Antibacterial protein targeting clostridium scintillator and preparation method and application thereof

Abstract

The invention provides antibacterial protein targeting clostridium scintillator as well as a preparation method and application of the antibacterial protein. Specifically, the invention provides an antibacterial protein targeting clostridium scintillator 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: 13; (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: 13 is 85% or more or 90% or more. The antibacterial protein disclosed by the invention can be used for efficiently killing clostridium scintillation and has specificity.

Description

Technical Field

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

[0002] Clostridium scindens is a Gram-positive, facultative anaerobic bacterium belonging to the genus Clostridium. Although Clostridium scindens is part of the healthy microbiome to a certain 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 effect of prostate cancer patients and increase the risk of hormone-resistant tumors. (2) Clostridium scindens produces secondary bile acids such as deoxycholic acid (DCA) through bile acid metabolism. These secondary bile acids play 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 inhibits the NFAT2 signaling pathway in CD8+ T cells by enhancing the activity of cell membrane calcium ion pumps and reducing the accumulation of intracellular calcium ions. 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 response. In summary, DCA produced 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 in turn affect the liver's anti-tumor immune response 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, the 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 promotes 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 C. scintillans have been used to reduce the number of this bacterium. In mouse experiments, the use of phages targeting C. scintillans reduced the concentration of intestinal DCA and effectively slowed the growth of tumors. This suggests that phages may become a tool for precise control of intestinal microorganisms. In addition, bile acid sequestrants have also shown some effect by reducing the production of DCA. In mouse models, this approach helped restore anti-tumor CD8+ T cell function. In some cases, researchers have reduced C. scintillans in the intestinal flora through antibiotic treatment.

[0004] At present, the main means of treating Clostridium scintillans infection are antibiotics and phage therapy, but these methods have some obvious problems: 1. Bacteria may develop resistance to phages and antibiotics through mutations, which reduces the effectiveness of phage or antibiotic treatment and easily leads to the further spread of resistant strains. 2. Non-specific effects. The use of antibiotics will not only kill Clostridium scintillans, but also affect the beneficial flora in the intestine, leading to an imbalance in the intestinal microecology. This non-targeted treatment will cause damage to the 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). 3. Immune-related side effects. For immunosuppressed patients, the treatment of Clostridium scintillans infection is particularly tricky. Antibiotics themselves may not be able to effectively control the infection in the case of low immune function, and further suppression of the immune system by drugs may aggravate the condition. In addition, the use of immunomodulatory therapy has side effects and uncertainties, and it is difficult to ensure its effectiveness in all patients.

[0005] In summary, antibiotic treatment of Clostridium scintillans faces problems such as drug resistance, microecological destruction and nonspecific side effects, and new effective means to deal with Clostridium scintillans infection need to be developed. Summary of the invention

[0006] The inventors of this case discovered a class of antibacterial proteins targeting Clostridium scintillans during their research, which can kill Clostridium scintillans efficiently and specifically.

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

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

[0009] (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);

[0010] (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: 13.

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

[0012] 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.

[0013] 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: 13 of the present invention 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 the amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:13 of the present invention contains a catalytic domain and a cell wall binding domain and their position information as shown in Table 1. The catalytic domain includes Amidase_2, Amidase_3, CHAP, Glyco_hydro_25, Lysozyme_like, and Glucosaminidase. The cell wall binding domain includes PG_binding_1, Choline_bind_1, Choline_bind_3, CW_7, SH3, and LysM. As can be seen from Table 1, LysCS13 has the same catalytic domain Amidase_3 as LysCS12 and LysCS04, while LysCS13 has the same cell wall binding domain Choline_bind_1 and Choline_bind_3 as LysCS12 and LysCS01.

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

[0042]

[0043] According to some specific embodiments of the present invention, the antibacterial protein targeting Clostridium scintillans 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, one or more of 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 targeting Clostridium scintillans or its chimera of the present invention, wherein the chimera 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 encoding the antibacterial protein targeting Clostridium scintillans of 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 one 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 of the present invention is a DNA molecule or an RNA molecule.

