Antibacterial protein targeting chicken enterococcus as well as preparation method and application of antibacterial protein

Abstract

The invention provides an antibacterial protein targeting chicken enterococcus as well as a preparation method and application of the antibacterial protein. Specifically, the invention provides an antibacterial protein targeting chicken enterococcus or a chimeric compound thereof, and the antibacterial protein comprises one or more of proteins formed by the following amino acid sequences: (a) an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 9; (b) a derivative sequence which is obtained by replacing, adding and / or deleting one or more amino acids from the amino acid sequence of (a) and has the same function as (a); and (c) a derived amino acid sequence of which the identity with any one sequence from SEQ ID NO: 1 to SEQ ID NO: 9 is 85% or more or 90% or more. The antibacterial protein disclosed by the invention can be used for efficiently killing chicken enterococcus and has specificity.

Description

Technical Field

[0001] The present invention provides an antibacterial protein targeting Enterococcus gallinarum and a preparation method and application thereof. Background Art

[0002] Enterococcus gallinarum is a Gram-positive, facultative anaerobic bacterium belonging to the genus Enterococcus. Enterococcus gallinarum is a Gram-positive bacterium that coexists with poultry and humans. Recent studies have found that when immunity is reduced or the intestinal barrier is damaged, the bacteria can migrate from the intestine to organs such as the liver, spleen, and lymph nodes, causing systemic infections or exacerbating autoimmune diseases. In addition, Enterococcus gallinarum has natural resistance to multiple antibiotics (such as vancomycin), making it difficult to treat in infection and disease management.

[0003] Infectious diseases associated with Enterococcus gallinarum: 1. Enterococcus gallinarum can be transmitted through blood and cause bacteremia, which is especially common in patients in intensive care or those with compromised immune systems (such as cancer and organ transplant patients). 2. The intestinal origin of Enterococcus gallinarum makes it easy to cause peritonitis and other abdominal infections after gastrointestinal surgery or trauma. 3. There are occasional reports of heart valve infections. Infectious endocarditis caused by Enterococcus gallinarum usually involves native valves or prosthetic valves. 4. Enterococcus gallinarum often causes urinary tract infections in the elderly and long-term catheter users. These infections are often resistant to multiple antibiotics, which increases the complexity of treatment.

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

[0005] Although research on E. gallinarum is still in its early stages, there is evidence that it plays an important role in infectious and non-infectious diseases. Treatment of E. gallinarum infection currently relies mainly on antibiotics, but drug resistance has made treatment more difficult. Future research should focus on developing new antimicrobial strategies to address the pathogenic effects of E. gallinarum in humans.

[0006] At present, the main means of treating Enterococcus gallus infection is broad-spectrum antibiotics, but these methods have some obvious problems. The main problems of existing treatment technologies are: 1. Antibiotic resistance. Enterococcus gallus gradually develops resistance to a variety of commonly used antibiotics, especially vancomycin. This reduces the effectiveness of treatment with broad-spectrum antibiotics and easily leads to the further spread of resistant strains. During treatment, doctors often face the difficulty of choosing a limited number of antibiotics. 2. Intestinal microecological destruction. The use of antibiotics will not only kill Enterococcus gallus, but also affect the beneficial flora in the intestine, leading to intestinal microecological imbalance. The destruction of intestinal flora may cause more serious secondary infections, such as Clostridium difficile-related infections, and may even aggravate the patient's condition. 3. Non-specific effects. Current antibiotic therapies lack specificity and often work on the entire bacterial population. This non-targeted treatment can cause damage to normal flora, especially in the case of long-term medication, increasing the risk of opportunistic infections by other pathogens (such as fungi and drug-resistant bacteria). 4. Immune-related side effects. For immunosuppressed patients, the treatment of Enterococcus gallus infection is particularly difficult. Antibiotics alone may not be effective in controlling infection in immunocompromised individuals, and further suppression of the immune system by drugs may exacerbate the condition. In addition, the use of immunomodulatory therapies has side effects and uncertainties, making it difficult to ensure their effectiveness in all patients.

