WO2022166736A1 - 噬菌体裂解酶、其嵌合物及应用 - Google Patents

噬菌体裂解酶、其嵌合物及应用 Download PDF

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WO2022166736A1
WO2022166736A1 PCT/CN2022/074221 CN2022074221W WO2022166736A1 WO 2022166736 A1 WO2022166736 A1 WO 2022166736A1 CN 2022074221 W CN2022074221 W CN 2022074221W WO 2022166736 A1 WO2022166736 A1 WO 2022166736A1
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seq
chimera
lyase
acnes
amino acid
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French (fr)
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阿萨夫·拉兹
米娅·丹达
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武汉乐斯吉生物科技有限公司
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Priority to JP2023548280A priority Critical patent/JP2024508694A/ja
Priority to EP22749015.8A priority patent/EP4289949A1/en
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Definitions

  • the invention relates to the field of medicine and biology, and in particular provides a phage lyase with anti-P. acnes activity, a chimera and applications thereof.
  • Acne is an inflammatory disease of the skin, especially on the face, neck, shoulders, chest, back and upper arms.
  • the pilosebaceous unit is inflamed due to the blockage of excessively secreted oil and the accumulation of dead skin cells caused by the proliferation of the pathogenic Cutibacterium acnes (C. acnes, the same below).
  • C. acnes Cutibacterium acnes
  • 80 percent of people between the ages of 11 and 30 have some degree of acne.
  • Acne usually begins in adolescence and may last into the forties and fifties. Different factors can cause acne breakouts, such as stress, changes in hormone levels, allergens, and more.
  • the 2010 Global Burden of Disease Study estimated that acne affects 9.4% of the global population and ranks it as the eighth most prevalent disease in the world.
  • the American Academy of Dermatology estimates that in 2013 the cost of acne patients related to treatment and lost productivity exceeded $1.2 billion. While rarely life-threatening by itself, it can cause lasting scarring on the skin and considerable emotional distress to patients.
  • Glanoids Current options for treating mild acne are over-the-counter topical medications that contain chemicals such as benzoyl peroxide, resorcinol, salicylic acid, and sulfur, as well as prescription topical medications such as antibiotics, benzoyl peroxide, Diacids, dapsone, corticosteroids, and derivatives of vitamin A called retinoids.
  • Moderate to severe disease can be treated with topical prescription or oral medications.
  • the purpose of the intervention is to remove dead skin cells and excess oil to open the follicle-sebaceous unit and reduce bacterial load. Since acne is a chronic disease, it requires prolonged treatment and sometimes prevention of recurrence.
  • none of the existing treatment options are suitable for long-term treatment.
  • P. acnes has been associated with a range of invasive infections, including postoperative shoulder infections and intervertebral disc infections. Among such infections, P. acnes exhibits chronic progressive biofilm-like infection. Bacteria found in biofilms are inherently resistant to antibiotics. A P. acnes fungicide with strong anti-biofilm properties is ideal for the treatment of such infections.
  • bacteriophage endolysin Phages use these enzymes to destabilize the cell wall for lysis, thereby releasing phage progeny from inside the cell. They are classified as peptidoglycan hydrolases and can break various bonds in bacterial peptidoglycans. With few exceptions, the target bacteria for each lyase belong to a single genus or species. Recombinant anti-Gram-positive lyases have been shown to be effective bactericides capable of causing hypotonic lysis (eg, PlyC against several Streptococcus species, PlyG against Bacillus anthracis, etc.).
  • lyases are under selective evolutionary pressure over billions of years in order to consistently successfully infect cells, the target peptidoglycan bonds are highly conserved and unlikely to be easily altered by bacteria. This feature makes the target bacteria less likely to develop resistance to the lyase, making it suitable for long-term therapeutic use. Bacterial resistance to the respective phage lyases has not been detected to date.
  • lyases against Gram-positive bacteria are composed of one or two N-terminal catalytic domains (CD) and a C-terminal cell wall binding domain (BD), with linkers of different lengths between the domains. ). Lyases act as modular proteins, and chimeric lyases can be created by pairing catalytic and binding domains from different lyases.
  • the length and sequence of the linker is also variable.
  • the main bottleneck in the development of lyases as antibacterial agents is the inability to soluble express highly active lyases.
  • no lyase that expresses high levels of soluble and can effectively kill P. acnes has been reported so far.
  • Patent CN102482655A discloses an antimicrobial agent, which is composed of an endolysin having the activity of degrading the cell wall of Gram-positive bacteria and an amphiphilic peptide segment fused to the endolysin at the N-terminal or C-terminal or both ends , can be used for the treatment or prevention of Gram-positive bacterial infections, as a diagnostic tool or as a cosmetic substance.
  • PA6 has activity against P. acnes DSMZ 1897 strain and DSMZ 16379 strain, it does not provide initial cfu/mL; in addition, PA6 cannot solve the problem of increasing the soluble expression level.
  • Phage lyase can specifically and effectively kill P. acnes while leaving other commensal bacteria intact, providing a safe and effective acne solution suitable for long-term use without high dysbiosis and acquired risk of drug resistance.
  • the present invention provides a phage lyase with anti-P. acnes activity, its chimera and application.
  • the specific invention is as follows:
  • the present invention provides the application of a phage lyase or its chimera in the preparation of medicines, cosmetics or medical equipment for preventing, treating or improving acne or infections caused by P. acnes or diseases related to P. acnes
  • the phage lyase includes phage lyase derived from Nocardioidaceae (Nocardioidaceae) and Propionibacteriaceae (Propionibacteriaceae) bacteria.
  • the Nocardioidaceae bacteria include Micropruina, Propionicimonas, and Propionicimonas Propionicicella, Friedmaniella bacteria; the Propionibacteriaceae bacteria include Propioniferax, Mariniluteicoccus, Granulicoccus , Naumannella, Propioniciclava, Auraticoccus, Microlunatus, Aestuariimicrobium, Luteococcus, four Tessaracoccus, Brooklawnia, Propionimicrobium, Propionibacterium, Cutibacterium, Propionibacterium Acidipropionibacterium, or Pseudopropionibacterium, preferably Propionibacterium, Cutibacterium, Acidipropionibacterium, or Pseudopropionibacterium bacteria.
  • the bacteriophage lyase includes sources from Propionibacterium acnes (Cutibacterium acnes), Propionibacterium humerusii (Propionibacterium humerusii), Propionibacterium avidum (Cutibacterium avidum), propionic acid granules Cutibacterium granulosum, Acidipropionibacterium thoenii, Acidipropionibacterium jensenii, Acidipropionibacterium acidipropionici, Aestuariimicrobium kwangyangense, Granulicoccus phenolivorans), Microlunatus phosphovorus, Pseudopropionibacterium propionicum, Tessaracoccus sp., Propionicicella superfundia, Pseudopropionibacterium (Pseudopropionibacterium propionicum) Propionibacterium freudenreichii), Propionicicella superfundia, Pseud
  • the bacteriophage lyase includes those derived from Acidipropionibacterium jensenii, Acidipropionibacterium thoenii, and Acidipropionibacterium acidipropionici , Acidipropionibacterium microaerophilum, Acidipropionibacterium olivae, Acidipropionibacterium damnosum, Cutibacterium acnes, Cutibacterium avidum , phage lyase of Cutibacterium granulosum and Pseudopropionibacterium propionicum bacteria.
  • the phage lyase has the amino acid sequence shown in any one of SEQ ID NO: 1 to SEQ ID NO: 28;
  • the phage lyase has the nucleotide sequence shown in any one of SEQ ID NO: 29 to SEQ ID NO: 56;
  • the phage lyase preferably has the PACL10 of the amino acid sequence shown in SEQ ID NO: 10;
  • the phage lyase preferably has the PACL10 of the nucleotide sequence shown in SEQ ID NO: 38.
  • the chimera comprises a catalytic domain derived from a phage lyase, or a combination of a catalytic domain and a binding domain derived from a phage lyase.
  • the catalytic domain has a full C-terminal linker, a half C-terminal linker, no C-terminal linker, or any part of the linker; the binding domain has A full N-terminal linker, a half N-terminal linker, no N-terminal linker, or any portion of the linker.
  • a synthetic linker can be used, which also includes a synthetic linker.
  • Non-limiting examples of possible linkers derived from phage lyases are shown in Table 3 below.
  • Non-limiting examples of synthetic linkers are shown in Table 4 below.
  • the chimera is formed by pairing catalytic domains and binding domains derived from different phage lyases.
  • the chimera comprises one or two catalytic domains and no binding domains;
  • the chimera includes one or more catalytic domains and one or more binding domains
  • the chimera may be one or more catalytic domains linked to a single binding domain
  • the chimera may be a single catalytic domain linked to a single binding domain
  • the chimera may be a single catalytic domain in the N-terminus of the lyase linked to a single binding domain in the C-terminus of the lyase.
  • one or more catalytic domains can be used in the absence of a binding domain.
  • one or more binding domains can be used in the absence of the catalytic domain and fused to a different molecule to facilitate detection.
  • molecules include, but are not limited to, biotin, flag tags, myc tags, avidin, streptavidin, ovalbumin, firefly luciferase, biotin, fluorescent molecules such as FITC, TRITC, Alexafluor, and the like.
  • the chimera further comprises a linker between the catalytic domain and the binding domain, and the linker includes:
  • the linker region derived from the parent lyase molecule has the amino acid sequence shown in any one of SEQ ID NO: 343 to SEQ ID NO: 367.
  • the linker region derived from the parent lyase molecule has the nucleotide sequence shown in any one of SEQ ID NO: 368 to SEQ ID NO: 392; or
  • n has the amino acid sequence shown in any of SEQ ID NO: 393 to SEQ ID NO: 406, or be (GGGS)n, (GGGGS)n, (GGGGGS)n, (Gly)3-8, (EAAAK)n , (Ala-Pro)n, A(EAAAK)nALEA(EAAAK)nA synthetic linker domains of amino acid sequences (wherein 1 ⁇ n ⁇ 15, n is an integer), by way of example, n can be 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • the chimera comprises that the catalytic domain has SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO: 71, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106
  • the binding domain has SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:68, SEQ ID NO:72, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO: 94, SEQ ID NO:96, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:134, SEQ ID NO:137, SEQ ID NO: 138, or the amino acid sequence shown in SEQ ID NO: 139.
  • the catalytic domain has SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 141 NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO: 157, SEQ ID NO: 160, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 168, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO: 140, SEQ ID NO: 141, S
  • the binding domains have SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:151, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:158 NO:162, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 192, SEQ ID NO: 195, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 202, SEQ ID NO:203, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:217, SEQ ID NO:220, SEQ ID NO:221, or the nu
  • the chimera has the amino acid sequence shown in any one of SEQ ID NO: 223 to SEQ ID NO: 282; preferably SEQ ID NO: 258, SEQ ID NO: 259 , the amino acid sequence shown in SEQ ID NO: 260, SEQ ID NO: 271 or SEQ ID NO: 272, more preferably the amino acid sequence shown in SEQ ID NO: 260 or SEQ ID NO: 271;
  • the chimera has the nucleotide sequence shown in any one of SEQ ID NO: 283 to SEQ ID NO: 342; preferably SEQ ID NO: 316, SEQ ID NO: : 317, SEQ ID NO: 318, SEQ ID NO: 329 or the nucleotide sequence shown in SEQ ID NO: 330; more preferably the nucleotide sequence shown in SEQ ID NO: 318 or SEQ ID NO: 329.
  • the infection caused by P. acnes includes invasive infection, postoperative infection and/or device-related infection.
  • the device-related infection includes joint prosthesis, shunt tube and artificial heart valve-related infection.
  • the infection includes bone and/or joint infection, especially post-operative shoulder infection, as well as oral cavity, eye, intervertebral disc and brain infection.
  • the conditions associated with P. acnes include prostatitis, SAPHO (synovitis, acne, impetigo, hypertrophy, osteitis) syndrome, which causes cancer, Sarcoidosis, or sciatica.
  • the medical device comprises any device for delivering the lyase or its chimeric to the affected area, preferably a clip or patch or spray applied to the surface of the skin, Devices that enhance skin penetration of lyase or chimeras using microneedles, fine needles used by cosmetic professionals to specifically apply lyase or chimeras to acne-affected hair follicles, or other similar devices.
  • the medical device comprises a prosthetic device for immobilizing a lyase or a chimera thereof in a position susceptible to P. acnes infection, preferably for acne particularly susceptible to infection Prosthetic implants in shoulder surgery with Propionibacterium.
  • the present invention also provides the phage lyase chimeras described in the aforementioned applications.
