Antibacterial protein targeting enterococcus gallinarum and preparation method and application thereof

Abstract

The present application provides an antibacterial protein targeting Enterococcus gallinarum and a preparation method and application thereof. Specifically, the present application provides an antibacterial protein targeting Enterococcus gallinarum or a chimera thereof, wherein the antibacterial protein comprises one or more of the following proteins formed by the following amino acid sequences: (a) an amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 9; (b) a derived sequence obtained by replacing, adding and / or deleting one or more amino acids from the amino acid sequence of (a) and having the same function as (a); and (c) a derived amino acid sequence having an identity of more than 85% or more than 90% with any one of SEQ ID NO: 1 to SEQ ID NO: 9. The antibacterial protein of the present application can efficiently kill Enterococcus gallinarum and has specificity.

Description

Technical Field

[0001] This invention provides an antimicrobial protein targeting Enterococcus maximus, its preparation method, and its application. Background Technology

[0002] *Enterococcus gallinarum* is a Gram-positive, facultative anaerobic bacterium belonging to the genus *Enterococcus*. It is a Gram-positive bacterium found in commensal colonies with poultry and humans. Recent studies have found that in cases of weakened immunity or impaired intestinal barrier function, this bacterium can migrate from the intestine to organs such as the liver, spleen, and lymph nodes, causing systemic infections or exacerbating autoimmune diseases. Furthermore, *Enterococcus gallinarum* exhibits natural resistance to multiple antibiotics (such as vancomycin), making its treatment in infection and disease management challenging.

[0003] Infectious diseases associated with Enterococcus garutus: 1. Enterococcus garutus can be transmitted through the bloodstream, causing bacteremia, especially common in critically ill patients or those with compromised immune systems (such as cancer and organ transplant patients). 2. The intestinal origin of Enterococcus garutus makes it prone to causing peritonitis and other intra-abdominal infections after gastrointestinal surgery or trauma. 3. Occasionally, there are reports of infective endocarditis caused by Enterococcus garutus, usually involving native or prosthetic valves. 4. Enterococcus garutus commonly causes urinary tract infections in the elderly and long-term catheter users; these infections are often resistant to multiple antibiotics, increasing the complexity of treatment.

[0004] Non-infectious diseases associated with Enterococcus garutus: Enterococcus garutus is also considered to play an important role in a variety of non-infectious diseases, especially in autoimmune diseases. Enterococcus garutus can migrate from the intestines to tissues such as the liver and lymph nodes, triggering an overreaction of the immune system. This migration is often accompanied by disruption of the intestinal barrier, leading to systemic inflammatory responses. This disruption of the intestinal barrier is not only the basis for infection but also an important contributing factor to autoimmune diseases. Studies have found that intestinal metastasis of Enterococcus garutus can induce or exacerbate autoimmune diseases such as systemic lupus erythematosus and autoimmune hepatitis.

[0005] Although research on Enterococcus fowleri is still in its early stages, evidence already suggests it plays an important role in both infectious and non-infectious diseases. Treatment for Enterococcus fowleri infections currently relies primarily on antibiotics, but drug resistance exacerbates the challenge. Future research should focus on developing novel antimicrobial strategies to address the pathogenicity of Enterococcus fowleri in humans.

[0006] Currently, the main treatment for Enterococcus faecium infection in chickens is broad-spectrum antibiotics, but these methods have some significant problems. The main issues with existing treatment techniques are: 1. Antibiotic resistance: Enterococcus faecium is gradually developing resistance to many commonly used antibiotics, especially vancomycin. This reduces the effectiveness of broad-spectrum antibiotic treatment and easily leads to the further spread of drug-resistant strains. During treatment, doctors often face the challenge of having a limited selection of antibiotics. 2. Disruption of the gut microbiota: Antibiotics not only kill Enterococcus faecium but also affect beneficial bacteria in the gut, leading to gut microbiota imbalance. Disruption of the gut microbiota can trigger more serious secondary infections, such as Clostridium difficile-associated infections, and may even worsen the patient's condition. 3. Non-specific effects: Current antibiotic therapies lack specificity and often act on the entire bacterial population. This non-targeted treatment can harm normal flora, especially with long-term use, increasing the risk of opportunistic infections by other pathogens (such as fungi and drug-resistant bacteria). 4. Immune-related side effects: Treatment of Enterococcus faecium infection is particularly challenging for immunosuppressed patients. Antibiotics themselves may not be effective in controlling infection in individuals with weakened immune systems, and further suppression of the immune system by these drugs may worsen the condition. Furthermore, the use of immunomodulatory therapies carries side effects and uncertainties, making it difficult to ensure their effectiveness in all patients.

[0007] In summary, antibiotic treatment of Enterococcus faecium in chickens faces challenges such as drug resistance, disruption of the gut microbiota, and nonspecific side effects. New and effective methods for dealing with Enterococcus faecium infections in chickens need to be developed, especially in the face of complex and multidrug-resistant infections. Summary of the Invention

[0008] The inventors in this case discovered a class of antimicrobial proteins that target Enterococcus faecium in chickens, which can effectively kill Enterococcus faecium from different sources, including antibiotic-resistant strains, and are specific.

[0009] Specifically, on one hand, the present invention provides an antimicrobial protein or a chimeric thereof targeting Enterococcus faecium in chickens, wherein the antimicrobial protein comprises one or more proteins formed from the following amino acid sequences:

[0010] (a) Any of the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:9;

[0011] (b) A derivative sequence of (a) with one or more amino acids replaced, added and / or deleted, and having the same function as (a);

[0012] (c) A derived amino acid sequence that is 85% or 90% or more identical to any one of the sequences SEQ ID NO:1 to SEQ ID NO:9.

