Antibacterial protein targeting harmless clostridium as well as preparation method and application of antibacterial protein

Abstract

The invention provides antibacterial protein targeting harmless clostridium as well as a preparation method and application of the antibacterial protein. Specifically, the invention provides an antibacterial protein targeting clostridium harmlessly 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: 8; (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: 8 is 85% or more or 90% or more. The antibacterial protein provided by the invention can efficiently kill harmless clostridium of different sources, including bacterial strains with antibiotic resistance, and has specificity.

Description

Technical Field

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

[0002] Clostridium innocuum is a Gram-positive anaerobic bacterium belonging to the genus Clostridium. Although its name implies harmlessness, recent studies have found that it may be associated with a variety of infectious and non-infectious diseases in the human body. Clostridium innocuum has gradually been recognized as an important pathogen, especially in people with weakened immune function and excessive use of antibiotics.

[0003] Infectious diseases associated with innocuous Clostridium include: 1. Innocuous Clostridium is found in antibiotic-associated diarrhea and enteritis, especially after the use of fluoroquinolone antibiotics. Some research reports show that innocuous Clostridium may coexist with Clostridium difficile, or fill its ecological niche when Clostridium difficile disappears, leading to persistent diarrhea or recurrence of diarrhea. 2. Sepsis. Although there are fewer cases of sepsis caused by innocuous Clostridium, it increases the risk of infection in immunosuppressed patients, especially after the use of broad-spectrum antibiotics. Innocuous Clostridium sepsis is common in patients receiving chemotherapy or bone marrow transplantation, suggesting that the bacteria are potentially pathogenic in critically ill patients. 3. Other infections. Individual cases have reported osteomyelitis, endocarditis, peritonitis, brain abscesses, and genitourinary system infections caused by innocuous Clostridium. Although these cases are rare, they suggest that innocuous Clostridium has the potential to cause a wide range of pathogens under specific circumstances.

[0004] Non-infectious diseases associated with Clostridium innocua: (1) Clostridium innocua interacts with lipid rafts of intestinal epithelial cells, activates the NOD2 pathway, and induces cytotoxic and inflammatory responses, which may play an important role in intestinal inflammation (such as inflammatory bowel disease) and related diseases. (2) The transfer and colonization of Clostridium innocua in patients with Crohn's disease may drive the formation of creeping fat, and may play a key role in the pathogenesis of Crohn's disease. It is a potential therapeutic target for Crohn's disease. (3) Clostridium innocua can convert progesterone into less active cycloprogesterone, thereby reducing the host's progesterone level. Long-term administration of Clostridium innocua leads to stagnation of ovarian follicle development in mice. In short, Clostridium innocua may be an important factor leading to female infertility by inactivating host intestinal progesterone and inducing stagnation of ovarian follicle development, and is a potential therapeutic target for female infertility. (4) In some studies, Clostridium innocua is considered to be an important indicator of intestinal flora imbalance after antibiotic use, which may aggravate intestinal inflammation. Clostridium innocua may affect the host's metabolic state through its excessive proliferation or metabolites in the intestine, and thus be associated with diseases such as metabolic syndrome.

[0005] Currently, the treatment for innocuous Clostridium infection is relatively limited, and most clinical programs still draw on the experience of other Clostridium infections. However, due to the increasing resistance of innocuous Clostridium to certain antibiotics, treatment has become more challenging. 1. Antibiotic treatment: Broad-spectrum antibiotics such as vancomycin, metronidazole, and teicoplanin are usually used to treat innocuous Clostridium infection. However, antibiotic use requires caution, especially in cases where drug resistance may occur. Studies have shown that the resistance of innocuous Clostridium to antibiotics such as vancomycin is gradually increasing.

[0006] Although research on Clostridium innocuum is still in its early stages, there is evidence that it plays an important role in infectious and non-infectious diseases. Treatment for Clostridium innocuum infection currently relies mainly on antibiotics, but the problem of drug resistance has exacerbated the difficulty of treatment. Future research should focus on developing new antimicrobial strategies to address the pathogenic effects of Clostridium innocuum in the human body.

