CN105085624B - Smooth Xenopus laevis antibacterial skin peptide and preparation method thereof and purposes - Google Patents
Smooth Xenopus laevis antibacterial skin peptide and preparation method thereof and purposes Download PDFInfo
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- 241000269368 Xenopus laevis Species 0.000 title claims abstract description 39
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
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- 230000000694 effects Effects 0.000 claims abstract description 26
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- 238000000034 method Methods 0.000 claims abstract description 8
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Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
A kind of smooth magainin and its preparation method and application, the antibacterial peptide are isolated from smooth Xenopus laevis skin secretion, its sequence is obtained after purified sequencing as Asp Glu Asp Asp Asp（DEDDD）, its molecular weight of Mass Spectrometer Method is 607.7Da.The present invention stimulates the skin of smooth Xenopus laevis using physical method, dramatically protects the structural intergrity and biological activity of antibacterial peptide.The antibacterial peptide has significant bacteriostatic activity, relatively low hemolytic activity and good anti-breast cancer activity.Its molecular weight is relatively low, reduces the difficulty of its chemical synthesis, substantially reduces cost.It has significant bacteriostasis to gram-positive bacteria and Gram-negative bacteria, and hemolytic activity is extremely low in the range of MIC value, and there is higher anti-breast cancer activity, available for a variety of medical directions such as the preventing of wound wound infection, antitumor, inhibiting-bacteria preparation, have a good application prospect.
Description
Technical field
The present invention relates to pharmaceutical chemical technical field, specifically a kind of smooth Xenopus laevis antibacterial skin peptide and its preparation side
Method and purposes.
Background technology
Antibacterial peptide（Antibacterial peptide）, also known as antimicrobial peptide is that having for immune system release is anti-
The substance of microbial activity, the part as congenital immunity ingredient are widely present in the organism of nature.Antibacterial peptide
It is a kind of polypeptides matter of small molecule, there is extensive biological activity, shows that it has sterilization, anti-inflammatory, antiviral, suppression
The effects that tumour processed is the important component of biological non-specific defense system.Antibacterial peptide has the antibacterial activity of wide spectrum, can
It is combined with the lipopolysaccharides of gram-negative bacteria outer membrane and the peptide glycan on gram positive bacteria surface, is formed so as to destroy after birth and wear film
Channel, cellular content leakage or death due to hypertonic rupture after ion outflow, are not likely to produce drug resistance.Therefore, antibacterial peptide is made
For a kind of small-molecular peptides that there is efficient bio-active and be not likely to produce drug resistance, there is a very wide range of application value.
The skin of amphibian is exposed to be directly in contact with living environment, without the protection of scale, crust and hair, but
On the surface of amphibian skin, the substance for often having mucus is covered in surface and forms protective barrier.Contain in this barrier
The macromolecular substances and antibacterial substance vdiverse in function for having a large amount of structure more complicated, antibacterial peptide as it is therein into
Point, it plays an important role.
Antibacterial peptide as antibacterial medicines research extensively and profoundly, wherein, the achievement in research of small molecule antibacterial peptide is particularly
Significantly, small molecule antibacterial peptide not only has efficient bacteriostatic activity, due also to its molecular structure is simple and to have a synthesis cost low,
There is no the advantages that immune response.In the Chinese patent of Publication No. 103641901A, disclosing a kind of sequence is
The antibacterial peptide of GLPLLISWIKRKRQQAGPGSKKPVPIIYCNRRTGKCQRM.It is special in the China of Publication No. 102140130A
In profit, the antibacterial peptide that a kind of sequence is KCRRRKVHGPMIRIRKK is disclosed.Above two antibacterial peptide since its sequence is longer,
It is higher so as to cause its synthesis difficulty, cost there are secondary structure.It is open in the Chinese patent of Publication No. 103524602A
A kind of sequence is 6 peptide antibacterial peptides of RRRWWC, but its virus activity is merely illustrated in the invention, its is antitumor without clear and definite
Deng other effects, and the patent does not study the hemolytic activity of the antibacterial peptide, greatly limits its application.
Invention content
The technical problem to be solved in the present invention is to provide a kind of smooth Xenopus laevis antibacterial skin peptide and preparation method thereof and purposes.
