| Goose parvovirus(GPV) belongs to Parvovirinae Dependovirus.GPV infection also known as goose plaque ,causes domestic goslings and Muscovy ducklings infected under the age of 20 days. GPV infection is a kind of acute high-contagious septico- infection disease. It has the feature of high mortality and high contagiousness,so it can still be one of the most jeopardized contagious disease.Owning to the hindrance to development of goose culturlal industry because of the disease,there needs accurate,convenient diagnostic method for early diagnosis avoiding the interference of maternal antibody.Meanwhile ,it needs monitor the level of maternal antibodies. Furthermore,it needs analysis of the viral antigen to select the suitable peptides be as subunits vaccine for desease prevent.GPV-VP1 is the main structural protein. It affects when virus infecting cells. It is the main antigen protein. It not only can help us comprehend the relations between its antigen constitution and its function by studying the epitides of GPV-VP1 with its monoclonal antibodies,but also has the significant meaning for diagnosing and designing the safe and effective genetic engineering epitide vaccine.In this study, we first contributed a recombinant protein GPV-VP1. Then expressed it in the system of E.coli Rosetta. After analyzing the Western blot with infected goose serum to the expression product, it confirmed that the expressed recombinant protein GPV-VP1 had good reaction activity. Then the BALB/c mice were immunized with purified GPV-VP1. then we use The purified GPV-VP as an antigen to detect the McAbs.Cells inosculation was carried out by standard method.The hybridoma supernatants were selected by indirect ELISA.The selected cells were subcloned three times by limiting dilution. Five hybridoma lines against GPV-VP1 were obtained , which were designated as 3A8,3G4,4B11,5G6,5C11.The subtypes of the McAbs were IgA,IgG1,IgG2a,IgG1,IgG1, All McAbs belonged toκchain. The five hybridoma lines can secrete antibodies steadily after culturing and saving for a long time.The average number of chromosomes was 91±7 pairs.For detecting the McAbs'specificity, These McAbs were used to react with purified antigen GPV-VP1 by Western blot. The results of Western blot showed that specific band was appeared obviously. This shows that the epitopes of these McAb were all linear. And they all had good performance of specificity with GPV-VP1 protein.For analyzing these five McAbs further, we used 19 recombinated proteins which were loppedly expressed by GPV-VP1 to detect them by Western blot. The results showed that 3 McAbs reacted with fusion peptide VP(81-136), 1 McAb reacted with fusion peptide VP(81-136) and VP(124-161), while 1 McAb reacted with fusion peptide VP(124-161). Then we used the futher loppedly expressed recombinated proteins of the reacted proteins to detect these five McAbs. Then we definited the main domains of the epitopes of these five McAbs. The over lapping region VP1(81-161) were then divided into fagments. On the basis of the Western blot results with multiclonal antibody serum once we had done, could also show these antigen epitopes had better dominantposition when it was infected by GPV.This study has prepared McABs against protein GPV-VP1, and detected the epitopes of them. It can help us to better comprehend the characteristics of its antigen, and has a significant meaning for diagnosing and designing the safe and effective genetic engineering epitide vaccine against GPV on the basis of the molecule level of epitopes. |
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