Polypeptide with lung injury repairing function and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a polypeptide with a lung injury repairing function and application thereof. The polypeptide provided by the invention comprises a sequence SEQ ID NO: 1 or a variant sequence thereof, wherein the variant sequence comprises an amino acid sequence as shown in a general formula I. The polypeptide shows remarkable anti-inflammatory activity in an LPS-induced mouse acute lung injury model and improves lung pathological injury caused by LPS, and experimental data shows that the polypeptide can remarkably inhibit overexpression of proinflammatory factors TNFa and IL-6 by adjusting a lung immune microenvironment, so that acute inflammatory infiltration of the lung is relieved, and the lung inflammatory activity is improved. And a necessary low-inflammation microenvironment is created for repairing the damaged lung. In combination with the characteristics of low dosage and high drug effect in an LPS model, the polypeptide provides a safe candidate drug scheme for clinical treatment of acute respiratory distress syndrome and acute lung injury caused by virus or bacterial infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to polypeptides with lung injury repair function and their applications. Background Technology

[0002] Serine proteases, as core enzymes regulating key physiological processes such as coagulation, inflammatory response, tissue remodeling, and tumor invasion, are crucial to the pathological mechanisms of major diseases such as thrombotic diseases (e.g., deep vein thrombosis, acute coronary syndrome), chronic inflammatory diseases (e.g., chronic obstructive pulmonary disease, rheumatoid arthritis), and malignant tumor metastasis. Currently, clinically used serine protease inhibitors are mainly small molecule compounds (e.g., warfarin, a thrombin inhibitor, and cevelex sodium, a neutrophil elastase inhibitor). However, these drugs generally suffer from poor target selectivity, significant off-target effects, low bioavailability, and cumulative toxicity with long-term use, failing to meet the clinical demand for highly safe and specific therapeutic drugs. Therefore, the development of novel inhibitors is urgently needed to overcome existing technological bottlenecks. Natural peptides, due to their precise compatibility with the active sites and allosteric sites of serine proteases, high biocompatibility (composed of natural amino acids, with non-toxic metabolites), low immunogenicity, and strong structural modifiability, have become a core direction for the research and development of serine protease inhibitors.

[0003] Lung diseases are a major cause of high mortality and disability rates. Acute lung injury and its severe stage, acute respiratory distress syndrome, are characterized by damage to the alveolar epithelium and capillary endothelium, diffuse pulmonary edema, and inflammatory cell infiltration. Furthermore, long-term chronic lung injury (such as fibrosis and chronic myeloid pulmonary disease) involves the continuous destruction and abnormal repair of alveolar structure, ultimately leading to irreversible loss of lung function.

[0004] Currently, clinical treatments for lung injury still have significant limitations. Existing treatments are mostly symptomatic and supportive, such as mechanical ventilation, oxygen therapy, and the use of glucocorticoids. However, mechanical ventilation may lead to ventilator-associated lung injury (VILI), while long-term use of hormones is accompanied by serious systemic side effects (such as immunosuppression and osteoporosis). Small molecule drugs are rapidly metabolized in the body and widely distributed, making it difficult to achieve effective therapeutic concentrations at the site of lung lesions, and they are also prone to extrapulmonary toxicity.

[0005] Therefore, developing a novel active polypeptide that can efficiently promote lung injury repair, inhibit inflammatory response, and has high safety is of great scientific research value and application prospects for the clinical treatment of lung injury, improving patient prognosis, and reducing mortality. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned shortcomings of existing technologies by providing peptides with lung injury repair functions and their applications. The peptides of this invention are convenient for recombinant expression and exhibit good lung injury repair functions. Drugs for repairing lung injury prepared using the peptides of this invention have promising clinical application prospects.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The first objective of this invention is to provide a polypeptide with lung injury repair function, the polypeptide comprising the sequence SEQ ID NO:1 or a variant sequence thereof, the variant sequence comprising the amino acid sequence of general formula I. The general formula I is: TPDEX1CX2LPRDRGX3CX4RGX5QWFYYNKETNECKPFMYGGCAGNMNRFRTTEECETVCM, where X1, X2, X3, X4, and X5 are amino acid substitution sites, corresponding to positions 5, 7, 14, 16, and 19 of SEQ ID NO:1. X1 is selected from any one of E, R, A, K, and P; X2 is selected from any one of A, E, D, V, and P; X3 is selected from any one of A, D, H, and K; X4 is selected from any one of A, E, H, and K; X5 is selected from any one of A, E, H, K, and P.