The present invention is adopted the technical scheme that solve technical problem present in known technology:
The smooth Xenopus laevis antibacterial skin peptide of the present invention is to include 4 molecule aspartic acids and the sequence of 1 molecule glutamic acid is
5 peptides of Asp-Glu-Asp-Asp-Asp, that is, DEDDD.
The present invention can also use following technical measures：
The sequence that the smooth Xenopus laevis antibacterial skin peptide of the present invention is made of 4 molecule aspartic acids and 1 molecule glutamic acid
For 5 peptides of Asp-Glu-Asp-Asp-Asp, that is, DEDDD, molecular weight 607.7Da.
The preparation method of the smooth Xenopus laevis antibacterial skin peptide of the present invention, includes the following steps：
A, smooth Xenopus laevis skin secretion is extracted；
B, preparative high performance liquid chromatography isolates and purifies；
C, the Activity determination at liquid phase separation peak is prepared；
D, analytic type high performance liquid chromatography separation purifies；
E, the Activity determination and Mass Spectrometric Identification at analysis liquid phase separation peak.
Smooth Xenopus laevis skin secretion after the smooth Xenopus laevis skin of physical stimulation by obtaining in the step A.
The physical stimulation be constant pressure electro photoluminescence, voltage 15-20V.
Application of the smooth Xenopus laevis antibacterial skin peptide of the present invention in antibacterials are prepared.
Application of the smooth Xenopus laevis antibacterial skin peptide of the present invention in the antibacterials as caused by bacterium are prepared.
The smooth Xenopus laevis antibacterial skin peptide application in preparation of anti-tumor drugs of the present invention.
Application of the smooth Xenopus laevis antibacterial skin peptide of the present invention in the drug for preparing treatment breast cancer.
The invention has the advantages and positive effects that:
The present invention stimulates skin using physical method, dramatically protect the structural intergrity of antibacterial peptide with
And biological activity；Secondly, antibacterial peptide disclosed by the invention is micromolecule polypeptide, is conducive to skin absorption, is also greatly reduced
The difficulty and cost of its chemical synthesis enhance the possibility of its application.Finally, antibacterial peptide disclosed by the invention has significant
Bacteriostatic activity, relatively low hemolytic activity and good anti-breast cancer cell activity, and without toxic side effect, be not easy to produce
Drug resistance, so as to greatly strengthen application range.In summary advantage, antibacterial peptide provided by the invention can be applied to industrial life
In production.
Description of the drawings
Fig. 1 is the chromatogram of the smooth Xenopus laevis antibacterial skin peptide of the present invention；
Fig. 2 is the mass spectrogram of the smooth Xenopus laevis antibacterial skin peptide of the present invention；
Fig. 3 is the synthesis in solid state chromatogram of the smooth magainin of the present invention；
Fig. 4 is the synthesis in solid state mass spectrogram of the smooth magainin of the present invention；
Fig. 5 is the hemolysis rate of the smooth magainin of the various concentration present invention；
Fig. 6 is inhibiting rate of the smooth magainin to breast cancer cell MCF-7 of the various concentration present invention.
Specific embodiment
The sequence that the smooth Xenopus laevis antibacterial skin peptide of the present invention is made of 4 molecule aspartic acids and 1 molecule glutamic acid
For 5 peptides of Asp-Glu-Asp-Asp-Asp, that is, DEDDD, molecular weight 607.7Da is named as XLAsp-P1.
1st, smooth Xenopus laevis antibacterial skin peptide of the invention is prepared using following methods：
The acquisition of 1.1 smooth Xenopus laevis skin secretions
100 smooth Xenopus laevis are taken, are cleaned up its skin surface with distilled water, remove impurity.Utilize the direct current of 15V
Electricity, the stimulation skin surface of discontinuity, induced skin secretion.10 min or so visible dermis secrets out of more water white transparency object
Matter, while it is mingled with white mucus shape substance.Above-mentioned secretion effect can be received using the direct current between voltage 15-20V,
Secretion is collected with ultra-pure water, 12000 rpm centrifuge 15 min, and removal precipitation takes supernatant.Supernatant after 0.45 μm of membrane filtration,
20 min are centrifuged with 12000 rpm of super filter tube of 10 KDa, to remove high molecular weight protein.With staphylococcus aureus S.aureus
ATCC29213 detect crude extract antibacterial activity, crude extract is concentrated using freeze dryer, after -40 DEG C preservation.
