CN103012558A - Cation symmetrical amphipathic self-assembled polypeptide and application thereof - Google Patents
Cation symmetrical amphipathic self-assembled polypeptide and application thereof Download PDFInfo
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- CN103012558A CN103012558A CN2012102196760A CN201210219676A CN103012558A CN 103012558 A CN103012558 A CN 103012558A CN 2012102196760 A CN2012102196760 A CN 2012102196760A CN 201210219676 A CN201210219676 A CN 201210219676A CN 103012558 A CN103012558 A CN 103012558A
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Abstract
The invention discloses a cation symmetrical amphipathic self-assembled polypeptide and an application of the cation symmetrical amphipathic self-assembled polypeptide. The cation symmetrical amphipathic self-assembled polypeptide has the formula of an amino acid sequence as follows: Ac-X1-Leu-I1e-X2-X3-Va1-X3-X2-I1e-Leu-X1-NH2, wherein X1, X2 and X3 are amino acids with positive charges which are symmetrical by using the Va1 as a central point. The amino acids in the sequence are L amino acids. The cation symmetrical amphipathic self-assembled polypeptide provided by the invention has a better bacteriostatic effect, and can be used for researching novel nanometer antibacterial agents. In addition, the cation symmetrical amphipathic self-assembled polypeptide provided by the invention can be used as a molecular sensor to detect change of a micro-environment.
Description
Technical field
The invention belongs to self-assembly polypeptide field, be specifically related to the application that the symmetrical amphipathic self-assembly polypeptide of a cationoid and conduct thereof suppress streptococcus aureus novel nano antibacterials and detect the molecule sensor of microenvironment variation.
Background technology
The peptide molecule self-assembling technique is study hotspot in recent years, can obtain the thousands of kinds of peptide molecules that structure is different by means such as molecular designing and solid phase synthesis, and the nano-sized materials that forms after the peptide molecule self-assembly has widely potential use.Existing polypeptide material is applied to the report of nano biological medical field both at home and abroad, comprise for 3D cell cultures, tissue repair, medicament transport and hemostasis etc., can self-assembly form nano-sized materials structurally has whole process or half way charge matching more for the polypeptide in above-mentioned field characteristics (Tang Lili, He Daohang but prior art is disclosed at present
*. Guangdong chemical industry, 2011,38 (1): 76-79.).For cationic polypeptide, be expected to overcome day by day serious clinically bacterial drug resistance as the polypeptide with anti-microbial effect, report multiplex in making up antiseptic-germicide both at home and abroad.The present invention has designed the symmetrical amphipathic self-assembly polypeptide of a cationoid, and their primary structure is different from aforementioned polypeptides, has the positively charged ion symmetry, has anti-microbial effect, can also be applied to detect microenvironment and change.
Summary of the invention
The object of the present invention is to provide the symmetrical Amphiphilic peptide of positively charged ion that a class can self-assembly.
The present invention's the second purpose is to provide the application of amphipathic self-assembly polypeptide in preparation inhibition streptococcus aureus novel nano antibacterials of positively charged ion symmetry.
The present invention's the 3rd purpose is to provide the application of amphipathic self-assembly polypeptide in the molecule sensor that preparation detection microenvironment changes of positively charged ion symmetry.
Purpose of the present invention is achieved through the following technical solutions:
The amphipathic self-assembly polypeptide of one cationoid symmetry, the general formula of its aminoacid sequence is as follows:
Ac-X
1-Leu-Ile-X
2-X
3-Val-X
3-X
2-Ile-Leu-X
1-NH
2, wherein Ac represents the acetylize of N end, NH
2The amination of expression C end, X
1, X
2, X
3Be the amino acid of polarity zone positive charge, and positive charge point both sides centered by Val are symmetrical, the amino acid in the sequence is L-type amino acid entirely.X
1, X
2, X
3For identical or different amino acid all can.
The amino acid of described polarity zone positive charge is Arg, Lys or His.
The amphipathic self-assembly polypeptid acid sequence of described positively charged ion symmetry is preferably Ac-RLIRKVKRILR-NH
2, Ac-KLIRKVKRILK-NH
2, Ac-KLIKKVKKILK-NH
2, Ac-HLIKKVKKILH-NH
2, Ac-HLIHKVKHILH-NH
2, Ac-HLIHRVRHILH-NH
2, Ac-HLIRRVRRILH-NH
2, Ac-KLIRRVRRILK-NH
2, Ac-KLIKRVRKILK-NH
2Or Ac-RLIRRVRRILR-NH
2
The application of the amphipathic self-assembly polypeptide of described positively charged ion symmetry in preparation inhibition streptococcus aureus novel nano antibacterials.
