Bacterial lyases and uses thereof

Abstract

The present invention provides an isolated bacterial lyase polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 4 or 5. The present invention also provides a polynucleotide encoding the polypeptide, a vector or a host cell comprising the polynucleotide, and a method of treating a disease associated with a bacterial infection, such as a Klebsiella pneumoniae (Klebsiella pneumoniae), with the polypeptide.

Description

Technical Field

[0001] The present invention relates to the fields of microbiology and molecular biology. In particular, the present invention relates to bacterial lytic enzymes from bacteriophages and their use in treating diseases associated with Klebsiella pneumoniae infection. Background Art

[0002] Bacteriophage is a bacterial virus that is widely found in places where bacteria gather, such as soil, oceans, rivers, and animal feces. Bacteriophages are composed of protein capsids and nucleic acids (DNA or RNA) and are about 20-200nm in length. Bacteriophages bind to hosts by recognizing specific receptors on the surface of bacteria and have strict specificity.

[0003] Due to the abuse of antibiotics, multi-drug resistant "super bacteria" continue to emerge. With the continuous emergence of multi-drug resistant bacteria, the treatment methods have gradually evolved from antibiotic therapy to phage-related therapy or phage-antibiotic combination therapy.

[0004] The phage genome encodes bacterial lytic enzymes. After the phage reproduces in the host bacteria, the bacterial lytic enzymes expressed by the phage can cause the host bacteria to lyse and release the phage therein. The bacterial lytic enzymes from the phage can also inhibit the growth of bacteria, thereby treating diseases related to bacterial infection.

[0005] Therefore, there remains a need to identify new bacterial lytic enzymes from bacteriophages, preferably bacterial lytic enzymes with improved ability to lyse bacteria and / or inhibit bacterial growth. Summary of the invention

[0006] In a first aspect, the present invention provides an isolated bacterial lytic enzyme polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 4 or 5.

[0007] In some embodiments, the polypeptide consists of the amino acid sequence of SEQ ID NO: 1, 4 or 5.

[0008] In a second aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of the first aspect of the present invention.

[0009] In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2, 3, 7, or 8. In some embodiments, the polynucleotide consists of the nucleotide sequence of SEQ ID NO: 2, 3, 7, or 8.

[0010] The present invention also provides a vector, preferably an expression vector, comprising the polynucleotide of the present invention.

[0011] The present invention also provides a recombinant cell, preferably a prokaryotic cell, comprising the polynucleotide or expression vector of the present invention.

[0012] In a third aspect, the present invention provides use of the polypeptide of the present invention in the preparation of a medicament for treating diseases associated with infection by bacteria such as Klebsiella pneumoniae and Escherichia coli.

[0013] In some embodiments, the Klebsiella pneumoniae and Escherichia coli are drug-resistant bacteria.

[0014] In some embodiments, the disease is selected from pneumonia, mastitis, metritis, meningitis and sepsis caused by Klebsiella pneumoniae. In some embodiments, the disease is selected from inflammation caused by Escherichia coli, such as enteritis and gastritis, and urogenital tract infections such as vaginitis and urethritis.

[0015] The present invention also provides a pharmaceutical composition comprising the polypeptide of the present invention and a pharmaceutically acceptable excipient. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The plaques of the isolated phages on Klebsiella pneumoniae KPP strain are shown.

[0017] Figure 2 Agarose gel electrophoresis showing the products of PCR amplification of nucleic acid encoding the recombinant bacterial lytic enzyme.

[0018] Figure 3 SDS-PAGE and Coomassie blue staining of recombinantly expressed bacterial lytic enzymes are shown.

[0019] Figure 4 Showing the inhibition of E. coli by recombinantly expressed bacterial lytic enzymes.

[0020] Figure 5 Showing inhibition of Klebsiella pneumoniae by recombinantly expressed bacterial lytic enzymes. DETAILED DESCRIPTION OF THE INVENTION

[0022] Although various embodiments and aspects of the present invention are shown and described herein, it is obvious to those skilled in the art that these embodiments and aspects are provided only by way of example. Without departing from the present invention, those skilled in the art will appreciate that many modifications, changes and substitutions are possible. It should be understood that the various alternatives of the embodiments of the present invention described herein may be used to implement the present invention.

[0023] I. Definition

[0024] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art. See, for example, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices and materials similar to or equivalent to those described herein can be used to implement the present invention. The following definitions are provided to facilitate the understanding of certain terms frequently used herein and are not intended to limit the scope of the present disclosure.

[0025] As used herein, the term "bacteriophage" refers to a virus that uses bacteria as a host and replicates in it. The large-scale reproduction of bacteriophages in bacterial cells will eventually lead to bacterial cell lysis, thereby killing the bacterial cells. Bacteriophages parasitize in living bacteria and have strict host specificity, which depends on the molecular structure and complementarity of the phage adsorption structure and the receptor on the surface of the recipient bacteria. Phages are the most common and widely distributed group of viruses, and are usually found in some environments where bacterial communities exist.

[0026] Bacterial "lytic enzymes" expressed by phages are proteins encoded by the phage genome with cell wall hydrolase activity, which can hydrolyze peptidoglycan in the cell wall, leading to the rupture of bacteria. Bacterial lytic enzyme polypeptides usually contain a LysM domain.

[0027] The "LysM domain" was first discovered in Bacillus phage lysozyme, which degrades the cell wall by degrading the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine. The typical LysM domain consists of 44 amino acid residues, of which the 16 amino acid residues at the N-terminus and the 10 amino acid residues at the C-terminus are relatively conservative, while the region between the two is less conservative. Except for the leucine / isoleucine at the 23rd and 30th amino acid positions and the asparagine at the 27th amino acid position, the amino acids at the remaining positions have relatively large changes (see Jiang et al., Chinese Bulletin of Botany, 2014, 49(2): 221–228).

[0028] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0029] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids, i.e., an alpha carbon bound to a hydrogen, carboxyl, amino, and R groups, such as homoserine, norleucine, methionine sulfoxide, methylsulfonium methionine. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of an amino acid but function in a manner similar to a natural amino acid.

[0030] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0031] "Conservatively modified variants" apply to amino acids and nucleic acid sequences. With respect to a particular nucleotide sequence, a conservatively modified variant refers to a nucleic acid encoding the same or substantially identical amino acid sequence, for example, by changing nucleotides in a region that does not encode the amino acid sequence. In addition, due to the degeneracy of the genetic code, a given protein can be encoded by a large number of functionally identical nucleic acids. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Those skilled in the art will recognize that individual codons in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan)) can be modified to produce functionally identical molecules.

[0032] Conservatively modified variants of nucleotide sequences also include nucleotide sequences that encode conservatively modified variants of the polypeptide encoded by the starting nucleotide sequence.

[0033] With respect to amino acid sequences, "conservatively modified variants" include peptides, polypeptides or proteins in which certain amino acid(s) are altered by replacing them with functionally / chemically similar amino acids (ie, conservative substitutions).

