Antibacterial protein targeting clostridium perfringens and preparation method and application thereof

Abstract

The present application provides an antibacterial protein targeting Clostridium scindens and a preparation method and application thereof. Specifically, the present application provides an antibacterial protein targeting Clostridium scindens 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: 13; (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: 13. The antibacterial protein of the present application can efficiently kill Clostridium scindens and has specificity.

Description

Technical Field

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

[0002] Clostridium scindens is a Gram-positive, facultative anaerobic bacterium belonging to the genus Clostridium. While Clostridium scindens is part of the healthy microbiome to some extent, its metabolic functions may be associated with some non-infectious diseases: (1) Clostridium scindens is known for its unique bile acid 7α-dehydrogenase activity, which enables it to convert cholesterol metabolites into androgen precursors such as 11β-hydroxyandrostenedione. These precursors may be further converted into active androgens in the human body, stimulating the growth of prostate cancer cells and leading to resistance to hormone deprivation therapy. Studies have found that these metabolic activities may affect the treatment outcomes of prostate cancer patients and increase the risk of hormone-resistant tumors. (2) Clostridium scindens metabolizes bile acids to produce secondary bile acids, such as deoxycholic acid (DCA), which plays a key role in the progression of colorectal cancer. Studies have shown that DCA inhibits the effector function of anti-tumor CD8+ T cells. Specifically, DCA reduces intracellular calcium ion accumulation by enhancing the activity of cell membrane calcium ion pumps, thereby inhibiting the NFAT2 signaling pathway in CD8+ T cells. This weakens the ability of T cells to produce interferon (IFN-γ) and tumor necrosis factor (TNF-α), leading to the failure of anti-tumor immune responses. In summary, DCA generated by Clostridium scintillans is an important factor in the occurrence and progression of colorectal cancer. (3) Clostridium scintillans can affect the accumulation of natural killer T cells (NKT cells) in the liver by regulating bile acid metabolism, which may affect the anti-tumor immune response in the liver and the development of liver cancer. Mouse experiments showed that colonization of Clostridium scintillans led to a rapid decrease in the level of liver NKT cells and an increase in the number of liver tumors. In addition, by treating mice with antibiotics, researchers found that removing Gram-positive bacteria (including Clostridium scintillans) could increase the accumulation of liver NKT cells and reduce liver tumor growth. These results indicate that Clostridium scintillans has a promoting effect on the development of liver cancer and is a potential therapeutic target. (4) Clostridium scintillans may play an important role in the occurrence and development of inflammatory bowel diseases such as chronic diarrhea and diarrhea-predominant irritable bowel syndrome by affecting bile acid metabolism, and may also play a role in the pathological process of non-alcoholic fatty liver disease.

[0003] Specific phages targeting *Clostridium scintillans* have been used to reduce the abundance of this bacterium. In mouse experiments, using phages targeting *Clostridium scintillans* reduced the concentration of delta-lactamase (DCA) in the gut and effectively slowed tumor growth. This suggests that phages may be a tool for precise control of the gut microbiota. Furthermore, bile acid sequestrants have also shown some effectiveness in reducing DCA production. In mouse models, this method helped restore the function of anti-tumor CD8+ T cells. In some cases, researchers have reduced *Clostridium scintillans* in the gut microbiota through antibiotic treatment.

[0004] Currently, the main treatments for Clostridium scintillans infection are antibiotics and phage therapy. However, these methods have several significant drawbacks: 1. Bacteria may develop resistance to phages and antibiotics through mutation, reducing the effectiveness of phage or antibiotic treatment and increasing the risk of further spread of resistant strains. 2. Non-specific effects: Antibiotics not only kill Clostridium scintillans but also affect beneficial gut flora, leading to intestinal microecological imbalance. 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). 3. Immune-related side effects: Treatment of Clostridium scintillans infection is particularly challenging for immunosuppressed patients. Antibiotics themselves may not effectively control the infection in immunocompromised individuals, and further suppression of the immune system by the drugs may exacerbate the condition. Furthermore, the use of immunomodulatory therapies carries side effects and uncertainties, making it difficult to ensure their effectiveness in all patients.

[0005] In summary, antibiotic treatment of Clostridium scintillans faces challenges such as drug resistance, disruption of the gut microbiota, and nonspecific side effects, necessitating the development of new and effective methods to combat Clostridium scintillans infection. Summary of the Invention

[0006] The inventors in this case discovered a class of antimicrobial proteins that target Clostridium scintillans, which can effectively kill Clostridium scintillans and are specific.

[0007] Specifically, on one hand, the present invention provides an antimicrobial protein or a chimera thereof targeting Clostridium scintillans, wherein the antimicrobial protein comprises one or more proteins formed from the following amino acid sequences:

[0008] (a) Any of the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:13;

[0009] (b) A derivative sequence of (a) with one or more amino acids replaced, added and / or deleted, and having the same function as (a);

[0010] (c) A derived amino acid sequence that is 85% or 90% or more identical to any of the sequences in SEQ ID NO:1 to SEQ ID NO:13.