[0007] At present, the main means of treating harmless Clostridium infection is broad-spectrum antibiotics, but these methods have some obvious problems. The main problems of existing treatment technologies: 1. Antibiotic resistance. Harmless Clostridium gradually develops resistance to a variety of commonly used antibiotics, especially fluoroquinolones, vancomycin, etc. This reduces the effectiveness of treatment with broad-spectrum antibiotics and easily leads to the further spread of resistant strains. During treatment, doctors often face the problem of limited selection of antibiotics. 2. Intestinal microecological destruction. The use of antibiotics will not only kill harmless Clostridium, but also affect the beneficial flora in the intestine, leading to intestinal microecological imbalance. The destruction of intestinal flora may cause more serious secondary infections, such as Clostridium difficile-related infections, and may even aggravate the patient's condition. 3. Non-specific effects. Current antibiotic therapies lack specificity and often work on the entire bacterial community. This non-targeted treatment can cause damage to normal flora, especially in the case of long-term medication, increasing the risk of opportunistic infections by other pathogens (such as fungi and drug-resistant bacteria). 4. Immune-related side effects. For immunosuppressed patients, the treatment of harmless Clostridium infection is particularly difficult. Antibiotics alone may not be effective in controlling infection in immunocompromised individuals, and further suppression of the immune system by drugs may exacerbate the condition. In addition, the use of immunomodulatory therapies has side effects and uncertainties, making it difficult to ensure their effectiveness in all patients.

[0008] In summary, antibiotic treatment of C. innocua faces problems such as drug resistance, microecological disruption, and nonspecific side effects. It is necessary to develop new and effective means to deal with C. innocua infections, especially in the face of complex and multidrug-resistant infections. Summary of the invention

[0009] The inventors of this case discovered a class of antibacterial proteins targeting harmless Clostridium in their research, which can effectively kill harmless Clostridium from different sources, including strains with antibiotic resistance, and are specific.

[0010] Specifically, in one aspect, the present invention provides an antimicrobial protein or a chimera thereof targeting innocuous Clostridium, wherein the antimicrobial protein comprises one or more of the proteins formed by the following amino acid sequences:

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

[0012] (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);

[0013] (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: 8.

[0014] 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: 8 of the present invention 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 the amino acid sequence shown in any one of SEQ ID NO:1 to SEQ ID NO:8 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, Lysozyme_like, CHAP, Glyco_hydro_25, Peptidase_M23 and Peptidase_M20. The cell wall binding domain includes SH3 and Zoocin_A, etc. As can be seen from Table 1, the lyase LysCI01 has the same cell wall binding domain SH3 as LysCI02 and LysCI06.

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

[0032]

[0033] According to some specific embodiments of the present invention, the antibacterial protein targeting innocuous Clostridium or its chimera of the present invention, wherein the chimera comprises one or more of the catalytic domains Amidase_2, Amidase_3, Lysozyme_like, CHAP, Glyco_hydro_25, Peptidase_M23 and Peptidase_M20 and the cell wall binding domains SH3 and Zoocin_A.

[0034] According to some specific embodiments of the present invention, the antibacterial protein targeting Clostridium innocua or its chimera according to the present invention, wherein the chimera comprises a catalytic domain CHAP, 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 encoding the antibacterial protein targeting Clostridium innocua of 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 one 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] Nucleic acid sequence of LysCI02 (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] LysCI06 nucleic acid sequence (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 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.

[0055] On the other hand, the present invention also provides a vector comprising the nucleic acid molecule of the present invention.

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

[0057] 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.

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

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

[0060] 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.

[0061] 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.

[0062] On the other hand, the present invention also provides the use of the antimicrobial protein or the antimicrobial drug composition in the preparation of a product targeting innocuous 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 preventing and treating diseases related to innocuous Clostridium;

[0064] According to a specific embodiment of the present invention, the conditions associated with Clostridium innocuum include one or more of the following conditions: antibiotic-associated diarrhea and enteritis, sepsis, osteomyelitis, endocarditis, peritonitis, brain abscess and / or urogenital system infection caused by Clostridium innocuum, non-infectious diseases associated with Clostridium innocuum 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 a specific embodiment 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 targeting innocuous Clostridium of the present invention can effectively kill innocuous Clostridium from different sources, including strains with antibiotic resistance, and has specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is the plasmid map of the constructed LysCI01 expression vector of the lytic enzyme.

