A chicken pd-1 polypeptide and applications thereof

Abstract

The present application relates to a kind of targeting chicken PD-1 polypeptide and its application, the amino acid sequence of the polypeptide is CLIEYGGADYKTINLK.This polypeptide can be combined with PBMCs, promote PBMCs proliferation;Can be combined with chicken PD-1 protein.The polypeptide gCK-16 can be combined with PD-1 and PD-L1 on the cellular level, and the signal pathway of blocking PD-1 and PD-L1 is blocked.Provide theoretical basis and technical support for the prevention and clinical treatment of immunosuppressive diseases.

Description

Technical Field

[0001] This invention relates to a targeted chicken PD-1 polypeptide and its applications, belonging to the fields of molecular pathology and immunology. Background Technology

[0002] Immune checkpoint molecules play a protective role in the autoimmune system. Examples include cytotoxic T-lymphocyte-associated antigen-4, T-cell immunoglobulins, programmed cell death receptor-1 (PD-1), and programmed cell death ligand-1 (PD-L1). PD-1 and PD-L1 regulate the immune system to maintain an appropriate state, preventing excessive activation of immune cells that could lead to autoimmune diseases. However, when immune checkpoint molecules are overexpressed or overactivated, immune function is suppressed. Therefore, when tumor cells are present in the body, they can overactivate immune checkpoint molecules, reducing the body's immune system function, allowing tumor cells to escape immunely and accelerating their proliferation.

[0003] PD-1 can be expressed on the surface of immune cells such as T cells and B cells. After T cell activation, PD-1 is expressed on the T cell surface. PD-L1, in addition to being expressed on the surface of tumor cells and participating in immune escape, is also expressed on antigen-presenting cells (DCs, macrophages, etc.) under IFN-γ stimulation. The binding of PD-1 and PD-L1 leads to phosphorylation of ITIM (immunoreceptor tyrosine repressor motif) and ITSM (immunoreceptor tyrosine switching motif) in the intracellular domain of PD-1. This phosphorylation recruits SHP-2 (tyrosine phosphatase) to regulate the PI3K / AKT and Ras / MAPK / ERK signaling pathways, ultimately leading to T cell exhaustion. Therefore, inhibiting the PD-1 / PD-L1 signaling pathway plays a positive role in immunotherapy. Blocking PD-1 / PD-L1 can restore immune function to exhausted T cells, promote T cell proliferation and the production of effector cytokines, and enhance the body's immune cells' ability to kill tumor cells.

[0004] Currently, blocking the PD-1 / PD-L1 signaling pathway mainly employs the following strategies: peptide small molecule inhibitors, non-peptide small molecule inhibitors, substances that inhibit PD-L1 expression, and PD-1 / PD-L1 antibodies. Monoclonal antibodies targeting PD-1 / PD-L1 remain the most effective strategy for blocking PD-1 / PD-L1. In 2014, nivolumab was approved as the first PD-1 monoclonal antibody drug for the treatment of melanoma. In recent years, anti-PD-L1 monoclonal antibodies have also shown positive responses in clinical trials for various malignant tumors, including melanoma, metastatic non-small cell lung cancer, and bladder cancer. However, antibody drugs suffer from immunogenicity issues and poor penetration into tumor tissues, resulting in a low overall response rate for PD-1 / PD-L1 antibody drugs. Furthermore, due to the high binding rate of antibody drugs, it is difficult to eliminate immunotoxic side effects.

[0005] Therefore, peptides targeting PD-1 or PD-L1 have become a novel strategy for blocking the PD-1 / PD-L1 signaling pathway. Several studies have demonstrated the effectiveness of peptides in blocking the PD-1 / PD-L1 signaling pathway. However, research on peptides targeting chicken PD-1 or PD-L1 is rarely reported. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the purpose of this invention is to provide a targeted chicken PD-1 polypeptide and its applications.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A target PD-1 polypeptide for chickens, wherein the amino acid sequence of the polypeptide is CLIEYGGADYKTINLK.

[0009] The polypeptide is located in the IgV region of the complete coding sequence of the chicken PD-L1 protein.

[0010] The polypeptide can bind to chicken PBMCs.

[0011] The polypeptide can bind to chicken PD-1 protein.

[0012] The application of the chicken PD-1 targeted peptide in improving the proliferation capacity of chicken PBMCs.

[0013] The application of the target chicken PD-1 peptide in directly blocking the interaction between chicken PD-1 and PD-L1.