  • the chimera has the amino acid sequence shown in any one of SEQ ID NO: 223 to SEQ ID NO: 282; preferably SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260 , the amino acid sequence shown in SEQ ID NO: 271 or SEQ ID NO: 272; more preferably the amino acid sequence shown in SEQ ID NO: 260 or SEQ ID NO: 271;
  • the chimera has the nucleotide sequence shown in any one of SEQ ID NO: 283 to SEQ ID NO: 342; preferably SEQ ID NO: 316, SEQ ID NO: : 317, SEQ ID NO: 318, SEQ ID NO: 329 or the nucleotide sequence shown in SEQ ID NO: 330; more preferably the nucleotide sequence shown in SEQ ID NO: 318 or SEQ ID NO: 329.
  • the present invention also provides a method for preparing the aforementioned chimera, comprising:
  • the method further comprises:
  • the Escherichia coli BL21(DE3) containing the recombinant chimeric lyase expression plasmid was cultured in autoinduction medium at 37°C and 300rpm until the OD 600 reached 0.6-0.8, and then continued to culture at 18°C and 300rpm for 16- 18 hours.
  • Cells were harvested by centrifugation, resuspended in 50 mM sodium phosphate pH 7.4, and lysed by high pressure homogenization. The lysate was centrifuged again to collect soluble crude lysate. The soluble fraction was mixed with an equal volume of 5M NaCl and the mixture was loaded onto a hydrophobic column.
  • lyase can be expressed in LB using different concentrations of IPTG at different temperatures, aeration and induction times. Cells can be resuspended in different buffers to increase solubility and lysed by mechanical or chemical means. Different methods of protein chromatography can be used to purify lyases. These variables can be optimized for better lyase protein yield.
  • the present invention also provides formulations containing the aforementioned chimeras further comprising antibiotics, other lyases, or inactive excipients.
  • the present invention also provides an amino acid sequence encoding the aforementioned chimera, which is 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more similar to the aforementioned sequence, or has the same amino acid sequence Alternative amino acid sequences for functional groups.
  • the present invention also provides 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% similarity to the aforementioned sequences %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence.
  • the replacement amino acid sequence is a conservative substitution of amino acids in the same group of amino acids, and the amino acid group includes:
  • Aromatic phenylalanine, tyrosine, tryptophan
  • Acidic and their amides aspartic acid, glutamic acid, asparagine, glutamine.
  • the present invention also provides a nucleotide sequence encoding the aforementioned chimera, or a synonymous codon sequence thereof.
  • the present invention also provides the use of the aforementioned chimeras as reagents for bacterial lysis for DNA extraction and typing using PCR-based kits.
  • the present invention also provides the use of the aforementioned chimeras as a disinfectant or sterilant on abiotic surfaces to prevent infection by removing P. acnes in planktonic forms or in biofilms,
  • the surgical equipment or prosthetic implant device is preferably sterilized during the procedure.
  • the present invention also provides the aforementioned chimeras, a single binding domain in the aforementioned chimera, or a tandem combination of similar or different binding domains in the aforementioned chimera in the preparation of a diagnostic tool for P. acnes , the lyase, chimeric or binding domain is used in combination with a detection marker.
  • the lyase, chimeric or binding domain forms a fusion with a signal molecule by genetic fusion or chemical coupling.
  • the fusion is used for direct detection of P. acnes on microscope slides by fluorescence or other means, for immunohistochemical labeling of P. acnes for use as detection reagents in ELISA assays, Use as a detection reagent on Western blots, for attachment to magnetic beads in MACS or other pull-down assays, or as a detection reagent in assays where antibodies are used as detection reagents.
  • the signaling molecule includes a protein or chemical fluorescent dye, a protein tag, an enzyme, avidin, streptavidin, ovalbumin, biotin, a tag sensitive to click chemistry labeling, a Peptides, or other molecules capable of causing the recruitment of secondary proteins or molecules that generate signals,
  • the fluorescent dye includes GFP, RFP, mCherry, FITC, TRITC, Alexafluor488, Cy3 or Cy5;
  • the protein tag includes a Flag-tag, myc-tag, halo-tag, his-tag, or any other tag capable of binding to an antibody or other high-affinity molecule to generate a signal;
  • the enzymes include firefly luciferase, beta-lactamase, alkaline phosphatase, horseradish peroxidase, or any enzyme that induces a reaction such as light, color change, substrate deposition, or other that can be detected in an assay. other enzymes.
  • the present invention also provides the application of the catalytic domain in the aforementioned chimera in the preparation of a medicament for treating P. acnes infection, wherein the catalytic domain is combined with a targeting module.
  • the present invention provides compositions and methods for preventing and treating P. acnes infection and skin colonization and acne lesions associated with such infection or colonization.
  • the present invention provides uses and uses of lyases having broad killing activity against skin-associated organisms involved in the development or progression of acne lesions and secondary infection of acne lesions , including but not limited to bacteria of the genus Propionibacterium, Cutibacterium, Acidipropionibacterium, Pseudopropionibacterium.
  • the present invention describes methods for the decolonization, dispersion and removal of established bacterial communities in the skin, with a particular focus on Propionibacterium acnes (Cutibacterium acnes, formerly known as Propionibacterium acnes).
  • the present invention also provides methods of producing chimeric lyases comprising catalytic and binding domains from different lyases resulting in various advantageous properties including, but not limited to, improved activity , host range, expression, solubility, stability or more suitable for commercialization.
  • sources derived from Propionibacterium, Cutibacterium, Acidipropionibacterium, Pseudopropionibacterium are used.
  • Bacteriophage lyases of bacteria are used. Table 2 provides the lyases and polypeptides used in the present invention.
  • the lyases shown in SEQ ID NO: 1 to SEQ ID NO: 28 are encoded by the nucleotide sequences shown in SEQ ID NO: 29 to SEQ ID NO: 56.
  • the present invention also provides a method for generating chimeras between the binding and catalytic domains of different phage lyases.
  • SEQ ID NO: 57-SEQ ID NO: 139 show a non-limiting sequence listing providing examples of polypeptide sequences encoding the catalytic and binding domains derived from phage lyases described herein.
  • the catalytic and binding domains set forth in SEQ ID NO:57 to SEQ ID NO:139 are encoded by the nucleotide sequences set forth in SEQ ID NO:140 to SEQ ID NO:222 .
  • the present invention also provides non-limiting examples of chimeric lyases produced by pairing the catalytic and binding domains of different lyases as described herein.
  • SEQ ID NO: 223-SEQ ID NO: 282 provide a non-limiting sequence listing of the anti-P. acnes active chimeric lyases produced by the methods described herein.
  • nucleotide sequences set forth in SEQ ID NO: 283 to SEQ ID NO: 342 are used to generate the chimeric lyases of the invention.
  • the polypeptide sequence is encoded by a nucleotide sequence inserted into the expression vector pET28.
  • the expression vector encoding the polypeptide of the present invention is expressed in E. coli BL21 (DE3) strain.
  • the present invention provides a method for screening and evaluating the activity of phage lyase against P. acnes.
  • the present invention demonstrates the activity of defined phage lyases against P. acnes.
  • the lyase used to treat Propionibacterium is PACL10.
  • the present invention provides a method for expressing and purifying PACL10.
  • the present invention proves that PACL10 has high antibacterial activity against P. acnes.
  • PACL10 The activity of PACL10 against a series of P. acnes strains of different clades was evaluated by minimum inhibitory concentration (MIC) assay and time kill assay, and the results consistently showed that PACL10 is highly effective.
  • MIC minimum inhibitory concentration
  • the phage lyase and its derivatives produced in the present invention are suitable for industrial scale production, can specifically and effectively kill Propionibacterium acnes, while keeping other symbiotic bacteria intact, and can provide a safe and effective solution for acne, suitable for Long-term use without a high risk of dysbiosis and acquired resistance.
  • the phage lyase and its derivatives produced in the present invention have the following advantages:
  • C. acnes is an organism that strongly secretes biofilms. Even for antibiotic-sensitive organisms, they become resistant to antibiotics when found in biofilm form. Lyases are highly effective against biofilms.
  • the present invention is more efficient - low MIC and high activity in other assays.
  • Non-limiting examples of natural linkers described in the present invention are as follows in Table 3:
  • PACL01 01linker1 SEQ ID NO: 343 SEQ ID NO: 368 PACL01 01linker2 SEQ ID NO: 344 SEQ ID NO: 369 PACL02 02linker SEQ ID NO: 345 SEQ ID NO: 370 PACL05 05linker1 SEQ ID NO: 346 SEQ ID NO: 371 PACL05 05linker2 SEQ ID NO: 347 SEQ ID NO: 372 PACL06 06linker1 SEQ ID NO: 348 SEQ ID NO: 373 PACL06 06linker2 SEQ ID NO: 349 SEQ ID NO: 374 PACL07 07linker SEQ ID NO: 350 SEQ ID NO: 375 PACL08 08linker SEQ ID NO: 351 SEQ ID NO: 376 PACL09 09linker SEQ ID NO: 352 SEQ ID NO: 377 PACL10 10linker SEQ ID NO: 353 SEQ ID NO: 378 PACL11 11linker SEQ ID NO:
  • Non-limiting examples of synthetic linkers described in the present invention are as follows in Table 4:
  • Example 3 of FIG. 1 The potent killing effect of selected lyases on P. acnes is shown in Example 3 of FIG. 1 .
  • the lyase was expressed in E. coli BL21 (DE3) on LB agar with IPTG and released by E. coli osmosis.
  • the clear zone or halo around E. coli indicates that the expressed lyase can inhibit growth or kill P. acnes embedded in the agar overlay.
  • Example 4 demonstrates that PACL10 can be soluble expressed and purified. All samples were run in a 5-12% triglycine gel for 50 min at 150 V and stained with Coomassie brilliant blue. PACL10 was expressed in E. coli BL21(DE3) using the pET expression system and purified by hydrophobic interaction. The elution fractions show purified PACL10 at 29.4 kDa.
  • Example 7 shows the kinetics of PACL10 reducing cfu/mL of P. acnes strain 34A.
  • the cfu/mL could drop below the detection limit within 3 hours and 2 hours, respectively.
  • Figure 5 shows a single-step purified lyase for MIC assay.
  • Lyase concentrations loaded onto 5-12% Tris-HCl SDS gels were: 0.62 mg/mL PACL10, 1.41 mg/mL PACL390, 2.12 mg/mL PACL391, 2.10 mg/mL PACL392, 1.49 mg/mL PACL403 and 1.60 mg/mL PACL404.
  • Figure 6 shows PACL392 purified by mixed mode and ion exchange chromatography.
  • Figure 7 shows the bactericidal activity of PACL392 relative to strain 10.
  • Figure 8 shows PACL403 purified by mixed mode chromatography.
  • Figure 9 shows the bactericidal activity - OD reduction of PACL403 relative to strain 10.
  • Figure 10 shows the bactericidal activity of PACL403 relative to strain 10 - CFU reduction.
  • Figure 11 shows the bactericidal activity of PACL403 relative to biofilm-associated strain 10.
  • P. acnes phage lyases that can be soluble expressed and purified.
  • the gene sequence for expressing phage lyase was synthesized by Sangon and ligated with the expression plasmid pET28. Each recombinant plasmid was then transformed into E. coli BL21(DE3) strain.
  • Chimeras were constructed as an N-terminal catalytic domain (CD) and a C-terminal binding domain (BD) with variable length linkers in between. Domains were identified by sequence analysis using the NCBI constant region database and the RaptorX web server for in silico protein structure prediction. Catalytic domains have full C-terminal, half C-terminal or no C-terminal linker. Likewise, binding domains have full N-terminal, half-N-terminal or no N-terminal linkages. Primers for amplifying the domain sequences were synthesized by GeneCreate. The domain sequences were amplified using Taq DNA polymerase PCR at an annealing temperature of 55°C. The PCR product was gel purified and ligated to the expression plasmid pET28. The recombinant plasmid was then transferred to E. coli BL21(DE3).
  • E. coli BL21(DE3) clones containing recombinant lyase plasmids were induced in liquid medium.
  • Cells were harvested by centrifugation and lysed by sonication in 50 mM sodium phosphate pH 7.4 buffer.
  • the lysate was centrifuged to separate soluble and insoluble components.
  • the soluble crude lysate was spotted on soft agar embedded with P. acnes. After incubation to grow P. acnes, the active lyase in the soluble crude lysate forms a clearance zone.
  • the experimental results are shown in Figure 2.
  • PACL10 was expressed and purified as follows. Briefly, E. coli BL21(DE3) containing recombinant PACL10 expression plasmid was cultured in autoinduction medium at 37°C and 300rpm until OD 600 reached 0.6-0.8, followed by 18°C, 300rpm for 16-18 Hour. Cells were harvested by centrifugation, resuspended in 50 mM sodium phosphate pH 7.4, and lysed by high pressure homogenization. The lysate was centrifuged again to collect soluble crude lysate. The soluble fraction was mixed with an equal volume of 5M NaCl and the mixture was loaded onto a hydrophobic column.