The application of the amphipathic self-assembly polypeptide of described positively charged ion symmetry in the molecule sensor that preparation detection microenvironment changes.
With respect to prior art, the present invention has following advantage and beneficial effect:
(1) self-assembling polypeptide of the present invention be its intermolecular by non-covalent interaction spontaneously be combined to form that a kind of structure is clear and definite, construction of stable, the molecule aggregates with certain physicochemical property or supramolecular structure.
(2) experiment shows, the amphipathic self-assembly polypeptide of positively charged ion symmetry of the present invention under different envrionment conditionss different structural changess can occur, and therefore, this peptide species can be used in the molecule sensor that preparation detection microenvironment changes.Different envrionment conditionss mainly refers to be different peptide concentrations, temperature, NaCl concentration, Na
2SO
4The solution of concentration, SDS concentration and TFE volume fraction.
(3) experiment shows, the amphipathic self-assembly polypeptide of positively charged ion symmetry of the present invention has preferably bacteriostatic action to streptococcus aureus, therefore, can be used for the novel nano antibacterials that development is suitable for using clinically.
(4) the invention provides the self-assembly polypeptide of a class novel texture, increased the type of self-assembly polypeptide.
(5) the invention provides the novel molecule sensor of a class, the novel antibacterials of a class, the prevention that is conducive to catch and treatment.
Description of drawings
Fig. 1 is the three-dimensional molecular structure schematic diagram of the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention.
Fig. 2 is high performance liquid phase (HPLC) color atlas of the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention, and the result shows that its purity is 97.4%.
Fig. 3 is mass spectrum (MS) figure of the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention, and the result shows that its molecular weight is 1491.9.
Fig. 4 is the atomic force microscope nanometer shape appearance figure of the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention in ultrapure water solution, wherein the symmetrical amphipathic self-assembly polypeptide P of positively charged ion
5VP
5Concentration be 0.4mg/ml.
Fig. 5 is the atomic force microscope nanometer shape appearance figure of the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention in 0.1M NaCl solution, wherein the symmetrical amphipathic self-assembly polypeptide P of positively charged ion
5VP
5Concentration be 0.4mg/ml.
Fig. 6 is circular dichroism (CD) spectrogram of the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention, and this figure shows that peptide concentration is to the symmetrical amphipathic self-assembly polypeptide P of positively charged ion
5VP
5The impact of structure.
Fig. 7 is circular dichroism (CD) spectrogram of the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention, and this figure shows that temperature is to the symmetrical amphipathic self-assembly polypeptide P of positively charged ion
5VP
5The impact of structure.
Fig. 8 is circular dichroism (CD) spectrogram of the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention, and this figure shows that different N aCl concentration is to the symmetrical amphipathic self-assembly polypeptide P of positively charged ion
5VP
5The impact of structure.
Fig. 9 is circular dichroism (CD) spectrogram of the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention, and this figure shows different N
a2SO
4Concentration is to the symmetrical amphipathic self-assembly polypeptide P of positively charged ion
5VP
5The impact of structure.
Figure 10 is circular dichroism (CD) spectrogram of the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention, and this figure shows that different sodium lauryl sulphate (SDS) concentration is to the symmetrical amphipathic self-assembly polypeptide P of positively charged ion
5VP
5The impact of structure.
Figure 11 is circular dichroism (CD) spectrogram of the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention, and this figure shows that different trifluoroethanols (TFE) volume fraction is to the symmetrical amphipathic self-assembly polypeptide P of positively charged ion
5VP
5The impact of structure.
Figure 12 be the symmetrical amphipathic self-assembly polypeptide of the positively charged ion of different concns of the present invention to the large logotype of inhibition zone of streptococcus aureus, among the figure, the symmetrical amphipathic self-assembly polypeptide P of the positively charged ion at 1,2 places
5VP
5Concentration be respectively 0.1mg/ml, 1mg/ml, 3 places are the blank aqueous solution.
Embodiment
In order to understand better the present invention, the invention will be further described below in conjunction with embodiment, but the scope of protection of present invention is not limited to the scope that embodiment represents.
