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Preparation And Identification Of Monoclonal Antibody Against Erns Protein Of Bovine Viral Diarrhea Virus

Posted on:2021-01-30Degree:MasterType:Thesis
Country:ChinaCandidate:W L ChenFull Text:PDF
GTID:2370330620474557Subject:Veterinary Medicine
Abstract/Summary:PDF Full Text Request
Bovine viral diarrhea/Mucosal disease is an important disease affecting cattle industry all over the world.The virus has many subtypes and a wide host range and animal infection with virus can cause immunosuppression that it can lead to secondary infection.BVDV is a RNA virus belonging to the genus Flaviviridae;BVDV is divided into 1 serotype,2 biotypes,and 3 genotypes.BVDV mainly causes bovine diarrhea,respiratory diseases,production performance decline,immunosuppression and other clinical symptoms.BVDV also seriously affect cattle-related products,such as infected cattle frozen essence,serum,antibodies,and have a serious economic impact on animal husbandry countries.Since 1980,the virus was detected in Changchun area of Jilin Province,china.The positive rate of infected animals increased year by year,and the latest data released by Bringer Ingelhan in2018 showed that the positive rate of BVDV in China had reached 46.7%,the persistent infection rate was 2.2%,and the statistical data were higher than that of other Asian countries.Therefore,it is of practical significance to explore BVDV positive animal detection methods,eliminate the source of infection from the root causes and reduce the loss of production.Based on this study,the following experiments were carried out and completed:1.BVDV Erns and E2 full-length gene cloning and sequence bioinformatics analysisThe MDBK cell proliferation BVDV,virus extraction RNA;design full-length primers and E2 genes to RT-PCR amplified genes;Sequencing validation after cloning vectors,than we are done bioinformatics analysis.The results showed that the full-length gene fragment Erns 681 bp was successfully amplified;The bioinformatics prediction Erns gene sequence encodes 227 amino acids with signal peptide and no transmembrane region,while the Erns protein is hydrophilic and secreted.Successful amplification of 1122 bp E2 full-length gene fragments;Bioinformatics prediction E2 gene sequences encoding 374 amino acids;signal peptide,no transmembrane and the E2 protein is hydrophilic and secreted.The prediction results provide theoretical basis for the subsequent construction of expression carriers.2.Analysis on the titer of Erns ? E2 protein immunized mouse polyclonal antibodyReference to the results of bioinformatics analysis,design Erns and E2 gene optimization primers;PCR amplified genes,ligation vectors,After bacterial fluid PCR and double enzyme digestion identification send to biological company sequencing,Recombinant plasmid transformation of Escherichia coli,IPTG method to induce its expression of target protein;SDS-PAGE validation of protein expression form and size;Freund adjuvant combined with recombinant protein to immunize mice to prepare polyclonal antibodies;Indirect ELISA determination of polyclonal antibody titer,comparison of polyclonal antibody titer levels prepared by Erns and E2 protein.Results: Both Erns and E2 recombinant proteins expressed by E.coli were not available Erns protein size 17 kDa,E2 protein size 32 kDa;Western blot,IFA confirmed that polyclonal antibody had good specific reactivity with recombinant protein and virus in cells,and proved that purified recombinant protein had good immunogenicity.The results showed that immunogen was screened for the subsequent preparation of monoclonal antibody.3.Preparation and identification of monoclonal antibody against Erns protein of bovine viral diarrhea virusUsing Erns protein as antigen,combined with Freund's adjuvant to immunize mice,the spleen was enlarged after 5 times of immunization,immune lymphocytes were prepared,hybridoma cells were prepared by using polyethylene glycol fusion of spleen cells and myeloma cells;Cultured cells were cultured by screening medium;cell morphology was observed by microscope,antibody titer was determined ELISA antibody activity was verified;Hybridoma cells were identified after 3 subclonal screening,positive holes were expanded after repeated determination;Sterile Kunming mice were injected into the abdominal cavity after counting;Ascites antibody was collected and identified after 20 d.biological activity of antibodies.As a result,three hybridoma cell lines were prepared,and the specificity of ascites antibody was good,ELISA the antibody titer was higher than 1:50000.
Keywords/Search Tags:Bovine viral diarrhea virus, Erns, E2, Polyclonal antibody, Monoclonal antibody
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