Lysozyme and methods of making and using same, methods of hydrolyzing bacteria

Abstract

This application discloses polypeptides and their uses. The amino acid sequence of the polypeptides is shown in SEQ ID NO:1. The polypeptides of this application have lysozyme activity; the method of this application can be scaled up and standardized for production, and the obtained polypeptides have lysozyme activity superior to that of natural lysozyme; compositions or kits containing the above-mentioned polypeptides can be used in medicine, food, and industry.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, specifically relating to lysozyme and its preparation method and uses, methods for hydrolyzing bacteria, and more specifically to a lysozyme obtained by screening based on artificial intelligence models and wet experimental iterative design and its uses. Background Technology

[0002] Lysozyme, also known as muramidase or N-acetylmuramide glycanohydrlase, is an alkaline enzyme that hydrolyzes the mucopolysaccharides in bacteria. As a natural, safe, and effective antibacterial agent, lysozyme has applications in multiple fields. For example, in medicine, it possesses antibacterial, antiviral, and tissue-repairing properties; clinically, it can be used to treat chronic rhinitis, acute and chronic pharyngitis, oral ulcers, chickenpox, herpes zoster, and flat warts. In food, it can be used as a food preservative. Industrially, it can be applied in cell engineering and bioengineering.

[0003] Currently, lysozyme can be extracted from egg white and eggshell membrane using common methods such as affinity chromatography, ion exchange resin methods, direct crystallization, and polyacrylic acid precipitation. Existing methods, such as affinity chromatography and ion exchange resin methods, are complex and require expensive equipment and reagents, resulting in high production costs and limiting large-scale production and application. Furthermore, most natural lysozymes have poor thermal stability, failing to meet the requirements of high-temperature pelleting processes in feed; and the activity of lysozyme may be lost during extraction and purification, affecting the quality of the final product.

[0004] Therefore, there is an urgent need to develop a new type of lysozyme. Summary of the Invention

[0005] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a polypeptide and its use as a lysozyme.

[0006] In a first aspect, this application provides a polypeptide. According to embodiments of this application, the amino acid sequence of the polypeptide is shown in SEQ ID NO:1. The polypeptide of this application has advantages such as high lysozyme activity.

[0007] In a second aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the polypeptide described in the first aspect. The nucleic acid molecule of this application can encode the polypeptide of the first aspect, which has advantages such as high lysozyme activity.

[0008] In a third aspect, this application provides an expression vector. According to an embodiment of this application, the expression vector carries the nucleic acid molecule described in the second aspect. The expression vector of this application, carrying the nucleic acid molecule described in the second aspect, can express the polypeptide of the first aspect, which has advantages such as high lysozyme activity.

[0009] In a fourth aspect, this application provides a recombinant cell. According to embodiments of this application, the recombinant cell carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect, or the recombinant cell expresses the polypeptide described in the first aspect. The recombinant cell of this application can express the polypeptide of the first aspect, which has advantages such as high lysozyme activity.

[0010] In a fifth aspect of this application, a method for preparing a polypeptide is provided. According to embodiments of this application, the amino acid sequence of the polypeptide is shown in SEQ ID NO:1; the method comprises: culturing the recombinant cells described in the fourth aspect to obtain the polypeptide. The method of this application can be scaled up and standardized for production, and the obtained polypeptide has lysozyme activity superior to that of natural lysozyme.

[0011] In a sixth aspect of this application, the use of the polypeptide described in the first aspect or the polypeptide prepared according to the method described in the fifth aspect as a lysozyme is proposed.

[0012] In a seventh aspect of this application, the present application proposes the use of the polypeptide described in the first aspect in the preparation of a product, said product being used for at least one of antibacterial, antiviral, tissue repair, disease treatment, food preservation, food freshness preservation, cell fusion promotion, and preparation of cell extracts.

[0013] In an eighth aspect of this application, a composition is provided. According to an embodiment of this application, the composition comprises the polypeptide described in the first aspect. As is known before, the polypeptide of the first aspect has excellent lysozyme activity and can be used as a pharmaceutical, food additive, or tool enzyme in bioengineering. Thus, the composition containing the above-mentioned polypeptide can be used in medicine, food, and industry.

[0014] In a ninth aspect of this application, a reagent or kit is provided. According to an embodiment of this application, the reagent or kit comprises the polypeptide described in the first aspect. As is previously known, the polypeptide of the first aspect possesses excellent lysozyme activity. Therefore, the reagent or kit containing the aforementioned polypeptide can be used as a food additive or as a tool enzyme in bioengineering, etc.

[0015] In a tenth aspect of this application, a method for hydrolyzing bacteria is provided. According to embodiments of this application, the method comprises: contacting a sample containing bacteria with a polypeptide described in the first aspect, a polypeptide prepared by the method described in the fifth aspect, a composition described in the eighth aspect, or a reagent or kit described in the ninth aspect.

[0016] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0017] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0018] Figure 1 This is a flowchart illustrating the protein design process in the embodiments of this application;

[0019] Figure 2 This is a flowchart illustrating the construction process of the preference dataset in this application embodiment;

[0020] Figure 3 This represents the ratio of the lysozyme lysis activity of each enzyme in the embodiments of this application to the baseline scheme. Detailed Implementation

[0021] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0022] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0023] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0024] In this document, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components comprising a polypeptide or its derivatives and / or with the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0025] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, including but not limited to pharmaceutically acceptable stabilizers, diluents, or other liquid excipients, suitable for a specific target dosage form. The use of any conventional excipients is also within the scope of consideration for this application, except for any range of incompatibilities with the polypeptides, pharmaceutical compositions, or drugs containing them, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition.

[0026] In addition to any conventional excipients, the use of peptides, pharmaceutical compositions, or drugs containing them that are incompatible with the present application, such as any adverse biological effects or harmful interactions with any other component of a pharmaceutically acceptable composition, is also within the scope of this application.

[0027] In this document, the term "treatment" refers to the administration of a drug to achieve a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and / or therapeutic in terms of partial or complete cure of a disease and / or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to disease but not yet diagnosed with it; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug containing a peptide or its derivative to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing the peptide described herein to an individual in need.