[0014] The application of the aforementioned chicken PD-1 targeted peptide in the preparation of drugs for poultry immunosuppressive diseases.

[0015] The application of the target chicken PD-1 peptide in the preparation of poultry vaccines.

[0016] The beneficial effects of this invention are:

[0017] The amino acid sequence of the polypeptide of this invention is CLIEYGGADYKTINLK, consisting of 16 amino acids, and named gCK-16. The short amino acid sequence of this polypeptide significantly reduces the difficulty and cost of synthesis. Once put into clinical use, it can reduce production costs for poultry farmers and maximize economic benefits.

[0018] The peptide gCK-16 of this invention has a strong affinity for PD-1 protein, with a Kd value of 3.37±0.66 nM, indicating that the peptide gCK-16 can bind to chicken PD-1 protein and block the PD-1 / PD-L1 immunosuppressive signaling pathway.

[0019] The polypeptide gCK-16 of this invention can promote the proliferation of PBMCs and has an immune-enhancing effect, providing a theoretical basis for the development of novel polypeptide immune enhancers, drugs and vaccines.

[0020] In vitro experiments have confirmed that the peptide gCK-16 of the present invention can block the binding of PD-1 / PD-L1 at the cellular level, block the PD-1 / PD-L1 signaling pathway, and reverse the immunosuppression caused by PD-1 / PD-L1 overexpression during chicken viral disease infection. This provides a theoretical basis and technical support for the clinical treatment of immunosuppressive diseases. Attached Figure Description

[0021] Figure 1 : The mass spectrum of the polypeptide gCK-16 of the present invention;

[0022] Figure 2 : High-performance liquid chromatogram of the polypeptide gCK-16 of the present invention;

[0023] Figure 3 Flow cytometry plot of peptide gCK-16 binding to PBMCs;

[0024] Wherein, A: scatter plot; B: flow cytometry plot of blank cells; C: flow cytometry plot of polypeptide gCK-16;

[0025] D: Irrelevant polypeptide flow cytometry;

[0026] Figure 4 Fluorescent image of peptide gCK-16 binding to chicken PBMCs (magnified 20 times);

[0027] A, DAPI is a marker for cell nuclei; B, FITC-labeled peptide gCK-16 binds to chicken PBMCs.

[0028] Combined chart of channels C, A, and B;

[0029] Figure 5 Western blotting assay was used to detect the expression of PD-1 protein in COS-7 cells transfected with pEGFP-PD-1.

[0030] Figure 6 : Results of pEGFP-PD-1 protein fluorescence intensity;

[0031] Figure 7 Results of MST detection of GFP fluorescence decay time and relative fluorescence intensity of PD-1 protein;

[0032] Figure 8 MST software automatically fits the Kd value of gCK-16 peptide binding to chicken PD-1 protein;

[0033] Figure 9 Scatter plot of the ability of peptide gCK-16 to promote the proliferation of chicken PBMCs;

[0034] Figure 10 CFSE fluorescence intensity results of the ability of peptide gCK-16 to promote the proliferation of PBMCs;

[0035] Figure 11 : CFSE positive control fluorescence intensity;

[0036] Figure 12 Flow cytometry plots of different concentrations of peptide gCK-16 blocking PD-1 / PD-L1;

[0037] Figure 13 Statistical chart of the blocking efficacy of peptide gCK-16 in blocking PD-1 / PD-L1 binding. Detailed Implementation

[0038] Example 1: Optimized Design of Peptides

[0039] Bioinformatics methods were used to analyze and predict the epitope information of the interaction between chicken PD-1 and its ligand PD-L1. A polypeptide fragment was designed in the IgV region of the complete coding sequence of chicken PD-1 / PD-L1 and named according to the first and last amino acids and the number of amino acids in the whole polypeptide chain. It was named gCK-16 and its amino acid sequence is CLIEYGGADYKTINLK (SEQ ID NO.1).

[0040] The peptides were synthesized by a professional peptide synthesis company using solid-phase peptide synthesis, and the synthesized peptides were analyzed by mass spectrometry. Figure 1 ) and high performance liquid chromatography (HPLC) Figure 2 The results showed that a single narrow peak appeared around the 12th minute, indicating that the peptide purity reached or exceeded the desalting level, suggesting that the synthesized peptide could be used for subsequent experiments.

