CN104877022B - The polypeptide extracted in a kind of nematoblast venom from fire medusa and its application - Google Patents
The polypeptide extracted in a kind of nematoblast venom from fire medusa and its application Download PDFInfo
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- CN104877022B CN104877022B CN201510148286.2A CN201510148286A CN104877022B CN 104877022 B CN104877022 B CN 104877022B CN 201510148286 A CN201510148286 A CN 201510148286A CN 104877022 B CN104877022 B CN 104877022B
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Abstract
The invention discloses the polypeptides extracted in a kind of nematoblast venom from fire medusa, and amino acid sequence is as shown in SEQ ID No.1.The invention also discloses the extracting methods of this polypeptide：Venom protein is extracted from fire medusa nematoblast venom, uses Histrap SP strong cat ion exchange columns and Histrap Q strong anion exchange columns point polypeptide from venom protein successively later.The invention also discloses application of this polypeptide in preparing the drug of angiogenesis of antitumor induction.The polypeptide of the present invention can inhibit umbilical vein endothelial cell HUVEC to grow, induction of cord venous endothelial cell HUVEC apoptosis.The extracting method that the present invention uses is easy to operate, and separating step is few, is suitable for quick separating；Separation condition is mild, can farthest preserve the antitumor activity of gained protein.
Description
Technical field
The present invention relates to marine biotechnologies and field of medicaments, and in particular to one kind is extracted from fire medusa nematoblast venom
Polypeptide and its application.
Background technology
North Sea fire medusa (Tamoya alata Uchida) is a kind of large-scale play for being distributed only over China's Beibu Bay
Malicious jellyfish touches wrist length and has a large amount of ecthoaeum bladder cell, the venom that main active is protein/polypeptide included, with people
Or the nematoblast can penetrate skin when animal contact, can be deep into dermal sites, and discharge the toxin stored into the cell, cause to sting
Wound.According to badly wounded degree difference, victim occurred immediately at bruising red swelling of the skin, shouting pain, bleed, exanthemv etc. it is existing
As severe patient is weak, generates heat, vomiting, or even dead.To find out its cause, jellyfish touches in the nematoblast of wrist containing a large amount of jellyfish poison
Element, the mainly protein matter of structure novel.
Species of jellyfish is different, physiological structure and toxic components contained in vivo and activity there is also larger difference, this
Just cause the work that isolates and purifies of the toxin of variety classes jellyfish to be difficult to imitate, can only grope to obtain by complicated experiment.Institute
There is prodigious difficulty with the work that isolates and purifies of medusocongestin.Inventor further studies fire medusa venom, from the venom
In can isolate a kind of polypeptide with notable antitumor activity, which has significant antitumor activity, corresponding activity
Ingredient is a kind of protein.
New vessels and lymphatic vessel in human tumor are realized by angiogenesis and lymphatic vessel generation respectively, and in tumour
Growth and diffusion (invasion and transfer) in play an important role.Tumor vascular fault of construction makes blood vessel have high-permeability,
It is one of the approach of metastases, different types of mesenchyma stroma of tumors blood vessel does not have essential distinction, but in form and quantity
It is different.Therefore, the technical issues of a kind of conservative therapy of searching is urgent need to resolve.
Invention content
It is excellent it is an object of the invention to solve at least the above and/or defect, and provide at least to will be described later
Point.
It is a still further object of the present invention to provide the polypeptides extracted in a kind of nematoblast venom from fire medusa, can inhibit
Umbilical vein endothelial cell HUVEC growths, induction of cord venous endothelial cell HUVEC apoptosis.
A further object of the invention provides a kind of extracting method of the polypeptide, and easy to operate, separating step is few, suitable
In quick separating；Separation condition is mild, can farthest preserve the antitumor activity of gained protein.
It is a still further object of the present invention to provide a kind of applications of the polypeptide.
In order to realize these purposes and other advantages according to the present invention, one kind is provided from fire medusa nematoblast venom
The polypeptide of extraction, amino acid sequence is as shown in SEQ ID No.1.