[0011] According to a specific embodiment of the present invention, the antimicrobial protein or its chimeric compound targeting Clostridium scintillans of the present invention, wherein the chimeric compound comprises:

[0012] 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), combined with 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.

[0013] 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:13 are named LysCS01 to LysCS13, respectively, and the specific amino acid sequences are as follows.

[0014] LysCS01 protein sequence (SEQ ID NO:3):

[0015] MNRLNCMNQKIKIFMQMCFAVLVIMTVMVLPVRAKETETAGASLGKETETEDAISFYASAVEGWVQTADGRWWYQYNDGSWPKTSWRKINGSWYYFDSNGYWVDNNVHESGSLKGIDVSQWQGNIDWQAVKDDGIQFALIRLGHGIHELDTYYQRNMQNANAAGIPVGVYFYSTAKSEEEAIADAQFVISNMKGYLVSYPVVIDLEDSSQASLSKTQLGKIAKAYCDEIHAAGYTPMLYCNENWYRNHIDISQIADVEMWVARYGGTYSTSIPRGIWQCCSTGRVNGIGGDVDIDFGYKDYTQIVTPRTDYAEGYVMTEGIWVKDGHGWWYRYFAGGYPSNTWKNIRGNWYWFDADGYMETGWHLIDGTWYYFNSSGAMVTGWQLIGNTWYYMDGSGAMATGWCLIGGTWYYFNGSGAMETGWHLIDGTWYYFNGSSGAMTTGWQLIGNTWYYLGTDGKMVTGLTTVTGAIYYLESSGAMATGWRQIDGIWYYFN

[0016] LysCS02 protein sequence (SEQ ID NO:4):

[0017] MAKSRQAVVNLVESWDGKKESNGSYKSIIDLYNDFFEKICAGKFPRGIRMRYDWAWCACTWSALAAALRYESIMPMEISCYYLIEAAKKMGCWQENDAYVPSPGDAILYDWQDNGISDNTGNPDHVGTVIEVHKESGYMVIEEGNYSNAVKKRTLSINGKFIRGFITPKYDDNTVAAPGLSKGKDIKTVAHDVIVGLWGSGENRKKLLTEYGYSYSEVQSMVNQILNGSAVTPSNTKQDQNQSISKKVVATCSAKQFNKAYAGEYKTTAVLYCRNDAGTNKKALCKIPAGTKVKCYGYYTMANGVKWLYIQFVLDGVQYTGFSSSAYLAK

[0018] LysCS03 protein sequence (SEQ ID NO:5):

[0019] MGWTEYRDVLDSWFGYSEARGQDDLIIDIYNSQRVESYKMSHQDPWCHATISAAGYQSGNQGRVPNTAYCPYGINWFKARGLWTGRYAGNYAPAVGDIIYYDWGGDGVSDHVGAIIKVSGNTLTVREGNRNDMVCDRTISKWSNLIMGYGRPNWGSATIIMPSPVVVESGSNGAYGIHRKDLIRQGQQHAINFTGVKIGVDGIRGPETKKAAIRCVQHAMNMDYNAGLKEDGIWGKKTDAAFAQHYVCEGETQYMVTAWEILLLLNGYDPNGVEHPGEFGSGCAAATRMFQGDKSLVQDGVAGRKSFLTAIN

[0020] LysCS04 protein sequence (SEQ ID NO:6):

[0021] MGTYNVHAGHCPQGQGASGAVGLLQESVEDRKVKNRVISALQSAGHVVYDCTDDSNCNVSQNLRNIVAKCNAHSVDLDVSIHLNAGGGTGVEVWCYDGKTANIASAICQNVSTALGISNRGVKYSTGLYVLRKTKAPALLVECCFVDNQNDYSHWNVEKCGDDIASAIAGKTVQGNASAPAQNPAPTPNAGFDFAGWVGRLQAECNAQEFSRQKVDRIPGPITLAGCPLIKRGASGNITRLVQERLNSLGFCCGVDGDYGRAPFHETYDAVIAYQRANDLVPNGIVGQKTWSKLLGLS

[0022] LysCS05 protein sequence (SEQ ID NO:7):

[0023] MKLVESILTKNPCYTAGRKITVKGLMLHSVGCPQPKASVFINSWNSPSYDNACVHGFIDGNDGTVYQTLPWNHRGWHCGSGSKGSGNNTHIGVEMCEPACIKYTSGANFTCSDTATAKAVAKRTYEAAVELFAMLCRQYSLNPTADGVIISHREGHSRGIASNHGDPEHLWTQLGMGYTMDTFRQAVKAAMDGGSSAETSGYTKIMGNAVATAEQMETYIQGKNPQVLQSVLDMIPLYLSEGQAEGVRGDIAFAQSCLETGNFTFSGSAVTLDQNNFCGMGVTSNGLKGNSFDTPQLGIRAQIQHLKAYASTDALKNDCVDPRFKYVTRGSAEYVEWLGQQENPQGKGWAAGAGYGEKILTILKNICGTAGGASGTADIWYRVRKTWADAKSQIGAFRVLENAKNCADENPGYRVFDVNGVNIYTPDTAAFSPYLVRVSITDLNIRKGPGTNYAKTGKFTGKGVFTIVEMQTGKGSDTGWGRLKSGAGWISLDYTEKIS

