CN119799685A - An antibacterial protein targeting Clostridium scintillans and its preparation method and application - Google Patents

Abstract

The present invention provides an antimicrobial protein targeting Clostridium scintillans and a preparation method and application thereof. Specifically, the present invention provides an antimicrobial protein targeting Clostridium scintillans or a chimera thereof, wherein the antimicrobial protein comprises one or more of the proteins formed by the following amino acid sequences: (a) any amino acid sequence shown in SEQ ID NO:1 to SEQ ID NO:13; (b) a derivative sequence having the same function as (a) by replacing, adding and/or deleting one or more amino acids in the amino acid sequence of (a); (c) a derivative amino acid sequence having an identity of more than 85% or more than 90% with any sequence of SEQ ID NO:1 to SEQ ID NO:13. The antimicrobial protein of the present invention can efficiently kill Clostridium scintillans and has specificity.

Description

Antibacterial protein of targeted clostridium scintinus and preparation method and application thereof

Technical Field

The invention provides an antibacterial protein targeting clostridium scintinus, a preparation method and application thereof.

Background

Clostridium scintillans (Clostridium scindens) is a gram-positive, facultative anaerobic bacterium belonging to the genus Clostridium. Although Clostridium scintillans are part of a healthy microbiota to some extent, their metabolic function may be associated with some non-infectious diseases (1) Clostridium scintillans are known for their unique bile acid 7α -dehydrogenase activity, which enables them to convert cholesterol metabolites into androgen precursors such as 11β -hydroxyandrostenedione. These precursors may be further converted to active androgens in humans, stimulating the growth of prostate cancer cells and leading to resistance to hormone deprivation therapy. It was found that these metabolic activities may affect the therapeutic effect of prostate cancer patients, increasing the risk of hormone-resistant tumors. (2) Clostridium scintillans produces secondary bile acids, such as deoxycholic acid (DCA), by bile acid metabolism, which play a key role in colorectal cancer progression. Studies have shown that DCA inhibits the effector function of anti-tumor cd8+ T cells. Specifically, DCA inhibits NFAT2 signaling in cd8+ T cells by enhancing cell membrane calcium ion pump activity, reducing intracellular calcium ion accumulation. This impairs the ability of T cells to produce interferon (IFN- γ) and tumor necrosis factor (TNF- α), resulting in failure of the anti-tumor immune response. In summary, clostridium scintillans produced DCA is an important factor in colorectal cancer development and progression. (3) Clostridium scintillans can affect natural killer T cell (NKT cell) accumulation in the liver by modulating bile acid metabolism, which in turn may affect the liver's anti-tumor immune response and the development of liver cancer. Mice experiments show that the colonization of clostridium scintinus leads to rapid decrease of liver NKT cell level, and increases the number of liver tumors. Furthermore, by treating mice with antibiotics, researchers found that removal of gram positive bacteria (including clostridium scintinus) increased liver NKT cell accumulation and reduced liver tumor growth. These results indicate that clostridium scintinus has a promoting effect in liver cancer development 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 play a role in the pathological process of non-alcoholic fatty liver disease.

Specific phages against clostridium scintinus have been used to reduce the number of bacteria. In mouse experiments, the use of phage targeting clostridium scintinus reduced the concentration of intestinal DCA and effectively slowed down the growth of the tumor. This suggests that phage may be a tool for precise control of intestinal microorganisms. In addition, bile acid sequestrants also show some effect by reducing DCA production. In a mouse model, this approach helps restore anti-tumor cd8+ T cell function. In some cases, researchers reduce clostridium scintinus in the intestinal flora by antibiotic treatment.

The main means of treating clostridium scintinus infection is antibiotic and phage therapy at present, but these methods have some obvious problems of 1. Bacteria may develop resistance to phage and antibiotics through mutation, which reduces the effectiveness of treatment with phage or antibiotics and easily leads to further spread of resistant strains. 2. The use of antibiotics not only kills clostridium scintinus but also affects the beneficial flora in the intestinal tract, resulting in an imbalance of intestinal microecology. Such non-targeted therapies can cause damage to normal flora, especially in the case of long-term administration, increasing the risk of opportunistic infections with other pathogenic bacteria such as fungi and drug-resistant bacteria. 3. Side effects associated with immunity are particularly troublesome for immunosuppressed patients in the treatment of clostridium scintillans infection. Antibiotics themselves may not be effective in controlling infections in cases of impaired immune function, while further inhibition of the immune system by drugs may exacerbate the condition. Furthermore, the use of immunomodulating therapies presents side effects and uncertainties that it is difficult to ensure their efficacy in all patients.

In summary, antibiotic treatment of clostridium scintinus is faced with problems of drug resistance, microecological destruction, non-specific side effects, etc., and new effective means for coping with clostridium scintinus infection needs to be developed.

Disclosure of Invention

The inventor discovers a class of antibacterial proteins targeting clostridium scintinus in research, can kill clostridium scintinus with high efficiency and has specificity.

In particular, in one aspect, the invention provides an antimicrobial protein or a chimeric thereof that targets clostridium scintillans, wherein the antimicrobial protein comprises one or more of the following amino acid sequence-forming proteins:

(a) The amino acid sequence shown in any one of SEQ ID NO.1 to SEQ ID NO. 13;

(b) A derivative sequence having the same function as (a) by substituting, adding and/or deleting one or more amino acids from the amino acid sequence of (a);

(c) A derivative amino acid sequence having a sequence identity of 85% or more or 90% or more to any one of SEQ ID NO 1 to SEQ ID NO 13.

According to a specific embodiment of the present invention, the antibacterial protein targeting clostridium scintinus or a chimeric thereof according to the present invention, wherein the chimeric comprises:

A chimeric protein comprising the amino acid sequence of the entire antimicrobial protein of (a), (b), and (c), the catalytic domain sequence of the antimicrobial protein, and a combination of two or more sequences of the cell wall binding domain sequence of the antimicrobial protein. For example, the protein may be a chimeric protein composed of the amino acid sequence of the entire antimicrobial protein of any one of (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, or may be a chimeric protein composed of a combination of 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.

According to a specific embodiment of the present invention, the proteins formed by the amino acid sequences shown in any one of SEQ ID NOs 1 to 13 of the present invention are designated LysCS to LysCS, respectively, and the specific amino acid sequences are as follows.

LysCS01 protein sequence (SEQ ID NO: 3):

MNRLNCMNQKIKIFMQMCFAVLVIMTVMVLPVRAKETETAGASLGKETETEDAISFYASAVEGWVQTADGRWWYQYNDGSWPKTSWRKINGSWYYFDSNGYWVDNNVHESGSLKGIDVSQWQGNIDWQAVKDDGIQFALIRLGHGIHELDTYYQRNMQNANAAGIPVGVYFYSTAKSEEEAIADAQFVISNMKGYLVSYPVVIDLEDSSQASLSKTQLGKIAKAYCDEIHAAGYTPMLYCNENWYRNHIDISQIADVEMWVARYGGTYSTSIPRGIWQCCSTGRVNGIGGDVDIDFGYKDYTQIVTPRTDYAEGYVMTEGIWVKDGHGWWYRYFAGGYPSNTWKNIRGNWYWFDADGYMETGWHLIDGTWYYFNSSGAMVTGWQLIGNTWYYMDGSGAMATGWCLIGGTWYYFNGSGAMETGWHLIDGTWYYFNGSSGAMTTGWQLIGNTWYYLGTDGKMVTGLTTVTGAIYYLESSGAMATGWRQIDGIWYYFN

LysCS02 protein sequence (SEQ ID NO: 4):

MAKSRQAVVNLVESWDGKKESNGSYKSIIDLYNDFFEKICAGKFPRGIRMRYDWAWCACTWSALAAALRYESIMPMEISCYYLIEAAKKMGCWQENDAYVPSPGDAILYDWQDNGISDNTGNPDHVGTVIEVHKESGYMVIEEGNYSNAVKKRTLSINGKFIRGFITPKYDDNTVAAPGLSKGKDIKTVAHDVIVGLWGSGENRKKLLTEYGYSYSEVQSMVNQILNGSAVTPSNTKQDQNQSISKKVVATCSAKQFNKAYAGEYKTTAVLYCRNDAGTNKKALCKIPAGTKVKCYGYYTMANGVKWLYIQFVLDGVQYTGFSSSAYLAK

