TLR4 binding peptide and application thereof

Abstract

The invention belongs to the field of polypeptides, and particularly relates to a TLR4 binding peptide and application thereof. The specific technical scheme is as follows: the nucleotide sequence of the polypeptide is edited as shown in SEQ ID NO: 1. The binding peptide containing 67 amino acids is screened out, is good in solubility, can be stably bound with TLR4, and is expected to become a new medicine for treating or relieving pain including chronic pain. The polypeptide can also be prepared into health care products, special medical foods and the like with the function of treating or relieving pain according to actual conditions.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of polypeptides, and particularly relates to a TLR4 binding peptide and application thereof. BACKGROUND

[0002] TLR4 is a related protein in the Toll-like receptor family (Toll-like receptors, TLR) of natural immune pattern recognition receptors, and is expressed in myeloid cells, microglia, astrocytes and peripheral primary sensory cells. The structure of TLR4 includes extracellular, transmembrane and intracellular three parts, the extracellular part is a repeated leucine sequence, which mediates the recognition of pathogen-associated molecules, and the intracellular part is a highly conserved sequence, which has homology with the intracellular fragment of interleukin 1 receptor. The extracellular domain of TLR4 forms a heterodimer with MD4, and after binding with the ligand, two TLR4-MD4 complexes are induced to form a homodimer, which causes the aggregation of the intracellular domain, and then activates the downstream response, and starts the downstream channel through the MYD88-dependent and independent two pathways, and promotes the expression of inflammatory cytokines and chemokines.

[0003] A large number of studies have shown that TLR4 is involved in the excitation of neuron cells, glial cells and inflammatory cells, and regulates the transmission of pain signals in the spinal cord, and plays an important role in the formation and maintenance of pain. TLR4 is considered as a target for analgesia, and targeting binding TLR4 can reduce many types of pain, including inflammatory pain, neuropathic pain and postoperative pain.

[0004] At present, the research on analgesic drugs targeting TLR4 is still in the early stage, and the existing antagonists are all aimed at TLR4-MD4 dimer, and there is no inhibitor directly targeting the extracellular domain structure of TLR4. Because the extracellular domain molecular structure is relatively stable, the antagonist targeting the extracellular domain of TLR4 can have better bioavailability and longer half-life, so as to achieve more persistent analgesic effect, and has great potential in the research and development of clinical drugs.

[0005] Therefore, if a new targeting binding peptide can be provided for TLR4 extracellular, it is expected to provide a theoretical basis for the research and development of drugs for treating chronic pain. SUMMARY

[0006] The present application aims to provide a TLR4 binding peptide and application thereof.

[0007] To achieve the above-mentioned purpose of the application, the technical scheme adopted by the present application is: a polypeptide, the nucleotide sequence of the polypeptide is shown in SEQ ID NO: 1.

[0008] Correspondingly, the application of the polypeptide in binding with TLR4.

[0009] Correspondingly, the polypeptide is used to prepare a medicine, a health product or a food.

[0010] Preferably, the medicine is a medicine that can relieve or treat pain.

[0011] Correspondingly, the polypeptide is used to prepare a medicine, a health product or a food.

[0012] Preferably, the medicine is a medicine that can relieve or treat pain.

[0013] The present application has the following beneficial effects: the present application screens a binding peptide containing 67 amino acids, which has good solubility and can be stably combined with TLR4, and is expected to become a new medicine for treating or relieving pain including chronic pain. The polypeptide can also be prepared into a health product, a special medical food and the like with the function of treating or relieving pain according to actual conditions. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a schematic diagram of spatial structure simulation of the binding peptide and structure simulation of combination with TLR4;

[0015] Figure 2 It is a schematic diagram of comparison of binding ability of the binding peptide with TLR4 protein at different concentrations. DETAILED DESCRIPTION

[0016] The present application provides a new TLR4 binding peptide, and the nucleotide sequence of the binding peptide is 3'-AATTCAAATTCCCAGAAGTTTCAGCCAGAAGAAACGGCTACAGCAACATCAAGAGAG TCACAGCTTCCCATCTGGCTTGGCAGCCTCTATTCTCCATTAAGACCTGAGCTTAGCCCAA AGGCAATTTACCTTGAAGGAAGTCCTCTGGATAAATGGAAACAGAATTTCTGCTCAAACAT GTTTCTATACAACAGATATATAA-5'(SEQ ID NO:1). The TLR4 binding peptide can be combined with TLR4 protein and has good stability.

