WO2011026447A1 - 一种含抗体模拟物的新型抗生素及其制备方法与应用 - Google Patents

一种含抗体模拟物的新型抗生素及其制备方法与应用 Download PDF

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WO2011026447A1
WO2011026447A1 PCT/CN2010/077351 CN2010077351W WO2011026447A1 WO 2011026447 A1 WO2011026447 A1 WO 2011026447A1 CN 2010077351 W CN2010077351 W CN 2010077351W WO 2011026447 A1 WO2011026447 A1 WO 2011026447A1
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peptide chain
antibody
colicin
antibiotic
mic
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PCT/CN2010/077351
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English (en)
French (fr)
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丘小庆
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畿晋庆三联（北京）生物技术有限公司
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Priority to AU2010291640A priority Critical patent/AU2010291640B2/en
Application filed by 畿晋庆三联（北京）生物技术有限公司 filed Critical 畿晋庆三联（北京）生物技术有限公司
Priority to UAA201202043A priority patent/UA102325C2/uk
Priority to SG2012011532A priority patent/SG178503A1/en
Priority to DK10813369.5T priority patent/DK2474558T3/en
Priority to EP10813369.5A priority patent/EP2474558B1/en
Priority to EA201200417A priority patent/EA028912B1/ru
Priority to ES10813369.5T priority patent/ES2538840T3/es
Priority to AP2012006185A priority patent/AP3302A/xx
Priority to NZ598593A priority patent/NZ598593A/xx
Publication of WO2011026447A1 publication Critical patent/WO2011026447A1/zh
Priority to ZA2012/01386A priority patent/ZA201201386B/en
Priority to IL218435A priority patent/IL218435A/en
Priority to HK12108839.4A priority patent/HK1168113A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1271—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Micrococcaceae (F), e.g. Staphylococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6817—Toxins
    • A61K47/6829—Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1214—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pseudomonadaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds

Definitions

  • the invention relates to the field of biomedicine, in particular to a novel antibiotic containing an antibody mimic and a preparation method and application thereof.
  • antibiotics such as penicillin have been used, not only meningococcus, but many other life-threatening pathogens such as Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Mycobacterium tuberculosis have been resistant to it. Medicinal. According to reports published by the Centers for Disease Control (CDC) over the years, these antibiotics may be completely ineffective within another 10 to 20 years.
  • CDC Centers for Disease Control
  • antibiotics mainly achieve antibacterial purposes by inhibiting cell wall synthesis, inhibiting or interfering with bacterial nucleic acid and protein metabolism and synthesis pathways.
  • these antibacterial methods are prone to induce bacterial mutations and develop drug resistance. Therefore, people have been working on the development of new types of antibiotics. It is one of the more promising directions to imitate the way in which allogeneic bacteria work together to develop new antibiotics.
  • colicin is an ideal ion channel antibiotic prototype, but wild type colicin can only act on allogeneic E. coli, so it is necessary to change its targeting to enable coliform transfer.
  • Porin is a channel protein present on the outer membrane of the bacterial plasma membrane, mitochondria and chloroplast outer membrane, which allows larger molecules to pass, has strong immunogenicity, can Inducing a specific high level of monoclonal antibody in the host, such as using an antibody against the Porin protein on the bacterial surface as a prototype, designing an antibody mimetic with better recognition performance, and using this mimic to change the target of colicin Directionality should be an ideal antibiotic development direction.
  • the present invention provides a novel antibiotic for the bactericidal ability of the antibiotic in the above field. It is more than a thousand times more commonly used antibiotics. Because of its special mechanism of action, it is difficult for pathogenic bacteria to produce drug resistance, and it will not harm normal human cells during sterilization.
  • a novel antibiotic comprising an antibody mimetic, consisting of a colicin El, Ia, Ib, A, B, N or an aqueous pore domain thereof and a peptide chain covalently linked to the colicin or its aqueous pore domain antibody mimics the carboxyl terminus configuration
  • said antibody mimetic is a carboxy terminus of an immunoglobulin V H CDR1 is connected to the amino terminus of V H FR2, V H FR2 carboxy terminal amino terminus VLCDR reconnection configuration; the immunoglobulins
  • the protein specifically recognizes the bacterial membrane porin.
  • the bacterium refers to meningococcus, which is a membrane pore protein PorA on the surface of meningococcus.
  • the immunoglobulin is an antibody protein obtained by covalently binding a peptide chain having an amino acid sequence of the PUBMED accession number of 2MPA_H to a peptide chain having the amino acid sequence of Accession No. 2MPA_L.
  • the colicin is Ia.
  • a peptide chain molecule characterized by comprising a peptide chain of colicin El, Ia, Ib, A, B, N or an aqueous channel domain thereof, and an antibody mimetic peptide chain covalently bound at a carboxy terminus thereof,
  • the antibody mimetic peptide chain is composed of the amino acid ends of the three regions VH CDR1, V H FR2, and V L CDR3 of the immunoglobulin, which are sequentially linked at the carboxy terminus to the amino terminus of the next region peptide chain; Specific recognition of bacterial membrane porin.
  • the peptide chain molecule has the amino acid sequence shown by Seq ID No. 6.
  • the nucleotide sequence is shown as Seq ID No. 5.
  • a recombinant expression vector comprising the above nucleotide sequence.
  • the preparation method of the above novel antibiotic refers to introducing the above recombinant expression vector into an expression system for expression; and isolating and purifying the expressed polypeptide to obtain an antibiotic.
  • the antibacterial drug refers to a drug against meningococcus, an anti-vancomycin-resistant enterococci, an anti-methicillin-resistant Staphylococcus aureus or a multi-drug resistant Pseudomonas aeruginosa.
  • the invention utilizes colicin to form an ion channel on the target bacterial membrane, and causes the cytoplasm in the target cell to leak and die, and the structure which plays a targeted role in the invention is the protection against the membrane pore protein of the bacterial surface, that is, the Porin protein.
  • Partial region of an immunoglobulin obtained after being reconstructed analog antibody was first complementary heavy chain variable region comprises determining regions of the immunoglobulin V H CDR1, a heavy chain variable region framework regions of a second V H FR2
  • the third complementarity determining region V L CDR3 of the light chain variable region, the three regions sequentially carboxy-terminally linked to the amino terminus of the next region constitutes a linear molecule of the VHCDR1-VHFR2-V L CDR3; it is well known that immunoglobulin recognizes
  • the active region of action is called the complementarity determining region, and the complementarity determining region is composed of only a few to a dozen amino acids.
  • the present invention Compared with the intact immunoglobulin or the currently reported single-chain antibody seFv, Fab and other artificially engineered antibodies, the present invention
  • the molecular weight is very small, the tissue permeability is good, and because of its simple structure, most of the framework structure and Fc segment of intact immunoglobulin molecules are removed, which greatly reduces the immunogenicity of the patient, and is very beneficial to lead colicin.
  • colicin is introduced to the surface of the cell membrane of the pathogenic bacteria, and colicin forms an ion channel on the target bacterial membrane, causing the cytoplasm in the target cell to leak and die, and has the same strain for the strain that has already developed resistance. Sterilization ability.