LysCS03 protein sequence (SEQ ID NO: 5):

MGWTEYRDVLDSWFGYSEARGQDDLIIDIYNSQRVESYKMSHQDPWCHATISAAGYQSGNQGRVPNTAYCPYGINWFKARGLWTGRYAGNYAPAVGDIIYYDWGGDGVSDHVGAIIKVSGNTLTVREGNRNDMVCDRTISKWSNLIMGYGRPNWGSATIIMPSPVVVESGSNGAYGIHRKDLIRQGQQHAINFTGVKIGVDGIRGPETKKAAIRCVQHAMNMDYNAGLKEDGIWGKKTDAAFAQHYVCEGETQYMVTAWEILLLLNGYDPNGVEHPGEFGSGCAAATRMFQGDKSLVQDGVAGRKSFLTAIN

LysCS04 protein sequence (SEQ ID NO: 6):

MGTYNVHAGHCPQGQGASGAVGLLQESVEDRKVKNRVISALQSAGHVVYDCTDDSNCNVSQNLRNIVAKCNAHSVDLDVSIHLNAGGGTGVEVWCYDGKTANIASAICQNVSTALGISNRGVKYSTGLYVLRKTKAPALLVECCFVDNQNDYSHWNVEKCGDDIASAIAGKTVQGNASAPAQNPAPTPNAGFDFAGWVGRLQAECNAQEFSRQKVDRIPGPITLAGCPLIKRGASGNITRLVQERLNSLGFCCGVDGDYGRAPFHETYDAVIAYQRANDLVPNGIVGQKTWSKLLGLS

LysCS05 protein sequence (SEQ ID NO: 7):

MKLVESILTKNPCYTAGRKITVKGLMLHSVGCPQPKASVFINSWNSPSYDNACVHGFIDGNDGTVYQTLPWNHRGWHCGSGSKGSGNNTHIGVEMCEPACIKYTSGANFTCSDTATAKAVAKRTYEAAVELFAMLCRQYSLNPTADGVIISHREGHSRGIASNHGDPEHLWTQLGMGYTMDTFRQAVKAAMDGGSSAETSGYTKIMGNAVATAEQMETYIQGKNPQVLQSVLDMIPLYLSEGQAEGVRGDIAFAQSCLETGNFTFSGSAVTLDQNNFCGMGVTSNGLKGNSFDTPQLGIRAQIQHLKAYASTDALKNDCVDPRFKYVTRGSAEYVEWLGQQENPQGKGWAAGAGYGEKILTILKNICGTAGGASGTADIWYRVRKTWADAKSQIGAFRVLENAKNCADENPGYRVFDVNGVNIYTPDTAAFSPYLVRVSITDLNIRKGPGTNYAKTGKFTGKGVFTIVEMQTGKGSDTGWGRLKSGAGWISLDYTEKIS

LysCS06 protein sequence (SEQ ID NO: 8):

MNGIDISNWQKGINLDAVPCDFVIMKATEGTWYVNPDCERAYQQAKNAGKCLGVYHYAEGKDAKAEADFFLKHIQGYIGEALIALDWEKENNSSFGKNDLNWVKQWLDYIYGKTGVRPLLYISQSIMGKFNGIGDYGLWVAQYANMNTTGYQDAPWNEGKYNCAIRQYSSCGRLSGYSGNLDLNKFYGDKTAWNRYAGKGNVTKPSTGTAASSTSSPGGTVLDLVVATLQGKYGNGDVRKTALGNRYIEVQNMINHIASASASTLAGEVKAGKYGNGDARKIALGSRYNEVQKIVNGSTGLAASYHTIKSGETLSGIAAKYGTTVAKLQSLNGIKNVNKIYAGSRIRVK

LysCS07 protein sequence (SEQ ID NO: 9):

MGWNEYNAKLQEWYGFTEAAGQDDIIIDAYNRQKEEAYTMSHQDPWCHATVSAAAAESGNRGRVPNTAYCPTGINWFKARGQWTGRYDTGYNPSVGDIIYYDWGGDGVSDHVGTIVGVNGNTLQVREGNKNDMLTDRYIQKGNTLIMGYGRPAWGGSVPVPSQQSGSIGRSWLQRGDKGEAVKDVQNKLIALGYSCGPDGADGDYGTNTIAAVKRFQADVGITVDGLAGEITRAKLNNAYNTGGVNKAGGSWVARLQAECNAQGFSTQKVDGLPGPNTLAGCPTLGRTSRGKITALMQERLISLGYSCGPCGADGINGAGTQAAIKAFQRDHGLAVDGIVGQKTWSKLLGLS

LysCS08 protein sequence (SEQ ID NO: 10):

MSMNGIDISNHQQGLDVSKVPCDFVIMKATEGTTFVDKYCDKFYQQAKKLGKKLGVYHFASGKSSGTAEADFFLKNIAGYVGEAILVLDWEGSAVNKGVGYAKEFLDRVYAKTGVRPLLYSYNNCINAYDWRSVAQADYGLWNAGYYAGYQTMGYNPNAPIKGGLGAFGSCAMYQYTSSGRLSGWAGNLDLDVFYGDSAAWDAYAKGSAIASPGGTPEPSQQPTNTSPSSQSMLNAQIHINNFTGSGIPEDGKNGRKTRKGVVMSLQHACNLDYKPNPVLKEDGLIGSKTNRVRGLHYVKRGETQYLVTFVEIGLTALGYYAGAVECPGVFGEGLEDAVHRFQHDMNLNEDKIAGRNVMDMMLRQLGCI

LysCS09 protein sequence (SEQ ID NO: 11):

MSNSSLVNCTVKSPNHSGARTHSIDRITPHCVVGQLSAESIGGCFTSPSREASCNYGIGTDGRVVLCVDEANRSWCSSSNANDQRAVTIECASDMTDPYAMTSAVYEKLVALCVDICQRNGKSKLIWFGDKDKSLNYSPKSNEMILTVHRWFANKACPGDWLYSRLGDLANRVTSQLGGSTTDSTSKTYKTGLYKVDVGDLNIRKGPGTNYGINGMITDRGTYTITEIQNGYWGKLKSGAGWISVHEAYCTYKGAASGESEEKPSSNFLVQVDIPDLYIRKGPGTNYGNNGFCPKGVYTIVEVKSGAGSDAGWGKLKSGAGWISLDYATRI

LysCS10 protein sequence (SEQ ID NO: 12):

MEKQEFIKQIAGYVKKYAARYGIKVHSPIIAQAILESGWGESKLAAVYHNYFGLKCGTKWTGKSVNLKTMEEYTPGTLTQIKDNFRVYDNMEEGVKGYFEFIQLKRYQNLKGITDPEEYLKTIKADGYATSSKYVENTMRIVTQYNLQKYDTKGEESMAKKASAVLSQARAWIGRKEANGTHREIIDVYNAHRPLARGYKVKYTDAWCATFVSAVAIKCGLTSIIPTECGCGQMIELFKKLGEWQESDSRTPKPGDVVFYDWDDTGTGNNTGWPDHVGIVESVSGGSITVIEGNKNNAVERRTLSVNGRYIRGYGVPKYDSEAGTGTTQPGKSVAEVAKEVIAGKWGNNPQRKERLEAAGYDYQAVQNQVNAILNGNAKPQKSVAEVAKEVIAGKWGNNPQRKERLEAAGYDYQAVQNKVNQLLK

LysCS11 protein sequence (SEQ ID NO: 13):

MNIIKHGLRFRGSFTYRNRTDEIIWHHAEANCTVEDIHSWHLNNGWIGCGYNLIIYKDGTVHEGRPLNAVGAHASGHNSRSVGVCCIGRYDVETMPKEQLDAAKQVQAYLKGLYPGAATKRHKDVNATSCPGKNFPFSEISGASAGSVDVTTSDVQAQSSDDWCARLQKECNAQKFSKQKVDNIPGPDTLAGCPTLGRKSRGKITALMQERLNALGYDCGAVDGINGTKTQAAIKAFQRDYGLVADGIVGPKTWSKLLGLS

LysCS12 protein sequence (SEQ ID NO: 1):