[0034] The term "conservative substitution", also known as substitution by a "homologous" amino acid residue, refers to substitutions in which the amino acid residue is replaced by an amino acid residue having a similar side chain, for example, amino acids with basic side chains (e.g., lysine, arginine and histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), non-charged polar side chain amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chain amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chain amino acids (e.g., threonine, valine, isoleucine) and aromatic side chain amino acids (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0035] Conservative amino acid substitutions generally have the least effect on the activity of the resulting protein. Such substitutions are described below. Conservative substitutions are replacements of an amino acid with an amino acid that is similar in size, hydrophobicity, charge, polarity, steric characteristics, aromaticity, etc. Such substitutions are generally conservative when it is desired to fine-tune the properties of a protein.

[0036] As used herein, "homologous" amino acid residues refer to amino acid residues with similar chemical properties, such as hydrophobicity, charge, polarity, steric characteristics, aromatic characteristics, etc. Examples of amino acids homologous to each other include positively charged lysine, arginine, histidine, negatively charged glutamic acid, aspartic acid, hydrophobic glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, polar serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, glutamine, aromatic phenylalanine, tyrosine, tryptophan, serine and threonine with chemically similar side chain groups, or glutamine and asparagine, or leucine and isoleucine.

[0037] Examples of conservative substitutions of amino acids in proteins include: Ser for Ala, Lys for Arg, Gln or His for Asn, Glu for Asp, Ser for Cys, Asn for Gln, Asp for Glu, Pro for Gly, Asn or Gln for His, Leu or Val for Ile, Ile or Val for Leu, Arg or Gln for Lys, Leu or Ile for Met, Met, Leu or Tyr for Phe, Thr for Ser, Ser for Thr, Tyr for Trp, Trp or Phe for Tyr, and Ile or Leu for Val.

[0038] "Nucleic acid" refers to a polymer of nucleotides (e.g., deoxyribonucleotides or ribonucleotides) in single-stranded, double-stranded or multi-stranded form. In the usual and customary sense, the terms "polynucleotide", "oligonucleotide", "oligonucleotide" and the like refer to a linear sequence of nucleotides. The length of an oligonucleotide is generally about 5, 6, 7, 8, 9, 10, 12, 15, 25, 30, 40, 50 or more nucleotides, up to about 100 nucleotides in length. A polynucleotide is a polymer of any length, including longer lengths, such as 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10000, 20000, 30000, 40000 and the like. Polynucleotides and oligonucleotides will typically contain phosphodiester bonds, although in some cases, including nucleic acid analogs, they may have alternative main chains, including, for example, phosphoramidate, phosphorothioate, phosphorodithioate or O-methylphosphoramidite bonds (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); and peptide nucleic acid main chains and bonds. Other analog nucleic acids include nucleic acids with positive main chains; non-ionic main chains and non-ribose main chains, including those described in U.S. Patents 5,235,033 and 5,034,506 and ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, eds Chapters 6 and 7. Nucleic acids containing one or more carbocyclic sugars are also included in a definition of nucleic acids. Modification of the ribose-phosphate main chain can be performed for a variety of reasons, for example, to increase the stability and half-life of such molecules in physiological environments or as probes on biochips. Mixtures of naturally occurring nucleic acids and analogs can be prepared. Alternatively, mixtures of different nucleic acid analogs can be prepared, as well as mixtures of naturally occurring nucleic acids and analogs.

[0039] The term "identity" or "sequence identity" is determined by comparing two optimally aligned sequences in a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (excluding additions or deletions) to achieve optimal alignment of the two sequences. Identity is usually expressed as a percentage. For example, the number of positions at which the same nucleic acid base or amino acid residue appears in the two sequences is determined to produce the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window sequence, and the result is multiplied by 100 to calculate the percentage to obtain the identity of the sequence.

[0040] In order to compare sequences, a sequence is usually used as a reference sequence to compare it with a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Preferably, default program parameters can be used, or alternative parameters can be specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence according to the program parameters.

[0041] As used herein, a "comparison window" includes a segment with reference to any one of a plurality of consecutive positions, wherein after the two sequences are optimally aligned, a sequence can be compared to a reference sequence of the same number of consecutive positions. For example, the comparison window includes about or at least about 20, 50, 100, 1000, 2500, 5000, 7500, 10000, 15000, 20000, 25000, 30000 or more consecutive positions.

[0042] Methods for sequence alignment are well known in the art, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0043] An example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. Those skilled in the art will appreciate that software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI).

[0044] As used herein, the term "coding" refers to a polynucleotide that directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or other start codons such as GTG and TTG and ends with stop codons such as TAA, TAG and TGA. The coding sequence can be a DNA, cDNA or recombinant nucleotide sequence.

[0045] As used herein, the term "hybridization" means that nucleotide sequences at least about 90%, preferably at least about 95%, more preferably at least about 96%, and more preferably at least 98% homologous to each other typically remain hybridized to each other under given stringent hybridization and washing conditions.

[0046] Those skilled in the art are aware of various conditions for hybridization, such as stringent hybridization conditions and highly stringent hybridization conditions. See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, John Wiley & Sons, NY.

[0047] As used herein, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0048] The term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or is modified to contain a naturally occurring nucleic acid segment. When the nucleic acid construct contains the control sequences required for expressing the coding sequence of the present invention, the term nucleic acid construct is synonymous with the term "expression cassette".

[0049] The term "expression vector" herein refers to a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide of the present invention, the polynucleotide being operably linked to additional nucleotides, e.g., control sequences, that provide for the expression of the polynucleotide. The expression vector includes a viral vector or a plasmid vector.

[0050] The term "control sequence" as used herein refers to all elements that are necessary or advantageous for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, a control sequence includes a promoter and transcription and translation stop signals.

[0051] For example, the control sequence can be a suitable promoter sequence, a nucleotide sequence that is recognized by a host cell to express a polynucleotide encoding a polypeptide of the present invention. The promoter sequence contains a transcriptional control sequence that mediates the expression of the polypeptide. The promoter can be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, for example, the lac operon of Escherichia coli. The promoter also includes mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0052] The term "operably linked" herein refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide sequence such that the control sequence directs the expression of the polypeptide coding sequence.

[0053] As used herein, the terms "transformation" and "transfection" refer to various art-recognized techniques for introducing exogenous nucleic acid (e.g., DNA) into host cells, as found in, for example, Sambrook et al., 1989, Davis et al., Basic Methods in Molecular Biology (1986), supra, and other laboratory manuals.

[0054] As used herein, "pharmaceutically acceptable excipients" and "pharmaceutically acceptable carriers" refer to substances that facilitate administration of active agents to subjects and absorption thereof, and can be included in the compositions of the present invention without causing significant adverse toxicological effects to patients. Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, water, physiological saline solutions, lactated Ringer's solution, standard sucrose, standard glucose, binders, fillers, disintegrants, lubricants, coatings, flavoring agents, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates (such as lactose, amylose or starch), fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and pigments, etc. These preparations can be sterilized and, if necessary, can be mixed with adjuvants that do not react adversely with bacteriophages, such as preservatives, stabilizers, wetting agents, emulsifiers, osmotic pressure regulators, buffers, colorants, and / or aromatic substances, etc.