[0017] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. If not specifically indicated, the technical means used in the embodiments are conventional means familiar to those skilled in the art. The obtained data are all average values obtained after at least 3 repetitions, and the data obtained in each repetition are all effective data.

[0018] Example 1: Obtaining and identifying TLR4 binding peptide

[0019] Naja naja atra was selected from Guangdong snake farm. The snake was placed at -80℃ for 2 hours, and the venom gland was removed after being killed, and placed in liquid nitrogen for 2 hours and then transferred to -80℃ for long-term preservation.

[0020] Total RNA was extracted from the venom gland using RNAisoPlus (Takara) and purified, and then immediately subjected to gel electrophoresis to verify the integrity of the extracted total RNA, and the concentration of the extracted total RNA was detected using nanodrop. Poly(A)+mRNA was separated from the total RNA using an Oligotex kit (QIAGEN). The sequence (SEQ ID NO: 2) AAGCAGTGGTATCAACGCAGAGTGAATTCGGG of the SMARTI Oligo and the sequence (SEQ ID NO: 3) ATTCTAGACAAGCTTGGACATG-d(T) were designed. 30 The CDS III Primer of VN (N=A, G, C, or T; V=A, G, or C) has an EcoRI enzyme digestion recognition sequence, and the CDS III Primer has a HindIII enzyme digestion recognition sequence. The mRNA was reverse transcribed into a first strand of cDNA containing the desired enzyme digestion recognition sequence at both ends using SMARTScribe MMLV Reverse Transcriptase (Takara). Double-stranded DNA was synthesized using Advantage2 Polymerase Mix (Takara). Finally, purification was performed using a purification kit (Beyotime), and a cDNA library of Naja naja atra venom gland was obtained. The product was stored at -20℃ for long-term preservation.

[0021] The TLR4 binding peptide described in the present application was obtained by screening from the library, and the nucleotide sequence thereof is shown in SEQ ID NO: 1. Blastn alignment of SEQ ID NO: 1 showed that the polypeptide base sequence did not match any information in the NCBI database. The amino acid sequence translated by the +1 reading frame of the polypeptide (i.e. the amino acid sequence of the TLR4 binding peptide) is: C'-NSNSQKFQPEETATATSRESQLPIWLGSLYSPLRPELSPKAIYLEGSPLDKWKQNFCS NMFLYNRYI-N' (SEQ ID NO: 4), indicating that the polypeptide is a completely new substance.

[0022] Example 2: Artificial synthesis of TLR4 binding peptide and determination of molecular weight

[0023] The binding peptide in dry powder state was synthesized by solid phase synthesis Fmoc method (SPPS). The purity of the synthesized binding peptide was 95.170% measured by high performance liquid chromatography (HPLC). [M+9H] 9+ , [M+8H] 8+ , [M+7H] 7+ , [M+6H] 6+ , [M+5H] 5+ The mass-to-charge ratios of the binding peptide were 866.00, 974.20, 1113.20, 1298.40, 1557.80, respectively.

[0024] According to the highest peak [M+8H] 8+ , the molecular weight of the polypeptide was calculated according to the following formula: wherein, MW is the molecular weight, m is the mass of the molecular ion peak, z is the number of ion charges, and C is the number of charges of the positive ionization reagent. According to the mass spectrometry results, the molecular weight of the binding peptide was 7785.60.

[0025] Example Three: Performance display of TLR4 binding peptide

[0026] 1. Stability and solubility.

[0027] The solubility of the binding peptide corresponding amino acid sequence was predicted using Protine-Sol, and the stability of the binding peptide corresponding amino acid sequence was predicted using Expasy-ProtParam tool. A solubility prediction score greater than 0.45 indicates that the predicted molecule has good solubility, and an unstable score less than 40 in the stability prediction result indicates that the predicted molecule has good stability.

