CN107365363A - Micromolecule polypeptide and its application - Google Patents

Micromolecule polypeptide and its application Download PDF

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Publication number
CN107365363A
CN107365363A CN201710855117.1A CN201710855117A CN107365363A CN 107365363 A CN107365363 A CN 107365363A CN 201710855117 A CN201710855117 A CN 201710855117A CN 107365363 A CN107365363 A CN 107365363A
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tubercle bacillus
micromolecule polypeptide
polypeptide
micromolecule
h37rv
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CN107365363B (en
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卫林
徐薇
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Suzhou University
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Suzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10033—Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory

Abstract

The present invention relates to a kind of micromolecule polypeptide, and the amino acid sequence of its mature peptide is as shown in SEQ ID No.1.The invention also discloses above-mentioned micromolecule polypeptide as the application in anti-mycobacterium tuberculosis medicine.The anti-mycobacterium tuberculosis micromolecule polypeptide in the mycobacteriophage source in the present invention has molecular weight small, it is low to synthesize cost, anti-mycobacterium tuberculosis H37Rv positive effect, also there is killing action to the H37Rv of rifampin-resistance, and other normal gram-positive bacteria, negative bacterium and fungies are not influenceed, there is preferable application prospect in antituberculotic is prepared.

Description

Micromolecule polypeptide and its application
Technical field
The present invention relates to biomedical sector, more particularly to a kind of micromolecule polypeptide and its application.
Background technology
Tuberculosis is listed in one of whole world serious infectious diseases.Tubercle bacillus is the pathogen for causing this sick, can be infected complete Body organ, but it is most commonly seen with pulmonary tuberculosis, it is a kind of serious infectious respiratory disease.Because conventional BCG vaccine is to pulmonary tuberculosis The ineffectivity of prevention, tuberculosis concurrent infection caused by the appearance of Drug Resistance for Tuberculosis bacterial strain and HIV, phthisical morbidity and dead Die and remain high at present, situation is extremely serious.There are 1/3 population infection tubercle bacillus, about 10% the infected performance in the whole world Go out clinical symptoms.It is annual all to have 9,000,000 newly-increased mycobacterium tuberculosis infection cases, there are 2,000,000 people to die from tuberculosis.Tuberculosis is still It is a current main infectious disease killer for threatening human health.
The World Health Organization, which advocates the line anti-tubercular drug used, at present isoniazid, rifampin, pyrazinamide, strepto- Element, ethambutol and thiacetazone etc..These medicines are although largely effective, but due to the use lack of standardization of antituberculotic in recent years Cause variation and the prevalence of drug resistant M bacterial strain, serious Mycobacterium tuberculosis drug-resistant sex chromosome mosaicism occur.Isoniazid mainly passes through Suppress somatic cells wall mycolic acid synthesis, cause cell wall defective and it is dead；Rifampin then with rely on DNA RNA polymerases β subunits strong bonded, prevent the enzyme to be connected with DNA, suppress the synthesis of bacteria RNA, so as to block rna transcription process, cause Make the synthesis of DNA and albumen stop and it is dead.Both Antibacterial Mechanisms suppress key cells knot in tubercle bacillus metabolic process The synthesis of structure component, this kind of Antibacterial Mechanism easily induce the drug resistance of microorganism.In in the past few decades, drug resistance knot is tackled The progress of core disease is very slow, only has a people to be come out by Accurate Diagnosis in every five drug resistance patients in global range.It is resistance to The appearance of medicine mycobacterium tuberculosis strain, extends the course of disease, and mortality prediction rises, it is also possible to can cause long-term infectiousness, expand drug resistance The chance of propagation, increase medical expense.With Resistance Mycobacterium Tuberculosis strain rapid growth and lack effective new anti-tubercular drug, urgently Biological species antituberculotic yet-to-be developed with unique bactericidal mechanism.