[0024] LysCS06 protein sequence (SEQ ID NO:8):

[0025] MNGIDISNWQKGINLDAVPCDFVIMKATEGTWYVNPDCERAYQQAKNAGKCLGVYHYAEGKDAKAEADFFLKHIQGYIGEALIALDWEKENNSSFGKNDLNWVKQWLDYIYGKTGVRPLLYISQSIMGKFNGIGDYGLWVAQYANMNTTGYQDAPWNEGKYNCAIRQYSSCGRLSGYSGNLDLNKFYGDKTAWNRYAGKGNVTKPSTGTAASSTSSPGGTVLDLVVATLQGKYGNGDVRKTALGNRYIEVQNMINHIASASASTLAGEVKAGKYGNGDARKIALGSRYNEVQKIVNGSTGLAASYHTIKSGETLSGIAAKYGTTVAKLQSLNGIKNVNKIYAGSRIRVK

[0026] LysCS07 protein sequence (SEQ ID NO:9):

[0027] MGWNEYNAKLQEWYGFTEAAGQDDIIIDAYNRQKEEAYTMSHQDPWCHATVSAAAAESGNRGRVPNTAYCPTGINWFKARGQWTGRYDTGYNPSVGDIIYYDWGGDGVSDHVGTIVGVNGNTLQVREGNKNDMLTDRYIQKGNTLIMGYGRPAWGGSVPVPSQQSGSIGRSWLQRGDKGEAVKDVQNKLIALGYSCGPDGADGDYGTNTIAAVKRFQADVGITVDGLAGEITRAKLNNAYNTGGVNKAGGSWVARLQAECNAQGFSTQKVDGLPGPNTLAGCPTLGRTSRGKITALMQERLISLGYSCGPCGADGINGAGTQAAIKAFQRDHGLAVDGIVGQKTWSKLLGLS

[0028] LysCS08 protein sequence (SEQ ID NO:10):

[0029] MSMNGIDISNHQQGLDVSKVPCDFVIMKATEGTTFVDKYCDKFYQQAKKLGKKLGVYHFASGKSSGTAEADFFLKNIAGYVGEAILVLDWEGSAVNKGVGYAKEFLDRVYAKTGVRPLLYSYNNCINAYDWRSVAQADYGLWNAGYYAGYQTMGYNPNAPIKGGLGAFGSCAMYQYTSSGRLSGWAGNLDLDVFYGDSAAWDAYAKGSAIASPGGTPEPSQQPTNTSPSSQSMLNAQIHINNFTGSGIPEDGKNGRKTRKGVVMSLQHACNLDYKPNPVLKEDGLIGSKTNRVRGLHYVKRGETQYLVTFVEIGLTALGYYAGAVECPGVFGEGLEDAVHRFQHDMNLNEDKIAGRNVMDMMLRQLGCI

[0030] LysCS09 protein sequence (SEQ ID NO:11):

[0031] MSNSSLVNCTVKSPNHSGARTHSIDRITPHCVVGQLSAESIGGCFTSPSREASCNYGIGTDGRVVLCVDEANRSWCSSSNANDQRAVTIECASDMTDPYAMTSAVYEKLVALCVDICQRNGKSKLIWFGDKDKSLNYSPKSNEMILTVHRWFANKACPGDWLYSRLGDLANRVTSQLGGSTTDSTSKTYKTGLYKVDVGDLNIRKGPGTNYGINGMITDRGTYTITEIQNGYWGKLKSGAGWISVHEAYCTYKGAASGESEEKPSSNFLVQVDIPDLYIRKGPGTNYGNNGFCPKGVYTIVEVKSGAGSDAGWGKLKSGAGWISLDYATRI

[0032] LysCS10 protein sequence (SEQ ID NO:12):

[0033] MEKQEFIKQIAGYVKKYAARYGIKVHSPIIAQAILESGWGESKLAAVYHNYFGLKCGTKWTGKSVNLKTMEEYTPGTLTQIKDNFRVYDNMEEGVKGYFEFIQLKRYQNLKGITDPEEYLKTIKADGYATSSKYVENTMRIVTQYNLQKYDTKGEESMAKKASAVLSQARAWIGRKEANGTHREIIDVYNAHRPLARGYKVKYTDAWCATFVSAVAIKCGLTSIIPTECGCGQMIELFKKLGEWQESDSRTPKPGDVVFYDWDDTGTGNNTGWPDHVGIVESVSGGSITVIEGNKNNAVERRTLSVNGRYIRGYGVPKYDSEAGTGTTQPGKSVAEVAKEVIAGKWGNNPQRKERLEAAGYDYQAVQNQVNAILNGNAKPQKSVAEVAKEVIAGKWGNNPQRKERLEAAGYDYQAVQNKVNQLLK