[0007] In summary, antibiotic treatment of Enterococcus gallinarum faces problems such as drug resistance, microecological destruction and nonspecific side effects. It is necessary to develop new and effective means to deal with Enterococcus gallinarum infection, especially in the face of complex and multidrug-resistant infections. Summary of the invention

[0008] The inventors of this case discovered a class of antibacterial proteins targeting Enterococcus gallinarum, which can effectively kill Enterococcus gallinarum from different sources, including strains with antibiotic resistance, and are specific.

[0009] Specifically, on the one hand, the present invention provides an antimicrobial protein or a chimera thereof targeting Enterococcus gallinarum, wherein the antimicrobial protein comprises one or more of the proteins formed by the following amino acid sequences:

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

[0011] (b) a derivative sequence of the amino acid sequence of (a) which has one or more amino acids substituted, added and / or deleted and has the same function as (a);

[0012] (c) a derived amino acid sequence having an identity of greater than 85% or greater than 90% with any one of SEQ ID NO: 1 to SEQ ID NO: 9.

[0013] According to a specific embodiment of the present invention, the antibacterial protein targeting Enterococcus gallinarum or its chimera of the present invention, wherein the chimera comprises:

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

[0015] According to a specific embodiment of the present invention, the proteins formed by the amino acid sequences shown in any one of SEQ ID NO: 1 to SEQ ID NO: 9 of the present invention 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 the amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:9 of the present invention contains a catalytic domain and a cell wall binding domain and its position information as shown in Table 1. The catalytic domain includes Amidase_2, Amidase_3, Amidase_5, CHAP, Glyco_hydro_25, Peptidase_M23 and Glucosaminidase. The cell wall binding domain includes SH3, CW_7 and PG_binding_1, etc. As can be seen from Table 1, the lyase LysEG01 has the same catalytic domain as LysEG07 and LysEG09, and the lyase LysEG01 has the same cell wall binding domain as LysEG03, LysEG05 and LysEG08.

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

[0036]

[0037] According to some specific embodiments of the present invention, the antibacterial protein targeting Gallinarum or its chimera according to the present invention, wherein the chimera comprises one or more of the catalytic domains and / or cell wall binding domains shown in Table 1, for example, comprises 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 antibacterial protein targeting Gallinarum or its chimera of the present invention, wherein the chimera 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 encoding the antibacterial protein targeting Gallinarum or its chimera of 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 one 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] LysEG04 nucleic acid sequence (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 of the present invention is a DNA molecule or an RNA molecule.

[0060] According to a specific embodiment of the present invention, the nucleic acid molecule of the present invention is modified or unmodified. The modification may be, for example, chemical modification: modifying the nucleic acid molecule by introducing chemical groups, such as phosphate groups, methyl groups, acetyl groups, etc., to change its stability, affinity, targeting and other properties. Enzyme modification: modifying the nucleic acid by using specific enzymes, such as treating the nucleic acid by restriction endonuclease cutting, ligase ligation, DNA methylation, RNA editing, etc. Nucleotide substitution: replacing or inserting a specific nucleotide sequence to improve the function of the nucleic acid molecule or increase its stability. For example, modified nucleotides (such as 5-methylcytosine) are used to regulate gene expression. Synthetic modification: using artificial synthesis methods to perform specific chemical modifications on nucleic acid molecules, such as by adding special modifying groups when synthesizing DNA or RNA to improve its stability in vivo or enhance its activity in a specific environment.

[0061] On the other hand, the present invention also provides a vector comprising the nucleic acid molecule of the present invention.

[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 (eg, DNA sequence) of the present invention.

[0063] On the other hand, the present invention also provides a host cell, which comprises the nucleic acid molecule or the vector of the present invention.

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

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

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

[0067] On the other hand, the present invention further provides an antibacterial pharmaceutical composition, which comprises: the antibacterial protein or the nucleic acid molecule or the vector described in the present invention, and a pharmaceutically acceptable excipient.

[0068] On the other hand, the present invention also provides the use of the antimicrobial protein or the antimicrobial drug composition in the preparation of a product targeting Gallinarum.