[0008] Furthermore, the variant sequence is the amino acid sequence shown in SEQ ID NO:19 in the sequence listing.

[0009] A second objective of this invention is to provide the application of the above-mentioned polypeptide in the preparation of lung injury repair products.

[0010] Furthermore, the product includes a pharmaceutical composition.

[0011] A third object of the present invention is to provide a pharmaceutical composition comprising the above-described polypeptide or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, and / or excipient.

[0012] Furthermore, a third objective of this invention is to provide a biomaterial with lung injury repair function, which is any one of B1 to B6 below: B1: A nucleic acid molecule encoding the polypeptide of claim 1; B2: An expression cassette containing the nucleic acid molecules described in B1; B3: A recombinant vector containing the nucleic acid molecules described in B1; B4: Recombinant microorganisms containing the nucleic acid molecules described in B1; B5: Recombinant microorganisms containing the expression cassette described in B2; B6: Recombinant microorganisms containing the recombinant vector described in B3.

[0013] Furthermore, the nucleic acid molecule is a DNA molecule encoding the polypeptide of claim 1.

[0014] Further, the nucleic acid molecule is: the DNA molecule shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, or SEQ ID NO.20 in the sequence listing.

[0015] A fourth objective of this invention is to provide the application of the above-mentioned biomaterials in the preparation of lung injury repair products.

[0016] The fifth objective of this invention is to provide a method for preparing a polypeptide with lung injury repair function, comprising the step of expressing the encoding gene of the above-mentioned polypeptide in a biological cell to obtain a polypeptide with lung injury repair function, wherein the biological cell is a microbial cell.

[0017] Compared with existing technologies, the beneficial effects of the technical solution provided by this invention are as follows: (1) The peptide with lung injury repair function provided by the present invention showed significant anti-inflammatory activity and improved LPS-induced lung pathological damage in LPS-induced mouse acute lung injury model. Experimental data showed that the peptide could regulate the lung immune microenvironment by inhibiting elastase, and significantly inhibit the expression of pro-inflammatory factors TNFα and IL-6. It not only reduced the acute inflammatory infiltration of the lung, but also created the necessary low-inflammatory microenvironment for the repair of damaged lungs.

[0018] (2) The peptides provided by this invention not only exhibit highly effective therapeutic activity, but also possess excellent biocompatibility and clinical translational potential. Combined with their low-dose, high-efficiency characteristics in the LPS model, the peptides of this invention provide a safe candidate drug regimen for the clinical treatment of acute respiratory distress syndrome and acute lung injury caused by viral or bacterial infections. Attached Figure Description