MAHLYVIAGHGAGDSGAVGNGYTEAERVRALASKIKQLGGDNVTLGDMNRNYYADNGISSLNIPKDWCIIELHMDSASASARGGHVIIKGTFSPDSHDLALASFVSAMFPGRANSIVGRNNLANPNRAAAKGYNYRLVEFGFISNATDVSIFNNNLDAIARGVLSCFGINTNTAKWVLDNVGWWYQRADGSYPKSQWLLLDCYYYFNDKGYALANEWLSYGGNWYWLKDDCRMATGWQYIDKHWYYLNPTGTKNKPVGAMLDGWQFIDGQWYYLRTKADGEHPHGSMVEGSVTVGEHDYYCREAGTDKNYPTGSMLMGWRKVTETAEDGTKKTKWFWYNKDSNCQPIGSMLKNHWLTTSNGKKYYLKDDGVMACDETMTISGKEYTFDASGALV

LysCS13 protein sequence (SEQ ID NO: 2):

MAHLYVIAGHGAGDSGAVGNGYTEAERVRALASKIKQLGGDNVTLGDMNRNYYADNGISSLNIPKDWCIIELHMDSASASARGGHVIIKGTFNPDSHDLALASFVSAMFPGRANSIVGRNNLANPNRAAAKGYNYRLVEFGFISNATDVSIFNSNLDAIARGVLSCFGINTNTAKWVLDNVGWWYQRADGSYPKSQWLLLDCYYYFNDKGYALANERLSYGGNWYWLKDDCRMATGWQYIDKHWYYLNPTGTKNKPVGAMLDGWQFIDGQWYYLRTKADGEHPHGSMVEGSVTVGEHDYYCREAGTDKNYPTGSMLMGWRKVTETAEDGTKKTKWFWYNKDSNCQPIGSMLKNHWLTTSNGKKYYLKDDGVMACDETMTISGKEYTFDASGALV

The protein formed by the amino acid sequences shown in any one of SEQ ID NO. 1 to SEQ ID NO. 13 contains a catalytic domain, a cell wall binding domain and position information thereof as shown in Table 1. Wherein the catalytic domain comprises Amidase _2, amidase_3, CHAP, glyco_hydro_25, lysozyme_like, glucosaminidase. Cell wall binding domains include PG_binding_1, choline_bind_1, choline_bind_3, CW_7, SH3, lysM, and the like. As can be seen from Table 1, lysCS and LysCS12, lysCS have the same catalytic domain Amidase _3, while LysCS and LysCS12, lysCS01 have the same cell wall binding domains Choline _bind_1 and Choline _bind_3.

TABLE 1 catalytic and cell wall binding domains of antibacterial proteins LysCS to LysCS13

According to some embodiments of the invention, the antibacterial protein targeting clostridium scintinus or a chimeric thereof according to the invention, wherein the chimeric comprises one or more of the catalytic domains shown in table 1 and/or the cell wall binding domains, e.g. one or more of catalytic domains Amidase _3, amidase _2, CHAP, glyco_hydro_25, lysozyme_ like, glucosaminidase and cell wall binding domains pg_binding_1, choline _bind_1, choline _bind_3, cw_7, SH3 and LysM, etc.

According to some embodiments of the invention, the antibacterial protein targeting clostridium scintinus or a chimeric thereof according to the invention, wherein the chimeric comprises catalytic domain Amidase _3 and/or cell wall binding domain Choline _bind_1 and/or Choline _bind_3.

In another aspect, the invention also provides a nucleic acid molecule encoding an antibacterial protein of the targeted clostridium for use according to the invention.

According to a specific embodiment of the invention, the nucleotide sequence of the nucleic acid molecule according to the invention comprises the sequence shown in any one of SEQ ID NO. 14 to SEQ ID NO. 26.

LysCS 01A 01 nucleic acid sequence (SEQ ID NO: 16):

ATGAATCGCTTAAATTGTATGAATCAAAAAATCAAAATTTTTATGCAGATGTGCTTCGCCGTTCTGGTGATTATGACAGTTATGGTGCTGCCTGTTCGCGCGAAAGAAACGGAAACAGCAGGTGCCTCACTGGGTAAAGAAACTGAAACCGAAGACGCAATTTCCTTCTACGCCTCGGCTGTGGAAGGTTGGGTGCAAACCGCCGACGGCCGTTGGTGGTATCAATATAACGATGGCAGTTGGCCGAAGACCAGCTGGCGCAAAATTAACGGGAGCTGGTATTATTTCGATTCGAATGGATACTGGGTCGACAACAACGTGCACGAATCCGGCAGCCTTAAAGGTATTGATGTGAGTCAATGGCAAGGAAACATCGATTGGCAGGCCGTAAAAGACGATGGAATCCAGTTCGCCTTGATTCGCCTTGGTCATGGCATTCACGAACTGGACACGTACTATCAGCGTAACATGCAAAACGCGAACGCCGCCGGTATTCCGGTGGGGGTCTATTTCTATTCGACTGCCAAATCGGAGGAAGAAGCAATTGCGGACGCGCAGTTCGTCATCTCCAACATGAAAGGTTATCTGGTTTCATATCCAGTCGTCATTGACCTTGAAGACTCGAGCCAGGCATCGCTGTCCAAAACCCAGCTGGGGAAGATCGCAAAGGCCTACTGCGACGAGATTCATGCAGCCGGTTATACCCCTATGTTGTATTGCAACGAGAACTGGTATCGCAACCATATTGATATCTCGCAGATTGCAGATGTTGAAATGTGGGTTGCTCGTTACGGCGGCACTTACTCAACGTCTATTCCGCGTGGTATCTGGCAATGTTGCTCCACTGGTCGTGTGAACGGCATTGGCGGGGATGTGGACATCGATTTCGGTTACAAAGACTACACCCAGATCGTCACACCGCGCACCGACTATGCAGAAGGGTACGTGATGACGGAAGGCATCTGGGTTAAAGATGGCCATGGTTGGTGGTACCGCTATTTTGCTGGAGGATATCCTTCTAATACCTGGAAAAACATTCGCGGTAACTGGTATTGGTTCGATGCCGACGGCTACATGGAGACTGGATGGCATCTGATTGATGGTACTTGGTACTATTTCAACTCCAGTGGCGCAATGGTCACCGGCTGGCAATTAATCGGTAACACCTGGTATTATATGGATGGTTCCGGCGCAATGGCAACCGGTTGGTGCCTGATTGGCGGTACCTGGTATTACTTTAATGGCAGCGGCGCCATGGAAACCGGCTGGCACCTGATCGATGGGACATGGTACTATTTCAATGGTTCTTCTGGTGCTATGACGACGGGTTGGCAGCTGATTGGCAACACTTGGTACTATTTGGGTACGGATGGCAAGATGGTGACGGGACTGACAACCGTTACGGGCGCGATCTACTACCTGGAAAGTAGTGGCGCCATGGCCACGGGCTGGCGCCAGATTGACGGAATCTGGTACTACTTCAACTAA

LysCS02 nucleic acid sequence (SEQ ID NO: 17):

ATGGCCAAATCACGCCAGGCTGTGGTTAATTTGGTCGAGAGTTGGGATGGCAAGAAGGAATCCAACGGTAGCTACAAATCGATCATCGATCTGTACAACGATTTTTTCGAAAAAATTTGCGCCGGTAAGTTTCCCCGTGGGATTCGTATGCGTTATGATTGGGCCTGGTGTGCGTGCACCTGGTCCGCTCTTGCGGCTGCATTACGTTATGAAAGTATTATGCCGATGGAGATCTCTTGCTATTACCTGATCGAAGCAGCCAAGAAAATGGGGTGCTGGCAGGAAAATGATGCCTATGTGCCGAGTCCGGGCGATGCTATCCTTTATGATTGGCAGGACAACGGCATCTCGGATAACACAGGCAATCCGGATCATGTTGGCACCGTGATTGAAGTACACAAGGAGTCGGGCTACATGGTTATTGAAGAAGGTAATTATTCAAATGCCGTGAAAAAACGTACCTTGTCGATCAACGGCAAATTTATTCGCGGTTTTATTACGCCCAAATATGACGACAACACCGTTGCCGCCCCGGGCCTGAGCAAGGGAAAAGATATTAAGACCGTCGCACACGATGTTATTGTAGGCCTGTGGGGCAGTGGCGAGAACCGCAAAAAGCTGCTGACGGAATATGGTTATTCATACTCTGAAGTCCAGAGTATGGTAAACCAGATTCTGAACGGTTCGGCCGTCACCCCAAGTAATACCAAACAGGATCAGAACCAAAGCATCAGTAAGAAGGTGGTCGCTACGTGCAGCGCCAAACAGTTCAACAAAGCATACGCAGGTGAATATAAAACGACAGCAGTGCTGTATTGCCGCAACGACGCCGGCACGAACAAGAAAGCTCTGTGCAAAATCCCCGCAGGGACCAAAGTGAAATGTTATGGTTATTATACCATGGCGAATGGAGTCAAATGGCTCTACATCCAATTCGTCCTGGATGGTGTGCAATACACCGGCTTTTCATCAAGCGCGTACCTGGCAAAATAA

