A c-terminal modified hybrid antibacterial protein and pharmaceutical composition and application thereof

Abstract

The application provides a C-terminal modified antibacterial hybrid protein, which has an amino acid sequence shown in formula (I): X-Y (I); wherein X is a modified hybrid antibacterial protein, including AB469 hybrid antibacterial protein or an analogue thereof; Y is a C-terminal modifier of X, and the C-terminal modifier includes a polypeptide containing 3-10 arginine residues. The C-terminal modified antibacterial hybrid protein has the advantages that the C-terminal modified arginine residue-containing helical amphiphilic polypeptide significantly improves the permeability of the hybrid antibacterial protein to the outer membrane of gram-negative bacteria, and significantly improves the bactericidal effect on gram-negative bacteria.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more particularly to a C-terminal modified hybrid antimicrobial protein, its pharmaceutical composition, and its application. Background Technology

[0002] Antimicrobial resistance has seriously threatened human antibiotic resources and public health safety. Multidrug-resistant bacteria can be detected in wastewater and feces from animal farms, as well as in farm soil, water sources, grains, and aquatic products. Reports predict that 10 million people will die from antimicrobial diseases by 2050. The best way to solve the problem of antimicrobial resistance is to develop new antibiotics; however, the development process is slow and expensive. Bacteriophages and their lysin preparations can efficiently, rapidly, and specifically lyse drug-resistant pathogens, are less likely to induce resistance, have good safety profiles, and are environmentally friendly.

[0003] Lyases primarily function by catalyzing peptidoglycan in bacterial cell walls to lyse cells. In Gram-positive bacteria, the peptidoglycan is located on the outermost layer of the cell wall, allowing lyases to directly interact with and lyse it. Lyase preparations are effective at killing Gram-positive bacteria, such as streptococci and Staphylococcus aureus, both in vivo and in vitro. However, the cell walls of Gram-negative bacteria differ from those of Gram-positive bacteria. Their outermost layer is an outer membrane composed of complexes of lipopolysaccharides, phospholipids, proteins, and lipoproteins. Outer membrane proteins are a collective term for proteins embedded within the membrane, including lipoproteins and microporous proteins. Bacteria exchange substances with the external environment through microporous proteins, but only small molecules (molecular weight less than 600) can pass through. The innermost layer of the cell wall is the cytoplasmic membrane. Between the outer membrane and the cytoplasmic membrane lies the periplasmic space, containing 2-3 nm of peptidoglycan. Lyases have a molecular weight of at least 30 kDa, therefore they cannot penetrate the outer membrane of Gram-negative bacteria to access the peptidoglycan in the periplasmic space, preventing them from directly lysing Gram-negative bacteria from outside the bacteria.

[0004] To address the issue of lysins' inability to penetrate the outer membrane of Gram-negative bacteria, chemical permeabilizing agents are generally used to assist lysins in their passage across the cell membrane. Permeabilizing agents are generally divided into two categories: the first category consists of polyvalent cationic compounds that competitively substitute for neighboring divalent anions linked to lipopolysaccharide molecules, such as polymyxins and their derivatives, lysine polymers, and aminoglycosides; the second category consists of chelating agents, with EDTA being the most commonly used, although protonated forms of weak organic acids can also be used as chelating agents. Multiple studies have shown that EDTA has the strongest synergistic effect with lysins in penetrating the cell wall, but due to its other pharmacological effects, it is not suitable for the treatment of systemic infections.

[0005] Chinese patent CN201910975220.9 discloses a hybrid antimicrobial protein AB469 with strong bactericidal effects. It exhibits very strong bactericidal activity against Gram-negative bacteria and their drug-resistant counterparts, and also shows considerable bactericidal activity against Gram-positive bacteria. Compared to wild-type ABgp46, the hybrid antimicrobial protein AB469 has an additional binding domain, significantly improving its antimicrobial activity in complex environments. Although the hybrid antimicrobial protein AB469 shows significantly enhanced antibacterial activity compared to wild-type lysins, and is an antimicrobial substance that can directly lyse bacteria, its inhibitory ability against Gram-negative bacteria still needs improvement, affecting its further clinical application. Summary of the Invention

[0006] To further improve the antibacterial effect against Gram-negative bacteria, this invention provides a C-terminal modified hybrid antibacterial protein. By modifying the C-terminus of a helical amphoteric polypeptide containing arginine residues, the permeability of the hybrid antibacterial protein to the outer membrane of Gram-negative bacteria is significantly improved, thereby significantly enhancing the bactericidal effect against Gram-negative bacteria.

[0007] The present invention also provides the encoding nucleic acid sequence of the C-terminal modified hybrid antimicrobial protein, the expression vector, the engineered bacteria, and the pharmaceutical composition containing the C-terminal modified hybrid antimicrobial protein and its application.

[0008] To achieve the above-mentioned objective, the present invention provides a C-terminal modified antibacterial hybrid protein having the amino acid sequence shown in formula (I):

[0009] XY (Ⅰ)

[0010] Wherein, X is the modified hybrid antimicrobial protein, including AB469 hybrid antimicrobial protein or its analogues;

[0011] Y is the C-terminal modification of X, wherein the C-terminal modification comprises a polypeptide containing 3-10 arginine residues.

[0012] Preferably, the AB469 hybrid antimicrobial protein analog includes proteins having at least 70% homology with the coding sequence of the AB469 hybrid antimicrobial protein, proteins having at least 70% homology with the coding sequence of the catalytic domain of the AB469 hybrid antimicrobial protein, proteins having at least 70% homology with the coding sequence of the binding domain of the AB469 hybrid antimicrobial protein, and proteins having at least 70% homology with the coding sequence of a protein formed by interchanging the positions of the catalytic and binding domains of the AB469 hybrid antimicrobial protein.

[0013] Preferably, the C-terminal modifier comprises a polypeptide containing 5-8 arginine residues.

[0014] Preferably, the C-terminal modifier comprises a polypeptide containing 3-10 consecutive arginine residues.

[0015] Preferably, the amino acid sequence of the C-terminal modified antibacterial hybrid protein is shown in SEQ ID NO.1 or SEQ ID NO.3.

[0016] Preferably, the C-terminal modified version Y also includes hydrophobic amino acid residues.

[0017] Preferably, the C-terminal modifier is an alternating arrangement of arginine residues and hydrophobic amino acid residues.

[0018] Preferably, the hydrophobic amino acid residue is an alanine residue.

[0019] Preferably, the amino acid sequence of the C-terminal modified antibacterial hybrid protein is shown in SEQ ID NO.5.

[0020] A second aspect of the present invention provides a nucleotide sequence including the coding sequence of the C-terminal modified antibacterial hybrid protein described in the above-described technical solutions.

[0021] Preferably, the nucleotide sequence is as shown in SEQ ID NO.2, SEQ ID NO.4 or SEQ ID NO.6.

[0022] A third aspect of the present invention provides an expression vector comprising the nucleotide sequence described above.

[0023] A fourth aspect of the present invention provides an engineered bacterium comprising the nucleotide sequence described in the foregoing technical solutions, or the expression vector described in the foregoing technical solutions.