A kind of extracting method of the polypeptide extracted in the nematoblast venom from fire medusa, is extracted from fire medusa nematoblast venom
Venom protein uses Histrap SP strong cat ion exchange columns and Histrap Q strong anions exchange columns from venom successively later
Polypeptide as described in claim 1 is detached in albumen.
Preferably, the extracting method of polypeptide is extracted in the nematoblast venom from fire medusa, it is further comprising the steps of：
Step 1: taking tactile wrist from fire medusa living, mixes, stir for 1: 1-1.5 according to volume ratio with 0-4 DEG C of deionized water
After mixing, standing, precipitation is taken to repeat 2-4 time, centrifuged for the first time, collection sediment；
Step 2: the sediment that step 1 is obtained and the phosphorus that total phosphate a concentration of 0.025mol/L, pH are 5.5-6.5
Sour disodium hydrogen-sodium dihydrogen phosphate buffer is mixed according to mass volume ratio 1g: 40-60mL, ultrasonic disruption, second from
The heart collects supernatant, obtains venom protein；
Step 3: step 2 is taken to obtain venom protein, the phosphorus for being 5.5-6.5 with total phosphate concentration 0.025mol/L, pH
Sour disodium hydrogen-sodium dihydrogen phosphate buffer dilutes 3-5 times, is allowed to slowly flow through Histrap SP strong cat ion exchange columns, press
It is the first phosphate buffer solution that 0.085-0.090mol/L, pH are 5.5-6.5 that sodium chloride concentration, which is added, according to 4-6 times of column volume
First time elution is carried out, it is 0.090-0.100mol/L, pH 5.5- that sodium chloride concentration then, which is added, according to 4-6 times of column volume
6.5 the second phosphate buffer solution carries out second and elutes, and collects leacheate and obtains the second leacheate；
Step 4: taking step 3 to obtain the second leacheate carries out buffer solution displacement, change buffer solution is total phosphate
A concentration of 0.025mol/L, pH are disodium hydrogen phosphate-sodium dihydrogen phosphate buffer of 7.5-8.5, are allowed to slowly flow through
Histrap Q strong anion exchange columns, it is that 0.080-0.090mol/L, pH are that sodium chloride concentration, which is added, according to 4-6 times of column volume
The third phosphate buffer solution of 7.5-8.5 carries out third time elution, and sodium chloride concentration then, which is added, according to 4-6 times of column volume is
0.090-0.100mol/L, pH are that the tetraphosphate buffer solution of 7.5-8.5 carries out the 4th elution, collect leacheate and obtain
4th leacheate；
Step 5: the 4th leacheate that step 4 obtains is taken to carry out third time centrifugal ultrafiltration concentration, supernatant, decompression are collected
Freeze-drying is to get the polypeptide.
Preferably, the extracting method of the polypeptide extracted in the nematoblast venom from fire medusa, further includes following step
Suddenly：
Step 1: taking tactile wrist from fire medusa living, mixed for 1: 1.3 according to volume ratio with 2 DEG C of deionized water, stirring,
After standing, precipitation is taken to be repeated 3 times, centrifuged for the first time, collects sediment；
Step 2: the phosphoric acid hydrogen that the sediment that step 1 is obtained is 6.0 with total phosphate a concentration of 0.025mol/L, pH
Disodium-sodium dihydrogen phosphate buffer is mixed according to mass volume ratio 1g: 50mL, ultrasonic disruption, second of centrifugation, in collection
Clear liquid obtains venom protein；
Step 3: step 2 is taken to obtain venom protein, the phosphoric acid hydrogen for being 6.0 with total phosphate concentration 0.025 mol/L, pH
Disodium-sodium dihydrogen phosphate buffer dilutes 4 times, is allowed to slowly flow through Histrap SP strong cat ion exchange columns, according to 5 times of columns
It is that the first phosphate buffer solution that 0.087mol/L, pH are 6.0 is once eluted that sodium chloride concentration, which is added, in volume, is then pressed
It is that the second phosphate buffer solution that 0.095mol/L, pH are 6.0 carries out second of leaching that sodium chloride concentration, which is added, according to 5 times of column volumes
It washes, collects leacheate and obtain the second leacheate；
Step 4: taking step 3 to obtain the second leacheate carries out buffer solution displacement, change buffer solution is total phosphate
Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer that a concentration of 0.025mol/L, pH are 8.0, be allowed to slowly to flow through by
Histrap Q strong anion exchange columns, it is the third that 0.085mol/L, pH are 8.0 that sodium chloride concentration, which is added, according to 5 times of column volumes
Phosphate buffer solution carries out third time elution, and it is that 0.095mol/L, pH are that sodium chloride concentration then, which is added, according to 5 times of column volumes
8.0 tetraphosphate buffer solution carries out the 4th elution, collects leacheate and obtains the 4th leacheate；
Step 5: the 4th leacheate that step 4 obtains is taken to carry out third time centrifugal ultrafiltration concentration, supernatant, decompression are collected
Freeze-drying is to get the polypeptide.
