Seneca virus swine monoclonal gene engineering antibody and preparation method and application thereof
A genetically engineered antibody and monoclonal technology, applied in genetic engineering, peptide preparation methods, viruses/phages, etc., can solve the problems of easy missed detection, poor sensitivity and specificity, and many false positives.
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[0045] The invention provides a preparation method of Seneca virus porcine monoclonal genetic engineering antibody, comprising the following steps:
[0046] 1) Infect pigs with Seneca virus strain, immunize pigs with the inactivated vaccine prepared by said Seneca virus strain after 21 days, collect peripheral blood of pigs 21-30 days after immunization, and separate PBMCs;
[0047] 2) Sorting the PBMCs by flow cytometry, and sorting out IgG + -SVA + cell;
[0048] 3) with the IgG + -SVA + The genome of the cell is reverse-transcribed as a template to obtain single-cell cDNA;
[0049] 4) using the single-cell cDNA as a template to perform nested PCR amplification to obtain the gene of the variable region of the heavy chain of pig IgG and the gene of the variable region of the light chain;
[0050] 5) constructing an IgG heavy chain expression vector and an IgG light chain expression vector with the gene of the porcine IgG heavy chain variable region and the gene of the li...
Embodiment 1
[0077] 1. Virion Labeling Protocol
[0078] 1.1 Biotin labeling of SVA virion antigen
[0079] Take 2 mg of the SVA virus (SVA / HN / 11 / 2017 Lanshouyan) particles to be labeled in 1 mL of BuphTm phosphate buffer, and calculate the dissolved millimoles. Calculate the ratio of protein amount / protein molecular weight according to the following formula I:
[0080] Protein amount (mg) / protein molecular weight (MW) = millimoles of protein Formula I.
[0081] Equilibrate biotin to room temperature, add 2mg Sulfo-NHS-Biotin to 100μL ultrapure water, add sufficient concentration of biotin, generally more than 12 times molecular number for 10mg / mL protein, and more than 12 times for 2mg / mL protein solution 20 times. After placing on ice for 2 hours, pre-wash the purification column with 30mL PBS solution (pH value 7.4), load the sample, add the same buffer solution as the amount to be collected, collect 1mL in a separate tube, add an equal volume of sterilized 100% Glycerol, stored bel...
Embodiment 2
[0152] Indirect immunofluorescence assay (IFA)
[0153] The SVA strain (SVA / HN / 11 / 2017 Lan Animal Research) was inoculated into a single layer of BHK-21 cells that had grown to 70% to 80% full. 2 Incubate for 6-8 hours in a cell culture incubator. Follow the steps below: (1) fix and discard the supernatant, gently rinse with phosphate buffered saline (PBS) for 3 times, 5min each time, fix with 4% paraformaldehyde solution at room temperature for 20min;
[0154] (2) Permeate and wash as above, and permeate with 2% Triton X-100 for 10 minutes;
[0155] (3) Closing and washing as above, and blocking with 5% BSA for 1 h;
[0156] (4) Incubate the primary antibody and wash as above, then add the purified antibody at a concentration of 5 μg / mL, and incubate at 37°C for 1 hour;
[0157] (5) Incubate the secondary antibody and wash as above, add the working concentration of goat anti-pig IgG-FITC fluorescent secondary antibody, and incubate at 37°C for 1 hour;
[0158] (6) Microsc...
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