Dissertation
Dissertation > Agricultural Sciences > Livestock, animal medicine,hunting,silkworm,bee > Animal Medicine ( Veterinary Medicine) > Livestock, poultry, wildlife diseases > Poultry > Goose

Preparation and Identification of the Monoclonal Antibodies of the Goose Parvovirus

Author CuiMingChao
Tutor ChaiTongJie
School Shandong Agricultural University
Course Veterinary
Keywords GPV Amplification clone and identification Hybridoma cell Monoclonalantibody ELISA
CLC S858.33
Type Master's thesis
Year 2012
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Goose parvovirus(GPV) infection is a fatal disease affecting gosling and Muscovy ducklings.Occasionally the disease accounts for mortality more than70%in susceptible flocks.The most typical lesions found in goslings or Muscovy ducklings is myositis in skeletal muscles and myocardium,but it is very difficult to diagnose the diseases pathologically.Diagnosis is based mainly on serological or virological methods because the symptoms and lesions of the disease are not pathognomonic.The purification of the GPV virus and VP3gene fragments were amplified,cloned and identified,the sepuence with the published GPV VP3sequence homology were compared,the results of homology between95.8%-99.5%,the coloned fragment is needed for the purpose of section.The Balb/c mice were immunized with purified antigen of GPV, and indirect enzyme linked immunosorbent assay (iELISA) used to screen hybridoma cells was developed. The best reaction conditions of iELISA were that the purified antigen diluted as1:1600and coated in4℃for one night, the negative and positive serum were diluted as1:200, the temperature of the reaction was37℃and the reaction time was60min. The spleen cells from immunized mice were fused with SP2/0and6potive hybridoma cells were obtained by iELISA. Limiting dilution method was performed to subclone positive hybridoma cells, and2potive hybridoma cells that could secret McAbs stably were obtained finally after3-4subcloning, named as5F11,3C11. The stability, chromosome number, subtype, titers of the two McAbs in the ascites and specificity were identified. The ability of secreting McAbs of the hybridoma cells were stable after serial passaging for20times,whick indicated that the stability of the two hybridoma cells were good. The chromosome numbers of the two hybridoma cells were between100and110, which were according with the chromosome number of the hybridoma cells. The subtype of5F11and3C11were both IgGl, and the titers of the McAbs in the ascites were1:819200,and1:1638400. All the two McAbs were specific for GPV.

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