[0041] Example 2: Detection of the binding ability of peptides to PBMCs

[0042] 1. Flow cytometry detection of peptide binding to PBMCs

[0043] The specific procedure for flow cytometry is as follows: 3-week-old healthy chickens are harvested, PBMCs are isolated and counted, and the cells are analyzed at a density of 5 × 10⁻⁶. 5 PBMCs were seeded into 6-well cell culture plates; PBMCs were stimulated with IFN-γ (200 ng / mL) for 30 min to induce the expression of chicken PD-1 protein; gCK-16-FITC (final concentration 0.1 mg / mL) and an irrelevant peptide were added, and the plates were incubated on ice for 2 h. The plates were washed three times with PBS for 10 min each time, and resuspended in 500 μL PBS after the last wash.

[0044] FITC fluorescence intensity was detected by flow cytometry, and 10,000 cells were counted.

[0045] See results Figure 3 In the figure, the horizontal axis represents fluorescence intensity, and the vertical axis represents cell number. 3A: Scatter plot; 3B: Flow cytometry plot of blank cells; 3C: Flow cytometry plot of peptide gCK-16; 3D: Flow cytometry plot of irrelevant peptides.

[0046] Figure 3 In A, 82.22% represents the proportion of the total number of cells to the total number of cells. Figure 3 In B, 0.01% represents the percentage of positive cells out of all cells; Figure 3 In C, 52.38% represents the proportion of positive cells to all cells, reflecting the strong binding ability of polypeptide gCK-16 to PBMCs. Figure 3 In D, 0% represents the proportion of positive cells among all cells, indicating that irrelevant peptides do not bind to PBMCs.

[0047] 2. Fluorescent detection of the binding affinity of peptides to PBMCs

[0048] The 6-well cell culture plates were pretreated with poly-L-lysine to encourage PBMCs to adhere to the bottom of the plates. PBMCs were then isolated using 1×10⁶ cells / well plate. 6 Cells / ml were seeded in 6-well plates and cultured for 24 h. After 30 min of stimulation with IFN-γ, the polypeptide gCK-16-FITC was added and incubated in the dark for 2 h. The cells were washed 3 times with PBS and stained with DAPI for 20 min. The fluorescence signals of FITC (green) and DAPI (nucleus) (blue) were observed under a fluorescence microscope.

[0049] The results showed that green fluorescence was present around the cell nucleus, indicating that the peptide gCK-16 has the ability to bind to PBMCs. A represents the cell nucleus, B represents gCK-16-FITC, and C represents the merging of DAPI and FITC channels. Figure 4 ).

[0050] Example 3: Identification of the binding of peptide gCK-16 to chicken PD-1 protein using the MST method

[0051] Micro-thermophoresis (MST) is a cutting-edge technique for quantitatively analyzing interactions between biomolecules. MST is used to quantitatively detect the affinity of peptide-protein interactions, thereby obtaining the equilibrium dissociation constant Kd. Using a constructed pEGFP-PD-1 eukaryotic expression vector, the pEGFP-PD-1 overexpression plasmid was transfected into COS-7 cells via Lipofectamine™ 2000 transfection reagent. After cell lysis, Western blotting experiments were performed, and the PD-1 target band was detected, indicating that the pEGFP-PD-1 plasmid was successfully transfected into COS-7 cells and expressed the PD-1 protein. Figure 5 ).

[0052] Thirty-six hours after transfection of COS-7 cells with pEGFP-PD-1 plasmid, protein samples obtained by lysing cells with RIPA lysis buffer (containing protease inhibitors) were used as the target protein. The synthesized peptide gCK-16 was used as the ligand, and the cells were serially diluted to concentrations of 1800 nM, 900 nM, 450 nM, 225 nM, 112.5 nM, 56.25 nM, 28.125 nM, 14.062 nM, 7.031 nM, 3.515 nM, 1.757 nM, 0.878 nM, 0.439 nM, 0.219 nM, 0.109 nM, and 0.054 nM. nM, using MST technology, was used to detect the affinity of the synthesized peptide gCK-16 to the fusion-expressed pEGFP-PD-1 protein in cell lysate. Finally, the binding force was determined based on the Kd value; the smaller the Kd value, the greater the affinity, and vice versa.