[0055] "Patient" or "subject in need thereof" refers to a human or non-human animal, such as a non-human mammal, who has or may have the indicated condition. Non-limiting examples of mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bush babies, monkeys, and apes), rabbits, dogs (e.g., companion dogs, service dogs, or working dogs), horses (e.g., race horses and working horses), cats (e.g., domestic cats), livestock (e.g., pigs, cattle, donkeys, mules, bison, goats, camels, and sheep), and deer.

[0056] The terms "subject," "patient," "individual," and the like are not intended to be limiting and are generally interchangeable. That is, an individual described as a "patient" does not necessarily have a disease but may simply be seeking medical advice.

[0057] As used herein, "treating" a condition, disease, or disorder, or symptoms associated with a condition, disease, or disorder refers to methods for obtaining beneficial or desired results, including clinical results. Beneficial or desirable clinical results may include, but are not limited to, alleviating or ameliorating one or more symptoms or conditions, reducing the extent of a condition, disorder, or disease, stabilizing the state of a condition, disease, or disorder, preventing the development of a condition, disease, or disorder, preventing the spread of a condition, disease, or disorder, delaying or slowing the progression of a condition, disease, or disorder, delaying or slowing the onset of a condition, disease, or disorder, alleviating or relieving the condition, disease, or disorder state, and eliminating the condition, disease, or disorder state, whether partially or completely. "Treatment" may also mean temporarily inhibiting the progression of a condition, disease, or disorder, temporarily slowing the progression of a condition, disease, or disorder, although in some cases, it involves permanently stopping the progression of a condition, disease, or disorder.

[0058] As used herein, the terms "treat" and "prevent" are not intended to be absolute terms. It should be understood that treatment does not necessarily refer to a cure or complete elimination of a disease, condition, or symptoms of a disease or condition. Treatment may refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, condition, or symptom of a disease or condition.

[0059] As used herein, the term "therapeutically effective amount" and the like refers to an amount of a drug / composition sufficient to alleviate a condition. For example, administration of a therapeutically effective amount of a drug will increase or decrease a given parameter by at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.

[0060] In this article, the "activity" of the bacterial lytic enzyme can be characterized by the ability of the bacterial lytic enzyme to inhibit bacterial growth or kill bacteria. For example, it can be calculated by detecting the OD value or CFU of the bacteria after incubating the lytic enzyme with the bacteria for a period of time, wherein a solvent (a solvent that dissolves the lytic enzyme but does not contain the lytic enzyme) can be used as a negative control to incubate the bacteria for the same time.

[0061] 2. Bacterial Lytic Enzyme Peptides

[0062] The present invention provides an isolated bacterial lytic enzyme polypeptide, which can hydrolyze cell walls, thereby inhibiting bacterial growth, causing bacterial cell rupture or killing bacteria.

[0063] In some embodiments, the bacterial lytic enzyme polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1.

[0064] In some embodiments, the polypeptide has one or more amino acid substitutions, deletions, insertions and / or additions compared to SEQ ID NO: 1. In some embodiments, the substituted, deleted and / or inserted amino acids are outside the LysM domain. In some embodiments, the polypeptide has one or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 1. In some embodiments, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 1. In some embodiments, the substituted amino acids are outside the LysM domain.

[0065] In some embodiments, the polypeptide comprises or consists of an amino acid sequence that is at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, wherein the bacterial lytic enzyme activity of the polypeptide is SEQ ID NO: 1. At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of NO:1.

[0066] In some embodiments, the polypeptide has one or more amino acid substitutions, deletions, insertions and / or additions compared to SEQ ID NO: 1, wherein the bacterial lytic enzyme activity of the polypeptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 1. In some embodiments, the substituted, deleted and / or inserted amino acids are outside the LysM domain. In some embodiments, the polypeptide has one or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 1, wherein the bacterial lytic enzyme activity of the polypeptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 1. In some embodiments, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 1, wherein the bacterial lytic enzyme activity of the polypeptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 1. In some embodiments, the substituted amino acid is outside the LysM domain.

[0067] For example, the inventors found that replacing the aspartic acid residue in SEQ ID NO: 1 with a lysine residue had no significant effect on the activity of the bacterial lytic enzyme.

[0068] Accordingly, in some embodiments, the bacterial lytic enzyme polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4.

[0069] In some embodiments, the polypeptide has one or more amino acid substitutions, deletions, insertions and / or additions compared to SEQ ID NO: 4. In some embodiments, the substituted, deleted and / or inserted amino acids are outside the LysM domain. In some embodiments, the polypeptide has one or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 4. In some embodiments, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 4. In some embodiments, the substituted amino acids are outside the LysM domain.

[0070] In some embodiments, the polypeptide comprises or consists of an amino acid sequence that is at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:4, wherein the bacterial lytic enzyme activity of the polypeptide is SEQ ID NO:4. At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of NO:4.

[0071] In some embodiments, the polypeptide has one or more amino acid substitutions, deletions, insertions and / or additions compared to SEQ ID NO: 4, wherein the bacterial lytic enzyme activity of the polypeptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 4. In some embodiments, the substituted, deleted and / or inserted amino acids are outside the LysM domain. In some embodiments, the polypeptide has one or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 4, wherein the bacterial lytic enzyme activity of the polypeptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 4. In some embodiments, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 4, wherein the bacterial lytic enzyme activity of the polypeptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 4. In some embodiments, the substituted amino acid is outside the LysM domain.

[0072] Those skilled in the art know that for the expression and purification of proteins, the polypeptide to be expressed can be connected to a tag, such as a 6*His tag, without affecting the function of the protein. Those skilled in the art also know that the tag can be removed after purification, and the removal of the tag will also result in the removal of the N-terminal residue M1.

[0073] The bacterial lytic enzyme polypeptide of the present invention may also be a fusion protein comprising a bacterial lytic enzyme.

[0074] In some embodiments, the fusion protein further comprises a polypeptide having the activity of penetrating the outer membrane of the cell wall. In some embodiments, the polypeptide having the activity of penetrating the outer membrane of the cell wall is fused to the N-terminus of the bacterial lytic enzyme polypeptide.

[0075] In some embodiments, the fusion protein further comprises an additional antimicrobial peptide. In some embodiments, the additional antimicrobial peptide is fused to the N-terminus of the bacterial lytic enzyme of the present invention. For example, the bacterial lytic enzyme of the present invention can be fused to the Cecropin A (CerA) polypeptide (SEQ ID NO: 6). Therefore, in some embodiments, the bacterial lytic enzyme polypeptide is a fusion protein of the bacterial lytic enzyme of the present invention and the CerA polypeptide.