  • the recognition site is an antigenic substance unique to the surface of the bacteria, such a recognition site does not exist on the cell membrane of the human tissue, so it is safe for the human body.
  • the antibiotic of the present invention does not need to pass through the membrane pore protein because its targeting antibody mimics only need to lead the colicin to the pathogenic bacteria.
  • the site acts as a lethal bacteria, but acts on the biofilm of the bacteria by colistin, causing the biofilm to produce ion channels leading to cell leakage and death.
  • Traditional bacterial resistance generally changes the structure of the membrane pore proteins to enter the bacterial cells.
  • the invention causes obstacles and produces drug resistance, and the present invention only needs the activity of the antibody recognition site of the membrane porin to reach the purpose of sterilization. It can be said that it is a strategy of squeezing the membrane, that is, identifying the membrane pore protein site, but Coliformin binds to another site-shaped ion channel lethal bacteria in the cell membrane.
  • the real site of action is not the Porin protein. Therefore, it is difficult for bacteria to morph or evolve to lose or change the structure necessary for the survival of the membrane pore protein. It is difficult to develop resistance to the antibiotic of the present invention.
  • the novel antibiotics obtained according to the design concept of the present invention are also diverse due to the diversity of the membrane pore proteins of different bacterial surfaces and the diversity of immunoglobulins recognized thereby.
  • Meningitis caused by meningococcal disease poses a great threat to the health of infants and adolescents at home and abroad, and its resistance to current common drugs is very obvious. In order to effectively inhibit the bacteria, the dosage is becoming more and more effective. High, this seriously jeopardizes the health of the patient. Therefore, the inventors have adopted the concept of the present invention, and the antibody against the membrane pore protein Porin A of Meningococcus, the PUBMED accession number of the heavy chain peptide chain is 2MPA_H, and the PUBMED of the light chain peptide chain The accession number is 2MPA_L, and reconstitution is performed to obtain an antibody mimetic, that is, an amino acid sequence as shown in Seq ID No.
  • the inventors used the novel antibiotic PMC-AM1 of the present invention to conduct tentative experiments on other pathogenic bacteria which are currently highly resistant, and found that PMC-AM1 is also resistant to multi-drug resistant Pseudomonas aeruginosa, vancomycin-resistant enterococci, and nail-resistant Oxyxocillin has a strong antibacterial effect.
  • PMC-AM1 has a stronger bactericidal effect against multi-drug resistant Pseudomonas aeruginosa than ceftazidime, levofloxacin, gentamicin and other active antibiotics 127 ⁇ 3800 More than double; PMC-AM1 has obvious inhibitory effect on vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus, as shown in Figures 6B and C.
  • the antibiotic of the present invention can be used for the preparation of an antibacterial agent, particularly for the preparation of a medicament against meningococcus, anti-Pseudomonas aeruginosa, vancomycin-resistant Enterococcus, and anti-methicillin-resistant Staphylococcus aureus.
  • the nucleotide encoding the antibiotic of the present invention can be cloned into an expression vector to construct a recombinant expression vector, which can express the fusion protein in a host, and isolate the fusion protein to obtain the antibiotic protein of the present invention.
  • nucleotide sequence encoding the antibiotic or antibody mimetic of the present invention is adjustable, and the nucleotide sequence can be adjusted according to the host cell's preference for codons, as long as the encoded amino acid remains unchanged. It is within the scope of the claimed invention.
  • the peptide chain of the antibody mimetic is linked to the carboxy terminus of the peptide chain of la, and the amino acid sequence of the antibody mimetic is as shown by Seq ID ⁇ 2.
  • the antibody mimetic peptide chain is ligated at the carboxy terminus of la, and the antibody mimetic peptide chain is: the carboxy-terminal linked heavy chain second framework region of the first complementarity determining region of the heavy chain variable region, and the third light chain variable region region Complementary decision region
  • the peptide chain carboxy terminal amino acid is linked to the carboxy terminal amino acid of the second backbone region of the heavy chain, as shown by Seq ID No. 4.
  • T and R are two signal recognition domains of coenzyme la at the amino terminus
  • Channel-forming is the formation of an ion channel domain at the carboxy terminus of colicin la; AM is an antibody mimetic.
  • the curves in the figure are from left to right as controls, 5 ⁇ ⁇ / ⁇ 1 ampicillin, 5 ⁇ ⁇ / ⁇ 1 PMC-AM2, 5 g/ml PMC-AMI, 10 /ml PMC-AMI.
  • the horizontal coordinate of this figure is the bacterial growth time in hours; the ordinate is the optical density of 600 nm in the culture medium, showing the amount of bacterial growth.
  • Plates display: (Con), blank control, (A), ceftazidime at 16 g/ml, (B), levofloxacin at 8 g/ml, (C), gentamicin greater than 512 g/ml, (D).
  • the minimum inhibitory concentration of the new antibiotic (PMC-AM1) against multi-drug resistant Pseudomonas aeruginosa is 8 g/ml.
  • FIG. 6 New antibiotics and common antibiotics of the present invention against methicillin-resistant Staphylococcus aureus (ATCC BAA-42), vancomycin-resistant enterococci (ATCC 700802), multi-drug resistant Pseudomonas aeruginosa (Huaxi Hospital clinical isolate 13078) And the menopausal diphtheria (Chinese Culture Preservation Center 29332)
  • the minimum inhibitory concentration of the experimental ordinate is the minimum inhibitory concentration (nMol);
  • FIG. 1 Novel antibiotics and wild-type colicin and anti-S. aureus polypeptides (ZL 01128836.1) Survival curves of inhibition of methicillin-resistant Staphylococcus aureus (ATCC BAA-42), vancomycin-resistant enterococci (ATCC 700802), multi-drug resistant Pseudomonas aeruginosa (Huaxi Hospital clinical isolate 13078)
  • the ordinate is the minimum inhibitory concentration (nMol);
  • FIG. 8 Survival curve of the in vivo protection test of the novel antibiotic of the present invention against animals infected with meningococcus.
  • the abscissa is the survival time of the mouse, and the unit is day; the ordinate is the number of animals surviving, and the unit is each mouse.
  • PMC-AM1 a novel antibiotic of the invention
  • Gen gentamicin
  • PEN penicillin
  • Con. control; all drug injection concentrations were 1.5 mg/kg.