Embodiment 1
Work as X
1Be Arg, X
2Be Arg, X
3During for Lys, self-assembly polypeptide 1. sequence is as follows:
CH
3CO-Arg-Leu-Ile-Arg-Lys-Val-Lys-Arg-Ile-Leu-Arg-NH
2
1. synthetic of self-assembly polypeptide:
1, material
Fmoc-Arg (pbf)-OH (N-fluorenes methoxy carbonyl acyl group-2,2,4,6,7-pentamethyl-Dihydrobenzofuranes-5-sulphonyl-arginine), Fmoc-Leu-OH(N-fluorenes methoxy carbonyl acyl group-leucine), Fmoc-Ile-OH(N-fluorenes methoxy carbonyl acyl group-Isoleucine), Fmoc-Lys (Boc)-OH (N-fluorenes methoxy carbonyl acyl group-N '-tertiary butyloxycarbonyl acyl group-Methionin), Fmoc-Val-OH(N-fluorenes methoxy carbonyl acyl group-α-amino-isovaleric acid), Rink Amide-MBHA Resin(resin), HBTU(O-benzotriazole-1-base-N, N, N, the N-tetramethyl-is urinated phosphofluoric acid fat) and the HOBT(1-hydroxy benzo triazole) available from Zhongtai Bio-Chem. Co., Ltd., Hangzhou; Piperidines, acetic anhydride, DMF(N, dinethylformamide), the TFA(trifluoroacetic acid), the NMM(N-methylmorpholine), ether, methyl alcohol, DCM(methylene dichloride) available from Tianjin Fu Yu Fine Chemical Co., Ltd.
2, preparation method
Employing Fmoc(fluorenes methoxy carbonyl acyl group) solid-phase synthesis of protection, its processing step is as follows:
(1) takes by weighing the Rink Amide-MBHA Resin of 20g 0.5mmol/g in the peptide synthesizer ware, get 200mlDMF swelling resin 30min, then suction filtration, take again the 300mlDMF washing resin, divide and carry out for three times, each washing time is 2min, add 100ml20% piperidines/DMF(V in the backward peptide synthesizer of suction filtration dry cleaning liquid: V) concussion reaction 30min, after reaction finishes, suction filtration goes out reaction solution again, divides washing resin four times with 400mlDMF again, washes the resin that takes a morsel after finishing and does triketohydrindene hydrate and detect test, resin is positive, and adds following raw material in the peptide synthesizer ware:
After above-mentioned raw materials adds, shake reaction 30min, reaction divides washing resin four times with 300mlDMF after finishing, and each washing time 2 minutes is got a little resin and done the ninhydrin reaction detection, and resin is negative.
(2) add 5ml 20% piperidines/DMF(V in the peptide synthesizer ware: V) 30min is reacted in concussion, suction filtration went out reaction solution after reaction finished, divide washing resin four times with 40ml DMF again, wash complete a little resin of getting and do the ninhydrin reaction detection, resin is positive as a result, adds following raw material in the reaction vessels:
（a）Fmoc-Leu-OH 14.14g
（b）HBTU 15.16g
（c）HOBT 5.40g
（d）NMM 4.39ml
（e）DMF 160ml
After above-mentioned raw materials adds, shake reaction 40min, reaction divides washing resin four times with 40mlDMF after finishing, and each washing time is 2min, gets a little resin and does the triketohydrindene hydrate detection, and resin is negative.
(3) (a) raw material in the shift step (2), (b) (c) (d) (e) raw material and add-on are constant, the operation of repeating step (2); (a) raw material replaces with Fmoc-Ile-OH(14.14g successively), Fmoc-Arg(pbf)-OH(25.95g), Fmoc-Lys (Boc)-OH(18.74g), Fmoc-Val-OH(13.58g), Fmoc-Lys (Boc)-OH(18.74g), Fmoc-Arg(pbf)-OH(25.95g), Fmoc-Ile-OH(14.14g), Fmoc-Leu-OH(14.14g), Fmoc-Arg(pbf)-OH(25.95g).Be the operation of a step of every repetition (2), conversion a kind of (a) raw material is until till all using above-mentioned raw materials once;
(4) repeat once the operation of (1) (2) (3) step, the raw material of each step and raw materials used amount are all constant again; Behind last Arg end of synthesis, deviate from the Fmoc-protecting group, add 20% piperidines/DMF(V: V) reaction 30min; eluted resin adds 160ml 50% acetic anhydride/DMF(V: V) reaction 30min, with 40ml DMF eluted resin; use again the methanol wash resin 8 times, remove DMF.After nitrogen dries up; use the TFA(trifluoroacetic acid)/DCM strong acid lysate; cracking 3 hours will be synthesized polypeptide cracking from the resin and be got off, and slough simultaneously all blocking groups; collection is dissolved with the lysate of synthetic peptide; then filtration under diminished pressure is collected filtrate, with the polypeptide of cold diethyl ether resolution of precipitate in filtrate; suction filtration obtains white solid again, namely gets the crude product of synthetic peptide.The synthetic peptide crude product is through the HPLC(high performance liquid chromatography) purifying, collect main peak, through after the lyophilize, namely obtain the target synthetic peptide.