[0053] MST experimental results showed that the fluorescence intensity of the cleaved pEGFP-PD-1 protein was uniform ( Figure 6 This ensured the accuracy of the experiment. The fluorescence intensity decay time was within a reasonable range. Figure 7 The software automatically fitted the Kd value, and the results showed that the Kd value of peptide gCK-16 and PD-1 protein was 3.37±0.66 nM, indicating that peptide gCK-16 has a strong binding force with chicken PD-1 protein. Figure 8 ).

[0054] Example 4: CFSE method for detecting the proliferation of PBMCs promoted by different peptides

[0055] Chicken PBMCs were isolated, resuspended in 500 μL PBS, and carboxyfluorescein succinimidyl ester (CFSE) was added to a final concentration of 5 μM. The cells were incubated at 37°C for 30 minutes to label them. Cells were washed five times with PBS, 5 min each time. The cell number was adjusted to 1 × 10⁶ cells / year using RPMI-1640 medium containing 10% fetal bovine serum, 5% chicken serum, and 1% penicillin-dextrose antibody. 6 Cells were seeded at a concentration of 0.1 mg / mL in 6-well cell culture plates, and gCK-16 was added to a final concentration of 0.1 mg / mL. ConA was then added to a final concentration of 10 mg / mL, and the plates were incubated at 37°C with 5% CO2 for 72 hours. After 72 hours, cells were collected, washed three times with PBS, and resuspended in 500 μL of PBS on the last wash. Flow cytometry was used to identify the main cell population, and 10,000 cells were counted. Figure 9 ).

[0056] The proliferation capacity of PBMCs was assessed by the intensity of CFSE fluorescence signals. The results showed that peptide gCK-16 significantly promoted PBMC proliferation, with a proliferation rate of 51.41%, indicating that peptide gCK-16 has a certain degree of immunomodulatory effect (see...). Figure 10 , Figure 11 ).

[0057] Example 5: Peptide gCK-16 directly blocks PD-1 / PD-L1 binding at the cellular level.

[0058] To investigate the effect of peptide gCK-16 on blocking PD-1 / PD-L1 binding at the cellular level, COS-7-chPD-1 cells were directly incubated with peptide gCK-16, followed by incubation with PD-L1-his protein and FITC-labeled mouse anti-his tag antibody at 37°C for 2 hours. Cells were washed three times with PBS, and fluorescence signals were detected by flow cytometry. The positive control group (without peptide, showing strong fluorescence) and the negative control group (COS-7 cells only) were used.

[0059] The blocking efficacy was calculated using the formula: Blocking efficacy % = (Average fluorescence intensity of positive control - Average fluorescence intensity of experimental group) / Average fluorescence intensity of positive control * 100%.

[0060] The calculation results showed that the peptide gCK-16 had the ability to block PD-1 / PD-L1 binding at 50 nM and 100 nM, with blocking efficiencies of 58.41 ± 2.33% and 66.68 ± 2.55%, respectively (see...). Figure 12 , Figure 13 ).

[0061] The polypeptide gCK-16 of this invention can bind to PBMCs stimulated by IFN-γ. In immunosuppressive diseases, such as infectious bursal disease (IBD) infection in chickens, in addition to the immunosuppression caused by the destruction of B lymphocytes in the bursa of Fabricius, the expression levels of IFN-γ and the immunosuppressive molecule PD-1 in PBMCs are also increased, which to some extent exacerbates the body's immunosuppression. Activation of the PD-1 / PD-L1 signaling pathway leads to immunosuppression. The polypeptide binds to the PD-1 protein expressed on the surface of PBMCs, blocking the PD-1 / PD-L1 signaling pathway. This has a reversible effect on the immunosuppression caused by PD-1 / PD-L1 binding in the prevention and treatment of viral diseases in chickens.

[0062] The polypeptide gCK-16 of this invention can promote the proliferation of PBMCs, which include T lymphocytes, B lymphocytes, natural killer lymphocytes, monocytes, dendritic cells, etc. The chicken immune system depends on the phenotype of PBMCs, CD8... + T lymphocytes, after recognizing pathogens, directly kill them; they are cytotoxic T cells. CD4 + After recognizing pathogens, T lymphocytes attract other immune cells to the site of infection by producing antibodies, thus eliminating the pathogens; they are helper T cells. The polypeptides of this invention can promote the proliferation of PBMCs and have an immune-enhancing effect, providing a theoretical basis for the development of novel immune enhancers.

Claims

1. A target PD-1 polypeptide for chickens, characterized in that, The amino acid sequence of the polypeptide is CLIEYGGADYKTINLK.

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