  • PCR amplification, amplification conditions denaturation 95 ° C, 35 seconds, annealing 53 ° C, 70 seconds, extension 68 ° C, 17 points, a total of 20 cycles;
  • the mutated plasmid 100 ng was incubated with the prepared BL-21 engineering bacteria competent cells for 40 minutes, heat shocked at 42 ° C for 30 seconds, placed in ice for 2 minutes, and added to SOC broth 160 ul, 220 rpm, 37 After shaking for 1 hour at °C, plate (LB PBS plus 1% agar, add 50 ug/ml ampicillin, overnight at 37 °C), pick up a large number of colonies to increase the bacteria;
  • Augmented bacteria 8-10 liters of FB, 250 rpm, 30 °C, 3-4 hours; warmed to 42 ° C, 250 rpm for 0.5 hours; cooled to 37 ° C, 250 rpm for 1.5 hours; 4 ° C, 6000g, centrifuge the cells for 20 minutes, take 60-100 ml suspension of cells at 4 ° C, 50 mM boric acid buffer (pH 9.0, 2 mM EDTA), add 50 ug of PMSF, and ultrasonically disrupt the cells (4 °C, 400W, 1 minute, repeat 4 to 5 times, intermittent 2-3 minutes to ensure the temperature of the bacterial liquid), high-speed centrifugation to precipitate the broken cells (4 ° C, 75,000g, 90 minutes), take the supernatant and add streptomycin sulfate 500 Million units of precipitated DNA (stirred at 4 °C for 1 hour), 10,000 g, 4 ° C, 10 minutes after centrifugation, the supernatant was taken into a 15,000 dia
  • the pellet was centrifuged again at 10,000 g, 4 ° C, 10 minutes, and the supernatant was applied to a CM ion exchange column. After thorough washing, 0.3 M NaCl + 50 mM boric acid buffer was eluted to obtain a novel antibiotic prepared.
  • two antibiotics, PMC-AM1 and PMC-AM2 were obtained, and the amino acid sequences thereof were as shown in Seq ID No. 6 and Seq ID ⁇ 8, respectively.
  • AMI is the first complementarity determining region of the heavy chain variable region, the second heavy chain region, and the third complementary region of the light chain variable region, and the three regions are sequentially linked to the amino terminus of the next region by the carboxy terminus, amino acid
  • the sequence is represented by Seq ID No. 2;
  • AM2 is the carboxy-terminally linked heavy chain second framework region of the first complementarity determining region of the heavy chain variable region, and the peptide chain carboxy terminus of the third complementarity determining region of the light chain variable region is The amino acid is linked to the carboxy terminal amino acid of the second framework region of the heavy chain, and the amino acid sequence is shown as Seq ID No. 4.
  • oligonucleotide primer sequences designed in the above preparation plasmids are as follows:
  • Gcc tgt ctt ata ttt tat tta TCC GAT CCA CAG TCC CTG ACC AGG TCT ctg ttt aat cca atg cag cca
  • Gcc tgt ctt ata ttt tat tta GGT TCT CGG CAC ATG CGT GGA CTG AGA tec gat cca cag tec ctg acc
  • Gcc tgt ctt ata ttt tat tta TCC GAT CCA CAG TCC CTG ACC AGG TCT ctg ttt aat cca atg cag cca
  • the bacteria is the Chinese mushroom preservation center 29332 meningitis double-sphere strain
  • 2 ⁇ L of the bacterial liquid (10 5 CFU/ml) is added to the rabbit blood chocolate culture solution 10 ml (beef extract 50 mg, tryptone 100 mg, KH 2 P0 4 In the 30 mg NaCl 50 mg defibrated rabbit blood 0.5-0.8 ml), a total of 5 groups were prepared.
  • the first group was added with 0.3 M NaCl + 50 mM boric acid buffer (ie, a blank preservation solution of the new antibiotic, the amount added to the experimental group).
  • the new antibiotics have the same amount of liquid.
  • the second group added 5 ⁇ ⁇ / ⁇ 1 ampicillin
  • the third group added 5 g/ml PMC-AM1
  • the fourth group added 5 g/ml PMC-AM2
  • the fifth group added 10 /ml PMC-AMI.
  • the minimum inhibitory concentration (MIC) of the new antibiotic was determined by agar double dilution method.
  • the bacteria were inoculated on agar plates containing different drug concentrations using a multi-point inoculation instrument (De ne ley A400), with a bacterial content of 10 5 CFU/ml per point, and incubation at 37 ° C for 18-24 hours.
  • the lowest concentration of the drug contained in the growth plate medium is the minimum inhibitory concentration (MIC value) of the drug against the bacteria.
  • the strain used was multi-drug resistant Pseudomonas aeruginosa (Huaxi Hospital clinical isolate 13078), and the medium was MH medium (per hundred milliliters: beef extract 500 mg, casein hydrolyzate 1.75 g, soluble starch 150 mg, agar) 1.7 g).
  • the new antibiotic (D) (PMC-AM1) has a minimum inhibitory concentration of 8 g/ml for multi-drug resistant Pseudomonas aeruginosa, 16 g/ml for ceftazidime (A), and levofloxacin (B).
  • D The new antibiotic
  • PMC-AM1 has a minimum inhibitory concentration of 8 g/ml for multi-drug resistant Pseudomonas aeruginosa, 16 g/ml for ceftazidime (A), and levofloxacin (B).
  • ) 8 g/ml
  • gentamicin (C) is greater than 512 g/ml.
  • the minimum inhibitory concentration of PMC-AM1 against multi-drug resistant Pseudomonas aeruginosa is 0.23 nMoL ceftazidime is 29.3 nMoK levofloxacin is 43.2 nMoK gentamicin is greater than 890 nMol; BP, PMC-AM1 is resistant to multiple The antibacterial efficacy of Pseudomonas aeruginosa is stronger than that of cephedrine, levofloxacin, gentamicin and other active antibiotics 127-3800 times.
  • the minimum inhibitory concentration (MIC) of the new antibiotic was determined by agar double dilution method.
  • the bacteria were inoculated on the surface of agar plates containing different drug concentrations using a multi-point inoculation instrument (Deneley A400), with a bacterial content of 10 5 CFU/ml per point, and incubation at 37 ° C for 18-24 hours to observe the results without bacterial growth.
  • the lowest concentration of the drug contained in the medium is the minimum inhibitory concentration (MIC value) of the drug to the bacteria.
  • the strain used was multi-drug resistant Pseudomonas aeruginosa (Huaxi Hospital clinical isolate 13078), MH medium (500 mg per 100 ml of beef extract, 1.75 g of casein hydrolysate, 150 mg of soluble starch, 1.7 g of agar) ; methicillin-resistant Staphylococcus aureus (ATCC BAA-42), BM medium (1 g per 100 ml of tryptone, 0.5 g of yeast, 0.1 g of glucose, 1 g of NaCl, 100 mg of KH 2 P0 4 , ag of 1 g) ;; vancomycin-resistant enterococci (ATCC 700802), MH medium; meningococcus (Chinese Culture Preservation Center 29332), Petri's same as Example 2 (plus Columbia blood agar base 3.9 g).
  • the minimum inhibitory concentration (MIC) of the new antibiotic was determined by agar double dilution method.
  • the lowest concentration of the drug contained in the bacteria-free growth plate medium is the minimum inhibitory concentration (MIC value) of the drug against the bacteria.
  • the strains used were multi-drug resistant Pseudomonas aeruginosa (Huaxi Hospital clinical isolate 13078), methicillin-resistant Staphylococcus aureus (ATCC BAA-42), vancomycin-resistant enterococci (ATCC 700802), MH medium; meningitis Diplococcus (Chinese Culture Preservation Center 29332), Petri.
