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Polypeptide sequence bound with classical swine fever virus Erns protein and application

A peptide sequence and swine fever virus technology, applied in peptides, material inspection products, instruments, etc., can solve the problems of reaction solvent limitations, peptide non-targetability, small peptide library capacity, etc., and achieve easy synthesis, modification, and screening The effect of low strength and short development cycle

Active Publication Date: 2021-04-09
龙湖现代免疫实验室
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] With the development of solid-phase combinatorial chemistry, high-throughput screening technology and phage surface display technology, it provides favorable conditions for large-scale construction and synthesis of peptide libraries, but the peptides screened by combinatorial chemical synthesis of peptide libraries are non-targeting. , time-consuming, and limitations of reaction solvents; the peptide library screened by phage display technology has disadvantages such as small peptide library capacity, peptide library diversity is affected by host cell selection of polypeptide types, and activity disappears after linear synthesis.

Method used

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  • Polypeptide sequence bound with classical swine fever virus Erns protein and application
  • Polypeptide sequence bound with classical swine fever virus Erns protein and application
  • Polypeptide sequence bound with classical swine fever virus Erns protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Molecular docking and screening of virtual peptide library

[0022] 1. Preparation of Erns protein

[0023] Search the crystal structure of bovine viral diarrhea virus Erns protein (PDB ID: 4DWC) from the PDB database, analyze the crystal structure with the help of computer programs, and select the docking active region (see figure 1 ) for molecular docking.

[0024] 2. Design of virtual peptide library

[0025] With the help of computer programs, the method of amino acid extension one by one is adopted, and the amino acid residues with the best docking situation are the core, and then amino acids are added one by one to realize batch generation of input target polypeptides to meet the automatic call and processing of molecular docking computer programs. The virtual polypeptides are all generated in the form of straight chains, and the polypeptides generated by the virtual polypeptide library preferably have 2-9 amino acid residues.

[0026] 3. Evaluation o...

Embodiment 2

[0028] Example 2P 1 Identification of Sequence and Affinity of Artificially Expressed Erns Protein

[0029] 1. Dilute the artificially expressed and purified CSF Erns protein with PBS solution and the matching chip activation buffer to 250 μg / mL (protein meter), and use the active ester method to first dilute 1-(3-dimethylaminopropyl) -3-Ethylcarbodiimide / N-hydroxysuccinimide (EDC / NHS, volume ratio 1:1) is injected into the SPR detector equipped with a carboxyl chip, and the carboxyl groups on the chip are fully displayed on the surface, and Activate the carboxyl group on the chip, and then inject the swine fever Erns protein into the SPR detector; ensure that the EDC / NHS and the protein interact with the carboxyl chip for 5 minutes, and implement the coupling of the swine fever Erns protein and the carboxyl chip. After the coupling is completed, the sensor chip is used to measure the swine fever Erns protein and P 1 Interactions between sequences.

[0030] 2. Inject 200 μL...

Embodiment 3

[0033] Example 3P 1 Sequence and ELISA identification of artificially expressed Erns protein

[0034] 1. Dilute the artificially expressed and purified CSF Erns protein with coating buffer solution, add 50 μL per well at a concentration of 10 μg / mL to coat the microtiter plate, incubate at 37°C for 2 hours, and wash the plate with PBST After four times, block with 5% skimmed milk PBST solution for 2h, then wash the plate with PBST for 4 times and set aside for use; in the same way, different viruses express purified proteins, namely bovine viral diarrhea virus Erns protein (BVDV-Erns), pseudo Rabies virus gD protein (PRV-gD), circovirus Cap protein (PCV-Cap), Japanese encephalitis virus E protein (JEV-E), and coated with 2% bovine serum albumin (BSA) As a negative control, PBS was used as a blank control.

[0035] 2. The artificially solid-phase synthesized P1 Dry powder, diluted with ultrapure water to a concentration of 10mg / ml, then diluted the peptide solution to 500μg / m...

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Abstract

The invention mainly relates to a classical swine fever virus Erns protein affinity polypeptide and application. The polypeptide is designed by a computer-simulated virtual molecular docking technology, and the sequence is WRHYIH. According to the invention, a polypeptide sequence P1 is obtained according to a virtual molecular screening technology by taking a swine fever Erns protein crystal structure obtained by homologous modeling as a template. After the polypeptide is artificially synthesized, an enzyme-linked immunosorbent assay test and a plasma resonance test are employed to identify the affinity of the interaction of the sequence and the swine fever Erns protein, and the result shows that the sequence P1 and the swine fever Erns protein have relatively high specificity and affinity; and the invention provides a reliable theoretical basis for affinity chromatography taking affinity peptide as an affinity ligand, and swine fever Erns protein can be rapidly detected by labeling the polypeptide.

Description

technical field [0001] The invention mainly relates to the design and application of a polypeptide capable of specifically binding to the Erns protein of the swine fever virus, and belongs to the design of an affinity polypeptide targeting the Erns protein of the swine fever virus and its application in the detection of the swine fever virus. Background technique [0002] Classical swine fever (CSF) is an acute, febrile and highly contagious disease caused by classical swine fever virus (CSFV). It is mainly characterized by high fever, internal bleeding, leukopenia in the body and neurological symptoms. It is distributed worldwide and is listed as a class A infectious disease by the International Veterinary Bureau. CSFV is a single-stranded positive-sense RNA virus of Flaviviridae and Pestivirus. The genome is about 12.3kb long, encoding 4 structural proteins and 7 non-structural proteins. The E0 protein, also known as Erns protein, is the first CSFV An envelope glycoprotei...

Claims

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Application Information

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IPC IPC(8): C07K7/06G01N33/569G01N33/68
CPCC07K7/06G01N33/56983G01N33/68G01N2333/183Y02A50/30
Inventor 张改平王爱萍王方雨刘东民周景明赵建国刘燕凯丁培杨冯景
Owner 龙湖现代免疫实验室
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