[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 preventing and treating diseases related to Enterococcus avium;

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

[0071] According to a specific embodiment of the present invention, an experiment was conducted using Enterococcus gallinarum DO103 isolated from the human intestine as an example. The Enterococcus gallinarum genome contains the vancomycin resistance gene VanC1XY and the tetracycline resistance gene tet (S), and is resistant to vancomycin and a variety of tetracycline antibiotics, which may cause bacteremia, abdominal infection, endometritis, urinary tract infection, and induce or aggravate autoimmune diseases such as systemic lupus erythematosus and autoimmune hepatitis through intestinal liver metastasis. The antimicrobial protein of the present invention can efficiently kill the Enterococcus gallinarum with antibiotic resistance.

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

[0073] The antibacterial protein targeting Enterococcus gallinarum of the present invention can effectively kill Enterococcus gallinarum from different sources, including strains with antibiotic resistance, and has specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 This is the plasmid map of the constructed LysEG01 expression vector of the lytic enzyme.

[0075] Figure 2 This is the electrophoresis diagram for the identification of LysEG01 heterologously expressed in E. coli.

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

[0077] Figure 4 Experimental results showing that LysEG01 can efficiently lyse Enterococcus gallinarum.

[0078] Figure 5 The results showed that LysEG01 has lytic activity against Enterococcus bacteria.

[0079] Figure 6The lytic activity of LysEG01 against intestinal commensal bacteria was shown. DETAILED DESCRIPTION

[0080] Before further describing the 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 terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention.

[0081] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

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

[0083] Example 1: Identification and protein sequence analysis of the lytic enzyme gene targeting Enterococcus gallinarum

[0084] Phage and prophage genome sequences targeting Enterococcus gallinarum were collected, and the lytic enzyme genes therein were identified by sequence alignment and domain alignment using BLAST and Interproscan. A total of more than 180 potential phage lytic enzyme gene sequences were found, and the present invention further identified a class of lytic enzymes from them, which respectively contained the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9, and were named LysEG01-LysEG09, respectively.

[0085] The nucleic acid sequences of the lytic enzyme after codon optimization for Escherichia coli are shown in SEQ ID NO: 10 to SEQ ID NO: 18.

[0086] Example 2: Heterologous expression and purification of lytic enzyme targeting Enterococcus gallinarum

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

[0088] The constructed plasmid containing the lytic enzyme LysEG01 gene was transformed into BL21 (DE3) competent cells, and then evenly spread on LB plates (containing 50μg / mL kanamycin sulfate), and then inverted in a 37°C incubator overnight. Single clones were selected from the transformed plates and inoculated into 1L LB medium (containing 50μg / mL kanamycin sulfate), and cultured until OD600 was 0.6-0.8, IPTG was added to the test tube culture medium to a final concentration of 0.1-1mM, and then placed at 16-37°C, 100rpm, 18h to induce protein expression.

[0089] Take the induced culture solution and centrifuge it at 12000rpm for 5min, remove the culture medium, add PBS solution to resuspend and wash the strain, and finally add SDS-PAGE loading buffer to heat the sample at 100℃ for 30min, centrifuge and take the supernatant for electrophoresis. 10min before electrophoresis, 100-150V constant voltage electrophoresis, after the bromophenol blue indicator enters the separation gel, 200V constant voltage electrophoresis until the bromophenol blue band migrates to 1cm from the bottom of the gel, take out the gel and stain it with Coomassie brilliant blue staining solution, then transfer it to the decolorizing solution and decolorize it until the background is clear.

[0090] The whole bacteria were added to PBS buffer and lysed by ultrasonic or high-pressure homogenization. At the same time, the Ni-IDA affinity chromatography column was equilibrated with PBS buffer for at least three times. Then, the target protein was eluted with equilibration buffer containing different concentrations of imidazole, and each elution fraction was collected for SDS-PAGE analysis. SDS results are shown in Figure 2 (In the figure, M is SDS-PAGE Protein marker, 1 is whole cells before induction, 2 is whole cells after induction, 3 is induced expression supernatant, and 4 is induced expression precipitate).

[0091] Figure 2 The electrophoresis diagram for the identification of LysEG01 heterologous expression in E. coli. Figure 2 It was shown that the lytic enzyme LysEG01 protein was expressed in a soluble form in Escherichia coli after fragmentation and separation.