[0013] According to a specific embodiment of the present invention, the antimicrobial protein or its chimeric compound targeting Enterococcus faecium of chickens of the present invention, wherein the chimeric compound comprises:

[0014] A chimeric protein comprising two or more sequences from the complete amino acid sequence of the antimicrobial protein described in (a), (b), and (c), the catalytic domain sequence of the antimicrobial protein, and the cell wall binding domain sequence of the antimicrobial protein. For example, it may be a chimeric protein composed of any one complete amino acid sequence of the antimicrobial protein from (a), (b), and (c), and the catalytic domain sequence and / or the cell wall binding domain sequence of the antimicrobial protein; or it may be a chimeric protein formed by combining at least two or more sequences from the catalytic domain sequence and the cell wall binding domain sequence of the antimicrobial protein.

[0015] According to a specific embodiment of the present invention, the proteins formed by any of the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:9 are named LysEG01 to LysEG9, respectively, and the specific amino acid sequences are as follows.

[0016] LysEG01 protein sequence (SEQ ID NO:1):

[0017] MKYINKSVCRGVAGRRARGWKPLGIVIHNDGGSMTPEAYVEWLAAKSTTQLQAGFAHYYGNRNTMARVEDTYNGAWHVANYNGNMDYVGYEVCESLKVSDKDFIANEEAVFQQAAKDMKDWGLTPSRTTVKLHRAFVPTDCPHRSWALHVGRGAADTTSNRNKLVDYFVAR IKHYMGGGSNNSNSGSKPNTGGNSGGNKITNTTKFNNGDKVKVLAKATNYQTGQSIPSFVKGQTYTVKDSKVVNQSVSKYAFLLDGVNSWFLAQDLQKVSGGSTGYAAKKVGDTVTVQSFASRYQTGEPIASFVKGKKYKIKQVKSVKQSKSKRAYLLDGIQSWVLEQDVK

[0018] LysEG02 protein sequence (SEQ ID NO:2):

[0019] MSIENMISWMTSKEGRVNYSMTSRLGPNSYDCSSAVFFAMIAGGFLPAGTMGNTETLFAMNGTVLKKISRAEVRRGDIFIAGTPGASHGSEGHTGIFLSNKSFIHCSYYWNGIHTDSHDSYMSTRLTHHFYRIVASGNADVNTDNSAQMIKLAIDGQWGPLVTLRLQEYYNTTRDKVISHQYKQKYNKNIYSAEFDITLIGSDVIRAIQMGLKARGYYSGSIDGLCGEATIKAMQKALGTTVDGIISPVSDMVKALQRALNNNKLPW

[0020] LysEG03 protein sequence (SEQ ID NO: 3):

[0021] MSIPKPVILDISEWQTPNSINYDKLASAVDGVIVRIQYGSRYIDKHYKTHIAEFQKRGVPVAVYAWVRGVSNSDMEKEATDFYNRAKAYNPTFWWLDVEEKSMSDMRTGIEKYRAKFKSLGAKKVGAYIANHLYASFNLDTSKFDGIWIPTYGSNNGQYNGSNPTATSNYDIHQYTSNGKLSGYSGPLDLNRIVRKGFEYFFGNSSTGSNTDNGSSSNSTTGGKIKMKTITLKANVNLRVSANTNSKIIATLKKGSKVEFNDIVTGSGYIWGVQPRTDSYKKGYIAIGKISDWGNII

[0022] LysEG04 protein sequence (SEQ ID NO: 4):

[0023] MTVSANTILTEARKYLGTVMGKTPHKNIVDKYNATKPLPVGYQVKYTDDWCDTFVSFIGIKTGATDLIGRECGVQRHIDIFKSKGIWIEDGKTTPKPGDIVVFNWDDVTQPNDGWADHIGFVESVSNGVITTIEGNYGRQVKRRTMPVGWGYIRGYARPAYGASTSSGGTAGQKTIETIAKEVINGAWGNGDDRKKKLTAAGYSYDAVQAKVTELLKGSSSSQASTNPFANIVIDSNWDGELNGYLQRYYGTTIDKVISGQIKGDWNAGMTGIKYGTGGSDLVAAIQKDLGLKVDRNMGPATIGSMQAKAGTAVDRKITRPSALAKQIKTNLKTKGKPW

[0024] LysEG05 protein sequence (SEQ ID NO:5):

[0025] MVKYNNSTSHRGHNAIVPGASGCGLKEHEVAQKIHDKFRAVTKAVDATDNVGRTANDNLYNIVKKMNAVGKSFHVSHHLNAFNGTANGFEVWYYAGNAQAKKLAEEICTAVCKVTGWVNRGAKATTSLYVIRASTGSAILIEWGFVDSQKDMDIMAKKMDAAVNATLAVFGYSANSSNNNNSSTSKPAASTSFKVGDKVKITDALYKDSTGAGRSTASRGKTGTIKRVVSGNKPYLIDSLGWAHKNDIQLATTTTTTTKKVGDTVTVQNHATKYQTGQNIPSWVKGKKYKIKQIKSVNQSKSKKAYLLDGINSWFLEQDVK

[0026] LysEG06 protein sequence (SEQ ID NO:6):