  • Meningococcal Choinese Center for Strain (Beijing Institute of Food and Drug Administration, Central Inspection Institute) 29332).
  • a total of 40 Kunming mice in the experimental group were divided into 4 experimental groups, 10 in each group.
  • ferrous ferrous solution (20 mg/kg) for 1 hour
  • 0.5 ml of bacterial solution was injected.
  • the bacterial liquid One case of meningococcal broth (CFU was 2.36 X 10 9 ) was composed of 1.5 parts of inactivated 5% dry yeast solution.
  • CFU meningococcal broth
  • One hour after intraperitoneal injection of a lethal dose of bacteria the drug was injected into the tail vein and the control saline (all drug injection concentrations were 1.5 mg/kg), and the results were observed every 2 hours for 8 consecutive days, with a positive result of mouse death.
  • 1 PMC-AM1 a novel antibiotic of the invention
  • 2 Gen gentamicin
  • 3 PEN penicillin
  • 4 Con control.
  • the mouse survival curve after intraperitoneal injection of lethal dose of meningococcus, 1), the control group died in 2 days, 2), the penicillin group died in 2 days, 3), gentamicin group
  • the 8-day survival rate was 50%, 4), and the 8-day survival rate of the novel antibiotic of the present invention was 90%.

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Description

技术领域
本发明涉及生物医药领域，特别涉及一种含抗体模拟物的新型抗生素及其制 备方法与应用。
背景技术
自 ： 1944年青霉素等抗生素投入使用以来， 不仅仅是脑膜炎双球菌， 其它很 多威胁生命的致病菌， 如金葡球菌、 肺炎链球菌、 绿脓杆菌、 结核杆菌都已对其 产生了耐药性。 据美国疾病控制中心 （CDC) 历年发表的有关报告预测， 再过 10年至 20年， 这些抗生素将可能完全失效。
目前常用的抗生素主要通过抑制细胞壁合成、抑制或干扰细菌的核酸和蛋白 质代谢与合成途径来达到抗菌目的。然而这些抗菌方式容易诱导细菌发生突变而 产生耐药性。因此人们一直在致力于开发新型的抗生素。模仿同种异株细菌之间 互相杀伤的工作方式来开发新型抗生素是比较有前途的方向之一。自然界中有不 少细菌毒素直接在细菌胞膜上形成离子通道来杀死细菌。其模式标本就是大肠杆 菌分泌的一种细菌毒素一大肠菌素。 其中大肠菌素 la自 1952年被 Jacob发现之 后， 经过数代人的努力， 1996年终于揭示了大肠菌素 la在人工脂质双分子膜上 所形成离子通道开放和关闭时的跨膜立体结构 （Qiu et al, Major transmemebrane movement soociated with colicin la channel gating. J. Gen. Physiology, 107:313-328 (1996) )， 为在分子水平上设计和制备新型的抗菌素奠定了理论基础。
如上所述， 大肠菌素是一种理想的离子通道抗生素原型， 但野生型大肠菌 素只能作用于同种异株的大肠杆菌， 因此必需改变它的靶向性， 才能使大肠菌素 转而攻击其它致病菌； 膜孔蛋白 Porin是存在于细菌质膜的外膜、 线粒体和叶 绿体的外膜上的通道蛋白,它们允许较大的分子通过,， 具有较强的免疫原性， 能在宿主体内诱导出特异性的高水平的单抗， 如能利用对抗细菌表面 Porin蛋 白的抗体作为原型， 设计出具有更理想识别性能的抗体模拟物， 用这个模拟物来 改变大肠菌素的靶向性， 应该是一种理想的抗生素开发方向。
发明内容
本发明针对上述领域的空白，提供一种的新型抗生素， 该抗生素的杀菌能力 是常用抗生素千倍以上， 由于其作用机理特殊， 因此致病菌很难产生耐药性， 杀 菌过程中不会伤害人体正常细胞。
一种含抗体模拟物的新型抗生素， 由大肠菌素 El、 Ia、 Ib、 A、 B、 N或其 水性孔道结构域及共价连接在所述大肠菌素或其水性孔道结构域的肽链羧基端 的抗体模拟物构成， 所述抗体模拟物是由免疫球蛋白的 VHCDR1 的羧基端连接 VHFR2的氨基端， VHFR2的羧基端再连接 VLCDR的氨基端构成；所述免疫球蛋 白特异性识别细菌膜孔蛋白。
所述细菌指脑膜炎双球菌， 所述膜孔蛋白指脑膜炎双球菌表面的膜孔蛋白 PorA。
所述免疫球蛋白指在具有 PUBMED 登录号为 2MPA_H所记载的氨基酸序 列的肽链与具有登录号为 2MPA_L所记载的氨基酸序列的肽链共价结合而得的 抗体蛋白。
所述大肠菌素为 Ia。
一种肽链分子， 其特征在于由大肠菌素 El、 Ia、 Ib、 A、 B、 N或其水性孔 道结构域的肽链及共价结合在其羧基端的抗体模拟物肽链连接而成，所述抗体模 拟物肽链由免疫球蛋白的三个区域 VHCDR1、 VHFR2、 VLCDR3的肽链顺次以羧 基端连接下一区域肽链的氨基端构成； 所述免疫球蛋白特异性识别细菌膜孔蛋 白。
所述肽链分子， 具有 Seq ID No.6所示氨基酸序列。
编码上述肽链分子的核苷酸序列。
所述核苷酸序列， 如 Seq ID No.5所示。
包含上述核苷酸序列的重组表达载体。
上述的新型抗生素的制备方法，指将上述的重组表达载体导入到表达***中 进行表达； 分离纯化表达的多肽获得抗生素。
所述的新型抗生素在制备抗细菌药物中的应用。
所述抗细菌药物指抗脑膜炎双球菌、抗耐万古霉素肠球菌、抗耐甲氧西林金 葡菌或抗多重耐药绿脓杆菌的药物。
