Polypeptide and its use, method, composition or kit for preparing polypeptide

Abstract

The present application discloses a polypeptide and its use, a method for preparing the polypeptide, a composition or a kit. The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or 2; the method comprises: culturing the recombinant cell described in the fourth aspect to obtain the polypeptide. The polypeptide of the present application has lysozyme activity and thermal stability; the method of the present application can be produced on a large scale and in a standardized manner, and the obtained polypeptide has lysozyme activity and thermal stability that are superior to natural lysozyme; the composition or kit containing the above polypeptide can be used in medicine, food and industry.

Description

Technical Field

[0001] The present application belongs to the field of biotechnology, and specifically relates to polypeptides and uses thereof, methods, compositions or kits for preparing polypeptides, and more specifically to a lysozyme obtained by screening based on an artificial intelligence model and uses thereof. Background Art

[0002] Lysozyme, also known as muramidase or N-acetylmuramide glycanohydrlase, is an alkaline enzyme that can hydrolyze mucopolysaccharides in bacteria. Lysozyme mainly breaks down the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, decomposing the insoluble mucopolysaccharides in the cell wall into soluble glycopeptides, causing the cell wall to rupture and the contents to escape, thereby dissolving the bacteria. Lysozyme can also directly bind to negatively charged viral proteins, forming complexes with DNA, RNA, and apoproteins to inactivate the virus. The enzyme is widely present in many tissues of the human body, and is also found in the egg white of birds and poultry, the tears, saliva, plasma, milk and other fluids of mammals, and microorganisms, among which egg white is the most abundant.

[0003] As people pay more and more attention to health, "green antibiotics" such as lysozyme are quietly rising. As a natural, safe and effective antibacterial agent, lysozyme meets the modern consumers' pursuit of health and environmental protection. Its application in many fields also demonstrates the potential of lysozyme, such as:

[0004] 1. Application in medicine:

[0005] Antibacterial effect: Lysozyme can destroy the peptidoglycan structure in the bacterial cell wall, causing the bacterial cell wall to rupture and the contents to leak out, thus causing the bacteria to dissolve and die;

[0006] Antiviral effect: Lysozyme can bind to viral coat proteins, preventing viruses from adsorbing and invading host cells, thus exerting an antiviral effect;

[0007] Tissue repair: Lysozyme can remove the debris of damaged tissues and cells, creating a good environment for the growth and repair of new cells;

[0008] Clinical application: Lysozyme is used to treat chronic rhinitis, acute and chronic pharyngitis, oral ulcers, chickenpox, herpes zoster and flat warts, etc.

[0009] 2. Application in food:

[0010] Food preservation: Lysozyme can be used as a preservative. It hydrolyzes bacterial cell walls, destroys the β-1,4 glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in the cell wall, decomposes the insoluble mucopolysaccharide in the cell wall into soluble glycopeptides, causes the cell wall to rupture and the contents to escape, thus dissolving the bacteria.

[0011] Food preservation: Adding lysozyme to dairy products, meat products, cakes, sake, cooking wine and beverages can extend the shelf life of food;

[0012] 3. Industrial application:

[0013] Cell engineering: Lysozyme is an essential tool enzyme for cell fusion operations in genetic engineering and cell engineering;

[0014] Bioengineering: Lysozyme can be used to prepare cell extracts, increase the yield of extracts, and shorten the preparation time of yeast extracts.

[0015] Commercial lysozymes are mainly hen egg white lysozymes and microbial lysozymes. Hen egg white lysozymes are the most common lysozymes and are widely used because of their early development and relatively mature extraction process. Hen egg white lysozymes are extracted from hen egg white and eggshell membrane by affinity chromatography, ion exchange resin, direct crystallization or polyacrylic acid precipitation. However, existing lysozymes are isolated from natural products. Such lysozymes obtained from natural products have some defects, such as low yield and output, poor thermal stability, immunogenicity, complex preparation process and high cost, difficulty in large-scale purification, enzyme activity loss, and the risk of virus and harmful substance contamination. In particular, most natural lysozymes have poor thermal stability and cannot meet the process requirements of high-temperature feed granulation; and during the extraction and purification process, the activity of lysozymes may be lost, affecting the quality of the final product.

[0016] Therefore, it is urgent to develop a new type of lysozyme. Summary of the invention

[0017] The present application aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present application provides a polypeptide and its use as lysozyme.

