CN110386972A - Antibacterial polypeptide Pb2-1 or PCL-1 and its preparation method and application - Google Patents
Antibacterial polypeptide Pb2-1 or PCL-1 and its preparation method and application Download PDFInfo
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Abstract
The invention discloses antibacterial polypeptide Pb2-1 or PCL-1 and its preparation method and application, and the amino acid sequence of antibacterial polypeptide Pb2-1 and PCL-1 are respectively as shown in SEQ ID NO:1 and 2.The present invention is from the peptide C ATHPb2 in the source Burma boa Python bivittatu, pass through structure activity study, obtain the high polypeptide Pb2-1 of alpha-helix degree, and it is covalently attached one section of 11 cyclic peptide with trypsin inhibition activity at its end N-, obtain the new construction polypeptide PCL-1 of 24 amino acid composition.The present invention prepares resulting polypeptide Pb2-1 and PCL-1, and the alpha-helix degree and hydrophobicity of drug effect module improve；In addition, 11 loop peptide sequences of PCL-1 connection make polypeptide while with good antibacterial activity in vitro, the stability of trypsase is also significantly improved, can be applied to the treatment of bacterium and fungal infectious disease.
Description
Technical field
The invention belongs to the polypeptide drugs technical fields in biochemistry, and in particular to antibacterial polypeptide Pb2-1 or PCL-1 and
Preparation method and application.
Background technique
Since nineteen twenty-eight penicillin is found, people progress into control and treat the epoch of bacterial infection disease.So
And the abuse of antibiotic accelerates the evolution of pathogenic bacteria, so that a large amount of drug-resistant bacterias and multiple drug resistant bacteria constantly went out in recent years
It is existing.Since the research and development of new antibiotic do not accelerate with increasing sharply for drug-resistant bacteria, and since antibiotic economy is imitated
Benefit is low, and declaration difficulty is big, and many drugmakers have all exited the research and development of antibiotic medicine, leads to the discovery of antibiotic medicine year by year
Slow down.The appearance of multiple drug resistant bacteria and the shortage of antibiotic medicine are so that searching novel antibacterial drug is extremely urgent.
Antibacterial peptide is the important component of most of organism innate immune defence mechanism, is generally deposited in nature
The antibacterial peptide number found from animal, plant, microorganism at present is up to more than 2900 kinds.Although antibacterial peptide source and sequence
It is different, but there are some general character in structure, and they are generally made of 10~60 amino acid residues, and natural antibacterial peptide is logical
Often contain lysine and arginine, peptide molecule electropositive can be assigned in conjunction with hydrogen ion in water, so common antibacterial peptide is equal
For cationic polypeptides, secondary structure is commonly divided into α-helixstructure, beta sheet structure and extended configuration.Most of antibacterials
Peptide has the antibacterial activity of wide spectrum, all has to Gram-negative bacteria, gram-positive bacteria, protozoon, fungi even virus good
Lethal effect.In addition to possessing good antibacterial activity, antibacterial peptide also has antitumor, immunological regulation and promotion wound healing etc.
Effect.The unique film of cationic antibacterial peptide destroys sterilization mechanism and is not easy inducing pathogen generation drug resistance, this is anti-relative to tradition
Raw element has very big advantage, is expected to the novel substitute as clinical treatment infectious diseases；But its poor stability, especially
It is to be restricted it in clinical application the unstability of trypsase.In recent years, the stability problem of polypeptide drug
Become the hot spot studied both at home and abroad.
Summary of the invention
Goal of the invention: in view of the problems of the existing technology, the present invention provides antibacterial polypeptide Pb2-1 or PCL-1, the present invention
Polypeptide have good inside and outside antibacterial activity, and to trypsase have preferable stability, can be applied to various
Common infection and obstinate infectious diseases, excellent alternative medicine or ancillary drug as existing antibiotic.
The present invention also provides the preparation method and application of antibacterial Pb2-1 or PCL-1.
Technical solution: to achieve the goals above, a kind of antibacterial polypeptide Pb2-1 as described herein, amino acid sequence
For SEQ ID NO:1:GFRKFMRRLKKFF.
Antibacterial polypeptide PCL-1 of the present invention, amino acid sequence are SEQ ID NO:2:
CWTKSIPPKPCGFRKFMRRLKKFF。
The preparation method of antibacterial polypeptide Pb2-1 of the present invention, includes the following steps:
Using peptide C ATHPb2 as template, the region of its sequence preservative and alpha-helix high concentration is intercepted, from the end N-
3rd starts interception and polypeptide amino acid number is reduced to 13 by 29, obtains polypeptide Pb2-1.
The preparation method of antibacterial polypeptide PCL-1 of the present invention, includes the following steps:
(1) using peptide C ATHPb2 as template, the region of its sequence preservative and alpha-helix high concentration is intercepted, from
The 3rd, the end N- starts interception and polypeptide amino acid number is reduced to 13 by 29, obtains polypeptide Pb2-1；
(2) it is 11 cyclic peptide of CWTKSIPPKPC (SEQ ID NO:3) by a sequence, covalently connects with the end N- of Pb2-1
It connects, obtains the polypeptide PCL-1 with intramolecular disulfide bond.