[0076] In some embodiments, the polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:5, or an amino acid sequence that is at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5.

[0077] In some embodiments, the polypeptide has one or more amino acid substitutions, deletions, insertions and / or additions compared to SEQ ID NO: 5. In some embodiments, the substituted, deleted and / or inserted amino acids are outside the LysM domain. In some embodiments, the polypeptide has one or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 5. In some embodiments, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 5. In some embodiments, the substituted amino acids are outside the LysM domain.

[0078] In some embodiments, the polypeptide comprises or consists of an amino acid sequence that is at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5, wherein the bacterial lytic enzyme activity of the polypeptide is SEQ ID NO:5. At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of NO:5.

[0079] In some embodiments, the polypeptide has one or more amino acid substitutions, deletions, insertions and / or additions compared to SEQ ID NO: 5, wherein the bacterial lytic enzyme activity of the polypeptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 5. In some embodiments, the substituted, deleted and / or inserted amino acids are outside the LysM domain. In some embodiments, the polypeptide has one or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO:5, wherein the bacterial lytic enzyme activity of the polypeptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO:5. In some embodiments, the polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 5, wherein the bacterial lytic enzyme activity of the polypeptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 5. In some embodiments, the substituted amino acid is outside the LysM domain.

[0080] 3. Polynucleotides encoding lysozyme polypeptides and expression of the polypeptides

[0081] The present invention also provides a polynucleotide encoding the bacterial lytic enzyme polypeptide of the present invention.

[0082] In some embodiments, the polypeptide encoded by the polynucleotide comprises or consists of the amino acid sequence of SEQ ID NO:1, 4 or 5, or an amino acid sequence that is at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:1, 4 or 5.

[0083] In some embodiments, the polypeptide encoded by the polynucleotide has one or more amino acid substitutions, deletions, insertions and / or additions compared to SEQ ID NO: 1, 4 or 5. In some embodiments, the substituted, deleted and / or inserted amino acids are outside the LysM domain. In some embodiments, the polypeptide encoded by the polynucleotide has one or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 1, 4 or 5. In some embodiments, the polypeptide encoded by the polynucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 1, 4 or 5. In some embodiments, the substituted amino acids are outside the LysM domain.

[0084] In some embodiments, the polypeptide encoded by the polynucleotide comprises or consists of an amino acid sequence that is at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, 4 or 5, wherein the bacterial lytic enzyme activity of the polypeptide is SEQ ID NO: 1, 4 or 5. At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of NO: 1, 4 or 5.

[0085] In some embodiments, the polypeptide encoded by the polynucleotide has one or more amino acid substitutions, deletions, insertions and / or additions compared to SEQ ID NO: 1, 4 or 5, wherein the bacterial lytic enzyme activity of the polypeptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 1, 4 or 5. In some embodiments, the substituted, deleted and / or inserted amino acids are outside the LysM domain. In some embodiments, the polypeptide encoded by the polynucleotide has one or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 1, 4 or 5, wherein the bacterial lytic enzyme activity of the polypeptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 1, 4 or 5. In some embodiments, the polypeptide encoded by the polynucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 1, 4 or 5, wherein the bacterial lytic enzyme activity of the polypeptide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 1, 4 or 5. In some embodiments, the substituted amino acid is outside the LysM domain.

[0086] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding a tag (eg, a 6*His tag).

[0087] In some embodiments, the polynucleotide comprises or consists of the nucleotide sequence of SEQ ID NO: 2, 3, 7 or 8, or a nucleotide sequence that is at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2, 3, 7 or 8.

[0088] In some embodiments, the polynucleotide has one or more nucleotide substitutions, deletions, insertions and / or additions compared to SEQ ID NO: 2, 3, 7 or 8. In some embodiments, the substituted, deleted and / or inserted nucleotides are outside the region encoding the LysM domain. In some embodiments, the polynucleotide has one or more nucleotide substitutions compared to SEQ ID NO: 2, 3, 7 or 8. In some embodiments, the polynucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide substitutions compared to SEQ ID NO: 2, 3, 7 or 8. In some embodiments, the substituted nucleotides are outside the region encoding the LysM domain.

[0089] In some embodiments, the polypeptide encoded by the polynucleotide comprises conservative substitutions of amino acids compared to SEQ ID NO: 1, 4 or 5.

[0090] In some embodiments, the polynucleotide comprises or consists of a nucleotide sequence that is at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85% or 90%, particularly preferably at least 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 2, 3, 7 or 8, wherein the bacterial lytic enzyme activity of the polypeptide encoded by the polynucleotide is SEQ ID NO: 2, 3, 7 or 8. At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of NO: 1, 4 or 5.

[0091] In some embodiments, the polynucleotide has one or more nucleotide substitutions, deletions, insertions and / or additions compared to SEQ ID NO: 2, 3, 7 or 8, wherein the bacterial lytic enzyme activity of the polypeptide encoded by the polynucleotide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 1, 4 or 5. In some embodiments, the substituted, deleted and / or inserted nucleotides are outside the region encoding the LysM domain. In some embodiments, the polynucleotide has one or more nucleotide substitutions compared to SEQ ID NO: 2, 3, 7 or 8, wherein the bacterial lytic enzyme activity of the polypeptide encoded by the polynucleotide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 1, 4 or 5. In some embodiments, the polynucleotide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid substitutions (e.g., conservative substitutions) compared to SEQ ID NO: 2, 3, 7 or 8, wherein the bacterial lytic enzyme activity of the polypeptide encoded by the polynucleotide is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200% or more of the activity of SEQ ID NO: 1, 4 or 5. In some embodiments, the substituted nucleotides are outside the region encoding the LysM domain.

[0092] In some embodiments, the polynucleotide is a degenerate variant of SEQ ID NO: 2, 3, 7, or 8.

[0093] It should be noted that SEQ ID NO: 7 and 8 do not contain a stop codon. Therefore, embodiments involving sequences consisting of these two sequences also encompass technical solutions in which a stop codon is added to the 3' end of SEQ ID NO: 7 and 8.

[0094] Nucleic acid molecules encompassing all or part of the nucleic acid sequences of the invention can be isolated by polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based on the sequence information contained in the sequences.

[0095] The polynucleotides of the present invention can be amplified using cDNA, mRNA or genomic DNA as templates and suitable oligonucleotide primers according to standard PCR amplification techniques. The nucleic acids amplified in this way can be cloned into suitable vectors and characterized by DNA sequence analysis.

[0096] The polynucleotides of the present invention can be prepared by standard synthetic techniques, for example, using an automated DNA synthesizer.

[0097] The present invention also relates to complementary strands of the nucleic acid molecules described herein.A nucleic acid molecule that is complementary to another nucleotide sequence is a molecule that is sufficiently complementary to that nucleotide sequence such that it can hybridize to the other nucleotide sequence to form a stable duplex.