[0018] In the first aspect of the present application, the present application provides a polypeptide. According to an embodiment of the present application, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or 2. The polypeptide of the present application has lysozyme activity and thermal stability, and can be mass-produced.

[0019] In the second aspect of the present application, the present application proposes a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the polypeptide described in the first aspect. The nucleic acid molecule of the present application can encode the polypeptide of the first aspect, which has lysozyme activity and thermal stability and can be mass-produced.

[0020] In the third aspect of the present application, the present application proposes an expression vector. According to an embodiment of the present application, the expression vector carries the nucleic acid molecule described in the second aspect. The expression vector of the present application carries the nucleic acid molecule described in the second aspect and can express the polypeptide of the first aspect, which has lysozyme activity and thermal stability and can achieve large-scale production.

[0021] In the fourth aspect of the present application, the present application proposes a recombinant cell. According to an embodiment of the present 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 the present application can express the polypeptide of the first aspect, which has lysozyme activity and thermal stability, and can be mass-produced.

[0022] In the fifth aspect of the present application, the present application proposes a method for preparing a polypeptide. According to an embodiment of the present application, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or 2; the method comprises: culturing the recombinant cell described in the fourth aspect to obtain the polypeptide. The method of the present application can be produced on a large scale and in a standardized manner, and the obtained polypeptide has lysozyme activity and thermal stability superior to natural lysozyme.

[0023] In the sixth aspect of the present application, the present application proposes the use of the polypeptide described in the first aspect as lysozyme.

[0024] In the seventh aspect of the present application, the present application proposes the use of the polypeptide described in the first aspect in preparing a product, wherein the product is used for at least one of antibacterial, antiviral, tissue repair, disease treatment, food preservation, food preservation, promotion of cell fusion, and preparation of cell extracts.

[0025] In the eighth aspect of the present application, the present application proposes a composition or a kit. According to an embodiment of the present application, the composition or the kit comprises the polypeptide described in the first aspect. As can be seen from the foregoing, the polypeptide of the first aspect has excellent lysozyme activity, which can be used as a medicine, a food additive, or a tool enzyme in bioengineering. Thus, the composition or the kit containing the above polypeptide can be used in medicine, food and industry.

[0026] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and / or additional aspects and advantages of the present application will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0028] Figure 1 A flowchart for protein design in the examples of this application;

[0029] Figure 2 This is the western blots band of lysozyme expression in the examples of this application. DETAILED DESCRIPTION

[0030] The embodiments of the present application are described in detail below. The embodiments described below are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0031] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0032] In this document, the terms "include" or "comprising" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0033] In this article, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with other ingredients comprising the polypeptide or its derivatives and / or the mammals treated therewith. Preferably, "pharmaceutically acceptable" as described in this application refers to those approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeias for use in animals, particularly humans.

[0034] In this article, the term "pharmaceutically acceptable excipient" may include any solvent, including but not limited to pharmaceutically acceptable stabilizers, diluents or other liquid excipients, etc., suitable for a specific target dosage form. In addition to any conventional excipients and the polypeptides, pharmaceutical compositions or drugs containing them in the present application, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, their use is also within the scope of consideration of the present application.

[0035] In addition to any conventional excipients, the use of excipients that are incompatible with the polypeptides, pharmaceutical compositions or drugs containing them of the present application, such as any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a harmful manner, is also within the scope of consideration of the present application.

[0036] As used herein, the term "treatment" refers to the use of drugs to obtain the desired pharmacological and / or physiological effects. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug of a polypeptide or a derivative thereof to an individual to treat, cure, alleviate, improve, mitigate or inhibit an individual's disease, including but not limited to administering a drug containing a polypeptide described herein to an individual in need.

[0037] The present application proposes a polypeptide and its use as lysozyme, which will be described in detail below.

[0038] Peptides

[0039] In the first aspect of the present application, the present application proposes a polypeptide. According to an embodiment of the present application, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or 2. The polypeptide of the present application is obtained by designing a lysozyme protein sequence different from a natural product through an artificial intelligence model and further screening, and has a lysozyme activity and thermal stability superior to that of natural lysozyme, can be used as a lysozyme, and can be mass-produced.