1. 2 preparative high performance liquid chromatographies isolate and purify
Semi-preparative C18 columns are connected with instrument, and by mobile phase A（Sample-loading buffer ,+0.05% TFA of water）, B
（Elution buffer ,+0.05% TFA of acetonitrile）Pump with instrument communicates.Adjusting instrument, and with A liquid balance systems, until absorption value
Reach balance.Then automatic loading is set 2 mL/ times, automatic 2 min/ of collection procedure pipes, flow velocity is 2 mL/min.And according to
Preliminary experiment as a result, setting elution program, as shown in Table 1 below.Multiple loading merges the component of same time, and laggard
Row concentration.
The elution program of 1 preparative high performance liquid chromatography of table
| Time（min） | Controller | Channel | Ratio（%） |
| 0.01 | Pumps | B.Conc | 0 |
| 8.00 | Pumps | B.Conc | 0 |
| 28.00 | Pumps | B.Conc | 30 |
| 90.00 | Pumps | B.Conc | 70 |
| 95.00 | Pumps | B.Conc | 70 |
| 96.00 | ControllerStop |
1. 3 prepare the Activity determination at liquid phase separation peak
Strain is used using staphylococcus aureus S.aureus ATCC29213 as Activity determination, utilizes susceptibility piece body of laws
The activity of outer detection detached peaks.Bacterium solution is diluted to the Bacteria Culture plate that 100 μ L is taken to be coated onto a diameter of 90mm after 0.5 Maxwell turbidity
On, when will air-dry, the susceptibility piece that each detached peaks make is attached to agar surface, 37 DEG C of incubator overnight incubations, according to
The activity of the presence or absence of inhibition zone and size judgement detached peaks whether there is and power, active detached peaks further separate.
1. 4 analytic type high performance liquid chromatography separations purify
In order to determine the active material in each Peak Activity, obtain the higher sample of purity consequently facilitating sequencing experiment into
Row, we utilize analytic type high performance liquid chromatography（Analytical High Performance Liquid
Chromatography, Analytical HPLC）Instrument makees Peak Activity further separation.First by the same instrument of analytic type C18 columns
Device is connected, and by mobile phase A（Sample-loading buffer ,+0.05% TFA of water）, B（Elution buffer, acetonitrile+0.05%TFA）Together
The pump correspondence of instrument communicates.Adjusting instrument, A pumps and B pumps purge, and use A liquid balance systems respectively, reach flat up to absorption value
Weighing apparatus.Setting flow velocity is 1mL/min, and manual loading 100uL/ times is collected manually according to absorption peak.Multiple loading merges identical peak
And it is concentrated.Elution program setting is as shown in table 2, and different samples are according to first time elution effect change elution program.
The elution program of 2 analytic type high performance liquid chromatography of table
| Time（min） | Flow velocity（mL/min） | Ratio（%B） |
| 0.01 | 1 | 0 |
| 5.00 | 1 | 0 |
| 20.00 | 1 | 5 |
| 60.00 | 1 | 40 |
| 70.00 | 1 | 70 |
The Activity determination and Mass Spectrometric Identification at 1.5 analysis liquid phase separation peaks
The method of Activity determination is the same as 1. 3.Using MALDI-TOF/TOF mass spectrographs to the detached peaks with bacteriostatic activity into
Row identification, including molecular weight and purity.The separation sample of 1 μ L and peptide standard are put respectively on polishing target first, after air-drying,
Again plus 1uL saturated alpha-CCA matrix（Solvent：+ 50% acetonitrile of 0.1% trifluoroacetic acid) in sample surfaces, it spontaneously dries.MALDL-
TOF/TOF is using cation and automatic obtaining mode gathered data.It first passes through and is corrected using poly saccharide peptide standard product, and set
Mass scan range for 0-4 KDa, so as to obtain sample first mass spectrometric figure.By analyzing mass spectrum, it is higher to obtain purity（It reaches
To 95%）Component can carry out the measure of amino acid sequence.Be finally recovered it is satisfactory be 4-9 peaks antibacterial peptide,
Chromatography and mass spectrogram are as shown in Fig. 1, Fig. 2.
