A specific affinity peptide for interleukin-10 and its uses

Abstract

This invention discloses a specific affinity peptide for interleukin-10 and its uses. The amino acid sequence of the affinity peptide is selected from one of SEQ ID No. 2. The affinity peptide can efficiently and specifically bind to interleukin-10, and diffracts bioactive fragments, polynucleotide sequences, and composite materials. The peptide of this invention has a high affinity for IL-10 and can efficiently recruit IL-10 protein, thereby inhibiting overactivated immune responses in inflamed tissues, providing more options for the suppression of inflammation and the treatment of diseases related to IL-10 deficiency.

Description

[0001] This invention application is a divisional application of the invention patent with application number 2022107246350 and application date of 2022-06-23. Technical Field

[0002] This invention relates to an affinity polypeptide for inflammatory pharmaceuticals and its uses in the field of biomedicine, specifically a novel affinity polypeptide capable of specifically binding to interleukin-10 (IL-10) and its uses. Background Technology

[0003] Phage display is a molecular biology technique that uses genetic engineering to insert exogenous peptide or protein genes into the phage coat protein gene. The peptide or protein encoded by the exogenous gene exists on the phage surface as a fusion protein. The displayed peptide or protein can maintain its relative spatial structure and biological activity, enabling it to bind to and recognize target molecules.

[0004] Interleukin-10 (IL-10) is an anti-inflammatory cytokine that plays a crucial role in the suppression and resolution of inflammation. It can inhibit the synthesis and release of inflammatory factors such as IL-1, IL-6, IL-8, and TNF-α, and enhance the release of anti-inflammatory factors, such as IL-1 receptor antagonists and lysing TNF-α receptors. Furthermore, it can also inhibit antigen presentation by monocytes and macrophages.

[0005] For inflammatory bowel disease (IBD), current treatment methods can be divided into two main categories: non-surgical IBD treatment, primarily involving traditional medications and emerging biologics, and surgical treatment. Traditional drug treatments have limited efficacy, high side effects, and significant dose dependence, while biologics are expensive and have numerous adverse reactions. These drugs are ineffective for some patients, and treatment may fail midway through. Potential immunosuppressive reactions can lead to adverse events such as infections, neurological disorders, and malignancies, further increasing the complexity of the disease. Furthermore, the relapse rate after discontinuing medication is relatively high. Surgical treatment, on the other hand, is prone to relapse and carries the risk of serious postoperative complications. Therefore, finding new treatment approaches has become an urgent priority. Summary of the Invention

[0006] To address the problems existing in the background art, the present invention provides a novel polypeptide sequence with high affinity for IL-10, which can specifically recruit IL-10. This polypeptide can be used to inhibit and resolve inflammatory diseases and to treat diseases related to IL-10 deficiency.

[0007] The present invention adopts the following technical solution.

[0008] 1. An affinity polypeptide, wherein the amino acid sequence of the polypeptide is selected from one of SEQ ID No. 1 to SEQ ID No. 5.

[0009] The amino acid sequences are as follows: HSGSSVFAQPVM; FPWPTPHWWHRS; HPSRRRDGNLPL; GHWKHHFRPPAP; EMFRELKNWTAA. See the table below:

[0010]

[0011] II. A bioactive substance comprising polypeptides, including but not limited to chemically coupled compounds, modified drugs, etc.

[0012] This bioactive substance can perform the same function as the affinity peptide, namely, recruiting the IL-10 protein.

[0013] 3. A polynucleotide sequence capable of encoding the polypeptide or the bioactive substance.

[0014] IV. A composite material comprising polypeptides, and also, but not limited to, nanoparticles, engineered bacteriophages, polymer materials, and other carrier materials for grafting polypeptides.

[0015] The composite material can perform the same function as the affinity peptide, namely, recruiting the IL-10 protein.

[0016] The application of the affinity peptide, the bioactive fragment, the polynucleotide sequence, or the composite material in the preparation of biomedical materials and drugs for inflammation. Specifically, its application in the preparation of biomedical materials and drugs for the specific release of the anti-inflammatory cytokine IL-10.