[0027] MNKKKYVLLVSLISSIGIQSTTALASMQEQPPYYISSIGQMDNYMQLSKNVNSSTVEKMDKLVKPEESTSPSEDKTEVKPEESTSPSEDKTEVKPEESTSPSEDKTEVKPEESTSPSEDKTEVKPEESTSPSEDKTEVKPEESTSPSEDKTEAFIPEKIINDNQFTDDANSKNNNFVDSYSVKVPNELTQEFIKKIGEIARKIGQEKDLYASVIIAQAILESGSGQSSLASAPNYNLFGIKGNYKGNSVSFLTFEDNGVGNLVSVKAIFRKYTSYKEALEDYANLMEYGLSHNKNFYHGAKKSNSKTYEEATKFLTGKYATDINYNKKLNQIIKTYDLTKYDHPKQKLILKKSSNINKKIFEVANPINNQLEDFILPLSDNYTISSHFGFRGPDHHDGIDLATSFGTPIYVSSEGQVINTGFDSSAGNYVIVKHPNGLYTNYFHMSETNVTVGQTIQIGEVIGYVGSTGNSTGPHLHFGISTNEWHNYLNPSDYLDFN

[0028] LysEG07 protein sequence (SEQ ID NO:7):

[0029] MVVSYSGIAGKRGKNPTKIVIHNDGGSQGATAAFYKGWLEHHQPELGFAHYYVASDGTYQAEVDGNSAWHCANYVGNRDYIGIEVCQSLGKESTFLENEQEAFKLAASLCKKYGLNPSVSVFPLHRELSATDCPHRSFALHGKANAGVKSYYVSQVQKYMGNTSNTGSNSSSKPSKPSASTGISIDGMWGIATTKRLQKYLGTTQDGVISHQYKQKYNQNIYSAQFDKTLIGSNVIKAMQKRLKAKGLYSGDLDGLCGQSTVKALQKAFGTTQDGVISPVSNMVSAMQKALNNNKLPF

[0030] LysEG08 protein sequence (SEQ ID NO:8):

[0031] MTIQSVHAGHGGKKNGNAWTDPGAVGNGYKEADVARTITDLMVKKTGAKNVTDNTSSTSNGIINNVAANINKCTDGWQISNHLNAFNGKATGVEVLYGSASNKATAAKVSAAIAKTLGLVDRGAKDGSWLGIARNSGSGKKVLLIEWGFIDNASDMKALFAKMDAAVNAALAVFGYSANSSSNNNTSTSKPAASTSFKVGDKVKIVDALYKDNTGAGRSTASRGKTGTIKRVASGNKPYLIDSLGWAHKNDIQLVTSSNATYKVGDTVTVQSFATNYQTGQKIPNWVKGQKYKIKKVKSVNQSRSKKAYLLDDVNSWFLEQDIK

[0032] LysEG9 protein sequence (SEQ ID NO:9):

[0033] MSFIKYEYIRINKFSRPGIKNYGIKGIIMHYTANNGGTARNHKDYFNNLNGVYASAHLFVDDNEAICIIPLDEVAYHANDTVRYNSDGSIYKPLYSQIGNANYGAIGVEMCLDRNGNITEKTFQNTVKAVKELIAKYPNITRNKIWRHYDVTGKNCPAPWVAKPSELERFKDAAFGKTSGSNSAAKPSTPSVKPNTNKISEDGMFGPSTANKAMQYEGITPDDEISHQYRQVCNKNLYAAQFDNTLKGSTLIRTWQKRLKAKGLYKGAIDGLCGTEMIKAMQRALKTTVDGIISPTSNMVKALQRALNNNKLPW

[0034] The protein formed by any of the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:9 of this invention contains catalytic and cell wall-binding domains and their location information, as shown in Table 1. The catalytic domains include Amidase_2, Amidase_3, Amidase_5, CHAP, Glyco_hydro_25, Peptidase_M23, and Glucosaminidase. The cell wall-binding domains include SH3, CW_7, and PG_binding_1, etc. As can be seen from Table 1, the lyase LysEG01 shares the same catalytic domain as LysEG07 and LysEG09, and the lyase LysEG01 shares the same cell wall-binding domain as LysEG03, LysEG05, and LysEG08.

[0035] Table 1: Catalytic and cell wall-binding domains of antimicrobial proteins LysEG01 to LysEG09

[0036]

[0037] According to some specific embodiments of the present invention, the antimicrobial protein or its chimeric compound targeting Enterococcus avianus of the present invention comprises one or more of the catalytic domains and / or cell wall binding domains shown in Table 1, for example, comprising one or more of the catalytic domains Amidase_2, Amidase_3, Amidase_5, CHAP, Glyco_hydro_25, Peptidase_M23, Glucosaminidase and the cell wall binding domains SH3, CW_7, PG_binding_1.

[0038] According to some specific embodiments of the present invention, the antimicrobial protein or its chimeric compound targeting Enterococcus avianus of the present invention comprises a catalytic domain Amidase_2 and / or a cell wall binding domain SH3.

[0039] On the other hand, the present invention also provides a nucleic acid molecule that encodes the antimicrobial protein or a chimeric thereof targeting Enterococcus chrysogenum as described in the present invention.

[0040] According to a specific embodiment of the present invention, the nucleotide sequence of the nucleic acid molecule of the present invention includes any of the sequences shown in SEQ ID NO:10 to SEQ ID NO:18.