[0024] A fifth aspect of the present invention provides a pharmaceutical composition comprising the C-terminal modified antibacterial hybrid protein described in the foregoing technical solutions, and pharmaceutically acceptable excipients.

[0025] Preferably, when the pharmaceutical composition is a topical preparation, the mass concentration of the C-terminal modified antibacterial hybrid protein does not exceed 0.5%.

[0026] The sixth aspect of the present invention provides the use of the C-terminal modified antibacterial hybrid protein described in the foregoing technical solutions, the nucleotide sequence described in the foregoing technical solutions, the expression vector described in the foregoing technical solutions, the engineered bacteria described in the foregoing technical solutions, or the pharmaceutical composition described in the foregoing technical solutions in the preparation of antibacterial drugs.

[0027] Preferably, the antibacterial drug is a drug that inhibits Gram-negative bacteria.

[0028] Preferably, the Gram-negative bacteria include one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella, and Escherichia coli.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] The hybrid antimicrobial protein AB469 is a hybrid antimicrobial protein synthesized by the applicant in the past, which has a strong bactericidal effect against Gram-negative bacteria. Based on this, the applicant unexpectedly discovered that modifying the C-terminus of AB469 with a polypeptide containing 3-10 arginine residues resulted in a more than 100% increase in the antibacterial effect against Gram-negative bacteria (MIC ≤ 50 μg / mL) compared to the C-terminal modified AB469 described in this invention (hereinafter referred to as "AB469A"), and a significantly enhanced bactericidal effect against Gram-negative bacteria. Attached Figure Description

[0031] Figure 1 This is an SDS-PAGE image of the AB469A5 protein.

[0032] Figure 2 This is an SDS-PAGE image of the AB469A8 protein. Detailed Implementation

[0033] The technical solution of the present invention will now be described with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them; and the structures shown in the accompanying drawings are merely illustrative and do not represent physical objects. It should be noted that all other embodiments obtained by those skilled in the art based on these embodiments of the present invention are within the scope of protection of this application.

[0034] In this invention, for convenience, the singular forms such as “a,” “an,” and “the” are often used; however, unless the context explicitly specifies or clearly indicates that it is only singular, the singular forms are intended to include the plural.

[0035] The term "coding sequence" refers to a nucleotide sequence that encodes a protein or peptide with corresponding protein or peptide activity, such as the nucleotide sequence of the AB469 heterozygous antimicrobial protein and its degenerate sequence. A degenerate sequence is a sequence in which one or more codons are replaced by degenerate codons encoding the same amino acid. The term also includes variations of open reading frame sequences in protein or peptide nucleotide sequences that encode the same protein or peptide function as the corresponding protein or peptide. These variations include, but are not limited to, deletions, insertions, and / or substitutions of several nucleotides, and the addition of several nucleotides at the 5' and / or 3' ends.

[0036] The term "analyte" refers to a polypeptide or nucleotide sequence that differs from the corresponding protein or polypeptide and its nucleotide sequence but retains its main properties. This analog can be naturally occurring or can be derived from one or more modifications (such as substitution, addition, and / or deletion) that result in a different polypeptide or nucleotide sequence, such as conserved analogs. This genus also includes protein modification products (such as methylation, acetylation, phosphorylation, ubiquitination, ADP ribosylation), conjugates (such as antibody conjugates, peptide conjugates, etc.), conjugates (such as drug conjugates, polymer conjugates, etc.), and all analogs that are functionally identical or similar to the corresponding protein or polypeptide.

[0037] The term "polypeptide" refers to a polymer composed of any number of amino acids, regardless of its size. Although the term "protein" is often used to refer to relatively large polypeptides, and "peptide" is often used to refer to small polypeptides, the terms "polypeptide," "protein," and "peptide" are often used interchangeably in this field. Unless otherwise noted, the term "polypeptide" generally refers to proteins, polypeptides, and peptides.

[0038] The percentage of homology of coding sequences can be analyzed using software known in the field, such as GAP analysis.

[0039] This invention provides a C-terminal modified antibacterial hybrid protein having the amino acid sequence shown in formula (I):

[0040] XY (Ⅰ)

[0041] Wherein, X is the modified hybrid antimicrobial protein, including AB469 hybrid antimicrobial protein or its analogues; Y is the C-terminal modified form of X, wherein the C-terminal modified form comprises a polypeptide containing 3-10 arginine residues. This invention shows that only by using a specific number of arginine residues to modify the C-terminus of AB469 can its bactericidal effect against Gram-negative bacteria be significantly improved; in the embodiments of this invention, the bactericidal effect against Gram-negative bacteria after C-terminal modification of the AB469 hybrid antimicrobial protein with lysine and histidine was actually reduced compared to AB469.

[0042] In this invention, the nucleotide and amino acid sequences of the AB469 hybrid antimicrobial protein, as well as the nucleotide and amino acid sequences of certain analogues of the AB469 hybrid antimicrobial protein (such as hybrid proteins similar to the AB469 catalytic domain sequence, hybrid proteins similar to the AB469 binding domain sequence, and hybrid proteins in which the AB469 catalytic and binding domains are interchanged), have been described in Chinese Patent CN201910975220.9, and will not be repeated here.

[0043] The AB469 hybrid antimicrobial protein analogs of the present invention include proteins having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, and 99.9% homology with the coding sequence of the AB469 hybrid antimicrobial protein, proteins having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, and 99.9% homology with the coding sequence of the catalytic domain of the AB469 hybrid antimicrobial protein, proteins having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, and 99.9% homology with the coding sequence of the binding domain of the AB469 hybrid antimicrobial protein, and proteins whose coding sequences are formed by interchanging the positions of the catalytic and binding domains of the AB469 hybrid antimicrobial protein, and proteins whose coding sequences have at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, and 99.9% homology.

[0044] In this invention, the C-terminal modifier comprises a polypeptide containing 3-10 arginine residues. C-terminal modifiers with more or less arginine residues than defined in this invention cannot significantly improve the bactericidal effect of AB469 against Gram-negative bacteria. Preferably, the C-terminal modifier comprises a polypeptide containing 5-8 arginine residues, and the AB469A hybrid antimicrobial protein including this C-terminal modifier can improve the bactericidal effect against Gram-negative bacteria by more than 8 times, with a MIC ≤ 12.5 μg / mL.

[0045] In some specific embodiments of the present invention, the C-terminal modifier comprises a polypeptide containing 3-10 consecutive arginine residues, the sequence of which may be as shown in SEQ ID NO.7-SEQ ID NO.14 (Table 1); preferably, the C-terminal modifier comprises a polypeptide containing 5-8 consecutive arginine residues, the sequence of which may be as shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12.

[0046] Table 1

[0047]

[0048] In some specific embodiments of the present invention, the C-terminal modification consists of a polypeptide of five consecutive arginine residues (as shown in SEQ ID NO. 9). The modified hybrid antibacterial protein is named "AB469A5", its amino acid sequence is shown in SEQ ID NO. 1, and its protein coding sequence is shown in SEQ ID NO. 2.