本发明利用大肠菌素能在靶细菌膜上形成离子通道，使靶细胞内胞质泄漏而 死， 而本发明中起靶向性作用的结构是对抗细菌表面膜孔蛋白即 Porin蛋白的免 疫球蛋白的部分区域被重构后获得的模拟抗体物，包含该免疫球蛋白的重链可变 区的第一互补决定区 VHCDR1、 重链可变区的第二骨架区 VHFR2、 轻链可变区 的第三互补决定区 VLCDR3，三个区域顺次以羧基端链接下一区域的氨基端构成 VHCDR1-VHFR2-VLCDR3 的线性分子； 众所周知， 免疫球蛋白起识别作用的活 性区域称为互补决定区， 互补决定区仅有几个到十几个氨基酸组成， 与完整的免 疫球蛋白或者目前有报道的单链抗体 seFv、 Fab等人工改造抗体相比， 本发明的 分子量极小， 组织透过性好， 同时由于其结构简单， 去除了完整免疫球蛋白分子 的大部分框架结构及 Fc段， 使其对患者的免疫原性大大降低， 非常利于带领大 肠菌素到达致病部位识别致病菌。在治疗中，大肠菌素被引向致病菌细胞膜表面， 大肠菌素在靶细菌膜上形成离子通道，使靶细胞内胞质泄漏而死， 对于目前已经 产生耐药性的菌株也具有相同的杀菌能力。由于其识别位点为细菌表面特有的抗 原物质， 人体组织细胞膜上不存在这样的识别位点， 因此其对于人体是安全的。 相对于其他易于产生耐药性的抗生素，本发明的抗生素， 由于其起靶向作用的抗 体模拟物仅仅需将大肠菌素引向致病菌就完成了任务，我们并不需要通过膜孔蛋 白位点作用致死细菌， 而是通过大肠菌素作用于细菌的生物膜， 使生物膜产生离 子通道导致细胞泄漏而死，传统的细菌耐药性一般通过改变膜孔蛋白的结构对抗 生素进入细菌细胞内造成障碍而产生耐药性，而本发明仅仅需要膜孔蛋白的抗体 识别位点的活性即可到达杀菌的目的， 可以说， 是一种声东击西的策略， 即识别 膜孔蛋白位点， 但大肠菌素结合在细胞膜的另外一个位点形离子通道致死细菌， 真正的作用位点不在 Porin蛋白， 因此细菌很难通过变异、 进化而丢掉或改变膜 孔蛋白这种生存必须的结构， 因此细菌很难对本发明的抗生素产生耐药性。 由于 本发明根据不同细菌表面膜孔蛋白的多样性及与其识别的免疫球蛋白的多样性， 根据本发明这一设计构思获得的新型抗生素也是多样的。
由于脑膜炎双球菌引起的脑膜炎对国内外的婴儿以及青少年的健康造成了 极大的威胁， 且其对目前常用药的抗药性表现非常明显， 为了有效抑制该菌而使 药剂量越来越高， 这严重危害患者健康， 因此， 发明人采用本发明的构思， 针对 脑膜炎双球菌的膜孔蛋白 Porin A 的抗体， 其重链肽链的 PUBMED 登录号为 2MPA_H,轻链肽链的 PUBMED 登录号为 2MPA_L，进行重构获得抗体模拟物， 即如 Seq ID No.2所示的氨基酸序列； 并可操作地连接在大肠菌素 la的肽链的羧 基端， 获得具有如 Seq ID No.6所示的氨基酸序列的新型抗生素 PMC-AM1。 如 图 7 所示， 在脑膜炎双球菌致死量感染小鼠的存活实验中， 注射本发明抗生素 PMC-AM1的小鼠的 8天存活率为 90%，说明本发明的抗生素比现用抗生素青霉 素、庆大霉素等具有无法比拟的抗菌活性和体内保护效果； 同时， 通过杀菌效果 比较， 如图 6 A所示， 比较本发明的抗生素 PMC-AM1与头孢它啶、 氨苄青霉 素对脑膜炎双球菌的最低抑菌浓度（MIC值）， 实验结果显示 PMC-AM1的 MIC 0.11 nMol, 头孢它啶的 MIC 3.02 nMol, 氨苄青霉素的 MIC 1.35 nMol; 说明其 杀菌能力明显高于目前常用于抑制脑膜炎双球菌的抗生素。
发明人用本发明的新型抗生素 PMC-AM1 对其他目前产生耐药性严重的致 病菌进行试探性实验，发现 PMC-AM1还对多重耐药绿脓杆菌、耐万古霉素肠球 菌、 耐甲氧西林金葡菌有极强的抗菌效力， 如图 5所示， PMC-AM1对多重耐药 绿脓杆菌的杀菌效果比头孢它啶、 左氧氟沙星、 庆大霉素等现用抗菌素强 127〜 3800倍以上； PMC-AM1对耐万古霉素肠球菌、耐甲氧西林金葡菌有明显的抑制 效果， 如图 6B、 C所示。
本发明的抗生素可用于制备抗菌药物，特别是在制备抗脑膜炎双球菌、抗绿 脓杆菌、 抗耐万古霉素肠球菌及抗耐甲氧西林金葡菌的药物中的应用。
可将编码本发明抗生素的核苷酸克隆到表达载体中构建重组表达载体，这些 载体可在宿主中表达融合蛋白， 分离融合蛋白获得本发明的抗生素蛋白。
根据密码子的兼并性，编码本发明的抗生素或者抗体模拟物的核苷酸序列是 可调整的， 可根据宿主细胞对密码子的偏好性调整核苷酸序列， 只要编码的氨基 酸不变， 都属于本发明要求保护的范围。 附图说明
图 1.含抗体模拟物和大肠菌素 la的重组质粒 pBHC-PorAl的结构
其中抗体模拟物的肽链连接在 la的肽链羧基端， 抗体模拟物的氨基酸序列 如 Seq ID Νο·2所示。
图 2.含抗体模拟物肽链和大肠菌素 la的重组质粒 pBHC-PorA2的结构
其中抗体模拟物肽链连接在 la的羧基端， 抗体模拟物肽链是： 重链可变区 的第一互补决定区的羧基端链接重链第二骨架区，轻链可变区的第三互补决定区 的肽链羧基端氨基酸链接在重链第二骨架区的羧基端氨基酸上， 如 Seq ID No.4 所示。
图 3.本发明新型抗生素的结构
其中 T和 R是大肠菌素 la位于氨基端的两个信号识别结构域；
channel-forming是大肠菌素 la位于羧基端的形成离子通道结构域; AM是抗体模 拟物。
图 4.为本发明新型抗生素 PMC-AM1对脑膜炎双球菌的抑制实验
图中曲线从左到右依次为对照， 5 μ§/ηι1氨苄青霉素， 5 μ§/ηι1 PMC-AM2, 5 g/ml PMC- AMI, 10 /ml PMC-AMI。
本图横座标为细菌生长时间， 单位为小时； 纵座标为培养液 600 nm光密度, 显示细菌生长数量。
图 5.琼脂二倍稀释法测定新型抗生素的最低抑菌浓度 （MIC)
平皿显示： （Con) , 空白对照， （A)， 头孢它啶为 16 g/ml、 （B)， 左氧氟沙 星为 8 g/ml、 （C)， 庆大霉素大于 512 g/ml、 （D)， 新型抗生素 （PMC-AM1 )对 多重耐药绿脓杆菌的最低抑菌浓度为 8 g/ml。
图 6.本发明新型抗生素与常用抗菌素对耐甲氧西林金葡菌 （ATCC BAA-42)、 耐 万古霉素肠球菌 （ATCC 700802)、 多重耐药绿脓杆菌 （华西医院临床分离株 13578) 和脑膜炎双球菌 （中国菌种保存中心 29332) 最小抑菌浓度的比较实验 纵座标为最小抑菌浓度 （nMol);
其中 A 图为脑膜炎双球菌， （1 ) PMC-AMI , MIC = 0.11 nMol, (2) 头孢它 啶， MIC = 3.02 nMol, (3) 氨苄青霉素， MIC = 1.35 nMol;
B图为耐万古霉素肠球菌， （1 ) PMC-AMI , MIC = 0.23 nMol, (2) 万古霉素， MIC = 21.54 nMol, (3) 氨苄青霉素， MIC = 10.78 nMol;