[0034] LysCS11 protein sequence (SEQ ID NO:13):

[0035] MNIIKHGLRFRGSFTYRNRTDEIIWHHAEANCTVEDIHSWHLNNGWIGCGYNLIIYKDGTVHEGRPLNAVGAHASGHNSRSVGVCCIGRYDVETMPKEQLDAAKQVQAYLKGLYPGAATKRHKDVNATSCPGKNFPFSEISGASAGSVDVTTSDVQAQSSDDWCARLQKECNAQKFSKQKVDNIPGPDTLAGCPTLGRKSRGKITALMQERLNALGYDCGAVDGINGTKTQAAIKAFQRDYGLVADGIVGPKTWSKLLGLS

[0036] LysCS12 protein sequence (SEQ ID NO:1):

[0037] MAHLYVIAGHGAGDSGAVGNGYTEAERVRALASKIKQLGGDNVTLGDMNRNYYADNGISSLNIPKDWCIIELHMDSASASARGGHVIIKGTFSPDSHDLALASFVSAMFPGRANSIVGRNNLANPNRAAAKGYNYRLVEFGFISNATDVSIFNNNLDAIARGVLSCFGINTNTAKWVLDNVGWWYQRADGSYPKSQWLLLDCYYYFNDKGYALANEWLSYGGNWYWLKDDCRMATGWQYIDKHWYYLNPTGTKNKPVGAMLDGWQFIDGQWYYLRTKADGEHPHGSMVEGSVTVGEHDYYCREAGTDKNYPTGSMLMGWRKVTETAEDGTKKTKWFWYNKDSNCQPIGSMLKNHWLTTSNGKKYYLKDDGVMACDETMTISGKEYTFDASGALV

[0038] LysCS13 protein sequence (SEQ ID NO:2):

[0039] MAHLYVIAGHGAGDSGAVGNGYTEAERVRALASKIKQLGGDNVTLGDMNRNYYADNGISSLNIPKDWCIIELHMDSASASARGGHVIIKGTFNPDSHDLALASFVSAMFPGRANSIVGRNNLANPNRAAAKGYNYRLVEFGFISNATDVSIFNSNLDAIARGVLSCFGINTNTAKWVLDNVGWWYQRADGSYPKSQW LLLDCYYYFNDKGYALANERLSYGGNWYWLKDDCRMATGWQYIDKHWYYLNPTGTKNKPVGAMLDGWQFIDGQWYYLRTKADGEHPHGSMVEGSVTVG EHDYYCREAGTDKNYPTGSMLMGWRKVTETAEDGTKKTKWFWYNKDSNCQPIGSMLKNHWLTTSNGKKYYLKDDGVMACDETMTISGKEYTFDASGALV

[0040] The protein formed by any of the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:13 of this invention contains catalytic and cell wall-binding domains and their positional information, as shown in Table 1. The catalytic domains include Amidase_2, Amidase_3, CHAP, Glyco_hydro_25, Lysozyme_like, and Glucosaminidase. The cell wall-binding domains include PG_binding_1, Choline_bind_1, Choline_bind_3, CW_7, SH3, and LysM, etc. As can be seen from Table 1, LysCS13 shares the same catalytic domain Amidase_3 with LysCS12 and LysCS04, while LysCS13 shares the same cell wall-binding domains Choline_bind_1 and Choline_bind_3 with LysCS12 and LysCS01.

[0041] Table 1: Catalytic and cell wall-binding domains of antimicrobial proteins LysCS01 to LysCS13

[0042]

[0043] According to some specific embodiments of the present invention, the antimicrobial protein or its chimera targeting Clostridium scintillans of the present invention comprises one or more of the catalytic domains and / or cell wall binding domains shown in Table 1, such as the catalytic domains Amidase_3, Amidase_2, CHAP, Glyco_hydro_25, Lysozyme_like, Glucosaminidase, and the cell wall binding domains PG_binding_1, Choline_bind_1, Choline_bind_3, CW_7, SH3, and LysM.

[0044] According to some specific embodiments of the present invention, the antimicrobial protein or its chimeric compound targeting Clostridium scintillans of the present invention comprises a catalytic domain Amidase_3 and / or a cell wall binding domain Choline_bind_1 and / or Choline_bind_3.

[0045] On the other hand, the present invention also provides a nucleic acid molecule that encodes the antimicrobial protein targeting Clostridium scintillans described in the present invention.

[0046] 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:14 to SEQ ID NO:26.