[0019] Figure 1 This is a diagram of the structure of a prokaryotic expression plasmid for the polypeptide. Figure 2 SDS-PAGE image of the prokaryotic expression profile of the peptide, lane 1 shows the fusion protein, and lane 2 shows the fusion protein after protease digestion. Figure 3For peptide separation, purification, and mass spectrometry identification; Figure 4 This is a graph showing the results of verifying the inhibitory activity of the peptide against elastase. Figure 5a Figure 1 shows the detection results of the inflammatory factor IL-6 in an LPS-induced cellular inflammation model. Figure 5b Figure 1 shows the detection results of the inflammatory factor TNFα in an LPS-induced cellular inflammation model. Figure 6 Image of HE-stained lung tissue from LPS-induced lung inflammation in mice. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0021] Definitions and explanations of terms in this invention: In the amino acid sequences defined in this invention, amino acids are represented by single-letter symbols. These single-letter and three-letter symbols are well known to those skilled in the art and have the following meanings: A (Ala) is alanine, C (Cys) is cysteine, D (Asp) is aspartic acid, E (Glu) is glutamic acid, F (Phe) is phenylalanine, G (Gly) is glycine, H (His) is histidine, I (Ile) is isoleucine, K (Lys) is lysine, L (Leu) is leucine, M (Met) is methionine, N (Asn) is asparagine, P (Pro) is proline, Q (Gln) is glutamine, R (Arg) is arginine, S (Ser) is serine, T (Thr) is threonine, V (Val) is valine, W (Trp) is tryptophan, and Y (Tyr) is tyrosine.

[0022] The polypeptide of the present invention comprises the sequence SEQ ID NO:1 or a variant thereof, wherein the variant sequence is a polypeptide having the same function as SEQ ID NO:1 and comprises the amino acid sequence of general formula I: TPDEX1CX2LPRDRGX3CX4RGX5QWFYYNKETNECKPFMYGGCAGNMNRFRTTEECETVCM, where X1, X2, X3, X4, and X5 are amino acid substitution sites, corresponding to positions 5, 7, 14, 16, and 19 of SEQ ID NO:1. X1 is selected from any one of E, R, A, K, and P; X2 is selected from any one of A, E, D, V, and P; X3 is selected from any one of A, D, H, and K; X4 is selected from any one of A, E, H, and K; X5 is selected from any one of A, E, H, K, and P.

[0023] Salts of polypeptides of the present invention. Such salts include, but are not limited to, acid addition salts and base addition salts. As used herein, a “pharmaceutically acceptable salt” of a polypeptide means a salt that retains the desired antimicrobial activity of the polypeptide and is suitable for administration to humans or animals. Methods for preparing salts of polypeptides are known in the art and generally involve mixing the polypeptide with a pharmaceutically acceptable acid or base, for example, by mixing the free acid or free base form of the product with one or more equivalents of a suitable acid or base in a solvent or medium in which the salt is insoluble or in a solvent (such as water, which is then removed by vacuum or lyophilization), or by exchanging the cation of an existing salt for another cation on a suitable ion exchange resin. Examples of pharmaceutically acceptable acids and bases include organic and inorganic acids such as formic acid, acetic acid, propionic acid, lactic acid, glycolic acid, oxalic acid, pyruvic acid, succinic acid, maleic acid, malonic acid, trifluoroacetic acid, cinnamic acid, sulfuric acid, hydrochloric acid, hydrobromic acid, nitric acid, perchloric acid, phosphoric acid, and thiocyanate, which form ammonium salts with the free amino group of polypeptides, and bases such as ethylamine, methylamine, dimethylamine, triethylamine, isopropylamine, diisopropylamine, and other monoalkyl, dialkyl, and trialkylamines, and ethylarylamine.

[0024] The peptides of the present invention can be prepared by various methods. For example, the peptides can be synthesized by commonly used solid-phase synthesis methods well known in the art, such as methods involving t-BOC or FMOC protection of the α-amino group. Here, an amino acid is subsequently added to the elongated amino acid chain. Such methods are described, for example, in "Solid Phase Peptide Synthesis," IRL Publishing, London. Solid-phase peptide synthesis methods are particularly suitable for the large-scale production of shorter peptides.

[0025] Alternatively, recombinant techniques well known in the art can be used to prepare the polypeptides of the present invention, wherein the nucleotide sequence encoding the polypeptide is expressed in a host cell. The present invention therefore provides a method for preparing the polypeptides of the present invention, comprising: Provide a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide of the present invention; transform a host cell with the nucleic acid molecule; culture the host cell under conditions that allow expression of the polypeptide; harvest the polypeptide from the cell; optionally modify the polypeptide at its N-terminus or C-terminus, for example by adding an N-terminal and / or C-terminal extension group.