[0040] PWAEEAAARLGVAPELVLAHAALESGWGRQPLRDPATGQDTHNYFGIKAGGTWRGEVARAATTEYEDGVPQAQTAAFRSYPDGASAFRDYARLLSTQPGYQGALNAGHDARAFAQGLVRGGYATDPDYADKLVRVARQLRAAAALF (SEQ ID NO: 1);

[0041] MLAPRTVAGGVAGAAMWDQVAQCESSGDWQNGFHGGLQFTQSTWETAFGGLYAPSADKASRPDLASRSQQIAVAERVLAGQGPGAWPNCGGRLP (SEQ ID NO: 2).

[0042] It should be noted that in the present application, "the amino acid sequence is as shown in SEQ ID NO: A" includes the amino acid sequence of SEQ ID NO: A, or the amino acid sequence of conservative modification of SEQ ID NO: A, or the amino acid sequence having at least 90% identity with SEQ ID NO: A, all of which are within the scope of protection of the present application. Exemplarily, "the amino acid sequence of the polypeptide is 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 conservative modification of SEQ ID NO: 1, or the polypeptide has the amino acid sequence of at least 90% identity with SEQ ID NO: 1, all of which are within the scope of protection of the present application. Exemplarily, "the amino acid sequence of the polypeptide is as shown in SEQ ID NO: 2" means that the polypeptide has the amino acid sequence of SEQ ID NO: 2, or the polypeptide has the amino acid sequence of conservative modification of SEQ ID NO: 2, or the polypeptide has the amino acid sequence of at least 90% identity with SEQ ID NO: 2, all of which are within the scope of protection of the present application.

[0043] As used herein, "conservatively modified forms of an amino acid sequence" refers to amino acid modifications that do not significantly affect or alter the activity of the lysozyme comprising the amino acid sequence, including amino acid substitutions, additions and deletions. Modifications can be introduced into the polypeptides of the present application by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with 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, histidine), amino acids with acidic side chains (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (such as threonine, valine, isoleucine) and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, histidine). Exemplarily, conservative modifications are in a number not exceeding 20% ​​of the total number, preferably not exceeding 10% of the total number.

[0044] As used herein, the terms "identity", "homology" or "similarity" are used to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, and the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences is 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 program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48:443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85:2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70:173-187 (1997); and BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215:403-410). Computer programs that utilize these algorithms are also available, and include, but are not limited to, ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul et al. (1990) J. Mol. Biol. 215:403-410). et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLAST Altschul et al., supra, FASTA, and TFASTA, available in the Genetics Computing Group (GCG) package, Version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0045] In this article, the term "having at least 90% sequence similarity" can be a sequence having at least 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 identity described in the application can be measured using sequence analysis software. For example, the computer program BLAST, especially BLASTP or TBLASTN using default parameters is used. The amino acid sequences described in the application are all shown in a manner from N to C.

[0046] Nucleic acid molecules, expression vectors and recombinant cells

[0047] In the second aspect of the present application, the present application proposes a nucleic acid molecule. According to an embodiment of the present application, the nucleic acid molecule encodes the polypeptide described in the first aspect. The nucleic acid molecule of the present application can encode the polypeptide of the first aspect, and the polypeptide has lysozyme activity and thermal stability, especially lysozyme activity and thermal stability better than natural lysozyme, and can achieve large-scale production.

[0048] According to an embodiment of the present application, the nucleic acid molecule is DNA.

[0049] It should be noted that, for the nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or two of the complementary double strands. For convenience, in this article, although only one strand is provided in most cases, the other strand complementary thereto is actually disclosed. In addition, the nucleic acid molecule sequence in this application includes a DNA form or an RNA form, and disclosing one of them means that the other is also disclosed.

[0050] In the third aspect of the present application, the present application proposes an expression vector. According to an embodiment of the present application, the expression vector carries the nucleic acid molecule described in the second aspect. When the above-mentioned nucleic acid molecule is connected to the expression vector, the above-mentioned nucleic acid molecule can be directly or indirectly connected to the control element on the expression vector, as long as these control elements can control the translation and expression of the above-mentioned nucleic acid molecule. Of course, these control elements can come directly from the expression vector itself, or they can be exogenous, that is, not from the expression vector itself. Of course, the above-mentioned nucleic acid molecule can be operably connected to the control element.