C图为耐甲氧西林金葡菌， （1 ) PMC-AMI , MIC = 0.06 nMol, (2) 氨苄青霉素， MIC = 21.55 nMol, (3) 苯唑西林， MIC = 14.1 nMol;
D图为多重耐药绿脓杆菌， （1 ) PMC-AMI , MIC = 0.91 nMol, (2) 左氧氟沙星， MIC = 43.2 nMol, (3) 头孢它啶， MIC = 29.3 nMol, (4) 庆大霉素， MIC > 889.4 nMol。
图 7.本发明新型抗生素与野生型大肠菌素、 抗金葡菌多肽 （ZL 01128836.1 ) 对 耐甲氧西林金葡菌 （ATCC BAA-42)、 耐万古霉素肠球菌 （ATCC 700802)、 多 重耐药绿脓杆菌 （华西医院临床分离株 13578 ) 抑制作用的比较之生存曲线
纵座标为最小抑菌浓度 （nMol );
其中 A 图为耐万古霉素肠球菌， （1 )抗金葡菌多肽， MIC = 0.91 nMol, (2) 野生 型大肠菌素 la, MIC = 0.91 nMol, (3) PMC- AMI， MIC = 0.23 nMol;
B图为耐甲氧西林金葡菌， （1 ) 抗金葡菌多肽， MIC = 0.06 nMol, (2) 野生型大 肠菌素 la, MIC = 0.23 nMol, (3) PMC- AMI， MIC = 0.06 nMol;
C图为多重耐药绿脓杆菌， （1 ) 抗金葡菌多肽， MIC = 0.91 nMol, (2) 野生型大 肠菌素 la, MIC = 0.91 nMol, (3) PMC- AMI， MIC = 0.23 nMol。
图 8.本发明新型抗生素对脑膜炎双球菌感染动物的体内保护试验之生存曲线 横座标为鼠存活时间， 单位为天； 纵座标为动物存活数， 单位为每只鼠
1) PMC-AM1, 本发明新型抗生素； 2) Gen， 庆大霉素； 3 ) PEN, 青霉素； 4) Con. , 对照； 所有药物注射浓度均为 1.5mg/kg。 具体实施方式
结合附图， 通过本发明较佳实施例的描述具体说明本发明。
【实施例 1】 表达新型抗生素的质粒的构建和新型抗生素制备
原始质粒为装载了大肠菌素和 immunity蛋白基因的 pSELECTTM-l质粒（8.3 kb)。 经双链寡聚核苷酸点突变技术（QuickChange™Kit， Strategene公司）将编 码抗体模拟物基因，如 Seq ID No. l和 Seq ID No.3所述的核苷酸序列分别***到 大肠菌素变构多肽基因的 626 位点上， 制备了新型抗生素的突变质粒 pBHC-PorAK PBHC-PorA2 (如图 1一图 2所示）。 突变质粒转染入 E.co BL-21 工程菌里制备新型抗生素。
突变禾呈序按 Strategene QuickChange SiteDirected Mutagenesis Kit (catalog #200518)药箱手册进行： 即
1.准备点突变反应物：
5 ul 10X buffer
2ul (10 ng) 装载了大肠菌素变构多肽和 immunity蛋白基因的原始质粒 1.25 ul (125 ng)设计的 5'— 3'寡聚核苷酸引物 （见所列引物序列） 1.25 ul (125 ng)设计的 3'— 5'寡聚核苷酸引物 （见所列引物序列） 1 ul dNTP
双蒸水 50 ul
1 ul pfu
(除质粒、 引物和双蒸水外， 均为药箱所备试剂）
行 PCR扩增，扩增条件：变性 95°C, 35秒，退火 53 °C, 70秒，延伸 68 °C, 17 分， 共 20个循环；
入 Dpn 1内切酶 1 ul 消化母体 DNA链后 （37 °C, 1小时）， 取 1 ul反应物与 XLl-Blue感受态细胞 50 ul冰孵 30分钟， 热冲击 42°C， 45秒， 再置入冰中 2分钟；
入 NZY培基 0.5 ml, 220 rpm, 37 °C摇菌 1小时， 取 50— 100 ul 反应物铺板
(LB培基加 1 %琼脂， 加 50 ug/ml氨苄青霉素， 37 °C过夜）；
8 小时后挑菌， 提取质粒后测序确定突变成功；
突变质粒 100 ng 与制备的 BL-21工程菌感受态细胞 40 ul冰孵 5分钟，热冲 击 42°C， 30秒， 再置入冰中 2分钟， 加入 SOC培基 160 ul, 220 rpm, 37 °C摇 菌 1小时后铺板（LB培基加 1 %琼脂， 加 50 ug/ml氨苄青霉素， 37 °C过夜）， 挑取单克隆菌落大量增菌；
量增菌， 8— 10升 FB培基， 250 rpm, 30 °C, 3— 4小时；升温至 42°C, 250 rpm 生长 0.5小时； 降温至 37 °C, 250 rpm生长 1.5小时； 4°C， 6000g, 20分钟离 心沉淀菌体， 取 4°C， 50 mM硼酸缓冲液 （pH 9.0, 2 mMEDTA) 80-100 ml悬 浮菌体， 加入 PMSF 50 ug 后超声破碎菌体（4 °C， 400W, 1分钟， 重复 4一 5 次， 间歇 2— 3分钟确保菌液温度）， 高速离心沉淀破碎的菌体（4°C， 75,000g, 90分钟）， 取上清加入硫酸链霉素 500万单位沉淀 DNA (4 °C搅拌 1小时)， 10,000g, 4°C， 10分钟离心沉淀后， 取上清装入分子量 15,000透析袋于 4 °C， 50 mM硼酸缓冲液 10升透析过夜后， 再次 10,000g, 4°C， 10分钟离心沉淀， 取上清上样于 CM离子交换柱，充分冲洗后， 0.3 M NaCl + 50 mM硼酸缓冲液 洗脱即可得到所制备的新型抗生素。 对应于以上 2 种质粒， 可分别获得 PMC-AM1和 PMC-AM2两种抗生素，其氨基酸序列分别如 Seq ID No.6 、 Seq ID Νο·8所示。 其中 AMI是重链可变区的第一互补决定区、 重链第二骨架区、 轻链可变 区第三互补决定区，三个区域顺次以羧基端链接下一区域的氨基端，氨基酸序 列如 Seq ID No.2所示； AM2是重链可变区的第一互补决定区的羧基端链接重 链第二骨架区，轻链可变区的第三互补决定区的肽链羧基端氨基酸链接在重链 第二骨架区的羧基端氨基酸上， 氨基酸序列如 Seq ID No.4所示。
将 PMC-AM2作为 PMC-AM1 的对照， 验证本发明设计的抗体模拟物的 氨基酸区段之间在不同连接方式下产生的抗生素的功能。
上述制备质粒中所设计的寡聚核苷酸引物序列如下：
5，一 3， ( SEQ ID N0.9)
gcg aat aag ttc tgg ggt att TCT TAT TGG CTG CAT TGG ATT AAA CAG taa ata aaa tat aag aca ggc
3，一5， ( SEQ ID NO.10)