[0098] To express the bacterial lytic enzyme polypeptides of the present invention, nucleic acid constructs and vectors, such as expression vectors, comprising the polynucleotides of the present invention are also provided.

[0099] The polynucleotide encoding the polypeptide of the present invention may be subjected to various manipulations to allow the polypeptide to be expressed. Manipulation of the polynucleotide may be desirable or necessary depending on the expression vector prior to its insertion into the vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

[0100] In order to identify and select host cells containing the expression vectors of the present invention, the vectors of the present invention preferably contain one or more selectable markers, which allow for simple selection of cells that have been transformed, transfected, transduced, etc. A selectable marker is a gene whose product provides biocide or viral resistance, heavy metal resistance, complementation of auxotrophic deficiencies, etc. For example, bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance.

[0101] The vector of the present invention can be integrated into the host cell genome or replicate autonomously in the cell independently of the genome. The elements required for integration into the host cell genome or autonomous replication are known in the art (see, for example, Sambrook et al., 1989, supra).

[0102] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques.

[0103] The present invention also relates to a recombinant host cell, preferably a prokaryotic host cell, comprising a polynucleotide of the present invention, which is advantageously used in the recombinant production of the bacterial lytic enzyme polypeptide. A vector comprising a polynucleotide of the present invention is introduced into a host cell, whereby the vector is retained as a chromosomal integrant or as a self-replicating extrachromosomal vector. Conventional vectors and host cells for expressing proteins are known to those skilled in the art.

[0104] In some embodiments, the host cell of the present invention is a bacterial cell, such as an E. coli cell, such as E. coli BL21 (DE3) pLysS. In some embodiments, the expression vector is pET-30a (+), pET24b or pET-SUMO.

[0105] IV. Bacterial Infections in Patients

[0106] The present invention provides a pharmaceutical composition comprising the bacterial lytic enzyme polypeptide of the present invention and a pharmaceutically acceptable excipient or carrier.

[0107] The present invention provides a method for treating a disease associated with bacterial infection in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the bacterial lytic enzyme polypeptide of the present invention or the pharmaceutical composition of the present invention.

[0108] The present invention also provides use of the bacterial lytic enzyme polypeptide or pharmaceutical composition of the present invention in the preparation of a medicament for treating a disease associated with bacterial infection in a subject in need thereof.

[0109] The present invention also provides the bacterial lytic enzyme polypeptide or pharmaceutical composition of the present invention, for use in treating a disease associated with bacterial infection in a subject in need thereof.

[0110] In some embodiments, the bacteria is Klebsiella pneumoniae or Escherichia coli. In some embodiments, the bacteria is a drug-resistant bacteria. In some embodiments, the disease is selected from pneumonia, mastitis, metritis, meningitis and sepsis caused by Klebsiella pneumoniae. In some embodiments, the disease is selected from inflammation caused by Escherichia coli, such as digestive tract inflammation, such as enteritis and gastritis, and urogenital tract infection such as vaginitis, urethritis.

[0111] V. Beneficial Effects of the Invention

[0112] The present invention provides novel bacterial lytic enzyme polypeptides, thereby providing a novel method for treating diseases associated with bacterial infection such as Klebsiella pneumoniae. Example

[0113] The purpose of this example is to separate bacteriophages with Klebsiella as host from sewage and obtain lytic enzymes capable of lysing the host from the bacteriophages.

[0114] strain

[0115] Klebsiella pneumoniae KPP strain was a clinical isolate donated by Huashan Hospital.

[0116] Example 1: Isolation of bacteriophage

[0117] The phages were isolated by the following procedure.

[0118] 1) Inoculate the bacteria into 10 mL of fresh LB liquid medium and culture at 37°C until OD600≈0.5;

[0119] 2) Add 1.3 mL of farm wastewater and 18 μL of 1 M CaCl to each well of a 96-well deep-well plate. 2 and MgCl 2 (final concentration 10 mM), 90 μL of the bacterial solution prepared in step 1) and 360 μL of 5× liquid medium, cultured overnight at 37°C;

[0120] 3) 200 μL of the culture was transferred to a well of a 96-well filter plate (Merck Millipore, MSHAS4510), and the filter plate was placed on a new 96-well shallow well plate, centrifuged at 900 g for 20 min at room temperature, and the filter plate was discarded;

[0121] 4) Add 225 μL liquid culture medium and 2.5 μL 1M CaCl 2 and MgCl 2 (final concentration 10 mM), 12.5 μL of the bacterial solution prepared in step 1) and 7.5 μL of the filtrate, cultured overnight at 37°C;

[0122] 5) Drop 2 mL of the bacterial solution prepared in step 1) onto a solid culture medium plate (Nunc, 242811), rotate the plate to cover the entire surface of the culture medium, tilt the plate and remove the remaining liquid, and dry it in a biosafety cabinet for 30 min;

[0123] 6) 200 μL of the overnight culture in step 4) was transferred into the wells of a 96-well filter plate, the filter plate was placed on top of the 96-well microplate, centrifuged at 900×g for 20 min at room temperature, and the filter plate was discarded;

[0124] 7) The filtrate was diluted with SM buffer (Sanggong, catalog number: B548130-0100) for 10 0 , 10 -2 , 10 -4 , 10 -6 Serial dilutions were performed, and 5 μL of each sample was dropped onto the dried plate in step 5) and cultured at 37°C overnight;

[0125] 8) When plaque formation is observed, use a 200 μL pipette tip to insert under the agar to pick up two plaques of different morphology, and resuspend them in 200 μL SM buffer;

[0126] 9) Use SM buffer for 10 0 , 10 -2 , 10 -4 For gradient dilution, 10 μL of each dilution was streaked on LB solid medium plates and dried in a biosafety cabinet for 30 min;

[0127] 10) Take 1 mL OD 600 = 0.5 bacteria were evenly spread on the plate, rotated until it touched the entire surface, tilted the plate and aspirated the remaining liquid, dried, and cultured at 37°C overnight;

[0128] 11) When plaque formation is observed, use a 200 μL pipette tip to insert under the agar to pick up a single plaque and resuspend it in 200 μL SM buffer;

[0129] 12) Add 100 μL of the resuspended phage to 10 mL of the bacterial solution (OD600=0.1) prepared in step 1), culture at 180 rpm and 37°C for 4-6 h, centrifuge at 8000 g for 5 min at room temperature, take the supernatant, filter using a 0.22 μm filter, and the filtrate is the phage solution.

[0130] Example 2: Quantification of bacteriophage

[0131] The phage titer was measured as follows.

[0132] 1) Inoculate the bacteria into 5 ml of fresh LB liquid medium at a dilution of 1:200 and culture until the OD600 is about 0.5;

[0133] 2) Take 1 ml of bacterial liquid culture and drop it on the plate, rotate it to make it contact the entire surface, deflect it and suck off the remaining liquid from one side, and dry it in a biosafety cabinet for 30 minutes;

[0134] 3) Use SM buffer and press 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 Dilute the phage sample;

[0135] 4) Take 5 μl of phage of each concentration and drop it on a dry bacterial plate. Repeat three times for each sample.