[0041] LysEG01 nucleic acid sequence (SEQ ID NO:10):

[0042]

[0043] Nucleic acid sequence of LysEG02 (SEQ ID NO:11):

[0044] ATGAGTATTGAGAATATGATCTCCTGGATGACGAGCAAGGAGGGTCGTGTTAACTATAGCATGACCAGCCGCTTAGGCCCCAACTCCTACGATTGTTCAAGTGCTGTTTTTTTTGCCATGATTGCCGGTGGATTTCTTCCTGCCGGGACAATGGGCAATACTGAAACCCTGTTTGCAATGAATGGTACGGTTTTAAAAAAAATTTCACGCGCTGAGGTGCGCCGCGGGGACATCTTTATCGCAGGCACCCCGGGAGCATCGCATGGTTCTGAAGGCCACACCGGTATTTTTCTCTCGAATAAGTCCTTTATCCATTGTTCATATTACTGGAATGGGATTCACACCGACAGCCATGATTCCTATATGAGCACCCGCTTGACGCACCATTTTTATCGTATCGTTGCATCGGGGAATGCCGACGTCAACACGGATAATAGCGCACAGATGATTAAACTGGCCATCGATGGCCAGTGGGGCCCATTGGTCACGCTGCGCCTGCAGGAATACTACAATACTACGCGCGACAAAGTGATCAGCCACCAGTATAAACAGAAATATAATAAAAACATTTATTCGGCTGAGTTCGATATCACTCTTATTGGGTCCGATGTTATTCGCGCAATCCAGATGGGGCTGAAAGCACGTGGTTACTACAGCGGTAGTATTGATGGTCTGTGTGGTGAAGCCACGATTAAGGCGATGCAGAAAGCTCTCGGCACAACCGTAGATGGTATCATTTCGCCAGTGTCCGACATGGTTAAGGCCCTGCAACGCGCCCTTAACAATAACAAGCTCCCGTGGTAA

[0045] Nucleic acid sequence of LysEG03 (SEQ ID NO:12):

[0046] ATGTCAATCCCGAAACCGGTGATCCTCGATATTTCTGAATGGCAGACACCGAATAGTATTAACTATGATAAACTGGCTTCTGCGGTTGATGGCGTAATCGTCCGTATCCAGTATGGGAGTCGCTATATTGATAAACATTATAAAACACATATTGCGGAATTCCAGAAGCGTGGGGTGCCCGTCGCAGTATATGCTTGGGTCCGTGGCGTGTCCAATTCAGATATGGAAAAGGAGGCCACCGATTTTTACAACCGTGCAAAAGCCTACAACCCGACTTTTTGGTGGTTAGATGTGGAGGAAAAATCCATGTCCGATATGCGCACGGGGATCGAAAAATACCGTGCAAAATTCAAATCGCTGGGCGCTAAGAAGGTTGGCGCATACATTGCCAATCATCTCTACGCGTCCTTTAACCTGGATACCAGCAAATTTGACGGCATTTGGATTCCGACGTATGGCTCCAACAACGGCCAATACAACGGTAGCAATCCTACTGCTACGAGCAATTATGACATCCACCAGTACACGAGTAACGGCAAACTCTCGGGTTATTCCGGTCCGTTAGACCTTAACCGTATTGTGCGCAAAGGGTTTGAATATTTTTTCGGTAACAGCTCCACCGGGTCCAACACTGATAATGGTTCAAGCAGCAACTCGACAACCGGTGGTAAAATCAAGATGAAGACTATTACGCTGAAAGCGAATGTAAACCTGCGCGTAAGCGCCAATACGAATTCTAAGATCATTGCAACCTTAAAGAAAGGTTCCAAAGTTGAGTTTAATGACATTGTCACGGGGAGCGGGTATATCTGGGGTGTTCAGCCGCGCACTGACAGTTATAAAAAAGGTTATATCGCTATCGGCAAAATTAGCGACTGGGGTAACATTATCTAA

[0047] Nucleic acid sequence of LysEG04 (SEQ ID NO: 13):

[0048]

[0049] LysEG05 nucleic acid sequence (SEQ ID NO:14):

[0050] ATGGTAAAATATAACAACTCGACCTCCCACCGCGGTCACAACGCTATTGTGCCGGGCGCTAGCGGCTGCGGGCTGAAGGAACATGAAGTCGCACAAAAAATCCACGACAAATTCCGTGCCGTCACGAAAGCCGTCGACGCCACCGACAATGTAGGCCGCACCGCCAATGACAACCTTTACAACATTGTCAAGAAGATGAACGCCGTGGGGAAATCTTTCCACGTGTCCCATCACCTGAACGCATTCAATGGTACGGCAAATGGCTTTGAAGTGTGGTATTATGCCGGGAACGCCCAAGCCAAAAAGTTAGCCGAAGAAATTTGCACTGCCGTATGCAAAGTCACTGGTTGGGTGAATCGCGGAGCAAAGGCTACCACCAGCCTGTACGTGATCCGCGCCAGCACGGGAAGCGCAATCCTCATTGAATGGGGCTTTGTTGATTCGCAAAAAGACATGGACATCATGGCAAAAAAAATGGACGCTGCCGTCAATGCCACCCTGGCCGTCTTCGGTTACTCTGCCAACTCTTCCAATAACAACAACAGCAGCACCAGCAAACCGGCGGCGAGTACGTCCTTTAAAGTGGGCGACAAAGTCAAAATTACCGATGCCCTTTATAAAGATAGCACCGGTGCGGGTCGCTCCACCGCTAGCCGCGGCAAAACGGGAACCATTAAGCGCGTGGTGAGCGGTAATAAACCGTACTTGATCGATAGTCTGGGATGGGCTCATAAGAACGATATCCAGTTAGCAACTACGACGACCACGACCACCAAAAAAGTGGGCGATACCGTGACCGTGCAGAATCATGCAACTAAATACCAAACCGGCCAGAACATCCCGTCATGGGTTAAGGGTAAAAAGTATAAAATCAAACAGATTAAGTCGGTTAACCAGAGTAAATCTAAGAAAGCATATCTGCTGGATGGCATTAATAGCTGGTTTTTAGAACAGGACGTTAAATAA