[0026] The present invention relates to a biomaterial with lung injury repair function, comprising: 1) a nucleic acid molecule encoding the polypeptide of claim 1; 2) an expression cassette containing the nucleic acid molecule of claim 1); 3) a recombinant vector containing the nucleic acid molecule of claim 1); 4) a recombinant microorganism containing the nucleic acid molecule of claim 1); 5) a recombinant microorganism containing the expression cassette of claim 1); and 6) a recombinant microorganism containing the recombinant vector of claim 1). The above biomaterial is described in detail below: Nucleic acid molecules can be naturally occurring or artificially designed or optimized sequences, including but not limited to codon optimization to suit the host expression system. Nucleic acid molecules may include promoters, terminators, and necessary regulatory elements to ensure correct transcription and translation in the expression system.

[0027] An expression cassette is a structural unit containing the aforementioned nucleic acid molecules, including necessary regulatory elements, inserted into a suitable vector. This expression cassette can be used for efficient expression of target peptides in various host cells. The expression cassette can be in the form of plasmids, viral vectors, or other suitable molecular vectors. The design of the expression cassette can be optimized according to the host type.

[0028] Recombinant microorganisms refer to transgenic microorganisms obtained by introducing the aforementioned nucleic acid molecules into a microbial host (such as Escherichia coli, yeast, or other microorganisms). These microorganisms are capable of expressing the polypeptides provided by this invention, thereby exerting lung injury repair functions in vitro or in vivo. Recombinant microorganisms can be obtained through conventional transformation, transfection, or infection methods, and can be appropriately screened to obtain high-yield expression strains.

[0029] In some embodiments, the expression cassette can be introduced into a microbial host to obtain a transgenic microorganism. Through regulatory elements within the expression cassette, the microorganism can stably and efficiently express the target peptide. It can be prepared using conventional molecular cloning and microbial culture methods, and the peptide can be extracted and purified in vitro.

[0030] In some embodiments, transgenic microorganisms can also be obtained by introducing a recombinant vector into a host microorganism. These microorganisms are capable of expressing the target polypeptide encoded in the vector. The microorganisms provided by this invention enable the large-scale production and application of polypeptides.

[0031] This invention also provides nucleic acid molecules comprising a nucleic acid sequence encoding the polypeptide of this invention, which are also referred to herein as nucleic acid molecules of this invention. The nucleic acid molecules or nucleic acid sequences of this invention, as used herein, comprise nucleotides, preferably DNA.

[0032] Another aspect of the invention is a pharmaceutical composition comprising at least one polypeptide described herein. The composition may also comprise a pharmaceutically or pharmacologically acceptable carrier. The phrase "pharmaceutically or pharmacologically acceptable" means a molecular entity or composition that, when properly administered to an animal or human, will not produce adverse, allergic, or other adverse reactions. The compositions of the invention may be aqueous compositions comprising an effective amount of the polypeptide dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

[0033] The term "pharmaceutically acceptable carrier" as used in this invention includes any and all solvents, dispersion media, coatings, isotonic agents, and absorption delay agents, etc. The use of such media and reagents for pharmaceutically active substances is well known in the art. Their use in therapeutic compositions is also considered, except that any conventional media or reagent may be incompatible with the active ingredient. Additional active ingredients may also be incorporated into the composition.