[0092] The cultured bacteria were centrifuged at 4000rpm for 10min to collect the bacteria. Then the bacteria were resuspended with Lysis buffer and broken. Then centrifuged at 18000rpm at 4℃ for 15min to collect the supernatant; the supernatant obtained above was incubated with Niresin at 4℃ for 2h; after the supernatant flowed through, the column was washed with Washbuffer with different concentration gradients of imidazole until the effluent did not turn blue; 10ml of Elute buffer was added for elution and SDS-PSGE detection was performed; the column was concentrated to 1ml, centrifuged at low temperature to remove impurities, and then passed through molecular sieves, proteins were collected, and SDS-PAGE gel was run for detection; the protein was concentrated, the concentration was determined, and the column was frozen. Figure 3 The electrophoresis diagram of the purified LysEG01 protein shows that highly pure lytic enzyme protein LysEG01 can be obtained through multiple purification steps.

[0093] Example 3: Determination of the lytic activity of lytic enzyme protein against Gallinarum

[0094] Enterococcus gallinarum DO103 (the strain was provided by the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, and has been publicly preserved and shared for public benefit at the General Microbiological Center of the China Microbiological Culture Collection Administration Committee) was inoculated into an anaerobic BHI liquid medium and cultured to OD600 = 0.6. The bacterial liquid was centrifuged at 4000rpm for 5min from the culture medium, washed twice with a buffer solution (50mMTris-HCl [pH 7.0], 100mM NaCl), and the bacteria were resuspended in the buffer solution after washing. The buffer solution containing the bacteria was added to a 96-well plate, and LysEG02 lyase protein was added to a final concentration of 5 micromolar, and the final volume was 200 microliters. After culturing for 3h, the experimental data was sorted and the lysis activity was calculated. The lysis rate calculation formula: [test ΔOD600 (lyase added)-ΔOD600 control (buffer only)] / initial OD600.

[0095] Figure 4 This indicates that LysEG01 can efficiently lyse Enterococcus gallinarum. After adding the lytic enzyme LysEG01, the OD of the culture solution decreased rapidly, proving that LysEG01 can effectively lyse Enterococcus gallinarum. After adding LysEG01 lytic enzyme, the absorbance OD600 had dropped by more than 60% in about 30 minutes, and after 90 minutes, the Enterococcus gallinarum was basically completely lysed, indicating that the lytic enzyme LysEG01 has a strong lysis activity against Enterococcus gallinarum.

[0096] Example 4: Targeted determination of lytic enzyme protein

[0097] The purified protein LysEG01 was used to determine its lytic activity against different symbiotic intestinal bacteria, and the method for determining the lytic activity was the same as in Example 3. In short: Different symbiotic intestinal bacteria were inoculated into anaerobic BHI liquid culture medium, and Enterococcus gallinarum DO103 was cultured to OD600 = 0.6. The bacterial liquid was centrifuged at 4000rpm for 5min from the culture medium, washed twice with buffer (50mM Tris-HCl [pH 7.0], 100mM NaCl), and the bacteria were resuspended with the buffer after washing. The buffer containing the bacteria was added to a 96-well plate, and LysEG01 lytic enzyme protein was added to a final concentration of 5 micromolar, and the final volume of each well was 200 microliters. It was placed in a 96-well plate and placed in an ELISA instrument to continuously measure the absorbance changes. At the same time, a control group without lytic enzyme was set up. After 2h, the absorbance changes were calculated. The lytic activity was calculated.

[0098] Figure 5 is the lytic activity of the lytic enzyme LysEG01 against different Enterococcus bacteria. The lysis rate is the proportion of cells lysed to the total cells within a specified time. The closer the ratio is to 1, the higher the lytic activity. Figure 5 It can be seen that LysEG01 exhibits efficient lysis activity against Enterococcus gallinarum, with a lysis rate close to 0.9; but for other Enterococcus bacteria, such as Enterococcus hirae, Enterococcus foetida, Enterococcus faecalis and Enterococcus faecium, LysEG01 does not show obvious lysis activity. This shows that LysEG01 has a high targeting ability and can eliminate Enterococcus gallinarum in the intestine, but has no lysis activity against other Enterococcus bacteria.