  • the chimeric lyase was expressed and purified in a manner similar to PACL10.
  • the Escherichia coli BL21(DE3) containing the recombinant chimeric lyase expression plasmid was cultured in autoinduction medium at 37°C and 300rpm until the OD 600 reached 0.6-0.8, and then continued to culture at 18°C and 300rpm for 16- 18 hours.
  • Cells were harvested by centrifugation, resuspended in 50 mM sodium phosphate pH 7.4, and lysed by high pressure homogenization. The lysate was centrifuged again to collect soluble crude lysate.
  • the soluble fraction was mixed with an equal volume of 5M NaCl and the mixture was loaded onto a hydrophobic column. After loading, the column was washed with 5 column volumes of 20 mM sodium phosphate (pH 7.4), 2.5 M NaCl. The recombinant chimeric lyase was then eluted with 10 mM sodium phosphate (pH 7.4).
  • the minimum inhibitory concentration (MIC) determination method is as follows.
  • the inoculum was first prepared by preparing a colony suspension in 0.9% saline to an OD600 of 0.05, equivalent to 107 colony forming units per milliliter ( cfu /mL).
  • the suspension was diluted 20-fold in agar-free reinforced Clostridium medium (RCM) to an inoculum of 5 *105 cfu/mL.
  • RCM Clostridium medium
  • the MIC is the lowest concentration of PACL10 or vancomycin at which the growth of P.
  • MBC Minimum bactericidal concentrations
  • Table 6-1 shows the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MIC) and minimum bactericidal concentrations ( MBC).
  • the time sterilization test method is as follows. Colonies of P. acnes strain 34A were resuspended in 0.9% saline to an OD600 of 0.05, equivalent to 107 cfu /mL. The suspension was diluted 10-fold to -106 cfu /mL in 50 mM sodium phosphate, pH 6.0. Strain 34A was spiked with two-fold serial dilutions of PACL10 at 0, 0.5x, 1x, 2x MIC, respectively. Ten-fold serial dilutions (0, 1, 2, 3 log) of the assay were plated on reinforced Clostridium agar (RCA) at each time point and incubated under anaerobic conditions at 37°C for 3 days. The experimental results are shown in Figure 4.
  • PACL 10, 390, 391, 392, 403 and 404 refer to the methods of Examples 1 to 4, and carry out a single-step purification as follows: E. coli BL21 (DE3) containing each recombinant expression plasmid was placed in autoinduction medium Incubate at 37°C, 300 rpm for 3 h, followed by 16-18 h at 18°C, 300 rpm. Cells were collected by centrifugation and lysed by high pressure homogenization. The clarified lysate from 1 L of E. coli medium expressing the protein of interest was loaded onto a 26 mm/200 mm column containing 70 mL of purified resin. The lyase was purified according to the conditions in Table 8-1, and the purity determination of the lyase after single-step purification is shown in Figure 5. These lyases were used to determine the minimum inhibitory concentration (MIC).
  • MIC minimum inhibitory concentration
  • the minimum inhibitory concentration (MIC) determination method is as follows. P. acnes on Fortified Clostridium Agar (Fortified Clostridium Medium RCM with 1.5% agar, RCM formulated according to revised recipe, per liter - 10g acid hydrolyzed casein, 10g beef extract, 3g yeast extract, 5gD - glucose, 5g sodium chloride, 3g sodium acetate, 0.5g L-cysteine hydrochloride) underlined. Plates were incubated anaerobically at 37°C for 72 hours.
  • Monoclones from RCA were first resuspended in 0.9% saline to an OD600 of 0.05 to prepare the inoculum for the MIC assay, corresponding to 107 colony forming units per milliliter ( cfu /mL).
  • the suspension was diluted 20-fold in RCM to an inoculum of 5 *105 cfu/mL.
  • 2-fold serial dilutions of each lyase were prepared in 0.9% saline and added no more than one tenth of the total culture volume.
  • the cultures were grown under anaerobic conditions at 37°C for 48-72 hours.
  • the MIC is the lowest concentration of lyase at which the growth of P. acnes cannot be observed with the naked eye.
  • the MIC results are shown in Table 8-2.
  • PACL392 was expressed in Escherichia coli BL21(DE3) containing the recombinant expression plasmid by placing in autoinduction medium at 37°C, 300rpm for 3h, followed by 18°C, 300rpm for 16-18h.
  • Cells were harvested by centrifugation and lysed by high pressure homogenization in 20 mM sodium phosphate, pH 7.4.
  • the lysate was centrifuged at 12,000 rpm at 4°C for 1 h.
  • the supernatant was mixed with an equal volume of 20 mM sodium phosphate, pH 7.4, 1 M ammonium sulfate, and centrifuged again at 12,000 rpm, 4°C for 1 h.
  • the supernatant was filtered through a 0.22 ⁇ m filter and loaded onto a mixed mode column (70 mL of purified resin on a 26 mm/200 mm column) equilibrated with 20 mM sodium phosphate, pH 7.4, 0.5 M ammonium sulfate. After loading, the column was washed with a series of buffers: 20 mM sodium phosphate, pH 7.4, 0.5 M ammonium sulfate; 20 mM, pH 7.4 sodium phosphate, 2.5 M NaCl; 20 mM, pH 7.4 sodium phosphate and 50 mM, pH 10.02 Piperazine, 50 mM NaCl.
  • PACL392 was eluted with 50 mM piperazine, pH 10.02 and 750 mM NaCl. The eluate was dialyzed against 50 mM sodium phosphate, pH 7.4 to neutralize pH and remove cations. The dialyzed sample was filtered and loaded onto an ion exchange chromatography column (70 mL of a 26 mm/200 mm column of purified resin). The final result of PACL392 purification is shown in Figure 6.
  • the bactericidal activity of PACL392 was enhanced by 50 mM, pH 7.0 in the presence of a range of additives such as NaCl, CaCl 2 , MgCl 2 , EDTA, DTT, Tween-20, Tween-80 and hyaluronic acid (concentrations shown in Figure 7). Determination of CFU reduction in HEPES.
  • PACL403 was expressed in E. coli BL21(DE3) containing the recombinant expression plasmid by placing in autoinduction medium at 37°C, 300rpm for 3h, followed by 18°C, 300rpm for 16-18h. Cells were harvested by centrifugation and lysed by high pressure homogenization in 20 mM sodium phosphate, pH 7.4. The lysate solution was adjusted to 20 mM sodium phosphate pH 7.4, 1 M NaCl and centrifuged at 12,000 rpm, 4°C for 1 h.
  • the supernatant was filtered through a 0.22 ⁇ m filter and loaded onto a mixed-mode column (26 mm/200 mm column of 70 mL of purified resin) equilibrated with 20 mM sodium phosphate, pH 7.4, 1 M NaCl. After loading, the column was washed with a series of buffers: 20 mM sodium phosphate, pH 7.4, 1 M NaCl; 20 mM sodium phosphate, pH 7.4, 2.5 M NaCl; 20 mM sodium phosphate, pH 7.4; 20 mM sodium phosphate, pH 7.4 , 0.1% Triton X-114 and again 20 mM sodium phosphate, pH 7.4.
