Antibacterial protein targeting clostridium innocua and preparation method and application thereof

Abstract

The present application provides an antibacterial protein targeting Clostridium innocua and a preparation method and application thereof. Specifically, the present application provides an antibacterial protein targeting Clostridium innocua 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: 8; (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); (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: 8. The antibacterial protein of the present application can efficiently kill Clostridium innocua of different sources, including strains with antibiotic resistance, and has specificity.

Description

Technical Field

[0001] This invention provides an antimicrobial protein that targets harmless Clostridium, along with its preparation method and application. Background Technology

[0002] Clostridium innocuum is a Gram-positive anaerobic bacterium belonging to the genus Clostridium. Despite its name suggesting harmlessness, recent studies have found that it may be associated with a variety of infectious and non-infectious diseases in humans. Clostridium innocuum is increasingly recognized as an important pathogen, particularly in immunocompromised populations and those with excessive antibiotic use.

[0003] Infectious diseases associated with *Clostridium perfringens* include: 1. *Clostridium perfringens* has been found in antibiotic-associated diarrhea and enteritis, especially after the use of fluoroquinolone antibiotics. Some studies have reported that *Clostridium perfringens* may coexist with *Clostridium difficile* or fill its niche when *Clostridium difficile* disappears, leading to persistent or recurrent diarrhea. 2. Sepsis. Although cases of sepsis caused by *Clostridium perfringens* are rare, its infection risk is increased in immunosuppressed patients, especially after the use of broad-spectrum antibiotics. *Clostridium perfringens* sepsis is common in patients undergoing chemotherapy or bone marrow transplantation, suggesting the potential pathogenicity of this bacterium in critically ill patients. 3. Other infections. Individual cases of osteomyelitis, endocarditis, peritonitis, brain abscess, and genitourinary infections caused by *Clostridium perfringens* have been reported. Although these cases are rare, they suggest that *Clostridium perfringens* may have potentially broad pathogenicity under certain circumstances.

[0004] Non-infectious diseases associated with Clostridium perfringens: (1) Clostridium perfringens may play an important role in intestinal inflammation (such as inflammatory bowel disease) and related diseases by activating the NOD2 pathway through interaction with lipid rafts of intestinal epithelial cells and inducing cytotoxic and inflammatory responses. (2) The migration and colonization of Clostridium perfringens in Crohn's disease patients may drive the formation of crawling fat and may play a key role in the pathogenesis of Crohn's disease, making it a potential therapeutic target for Crohn's disease. (3) Clostridium perfringens can convert progesterone into less active cyclooxyprogesterone, thereby reducing the host's progesterone levels. Long-term administration of Clostridium perfringens leads to follicular arrest in mice. In conclusion, Clostridium perfringens may be an important factor leading to female infertility by inactivating host intestinal progesterone and inducing ovarian follicular arrest, making it a potential therapeutic target for female infertility. (4) In some studies, harmless Clostridium is considered an important indicator of gut microbiota imbalance after antibiotic use, which may aggravate intestinal inflammation; harmless Clostridium may affect the host's metabolic state through its excessive proliferation or metabolites in the gut, and thus be associated with diseases such as metabolic syndrome.

[0005] Currently, treatment options for Clostridium perfringens infections are limited, with most clinical protocols drawing on experience with other Clostridium species. However, treatment is becoming more challenging due to increasing resistance in Clostridium perfringens to certain antibiotics. 1. Antibiotic Treatment: Broad-spectrum antibiotics, such as vancomycin, metronidazole, and teicoplanin, are commonly used to treat Clostridium perfringens infections. However, antibiotic use requires caution, especially in cases where resistance may develop. Studies have shown that resistance to antibiotics such as vancomycin is gradually increasing in Clostridium perfringens.

[0006] Although research on Clostridium perfringens is still in its early stages, evidence already suggests it plays an important role in both infectious and non-infectious diseases. Treatment for Clostridium perfringens infections currently relies primarily on antibiotics, but drug resistance exacerbates the challenge. Future research should focus on developing novel antimicrobial strategies to address the pathogenicity of Clostridium perfringens in humans.

[0007] Currently, the main treatment for Clostridium perfringens infections is broad-spectrum antibiotics, but these methods have several significant problems. The main issues with existing treatment techniques are: 1. Antibiotic resistance: Clostridium perfringens is gradually developing resistance to many commonly used antibiotics, especially fluoroquinolones and vancomycin. This reduces the effectiveness of broad-spectrum antibiotic treatment and easily leads to the further spread of drug-resistant strains. During treatment, doctors often face the challenge of having a limited selection of antibiotics. 2. Disruption of the gut microbiota: Antibiotics not only kill harmless Clostridium perfringens but also affect beneficial bacteria in the gut, leading to gut microbiota imbalance. Disruption of the gut microbiota can trigger more serious secondary infections, such as Clostridium difficile-associated infections, and may even worsen the patient's condition. 3. Non-specific effects: Current antibiotic therapies lack specificity and often act on the entire bacterial population. This non-targeted treatment can harm normal flora, especially with long-term use, increasing the risk of opportunistic infections by other pathogens (such as fungi and drug-resistant bacteria). 4. Immune-related side effects: Treatment of Clostridium perfringens infections is particularly challenging for immunosuppressed patients. Antibiotics themselves may not be effective in controlling infection in patients with weakened immune systems, and further suppression of the immune system by drugs may worsen the condition. Furthermore, the use of immunomodulatory therapies carries side effects and uncertainties, making it difficult to ensure their effectiveness in all patients.