[0017] The inflammation mentioned refers to inflammation in inflammatory diseases such as inflammatory bowel disease, psoriasis, and arthritis.

[0018] The IL-10 affinity peptide obtained in this invention has a high affinity for IL-10, can specifically recruit IL-10, inhibit the release of pro-inflammatory cytokines, and increase the level of anti-inflammatory cytokines, thereby inhibiting and reducing inflammation, and suppressing overactive immune responses in inflamed tissues. It has broad application prospects in the treatment of various inflammatory diseases, such as inflammatory bowel disease, psoriasis, and arthritis.

[0019] This invention yields a polypeptide sequence with specific affinity for IL-10, targeting the IL-10 protein. Addressing the limitations of existing biological agents, such as limited efficacy and significant side effects, this polypeptide, possessing the ability to specifically bind to the IL-10 protein, offers a new option for inhibiting inflammatory responses and treating IL-10 deficiency-related diseases in the biomedical field.

[0020] The present invention adopts the following technical solution:

[0021] 1) Using Ph.D.-12 TM Phage peptide libraries were screened for IL-10 to obtain phages that have a specific affinity for IL-10;

[0022] 2) Perform DNA sequencing on the IL-10 affinity phage from step (1) and deduce the amino acid sequence of the affinity peptide based on the corresponding DNA sequence;

[0023] 3) Verify the affinity of the peptide obtained in step (2) for IL-10. Attached Figure Description

[0024] Figure 1 In Example 1, Ph.D.-12 was used TM The logarithmic ratio of output to input in each round of screening of IL-10 protein using a phage peptide library.

[0025] Figure 2 These are a series of high-frequency sequences obtained after analyzing and translating the DNA sequencing results in Example 2.

[0026] Figure 3 In Example 3, an enzyme-linked immunosorbent assay (ELISA) was used to test the affinity of phages with five different polypeptide sequences for the IL-10 protein. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The following embodiments are preferred embodiments of the present invention, but the present invention is not limited to the following embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0028] The embodiments of the present invention are as follows:

[0029] Example 1: Using Ph.D.-12 TM Phage peptide libraries were screened four times for the IL-10 protein.

[0030] 1) Coating IL-10 protein: Add 150 μL of IL-10 protein solution to a 24-well plate, place the plate in a humidified chamber, and incubate overnight on a shaker at 4°C.

[0031] 2) Blocking: Absorb the liquid from the plate, invert the plate onto a sterile paper towel and shake vigorously to remove any remaining solution. Then fill the well plate with 5% BSA blocking solution and gently shake at room temperature for 2 hours;

[0032] 3) Background removal: Take a new 24-well plate and add Ph.D.-12 TM Phage peptide library, incubated in a shaker at room temperature for 1 hour;

[0033] 4) Washing: Aspirate the blocking solution, invert the plate onto a sterile paper towel and shake vigorously to remove any remaining solution. Then wash the plate rapidly 6 times with PBST buffer, rotating it each time to ensure the bottom and edges of the wells are washed. Pour off the buffer solution, invert the plate onto a sterile paper towel and shake vigorously to remove any remaining solution.

[0034] 5) Binding: Add the phage solution from step 3) to the well plate treated in step 4), and incubate on a shaker for 1 hour;

[0035] 6) Washing: Discard any unbound phages, invert the plate onto a sterile paper towel and shake vigorously to remove any remaining solution. Wash the plate 10 times with PBST buffer as described in step 4), gently shaking for 10 minutes each time, and shake with a clean paper towel after each wash to avoid cross-contamination;

[0036] 7) Elution: First add 100 μL of elution buffer, shake gently at room temperature for 8 minutes, then add 15 μL of neutralization buffer, and collect the mixture into an EP tube;

[0037] 8) Plate Spreading: Preheat the prepared LB / IPTG / Xgal plates in a 37°C incubator. Dilute a portion of the phage solution collected in step 7) to an appropriate concentration. Take 10 μL of the diluted phage solution and incubate it with 200 μL of activated E. coil and ER2738 bacterial suspension for 15 minutes. Transfer the incubated phage / bacteria mixture to LB / IPTG / Xgal plates, spread evenly with a disposable sterile spreader, and incubate overnight at 37°C. Count the number of blue phage spots the next day.