[0028] This application discloses a polypeptide and its use as a lysozyme, which will be described in detail below.

[0029] polypeptide

[0030] In a first aspect, this application proposes a polypeptide. According to embodiments of this application, the amino acid sequence of the polypeptide is shown in SEQ ID NO:1. The polypeptide of this application was obtained by designing a lysozyme protein sequence different from that of natural products using an artificial intelligence model and further screening it. It possesses superior lysozyme activity compared to natural lysozyme and can be used as a lysozyme.

[0031] MWERVAACESGGRWHLDTGNGYFGGLQLAKATWAAFGGTGSAAEHSRARQIEIAKQVRAARGTRPWPRCTAGLGLAGG (SEQ ID NO: 1).

[0032] It should be noted that in this application, "amino acid sequence as shown in SEQ ID NO:A" includes the amino acid sequence of SEQ ID NO:A, or the amino acid sequence of a conservatively modified form of SEQ ID NO:A, or an amino acid sequence having at least 80% identity with SEQ ID NO:A, all of which are within the scope of protection of this application. For example, "amino acid sequence of polypeptide as shown in SEQ ID NO:1" means that the polypeptide has the amino acid sequence of SEQ ID NO:1, or the polypeptide has the amino acid sequence of a conservatively modified form of SEQ ID NO:1, or the polypeptide has the amino acid sequence having at least 80% identity with SEQ ID NO:1, all of which are within the scope of protection of this application.

[0033] In this document, "conserved modified amino acid sequences" refers to amino acid modifications that do not significantly affect or alter the lysozyme activity containing that amino acid sequence. These modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the peptides of this application using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine). Exemplarily, conservative modifications are limited to no more than 20% of the total number, preferably no more than 10%. For example, the number of conserved modified amino acids in the conserved modified form of SEQ ID NO:1 does not exceed 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20), preferably not exceeding 10, and more preferably not exceeding 5.

[0034] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including: Needleman et al. (1970) J. Mol. Biol. 48: 443, a homology alignment algorithm; Smith et al. (1981) Adv. Appl. Math. 2: 482, a local homology algorithm; Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444, a similarity search method; and the Smith-Waterman algorithm (Meth. Mol. Biol). .70:173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J.Mol.Biol. 215:403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al., Meth.Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0035] In this document, the term "having at least 80% sequence similarity" can mean having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence similarity. The sequence similarity described in this application can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. All amino acid sequences mentioned in this application are shown from the N-terminus to the C-terminus.

[0036] According to embodiments of this application, the polypeptide has an amino acid sequence as shown in SEQ ID NO:1 or an amino acid sequence having at least 90% sequence similarity thereto.

[0037] Nucleic acid molecules, expression vectors and recombinant cells

[0038] In a second aspect of this application, a nucleic acid molecule is provided. According to embodiments of this application, the nucleic acid molecule encodes the polypeptide described in the first aspect. The nucleic acid molecule of this application can encode the polypeptide of the first aspect, which has high lysozyme activity, particularly superior to that of natural lysozyme.

[0039] According to an embodiment of this application, the nucleic acid molecule is DNA.

[0040] It should be noted that those skilled in the art will understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases, the complementary strand is also disclosed. Furthermore, the nucleic acid molecule sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

[0041] In a third aspect, this application provides an expression vector. According to an embodiment of this application, the expression vector carries the nucleic acid molecule described in the second aspect. When linking the nucleic acid molecule to the expression vector, the nucleic acid molecule can be directly or indirectly connected to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself, or they can be exogenous, i.e., not derived from the expression vector itself. Naturally, the connection between the nucleic acid molecule and the control elements only needs to be operably established.

[0042] In this document, "operably ligated" refers to ligating a foreign gene to an expression vector, enabling the control elements within the expression vector, such as transcriptional and translational control sequences, to perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used expression vectors include plasmids and bacteriophages. According to some specific embodiments of this application, after the expression vector is introduced into suitable recipient cells, the aforementioned polypeptide can be effectively expressed under the mediation of a regulatory system, thereby achieving the large-scale in vitro production of the polypeptide.

[0043] In some specific embodiments of this application, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

[0044] In some specific embodiments of this application, the expression vector is a lentiviral vector.

[0045] In one optional embodiment of this application, the expression vector is a plasmid expression vector.

[0046] In a fourth aspect, this application provides a recombinant cell. According to embodiments of this application, the recombinant cell carries the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect, or the recombinant cell expresses the polypeptide described in the first aspect. Using this recombinant cell under suitable conditions, the aforementioned polypeptide can be effectively expressed within the recombinant cell.

[0047] According to embodiments of this application, the recombinant cells are obtained by introducing the expression vector described in the third aspect into the host cell.

[0048] It should be noted that the host cell in this application is not particularly limited and can be a prokaryotic cell, a eukaryotic cell, or a bacteriophage. The prokaryotic cell can be *Escherichia coli*, *Bacillus subtilis*, *Streptomyces*, or *Proteus mirabilis*, etc. The aforementioned eukaryotic cells include fungi such as *Pichia pastoris*, *Saccharomyces cerevisiae*, *Schizosaccharomyces cerevisiae*, and *Trichoderma*, insect cells such as armyworms, plant cells such as tobacco, and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

[0049] According to an embodiment of this application, the host cell is a eukaryotic cell.

[0050] According to embodiments of this application, the host cell is a mammalian cell, including but not limited to BHK cells, CHO cells, NSO cells or COS cells, and does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0051] It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the peptide described in this application. Those skilled in the art will readily understand that suitable conditions for peptide expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for peptide expression based on the specific environment of their laboratory.

[0052] Methods for preparing peptides

[0053] In a fifth aspect of this application, a method for preparing a polypeptide is provided. According to embodiments of this application, the amino acid sequence of the polypeptide is shown in SEQ ID NO:1; the method comprises: culturing the recombinant cells described in the fourth aspect to obtain the polypeptide. The method of this application can be scaled up and standardized for production, and the obtained polypeptide has lysozyme activity superior to that of natural lysozyme.