[0049] SEQ ID NO.1:

[0050] MAILTKDGFGIIRNELFGGKLDQTQVDAINFIVEKATESGLSYPEAAYLLATIYHETGLP 60

[0051] SGYRTMQPIKEAGSDNYLRSKKYYPYIGYGYVQLTWKENYGRIGKLIGIDLIKNPEKALE 120

[0052] PLIAIQIAIKGMLNGWFTGVGFRRKRPVSKYNKQQYIAARNIINGKDKAELIAKYAIIFE 180

[0053] RALRSLGSNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKG 240

[0054] AKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKLRRRRR

[0055] SEQ ID NO.2:

[0056] ccatggctatcctgaccaaagatggctttggcatcattcgcaacgaactgtttggcggca 60

[0057] aactggatcagacacaggtggatgccattaattttattgttgaaaaagctaccgaatctg 120

[0058] ggttaagttatccggaagcggcctatctgttagcgacgatctatcatgaaacgggtctgc 180

[0059] cgagcggttatcgtaccatgcagccaatcaaagaagccggtagtgataattacctccgct 240

[0060] ctaaaaaatattatccgtatatcggctatggctatgttcagctgacgtggaaagaaaatt 300

[0061] atggtcgtattggtaaactgatcggcatcgacttgatcaaaaatccggaaaaagccttag 360

[0062] aaccgctgattgcgattcagattgccatcaaaggtatgctgaatggttggtttacaggtg 420

[0063] tgggctttcgtcgcaaacgtccagtgagtaaatataacaagcagcagtatattgctgcac 480

[0064] gcaatatcattaatggtaaagataaagcagaactgatcgcgaaatatgccatcatctttg 540

[0065] aacgcgccctgcgttctctgggctccaatagtacctctaatagtagcaccaattcaggct 600

[0066] ctaccggtaaagtgagtctgccgaatcgtgtgattcgcgtgaccaaaccgattgttcatg 660

[0067] gtagcgatgtgctggcaattcagaaagcactgtcaagcctgtatttttatccggaaaaag 720

[0068] gtgccaaagataatggttccgatagctattatggtccgaaaaccgccaatgccgttaaac 780

[0069] gctttcagtctgttaatggcttagttgcagatggcgtgtatggcccgaaaacccgcgcag 840

[0070] ccattctgaaaaaactgcgtcgtcgtcgtcgttaagctt

[0071] In some specific embodiments of the present invention, the C-terminal modification consists of a polypeptide of eight consecutive arginine residues (as shown in SEQ ID NO. 12). The modified hybrid antibacterial protein is named "AB469A8", its amino acid sequence is shown in SEQ ID NO. 3, and its protein coding sequence is shown in SEQ ID NO. 4.

[0072] SEQ ID NO.3:

[0073] MAILTKDGFGIIRNELFGGKLDQTQVDAINFIVEKATESGLSYPEAAYLLATIYHETGLP 60

[0074] SGYRTMQPIKEAGSDNYLRSKKYYPYIGYGYVQLTWKENYGRIGKLIGIDLIKNPEKALE 120

[0075] PLIAIQIAIKGMLNGWFTGVGFRRKRPVSKYNKQQYIAARNIINGKDKAELIAKYAIIFE 180

[0076] RALRSLGSNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKG 240

[0077] AKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKLRRRRRRRR

[0078] SEQ ID NO.4:

[0079] ccatggctatcctgaccaaagatggctttggcatcattcgcaacgaactgtttggcggca 60

[0080] aactggatcagacacaggtggatgccattaattttattgttgaaaaagctaccgaatctg 120

[0081] ggttaagttatccggaagcggcctatctgttagcgacgatctatcatgaaacgggtctgc 180

[0082] cgagcggttatcgtaccatgcagccaatcaaagaagccggtagtgataattacctccgct 240

[0083] ctaaaaaatattatccgtatatcggctatggctatgttcagctgacgtggaaagaaaatt 300

[0084] atggtcgtattggtaaactgatcggcatcgacttgatcaaaaatccggaaaaagccttag 360

[0085] aaccgctgattgcgattcagattgccatcaaaggtatgctgaatggttggtttacaggtg 420

[0086] tgggctttcgtcgcaaacgtccagtgagtaaatataacaagcagcagtatattgctgcac 480

[0087] gcaatatcattaatggtaaagataaagcagaactgatcgcgaaatatgccatcatctttg 540

[0088] aacgcgccctgcgttctctgggctccaatagtacctctaatagtagcaccaattcaggct 600

[0089] ctaccggtaaagtgagtctgccgaatcgtgtgattcgcgtgaccaaaccgattgttcatg 660

[0090] gtagcgatgtgctggcaattcagaaagcactgtcaagcctgtatttttatccggaaaaag 720

[0091] gtgccaaagataatggttccgatagctattatggtccgaaaaccgccaatgccgttaaac 780

[0092] gctttcagtctgttaatggcttagttgcagatggcgtgtatggcccgaaaacccgcgcag 840

[0093] ccattctgaaaaaactgcgtcgtcgtcgtcgtcgtcgtcgttaagctt

[0094] In some specific embodiments of the present invention, the C-terminal modifier may include hydrophobic amino acid residues in addition to arginine residues. In the present invention, the hydrophobic amino acid is selected from one or more of glycine, alanine, valine, leucine, isoleucine, phenylalanine, and proline; in some preferred embodiments of the present invention, the hydrophobic amino acid is alanine.

[0095] In some specific embodiments of the present invention, the arginine in the C-terminal modified body of the present invention may also be arranged discontinuously, such as inserting 1-2 hydrophobic amino acids between two arginine residues.

[0096] In some specific embodiments of the present invention, the C-terminal modified body is a polypeptide comprising 6 arginine residues with 2 alanine residues inserted between any two arginine residues (as shown in SEQ ID NO. 15). The modified hybrid antimicrobial protein is named "AB469A11", its amino acid sequence is shown in SEQ ID NO. 5, and its protein coding sequence is shown in SEQ ID NO. 6.

[0097] SEQ ID NO.15:

[0098] RAARAARAARAARAAR

[0099] SEQ ID NO.5:

[0100] MAILTKDGFGIIRNELFGGKLDQTQVDAINFIVEKATESGLSYPEAAYLLATIYHETGLP 60

[0101] SGYRTMQPIKEAGSDNYLRSKKYYPYIGYGYVQLTWKENYGRIGKLIGIDLIKNPEKALE 120

[0102] PLIAIQIAIKGMLNGWFTGVGFRRKRPVSKYNKQQYIAARNIINGKDKAELIAKYAIIFE 180

[0103] RALRSLGSNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKG 240

[0104] AKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKLRAARAARAARAARAA 300

[0105] R

[0106] SEQ ID NO.6:

[0107] ccatggctatcctgaccaaagatggctttggcatcattcgcaacgaactgtttggcggca 60

[0108] aactggatcagacacaggtggatgccattaattttattgttgaaaaagctaccgaatctg 120

[0109] ggttaagttatccggaagcggcctatctgttagcgacgatctatcatgaaacgggtctgc 180

[0110] cgagcggttatcgtaccatgcagccaatcaaagaagccggtagtgataattacctccgct 240

[0111] ctaaaaaatattatccgtatatcggctatggctatgttcagctgacgtggaaagaaaatt 300

[0112] atggtcgtattggtaaactgatcggcatcgacttgatcaaaaatccggaaaaagccttag 360

[0113] aaccgctgattgcgattcagattgccatcaaaggtatgctgaatggttggtttacaggtg 420

[0114] tgggctttcgtcgcaaacgtccagtgagtaaatataacaagcagcagtatattgctgcac 480

[0115] gcaatatcattaatggtaaagataaagcagaactgatcgcgaaatatgccatcatctttg 540

[0116] aacgcgccctgcgttctctgggctccaatagtacctctaatagtagcaccaattcaggct 600

[0117] ctaccggtaaagtgagtctgccgaatcgtgtgattcgcgtgaccaaaccgattgttcatg 660

[0118] gtagcgatgtgctggcaattcagaaagcactgtcaagcctgtatttttatccggaaaaag 720

[0119] gtgccaaagataatggttccgatagctattatggtccgaaaaccgccaatgccgttaaac 780

[0120] gctttcagtctgttaatggcttagttgcagatggcgtgtatggcccgaaaacccgcgcag 840

[0121] ccattctgaaaaaactgcgtgatgatcgtgatgatcgtgatgatcgtgatgatcgtgatg 900

[0122] atcgttaagctt

[0123] A second aspect of the present invention provides a nucleotide sequence including the coding sequence of the C-terminal modified antibacterial hybrid protein described in the above-described technical solutions. In some specific embodiments of the present invention, the nucleotide sequence may be as shown in SEQ ID NO.2, SEQ ID NO.4, or SEQ ID NO.6.

[0124] A third aspect of the present invention provides an expression vector comprising the nucleotide sequence described above. In some specific embodiments of the present invention, the expression vector may be a plasmid containing the AB469A coding sequence.

[0125] A fourth aspect of the present invention provides an engineered bacterium comprising the nucleotide sequence described in the foregoing technical solutions, or the expression vector described in the foregoing technical solutions. In some specific embodiments of the present invention, the engineered bacterium may be yeast, Escherichia coli, etc., comprising a plasmid vector containing the AB469A coding sequence.

[0126] A fifth aspect of the present invention provides a pharmaceutical composition comprising the C-terminal modified antibacterial hybrid protein described in the foregoing technical solutions, and pharmaceutically acceptable excipients. The pharmaceutically acceptable carriers of the present invention include, but are not limited to, one or more of stabilizers, excipients, fillers, binders, dispersants, solvents, and flavoring agents. The pharmaceutical composition can be formulated into modified release dosage forms, including delayed release, extended release, prolonged release, sustained release, pulsatile release, controlled release, accelerated release, rapid release, targeted release, programmed release, and gastric retention dosage forms. These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art.

[0127] In this invention, the mass percentage of AB469A hybrid antimicrobial protein in the pharmaceutical composition can be 0.1-99.99%. In some specific embodiments of this invention, when the pharmaceutical composition is a topical preparation, the mass concentration of the C-terminal modified antimicrobial hybrid protein does not exceed 0.5%; preferably 0.00001-0.5%.

[0128] The sixth aspect of the present invention provides the use of the C-terminal modified antibacterial hybrid protein, the nucleotide sequence, the expression vector, the engineered bacteria, or the pharmaceutical composition described in the foregoing technical solutions in the preparation of antibacterial foods, pharmaceuticals, or health products. Preferably, the antibacterial food, pharmaceutical, or health product is a food, pharmaceutical, or health product that inhibits Gram-negative bacteria; preferably, the Gram-negative bacteria include, but are not limited to, one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella, and Escherichia coli.

[0129] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0130] Example 1: AB469A5 hybrid antimicrobial protein

[0131] 1. Construction of engineered bacteria

[0132] The amino acid sequence of AB469:

[0133] MAILTKDGFG IIRNELFGGK LDQTQVDAIN FIVEKATESG LSYPEAAYLL 51

[0134] ATIYHETGLP SGYRTMQPIK EAGSDNYLRS KKYYPYIGYG YVQLTWKENY 101

[0135] GRIGKLIGID LIKNPEKALE PLIAIQIAIK GMLNGWFTGV GFRRKRPVSK 151

[0136] YNKQQYIAAR NIINGKDKAE LIAKYAIIFE RALRSLGSNS TSNSSTNSGS 201

[0137] TGKVSLPNRV IRVTKPIVHG SDVLAIQKAL SSLYFYPEKG AKDNGSDSYY 251

[0138] GPKTANAVKR FQSVNGLVAD GVYGPKTRAA ILKKL

[0139] Adding the "RRRRR" polypeptide, as shown in SEQ ID NO.9, to the C-terminus of the AB469 amino acid sequence yields the amino acid sequence of AB469A5 (as shown in SEQ ID NO.1). Following the method described in Example 1 of Chinese Patent CN201910975220.9, the coding sequence of AB469A5 (as shown in SEQ ID NO.2) is inserted into the pET28a plasmid to obtain the recombinant plasmid pET28a-AB469A5.

[0140] SEQ ID NO.1:

[0141] MAILTKDGFGIIRNELFGGKLDQTQVDAINFIVEKATESGLSYPEAAYLLATIYHETGLP 60

[0142] SGYRTMQPIKEAGSDNYLRSKKYYPYIGYGYVQLTWKENYGRIGKLIGIDLIKNPEKALE 120

[0143] PLIAIQIAIKGMLNGWFTGVGFRRKRPVSKYNKQQYIAARNIINGKDKAELIAKYAIIFE 180

[0144] RALRSLGSNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKG 240

[0145] AKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKLRRRRR

[0146] SEQ ID NO.2:

[0147] ccatggctatcctgaccaaagatggctttggcatcattcgcaacgaactgtttggcggca 60

[0148] aactggatcagacacaggtggatgccattaattttattgttgaaaaagctaccgaatctg 120

[0149] ggttaagttatccggaagcggcctatctgttagcgacgatctatcatgaaacgggtctgc 180

[0150] cgagcggttatcgtaccatgcagccaatcaaagaagccggtagtgataattacctccgct 240

[0151] ctaaaaaatattatccgtatatcggctatggctatgttcagctgacgtggaaagaaaatt 300

[0152] atggtcgtattggtaaactgatcggcatcgacttgatcaaaaatccggaaaaagccttag 360

[0153] aaccgctgattgcgattcagattgccatcaaaggtatgctgaatggttggtttacaggtg 420

[0154] tgggctttcgtcgcaaacgtccagtgagtaaatataacaagcagcagtatattgctgcac 480

[0155] gcaatatcattaatggtaaagataaagcagaactgatcgcgaaatatgccatcatctttg 540

[0156] aacgcgccctgcgttctctgggctccaatagtacctctaatagtagcaccaattcaggct 600

[0157] ctaccggtaaagtgagtctgccgaatcgtgtgattcgcgtgaccaaaccgattgttcatg 660

[0158] gtagcgatgtgctggcaattcagaaagcactgtcaagcctgtatttttatccggaaaaag 720

[0159] gtgccaaagataatggttccgatagctattatggtccgaaaaccgccaatgccgttaaac 780

[0160] gctttcagtctgttaatggcttagttgcagatggcgtgtatggcccgaaaacccgcgcag 840

[0161] ccattctgaaaaaactgcgtcgtcgtcgtcgttaagctt

[0162] Using recombinant plasmid pET28a-AB469A5 as a template, the entire coding region was amplified using AB469A5-specific primers (upstream primer shown in SEQ ID NO.16, downstream primer shown in SEQ ID NO.17). The PCR reaction program was set as follows: ① 94℃ pre-denaturation for 5 min, ② 94℃ denaturation for 30 s, ③ 55℃ annealing for 30 s, ④ 72℃ extension for 1 min, ⑤ 72℃ final extension for 5 min. Steps ②, ③, and ④ were repeated for 30 cycles. The PR product was purified and recovered, and ligated into the pET28a vector. The ligation product was transformed into E. coli BL21(DE3), and three single colonies were selected and inoculated into LB liquid medium and sent to a sequencing company for sequencing to verify the correctness of the reading frame.