[0038] 9) Amplification to form a daughter library: Add the remaining phage solution from step 7) to 20 mL of E. coil and ER2738 bacterial culture in the logarithmic growth phase. Incubate for 20 minutes, then place in a shaker at 37°C and shake at 220 rpm for 4.5 hours. After centrifuging to remove bacteria from the phage / bacteria mixture, add one-fifth volume of PEG / NaCl solution to the supernatant and allow to settle overnight at 4°C. The next day, centrifuge again, retain the precipitate, and resuspend the precipitate in sterile PBS to complete one round of phage purification. Repeat the purification process twice; the resulting phage daughter library will be used for the next round of amplification.

[0039] 10) Repeat steps 1) to 9) above 4 times to ensure that the amount of phage input is consistent in each round and complete the screening process.

[0040] Test 1: Calculate the input-output ratio

[0041] The input-output ratio for each round of screening can be obtained by dividing the number of phage blue spots counted in step 8) by the number of phages input. Figure 1 The graph shows the input-output ratio results for screening IL-10 affinity peptides. The logarithm of the input-output ratio is used to make the bar chart more visually appealing. According to the screening results, the input-output ratio increases with each round, indicating that phages displaying IL-10 affinity peptides have undergone significant enrichment.

[0042] Test 2: DNA sequencing and translation of the screened bacteriophages.

[0043] 1) Preservation and amplification of phages with specific affinity for IL-10: 80 blue spots were randomly selected from the plates from the fourth round of screening and added to shake tubes containing LB medium for amplification. After 24 hours, a portion of the bacterial culture was stored at -80°C, and the remaining culture was used for DNA sequencing.

[0044] 2) Sequencing Result Analysis: In the DNA sequencing results, the inserted foreign gene sequence was identified, the codons were translated into amino acid sequences, and the repetitive polypeptide sequences and their frequencies were statistically analyzed. The results are as follows: Figure 2 As shown.

[0045] Test 3: The IL-10 affinity of the screened bacteriophages was tested by enzyme-linked immunosorbent assay.

[0046] 1) Amplification and purification of IL-10 affinity phage: After thawing the five preserved phage samples and the wild-type phage sample at room temperature, 200 μL was added to 20 mL of E. coil and ER2738 bacterial culture in the logarithmic growth phase. After incubation for 20 minutes, the mixture was placed in a shaker at 37°C and shaken at 220 rpm for 4.5 hours. After centrifugation to remove bacteria from the phage / bacteria mixture, one-fifth volume of PEG / NaCl solution was added to the supernatant, and the mixture was allowed to settle overnight at 4°C. The next day, after centrifugation, the precipitate was retained and resuspended in sterile PBS, thus completing one stage of phage purification. The purification process was repeated twice, and the resulting phage solution was used for IL-10 affinity verification.

[0047] The five affinity phages are specifically those displaying the HSGSSVFAQPVM sequence, FPWPTPHWWHRS sequence, HPSRRRDGNLPL sequence, GHWKHHFRPPAP sequence, and EMFRELKNWTAA sequence.

[0048] 2) Coating IL-10 protein: Add 50 μL of protein at a concentration of 4 μg / mL to a 96-well microplate, place the microplate in a humidified chamber, and incubate overnight on a shaker at 4°C.

[0049] 3) Blocking: Aspirate the liquid from the wells, invert the plate onto a sterile paper towel, and shake vigorously to remove any remaining solution. Add 200 μL of 5% BSA blocking buffer to the wells and incubate for 1 hour.