gcc tgt ctt ata ttt tat tta CTG TTT AAT CCA ATG CAG CCA ATA AGA aat acc cca gaa ctt att cgc
5，一3， ( SEQ ID NO.11 )
tgg ctg cat tgg att aaa cag AGA CCT GGT CAG GGA CTG TGG ATC GGA taa ata aaa tat aag aca ggc
3，一5， ( SEQ ID NO.12)
gcc tgt ctt ata ttt tat tta TCC GAT CCA CAG TCC CTG ACC AGG TCT ctg ttt aat cca atg cag cca
5，一3， ( SEQ ID NO.13 )
ggt cag gga ctg tgg ate gga TCT CAG TCC ACG CAT GTG CCG AGA ACC taa ata aaa tat aag aca ggc
3，一5， ( SEQ ID NO.14)
gcc tgt ctt ata ttt tat tta GGT TCT CGG CAC ATG CGT GGA CTG AGA tec gat cca cag tec ctg acc
pBHC-PorA 2
5，一3， ( SEQ ID NO.15 )
gcg aat aag ttc tgg ggt att TCT TAT TGG CTG CAT TGG ATT AAA CAG taa ata aaa tat aag aca ggc 3，一5， ( SEQ ID NO.16)
gcc tgt ctt ata ttt tat tta CTG TTT AAT CCA ATG CAG CCA ATA AGA aat acc cca gaa ctt att cgc
5，一3， ( SEQ ID NO.17 )
tgg ctg cat tgg att aaa cag AGA CCT GGT CAG GGA CTG TGG ATC GGA taa ata aaa tat aag aca ggc
3，一5， ( SEQ ID NO.18 )
gcc tgt ctt ata ttt tat tta TCC GAT CCA CAG TCC CTG ACC AGG TCT ctg ttt aat cca atg cag cca
5，一3， ( SEQ ID NO.19)
ggt cag gga ctg tgg ate gga ACC AGA CCG GTG CAT ACG TCC CAG TCT taa ata aaa tat aag aca ggc
3，一 5， ( SEQ ID NO.20)
gcc tgt ctt ata ttt tat tta AGA CTG GGA CGTATG CAC CGG TCT GGT tec gat cca cag tec ctg acc
【实施例 2】 新型抗生素对脑膜炎双球菌的抑制作用
细菌为中国菌种保存中心 29332 脑膜炎双球菌株， 菌液 2 微升 （105 CFU/ml)加入兔血巧克力培养液 10毫升（牛肉浸膏 50 mg、胰蛋白胨 100 mg、 KH2P04 30 mg NaCl 50 mg 脱纤维兔血 0.5-0.8 ml) 中， 共准备 5组， 第一 组加入 0.3 M NaCl + 50 mM硼酸缓冲液 （即新型抗生素的空白保存液， 量与 实验组中加入的新型抗生素液体量相同）作为对照， 第二组加入 5 μ§/ηι1氨苄 青霉素，第三组加入 5 g/ml PMC-AM1, 第四组加入 5 g/ml PMC-AM2,第五 组加入 10 /ml PMC-AMI。
上述各组液体分别置于 100毫升三角烧瓶中， 200 rpm, 37 °C生长， 每小时 采样 100微升加入 96孔酶表板中经分光光度计 （A 595nm) 比色测试细菌生 长浊度， 画出细菌生长曲线来比较新型抗生素的抑菌效力， 结果如图 4所示， 显示所试脑膜炎双球菌只能被 PMC-AM1所抑制。 【实施例 3】 新型抗生素对多重耐药绿脓杆菌的最低抑菌浓度与现用抗菌素的 最低抑菌浓度之比较。
采用琼脂二倍稀释法测定新型抗生素的最低抑菌浓度（MIC)。用多点接种 仪 （Deneley A400)将细菌接种于含不同药物浓度的琼脂平皿表面， 每点含菌量 为 105 CFU/ml， 37°C孵育 18-24小时观察结果， 以无细菌生长平皿培养基中所含 药物的最低浓度为药物对该菌的最低抑菌浓度 （MIC值）。
所用菌种为多重耐药绿脓杆菌 （华西医院临床分离株 13578)， 培养基为 MH 培养基（每百毫升： 牛肉浸膏 500 mg、酪蛋白酸水解物 1.75 g、可溶性淀粉 150 mg、 琼脂 1.7 g)。
结果如图 5所示， 新型抗生素 (D) (PMC-AM1 ) 对多重耐药绿脓杆菌的最低 抑菌浓度为 8 g/ml、 头孢它啶 (A)为 16 g/ml、 左氧氟沙星 (B) 为 8 g/ml、 庆大 霉素 (C) 大于 512 g/ml。若以分子量标化， PMC-AM1对多重耐药绿脓杆菌的最 低抑菌浓度为 0.23 nMoL 头孢它啶为 29.3 nMoK 左氧氟沙星为 43.2 nMoK 庆 大霉素大于 890 nMol; BP , PMC-AM1对多重耐药绿脓杆菌的抗菌效力强过头 孢它啶、 左氧氟沙星、 庆大霉素等现用抗菌素 127— 3800余倍。
【实施例 4】新型抗生素的体外抗菌活性与现用抗菌素的比较
采用琼脂二倍稀释法测定新型抗生素的最低抑菌浓度（MIC)。用多点接种仪 (Deneley A400)将细菌接种于含不同药物浓度的琼脂平皿表面， 每点含菌量为 105 CFU/ml, 37°C孵育 18-24小时观察结果， 以无细菌生长平皿培养基中所含药 物的最低浓度为药物对该菌的最低抑菌浓度 （MIC值）。
所用菌种为多重耐药绿脓杆菌（华西医院临床分离株 13578)， MH培养基（每 百毫升含牛肉浸膏 500 mg、酪蛋白酸水解物 1.75 g、可溶性淀粉 150 mg、琼脂 1.7 g); 耐甲氧西林金葡菌 （ATCC BAA-42) , BM 培养基 （每百毫升含胰蛋白 胨 1 g、 酵母 0.5 g、 葡萄糖 0.1 g、 NaCl 1 g、 KH2P04 100 mg、 琼脂 1 g); 耐 万古霉素肠球菌 （ATCC 700802), MH培养基； 脑膜炎双球菌 （中国菌种保存 中心 29332)， 培基同实施例 2 (另加哥伦比亚血琼脂基础 3.9 g)。