The unique antimicrobial mechanism of antibacterial peptide, become the focus of novel anti-infection medicament research and development on our times.Antibacterial Peptide is mainly directly incorporated in microbial cell film surface by its amphipathic helical structure and net positive charge interaction, changes Become the permeability of microbial cell film, cause microbial cell osmotic pressure to change, form perforation and cause cytoplasm to outflow. The sterilization mechanism is different from conventional antibiotic by suppressing the modes such as microbial metabolism, protein synthesis, therefore its sterilization speed Hurry up (what is had in the antibacterial peptide reported at present can play bactericidal effect in less than one minute, and as the anti-of positive control Raw element needs at least 3 hours), shorter sterilizing time hardly results in microorganism resistance.Antibiotic induces Microbiological release endogenous toxic material Element is so as to cause pyemic generation, and antibacterial peptide does not induce endotoxic release not only, can also neutralize endotoxin, does not induce purulence Toxication.Therefore, with antibacterial peptide suppress mycobacterium tuberculosis possess sterilization directly, speed is fast, is not easy the good spy of in-ductive drug -tolerance Property.
For bacteriophage (Bacteriophage) as a kind of obligatory parasitism in the virus of bacterium, having evolved one kind can be with With the unique mechanism of host bacteria interaction, abundant bioactive substance, including many active peptides and albumen are rich in, It is the good source storehouse for screening anti-mycobacterium tuberculosis polypeptide.Have been reported that at present from phage-coded polypeptide and egg In vain, bacterium can quickly be killed.Also the polypeptide in some bacteriophage sources and albumen can targeting in the metabolism of bacterium Journey, the effect of bacteria growing inhibiting.The genome of 26 kinds of pseudomonas aeruginosa bacteriophages is sequenced China researcher, 31 polypeptides are identified, these polypeptides can be with targeting in DNA duplication and RNA transcription, so as to suppress the life of bacterium Long and propagation.Need to find a variety of polypeptides and albumen for acting on tubercle bacillus at present, particularly find that some are effective small point Sub- polypeptide, outstanding template molecule is provided for research and development new bio class treating tuberculosis (auxiliary) medicine, while reduce polypeptide drugs research and development Cost.
The content of the invention
In order to solve the above technical problems, it is an object of the invention to provide a kind of micromolecule polypeptide and its application, the small molecule Polypeptide molecular weight is small, and synthesis cost is low, anti-mycobacterium tuberculosis positive effect, other bacteriums and fungi is not influenceed, anti-preparing There is preferable application prospect in tubercular drugs.
A kind of micromolecule polypeptide of the present invention, its amino acid sequence is as shown in SEQ ID No.1.Its molecular weight is 1827.25Da isoelectric point 12.03.It is named as AK15.
Further, the amino acid in sequence is L-type.
Further, AK15 micromolecule polypeptides derive from mycobacteriophage.
Further, mycobacteriophage is mycobacteriophage Che12 or mycobacteriophage Adzzy.
Further, AK15 micromolecule polypeptides are by mycobacteriophage Che12 genomes (NC_008203.1) Gp65 genes (GeneID:4156925) coding or mycobacteriophage Adzzy genomes (NC_022058.1) in ADZZY_66 genes (GeneID:16546204) encode.
Further, the precursor of AK15 micromolecule polypeptides forms (YP_655644.1, YP_ by 47 amino acid residues 008409360.1)。
The invention also discloses application of the above-mentioned AK15 micromolecule polypeptides in anti-mycobacterium tuberculosis medicine is prepared.
Further, tubercle bacillus is tubercle bacillus H37Rv or H37Ra.AK15 to tubercle bacillus toxic strain, avirulent strain all Show extremely strong antibacterial activity.The tubercle bacillus H37Rv that AK15 is resistant to rifampin also has antibacterial activity.
Further, AK15 does not influence on bacterium and fungi.Bacterium is staphylococcus aureus ATCC 6538, golden yellow The gram-positive bacterias such as color staphylococcus A TCC 25923, bacillus subtilis ATCC 6633 and Escherichia coli ATCC 25922nd, the Gram-negative bacteria such as Escherichia coli ATCC 35218, pseudomonas aeruginosa ATCC 27853.Fungi is Candida albicans Bacterium ATCC 10231, Candida albicans ATCC 2002 etc..