[0163] Specific primers for AB469A5:

[0164] SEQ ID NO.16: 5'-tataccatggctatcctgaccaaag-3'

[0165] SEQ ID NO.17: 5'-ccgcaagcttaacgacgacgacgacgcagttttttcagaatggctgcgcgggtttt-3'

[0166] 2. Expression and purification of recombinant proteins:

[0167] Single clones of the engineered bacteria were inoculated into LB broth (containing 30 mg / L kanamycin) and cultured overnight at 30°C with shaking. The inoculation was then carried out at 1% in the same LB broth and cultured at 30°C with shaking until the optical density (wavelength 600 nm) ≈ 0.6. IPTG was then added to a final concentration of 0.05 mM to induce protein expression. The culture was continued at 30°C with shaking for about 4 hours. The fermentation supernatant was then collected by centrifugation.

[0168] The fermentation supernatant was purified in two steps: cation exchange and gel filtration. The purified sample was stored at -20°C for later use. The size and purity of the recombinant protein sample were determined by 15% SDS-PAGE, and the electrophoresis results are shown below. Figure 1 As shown, the main band of the hybrid antimicrobial protein AB469A5 is around 32 kDa on electrophoresis.

[0169] Example 2: AB469A8 hybrid antimicrobial protein

[0170] The recombinant plasmid pET28a-AB469A8 was constructed according to the method in Example 1. The amino acid sequence of AB469A8 is shown in SEQ ID NO.3, and the coding sequence is shown in SEQ ID NO.4. After PCR amplification and verification of pET28a-AB469A8 using AB469A8-specific primers (upstream primer shown in SEQ ID NO.18, downstream primer shown in SEQ ID NO.19), the recombinant plasmid pET28a-AB469A8 was introduced into E. coli for recombinant protein expression and purification.

[0171] The size and purity of the recombinant protein sample were determined using 15% SDS-PAGE, and the electrophoresis results are shown below. Figure 2 As shown, the main band of the hybrid antimicrobial protein AB469A8 is around 32 kDa on electrophoresis.

[0172] SEQ ID NO.3:

[0173] MAILTKDGFGIIRNELFGGKLDQTQVDAINFIVEKATESGLSYPEAAYLLATIYHETGLP 60

[0174] SGYRTMQPIKEAGSDNYLRSKKYYPYIGYGYVQLTWKENYGRIGKLIGIDLIKNPEKALE 120

[0175] PLIAIQIAIKGMLNGWFTGVGFRRKRPVSKYNKQQYIAARNIINGKDKAELIAKYAIIFE 180

[0176] RALRSLGSNSTSNSSTNSGSTGKVSLPNRVIRVTKPIVHGSDVLAIQKALSSLYFYPEKG 240

[0177] AKDNGSDSYYGPKTANAVKRFQSVNGLVADGVYGPKTRAAILKKLRRRRRRRR

[0178] SEQ ID NO.4:

[0179] ccatggctatcctgaccaaagatggctttggcatcattcgcaacgaactgtttggcggca 60

[0180] aactggatcagacacaggtggatgccattaattttattgttgaaaaagctaccgaatctg 120

[0181] ggttaagttatccggaagcggcctatctgttagcgacgatctatcatgaaacgggtctgc 180

[0182] cgagcggttatcgtaccatgcagccaatcaaagaagccggtagtgataattacctccgct 240

[0183] ctaaaaaatattatccgtatatcggctatggctatgttcagctgacgtggaaagaaaatt 300

[0184] atggtcgtattggtaaactgatcggcatcgacttgatcaaaaatccggaaaaagccttag 360

[0185] aaccgctgattgcgattcagattgccatcaaaggtatgctgaatggttggtttacaggtg 420

[0186] tgggctttcgtcgcaaacgtccagtgagtaaatataacaagcagcagtatattgctgcac 480

[0187] gcaatatcattaatggtaaagataaagcagaactgatcgcgaaatatgccatcatctttg 540

[0188] aacgcgccctgcgttctctgggctccaatagtacctctaatagtagcaccaattcaggct 600

[0189] ctaccggtaaagtgagtctgccgaatcgtgtgattcgcgtgaccaaaccgattgttcatg 660

[0190] gtagcgatgtgctggcaattcagaaagcactgtcaagcctgtatttttatccggaaaaag 720

[0191] gtgccaaagataatggttccgatagctattatggtccgaaaaccgccaatgccgttaaac 780

[0192] gctttcagtctgttaatggcttagttgcagatggcgtgtatggcccgaaaacccgcgcag 840

[0193] ccattctgaaaaaactgcgtcgtcgtcgtcgtcgtcgtcgttaagctt

[0194] SEQ ID NO.18: 5'-tataccatggctatcctgaccaaag-3'

[0195] SEQ ID NO.19: 5'-ccgcaagcttaacgacgacgacgacgacgacgacgcagttttttcagaatggctgcgcgggtttt-3'

[0196] Example 3

[0197] Following the methods described in Examples 1 and 2, the hybrid antimicrobial proteins shown in the table below were constructed:

[0198] Table 2

[0199]

[0200] Example 4: Comparative Experiment on the Antibacterial Activity of Hybrid Antibacterial Proteins against Gram-Negative Bacteria

[0201] (1) Test strain: Acinetobacter baumannii (ATCC 19606) was obtained from the U.S. Culture Collection Center.

[0202] (2) Culture of strains: The strains were streaked onto LB agar plates using glycerol tubes. Acinetobacter baumannii was preserved in LB liquid medium: The LB liquid medium used to culture Acinetobacter baumannii was mixed with 30% glycerol at a 1:1 ratio and then stored at -80℃.

[0203] (3) Two-fold dilution micropore method:

[0204] The AB469 hybrid antimicrobial protein, the hybrid antimicrobial proteins AB469A1-AB469A11 and AB469B1-AB469B7 prepared in Examples 1-3 were diluted with LB liquid medium to appropriate starting concentrations (200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, 3.125 μg / mL, and 1.56 μg / mL, respectively). An appropriate amount of Acinetobacter baumannii from the plate was scraped into 2 mL of LB liquid medium, mixed well, and a 0.5 McFarland concentration bacterial suspension was prepared. This suspension was then diluted 100-fold with LB liquid medium (approximately 10...). 6 (cfu / mL).

[0205] 96-well plate sample loading procedure:

[0206] For the negative control, add 200 μL of LB liquid medium to each well.