Wherein, the sequence C WTKSIPPKPC is disulfide bond cyclization, 11 cyclic peptide with trypsin inhibition activity.
11 cyclic peptide of the invention only have trypsin inhibition activity, without any antibacterial activity.
The antibacterial polypeptide Pb2-1 of the present invention or antibacterial polypeptide PCL-1 is preparing anti-microbial pathogen infection medicine
Application in object.
Wherein, the anti-microbial pathogen infection medicine includes antibacterium and fungal infection drug.
Further, the bacterium is staphylococcus aureus, methicillin-resistant staphylococcus aureus (MRSA), epidermis
Staphylococcus, micrococcus scarlatinae, streptococcus pneumonia, pseudomonas aeruginosa, escherichia coli, shigella dysenteriae and kerekou pneumonia primary
Bacterium；Fungi is Candida albicans and neogenesis cryptococcus.
Wherein the synthesis of antibacterial polypeptide Pb2-1 and PCL-1 is using conventional polypeptide solid-state reaction method synthesis.
Pathogenic bacteria and pathogenic epiphyte of the present invention, comprising: the reference culture in the source ATCC and be clinically separated
Conventional bacterial strain.
Antibacterial polypeptide Pb2-1 or PCL-1 of the invention with peptide C ATHPb2 (SEQ ID NO:4:
It KRNGFRKFMRRLKKFFAGGGSSIAHIKLH is) template, CATHPbs is by Burma's boa Python bivittatu lung tissue
The cathelicidins family cationic polypeptide of middle extraction, totally six polypeptides, CATHPb2 is wherein one, by 29 amino acid
Composition has antibacterial effect.The present invention passes through structure activity study, 13 amino acid preferable to the alpha-helix degree at its end N-
Residue is intercepted, though resulting polypeptide Pb2-1 positive changes are reduced, alpha-helix degree, hydrophobicity and hydrophobic torque are improved,
Amino acid quantity is greatly decreased, and reduces the cost of Peptide systhesis, while having stronger antibacterial effect compared to CATHPb2；It is logical
It crosses and is covalently attached the end N- of 11 cyclic peptide and Pb2-1, it is more that the present invention also obtains the antibacterial that trypsinase stability is substantially improved
Peptide PCL-1.Polypeptide PCL-1 in the present invention not only has good antibacterial activity in vitro, but also has to trypsase preferable
Stability, can be applied to various common infections and obstinate infectious diseases, the excellent alternative medicine as existing antibiotic
Or ancillary drug.
Antibacterial polypeptide Pb2-1 of the invention is by intercept to its n terminal amino acid using peptide C ATHPb2 as template
It arrives, while being covalently attached one section of 11 cyclic peptide with trypsin inhibition activity in the end N- of Pb2-1 and obtaining PCL-1, use
Solid-phase synthesis obtains polypeptide Pb2-1 and PCL-1.Antibacterial polypeptide Pb2-1 and PCL-1 of the present invention, amino acid sequence such as SEQ ID
Shown in NO.1 and 2.
The transformation of antibacterial polypeptide PCL-1 of the invention is the design carried out using CATHPb2 as template, from the N- of CATHPb2
It holds the 3rd to start 13 amino acid of interception and first obtains Pb2-1, and be covalently attached one section at the end sequence N- of interception there is tryptose
11 cyclic peptide of enzyme inhibition activity are finally reached the purpose that trypsinase stability is improved while maintaining antibacterial activity.Total order
It is classified as cysteine-tryptophan-threonine-lysine-serine-isoleucine-valine-proline-lysine-dried meat ammonia
Acid-Cysteine-Glycine-Phe-A taug-lysine-phenylalanine-methionine-Arg-Arg-is bright
Propylhomoserin-lysine-lysine-phenylalanine-phenylalanine.
In vitro Bactericidal Experiments the result shows that, antibacterial polypeptide Pb2-1 of the invention passes through interception 13 of the end CATHPb2-N
Amino acid obtains, and has better broad spectrum antibiotic activity compared to female Peptide C ATHPb2, and cost substantially reduces.Of the invention is anti-
Bacterium polypeptide PCL-1 is remained under conditions of intercepting 13 amino acid at the end CATHPb2 N- and being covalently attached with 11 cyclic peptide
Keep preferable broad spectrum antibiotic activity.The antibacterial polypeptide PCL-1 phase that 11 cyclic peptide obtain is connect with the N- terminal covalent in Pb2-1
Than connecting the polypeptide Pb2-1TI (GFRKFMRRLKKFFCWTKSIPPKPC) that 11 cyclic peptide obtain in the C- terminal covalent of Pb2-1
It is weaker to the antibacterial activity of each strain bacterium or fungi.Simultaneously killing curve the result shows that, polypeptide PCL-1 is to Staphylococcus aureus
Bacterium, escherichia coli and neogenesis cryptococcus clinical separation strain all show good bactericidal activity, can be uncommon by large intestine angstrom in 8h
Bacterium and neogenesis cryptococcus are killed, and more significant to the bactericidal effect of staphylococcus aureus, can be killed its whole in 1h
It goes out.