[0136] 5) Count the number of plaques on the plate and calculate the titer of the sample phage according to the following formula:

[0137] Titer = (x1+x2+x3) / 3*2*10 (y+2) / ml

[0138] where x1, x2 and x3 are the number of plaques in each replicate and y is the dilution. For example, for 10 -6 Diluted phage sample, y=6.

[0139] The plaques of phage KPP18NQ3 are shown in Figure 2. Figure 1 As shown. After quantification, the phage titer obtained was 2.6×10 9 PFU / ml. This indicates that the phage can replicate efficiently and lyse Klebsiella pneumoniae KPP strain.

[0140] Example 3. Phage genome sequencing and analysis

[0141] After titer determination, phage DNA is extracted and sequenced. Specifically:

[0142] 1) Take 600 μL of phage, add DNase I and RNase A to a final concentration of 1 μg / ml, and incubate at 37°C for 2 h;

[0143] 2) Add 24 μL of 0.5 M EDTA (final concentration 20 mM), 1.5 μL of 20 mg / mL proteinase K (final concentration 50 ug / mL), and 30 μL of 10% SDS (final concentration 0.5%), and incubate in a 56°C water bath for 1 h;

[0144] 3) Extract phage genomic DNA using a blood / tissue genomic DNA extraction kit (Qiagen catalog number: 69504) according to the manufacturer's instructions;

[0145] 4) The samples were sent to the sequencing platform of the School of Life Sciences of Fudan University for next-generation sequencing to obtain the phage genome sequence.

[0146] 5) Analysis of genomic sequences to identify genes encoding proteins containing the LysM domain.

[0147] Analysis of the genome sequence showed that its genome contained a nucleotide sequence of SEQ ID NO: 2, encoding a protein containing a LysM domain (positions 7-54 of SEQ ID NO: 1 shown in bold) of SEQ ID NO: 1. This indicates that the genome of bacteriophage KPP18NQ3 encodes a bacterial lytic enzyme of SEQ ID NO: 1, which is capable of lysing Klebsiella pneumoniae.

[0148] Example 4. Modification and expression of lyase

[0149] The nucleotide sequence encoding the lytic enzyme was optimized according to the codon preference of E. coli (SEQ ID NO: 3) and synthesized by Qingke Biotechnology Co., Ltd.

[0150] By primer

[0151] SUMO-LYS18NQ3-F:

[0152] CACAGAGAACAGATTGGTGGAATGAAACTGGTGAACCAGAA (SEQ ID NO: 11), and

[0153] SUMO-LYS18NQ3-R:

[0154] GTGGTGGTGGTGGTGCTCGAGTTACGGCAGAGTCTTAAGAA(SEQ ID NO:12)

[0155] The synthesized sequence was amplified by PCR, and the amplified product was separated and recovered by agarose gel electrophoresis ( Figure 2 Left), using the seamless cloning method ( -Basic Seamless Cloning and Assembly Kit, full-form gold) to connect the fragment into the pET-SUMO vector, which was digested with BamHI and XhoI. The seamless cloning product was passed through CaCl 2 The method was used to transform Escherichia coli DH5α competent cells, and the transformed cells were spread on LB solid plates containing 50μg / ml kanamycin (Km), inverted and cultured overnight at 37℃, and single colonies on the plates were randomly picked and inoculated into LB liquid medium containing 50μg / ml Km for culture. The plasmid was extracted and verified by NdeI and XhoI restriction enzymes, and the positive clones were sent to Qingke Biotechnology Co., Ltd. for sequencing verification. The results showed that the gene of the recombinant expression vector pET-SUMO-LYS18NQ3 was consistent with the target gene sequence. The recombinant expression plasmid pET-SUMO-LYS18NQ3 with correct sequencing was transformed into Escherichia coli BL21 (DE3) pLysS competent cells, and the single clone was stored at -80℃ as an engineering bacterium for recombinant expression of LYS18NQ3 lyase.

[0156] Furthermore, the lyase sequence was modified by i) replacing aspartic acid with lysine in the protein sequence (LYS18NQ3-DK, SEQ ID NO: 4), and ii) adding Cecropin A (CerA) polypeptide to the N-terminus of the protein sequence (LYS18NQ3+CerA, SEQ ID NO: 5).

[0157] The nucleic acid encoding the above protein (SEQ ID NO: 9 and 10, including a sequence encoding a His tag) was synthesized by Qingke Biotechnology and amplified, and the amplified product was separated and recovered by agarose gel electrophoresis ( Figure 2 The amplified product was connected to the pET-24b vector using the method described above. The obtained vector was verified by restriction digestion with BamHI and XhoI, and sequenced. The recombinant expression plasmids with correct sequencing (named pET24b-LYS18NQ3-DK and pET24b-LYS18NQ3+CerA, respectively) were transformed into Escherichia coli BL21 (DE3) pLysS competent cells to obtain engineered bacteria that recombinantly expressed the modified lyase.

[0158] The engineered bacteria of BL21(DE3)pLysS containing the recombinant expression vectors pET-SUMO-LYS18NQ3, pET24b-LYS18NQ3-DK, and pET24b-LYS18NQ3+CerA were inoculated into 5 mL of liquid LB medium containing 50 μg / mL Km and 25 μg / ml chloramphenicol (Cm), and cultured overnight at 37°C in a shaking incubator. The bacterial suspension was inoculated into 200 ml of liquid LB medium containing 50 μg / mL Km and 25 ug / ml Cm at a volume ratio of 1:100, and cultured at 37°C in a shaking incubator until OD600 = 0.6, IPTG was added to a final concentration of 0.1 mM, and the induction culture was continued at 25°C in a shaking incubator for 16 hours. The cells were collected by centrifugation (5,000 g, 30 min, 4°C), and 10 ml of lysis buffer (20 mM Na 2 HPO 4 , 500mM NaCl, 10mM imidazole, pH=7.4) to resuspend the cells, and lyse the cells on ice by ultrasound; centrifuge at 4°C, 7000g for 1h, and purify the supernatant with Ni-NTA column, gradient wash (10mM, 20mM, 40mM, 60mM, 80mM imidazole, 20mM Na2HPO4, 500mM NaCl, pH=7.4), and use elution buffer (20mM Na 2 HPO 4 , 500 mM NaCl, 500 mM imidazole, pH = 7.4), and dialyzed with 10 mM PBS (pH 7.4) buffer at 4 ° C to obtain the lysase recombinant proteins His-SUMO-LYS18NQ3, His-LYS18NQ3-DK, and His-LYS18NQ3+CerA; the obtained protein solution was subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining detection ( Figure 3 ).

[0159] Example 5. Antibacterial activity of recombinant lytic enzyme

[0160] The purpose of this example is to detect the antibacterial activity of the recombinantly expressed lytic enzyme.