[0054] use

[0055] In a sixth aspect of this application, the use of the polypeptide described in the first aspect or prepared according to the method described in the fifth aspect as a lysozyme is proposed. As is known prior, the polypeptide of the first aspect possesses excellent lysozyme activity. Therefore, the aforementioned polypeptide can be used as a lysozyme in the production of pharmaceuticals, food additives, food preservatives, or as a tool enzyme in cell engineering / bioengineering, etc.

[0056] In a seventh aspect of this application, the use of the polypeptide described in the first aspect in the preparation of a product for at least one of the following: antibacterial, antiviral, tissue repair, disease treatment, food preservation, food freshness, cell fusion promotion, and preparation of cell extracts. As is known prior, the polypeptide of the first aspect possesses excellent lysozyme activity and can be used as a pharmaceutical, food additive, or tool enzyme in cell engineering / bioengineering. Therefore, products containing the aforementioned polypeptide can be used in medicine (e.g., for antibacterial, antiviral, tissue repair, and disease treatment), in food (e.g., for food preservation and food freshness), and in industry (e.g., for promoting cell fusion and preparing cell extracts).

[0057] According to embodiments of this application, the diseases include chronic rhinitis, acute and chronic pharyngitis, oral ulcers, chickenpox, herpes zoster, and flat warts.

[0058] According to embodiments of this application, the product is selected from pharmaceuticals, food additives, food preservatives, and reagents.

[0059] According to embodiments of this application, the product is selected from pharmaceuticals. It can thus be used for antibacterial, antiviral, tissue repair, and disease treatment.

[0060] According to embodiments of this application, the product is selected from food additives and food preservatives. Therefore, it can be used for food preservation and food anti-corrosion.

[0061] According to embodiments of this application, the product is selected from reagents. This can promote cell fusion, prepare cell extracts, and be applied in bioengineering, such as for cell fusion in genetic engineering and cell engineering, or for preparing cell extracts, increasing the yield of extracts, and shortening the preparation time of yeast extracts.

[0062] Composition, reagent or kit

[0063] In an eighth aspect of this application, a composition is provided. According to an embodiment of this application, the composition comprises the polypeptide described in the first aspect. As is known before, the polypeptide of the first aspect has excellent lysozyme activity and can be used as a pharmaceutical, food additive, or tool enzyme in bioengineering. Thus, the composition containing the above-mentioned polypeptide can be used in medicine, food, and industry.

[0064] According to embodiments of this application, the composition is a pharmaceutical composition, a feed composition, or a food composition.

[0065] According to embodiments of this application, the composition is a pharmaceutical composition, and the composition further comprises pharmaceutically acceptable excipients.

[0066] In a ninth aspect of this application, a reagent or kit is provided. According to an embodiment of this application, the reagent or kit comprises the polypeptide described in the first aspect. As is previously known, the polypeptide of the first aspect possesses excellent lysozyme activity. Therefore, the reagent or kit containing the aforementioned polypeptide can be used as a food additive or as a tool enzyme in bioengineering, etc.

[0067] Methods of hydrolyzing bacteria

[0068] In a tenth aspect of this application, a method for hydrolyzing bacteria is proposed. According to embodiments of this application, the method comprises: contacting a sample containing bacteria with a polypeptide described in the first aspect, a polypeptide prepared by the method described in the fifth aspect, a composition described in the eighth aspect, or a reagent or kit described in the ninth aspect. This method is particularly suitable for in vitro hydrolysis of bacteria.

[0069] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. 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 the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0070] Example 1: Peptide Design and Screening

[0071] like Figure 1 The flowchart shown encompasses a series of steps from protein design and sequence generation to screening and optimization. For an example, see patent application number 2025103739070, filed on March 27, 2015, entitled "Optimization Method, Apparatus, Electronic Device, and Storage Medium for Protein Language Models." It uses a Protein Design Agent (PDA) to output prompts to a Protein Generation Model (PGM). The PGM generates lysozyme protein sequences, and a Quality Control Pipeline (QCP) screens these generated lysozyme protein sequences to select those that meet quality standards. Based on the selected protein sequence scheme, protein-coding DNA is synthesized and expressed in a protein expression system. The lysozyme activity of the expressed protein is then detected using a wet-lab assay.

[0072] Furthermore, the experimental results were fed back into the PGM model using reinforcement learning for optimization. Then, a new round of protein design was performed, the designed protein was resynthesized into DNA, and expressed in a protein expression system. The lysozyme activity of the expressed protein was then compared with that of the previously designed protein using biological experiments. The model optimization process based on the experimental results is as follows:

[0073] Step 1: Use the PGM model (denoted as π) θ (θ is the network structure parameter that can be optimized) to generate a large number of candidate protein sequences.

[0074] Step 2: Predict the enzyme activity of candidate protein sequences using the Unified Sequence Model (USM) in the quality control process screening, and perform virtual screening to obtain a small number of protein sequences for wet experiments.

[0075] Step 3: Perform a wet experiment to obtain feedback on the generated sequence.

[0076] For example, a wet laboratory test includes the following steps:

[0077] 3-1 Synthesis of the target lysozyme sequence

[0078] 1) Plasmid construction

[0079] Genes for synthesizing the target proteins were amplified and synthesized separately via homologous recombination. Hieff Clone was then used. TM The One Step Cloning Kit inserts the target gene into the corresponding pJL1 expression vector via restriction enzyme digestion and ligation. The expression vector is sequenced to confirm correct insertion. The expression vector containing the target gene is transformed into DH5α competent cells. Single clones are selected for vector amplification. Plasmids are extracted using a kit similar to the Pure Strand HQ 96 Plasmid DNA Purification Kit (ThermoCat#K210096).

[0080] 2) No cell protein synthesis

[0081] The plasmid extracted in step 1) was added to an EP tube containing the cell-free expression reaction system. The reaction was maintained in the EP tube at 30°C for 3 hours by an orbital oscillator with appropriate stirring speed.