[0047] LysCS01 nucleic acid sequence (SEQ ID NO:16):

[0048]

[0049] LysCS02 nucleic acid sequence (SEQ ID NO:17):

[0050] ATGGCCAAATCACGCCAGGCTGTGGTTAATTTGGTCGAGAGTTGGGATGGCAAGAAGGAATCCAACGGTAGCTACAAATCGATCATCGATCTGTACAACGATTTTTTCGAAAAAATTTGCGCCGGTAAGTTTCCCCGTGGGATTCGTATGCGTTATGATTGGGCCTGGTGTGCGTGCACCTGGTCCGCTCTTGCGGCTGCATTACGTTATGAAAGTATTATGCCGATGGAGATCTCTTGCTATTACCTGATCGAAGCAGCCAAGAAAATGGGGTGCTGGCAGGAAAATGATGCCTATGTGCCGAGTCCGGGCGATGCTATCCTTTATGATTGGCAGGACAACGGCATCTCGGATAACACAGGCAATCCGGATCATGTTGGCACCGTGATTGAAGTACACAAGGAGTCGGGCTACATGGTTATTGAAGAAGGTAATTATTCAAATGCCGTGAAAAAACGTACCTTGTCGATCAACGGCAAATTTATTCGCGGTTTTATTACGCCCAAATATGACGACAACACCGTTGCCGCCCCGGGCCTGAGCAAGGGAAAAGATATTAAGACCGTCGCACACGATGTTATTGTAGGCCTGTGGGGCAGTGGCGAGAACCGCAAAAAGCTGCTGACGGAATATGGTTATTCATACTCTGAAGTCCAGAGTATGGTAAACCAGATTCTGAACGGTTCGGCCGTCACCCCAAGTAATACCAAACAGGATCAGAACCAAAGCATCAGTAAGAAGGTGGTCGCTACGTGCAGCGCCAAACAGTTCAACAAAGCATACGCAGGTGAATATAAAACGACAGCAGTGCTGTATTGCCGCAACGACGCCGGCACGAACAAGAAAGCTCTGTGCAAAATCCCCGCAGGGACCAAAGTGAAATGTTATGGTTATTATACCATGGCGAATGGAGTCAAATGGCTCTACATCCAATTCGTCCTGGATGGTGTGCAATACACCGGCTTTTCATCAAGCGCGTACCTGGCAAAATAA

[0051] LysCS03 nucleic acid sequence (SEQ ID NO:18):

[0052] ATGGGTTGGACCGAATATCGTGATGTCCTTGACTCGTGGTTCGGTTATAGTGAAGCCCGCGGTCAAGATGATCTGATCATTGATATTTACAATAGCCAGCGTGTCGAGTCCTACAAAATGTCTCACCAGGACCCGTGGTGTCATGCAACGATCTCCGCTGCGGGCTATCAGTCTGGCAATCAGGGTCGCGTTCCCAACACTGCTTATTGTCCTTATGGGATCAACTGGTTCAAAGCCCGTGGACTGTGGACGGGCCGCTACGCAGGTAACTACGCCCCGGCTGTCGGCGACATCATCTATTATGATTGGGGCGGCGACGGCGTCTCTGATCACGTAGGCGCTATCATCAAAGTGTCTGGAAACACGTTGACCGTGCGTGAAGGTAATCGTAACGATATGGTTTGCGATCGCACGATTTCTAAATGGTCTAACCTGATTATGGGCTACGGTCGTCCAAACTGGGGCAGTGCAACGATTATCATGCCATCTCCGGTTGTAGTTGAGTCAGGCAGTAACGGAGCGTACGGTATCCATCGTAAGGACTTAATCCGTCAGGGACAGCAGCATGCGATTAACTTTACTGGGGTTAAAATTGGCGTGGACGGTATCCGCGGCCCTGAAACCAAGAAAGCGGCTATCCGCTGTGTCCAACATGCAATGAACATGGACTATAATGCCGGTCTGAAAGAAGACGGCATTTGGGGAAAAAAAACGGATGCAGCCTTCGCCCAACATTATGTATGCGAGGGCGAAACGCAATATATGGTGACAGCTTGGGAAATTTTACTGCTCTTAAACGGCTACGATCCAAATGGTGTAGAACATCCGGGCGAATTTGGCAGTGGCTGTGCCGCAGCCACACGTATGTTCCAAGGCGATAAATCCCTGGTCCAGGATGGCGTGGCAGGGCGTAAATCATTTCTGACGGCCATCAACTGA

[0053] LysCS04 nucleic acid sequence (SEQ ID NO:19):