[0028] The results showed that the solubility prediction score of the binding peptide was 0.566, and the unstable score was 63.32, indicating that it had good solubility and stability.

[0029] 2. Binding ability to TLR4.

[0030] The binding score of the binding peptide corresponding amino acid sequence to TLR4 protein was predicted using HDOCK Server, and a molecular docking confidence score greater than 0.7 indicates that the two molecules have good binding. The predicted binding score was 0.9261, indicating that the binding peptide can stably bind to TLR4.

[0031] The tertiary structure of the polypeptide was predicted using homology modeling, as shown in the left and middle graphs of Figure 1 (left graph in cartoon form, middle graph in surface form). Figure 1 The right graph of

[0032] The binding ability of the binding peptide to TLR4 was further verified by surface plasmon resonance using a molecular interaction detection instrument Biacore T200 (Uppsala, Sweden).

[0033] According to the formula The coupling amount of the protein target was calculated.

[0034] Wherein, Rmax is the maximum binding capacity of the chip surface, set to 100 RU, analyte MW and ligand MW are the molecular weight of TLR4 protein and the molecular weight of the binding peptide, respectively, Sm is the stoichiometric ratio, set to 1, RL is the ligand coupling level, and the actual coupling amount is 1.5 times RL. According to the pre-enrichment experiment, the optimal coupling conditions of the protein were determined as follows: a sodium acetate solution with pH = 4.0, the maximum corresponding concentration of 2500 mM, and the optimal conditions for chip regeneration were a Glycine-HCl solution with pH = 2.0. The TLR4 protein solution was diluted to 20 μg / mL with a sodium acetate solution with pH = 4.0 for coupling, and the CM5 chip (GE Healthcare) was activated by EDC and NHS for TLR4 protein coupling. The TLR4 protein was coupled to the CM5 chip, and the concentration gradient of the binding peptide was set to 0 nM, 156.25 nM, 312.5 nM, 625 nM, 1250 nM, 2500 nM, 5000 nM, and 10000 nM, which were sequentially flowed through the chip from low to high. After coupling, the chip surface was blocked with ethanolamine, and a channel in the CM5 chip was used as a blank reference without TLR4 protein, and the rest was treated in the same manner. After binding, the chip was regenerated with a Glycine-HCl solution with pH = 2.0, and each concentration was repeated three times. The response value of the experimental channel minus the response value of the blank channel was the result of the binding force between the binding peptide and TLR4 protein.

[0035] The results are shown in Figure 2 Figure 2 From top to bottom, the SPR binding values of the polypeptide and TLR4 at different concentrations are represented, respectively, with blue for 10000 nM, red for 5000 nM, cyan for 2500 nM, green for 1250 nM, yellow for 625 nM, orange for 312.5 nM, and light blue for 156.25 nM. The results show that the binding peptide can bind to TLR4 protein well, and there is an obvious concentration dependence; the higher the concentration of the binding peptide, the stronger the binding ability to TLR4. The results show that the binding peptide can specifically bind to TLR4, proving that the binding peptide can act on TLR4 receptor, and is expected to play a role in analgesia, and has the potential to prepare analgesic drugs.

[0036] ​The above described embodiments are only to illustrate the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification, variation, modification, and replacement of the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope determined by the claims of the present application.

Claims

1. A polypeptide, characterized in that: The nucleotide sequence of the polypeptide is shown in SEQ ID NO:

1.

2. A polypeptide, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO:

4.

3. The use of the polypeptide of claim 1 or 2 in binding with TLR4.

4. Drugs, health products, and food prepared using the polypeptide described in claim 1.

5. The drug, health product, or food according to claim 4, characterized in that: The medication in question is a drug that can relieve or treat pain.

6. Pharmaceuticals, health products, and food products including the polypeptide described in claim 1.

7. The drug according to claim 6, characterized in that: The medication in question is a drug that can relieve or treat pain.

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