Expression Of Gosling Plague Virus VP3Gene And Development Of Monoclonal Antibodies Against GPV

Goose parvovirus infection known as gosling plague is caused by goose parvovirus (GPV). It is an acute, highly contagious and septic infectious diseases that is susceptible to less than30days old goslings and Muscovy Duck. It is characterized by the substance of the exudative enteritis, liver, kidney, heart and other organs inflammation. Gosling plague that has strong pathogenicity and high mortality is one of the major diseases that endanger the goose industry. In this study, we cloned and sequenced VP3gene of the GPV vaccine strain SYG61and field strain P.I, and analyzed the sequences. We compared some biological characteristics of the two GPV strains. VP3capsid protein of GPV vaccine strain SYG61was expressed by a baculovirus expression system. After that two monoclonal antibodies against GPV were developed by cell fusion between sp2/0cells and spleen cells from BalB/c mice immunized with GPV SYG61. This study provides good reagents for further detection and prevention of gosling plague.1. Cloning and expression of goose parvovirus VP3gene.In this study, we cloned and sequenced VP3gene of the GPV vaccine strain SYG61and field strain P.1, respectively.The result showed that both of the VP3genes from two different GPV strains contain1605bp which encode534amino acids. Nucleotide and Amino acid sequences homology of VP3gene were95.8%and98.1%between GPV SYG61and GPV P.1strain. When compared with the other GPV stains published in GenBank, the nucleotide and amino acid homology of VP3sequences were93.6%-99.9%and96.3%～99.8%. In order to study and compare biological characteristics of the two different GPV strains SYG61and P.1, we detected ELD50and LD50respectively. The ELD50and LD50of GPV P.1were10-5.29/0.2mL and10-2.33/0.2mL. The ELD50and LD50of GPV SYG61were10-6.0/0.2mL and10-3.45/0.2mL. The results showed that the virulence of GPV SYG61strain was slightly stronger than GPV P.1strain. At the same time, we prepared two kinds of antiserum by gosling immunization with GPV SYG61and GPV P.1, respectively. PD50of the two kinds of antiserum was tested by goose embryo neutralization. The results showed that the antigenicity of correlation coefficient R is85.41%, which indicated that the two different GPV strains belonged to the same serotype. In order to express the VP3capsid protein by Bac-to-Bac expression system, VP3fragment was digested from the recombinant plasmid pGEM-T-VP3by BamHI and XhoI enzymes and cloned into the baculovirus transfer vector pFastBac1. Then pFastBac1was recombined with the baculovirus shuttle vector Bacmid by transforming DHlOBac competent cells. The positive recombinant was transfected into Sf9cell and the recombinant baculovirus was obtained, named rBac-VP3. PCR analysis showed that VP3gene had been inserted into Bacmid. The VP3protein expression in rBacmid-VP3was found in indirect fluorescent assay (IFA) with polyclonal antibody against GPV,2. Development and identification of monoclonal antibodies against GPVBalB/c mice were immunized with GPV SYG61goose embryo allantoic fluid. After boost three times the spleen cells of immunized mice were fused with SP2/0myeloma cells. Hybridoma cells were screened by indirect fluorescent assay (IFA). Two positive monoclonal antibodies were obtained, named as GPV-Mab-2C5and GPV-Mab-4G9. The immunoglobulin subtypes of monoclonal antibodies were identified with a commercial capture-ELISA kit. The results showed that both of the two monoclonal antibodies belonged to IgM subgroup. The ascite titer of GPV-Mab-2C5monoclonal antibody was1:3200and its cell supernatant titer was1:256. The ascite and cell supernatant titers of GPV-Mab-4G9monoclonal antibody were1:800and1:64respectively. The results of agar preciptation test showed that the two monoclonal antibody ascites could react with gosling plague antigen to produce significant precipitiation line. The two monoclonal antibodies also could recognize VP3protein expression in rBacmid-VP3systerm, in which specific bright green fluorescence was observed under fluorescence microscope by indirect immunofluorescent assay. These monoclonal antibodies would be potential tools for the development of diagnostic methods for detecting GPV in the future.