3, other the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention can by replacing the amino acid in above-mentioned 1, obtain according to above-mentioned 2 described preparation method's synthesizing and purifyings.Compound experiment parameters different in this process is as shown in table 1 below:
The different compound experiment parameters of the symmetrical amphipathic self-assembly polypeptide of table 1 partial cation of the present invention
Embodiment 2: the self-assembly polypeptide is CH 1.
3CO-Arg-Leu-Ile-Arg-Lys-Val-Lys-Arg-Ile-Leu-Arg-NH
2High performance liquid chromatography and mass spectrometric detection and three-dimensional molecular modeling rendering
1. adopt high performance liquid chromatography (HPLC) to detect the self-assembly polypeptide of embodiment 1 preparation, detected result is seen Fig. 2, determines that according to the result among Fig. 2 its purity reaches 97.4%.
1. adopt mass spectrum (MS) to detect the self-assembly polypeptide of embodiment 1 preparation, detected result is seen Fig. 3, and the result shows that its molecular weight is 1491.9.
To the self-assembly polypeptide of embodiment 1 preparation 1. through molecular simulation namely based on minimum energy principle drawing three-dimensional molecular model schematic diagram; the schematic diagram of drawing as shown in Figure 1; among the figure as seen; polypeptide N holds acetylize; C holds amination, and amino-acid residue and positive charge point both sides centered by Val are symmetrical in the sequence.By this figure spatial distribution of its amino acid side chain as can be known.
High performance liquid chromatography and the mass spectrometric detection data of the symmetrical amphipathic self-assembly polypeptide of partial cation of the present invention see Table 2:
Purity and the molecular weight of the symmetrical amphipathic self-assembly polypeptide of table 2 partial cation of the present invention
Embodiment 3: atomic force microscope (AFM) detects self-assembly polypeptide nanostructure 1.
1, atomic force microscope (AFM) detect nanostructure that 1. the self-assembly polypeptide form in ultrapure water solution get be stored in 4 ℃ the self-assembly polypeptide 1. sample preparation to become concentration be the solution of 1.0mg/ml, (18.2M Ω) is diluted to 0.4mg/ml with the Milli-Q ultrapure water, carries out AFM and detects.
(1) getting 10 μ l sample solutions evenly places and newly takes off on the sheet mica;
(2) each sample stops approximately 30～60s so that absorption on sheet mica surface;
(3) use 100 μ l Milli-Q ultrapure waters flushing sheet mica surface to remove not adsorption sample;
(4) place the petri dish Air dry, avoid simultaneously polluting, placement is spent the night;
(5) at room temperature, use Nanoscope Illa (Digital Instruments, USA) under the pattern of rapping, to scan the nanotopography that mica surface is observed sample.
The aspect graph of AFM is seen Fig. 4, shows that 1. the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention can form sphere aggregates in ultrapure water solution.
2, atomic force microscope (AFM) detects the self-assembly polypeptide nanostructure that 1. self-assembly forms in salts solution
Experimental implementation such as above-mentioned 1 just adds a certain amount of NaCl in test soln, so that NaCl concentration is 0.1M in the final solution, the concentration of polypeptide is 0.4mg/ml.
The aspect graph of AFM is seen Fig. 5, shows that 1. the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention can self-assembly form the ball shaped nano grain in 0.1M NaCl solution.
Embodiment 4: the self-assembly polypeptide is the application in the molecule sensor that preparation detection microenvironment changes 1.