Preferably, the extracting method of the polypeptide extracted in the nematoblast venom from fire medusa, the step 5 are super
The molecular cut off of super filter tube used in filter is 30K.
Preferably, the extracting method of the polypeptide extracted in the nematoblast venom from fire medusa, the primary centrifugation
Operating condition be：Centrifugal force 1000G, 4 DEG C, 15min；The operating condition of the secondary centrifuging is：Centrifugal force 13000G, 4 DEG C,
2h；The operating condition centrifuged three times is：Centrifugal force 5000G, 4 DEG C, 1h.
Preferably, the extracting method of the polypeptide extracted in the nematoblast venom from fire medusa, first phosphorus
Hydrochlorate buffer solution and the second phosphate buffer solution are disodium hydrogen phosphate-phosphoric acid of a concentration of 0.025mol/L of total phosphate
Sodium dihydrogen buffer solution；Third phosphate buffer solution and tetraphosphate buffer solution are that total phosphate is a concentration of
Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer of 0.025mol/L.
The polypeptide extracted in a kind of nematoblast venom from fire medusa is in preparing the drug of angiogenesis of antitumor induction
Application.
The present invention includes at least following advantageous effect：
The first, the present invention extracts a kind of new polypeptide from fire medusa nematoblast venom, is to pass through Histrap SP successively
Strong cat ion exchange column and Histrap Q strong anion exchange columns, it is isolated from venom protein, it is that there is antitumor lure
The new albumen for the angiogenic activity led, belongs to pioneer invention；
The second, tactile wrist is immersed in 1: 1-1.5 volume ratio in 0-4 DEG C of deionized water, prevents temperature height from causing to touch wrist
Albuminous degeneration and convenient under low temperature touch wrist self solve, stirring stand destroying for times and decompose touch wrist collagen and protective layer；
Third, primary centrifugation are in 1000G, 4 DEG C, are carried out under 15min, need tactile wrist being completely dissolved as touch cells；It is secondary
Centrifugation is in 13000G, 4 DEG C, is carried out under 2h, need by touch cells it is broken after venom protein all dissolve out, ultrasonic disruption cell is residual
The centrifugal force of the smaller needs of piece grain size is bigger；It is centrifuged three times in 5000G, 4 DEG C, is carried out under 1h, needing will be after layers-separated
Albumen is obtained to be concentrated；
4th, venom protein is passed through into Histrap SP strong cat ion exchange columns, weakly acidic buffer solution makes big portion
Divide venom protein and H+In conjunction with then it is positively charged, strong cat ion exchange column adsorbs positively charged venom protein, uses chlorine successively
The first phosphate buffer solution and sodium chloride concentration that change na concn is 0.085-0.090mol/L are 0.090-0.100mol/L
The second phosphate buffer solution elution, abandon a part of unwanted albumen, then exchange buffering solution be 0.025mol/L,
PH is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer of 7.5-8.5, and weakly alkaline buffer solution makes most of protein belt
Negative electrical charge, using Histrap Q strong anion exchange columns, weakly alkaline buffer solution makes strong anion exchange column adsorb band
The albumen of negative electrical charge, successively with sodium chloride concentration be 0.080-0.090mol/L third phosphate buffer solution and sodium chloride it is dense
The tetraphosphate buffer solution that degree is 0.090-0.100mol/L elutes, and obtains target protein；
5th, the polypeptide that the present invention obtains inhibits it was proved that with anti-breast ductal cancer activity in umbilical vein
Chrotoplast HUVEC growths, induction of cord venous endothelial cell HUVEC apoptosis；Method using the present invention can be with mass disposal
Fire medusa nematoblast venom, isolates the anti-tumor active protein wherein contained.Easy to operate, separating step is few, is suitable for fast
Speed separation；Separation condition is mild, can farthest preserve the antitumor activity of gained protein.