LysCS03 nucleic acid sequence (SEQ ID NO: 18):

ATGGGTTGGACCGAATATCGTGATGTCCTTGACTCGTGGTTCGGTTATAGTGAAGCCCGCGGTCAAGATGATCTGATCATTGATATTTACAATAGCCAGCGTGTCGAGTCCTACAAAATGTCTCACCAGGACCCGTGGTGTCATGCAACGATCTCCGCTGCGGGCTATCAGTCTGGCAATCAGGGTCGCGTTCCCAACACTGCTTATTGTCCTTATGGGATCAACTGGTTCAAAGCCCGTGGACTGTGGACGGGCCGCTACGCAGGTAACTACGCCCCGGCTGTCGGCGACATCATCTATTATGATTGGGGCGGCGACGGCGTCTCTGATCACGTAGGCGCTATCATCAAAGTGTCTGGAAACACGTTGACCGTGCGTGAAGGTAATCGTAACGATATGGTTTGCGATCGCACGATTTCTAAATGGTCTAACCTGATTATGGGCTACGGTCGTCCAAACTGGGGCAGTGCAACGATTATCATGCCATCTCCGGTTGTAGTTGAGTCAGGCAGTAACGGAGCGTACGGTATCCATCGTAAGGACTTAATCCGTCAGGGACAGCAGCATGCGATTAACTTTACTGGGGTTAAAATTGGCGTGGACGGTATCCGCGGCCCTGAAACCAAGAAAGCGGCTATCCGCTGTGTCCAACATGCAATGAACATGGACTATAATGCCGGTCTGAAAGAAGACGGCATTTGGGGAAAAAAAACGGATGCAGCCTTCGCCCAACATTATGTATGCGAGGGCGAAACGCAATATATGGTGACAGCTTGGGAAATTTTACTGCTCTTAAACGGCTACGATCCAAATGGTGTAGAACATCCGGGCGAATTTGGCAGTGGCTGTGCCGCAGCCACACGTATGTTCCAAGGCGATAAATCCCTGGTCCAGGATGGCGTGGCAGGGCGTAAATCATTTCTGACGGCCATCAACTGA

LysCS04 nucleic acid sequence (SEQ ID NO: 19):

ATGGGGACATATAACGTCCATGCGGGCCATTGCCCACAAGGGCAAGGCGCGTCTGGCGCCGTAGGTTTGCTTCAAGAATCCGTGGAAGACCGTAAGGTGAAAAACCGCGTGATCTCCGCGCTGCAGTCGGCAGGTCACGTGGTGTACGATTGCACCGATGACTCAAATTGTAACGTGAGTCAGAATTTACGCAACATTGTTGCTAAATGCAACGCTCACTCCGTGGATCTGGACGTTTCGATTCATCTGAATGCCGGGGGTGGTACGGGCGTTGAAGTTTGGTGTTATGATGGCAAAACGGCCAACATTGCCTCCGCTATTTGTCAAAACGTGTCCACCGCTCTGGGCATTAGCAACCGTGGAGTGAAATATTCCACGGGCCTGTACGTTCTGCGCAAAACCAAAGCTCCTGCCTTATTAGTCGAATGTTGTTTCGTGGATAATCAGAATGATTATTCACATTGGAACGTTGAAAAATGTGGCGACGATATTGCCTCAGCTATTGCCGGCAAGACCGTGCAGGGCAATGCCTCGGCCCCGGCACAGAATCCAGCACCAACCCCTAACGCTGGCTTTGATTTCGCTGGCTGGGTTGGACGCCTGCAGGCTGAATGTAACGCCCAGGAATTTTCCCGCCAGAAAGTCGATCGTATCCCGGGTCCGATTACCCTGGCTGGCTGCCCGCTCATCAAGCGCGGAGCCAGCGGCAATATTACCCGCTTAGTGCAAGAACGCCTGAATAGCTTAGGCTTTTGTTGCGGCGTGGACGGCGACTATGGACGCGCACCGTTTCATGAAACCTACGACGCAGTCATCGCCTATCAGCGTGCCAATGATCTGGTACCAAATGGGATTGTAGGTCAAAAAACATGGTCGAAACTGCTGGGCCTGAGCTAA

LysCS05 nucleic acid sequence (SEQ ID NO: 20):

ATGAAGCTTGTTGAGAGTATTCTGACCAAGAACCCGTGCTATACCGCCGGTCGCAAAATCACCGTAAAGGGCCTGATGTTACATAGCGTCGGATGCCCACAGCCGAAAGCTAGCGTGTTTATCAACTCATGGAATTCACCGTCTTACGATAACGCATGTGTGCACGGCTTTATTGACGGTAACGATGGCACGGTGTACCAGACGCTGCCGTGGAACCATCGCGGATGGCATTGCGGCTCCGGTAGCAAGGGTTCAGGTAACAACACCCATATTGGCGTGGAAATGTGCGAGCCTGCCTGCATCAAATATACTAGCGGCGCGAACTTCACTTGTTCGGACACTGCAACCGCAAAAGCCGTAGCGAAACGCACCTATGAAGCTGCCGTAGAACTGTTTGCCATGCTGTGTCGCCAGTACAGCTTAAACCCAACGGCCGATGGCGTAATTATTAGCCACCGCGAAGGGCATAGCCGTGGTATCGCTAGTAACCACGGCGATCCGGAGCACTTATGGACACAGCTGGGCATGGGTTATACAATGGATACGTTTCGTCAGGCAGTTAAAGCAGCTATGGATGGAGGAAGTAGTGCCGAAACCTCTGGCTACACAAAAATTATGGGCAACGCCGTAGCGACAGCCGAGCAGATGGAAACGTACATCCAAGGTAAAAATCCGCAGGTCCTCCAGTCAGTCTTAGACATGATCCCGCTGTATTTGTCAGAAGGACAAGCCGAAGGCGTACGCGGGGATATTGCATTCGCCCAAAGTTGCCTGGAGACCGGCAACTTCACTTTCAGTGGTTCGGCTGTCACATTGGACCAGAACAACTTCTGTGGAATGGGCGTCACCAGCAATGGTCTGAAAGGTAATAGCTTTGATACGCCGCAGCTTGGGATTCGTGCACAGATCCAGCATCTTAAGGCCTATGCTTCGACGGATGCTCTTAAAAACGATTGCGTGGATCCGCGCTTTAAATACGTGACACGTGGTAGTGCAGAATACGTCGAATGGCTGGGCCAGCAAGAAAATCCTCAGGGGAAAGGCTGGGCAGCAGGTGCAGGCTATGGCGAAAAAATCCTGACTATTTTAAAAAACATTTGTGGAACGGCTGGCGGTGCCTCGGGCACTGCTGACATTTGGTATCGCGTGCGTAAGACGTGGGCTGATGCAAAAAGCCAGATTGGCGCTTTCCGTGTGTTGGAAAACGCAAAAAACTGTGCTGATGAGAATCCGGGTTATCGCGTGTTTGATGTTAACGGAGTGAATATTTATACACCTGATACTGCAGCCTTCTCTCCGTATCTGGTACGTGTGAGTATCACTGACCTTAACATCCGCAAGGGACCGGGCACTAACTATGCCAAGACCGGCAAATTTACCGGAAAAGGGGTCTTTACGATTGTTGAAATGCAGACGGGAAAAGGCAGCGACACAGGTTGGGGTCGTCTGAAATCGGGAGCGGGCTGGATTTCCCTGGATTATACCGAGAAAATTTCGTGA

LysCS06 nucleic acid sequence (SEQ ID NO: 21):

