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An affinity peptide capable of binding to classical swine fever virus e2 protein and its application

A technology of swine fever virus and affinity peptides, applied in the field of affinity peptides, can solve the problems of non-standard feeding conditions for pigs, uneven vaccine quality, unscientific immunization procedures, etc., and achieve non-immunogenicity, easy synthesis and Modification, good inhibitory effect

Active Publication Date: 2021-04-27
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disease has been prevalent in my country, and it is mainly sporadic in regions; although major farms have paid great attention to CSF, due to unscientific immunization procedures, uneven vaccine quality and non-standard feeding conditions of pigs And so on, resulting in the current CSF is still a serious threat to my country's pig industry

Method used

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  • An affinity peptide capable of binding to classical swine fever virus e2 protein and its application
  • An affinity peptide capable of binding to classical swine fever virus e2 protein and its application
  • An affinity peptide capable of binding to classical swine fever virus e2 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 E2 protein

[0024] The crystal structure (4JNT) of bovine epidemic diarrhea virus E2 protein was searched from the PDB database, and the crystal structure was analyzed with the help of computer programs, and the 800th to 900th amino acid residues were selected as the docking setting region for molecular docking.

[0025] 2. Design of virtual peptide library

[0026] The spatial structure of different amino acid residues is established, and with the help of computer programs, the batch generation of input target polypeptides is realized to meet the automatic calling and processing of molecular docking computer programs. The virtual peptide libraries are all generated in the form of straight chains without modification of any side chains and head and tail amino and carboxyl groups. It is advisable to generate a single virtual polypeptide library with no more than four amino...

Embodiment 2

[0029] Example 2, P 7 Affinity identification (SPR) with artificially expressed E2 protein

[0030] 1. Dilute the purified CSFV E2 protein with PBS buffer to 1 μg / mL (protein amount), and use the EDC / NHS active ester method to prepare 1-(3-dimethylaminopropyl)-3-ethyl carbon Diimide / N-hydroxysuccinimide (EDC / NHS) and CSFVE2 proteins were injected into the SPR sensor equipped with an amino chip to ensure that the EDC / NHS and E2 proteins interacted with the amino chip for 5 min respectively, and the CSFV E2 protein and amino Chip coupling. After coupling, the sensor can be used to measure CSFVE2 protein and P 7 interaction between.

[0031] 2. Inject 250 μL of PBS buffer (pH 7.4) into the sensor, run the PBS buffer at the maximum flow rate (150 μL / min) to reach the signal baseline, and reduce the flow rate of the PBS buffer to 20 μL / min to obtain a more stable baseline .

[0032] 3. The synthesized P 7 Dilute to different concentrations with PBS buffer, inject 250 μL polyp...

Embodiment 3

[0034] Example 3, P 7 ELISA identification with artificially expressed E2 protein

[0035] 1. Coat the artificially expressed and purified CSFV E2 protein with 1 μg / mL (protein amount); in the same way, express the purified protein of different viruses, namely bovine viral diarrhea virus E2 protein (BVDV-E2) , Japanese encephalitis virus E2 protein (JEV-E2), circovirus Cap protein (PCV-Cap), pseudorabies virus gE protein (PRV-gD), and bovine serum albumin (BSA) with a mass fraction of 2%, PBS buffer solution was used to coat the microtiter plate as a control. Among them, the coated antigen was diluted with carbonate (CBS) buffer, 50 μL per well was added to a 96-well microtiter plate, placed at 4°C overnight, washed 5 times with PBST buffer, and then the mass fraction 2% BSA solution for blocking.

[0036]2. Artificially synthesized and biotinylated P at the amino terminal 7 The dry powder was diluted with PBS buffer (pH 7.4) to a concentration of 500 ng / mL, added to the a...

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Abstract

The present invention mainly relates to an affinity peptide that can be combined with classical swine fever virus E2 protein and its application. The sequence of the affinity peptide is HRKWKSKWK(P 7 ). The present invention uses the crystal structure of the E2 protein of bovine viral diarrhea virus as a template, and obtains the three-dimensional structure of the E2 protein of classical swine fever virus through homology modeling, and finally obtains a polypeptide sequence with a high score value, which is P 7 ; Synthetic P 7 ELISA and SPR tests were used to identify the affinity and specificity of its interaction with E2 protein, and the results showed that P 7 It has a high affinity and specific binding to E2; then its activity of inhibiting virus infection is verified by qRT‑PCR and IPMA tests, and the results show that P 7 The sequence has better activity of inhibiting the infection of PK‑15 cells by classical swine fever virus. The invention can provide a reliable theoretical basis and new ideas for further research on viral receptors and antiviral drug design.

Description

technical field [0001] The invention relates to an affinity peptide that can bind to the E2 protein of classical swine fever virus and its application, in particular to the design of an affinity peptide targeting the E2 protein of classical swine fever virus and the research on its anti-infective activity of classical swine fever virus, belonging to the design of polypeptides and the The field of virus infection research. Background technique [0002] With the rapid development of genomics and proteomics, more and more three-dimensional structures of biological macromolecules have been analyzed. As of January 2017, more than 120,000 crystal structures have been imported into the protein database. Further in-depth research on the homology modeling method based on amino acid sequence analysis, for some biological macromolecules whose structures cannot be resolved under the current technical level, the three-dimensional structure can also be analyzed by modeling, which is It h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06G01N33/569A61K38/08A61P31/14
CPCA61K38/00A61P31/14C07K7/06G01N33/56983G01N2333/183
Inventor 张改平王方雨邓瑞广余秋颖卢清侠孙亚宁郝俊芳赵东杨继飞
Owner HENAN ACAD OF AGRI SCI
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