Part is illustrated to embody by further advantage, target and the feature of the present invention by following, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
The term definition involved in the present invention arrived
Unless otherwise defined, otherwise all technologies used herein and scientific terminology all have with it is of the art
Those of ordinary skill usually understands identical meaning.Although the usable and described herein in the practice or test of the present invention
Similar or equivalent any method, apparatus and material, but preferred method, device and material will now be described.
Term " amino acid " means to constitute the base unit of protein, assigns the specific molecular morphosis of protein, make
Its molecule has biochemical activity.Protein is bioactive molecule important in organism, includes the enzyme of catalysis metabolism, two
Or more than two chemistry of amino acids aggregate into peptide, the original segments of a protein are proteinogenous precursor, amino acid
It broadly refers to not only contain there are one basic amine group but also contain the organic compound there are one acidic carboxypolymer, as described in its name
Like that.But general amino acid refers to then the structural units for constituting protein.In living nature, the ammonia of native protein is constituted
There is base acid its specific design feature, i.e. its amino to be connected directly between on alpha -carbon atom, and this amino acid is referred to as alpha-amido
Acid.More than 300 kinds of amino acid, wherein 21 kinds of a-amino acid are shared in nature, a-amino acid is the component point of peptide and protein
Son, and constitute one of the basic masonry of life mansion.
Term " polypeptide " means a-amino acid with the compound that peptide bond links together and is formed, it is also protein hydrolysis
Intermediate product.Compound is called dipeptides made of two amino acid molecular dehydrating condensations, similarly analogizes also tripeptides, four
Peptide, pentapeptide etc., the usually compound made of 10-100 amino acid molecular dehydrating condensations are less than polypeptide, their molecular weight
10000 dalton can penetrate semi-permeable membrane, not precipitated by trichloroacetic acid and ammonium sulfate, also have document by 2-10 amino acid
The peptide of composition is known as oligopeptides (small-molecular peptides)；The peptide of 10-50 amino acid composition is known as polypeptide；By 50 or more amino acid groups
At peptide be known as protein.
Description of the drawings
Fig. 1 is structural schematic diagram of the polypeptide of the present invention to umbilical vein endothelial cell HUVEC.
The design sketch of apoptosis occurs for Fig. 2 polypeptid induction umbilical vein endothelial cell HUVEC of the present invention.
Specific implementation mode
Invention is described in further detail with reference to embodiment, to enable those skilled in the art with reference to specification text
Word can be implemented according to this.
Embodiment 1：
A kind of peptide molecule, amino acid sequence is as shown in SEQ ID No.1；
SEQ ID No.1：
Embodiment 2：
The extraction of polypeptide of the present invention：
Step 1: taking tactile wrist from fire medusa living, is mixed for 1: 1 according to volume ratio with 0 DEG C of deionized water, stir, is quiet
It postpones, precipitation is taken to be repeated 2 times, centrifuge for the first time, centrifugal condition is：Centrifugal force 1000G, collects sediment by 4 DEG C, 15min；
Step 2: the phosphoric acid hydrogen two that the sediment that step 1 is obtained is 5.5 with total phosphate concentration 0.025mol/L, pH
Sodium-sodium dihydrogen phosphate buffer is mixed according to mass volume ratio 1g: 40mL, ultrasonic disruption, second of centrifugation, centrifugal condition
For：Centrifugal force 13000G, collects supernatant, obtains venom protein by 4 DEG C, 2h；
Step 3: step 2 is taken to obtain venom protein, the phosphoric acid hydrogen for being 5.5 with total phosphate concentration 0.025mol/L, pH
Disodium-sodium dihydrogen phosphate buffer dilutes 3 times, is allowed to slowly flow and passes through Histrap SP strong cat ion exchange columns, according to 4 times
Column volume be added sodium chloride concentration be 0.085mol/L, pH 5.5, the phosphoric acid hydrogen two of a concentration of 0.025mol/L of total phosphate