[0074] 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.

[0075] On the other hand, the present invention also provides a vector comprising the nucleic acid molecule of the present invention.

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

[0077] 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.

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

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

[0080] 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.

[0081] 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.

[0082] On the other hand, the present invention also provides the use of the antibacterial protein or the antibacterial drug composition in the preparation of a product 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 preventing and treating diseases related to Clostridium scintillans;

[0084] According to a specific embodiment of the present invention, the disorders associated with Clostridium fulgidus include one or more of the following disorders:

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

[0086] 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.

[0087] The antibacterial protein targeting Clostridium scintillans of the present invention can kill Clostridium scintillans efficiently and has specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 This is the plasmid map of the LysCS12 expression vector of the constructed lytic enzyme.

[0089] Figure 2 The electrophoretic diagrams are for identification of LysCS12 and LysCS13 heterologously expressed in E. coli.

[0090] Figure 3 The electrophoresis diagram of the purified LysCS12 and LysCS13 proteins.

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

[0092] Figure 5 The lytic activity of LysCS13 against different intestinal commensal Clostridium bacteria was shown.

[0093] Figure 6 The lytic activity of the lytic enzyme LysCS13 against different intestinal commensal bacteria is shown. DETAILED DESCRIPTION

[0094] 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.

[0095] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both 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.

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

[0097] Example 1: Identification and protein sequence analysis of the lytic enzyme gene targeting Clostridium fulgidus

[0098] Phage and prophage genome sequences targeting Clostridium scintillans were collected, and the lytic enzyme genes therein were identified by sequence alignment and domain alignment using BLAST and Interproscan. More than 70 potential phage lytic enzyme gene sequences were found, and the present invention further identified a class of lytic enzymes from them, comprising the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 13, respectively named LysCS01-LysCS13.

[0099] The nucleic acid sequences of the lytic enzyme after codon optimization for Escherichia coli are shown in SEQ ID NO: 14 to SEQ ID NO: 26.

[0100] Example 2. Heterologous expression and purification of lytic enzyme targeting Clostridium fulgidus

[0101] The identified lytic enzyme genes were codon-optimized for E. coli, and then the corresponding genes were cloned into pET32a 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 LysCS12 expression vector of the lytic enzyme.

[0102] The constructed plasmid containing the lytic enzyme LysCS12 gene was transformed into BL21 (DE3) competent cells, and then evenly spread on LB plates (containing 50 μg / 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 50 μg / 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, for 18h to induce protein expression.

[0103] 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.

[0104] 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).

[0105] Figure 2 The electrophoretic diagrams are for identification of LysCS12 and LysCS13 heterologously expressed in E. coli. Figure 2 It was shown that the lytic enzymes LysCS12 and LysCS13 proteins were expressed in soluble form in Escherichia coli after fragmentation and separation.

[0106] 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 Ni resin at 4℃ for 2h; after the supernatant flowed through, the column was washed with Wash buffer 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, the protein was collected and tested by SDS-PAGE gel; the protein was concentrated, the concentration was determined and frozen. Figure 3 The electrophoresis diagram of the purified proteins LysCS12 and LysCS13 shows that highly pure lytic enzyme proteins LysCS12 and LysCS13 can be obtained through multiple purification steps.

[0107] Example 3: Determination of the lytic activity of lytic enzyme protein against Clostridium scintillans