ATGAACGGAATCGATATTTCAAATTGGCAGAAAGGCATCAATCTGGATGCTGTCCCGTGCGATTTCGTCATTATGAAAGCCACCGAGGGTACTTGGTACGTCAATCCCGACTGCGAACGTGCATATCAGCAGGCAAAAAATGCGGGGAAATGTCTGGGCGTGTACCATTATGCAGAAGGCAAAGATGCCAAAGCCGAGGCAGACTTTTTCTTAAAACATATCCAAGGCTACATCGGTGAAGCGCTGATCGCCCTGGATTGGGAAAAAGAAAACAACTCATCATTCGGTAAAAATGATCTGAACTGGGTGAAACAGTGGCTGGATTATATTTACGGTAAAACGGGCGTGCGTCCATTACTGTATATCTCGCAGTCCATCATGGGGAAATTTAATGGCATCGGCGATTATGGTCTGTGGGTTGCCCAGTATGCCAATATGAACACCACCGGATATCAGGACGCCCCGTGGAATGAGGGGAAATATAACTGTGCGATTCGCCAGTATTCTTCATGCGGTCGTCTGAGTGGTTATTCAGGCAATTTAGATCTGAATAAATTTTACGGCGATAAGACCGCTTGGAATCGCTATGCCGGAAAAGGTAATGTCACTAAACCGTCTACGGGTACTGCCGCGAGTTCGACAAGTTCCCCTGGGGGCACCGTCCTTGATCTGGTTGTTGCAACCCTGCAGGGAAAATACGGCAATGGTGATGTGCGCAAAACGGCCTTGGGTAATCGCTATATCGAAGTGCAGAACATGATTAACCATATTGCATCCGCCAGTGCCAGTACGCTGGCCGGTGAAGTGAAAGCAGGTAAATATGGCAATGGGGATGCCCGCAAGATCGCCCTGGGCTCGCGTTACAATGAGGTTCAGAAAATCGTTAACGGCTCAACGGGTCTGGCAGCTAGTTATCATACAATTAAGAGTGGAGAGACGTTAAGCGGGATTGCCGCAAAATACGGCACGACCGTCGCAAAACTGCAAAGTCTCAACGGCATTAAAAACGTTAACAAAATCTACGCGGGTTCTCGTATCCGTGTCAAGTGA

LysCS07 nucleic acid sequence (SEQ ID NO: 22):

ATGGGTTGGAACGAGTACAATGCAAAACTGCAGGAGTGGTACGGTTTCACCGAAGCCGCAGGTCAGGATGACATTATTATTGACGCATACAATCGCCAGAAAGAAGAGGCGTACACCATGTCTCATCAGGATCCTTGGTGCCATGCGACTGTCTCCGCGGCTGCTGCCGAAAGTGGCAACCGTGGTCGTGTCCCCAATACAGCCTACTGCCCTACAGGGATCAACTGGTTCAAGGCCCGTGGTCAGTGGACGGGTCGCTATGATACAGGATATAACCCCTCAGTTGGCGATATTATCTATTACGATTGGGGCGGCGATGGTGTCAGTGACCATGTTGGCACTATTGTCGGCGTCAACGGAAACACGCTTCAAGTACGCGAAGGTAATAAAAATGATATGTTGACCGACCGTTACATTCAGAAAGGTAACACCCTCATTATGGGATATGGCCGCCCGGCCTGGGGTGGCTCAGTACCGGTGCCGAGCCAGCAGAGTGGTAGTATCGGTCGCAGTTGGCTGCAGCGTGGTGATAAAGGCGAGGCTGTTAAGGACGTTCAGAATAAACTGATCGCACTTGGCTATAGCTGCGGCCCGGATGGCGCCGATGGTGATTATGGGACCAACACCATTGCTGCCGTTAAACGTTTCCAAGCAGATGTGGGCATTACCGTAGACGGTCTGGCCGGTGAAATTACCCGCGCCAAACTGAACAATGCATATAACACGGGGGGCGTTAACAAAGCCGGTGGTTCGTGGGTTGCGCGCCTGCAGGCCGAGTGCAATGCACAGGGATTCAGCACGCAAAAGGTGGACGGATTACCAGGACCGAACACCCTGGCCGGTTGCCCGACCTTAGGGCGTACCTCTCGTGGAAAAATCACGGCGTTGATGCAGGAACGCTTAATCTCTCTGGGTTACAGCTGTGGGCCGTGCGGTGCCGACGGAATCAATGGTGCTGGTACGCAGGCTGCGATTAAGGCTTTTCAGCGCGATCATGGCCTCGCCGTCGACGGAATTGTGGGCCAGAAAACATGGAGCAAACTTCTGGGCCTGTCATGA

LysCS08 nucleic acid sequence (SEQ ID NO: 23):

ATGAGTATGAACGGCATTGACATTTCTAACCACCAGCAGGGGTTAGATGTAAGCAAAGTCCCATGTGATTTCGTCATCATGAAAGCCACCGAGGGCACCACCTTTGTGGATAAATATTGCGATAAATTTTATCAACAGGCGAAAAAACTGGGCAAAAAGCTGGGCGTGTATCACTTTGCCAGCGGCAAATCCAGTGGGACCGCTGAAGCCGACTTTTTTCTGAAAAACATTGCAGGCTATGTCGGGGAAGCCATTCTGGTGCTCGATTGGGAAGGCTCTGCTGTTAATAAAGGTGTTGGTTACGCAAAAGAGTTTTTAGACCGTGTTTACGCCAAAACTGGGGTTCGCCCGCTGCTTTACAGCTATAACAATTGCATCAATGCGTATGATTGGCGCAGTGTTGCCCAAGCTGATTATGGATTATGGAATGCTGGCTATTATGCTGGTTATCAGACCATGGGCTATAATCCTAATGCGCCGATTAAAGGTGGCCTCGGCGCATTCGGGAGCTGCGCGATGTACCAGTACACAAGCTCTGGACGCTTATCAGGCTGGGCGGGGAACCTTGACCTCGATGTCTTCTATGGCGATAGCGCCGCGTGGGATGCATACGCAAAAGGTAGTGCGATCGCATCGCCGGGCGGTACGCCTGAACCGTCCCAACAGCCGACCAATACCAGTCCGAGCAGCCAGAGCATGCTGAACGCACAGATCCACATTAACAACTTCACCGGCAGCGGCATTCCAGAGGACGGGAAAAACGGACGCAAGACCCGTAAAGGTGTTGTGATGTCGCTGCAGCATGCTTGTAACTTGGATTATAAACCTAACCCTGTGTTGAAAGAAGACGGCTTAATCGGGAGTAAGACGAACCGCGTCCGCGGCCTGCATTACGTTAAGCGTGGTGAAACCCAGTACCTCGTAACCTTTGTCGAAATTGGGTTAACCGCATTGGGCTACTATGCCGGTGCAGTGGAATGTCCTGGTGTGTTCGGGGAAGGTCTGGAAGACGCCGTCCATCGCTTCCAGCACGACATGAATCTTAACGAAGATAAAATTGCAGGCCGCAATGTCATGGACATGATGCTGCGTCAGCTGGGGTGCATTTGA

LysCS09 nucleic acid sequence (SEQ ID NO: 24):

ATGAGTAACTCAAGTCTGGTGAACTGTACCGTGAAATCCCCGAACCATAGCGGCGCACGTACACACTCAATTGACCGCATCACCCCCCATTGTGTGGTGGGCCAGCTGTCTGCGGAGTCTATCGGTGGCTGCTTCACGAGCCCATCCCGTGAAGCATCGTGTAATTACGGAATCGGGACGGACGGACGCGTCGTTTTGTGTGTTGACGAAGCAAATCGCAGCTGGTGCTCCAGTTCAAACGCCAACGATCAGCGTGCTGTGACCATCGAGTGCGCAAGCGATATGACGGACCCCTATGCTATGACCTCCGCGGTGTATGAGAAACTTGTCGCTTTATGCGTGGACATCTGCCAACGCAACGGTAAATCTAAACTGATTTGGTTTGGCGATAAGGATAAAAGTTTAAATTACTCTCCGAAAAGCAACGAGATGATTTTAACTGTGCACCGCTGGTTTGCAAATAAGGCCTGCCCGGGTGACTGGTTGTATAGCCGTCTGGGAGATCTGGCAAACCGTGTAACGTCACAGCTGGGAGGTTCTACTACCGATTCCACATCAAAGACATATAAAACCGGGTTATATAAAGTCGATGTTGGGGATCTGAACATCCGCAAAGGCCCGGGCACCAACTATGGGATTAATGGCATGATTACCGATCGCGGCACCTATACTATTACGGAAATTCAAAACGGCTACTGGGGCAAATTGAAATCGGGCGCAGGCTGGATTTCCGTGCACGAAGCTTATTGCACGTACAAGGGTGCCGCAAGTGGCGAATCCGAAGAGAAACCGAGTAGTAACTTTCTGGTGCAGGTTGATATTCCCGATCTCTACATCCGTAAGGGCCCGGGAACCAACTATGGCAACAACGGCTTTTGTCCTAAAGGCGTCTATACCATTGTTGAAGTGAAAAGTGGTGCAGGTTCCGATGCGGGCTGGGGCAAACTGAAAAGCGGTGCCGGCTGGATCAGTCTGGATTATGCCACTCGCATCTAA