In vitro toxicity studies have shown that antibacterial polypeptide Pb2-1 and PCL-1 of the invention is low to sheep red blood cell (SRBC) hemolytic activity,
Hemolytic activity can be ignored in the effective range of Pb2-1 and PCL-1.
The vitro stability of antibacterial polypeptide Pb2-1 and PCL-1 prepared by the present invention is studied by HPLC and antibacterial tests.Knot
Fruit shows that under condition of different pH and different temperatures, the surplus of Pb2-1 and PCL-1 can protect all there is no significantly changing
Structural intergrity is held, illustrates it with pH stability and thermal stability；When NaCl concentration 200mM and it is following when, Pb2-1 and
PCL-1 remains unchanged the antibacterial activity of escherichia coli, illustrates that Pb2-1 and PCL-1 also can under high salt concentration ionic conditions
It is preferable to retain antibacterial activity；PCL-1 after being covalently attached with 11 cyclic peptide still has higher after being incubated for 1h with trypsase
Integrality, 4 times are improved to the MIC value of escherichia coli.
The utility model has the advantages that compared with prior art, the present invention has the advantage that
1, antibacterial polypeptide Pb2-1 prepared by the present invention has better broad spectrum antibiotic activity relative to peptide C ATHPb2, and
Cost substantially reduces.
2, antibacterial polypeptide PCL-1 prepared by the present invention is while with broad spectrum antibiotic activity, compared to mutant peptide Pb2-
1, trypsinase stability significantly improves.
3, the design preparation method of antibacterial polypeptide Pb2-1 and PCL-1 of the present invention is simple and convenient, novel in design, raw material sources
It is easy to get, it can be with industrialized production and application.
4, antibacterial polypeptide Pb2-1 and PCL-1 prepared by the present invention can be applied in preparation antibacterium and anti-fungal infection medicine
In object, there is good inside and outside antibacterial activity；Wherein, have trypsin inhibition activity living without antibacterial at one section of connection
Property 11 cyclic peptide after, PCL-1's still has excellent antibacterial activity, while having obtained to the stability of trypsase very big
Promotion, can be applied to various common infections and obstinate infectious diseases, as existing antibiotic excellent alternative medicine or
Ancillary drug.
Detailed description of the invention
Fig. 1 is the HPLC map of antibacterial polypeptide PCL-1 of the present invention；
Fig. 2 is the mass spectrogram of PCL-1；
Fig. 3 is the HPLC map of antibacterial polypeptide Pb2-1 of the present invention；
Fig. 4 is the mass spectrogram of Pb2-1；
Fig. 5 is the killing curve figure of PCL-1, Pb2-1 to staphylococcus aureus；
Fig. 6 is the killing curve figure of PCL-1, Pb2-1 to escherichia coli；
Fig. 7 is the killing curve figure of PCL-1, Pb2-1 to neogenesis cryptococcus；
Fig. 8 is the hemolytic activity figure of PCL-1, Pb2-1 to sheep red blood cell (SRBC)；
Fig. 9 is the stability quantization figure of PCL-1, Pb2-1 to temperature；
Figure 10 is the stability quantization figure of PCL-1, Pb2-1 to pH；
Figure 11 is the stability quantization figure of PCL-1, Pb2-1 to trypsase；
Figure 12 is influence of the PCL-1 to bacterial number in infection due to Escherichia coli mouse blood.
Specific embodiment
The invention will be further described with attached drawing with reference to embodiments.
Embodiment 1
The preparation of antibacterial polypeptide Pb2-1, PCL-1:
(1) using peptide C ATHPb2 as template, the region of its sequence preservative and alpha-helix high concentration is intercepted, from
The 3rd, the end N- starts interception and polypeptide amino acid number is reduced to 13 by 29, obtains polypeptide Pb2-1；
It (2) is CWTKSIPPKPC by a sequence, disulfide bond cyclization, 11 cyclic peptide with trypsin inhibition activity,
It is covalently attached with the end N- of Pb2-1, obtains the polypeptide PCL-1 with intramolecular disulfide bond.
(3) solid-phase synthesis synthesis polypeptide Pb2-1 and PCL-1.
The amino acid sequence of the Pb2-1 of preparation are as follows: Gly-Phe-arginine-lysine-phenylalanine-first sulphur
Propylhomoserin-arg-arg-leu-lysine-lysine-phenylalanine-phenylalanine.
The amino acid sequence of the PCL-1 of preparation are as follows: the cysteine-different bright ammonia of tryptophan-threonine-lysine-serine-
Acid-Pro-Pro-Lys-Pro-Cysteine-Glycine-Phe-A taug-lysine-phenylpropyl alcohol ammonia
Acid-methionine-arg-arg-leu-lysine-lysine-phenylalanine-phenylalanine.