[0099] Figure 6 The lytic activity of LysEG01 and lysozyme (from chicken egg white, CAS: 12650-88-3) on different intestinal symbiotic bacteria. Figure 6 As shown in Figure A, LysEG01 exhibited efficient lysis activity against Enterococcus gallinarum; but for the other 11 intestinal symbiotic bacteria tested (the information and taxonomic status of the test strains are shown in Table 2), LysEG01 did not show lysis activity. These intestinal symbiotic bacteria tested came from five common intestinal symbiotic bacterial phyla, including Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Fusobacteria. This shows that the lytic enzyme LysEG01 has a high degree of targeting and can target and eliminate Enterococcus gallinarum in the intestine without affecting other intestinal symbiotic bacteria. Lysozyme derived from egg white has a certain lysis activity against Enterococcus gallinarum (the lysis rate is about 0.56, and the lysis ability is much lower than that of LysEG01), but lysozyme derived from egg white will also cause the lysis of multiple intestinal symbiotic bacteria at the same time, and there is no obvious targeting ( Figure 6 Figure B).

[0100] Table 2: Information on intestinal commensal bacteria used for targeting test of lysase LysEG01

[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 Actinobacteria Collinsella aerofaciens DA394 Actinobacteria Escherichia coli DH5α Proteobacteria Fusobacterium varium DA690 Fusobacteria

[0102] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and 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 present invention should be within the scope of protection of the present invention.

Claims

1. An antimicrobial protein targeting Enterococcus gallinarum or a chimera thereof, wherein: The antibacterial protein includes one or more of the following proteins formed by amino acid sequences: (a) the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 9; (b) a derivative sequence of the amino acid sequence of (a) which has one or more amino acids substituted, added and / or deleted and has the same function as (a); (c) a derived amino acid sequence having an identity of greater than 85% or greater than 90% with any one of SEQ ID NO: 1 to SEQ ID NO:

9.

2. The antimicrobial protein targeting Enterococcus gallinarum or its chimera according to claim 1, wherein: The chimera comprises: A chimeric protein comprising the amino acid sequence of the complete antimicrobial protein of claim 1 (a), (b), (c), the catalytic domain sequence of the antimicrobial protein, and the cell wall binding domain sequence of the antimicrobial protein; Preferably, the chimera comprises 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; More preferably, the chimera comprises the catalytic domain Amidase-2 and / or the cell wall binding domain SH3.

3. A nucleic acid molecule encoding the antibacterial protein targeting Enterococcus gallinarum according to claim 1 or 2 or a chimera thereof; Preferably, the nucleotide sequence of the nucleic acid molecule comprises any one of SEQ ID NO: 10 to SEQ ID NO: 18; More specifically, the nucleic acid molecule is modified or unmodified. A vector comprising the nucleic acid molecule according to claim 3 . A host cell comprising the nucleic acid molecule according to claim 3 or the vector according to claim 4.

6. A method for preparing the antimicrobial protein or its chimera according to claim 1 or 2, the method comprising: The antibacterial protein or its chimera according to claim 1 or 2 is prepared by in vitro transcription using the nucleic acid molecule according to claim 3, the vector according to claim 4, or the host cell according to claim 5.

7. An antibacterial pharmaceutical composition, comprising: the antibacterial protein or chimera thereof according to claim 1 or 2, the nucleic acid molecule according to claim 3, or the vector according to claim 4, and a pharmaceutically acceptable excipient.

8. Use of the antimicrobial protein or chimera thereof according to claim 1 or 2 or the antimicrobial pharmaceutical composition according to claim 7 in the preparation of a product targeting Enterococcus gallinarum.

9. Use of the antimicrobial protein or chimera thereof according to claim 1 or 2 or the antimicrobial pharmaceutical composition according to claim 7 in the preparation of a product for preventing and treating diseases associated with Enterococcus gallus; Preferably, the disease comprises one or more of the following diseases: bacteremia, peritonitis and other abdominal infections caused by gastrointestinal surgery or trauma, infective endocarditis caused by Enterococcus gallinarum, urinary tract infection caused by Enterococcus gallinarum in the elderly and long-term catheter users, non-infectious diseases associated with Enterococcus gallinarum, autoimmune diseases such as systemic lupus erythematosus and autoimmune hepatitis induced or aggravated by intestinal metastasis of Enterococcus gallinarum.

10. The use according to any one of claims 8 to 9, wherein: The product is a pharmaceutical preparation, a disinfectant preparation or a bactericide.

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