2nd, the measure of smooth Xenopus laevis antibacterial skin peptide amino acid sequence
By isolating and purifying and Mass Spectrometric Identification, amino acid sequencing is carried out to the antibacterial peptide for reaching sequencing requirement.491 types of ABI
The principle of amino acid sequence analysis instrument is Edman edman degradation Edmans：It is that isothiocyanic acid benzene fat homopolypeptide N- ends residue is coupled first
Reaction, forms PTC peptide（PTC- peptides）；Then PTC- peptides form thiazole purine ketone benzene after being cyclized cracking reaction
Ammonia amino acid（ATZ-AA）；Finally using conversion reaction, ATZ-AA is converted into benzene isothiourea amino acid（PTH-AA）.By micro-
Gradient delivery system（Capillary HPLC）Retention time with the PTH-AA of standard is resisted into comparison using Data Analysis Software
The amino acid sequence of bacterium peptide.
3rd, the bioinformatic analysis of smooth Xenopus laevis antibacterial skin peptide sequence
Pass through bioinformatic database NCBI（http://www.ncbi.nlm.nih.gov/）And
Antimicrobial Peptide Database（APD, http://aps.unmc.edu/AP/main.html）Two big datas
Library scans for comparing, and determines that obtained smooth Xenopus laevis antibacterial skin peptide sequence is differed with known any sequence, therefore
It can determine that as new sequence, be shown in Table 3 with known species homology analysis.
The biological information analytical table of 3 smooth Xenopus laevis antibacterial skin peptide sequence of table
4th, the synthesis in solid state of smooth magainin
The synthesis in solid state of 4.1 polypeptides
Difficulty is carried out to the amino acid sequence of synthetic antibacterial peptide first using the synthesis difficulty forecasting software of Peptide synthesizer
Prediction.According to prediction as a result, setting synthesis program in control software is synthesized.By synthesis cut after polypeptide product according to
Following method is handled：Remove the polypeptide products being placed in 15 mL centrifuge tubes, ice bath 30min；Concentration production is dried up using nitrogen
Product are to 2mL, then according to 5：1 adds in the ether of precooling, ice bath 3min；Overturning centrifuge tube repeatedly makes Precipitation, is then centrifuged for,
2000rpm, 3min.It discards supernatant, precipitation is washed with ether, supernatant is abandoned in centrifugation again.It is repeated 5 times；Test tube is placed in draught cupboard
In so that ether volatilization, -80 DEG C of solid product saves backup.
The purifying and identification of 4.2 polypeptides
The polypeptide products of synthesis are dissolved with ultra-pure water, are then purified using high performance liquid chromatograph.It will be partly
It prepares the reversed columns of C-18 with analytic type high performance liquid chromatograph to be connected, debugging machine is simultaneously balanced with A liquid.Setting flow velocity be
2 mL/min, manual 200 μ of loading L/ times, are collected manually according to absorption peak.Multiple loading merges identical peak and is concentrated.
Elution program setting changes elution program with table 2, different samples according to first time separating effect.The sample of each component is carried out
Concentration, and be detected according to 1.3 bacteriostatic activity detection method.Active separation component is likely to what we synthesized
Target peptide fragment, however, to ensure that accuracy, active separation component is pressed using MALDL-TOF/TOF biomass spectrometers
The identification of molecular weight is carried out according to 1.5 method, chromatogram and mass spectrogram such as Fig. 3 and Fig. 4 as a result, mass spectrogram and purified polypeptide
Mass spectrogram coincide, it was demonstrated that its component makes the target peptide fragment that we synthesize.
5th, the minimal inhibitory concentration of smooth Xenopus laevis antibacterial skin peptide（Minimal inhibitory concentration,
MIC）Measure
1. test bacterium is cultivated in TSB culture mediums to debita spissitudo.Concentration is adjusted to 0.5 wheat than turbid instrument first with Maxwell
Family name's unit, about 108 Then CFU/mL is diluted to 2 × 10 again5 CFU/mL。
2. according to the sample concentration of setting, sample is diluted using the culture medium of fresh high pressure.
3. the diluted 50 μ l of sample mixed liquor of the 50 μ L of bacterium solution and fresh culture that adjust concentration are added in into the training of 96 holes
It supports in plate, is uniformly mixed.The concentration of sample sets certain gradient.
4. no sample effect group is negative control.
