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Hog fever virus csfv E2 protein ligand epitope polypeptide and application thereof

A technology of protein ligands and epitope polypeptides, applied in the fields of molecular pathology and immunology, can solve problems such as lack of detailed reports

Active Publication Date: 2011-12-07
XINXIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no detailed report on which amino acid sequences of these three proteins serve as viral ligands to bind to viral receptors.

Method used

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  • Hog fever virus csfv E2 protein ligand epitope polypeptide and application thereof
  • Hog fever virus csfv E2 protein ligand epitope polypeptide and application thereof
  • Hog fever virus csfv E2 protein ligand epitope polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1, Design and synthesis of CSFV E2 protein polypeptide and Sulfo-NHS-Biotin labeled polypeptide

[0065] 1.1 Design of peptides

[0066] Using DNASIS (Ver. 2.5) software to analyze the amino acid sequence of CSFV E2 protein, design a polypeptide, which contains part of the amino acid sequence of E2 protein, and Cys is added to the N-terminal of the polypeptide, so as to be coupled to the carrier with SMCC bifunctional reagent . Use the peptide program to analyze the difficulty of synthesis; design the synthesis (cleavage) program; edit the peptide chain; calculate and prepare solutions and reagents; start the synthesis program.

[0067] 1.2 Synthesis of peptides

[0068] Use the Symphony 12-channel peptide synthesizer to synthesize peptides on Fmoc-Amino Acids attached to Wang Resin (Shanghai Jier) by solid-phase peptide synthesis (solid-phase peptide synthesis), and the peptides are synthesized according to standard operating procedures conduct. T...

Embodiment 2

[0075] Example 2 , Identification of Biotin-SE24 Polypeptide Binding to PK-15 Cells by Cell Immunochemical Staining

[0076] (1) Culture PK-15 cells at 37°C, 5% CO 2 After culturing in the incubator for 24 hours, wait for the cells to cover the 96-well cell culture plate;

[0077] (2) Pour off the culture supernatant, wash the cultured monolayer cells with pre-cooled PBS for 3 times, and add 0.2mmol / L, 0.1mmol / L, 0.05mmol / L and 0.025mmol / L to the culture plate respectively. L of Biotin-labeled polypeptide Biotin-SE24, 100 μL / well, do 3 replicates for each dilution, incubate at 4°C for 1 hour to fully combine the Biotin-labeled polypeptide with PK-15 cells, set 6 wells without adding Biotin-labeled polypeptide Normal cells were used as a control, and Biotin-labeled BSA was used as an irrelevant polypeptide control;

[0078] (3) Gently wash the cells 3 times with pre-cooled PBS to wash away unbound peptides and stop the peptide binding test;

[0079] (4) Fixed, adding 0.3...

Embodiment 3

[0084] Example 3, Biotin-SE24 polypeptide binding test and virus blocking test were analyzed by flow cytometry

[0085] The following experiments were performed simultaneously with PK-15 cells. The test is divided into 3 groups, test group A: Biotin-labeled polypeptide binding PK-15 cell test; test group B: Biotin-BSA binding PK-15 cell test, as a negative control; test group C: CSFV blocking PK-15 cell binding site After spotting, the Biotin-labeled polypeptide binding PK-15 cell test was carried out as a virus blocking test. The specific test steps are as follows:

[0086] (1) Culture PK-15 cells, after 24 hours of culture, the cells grow into a monolayer;

[0087] (2) Wash the cells 3 times with pre-cooled PBS, digest the cells with trypsin for about 20 minutes, blow the cells from the cell flask with a 10ml pipette, mix well to reduce the large cell clumps, centrifuge at 1500r / m for 5 minutes, discard decellularized supernatant;

[0088](3) Add new pre-cooled PBS...

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Abstract

The invention relates to a (classical swine fever virus) CSFV E2 protein ligand epitope peptide and application thereof. The peptide has an amino acid sequence of VHASDERLGPMPCRPKEIGSSAGPVRKTSCTFNYAKTGKNKYYEPRDSYF and a molecular weight of 5.73kDa. With the PK-15 cell as the target cell, and by means of infection and harvest of the CSFV, TCID50 and the MOI (multiplicity of infection) of the CSFV can be determined. A binding test of a synthetic peptide and the target cell, a virus blocking test and a test for the peptide to block the CSFV from infecting the target cell shows that the screened specific binding target cell can inhibit the virus from infecting the ligand epitope peptide, and ligand epitope of the CSFV is positioned accurately. The peptide SE24 of the invention can inhibit the CSFV from infecting the PK-15 cell, and with the increase of the peptide concentration, the infection rate of the PK-15 cell is reduced. When the peptide concentration is 0.2mmol / L, the CSFV can be completely inhibited from infecting the PK-15 cell. The ligand epitope peptide of the invention provides the theoretical basis for a further study of the virus ligand epitope from the aspect of interactions between virus ligands and virus receptors.

Description

technical field [0001] The invention belongs to the technical field of molecular pathology and immunology, and in particular relates to a ligand epitope polypeptide of CSFV E2 protein and application thereof. Background technique [0002] CSF is a highly contagious infectious disease of pigs caused by CSFV, which has caused serious economic losses to the pig industry. CSFV is a member of the Flaviviridae family and the Pestivirus genus. During virus infection, the combination of receptors on the cell membrane and viral ligands is the key factor that mediates virus invasion into host cells, and is also the key to whether the virus can infect cells. Therefore, the study of the interaction between viral receptors and viral ligands has become one of the hot issues in the study of viral pathogenic mechanisms, because taking any one of them as a drug target can block the combination of the virus and the target cell, thereby Inhibits viral infection. Regarding the study of CSFV ...

Claims

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Application Information

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IPC IPC(8): C07K14/18C12Q1/02C12Q1/70G01N1/30A61K38/16A61P31/14C12R1/93
Inventor 王选年朱艳平岳锋贾文科宁红梅银梅张艳芳李鹏孙国鹏
Owner XINXIANG UNIV
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