[0108] The scintillans DA266 (the strain was provided by the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, and has been publicly preserved and shared for public benefit at the General Microbiology Center of the China Microbiological Culture Collection Administration Committee. The strain is also a strain recorded 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, 9April 2024, Pages876-889.e11, https: / / doi.org / 10.1016 / j.immuni.2024.02.014) was inoculated into anaerobic BHI liquid medium and cultured to OD600 = 0.6. The bacterial liquid was centrifuged from the culture medium at 4000rpm for 5min, washed twice with buffer (50mM Tris-HCl [pH 7.0], 100mM NaCl), and then resuspended with the buffer after washing. Add the buffer containing the bacteria into a 96-well plate, add LysCS12 or LysCS13 lyase protein to a final concentration of 5 micromolar, and the final volume is 200 microliters. After 3 hours of incubation, organize the experimental data and calculate the lysis activity. The lysis rate calculation formula is: [test ΔOD600 (add 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 lyase, the OD of the culture solution decreased rapidly, proving that LysCS12 and LysCS13 can effectively lyse Clostridium scintillans. After adding LysCS12 and LysCS13 lyases, the absorbance OD600 had dropped by more than 50% in about 50 minutes, indicating that the lyases LysCS12 and LysCS13 have strong lysing activity against Clostridium scintillans.

[0110] Example 4: Targeted determination of lytic enzyme protein

[0111] The purified protein LysCS13 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 Clostridium scintillans was cultured to OD600 = 0.6. The bacterial solution was centrifuged at 4000rpm for 5min from the culture medium, washed twice with buffer (50mMTris-HCl [pH 7.0], 100mM NaCl), and the bacteria were resuspended in the buffer after washing. The buffer containing the bacteria was added to a 96-well plate, and LysCS13 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 change in its absorbance. At the same time, a control group without lytic enzyme was set up. After 2h, the change in its absorbance was calculated. Calculate the lytic activity.

[0112] Figure 5 The lytic activity of the lytic enzyme LysCS13 on different intestinal symbiotic Clostridium bacteria. LysCS13 showed efficient lytic activity against Clostridium scintillans; but for other Clostridium bacteria, such as symbiotic Clostridium, Clostridium butyricum, Clostridium scintillans and Clostridium innocua, LysCS13 did not show lytic activity. This shows that LysCS13 has a high degree of targeting and can target Clostridium scintillans in the intestine without destroying other symbiotic intestinal Clostridium bacteria.

[0113] Figure 6 The lytic activity of LysCS13 and lysozyme (from chicken egg white, CAS: 12650-88-3) on different intestinal symbiotic bacteria. Figure 6As shown in Figure A, LysCS13 exhibited efficient lytic activity against Clostridium scintillans; but for the other 11 intestinal symbiotic bacteria tested (the information and taxonomic status of the test strains are shown in Table 2), LysCS13 only had lytic activity against strain DA394, and did not show lytic activity against other strains. These intestinal symbiotic bacteria tested came from five common intestinal symbiotic bacterial phyla, including Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Fusobacterium. This shows that the lytic enzyme LysCS13 has a high degree of targeting and can target and eliminate Clostridium scintillans in the intestine. In addition, although the lysozyme derived from egg white has a certain lytic activity against Clostridium scintillans, it will also cause the lysis of a variety of intestinal symbiotic bacteria, and there is no obvious targeting ( Figure 6 Figure B).

[0114] Table 2: Information on intestinal commensal bacteria used for targeting of LysCS13

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

[0116] 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 or a chimera thereof targeting Clostridium fulgidus, 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: 13; (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:

13.

2. The antimicrobial protein targeting Clostridium fulgidus 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_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; More preferably, the chimera comprises the catalytic domain Amidase_3 and / or the cell wall binding domains Choline_bind_1 and / or Choline_bind_3.

3. A nucleic acid molecule encoding the antibacterial protein targeting Clostridium fulgidus according to claim 1; Preferably, the nucleotide sequence of the nucleic acid molecule comprises any one of SEQ ID NO: 14 to SEQ ID NO: 26; 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 according to claim 1 or 2, the method comprising: The antibacterial protein 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 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 according to claim 1 or 2 or the antimicrobial pharmaceutical composition according to claim 7 in the preparation of a product targeting Clostridium scintillans.

9. Use of the antimicrobial protein 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 Clostridium fulgidus; Preferably, the disease comprises one or more of the following diseases: Clostridium fulgidus infection, inflammatory bowel diseases such as chronic diarrhea and diarrhea-predominant irritable bowel syndrome, non-alcoholic fatty liver disease, tumors such as colorectal cancer, prostate cancer and liver cancer.

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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