[0008] In summary, antibiotic treatment of harmless Clostridium faces challenges such as drug resistance, disruption of the microecology, and nonspecific side effects. New and effective means of dealing with harmless Clostridium infections need to be developed, especially in the face of complex and multidrug-resistant infections. Summary of the Invention

[0009] The inventors in this case discovered a class of antimicrobial proteins that target harmless Clostridium botulinum, which can effectively kill harmless Clostridium botulinum from different sources, including antibiotic-resistant strains, and are specific.

[0010] Specifically, on one hand, the present invention provides an antimicrobial protein or a chimeric thereof targeting harmless Clostridium, wherein the antimicrobial protein comprises one or more proteins formed from the following amino acid sequences:

[0011] (a) Any of the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:8;

[0012] (b) A derivative sequence of (a) with one or more amino acids replaced, added and / or deleted, and having the same function as (a);

[0013] (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:8.

[0014] 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:8 are named LysCI01 to LysCI08, respectively, and the specific amino acid sequences are as follows.

[0015] LysCI01 protein sequence (SEQ ID NO:2):

[0016] MTMSKKGIDISEHNGNAETAIKGADFVMIRSSWGHFAIDKKLEDNVKLCKKHGKPYGFYHFSYARNYKEAKDEAHKCMNLISRYGNTYPIALDLEWDDGANWKKNNGITYASEMEVLKAWKEVVEQECKTYLL LYCNRSFYNQLKAVNEARLKSVDLWLAEWGVSEPSIPCGMWQYRGDPLDLDVAYYDYPTLLKGLHKGNSQKPTTEIKVGDKVSPKEAVNYDGVKLIADVKGMKLDVIEISGDRIVVSYPTGGTEAFAKSNLKK

[0017] LysCI02 protein sequence (SEQ ID NO:1):

[0018] MKANDFLKTTYGKYYDIDGYYGAQCWDYFAYLCTVIGSKIINCTSTGYVIDVWNNRKNNGVLDKFKEVPVSSLQNGDVVVFKNGGSLTPLSHIGVFAGWLNKGSTFTLQAQNQYGSASVNKGLMYVSDIAGCLRPKVWDNKSPDLPIKSKGKASAKYDYIRVRNKPSLDNSALTGDWYNTGMKLNYQNVVKSDGLYWLEYVGSNTNKKHYVAYGTTDGKTVYWKID

[0019] LysCI03 protein sequence (SEQ ID NO:3):

[0020] MKKILLLLLLVPILIFICIFGLLQQIGNIFANEDSQTTVVKTCKYDWQTKFEDEEIEKMRKTAEQYLIDKKYVNAVLGIYSMDKKQDVAEIGKKINKLIEYAMEKKMDYDVMTYIQAYKFTADYFDYLRNNNMEHSLATAKTYQELDRNKDKYKENDYQFAQLALVSMSADCQMGQGGLPLDKNTYSVTSGFPYRSDGSFHAGLDMGVKYGSRTYAIEDGEVMIAGTGCPPDGGYFGNQCGMDDMYGAGNYVLYKVQKGMDTIYILQCHLSEVRVKKGEKITQGQLIGLSGNSGNSSGAHLHLEIHKNVVAIGADKNLVNPCEYIKGLCEVKNEELysCI04 protein sequence (SEQ ID NO:4):

[0021] MLEVHFNAFNGSGHGTEIFVTDSEQYTDVEQAIMNKLGKHFVKRGGSGVKVTNWLVIYTCKCLGISSALLETCFIDNKADMAEYQAHKESVAQGIVDGIAEGFQLKANSAEQKPGNKPAAQNKPSKPSKPAQPDQILHKGEYFVIPGVHSVDQVLANMDSIWCEEMTGNGGNS

[0022] LysCI05 protein sequence (SEQ ID NO:5):