[0051] LysEG06 nucleic acid sequence (SEQ ID NO:15):

[0052]

[0053] LysEG07 nucleic acid sequence (SEQ ID NO:16):

[0054] ATGGTTGTTTCATATAGCGGGATTGCTGGCAAACGCGGTAAAAACCCGACCAAAATTGTTATTCATAATGATGGTGGTTCCCAAGGTGCAACCGCCGCGTTCTATAAGGGCTGGTTAGAGCATCACCAGCCGGAACTGGGCTTCGCCCATTACTATGTTGCTAGTGATGGCACGTATCAGGCCGAAGTTGATGGCAACTCAGCTTGGCATTGTGCAAACTACGTAGGCAACCGTGATTATATTGGGATCGAAGTTTGTCAGAGCCTTGGTAAGGAAAGTACCTTCTTAGAAAACGAACAAGAAGCTTTTAAACTGGCGGCCTCCTTATGTAAAAAATATGGTCTCAACCCATCCGTGAGCGTGTTCCCATTACACCGTGAGTTGTCCGCAACCGATTGTCCTCATCGCAGTTTTGCTCTCCATGGAAAAGCTAATGCAGGTGTCAAATCCTATTATGTTTCCCAAGTACAGAAATATATGGGGAACACCAGTAATACAGGAAGTAATTCTTCTTCCAAACCGTCCAAGCCATCGGCGAGCACTGGTATTTCCATCGATGGCATGTGGGGTATCGCAACAACCAAACGTCTGCAGAAATACCTGGGTACGACCCAGGATGGGGTTATTAGCCATCAGTACAAACAGAAATACAATCAGAATATCTATTCCGCACAGTTTGATAAAACCCTGATTGGGTCTAACGTTATCAAAGCCATGCAAAAACGTCTGAAAGCTAAAGGCCTTTACAGCGGCGACCTGGATGGCTTGTGTGGGCAGAGCACCGTCAAAGCTTTACAAAAAGCCTTTGGAACCACGCAAGACGGTGTCATCTCACCAGTGAGCAACATGGTGAGCGCCATGCAGAAAGCGTTGAATAATAACAAACTGCCGTTCTAA

[0055] LysEG08 nucleic acid sequence (SEQ ID NO:17):

[0056] ATGACCATTCAGTCAGTGCACGCAGGCCATGGGGGTAAAAAAAACGGTAATGCGTGGACCGATCCGGGTGCAGTCGGGAACGGCTACAAAGAAGCTGACGTGGCCCGCACCATTACCGATCTTATGGTTAAAAAGACCGGCGCCAAGAACGTGACCGACAACACCAGTAGTACCTCTAACGGCATTATCAACAACGTTGCTGCCAATATCAACAAATGTACAGACGGCTGGCAGATCAGTAACCATCTGAACGCCTTCAACGGTAAAGCCACGGGTGTGGAAGTACTGTACGGCTCGGCATCTAATAAAGCGACCGCCGCGAAGGTGTCGGCCGCCATTGCCAAGACCCTCGGACTCGTGGATCGCGGTGCCAAAGATGGCTCCTGGCTCGGTATCGCACGCAACAGCGGCAGCGGTAAAAAAGTTCTGCTTATTGAATGGGGGTTCATCGACAATGCTAGTGACATGAAAGCACTGTTCGCTAAAATGGACGCCGCAGTCAACGCTGCGTTGGCCGTTTTCGGCTACAGCGCGAACTCCTCAAGTAATAATAATACCAGCACCTCCAAACCTGCTGCATCTACGAGCTTCAAGGTAGGGGATAAAGTCAAAATCGTCGATGCACTCTACAAAGATAACACGGGTGCCGGTCGCTCCACCGCCTCTCGTGGTAAGACCGGGACTATCAAACGTGTCGCATCCGGTAACAAGCCATACCTGATCGATAGTCTGGGTTGGGCCCATAAAAATGACATCCAATTAGTCACGTCGTCCAACGCTACCTATAAAGTAGGCGATACAGTCACCGTTCAATCCTTCGCAACTAATTATCAGACGGGGCAAAAAATCCCGAACTGGGTGAAAGGCCAGAAATATAAGATTAAGAAAGTGAAATCTGTGAACCAATCCCGTAGCAAGAAAGCCTACCTGTTGGATGACGTTAACAGTTGGTTCTTGGAGCAGGATATTAAGTAA

[0057] LysEG09 nucleic acid sequence (SEQ ID NO: 18):