  • PACL403 was eluted with 50 mM piperazine, pH 9.5, 1 M NaCl. High pH was neutralized by adding one-fifth volume of 500 mM sodium phosphate, pH 7.4. The eluate was then dialyzed against 50 mM sodium phosphate, pH 7.4, to remove cations. The dialysis sample was filtered. The final result of PACL403 purification is shown in Figure 8.
  • the bactericidal activity of PACL403 was determined by the decrease in OD in 50 mM sodium phosphate, pH 7.0, in the presence of Tween-20 and Tween-80 at the concentrations shown in Figure 9 .
  • the bactericidal activity of PACL403 was determined by CFU reduction in 50 mM HEPES pH 7.0 in the presence of Tween-20 and Tween-80 at the concentrations shown in Figure 10.
  • PACL403 also killed biofilm-associated P. acnes (Figure 11). Strain 10 with an OD600 of 1.0 was added to 96-well polystyrene plates to inoculate biofilms in an anaerobic gas mixture overnight. The supernatant was removed and the wells were gently rinsed with 0.9% NaCl. Biofilm formation medium (RCM + 5% glucose) was added to allow the biofilm to grow overnight in the anaerobic gas mixture. The medium was removed and the wells were gently rinsed with 0.9% NaCl. Biofilms were then treated with buffer or PACL403 overnight at 25°C under anaerobic conditions. The buffer or PACL403 was removed and the wells were rinsed with 0.9% NaCl. Resuspend in 0.9% NaCl, plate on RCM agar, and count biofilm-associated CFU.

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Abstract

提供了噬菌体裂解酶或噬菌体裂解酶嵌合物在制备预防、治疗或改善痤疮或由痤疮丙酸杆菌引起的感染或与痤疮丙酸杆菌有关的病症的药物、化妆品或治疗设备中的应用。还提供了噬菌体裂解酶嵌合物。

Description

噬菌体裂解酶、其嵌合物及应用
本申请主张如下优先权:CN202110187030.8,申请日2021.02.08。
技术领域
本发明涉及医药生物领域,具体提供一种具有抗痤疮丙酸杆菌活性的噬菌体裂解酶、其嵌合物及应用。
背景技术
痤疮是一种皮肤炎症性疾病,尤其是在面部、颈部、肩膀、胸部、背部和上臂。毛囊皮脂腺单元由于被过度分泌的油脂堵塞及致病性痤疮丙酸杆菌(Cutibacterium acnes,简称C.acnes,下同)的增殖导致的死皮细胞积累而产生炎症。根据美国国家健康研究所的数据,11到30岁的人群中有80%都有不同程度的痤疮。痤疮通常开始于***,可能持续到四五十岁。不同因素可能会引起痤疮的爆发,例如压力、激素水平的变化、过敏原等。2010年的全球疾病负担研究估计,痤疮影响了全球9.4%的人口,并将其列为世界第八大最流行的疾病。据美国皮肤学会估计,在2013年痤疮患者治疗和生产力损失相关的费用超过了12亿美元。虽然它本身很少危及生命,但它会造成皮肤上持久的疤痕和患者相当大的情绪困扰。
目前治疗轻度痤疮的选择是使用含有过氧化苯甲酰、间苯二酚、水杨酸和硫等化学物质的非处方外用药物,以及处方外用药物,如抗生素、过氧化苯甲酰、壬二酸、氨苯砜、皮质类固醇和称为类视黄醇(retinoids)的维生素A衍生物。中至重度病情可采用外用处方药或口服药物治疗。干预的目的是为了去除死皮细胞和多余的油脂,以使毛囊皮脂腺单元畅通并且减少细菌负荷。由于痤疮是一种慢性疾病,需要长时间的治疗并且有时需要防止复发。然而,现有的治疗方案没有一种适合长期治疗。当延长这些药物的使用时间,即使是最温和的化学物质也会引起皮肤的 过敏反应和刺激,导致皮肤干燥、起皮、龟裂等。由于耐药性痤疮丙酸杆菌在世界上越来越普遍,抗生素治疗选择也越来越局限。此外,长期使用抗生素已被证明会通过对耐药细菌种类施加选择压力而干扰微生物群的稳定状态。这转而会引发无数的并发症,例如菌群失调和免疫异常。有文献表明,在抗生素治疗过程中痤疮患者的皮肤葡萄球菌菌群会由大多数抗生素敏感转化为大多数耐药。不仅是患者本人,患者的密切接触者也会受到多药耐药性细菌的影响。值得注意的是,长期接受抗生素治疗的痤疮患者患上呼吸道感染的可能性增加两倍。因此,现在迫切需要一种更安全、更有效、更适合长期使用的治疗方案。一种这样的方案是病原体特异性抗菌药,其能够特异性杀死痤疮丙酸杆菌而不会影响其他共生菌,无需考虑抗生素耐药性。
此外,痤疮丙酸杆菌还与一系列侵袭性感染有关,包括术后肩部感染、椎间盘感染等。在此类感染中,痤疮丙酸杆菌表现出慢性进展性生物膜类感染。在生物膜中发现的细菌对抗生素具有固有耐药性。一种具有强烈抗生物膜特性的痤疮丙酸杆菌杀菌剂是治疗此类感染的理想药物。
一种此类病原体特异性抗菌药的备选药物是噬菌体细胞内溶素。噬菌体用这类酶破坏细胞壁稳定性进行裂解,从而从细胞内部释放噬菌体子代。它们被归类为肽聚糖水解酶,可以断裂细菌肽聚糖中的各种键。除了少数例外,每种裂解酶的目标细菌都属于单一的属或种。已经证明,重组的抗革兰氏阳性菌裂解酶是能够引起低渗裂解的有效杀菌剂(如抗若干种链球菌的PlyC,抗炭疽芽孢杆菌的PlyG等)。考虑到裂解酶为了持续成功感染细胞在数十亿年中受到选择性进化压力,目标肽聚糖键高度保守,不太可能被细菌轻易改变。这个特性导致目标细菌对裂解酶产生耐药性的可能很小,因此适合长期治疗使用。迄今为止还未检测出对各自的噬菌体裂解酶的细菌耐药性。通常,抗革兰氏阳性菌的裂解酶是由一个或两个N-末端催化结构域(CD)和一个C-末端细胞壁结合结构域(BD)组成,结构域间有不同长度的接头(linkers)。裂解酶作为模块蛋白质,可以通过配对来自不同裂解酶的催化结构域和结合结构域来产生嵌 合裂解酶。接头的长度和序列也是可变的。然而,开发作为抗菌剂的裂解酶的主要瓶颈是不能可溶性表达高活性裂解酶。事实上,到目前为止没有报道过高水平可溶性表达并能有效杀死痤疮丙酸杆菌的裂解酶。
专利CN102482655A公开了一种抗微生物剂,其由具有降解***细胞壁的活性的细胞内溶素和在N端或C端或两端融合于该细胞内溶素的两亲肽段构成,能够用于治疗或预防***感染,作为诊断手段或作为美容用物质的用途。但是,该专利中虽然描述了PA6具有抗P.acnes DSMZ 1897株和DSMZ 16379株活性,但并未提供初始cfu/mL;此外,PA6不能解决提高可溶性表达水平的问题。
综上所述,对治疗痤疮丙酸杆菌的新治疗方式显然还有很大的需求,并且迄今为止没有报道过适合工业规模生产的高痤疮丙酸杆菌杀伤活性的噬菌体裂解酶。噬菌体裂解酶能够特异性、有效地杀死痤疮丙酸杆菌,同时保持其他共生细菌完整无损,可以提供一种安全有效的痤疮解决方案,适合长期使用,而不会有很高的生态失调和获得性耐药风险。
发明内容
针对以上技术现状,本发明提供了具有抗痤疮丙酸杆菌活性的噬菌体裂解酶、其嵌合物及应用。具体发明如下:
本发明提供了一种噬菌体裂解酶或其嵌合物在制备预防、治疗或改善痤疮或由痤疮丙酸杆菌引起的感染或与痤疮丙酸杆菌有关的病症的药物、化妆品或医疗设备中的应用,所述噬菌体裂解酶包括来源于类诺卡氏菌科(Nocardioidaceae)和丙酸杆菌科(Propionibacteriaceae)细菌的噬菌体裂解酶。
本发明的应用中,作为实施方案之一,所述类诺卡氏菌科(Nocardioidaceae)细菌包括微白霜菌属(Micropruina)、产丙酸单孢菌属(Propionicimonas)、产丙酸细胞菌属(Propionicicella)、弗莱德门氏菌属(Friedmanniella)细菌;所述丙酸杆菌科(Propionibacteriaceae)细菌包括产丙酸菌属(Propioniferax)、海马球菌属(Mariniluteicoccus)、颗粒 球菌属(Granulicoccus)、瑙曼氏菌属(Naumannella)、棒状丙酸菌属(Propioniciclava)、光球菌属(Auraticoccus)、小月菌属(Microlunatus)、河口微菌属(Aestuariimicrobium)、黄球菌属(Luteococcus)、四合球菌属(Tessaracoccus)、布克劳氏菌属(Brooklawnia)、产丙酸微球菌属(Propionimicrobium)、丙酸杆菌属(Propionibacterium)、痤疮丙酸杆菌属(Cutibacterium)、酸性丙酸杆菌属(Acidipropionibacterium)、或假丙酸杆菌属(Pseudopropionibacterium)细菌,优选丙酸杆菌属(Propionibacterium)、痤疮丙酸杆菌属(Cutibacterium)、酸性丙酸杆菌属(Acidipropionibacterium)、或假丙酸杆菌属(Pseudopropionibacterium)细菌。
本发明的应用中,作为实施方案之一,所述噬菌体裂解酶包括来源于痤疮丙酸杆菌(Cutibacterium acnes)、肱丙酸杆菌(Propionibacterium humerusii)、贪婪丙酸杆菌(Cutibacterium avidum)、颗粒丙酸杆菌(Cutibacterium granulosum)、特氏丙酸杆菌(Acidipropionibacterium thoenii)、詹氏丙酸杆菌(Acidipropionibacterium jensenii)、产丙酸丙酸杆菌(Acidipropionibacterium acidipropionici)、潮汐滩菌(Aestuariimicrobium kwangyangense)、细粒球菌(Granulicoccus phenolivorans)、积磷小月菌(Microlunatus phosphovorus)、丙酸丙酸杆菌(Pseudopropionibacterium propionicum)、四合球菌(Tessaracoccus sp.)、极底产丙酸细胞菌(Propionicicella superfundia)、费氏丙酸杆菌(Propionibacterium freudenreichii)、费氏丙酸杆菌费氏亚种(Propionibacterium freudenreichii subsp.freudenreichii)、费氏丙酸杆菌舍氏亚种(Propionibacterium freudenreichii subsp.shermanii)、产酸丙酸杆菌(Propionibacterium acidifaciens)、嗜淋巴丙酸杆菌(Propionibacterium lymphophilum)、丙酸杆菌属细菌(Propionibacteriaceae bacterium)、丙酸杆菌分类群192(Propionibacterium sp.oral taxon 192)、无毒产丙酸菌(Propioniferax innocua)、盐藻瑙曼氏菌(Naumannella halotolerans)、慢生棒状丙酸菌(Propioniciclava tarda)、糖原微白霜菌(Micropruina glycogenica)、淡水产丙酸单孢菌(Propionicimonas paludicola)、纪念性光球菌(Auraticoccus  monumenti)、日本黄球菌(Luteococcus japonicus)、油田四合球菌(Tessaracoccus oleiagri)、本迪戈四合球菌(Tessaracoccus bendigoensis)、拉帕迪卡四合球菌(Tessaracoccus lapidicaptus)、微嗜气丙酸杆菌(Acidipropionibacterium microaerophilum)、橄榄体丙酸杆菌(Acidipropionibacterium olivae)、丹诺氏丙酸杆菌(Acidipropionibacterium damnosum)细菌的噬菌体裂解酶。
本发明的应用中,作为实施方案之一,所述噬菌体裂解酶包括来源于詹氏丙酸杆菌(Acidipropionibacterium jensenii)、特氏丙酸杆菌(Acidipropionibacterium thoenii)、产丙酸丙酸杆菌(Acidipropionibacterium acidipropionici)、微嗜气丙酸杆菌(Acidipropionibacterium microaerophilum)、橄榄体丙酸杆菌(Acidipropionibacterium olivae)、丹诺氏丙酸杆菌(Acidipropionibacterium damnosum)、痤疮丙酸杆菌(Cutibacterium acnes)、贪婪丙酸杆菌(Cutibacterium avidum)、颗粒丙酸杆菌(Cutibacterium granulosum)、丙酸丙酸杆菌(Pseudopropionibacterium propionicum)细菌的噬菌体裂解酶。
