Anti-waterfowl parvovirus VP3 protein monoclonal antibody
A waterfowl parvovirus and monoclonal antibody technology, applied in the direction of antiviral immunoglobulin, antiviral agent, antibody, etc., can solve problems such as loss in waterfowl breeding industry, and achieve the effects of simple preparation method, strong specificity and high purity
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Embodiment 1
[0024] The preparation of embodiment 1.MDPV VP3 recombinant protein
[0025] Design primers according to the MDPV sequence, PCR amplify the MDPV VP3 gene, clone it into the prokaryotic expression vector PET-30a, transform Escherichia coli BL21(DE3), and induce the expression of MDPV VP3 protein by IPTG; collect the expressing bacteria, freeze-thaw three times , centrifuged to separate the inclusion body, cleaved the precipitate with urea at pH 8.0, purified by nickel affinity chromatography, and obtained MDPV VP3 recombinant protein, and finally the protein was refolded by gradient dialysis with 6M urea.
[0026] 1). Construction of VP3 recombinant expression plasmid
[0027] According to the MDPV VP3 nucleotide sequence (SEQ ID NO: 1) design synthetic specific primers, upstream primer pVP3F: 5'-TTT GGATCC ATGGCAGAGGGAGGAAGCGGAGCTA-3' (SEQ ID NO: 2, the underline is the Bam H1 site), downstream primer pVP3R: 5'-GGG GTC GAC TTACAGATTCTGAGTC-3' (SEQ ID NO: 3, the Sal I site...
Embodiment 2
[0030] Example 2. Preparation of anti-VP3 protein monoclonal antibody
[0031] Emulsify and purify 100ug VP3 recombinant protein with an equal amount of Freund's adjuvant (Sigma Company), immunize 6-week-old female BALB / c mice, and emulsify an equal volume of protein with incomplete Freund's adjuvant (Sigma Company) for a second time 15 days later. Immunization, after the second immunization, the tail-docked blood was collected to detect the mouse serum titer by ELISA method. At this time, the mouse serum titer had increased significantly. After the third immunization, the mouse serum titer was tested, and the positive value reached more than 1.0 , when P / N is greater than 10, get its splenocytes and myeloma cell SP2 / 0 to carry out cell fusion under the effect of polyethylene glycol (Invitrogen Company), carry out selective culture with HAT medium (purchased from Invitrogen Company); The VP3 recombinant protein and His protein (purchased from KPL) were used as detection antige...
Embodiment 3
[0045] Example 3. Characterization of anti-VP3 protein monoclonal antibody 2D5
[0046] The ascites titer of anti-VP3 protein monoclonal antibody 2D5 was determined by indirect ELISA method with purified VP3 recombinant protein as detection antigen; the reactivity and specificity of anti-VP3 protein monoclonal antibody 2D5 and MDPV / GPV viral protein were identified by indirect immunofluorescence; Ig subtypes of the heavy and light chains of the anti-VP3 protein monoclonal antibody 2D5 were identified using the Zymed Laboratories, Inc. Subtype Determination Kit.
[0047] i). Reactivity and specificity identification of anti-VP3 protein monoclonal antibody 2D5 and waterfowl parvovirus
[0048] The reactivity and specificity of anti-VP3 protein monoclonal antibody to MDPV and GPV viral proteins were determined by indirect immunofluorescence. Chicken embryo fibroblasts DF1 were infected with MDPV and GPV viruses (provided by Harbin Veterinary Research Institute) for 24 hours, was...
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