[0058] ATGAGCTTCATCAAATACGAATATATCCGTATCAACAAATTCAGTCGTCCTGGTATTAAGAACTATGGTATCAAGGGTATTATCATGCATTATACGGCGAACAATGGTGGCACAGCTCGTAATCATAAAGATTATTTCAACAACTTGAACGGTGTCTACGCTTCAGCCCACCTGTTCGTTGATGATAATGAGGCCATCTGTATCATCCCGCTCGATGAAGTTGCCTATCATGCCAACGACACCGTCCGTTACAATTCGGACGGTAGTATTTACAAGCCGCTGTACAGCCAGATTGGTAACGCAAATTACGGTGCTATTGGCGTTGAGATGTGTTTAGATCGTAACGGCAATATCACCGAAAAAACTTTCCAGAACACCGTTAAGGCCGTGAAAGAGTTGATTGCCAAATATCCGAATATCACCCGTAACAAAATTTGGCGCCACTACGATGTGACGGGCAAAAATTGCCCGGCCCCATGGGTCGCTAAACCAAGCGAATTGGAGCGCTTTAAAGACGCGGCATTCGGTAAAACCTCTGGCTCTAATTCCGCCGCAAAGCCGTCGACCCCGTCCGTAAAGCCAAACACCAATAAAATCAGCGAAGACGGAATGTTCGGCCCCAGTACTGCCAACAAAGCTATGCAGTATGAAGGTATCACGCCCGATGATGAGATTTCGCATCAATATCGTCAAGTGTGTAACAAGAACTTGTACGCCGCCCAATTCGATAACACACTGAAAGGCAGTACGCTGATCCGCACCTGGCAAAAGCGTCTTAAAGCAAAAGGCCTTTACAAGGGTGCCATTGACGGACTGTGTGGAACTGAAATGATCAAAGCCATGCAGCGCGCGTTAAAAACGACCGTCGACGGCATTATCAGTCCGACTAGCAACATGGTCAAGGCGCTGCAGCGCGCACTGAACAATAATAAGCTGCCGTGGTAA

[0059] According to a specific embodiment of the present invention, the nucleic acid molecule described herein is a DNA molecule or an RNA molecule.

[0060] According to specific embodiments of the present invention, the nucleic acid molecules described herein may be modified or unmodified. The modifications may include, for example, chemical modification: modifying nucleic acid molecules by introducing chemical groups, such as phosphate groups, methyl groups, acetyl groups, etc., to alter their stability, affinity, targeting, and other properties. Enzymatic modification: modifying nucleic acids by using specific enzymes, such as processing nucleic acids through restriction endonuclease cleavage, ligase ligation, DNA methylation, RNA editing, etc. Nucleotide substitution: replacing or inserting specific nucleotide sequences to improve the function of nucleic acid molecules or enhance their stability. For example, using modified nucleotides (such as 5-methylcytosine) to regulate gene expression. Synthetic modification: using artificial synthesis methods to perform specific chemical modifications on nucleic acid molecules, such as adding special modifying groups during DNA or RNA synthesis to improve their stability in vivo or enhance their activity in specific environments.

[0061] On the other hand, the present invention also provides a carrier comprising the nucleic acid molecules described herein.

[0062] According to a specific embodiment of the present invention, the vector may be a pET28a(+) or pCold II vector recombinant plasmid loaded with the nucleic acid molecule (e.g., DNA sequence) described in the present invention.

[0063] On the other hand, the present invention also provides a host cell comprising the nucleic acid molecules or the vectors described herein.

[0064] According to a specific embodiment of the present invention, the host cell can be a competent Escherichia coli cell, such as BL21(DE3).

[0065] On the other hand, the present invention also provides a method for preparing the aforementioned antimicrobial protein, the method comprising:

[0066] The antimicrobial protein of this invention is prepared by in vitro transcription using the nucleic acid molecule, the vector, or the host cell described in this invention.

[0067] On the other hand, the present invention also provides an antibacterial pharmaceutical composition comprising: the antibacterial protein or the nucleic acid molecule or the carrier described herein, and a pharmaceutically acceptable excipient.

[0068] On the other hand, the present invention also provides the use of the aforementioned antimicrobial protein or the aforementioned antimicrobial drug composition in the preparation of products targeting Enterococcus chrysogenum.

[0069] On the other hand, the present invention also provides the use of the antimicrobial protein or the antimicrobial drug composition in the preparation of products for the prevention and treatment of diseases related to Enterococcus faecium in chickens;

[0070] According to specific embodiments of the present invention, diseases related to Enterococcus foetida include one or more of the following: bacteremia, peritonitis and other intra-abdominal infections following gastrointestinal surgery or trauma, infective endocarditis caused by Enterococcus foetida, urinary tract infections caused by Enterococcus foetida in the elderly and long-term catheter users, non-infectious diseases related to Enterococcus foetida, and autoimmune diseases such as systemic lupus erythematosus and autoimmune hepatitis induced or exacerbated by intestinal metastasis of Enterococcus foetida.

[0071] According to a specific embodiment of the present invention, experiments were conducted using *Enterococcus gallinarum* DO103, isolated from the human intestine, as an example. This *Enterococcus gallinarum* genome contains the vancomycin resistance gene *VanC1XY* and the tetracycline resistance gene *tet(S)*, exhibiting resistance to vancomycin and various tetracycline antibiotics. This can potentially cause bacteremia, abdominal infection, endometritis, urinary tract infection, and, through enterohepatic metastasis, induce or exacerbate autoimmune diseases such as systemic lupus erythematosus and autoimmune hepatitis. The antimicrobial protein of the present invention can effectively kill this antibiotic-resistant *Enterococcus gallinarum*.

[0072] According to specific embodiments of the present invention, the product of the present invention can be a pharmaceutical preparation, a disinfectant preparation, or a bactericide.

[0073] The antimicrobial protein of the present invention, which targets Enterococcus fowleri, can effectively kill Enterococcus fowleri from different sources, including antibiotic-resistant strains, and is specific. Attached Figure Description

[0074] Figure 1 Map of the LysEG01 expression vector plasmid for the constructed lysin.

[0075] Figure 2 Electrophoresis diagram for identifying heterologous expression of LysEG01 in Escherichia coli.

[0076] Figure 3 This is an electrophoresis image of the purified LysEG01 protein.

[0077] Figure 4 Experimental results show that LysEG01 can efficiently lyse Enterococcus faecium in chickens.