本发明的应用中,作为实施方案之一,所述噬菌体裂解酶具有SEQ ID NO:1~SEQ ID NO:28中任一所示的氨基酸序列;
本发明的应用中,作为实施方案之一,所述噬菌体裂解酶具有SEQ ID NO:29~SEQ ID NO:56中任一所示的核苷酸序列;
本发明的应用中,作为实施方案之一,所述噬菌体裂解酶优选具有SEQ ID NO:10所示的氨基酸序列的PACL10;
本发明的应用中,作为实施方案之一,所述噬菌体裂解酶优选具有SEQ ID NO:38所示的核苷酸序列的PACL10。
本发明的应用中,作为实施方案之一,所述嵌合物包含来源于噬菌体裂解酶的催化结构域,或包含来源于噬菌体裂解酶的催化结构域和结合结构域的组合。
本发明的应用中,作为实施方案之一,所述催化结构域具有全C-末端接头、半C-末端接头、无C-末端接头、或所述接头的任何部分;所述 结合结构域具有全N-末端接头、半N-末端接头、无N-末端接头、或所述接头的任何部分。
本发明的应用中,作为实施方案之一,可以用合成接头还包括合成接头。来源于噬菌体裂解酶的可能的接头的非限制性实例见下表3。合成接头的非限制性实例见下表4。
本发明的应用中,作为实施方案之一,所述嵌合物由来源于不同噬菌体裂解酶的催化结构域和结合结构域配对而成。
本发明的应用中,作为实施方案之一,所述嵌合物包括一个或两个催化结构域而没有结合结构域;
作为实施方案之一,所述嵌合物包括一个或多个催化结构域和一个或多个结合结构域
作为实施方案之一,所述嵌合物可以为一个或多个催化结构域与单个结合结构域连接;
作为实施方案之一,所述嵌合物可以为单个催化结构域与单个结合结构域连接;
作为实施方案之一,所述嵌合物可以为裂解酶N-末端中的单个催化结构域与裂解酶C-末端中的单个结合结构域连接。
此外,可以在不存在结合结构域的情况下使用一个或多个催化结构域。进一步地,对于诸如检测的专门应用,可在不存在催化结构域的情况下使用一个或多个结合结构域,并将其融合至不同分子以促进检测。此类分子包括但不限于生物素、flag标签、myc标签、抗生物素蛋白、链霉亲和素、卵清蛋白、萤火虫荧光素酶、生物素、荧光分子如FITC,TRITC,Alexafluor等。
本发明的应用中,作为实施方案之一,所述嵌合物进一步包含催化结构域和结合结构域之间的接头,所述接头包括:
1)来源于所述亲本裂解酶分子的接头区或其任何部分,所述催化结构域来源于该亲本裂解酶分子;
本发明的应用中,作为实施方案之一,所述来源于所述亲本裂解酶分子的接头区具有SEQ ID NO:343~SEQ ID NO:367任一所示的氨基酸 序列。
2)来源于所述亲本裂解酶分子的接头区或其任何部分,所述结合结构域来源于该亲本裂解酶分子;
本发明的应用中,作为实施方案之一,所述来源于所述亲本裂解酶分子的接头区具有SEQ ID NO:368~SEQ ID NO:392任一所示的核苷酸序列;或
3)具有SEQ ID NO:393~SEQ ID NO:406任一所示的氨基酸序列,或为(GGGS)n、(GGGGS)n、(GGGGGS)n、(Gly)3-8、(EAAAK)n、(Ala-Pro)n、A(EAAAK)nALEA(EAAAK)nA氨基酸序列(其中1≤n≤15,n为整数)的合成接头结构域,作为示例性说明,n可以为1、2、3、4、5、6、7、8、9、10、11、12、13、14或15。
本发明的应用中,作为实施方案之一,所述嵌合物包括所述催化结构域具有SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:70、SEQ ID NO:71、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:77、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:114、SEQ ID NO:117、SEQ ID NO:118、SEQ ID NO:121、SEQ ID NO:122、SEQ ID NO:125、SEQ ID NO:127、SEQ ID NO:128、SEQ ID NO:131、SEQ ID NO:132、SEQ ID NO:133、SEQ ID NO:135或SEQ ID NO:136中所示的氨基酸序列;
所述结合结构域具有SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:68、SEQ ID NO:72、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:94、SEQ ID  NO:96、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:109、SEQ ID NO:112、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:119、SEQ ID NO:120、SEQ ID NO:123、SEQ ID NO:124、SEQ ID NO:126、SEQ ID NO:129、SEQ ID NO:130、SEQ ID NO:134、SEQ ID NO:137、SEQ ID NO:138、或SEQ ID NO:139所示的氨基酸序列。
本发明的应用中,作为实施方案之一,所述催化结构域具有SEQ ID NO:140、SEQ ID NO:141、SEQ ID NO:142、SEQ ID NO:144、SEQ ID NO:146、SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149、SEQ ID NO:150、SEQ ID NO:152、SEQ ID NO:153、SEQ ID NO:154、SEQ ID NO:156、SEQ ID NO:157、SEQ ID NO:160、SEQ ID NO:163、SEQ ID NO:164、SEQ ID NO:168、SEQ ID NO:169、SEQ ID NO:170、SEQ ID NO:174、SEQ ID NO:176、SEQ ID NO:178、SEQ ID NO:180、SEQ ID NO:181、SEQ ID NO:182、SEQ ID NO:183、SEQ ID NO:185、SEQ ID NO:187、SEQ ID NO:188、SEQ ID NO:189、SEQ ID NO:190、SEQ ID NO:191、SEQ ID NO:193、SEQ ID NO:194、SEQ ID NO:196、SEQ ID NO:197、SEQ ID NO:200、SEQ ID NO:201、SEQ ID NO:204、SEQ ID NO:205、SEQ ID NO:208、SEQ ID NO:210、SEQ ID NO:211、SEQ ID NO:214、SEQ ID NO:215、SEQ ID NO:216、SEQ ID NO:218、SEQ ID NO:219、所示的核苷酸序列;
所述结合结构域具有SEQ ID NO:143、SEQ ID NO:145、SEQ ID NO:151、SEQ ID NO:155、SEQ ID NO:158、SEQ ID NO:159、SEQ ID NO:161、SEQ ID NO:162、SEQ ID NO:165、SEQ ID NO:166、SEQ ID NO:167、SEQ ID NO:171、SEQ ID NO:172、SEQ ID NO:173、SEQ ID NO:175、SEQ ID NO:177、SEQ ID NO:179、SEQ ID NO:184、SEQ ID NO:186、SEQ ID NO:192、SEQ ID NO:195、SEQ ID NO:198、SEQ ID NO:199、SEQ ID NO:202、SEQ ID NO:203、SEQ ID NO:206、SEQ ID NO:207、SEQ ID NO:209、SEQ ID NO:212、SEQ ID NO:213、SEQ ID NO:217、SEQ ID NO:220、SEQ ID NO:221、或SEQ ID NO:222所示的核苷酸序列。
本发明的应用中,作为实施方案之一,所述嵌合物具有SEQ ID NO:223~SEQ ID NO:282中任一所示的氨基酸序列;优选SEQ ID NO:258、SEQ ID NO:259、SEQ ID NO:260、SEQ ID NO:271或SEQ ID NO:272所示的氨基酸序列,更优选SEQ ID NO:260或SEQ ID NO:271所示的氨基酸序列;
本发明的应用中,作为实施方案之一,所述嵌合物具有SEQ ID NO:283~SEQ ID NO:342中任一所示的核苷酸序列;优选SEQ ID NO:316、SEQ ID NO:317、SEQ ID NO:318、SEQ ID NO:329或SEQ ID NO:330所示的核苷酸序列;更优选SEQ ID NO:318或SEQ ID NO:329所示的核苷酸序列。
本发明的应用中,作为实施方案之一,所述由痤疮丙酸杆菌引起的感染包括侵入性感染、术后感染和/或器械相关的感染。
本发明的应用中,作为实施方案之一,所述器械相关的感染包括关节假体、分流管和人工心脏瓣膜相关的感染。
本发明的应用中,作为实施方案之一,所述感染包括骨骼和/或关节的感染,尤其是术后肩部感染,以及口腔、眼、椎间盘和脑部的感染。
本发明的应用中,作为实施方案之一,所述与痤疮丙酸杆菌有关的病症包括导致癌症的***炎、SAPHO(滑膜炎、痤疮、脓疱病、肥大、骨炎)综合征、结节病、或坐骨神经痛。
本发明的应用中,作为实施方案之一,所述医疗设备包括用于将裂解酶或其嵌合物释放到受影响区域的任何设备,优选施加至皮肤表面的夹具或贴剂或喷雾剂,使用微针增强裂解酶或其嵌合物的皮肤渗透性的设备,美容专业人员使用的将裂解酶或其嵌合物专门施加至受痤疮影响的毛囊上的细针,或其他类似的设备。
本发明的应用中,作为实施方案之一,所述医疗设备包括将将裂解酶或其嵌合物固定在易于感染痤疮丙酸杆菌的位置中的假体装置上,优选用于特别容易感染痤疮丙酸杆菌的肩部手术中的假体植入物。
本发明还提供了前述应用中所述的噬菌体裂解酶嵌合物。作为实施方案之一,所述嵌合物具有SEQ ID NO:223~SEQ ID NO:282中任一所示 的氨基酸序列;优选SEQ ID NO:258、SEQ ID NO:259、SEQ ID NO:260、SEQ ID NO:271或SEQ ID NO:272所示的氨基酸序列;更优选SEQ ID NO:260或SEQ ID NO:271所示的氨基酸序列;
本发明的应用中,作为实施方案之一,所述嵌合物具有SEQ ID NO:283~SEQ ID NO:342中任一所示的核苷酸序列;优选SEQ ID NO:316、SEQ ID NO:317、SEQ ID NO:318、SEQ ID NO:329或SEQ ID NO:330所示的核苷酸序列;更优选SEQ ID NO:318或SEQ ID NO:329所示的核苷酸序列。
本发明还提供了一种前述嵌合物的制备方法,包括:
(1)合成结构域序列及扩增结构域序列的引物;
(2)采用Taq DNA聚合酶PCR扩增结构域序列;
(3)PCR产物凝胶纯化,与表达质粒pET28连接;
(4)重组质粒转移到大肠杆菌BL21(DE3);
(5)培养含有重组质粒的大肠杆菌BL21(DE3),诱导表达,离心收集细胞,裂解,纯化得到所述嵌合物。
本发明的制备方法中,作为实施方案之一,所述方法进一步包括:
将含有重组嵌合裂解酶表达质粒的大肠杆菌BL21(DE3)置于自诱导培养基中在37℃、300rpm下培养至OD 600达到0.6-0.8,随后在18℃、300rpm下,持续培养16-18小时。离心收集细胞,重悬于50mM pH 7.4的磷酸钠中,高压均质裂解。裂解液再次离心以收集可溶性粗裂解液。将可溶性组分与等体积的5M NaCl混合,混合物上样到疏水柱中。上样后,用5倍柱体积的20mM磷酸钠(pH 7.4)、2.5M NaCl洗涤柱子。然后用10mM磷酸钠(pH 7.4)洗脱重组嵌合裂解酶。可选择地,本领域技术人员可以对表达和纯化进行优化,可以在不同的温度、通气和诱导时间下使用不同浓度的IPTG在LB中表达裂解酶。可以将细胞重悬于不同的缓冲液中以提高溶解度,并通过机械或化学手段进行裂解。可以采用不同的蛋白质色谱方法来纯化裂解酶。可以优化这些变量以获得更好的裂解酶蛋白产量。
本发明还提供了含有前述嵌合物的制剂,所述制剂进一步包含抗生 素、其他裂解酶、或非活性赋形剂。
本发明还提供了编码前述嵌合物的氨基酸序列,与前述序列相似度80%及以上、85%及以上、90%及以上、95%及以上或99%及以上的氨基酸序列,或具有相同官能团的替代氨基酸序列。作为示例性的说明,本发明还提供了与前述序列相似度80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%的氨基酸序列。
作为实施方案之一,所述替代氨基酸序列为使用同一组氨基酸中的氨基酸的保守替换,所述氨基酸组包括:
脂肪族的:甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸;
含羟基或硫/硒的:丝氨酸、半胱氨酸、苏氨酸、甲硫氨酸;
环状的:脯氨酸;
芳族的:苯基丙氨酸、酪氨酸、色氨酸;
碱性的:组氨酸、赖氨酸、精氨酸;
酸性的以及它们的酰胺:天冬氨酸、谷氨酸、天冬酰胺、谷氨酰胺。
本发明还提供了编码前述嵌合物的核苷酸序列,或其同义密码子序列。
本发明还提供了前述嵌合物用作细菌裂解的试剂的应用,所述细菌裂解用于使用基于PCR的试剂盒进行DNA提取和分型。
本发明还提供了前述嵌合物用作非生物表面上的消毒剂或灭菌剂的应用,所述消毒剂或灭菌剂通过去除浮游形式或生物膜中的痤疮丙酸杆菌以防止感染,优选在手术过程中对手术设备或假体植入装置进行消毒。
本发明还提供了前述嵌合物、前述嵌合物中的单个结合结构域,或前述嵌合物中的相似或不同的结合结构域串联的组合在制备用于痤疮丙酸杆菌的诊断工具中的应用,所述裂解酶、嵌合物或结合结构域与检测标志物联合使用。
作为实施方案之一,所述裂解酶、嵌合物或结合结构域与信号分子通过基因融合或化学偶联形成融合物。
作为实施方案之一,所述融合物用于通过荧光或其他手段直接检测 显微镜载玻片上的痤疮丙酸杆菌,用于通过免疫组织化学标记痤疮丙酸杆菌,用作ELISA测定中的检测试剂,用作Western blot上的检测试剂,用于与在MACS或其他pull-down测定中的磁珠连接,或用于抗体作为检测试剂的测定中的检测试剂。作为实施方案之一,所述信号分子包括蛋白质或化学荧光染料、蛋白质标签、酶、抗生物素蛋白、链霉亲和素、卵清蛋白、生物素、对点击化学标记敏感的标签、内含肽、或能够引起产生信号的二级蛋白质或分子募集的其他分子,