The synthesis of linear polypeptide Pb2-1 or PCL-1: after C-terminal to N-terminal carries out one by one.Rink amide AM Resin is used
Methylene chloride impregnates 15min and pumps methylene chloride to resin expansion；Hexahydropyridine/DMF solution that volume ratio is 1:4 is added
(every gram of resin 10ml), is agitated using nitrogen, is reacted 2 times, and the time is 5min and 15min, washs resin with DMF after reaction
9 times.Take 20~40 resins that each 2-3 drop of toner ABC (A liquid: ninhydrin/ethanol solution is added；B liquid: pyridine；C liquid:
Phenol/ethanol solution, A, B, C liquid add 2-3 drop) the heat together 3min at 100 DEG C, solution and color of resin become blue
Color, to remove amido protecting.The Fmoc-Phe-OH and HOBT of twice of molal quantity of excessive response is added, with every gram of resin 10ml's
DMF dissolution, is added the DIC and Collidine of twice of molal quantity, and nitrogen is agitated, and reacts 1h.Tree is washed with DMF after reaction
Rouge 6 times, repeat condensation reaction, be sequentially connected each Fmoc protected amino acid, complete the synthesis of linear sequence, by resin with two
Chloromethanes and ether are drained after impregnating.TFA is added, reacts 2h, shaking speed 110r/min in constant-temperature table, 25 DEG C of temperature.
Resin is filtered off, anhydrous ether is added into filtrate, solid is obtained after centrifugation, anhydrous ether washing is added, then be centrifuged, is repeated several times
Drying can be obtained crude product polypeptide afterwards.
The cyclisation of polypeptide PCL-1 disulfide bond: crude product polypeptide is dissolved with pure water, concentration 1mg/ml.It is adjusted with weak aqua ammonia
PH to 8-9, normal-temperature reaction is for 24 hours.Reaction end is detected with HPLC and MASS.
The purifying of polypeptide: a certain amount of polypeptide Pb2-1 or PCL-1 aqueous solution filter is taken to remove large granular impurity.Together
Shi Caiyong preparative liquid chromatography instrument crosses column, and sample is collected in segmentation.Gradient analysis is done with analytical chromatograph, is up to required purity
Sample is retained.Then it carries out freeze-drying process and obtains polypeptide Pb2-1 or PCL-1.
Embodiment 2
The purity testing (HPLC method) and mass spectrometry results of polypeptide Pb2-1 and PCL-1:
Finished product is obtained through purifying after embodiment 1 polypeptide Pb2-1 and PCL-1 synthesis, finished product is through high performance liquid chromatography and mass spectrum
It is identified.
Liquid-phase chromatographic analysis condition: C18 chromatographic column (4.6 × 250mm, 5 μm)；Mobile phase A is containing 0.1% trifluoroacetic acid
Acetonitrile solution, Mobile phase B are the pure water containing 0.1% trifluoroacetic acid.Detection wavelength is 220nm；Flow velocity is 1.0ml/min；Into
10 μ l of sample amount carries out gradient elution.Elution requirement is linear gradient increasing concen-trations: when organic phase initial concentration is 27%, 25min
It is incremented to 52%, 25min to 30min and rinses chromatographic column with 100% organic phase.
The purity of polypeptide PCL-1 and Pb2-1 that it can be seen from attached drawing 1,3 prepared by the embodiment of the present invention 1 are all larger than
98%.
It is 1761.23 by the molecular weight that the molecular weight of 2,4 it was determined that PCL-1 of attached drawing is 2999.78, Pb2-1, with reason
It matches by value.
Embodiment 3
The measurement of polypeptide Pb2-1 and PCL-1 antibacterial activity in vitro of the present invention
(1) recovery and activation of strain
By freeze in -20 DEG C experiment with strain from glycerol tube be forwarded to corresponding agar slant culture-medium (bacterium transfer
Sabouraud's dextrose agar inclined-plane is forwarded to nutrient agar slopes, fungi) in.Bacterium is placed in 37 DEG C of cultures for 24 hours, and fungi is placed in 28
DEG C culture 48h, is placed spare in 4 DEG C of refrigerator.
(2) preparation of bacterium solution
The thallus on a small amount of inclined-plane is taken, is forwarded in the corresponding fluid nutrient medium of 2ml, bacterium is placed in 37 DEG C, fungi is placed in
28 DEG C of incubator culture 8h, are diluted to 10 with respective liquid culture medium5The bacteria suspension of CFU/ml, it is spare.
(3) preparation of drug
The polypeptide Pb2-1 or PCL-1 for weighing the preparation of embodiment 1 are dissolved separately in physiological saline, it is configured to 1024 μ
The mother liquor of g/ml, through 0.22 μm of water phase filter filtration sterilization, the packing of EP pipe is stored in -20 DEG C, spare.
(4) polypeptide Pb2-1 and PCL-1 is to the minimum inhibitory concentration (MIC) of test strain and minimum bactericidal concentration (MBC)
Measurement.