[0207] Experimental group: Add 100 μL of different diluted protein sample solutions to each well in sequence, then add 50 μL of LB liquid culture medium to each well in sequence, and finally add 50 μL of the above diluted Acinetobacter baumannii bacterial solution to each well. Set up two replicates.

[0208] Incubate at 37℃ for 24 hours and observe whether the bacterial solution in the wells becomes clear. If the first well is turbid, the MIC is considered to be >100 μg / mL. The experimental results are shown in Table 3.

[0209] Table 3. MIC values ​​of AB469 with different C-terminal modifications against Acinetobacter baumannii

[0210]

[0211] As shown in Table 3, when the C-terminal modified protein contains 3-10 consecutive or discontinuous arginine residues, its antibacterial activity against the Gram-negative bacterium Acinetobacter baumannii is increased by 1-23 times compared with the AB469 heterozygous protein, with a MIC value below 50 μg / mL. The MIC value of AB469A6 against Acinetobacter baumannii reaches 3.125 μg / mL. This indicates that the present invention significantly enhances the antibacterial activity of AB469 against Gram-negative bacteria through C-terminal modification.

[0212] When the C-terminal modifiers were 2, 11, or 12 arginine residues, the MIC values ​​were all >100 μg / mL. When the C-terminal modifiers were lysine (K) and histidine (H), although both are basic amino acids, their modification at the C-terminus not only failed to enhance the antibacterial activity of AB469 against Gram-negative bacteria, but actually resulted in a worse antibacterial activity. These comparative experiments demonstrate that only by modifying the C-terminus with the specified number of arginine residues can the inhibitory activity of AB469 against Gram-negative bacteria be significantly improved.

[0213] Example 5: Antibacterial activity of C-terminal modified hybrid antibacterial protein against different Gram-negative bacteria

[0214] Referring to the method shown in Example 4, the antibacterial activities of AB469A5 and AB469A8 against clinically isolated drug-resistant and drug-free Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae were tested. The specific results are shown in Table 4 below:

[0215] Table 4. MIC values ​​(μg / mL) of AB469A5 and AB469A8 against different Gram-negative bacteria.

[0216]

[0217] As can be seen from the data in Table 4, AB469A5 and AB469A8 have significant antibacterial effects against both drug-resistant and drug-free Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae, indicating that the C-terminal modified hybrid antibacterial protein AB469A provided by this invention has significant antibacterial activity against both drug-resistant and drug-free Gram-negative bacteria.

[0218] Example 5: In vitro antibacterial drug containing AB469A5

[0219] The formulation for manufacturing 100 ml of a biological antibacterial preparation containing AB469A5 is as follows:

[0220] AB469A5 0.01g

[0221] HPMC 1.00g

[0222] 0.16g of anhydrous potassium dihydrogen phosphate

[0223] 0.12g of anhydrous disodium hydrogen phosphate

[0224] 3ml of glycerin

[0225] Add water to make up to 100ml

[0226] Preparation method:

[0227] a) Calculate the required amount of excipients according to the prescription and total volume of liquid to be prepared, and weigh them accurately into a clean container.

[0228] b) Add 70% of the total prepared solution to water in the mixing vessel, first dissolve HPMC, disodium hydrogen phosphate and potassium dihydrogen phosphate, and after they are fully dissolved, add glycerin, mix well, then add AB469, and finally dilute to volume with water and mix well.

[0229] c) Sterilization filtration: The prepared disinfectant is passed through a sterilization filter, and the outflowing disinfectant is connected to a sterile container.

[0230] d) Filling: Fill the sterile disinfectant into plastic or glass bottles.

[0231] This antibacterial agent can be used to disinfect wounds or lesions, 1-2 times daily.