[0034] Specifically, the carrier can be an excipient widely used in the pharmaceutical manufacturing field. The carrier primarily serves to provide a safe, stable, and functional pharmaceutical composition, and can also provide a method for dissolving the active ingredient at a desired rate after administration to a subject, or for promoting the effective absorption of the active ingredient after administration to a subject. The pharmaceutical excipient can be an inert filler, or provide a function such as stabilizing the overall pH of the composition or preventing the degradation of the active ingredient. The carrier may include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0035] The pharmaceutical compositions of the present invention can be prepared using any method known to those skilled in the art, based on the disclosure. Examples include conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes. The effective dose range of the active substance can be wide, and it is usually administered at a pharmaceutically effective amount. However, it is understood that the actual amount of compound administered is usually determined by the physician based on relevant circumstances, including the condition being treated, the chosen route of administration, the actual compound administered, the patient's age, weight, and response, and the severity of the patient's symptoms.

[0036] Specifically, the pharmaceutical compositions of this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or delayed-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0037] The invention has now been generally described, and will be more readily understood by referring to the following embodiments, which are provided by way of example and not by way of limitation.

[0038] Example 1 Screening for peptide activity.

[0039] Elastase is a serine protease with a highly conserved catalytic triplet (Asp102-His57-Ser195) and a specific substrate-binding pocket (S1 pocket with Val216 and Thr226 as key residues, favoring small side-chain amino acids) at its active site. It plays a crucial role in inflammatory responses, tissue remodeling, and diseases such as chronic obstructive pulmonary disease and cystic fibrosis. High-resolution (≤2.5 Å) elastase crystal structures (1 EAU) were downloaded from the RCSB PDB database (https: / / www.rcsb.org / ). Virtual molecular docking screening was performed with 50 toxin peptides in AutoDock Vina. The 20 peptides with the optimal binding energy were further screened using molecular dynamics. Finally, 6 candidate peptides were selected, and their activity was detected using a fluorescent substrate assay. The results are as follows: Figure 1 As shown.

[0040] Table 1. Structural table for activity detection of 6 candidate peptides.

[0041]

[0042] Among them, the natural polypeptide HNSF-TY59 has relatively high activity. In order to improve the in vitro and in vivo stability of HNSF-TY59 compared with the natural polypeptide and to enhance its activity, thereby better exerting its anti-inflammatory effect, single or multiple amino acids at sequence positions 5, 7, 14, 16 and 19 of the HNSF-TY59 polypeptide were replaced, as follows: Position 5 is selected from any one of E, R, A, K, and P; Position 7 is selected from any one of A, E, D, V, and P; Position 14 is selected from any one of A, D, H, and K; Position 16 is selected from any one of A, E, H, and K; Position 19 is selected from any one of A, E, H, K, and P.

[0043] Example 2 Preparation of polypeptides.

[0044] Step S1: Based on the codon usage preferences of *E. coli*, the expressed gene series was optimized. The gene sequences shown in Table 2 (Shanghai Bioengineering Co., Ltd.) were synthesized using chemical methods. After double digestion with Hind III and EcoRI, they were ligated into the prokaryotic expression vector pET-32a (purchased from Novagen). The recombinant plasmid was then sequenced. Figure 1 The diagram shown is a structural diagram of the plasmid represented by HNSF-TY59.

[0045] Table 2. Sequence information table of HNSF-TY59 and its variants.

[0046]

[0047] Step S2: The coding gene of the polypeptide obtained in step S1 is cloned into the plasmid vector pET-32a to obtain the recombinant plasmid pET-32a-target polypeptide. According to the codon usage preference of E. coli, the expressed target gene sequence is optimized. In order to enable the TEV enzyme to effectively cleave the recombinant protein to remove the fusion tag, the specific recognition coding sequence ENLYFQG is introduced at the N-terminus of the polypeptide.