MIC with the MBC detection method of two polypeptides is identical.I.e. polypeptide drugs are with fluid nutrient medium doubling dilution at different dense
After spending gradient, respectively take 50 μ l in 96 orifice plates, and be inoculated with the 10 of 50 μ l in equal volume5The bacteria suspension of CFU/ml makes drug final concentration
Respectively 256,128,64,32,16,8,4,2,1 μ g/ml, while the fluid nutrient medium of 100 μ l is set as blank control group, 50 μ
Isometric fluid nutrient medium is added as negative control group in l bacteria suspension.Bacterium sets 37 DEG C of 16~18h of culture, and fungi sets 25 DEG C
48h is cultivated, microplate reader detects the absorbance value in each hole at 595nm.With can completely inhibit bacterium or fungi growth it is minimum
Drug concentration is minimum inhibitory concentration (MIC).Simultaneously as a comparison with Pb2-1TI and CATHPb2, Pb2-1TI and CATHPb2 are more
The synthesis of peptide and the preparation of drug and its measurement of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) and polypeptide
Pb2-1 is consistent with PCL-1.
The 100 μ l of culture for having no bacterium or fungi growth in 96 orifice plates of above-mentioned MIC measurement is taken, it is sterile that 900 μ l are added
It after culture medium dilution, is transferred in sterile petri dish, addition about 10.0ml temperature is 55 DEG C of culture mediums, and bacterium sets 37 DEG C of cultures
For 24 hours, fungi sets 28 DEG C of culture 48h, observes and records the clump count on plate, lowest concentration of drug of the clump count less than 5, note
For MBC value.It the results are shown in Table 1:
The MIC value and MBC value of table 1 antibacterial polypeptide PCL-1, Pb2-1TI and Pb2-1 to different bacterium and fungi
Continued 1:
aAmoxicillin antibody-resistant bacterium；bGentamicin antibody-resistant bacterium.
As seen from Table 1, Pb2-1 has better broad spectrum antibiotic activity relative to female Peptide C ATHPb2, to standard and clinic
Bacterium and fungi show preferable antibacterial activity, for MIC value in the range of 4~32 μ g/ml, MBC value is in 4~32 μ g/
In the range of ml；Have trypsin inhibition activity without the 11 of antibacterial activity by connecting one section at the end N- of Pb2-1
The polypeptide PCL-1 that cyclic peptide obtains, though being declined slightly compared to Pb2-1 antibacterial activity, it still has preferably bacterium and fungi
Antibacterial activity, to the MIC value of standard and clinical bacterium and fungi in the range of 8~64 μ g/ml, MBC value is in 8~128 μ
In the range of g/ml.And Pb2-1TI is weaker to the antibacterial activity of bacterium and fungi, the MIC value to bacterium is 4 times of PCL-1.
(5) killing curve of polypeptide Pb2-1 and PCL-1
Nutrient broth or the diluted bacteria suspension of Sabouraud dextrose fluid nutrient medium are adjusted to bacterial concentration about 105CFU/
Ml is added polypeptide PCL-1, Pb2-1 (final concentration: 4 × MIC) or Amoxicillin (final concentration: 1024 μ g/ml), sets 37 DEG C of constant temperature
Culture.0,1,2,4,8,12, for 24 hours when take out 200 μ l culture solutions and carry out gradient dilution, then carry out plate count.Physiology salt
Water group is set as negative control group, and polymyxins group is set as positive controls, and each dilution gradient is all provided with three in parallel, with bacterium colony pair
Numerical value is ordinate, and polypeptide action time is abscissa, draws killing curve.The results are shown in attached figure 5,6 and 7.
Attached drawing 5,6 and 7 respectively indicates the bactericidal effect of polypeptide PCL-1, Pb2-1 to each experimental strain, PCL-1 bactericidal effect
Significantly, escherichia coli and neogenesis cryptococcus can be killed in 8h, and more to the bactericidal effect of staphylococcus aureus
Significantly, it can all be killed in 1h.The result shows that antibacterial polypeptide PCL-1 has good bactericidal effect.
Embodiment 4
Influence of the antibacterial polypeptide Pb2-1 and PCL-1 to sheep red blood cell (SRBC) hemolytic activity
De- fiber Sheep Blood is taken to add in sterile EP tube, 3000rpm is centrifuged 10min, washes 3 times with PBS (pH 7.4) and lays equal stress on
It is outstanding, the red cell suspension of 3% (V/V) is made.The sheep red blood cell (SRBC) suspension for drawing 50 μ l respectively is added in 96 orifice plates, and addition etc.