[0232] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention. sequence list <110> Shanghai Gaoke Biotechnology Co., Ltd. <120> A C-terminal modified hybrid antimicrobial protein, its pharmaceutical composition and application <130> 210426 <140> 202111659134.0 <141> 2021-12-30 <160> 26 <170> SIPOSequenceListing 1.0 <210> 1 <211> 290 <212> PRT <213> Artificial sequence <400> 1 Met Ala Ile Leu Thr Lys Asp Gly Phe Gly Ile Ile Arg Asn Glu Leu 1 5 10 15 Phe Gly Gly Lys Leu Asp Gln Thr Gln Val Asp Ala Ile Asn Phe Ile 20 25 30 Val Glu Lys Ala Thr Glu Ser Gly Leu Ser Tyr Pro Glu Ala Ala Tyr 35 40 45 Leu Leu Ala Thr Ile Tyr His Glu Thr Gly Leu Pro Ser Gly Tyr Arg 50 55 60 Thr Met Gln Pro Ile Lys Glu Ala Gly Ser Asp Asn Tyr Leu Arg Ser 65 70 75 80 Light Light Tyr Tyr Pro Tyr Ile Gly Tyr Gly Tyr Val Gln Leu Thr Trp 85 90 95 Lys Glu Asn Tyr Gly Arg Ile Gly Lys Leu Ile Gly Ile Asp Leu Ile 100 105 110 Lys Asn Pro Glu Lys Ala Leu Glu Pro Leu Ile Ala Ile Gln Ile Ala 115 120 125 Ile Lys Gly Met Leu Asn Gly Trp Phe Thr Gly Val Gly Phe Arg Arg 130 135 140 Lys Arg Pro Val Ser Lys Tyr Asn Lys Gln Gln Tyr Ile Ala Ala Arg 145 150 155 160 Asn Ile Ile Asn Gly Lys Asp Lys Ala Glu Leu Ile Ala Lys Tyr Ala 165 170 175 Ile Ile Phe Glu Arg Ala Leu Arg Ser Leu Gly Ser Asn Ser Thr Ser 180 185 190 Asn Ser Ser Thr Asn Ser Gly Ser Thr Gly Lys Val Ser Leu Pro Asn 195 200 205 Arg Val Ile Arg Val Thr Lys Pro Ile Val His Gly Ser Asp Val Leu 210 215 220 Ala Ile Gln Lys Ala Leu Ser Ser Leu Tyr Phe Tyr Pro Glu Lys Gly 225 230 235 240 Ala Lys Asp Asn Gly Ser Asp Ser Tyr Tyr Gly Pro Lys Thr Ala Asn 245 250 255 Ala Val Lys Arg Phe Gln Ser Val Asn Gly Leu Val Ala Asp Gly Val 260 265 270 Tyr Gly Pro Lys Thr Arg Ala Ala Ile Leu Lys Lys Leu Arg Arg Arg 275 280 285 Arg Arg 290 <210> 2 <211> 879 <212> DNA <213> Artificial sequence <400> 2 ccatggctat cctgaccaaa gatggctttg gcatcattcg caacgaactg tttggcggca 60 aactggatca gacacaggtg gatgccatta attttattgt tgaaaaagct accgaatctg 120 ggttaagtta tccggaagcg gcctatctgt tagcgacgat ctatcatgaa acgggtctgc 180<00005*** cgagcggtta tcgtaccatg cagccaatca aagaagccgg tagtgataat tacctccgct 240 ctaaaaaata ttatccgtat atcggctatg gctatgttca gctgacgtgg aaagaaaatt 300 atggtcgtat tggtaaactg atcggcatcg acttgatcaa aaatccggaa aaagccttag 360 aaccgctgat tgcgattcag attgccatca aaggtatgct gaatggttgg tttacaggtg tgggctttcg tcgcaaacgt ccagtgagta fathercaa gcagcagtat attgctgcac 480 gcaatatcat taatggtaaa gataaagcag aactgatcgc gaataatgcc atcatctttg aacgcgccct gcgttctctg ggctccaata gtacctctaa tagtagcacc aattcaggct ctaccggtaa agtgagtctg ccgaatcgtg tgattcgcgt gaccaaaccg attgttcatg gtagcgatgt gctggcaatt cagaaagcac tgtcaagcct gtatttttat ccggaaaaag gtgccaaaga taatggttcc gatagctatt atggtccgaa aaccgccaat gccgttaaac gctttcagtc tgttaatggc ttagttgcag atggcgtgta tggcccgaaa acccgcgcag 840 ccattctgaa aaaactgcgt cgtcgtcgtc gttaagctt <210> 3 <211> 293 <212> PRT <213> The snowstorm <400> 3 Met Ala Ile Leu Thr Lys Asp Gly Phe Gly Ile Ile Arg Asn Glu Leu 1 5 10 15 Gly Gly Lys Leu Asp Gln Thr Gln Val Asp Ala Ile Asn Phe Ile Free Mp3 Download 20 25 30 Val Glu Lys Ala Thr Glu Ser Gly Leu Ser Tyr Pro Glu Ala Ala Tyr 35 40 45 Leu Leu Ala Thr Ile Tyr His Glu Thr Gly Leu Pro Ser Gly Tyr Arg 50 55 60 Thr Met Gln Pro Ile Lys Glu Ala Gly Ser Asp Asn Tyr Leu Arg Ser 65 70 75 80 Lys Lys Tyr Tyr Pro Tyr Ile Gly Tyr Gly Tyr Val Gln Leu Thr Trp 85 90 95 Lys Glu Asn Tyr Gly Arg Ile Gly Lys Leu Ile Gly Ile Asp Leu Ile 100 105 110 Lys Asn Pro Glu Lys Ala Leu Glu Pro Leu Ile Ala Ile Gln Ile Ala 115 120 125 Ile Lys Gly Met Leu Asn Gly Trp Phe Thr Gly Val Gly Phe Arg Arg 130 135 140 Lys Arg Pro Val Ser Lys Tyr Asn Lys Gln Gln Tyr Ile Ala Ala Arg 145 150 155 160 Asn Ile Ile Asn Gly Lys Asp Lys Ala Glu Leu Ile Ala Lys Tyr Ala 165 170 175 Ile Ile Phe Glu Arg Ala Leu Arg Ser Leu Gly Ser Asn Ser Thr Ser 180 185 190 Asn Ser Ser Thr Asn Ser Gly Ser Thr Gly Lys Val Ser Leu Pro Asn 195 200 205 Arg Val Ile Arg Val Thr Lys Pro Ile Val His Gly Ser Asp Val Leu 210 215 220 Ala Ile Gln Lys Ala Leu Ser Ser Leu Tyr Phe Tyr Pro Glu Lys Gly 225 230 235 240 Ala Lys Asp Asn Gly Ser Asp Ser Tyr Tyr Gly Pro Lys Thr Ala Asn 245 250 255 Ala Val Lys Arg Phe Gln Ser Val Asn Gly Leu Val Ala Asp Gly Val 260 265 270 Tyr Gly Pro Lys Thr Arg Ala Ala Ile Leu Lys Lys Leu Arg Arg Arg 275 280 285 Arg Arg Arg Arg Arg 290 <210> 4 <211> 888 <212> DNA <213> Artificial sequence <400> 4 ccatggctat cctgaccaaa gatggctttg gcatcattcg caacgaactg tttggcggca 60 aactggatca gacacaggtg gatgccatta attttattgt tgaaaaagct accgaatctg 120 ggttaagtta tccggaagcg gcctatctgt tagcgacgat ctatcatgaa acgggtctgc 180 cgagcggtta tcgtaccatg cagccatca aagaagccgg tagtgataat tacctccgct ctaaaaata ttatccgtat atcggctatg gctatgttca gctgacgtgg aaagaaaatt atggtcgtat tggtaaactg atcggcatcg acttgatcaa aaatccggaa aaagccttag aaccgctgat tgcgattcag attgccatca aaggtatgct gaatggttgg tttacaggtg tgggctttcg tcgcaaacgt ccagtgagta fathercaa gcagcagtat attgctgcac 480 gcaatatcat taatggtaaa gataaagcag aactgatcgc gaataatgcc atcatctttg aacgcgccct gcgttctctg ggctccaata gtacctctaa tagtagcacc aattcaggct ctaccggtaa agtgagtctg ccgaatcgtg tgattcgcgt gaccaaaccg attgttcatg gtagcgatgt gctggcaatt cagaaagcac tgtcaagcct gtatttttat ccggaaaaag gtgccaaaga taatggttcc gatagctatt atggtccgaa aaccgccaat gccgttaaac gctttcagtc tgttaatggc ttagttgcag atggcgtgta tggcccgaaa acccgcgcag 840 ccattctgaa aaaactgcgt cgtcgtcgtc gtcgtcgtcg ttaagctt 888 <210> 5 <211> 301 <212> PRT <213> Artificial Sequence <400> 5 Met Ala Ile Leu Thr Lys Asp Gly Phe Gly Ile Ile Arg Asn Glu Leu 1 5 10 15 Phe Gly Gly Lys Leu Asp Gln Thr Gln Val Asp Ala Ile Asn Phe Ile 20 25 30 Val Glu Lys Ala Thr Glu Ser Gly Leu Ser Tyr Pro Glu Ala Ala Tyr 35 40 45 Leu Leu Ala Thr Ile Tyr His Glu Thr Gly Leu Pro Ser Gly Tyr Arg 50 55 60 Thr Met Gln Pro Ile Lys Glu Ala Gly Ser Asp Asn Tyr Leu Arg Ser 65 70 75 80 Lys Lys Tyr Tyr Pro Tyr Ile Gly Tyr Gly Tyr Val Gln Leu Thr Trp 85 90 95 Lys Glu Asn Tyr Gly Arg Ile Gly Lys Leu Ile Gly Ile Asp Leu Ile 100 105 110 Lys Asn Pro Glu Lys Ala Leu Glu Pro Leu Ile Ala Ile Gln Ile Ala 115 120 125 Ile Lys Gly Met Leu Asn Gly Trp Phe Thr Gly Val Gly Phe Arg Arg 130 135 140 Lys Arg Pro Val Ser Lys Tyr Asn Lys Gln Gln Tyr Ile Ala Ala Arg 145 150 155 160 Asn Ile Ile Asn Gly Lys Asp Lys Ala Glu Leu Ile Ala Lys Tyr Ala 165 170 175 Ile Ile Phe Glu Arg Ala Leu Arg Ser Leu Gly Ser Asn Ser Thr Ser 180 185 190 Asn Ser Ser Thr Asn Ser Gly Ser Thr Gly Lys Val Ser Leu Pro Asn 195 200 205 Arg Val Ile Arg Val Thr Lys Pro Ile Val His Gly Ser Asp Val Leu 210 215 220 Ala Ile Gln Lys Ala Leu Ser Ser Leu Tyr Phe Tyr Pro Glu Lys Gly 225 230 235 240 Ala Lys Asp Asn Gly Ser Asp Ser Tyr Tyr Gly Pro Lys Thr Ala Asn 245 250 255 Ala Val Lys Arg Phe Gln Ser Val Asn Gly Leu Val Ala Asp Gly Val 260 265 270 Tyr Gly Pro Lys Thr Arg Ala Ala Ile Leu Lys Lys Leu Arg Ala Ala 275 280 285 Arg Ala Ala Arg Ala Ala Arg Ala Ala Arg Ala Ala Arg 290 295 300 <210> 6 <211> 912 <212> DNA <213> Artificial sequence <400> 6 ccatggctat cctgaccaaa gatggctttg gcatcattcg caacgaactg tttggcggca 60 aactggatca gacacaggtg gatgccatta attttattgt tgaaaaagct accgaatctg 120 ggttaagtta tccggaagcg gcctatctgt tagcgacgat ctatcatgaa acgggtctgc 180 cgagcggtta tcgtaccatg cagccaatca aagaagccgg tagtgataat tacctccgct 240 ctaaaaaata ttatccgtat atcggctatg gctatgttca gctgacgtgg aaagaaaatt 300 atggtcgtat tggtaaactg atcggcatcg acttgatcaa aaatccggaa aaagccttag 360 aaccgctgat tgcgattcag attgccatca aaggtatgct gaatggttgg tttacaggtg 420 tgggctttcg tcgcaaacgt ccagtgagta aatataacaa gcagcagtat attgctgcac 480 gcaatatcat taatggtaaa gataaagcag aactgatcgc gaaatatgcc atcatctttg 540 aacgcgccct gcgttctctg ggctccaata gtacctctaa tagtagcacc aattcaggct ctaccggtaa agtgagtctg ccgaatcgtg tgattcgcgt gaccaaaccg attgttcatg gtagcgatgt gctggcaatt cagaaagcac tgtcaagcct gtatttttat ccggaaaaag gtgccaaaga taatggttcc gatagctatt atggtccgaa aaccgccaat gccgttaaac gctttcagtc tgttaatggc ttagttgcag atggcgtgta tggcccgaaa acccgcgcag 840 ccattctgaa aaaactgcgt gatgatcgtg atgatcgtga tgatcgtgatgatcgtgatg atcgttaagc tt <210> 7 <211> 3 <212> PRT <213> The snowstorm <400> 7 Arg Arg Arg 1 <210> 8 <211> 4 <212> PRT <213> The snowstorm <400> 8 Arg Arg Arg 1 <210> 9 <211> 5 <212> PRT <213> The snowstorm <400> 9 Arg Arg Arg Arg 1 5 <210> 10 <211> 6 <212> PRT <213> Artificial sequence <400> 10 Arg Arg Arg Arg Arg Arg 1 5 <210> 11 <211> 7 <212> PRT <213> Artificial sequence <400> 11 Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 12 <211> 8 <212> PRT <213> Artificial sequence <400> 12 Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 13 <211> 9 <212> PRT <213> Artificial sequence <400> 13 Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 <210> 14 <211> 10 <212> PRT <213> Artificial sequence <400> 14 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 <210> 15 <211> 16 <212> PRT <213> Artificial sequence <400> 15 Arg Ala Ala Arg Ala Ala Arg Ala Ala Arg Ala Ala Arg Ala Ala Arg 1 5 10 15 <210> 16 <211> 25 <212> DNA <213> Artificial sequence <400> 16 tataccatgg ctatcctgac caaag 25 <210> 17 <211> 56 <212> DNA <213> Artificial sequence <400> 17 ccgcaagctt aacgacgacg acgacgcagt tttttcagaa tggctgcgcg ggtttt 56 <210> 18 <211> 25 <212> DNA <213> Artificial sequence <400> 18 tataccatgg ctatcctgac caaag 25 <210> 19 <211> 65 <212> DNA <213> Artificial sequence <400> 19 ccgcaagctt aacgacgacg acgacgacga cgacgcagtt ttttcagaat ggctgcgcgg 60 gtttt 65 <210> 20 <211> 5 <212> PRT <213> Artificial sequence <400> 20 Lys Lys Lys Lys Lys 1 5 <210> twenty one <211> 8 <212> PRT <213> Artificial sequence <400> twenty one Lys Lys Lys Lys Lys Lys Lys Lys 1 5 <210> twenty two <211> 4 <212> PRT <213> Artificial sequence <400> twenty two His His His His 1 <210> twenty three <211> 8 <212> PRT <213> Artificial sequence <400> twenty three His His His His His His 1 5 <210> twenty four <211> 2 <212> PRT <213> Artificial sequence <400> twenty four Arg Arg 1 <210> 25 <211> 11 <212> PRT <213> Artificial sequence <400> 25 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 <210> 26 <211> 12 <212> PRT <213> Artificial sequence <400> 26 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10