[0078] Figure 5 LysEG01 exhibits lytic activity against Enterococcus spp.

[0079] Figure 6LysEG01 exhibits lytic activity against gut symbiotic bacteria. Detailed Implementation

[0080] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0081] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0082] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0083] Example 1: Identification and protein sequence analysis of lysin genes targeting Enterococcus fowleri.

[0084] Genomic sequences of bacteriophages and prephages targeting Enterococcus jirovecii were collected, and lyase genes were identified using BLAST and Interproscan through sequence alignment and domain alignment. More than 180 potential bacteriophage lyase gene sequences were discovered. This invention further identified a class of lyases containing the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:9, named LysEG01-LysEG09 respectively.

[0085] The codon-optimized nucleic acid sequences of the lysin for E. coli are shown in SEQ ID NO:10 to SEQ ID NO:18.

[0086] Example 2: Heterologous expression and purification of lysin targeting Enterococcus faecium in chickens

[0087] The identified lyase genes were codon-optimized in E. coli, and then the corresponding genes were cloned into pET28a(+) or pCold II vectors. A 6x-His tag was added to the C-terminus or N-terminus, and the genes were controlled by the lactose operon. Figure 1 This is a map of the LysEG01 expression vector plasmid for the constructed lysin.

[0088] The constructed plasmid containing the LysEG01 lysin gene was transformed into BL21(DE3) competent cells, then evenly spread onto LB agar plates (containing 50 μg / mL kanamycin sulfate), and incubated overnight at 37°C. Single clones were selected from the transformed plates and inoculated into 1L of LB medium (containing 50 μg / mL kanamycin sulfate). After incubation until the OD600 reached 0.6-0.8, IPTG was added to the culture medium to a final concentration of 0.1-1 mM, and the cells were incubated at 16-37°C and 100 rpm for 18 h to induce protein expression.

[0089] Centrifuge the induced culture medium at 12000 rpm for 5 min to remove the culture medium. Resuspend the strain in PBS to wash it. Finally, add SDS-PAGE loading buffer and heat the sample at 100℃ for 30 min. Centrifuge and collect the supernatant for electrophoresis. For the first 10 min of electrophoresis, maintain a constant voltage of 100-150 V. After the bromophenol blue indicator enters the separating gel, maintain a constant voltage of 200 V until the bromophenol blue band migrates to 1 cm from the bottom of the gel. Remove the gel and stain it with Coomassie Brilliant Blue staining solution. Then transfer it to destaining solution and destain until the background is clear.

[0090] Whole bacteria were added to PBS buffer and homogenized by sonication or high-pressure grinding. Simultaneously, the Ni-IDA affinity chromatography column was equilibrated with PBS buffer at least three times. The target protein was then eluted with equilibration buffers containing different concentrations of imidazole, and each eluted fraction was collected for SDS-PAGE analysis. SDS results are shown below. Figure 2 (In the figure, M is the SDS-PAGE protein marker, 1 is the whole cell before induction, 2 is the whole cell after induction, 3 is the supernatant of induction expression, and 4 is the precipitate of induction expression).

[0091] Figure 2 Electrophoresis diagram for identifying heterologous expression of LysEG01 in Escherichia coli. Figure 2 The results showed that, after cleavage and separation, the lyase LysEG01 protein was expressed in a soluble form in E. coli.

[0092] The cultured bacterial cells were centrifuged at 4000 rpm for 10 min and collected. The cells were then resuspended in Lysis buffer and lysed. The cells were then centrifuged at 18000 rpm at 4°C for 15 min, and the supernatant was collected. The supernatant was incubated with Niresin at 4°C for 2 h. After the supernatant flowed through, the column was washed with washbuffer containing imidazole at different concentrations until the Bradford reagent no longer turned blue. 10 ml of Elute buffer was added for elution, and SDS-PAGE analysis was performed. The volume was concentrated to 1 ml, centrifuged at low temperature to remove impurities, passed through a molecular sieve, and the protein was collected and analyzed by SDS-PAGE. The protein was concentrated, its concentration determined, and it was frozen. Figure 3 The image shows the purified LysEG01 protein electrophoresis pattern, demonstrating that high-purity LysEG01 lysin protein can be obtained through multiple purification steps.

[0093] Example 3: Determination of the lysing activity of lysin protein against Enterococcus faecium in chickens

[0094] Enterococcus DO103 (provided by the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, and publicly deposited and shared by the China General Microbiological Culture Collection Center) was inoculated into anaerobic BHI liquid medium and cultured until OD600 = 0.6. The bacterial suspension was centrifuged at 4000 rpm for 5 min, washed twice with buffer (50 mM Tris-HCl [pH 7.0], 100 mM NaCl), and then resuspended in the same buffer. The buffer containing the bacterial cells was added to 96-well plates, and LysEG02 lyase protein was added to a final concentration of 5 μmol, with a final volume of 200 μL. After culturing for 3 h, the experimental data were compiled, and the lysis activity was calculated. The lysis rate was calculated using the formula: [ΔOD600 (with added lyase) - ΔOD600 (with buffer only)] / initial OD600.

[0095] Figure 4 This indicates that LysEG01 can efficiently lyse Enterococcus faecium in chickens. After adding the lysin LysEG01, the OD of the culture medium decreased rapidly, proving that LysEG01 can effectively lyse Enterococcus faecium. With the addition of LysEG01 lysin, the absorbance OD600 decreased by more than 60% in about 30 minutes, and after 90 minutes, the Enterococcus faecium was almost completely lysed, indicating that the lysin LysEG01 has strong lysing activity against Enterococcus faecium in chickens.