Further, AK15 micromolecule polypeptides are 37.5 μ g/ml to the minimal inhibitory concentration of tubercle bacillus.AK15 is to sharp good fortune The tubercle bacillus H37Rv of flat tolerance minimal inhibitory concentration is also 37.5 μ g/ml.
Further, AK15 micromolecule polypeptides play bactericidal effect when concentration is 37.5-200 μ g/ml.
Further, sterilizing time is less than 14 days.
Further, micromolecule polypeptide does not have hemolytic activity, to small in 0-200 μ g/ml concentration range to red blood cell Mouse peritoneal macrophage, RAW264.7 and HeLa cells do not have cytotoxicity.
By such scheme, the present invention at least has advantages below：
1st, the AK15 in the present invention is identified on the basis of infecting mycobacteria strategy based on mycobacteriophage Micromolecule polypeptide, source are natural.
2nd, the anti-mycobacterium tuberculosis micromolecule polypeptide AK15 in mycobacteriophage source has that molecular weight is small, and synthesis cost is low The advantages of.
3rd, the application the invention also discloses above-mentioned micromolecule polypeptide AK15 in as anti-mycobacterium tuberculosis medicine, it can Specific killing tubercle bacillus, it is equally effective to drug resistant M bacillus, and other normal bacterias and fungi are not influenceed, energy Enough change the form of tubercle bacillus cell membrane, membrane passage is changed, to mammalian cell safety, prepare resistive connection There is preferable application prospect in nuclear pharmaceuticals.
Described above is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention, And can be practiced according to the content of specification, below with presently preferred embodiments of the present invention and coordinate accompanying drawing describe in detail as after.
Brief description of the drawings
Fig. 1 is the test result that micromolecule polypeptide AK15 of the present invention is combined with tubercle bacillus cell；
Fig. 2 is morphology influence results of the micromolecule polypeptide AK15 of the present invention to tubercle bacillus cell membrane；
Fig. 3 is the laser co-focusing test knot that micromolecule polypeptide AK15 of the present invention influences on tubercle bacillus permeability of cell membrane Fruit.
Embodiment
With reference to the accompanying drawings and examples, the embodiment of the present invention is described in further detail.Implement below Example is used to illustrate the present invention, but is not limited to the scope of the present invention.
The AK15 of embodiment 1 preparation
Find coding mycobacteriophage Che12 (Mycobacterium phage Che12) genome (NC_ 008203.1) gene (GeneID of the gp65 in：Or mycobacteriophage Adzzy (Mycobacterium 4156925) Phage Adzzy) ADZZY_66 genes (GeneID in genome (NC_022058.1):16546204) one kind encoded is not Know the straight-chain polypeptide of function, its precursor forms (Mycobacterium phage Che12 by 47 amino acid residues：YP_ 655644.1 Mycobacterium phage Adzzy：YP_008409360.1), bioinformatics method pair is further utilized The polypeptide precursor carries out analysis prediction, and the complete of AK15 is synthesized with automatic Peptide synthesizer (433A, Applied Biosystems) Sequence, pass through HPLC reversed phase column chromatography desalting and purifyings.Using high-efficient liquid phase chromatogram HPLC method identification AK15 purity, using base Matter Assisted Laser Desorption ionization time of flight mass spectrometry (MALDI-TOF) determines its molecular weight, is determined with automatic Protein Sequencer Its amino acid sequence.
AK15 sequence is made up of 15 amino acid, and molecular weight is 1827.25 dalton, isoelectric point 12.03, its total order Row primary structure is as shown in SEQ ID No.1, wherein all amino acid are L-type.