所述荧光染料包括GFP、RFP、mCherry、FITC、TRITC、Alexafluor488、Cy3或Cy5;
所述蛋白质标签包括Flag-标签、myc-标签、halo-标签、his-标签、或能够与抗体或其他高亲和力分子结合产生信号的任何其他标签;
所述酶包括萤火虫荧光素酶、β-内酰胺酶、碱性磷酸酶、辣根过氧化物酶、或引起诸如光、颜色变化、底物沉积或其他能够在试验中检测到的反应的任何其他的酶。
本发明还提供了前述嵌合物中的催化结构域在制备治疗痤疮丙酸杆菌感染的药物中的应用,所述催化结构域与寻靶模块结合。
本发明提供了用于预防和治疗痤疮丙酸杆菌感染和皮肤定植以及与这种感染或定植相关的痤疮病变的组合物和方法。在广泛的方面,本发明提供了裂解酶的用途和应用,所述裂解酶具有对皮肤相关的生物体的广泛杀伤活性,所述生物体涉及痤疮病变的发生或恶化以及痤疮病变的继发感染,包括但不限于丙酸杆菌属(Propionibacterium)、痤疮丙酸杆菌属(Cutibacterium)、酸性丙酸杆菌属(Acidipropionibacterium)、假丙酸杆菌属(Pseudopropionibacterium)的细菌。特别地,本发明描述了用于皮肤中已建立的细菌群落的去定植、分散和去除的方法,特别着重于痤疮丙酸杆菌(Cutibacterium acnes,以前称作Propionibacterium acnes)。此外,本发明还提供了生产嵌合裂解酶的方法,所述嵌合裂解酶包含来自不同裂解酶的催化结构域和结合结构域,从而产生各种有利的特性,包括但不限于改善的活性、宿主范围、表达、溶解性、稳定性或更适合于商品化。
根据本发明,在本发明的方法和应用中使用了来源于丙酸杆菌属(Propionibacterium)、痤疮丙酸杆菌属(Cutibacterium)、酸性丙酸杆菌属(Acidipropionibacterium)、假丙酸杆菌属(Pseudopropionibacterium)细菌的噬菌体裂解酶。表2提供了本发明中使用的裂解酶和多肽。
在本发明的一个方面,SEQ ID NO:1~SEQ ID NO:28中所示的裂解酶由SEQ ID NO:29~SEQ ID NO:56中所示的核苷酸序列编码。
本发明还提供了一种在不同噬菌体裂解酶的结合结构域和催化结构域之间产生嵌合体的方法。SEQ ID NO:57~SEQ ID NO:139示出了提供编码本发明所述来源于噬菌体裂解酶的催化结构域和结合结构域的多肽序列的实例的非限制性序列表。
在本发明的一个方面,SEQ ID NO:57~SEQ ID NO:139中所示的催化结构域和结合结构域由SEQ ID NO:140~SEQ ID NO:222中示出的核苷酸序列编码。
本发明还提供了如本发明所述的通过配对不同裂解酶的催化结构域和结合结构域而产生的嵌合裂解酶的非限制性实例。SEQ ID NO:223~SEQ ID NO:282提供了由本发明所述方法产生的抗痤疮丙酸杆菌活性的嵌合裂解酶的非限制性序列表。
在本发明的一个方面,使用SEQ ID NO:283~SEQ ID NO:342中所述的核苷酸序列产生本发明所述的嵌合裂解酶。
在本发明的一个方面,所述多肽序列由***到表达载体pET28中的核苷酸序列编码。
在本发明的一个方面,编码本发明所述多肽的表达载体在大肠杆菌(E.coli)BL21(DE3)菌株中表达。
此外,本发明提供了一种筛选和评价噬菌体裂解酶抗痤疮丙酸杆菌活性的方法。
此外,本发明证明了确定的噬菌体裂解酶抗痤疮丙酸杆菌的活性。
在本发明的一个方面,用于治疗丙酸杆菌的裂解酶为PACL10。本发明提供了一种表达和纯化PACL10的方法。此外,本发明证明PACL10对痤疮丙酸杆菌具有较高的抗菌活性。
通过最小抑菌浓度(MIC)试验和时间杀菌试验评价了PACL10对一系列不同进化枝的痤疮丙酸杆菌株的活性,结果一致表明PACL10具有很高的有效性。
本发明中产生的噬菌体裂解酶及其衍生物适合工业规模生产,能够特异性、有效地杀死痤疮丙酸杆菌,同时保持其他共生细菌完整无损,可以提供一种安全有效的痤疮解决方案,适合长期使用,而不会有很高的生态失调和获得性耐药风险。本发明中产生的噬菌体裂解酶及其衍生物具有如下优点:
(1)能够杀死痤疮丙酸杆菌(C.acnes)而不损伤天然菌群。
(2)与需要很长时间才能起效的抗生素相比,活性迅速(几分钟之内)。
(3)没有耐药性。细菌会迅速对抗生素产生耐药性,因此裂解酶是一种解决方案。C.acnes是一种强烈分泌生物膜的生物。即使对于对抗生素敏感的生物,当以生物膜形式发现它们时,它们会对抗生素产生耐药性。裂解酶对生物膜高度有效。
(4)与其他裂解酶相比,本发明更有效——在其他测定中MIC低,活性高。
(5)能够可溶性表达,其产量适于大规模和工业生产。
(6)对所有进化枝的P.acnes.都有效。
定义
由于Scholz等对丙酸杆菌属(Propionibacterium)的重新分类,新旧名称对照的非限制性实例如下表1所示:
表1
Figure PCTCN2022074221-appb-000001
Figure PCTCN2022074221-appb-000002
本领域人员应该理解,细菌的重新分类、新旧名称、中文名称等不应影响对该细菌的认定,本发明无论采用新名称或旧名称,其代表为同一菌种。
本发明涉及序列的非限制性实例如下表2:
表2
Figure PCTCN2022074221-appb-000003
Figure PCTCN2022074221-appb-000004
Figure PCTCN2022074221-appb-000005
Figure PCTCN2022074221-appb-000006
Figure PCTCN2022074221-appb-000007
Figure PCTCN2022074221-appb-000008
本发明中所述天然接头的非限制性示例如下表3:
表3
原始裂解酶 接头名称 氨基酸序列 核酸序列
PACL01 01linker1 SEQ ID NO:343 SEQ ID NO:368
PACL01 01linker2 SEQ ID NO:344 SEQ ID NO:369
PACL02 02linker SEQ ID NO:345 SEQ ID NO:370
PACL05 05linker1 SEQ ID NO:346 SEQ ID NO:371
PACL05 05linker2 SEQ ID NO:347 SEQ ID NO:372
PACL06 06linker1 SEQ ID NO:348 SEQ ID NO:373
PACL06 06linker2 SEQ ID NO:349 SEQ ID NO:374
PACL07 07linker SEQ ID NO:350 SEQ ID NO:375
PACL08 08linker SEQ ID NO:351 SEQ ID NO:376
PACL09 09linker SEQ ID NO:352 SEQ ID NO:377
PACL10 10linker SEQ ID NO:353 SEQ ID NO:378
PACL11 11linker SEQ ID NO:354 SEQ ID NO:379
PACL12 12linker SEQ ID NO:355 SEQ ID NO:380
PACL13 13linker SEQ ID NO:356 SEQ ID NO:381
PACL14 14linker SEQ ID NO:357 SEQ ID NO:382
PACL15 15linker SEQ ID NO:358 SEQ ID NO:383
PACL16 16linker SEQ ID NO:359 SEQ ID NO:384
PACL19 19linker SEQ ID NO:360 SEQ ID NO:385
PACL20 20linker SEQ ID NO:361 SEQ ID NO:386
PACL21 21linker SEQ ID NO:362 SEQ ID NO:387
PACL22 22linker SEQ ID NO:363 SEQ ID NO:388
PACL23 23linker SEQ ID NO:364 SEQ ID NO:389
PACL25 25linker SEQ ID NO:365 SEQ ID NO:390
PACL26 26linker SEQ ID NO:366 SEQ ID NO:391
PACL27 27linker SEQ ID NO:367 SEQ ID NO:392
本发明中所述合成接头的非限制性示例如下表4:
表4
序号 接头名称 氨基酸序列
1 (GGGS)n,1≤n≤8,n为整数
2 (GGGGS)n,1≤n≤8,n为整数
3 (GGGGGS)n,1≤n≤8,n为整数
4 (Gly) 3-8
5 (EAAAK)n,1≤n≤8,n为整数
6 PAPAP SEQ ID NO:393
7 AEAAAKEAAAKA SEQ ID NO:394
8 (Ala-Pro) n,1≤n≤15,n为整数
9 A(EAAAK) nALEA(EAAAK) nA,1≤n≤8,n为整数
10 VSQTSKLTRAETVFPDV SEQ ID NO:395
11 PLG LWA SEQ ID NO:396
12 RVLAEA SEQ ID NO:397
13 EDVVCCSMSY SEQ ID NO:398
14 GGIEGRGS SEQ ID NO:399
15 TRHRQPRGWE SEQ ID NO:400
16 AGNRVRRSVG SEQ ID NO:401
17 RRRRRRRRR SEQ ID NO:402
18 GFLG SEQ ID NO:403
19 KESGSVSSEQLAQFRSLD SEQ ID NO:404
20 EGKSSGSGSESKST SEQ ID NO:405
21 GSAGSAAGSGEF SEQ ID NO:406
附图说明
图1实施例3中示出了所选裂解酶对痤疮丙酸杆菌的有效杀伤作用。裂解酶在含有IPTG的LB琼脂上的大肠杆菌BL21(DE3)中表达,并通过大肠杆菌渗透释放。大肠杆菌周围的透明区或晕圈表明,表达的裂解酶可以抑制生长或杀死嵌入在琼脂覆盖层中的痤疮丙酸杆菌。
图2实施例3中示出了由含有诱导的所选裂解酶的大肠杆菌粗裂解液在痤疮丙酸杆菌覆盖层上形成的清除区。
图3实施例4中证明PACL10可以可溶性表达和纯化。所有样品在5-12%的三甘氨酸凝胶中在150V运行50分钟,并用考马斯亮蓝染色。PACL10使用pET表达***在大肠杆菌BL21(DE3)中表达,并通过疏水作用纯化。洗脱级分示出了在29.4kDa处的纯化的PACL10。
图4实施例7示出了PACL10降低痤疮丙酸杆菌34A菌株的cfu/mL的动力学。当MIC为6.4μg/mL、2x MIC为12.8μg/mL时,cfu/mL可分别在3小时和2小时内降至检测限以下。
图5示出了用于MIC测定的单步纯化的裂解酶。上样至5-12%Tris-HCl SDS凝胶的裂解酶浓度为:0.62mg/mL PACL10,1.41mg/mL PACL390,2.12mg/mL PACL391,2.10mg/mL PACL392,1.49mg/mL PACL403和1.60mg/mL PACL404。
图6示出了通过混合模式和离子交换色谱法纯化的PACL392。
图7示出了PACL392相对于菌株10的杀菌活性。
图8示出了通过混合模式色谱法纯化的PACL403。
图9示出了PACL403相对于菌株10的杀菌活性—OD减少。
图10示出了PACL403相对于菌株10的杀菌活性—CFU减少。
图11示出了PACL403相对于生物膜相关的菌株10的杀菌活性。
具体实施方式
以下实施例和/或实验例仅用于进一步阐述本发明,但不以任何的方式限制本发明的有效范围。采用标准实验方法进行实验得出实施例所示结果。
实施例1
通过分析丙酸杆菌科细菌基因组中前噬菌体的序列,我们鉴定出可以可溶性表达和纯化的痤疮丙酸杆菌噬菌体裂解酶。表达噬菌体裂解酶的基因序列由Sangon合成,并与表达质粒pET28连接。然后将每种重组质粒转入大肠杆菌BL21(DE3)菌株。
实施例2
嵌合物构建为N-末端催化结构域(CD)和C-末端结合结构域(BD),其间有可变长度的接头(linker)。通过使用NCBI恒定区数据库和用于计算机模拟蛋白质结构预测的RaptorX网络服务器进行序列分析来鉴别结构域。催化结构域具有全C-末端、半C-末端或无C-末端接头。同样的,结合结构域具有全N-末端、半N-末端或无N-末端连接。扩增结构域序列的引物由GeneCreate合成。采用Taq DNA聚合酶PCR在55℃退火温度下扩增结构域序列。PCR产物经凝胶纯化,与表达质粒pET28连接。然后将重组质粒转移到大肠杆菌BL21(DE3)。
实施例3
痤疮丙酸杆菌覆盖试验证实了裂解酶的抗菌活性。简单地说,将含有重组裂解酶质粒的大肠杆菌BL21(DE3)克隆置于含IPTG的LB琼脂上进行诱导。一旦裂解酶过表达,大肠杆菌克隆就会渗透释放裂解酶。包埋有痤疮丙酸杆菌的软琼脂被覆盖并培养以使痤疮丙酸杆菌生长。活性裂解酶的存在会在大肠杆菌周围形成透明区或晕圈。实验结果见图1。
在类似的试验中,含有重组裂解酶质粒的大肠杆菌BL21(DE3)克隆在液体培养基中诱导。离心收集细胞,在50mM磷酸钠pH 7.4缓冲液中超声裂解。裂解液离心分离可溶和不溶性组分。将可溶性粗裂解液在包埋有痤疮丙酸杆菌的软琼脂上点样。在培养以使痤疮丙酸杆菌生长后,可溶性粗裂解物中的活性裂解酶形成了清除区。实验结果见图2。
实施例4
PACL10的表达和纯化
PACL10表达及纯化如下。简单地说,将含有重组PACL10表达质粒的大肠杆菌BL21(DE3)置于自诱导培养基中在37℃、300rpm下培养至OD 600达到0.6-0.8,随后18℃、300rpm,持续培养16-18小时。离心收集细胞,重悬于50mM pH 7.4的磷酸钠中,高压均质裂解。裂解液再次离心以收集可溶性粗裂解液。将可溶性组分与等体积的5M NaCl混合,混合物上样到疏水柱中。上样后,用5倍柱体积的20mM磷酸钠(pH 7.4)、2.5M NaCl洗涤柱子。然后用10mM磷酸钠(pH 7.4)洗脱PACL10。实验结果见图3。
实施例5