[0048] The coding gene of the polypeptide and the plasmid vector pET-32a were digested with NcoI and XhoI, the target fragment was recovered, and the gene fragment of the polypeptide and the pET-32a vector fragment were ligated with T4 ligase. The resulting ligation product was a recombinant plasmid, and the recombinant plasmid was subjected to DNA sequencing. Step S3: The recombinant plasmid pET-32a-target polypeptide obtained in step S2 is introduced into the host bacteria. The pET-32a-target polypeptide expression plasmid successfully constructed in step S2 is transformed into competent cells of Escherichia coli BL21(DE3) strain (purchased from Beijing Qingke Biotechnology Co., Ltd.). The cells are incubated overnight at 37°C with the cells inverted. The next day, a single colony is picked and cultured in 1 mL of medium containing the corresponding antibiotic to obtain the genetically engineered bacteria. Step S4: The host bacteria obtained in Step S3 were inoculated into LB medium containing ampicillin and cultured with shaking. After IPTG induction, the soluble recombinant protein Trx-target polypeptide was efficiently expressed. The genetically engineered bacteria obtained in Step S3 were cultured on a large scale with shaking. When the OD600 value reached approximately 0.8, IPTG was added to a final concentration of 1 mg / mL, and the culture was carried out at 28℃ and 120 rpm for 16 hours with shaking. After induction, the bacterial cells were enriched by refrigerated centrifugation, and after resuspending the bacterial cells, the soluble recombinant protein was obtained by high-pressure homogenization. After high-speed centrifugation, the His-tagged soluble recombinant protein Trx-target polypeptide was separated from the supernatant using nickel ion exchange resin. Step S5: Extract and purify the soluble recombinant protein Trx-target polypeptide obtained in step S4, and cleave Trx with TEV enzyme to obtain the polypeptide; elute impurities with 10mM and 30mM imidazole, wash the target protein off the column with 250mM imidazole, add an appropriate amount of TEV enzyme to the target protein, digest at 16℃ for 12 hours, dialyze to remove imidazole and other impurities, perform chromatographic purification and separation, collect the target solution, and freeze-dry to obtain the target polypeptide.

[0049] The gel electrophoresis identification results of the recombinant expression of the target polypeptide with sequence SEQ ID NO.19 are as follows: Figure 2 As shown, the recombinant protein can be observed to be cleaved before and after enzyme digestion, such as... Figure 3 The image shown is a mass spectrometry pattern of the target polypeptide with sequence SEQ ID NO.19.

[0050] Example 3 Study on the elastinase inhibitory activity of peptides.

[0051] The lyophilized peptides were subjected to elastin enzyme activity assays. Peptide concentrations of 20, 40, 80, 160, 320, 640, and 1280 nM were used, with an elastin enzyme concentration of 1 µg / mL. Ultrapure water was used as a control. The mixture was incubated at room temperature for 5 min to allow for complete enzyme-sample reaction. Immediately after incubation, reaction solution containing the substrate was added. The absorbance at 405 nm was dynamically recorded using a microplate reader every 30 s for a total of 30 min. Each reaction was repeated three times. The half-maximal inhibitory concentration (IC50) of the peptide against elastin was calculated using a nonlinear fitting method based on the concentration-activity relationship of the peptide in elastin enzyme kinetics (see Table 3). Figure 4 ).

[0052] Table 3. Results of Elastase Inhibition Activity of Each Peptide.

[0053]

[0054] Example 4 Study on the anti-inflammatory activity of peptides.

[0055] Using a medium containing 10% culture medium, resuscitated and passaged stable RAW.264.7 cells were seeded in 12-well plates at a cell density of 0.5 × 10⁻⁶ cells / well. 6 After 24 hours of incubation, the supernatant was discarded, and inflammation was induced again using 2% serum and 100 ng / ml LPS. After 24 hours, the supernatant was discarded, and the mixture was incubated for 24 hours in medium containing different concentrations of drugs (25 µM, 5 µM) with 2% serum. The supernatant was then collected for ELISA detection, including the detection of inflammatory factors such as TNF-α and IL-6. Figure 5a and Figure 5b It can be seen that the polypeptide of SEQ ID NO.19 has a good ameliorative effect on LPS-induced cellular inflammation and significantly reduces the expression of TNFα and IL-6.