Detect discovery by circular dichroism (CD), the structure of the amphipathic self-assembly polypeptide of positively charged ion symmetry of the present invention is relatively more responsive to the variation of solution environmental, under different envrionment conditionss different structural changess can occur, therefore can be used for preparing the molecule sensor that detects the microenvironment variation.CD detecting step and result thereof under the different solutions environment are as follows:
1, utilize CD to detect the 1. structural changes in different peptide concentration solution of self-assembly polypeptide
Adopt circular dichroism spectrograph (Chirascan Circular Dichroism Spectrometer) to 1. CH of the self-assembly polypeptide of embodiment 1 preparation
3CO-Arg-Leu-Ile-Arg-Lys-Val-Lys-Arg-Ile-Leu-Arg-NH
2Carry out CD and detect, step is as follows:
(1) get be stored in 4 ℃ the self-assembly polypeptide 1. sample preparation to become concentration be the solution of 1.0mg/ml, (18.2M Ω) is diluted to different concentration, concentration range 0.01～1.0mg/ml with the Milli-Q ultrapure water;
The sample solution that adds the preparation of 300 μ l steps (1) when (2) at every turn measuring in the quartz curette is swept first the baseline spectrum that only contains damping fluid under the background of sky cuvette and the identical conditions, to deduct baseline before the specimen;
(3) under 25 ℃ of conditions, carry out CD scanning, data gathering scope 190-260nm.Use the 1mm cuvette, bandwidth 0.5nm, step-length 1.0nm uses scanning mean value 3 times.
Measurement result shows the rising along with peptide concentration as shown in Figure 6, and 1. the self-assembly polypeptide presents the variation tendency of beta sheet gradually.
2, utilize CD to detect the 1. structural changes under differing temps of self-assembly polypeptide
Adopt circular dichroism spectrograph (Chirascan Circular Dichroism Spectrometer) to 1. CH of the self-assembly polypeptide of embodiment 1 preparation
3CO-Arg-Leu-Ile-Arg-Lys-Val-Lys-Arg-Ile-Leu-Arg-NH
2Carry out CD and detect, step is as follows:
(1) get be stored in 4 ℃ the self-assembly polypeptide 1. sample preparation to become concentration be the solution of 1.0mg/ml, (18.2M Ω) is diluted to 0.4mg/ml with the Milli-Q ultrapure water;
The sample solution that adds the preparation of 300 μ l steps (1) when (2) at every turn measuring in the quartz curette is swept first the baseline spectrum that only contains damping fluid under the background of sky cuvette and the identical conditions, to deduct baseline before the specimen;
(3) the data gathering scope of CD scanning is 190-260nm.Use the 1mm cuvette, bandwidth 0.5nm, step-length 1.0nm, the speed intensification with 2 ℃/min scans in 25～95 ℃ of temperature ranges, per 10 ℃ of run-downs, the starting time of permission 30s under each temperature, 0.1 ℃ of error.After the intensification, 25 ℃ of again tests fall back in cooling.
Measurement result shows the rising along with temperature as shown in Figure 7, and it is more orderly that 1. the self-assembly polypeptide becomes gradually.
3, utilize CD to detect the 1. structural changes in different N aCl concentration solution of self-assembly polypeptide
Adopt circular dichroism spectrograph (Chirascan Circular Dichroism Spectrometer) to 1. CH of the self-assembly polypeptide of embodiment 1 preparation
3CO-Arg-Leu-Ile-Arg-Lys-Val-Lys-Arg-Ile-Leu-Arg-NH
2Carry out CD and detect, step is as follows:
(1) get be stored in 4 ℃ the self-assembly polypeptide 1. sample preparation to become concentration be the solution of 1.0mg/ml;
(2) be mixed with first the NaCl solution of 0.5mol/l with volumetric flask, adding Milli-Q ultrapure water (18.2M Ω) dilutes again, mix with the polypeptide sample solution, be made into following concentration: 0.01mol/l, 0.02mol/l, 0.05mol/l, 0.1mol/l, 0.2mol/l and 0.3mol/l, the concentration of polypeptide sample is 0.4mg/ml in the solution of different N aCl concentration;
The sample solution that adds the preparation of 300 μ l steps (2) when (3) at every turn measuring in the quartz curette is swept first the baseline spectrum that only contains damping fluid under the background of sky cuvette and the identical conditions, to deduct baseline before the specimen;
(4) under 25 ℃ of conditions, carry out CD scanning, data gathering scope 190-260nm.Use the 1mm cuvette, bandwidth 0.5nm, step-length 1.0nm uses scanning mean value 3 times.
Measurement result as shown in Figure 8, show that the NaCl of different concns is (such as 0.01mol/l, 0.02mol/l, 0.05mol/l, 0.1mol/l, 0.2mol/l and 0.3mol/l) not obvious on the impact of the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention conformational change 1., that is to say, self-assembly polypeptide secondary structure 1. is relatively stable under the effect of NaCl.