以类似于PACL10的方式表达和纯化嵌合裂解酶。将含有重组嵌合裂解酶表达质粒的大肠杆菌BL21(DE3)置于自诱导培养基中在37℃、300rpm下培养至OD 600达到0.6-0.8,随后在18℃、300rpm下,持续培养16-18小时。离心收集细胞,重悬于50mM pH 7.4的磷酸钠中,高压均质裂解。裂解液再次离心以收集可溶性粗裂解液。将可溶性组分与等体积的5M NaCl混合,混合物上样到疏水柱中。上样后,用5倍柱体积的20mM磷酸钠(pH 7.4)、2.5M NaCl洗涤柱子。然后用10mM磷酸钠(pH 7.4)洗脱重组嵌合裂解酶。
实施例6
最小抑菌浓度测定
最小抑菌浓度(MIC)测定方法如下。首先在0.9%生理盐水中制备菌落悬浮液至OD 600为0.05以制备接种物,相当于每毫升10 7菌落形成单位(cfu/mL)。悬浮液在无琼脂强化梭菌培养基(RCM)中稀释20倍至接种物为5*10 5cfu/mL。用0.9%的生理盐水制备2倍连续稀释的PACL10或万古霉素,并且加入不超过总培养物的十分之一。培养物在厌氧条件下于37℃培养3天。MIC为肉眼观察不到痤疮丙酸杆菌的生长时的PACL10或万古霉素的最低浓度。通过将上述培养物在含有0.1%吐温-80的强化 梭菌琼脂(RCA)平板上传代培养来测定最小杀菌浓度(MBC)。MBC为在强化梭菌琼脂(RCA)平板上未见痤疮丙酸杆菌菌落时的PACL10或万古霉素的最低浓度。
实验结果:表6-1示出了PACL10与万古霉素相比,对来自IA 1、IA 2、II进化枝的12株痤疮丙酸杆菌菌株的最小抑菌浓度(MIC)和最小杀菌浓度(MBC)。
表6-1
Figure PCTCN2022074221-appb-000009
实施例7
时间杀菌试验
时间杀菌试验方法如下。将痤疮丙酸杆菌34A菌株菌落重新悬浮于0.9%生理盐水中至OD 600为0.05,相当于10 7cfu/mL。悬浮液在50mM磷酸钠(pH 6.0)中稀释10倍至~10 6cfu/mL。34A菌株分别加入0、0.5x、1x、2x MIC的两倍连续稀释的PACL10。在每个时间点将试验中的十倍系列稀释液(0,1,2,3log)铺板在强化梭菌琼脂(RCA)上,并在厌氧条件下于37℃培养3天。实验结果见图4。
实施例8
单步纯化后所选裂解酶的最小抑菌浓度
表达PACL 10、390、391、392、403和404参照实施例1~4的方法,并进行如下的单步纯化:将含有每种重组表达质粒的大肠杆菌BL21(DE3)置于自诱导培养基中在37℃、300rpm下培养3h,随后在18℃、300rpm下培养16-18小时。离心收集细胞,高压均质裂解。来自1L表达目标蛋白的大肠杆菌培养基的澄清裂解液上样至含有70mL纯化树脂的26mm/200mm柱。根据表8-1中的条件纯化裂解酶,单步纯化后的裂解酶的纯度测定如图5所示。这些裂解酶用于测定最小抑菌浓度(MIC)。
最小抑菌浓度(MIC)测定方法如下。痤疮丙酸杆菌在强化梭菌琼脂(具有1.5%琼脂的强化梭菌培养基RCM,根据修订后的配方配制RCM,每升-10g酸水解酪蛋白、10g牛肉提取物、3g酵母提取物、5gD-葡萄糖、5g氯化钠、3g醋酸钠、0.5g L-半胱氨酸盐酸盐)上划线。平板在37℃下厌氧培养72小时。首先在0.9%生理盐水中重悬来自RCA的单克隆至OD 600为0.05以制备用于MIC测定的接种物,相当于每毫升10 7菌落形成单位(cfu/mL)。悬浮液在RCM中稀释20倍至接种物为5*10 5cfu/mL。用0.9%的生理盐水制备2倍连续稀释的每种裂解酶,并且加入不超过总培养物体积的十分之一。培养物在厌氧条件下于37℃培养48-72小时。MIC为肉眼观察不到痤疮丙酸杆菌的生长时的裂解酶的最低浓度。MIC结果如表8-2所示。
表8-1所选裂解酶的纯化条件
Figure PCTCN2022074221-appb-000010
Figure PCTCN2022074221-appb-000011
表8-2所选裂解酶相对痤疮丙酸杆菌的MIC(μg/mL)
Figure PCTCN2022074221-appb-000012
实施例9
PACL392的纯化和杀菌活性
PACL392在含有重组表达质粒的大肠杆菌BL21(DE3)中通过置于自诱导培养基中在37℃、300rpm下培养3h,随后18℃、300rpm培养16-18小时进行表达。离心收集细胞,在20mM、pH 7.4的磷酸钠中高压均质裂解。裂解液在12,000rpm、4℃下离心1h。将上清液与等体积的20mM、pH 7.4的磷酸钠、1M硫酸铵混合,在12,000rpm、4℃下再次离心1h。上清液用0.22μm滤膜过滤,上样到用20mM、pH 7.4的磷酸钠、0.5M硫酸铵平衡的混合模式色谱柱(70mL纯化树脂的26mm/200mm柱)。上 样后,用一系列缓冲液洗涤柱:20mM、pH 7.4的磷酸钠,0.5M硫酸铵;20mM、pH 7.4的磷酸钠,2.5M NaCl;20mM、pH 7.4的磷酸钠和50mM、pH 10.02的哌嗪,50mM NaCl。用50mM、pH 10.02的哌嗪和750mM NaCl洗脱PACL392。洗脱液用50mM、pH 7.4的磷酸钠透析以中和pH并去除阳离子。透析样品过滤并上样到离子交换色谱柱(70mL纯化树脂的26mm/200mm柱)。PACL392纯化的最终结果如图6所示。
PACL392的杀菌活性通过在一系列添加剂如NaCl、CaCl 2、MgCl 2、EDTA、DTT、吐温-20、吐温-80和透明质酸(浓度如图7所示)存在下的50mM、pH 7.0的HEPES中的CFU减少来测定。
实施例10
PACL403的纯化和杀菌活性
PACL403在含有重组表达质粒的大肠杆菌BL21(DE3)中通过置于自诱导培养基中在37℃、300rpm下培养3h,随后18℃、300rpm培养16-18小时进行表达。离心收集细胞,在20mM、pH 7.4的磷酸钠中高压均质裂解。将裂解物溶液调节至20mM、pH 7.4的磷酸钠、1M NaCl,并在12,000rpm、4℃下离心1h。上清液用0.22μm滤膜过滤,上样到用20mM、pH 7.4的磷酸钠、1M NaCl平衡的混合模式色谱柱(70mL纯化树脂的26mm/200mm柱)。上样后,用一系列缓冲液洗涤柱:20mM、pH 7.4的磷酸钠,1M NaCl;20mM、pH 7.4的磷酸钠,2.5M NaCl;20mM、pH 7.4的磷酸钠;20mM、pH 7.4的磷酸钠,0.1%Triton X-114和再次加入20mM、pH 7.4的磷酸钠。用50mM、pH 9.5的哌嗪、1M NaCl洗脱PACL403。通过加入五分之一体积的500mM、pH 7.4的磷酸钠来中和高pH。然后将洗脱液用50mM、pH 7.4的磷酸钠透析以去除阳离子。透析样品过滤。PACL403纯化的最终结果如图8所示。
PACL403的杀菌活性通过在如图9所示浓度的吐温-20和吐温-80存在下的50mM、pH 7.0的磷酸钠中的OD减少来测定。
PACL403的杀菌活性通过在如图10所示浓度的吐温-20和吐温-80存在下的50mM、pH 7.0的HEPES中的CFU减少来测定。
PACL403还能杀死生物膜相关的痤疮丙酸杆菌(图11)。将OD 600为1.0的菌株10加入96孔聚苯乙烯平板,以在厌氧气体混合物中过夜接种生物膜。去除上清液,孔用0.9%NaCl轻轻冲洗。加入生物膜形成培养基(RCM+5%葡萄糖),以使生物膜在厌氧气体混合物中过夜生长。去除培养基,孔用0.9%NaCl轻轻冲洗。然后用缓冲液或PACL403在25℃、厌氧下过夜处理生物膜。去除缓冲液或PACL403,孔用0.9%NaCl冲洗。重悬于0.9%NaCl中,在RCM琼脂上铺板,对生物膜相关的CFU进行计数。

Claims (38)

  1. 一种噬菌体裂解酶或其嵌合物在制备预防、治疗或改善痤疮或由痤疮丙酸杆菌引起的感染或与痤疮丙酸杆菌有关的病症的药物、化妆品或医疗设备中的应用,其特征在于,所述噬菌体裂解酶包括来源于类诺卡氏菌科(Nocardioidaceae)或丙酸杆菌科(Propionibacteriaceae)细菌的噬菌体裂解酶。
  2. 根据权利要求1所述的应用,其特征在于,所述类诺卡氏菌科(Nocardioidaceae)细菌包括微白霜菌属(Micropruina)、产丙酸单孢菌属(Propionicimonas)、产丙酸细胞菌属(Propionicicella)、弗莱德门氏菌属(Friedmanniella)细菌;所述丙酸杆菌科(Propionibacteriaceae)细菌包括产丙酸菌属(Propioniferax)、海马球菌属(Mariniluteicoccus)、颗粒球菌属(Granulicoccus)、瑙曼氏菌属(Naumannella)、棒状丙酸菌属(Propioniciclava)、光球菌属(Auraticoccus)、小月菌属(Microlunatus)、河口微菌属(Aestuariimicrobium)、黄球菌属(Luteococcus)、四合球菌属(Tessaracoccus)、布克劳氏菌属(Brooklawnia)、产丙酸微球菌属(Propionimicrobium)、丙酸杆菌属(Propionibacterium)、痤疮丙酸杆菌属(Cutibacterium)、酸性丙酸杆菌属(Acidipropionibacterium)、或假丙酸杆菌属(Pseudopropionibacterium)细菌;
    优选丙酸杆菌属(Propionibacterium)、痤疮丙酸杆菌属(Cutibacterium)、酸性丙酸杆菌属(Acidipropionibacterium)、或假丙酸杆菌属(Pseudopropionibacterium)细菌。
  3. 根据权利要求1所述的应用,其特征在于,所述噬菌体裂解酶包括来源于痤疮丙酸杆菌(Cutibacterium acnes)、肱丙酸杆菌(Propionibacterium humerusii)、贪婪丙酸杆菌(Cutibacterium avidum)、颗粒丙酸杆菌(Cutibacterium granulosum)、特氏丙酸杆菌(Acidipropionibacterium thoenii)、詹氏丙酸杆菌(Acidipropionibacterium jensenii)、产丙酸丙酸杆菌(Acidipropionibacterium acidipropionici)、潮汐滩菌(Aestuariimicrobium kwangyangense)、细粒球菌(Granulicoccus  phenolivorans)、积磷小月菌(Microlunatus phosphovorus)、丙酸丙酸杆菌(Pseudopropionibacterium propionicum)、四合球菌(Tessaracoccus sp.)、极底产丙酸细胞菌(Propionicicella superfundia)、费氏丙酸杆菌(Propionibacterium freudenreichii)、费氏丙酸杆菌费氏亚种(Propionibacterium freudenreichii subsp.freudenreichii)、费氏丙酸杆菌舍氏亚种(Propionibacterium freudenreichii subsp.shermanii)、产酸丙酸杆菌(Propionibacterium acidifaciens)、嗜淋巴丙酸杆菌(Propionibacterium lymphophilum)、丙酸杆菌属细菌(Propionibacteriaceae bacterium)、丙酸杆菌分类群192(Propionibacterium sp.oral taxon 192)、无毒产丙酸菌(Propioniferax innocua)、盐藻瑙曼氏菌(Naumannella halotolerans)、慢生棒状丙酸菌(Propioniciclava tarda)、糖原微白霜菌(Micropruina glycogenica)、淡水产丙酸单孢菌(Propionicimonas paludicola)、纪念性光球菌(Auraticoccus monumenti)、日本黄球菌(Luteococcus japonicus)、油田四合球菌(Tessaracoccus oleiagri)、本迪戈四合球菌(Tessaracoccus bendigoensis)、拉帕迪卡四合球菌(Tessaracoccus lapidicaptus)、微嗜气丙酸杆菌(Acidipropionibacterium microaerophilum)、橄榄体丙酸杆菌(Acidipropionibacterium olivae)、或丹诺氏丙酸杆菌(Acidipropionibacterium damnosum)细菌的噬菌体裂解酶。
  4. 根据权利要求1所述的应用,其特征在于,所述噬菌体裂解酶包括来源于詹氏丙酸杆菌(Acidipropionibacterium jensenii)、特氏丙酸杆菌(Acidipropionibacterium thoenii)、产丙酸丙酸杆菌(Acidipropionibacterium acidipropionici)、微嗜气丙酸杆菌(Acidipropionibacterium microaerophilum)、橄榄体丙酸杆菌(Acidipropionibacterium olivae)、丹诺氏丙酸杆菌(Acidipropionibacterium damnosum)、痤疮丙酸杆菌(Cutibacterium acnes)、贪婪丙酸杆菌(Cutibacterium avidum)、颗粒丙酸杆菌(Cutibacterium granulosum)、或丙酸丙酸杆菌(Pseudopropionibacterium propionicum)细菌的噬菌体裂解酶。
  5. 根据权利要求1所述的应用,其特征在于,所述噬菌体裂解酶具 有SEQ ID NO:1~SEQ ID NO:28任一所示的氨基酸序列。
  6. 根据权利要求1所述的应用,其特征在于,所述噬菌体裂解酶具有SEQ ID NO:10所示的氨基酸序列。
  7. 根据权利要求1所述的应用,其特征在于,所述噬菌体裂解酶具有SEQ ID NO:29~SEQ ID NO:56任一所示的核苷酸序列。
  8. 根据权利要求1所述的应用,其特征在于,所述噬菌体裂解酶具有SEQ ID NO:38所示的核苷酸序列。
  9. 根据权利要求1所述的应用,其特征在于,所述嵌合物包含噬菌体裂解酶的催化结构域,或包含噬菌体裂解酶的催化结构域和结合结构域的组合。
  10. 根据权利要求9所述的应用,其特征在于,所述催化结构域具有全C-末端接头、半C-末端接头、无C-末端接头、或所述接头的任何部分;所述结合结构域具有全N-末端接头、半N-末端接头、无N-末端接头、或所述接头的任何部分。
  11. 根据权利要求9所述的应用,其特征在于,所述催化结构域包括一个、两个或两个以上的催化结构域。
  12. 根据权利要求9所述的应用,其特征在于,所述结合结构域包括一个、两个或两个以上的结合结构域。
