Preparation And Application Of Monoclonal Antibodies To Nucleocapsid Protein Of Duck-origin Newcastle Disease Virus

Duck-origin Newcastle disease is an acute septic infectious disease that caused by duck-origin Newcastle disease virus. It has high morbidity and mortality and has serious harm to the duck industry. The effectively diagostic method is one of the effective measures to prevent and control the disease. The Nucleocapsid protein (i.e. NP protein), the highest content of structral protein, has high conservation and immunogenicity. Therefore, prokaryotic expression vector of NP protein was constructed and expressed. Then, the monoclonal antibody was preparated after NP protein immunizing BALB/c mice.Finally, those were established that the indirect ELISA for detecting antibody and the sandwich ELISA for detection of NDV.According to nucleotide sequence of NDV SDWF02strain, a pair of primers was designed and synthesized. The complete NP gene of a domestic isolate SDFC02strain of Duck-origin NDV was amplified by RT-PCR.The fragment was insert into prokaryotic expression vector pET28a to construct recombinant plasmids pET28a-NP.The recombinant plasmid was transformed into Escherichia coli Rosetta for expression. SDS-PAGE indicated that the NP protein was expressed in inclusion bodies with weight of54KD and existed in the shape of cytorrhyctes. Western blot analysis showed that NP protein had the well immunogenicity.Based on the antigenic analysis of NP protein, an alternative indirect enzyme-linked immunosorbent assay (ELISA) was established to detect antibody against Duck-origin NDV by using the purified fusion protein as the coating antigen.In the NP-ELISA, the optimal concentration of the recombinant NP for coating was10ng/well,the optimal dilution of serum sample was1:1000, and the best buffer was CB.The detection results revealed that the NP-ELISA assay was confirmed to have good diagnostic sensitivity and specificity.The method is simpler to produce and perform, time-saving and suitable for large scale surveys of Duck-origin NDV antibody at low cost.Eight-week-old female BALB/c mice were immunized with purified NP protein to prepare monoclonal antibodies (MAb) against Duck-origin NDV. Two hybridoma cell lines of A1and B2were produced by using hybridoma technique and the MAbs were identified and analyzed by indirect ELISA and western blot. Titers of the both strains in cell culture medium were212, while those of Al and B2strains in ascetic fluids were respectively107and106.The results of western blot and IFA showed, MAb Al recognize only linear epitopes located on NP protein.The sandwich ELISA, purified monoclonal antibody Al as the capture antibody andwas developed to detecte NDV. The optimal conditions were determined as follows:the purified ascites fuild of Al was used as coating antibody, and the polyclonal antibody against Duck-origin NDV was the seconary antibody; anti-sera was diluted to1:1600,the specificity MAb was diluted to1:1600; the best coating buffer was CB. The sandwich ELISA also had excellent specificity and repeatability.