Sodium-sodium dihydrogen phosphate buffer carries out first time elution, and sodium chloride concentration then, which is added, according to 4 times of column volumes is
Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer of a concentration of 0.025mol/L of 0.090mol/L, pH 5.5, total phosphate into
Second of elution of row, collects leacheate and obtains the second leacheate；
Step 4: taking step 3 to obtain the second leacheate carries out buffer solution displacement, change buffer solution is total phosphate
A concentration of 0.025mol/L, pH are disodium hydrogen phosphate-sodium dihydrogen phosphate buffer of 7.5-8.5, are allowed to slowly flow through
Histrap Q strong anion exchange columns, according to 4-6 times of column volume be added sodium chloride concentration be 0.080mol/L, pH 7.5, it is total
Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer that phosphate concn is 0.025mol/L carries out third time elution, then according to 4
Times column volume be added sodium chloride concentration be 0.090mol/L, pH 7.5, the phosphoric acid hydrogen two of a concentration of 0.025mol/L of total phosphate
Sodium-sodium dihydrogen phosphate buffer carries out the 4th elution, collects leacheate and obtains the 4th leacheate；
Step 5: the 4th leacheate for taking step 4 to obtain, the super filter tube for being 30K with molecular cut off carry out third time from
The heart is concentrated by ultrafiltration, and centrifugal condition is：Centrifugal force 5000G, collects supernatant, decompression freeze-drying is to get described more by 4 DEG C, 1h
Peptide.
Embodiment 3：
The extraction of polypeptide of the present invention：
Step 1: taking tactile wrist from fire medusa living, mixed for 1: 1.5 according to volume ratio with 4 DEG C of deionized water, stirring,
After standing, precipitation is taken to be repeated 4 times, centrifuged for the first time, centrifugal condition is：Centrifugal force 1000G, collects sediment by 4 DEG C, 15min；
Step 2: the phosphoric acid hydrogen two that the sediment that step 1 is obtained is 6.5 with total phosphate concentration 0.025mol/L, pH
Sodium-sodium dihydrogen phosphate buffer is mixed according to mass volume ratio 1g: 60mL, ultrasonic disruption, second of centrifugation, centrifugal condition
For：Centrifugal force 13000G, collects supernatant, obtains venom protein by 4 DEG C, 2h；
Step 3: step 2 is taken to obtain venom protein, the phosphoric acid hydrogen for being 6.5 with total phosphate concentration 0.025mol/L, pH
Disodium-sodium dihydrogen phosphate buffer dilutes 5 times, is allowed to slowly flow through Histrap SP strong cat ion exchange columns, according to 6 times of columns
Volume be added sodium chloride concentration be 0.090mol/L, pH 6.5, the disodium hydrogen phosphate-of a concentration of 0.025mol/L of total phosphate
Sodium dihydrogen phosphate buffer carries out first time elution, be then 0.100mol/L according to 6 times of column volumes addition sodium chloride concentrations,
Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer that pH is 6.5, a concentration of 0.025mol/L of total phosphate carries out second and drenches
It washes, collects leacheate and obtain the second leacheate；
Step 4: taking step 3 to obtain the second leacheate carries out buffer solution displacement, change buffer solution is total phosphate
Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer that a concentration of 0.025mol/L, pH are 8.5, is allowed to slowly flow through Histrap Q
Strong anion exchange column, according to 6 times of column volumes be added sodium chloride concentrations be 0.090mol/L, pH 8.5, total phosphate it is a concentration of
The disodium hydrogen phosphate of 0.025mol/L-sodium dihydrogen phosphate buffer carries out third time elution, is then added according to 6 times of column volumes
Sodium chloride concentration is 0.100mol/L, pH 8.5, disodium hydrogen phosphate-biphosphate of a concentration of 0.025mol/L of total phosphate
Sodium buffer solution carries out the 4th elution, collects leacheate and obtains the 4th leacheate；
Step 5: the 4th leacheate for taking step 4 to obtain, the super filter tube for being 30K with molecular cut off carry out third time from
The heart is concentrated by ultrafiltration, and centrifugal condition is：Centrifugal force 5000G, collects supernatant, decompression freeze-drying is to get described more by 4 DEG C, 1h
Peptide.
