The Preparation Of Monoclonal Antibodies Against Mink Enteritis Virus VP2 Protein And Immunocolloidal Gold Strip

Mink viral enteritis(MEV) is a highly contagious and lethal disease caused by mink enteritis virus(MEV). The disease displays clinical syndrome acute diarrhea in natural conditons, particularly the newborn mink with high morbidity and mortality. With the rapid development of fur animal farming industry, MVE has been one of the three serious diseases of mink farming industry, bring greate conomic loss in the world.Gold immunochromatography assay(GICA) is a combination of immunochromatograghy,immunogold labelling technique and immunoreaction. Colloidal gold is a new detection technology as marker for the detection of antigen and antibody with advantages of simplicity, celerity, clearness, high sensitivity, pollution-free and convenient. Therefore, the technology is developing rapidly in the veterinary field.In this study, a colloidal gold test strip of MEV was established based on the principle of competitive immunoassay. The diameter of 20 nm colloidal gold particle was prepared by sodium citrate reduction, then the colloidal gold particle was conjugated with the McAb 10 of MEV. The MEV VP2 protein was dispensed on the bottom of the NC membrane as the test line(T line), while goat anti-mouse IgG was dispensed at the upper position as the control line(C line), assembling the two lines into colloidal gold chromatography strips.In this study, MEV VP2 structural protein gene was amplified by using polymerase chain reaction(PCR), the target gene sequence was 1 773 bp. The gene was inserted into cloning vector, then the target gene was inserted into prokaryotic expression plasmid PET-30 a to construct the prokaryotic expression recombinant plasmid PET-30a-VP2. This recombinant plasmid was transformed into Escherichia coil BL21(DE3). The recombinant plasmid was identified by enzyme digestion and sequencing analysis. The strains with recombinant plasmid of PET-30a-VP2 were induced by IPTG, the expression of recombinant protein was detected by SDS-PAGE and Western blotting. This result showed the recombinant protein could express with the induction of 37 ?C, IPTG concentration of 1.0 mmol/L. The recombinant protein was characterized by inclusion body, about 70 KD, pured fusion protein could be obtained after the nickel column purification.MEV cell culture was purified by sucrose density gradient ultracentrifugation. After immunization of BALB/c mice with purified MEV, were developed after fusion between SP2/0 myeloma cells and the stimulated splenocytes. The stable secrete antibody hybridomas were obtained by indirect ELISA methods, which were named 4, 6, 8, 10, 13 and 14, respectively. These six monoclonal antibodies were determined ascites titers respectively, monoclonal antibody 4 and 13, 6 and 14, 8 and 10 ascites titers were 1.28×105, 2.56×105 and 5.12×105, respectively. The specificity test proved that all the monoclonal antibodies were reacted with canine parvovirus(CPV) and mink enteritis virus(MEV), nevertheless they did not react with canine distemper virus(CDV), aleutian disease of mink(AMDV) and canine adenovirus(CAV). Monoclonal antibody 4, 10, 6, 8, 13 and 14 isotype belongs to IgG2 b, IgG1, IgG2 arespectively. Indirect immunofluorescence assay proved that all the six monoclonal antibodies induced bright green fluorescence in F81. Specific reaction zone 70 KD were tested by Western blotting analysis.The study laid the foundation for further study of MEV and establishment the quick diagnostic methods for MEV.Twenty nm colloidal gold was prepared through deoxidization of trisodium citrate, and anti-MEV monoclonal antibody 10 labeled with colloidal gold was used as a detector. The 0.8 mg/mL MEV VP2 protein was peridiumed in the NC membrane peridiumas test line(T line), 1 mg/mL goat anti-mouse IgG as control line(C line). The sensitivity of colloidal gold test strip was 1:64. The specificity test of colloidal gold test strip proved that CPV and MEV were positive results, nevertheless CDV, AMDV and CAV were negative results. The specificity of colloidal gold test strip was good. The accordance rate of CPV colloidal gold immunochromatography test strip detection method is up to 94.1%. Stability test indicated that the immunochromatography strips were stable for six months at 4 ? C and 25 ? C,sensitivity and specificity did not change significantly. This has very important practical significance for the early diagnosis, the prevention and control of MEV in our country.