Establishment Of A Competitive ELISA Assay For Anti-NDV Antibody In Chicken Serum Based On Nanobody-HRP Fusion Protein

Newcastle disease is a poultry infectious disease caused by Newcastle disease virus(NDV),which seriously endangers the healthy development of the poultry industry and belongs to the first class of infectious diseases.Nucleocapsid protein(NP),which is the main structural protein of NDV,is the main target antigen established by anti-NDV antibody detection method.Compared to conventional antibodies,nanobodies have the advantage of being small in molecular weight and easy to genetically engineer.Based on the advantages of nano-antibodies,we applied them to the development of new diagnostic technologies for NDV,in order to establish a new diagnostic technology for NDV,.In this study,the NP protein of NDV was expressed by prokaryotic expression system,and the NDV soluble NP recombinant protein was used to immunize Bactrian camel.Then,phage display technology was used to screen 9 specific Nanobodies against NP protein through 3 rounds of panning.Subsequently,based on the Nano-body against NDV-NP protein,a Nanobody-horseradish peroxidase(HRP)fusion protein preparation platform was established,and then the fusion protein was used to develop a competitive ELISA for anti-NDV antibodies in chicken serum(cELISA)detection method.The main experimental contents and results are as follows:1.Expression and purification of NDV-NP recombinant proteinThe constructed recombinant plasmid pET-28a-NP was transfected into Trans5?competent cells,and 56 kDa soluble NP recombinant protein was obtained after induction by IPTG.After purification by Ni-column chromatography,a higher purity NP recombinant protein was obtained.It was confirmed by Western blot that the target protein has good antigenicity.2.Construction of anti-NDV-NP protein nanobody library and screening of specific NanobodiesAfter the purified NP recombinant protein is fully emulsified with Freund's adjuvant,use the protein immunize Bactrian camel.Six times later,the specific antibody titer against NP recombinant protein in serum is measured,and its titer can reach 1:256000.First,we isolated the Lymphocytes and extracted the total RNA,and then reverse transcribed them into cDNA.The heavy chain variable region(VHH)gene of the Nanobody was amplified by nested PCR;then,the VHH phage display library against NDV-NP protein was obtained by restriction enzyme digestion,ligation and transformation,and the library capacity was 3×10~8pfu/mL,this libraries has a good diversity identified Gene sequencing.The NP recombinant protein was used as the coating antigen for the specific nano antibody panning.After 3rounds of panning,9 specific Nano-antibodies against NP recombinant protein were finally screened out.3.Establishment of a competitive ELISA assay for anti-NDV antibodies in chicken serum based on Nanobody-HRP fusion proteinFirstly,pEGFP-NDV-NP-Nb5 eukaryotic expression vector was constructed by using different gene originals.After transfection of HEK293T cells with recombinant plasmid,IFA and Western blot showed that NDV-Nb5-HRP was successfully expressed in HEK293T cells.Subsequently,a competitive ELISA assay for anti-Newcastle disease antibodies in chicken serum was successfully established.