[0161] To this end, clinically isolated Escherichia coli strains and Klebsiella pneumoniae strains (donated from Huashan Hospital) were selected for in vitro antibacterial experiments. Escherichia coli strains and Klebsiella pneumoniae strains were cultured to the logarithmic phase (OD600 = 0.4-0.6), centrifuged at 4 ° C, 6000g, and collected the cells. After washing with PBS three times, the cells were resuspended in PBS (OD600 = 0.5), and 40 μL of recombinant lysing enzyme (His-SUMO-LYS18NQ3, His-LYS18NQ3-DK, His-LYS18NQ3 + CerA) solution was mixed with 40 μl of bacterial solution, and the final concentration of lysing enzyme was 175 μg / ml. The mixture of equal amounts of PBS and bacterial solution was used as a negative control (n = 3). Incubated at 37 ° C in a microplate reader, OD600 was measured every 10 minutes. Compared with the negative control, the three types of lytic enzyme recombinant proteins can reduce the turbidity of Escherichia coli X2, X4, Klebsiella pneumoniae strains KPW, KPS, KPT bacterial liquid ( Figure 3 OD600 values ​​are shown as data measured by a microplate reader). There was no significant difference in the antibacterial effects of the three recombinant lytic enzymes.

[0162] sequence

[0163] SEQ ID NO:1 Amino acid sequence of lyase:

[0164] MKLVNQKVYIVKAGDTLSEIANHFCGTPAIGYDIAEDNGIINAGLIYQGQRIWIREEFLKEE

[0165] FRHGVKEEIPAPSIRSGGLIKITESLIVERFDGVNITEADFCSAKTREIGILDSFFYVVSTK

[0166] EELDRVPGTVVVNNKGSYFFLVLNGVGAVVSFYPAERLNLGFTVSVEGSLRVLKTLP

[0167] SEQ ID NO:2 Nucleotide sequence encoding lytic enzyme:

[0168] ATGAAACTCGTTAACCAAAAAGTTTATATTGTTAAGGCAGGTGATACGCTGTCAGAGATAGC

[0169] AAACCATTTTTGTGGGACTCCTGCTATCGGATACGATATAGCTGAGGACAACGGCATTATCA

[0170] ACGCCGGACTCATCTATCAAGGGCAAAGAATCTGGATAAGGGAGGAATTCTTAAAAGAAGAA

[0171] TTTCGTCATGGGGTTAAGGAAGAAATTCCAGCACCTTCTATTCGCAGTGGCGGGTTAATAAA

[0172] AATAACCGAAAGTCTTATTGTTGAAAGGTTTGATGGGGTCAACATTACAGAAGCCGATTTCT

[0173] GTAGCGCCAAAACTCGTGAGATCGGTATCTTAGACAGTTTCTTTTACGTCGTGTCGACAAAA

[0174] GAGGAACTGGACCGCGTCCCAGGAACAGTCGTGGTTAACAACAAAGGATCCTATTTCTTTCT

[0175] GGTATTAAATGGCGTCGGGGCCGTAGTATCGTTTTATCCAGCCGAACGACTTAACCTCGGGT

[0176] TTACGGTTAGTGTAGAAGGTTCTTTACGAGTGCTGAAGACCTTACCCTAA

[0177] Nucleotide sequence of codon-optimized coding lysis enzyme, SEQ ID NO:3

[0178] ATGAAACTGGTGAACCAGAAGGTGTACATCGTTAAAGCTGGTGACACTCTGTCTGAAATCGC

[0179] GAACCACTTCTGCGGTACTCCGGCTATCGGTTACGACATCGCTGAGGACAACGGTATCATCA

[0180] ACGCTGGTCTGATCTACCAGGGTCAGCGTATCTGGATTCGTGAAGAATTTCTGAAAGAAGAA

[0181] TTTCGTCACGGTGTTAAAGAAGAAATTCCGGCTCCGTCTATCCGTTCTGGTGGTCTGATCAA

[0182] GATCACCGAATCTCTGATCGTTGAACGTTTCGACGGTGTTAACATCACCGAAGCAGACTTCT

[0183] GCTCTGCTAAGACTCGTGAAATCGGTATCTTGGACTCTTTCTTCTACGTGGTTTCTACCAAA

[0184] GAAGAGTTGGACCGTGTACCAGGTACTGTTGTTGTGAACAACAAAGGTTCTTACTTCTTTCT

[0185] GGTTCTGAACGGTGTTGGTGCGGTTGTTAGCTTCTATCCGGCAGAACGTCTGAACCTGGGTT

[0186] TCACCGTTTCTGTTGAAGGTTCTCTGCGTGTTCTTAAGACTCTGCCGTAA

[0187] Amino acid sequence of the modified lyase (LYS18NQ3-DK) of SEQ ID NO:4:

[0188] MKLVNQKVYIVKAGDTLSEIANHFCGTPAIGYDIAEDNGIINAGLIYQGQRIWIREEFLKEE

[0189] FRHGVKEEIPAPSIRSGGLIKITESLIVERFKGVNITEAKFCSAKTREIGILKSFFYVVSTK

[0190] EELKRVPGTVVVNNKGSYFFLVLNGVGAVVSFYPAERLNLGFTVSVEGSLRVLKTLP

[0191] Amino acid sequence of the modified lyase (LYS18NQ3-CerA) of SEQ ID NO:5:

[0192] MGGLKKLGKKLEGAGKRVFNAAEKALPVVAGAKALRKGAGAGAMKLVNQKVYIVKAGDTLSE

[0193] IANHFCGTPAIGYDIAEDNGIINAGLIYQGQRIWIREEFLKEEFRHGVKEEIPAPSIRSGGL

[0194] IKITESLIVERFDGVNITEADFCSAKTREIGILDSFFYVVSTKEELDRVPGTVVVNNKGSYF

[0195] FLVLNGVGAVVSFYPAERLNLGFTVSVEGSLRVLKTLP

[0196] SEQ ID NO:6 Amino acid sequence of CerA peptide

[0197] GGLKKLGKKLEGAGKRVFNAAEKALPVVAGAKALRK

[0198] SEQ ID NO:7 Nucleotide sequence encoding LYS18NQ3-DK (excluding his tag, stop codon, restriction site):