[0082] 3) Protein purification

[0083] Pretreatment: The precipitate in the solution obtained in step 2) was removed by centrifugation, and the cell-free protein synthesis (CFPS) supernatant was collected for protein purification.

[0084] Protein purification: The target protein is extracted from the CFPS supernatant using affinity chromatography or a combination of chromatographic methods. After each purification step, the crude sample is buffer-replaced before proceeding to the next purification step. The final purified product is then transferred to a storage buffer.

[0085] Protein quality assessment: The purity and content of the crude samples after each purification step and the final protein product were assessed using methods such as SDS-PAGE and UV spectroscopy (OD280). Western blot analysis was also performed to detect the expressed lysozyme.

[0086] 3-2. Detection of lysozyme activity

[0087] The small number of protein sequences (i.e., each enzyme to be tested) obtained through virtual screening in step 2 above for wet experiments were analyzed using a lysozyme activity assay kit (Thermos Fisher EnzChek). TM The Lysozyme Assy Kit was used to test lysozyme activity in reaction systems with different pH levels and under 37°C conditions.

[0088] This embodiment exemplifies the detection results of lysozyme TX-L6, whose amino acid sequence is shown in SEQ ID NO:2, and compares it with commercially available chicken lysozyme under the same test conditions. The results are shown in Table 1. The results show that the lysozyme activity of lysozyme TX-L6 is superior to that of chicken lysozyme under all pH conditions.

[0089] MLAPRTVAGGVAGAAMWDQVAQCESSGDWQNGFHGGLQFTQSTWETAFGGLYAP SADKASRPDLASRSQQIAVAERVLAGQGPGAWPNCGGRLP (SEQ ID NO: 2).

[0090] Table 1:

[0091]

[0092] In Table 1, the lysozyme activity U is determined by EnzChek TM The lysozyme assay kit defines the components of the detection buffer as follows: 80 mM NaCl, 0.1% Tween 20, PB / Tris Buffer, and NA indicates that no activity was detected in the reaction system.

[0093] Step 4: Using the successful sequences from Step 3 and selecting a subset of sequences from Step 2 to form a preference dataset, iteratively train the PGM model using the Differential Population Optimization (DPO) algorithm, update the parameters of the PGM model, and obtain a new π. θ .

[0094] For example, the "preference dataset" is generated by screening protein sequences with the desired function from the protein sequences generated in step 2 through wet laboratory testing, and using these sequences as preference responses to construct the training dataset. For example, the construction of the preference dataset includes the following steps:

[0095] 4-1: Generate the initial dataset S0

[0096] Using the current PLM, generate a series of protein sequences (this dataset is denoted as S0) based on the given cue x (which could be "generate a highly thermally stable lysozyme").

[0097] 4-2: Virtual screening was conducted through dry experiments to obtain the candidate sequence set S1 and the remaining sequence set S2.

[0098] For each protein column in the generated protein sequence dataset S0, a series of indicators, such as structural stability and functional prediction score, are calculated. Based on pre-set screening criteria (such as a specific functional score threshold), a subset of candidate sequences are selected. The set of these candidate sequences is denoted as S1, and the set of sequences that are rejected is denoted as S2.

[0099] 4-3: Conduct wet experiments

[0100] The candidate sequences S1 obtained from the virtual screening were then tested in a wet assay (see step 3 for detailed steps of the wet assay). The wet assay is the ultimate standard for verifying protein functionality, determining the function and activity of these sequences through actual biological experiments. A series of wet assays were designed and performed to test the functionality and activity of the candidate sequences. The experimental results for each candidate sequence were recorded to determine whether it demonstrated the expected function.

[0101] 4-4: Constructing a Preference Dataset

[0102] The sequences that exhibit the expected function in the wet experiment are labeled as the preference response set. These sequences are considered to be positive feedback sequences.

[0103] From the sequences rejected in the dry experiment, sequences with low reference model loss (high confidence) and low pLDDT scores are selected and marked as the unfavorable response set. These sequences are considered negative feedback sequences. The number of negative feedback sequences is approximately 5-10 times that of positive feedback sequences.

[0104] The final dataset is constructed as follows: in Each element of the dataset contains a cue x and a positive feedback sequence y. w and negative feedback sequence y l .

[0105] For example, "iterative training" refers to using the DPO algorithm, combined with length regularization, to iteratively optimize the PGM model so that the PGM model generates protein sequences that better match the target properties. For example, the iterative optimization framework is shown below:

[0106] Given a preference dataset DPO optimizes the following loss function L DPO Update the model so that it prefers to output positive feedback samples y. w And not favoring negative feedback samples y l .

[0107]

[0108] Where, π θIt is a protein language model, where θ is π. θ Updatable parameters in the model architecture, π θ (y|x) represents the probability that the model will output y given a prompt x. π ref It is a reference protein language model, which is related to the initial π. θ It is copied, but does not participate in the update.

[0109] σ is the sigmoid function, and β is a hyperparameter used to adjust the magnitude of model updates. The larger β is, the more π... θ It is necessary to maintain and π to a greater extent ref The update magnitude is smaller, and the model is closer to the target value. Furthermore, by minimizing this loss function, the updated model can achieve maximum efficiency. and minimized This means that the updated model output y w The probability increases, so the output y l The probability decreases, thus aligning with the feedback from wet experiments.

[0110] A length-controlled regularization term is added during the wet experiment feedback process to reinforce the model's preference for outputting a specific length. The length-controlled regularization term is R(y) = min(|y| - l). max ,0)+l max Where |y| represents the length of sequence y, l max This indicates the maximum desired protein sequence length. When the output protein sequence length is greater than 1... max When R(y) = |y|, the length of the output protein sequence is small. max At that time, R(y) = l max It is independent of y and will not affect the optimization of the model.

[0111] The loss function with length control is:

[0112]

[0113] Here, α is a hyperparameter that controls the strength of the length regularization term. In practice, β = 0.1 and α = 0.5 are usually chosen.

[0114] Step 5: Repeat steps 1-4 until the wet experiment in step 3 yields satisfactory results.