[0023] MNITKMLAPVIYSGARSGIKRTKNGGVTIHNTDNFKAGAGAKNHGTYLQNSGSTLQASWHYAVDDKMITQSIPDNEVAWHAGDGYGNGNMTTIAIEICVNPDSNLELATDNAAWLAAKLLKAQGLDHQSLYQHHDWSGKNCPSQIRANKPYSWAKFVSKVKGYLQQNNASSKPKPDQILHKGEYFVIPGVHSVDQVLANMDSIWCEEMTGNGGNSIQAGPLTKCDKNGKKTKSQVFSVGDYWKCDKKFKVLAVDKPTNSVQANVGGRKIWLYAGPLREV

[0024] LysCI06 protein sequence (SEQ ID NO:6):

[0025] MAYTNSKMVAYTKLSPNHSGQRTHSIDRITPHCVVGQCTAEGLGDWFAKASTQASSNYGIDKNGRVGLYVEEKNRSWCSSSNANDQRAVTIECASDTKEPYWMNDKVYASLIKLCVDICKRNGKKKLLWFANKDKTLNYVPKSDEMVLTVHRWFANKSCPGDWLYARLGDLATKVTAELSGTTSGSGSTAPTTQMYRVRKSWSDAKSQIGAYKVLDNANKKVDENSGYKVFDASGNVVYPVASTPAPTPSKDTSYKVQVSIANLNIRKGPGTNYDRTGQFTGKGIFTIVQESKGAGATLWGKLKSGAGWISLDFAKKL

[0026] LysCI7 protein sequence (SEQ ID NO:7):

[0027] MKKQLKHYLQEHIEEYKYLVHDLYEHPEIGNQEFRSMRVLCKLLQKEGFEVTEEYVVPTGFLAVWKSEKPGPVIAYMCEYDALPEVGHGCGHNLIAGMSLAAGCALKSILSAIGGEVRVIGTPAEENFGGKVSMAAAHVFDDVDAALMLHPDTKNSLGGRTLAIYPLRFEFFGQNAHACTPQNGKSALDAAVM SYLSINLLRQFAEPNTFIHGVIAHGGEAANVIPAYASLEYYFRGETMEYVKELCEKAKACVEGACTMSGCTNRITTYECPYDDCVINYTLADMLQDEYEDLGYVWEGVDEVAQGSSDVGSVSYCCPTLQGYIKIADSCVNGHSREMAAATISQEGSRALLDGAFALADIGRRLIMEPDTLKKAWKEFNSTVKES

[0028] LysCI8 protein sequence (SEQ ID NO:8):

[0029] MAAEGPFNKKYPKVPNGIRDPEYSIDCGIQELRSVLKKAGVKSGEDKERIKVALAGYNFGSGYIEWVDANKGGKWTLENAKEFSAMMAAKMGWSVYGDPNYVDKVMEYYETAAIGIEGKDAFLVPMKSYTLTSSVGQ RSLGDYHYGTDIDGGYGANIYAPAAGIVYEVSNDCPPSDGYLGNSCPYSGGYIGGGNYVMLKVTNKKEDYYIFLCHMKRTLVSKGQKVKKGQKIGEQGHSGNSTASHLHIEIHKGTAHIATKDGLVDPEKIMNFKKR

[0030] The protein formed by any of the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:8 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, Lysozyme_like, CHAP, Glyco_hydro_25, Peptidase_M23, and Peptidase_M20. The cell wall-binding domains include SH3 and Zoocin_A, etc. As can be seen from Table 1, the lyase LysCI01 shares the same cell wall-binding domain SH3 as LysCI02 and LysCI06.

[0031] Table 1: Catalytic and cell wall-binding domains of antimicrobial proteins LysCI01 to LysCI08

[0032]

[0033] According to some specific embodiments of the present invention, the antimicrobial protein or its chimeric compound targeting harmless Clostridium of the present invention comprises one or more of the following: catalytic domains Amidase_2, Amidase_3, Lysozyme_like, CHAP, Glyco_hydro_25, Peptidase_M23 and Peptidase_M20, and cell wall binding domains SH3 and Zoocin_A.

[0034] According to some specific embodiments of the present invention, the antimicrobial protein or its chimeric compound targeting harmless Clostridium of the present invention comprises a catalytic domain CHAP, a Glyco-hydro-25 and / or a cell wall binding domain SH3.

[0035] On the other hand, the present invention also provides a nucleic acid molecule that encodes the antimicrobial protein targeting harmless Clostridium as described in the present invention.

[0036] 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:9 to SEQ ID NO:16.