[0054] ATGGGGACATATAACGTCCATGCGGGCCATTGCCCACAAGGGCAAGGCGCGTCTGGCGCCGTAGGTTTGCTTCAAGAATCCGTGGAAGACCGTAAGGTGAAAAACCGCGTGATCTCCGCGCTGCAGTCGGCAGGTCACGTGGTGTACGATTGCACCGATGACTCAAATTGTAACGTGAGTCAGAATTTACGCAACATTGTTGCTAAATGCAACGCTCACTCCGTGGATCTGGACGTTTCGATTCATCTGAATGCCGGGGGTGGTACGGGCGTTGAAGTTTGGTGTTATGATGGCAAAACGGCCAACATTGCCTCCGCTATTTGTCAAAACGTGTCCACCGCTCTGGGCATTAGCAACCGTGGAGTGAAATATTCCACGGGCCTGTACGTTCTGCGCAAAACCAAAGCTCCTGCCTTATTAGTCGAATGTTGTTTCGTGGATAATCAGAATGATTATTCACATTGGAACGTTGAAAAATGTGGCGACGATATTGCCTCAGCTATTGCCGGCAAGACCGTGCAGGGCAATGCCTCGGCCCCGGCACAGAATCCAGCACCAACCCCTAACGCTGGCTTTGATTTCGCTGGCTGGGTTGGACGCCTGCAGGCTGAATGTAACGCCCAGGAATTTTCCCGCCAGAAAGTCGATCGTATCCCGGGTCCGATTACCCTGGCTGGCTGCCCGCTCATCAAGCGCGGAGCCAGCGGCAATATTACCCGCTTAGTGCAAGAACGCCTGAATAGCTTAGGCTTTTGTTGCGGCGTGGACGGCGACTATGGACGCGCACCGTTTCATGAAACCTACGACGCAGTCATCGCCTATCAGCGTGCCAATGATCTGGTACCAAATGGGATTGTAGGTCAAAAAACATGGTCGAAACTGCTGGGCCTGAGCTAA

[0055] LysCS05 nucleic acid sequence (SEQ ID NO:20):

[0056]

[0057] LysCS06 nucleic acid sequence (SEQ ID NO:21):

[0058]

[0059] LysCS07 nucleic acid sequence (SEQ ID NO:22):

[0060]

[0061] LysCS08 nucleic acid sequence (SEQ ID NO:23):

[0062]

[0063] LysCS09 nucleic acid sequence (SEQ ID NO:24):

[0064] ATGAGTAACTCAAGTCTGGTGAACTGTACCGTGAAATCCCCGAACCATAGCGGCGCACGTACACACTCAATTGACCGCATCACCCCCCATTGTGTGGTGGGCCAGCTGTCTGCGGAGTCTATCGGTGGCTGCTTCACGAGCCCATCCCGTGAAGCATCGTGTAATTACGGAATCGGGACGGACGGACGCGTCGTTTTGTGTGTTGACGAAGCAAATCGCAGCTGGTGCTCCAGTTCAAACGCCAACGATCAGCGTGCTGTGACCATCGAGTGCGCAAGCGATATGACGGACCCCTATGCTATGACCTCCGCGGTGTATGAGAAACTTGTCGCTTTATGCGTGGACATCTGCCAACGCAACGGTAAATCTAAACTGATTTGGTTTGGCGATAAGGATAAAAGTTTAAATTACTCTCCGAAAAGCAACGAGATGATTTTAACTGTGCACCGCTGGTTTGCAAATAAGGCCTGCCCGGGTGACTGGTTGTATAGCCGTCTGGGAGATCTGGCAAACCGTGTAACGTCACAGCTGGGAGGTTCTACTACCGATTCCACATCAAAGACATATAAAACCGGGTTATATAAAGTCGATGTTGGGGATCTGAACATCCGCAAAGGCCCGGGCACCAACTATGGGATTAATGGCATGATTACCGATCGCGGCACCTATACTATTACGGAAATTCAAAACGGCTACTGGGGCAAATTGAAATCGGGCGCAGGCTGGATTTCCGTGCACGAAGCTTATTGCACGTACAAGGGTGCCGCAAGTGGCGAATCCGAAGAGAAACCGAGTAGTAACTTTCTGGTGCAGGTTGATATTCCCGATCTCTACATCCGTAAGGGCCCGGGAACCAACTATGGCAACAACGGCTTTTGTCCTAAAGGCGTCTATACCATTGTTGAAGTGAAAAGTGGTGCAGGTTCCGATGCGGGCTGGGGCAAACTGAAAAGCGGTGCCGGCTGGATCAGTCTGGATTATGCCACTCGCATCTAA

[0065] LysCS10 nucleic acid sequence (SEQ ID NO:25):

[0066]

[0067] LysCS11 nucleic acid sequence (SEQ ID NO: 26):

[0068] ATGAACATTATCAAACATGGTTTGCGTTTCCGCGGCAGCTTTACGTACCGTAATCGTACGGACGAAATTATTTGGCACCATGCCGAGGCGAACTGTACTGTTGAAGATATTCACAGTTGGCACCTGAACAATGGTTGGATTGGTTGTGGGTATAACCTCATCATCTACAAAGATGGAACTGTGCACGAAGGTCGCCCACTGAACGCTGTCGGCGCCCACGCTTCGGGGCATAATTCCCGTTCTGTTGGCGTGTGTTGTATTGGCCGCTATGATGTGGAAACCATGCCTAAAGAACAACTCGACGCCGCCAAGCAAGTGCAGGCTTACCTTAAAGGTTTGTATCCAGGTGCTGCCACCAAGCGCCACAAAGATGTCAATGCGACATCATGCCCGGGCAAGAACTTTCCCTTCTCGGAGATTTCGGGTGCCTCGGCCGGCAGCGTCGACGTGACTACGAGCGATGTTCAGGCCCAAAGCTCAGATGACTGGTGCGCACGCCTCCAAAAAGAGTGTAACGCACAGAAATTCAGTAAACAGAAAGTGGACAATATCCCCGGCCCGGACACCCTGGCTGGTTGTCCTACGCTGGGTCGTAAATCCCGTGGGAAAATTACAGCTCTGATGCAGGAACGCCTCAATGCTCTGGGTTACGACTGTGGTGCAGTTGACGGTATTAATGGCACCAAAACCCAGGCTGCTATCAAGGCGTTCCAGCGTGATTACGGTTTAGTGGCCGACGGTATTGTGGGTCCGAAGACTTGGTCAAAACTTCTTGGCTTAAGTTAA