结果如图 6所示， A 图为脑膜炎双球菌， （1 ) PMC-AM1， MIC = 0.11 nMol, (2) 头孢它啶， MIC = 3.02 nMol, (3) 氨苄青霉素， MIC = 1.35 nMol; B图为耐万古霉 素肠球菌， ( 1 )PMC-AM1 , MIC = 0.23 nMol, (2) 万古霉素， MIC = 21.54 nMol, (3) 氨苄青霉素， MIC = 10.78 nMol; C图为耐甲氧西林金葡菌， （1 ) PMC- AMI , MIC = 0.06 nMol, (2) 氨苄青霉素， MIC = 21.55 nMol, (3) 苯唑西林， MIC = 14.1 nMol; D图为多重耐药绿脓杆菌， （1 ) PMC-AMI , MIC = 0.91 nMol, (2) 左氧 氟沙星， MIC = 43.2 nMol, (3) 头孢它啶， MIC = 29.3 nMol, (4) 庆大霉素， MIC > 889.4 nMol。
【实施例 5】新型抗生素的体外抗菌活性与抗金葡菌多肽和野生型大肠菌素 la 的比较
采用琼脂二倍稀释法测定新型抗生素的最低抑菌浓度 （MIC)。 以无细菌生 长平皿培养基中所含药物的最低浓度为药物对该菌的最低抑菌浓度 （MIC值）。
所用菌种为多重耐药绿脓杆菌 （华西医院临床分离株 13578)， 耐甲氧西林 金葡菌 （ATCC BAA-42) ，耐万古霉素肠球菌 （ATCC 700802)， MH培养基； 脑 膜炎双球菌 （中国菌种保存中心 29332)， 培基同实施例 4。
结果如图 7所示， A 图为耐万古霉素肠球菌， （1 )抗金葡菌多肽， MIC = 0.91 nMol, (2) 野生型大肠菌素 Ia， MIC = 0.91 nMol, (3) PMC- AMI , MIC = 0.23 nMol; B图为耐甲氧西林金葡菌， （1 ) 抗金葡菌多肽， MIC = 0.06 nMol, (2) 野生型大 肠菌素 la, MIC = 0.23 nMol, (3) PMC- AMI , MIC = 0.06 nMol; C图为多重耐药 绿脓杆菌， （ 1 )抗金葡菌多肽， MIC = 0.91 nMol, (2) 野生型大肠菌素 la, MIC = 0.91 nMol, (3) PMC-AMI， MIC = 0.23 nMol。
【实施例 6】新型抗生素的脑膜炎双球菌感染动物的体内保护试验
一.实验材料
( 1 ) 药物
PMC-AMK 庆大霉素、 青霉素。
(2) 细菌
脑膜炎双球菌 （中国菌种中心 （北京天坛国家药监局中检所） 29332)。
二.实验方法
如图 7所示， 实验组共 40只昆明小鼠， 分为 4个实验组， 每组 10只。 腹腔注射葡萄糖亚铁溶液 （20mg/kg) 1个小时后， 再注射 0.5 ml菌液。 该菌液由 1份脑膜炎双球菌培养液 （CFU为 2.36 X 109 ) 比 1.5份灭活 5%干酵母溶液组成。 腹腔注射致死剂量的细菌 1小时后，尾静脉注射药物以及对照生理盐水（所有药 物注射浓度均为 1.5mg/kg)， 每 2小时观察结果， 连续 8天， 以小鼠死亡为阳性 结果。
如图 7所示：1 PMC-AM1, 本发明新型抗生素 ； 2 Gen，庆大霉素； 3 PEN, 青霉素 ; 4 Con.， 对照。
三.结果
如图 7所示小鼠生存曲线， 腹腔注射致死剂量脑膜炎双球菌后， 1 )， 对照组 在 2天内全部死亡， 2)， 青霉素组在 2天内全部死亡， 3)， 庆大霉素组 8天存活 率为 50%， 4)， 本发明新型抗生素的 8天存活率为 90%。
结果显示， 对脑膜炎双球菌致死性感染， 本发明的新型抗生素 PMC- AMI 表现出了所试现用抗生素无法比拟的抗菌活性。

Claims

1. 一种含抗体模拟物的新型抗生素， 由大肠菌素 El、 Ia、 Ib、 A、 B、 N或其水 性孔道结构域及共价连接在所述大肠菌素或其水性孔道结构域的肽链羧基端 的抗体模拟物构成， 所述抗体模拟物是由免疫球蛋白的 VHCDR1 的羧基端连 接 VHFR2的氨基端， VHFR2的羧基端再连接 VLCDR的氨基端构成； 所述免 疫球蛋白特异性识别细菌膜孔蛋白。
2.根据权利要求 1所述的新型抗生素， 所述细菌指脑膜炎双球菌， 所述膜孔蛋白 指脑膜炎双球菌表面的膜孔蛋白 PorA。
3.根据权利要求 2所述的新型抗生素， 所述免疫球蛋白指在具有 PUBMED 登录 号为 2MPA_H所记载的氨基酸序列的肽链与具有登录号为 2MPA_L所记载的 氨基酸序列的肽链共价结合而得的抗体蛋白。
4. 根据权利要求 1或 2或 3所述的新型抗生素， 所述大肠菌素为 Ia。
5.—种肽链分子， 其特征在于由大肠菌素 El、 Ia、 Ib、 A、 B、 N或其水性孔道结 构域的肽链及共价结合在其羧基端的抗体模拟物肽链连接而成，所述抗体模拟 物肽链由免疫球蛋白的三个区域 VHCDR1、 VHFR2、 VLCDR3的肽链顺次以羧 基端连接下一区域肽链的氨基端构成；所述免疫球蛋白特异性识别细菌膜孔蛋 白。
6.根据权利要求 5所述肽链分子， 具有 Seq ID No.6所示氨基酸序列。
7.编码权利要求 5或 6所述肽链分子的核苷酸序列。
8.根据权利要求 7所述核苷酸序列， 如 Seq ID No.5所示。
9.包含权利要求 7或 8所述核苷酸序列的重组表达载体。
10.权利要求 1至 4任一所述的新型抗生素的制备方法， 指将权利要求 9所述的 重组表达载体导入到表达***中进行表达； 分离纯化表达的多肽获得抗生素。
11.权利要求 1至 4任一所述的新型抗生素在制备抗细菌药物中的应用。
12.根据权利要求 11所述的应用， 所述抗细菌药物指抗脑膜炎双球菌、 抗耐万古 霉素肠球菌、 抗耐甲氧西林金葡菌或抗多重耐药绿脓杆菌的药物。
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NZ598593A (en) 2013-07-26
EP2474558A4 (en) 2013-03-27
ZA201201386B (en) 2013-08-28
ES2538840T3 (es) 2015-06-24
MY161926A (en) 2017-05-15
SG178503A1 (en) 2012-03-29
CN101633699B (zh) 2012-02-15
IL218435A (en) 2016-04-21
AP2012006185A0 (en) 2012-04-30
CR20120094A (es) 2012-06-05
IL218435A0 (en) 2012-04-30
UA102325C2 (uk) 2013-06-25
EA201200417A1 (ru) 2012-07-30
HK1168113A1 (zh) 2012-12-21
AP3302A (en) 2015-06-30
EP2474558A1 (en) 2012-07-11
AU2010291640B2 (en) 2013-02-07
DK2474558T3 (en) 2015-06-15
EA028912B1 (ru) 2018-01-31
AU2010291640A1 (en) 2012-03-22
CN101633699A (zh) 2010-01-27

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