[0037] LysCI01 nucleic acid sequence (SEQ ID NO:10):

[0038] ATGACCATGAGCAAGAAAGGCATCGATATCAGCGAACATAATGGCAATGCGGAAACCGCGATTAAAGGCGCGGATTTTGTGATGATTCGCAGCAGCTGGGGCCATTTTGCGATTGATAAAAAGCTGGAGGACAATGTGAAGCTGTGCAAAAAACACGGCAAACCGTATGGCTTTTACCATTTTAGCTATGCGCGCAATTATAAAGAAGCGAAGGACGAAGCGCATAAATGCATGAATCTGATTAGCCGCTATGGCAATACCTATCCGATTGCGCTGGATCTGGAATGGGATGATGGCGCGAATTGGAAAAAAAACAACGGCATTACCTATGCGAGCGAAATGGAAGTGCTGAAAGCGTGGAAAGAAGTGGTGGAACAGGAATGCAAAACCTATCTGCTGCTGTATTGCAATCGCAGCTTCTATAATCAGCTGAAAGCGGTGAATGAAGCGCGCCTGAAAAGCGTGGATCTGTGGTTGGCGGAATGGGGTGTTAGCGAACCAAGCATTCCGTGCGGCATGTGGCAATATCGTGGCGATCCGCTGGATCTGGATGTGGCGTATTATGATTATCCGACCCTGCTGAAAGGCCTGCATAAAGGCAATAGCCAGAAACCGACCACCGAAATTAAAGTGGGCGATAAAGTGAGCCCGAAAGAAGCGGTGAATTATGATGGCGTGAAACTGATTGCGGATGTGAAAGGCATGAAACTGGACGTGATTGAAATTAGCGGCGATCGCATTGTGGTGAGCTATCCAACCGGTGGCACCGAAGCGTTTGCGAAAAGCAACCTGAAGAAGTGA

[0039] LysCI02 nucleic acid sequence (SEQ ID NO:9):

[0040] ATGAAGGCGAACGATTTTCTGAAAACCACCTACGGCAAGTATTATGACATCGACGGCTATTATGGCGCCCAGTGCTGGGATTATTTTGCGTATCTGTGCACCGTGATTGGCAGCAAAATCATTAATTGCACCAGCACCGGCTATGTGATTGATGTGTGGAACAACCGCAAAAATAATGGCGTGCTGGACAAGTTTAAAGAAGTGCCGGTGAGCAGCTTGCAGAATGGCGATGTGGTGGTGTTTAAAAATGGCGGCAGCCTGACCCCGTTAAGCCATATTGGCGTGTTTGCGGGCTGGCTGAATAAAGGCAGCACCTTTACCCTGCAGGCGCAGAATCAATATGGCAGCGCGAGCGTGAATAAAGGCCTGATGTACGTGAGCGATATTGCGGGTTGCCTGCGCCCAAAAGTGTGGGATAATAAAAGCCCGGATCTGCCGATTAAAAGCAAAGGCAAAGCGAGCGCGAAATATGATTATATTCGCGTGCGCAATAAACCGAGCCTGGATAATAGTGCGCTGACCGGCGATTGGTATAATACCGGCATGAAGCTGAATTATCAGAACGTGGTGAAAAGCGATGGCCTGTATTGGCTGGAATATGTGGGCAGCAACACCAATAAAAAACACTACGTGGCGTATGGTACCACCGATGGCAAAACCGTGTATTGGAAGATCGACTGA

[0041] Nucleic acid sequence of LysCI03 (SEQ ID NO:11):

[0042]

[0043] LysCI04(SEQ ID NO:12):

[0044] ATGCTGGAAGTTCATTTTAATGCATTTAACGGAAGCGGCCATGGAACTGAAATCTTCGTTACGGATAGTGAACAATATACCGATGTTGAACAGGCAATCATGAACAACTGGGCAAGCATTTCGTAAAAC GTGGCGGAAGCGGCGTGAAGGTCACGAACTGGTTGGTCATCTACACCTGTAAATGCTTGGGCATCTCATCCGCTCTGTTGGAAACTTGTTTCATCGATAACAAGGCGGATATGGCTGAATACCAAGCCCAT AAAGAATCCGTTGCCCAGGGCATTTGTAGACGGAATCGCGGAGGGTTTTCAAATAAAGCGAACTCTGCTGAACAAAAACCAGGGAATAAACCAGGCTCAGAACAAACCATCTAAACCGAGTAAGCCAG CTCAGCCGGATCAGATTCTGCATAAAGGGGAGTATTTTGTTATCCCGGGGGTACATAGTGGACCAGGTCTTGGCAAACATGGACAGCATTTGGTGTGAGGAAATGACTGGCAACGGAGGAAACTCCTAA

[0045] LysCI05(SEQ ID NO:13):