[0115] The final amino acid sequence of lysozyme TXRL15, as shown in SEQ ID NO:1, was obtained. In the second stage, lysozyme TX-RL15, generated through wet-lab iterative optimization of the model, was tested for its lysinic activity using a wet-lab experiment (see step 3 for detailed wet-lab testing steps). The ratio of the lysinic activity of lysozyme TX-RL15 to that of lysozyme TX-L6 is shown below. Figure 3 As shown in the figure, the effect of lysozyme TX-RL15 is illustrated in the bar chart within the orange box, and its lysis activity is 66 times that of lysozyme TX-L6.

[0116] Example 2:

[0117] This embodiment aims to evaluate the in vitro tissue repair effect of lysozyme TX-RL15 in Example 1, and to verify its therapeutic effects on chronic rhinitis, acute and chronic pharyngitis, oral ulcers, chickenpox, herpes zoster, and flat warts. The specific steps are as follows:

[0118] First, the in vitro cell assay of lysozyme TX-RL15 promoting tissue repair, the specific experimental steps are as follows:

[0119] 1. Cell Culture and Damage Induction

[0120] Cells (commonly fibroblasts (such as 3T3 cells), keratinocytes (HaCaT), or human umbilical vein endothelial cells (HUVEC)) are seeded in 6-well plates and cultured until 80% confluence. Damage areas are created using pipette tips or mechanical scratching to establish an in vitro trauma model.

[0121] Cells were washed with PBS to remove suspension, and cultured in medium containing lysozyme for 24-72 hours. Lysozyme concentration gradients were set at 0 (control), 10, 50, and 100 μg / mL. Furthermore, synergistic effects were verified by combining the cells with antibiotics (e.g., clarithromycin) or growth factors (e.g., bFGF).

[0122] 2. Lysozyme activity detection

[0123] Lysis zone method: Make holes in an agar plate containing Micrococcus lysate, add lysozyme solution, and quantify enzyme activity by measuring the diameter of the lysis zone.

[0124] Fluorescent substrate method: 4-Methylumbelliferyl-β-DN,N',N”-triacetylchitotriose (MUNAC) was used as the substrate to detect the fluorescence intensity of the lysozyme hydrolysis products.

[0125] 3. Assessment of cell proliferation and migration

[0126] MTT / CCK-8 assay: detects cellular metabolic activity and reflects proliferative capacity.

[0127] Transwell migration assay: to assess the ability of cells to migrate to damaged areas.

[0128] 4. Detection of inflammatory and repair factors

[0129] ELISA assay: Measure the expression levels of TNF-α, IL-6 (inflammatory factors) and VEGF, TGF-β (pro-repair factors).

[0130] qPCR / Western Blot: Analyze the gene and protein expression of MMP-9 (matrix metalloproteinase) and TIMP-1 (tissue inhibitor of metalloproteinases) to assess extracellular matrix remodeling.

[0131] 5. Results

[0132] 5.1 Synergistic Antibacterial Effect

[0133] Lysozyme significantly reduces the bacterial load (such as Escherichia coli and Staphylococcus aureus) on wounds and lowers the intensity of inflammatory responses by hydrolyzing peptidoglycan in the cell walls of Gram-positive bacteria.

[0134] When used in combination with antibiotics, it can enhance the clearance effect against drug-resistant bacteria (such as MRSA) and reduce endotoxin release.

[0135] 5.2 Promoting Cell Proliferation and Migration

[0136] The cell proliferation rate of the lysozyme-treated group (50-100 μg / mL) was higher than that of the control group, and the scratch closure speed was faster.

[0137] The mechanism involves activating the EGFR / ERK signaling pathway and upregulating PCNA (proliferating cell nuclear antigen) expression.

[0138] 5.3 Anti-inflammatory and immune modulation

[0139] Compared to the control group, the lysozyme treatment group could inhibit the secretion of TNF-α and IL-6, and reduce tissue damage caused by excessive activation of macrophages.

[0140] Compared to the control group, the lysozyme treatment group can promote the release of IL-10 (anti-inflammatory factor), regulate the Th1 / Th2 balance, and enhance local immune homeostasis.

[0141] 5.4 Expression of tissue repair markers

[0142] Compared with the control group, the expression levels of VEGF and TGF-β in the lysozyme treatment group were significantly increased (2-3 times and 1.5-2 times, respectively), promoting angiogenesis and collagen deposition.

[0143] Compared to the control group, the expression of epithelial-mesenchymal transition (EMT) related genes (such as Snail and Slug) was downregulated in the lysozyme treatment group, maintaining the integrity of the epithelial barrier.

[0144] Second, the in vitro cell assay for lysozyme TX-RL15 in the treatment of chronic rhinitis, the specific experimental steps are as follows:

[0145] 1. Cells (human nasal mucosal epithelial cell lines such as HNEpC) or primary cultured cells were seeded and cultured. Cells were stimulated with lipopolysaccharide (LPS, 1-10 μg / mL) or inflammatory factors (TNF-α 10-50 ng / mL) to induce a chronic inflammatory state. Lysozyme concentration gradients were set at 0 (control), 10, 50, and 100 μg / mL for treatment for 24-72 hours. Furthermore, the synergistic effect was verified by combining dexamethasone (1 μM) or antibiotics (such as clarithromycin).

[0146] 2. Inflammatory response assessment

[0147] ELISA assay: The secretion levels of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α were measured using conventional methods, and the inhibitory effect of lysozyme on inflammatory pathways was analyzed.

[0148] NF-κB pathway detection: p65 phosphorylation level and IκBα degradation were analyzed by Western blotting to verify the regulation of nuclear transcription factors by lysozyme.

[0149] 3. Oxidative stress detection

[0150] ROS level: Intracellular reactive oxygen species production was detected using the DCFH-DA fluorescent probe.

[0151] Antioxidant enzyme activity: SOD and GSH-Px activities were measured to evaluate the mitigating effect of lysozyme on oxidative damage.

[0152] 4. Analysis of mucosal barrier function

[0153] Tight junction protein detection: The expression of ZO-1, occludin, and claudin-1 was analyzed by qPCR / Western Blot to assess the integrity of the epithelial barrier.

