| Newcastle disease (ND) is one of the most important diseases of poultry, caused by Newcastle disease virus (NDV). It is highly contagious and can cause high mortalities in susceptible birds. Since it was isolated in 1926, NDV are still occurring around the world, causing serious economic loss in the poultry industry. The nucleocapsid protein (NP) is composed of the most important structural protein in NDV; it may be stimulate the body to produce cellular immune response. Its main function is binding with the P&L protein and package genomic RNA to form a helical ribonucleoprotein (RNP) complex. It plays an important role in the genome RNA's transcription and replication. In this study, NP gene of NDV F48E8 strain was amplified by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) with a pair of primers designed according to NP gene sequence of NDV SRZ03 strain published in Genbank (EU167540). The NP gene was cloned into T-easy vector and sequenced. Monoclonal antibodies against NP protein was successfully developed with the NP protein expressed in E.coli BL21.1. Clone and sequence analysis of nucleocapsid protein gene of F48E8 Newcastle disease virusTotal RNA from Chick embryo Allantoic fluid infected with NDV F48E8 strain was extracted with a total RNA extraction kit (AXYGEN German). According to the NDV SRZ03 strain gene sequence (EU167540), a pair of specific primers was designed and synthesized. NP gene of F48E8 was amplified by RT-PCR. The PCR product was cloned into pGEM-T Easy Vector and sequence conformed. The result showed that NP gene is consist of 1470bp which encodes 489 amino acids. Comparing with other NP gene sequences of virulent strains published in Genbank, the homology was 77.4%~99.8% at nucleotide sequence and 91.2%~99.2% at deduced amino acid. Comparing with the NP gene sequence of mesogenic strain and avirulent strains, the homology was 87.3% and 84.9%~86.6% at nucleotide sequence, 93.9% and 90.2%~93.5% at deduced amino acid respectively2. Expression of NP gene of Newcastle disease virus in E.coliNP gene from T- vector, pGEM-T-NP, was subcloned into pGEX-6P-1 by EcoRI and XhoI double digestion, and transformed into competent E.coli BL21 strain. The positive recombinant clones pGEX-6p-1-NP/BL21 were selected by double-digestion and then induced by IPTG. SDS-PAGE and Western-blot analysis results indicated that the expressed fusion protein was in the form of inclusion bodies and molecular weight of the protein was 79kDa. In order to obtain soluble expression of the NP protein, CFP10 of Mycobacterium tuberculosis was inserted into pGEX-6p-1-NP to construct a fusion expression plasmid pGEX-6p-1-CFP10-NP. The fusion protein CFP10-NP-GST was induced with IPTG at different concentrations and at three different temperature, 25℃, 28℃and 37℃. The expression protein were analyzed by SDS-PAGE and Western-blot. The best results could be reached at 0.75mmol / L of the concentration of IPTG for induced 5 hours at 28℃. The expression fusion protein was a soluble form and molecular weight was 89kDa which was the same as the predicted size. The soluble fusion protein was purified by GST-agarose affinity column. An indirect ELISA for detection the antibodies to Newcastle disease virus was established with the purified NP proteins. The results showed that indirect ELISA was consistent with the agglutination inhibition (HI), but not with serum neutralization (SN).3. Development monoclonal antibodies against NP Protein of NDVBALB/C mice were immunized with recombinant NP protein and F48E8 NDV allantoic fluid respectively. After boost five times the spleen cells of immunized mice were fused with SP2 / 0 myeloma cell. Hybridoma cells were screened by indirect enzyme linked immunosorbent assay (ELISA) and indirect fluorescent assay (IFA). Seven positive monoclonal antibodies (McAbs) strains were obtained, named as NDV-NP-2G4, NDV-NP-2E9, NDV-NP-1F8, NDV-NP-1E9, NDV-NP-4G9, NDV-NP-5B9, NDV-NP-6F5. All the McAbs belonged to IgM subgroup except NDV-NP-6F5 which belonged to mouse IgG2a subtype. Western-blot results confirmed that the McAbs were against the NP of NDV. These McAbs were specific only to NDV. There were no cross reaction with other avian viruses. These McAbs could react with NDV strains from different birds such as chicken, pigeon, duck and goose. |