LysCS10 nucleic acid sequence (SEQ ID NO: 25):

ATGGAAAAGCAGGAATTTATTAAGCAGATCGCTGGGTATGTCAAGAAGTACGCCGCACGTTATGGGATCAAGGTGCATTCTCCTATTATTGCACAGGCAATCCTGGAAAGTGGCTGGGGCGAATCAAAGCTTGCAGCTGTATACCATAACTATTTCGGTCTGAAGTGCGGTACCAAATGGACAGGTAAGTCCGTGAACCTCAAAACTATGGAAGAATATACCCCTGGCACCTTAACCCAGATCAAAGATAATTTTCGCGTATATGATAATATGGAGGAAGGCGTGAAAGGCTACTTCGAATTCATCCAACTGAAACGTTATCAGAATCTGAAAGGCATCACCGATCCGGAAGAGTATCTGAAAACAATCAAGGCGGACGGCTACGCAACCTCTTCTAAATACGTTGAGAACACAATGCGTATTGTCACGCAATATAATCTTCAAAAATATGATACGAAAGGCGAAGAATCAATGGCAAAAAAAGCTAGTGCCGTTCTGTCACAGGCCCGTGCCTGGATTGGCCGTAAAGAAGCCAATGGGACACATCGCGAAATCATCGATGTATACAATGCACACCGCCCGCTGGCCCGTGGCTACAAAGTTAAATACACCGACGCCTGGTGCGCAACCTTTGTTTCTGCAGTAGCCATCAAATGTGGTCTTACCTCCATCATCCCGACCGAATGCGGGTGTGGCCAAATGATCGAGCTGTTTAAAAAACTGGGTGAATGGCAGGAGTCTGACTCGCGTACGCCGAAACCAGGTGATGTTGTTTTTTATGATTGGGATGACACTGGTACCGGCAACAACACCGGGTGGCCGGATCACGTTGGTATTGTGGAAAGCGTAAGTGGCGGCAGTATTACAGTAATTGAAGGCAATAAGAATAACGCAGTAGAACGTCGTACCTTATCAGTGAACGGCCGCTATATTCGCGGCTATGGGGTGCCGAAATATGATAGTGAAGCCGGCACCGGTACCACGCAGCCCGGGAAATCTGTGGCAGAGGTTGCAAAGGAAGTCATTGCCGGTAAATGGGGTAATAATCCGCAACGTAAAGAACGCCTGGAAGCCGCAGGTTATGATTATCAGGCCGTTCAGAACCAAGTTAACGCAATTCTGAACGGCAATGCTAAACCGCAGAAGAGCGTTGCAGAAGTGGCCAAAGAGGTTATTGCAGGGAAATGGGGCAATAATCCTCAGCGTAAAGAGCGCCTGGAGGCCGCGGGCTACGATTACCAAGCCGTGCAAAACAAAGTGAACCAACTGCTGAAATAA

LysCS11 nucleic acid sequence (SEQ ID NO: 26):

ATGAACATTATCAAACATGGTTTGCGTTTCCGCGGCAGCTTTACGTACCGTAATCGTACGGACGAAATTATTTGGCACCATGCCGAGGCGAACTGTACTGTTGAAGATATTCACAGTTGGCACCTGAACAATGGTTGGATTGGTTGTGGGTATAACCTCATCATCTACAAAGATGGAACTGTGCACGAAGGTCGCCCACTGAACGCTGTCGGCGCCCACGCTTCGGGGCATAATTCCCGTTCTGTTGGCGTGTGTTGTATTGGCCGCTATGATGTGGAAACCATGCCTAAAGAACAACTCGACGCCGCCAAGCAAGTGCAGGCTTACCTTAAAGGTTTGTATCCAGGTGCTGCCACCAAGCGCCACAAAGATGTCAATGCGACATCATGCCCGGGCAAGAACTTTCCCTTCTCGGAGATTTCGGGTGCCTCGGCCGGCAGCGTCGACGTGACTACGAGCGATGTTCAGGCCCAAAGCTCAGATGACTGGTGCGCACGCCTCCAAAAAGAGTGTAACGCACAGAAATTCAGTAAACAGAAAGTGGACAATATCCCCGGCCCGGACACCCTGGCTGGTTGTCCTACGCTGGGTCGTAAATCCCGTGGGAAAATTACAGCTCTGATGCAGGAACGCCTCAATGCTCTGGGTTACGACTGTGGTGCAGTTGACGGTATTAATGGCACCAAAACCCAGGCTGCTATCAAGGCGTTCCAGCGTGATTACGGTTTAGTGGCCGACGGTATTGTGGGTCCGAAGACTTGGTCAAAACTTCTTGGCTTAAGTTAA

LysCS12 nucleic acid sequence (SEQ ID NO: 14):

ATGGCCCATCTGTACGTTATTGCCGGTCACGGTGCGGGTGACTCCGGTGCCGTGGGCAACGGTTATACCGAGGCCGAACGTGTTCGCGCCCTGGCCTCCAAGATCAAACAGTTGGGCGGAGATAATGTTACGCTGGGGGACATGAACCGCAACTATTACGCAGATAATGGTATCTCATCGCTGAACATCCCAAAAGATTGGTGCATTATCGAACTCCACATGGATTCAGCCAGCGCTTCCGCTCGCGGTGGCCACGTGATTATTAAAGGTACCTTTAGCCCTGATAGTCACGACTTAGCCCTTGCCTCCTTTGTCTCAGCCATGTTCCCGGGCCGCGCAAATAGCATTGTCGGCCGTAATAATCTCGCCAATCCGAACCGCGCTGCAGCAAAAGGCTATAACTACCGTCTGGTGGAATTTGGCTTTATTTCGAATGCTACCGACGTCAGCATTTTTAACAATAATCTGGATGCAATCGCACGCGGTGTGCTGAGCTGCTTTGGCATCAATACGAACACTGCGAAGTGGGTACTTGATAATGTTGGATGGTGGTATCAACGTGCGGATGGTAGCTATCCGAAGTCGCAATGGTTACTGCTCGATTGCTATTATTACTTTAACGACAAAGGCTACGCTCTGGCTAATGAGTGGCTGAGCTATGGGGGCAACTGGTACTGGCTGAAGGACGATTGTCGCATGGCTACCGGCTGGCAATACATTGACAAACATTGGTATTACCTGAATCCGACCGGTACCAAAAATAAACCAGTGGGCGCGATGCTCGATGGATGGCAGTTTATCGATGGCCAGTGGTATTATTTGCGCACCAAAGCCGATGGCGAGCATCCGCACGGCTCCATGGTCGAAGGAAGCGTAACCGTGGGCGAACATGATTACTATTGCCGCGAGGCCGGTACGGACAAAAACTACCCGACCGGGAGTATGTTAATGGGGTGGCGCAAAGTTACCGAAACCGCCGAAGACGGCACCAAAAAAACGAAATGGTTTTGGTATAATAAGGACAGCAATTGTCAACCGATTGGGAGTATGTTAAAAAATCACTGGCTGACGACGAGTAACGGCAAAAAATACTACTTAAAAGATGACGGCGTGATGGCATGTGATGAAACTATGACGATTAGTGGGAAAGAATACACTTTTGATGCTAGCGGCGCATTAGTGTAA

LysCS13 nucleic acid sequence (SEQ ID NO: 15):

