Preparation And Application Of Monoclonal Antibodies Against VP2 Of Infectious Bursal Disease Virus

Infectious bursal disease(IBD)is a highly contagious infectious disease of chickens and turkeys caused by the infectious bursal disease virus(IBDV),which mainly infects chickens of 3-8 weeks old,causing a series of pathological changes such as bursal swelling,hemorrhage,necrosis and hemorrhage of the leg muscles and pectoral muscles.It can also cause serious immunosuppression and major economic losses in the global chicken industry.VP2 is the main structural protein and neutralizing antigen of IBDV.The recombinant vaccine expressing VP2 protein with the vector of herpesvirus of turkey(HVT)is widely used in China.The Monoclonal antibodies against IBDV VP2 was successfully prepared in this study,providing a powerful tool for the diagnosis and detection of IBDV1.Cloning and expressing of VP2 geneThe IBDV SNJ93 strain was inoculated into the chicken embryo chorioallantoic membrane(CAM)and the infected CAM was harvested after 72 h incubation in 37?.Total RNA was extracted and VP2 gene was amplified by RT-PCR.The cloned VP2 gene was sequenced and compared to VP2 gene sequences of other reference IBDV strains,which had been published in GenBank.According to the constructed phylogenetic tree based on VP2 gene,the IBDV SNJ93 strain could be classified as a very virulent strain.The amino acid sequence deduced from the VP2 gene of SNJ93 strain was analyzed by DNAStar software.And we found that the fragment 2 to 57 which with a better antigenicity than others and relatively conserved among different strains can be selected for prokaryotic expression.Then we designed corresponding primers to obtain this target gene.And this target gene was cloned into His-tagged pET-32a(+)and GST-tagged pGEX-6p-1 expression vectors respectively to obtaine recombinant expression plasmid.The two recombinant plasmids were transformed into expression bacteria BL21(DE3)and BL21 respectively and were induced expression by IPTG.The sizes of the recombinant proteins VP2-His and VP2-GST were 27 kD and 31 kD respectively.Western blot analysis showed that the recombinant proteins VP2-His and VP2-GST can both interact with IBDV positive serum,which indicated that both of recombinant proteins have good immunogenicity.2.Preparation for Monoclonal Antibody of VP2 ProteinThe VP2-His protein was used as the antigen to develop monoclonal antibodies against IBDV VP2 protein by hybridoma technology,and VP2-GST protein was used as antigen by the indirect ELISA.The BALB/c mice were immunized with the VP2-His protein.Each mouse was immunized with a dose of 50 ?g protein for 4 immunizations.The first immunization was performed with an equal volume of Freund's complete adjuvant mixed with VP2-His protein when emulsified then injected into the skin subcutaneously at multiple different points.Subsequent immunizations were performed with an equal volume of Freund's complete adjuvant mixed with VP2-His protein and injected into the skin subcutaneously at multiple different points.After 4 times of immunization,the titer of mouse serum antibody reached 1:10000 through indirect ELISA assay.The spleen cells of immunized mice were fused with mouse myeloma cells(SP2/0).The ELISA plate which coated with VP2-GST protein,used to screene and subclone of hybridoma cells by indirect ELISA.Finally,obtained 4 monoclonal cells can stably secrete VP2 protein-antibodies,named 3B2,3C12,4A3,5A6 respectively.Western blot analysis showed that four monoclonal antibodies can specifically bind to VP2 protein.3.3C12 monoclonal antibody applied in the detection of IBDVIndirect immunofluorescence assay(IFA)and agar diffusion test(AGPT)are commonly used for IBDV assays.In this study,IFA for detecting IBDV was established using 3C12 monoclonal antibodies with good sensitivity and specificity.The virus titer of the HVT-VP2 vaccine can be determined using the established IFA.That the HVT-VP2 vaccine was diluted 10-10-10 in a total of 10 dilutions was inoculated into 96-well cell culture plates filed with monolayer CEF cells.8 replicates were performed for each dilution.3 days later,IFA was carried out and the number of virus plaques was recorded under a fluorescence microscope.The virus titer was calculated to be 7×106 PFU/mL by this method.Compared with the common plaque counting method,IFA is less time consuming and enables more accurate counting of HVT-VP2 virus plaques.10 samples of bursa of suspected bursal disease infected with IBD were collected from the Animal Hospital of Yangzhou University.The samples were ground and added to PBS containing 1%penicillin-streptomycin solution to make a suspension.The supernatant was collected after repeated freeze-thaw centrifugation,total RNA was extracted from the supernatant and IBDV VP2 gene was detected by RT-PCR.Eight of these samples were positive.This study explored the possibility of using monoclonal antibodies for AGPT to detect IBDV.The 8 positive samples detected by RT-PCR were used as the antigens to be tested.AGPT was performed with 3C12 monoclonal antibody and IBDV positive serum was used as a control.The results detected by monoclonal antibodies were in accordance with the positive serum.Because the monoclonal antibodies have the advantages of stable source and uniform quality,they can replace positive serum in AGPT.