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High-affinity polypeptide sequence for porcine circovirus type 2 cap protein and its application

A porcine circovirus and affinity technology, applied in the direction of viral peptides, viruses, viruses/phages, etc., can solve the problems of economic losses in the pig industry, and achieve the effects of rapid artificial synthesis, simple operation, and rapid cost

Active Publication Date: 2021-11-26
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Porcine circovirus type 2 (PCV2) is the primary pathogen causing porcine circovirus-associated systemic disease (PCV2-systemic disease, PCV2-SD), and it is often mixed with other pathogens to cause subclinical disease in pigs. Infection, causing huge economic losses to the pig industry

Method used

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  • High-affinity polypeptide sequence for porcine circovirus type 2 cap protein and its application
  • High-affinity polypeptide sequence for porcine circovirus type 2 cap protein and its application
  • High-affinity polypeptide sequence for porcine circovirus type 2 cap protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Molecular docking and screening of virtual peptide library

[0023] 1. Preparation of Cap protein crystal structure

[0024] The amino acid sequence and crystal structure of porcine circovirus type 2 Cap protein were analyzed with the help of computer-aided design software, and the 63rd to 120th amino acid residues were selected as the docking region for molecular docking.

[0025] 2. Design of virtual peptide library

[0026] The present invention adopts the method of extending amino acid residues one by one. First, several amino acid libraries with the highest scores are docked with the structure of the target protein one by one. Until the best docking result is achieved. The peptide sequence generated by the virtual peptide library preferably has 2-9 amino acid residues.

[0027] 3. Evaluation of docking results

[0028] Calculate the free energy of peptide-protein binding, hydrogen chain, van der Waals force and other mechanical parameters for comprehe...

Embodiment 2

[0029] Example 2 Affinity Identification of L6-13 Sequence and Artificially Expressed Cap Protein

[0030] 1. The first is the preparation of the Cap protein chip, using the EDC / NHS method to covalently couple the PCV2Cap protein (5 μg / mL) to the carboxyl chip of LSPR. When the LSPR Single≥500pm, it indicates that the Cap protein coupling is successful, and the sensor can be used to measure the interaction between the PCV2Cap protein and the L6-13 sequence.

[0031] 2. The second is to run the sensor to obtain a stable signal baseline. Start running PBS buffer (pH 7.4) at the highest flow rate of 150 μL / min until the signal baseline of the sensor reaches a stable level. Then, reduce the flow rate to 20 μL / min and prepare for loading. Sample.

[0032] 3. Use PBS buffer to pre-preparate the dry powder of L6-13 synthesized in solid phase and modified by biotinylation at the amino terminal to different concentrations, and then inject 300 μL of L6-11 solutions of different concent...

Embodiment 3

[0034] Example 3 ELISA Identification of L6-13 Sequence and Artificially Expressed Cap Protein

[0035] 1. Dilute the purified PCV2Cap protein with CBS solution (pH9.6) to 10 μg / mL, and add it to an ELISA 96-well plate at a volume of 50 μL / well; express the purified protein from different viruses in the same way, that is, classical swine fever virus E2 protein (CSFV-E2), porcine epidemic diarrhea virus S protein (PEDV-S) and 2% bovine serum albumin (BSA), PBS buffer for ELISA 96-well plate coating, as a control, overnight coating at 4 °C .

[0036] 2. The ELISA plate was washed 5 times with PBST buffer, and blocked with 1% BSA solution at 37°C for 1 hour.

[0037] 3. Use PBS buffer to dilute the solid-phase synthesized L6-13 polypeptide to a working concentration of 1 μg / mL, add it to the above ELISA plate at a volume of 50 μL / well and mix well, wash the ELISA plate with PBST buffer at 37°C for 30 minutes 5 times.

[0038] 4. Dilute horseradish peroxidase-labeled avidin (SA...

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Abstract

The invention discloses a high-affinity polypeptide sequence for porcine circovirus type 2 Cap protein and application thereof, and the polypeptide sequence is KWWQSWEW. The present invention is based on the crystal structure of porcine circovirus type 2 Cap protein (PDB ID: 3R0R), through molecular docking virtual screening technology, obtains a polypeptide sequence that can specifically bind to porcine circovirus type 2 Cap protein, and its polypeptide sequence It is KWWQSWEW, which is L6‑13. Solid-phase synthesis of L6‑13 and its affinity identification with porcine circovirus type 2 Cap protein, the equilibrium dissociation constant K of the interaction between L6‑13 sequence and artificially expressed PCV2 Cap protein D 1.28×10 ‑7 M, that is, 128nM, shows that the affinity is better. The L6-13 sequence designed by the present invention can be combined with artificially expressed PCV2 Cap protein and artificially inoculated with PCV2, and does not cross-react with other viral proteins, and has good specificity.

Description

technical field [0001] The invention relates to a high-affinity polypeptide sequence for porcine circovirus type 2 Cap protein and application thereof, and belongs to the fields of polypeptide design and target protein separation and purification. Background technique [0002] With the continuous development of science and technology, structure-based molecular docking virtual screening technology, as an important means of rational design and screening of affinity peptides, has become a research hotspot in recent years. This method mainly realizes the one-by-one docking of small molecular polypeptides on the active site of the target protein by means of the rapid operation of computer software. These small molecular polypeptides come from the virtual peptide library prepared in advance, and then continuously optimize the spatial conformation, In terms of amino acid residue side chains, etc., find the optimal binding conformation of the small molecule polypeptide and the targe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06G01N33/68G01N33/569C07K14/01C07K1/14
CPCC07K7/06C07K14/005C12N2750/10022C12N2750/10051G01N33/56983G01N33/68G01N2333/01G01N2469/10
Inventor 张改平王方雨邓瑞广邢广旭郝俊芳卢清侠赵东滕蔓柴书军贾蕊张浩
Owner HENAN ACAD OF AGRI SCI
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