[0068] Figure 2 This is the electrophoresis diagram for the identification of LysCI01 and LysCI02 heterologously expressed in Escherichia coli.

[0069] Figure 3 The electrophoresis diagram of the purified proteins LysCI01 and LysCI02.

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

[0071] Figure 5 The lytic activity of LysCI02 against different strains of C. innocua is shown.

[0072] Figure 6 The lytic activities of the lytic enzymes LysCI01 and LysCI02 against different Clostridium bacteria are shown.

[0073] Figure 7 The lytic activity of the lytic enzyme LysCI02 against different intestinal commensal bacteria.

[0074] Figure 8 The results show that the lytic enzyme LysCI02 can alleviate the cytotoxicity caused by infection with Clostridium difficile. DETAILED DESCRIPTION

[0075] 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.

[0076] 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.

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

[0078] Example 1. Identification and protein sequence analysis of lytic enzyme genes targeting Clostridium innocua

[0079] Phage and prophage genome sequences targeting Clostridium innocua were collected, and the lytic enzyme genes therein were identified by sequence alignment and domain alignment using BLAST and Interproscan. More than 200 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: 8, respectively named LysCI01-LysCI08.

[0080] The nucleic acid sequences of the lytic enzyme after codon optimization for Escherichia coli are shown in SEQ ID NO:9 to SEQ ID NO:16.

[0081] Example 2. Heterologous expression and purification of lytic enzyme targeting Clostridium innocua

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

[0083] The constructed plasmids containing the lytic enzyme LysCI01 and LysCI02 genes were 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 TB culture medium (containing 50 μg / mL kanamycin sulfate). After culturing to OD600 of 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.

[0084] 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.

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

[0086] Figure 2 Figure 2 is the electrophoresis diagram of the identification of LysCI01 and LysCI01 heterologous expression in E. coli. Figure 3 It was shown that the lytic enzymes LysCI01 and LysCI02 proteins were expressed in soluble form in Escherichia coli after fragmentation and separation.

[0087] 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 LysCI01 and LysCI02 shows that high-purity lytic enzyme proteins LysCI01 and LysCI02 can be obtained through multiple purification steps.

[0088] Example 3: Determination of the lytic activity of lytic enzyme protein against Clostridium innocua

[0089] Inoculate the innocuous Clostridium (standard strain) into anaerobic BHI liquid culture medium and culture the innocuous Clostridium to OD600 = 0.6. Centrifuge the bacterial solution from the culture medium at 4000rpm for 5min, wash twice with buffer (50mM Tris-HCl [pH 7.0], 100mM NaCl), and resuspend the bacteria with the buffer after washing. Add the buffer containing the bacteria to a 96-well plate, add LysCI01 and LysCI02 lyase proteins to a final concentration of 5 micromolar, and the final volume is 200 microliters. After culturing for 3h, organize the experimental data and calculate the lysis activity. The lysis rate calculation formula is: [test ΔOD600 (lyase added)-ΔOD600 control (buffer only)] / initial OD600.

[0090] Figure 4 It shows that LysCI01 and LysCI02 can efficiently lyse Clostridium innocuum. After adding the lytic enzymes LysCI01 and LysCI02, the OD of the culture solution decreased rapidly, proving that LysCI01 and LysCI02 can effectively lyse Clostridium innocuum. Among them, the absorbance OD600 of the group added with LysCI01 lytic enzyme had decreased by 50% at around 150min; while the absorbance OD600 of the group added with LysCI02 lytic enzyme decreased more rapidly, and had decreased by ~90% at around 30min. This shows that there are differences in the lytic activity of the lytic enzymes LysCI01 and LysCI02 against Clostridium innocuum, and the lytic enzyme LysCI02 has a stronger lytic activity against Clostridium innocuum.