The AK15 of embodiment 2 determines to tubercle bacillus minimal inhibitory concentration (MIC)
By tubercle bacillus H37Rv (ATCC 27294) and H37Ra bacterial strains containing the oleic acid of 0.05%Tween 80 and 10%, Albumin, glucose and catalase (oleic acid, albumin, dextrose, and catalase, OADC) increase bacterium Culture about 4 weeks, treat tuberculosis in the Middlebrook 7H9 broth bouillons (being purchased from Qingdao Rishui Biotechnology Co., Ltd.) of liquid Bacillus density growth reaches Ma Kefalanshi turbidity standard McFarland No.1 (about 3 × 107CFU/ml), bacterium solution is with containing 0.05%Tween 80 and 10%OADC enrichment liquids Middlebrook 7H9 broth bouillons are diluted to 1 × 105CFU/ml。
100 μ l 7H9 fluid nutrient mediums are previously added in sterile each hole of 96 orifice plate, 100 μ are then added in the first hole L is diluted to 800 μ g/ml AK15 sample solutions with 7H9 fluid nutrient mediums, takes 100 μ l to add the 2nd hole after mixing, successively multiple proportions Dilution suctions out 100 μ l (referring to table 1), from the 9th hole and discarded, the 10th hole system control tube.
The dilution process of table 1.
After the doubling dilution of AK15 polypeptide samples is good, the good tubercle bacillus bacterium solution of the 100 above-mentioned dilutions of μ l is added, by 96 orifice plates Place and cultivated 7 days at 37 DEG C, visually observe or light absorbs are determined at 600nm wavelength.Minimal inhibitory concentration is invisible thin The minimum sample concentration of bacteria growing.As a result it is as shown in table 2.
Minimal inhibitory concentrations of the mycobacteriophage polypeptide A K15 of table 2. to tubercle bacillus
From table 2, AK15 shows extremely strong antibacterial activity to tubercle bacillus toxic strain, avirulent strain, to toxic strain H37Rv minimal inhibitory concentration is 37.5 μ g/ml, and the minimal inhibitory concentration to avirulent strain H37Ra is 37.5 μ g/ml.
The AK15 of embodiment 3 determines to when m- bactericidal effect, the concentration-bactericidal effect of tubercle bacillus
Tubercle bacillus type strain H37Rv (ATCC27294) is inoculated into containing 0.05%Tween's 80 and 10%OADC In Middlebrook 7H9 broth bouillon, cultivated in 37 DEG C of incubator about 4 weeks and arrive exponential phase.With Bacterium solution is diluted to 1 × 10 by Middlebrook 7H9 broth bouillons6CFU/ml, sample is added in the bacterium solution diluted, made Its final concentration of 0.5 ×, 1 × and 2 × MIC, negative control (Control) adds isometric physiological saline.Add sample Bacterium solution is put into rapidly in 37 DEG C of incubators and cultivated, the physiology salt for taking 10 μ l bacterium solutions to sterilize respectively at 0 day, 3 days, 7 days and 21 days Water dilutes 1000 times, takes on 50 μ l coating Middlebrook 7H9 agar mediums, culture about 4 is inverted in 37 DEG C of incubators Week to bacterium colony is grown, and is taken out bacterium colony and is counted.
According to document, compared with the clump count (CFUs) when inoculation, if CFUs decrement >=3 × log after processing10, it is fixed Justice is the effect with sterilization.As a result as shown in table 3, concentration is 1 × MIC and 2 × MIC AK15 respectively at the 14th day and the 7th It reaches the effect of sterilization to tubercle bacillus.
The AK15 of the various concentrations of table 3. is incubated the CFUs countings after different time with tubercle bacillus altogether
The AK15 of embodiment 4 and the measure of tubercle bacillus cell binding ability
Incubated altogether at 37 DEG C with H37Ra with the control peptide of AK15 (FITC-AK15,2 μ g/ml) or the FITC mark of FITC marks After educating 5 minutes, free FITC-AK15 or FITC-control peptide are washed away with physiological saline, then use fluidic cell Instrument (BD FACSCanto TM II, BD Biosciences) detects.
