| Mink Parvovirus enteritis virus is also called the mink small enteritis virus. The disease, which was firstly reported by Schofield, is one of the major diseases in mink-breeding industry that can causes severe diarrhea in mink, sometimes accompanies by vomiting, resulting huge economic losses reported by. It mainly At present the disease has no effective treatment, and the vaccination is the primary preventive measure. Wu Wei conducted different strains of mink parvovirus in various regions for a systematic classification with monoclonal antibodies, and had bred a strain with good immunogenicity and stable viral virulence heredity, and based on which he developed a mink parvovirus inactivated vaccine with good preventive effect, the disease was under control to some extent. But in recent years, there were still tiny outbreaks occurred in some sable field. Its main cause is that the mink enteritis virus gene is a single strand of linear DNA with high gene mutation frequency, and the existing vaccine could not offer the full immune protection against the new mutant virus, which resulted in the immune failure. Therefore, we worked for the research of mink small enteritis virus, and on this basis to develop a rapid diagnostic kit at the beginning of the mink parvo infection.VP2protein is the major structural protein of the mink parvovirus, it is the major component of the capsid, which is exposed to the capsid protein surface, and the main antigenic sites inducing neutralizing antibodies. The nucleotide sequence which deter mining the virus host range is also on the VP2gene. Several nucleotide mutations can seriously affect some biological characteristics of the virus. Studies have shown that, MEV is gradually evolved by FPV with genetic mutation that adapting to a new host. We analyzed the major antigenic sites on VP2gene of mink parvovirus, and designed three pairs of gene-specific primers to amplify the major antigenic sites. Then the gene was inserted into the expression vector pET-32a, and transformed into BL21(DE3) PLysS competent cells, then induced with IPTG, the samples were centrifuged after sonication treatment for the SDS-PAGE electrophoresis. The results showed that the truncated genes of VP2got soluble expression, and the size of recombinant proteins is32KDa fused with a22kDa tag, and the target protein is1OKDa. After identification by the indirect ELIS A, the recombinant protein expressed in the E.coli prokaryotic expression system has good antigenicity comparing with MEV specific positive sera. In subsequent experiments, we use purified recombinant protein as coating antigen to screen monoclonal antibody.Take the MEV-ZYL-1strain was propagated in FK81cat kidney cells and concentrated at26000rpm for2h, the5-week-old female BALB/c mice were immunized with the concentrated virus, after the third immunization measured the serum samples of mice were assayed by indirect ELISA, when the titers reached to1:1×105, the cell fusion was performed. Using limiting dilution method to subclone the hybridoma cells until positive cells are selected, we final select5strains hybridized cells which can secret the MEV monoclonal antibody By the specific experiments. Were named1A6,1H9,2C3,2D4,3E8, which1A6,1H9against Loopl epitopes,2C3,2D4epitope for Loop2area,3E8against Loop3epitope region. These5strains monoclonal antibodies do not interact with the Aleutian virus, but react with canine parvovirus. Prepared monoclonal antibodies can be used for MEV colloidal gold strip or the development of ELISA test kit. |
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