[0091] Example 4: Lytic activity of lytic enzyme protein against drug-resistant innocuous Clostridium from different sources

[0092] Expression and purification of LysCI01 and LysCI02 were performed as described in Examples 2 and 3. In brief, E. coli BL21 (DE3) containing each recombinant expression plasmid was cultured in LB medium for 2-3 h. 600 =0.6, then IPTG was added to a final concentration of 0.1 mM, and cultured at 16°C, 100 rpm for 18 h. The cells were collected by centrifugation, lysed by ultrasound, and purified by affinity chromatography. Finally, the purified protein was used to determine its lysis activity against innocuous Clostridium from different sources, and the determination method of lysis activity was the same as that in Example 3.

[0093] Figure 5 is the lytic activity of LysCI02 against different innocuous Clostridium strains. Among them, innocuous Clostridium strains 1-8 (strain 1 is a standard strain, and the other strains are provided by the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, and shared for public welfare) are innocuous Clostridium isolates derived from different human feces, including innocuous Clostridium strain 1 (ATCC14501), innocuous Clostridium strain 2 (DA45), innocuous Clostridium strain 3 (DA130), innocuous Clostridium strain 4 (DA461), innocuous Clostridium strain 5 (DA537), innocuous Clostridium strain 6 (DA1262), innocuous Clostridium strain 7 (DA1545), innocuous Clostridium strain 8 (DA664), among which strain 1 is a standard strain of innocuous Clostridium, and the other seven strains are isolated from fecal samples of different individuals, and all 8 strains are vancomycin-resistant strains. Through the lysis activity test, it was found that LysCI02 had a good lysis effect on different harmless Clostridium strains, and the lysis rate of all 8 strains was greater than 90%, and the lysis activity of some strains even reached 100%. It should be noted that according to the guidelines of the Clinical and Laboratory Standards Institute, the minimum inhibitory concentration of vancomycin for 8 harmless Clostridium strains isolated from different individuals was determined by the standard agar dilution method. The results showed that the minimum inhibitory concentration of vancomycin resistance for all 8 strains was 16 μg / ml, all of which were vancomycin-resistant strains. It is shown that the lytic enzyme obtained by the present invention can effectively lyse multiple different vancomycin-resistant harmless Clostridium strains.

[0094] Example 5: Targeted determination of lytic enzyme protein

[0095] Purified proteins LysCI01 and LysCI02 were used to determine their lytic activity against different symbiotic intestinal bacteria, and the method for determining lytic activity was consistent with Example 4. In short: Different symbiotic intestinal bacteria were inoculated into anaerobic BHI liquid culture medium, and the harmless Clostridium was cultured to OD600 = 0.6. The bacterial solution was centrifuged at 4000rpm for 5min from the culture medium, washed twice with buffer (50mM Tris-HCl [pH 7.0], 100mM NaCl), and the bacteria were resuspended in the buffer after washing. The buffer containing the bacteria was added to a 96-well plate, and LysCI01 and LysCI02 lytic enzyme proteins were added to a final concentration of 5 micromolar, and the final volume of each well was 200 microliters. The 96-well plate was placed in an ELISA instrument to continuously measure the absorbance changes. At the same time, a control group without lytic enzyme was set up. After 3h, the absorbance changes were calculated. Calculate the lytic activity.

[0096] Figure 6 The lytic activity of the lytic enzymes LysCI01 and LysCI02 against different intestinal symbiotic Clostridium bacteria. LysCI02 did not show obvious lytic activity against other intestinal symbiotic Clostridium bacteria, but LysCI01 showed certain lytic activity against Clostridium butyricum and Clostridium scintillans. This shows that LysCI02 has higher targeting and can eliminate harmless Clostridium in the intestine without destroying other symbiotic intestinal bacteria.

