Eukaryotic expression single-chain antibody of porcine epidemic diarrhea virus N protein, and preparation method and application thereof
A porcine epidemic diarrhea and single-chain antibody technology, applied in the field of genetic engineering, can solve the problems of rapid mutation, economic loss, and high incidence of PEDV
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0033] Construction of swine-derived anti-porcine epidemic diarrhea virus N protein single-chain antibody library, including:
[0034] Step 1. Immunize pigs with recombinantly expressed porcine epidemic diarrhea virus N protein. When the serum antibody titer reaches 1:64000 detected by indirect ELISA, the spleen tissue of the pig is taken and the total RNA is extracted by Trizol method (TRIzol reagent is purchased from Invitrogen Company). . Using the extracted total RNA as a template, using Oligo primer, according to the operation steps of the product instructions of the reverse transcription kit (reverse transcription cDNA synthesis kit purchased from TaKaRa Company), cDNA was synthesized.
[0035] Step 2. Analyze the variable region sequence of the porcine antibody coding gene in the published literature, and design primers for amplifying the variable region of the heavy chain and light chain of the antibody according to the sequence, as shown in Table 1), where VH-B and VH...
Embodiment 2
[0044] Indirect ELISA screening of porcine-derived single-chain antibody against porcine epidemic diarrhea virus N protein, specifically: coating the purified recombinantly expressed porcine epidemic diarrhea virus N protein in a 96-well plate overnight at 4°C; Blocked with 4% BSA-containing blocking solution at 37°C for 2h; washed 3 times with PBST buffer, added the periplasmic luminal protein of the extracted colonies to each well of the 96-well plate, reacted at 37°C for 2h; washed 3 times with PBST buffer, Add anti-Myctag mouse monoclonal antibody 100 μL (1:3000 dilution), react at 37°C for 2 h; wash 3 times with PBST buffer, add HRP-labeled goat anti-mouse IgG (H+L) 100 μL (1:6000 dilution), React at 37°C for 1 h; wash with PBST buffer for 3 times, add TMB chromogenic solution, and protect from light for 15 min; add 2 mol / L sulfuric acid to stop the reaction, and read the OD450 value with a microplate reader. The periplasmic luminal protein extracted from the empty plasmi...
Embodiment 3
[0046] Sequence analysis of the recombinant scFv, specifically: performing DNA sequencing on the obtained positive cloned single-chain antibody encoding gene to prove that it consists of 723 nucleotides and 241 amino acids deduced accordingly, and the nucleotide sequence is as shown in SEQ ID As shown in No.4, the amino acid sequence is shown in SEQ ID No.3.
PUM
Property | Measurement | Unit |
---|---|---|
molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com