[0154] Transepithelial resistance (TEER): Measures the resistance of a cell monolayer, reflecting changes in barrier permeability.

[0155] 5. Antibacterial and anti-biofilm effects

[0156] Bacterial adhesion experiment: Staphylococcus aureus or Streptococcus pneumoniae were co-cultured with cells to observe the inhibitory effect of lysozyme on bacterial adhesion.

[0157] Biofilm staining: Crystal violet staining was used to detect the destructive effect of lysozyme on bacterial biofilm formation.

[0158] 6. Cell migration and repair capabilities

[0159] Transwell migration assay: to evaluate the migration rate of epithelial cells after lysozyme treatment.

[0160] CCK-8 proliferation assay: to analyze the promoting effect of lysozyme on cell proliferation.

[0161] 7. Results Analysis

[0162] 7.1 Anti-inflammatory effects

[0163] The levels of IL-6 and TNF-α in the lysozyme treatment group (50-100 μg / mL) were lower than those in the control group, suggesting that it reduces the inflammatory response by inhibiting the NF-κB pathway.

[0164] It reduces the expression of M1 macrophage marker (iNOS), increases the expression of M2 marker (Arg-1), and promotes the formation of an anti-inflammatory microenvironment.

[0165] 7.2 Antioxidant stress

[0166] Lysozyme can significantly reduce intracellular ROS levels while increasing SOD activity and alleviating oxidative damage.

[0167] 7.3 Mucosal barrier repair

[0168] Compared with the control group, the expression levels of ZO-1 and occludin proteins were increased in the lysozyme treatment group, indicating improved barrier function.

[0169] 7.4 Antibacterial and anti-biofilm

[0170] Lysozyme treatment can reduce bacterial adhesion rate and biofilm thickness, suggesting that it inhibits pathogen colonization.

[0171] 7.5 Cell repair capacity

[0172] Transwell assays showed an increase in the number of migrating cells in the lysozyme-treated group, and CCK-8 assays showed an increased proliferation rate in the lysozyme-treated group, promoting mucosal repair.

[0173] Third, the in vitro cell assay for lysozyme TX-RL15 in the treatment of acute and chronic pharyngitis, the specific experimental steps are as follows:

[0174] 1. Cells (human laryngeal epithelial cells (Hep-2) or human pharyngeal fibroblasts (e.g., HNEpC)) were seeded and cultured. Cells were stimulated with lipopolysaccharide (LPS, 1-10 μg / mL) or inflammatory factors (TNF-α 10-50 ng / mL) to induce a chronic inflammatory state. Lysozyme concentration gradients were set at 0 (control), 10, 50, and 100 μg / mL for 24-72 hours. Furthermore, the synergistic effect was verified by combining antibiotics (e.g., penicillin) or antiviral drugs (e.g., ribavirin).

[0175] 2. Inflammatory response assessment

[0176] ELISA assay: Measure the secretion levels of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α, and analyze the inhibitory effect of lysozyme on inflammatory pathways.

[0177] NF-κB pathway detection: p65 phosphorylation level and IκBα degradation were analyzed by Western blotting to verify the regulation of nuclear transcription factors by lysozyme.

[0178] 3. Antibacterial and antiviral experiments

[0179] Bacterial adhesion experiment: Streptococcus pyogenes (a common pathogen of pharyngitis) was co-cultured with cells to observe the inhibitory effect of lysozyme on bacterial adhesion.

[0180] Virus neutralization assay: Cells were infected with rhinovirus (RV) or adenovirus (AdV), and the inhibition rate of lysozyme on viral replication was detected.

[0181] 4. Analysis of mucosal barrier function

[0182] Tight junction protein detection: ZO-1 and occludin expression were analyzed by qPCR / Western Blot to assess epithelial barrier integrity.

[0183] Mucus secretion detection: The amount of mucus secretion was determined by Alsin blue staining to verify the ability of lysozyme to decompose mucin.

[0184] 5. Assessment of cell repair capacity

[0185] CCK-8 proliferation assay: to analyze the promoting effect of lysozyme on the proliferation of damaged cells.

[0186] Scratch assay: to assess the migration rate of epithelial cells after lysozyme treatment.

[0187] 6. Results Analysis

[0188] 6.1 Anti-inflammatory effect

[0189] The levels of IL-6 and TNF-α in the lysozyme treatment group (50-100 μg / mL) were lower than those in the control group, suggesting that it reduces the inflammatory response by inhibiting the NF-κB pathway.

[0190] It reduces the expression of M1 macrophage marker (iNOS), increases the expression of M2 marker (Arg-1), and promotes the formation of an anti-inflammatory microenvironment.

[0191] 6.2 Antibacterial and antiviral effects

[0192] Lysozyme can inhibit Streptococcus pyogenes and significantly reduce the presence of pathogens in the pharynx.

[0193] Lysozyme can inhibit rhinovirus replication by disrupting viral capsid proteins or interfering with host receptor binding.

[0194] 6.3 Mucosal barrier repair

[0195] Compared with the control group, the expression levels of ZO-1 and occludin proteins were increased in the lysozyme treatment group, indicating improved barrier function.

[0196] Compared to the control group, the lysozyme treatment group showed reduced mucus secretion and downregulated expression of mucin MUC5B, thus alleviating the sticky feeling in the throat.

[0197] 6.4 Cell Repair and Protection

[0198] Transwell assays showed an increase in the number of migrating cells in the lysozyme-treated group, and CCK-8 assays showed an increase in the proliferation rate of the lysozyme-treated group.

[0199] TUNEL staining revealed a decreased apoptosis rate in the lysozyme-treated group, suggesting that it protects cells by activating the PI3K / Akt pathway.