[0096] Example 4: Targeting assay of lysin protein

[0097] The purified protein LysEG01 was used to determine its lytic activity against different symbiotic gut bacteria. The method for determining lytic activity was the same as in Example 3. In short: Different symbiotic gut bacteria were inoculated into anaerobic BHI liquid medium, and Enterococcus jirovecii was cultured to DO103 until OD600 = 0.6. The bacterial suspension was centrifuged from the medium at 4000 rpm for 5 min, washed twice with buffer (50 mM Tris-HCl [pH 7.0], 100 mM NaCl), and then resuspended in the same buffer. The buffer containing the bacterial cells was added to a 96-well plate, and LysEG01 lyase protein was added to a final concentration of 5 μmol, with a final volume of 200 μL for each well. The plate was placed in a 96-well plate and the absorbance was continuously measured using a microplate reader. A control group without lyase was also set up. The absorbance changes were calculated after 2 h. The lytic activity was calculated.

[0098] Figure 5 The lytic activity of the lysin LysEG01 against different Enterococcus species is represented. The lysis rate is the proportion of cells lysed within a specified time period relative to the total cell count; a ratio closer to 1 indicates higher lytic activity. Figure 5 As can be seen, LysEG01 exhibits highly efficient lytic activity against Enterococcus faecium, with a lysis rate close to 0.9%. However, it did not show significant lytic activity against other Enterococcus species, such as Enterococcus haematobium, Enterococcus faecalis, Enterococcus faecium, and Enterococcus fugax. This indicates that LysEG01 has high targeting specificity and can effectively eliminate Enterococcus faecium in the intestines, while lacking lytic activity against other Enterococcus species.

[0099] Figure 6 The lysing activities of the lysin LysEG01 and lysozyme (egg white source, CAS: 12650-88-3) against different intestinal commensal bacteria were studied. Figure 6 As shown in Figure A, LysEG01 exhibits highly efficient lytic activity against Enterococcus philus; however, it did not show lytic activity against the other 11 tested intestinal commensal bacteria (information and taxonomic status of the tested strains are shown in Table 2). These tested intestinal commensal bacteria came from five common intestinal commensal bacterial phyla, including Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Fusobacterium. This indicates that the lysin LysEG01 has high targeting specificity, capable of targeting and clearing Enterococcus philus in the gut without affecting other intestinal commensal bacteria. Lysozyme derived from egg white has some lytic activity against Enterococcus philus (lysis rate around 0.56, lytic ability far lower than LysEG01), but lysozyme derived from egg white also causes lysis of multiple intestinal commensal bacteria simultaneously, lacking obvious targeting specificity. Figure 6 Image B in the picture.

[0100] Table 2: Information on gut symbiotic bacteria used for the LysEG01 lysin targeting assay

[0101] Bacterial species name Bacterial strain name Bacterial classification Bacteroides uniformis ATCC8492 Bacteroidetes Parabacteroides distasonis ATCC8503 Bacteroidetes Blautia obeum DA69 Firmicutes Dorea longicatena DA136 Firmicutes Clostridium symbiosum DA229 Firmicutes Anaerostipes hadrus DA538 Firmicutes Faecalibacterium prausnitzii DA726 Firmicutes Bifidobacterium adolescentis DA06 Phylum Actinobacteria Collinsella aerofaciens DA394 Phylum Actinobacteria Escherichia coli DH5α Proteobacteria Fusobacterium varium DA690 Fusobacteria

[0102] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The use of an antimicrobial protein or antimicrobial drug composition in the preparation of a product targeting Enterococcus faecium in chickens; wherein The amino acid sequence of the antimicrobial protein is shown in SEQ ID NO:1; The antimicrobial pharmaceutical composition comprises: a protein with an amino acid sequence as shown in SEQ ID NO:1, a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:1, or a carrier comprising a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:1, and a pharmaceutically acceptable excipient.

2. The use according to claim 1, wherein, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

10.

3. The use according to claim 2, wherein, The nucleic acid molecules may be modified or unmodified.

4. The use of an antimicrobial protein or antimicrobial drug composition in the preparation of products for the prevention and treatment of diseases related to Enterococcus faecium in chickens; wherein The amino acid sequence of the antimicrobial protein is shown in SEQ ID NO:1; The antimicrobial pharmaceutical composition comprises: a protein with an amino acid sequence as shown in SEQ ID NO:1, a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:1, or a carrier comprising a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:1, and a pharmaceutically acceptable excipient.

5. Use according to claim 4, wherein, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

10.

6. Use according to claim 5, wherein, The nucleic acid molecules may be modified or unmodified.

7. The use according to any one of claims 4 to 6, wherein, The conditions include one or more of the following: bacteremia caused by Enterococcus fowleri, peritonitis and other intra-abdominal infections caused by Enterococcus fowleri following gastrointestinal surgery or trauma, infective endocarditis caused by Enterococcus fowleri, urinary tract infections caused by Enterococcus fowleri in the elderly and long-term catheter users, and non-infectious diseases associated with Enterococcus fowleri.

8. Use according to claim 7, wherein, The non-infectious diseases associated with Enterococcus fowleri are systemic lupus erythematosus and autoimmune hepatitis induced or exacerbated by intestinal metastasis of Enterococcus fowleri.

9. The use according to any one of claims 1 to 6, wherein, The product is a pharmaceutical preparation.

10. The use according to any one of claims 1 to 6, wherein, The product is a disinfectant.

11. The use according to any one of claims 1 to 6, wherein, The product is a bactericide.

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