[0051] Herein, "operably linked" means connecting the exogenous gene to the expression vector so that the control elements in the expression vector, such as transcription control sequences and translation control sequences, etc., can play their intended functions of regulating the transcription and translation of the exogenous gene. Commonly used expression vectors can be, for example, plasmids, bacteriophages, etc. After the expression vectors according to some specific embodiments of the present application are introduced into suitable recipient cells, the expression of the aforementioned polypeptide can be effectively achieved under the mediation of the regulatory system, thereby achieving large-scale in vitro acquisition of the polypeptide.

[0052] In some specific embodiments of the present application, the expression vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0053] In some specific embodiments of the present application, the expression vector is a lentiviral vector.

[0054] In an optional embodiment of the present application, the expression vector is a plasmid expression vector.

[0055] In the fourth aspect of the present application, the present application proposes a recombinant cell. According to an embodiment of the present 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 can be used to effectively express the aforementioned polypeptide in the recombinant cell under suitable conditions.

[0056] According to an embodiment of the present application, the recombinant cell is obtained by introducing the expression vector described in the fourth aspect into a host cell.

[0057] It should be noted that the host cell of the present 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 pombe, Trichoderma, insect cells such as armyworm, plant cells such as tobacco, mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells, etc.

[0058] According to an embodiment of the present application, the host cell is a eukaryotic cell.

[0059] According to an embodiment of the present 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.

[0060] It should be noted that the "suitable conditions" described in this application refer to conditions suitable for the expression of the polypeptides described in this application. It is easy for those skilled in the art to understand that conditions suitable for polypeptide expression include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell states, suitable cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the most suitable conditions for polypeptide expression according to the specific environment of the laboratory.

[0061] Method for preparing polypeptide

[0062] In the fifth aspect of the present application, the present application proposes a method for preparing a polypeptide. According to an embodiment of the present application, the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or 2; the method comprises: culturing the recombinant cell described in the fourth aspect to obtain the polypeptide. The method of the present application can be produced on a large scale and in a standardized manner, and the obtained polypeptide has lysozyme activity and thermal stability superior to natural lysozyme.

[0063] use

[0064] In the sixth aspect of the present application, the present application proposes the use of the polypeptide described in the first aspect as lysozyme. As can be seen from the foregoing, the polypeptide of the first aspect has excellent lysozyme activity and good thermal stability. Therefore, the above polypeptide can be used as lysozyme to prepare medicines, food additives, food preservatives, or tool enzymes in cell engineering / bioengineering, etc.

[0065] In the seventh aspect of the present application, the present application proposes the use of the polypeptide described in the first aspect 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 fresh-keeping, promotion of cell fusion, and preparation of cell extracts. As can be seen from the foregoing, the polypeptide of the first aspect has excellent lysozyme activity, and it can be used as a drug, food additive, or tool enzyme in cell engineering / bioengineering. Therefore, the product containing the above polypeptide can be used in medicine (for example, for antibacterial, antiviral, tissue repair, disease treatment), in food (for example, for food preservation, food fresh-keeping), and in industry (for example, for promoting cell fusion and preparing cell extracts).

[0066] According to an embodiment of the present application, the diseases include chronic rhinitis, acute and chronic pharyngitis, oral ulcers, chickenpox, herpes zoster, and flat warts.

[0067] According to an embodiment of the present application, the product is selected from medicines, food additives, food preservatives, and reagents.

[0068] According to an embodiment of the present application, the product is selected from medicines, and can be used for antibacterial, antiviral, tissue repair, and disease treatment.

[0069] According to an embodiment of the present application, the product is selected from food additives and food preservatives, and can be used for food preservation and food antiseptic.

[0070] According to an embodiment of the present application, the product is selected from a reagent, which can promote cell fusion, prepare cell extracts, and be applied to bioengineering, such as cell fusion in genetic engineering and cell engineering, or be used to prepare cell extracts, increase the yield of extracts, and shorten the preparation time of yeast extracts.

[0071] Composition or kit

[0072] In the eighth aspect of the present application, the present application proposes a composition or a kit. According to an embodiment of the present application, the composition or the kit comprises the polypeptide described in the first aspect. As can be seen from the foregoing, the polypeptide of the first aspect has excellent lysozyme activity, which can be used as a medicine, a food additive, or a tool enzyme in bioengineering. Thus, the composition or the kit containing the above polypeptide can be used in medicine, food and industry.

[0073] According to an embodiment of the present application, the composition is a pharmaceutical composition or a feed composition.