As shown in figure 1, compared with PBS groups, the fluorescent absorption peak for the tubercle bacillus that FITC-AK15 is incubated altogether moves to right, Show that AK15 can be combined with tubercle bacillus cell, and the fluorescent absorption peak of FITC-control peptide treatment groups does not occur Substantially move to right, show that control peptide not can be incorporated on tubercle bacillus cell.
The AK15 of embodiment 5 destroys the form of tubercle bacillus cell
Tubercle bacillus H37Rv is transferred to the Middlebrook 7H9 meat containing 0.05%Tween 80 Yu 10%OADC In soup culture medium, cultivated in 37 DEG C of incubators and arrive exponential phase.1000g is centrifuged 5 minutes, with Middlebrook 7H9 meat Soup culture medium, which is washed twice and suspended, removes the secondary metabolite of tubercle bacillus.AK15 is added into bacterium solution makes its final concentration of 5 × MIC, continue culture 6 hours in 37 DEG C.Processing terminates rear 1000g and centrifuged 10 minutes, discards supernatant, precipitation is rapidly added 2.5% glutaraldehyde solution, gently blown and beaten with pipettor, the thalline for the precipitation that suspends, 4 DEG C fixed overnight.Consolidated again with 1% osmium tetroxide Determine 4 DEG C of liquid and fix 2 hours, take one group of thalline to be replaced after Gradient elution using ethanol in isoamyl acetate, critical point drying, vacuum Metal spraying plated film, uses scanning electron microscopic observation.
As a result as shown in Fig. 2 control group Control (PBS treatment groups) tubercle bacillus cellular morphology rule is clear, completely Cell regularly flocks together, and after AK15 processing tubercle bacillus H37Rv cells, tubercle bacillus cell membrane form generation changes Become, a large amount of tubercle bacillus cellular morphologies are fuzzy, thalline is shrivelled, eucaryotic cell structure collapses, thalline flocks together at random.
Changes of the AK15 of embodiment 6 to tubercle bacillus permeability of cell membrane
It is incubated altogether with FITC- glucans (250 μ g/ml, 150kDa) and AK15 (10 × MIC) and tubercle bacillus H37Ra, point After 30 minutes and 60 minutes, free FITC- glucans Fu Yu not be removed, be then total to laser with brines three times Focus on whether (Nikon A1) observation FITC- glucans enter bacterium intracellular.If FITC- glucans enter in bacterial cell, Show that tubercle bacillus membrane passage changes, FITC fluorescence intensity reflects what cell membrane penetration sexually revised indirectly The order of severity.
As a result as shown in figure 3, the green fluorescence that FITC- glucans are sent can be seen from figure.With control group Control The tubercle bacillus H37Ra of (PBS processing) is compared, and AK15 is promoted FITC- glucans in a manner of time dependent and enters tuberculosis bar Bacterium is intracellular.Fluorescence signal can be observed when being incubated 30 minutes altogether in tubercle bacillus H37Ra and AK15, and is observed at 60 minutes To stronger fluorescence signal, illustrate there are substantial amounts of FITC- glucans intracellular into tubercle bacillus, show tubercle bacillus H37Ra Membrane passage changed by AK15.
Comparisons of the tubercle bacillus H37Rv of embodiment 7 to rifampin and AK15 tolerances
Tubercle bacillus H37Rv is transferred to the Middlebrook 7H9 meat containing 0.05%Tween 80 Yu 10%OADC In soup culture medium, the AK15 or tubercle bacillus clinical first line therapy medicine of sub-MIC (1/8MIC) concentration are added in culture medium simultaneously Thing rifampin, cultivate in 37 DEG C of incubators, when exponential phase is arrived in tubercle bacillus culture (about 4 weeks), then transfer containing sub- Continue to cultivate in the AK15 of MIC (1/8MIC) concentration or the broth bouillon of rifampin.Under identical condition, turn by 16 times Connect, while control group is set, control group adds the DMSO of isometric dissolving polypeptide PBS and dissolving rifampin.