[0046] ATGAATATCACCAAAATGCTGGCCCCTGTTATTTACTCGGGTGCACGCTCAGGCATTAAACGCACGAAGAACGGCGGTGTCACTATTCACAACACTGATAATTTCAAAGCCGGCGCTGGCGCCAAAAATCACGGCACCTACCTGCAGAATTCGGGAAGCACACTTCAAGCGAGCTGGCACTACGCCGTCGATGACAAAATGATTACCCAATCGATTCCGGATAATGAAGTGGCCTGGCACGCCGGCGACGGCTATGGAAACGGTAACATGACTACCATCGCAATTGAAATCTGCGTTAACCCGGACAGCAATTTGGAATTAGCAACAGACAATGCGGCATGGCTGGCTGCCAAACTTCTTAAAGCGCAGGGTCTGGATCACCAAAGTTTGTACCAGCATCATGACTGGAGTGGTAAAAACTGCCCCTCGCAGATTCGTGCTAATAAACCGTATTCGTGGGCCAAATTTGTATCAAAAGTAAAGGGATACTTACAGCAGAACAACGCTAGTAGTAAACCGAAGCCGGATCAGATCCTGCATAAAGGTGAGTATTTCGTGATCCCGGGTGTACACAGCGTGGACCAGGTTTTGGCAAATATGGACAGCATCTGGTGTGAAGAGATGACTGGCAATGGCGGGAATTCGATCCAGGCGGGCCCGCTGACTAAGTGCGATAAGAACGGAAAAAAGACGAAAAGCCAAGTCTTTTCGGTAGGGGATTACTGGAAGTGCGACAAGAAGTTCAAAGTTTTAGCCGTCGACAAACCAACTAACAGCGTGCAAGCTAATGTGGGTGGTCGTAAAATCTGGTTATATGCAGGCCCCCTGCGTGAGGTCTAA

[0047] Nucleic acid sequence of LysCI06 (SEQ ID NO:14):

[0048] ATGGCATACACCAATTCGAAAATGGTAGCTTATACCAAATTGTCACCGAATCATTCCGGCCAGCGCACGCATTCGATCGACCGTATTACCCCCCACTGCGTCGTAGGCCAATGCACAGCAGAAGGCCTGGGCGATTGGTTTGCTAAAGCCTCCACGCAGGCAAGTTCTAACTACGGGATTGACAAAAATGGCCGTGTGGGTCTTTATGTGGAAGAAAAAAATCGCAGCTGGTGCAGTTCTTCCAATGCGAACGACCAGCGTGCAGTTACAATTGAATGCGCATCGGACACGAAGGAACCGTATTGGATGAACGATAAAGTGTACGCCTCCCTGATTAAGCTGTGTGTGGATATCTGCAAGCGCAATGGCAAAAAAAAATTACTCTGGTTTGCAAATAAGGATAAAACCCTGAACTATGTTCCTAAATCAGATGAAATGGTCTTGACTGTACATCGCTGGTTCGCAAATAAATCCTGCCCGGGAGACTGGCTCTATGCACGTTTGGGCGACTTAGCAACTAAAGTCACCGCAGAACTGAGCGGGACAACGAGCGGCAGCGGCAGCACCGCCCCAACTACCCAGATGTACCGCGTTCGCAAGAGCTGGAGCGATGCCAAAAGTCAGATCGGGGCTTATAAGGTACTCGACAACGCCAACAAAAAAGTCGATGAGAACTCAGGTTATAAAGTTTTTGACGCCTCGGGGAACGTGGTGTACCCCGTTGCAAGCACGCCGGCTCCGACTCCGAGCAAGGACACTAGTTATAAAGTTCAGGTGTCAATCGCAAACCTGAACATTCGCAAGGGGCCGGGGACTAATTATGATCGTACCGGCCAGTTCACCGGCAAAGGTATCTTTACAATTGTGCAGGAAAGTAAAGGGGCAGGTGCAACCTTGTGGGGAAAGCTGAAATCCGGGGCGGGTTGGATCTCCCTGGATTTCGCAAAAAAACTGTGA

[0049] LysCI07 nucleic acid sequence (SEQ ID NO:15):

[0050]

[0051] LysCI08 nucleic acid sequence (SEQ ID NO:16):

[0052] ATGGCCGCTGAGGGACCATTCAACAAAAAATACCCGAAAGTGCCGAACGGTATCCGTGATCCGGAATATAGCATCGACTGCGGAATTCAGGAACTGCGCAGTGTCCTGAAGAAAGCTGGTGTGAAATCTGGTGAAGATAAAGAACGTATCAAAGTGGCTCTGGCAGGGTATAACTTTGGTTCAGGTTATATCGAATGGGTGGATGCCAATAAGGGCGGGAAATGGACCTTGGAAAATGCAAAGGAATTTAGCGCAATGATGGCTGCCAAAATGGGTTGGTCAGTATACGGCGATCCAAATTACGTTGATAAAGTCATGGAGTACTATGAGACGGCTGCTATCGGCATCGAAGGCAAAGATGCTTTCTTGGTCCCGATGAAGTCGTATACGTTAACCAGCAGTGTCGGCCAGCGCTCTCTGGGCGACTACCATTATGGCACCGATATCGATGGGGGTTATGGTGCCAATATTTACGCGCCGGCTGCCGGCATTGTATATGAAGTGAGCAACGATTGCCCTCCGAGTGACGGCTATTTGGGCAATTCCTGCCCGTATTCGGGTGGCTACATCGGCGGTGGCAACTATGTCATGCTGAAGGTGACGAACAAAAAAGAAGATTATTATATCTTCTTATGCCATATGAAGCGTACCCTGGTGTCCAAAGGTCAGAAGGTGAAAAAAGGCCAAAAAATCGGCGAGCAGGGCCATAGCGGTAACAGTACTGCCAGCCACTTACACATTGAGATTCATAAAGGCACGGCTCACATCGCAACGAAAGATGGGTTAGTCGATCCAGAAAAGATTATGAATTTTAAGAAGCGCTAA