[0074] According to an embodiment of the present application, the composition is a pharmaceutical composition, and the composition further includes a pharmaceutically acceptable excipient.

[0075] The scheme of the present application will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If no specific technology or conditions are indicated in the embodiments, the technology or conditions described in the document in this area or the product specification are carried out. The reagents used or the instruments that do not indicate the manufacturer are all conventional products that can be obtained by commercial purchase.

[0076] Embodiment 1:

[0077] The schematic diagram of the polypeptide design and screening process of the present application is shown in FIG1 , which covers a series of steps from the generation of lysozyme protein sequence to screening optimization.

[0078] See also Figure 1The design and screening mainly involve generating lysozyme protein sequences through a protein generation model (PGM), screening out target (quality qualified) lysozyme protein sequences from the generated lysozyme protein sequences through a quality control pipeline (QCP), synthesizing protein encoding DNA based on the screened target lysozyme protein sequences, expressing them in a protein expression system, and expressing the lysozyme capacity and thermal stability of the protein through a wet-lab assay.

[0079] 1. Generation of lysozyme protein sequence

[0080] For example, a protein generative model is used to generate a lysozyme protein sequence with specific functions and structures through computational methods. The "protein generative model" is usually based on deep learning techniques such as diffusion models, language models and graph neural networks, which can handle complex molecular systems.

[0081] 2. Screening of target lysozyme protein sequences

[0082] Exemplarily, a quality control process screening is used to screen out target lysozyme protein sequences of qualified quality from the generated lysozyme protein sequences. The quality control process screening includes screening of the function domain, sequence identity, and structure identity of the lysozyme protein sequence, as well as screening using a unified sequence model (USM).

[0083] The “functional region” is used to indicate the protein function of the lysozyme protein sequence in the organism to which it belongs, for example, the protein function of the lysozyme protein sequence in at least one description dimension of cellular elements, biological processes, and molecular functions.

[0084] "Sequence signature" is used to indicate the amino acid sequence included in the lysozyme protein sequence.

[0085] The "structural feature" is used to indicate the geometric structure of the lysozyme protein sequence in three-dimensional space, for example, at least one level category of the lysozyme protein sequence in a class, fold, superfamily, or family in structural classification.

[0086] The "unified sequence model" is a model that is compatible with the functions of the single sequence model and the multiple sequence alignment (MSA) model. The unified sequence model is not only a universal biological language model that can be applied to the screening task of lysozyme protein sequences, but also a universal multi-sequence model that can support both single sequence and multi-sequence alignment data, unifying single sequence and multi-sequence modes (for example: the corresponding processing of molecular formula data and amino acid sequence data is compatible), and is universal.

[0087] 3. Synthesis of target lysozyme sequence

[0088] 1) Plasmid construction

[0089] Genes for synthesizing the target proteins (lysozyme 1 with amino acid sequence as shown in SEQ ID NO:1, lysozyme 2 with amino acid sequence as shown in SEQ ID NO:2) were amplified by homologous recombination. The target genes were inserted into the corresponding pJL1 expression vectors by restriction digestion and ligation using Hieff Clone™ One Step Cloning Kit. The expression vectors were sequenced to confirm the correct insertion of the target genes. The expression vectors with the target genes were transformed into DH5α competent cells. Single clones were selected for vector amplification. Plasmids were extracted using a kit similar to the PureStrand HQ 96 Plasmid DNA Purification Kit (ThermoCat# K210096).

[0090] 2) Cell-free protein synthesis

[0091] The plasmid extracted in step 1) was added to the EP tube containing the cell-free expression reaction system. The reaction was maintained at 30°C for 3 hours in the EP tube with an orbital shaker at an appropriate stirring speed.

[0092] 3) Protein purification

[0093] 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.

[0094] Protein purification: The target protein is extracted from the CFPS supernatant by affinity chromatography or a combination of multiple chromatography methods. After each purification step, the crude sample is buffer exchanged and then subjected to the next purification step. The final purified product is exchanged into the storage buffer.

[0095] Protein quality assessment: Purity and content of crude samples after each purification step as well as the final protein product were assessed using methods such as SDS-PAGE and UV spectroscopy (OD280).