[0200] Fourth, the in vitro cell experiment of lysozyme TX-RL15 for the treatment of oral ulcers, the specific experimental steps are as follows:

[0201] 1. Cells (Hep-2 cells (human laryngeal epidermoid carcinoma cells) or primary oral mucosal epithelial cells) were inoculated and cultured. Cells were first stimulated with lipopolysaccharide (LPS, 1-10 μg / mL) or inflammatory factors (TNF-α 10-50 ng / mL) to induce a chronic inflammatory state. Then, cell layer defects were created using 5% glacial acetic acid or mechanical scratching to simulate ulcer formation. Lysozyme concentration gradients were set at 0 (control), 10, 50, and 100 μg / mL for 24-72 hours. Furthermore, the synergistic effect was verified by combining dexamethasone (1 μM) or an antibacterial agent (such as nystatin).

[0202] 2. Inflammation and Infection Assessment

[0203] ELISA assay: Measure the secretion levels of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α, and analyze the inhibitory effect of lysozyme on inflammatory pathways.

[0204] Bacterial / fungal adhesion assay: Streptococcus mutans and Candida albicans were co-cultured with cells to detect the effect of lysozyme on reducing the adhesion rate of pathogens.

[0205] 3. Antibacterial and antiviral experiments

[0206] Bactericidal rate determination: The bactericidal rate of lysozyme against oral pathogens (such as Staphylococcus aureus and Candida albicans) was assessed by crystal violet staining or MTT method.

[0207] Virus neutralization assay: Cells were infected with herpes simplex virus (HSV-1), and the inhibition rate of lysozyme on viral replication was detected.

[0208] 4. Analysis of Mucosal Barrier Repair

[0209] Tight junction protein detection: ZO-1 and occludin expression were analyzed by qPCR / Western Blot to assess epithelial barrier integrity.

[0210] Transepithelial resistance (TEER): Measures the resistance of a single cell layer, reflecting the recovery of barrier permeability.

[0211] 5. Assessment of cell proliferation and migration

[0212] CCK-8 / MTT assay: to analyze the promoting effect of lysozyme on the proliferation of damaged cells.

[0213] Scratch assay: to assess the migration rate of epithelial cells after lysozyme treatment.

[0214] 6. Tissue repair factor detection

[0215] VEGF / TGF-β expression: The upregulation of pro-angiogenic and collagen synthesis-related factors was detected by ELISA or qPCR.

[0216] 7. Results Analysis

[0217] 7.1 Anti-infective effects

[0218] Lysozyme has a high killing rate against Streptococcus mutans and Candida albicans, significantly reducing the risk of secondary infection of oral ulcers.

[0219] Lysozyme can inhibit HSV-1 replication, reduce viral load, and destroy viral capsid proteins.

[0220] 7.2 Anti-inflammatory and immune modulation

[0221] The levels of IL-6 and TNF-α in the lysozyme treatment group (50-100 μg / mL) were lower than those in the control group, which reduced inflammation by inhibiting the NF-κB pathway.

[0222] Compared with the control group, the lysozyme treatment group reduced the expression of M1 macrophage marker (iNOS), increased the expression of M2 marker (Arg-1), and promoted the formation of an anti-inflammatory microenvironment.

[0223] 7.3 Mucosal barrier repair

[0224] Compared to the control group, the expression levels of ZO-1 and occludin proteins were increased in the lysozyme treatment group, the TEER value returned to normal levels, and mucosal permeability was reduced.

[0225] It reduces the expression of mucin MUC5B, alleviates the stickiness on the ulcer surface, and accelerates surface debridement.

[0226] 7.4 Cell Repair Capacity

[0227] Transwell assays showed that the number of migrating cells increased twofold in the lysozyme-treated group, and CCK-8 assays showed that the proliferation rate of the lysozyme-treated group was increased.

[0228] TUNEL staining showed that the apoptosis rate of cells in the lysozyme-treated group was reduced, indicating that the PI3K / Akt pathway was activated to protect cells.

[0229] Fifth, the in vitro cell assay of lysozyme TX-RL15 for treating chickenpox, the specific experimental steps are as follows:

[0230] 1. Human skin fibroblasts (HSF) or human amniotic cells (HFL) were inoculated and cultured. The cells were then infected with VZV virus solution (MOI = 0.1–1) to establish an in vitro varicella model. Lysozyme concentration gradients were set at 0 (control), 10, 50, and 100 μg / mL for treatment for 24–72 hours. Furthermore, the synergistic effect was verified by combining acyclovir (5 μM).

[0231] 2. Viral infection and lysozyme treatment

[0232] VZV-infected HSF cells were divided into different treatment groups, and lysozyme of varying concentrations was added and incubated continuously.

[0233] 3. Viral replication inhibition assessment

[0234] Viral titer determination: The viral load in the cell culture supernatant was detected by the TCID50 method, and the inhibition rate of lysozyme on viral replication was calculated.

[0235] Viral protein detection: Western blotting analysis of viral glycoprotein (such as gE and gB) expression levels to assess the degree of viral assembly inhibition.

[0236] 4. Cell protection and apoptosis inhibition

[0237] CCK-8 / MTT assay: to determine cell viability and analyze the alleviating effect of lysozyme on VZV-induced cytopathic effect (CPE).

[0238] TUNEL staining: to detect the inhibitory effect of lysozyme on apoptosis of infected cells.

[0239] 5. Inflammatory factors and immune regulation

[0240] ELISA assay: Measure the secretion levels of pro-inflammatory factors such as IL-6, TNF-α, and IFN-γ, and analyze the anti-inflammatory effect of lysozyme.

[0241] Immunofluorescence staining: to observe the inhibitory effect of lysozyme on virus-induced NF-κB nuclear translocation.

[0242] 6. Virus adsorption and invasion blocking

[0243] Virus adsorption assay: Cells were infected after pretreatment with lysozyme, and the viral DNA load in the cells was detected by qPCR to evaluate the inhibitory effect during the adsorption phase.

[0244] 7. Results Analysis

[0245] 7.1 Antiviral effects

[0246] The lysozyme-treated group showed significantly better inhibition of viral replication than the control group.

[0247] Western blotting showed reduced expression of viral gE protein in the lysozyme-treated group, suggesting that it interferes with viral particle assembly.

[0248] 7.2 Cell Protection and Apoptosis Regulation

[0249] Compared with the control group, the cell survival rate of the lysozyme treatment group (100 μg / mL) was increased and CPE was significantly reduced.