[0056] Example 6 A study on the improvement of LPS-induced lung inflammation in mice by peptides.

[0057] A mouse pathological model was established using LPS (lipopolysaccharide) induction, suitable for preliminary screening and evaluation of the anti-inflammatory and lung injury repair effects of drugs. Balb / c female mice were used as experimental animals, with 6 groups of 3 mice per group to ensure data reproducibility. Mice were anesthetized with isoflurane. Group G1 was infused with 50 μL of PBS (D-PBS, E607009), and groups G2-G4 were infused with an equal volume of LPS (lipopolysaccharide, derived from E. coli O111:B4, Sigma-Aldrich) solution (5 mg / kg). Two hours after model establishment, mice were injected via the tail vein: groups G1 and G2 received PBS; group G3 received 10 mg / kg of Sivelestat (HY-17443, MCE) solution; and group G4 received 10 mg / kg of the peptide solution SEQ ID NO.19. Twenty-four hours after drug treatment, bronchoalveolar lavage was performed via tracheal instillation with PBS to obtain bronchoalveolar lavage fluid (BALF), which was then used for cell counting. (See Table 4 and...) Figure 6 It can be seen that peptide treatment significantly improved the degree of damage and inflammation in the lung lavage fluid and pathological sections of animal models.

[0058] Table 4. Inflammatory cell count in bronchoalveolar lavage fluid.

[0059]

[0060] Where there is no conflict, the above embodiments and features described herein can be combined with each other.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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.

Claims

1. A polypeptide with lung injury repair function, characterized in that, The polypeptide comprises the sequence SEQ ID NO:1 or a variant thereof, the variant sequence comprising the amino acid sequence of general formula I. The general formula I is: TPDEX1CX2LPRDRGX3CX4RGX5QWFYYNKETNECKPFMYGGCAGNMNRFRTTEECETVCM, where X1, X2, X3, X4, and X5 are amino acid substitution sites, corresponding to positions 5, 7, 14, 16, and 19 of SEQ ID NO:

1. X1 is selected from any one of E, R, A, K, and P; X2 is selected from any one of A, E, D, V, and P; X3 is selected from any one of A, D, H, and K; X4 is selected from any one of A, E, H, and K; X5 is selected from any one of A, E, H, K, and P.

2. The polypeptide according to claim 1, characterized in that, The variant sequence is the amino acid sequence shown in SEQ ID NO:19 in the sequence listing.

3. The use of the polypeptide according to any one of claims 1-2 in the preparation of lung injury repair products.

4. The application according to claim 3, characterized in that, The product includes a pharmaceutical composition.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the polypeptide of claim 1 or 2 or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, and / or excipient.

6. A biomaterial with lung injury repair function, characterized in that, It is any one of B1 to B6 below: B1: A nucleic acid molecule encoding the polypeptide of claim 1; B2: An expression cassette containing the nucleic acid molecules described in B1; B3: A recombinant vector containing the nucleic acid molecules described in B1; B4: Recombinant microorganisms containing the nucleic acid molecules described in B1; B5: Recombinant microorganisms containing the expression cassette described in B2; B6: Recombinant microorganisms containing the recombinant vector described in B3.

7. The biomaterial according to claim 6, characterized in that, The nucleic acid molecule is a DNA molecule encoding the polypeptide of claim 1.

8. The biomaterial according to claim 7, characterized in that, The nucleic acid molecule is the DNA molecule shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO.14, SEQ ID NO.16, SEQ ID NO.18, or SEQ ID NO.20 in the sequence listing.

9. The use of the biomaterial as described in any one of claims 6-8 in the preparation of lung injury repair products.

10. A method for preparing polypeptides with lung injury repair function, characterized in that, The method includes the step of expressing the gene encoding the polypeptide of claim 1 in a biological cell to obtain a polypeptide with lung injury repair function, wherein the biological cell is a microbial cell.

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