[0053] According to the specific embodiments of the present invention, the nucleic acid molecule of the present invention is a DNA molecule or an RNA molecule.

[0054] 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.

[0055] On the other hand, the present invention also provides a carrier comprising the nucleic acid molecules described herein.

[0056] 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.

[0057] On the other hand, the present invention also provides a host cell comprising the nucleic acid molecules or the vectors described herein.

[0058] According to a specific embodiment of the present invention, the host cell can be a competent Escherichia coli cell, such as BL21(DE3).

[0059] On the other hand, the present invention also provides a method for preparing the aforementioned antimicrobial protein, the method comprising:

[0060] 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.

[0061] 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.

[0062] 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 harmless Clostridium.

[0063] On the other hand, the present invention also provides the use of the antimicrobial protein or the antimicrobial drug composition in the preparation of products for the prevention and treatment of diseases related to harmless Clostridium.

[0064] According to specific embodiments of the present invention, Clostridium perfringens-related conditions include one or more of the following conditions: antibiotic-associated diarrhea and enteritis, sepsis, osteomyelitis, endocarditis, peritonitis, brain abscess and / or genitourinary infections caused by Clostridium perfringens, non-infectious diseases associated with Clostridium perfringens such as inflammatory bowel disease, Crohn's disease, and female infertility caused by metabolizing host intestinal progesterone and preventing ovarian follicle development.

[0065] 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.

[0066] The antimicrobial protein of this invention, which targets harmless Clostridium, can efficiently kill harmless Clostridium from various sources, including antibiotic-resistant strains, and is specific. Attached Figure Description

[0067] Figure 1 The plasmid map of the LysCI01 expression vector for the constructed lysin.

[0068] Figure 2 Electrophoresis diagram for identifying heterologous expression of LysCI01 and LysCI02 in Escherichia coli.

[0069] Figure 3 Electrophoresis images of purified LysCI01 and LysCI02 proteins.

[0070] Figure 4 Experimental results show that LysCI01 and LysCI02 can efficiently lyse harmless Clostridium.

[0071] Figure 5 The study demonstrated the lytic activity of LysCI02 against different harmless Clostridium strains.

[0072] Figure 6 The lysing activities of lyases LysCI01 and LysCI02 on different Clostridium species were demonstrated.

[0073] Figure 7 The lysing activity of the lysin LysCI02 against different intestinal symbiotic bacteria was measured.

[0074] Figure 8 The study demonstrated the mitigating effect of the lyase LysCI02 on cytotoxicity caused by harmless Clostridium infection. Detailed Implementation

[0075] 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.

[0076] 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.

[0077] Unless otherwise stated, the experimental methods, detection methods and preparation methods disclosed in this invention all adopt conventional techniques in this technical field.

[0078] Example 1: Identification and protein sequence analysis of lysin genes targeting harmless Clostridium.

[0079] Genomic sequences of bacteriophages and pre-phages targeting harmless Clostridium were collected, and lyase genes were identified using BLAST and Interproscan through sequence alignment and domain alignment. More than 200 potential bacteriophage lyase gene sequences were discovered. This invention further identified a class of lyases containing the amino acid sequences shown in SEQ ID NO:1 to SEQ ID NO:8, named LysCI01-LysCI08, respectively.

[0080] The codon-optimized nucleic acid sequences of the lysin for E. coli are shown in SEQ ID NO:9 to SEQ ID NO:16.

[0081] Example 2: Heterologous expression and purification of lysin targeting harmless Clostridium difficile

[0082] The identified lyase genes were codon-optimized in E. coli, and then the corresponding genes were cloned into pET28a(+) or pCold II vectors. A 6x-His tag was added to the C-terminus or N-terminus, and the genes were controlled by the lactose operon. Figure 1 This is a map of the LysCI01 expression vector plasmid for the constructed lysin.

[0083] The constructed plasmids containing the LysCI01 and LysCI02 lysin genes were transformed into BL21(DE3) competent cells, and 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 TB 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 then incubated at 16-37°C and 100 rpm for 18 h to induce protein expression.

[0084] 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.

[0085] 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 3 (In the figure, M is the SDS-PAGE protein marker, 0 is the control, 1 is the low temperature induction for 16-24h, 2 is the supernatant of induced expression, and 3 is the precipitate of induced expression).