Claims

1. A C-terminal modified antibacterial hybrid protein, characterized in that, Its amino acid sequence is shown in formula (Ⅰ): XY (I) Wherein, X is the modified hybrid antimicrobial protein, which is the AB469 hybrid antimicrobial protein; Y is the C-terminal modification of X, wherein the C-terminal modification is a polypeptide of 5-8 consecutive arginine residues or the amino acid sequence shown in SEQ ID NO.

15.

2. The antibacterial hybrid protein according to claim 1, characterized in that, The amino acid sequence of the C-terminal modified antibacterial hybrid protein is shown in SEQ ID NO.1 or SEQ ID NO.

3.

3. The antibacterial hybrid protein according to claim 1, characterized in that, The amino acid sequence of the C-terminal modified antibacterial hybrid protein is shown in SEQ ID NO.

5.

4. A polynucleotide, characterized in that, The coding sequence of the C-terminal modified antibacterial hybrid protein as described in any one of claims 1-3.

5. The polynucleotide according to claim 4, characterized in that, The nucleotide sequences are shown in SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO.

6.

6. An expression carrier, characterized in that, Includes the polynucleotides as described in claim 4 or 5.

7. An engineered bacterium, characterized in that, Includes the polynucleotide of claim 4 or 5, or the expression vector of claim 6.

8. A pharmaceutical composition, characterized in that, It includes the C-terminal modified antimicrobial hybrid protein as described in any one of claims 1-3, and pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, characterized in that, When the pharmaceutical composition is a topical preparation, the mass concentration of the C-terminal modified antibacterial hybrid protein does not exceed 0.5%.

10. The use of the C-terminal modified antibacterial hybrid protein of any one of claims 1-3, the polynucleotide of any one of claims 4-5, the expression vector of claim 6, the engineered bacteria of claim 7, or the pharmaceutical composition of any one of claims 8-9 in the preparation of antibacterial drugs.

11. The application according to claim 10, characterized in that, The antibacterial drug is a drug that inhibits Gram-negative bacteria.

12. The application according to claim 11, characterized in that, The Gram-negative bacteria include one or more of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella, and Escherichia coli.

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