[0096] The results of western blots for the expressed lysozyme can be found in Figure 2 ; The triangle mark is the lysozyme protein band, the one circled in the blue box is the protein expression band of lysozyme 1 (the amino acid sequence is shown in SEQ ID NO: 1), and the one circled in the yellow box is the protein expression band of lysozyme 2 (the amino acid sequence is shown in SEQ ID NO: 2).

[0097] 4. Detection of lysozyme capacity and thermal stability

[0098] 4.1 Lysozyme activity detection:

[0099] Each enzyme to be tested (lysozyme obtained in step 3 of this example) was tested using a lysozyme activity detection kit (ThermosFisher EnzChek TM The lysozyme activity was tested in reaction systems with different pH values ​​and at 37°C using the Lysozyme Assy Kit, and compared with commercially available chicken lysozyme under the same test conditions. The results are shown in Table 1. The results showed that the lysozyme activity of lysozyme 2 was better than that of chicken lysozyme under all pH conditions, and the lysozyme activity of lysozyme 1 was better than that of chicken lysozyme under neutral and acidic conditions (pH <= 7.0).

[0100] Table 1:

[0101]

[0102] In Table 1, the lysozyme activity U is defined by the EnzChek™ Lysozyme Assay Kit. The components of the assay buffer are: 80 mM NaCl, 0.1% Tween20, PB / Tris Buffer. NA means no activity was detected in the reaction system.

[0103] 4.1 Study on the thermal stability of lysozyme

[0104] Each enzyme to be tested was placed in a 4°C refrigerator and in a metal bath at the temperature to be tested (such as 80°C, 90°C, etc.) for incubation, and then the enzyme solution was transferred to a 4°C refrigerator and allowed to stand for cooling for 20 minutes. According to the enzyme activity detection method, the residual enzyme activity of different treatments was detected. The enzyme activity of the enzyme treated by standing in a 4°C refrigerator was 100%, and the remaining percentage of the enzyme activity treated by incubation in a metal bath was calculated. Under the same test conditions, it was compared with commercially available chicken lysozyme, where chicken lysozyme is a lysozyme with very good thermal stability, and the critical point of thermal denaturation is 77°C. Below this temperature, the thermal denaturation of chicken lysozyme is reversible. The results are shown in Tables 2 and 3, where the values ​​in Table 2 are the percentage of proteins with lysozyme activity in the total protein amount after heating at 80°C for 1-3 hours; the values ​​in Table 3 are the percentage of proteins with lysozyme activity in the total protein amount after heating at 90°C for 1-3 hours.

[0105] The results showed that the thermal stability of lysozyme 1 and lysozyme 2 was better than that of chicken lysozyme after heating at 80℃ and 90℃ for different time periods.

[0106] Table 2:

[0107]

[0108] Table 3:

[0109]

[0110] In summary, in different acidic and alkaline environments, the lysozyme activity and thermal stability of the two lysozymes of the present application are better than those of chicken lysozyme.

[0111] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0112] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or 2.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the polypeptide of claim 1.

3. An expression vector, characterized in that: The expression vector carries the nucleic acid molecule of claim 2.

4. A recombinant cell, characterized in that The recombinant cell carries the nucleic acid molecule of claim 2 or the expression vector of claim 3, or The recombinant cell expresses the polypeptide of claim 1.

5. A method for preparing a polypeptide, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1 or 2; The method comprises: The recombinant cell according to claim 4 is cultured to obtain the polypeptide.

6. Use of the polypeptide according to claim 1 in preparing lysozyme.

7. Use of the polypeptide according to claim 1 in preparing a product, wherein the product is used for at least one of antibacterial, antiviral, disease treatment, food preservation, food freshness preservation, cell fusion promotion, and preparation of cell extracts.

8. The use according to claim 7, characterized in that The product is used to perform tissue repair.

9. The use according to claim 7, characterized in that: The use includes one or more of the following conditions: The diseases include chronic rhinitis, acute and chronic pharyngitis, oral ulcers, chickenpox, herpes zoster, and flat warts; The product is selected from medicines and reagents.

10. The use according to claim 7, characterized in that The product is selected from food additives and food preservatives.

11. A composition or a kit, characterized in that: Comprising the polypeptide of claim 1.

12. The composition or kit according to claim 11, characterized in that The composition is a pharmaceutical composition or a feed composition.

13. The composition or kit according to claim 11, characterized in that The composition is a pharmaceutical composition, and the composition further comprises a pharmaceutically acceptable excipient.

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