4, utilize CD to detect the self-assembly polypeptide 1. at different N a
2SO
4Structural changes in the concentration solution
Adopt circular dichroism spectrograph (Chirascan Circular Dichroism Spectrometer) to 1. CH of the self-assembly polypeptide of embodiment 1 preparation
3CO-Arg-Leu-Ile-Arg-Lys-Val-Lys-Arg-Ile-Leu-Arg-NH
2Carry out CD and detect, step is as follows:
(1) get be stored in 4 ℃ the self-assembly polypeptide 1. sample preparation to become concentration be the solution of 1.0mg/ml;
(2) be mixed with first the Na of 0.5mol/l with volumetric flask
2SO
4Solution adds Milli-Q ultrapure water (18.2M Ω) again and dilutes, and mixes with the polypeptide sample solution, is made into following concentration: 0.01mol/l, 0.02mol/l, 0.05mol/l, 0.1mol/l, 0.2mol/l and 0.3mol/l, different N a
2SO
4The concentration of polypeptide sample is 0.4mg/ml in the solution of concentration;
The sample solution that adds the preparation of 300 μ l steps (2) when (3) at every turn measuring in the quartz curette is swept first the baseline spectrum that only contains damping fluid under the background of sky cuvette and the identical conditions, to deduct baseline before the specimen;
(4) under 25 ℃ of conditions, carry out CD scanning, data gathering scope 190-260nm.Use the 1mm cuvette, bandwidth 0.5nm, step-length 1.0nm uses scanning mean value 3 times.
Measurement result shows the Na of different concns as shown in Figure 9
2SO
4(such as 0.01mol/l, 0.02mol/l, 0.05mol/l, 0.1mol/l, 0.2mol/l and 0.3mol/l) is not obvious on the impact of the symmetrical amphipathic self-assembly polypeptide of positively charged ion conformational change 1., that is to say, at Na
2SO
4Effect under self-assembly polypeptide secondary structure 1. relatively stable.
5, utilize CD to detect the 1. structural changes in different sodium lauryl sulphate (SDS) concentration solution of self-assembly polypeptide
Adopt circular dichroism spectrograph (Chirascan Circular Dichroism Spectrometer) to 1. CH of the self-assembly polypeptide of embodiment 1 preparation
3CO-Arg-Leu-Ile-Arg-Lys-Val-Lys-Arg-Ile-Leu-Arg-NH
2Carry out CD and detect, step is as follows:
(1) get be stored in 4 ℃ the self-assembly polypeptide 1. sample preparation to become concentration be the solution of 1.0mg/ml;
(2) be mixed with first the SDS solution of 0.3mol/l with volumetric flask, adding Milli-Q ultrapure water (18.2M Ω) dilutes again, mix with the polypeptide sample solution, be made into following concentration: 1mmol/l, 2mmol/l, 4mmol/l, 8mmol/l, 10mmol/l, 30mmol/l, 60mmol/l and 180mmol/l, the concentration of polypeptide sample is 0.4mg/ml in the solution of different SDS concentration;
The sample solution that adds the preparation of 300 μ l steps (2) when (3) at every turn measuring in the quartz curette is swept first the baseline spectrum that only contains damping fluid under the background of sky cuvette and the identical conditions, to deduct baseline before the specimen;
(4) under 25 ℃ of conditions, carry out CD scanning, data gathering scope 190-260nm.Use the 1mm cuvette, bandwidth 0.5nm, step-length 1.0nm uses scanning mean value 3 times.
Measurement result as shown in figure 10, show in the middle of the SDS of 1mmol/l solution, 1. the symmetrical amphipathic self-assembly polypeptide of described positively charged ion mainly exists with the form of random coil, along with SDS concentration raises, 1. the self-assembly polypeptide random coil occurs to the structural changes of a-spiral, and in the SDS of different high densitys solution, self-assembly polypeptide a-spiral secondary structure spectral line shape 1. is to some extent difference also, illustrate that the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention conformational change 1. is very sensitive to SDS, can resist SDS to the Denaturation of albumen simultaneously.
6, utilize CD to detect the 1. structural changes in different trifluoroethanols (TFE) concentration solution of self-assembly polypeptide
Adopt circular dichroism spectrograph (Chirascan Circular Dichroism Spectrometer) to 1. CH of the self-assembly polypeptide of embodiment 1 preparation
3CO-Arg-Leu-Ile-Arg-Lys-Val-Lys-Arg-Ile-Leu-Arg-NH
2Carry out CD and detect, step is as follows:
(1) get be stored in 4 ℃ the self-assembly polypeptide 1. sample preparation to become concentration be the solution of 1.0mg/ml;
(2) the TFE solution of absorption different volumes, adding Milli-Q ultrapure water (18.2M Ω) dilutes again, mix with the polypeptide sample solution, be made into following different volume fraction: 10%, 20%, 30%, 40%, 50% and 60%, the concentration of polypeptide sample is 0.4mg/ml in the solution of different TFE volume fractions;
The sample solution that adds the preparation of 300 μ l steps (2) when (3) at every turn measuring in the quartz curette is swept first the baseline spectrum that only contains damping fluid under the background of sky cuvette and the identical conditions, to deduct baseline before the specimen;
(4) under 25 ℃ of conditions, carry out CD scanning, data gathering scope 190-260nm.Use the 1mm cuvette, bandwidth 0.5nm, step-length 1.0nm uses scanning mean value 3 times.