[0097] Figure 7 The lytic activity of LysClO2 and lysozyme (from chicken egg white, CAS: 12650-88-3) on different intestinal symbiotic bacteria. Figure 7 As shown in Figure A, LysCI02 exhibited efficient lytic activity against innocuous Clostridium; but for the other 11 intestinal symbiotic bacteria tested (the information and taxonomic status of the test strains are shown in Table 2), LysCI02 did not show lytic activity. These intestinal symbiotic bacteria tested came from five common intestinal symbiotic bacterial phyla, including Bacteroidetes, Firmicutes, Actinobacteria, Proteobacteria, and Fusobacteria. This shows that the lytic enzyme LysCI02 has a high degree of targeting and can target and eliminate innocuous Clostridium in the intestine without affecting other intestinal symbiotic bacteria. In addition, lysozyme derived from egg white has no lytic activity against innocuous Clostridium, but lysozyme derived from egg white will cause the lysis of a variety of intestinal symbiotic bacteria, and there is no obvious targeting ( Figure 7 Figure B).

[0098] Table 2: Information on intestinal commensal bacteria used for targeting test of lysase LysCI02

[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 Actinobacteria Collinsella aerofaciens DA394 Actinobacteria Escherichia coli DH5a Proteobacteria Fusobacterium varium DA690 Fusobacteria

[0100] Example 6: The mitigating effect of lytic enzyme protein on cytotoxicity caused by innocuous Clostridium infection

[0101] Purified protein LysCI02 was used to determine its mitigating effect on cytotoxicity of cells infected with innocuous Clostridium. Cytotoxicity determination method: The revived and passaged HT29 or Hela cells were plated on a 96-well plate, the culture medium and suspended cells were removed after fixation, washed with PBS, and then the cultured resuspended bacteria and / or lytic enzymes were added. After incubation for 24 hours, the cells were washed with PBS and lysed with TritonX-100, and the lysate was collected to determine the lactate dehydrogenase activity, so as to evaluate the mitigating effect of lytic enzymes on cytotoxicity caused by bacterial infection. The specific steps are as follows: revive the cells and passage them to a density that can be plated, then plate the cells on a 96-well plate and fix them for 18 hours; remove the suspended cells and culture medium, and rinse twice with PBS; centrifuge the bacteria cultured overnight with BHI, remove the BHI culture medium, and resuspend them with DMEM to OD600 = 0.25; add bacterial resuspension and / or lytic enzyme suspension, incubate for 24 hours, remove the culture medium, rinse twice with PBS, and then treat with 1% TritonX-100 for 1 hour to lyse the cells; collect 80 μL of cell lysate each, and determine the lactate dehydrogenase activity to evaluate the effect of lytic enzyme treatment.

[0102] Figure 8 The results show that the treatment of lyase LysCI02 can significantly reduce the cytotoxicity of HT29 cells and Hela cells, indicating that the lyase protein in the present invention can treat the cytotoxicity caused by innocuous Clostridium infection, which is of great significance for the application of lyase in intestinal microecological regulation.

[0103] 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 innocuous Clostridium, 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: 8; (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:

8.

2. The antimicrobial protein targeting innocuous Clostridium or its chimera according to claim 1, wherein: The chimera comprises: A chimeric protein comprising the amino acid sequence of the complete antimicrobial protein of claim 1 (a), (b), (c), the catalytic domain sequence of the antimicrobial protein, and the cell wall binding domain sequence of the antimicrobial protein; Preferably, the chimera comprises one or more of the catalytic domains Amidase_2, Amidase_3, Lysozyme_like, CHAP, Glyco_hydro_25, Peptidase_M23 and Peptidase_M20 and the cell wall binding domains SH3 and Zoocin_A; More preferably, the chimera comprises the catalytic domains CHAP, Glyco_hydro_25 and / or the cell wall binding domain SH3.

3. A nucleic acid molecule encoding the antibacterial protein targeting Clostridium innocua according to claim 1 or 2; Preferably, the nucleotide sequence of the nucleic acid molecule comprises any one of SEQ ID NO:9 to SEQ ID NO:16; 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 innocuous Clostridium.

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 innocua; Preferably, the condition comprises one or more of the following conditions: antibiotic associated diarrhea and enteritis, sepsis, osteomyelitis, endocarditis, peritonitis, brain abscess and / or genitourinary system infection caused by Clostridium innocua, non-infectious diseases associated with Clostridium innocua such as inflammatory bowel disease, Crohn's disease and female infertility caused by metabolizing host intestinal progesterone and preventing ovarian follicle development.

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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