AK15 is as shown in table 4 to the exercising result of drug resistant M bacillus, by 16 switchings, control group PBS treatment groups Still sensitive to AK15 with the H37Rv of AK15 treatment groups, MIC does not change, is still 37.5 μ g/ml.Control group DMSO treatment groups H37Rv it is still sensitive to rifampin, MIC does not change.And the H37Rv of rifampin treatment group MIC increases from 0.012 μ g/ml The ratio for being added to 1.5625 μ g/ml, MIC (16)/MIC (0) is 128, shows H37Rv after rifampin induces, rifampin pair H37Rv minimal inhibitory concentration adds 128 times, the results showed that the tubercle bacillus of rifampin processing generates significantly to rifampin Drug resistance, and the H37Rv of AK15 treatment groups does not produce drug resistance to AK15.
Table 4.AK15 and the H37Rv of rifampin induction drug resistance compare
aMIC (0) refers to that AK15 and the rifampin MIC to untreated H37Rv, MIC (16) refer to AK15 and rifampin pair PBS, AK15, DMSO or rifampin handle 16 times after H37Rv MIC.
The Determination of Antibacterial Activity for the drug resistant M bacillus H37Rv that the AK15 of embodiment 8 is induced rifampin
According to the method in embodiment 2, AK15 is determined to bar described in embodiment 7 using minimal inhibitory concentration (MIC) method What is induced under part is resistant to tubercle bacillus H37Rv antibacterial functions to rifampin.
As shown in Table 5, the MIC value for the tubercle bacillus H37Rv that AK15 is resistant to rifampin is sensitive with rifampin for experimental result H37Rv it is identical, also show for 37.5 μ g/ml, the result, rifampin tolerance tubercle bacillus bacterial strain and rifampin it is untreated Mycobacterium tuberculosis strain is equally sensitive to AK15.
Minimal inhibitory concentrations of the table 5.AK15 to the tubercle bacillus H37Rv of rifampin-resistance
The specific measure of AK15 anti-mycobacterium tuberculosis of embodiment 9
AK15 derives from tubercle bacillus bacteriophage, and tubercle bacillus bacteriophage is using tubercle bacillus as host, in order to clear and definite Whether specific effect is in tubercle bacillus for the polypeptide in tubercle bacillus bacteriophage source, and AK15 is to gram as shown in table 6 for detection Positive bacteria, Gram-negative bacteria and the antibacterial activity of fungi.By test strain Luria-Bertani fluid nutrient mediums (Qingdao Day aquatic organism Technology Co., Ltd.) it is incubated overnight at 37 DEG C to logarithmic phase, then with fresh Mueller-Hinton liquid training Foster base is diluted to 1 × 105CFU/ml。
100 μ l Mueller-Hinton fluid nutrient mediums are previously added in sterile each hole of 96 orifice plate, then first The AK15 sample solutions that 100 μ l are diluted to 800 μ g/ml with Mueller-Hinton fluid nutrient mediums are added in hole, are taken after mixing 100 μ l add the 2nd hole, successively doubling dilution (referring to table 1), and suctioning out 100 μ l from the 9th hole discards, the 10th hole system control tube.
After the doubling dilution of AK15 polypeptide samples is good, the good tubercle bacillus bacterium solution of the 100 above-mentioned dilutions of μ l is added, by 96 orifice plates 37 DEG C of culture 18h are placed, visually observes or light absorbs is determined at 600nm wavelength.Minimal inhibitory concentration is invisible bacterium The minimum sample concentration of growth.
As a result as shown in table 6, AK15 concentration is blue to the gram-positive bacteria in table 6, leather in the range of 0-200 μ g/ml Family name's negative bacterium and fungi do not detect antibacterial activity.Result above shows the AK15 polypeptid specificities in mycobacteriophage source Anti-mycobacterium tuberculosis.