Measurement result as shown in figure 11, show in the middle of 10% TFE solution, 1. the symmetrical amphipathic self-assembly polypeptide of described positively charged ion mainly exists with the form of random coil, rising along with the TFE volume fraction, 1. the self-assembly polypeptide random coil occurs to the structural changes of a-spiral, illustrates that the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention conformational change 1. is responsive to TFE.
Above-mentioned all measurement result promptings, 1. the symmetrical amphipathic self-assembly polypeptide of described positively charged ion has certain stability in salts solution, can not saltout, change relatively more responsive to other solution environmental simultaneously, can different structural changess occur according to different solution environmentals, therefore, this peptide species is expected to use in the molecule sensor that preparation detection microenvironment changes.
Embodiment 5: the application of the symmetrical amphipathic self-assembly polypeptide of positively charged ion in preparation novel nano antibacterials
1,1. CH of self-assembly polypeptide
3CO-Arg-Leu-Ile-Arg-Lys-Val-Lys-Arg-Ile-Leu-Arg-NH
2Application in preparation novel nano antibacterials
(1) test strain
Streptococcus aureus CMCC26003(Guangdong microorganism germ plasma resources bank).
(2) test method
Adopt the Oxford agar diffusion method to measure the symmetrical amphipathic self-assembly polypeptide of positively charged ion of the present invention to the inhibition zone of tested bacterium, judge that according to the inhibition zone size its bacteriostatic activity is strong and weak.Antibacterial circle diameter 〉=12mm shows that sample has anti-microbial activity.
In this test used self-assembly polypeptide 1. solution use the fresh preparation of aseptic Milli-Q ultrapure water (18.2M Ω), concentration is respectively 0.1mg/ml, lmg/ml, and is for subsequent use.
Used substratum is beef-protein medium in this test, and tested bacterium liquid final concentration is about 10
6CFU/ml, for subsequent use.
(3) inhibition zone of the tested bacterium of mensuration
The about beef-protein medium of 15ml is poured in the sterilization culture dish that diameter is 9cm, after cooling, draw the tested bacterium liquid of preparation in the 0.1ml above-mentioned steps (2) with liquid-transfering gun, add in the culture dish, be applied in equably gently each agar plate surface with spreading rod.After bacterium liquid was done, put diameter on the agar plate surface be the stainless steel Oxford cuvette of 6mm (internal diameter) and carry out mark.Draw the above-mentioned given the test agent solution for preparing of 200 μ l with liquid-transfering gun, be added dropwise to respectively in the cup of Oxford, each concentration is made three Duplicate Samples, with aseptic Milli-Q ultrapure water as blank.
After sample adds, each agar plate is built, placed 37 ℃ to hatch 24 hours, each tested liquid of observing different concns has or not inhibition zone on every side, each inhibition zone is measured its diameter by three different directions, and the mean value of three measurements is the size of this antibacterial circle diameter.After three groups of data statisticss of each medicine are complete, get the mean value of its inhibition zone size and judge numerical value as the bacteriostasis size of tested liquid under this concentration.Press drug sensitivity assay criterion and given the test agent self-assembly polypeptide inhibition zone size 1. and judge that its bacteriostatic activity is strong and weak.
(4) judging criterion
Occur clear inhibition zone around the testing sample, antibacterial circle diameter 〉=12mm is responsive, shows that sample has anti-microbial activity.
Drug sensitivity assay criterion sees Table 3.
