Prokaryotic Expression Of GPV VP3and Development Of Monoclonal Antibodies Against GPV

Gosling Plague was a kind of acute infectious disease that damaged young goose.It has high morbility and mortality, it seriously endangered geese industry. After the goose parvovirus was first isolated from the goose develping-embryo by Fang Ding-Yi in China in1956, related study on the goose parvovirus was developed in the world and had got some achievements. Many scholars reported some specific diaglosis methods successively. Because the diagnostic methods to GPV were very important for the development of geese production, they were paid common attention to in the world.Gosling plague virus is a member of Parvoviridae, Parvovirus.The virus genome is a single stranded, linear DNA, the length of nucleotide sequence of genome is5106bp. It has three kinds of structural proteins,the VP1、VP2、VP3. VP3is the major capsid protein and a major immune protective antigen, and can induce neutralization antibody. For this reason, VP3gene of GPV played a major role in immune diagnosis and prevention of GPV infection, VP3caspid protein is the preferred protein of genetic engineering vaccine research.Based on the expression of VP3in vitro and development of monoclonal antibodies against VP3, we set up double antibodies sandwich ELISA method to diagnose GPV in this study. Identifying epitopes of mAbs against VP3was in favor of founding antigen epitopes test method to diagnose GPV.1. Prokaryotic expression of VP3gene of Goose Parvovirus in E.coliA pair of the primes based on the sequence of B strain in GenBank was designed to amplify the VP3gene by PCR. The PCR products were cloned into the prokaryotic expression vector pCold-TF to construct recombinant plasmid pCold-TF-VP3, which was1.6kb, and then pCold-TF-VP3was transformed into E.coli BL21(DE3). After IPTG induce, the recombinant pCold-TF-VP3in E.coli BL21(DE3) expressed the soluble VP3protein with106kDa by SDS-PAGE and The purified VP3protein was recognized by the mouse serum in Western-blotting analysis..This indicated that VP3protein was reactogenicity.2. Development and application of monoclonal antibodies against GPVMonoclonal antibody against GPV was prepared to diagnose Goose Parvovirus quickly and simply. We chose PEG to concentrate the virus, and purified through sucrose density gradient centrifugation. The purified antigen was used to immunize6weeks old Balb/C mice. Monoclonal antibodies were typically made by fusing SP2/0myeloma cells and spleen lymphocyte of immuned Balb/C mice. Nine Monoclonal antibodies against GPV were chosen by indirect ELISA. Positive hybridomas were cloned three times by technique of limiting dilution, and three hybridomas cell lines were obtained finally,which was named1C4、1C6、3E8. The indirect ELISA titers of the ascites were1:128000、1:256000、1:64000.The result of Western-blotting showed that the Monoclonal bodies can react with purfied virus typically and had no cross reaction with DHV、DPV、NDV. Because of high specificity of the antibodies and sensitivity and stability of biotin advin system, the purified mAb3E8was labled with ELISA titers of1:256000. A double mAb-mediated sandwich ELISA was developed using1C4as capture antibody and biotinylated mAb3E8as detection antibody. In a checker-board analysis, the optical concentration of the capture mAb1C4was2μg/mL, and Biotin-3E8was50ng/mL With this system, it was possible to detect GPV concentration as low as88ng/mL. In addition,15possible GPV infection cases with typicalpathological changes were tested through sandwich ELISA, in which8samples were detected positive. Confirmed with PCR results, we find86.6%of them were matched. It is suggested that the double mAbs-mediated sandwich ELISA was a valuable method for specific detection of GPV.3Epitope analysis of monoclonal antibodies against GPVPCR strategy was applied to obtain four fragments of GPV VP3, respectively. And named them as A、B、C、D. Their ami no acid sites lie in1～152aa、135～332aa、323～535aa、1～535aa.The PCR product cloned into eukaryotic expression vector pCAGGS. The result showed that recombinant plasmids were constructed successfully. The four recombinant plasmids were transfected to COS-1cell line with LipofectamineTM LTX Reagent respectively. The expression of pCAGGS-VP3was detected by indirect immunofluorescence test.The result showed that the recombinant plasmid containing correctly expressed in COS-1cell line.Specific fluorescence was detected by indirect immunofluorescence test, indicating that the cell line was GPV specific monoclonal antibody. It determined primarily that the linear B-cell epitope located at the C-terminus322～332aa.