[0069] LysCS12 nucleic acid sequence (SEQ ID NO: 14):

[0070]

[0071] LysCS13 nucleic acid sequence (SEQ ID NO:15):

[0072]

[0073] According to a specific embodiment of the present invention, the nucleic acid molecule described herein is a DNA molecule or an RNA molecule.

[0074] 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.

[0075] On the other hand, the present invention also provides a carrier comprising the nucleic acid molecules described herein.

[0076] 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.

[0077] On the other hand, the present invention also provides a host cell comprising the nucleic acid molecules or the vectors described herein.

[0078] According to a specific embodiment of the present invention, the host cell can be a competent Escherichia coli cell, such as BL21(DE3).

[0079] On the other hand, the present invention also provides a method for preparing the aforementioned antimicrobial protein, the method comprising:

[0080] 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.

[0081] 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.

[0082] On the other hand, the present invention also provides the use of the said antimicrobial protein or the said antimicrobial drug composition in the preparation of products targeting Clostridium scintillans.

[0083] 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 Clostridium scintillans-related diseases;

[0084] According to a specific embodiment of the present invention, Clostridium scintillans-related diseases include one or more of the following:

[0085] Clostridium scintillans infection, inflammatory bowel diseases such as chronic diarrhea and diarrhea-predominant irritable bowel syndrome, non-alcoholic fatty liver disease, and tumors such as colorectal cancer, prostate cancer, and liver cancer.

[0086] 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.

[0087] The antimicrobial protein of the present invention, which targets Clostridium scintillans, can efficiently kill Clostridium scintillans and is specific. Attached Figure Description

[0088] Figure 1 Map of the LysCS12 expression vector plasmid for the constructed lysin.

[0089] Figure 2 Electrophoresis diagram for identifying heterologous expression of LysCS12 and LysCS13 in Escherichia coli.

[0090] Figure 3 Electrophoresis images of purified LysCS12 and LysCS13 proteins.

[0091] Figure 4 Experimental results showing that LysCS12 and LysCS13 can efficiently lyse Clostridium scintillans.

[0092] Figure 5 The study demonstrated the lytic activity of LysCS13 against different Clostridium species of gut symbiotic bacteria.

[0093] Figure 6 The study demonstrated the lytic activity of the lyase LysCS13 against different gut symbiotic bacteria. Detailed Implementation

[0094] 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.

[0095] 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.

[0096] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0097] Example 1: Identification and protein sequence analysis of lyase genes targeting Clostridium scintillans.

[0098] Genomic sequences of phages and prephages targeting Clostridium scintillans were collected, and lyase genes were identified using BLAST and Interproscan through sequence alignment and domain alignment. More than 70 potential phage 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:13, named LysCS01-LysCS13, respectively.

[0099] The codon-optimized nucleic acid sequences of the lysin for E. coli are shown in SEQ ID NO:14 to SEQ ID NO:26.

[0100] Example 2: Heterologous expression and purification of lysin targeting Clostridium scintillans

[0101] The identified lyase gene was codon-optimized in E. coli, and then the corresponding gene was cloned into the pET32a or pCold II vector, with a 6x-His tag added to the C-terminus or N-terminus. The gene is controlled by the lactose operon. Figure 1 This is a map of the LysCS12 expression vector plasmid for the constructed lysin.

[0102] The constructed plasmid containing the LysCS12 gene lysin 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.

[0103] 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.

[0104] 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).

[0105] Figure 2 Electrophoresis diagrams for identifying heterologous expression of LysCS12 and LysCS13 in Escherichia coli. Figure 2 The results showed that, after cleavage and separation, the lysin LysCS12 and LysCS13 proteins were expressed in soluble form in E. coli.

[0106] 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 column was then centrifuged at 18000 rpm at 4°C for 15 min, and the supernatant was collected. The supernatant was incubated with Ni resin at 4°C for 2 h. After the supernatant flowed through, the column was washed with wash buffer containing imidazole at different concentrations until the Bradford reagent no longer turned blue. 10 ml of elute buffer was added for elution, and the sample was detected by SDS-PAGE. The sample 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 the sample was frozen. Figure 3 Electrophoresis images of purified LysCS12 and LysCS13 proteins show that high-purity lysin proteins LysCS12 and LysCS13 can be obtained through multiple purification steps.

