Pig PD-L14QN-AF epitope polypeptide and application thereof

A PD-L14QN-AF, PD-L14QN-F technology, applied in the field of animal molecular immunology, can solve the problems of small toxic and side effects, high price, adverse reactions, etc.

Inactive Publication Date: 2021-05-18
XINXIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Blocking the PD-1/PD-L1 pathway by antibodies is the main field of research at present, but antibody drugs are prone to cause immune-related adverse reactions and are expensive, while s

Method used

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  • Pig PD-L14QN-AF epitope polypeptide and application thereof
  • Pig PD-L14QN-AF epitope polypeptide and application thereof
  • Pig PD-L14QN-AF epitope polypeptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Screening and Length Optimization of Polypeptide PD-L14

[0038] Screening of the polypeptide PD-L14: According to the reconstructed porcine PD-1 / PD-L1 complex structure and the analysis results of the action surface, the original space structure of the polypeptide was analyzed, and the polypeptide PD-L14 was screened. The sequence of the polypeptide PD-L14 is located in the FG loop region of the porcine PD-L1 protein, starting from the 106th amino acid Ala to the 126th amino acid Lys, covering a complete loop region and including hotspot amino acids Ala121, Asp122, Tyr123 and Arg125, the amino acid sequence is: AQINECLISYGGASYPRITLK (SEQ ID NO: 1).

[0039] Optimization of the polypeptide PD-L14: According to the protein interaction surface analysis, the fragment of the polypeptide PD-L14 forms a hairpin secondary structure, and Tyr118, Gly119, Gly120, Lys131, and Ala121 form a β-turn, and the center of the turning point is located at Gly119. Taking Gly119 a...

Embodiment 2

[0041] Embodiment 2 Amino acid residue mutation method of polypeptide PD-L14

[0042] PD-1 and PD-L1 are expressed in multiple species. In this experiment, the amino acids of some representative species were selected for sequence comparison. They are derived from humans (Homo sapiens), pigs (Sus scrofa), cattle (Bos taurus), mice (Mus musculus), domestic cats (Felis catus), dogs (Canis lupus familiaris), and are helpful for us to understand pig PD. The research on the structure, function, properties and homology of PD-1 and PD-L1 provides ideas for the optimization of peptide design. Expresso is a unique comparison tool for proteins under the T-Coffee website. Using Expresso to perform multiple sequence alignment of 6 PD-L1 protein sequences, the results are as follows figure 2 shown.

[0043] The analysis shows that the benzene ring of Tyr123 of porcine PD-L1 protein is embedded in the groove of the hydrophobic active center of PD-1 protein, and forms a π-π conjugate with...

Embodiment 3

[0047] Embodiment 3 Fluorescence quantitative PCR detects the establishment of PRRSV method

[0048] (1) Infection of porcine PBMCs with PPRSV in vitro

[0049] Separation of PBMC from porcine peripheral blood: extract 4mL of blood from the anterior vena cava of healthy pigs, add sodium citrate anticoagulant, the ratio of whole blood to anticoagulant is 10:1, gently mix up and down, add PBS solution at a ratio of 1:1 Dilute whole blood. Add the same volume of lymphocyte separation medium into a sterile centrifuge tube, tilt the centrifuge tube, and use a sterile disposable plastic test tube to gently spread the diluted pig blood along the tube wall above the liquid surface of the separation medium. Use a horizontal centrifuge to centrifuge at a speed of 500-1000g for about 20-30min. Note that the speed of centrifugation should not exceed 1200g. Gently take out the centrifuge tube, and observe that there is a thin buffy coat layer between the plasma layer and the separation l...

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Abstract

The invention relates to a pig PD-L14QN-AF epitope polypeptide, and the amino acid sequence of the pig PD-L14QN-AF epitope polypeptide is QDAQINECLIAYGGASFPRITLKVN. The polypeptide PD-L14QN-AF disclosed by the invention has a spatial structure highly consistent with a corresponding sequence on a PD-L1 protein, so that the improved polypeptide PD-L14QN-AF is ensured to have more effective receptor recognition capability. Two key hydrophilic amino acids are mutated into hydrophobic amino acids with similar structures, and the affinity with PD-1 protein is increased by enhancing hydrophobic acting force, so that a more remarkable immune effect is exerted. In a PBMC cell proliferation experiment, the cell proliferation percentage of the epitope polypeptide PD-L14QN-AF is 53.5%, and the epitope polypeptide PD-L14QN-AF can significantly slow down the PRRSV proliferation efficiency, down-regulate the expression of PD-1 and significantly up-regulate the expression and secretion of IFN-gamma and IL-2 genes.

Description

technical field [0001] The invention relates to a porcine PD-L14QN-AF epitope polypeptide and an application thereof, belonging to the field of animal molecular immunology. Background technique [0002] At present, various viral diseases such as swine fever (CSF), porcine multisystemic wasting syndrome (PMWS) and porcine reproductive and respiratory syndrome (PRRS) have caused huge economic losses to my country's pig industry. Many academic studies believe that the activation of porcine PD-1 / PD-Ls pathway is one of the important pathogenic mechanisms of immune function injury or immunosuppression caused by CSFV, PCV2 or PRRSV infection. Such as lymphocytic choroid plexus encephalitis virus (LCMV), human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), etc., during a variety of chronic or persistent viral infections, PD-1 excess Expression and activation of immune negative regulatory signaling pathways, resulting in immune function damage or im...

Claims

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Application Information

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IPC IPC(8): C07K14/705A61K38/17A61P37/04
CPCC07K14/70532A61P37/04A61K38/00
Inventor 岳锋王选年史叶萍周娟娟李鹏朱艳平孙国鹏郭东光张欣欣
Owner XINXIANG UNIV
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