[0086] Figure 2 Electrophoresis images for identifying LysCI01 and its heterologous expression in E. coli. Figure 3 The results showed that, after cleavage and separation, the lysin LysCI01 and LysCI02 proteins were expressed in soluble form in E. coli.

[0087] 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 LysCI01 and LysCI02 show that high-purity lysin proteins LysCI01 and LysCI02 can be obtained through multiple purification steps.

[0088] Example 3: Determination of the lysing activity of lysin protein against harmless Clostridium.

[0089] Harmless Clostridium (standard strain) was inoculated into anaerobic BHI liquid medium and cultured until OD600 = 0.6. The bacterial suspension was centrifuged at 4000 rpm for 5 min and washed twice with buffer (50 mM Tris-HCl [pH 7.0], 100 mM NaCl), and then resuspended in the same buffer. The buffer containing the bacterial cells was added to 96-well plates, and LysCI01 and LysCI02 lysin proteins were added to a final concentration of 5 μmol, with a final volume of 200 μL for each. After culturing for 3 h, the experimental data were compiled, and the lysis activity was calculated. The lysis rate was calculated using the formula: [ΔOD600 (with added lysin) - ΔOD600 (with buffer only)] / initial OD600.

[0090] Figure 4 This indicates that LysCI01 and LysCI02 can efficiently lyse harmless Clostridium. After adding the lyases LysCI01 and LysCI02, the OD of the culture medium decreased rapidly, proving that LysCI01 and LysCI02 can effectively lyse harmless Clostridium. Specifically, in the group with LysCI01 lyase, the absorbance OD600 decreased by 50% at approximately 150 minutes; while in the group with LysCI02 lyase, the absorbance OD600 decreased more rapidly, decreasing by approximately 90% at approximately 30 minutes. This indicates that there is a difference in the lysogenic activity of lyases LysCI01 and LysCI02 against harmless Clostridium, with LysCI02 exhibiting stronger lysogenic activity.

[0091] Example 4: Lytic activity of lysin protein against drug-resistant but harmless Clostridium difficile from different sources.

[0092] Expression and purification of LysCI01 and LysCI02 were performed according to Examples 2 and 3. In short: *E. coli* BL21(DE3) containing each recombinant expression plasmid was cultured in LB medium for 2-3 hours until OD... 600 =0.6, then IPTG was added to a final concentration of 0.1 Mm, and the cells were cultured at 16°C and 100 rpm for 18 h. Cells were collected by centrifugation, lysed by sonication, and purified by affinity chromatography. Finally, the lytic activity of the purified protein against harmless Clostridium species from different sources was determined using the same method as in Example 3.

[0093] Figure 5 The study investigated the lytic activity of LysCI02 against different harmless Clostridium strains. Harmless Clostridium strains 1-8 (strain 1 was the standard strain, and the other strains were provided by the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, for public sharing) were harmless Clostridium isolates from different human feces. These included harmless Clostridium strain 1 (ATCC14501), harmless Clostridium strain 2 (DA45), harmless Clostridium strain 3 (DA130), harmless Clostridium strain 4 (DA461), harmless Clostridium strain 5 (DA537), harmless Clostridium strain 6 (DA1262), harmless Clostridium strain 7 (DA1545), and harmless Clostridium strain 8 (DA664). Strain 1 was the standard strain of harmless Clostridium, and the other seven strains were isolated from fecal samples from different individuals. All eight strains were vancomycin-resistant. LysCI02 exhibited excellent lytic activity against various harmless Clostridium strains, with lysis rates exceeding 90% for all eight strains and reaching 100% for some. Notably, following guidelines from the Clinical and Laboratory Standards Institute (CSIS), the minimum inhibitory concentration (MIC) of vancomycin for the eight harmless Clostridium strains isolated from different individuals was determined using the standard agar dilution method. The results showed that the MIC for all eight strains was 16 μg / ml, indicating that they were all vancomycin-resistant strains. This demonstrates that the lysin obtained in this invention can effectively lyse multiple different vancomycin-resistant harmless Clostridium strains.

[0094] Example 5: Targeting assay of lysin protein

[0095] The lytic activities of purified proteins LysCI01 and LysCI02 against different symbiotic gut bacteria were determined using the same method as in Example 4. In short: Different symbiotic gut bacteria were inoculated into anaerobic BHI liquid medium, and harmless Clostridium was cultured to an OD600 of 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 LysCI01 and LysCI02 lysin proteins were 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 lysins was also set up. The absorbance changes were calculated after 3 h. The lytic activity was then calculated.

[0096] Figure 6 The lytic activities of the lysins LysCI01 and LysCI02 against different symbiotic Clostridium species in the gut were evaluated. LysCI02 did not show significant lytic activity against other symbiotic Clostridium species, but LysCI01 showed some lytic activity against Clostridium butyricum and Clostridium scintillans. This indicates that LysCI02 has higher targeting specificity, enabling it to target and eliminate harmless Clostridium species in the gut without damaging other symbiotic gut bacteria.