The criterion of table 3 drug sensitivity assay
(5) to the test result of streptococcus aureus
Figure 12 be the symmetrical amphipathic self-assembly polypeptide of the positively charged ion of different concns to the large logotype of inhibition zone of streptococcus aureus, among the figure, the symmetrical amphipathic self-assembly polypeptide P of the positively charged ion at 1,2 places
5VP
5Concentration be respectively 0.1mg/ml, lmg/ml, 3 places are the blank aqueous solution.As shown in figure 12, according to the inhibition zone size as can be known, 1. the self-assembly polypeptide of different concns all has very strong anti-microbial activity to streptococcus aureus CMCC26003.The self-assembly polypeptide is when 1. concentration is 0.1mg/ml, and its antibacterial circle diameter to streptococcus aureus is 21mm; The self-assembly polypeptide is when 1. concentration is 1mg/ml, and its antibacterial circle diameter to streptococcus aureus is 25mm.The result shows, the self-assembly polypeptide 1. bacteriostatic action to tested streptococcus aureus is very strong.When self-assembly polypeptide concentration 1. is O.Omg/ml, when namely testing as blank with aseptic Milli-Q ultrapure water, clearly inhibition zone does not appear in tested streptococcus aureus.
2,2. CH of self-assembly polypeptide
3CO-Lys-Leu-Ile-Arg-Lys-Val-Lys-Arg-Ile-Leu-Lys-NH
2Application in preparation novel nano antibacterials
2. 1. bacteriostatic activity experimental implementation such as above-mentioned 1 be changed to the self-assembly polypeptide with the self-assembly polypeptide, tests its inhibition zone size.
According to the inhibition zone size as can be known, 2. the self-assembly polypeptide of different concns all has stronger anti-microbial activity to streptococcus aureus CMCC26003.
The self-assembly polypeptide is when 2. concentration is 0.1mg/ml, and its antibacterial circle diameter to streptococcus aureus is 20mm; The self-assembly polypeptide is when 2. concentration is 1mg/ml, and its antibacterial circle diameter to streptococcus aureus is 23mm.The result shows, 2. the self-assembly polypeptide has stronger bacteriostatic action to tested streptococcus aureus.
When self-assembly polypeptide concentration 2. is 0.0mg/ml when namely testing as blank with aseptic Milli-Q ultrapure water, clearly inhibition zone does not appear in tested streptococcus aureus.
3, the symmetrical amphipathic self-assembly polypeptide of partial cation the results are shown in Table 4 to the symmetrical amphipathic self-assembly polypeptide of the antibacterial susceptibility partial cation of streptococcus aureus to the antibacterial susceptibility of streptococcus aureus.
The symmetrical amphipathic self-assembly polypeptide of table 4 partial cation is to the antibacterial susceptibility of streptococcus aureus
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1. the amphipathic self-assembly polypeptide of a cationoid symmetry is characterized in that, the general formula of its aminoacid sequence is as follows:
Ac-X
1-Leu-Ile-X
2-X
3-Val-X
3-X
2-Ile-Leu-X
1-NH
2, wherein Ac represents the acetylize of N end, NH
2The amination of expression C end, X
1, X
2, X
3Be amino acid identical or that dissimilar polarity is positively charged, and positive charge point both sides centered by Val are symmetrical, the amino acid in the sequence is L-type amino acid entirely.
2. the amphipathic self-assembly polypeptide of positively charged ion symmetry according to claim 1 is characterized in that, the amino acid of described polarity zone positive charge is Arg, Lys or His.
3. the amphipathic self-assembly polypeptide of positively charged ion symmetry according to claim 1 is characterized in that, its aminoacid sequence is preferably Ac-RLIRKVKRILR-NH
2, Ac-KLIRKVKRILK-NH
2, Ac-KLIKKVKKILK-NH
2, Ac-HLIKKVKKILH-NH
2, Ac-HLIHKVKHILH-NH
2, Ac-HLIHRVRHILH-NH
2, Ac-HLIRRVRRILH-NH
2, Ac-KLIRRVRRILK-NH
2, Ac-KLIKRVRKILK-NH
2Or Ac-RLIRRVRRILR-NH
2
4. the application of the amphipathic self-assembly polypeptide of the positively charged ion symmetry described in the claim 1 in preparation inhibition streptococcus aureus novel nano antibacterials.
5. the application of the amphipathic self-assembly polypeptide of the positively charged ion symmetry described in the claim 1 in the molecule sensor that preparation detection microenvironment changes.
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| CN102199195A (en) * | 2011-03-07 | 2011-09-28 | 华南理工大学 | Half-path charge matching amphiphilic self assembling short peptide, and its application as nanometer hemostatic material and hydrophobic drug carrier |
| CN102827250A (en) * | 2012-06-28 | 2012-12-19 | 华南理工大学 | Fatty acid-modificatory amphiphilic cation self-assembling peptide and application thereof |
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| CN102827250A (en) * | 2012-06-28 | 2012-12-19 | 华南理工大学 | Fatty acid-modificatory amphiphilic cation self-assembling peptide and application thereof |
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