[0107] Example 3: Determination of the lysing activity of lysin protein against Clostridium scintillans

[0108] Clostridium scintillans DA266 (this strain was provided by the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, and has been publicly deposited and shared by the China General Microbiological Culture Collection Center; this strain is also the one described in the following literature: Bile acids modified by the intestinal microbiota promote colorectal cancer growth by suppressing CD8+T cell effector functions, Volume 57, Issue 4, 9 April 2024, Pages 876-889.e11, https: / / doi.org / 10.1016 / j.immuni.2024.02.014) was inoculated into anaerobic BHI liquid medium and cultured 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. Add the bacterial cell-containing buffer to a 96-well plate, then add LysCS12 or LysCS13 lyase protein to a final concentration of 5 μmol, with a final volume of 200 μL for both. After culturing for 3 hours, compile the experimental data and calculate the lysis activity. The lysis rate is calculated using the formula: [ΔOD600 tested (with added lyase) - ΔOD600 control (buffer only)] / initial OD600.

[0109] Figure 4This indicates that LysCS12 and LysCS13 can efficiently lyse Clostridium scintillans. After the addition of the lysin, the OD of the culture medium decreased rapidly, proving that LysCS12 and LysCS13 can effectively lyse Clostridium scintillans. With the addition of LysCS12 and LysCS13 lysins, the absorbance OD600 decreased by more than 50% within approximately 50 minutes, indicating that the lysins LysCS12 and LysCS13 have strong lysogenic activity against Clostridium scintillans.

[0110] Example 4: Targeting assay of lysin protein

[0111] The purified protein LysCS13 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 Clostridium scintillans was cultured to 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 a 96-well plate, and LysCS13 lyase protein was added to a final concentration of 5 μmol, with a final volume of 200 μL in each well. The plate was placed in a 96-well microplate reader, and the absorbance changes were continuously measured. A control group without the lyase was also set up. The absorbance changes were calculated after 2 h. The lytic activity was calculated.

[0112] Figure 5 The lytic activity of the lyase LysCS13 against different symbiotic Clostridium species in the gut was measured. LysCS13 exhibited highly efficient lytic activity against *Clostridium scintillans*; however, it did not show lytic activity against other *Clostridium* species, such as symbiotic *Clostridium*, *Clostridium butyricum*, *Clostridium scintillans*, and harmless *Clostridium*. This indicates that LysCS13 has high targeting specificity, enabling it to target and eliminate *Clostridium scintillans* in the gut without damaging other symbiotic *Clostridium* species.

[0113] Figure 6 The lytic activities of the lysin LysCS13 and lysozyme (egg white source, CAS: 12650-88-3) against different intestinal commensal bacteria were studied. Figure 6As shown in Figure A, LysCS13 exhibits highly efficient lytic activity against *Clostridium scintillans*; however, for the other 11 tested gut commensal bacteria (information and taxonomic status of the tested strains are shown in Table 2), LysCS13 only showed lytic activity against strain DA394, and did not show lytic activity against other strains. These tested gut commensal bacteria came from five common gut commensal bacterial phyla, including Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Fusobacterium. This indicates that the lysin LysCS13 has high targeting specificity and can target and clear *Clostridium scintillans* in the gut. In addition, although egg white-derived lysozyme has some lytic activity against *Clostridium scintillans*, it also causes the lysis of multiple gut commensal bacteria, showing no obvious targeting specificity. Figure 6 Image B in the picture.

[0114] Table 2: Information on gut symbiotic bacteria used for the LysCS13 lysin targeting assay

[0115] 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

[0116] 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. An antibacterial protein targeting Flavobacterium that is targeted to the outer membrane of the bacteria, wherein, The antibacterial protein comprises one or more of the following proteins formed by the following amino acid sequences: an amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO:

2.

2. A nucleic acid molecule encoding the antibacterial protein targeting Flavobacterium that is claimed in claim 1.

3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of the nucleic acid molecule comprises a sequence shown in any one of SEQ ID NO: 14 to SEQ ID NO:

15.

4. The nucleic acid molecule of claim 2, wherein the nucleic acid molecule is modified or unmodified.

5. A vector comprising the nucleic acid molecule of any one of claims 2 to 4.

6. A host cell comprising the nucleic acid molecule of any one of claims 2 to 4 or the vector of claim 5.

7. A method of preparing the antibacterial protein of claim 1, the method comprising: transcribing in vitro the nucleic acid molecule of any one of claims 2 to 4 or the vector of claim 5 or the host cell of claim 6 to prepare the antibacterial protein of claim 1.

8. An antibacterial pharmaceutical composition comprising: the antibacterial protein of claim 1 or the nucleic acid molecule of any one of claims 2 to 4 or the vector of claim 5, and a pharmaceutically acceptable excipient.

9. Use of the antibacterial protein of claim 1 or the antibacterial pharmaceutical composition of claim 8 in the preparation of a medicament for targeting and lysing Flavobacterium.

10. Use according to claim 9, wherein, The medicament is a disinfectant.

11. Use according to claim 9, wherein, The medicament is a bactericide.

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