[0199] ATGAAACTGGTTAACCAGAAAGTTTACATCGTTAAAGCGGGTGACACTCTGTCTGAAATCGC

[0200] GAACCACTTCTGCGGTACTCCGGCGATCGGTTACGACATCGCGGAAGATAACGGTATCATCA

[0201] ACGCGGGTCTGATCTACCAGGGTCAGCGTATCTGGATTCGTGAAGAGTTCTTGAAAGAAGAA

[0202] TTTCGTCACGGTGTTAAAGAAGAAATTCCAGCGCCGTCTATCCGTTCTGGTGGTCTGATCAA

[0203] GATCACCGAATCTCTGATCGTTGAACGTTTCAAAGGTGTTAACATCACCGAAGCGAAATTCT

[0204] GCTCTGCAAAGACTCGTGAAATCGGCATTCTCAAGAGCTTCTTCTACGTTGTTTCCACCAAA

[0205] GAAGAACTGAAACGTGTTCCGGGCACCGTTGTTGTGAACAACAAAGGTAGCTACTTCTTCTT

[0206] GGTTCTGAACGGTGTTGGTGCGGTTGTTTCTTTCTATCCGGCGGAACGTCTGAACCTGGGTT

[0207] TCACCGTTTCTGTTGAAGGTAGCCTGCGTGTTCTTAAGACCTTGCCG

[0208] Nucleotide sequence encoding LYS18NQ3 + CerA of SEQ ID NO:8 (excluding his tag, stop codon, restriction sites):

[0209] ATGGGTGGTCTGAAGAAACTGGGTAAGAAACTGGAAGGTGCTGGTAAACGTGTGTTCAACGC

[0210] GGCGGAGAAAGCTCTGCCGGTTGTTGCGGGTGCGAAAGCGCTGCGTAAAGGAGCCGGAGCAG

[0211] GCGCTATGAAACTGGTGAACCAGAAGGTGTACATCGTTAAAGCTGGTGACACTCTGTCTGAA

[0212] ATCGCGAACCACTTCTGCGGTACTCCGGCTATCGGTTACGACATCGCTGAGGACAACGGTAT

[0213] CATCAACGCTGGTCTGATCTACCAGGGTCAGCGTATCTGGATTCGTGAAGAATTTCTGAAAG

[0214] AAGAATTTCGTCACGGTGTTAAAGAAGAAATTCCGGCTCCGTCTATCCGTTCTGGTGGTCTG

[0215] ATCAAGATCACCGAATCTCTGATCGTTGAACGTTTCGACGGTGTTAACATCACCGAAGCAGA

[0216] CTTCTGCTCTGCTAAGACTCGTGAAATCGGTATCTTGGACTCTTTCTTCTACGTGGTTTCTA

[0217] CCAAAGAAGAGTTGGACCGTGTACCAGGTACTGTTGTTGTGAACAACAAAGGTTCTTACTTC

[0218] TTTCTGGTTCTGAACGGTGTTGGTGCGGTTGTTAGCTTCTATCCGGCAGAACGTCTGAACCT

[0219] GGGTTTCACCGTTTCTGTTGAAGGTTCTCTGCGTGTTCTTAAGACTCTGCCG

[0220] SEQ ID NO:9 nucleotide sequence encoding HIS-18NQ3-DK (including his tag, stop codon, restriction site): ATGAAACTGGTTAACCAGAAAGTTTACATCGTTAAAGCGGGTGACACTCTGTCTGAAATCGCGAACCACTTCTGCGGTACTCCGGCGATCGGTTACGACATCGCGGAAGATAACGGTATCATCAACGCGGGTCTGATCTACCAGGGTCAGCGTATCTGGATTCGTGAAGAGTTCTTGAAAGAAGAATTTCGTCACGGTGTTAAAGAAGAAATTCCAGCGCCGTCTATCCGTTCTGGTGGTCTGATCAAGATCACCGAATCTCTGATCGTTGAACGTTTCAAAGGTGTTAACATCACCGAAGCGAAATTCTGCTCTGCAAAGACTCGTGAAATCGGCATTCTCAAGAGCTTCTTCTACGTTGTTTCCACCAAAGAAGAACTGAAACGTGTTCCGGGCACCGTTGTTGTGAACAACAAAGGTAGCTACTTCTTCTTGGTTCTGAACGGTGTTGGTGCGGTTGTTTCTTTCTATCCGGCGGAACGTCTGAACCTGGGTTTCACCGTTTCTGTTGAAGGTAGCCTGCGTGTTCTTAAGACCTTGCCGCTCGAGCACCACCATCATCACCACTGA

[0221] Nucleotide sequence encoding His-LYS18NQ3+CerA (including his tag and stop codon): ATGGGTGGTCTGAAGAAACTGGGTAAGAAACTGGAAGGTGCTGGTAAACGTGTGTTCAACGCGGCGGAGAAAGCTCTGCCGGTTGTTGCGGGTGCGAAAGCGCTGCGTAAAGGAGCCGGAGCAGGCGCTATGAAACTGGTGAACCAGAAGGTGTACATCGTTAAAGCTGGTGACACTCTGTCTGAAATCGCGAACCACTTCTGCGGTACTCCGGCTATCGGTTACGACATCGCTGAGGACAACGGTATCATCAACGCTGGTCTGATCTACCAGGGTCAGCGTATCTGGATTCGTGAAGAATTTCTGAAAGAAGAATTTCGTCACGGTGTTAAAGAAGAAATTCCGGCTCCGTCTATCCGTTCTGGTGGTCTGATCAAGATCACCGAATCTCTGATCGTTGAACGTTTCGACGGTGTTAACATCACCGAAGCAGACTTCTGCTCTGCTAAGACTCGTGAAATCGGTATCTTGGACTCTTTCTTCTACGTGGTTTCTACCAAAGAAGAGTTGGACCGTGTACCAGGTACTGTTGTTGTGAACAACAAAGGTTCTTACTTCTTTCTGGTTCTGAACGGTGTTGGTGCGGTTGTTAGCTTCTATCCGGCAGAACGTCTGAACCTGGGTTTCACCGTTTCTGTTGAAGGTTCTCTGCGTGTTCTTAAGACTCTGCCGCTCGAGCACCACCATCATCACCACTGA

Claims

1. An isolated bacterial lytic enzyme polypeptide comprising the amino acid sequence of SEQ ID NO: 1, 4 or 5.

2. The polypeptide of claim 1, which consists of the amino acid sequence of SEQ ID NO: 1, 4 or 5.

3. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide of claim 1 or 2. The polynucleotide of claim 3 , comprising the nucleotide sequence of SEQ ID NO: 2, 3, 7 or 8.

5. A vector, preferably an expression vector, comprising the polynucleotide of claim 3 or 4.

6. A recombinant cell, preferably a prokaryotic cell, comprising the polynucleotide of claim 3 or 4 or the expression vector of claim 5.

7. Use of the polypeptide of claim 1 or 2 in the preparation of a medicament for treating diseases associated with infection by bacteria such as Klebsiella pneumoniae and Escherichia coli.

8. The use according to claim 7, wherein the Klebsiella pneumoniae or Escherichia coli is a drug-resistant bacterium.

9. The method according to claim 7 or 8, wherein the disease is selected from pneumonia, mastitis, metritis, meningitis and sepsis caused by Klebsiella pneumoniae, or inflammation caused by Escherichia coli, such as enteritis and gastritis, and urogenital tract infections such as vaginitis and urethritis.

10. A pharmaceutical composition comprising the polypeptide of claim 1 or 2 and a pharmaceutically acceptable excipient.

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