ATGGCCCATCTGTATGTGATCGCCGGCCATGGCGCTGGTGACTCTGGCGCGGTGGGGAATGGTTATACTGAAGCTGAGCGTGTTCGTGCCTTGGCGTCAAAGATTAAGCAGCTCGGCGGTGACAACGTTACGCTGGGGGACATGAACCGTAACTATTATGCTGACAATGGCATCAGCAGTCTTAATATTCCGAAAGATTGGTGCATCATTGAACTGCATATGGACTCCGCAAGCGCATCCGCACGCGGCGGGCACGTTATCATCAAGGGAACCTTCAACCCGGATTCTCACGATTTAGCACTGGCAAGCTTTGTTTCGGCTATGTTCCCGGGACGCGCTAACAGCATTGTCGGCCGCAATAACCTGGCTAATCCTAACCGTGCCGCAGCCAAAGGCTATAACTACCGTCTGGTGGAATTTGGTTTTATTAGCAACGCAACCGATGTGAGTATCTTTAATAGTAATTTGGATGCAATTGCGCGTGGTGTTCTGAGTTGTTTTGGGATTAATACCAATACGGCGAAGTGGGTCCTGGATAATGTGGGCTGGTGGTACCAACGCGCCGACGGCTCTTATCCGAAAAGCCAATGGCTTCTGTTAGATTGTTATTATTACTTCAACGATAAAGGTTATGCATTAGCAAACGAACGTTTGTCCTATGGTGGCAATTGGTACTGGTTAAAAGATGATTGCCGTATGGCGACCGGTTGGCAGTATATTGACAAACATTGGTATTATCTTAATCCGACCGGGACAAAAAATAAACCCGTTGGAGCAATGCTTGATGGTTGGCAGTTCATTGACGGCCAGTGGTATTATCTGCGTACGAAAGCAGACGGTGAACATCCTCATGGTTCGATGGTCGAAGGTAGCGTCACGGTTGGCGAACACGACTACTACTGTCGTGAGGCTGGGACCGATAAAAACTATCCGACTGGTTCGATGCTGATGGGGTGGCGCAAAGTGACGGAGACGGCTGAAGACGGCACTAAAAAAACTAAATGGTTCTGGTATAACAAAGACAGCAACTGTCAGCCGATCGGTAGCATGTTGAAAAATCACTGGTTAACAACATCTAACGGTAAAAAATACTACTTGAAAGACGATGGGGTAATGGCGTGTGACGAAACGATGACCATTAGTGGCAAAGAATATACTTTCGATGCCTCGGGCGCTTTAGTTTGA

according to a specific embodiment of the invention, the nucleic acid molecule according to the invention is a DNA molecule or an RNA molecule.

According to a specific embodiment of the invention, the nucleic acid molecules according to the invention are modified or unmodified. The modification may be, for example, chemical modification by introducing chemical groups such as phosphate groups, methyl groups, acetyl groups, etc., to modify the nucleic acid molecule to alter its stability, affinity, targeting, etc. Enzymatic modification by modification of nucleic acids with specific enzymes, such as by restriction endonuclease cleavage, ligase ligation, DNA methylation, RNA editing, and the like. Nucleotide substitutions-substitution or insertion of a specific nucleotide sequence to improve the function or to increase the stability of the nucleic acid molecule. For example, modified nucleotides (e.g., 5-methylcytosine) are used to regulate gene expression. Synthetic modification-specific chemical modification of nucleic acid molecules using synthetic methods, for example by adding specific modifying groups during the synthesis of DNA or RNA, to improve their stability in vivo or to enhance their activity in specific environments.

In another aspect, the invention also provides a vector comprising a nucleic acid molecule according to the invention.

According to a specific embodiment of the invention, the vector may be a pET28a (+) or pCold II vector recombinant plasmid loaded with a nucleic acid molecule (e.g., DNA sequence) according to the invention.

In another aspect, the invention provides a host cell comprising a nucleic acid molecule according to the invention or a vector according to the invention.

According to a specific embodiment of the invention, the host cell may be an E.coli competent cell, such as BL21 (DE 3).

In another aspect, the present invention also provides a method for preparing the antibacterial protein, which comprises:

the antibacterial protein is prepared by in vitro transcription by using the nucleic acid molecule or the vector or the host cell.

In another aspect, the invention also provides an antibacterial pharmaceutical composition comprising the antibacterial protein or the nucleic acid molecule or the carrier of the invention and a pharmaceutically acceptable excipient.

On the other hand, the invention also provides application of the antibacterial protein or the antibacterial pharmaceutical composition in preparing products targeting clostridium scintinus.

In another aspect, the invention also provides the use of the antimicrobial protein or the antimicrobial pharmaceutical composition in the preparation of a product for the prevention and treatment of clostridium scintillans-associated disorders;

According to particular embodiments of the invention, the clostridium scintillans-associated disorder includes one or more of the following:

clostridium infection, chronic diarrhea, diarrhea-predominant irritable bowel syndrome and other inflammatory bowel diseases, nonalcoholic fatty liver, tumors such as colorectal cancer, prostate cancer, liver cancer and the like.

According to a specific embodiment of the invention, the product according to the invention may be a pharmaceutical, disinfectant or bactericide.

The antibacterial protein targeting clostridium scintinus can kill clostridium scintinus with high efficiency and has specificity.

Drawings

FIG. 1 is a map of a LysCS expression vector plasmid for a constructed lyase.

FIG. 2 is an identification electrophoretogram of heterologous expression of LysCS and LysCS13 in E.coli.

FIG. 3 is an electrophoretogram of LysCS and LysCS13 purified proteins.

Fig. 4 shows the experimental results that LysCS and LysCS13 were able to lyse clostridium scintinus efficiently.

FIG. 5 shows LysCS the lytic activity of LysCS against different gut-associated Clostridium bacteria.

FIG. 6 shows the lytic activity of lyase LysCS against various gut-associated bacteria.

Detailed Description

Before further describing embodiments of the invention, it is to be understood that the scope of the invention is not limited to the specific embodiments described below, and that the terminology used in the examples of the invention is intended to be in the nature of specific embodiments and is not intended to be limiting of the scope of the invention.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all employ techniques conventional in the art.

Example 1 identification of a Clostridium scintillans-targeted lyase Gene and protein sequence analysis

Phage and prophage genomic sequences targeting clostridium scintinus were collected and their lyase genes were identified by sequence alignment and domain alignment using BLAST and Interproscan. More than 70 potential phage lyase gene sequences are found, and the invention further identifies a class of lyase from the gene sequences, which comprise amino acid sequences shown in SEQ ID NO. 1-SEQ ID NO. 13, and are respectively named LysCS 01-LysCS.

The nucleic acid sequence of the lyase after codon optimization for escherichia coli is shown as SEQ ID NO. 14-SEQ ID NO. 26.

Example 2 heterologous expression and purification of Clostridium scintillans-targeted lytic enzymes

The identified lyase genes were codon optimized for E.coli, and then the corresponding genes were cloned into pET32a or pCold II vectors, with 6x-His tags added at the C-or N-terminus, and the genes were under the control of lactose operon. FIG. 1 is a map of a LysCS expression vector plasmid for a constructed lyase.

The constructed plasmid containing the gene LysCS of lyase was transformed into BL21 (DE 3) competent cells, then spread uniformly on LB plates (containing 50. Mu.g/mL kanamycin sulfate), and then placed in a 37℃incubator overnight. From the transformed plates, single clones were selected, inoculated into 1L of LB medium (containing 50. Mu.g/mL kanamycin sulfate), cultured until OD600 was 0.6-0.8, IPTG was added to the tube culture medium to a final concentration of 0.1-1mM, and then placed at 16-37℃respectively at 100rpm for 18h to induce protein expression.

Centrifuging the induced culture solution at 12000rpm for 5min, removing the culture medium, adding PBS solution to resuspend and clean the strain, adding SDS-PAGE loading buffer, heating the sample at 100deg.C for 30min, centrifuging, and collecting supernatant. And (3) performing voltage-stabilized electrophoresis at 100-150V 10min before electrophoresis, performing voltage-stabilized electrophoresis at 200V after the bromophenol blue indicator enters the separation gel until the bromophenol blue band migrates to 1cm away from the bottom of the gel, taking out the gel, dyeing the gel with coomassie brilliant blue staining solution, and transferring the gel into a decolorizing solution to decolorize until the background is clear.