Antibacterial functions of the mycobacteriophage polypeptide A K15 of table 6. to other bacterial strains
Measure of the AK15 of embodiment 10 to mammalian cell security
1st, hemolytic activity determines
People, rabbit and the mouse blood brine of fresh collection twice and dilute, by physiological saline (blank control), A series of AK15 polypeptides that Qula leads to (Triton X-100, positive control) and doubling dilution be added separately to have diluted it is red carefully In born of the same parents' suspension, 30min, 1000rpm centrifugation 5min are incubated in 37 DEG C of incubators, supernatant detects absorbance value in 540nm, according to Equation below calculates AK15 hemolysis rate：H%=(ASample-ABlank control) * 100%/APositive control。
As a result show, in 0-200 μ g/ml concentration range, AK15 does not detect molten to the red blood cell of people, rabbit and mouse Blood activity.
2nd, CTA
C57BL/6 Turnover of Mouse Peritoneal Macrophages, RAW264.7 and HeLa cells are laid in 96 well culture plates respectively (2 × 104Cells/well), with the cell culture added with 2% hyclone, 100U/ml ampicillins and 100 μ g/ml streptomycin sulphates Base (100 μ l/ holes, buying from Gbico companies of the U.S.) is cultivated, wherein peritoneal macrophage and RAW264.7 cells RMPI- 1640, HeLa cells are cultivated with DMEM, after cell attachment, are separately added into a series of AK15 polypeptides of doubling dilution, are co-cultured After 24 hours, cell propagation is added per hole and (10 μ l/ holes, is bought fertile more limited than gloomy science and technology from Beijing with toxicity detection reagent C CK-8 Company), after being incubated 4 hours, light absorbs at 450nm are detected, the cell viability that the solvent (PBS) for dissolving polypeptide is handled is defined For 100%, and calculate the cell viability after various concentrations AK15 processing.
As a result show, AK15 concentration is thin to Turnover of Mouse Peritoneal Macrophages, RAW264.7 and HeLa in 0-200 μ g/ml The cell of born of the same parents is not detected by cytotoxicity.
Above AK15 hemolytic activity and the measurement result of cytotoxicity show, in the scope that concentration is 0-200 μ g/ml Interior, AK15 is safe to mammalian cell.
Described above is only the preferred embodiment of the present invention, is not intended to limit the invention, it is noted that for this skill For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is some improvement and Modification, these improvement and modification also should be regarded as protection scope of the present invention.
Sequence table
<110>University Of Suzhou
<120>Micromolecule polypeptide and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213>（Artificial sequence）
<400> 1
Ala Lys Lys Lys Leu Ser Arg Trp Trp Leu Arg Gly Ala Val Lys
1 5 10 15

Claims (10)

  1. A kind of 1. micromolecule polypeptide, it is characterised in that：Its amino acid sequence is as shown in SEQ ID No.1.
  2. 2. micromolecule polypeptide according to claim 1, it is characterised in that：Amino acid in sequence is L-type.
  3. 3. micromolecule polypeptide according to claim 1, it is characterised in that：The AK15 micromolecule polypeptides derive from branch bar Bacterium bacteriophage.
  4. 4. micromolecule polypeptide according to claim 3, it is characterised in that：The mycobacteriophage is bitten for mycobacteria Thalline Che12 or mycobacteriophage Adzzy.
  5. 5. application of the micromolecule polypeptide in anti-mycobacterium tuberculosis medicine is prepared according to any one of claim 1-4.
  6. 6. application according to claim 5, it is characterised in that：The tubercle bacillus is tubercle bacillus H37Rv or H37Ra.
  7. 7. the application according to claim 5 or 6, it is characterised in that：The micromolecule polypeptide presses down to the minimum of tubercle bacillus Bacteria concentration is 37.5 μ g/ml.
  8. 8. application according to claim 6, it is characterised in that：The micromolecule polypeptide is 37.5-200 μ g/ml in concentration Shi Fahui bactericidal effects.
  9. 9. application according to claim 8, it is characterised in that：Sterilizing time is less than 14 days.
  10. 10. application according to claim 5, it is characterised in that：The concentration of the micromolecule polypeptide is below 200 μ g/ml When, there is no hemolytic activity to red blood cell.
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CN108218962A (en) * 2017-12-29 2018-06-29 广西中医药大学 Micromolecule polypeptide and application thereof

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