 26.39%,and suspicious when the blocking rate was between 33.53%and 26.39%.The results of the specificity test showed that this method specifically identifies NGPV-positive duck sera.The results of intra-and inter-batch reproducibility experiments of this method showed that the coefficients of variation were less than 10%,with good reproducibility.The results of the compliance experiments showed that the compliance rate of this method could reach 96%.A total of 26 positive samples,11 suspicious samples and 106 negative samples were detected by the established method on 143 sera collected from the clinic.The established method provides an important tools for the detection of NGPV antibodies in China.In summary,in this study,six anti-NGPV VP2 monoclonal antibodies were successfully prepared,and three antigenic epitopes located on the VP2 protein were successfully identified using the monoclonal antibodies;and a blocking ELISA method for NGPV antibody detection was successfully established based on the monoclonal antibodies,which provides an important test for the epidemiological investigation of duck short beak and dwarfism syndrome and the evaluation of vaccine immunization effect.