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Polypeptide sequence specifically combined with porcine circovirus type-2 Cap protein and application of polypeptide sequence

A porcine circovirus and peptide sequence technology, applied in the direction of viral peptides, peptides, peptide sources, etc., can solve problems such as economic losses in the pig industry, and achieve the effects of rapid artificial synthesis, low detection cost, and simple operation.

Active Publication Date: 2019-05-17
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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  • Polypeptide sequence specifically combined with porcine circovirus type-2 Cap protein and application of polypeptide sequence
  • Polypeptide sequence specifically combined with porcine circovirus type-2 Cap protein and application of polypeptide sequence
  • Polypeptide sequence specifically combined with porcine circovirus type-2 Cap protein and application of polypeptide sequence

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] Separately calculate the free energy of peptide-protein binding, hydrogen chain, van der Waals force and other mechanical parameters f...

Embodiment 2

[0029] Example 2 Affinity Identification of L6-11 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 Cap protein coupling is successful, and this sensor can be used to measure the interaction between PCV2Cap protein and L6-11 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 L6-11 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 concentrations int...

Embodiment 3

[0034] Example 3 ELISA Identification of L6-11 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 1% 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 incubated with 1% BSA solution at 37°C for 1 hour.

[0037] 3. Use PBS buffer to dilute the solid-phase synthesized L6-11 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 (...

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Abstract

The invention discloses a polypeptide sequence specifically combined with porcine circovirus type-2 Cap protein and application of the polypeptide sequence. The polypeptide sequence is DYWWQSWE. On the basis of a crystal structure (PDB ID: 3R0R) of the porcine circovirus type-2 Cap protein, a polypeptide sequence which is specifically combined with the porcine circovirus type-2 Cap protein is obtained through a molecular docking virtual screening technology, wherein the polypeptide sequence is DYWWQSWE, namely L6-11. Solid-phase synthesis of the L6-11 is carried out, and affinity analysis withthe porcine circovirus type-2 Cap protein is conducted on the L6-11. The equilibrium dissociation constant (KD) of the interaction between the L6-11 and the PCV2 Cap protein is 1.03*10<-8> M, namely10.3 nM, which indicates that the affinity is high. The designed L6-11 sequence can be combined with artificially expressed PCV2 Cap protein and artificially inoculated PCV2 without causing cross reactions with other viral proteins, and the specificity is high.

Description

technical field [0001] The invention relates to a polypeptide sequence specifically combined with 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06C07K14/01G01N33/68G01N33/569
Inventor 王方雨张改平邢广旭郝俊芳罗俊赵东郝慧芳柴书军郑关民牛艳
Owner HENAN ACAD OF AGRI SCI
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