African swine fever virus p72 recombinant protein, monoclonal antibody and test paper
An African swine fever virus, monoclonal antibody technology, applied in the direction of antiviral immunoglobulin, virus/phage, virus, etc., can solve the problems of weak immunogenicity, short reaction time, false positive, etc., to achieve high specificity and Sensitive, immunogenic, and specific effects
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Embodiment 1
[0039] Example 1 Preparation of African swine fever virus p72 recombinant protein monoclonal antibody
[0040] 1. African swine fever virus p72 recombinant protein gene cloning and recombinant expression vector construction
[0041] According to the African swine fever virus p72 gene sequence published by Genbank (sequence number: AAT84439.1) as a reference, the optimized gene sequence was designed to make the N / C terminal epitope of the antigen more easily exposed and show more advantages. Thus, a relatively suitable monoclonal antibody is screened. The present invention analyzes the dominant epitope region of the protein sequence through DNAstar and IEDB databases, and uses GGGGS as a linker to express the two major epitope regions in tandem. The two epitope regions The amino acid sequences of the regions are respectively SEQ ID No.1 and SEQ ID No.2, which minimize the influence of steric hindrance while retaining more amino acids. The nucleotide sequence of the optimized A...
Embodiment 2
[0082] Example 2 Preparation of Latex Microsphere-labeled African Swine Fever Virus p72 Recombinant Protein Monoclonal Antibody
[0083] Dilute the prepared African swine fever virus p72 recombinant protein monoclonal antibody (7A7) with diluent and label it with latex microspheres. The specific operation is as follows:
[0084] 1. Cleaning of latex microspheres: Measure a certain volume of latex microspheres with a particle size of 300nm (purchased from Suzhou Weidu Biotechnology Co., Ltd., item number DR05C), pour them into a clean centrifuge tube, and add Proportional to 900 µL of labeling buffer (50 mM MES, pH 6.0) was added to the labeling buffer. Centrifuge at 17000 r / min for 10 min, remove the supernatant, then add 1000 μL of labeling buffer, centrifuge at 17000 r / min for 10 min, remove the supernatant, add 1000 μL of labeling buffer to resuspend the microspheres.
[0085] 2. Activation of latex microspheres: Weigh 20 mg of NHS and EDC each, dissolve in 1 mL of labelin...
Embodiment 3
[0088] The preparation of embodiment 3 latex microsphere cushion
[0089] 1. Take out the latex microsphere-labeled African swine fever virus p72 recombinant protein monoclonal antibody solution from the 4°C refrigerator and return to room temperature.
[0090] 2. Take a 20cm×30cm piece of glass fiber and cut it into 0.6cm×30cm size with an auxiliary material cutting machine.
[0091] 3. Spread a layer of plastic wrap on the table, place the cut glass fibers on the plastic wrap, and use a pipette to absorb 1 mL of latex microsphere-labeled African swine fever virus p72 recombinant protein monoclonal antibody solution to evenly wet A sheet of cut fiberglass. Dry at room temperature for 12-16 hours, then transfer to a 40°C oven for 1 hour, then place in a sealed bag with a desiccant, seal it, and set it aside.
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