[0050] 4) Washing: Discard the blocking solution, invert the ELISA plate on a sterile paper towel and shake vigorously to remove any remaining solution. Add 200 μL of PBST buffer and wash the plate 6 times, incubating on a shaker for 6 minutes each time. After each wash, shake vigorously on a sterile paper towel to remove any remaining solution.

[0051] 5) Binding: Add 50 μL of the phage amplified in step 1), maintaining a consistent concentration for each phage. Incubate at room temperature for 1 hour. Also, design groups containing PBS and wild-type phage for control purposes.

[0052] 6) Washing: Aspirate the phage solution and wash 6 times with PBST buffer as in step 4);

[0053] 7) Add primary antibody: Add 100 μL of antiphage capsid protein g8p antibody diluted to the appropriate multiple and incubate at room temperature for 1 hour;

[0054] 8) Washing: Aspirate the primary antibody and wash three times with PBST buffer as per step 4);

[0055] 9) Add secondary antibody: Add 100 μL of HRP-conjugated goat anti-mouse secondary antibody diluted to the appropriate multiple, and incubate at room temperature for 1 hour;

[0056] 10) Washing: Aspirate the secondary antibody and wash three times with PBST buffer as per step 4);

[0057] 11) Add TMB colorimetric solution: Add 100 μL of TMB colorimetric solution and incubate at 37°C for 10-20 minutes;

[0058] 12) Add stop solution: After step 11), when the color turns blue, add 100 μL of stop solution 2MH2SO4 to stop the reaction;

[0059] 13) Measure absorbance: Measure absorbance at 450 nm using an ELISA reader.

[0060] An experiment was conducted based on the results of this embodiment. The experimental procedure is as follows:

[0061] Test: The IL-10 affinity of the screened bacteriophages was tested by enzyme-linked immunosorbent assay.

[0062] 1) Amplification and purification of IL-10 affinity phage: After thawing the five preserved phage samples and the wild-type phage sample at room temperature, 200 μL was added to 20 mL of E. coil and ER2738 bacterial culture in the logarithmic growth phase. After incubation for 20 minutes, the mixture was placed in a shaker at 37°C and shaken at 220 rpm for 4.5 hours. After centrifugation to remove bacteria, one-fifth volume of PEG / NaCl solution was added to the supernatant, and the mixture was allowed to settle overnight at 4°C. The next day, after centrifugation, the precipitate was retained and resuspended in sterile PBS, thus completing one stage of phage purification. The purification process was repeated twice, and the resulting phage solution was used for IL-10 affinity verification.

[0063] The five affinity phages are specifically those displaying the HSGSSVFAQPVM sequence, FPWPTPHWWHRS sequence, HPSRRRDGNLPL sequence, GHWKHHFRPPAP sequence, and EMFRELKNWTAA sequence.

[0064] 2) Coating IL-10 protein: Add 50 μL of protein at a concentration of 4 μg / mL to a 96-well microplate, place the microplate in a humidified chamber, and incubate overnight on a shaker at 4°C.

[0065] 3) Blocking: Aspirate the liquid from the wells, invert the plate onto a sterile paper towel, and shake vigorously to remove any remaining solution. Add 200 μL of 5% BSA blocking buffer to the wells and incubate for 1 hour.

[0066] 4) Washing: Discard the blocking solution, invert the ELISA plate on a sterile paper towel and shake vigorously to remove any remaining solution. Add 200 μL of PBST buffer and wash the plate 6 times, incubating on a shaker for 6 minutes each time. After each incubation, shake vigorously on a sterile paper towel to remove any remaining solution.

[0067] 5) Binding: Add 50 μL of the phage amplified in step 1), maintaining a consistent concentration for each phage. Incubate at room temperature for 1 hour. Simultaneously, design groups containing PBS and wild-type phage for control purposes.

