Preparation Of Monoclonal Antibodies To Hemagglutinin-neuraminidase Protein Of Newcastle Disease Virus And Establishment Of An Indirect ELISA

Duck-origin Newcastle disease, also known as Duck paramyxovirus disease(DPMV) is an acute type I by avian paramyxovirus(APMV-I), which caused by highly contagious disease. It caused massive death of ducks and serious losses to the poultry industry. Newcastle disease virus(NDV) particle envelope contains two types of surface glycoproteins:hemagglutinin-neuraminidase protein(HN) and fusion protein(F). The HN protein is the major protective antigen which closely related to the virulence and pathogenicity. In this study, HN gene of NDV SDWF02 train was amplified by Reverse Transcription-Polymerase Chain Reaction(RT-PCR) with a pair of primers designed according to HN gene sequence of NDV SDWF02 strain published in Genbank(accession number: HM188399). The HN gene was cloned into p MD18-T vector and sequenced. Monoclonal antibodies against HN protein was successfully developed with the HN protein expressed in E.coli Rosetta, provides a new way for the further research and clinical testing of DPMV.1. Clone and sequence analysis of HN protein gene of SDWF02The RNA was extracted from the allantoic fluid using TRIzol Reagent according to the manufacturer’s instructions. According to the HN gene of DPMV sequences published in Gen Bank(accession number: HM188399), a pair of specific primers was designed and synthesized. HN gene of NDV SDWF02 was amplified by RT-PCR. The PCR product was cloned into p MD18-T vector and sequence conformed. The result showed that HN gene is consist of 822 bp which encodes 274 amino acids, which consistent with NDV SDWF02 strains.2. Expression of HN gene of DPMV in E.coli establishment of an indirect ELISAHN gene fragment was subcloned into the expression vector p ET-28 a by Sac I and Hind III double digestion from p MD18-T-HN vector, and transformed into E. coli Rosetta. The positive recombinant plasmid p ET-28a-HN were induced by IPTG to express the fusion protein. SDS-PAGE analysis results indicated that the expressed fusion protein was 30.5k Da,which consistent with the expected size. Ni-NTA Resin was used for the purification of the protein. Western blot showed that the protein is capable of specifically recognizing the DPMV serum, shows a good immunogenicity. An indirect ELISA for detection the antibodies to DPMV was established with the purified HN proteins.3. Development monoclonal antibodies against HN protein of DPMVBalb / c mice were immunized three times with the purified HN protein. After booster,the spleen cells of immunized mice were fused with SP2 / 0 myeloma cell. Indirect ELISA was used for screening the Hybridoma cells. Limiting dilution method was used to subclone,and one positive monoclonal antibody(Mc Ab) strain was obtained, named as B1F12. The identification of Mc Ab subclasses results revealed that the Mc Ab which secreting by hybridoma cells belonged to Ig G2 b subtype. A large amount of ascites was produced through the production system in mice, and purified with caprylic acid-ammonium sulfate method.