[0097] Figure 7 The lysing activities of the lysin LysClO2 and lysozyme (egg white source, CAS: 12650-88-3) against different intestinal symbiotic bacteria were studied. Figure 7 As shown in Figure A, LysCI02 exhibited highly efficient lytic activity against harmless Clostridium; however, it did not show lytic activity against the other 11 tested gut commensal bacteria (information and taxonomic status of the tested strains are shown in Table 2). 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 LysCI02 has high targeting specificity, capable of targeting and clearing harmless Clostridium in the gut without affecting other gut commensal bacteria. Furthermore, lysozyme derived from egg white showed no lytic activity against harmless Clostridium, but it caused lysis of multiple gut commensal bacteria, indicating no significant targeting specificity. Figure 7 Image B in the picture.

[0098] Table 2: Information on gut symbiotic bacteria used for the LysCI02 lysin targeting assay

[0099] Bacterial species name Bacterial strain name Bacterial classification Bacteroides uniformis ATCC8492 Bacteroidetes Parabacteroides distasonis ATCC8503 Bacteroidetes Blautiaobeum DA69 Firmicutes Dorea longicatena DA136 Firmicutes Clostridium symbiosum DA229 Firmicutes Anaerostipes hadrus DA538 Firmicutes Faecalibacterium prausnitzii DA726 Firmicutes Bifidobacterium adolescentis DA06 Phylum Actinobacteria Collinsellaaerofaciens DA394 Phylum Actinobacteria Escherichia coli DH5a Proteobacteria Fusobacterium varium DA690 Fusobacteria

[0100] Example 6: The mitigating effect of lysin protein on cytotoxicity caused by harmless Clostridium infection.

[0101] The purified protein LysCI02 was used to assess its alleviating effect on cytotoxicity following infection by harmless Clostridium difficile. The cytotoxicity assay involved seeding resuscitated and passaged HT29 or HeLa cells onto 96-well plates, fixing them, removing the culture medium and cell suspension, washing with PBS, adding cultured resuspended bacteria and / or lysin, incubating for 24 hours, washing with PBS, and lysing the cells with Triton X-100. The lysate was collected and lactate dehydrogenase activity was measured to evaluate the alleviating effect of the lysin on bacterial infection-induced cytotoxicity. The specific steps are as follows: Resuscitate and passage the cells to a plate-coated density, then plate the cells in a 96-well plate and fix for 18 hours; remove the suspended cells and culture medium, and wash twice with PBS; centrifuge the bacteria cultured overnight with BHI, remove the BHI medium, and resuspend in DMEM to OD600 = 0.25; add bacterial resuspension and / or lyase suspension, incubate for 24 hours, remove the culture medium, wash twice with PBS, and then treat with 1% Triton X-100 for 1 hour to lyse the cells; collect 80 μL of cell lysates and measure lactate dehydrogenase activity to evaluate the effect of lyase treatment.

[0102] Figure 8 This study investigated the mitigating effect of the lyase LysCI02 on cytotoxicity caused by harmless Clostridium infection. Results showed that treatment with LysCI02 significantly reduced cytotoxicity in both HT29 and HeLa cells, indicating that the lyase protein in this invention can treat cytotoxicity caused by harmless Clostridium infection. This has significant implications for the application of lyases in the regulation of the intestinal microecology.

[0103] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The use of an antimicrobial protein or antimicrobial drug composition in the preparation of a product targeting harmless Clostridium; in, The antimicrobial protein is a protein formed by the amino acid sequence shown in SEQ ID NO:1; The antimicrobial pharmaceutical composition comprises: a protein formed from the amino acid sequence shown in SEQ ID NO:1, a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:1, or a carrier comprising a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:1, and a pharmaceutically acceptable excipient.

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

9.

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

4. The use of an antimicrobial protein or antimicrobial drug composition in the preparation of a product for the prevention and treatment of diseases related to Clostridium perfringens; said diseases being selected from one or more of the following: osteomyelitis, endocarditis, peritonitis, brain abscess and / or genitourinary infections caused by Clostridium perfringens; in, The antimicrobial protein is a protein formed by the amino acid sequence shown in SEQ ID NO:1; The antimicrobial pharmaceutical composition comprises: a protein formed from the amino acid sequence shown in SEQ ID NO:1, a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:1, or a carrier comprising a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO:1, and a pharmaceutically acceptable excipient.

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

9.

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

7. The application according to any one of claims 1-6, wherein, The product is a pharmaceutical preparation.

8. The application according to any one of claims 1-6, wherein, The product is a disinfectant.

9. The application according to any one of claims 1-6, wherein, The product is a bactericide.

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