Single-chain antibody of chicken Newcastle disease virus P protein, preparation method and use of single-chain antibody
A technology of chicken Newcastle disease virus and single-chain antibody, which is applied in the direction of antiviral immunoglobulin, antiviral agent, botany equipment and method, etc., and can solve the problem of no specific therapeutic drug, etc.
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Embodiment 1
[0022] Example 1 Preparation of chicken-derived single-chain antibody against chicken Newcastle disease virus
[0023] 1: The chicken Newcastle disease vaccine (Bio ND VG / GA purchased from Merial Group) was used to immunize chickens (Roman hen, 4 months old, weight 1.5kg, purchased from Shanghai Sitian Poultry Breeding Professional Cooperative) according to the instructions for use. FM Osberg et al. "Comprehensive Molecular Biology Experiment Guide") ELISA method detects serum antibody titer greater than 1:20000, harvest chicken spleen, homogenize and grind, and extract with Trizol method (TRIZOL Reagent purchased from TaKaRa) Total RNA. Take the extracted total RNA as a template, use Oligo primer, and synthesize the first strand cDNA according to the product instructions of the reverse transcription kit (cDNA first strand synthesis kit purchased from TaKaRa).
[0024] 2: According to the FR region of the chicken antibody coding gene variable region sequence (AJ298107.1) published...
Embodiment 2
[0036] Example 2 Indirect ELISA screening of antigen-specific ScFv
[0037] Take the purified prokaryotic expression NDV P protein (provided by Changzhou Ambisen Biotechnology Co., Ltd.), coat it with 50mmol / L sodium bicarbonate salt solution (pH 9.6) at 4℃ overnight, and seal with 5% skimmed milk powder solution for 1h. Wash with PBST (containing 0.1% Tween20, the same below) for 3 times; take 50μL of TES-treated bacterial supernatant, mix with 50μL of 4% skimmed milk powder solution and add the above-mentioned coated P protein, react at 37°C for 2h, and wash with PBST; Add Myc-Tag Mouse mAb (Myc-tag mouse monoclonal antibody purchased from Ray Biotech) 100μL (1:2000), react at 37°C for 2h, wash with PBST; add Peroxidase-conjugated Affinipure Goagt Anti-Mouse IgG (purchased from Abmart) 100μL (1:4000), react at 37℃ for 1h, wash with PBST; TMB develops color, 2mol / L sulfuric acid terminates the reaction, reads the OD450 value with a microplate reader, and sets the supernatant of ...
Embodiment 3
[0038] In Example 3, the obtained single-chain antibody coding gene was sequenced, and it was proved that it consisted of 726 nucleotides and 244 amino acids inferred therefrom. The nucleotide sequence is shown in SEQ ID No. 4, and the amino acids The sequence is shown in SEQ ID No.3.
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