Expression Of Glycoprotein Genes Of Goose-host Paramyxovirus Strain NA-1 In The Prokaryotic And Eukaryotic Expression Systems

Newcastle disease(ND),caused by Newcastle disease virus(NDV), is an acute and highly contacted contiguous disease mainly infecting avian. ND is classified as a violent contagion by OIE. NDV is a member of the genus Paramyxovirinae,order Paramyxoviridae. NDV is a negative RNA virus with envelop. It exists in various tissues, eg. apparatus, body fluid, secretion, and excrement. ND is a serious avian disease in the worldwide. ND causes severe economic losses in the poultry indusity. Avian Paramyxovirus consisted of nine serotypes, APMV1-9, among which the APMV-1 serotype includes only NDV. The difference of virulence is tremendous in different hosts, in which chicken is the most sensitive,pigeon and parrot also could be infected and falled ill. Waterfowls such as geese or ducks constantly were not considered to be infected by NDV. The outbreak of ND in goose flocks in Southern and Eastern China have been reported frequentaly after the year 1997. This pathogen has posed a great threat to goose and other poultry industries. The pathogen, named Goose Paramyxovirus(GPMV), belongs to APMV-1 based on biologic activity and aetiology identification. Uninterruptly expanding of the host infection spectrum of NDV,chicken, pigeon, parrot, some rare birds as well as waterfowls were infected. The outbreak of paramyxovirus disease in goose broke down the traditional theory that waterfowls can not be infected with NDV. The study about genetic variation of antigen variation strain GPMV are necessary for studing furtherly the interspecies transmission mechanism of NDV.GPMV genome is a single-strand negative-sense RNA and comprises six genes, which encode the nucleocapsid protein (NP), phosphoprotein(P), matrix protein(M), fusion protein(F), haemagglutinin-neuraminidase(HN) and Large protein (L). Glycoprotein in envelop of virus correlated to pathogenicity and immunogenicity. F protein contributes to the fusion between virus envelop and susceptible cell, which is necessary to infection of NDV. F protein is the mainly protective antigen of NDV, which caused immune response and pathopoiesis. HN protein participates in the process of viral penetration, cell fusion, haemolysis. Glycoprotein gene could be taken to protective antigen of NDV genetically engineering vaccine. Therefore, glycoprotein genes were expressed in the prokaryotic and eucaryotic expression systems by molecular biology methods.Goose paramyxovirus strain NA-1 was separated and purified, and then extracted RNA genome of virus. Based on the complete genome sequence of goose paramyxovirus strain ZJ1 logged in GenBank and then two pairs specific primers were designed to amplified F and HN genes fragment by the method of RT-PCR. Meanwhile,the target gene fragments were respectively cloned into the pGEM-T vectors and were identificated by restriction enzyme digestion and sequencing analysis. The positive recombinant plasmids were named pT-F and pT-HN after identification by restriction enzyme digestion. The results showed that the complete open reading frame(ORF)of F gene comprised 1 662 bp and encoded 553 amino acids. Compared F gene of GPMV strain NA-1 with other published NDV strains,the homology of the nucleotide are 84.6%-98.3%,and the homology deduced amino acids are 88.4%-99.1%. The results showed that the ORF of HN gene comprised 1 716 bp and encoded 571 amino acids. Compared HN gene of GPMV strain NA-1 with other published NDV strains,the homology of the nucleotide are 82.1%-96.6%,and the homology deduced amino acids are 87.8%-97.6%. These numbers showed that the Fgene and HN gene of GPMV strain NA-1, had highly genetic stability, varid from traditional NDV.The recombinant plasmids pET-F and pET-HN were constructed by inserting the F gene and HN gene into the pET-28a vector, respectively. And the expression protein were induced by IPTG in E.coli BL21(DE3). SDS-PAGE analysis showed that F protein amounted to 31.8% of the total protein of E.coli BL21(DE3)after induced by IPTG(1 mmol/L)at 37℃for 4 hours,and HN protein amounted to 33.6% for 4 hours. A great of soluble proteins were producted after induced by IPTG(1 mmol/L)at 25℃in a low speed for overnight. The molecular weight of the expressed F protein and HN protein were about 59 Ku, 63 Ku. Western blot analysis showed that the proteins were specifically recognized by polyclonal antibody against GPMV.The recombinant plasmids pCI-F and pCI-HN were constructed by inserting the F gene and HN gene into the pCI-neo vector respectively. Then the recombinant plasmids were amplified,purified and transfected into Vero cells. The green fluorescence were observed under fluorescent microscope by the indirect immunofluorescent test, The results indicated that the recombinant plasmids could be successfully expressed. The positive Vero cells were collected and disrupted. Western blot indicated that the molecular weight of expressed protein about 59ku and 63Ku. Eukaryotic expression protein have favourable immunogenicity and biologic activity of native protein. Immune animals were protected from violent virus for high-level specific antibody.F gene and HN gene were coloned into in the prokaryotic and eukaryotic expression systems by DNA recombination technology. This research may contribute to the furtherly studying for extraorgan cell expression and gene vaccine. In addition,expressed protein could be purified to prepare highly specific monoclonal antibody. Combination of several structural proteins had a important significance to antigenic specificity,pathopoiesis mechanism and epidemiology.