  13. 根据权利要求9所述的应用,其特征在于,所述嵌合物进一步包含催化结构域和结合结构域之间的接头,所述接头包括:
    1)SEQ ID NO:343~SEQ ID NO:367任一所示的氨基酸序列;
    2)SEQ ID NO:368~SEQ ID NO:392任一所示的氨基酸序列;
    3)SEQ ID NO:393~SEQ ID NO:406任一所示的氨基酸序列;或
    4)(GGGS)n、(GGGGS)n、(GGGGGS)n、(Gly)3-8、(EAAAK)n、(Ala-Pro)n、或A(EAAAK)nALEA(EAAAK)nA,其中1≤n≤15,n为整数。
  14. 根据权利要求9所述的应用,其特征在于,所述嵌合物包括所述催化结构域具有SEQ ID NO:57、SEQ ID NO:58、SEQ ID NO:59、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ ID NO:67、SEQ ID NO:69、SEQ ID NO:70、SEQ ID  NO:71、SEQ ID NO:73、SEQ ID NO:74、SEQ ID NO:77、SEQ ID NO:80、SEQ ID NO:81、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:91、SEQ ID NO:93、SEQ ID NO:95、SEQ ID NO:97、SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100、SEQ ID NO:102、SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、SEQ ID NO:107、SEQ ID NO:108、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:113、SEQ ID NO:114、SEQ ID NO:117、SEQ ID NO:118、SEQ ID NO:121、SEQ ID NO:122、SEQ ID NO:125、SEQ ID NO:127、SEQ ID NO:128、SEQ ID NO:131、SEQ ID NO:132、SEQ ID NO:133、SEQ ID NO:135、或SEQ ID NO:136中所示的氨基酸序列;
    所述结合结构域具有SEQ ID NO:60、SEQ ID NO:62、SEQ ID NO:68、SEQ ID NO:72、SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:78、SEQ ID NO:79、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:90、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:96、SEQ ID NO:101、SEQ ID NO:103、SEQ ID NO:109、SEQ ID NO:112、SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:119、SEQ ID NO:120、SEQ ID NO:123、SEQ ID NO:124、SEQ ID NO:126、SEQ ID NO:129、SEQ ID NO:130、SEQ ID NO:134、SEQ ID NO:137、SEQ ID NO:138、或SEQ ID NO:139所示的氨基酸序列。
  15. 根据权利要求9所述的应用,其特征在于,所述催化结构域具有SEQ ID NO:140、SEQ ID NO:141、SEQ ID NO:142、SEQ ID NO:144、SEQ ID NO:146、SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149、SEQ ID NO:150、SEQ ID NO:152、SEQ ID NO:153、SEQ ID NO:154、SEQ ID NO:156、SEQ ID NO:157、SEQ ID NO:160、SEQ ID NO:163、SEQ ID NO:164、SEQ ID NO:168、SEQ ID NO:169、SEQ ID NO:170、SEQ ID NO:174、SEQ ID NO:176、SEQ ID NO:178、SEQ ID NO:180、SEQ ID NO:181、SEQ ID NO:182、SEQ ID NO:183、SEQ ID NO:185、SEQ ID NO:187、SEQ ID NO:188、SEQ ID NO:189、SEQ ID NO:190、SEQ ID NO:191、SEQ ID NO:193、SEQ ID NO:194、SEQ ID NO:196、 SEQ ID NO:197、SEQ ID NO:200、SEQ ID NO:201、SEQ ID NO:204、SEQ ID NO:205、SEQ ID NO:208、SEQ ID NO:210、SEQ ID NO:211、SEQ ID NO:214、SEQ ID NO:215、SEQ ID NO:216、SEQ ID NO:218、SEQ ID NO:219所示的核苷酸序列;
    所述结合结构域具有SEQ ID NO:143、SEQ ID NO:145、SEQ ID NO:151、SEQ ID NO:155、SEQ ID NO:158、SEQ ID NO:159、SEQ ID NO:161、SEQ ID NO:162、SEQ ID NO:165、SEQ ID NO:166、SEQ ID NO:167、SEQ ID NO:171、SEQ ID NO:172、SEQ ID NO:173、SEQ ID NO:175、SEQ ID NO:177、SEQ ID NO:179、SEQ ID NO:184、SEQ ID NO:186、SEQ ID NO:192、SEQ ID NO:195、SEQ ID NO:198、SEQ ID NO:199、SEQ ID NO:202、SEQ ID NO:203、SEQ ID NO:206、SEQ ID NO:207、SEQ ID NO:209、SEQ ID NO:212、SEQ ID NO:213、SEQ ID NO:217、SEQ ID NO:220、SEQ ID NO:221、或SEQ ID NO:222所示的核苷酸序列。
  16. 根据权利要求9所述的应用,其特征在于,所述嵌合物具有SEQ ID NO:223~SEQ ID NO:282中任一所示的氨基酸序列;优选SEQ ID NO:258、SEQ ID NO:259、SEQ ID NO:260、SEQ ID NO:271或SEQ ID NO:272所示的氨基酸序列;更优选SEQ ID NO:260或SEQ ID NO:271所示的氨基酸序列。
  17. 根据权利要求9所述的应用,其特征在于,所述嵌合物具有SEQ ID NO:283~SEQ ID NO:342中任一所示的核苷酸序列;优选SEQ ID NO:316、SEQ ID NO:317、SEQ ID NO:318、SEQ ID NO:329或SEQ ID NO:330所示的核苷酸序列;更优选SEQ ID NO:318或SEQ ID NO:329所示的核苷酸序列。
  18. 根据权利要求1所述的应用,其特征在于,所述由痤疮丙酸杆菌引起的感染包括侵入性感染、术后感染和/或器械相关的感染。
  19. 根据权利要求18所述的应用,其特征在于,所述器械相关的感染包括关节假体、分流管和人工心脏瓣膜相关的感染。
  20. 根据权利要求18所述的应用,其特征在于,所述感染包括骨骼 和/或关节的感染,尤其是术后肩部感染,以及口腔、眼、椎间盘和脑部的感染。
  21. 根据权利要求1所述的应用,其特征在于,所述与痤疮丙酸杆菌有关的病症包括导致癌症的***炎、SAPHO(滑膜炎、痤疮、脓疱病、肥大、骨炎)综合征、结节病、或坐骨神经痛。
  22. 根据权利要求1所述的应用,其特征在于,所述医疗设备包括用于将裂解酶或其嵌合物释放到受影响区域的任何设备,优选施加至皮肤表面的夹具或贴剂或喷雾剂,使用微针增强裂解酶或其嵌合物的皮肤渗透性的设备,美容专业人员使用的将裂解酶或其嵌合物专门施加至受痤疮影响的毛囊上的细针,或其他类似的设备。
  23. 根据权利要求1所述的应用,其特征在于,所述医疗设备包括将将裂解酶或其嵌合物固定在易于感染痤疮丙酸杆菌的位置中的假体装置上,优选用于特别容易感染痤疮丙酸杆菌的肩部手术中的假体植入物。
  24. 一种权利要求1~23任一所述应用中的噬菌体裂解酶嵌合物,所述嵌合物具有SEQ ID NO:223~SEQ ID NO:282中任一所示的氨基酸序列;优选SEQ ID NO:258、SEQ ID NO:259、SEQ ID NO:260、SEQ ID NO:271或SEQ ID NO:272所示的氨基酸序列;更优选SEQ ID NO:260或SEQ ID NO:271所示的氨基酸序列。
  25. 根据权利要求24所述的嵌合物,其特征在于,所述嵌合物具有SEQ ID NO:283~SEQ ID NO:342中任一所示的核苷酸序列;优选SEQ ID NO:316、SEQ ID NO:317、SEQ ID NO:318、SEQ ID NO:329或SEQ ID NO:330所示的核苷酸序列;更优选SEQ ID NO:318或SEQ ID NO:329所示的核苷酸序列。
  26. 一种权利要求1~23任一所述应用中的或权利要求24~25任一所述的嵌合物的制备方法,其特征在于,所述方法包括:
    (1)合成结构域序列及扩增结构域序列的引物;
    (2)采用Taq DNA聚合酶PCR扩增结构域序列;
    (3)PCR产物凝胶纯化,与表达质粒pET28连接;
    (4)重组质粒转移到大肠杆菌BL21(DE3);
    (5)培养含有重组质粒的大肠杆菌BL21(DE3),诱导表达,离心收集细胞,裂解,纯化得到所述嵌合物。
  27. 根据权利要求26所述的制备方法,其特征在于,所述方法进一步包括:
    将含有重组嵌合裂解酶表达质粒的大肠杆菌BL21(DE3)置于自诱导培养基中在37℃、300 rpm下培养至OD 600达到0.6-0.8,随后在18℃、300rpm下,持续培养16-18小时;离心收集细胞,重悬于50mM pH 7.4的磷酸钠中,高压均质裂解;裂解液再次离心以收集可溶性粗裂解液。将可溶性组分与等体积的5M NaCl混合,混合物上样到疏水柱中;上样后,用5倍柱体积的20mM磷酸钠(pH 7.4)、2.5M NaCl洗涤柱子;然后用10mM磷酸钠(pH 7.4)洗脱重组嵌合裂解酶。
  28. 含有权利要求1~23任一所述应用中的噬菌体裂解酶或其嵌合物,或权利要求24~25任一所述的嵌合物的制剂,其特征在于,所述制剂进一步包含抗生素、其他裂解酶、或非活性赋形剂。
  29. 编码权利要求1~23任一所述应用中的噬菌体裂解酶或其嵌合物,或权利要求24~25任一所述的嵌合物,或权利要求26制备的嵌合物的氨基酸序列,或与前述序列相似度80%及以上、85%及以上、90%及以上、95%及以上或99%及以上的氨基酸序列,或具有相同官能团的替代氨基酸序列。
  30. 根据权利要求29所述的氨基酸序列,其特征在于,所述替代氨基酸序列为使用同一组氨基酸中的氨基酸的保守替换,所述氨基酸组包括:
    脂肪族的:甘氨酸、丙氨酸、缬氨酸、亮氨酸或异亮氨酸;
    含羟基或硫/硒的:丝氨酸、半胱氨酸、苏氨酸或甲硫氨酸;
    环状的:脯氨酸;
    芳族的:苯基丙氨酸、酪氨酸或色氨酸;
    碱性的:组氨酸、赖氨酸或精氨酸;或
    酸性的以及它们的酰胺:天冬氨酸、谷氨酸、天冬酰胺、谷氨酰胺。
  31. 编码权利要求1~23任一所述应用中的噬菌体裂解酶或其嵌合 物,或权利要求24~25任一所述的嵌合物,或权利要求26制备的嵌合物的核苷酸序列,或其同义密码子序列。
  32. 权利要求1~23任一所述应用中的噬菌体裂解酶或其嵌合物,或权利要求24~25任一所述的嵌合物,或权利要求26制备的嵌合物用作细菌裂解的试剂的应用,其特征在于,所述细菌裂解用于使用基于PCR的试剂盒进行DNA提取和分型。
  33. 权利要求1~23任一所述应用中的噬菌体裂解酶或其嵌合物,或权利要求24~25任一所述的嵌合物,或权利要求26制备的嵌合物用作非生物表面上的消毒剂或灭菌剂的应用,其特征在于,所述消毒剂或灭菌剂通过去除浮游形式或生物膜中的痤疮丙酸杆菌以防止感染,优选在手术过程中对手术设备或假体植入装置进行消毒。
  34. 权利要求1~23任一所述应用中的噬菌体裂解酶或其嵌合物,或权利要求24~25任一所述的嵌合物,或权利要求26制备的嵌合物,或上述分子中的单个结合结构域,或上述分子中的相似或不同的结合结构域串联的组合,在制备用于痤疮丙酸杆菌的诊断工具中的应用,其特征在于,所述裂解酶、嵌合物或结合结构域与信号分子联合使用。
  35. 根据权利要求34所述的应用,其特征在于,所述裂解酶、嵌合物或结合结构域与信号分子通过基因融合或化学偶联形成融合物。
  36. 根据权利要求35所述的应用,其特征在于,所述融合物用于通过荧光或其他手段直接检测显微镜载玻片上的痤疮丙酸杆菌,用于通过免疫组织化学标记痤疮丙酸杆菌,用作ELISA测定中的检测试剂,用作Western blot上的检测试剂,用于与在MACS或其他pull-down测定中的磁珠连接,或用于抗体作为检测试剂的测定中的检测试剂。
  37. 根据权利要求34所述的应用,其特征在于,所述信号分子包括蛋白质或化学荧光染料、蛋白质标签、酶、抗生物素蛋白、链霉亲和素、卵清蛋白、生物素、对点击化学标记敏感的标签、内含肽、或能够引起产生信号的二级蛋白质或分子募集的其他分子,
    所述荧光染料包括GFP、RFP、mCherry、FITC、TRITC、Alexafluor 488、Cy3或Cy5;
    所述蛋白质标签包括Flag-标签、myc-标签、halo-标签、his-标签、或能够与抗体或其他高亲和力分子结合产生信号的任何其他标签;
    所述酶包括萤火虫荧光素酶、β-内酰胺酶、碱性磷酸酶、辣根过氧化物酶、或引起诸如光、颜色变化、底物沉积或其他能够在试验中检测到的反应的任何其他的酶。
  38. 权利要求1~23任一所述应用中的噬菌体裂解酶或其嵌合物,或权利要求24~25任一所述的嵌合物,或权利要求26制备的嵌合物中的催化结构域在制备治疗痤疮丙酸杆菌感染的药物中的应用,其特征在于,所述催化结构域与寻靶模块结合。
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