Adding whole bacteria into PBS buffer solution, performing ultrasonic or high-pressure homogenization and cleavage, balancing Ni-IDA affinity chromatography column with PBS buffer solution for at least three times, eluting target protein with balancing buffer solution of imidazole with different concentrations, and collecting each eluting component for SDS-PAGE analysis and detection. SDS results are shown in FIG. 2 (M is SDS-PAGE Protein marker,1 is whole cells before induction, 2 is whole cells after induction, 3 is induced expression supernatant, 4 is induced expression pellet).

FIG. 2 is an identification electrophoretogram of heterologous expression of LysCS and LysCS13 in E.coli. As shown in FIG. 2, the lytic enzymes LysCS and LysCS proteins were expressed in soluble form in E.coli after disruption and isolation.

The cultured cells were centrifuged at 4000rpm for 10min to collect the cells. The bacteria were then resuspended in Lysis buffer and disrupted. Centrifuging at 18000rpm and at 4deg.C for 15min, collecting supernatant, incubating the supernatant with Ni resin at 4deg.C for 2 hr, washing the column with Wash buffer with imidazole of different concentration gradients until the eluate is no longer blue, adding 10ml of Elute buffer, eluting, detecting with SDS-PSGE, concentrating to 1ml, centrifuging at low temperature to remove impurities, sieving, collecting protein, detecting with SDS-PAGE gel, concentrating, determining concentration, and freeze-preserving. FIG. 3 is an electrophoretogram of LysCS and LysCS13 of purified proteins showing that high purity of the lyase proteins LysCS and LysCS can be obtained by multiple purification steps.

Example 3 determination of lytic Activity of lytic enzyme proteins against Clostridium scintinus

Clostridium scintillans DA266 (this strain was offered by the advanced technical institute of Shenzhen, national academy of sciences, which was publicly deposited and shared by the general microbiological center of the national institutes of microbiological culture, strain ：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) described in the following document was also inoculated into anaerobic BHI broth, clostridium scintillans was cultured until od600=0.6, the broth was centrifuged at 4000rpm for 5min from the broth, washed twice with buffer (50 mM Tris-HCl [ pH 7.0], 100mM NaCl), and the cells were resuspended with this buffer after washing, the buffer containing the cells was added to a 96-well plate, lysCS or LysCS13 lytic enzyme protein was added to a final concentration of 5 micromolar, and the final volumes were 200 μl, after 3h of culture, experimental data were collated, and the lytic activity was calculated.

FIG. 4 shows that LysCS and LysCS13 are capable of highly efficient lysis of Clostridium scintinus. After addition of the lyase, the OD of the broth decreased rapidly, demonstrating that LysCS and LysCS13 were able to lyse clostridium scintinus effectively. By adding LysCS and LysCS lyase, the absorbance OD600 is reduced by more than 50% within about 50min, which indicates that the lyase LysCS and LysCS has stronger cleavage activity on clostridium scintinus.

Example 4 Targeted assay of lyase proteins

Using purified protein LysCS, its lytic activity against different commensal intestinal bacteria was determined in the same manner as in example 3. Briefly, different symbiotic enterobacteria were individually inoculated into anaerobic BHI broth and clostridium scintinus was cultured to od600=0.6. The bacterial solution was centrifuged at 4000rpm for 5min from the culture medium, washed twice with a buffer (50 mM Tris-HCl [ pH 7.0], 100mM NaCl), and the bacterial cells were resuspended in the buffer. The buffer containing the thalli is added into a 96-well plate, lysCS micromoles of the lyase protein are added to a final concentration of 5 micromoles, the final volume of each well is 200 micromoles, and the mixture is placed into the 96-well plate and placed into an enzyme-labeled instrument to continuously measure the absorbance change. A control group without lyase was also set. And calculating the absorbance change after 2 hours. The cleavage activity was calculated.

FIG. 5 shows the lytic activity of lyase LysCS against various intestinal symbiotic Clostridium bacteria. LysCS13 shows a high lytic activity against Clostridium scintinus, but LysCS does not show lytic activity against other Clostridium bacteria, such as Clostridium symbiotic, clostridium butyricum, clostridium scintinus and Clostridium harmlessly. This demonstrates that LysCS has a higher targeting, which can target the removal of clostridium scintinus in the gut without destroying other commensal clostridium enterobacteria.

FIG. 6 shows the lytic activity of lyase LysCS and lysozyme (egg white source, CAS: 12650-88-3) against various gut-associated bacteria. As shown in panel A of FIG. 6, lysCS.sup.13 showed high lytic activity against Clostridium scintinus, but LysCS.sup.13 showed only lytic activity against strain DA394 and not against other strains for the other 11 enteric symbiotic bacteria tested (information and taxonomic status of the test strains are shown in Table 2). These tested gut symbiotic bacteria were from 5 common gut symbiotic bacteria phylum including bacteroidetes, firmicutes, actinomycetes, proteus and fusobacterium bacteria. The result shows that the lyase LysCS has higher targeting property and can target and clear clostridium scintinus in intestinal tracts. In addition, although egg white-derived lysozyme has a certain cleavage activity on clostridium scintinus, it also resulted in the cleavage of various intestinal symbiotic bacteria without obvious targeting (panel B in fig. 6).

TABLE 2 information on intestinal commensal bacteria for the lyase LysCS13 targeting test

Bacterial species designation	Bacterial strain designation	Bacterial classification
			Bacteroides uniformis	ATCC8492	Bacteroides door
Parabacteroides distasonis	ATCC8503	Bacteroides door
			Blautia obeum	DA69	Thick wall fungus door
Dorea longicatena	DA136	Thick wall fungus door
			Clostridium symbiosum	DA229	Thick wall fungus door
Anaerostipes hadrus	DA538	Thick wall fungus door
			Faecalibacterium prausnitzii	DA726	Thick wall fungus door
Bifidobacterium adolescentis	DA06	Actinomycetes door
			Collinsella aerofaciens	DA394	Actinomycetes door
Escherichia coli	DH5α	Proteus gate
			Fusobacterium varium	DA690	Fusobacterium door

The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.

Claims (10)

1. An antibacterial protein targeting clostridium scintinus or a chimeric thereof, wherein the antibacterial protein comprises one or more of the following amino acid sequence-forming proteins:

(a) The amino acid sequence shown in any one of SEQ ID NO.1 to SEQ ID NO. 13;

(b) A derivative sequence having the same function as (a) by substituting, adding and/or deleting one or more amino acids from the amino acid sequence of (a);

(c) A derivative amino acid sequence having a sequence identity of 85% or more or 90% or more to any one of SEQ ID NO 1 to SEQ ID NO 13.

2. The clostridium-targeted antimicrobial protein or chimera thereof according to claim 1 wherein the chimera comprises:

A chimeric protein comprising the amino acid sequence of the intact antimicrobial protein of claims 1 (a), (b), (c), the catalytic domain sequence in the antimicrobial protein, the cell wall binding domain sequence in the antimicrobial protein, and combinations of two or more sequences;

Preferably, the chimeric comprises one or more of catalytic domains Amidase _3, amidase _2, CHAP, glyco_hydro_25, lysozyme_ like, glucosaminidase, and cell wall binding domains pg_binding_1, choline _bind_1, choline _bind_3, cw_7, SH3, and LysM;

more preferably, the chimeric comprises catalytic domain Amidase _3 and/or cell wall binding domain Choline _bind_1 and/or Choline _bind_3.

3. A nucleic acid molecule encoding the clostridium scintillans-targeted antimicrobial protein of claim 1;

Preferably, the nucleotide sequence of the nucleic acid molecule comprises any one of the sequences shown in SEQ ID NO. 14 to SEQ ID NO. 26;

More specifically, the nucleic acid molecule is modified or unmodified.

4. A vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the vector of claim 4.

6. A method of preparing the antimicrobial protein of claim 1 or 2, the method comprising:

The antimicrobial protein of claim 1 or 2 is prepared by in vitro transcription using the nucleic acid molecule of claim 3 or the vector of claim 4 or the host cell of claim 5.

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

8. Use of an antimicrobial protein according to claim 1 or 2 or an antimicrobial pharmaceutical composition according to claim 7 for the preparation of a clostridium scintillans targeted product.

9. Use of an antimicrobial protein according to claim 1 or 2 or an antimicrobial pharmaceutical composition according to claim 7 for the preparation of a product for the control of a clostridium scintillans-related disorder;

Preferably, the condition comprises one or more of clostridium scintillans infection, inflammatory bowel disease 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. Use according to any one of claims 8-9, wherein the product is a pharmaceutical, disinfectant or bactericide.