The polypeptide solution of volume doubling dilution, making the final concentration of polypeptide is respectively 512,256,128,64,32,16,8 μ g/ml.Separately set
PBS processing group is negative control, and final concentration 1%Triton X-100 processing group is positive control, every group of 3 multiple holes.At dosing
It after reason, is taken out after 96 orifice plates are placed in 37 DEG C of constant temperature incubation 1h, 3000rpm is centrifuged 10min.Supernatant is drawn, is read with microplate reader
The OD value in each hole at 450nm, and hemolysis rate is calculated, formula is as follows:
Hemolysis rate=(dosing group OD450Negative control group OD450)/(positive controls OD450Negative control group OD450)×
100%
It can be seen that by attached drawing 8, even if Pb2-1 and PCL-1 are to sheep red blood cell (SRBC) when peptide concentration is up to 512 μ g/ml
Hemolysis rate is still very low, is no more than 5%.The range of comprehensive MIC value can be seen that PCL-1 in effective sphere of action, and haemolysis is living
Property ratio Pb2-1 it is lower, 1% is lower than to the hemolysis rate of red blood cell below the concentration of 256 μ g/ml.
Embodiment 5
The stabilization in vitro Journal of Sex Research of antibacterial polypeptide Pb2-1 and PCL-1 of the present invention
(1) thermal stability
The polypeptide that concentration is 1024 μ g/ml is mixed with isometric PBS (pH 7.4), be respectively placed in 0,20,37,50,
70,90 DEG C of water bath with thermostatic control 1h.Sample after incubation is detected with RT-HPLC, liquid-phase chromatographic analysis condition: C18 chromatographic column (4.6
×250mm,5μm)；Mobile phase A is the acetonitrile solution containing 0.1% trifluoroacetic acid, and Mobile phase B is containing the pure of 0.1% trifluoroacetic acid
Water purification.Detection wavelength is 220nm；Flow velocity is 1.0ml/min；20 μ l of sample volume carries out gradient elution.Elution requirement is linear ladder
Increasing concen-trations are spent, wherein the elution requirement of Pb2-1 specifically: organic phase initial concentration is incremented to 42% when being 22%, 20min,
20min to 25min rinses chromatographic column with 100% organic phase；The elution requirement of PCL-1 specifically: organic phase initial concentration is
It is incremented to 45%, 20min to 25min when 25%, 20min and rinses chromatographic column with 100% organic phase.The results are shown in attached figure 9.
After being incubated for 1h at different temperatures by PCL-1 and Pb2-1 it can be seen from attached drawing 9, compared with 0 DEG C of group, polypeptide it is surplus
For surplus all there is no decline, even incubation temperature is up to 90 DEG C, two polypeptides are still able to maintain very high integrality, illustrate two
A polypeptide thermal stability all with higher.
(2) pH stability
The solution that pH 1,3,5 is prepared with 1M HCl prepares the solution of pH 9,11 with 1M NaOH.With the solution of different pH
Polypeptide solution is prepared, makes the final concentration of 512 μ g/ml of polypeptide, is placed in 37 DEG C of constant-temperature incubations for 24 hours.By the sample RT- after incubation
HPLC detection, liquid-phase chromatographic analysis condition is the same as embodiment 5 (1).The results are shown in attached figure 10.
Be incubated for for 24 hours in the extreme acid condition that pH is 1 it can be seen from attached drawing 10, the surplus of PCL-1 there is no
Apparent decline, and Pb2-1 still has 90% holding complete, illustrates that PCL-1 can stablize in people's gastric acid (pH is about 1.5) and deposits
, and Pb2-1 then have in gastric acid it is slight unstable.And when pH is up to 11, there is no occur for the surplus of two polypeptides
It is decreased obviously, illustrates that two polypeptides can keep very high integrality in different pH environment.
(3) salt-stable
Bacterium solution is diluted by escherichia coli culture to logarithmic growth phase, and with the MHB culture medium of the NaCl containing various concentration
To 105CFU/ml.By polypeptide with MH culture medium doubling dilution at various concentration gradient after, respectively take 50 μ l in 96 orifice plates, and wait
Volume is inoculated with the 10 of 50 μ l NaCl containing various concentration5The bacteria suspension of CFU/ml, make drug final concentration be respectively 256,128,64,
32,16,8,4,2,1 μ g/ml, NaCl final concentration is respectively 0,50,100,150,200 and 400mM.Containing not for 100 μ l is set simultaneously
For MHB culture medium with concentration NaCl as blank control group, the MHB of isometric NaCl containing various concentration is added in 50 μ l bacteria suspensions
Culture medium sets 37 DEG C of 16~18h of culture, detects different salt ionic concentrations to the anti-escherichia coli of polypeptide as negative control group
The influence of MIC value.It the results are shown in Table 2:
The active influence of 2 various concentration NaCl of table E.coli anti-on polypeptide
As can be seen from Table 2, when NaCl concentration is up to 200mM, PCL-1 and Pb2-1 to the MIC value of escherichia coli simultaneously
It does not change, and normal NaCl is about 150mM in human body, is illustrated when NaCl concentration is higher than normal physiologic concentrations, two
A polypeptide is also able to maintain good antibacterial activity, two polypeptides salt tolerance all with higher.