[0068] 6) Washing: Aspirate the phage solution and wash 6 times with PBST buffer as in step 4);

[0069] 7) Add primary antibody: Add 100 μL of antiphage capsid protein g8p antibody diluted to the appropriate multiple and incubate at room temperature for 1 hour;

[0070] 8) Washing: Aspirate the primary antibody and wash three times with PBST buffer as per step 4);

[0071] 9) Add secondary antibody: Add 100 μL of HRP-conjugated goat anti-mouse secondary antibody diluted to the appropriate multiple, and incubate at room temperature for 1 hour;

[0072] 10) Washing: Aspirate the secondary antibody and wash three times with PBST buffer as per step 4);

[0073] 11) Add TMB colorimetric solution: Add 100 μL of TMB colorimetric solution and incubate at 37°C for 10-20 minutes;

[0074] 12) Add stop solution: After step 11), when the color turns blue, add 100 μL of stop solution 2MH2SO4 to stop the reaction;

[0075] 13) Measure absorbance: Measure the absorbance at 450 nm using a microplate reader. For example... Figure 3 As shown, the affinity of the five selected phages for IL-10 protein was higher than that of the wild-type phage and the PBS negative control group. Among them, the phage displaying the HSGSSVFAQPVM sequence showed a significantly stronger affinity for IL-10 protein than the other phages, and showed a significant difference from the two control groups (***p<0.001).

[0076] Results Explanation: ELISA (Enzyme-Linked Immunosorbent Assay) is a commonly used experimental technique for detecting the content of biomolecules such as proteins, antibodies, and antigens. ELISA utilizes the principle of enzyme labeling and antibody binding to quantitatively or qualitatively detect the presence of specific molecules in a sample. In this experiment, antibodies were used to bind to the coat protein g8p of bacteriophages. By detecting the absorbance at 450 nm, the binding amount of bacteriophages displaying different polypeptide sequences to the IL-10 protein could be reflected. Higher absorbance values ​​indicate a greater number of bacteriophages with specific polypeptides that bind strongly to the IL-10 protein, and a stronger affinity for that polypeptide. Figure 3 As shown, the affinity of the five selected phages for IL-10 protein was higher than that of the wild-type phage and the PBS negative control group. Among them, the phage displaying the HSGSSVFAQPVM sequence showed a significantly stronger affinity for IL-10 protein than the other phages, and showed a significant difference from the two control groups (***p<0.001).

[0077] The phage displaying the FPWPTPHWWHRS sequence showed a significant affinity for the IL-10 protein, exhibiting a statistically significant difference compared to the two control groups (**p<0.01, ***p<0.001).

[0078] Therefore, as follows Figure 3 As shown, the affinity of the five selected phages for IL-10 protein was higher than that of the wild-type phage and the PBS negative control group. Among them, the phage displaying the HSGSSVFAQPVM sequence showed a significantly stronger affinity for IL-10 protein than the other phages.

[0079] The amino acid sequence involved in this invention is as follows:

[0080] SEQ ID No.1;

[0081] Name: Amino acid sequence of affinity peptide 1

[0082] Source: Artificial Sequence

[0083] HSGSSVFAQPVM

[0084] ID No. 2;

[0085] Name: Amino acid sequence of affinity peptide 2

[0086] Source: Artificial Sequence

[0087] FPWPTPHWWHRS

[0088] ID No. 3;

[0089] Name: Amino acid sequence of affinity peptide 3

[0090] Source: Artificial Sequence

[0091] HPSRRRDGNLPL

[0092] ID No. 4;

[0093] Name: Amino acid sequence of affinity peptide 4

[0094] Source: Artificial Sequence

[0095] GHWKHHFRPPAP

[0096] ID No. 5;

[0097] Name: Amino acid sequence of affinity peptide 5

[0098] Source: Artificial Sequence

[0099] EMFRELKNWTAA.

Claims

1. A polypeptide with affinity for interleukin-10, characterized in that: The amino acid sequence of the polypeptide is SEQ ID No.

2.

2. The application of the affinity polypeptide according to claim 1, characterized in that: Its application in the preparation of drugs for the treatment of inflammatory bowel disease, psoriasis, and arthritis.

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