5. after 16~18 h of biochemical cultivation case culture, OD600 nm detection absorption values.Estimate the state in each hole, Kong Zhongwu
Misty suspension state is considered as growth inhibition, using the minimum concentration significantly inhibited as MIC value.
MIC value is shown in Table 4, the results showed that, antibacterial peptide prepared by the present invention makees experimental strain with preferable growth inhibition
With, and have good bacteriostatic activity to Gram-positive and Gram-negative bacteria.
4 smooth Xenopus laevis antibacterial skin peptide of table is to the MIC value of experimental bacteria
6th, smooth Xenopus laevis antibacterial skin peptide hemolytic activity detection
The hemolytic activity of antibacterial peptide as antibacterial peptide for one of side effect index of clinical treatment, has and its important
Effect.
1. acquiring rabbit new blood using anti-coagulants, 1000 rpm centrifuge 5 min, removal supernatant, blood platelet and leucocyte
Layer.
2. the PBS buffer solution using PH7.4 is washed three times, supernatant leaving layer red blood cell layer is abandoned.It is preceding to wash 1000 twice
Rpm centrifuges 10 min.After second is washed, directly red blood cell will be taken with 1 mL pipettors, in removal leucocyte and blood
During platelet layer, rough quantify can also be carried out to red blood cell.Third time washing centrifuges 10 min for 2000 rpm, abandons supernatant.
3. taking a certain amount of red blood cell, it is diluted using PBS, until final concentration of 8%.
4. carrying out doubling dilution to sample using PBS, the sample of maximum concentration is obtained first, then carry out doubling dilution.First
The 100 μ L of sample of each concentration are put into centrifuge tube, label is clear.
5. take 100 μ L red cell suspensions after dilution（8%）It is mixed with（Red blood cell final volume is 4%）.37 DEG C of water-baths
1h.0.1%Triton X-100 are positive control, and PBS buffer solution is negative control.
6. 1000 rpm centrifuge 5 min, 100 μ L of supernatant are drawn, supernatant extinction is detected under 535 nm of wavelength with microplate reader
Degree（Detect the release of ferroheme）.Minimum hemolytic concentration（Minimal hemolytic concentration, MHC）To cause
The minimum concentration of 10% haemolysis.
7. the calculation formula of percentage of hemolysis is
Haemolysis %=100 × (（Asample-Ablank）/(Atriton-Ablank)〕
As shown in figure 5, when being as a result shown in 64 μ g/mL of antibacterial peptide concentration, hemolysis rate only has 6.2%, shows system of the present invention
Standby antibacterial peptide has relatively low hemolytic activity in the range of MIC value, unlimited to its application in medical treatment.
7th, tumor growth inhibiting activity detects
Compared with traditional antitumor drug, some antibacterial peptides pass through unique tumor cytotoxicity mechanism and coating dissolving machine
System, so as to antitumor activity.So the research about antibacterial peptide Anticancer Effect and Mechanism has become hot spot in recent years.This
It tests and preliminary research carries out its tumor growth inhibiting activity using mtt assay to newfound antibacterial peptide, be as follows：
1. MCF-7 cells are cultivated, until exponential phase.
2. being digested using trypsase to cell, and carry out cell count.Diluting cells suspension is by 0.5 × 104A/
ML to cell concentration be 5 × 104A/mL.
3. plating cells, per 100 μ L of hole, cell number is 5000/hole.
4. 37 DEG C, 5% CO2 cultivates 12 h.The antibacterial peptide of various concentration is set, then carries out loading.
5. continuing to cultivate 24 h after loading, 10 μ L MTT are then added in per hole under conditions of being protected from light, are incubated 4 h.
6. discarding supernatant culture medium, action is light.Then 100 μ LDMSO are added in per hole, it is dissolving crystallized.
7. 37 DEG C, 80 rpm, shaking table is incubated 20 min, until crystallization fully dissolving.
8. light absorption value is detected under 535nm using microplate reader.
9. inhibiting rate calculates：
Inhibiting rate %=(1-（A sample-A blank）/（A feminine gender-A blank）〕×100%
As shown in Figure 6, the results showed that, antibacterial peptide prepared by the present invention is respectively provided with MCF-7 preferable growth inhibition effect.