(4) trypsinase stability
Stability first with liquid chromatography detection polypeptide in trypsin solution.It is 2048 μ g/ml's by concentration
Polypeptide mixes in equal volume with the trypsase (>=250NFu/mg) that concentration is 0.02mg/ml, is placed in 37 DEG C of constant-temperature incubations
0.5,1,2h, while not enzyme group is set as blank control.Then addition and the isometric acetonitrile of incubation system, are placed in 4 DEG C of preservations
2h makes trypsase be denaturalized and precipitate, and sample 10000rpm is centrifuged 10min later, takes supernatant to filter, then examined with RT-HPLC
It surveys, liquid-phase chromatographic analysis condition is the same as embodiment 5 (1).The results are shown in attached figure 11.
Except the influence with liquid chromatography detection trypsase to polypeptide, also further detect after trypsin treatment
The variation of polypeptide antibacterial activity.By escherichia coli culture to logarithmic growth phase, 10 are diluted to MH culture medium5CFU/ml.It will
300:1 is mixed polypeptide in molar ratio with trypsase (>=250NFu/mg), be placed in 37 DEG C of constant-temperature incubations 0.5,1,2,4,
8h, while not enzyme group is set as blank control.Then boiling water bath 5min makes trypsin inactivation, by each group polypeptide MH culture medium
It after doubling dilution is at various concentration gradient, respectively takes 50 μ l in 96 orifice plates, and is inoculated with the 10 of 50 μ l in equal volume5The bacterium of CFU/ml is outstanding
Liquid, making drug final concentration is respectively 256,128,64,32,16,8,4,2,1 μ g/ml, while the fluid nutrient medium for setting 100 μ l is made
For blank control group, 50 μ l bacteria suspensions are added isometric fluid nutrient medium as positive controls and set 37 DEG C of 16~18h of culture,
Detect influence of the trypsase to the MIC value of the anti-escherichia coli of polypeptide.It the results are shown in Table 3.
3 trypsase of table is on the active influence of the anti-escherichia coli of polypeptide
It can be seen from attached drawing 11 after being incubated for 0.5h altogether with trypsase, polypeptide PCL-1 still has 90% holding complete,
When incubation time extends to 1h, PCL-1 has decomposed about 40%, and when incubation time is up to 2h, polypeptide PCL-1 is also
25% keeps complete, illustrates still have part of polypeptide not to be degraded.
Meanwhile as can be seen from Table 3, after trypsin treatment 0.5h, polypeptide PCL-1 increases escherichia coli MIC
2 times, there is preferable trypsinase stability.
It can be seen that stability of the PCL-1 in trypsase has obtained great promotion, it is possible to prevente effectively from tryptose
Enzyme is on the active influence of the anti-escherichia coli of polypeptide.
Embodiment 6
Protective effect of the antibacterial polypeptide PCL-1 of the present invention to bacteremia model mice
It takes healthy weight in the Female ICR mice of 20~22g, is randomly divided into 6 groups, every group 13.The large intestine for taking MLD bacterium dense
In angstrom uncommon bacterium bacterium solution intraperitoneal injection to Mice Body, every 0.5ml establishes the model of septicemia.Administration group is divided into 5 groups, polypeptide
PCL-1 high dose group (10mg/kg), polypeptide PCL-1 middle dose group (5.0mg/kg), polypeptide PCL-1 low dose group (2.5mg/
Kg), polymyxins group (2.5mg/kg), physiological saline group, wherein PCL-1 is the preparation of embodiment 1.Polypeptide administration group, physiology salt
0.5h, 2h distinguish intraperitoneal administration twice to water group after infection, and polymyxins group is primary in infection 2h rear molding intravenously administrable.It sets simultaneously
Setting not is blank control group to bacterium group.
For mouse after infecting 8h, eye socket takes about 400 μ l of blood, anticoagulant with heparin sodium.Different gradients are diluted to, then take each dilution
Liquid 1.0ml is added in sterile petri dish, and maconkey agar culture medium is added, and is placed in 37 DEG C of constant temperature incubations 18~for 24 hours, observe and
The clump count on plate is recorded, the amount of survival of bacterium in mouse blood is calculated.The results are shown in attached figure 12.
After infecting 8h it can be seen from attached drawing 12, number of bacteria is about 10 in mouse blood8CFU/ml, polymyxins energy
The bacterial population in mouse blood is significantly reduced, relative to model group, polypeptide PCL-1 middle dose group bacterial population reduces 1.6 log
Value, and high dose group reduces nearly 2.5 log values, there is significant difference compared with model group, illustrates polypeptide PCL-1 energy
Remove to conspicuousness the bacterium in infecting mouse blood.