[0250] TUNEL staining showed that the proportion of apoptotic cells was reduced in the lysozyme-treated group, indicating that the PI3K / Akt pathway was activated to protect host cells.

[0251] 7.3 Anti-inflammatory and Immune Modulation

[0252] Compared to the control group, the lysozyme treatment group showed decreased levels of IL-6 and TNF-α, reduced NF-κB nuclear translocation, and blocked the "cytokine storm".

[0253] Compared to the control group, the lysozyme treatment group showed increased IFN-γ secretion, which enhanced the antiviral immune response.

[0254] 7.4 Virus Adsorption and Invasion Blocking

[0255] Pretreatment of lysozyme can reduce the viral DNA load in cells, suggesting that it blocks the binding of the virus to the host receptor.

[0256] Sixth, the in vitro cell assay of lysozyme TX-RL15 for treating flat warts, the specific experimental steps are as follows:

[0257] 1. Human skin fibroblasts (HSF) or human keratinocytes (HaCaT) were inoculated and cultured. The cells were then infected with VZV virus solution (e.g., HPV-3, HPV-10 subtypes, MOI = 0.1-1) to establish an in vitro model of flat warts. Lysozyme concentration gradients were set at 0 (control), 10, 50, and 100 μg / mL for treatment for 24-72 hours. Furthermore, the synergistic effect was verified by combining retinoic acid cream (0.1%) or liquid nitrogen cryotherapy to simulate physical therapy.

[0258] 2. Viral infection and lysozyme treatment

[0259] HPV-infected HSF cells were divided into different treatment groups, and lysozyme of varying concentrations was added and incubated continuously.

[0260] 3. Viral replication inhibition assessment

[0261] Viral DNA quantification: Intracellular HPV DNA load was detected by qPCR, and the inhibition rate of lysozyme on viral replication was calculated.

[0262] Viral capsid protein detection: Western blotting analysis of the expression level of L1, the main HPV capsid protein, to assess the degree of inhibition of viral particle assembly.

[0263] 4. Cell protection and apoptosis inhibition

[0264] CCK-8 / MTT assay: to determine cell viability and analyze the alleviating effect of lysozyme on HPV-induced cytopathic effect (CPE).

[0265] TUNEL staining: to detect the inhibitory effect of lysozyme on apoptosis of infected cells.

[0266] 5. Inflammatory factors and immune regulation

[0267] ELISA assay: Measure the secretion levels of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α, and analyze the anti-inflammatory effect of lysozyme.

[0268] Immunofluorescence staining: to observe the inhibitory effect of lysozyme on virus-induced NF-κB nuclear translocation.

[0269] 6. Virus adsorption and invasion blocking

[0270] Virus adsorption assay: Cells were infected after pretreatment with lysozyme, and the viral DNA load in the cells was detected by qPCR to evaluate the inhibitory effect during the adsorption phase.

[0271] 7. Results Analysis

[0272] 7.1 Antiviral effects

[0273] After 48 hours of lysozyme treatment, the HPV DNA load in the lysozyme-treated group decreased significantly, which was much better than that in the control group.

[0274] Compared with the control group, Western blot showed that the expression level of HPV L1 protein was reduced in the lysozyme-treated group, suggesting that it interfered with viral particle maturation.

[0275] 7.2 Cell Protection and Apoptosis Regulation

[0276] Compared with the control group, the cell survival rate of the lysozyme treatment group was improved and CPE was significantly reduced.

[0277] Compared with the control group, TUNEL staining showed that the proportion of apoptotic cells was reduced in the lysozyme treatment group, and the PI3K / Akt pathway was activated to protect host cells.

[0278] 7.3 Anti-inflammatory and Immune Modulation

[0279] Compared to the control group, the lysozyme treatment group showed decreased levels of IL-6 and TNF-α, reduced NF-κB nuclear translocation, and blocked the "cytokine storm".

[0280] Compared to the control group, the lysozyme treatment group showed increased IFN-γ secretion, which enhanced the antiviral immune response.

[0281] 7.4 Virus Adsorption and Invasion Blocking

[0282] Pretreatment of lysozyme reduced the viral DNA load in cells, suggesting that it blocked the binding of the virus to the host receptor.

[0283] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0284] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

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

1.

2. A nucleic acid molecule, an expression vector, and a recombinant cell, characterized in that, The nucleic acid molecule encodes the polypeptide of claim 1; The expression vector carries the aforementioned nucleic acid molecules; The recombinant cell carries the aforementioned nucleic acid molecule or the aforementioned expression vector, or the recombinant cell expresses the polypeptide of claim 1.

3. A method for preparing polypeptides, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:1; The method includes: The recombinant cells of claim 2 are cultured to obtain the polypeptide.

4. Use of the polypeptide of claim 1 or the polypeptide prepared by the method of claim 3 as a lysozyme.

5. The use of the polypeptide of claim 1 in the preparation of a product, wherein the product is used for at least one of antibacterial, antiviral, tissue repair, disease treatment, food preservation, food freshness preservation, cell fusion promotion, and preparation of cell extracts.

6. The use according to claim 5, characterized in that, The intended use includes one or more of the following conditions: The diseases mentioned include chronic rhinitis, acute and chronic pharyngitis, oral ulcers, chickenpox, herpes zoster, and flat warts; The products are selected from pharmaceuticals, food additives, food preservatives, and reagents.

7. A composition, characterized in that, Includes the polypeptide described in claim 1.

8. The composition according to claim 7, characterized in that, The composition is a pharmaceutical composition, a feed composition, or a food composition; The composition is a pharmaceutical composition, and the composition further comprises pharmaceutically acceptable excipients.

9. A reagent or kit, characterized in that, Includes the polypeptide described in claim 1.

10. A method for hydrolyzing bacteria, characterized in that, include: The sample contains bacteria and is contacted with the polypeptide of claim 1, the polypeptide prepared by the method of claim 3, the composition of claim 7 or 8, or the reagent or kit of claim 9.

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