It should be noted that various experimental implementations involved in the present invention, are the ordinary skill in the art, if in the text
It is not particularly illustrated, then various common tool books before those of ordinary skill in the art are referred to the present patent application day,
Scientific and technical literature or relevant specification, handbook etc. are implemented.
Involved various experimental articles herein（Including but not limited to：Chemical reagent, biological products, cell, biology
Body, instrument etc.）Among, for those are special or should not obtain, Wen Zhongjun has indicated manufacturer, bibliography or in detail
Thin preparation method；It is routine experiment articles for use without special instruction, it, can be by various before the present patent application day
Mode（Such as it buys, voluntarily prepare）Easily obtain.
It should be appreciated that without departing from the spirit and scope of the present invention, those of ordinary skill in the art can be with
Various changes and improvement are made to it in form and details, and these are all considered to fall into protection scope of the present invention.
Claims (4)
1. a kind of preparation method of smooth Xenopus laevis antibacterial skin peptide, which is characterized in that include the following steps：
A, smooth Xenopus laevis skin secretion is extracted：Secretion is secreted out of by the smooth Xenopus laevis skin induced skin of physical stimulation, with super
After the above-mentioned secretion of pure water collection, centrifugation removal precipitation takes supernatant, and supernatant after filtering, centrifuges again, crude extract is made, with gold
Staphylococcus aureus S.aureusATCC29213 detects the antibacterial activity of crude extract, and crude extract is concentrated using freeze dryer
Freezen protective afterwards；
By being obtained after the smooth Xenopus laevis skin of physical stimulation, physical stimulation pierces smooth Xenopus laevis skin secretion for constant pressure electricity in step A
Swash, voltage 15-20V；Rotating speed during centrifugation is 12000rpm；
B, preparative high performance liquid chromatography isolates and purifies；And according to following table curriculum offering elution program：
C, the Activity determination at liquid phase separation peak is prepared；Wherein test bacterium is staphylococcus aureus, and method is used to live for inhibition zone
Property detection；
D, analytic type high performance liquid chromatography separation purifies；And according to following table curriculum offering elution program；
E, the Activity determination and Mass Spectrometric Identification at analysis liquid phase separation peak, activity test method are identical with the detection method in step C；
The smooth Xenopus laevis antibacterial skin peptide prepared is that include the sequence of 4 molecule aspartic acids and 1 molecule glutamic acid be Asp-Glu-
5 peptides of Asp-Asp-Asp, that is, DEDDD.
2. according to the preparation method of the smooth Xenopus laevis antibacterial skin peptide described in claim 1, it is characterised in that：What is prepared is smooth
The molecular weight of Xenopus laevis antibacterial skin peptide is 607.7Da.
3. application of the smooth Xenopus laevis antibacterial skin peptide in antibacterials are prepared described in claims 1 or 2.
4. application of the smooth Xenopus laevis antibacterial skin peptide in the drug for preparing treatment breast cancer described in claims 1 or 2.
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Citations (4)
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|---|---|---|---|---|
| WO2007042010A3 (en) * | 2005-10-12 | 2008-02-21 | Goerne Herbert | Synergistic pharmaceutical composition containing a peptide with 2 to 5 amino acids |
| CN101186921A (en) * | 2007-12-12 | 2008-05-28 | 山东大学 | Novel gene Xsynuclein for xenopus laevis GRK family |
| WO2008083678A2 (en) * | 2007-01-10 | 2008-07-17 | Trojanon Gmbh & Co. Kg | Pharmaceutically active compounds |
| CN102304178A (en) * | 2011-09-14 | 2012-01-04 | 杨阳 | Preparation method and application of African clawed toad antitumor polypeptide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007042010A3 (en) * | 2005-10-12 | 2008-02-21 | Goerne Herbert | Synergistic pharmaceutical composition containing a peptide with 2 to 5 amino acids |
| WO2008083678A2 (en) * | 2007-01-10 | 2008-07-17 | Trojanon Gmbh & Co. Kg | Pharmaceutically active compounds |
| CN101186921A (en) * | 2007-12-12 | 2008-05-28 | 山东大学 | Novel gene Xsynuclein for xenopus laevis GRK family |
| CN102304178A (en) * | 2011-09-14 | 2012-01-04 | 杨阳 | Preparation method and application of African clawed toad antitumor polypeptide |
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