Sequence table
<110>China Medicine University
<120>antibacterial polypeptide Pb2-1 or PCL-1 and its preparation method and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 13
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Gly Phe Arg Lys Phe Met Arg Arg Leu Lys Lys Phe Phe
1 5 10
<210> 2
<211> 24
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Cys Trp Thr Lys Ser Ile Pro Pro Lys Pro Cys Gly Phe Arg Lys Phe
1 5 10 15
Met Arg Arg Leu Lys Lys Phe Phe
20
<210> 3
<211> 11
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Cys Trp Thr Lys Ser Ile Pro Pro Lys Pro Cys
1 5 10
<210> 4
<211> 29
<212> PRT
<213>Burma boa (Python bivittatu)
<400> 4
Lys Arg Asn Gly Phe Arg Lys Phe Met Arg Arg Leu Lys Lys Phe Phe
1 5 10 15
Ala Gly Gly Gly Ser Ser Ile Ala His Ile Lys Leu His
20 25
Claims (8)
1. a kind of antibacterial polypeptide Pb2-1, which is characterized in that its amino acid sequence is SEQ ID NO:1:GFRKFMRRLKKFF.
2. a kind of antibacterial polypeptide PCL-1, which is characterized in that its amino acid sequence is SEQ ID NO:2:
CWTKSIPPKPCGFRKFMRRLKKFF。
3. a kind of preparation method of antibacterial polypeptide Pb2-1 described in claim 1, which comprises the steps of:
Using peptide C ATHPb2 as template, the region of its sequence preservative and alpha-helix high concentration is intercepted, from the end N- the 3rd
Position starts interception and polypeptide amino acid number is reduced to 13 by 29, obtains polypeptide Pb2-1.
4. a kind of preparation method of antibacterial polypeptide PCL-1 as claimed in claim 2, which comprises the steps of:
(1) using peptide C ATHPb2 as template, the region of its sequence preservative and alpha-helix high concentration is intercepted, from the end N-
3rd starts interception and polypeptide amino acid number is reduced to 13 by 29, obtains polypeptide Pb2-1；
(2) 11 cyclic peptide for being CWTKSIPPKPC by a sequence are covalently attached with the end N- of Pb2-1, and obtaining one has point
The polypeptide PCL-1 of disulfide bond in son.
5. the preparation method according to claim 4, which is characterized in that the sequence C WTKSIPPKPC be disulfide bond cyclization,
11 cyclic peptide with trypsin inhibition activity.
6. prepared by a kind of antibacterial polypeptide Pb2-1 described in claim 1 or antibacterial polypeptide PCL-1 as claimed in claim 2
Application in anti-microbial pathogen infection medicine.
7. application according to claim 6, which is characterized in that the anti-microbial pathogen infection medicine includes antibacterium and fungi
Infection medicine.
8. application according to claim 6, which is characterized in that the bacterium is preferably staphylococcus aureus, resistance to methoxy
It is XiLin staphylococcus aureus (MRSA), staphylococcus epidermis, micrococcus scarlatinae, streptococcus pneumonia, pseudomonas aeruginosa, big
The uncommon bacterium of intestines angstrom, shigella dysenteriae and Klebsiella Pneumoniae；Fungi is preferably Candida albicans and neogenesis cryptococcus.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101024672A (en) * | 2007-02-12 | 2007-08-29 | 中国科学院昆明动物研究所 | Novel ring-shape small-peptide BA and its use |
| CN104761629A (en) * | 2015-03-05 | 2015-07-08 | 大连理工大学 | A broadspectrum efficient antimicrobial peptide Pb-CATH-OH1, a gene thereof, a preparing method of the peptide and applications of the peptide |
| CN105085647A (en) * | 2015-09-07 | 2015-11-25 | 大连理工大学 | Natural peptide Pb-CATH2 having anti-infection and antioxidant functions as well as gene and application of peptide |
-
2019
- 2019-08-23 CN CN201910787413.1A patent/CN110386972B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101024672A (en) * | 2007-02-12 | 2007-08-29 | 中国科学院昆明动物研究所 | Novel ring-shape small-peptide BA and its use |
| CN104761629A (en) * | 2015-03-05 | 2015-07-08 | 大连理工大学 | A broadspectrum efficient antimicrobial peptide Pb-CATH-OH1, a gene thereof, a preparing method of the peptide and applications of the peptide |
| CN105085647A (en) * | 2015-09-07 | 2015-11-25 | 大连理工大学 | Natural peptide Pb-CATH2 having anti-infection and antioxidant functions as well as gene and application of peptide |
Non-Patent Citations (5)
| Title |
|---|
| DAYEONG KIM等: "Genomewide Analysis of the Antimicrobial Peptides in Python bivittatus and Characterization of Cathelicidins with Potent Antimicrobial Activity and Low Cytotoxicity", 《ANTIMICROBIAL AGENTS AND CHEMOTHERAPY》 * |
| JIANXU LI等: "Trypsin inhibitory loop is an excellent lead structure to design serine protease inhibitors and antimicrobial peptides", 《THE FASEB JOURNAL 》 * |
| SHASHA CAI等: "Python Cathelicidin CATHPb1 Protects against Multidrug-Resistant Staphylococcal Infections by Antimicrobial-Immunomodulatory Duality", 《J. MED. CHEM.》 * |
| 王晨: "蛋白酶稳定的新型肽类抗生素-Cathelicidin-TI环轭合物的设计与优化", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
| 王晨等: "Cathelicidins结构与功能的关系及其分子设计研究进展", 《生物工程学报》 * |
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