Embodiment 4：
The extraction of polypeptide of the present invention：
Step 1: taking tactile wrist from fire medusa living, mixed for 1: 1.3 according to volume ratio with 2 DEG C of deionized water, stirring,
After standing, precipitation is taken to be repeated 3 times, centrifuged for the first time, centrifugal condition is：Centrifugal force 1000G, collects sediment by 4 DEG C, 15min；
Step 2: the phosphoric acid hydrogen two that the sediment that step 1 is obtained is 6.0 with total phosphate concentration 0.025mol/L, pH
Sodium-sodium dihydrogen phosphate buffer is mixed according to mass volume ratio 1g: 50mL, ultrasonic disruption, second of centrifugation, centrifugal condition
For：Centrifugal force 13000G, collects supernatant, obtains venom protein by 4 DEG C, 2h；
Step 3: step 2 is taken to obtain venom protein, the phosphoric acid hydrogen for being 6.0 with total phosphate concentration 0.025mol/L, pH
Disodium-sodium dihydrogen phosphate buffer dilutes 4 times, is allowed to slowly flow through Histrap SP strong cat ion exchange columns, according to 5 times of columns
Volume be added sodium chloride concentration be 0.087mol/L, pH 6.0, the disodium hydrogen phosphate-of a concentration of 0.025mol/L of total phosphate
Sodium dihydrogen phosphate buffer carries out first time elution, be then 0.095mol/L according to 5 times of column volumes addition sodium chloride concentrations,
Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer that pH is 6.0, a concentration of 0.025mol/L of total phosphate carries out second and drenches
It washes, collects leacheate and obtain the second leacheate；
Step 4: taking step 3 to obtain the second leacheate carries out buffer solution displacement, change buffer solution is total phosphate
Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer that a concentration of 0.025mol/L, pH are 8.0, is allowed to slowly flow through Histrap Q
Strong anion exchange column, according to 5 times of column volumes be added sodium chloride concentrations be 0.085mol/L, pH 8.0, total phosphate it is a concentration of
The disodium hydrogen phosphate of 0.025mol/L-sodium dihydrogen phosphate buffer carries out third time elution, is then added according to 5 times of column volumes
Sodium chloride concentration is 0.095mol/L, pH 8.0, disodium hydrogen phosphate-biphosphate of a concentration of 0.025mol/L of total phosphate
Sodium buffer solution carries out the 4th elution, collects leacheate and obtains the 4th leacheate；
Step 5: the 4th leacheate for taking step 4 to obtain, the super filter tube for being 30K with molecular cut off carry out third time from
The heart is concentrated by ultrafiltration, and centrifugal condition is：Centrifugal force 5000G, collects supernatant, decompression freeze-drying is to get described more by 4 DEG C, 1h
Peptide.
Embodiment 5：
Polypeptide of the present invention carries out following anti-tumor experiment：
One, function and effect of the polypeptide of the present invention to umbilical vein endothelial cell HUVEC are detected using mtt assay
Experimental method：
It is 10 with culture medium adjustment concentration of cell suspension Step 1: collecting logarithmic phase HUVEC cells5A cells/ml,
96 orifice plates are taken, 100 μ L of cell suspension are added per hole, edge is filled with sterile water；
Step 2: 5%CO2, cultivating 24 hours for 37 DEG C keeps cell adherent, and the target polypeptides that various concentration is added carry out in fact
It tests.5-7 gradient is set, and per 100 μ L of hole, 3-5 Duplicate Samples are arranged in each concentration gradient；
Step 3: 5%CO2, 37 DEG C are cultivated 48 hours, and 20 μ LMTT solution (5mg/mL) are added per hole, and it is small to continue culture 4
When after terminate culture, carefully suck culture solution in hole；
Step 4: 150 μ L dimethyl sulfoxides are added per hole, it is placed in low-speed oscillation 10min on shaking table, makes MTT and living cells line
The first a ceremonial jade-ladle, used in libation crystallization fully dissolving that plastochondria lactic dehydrogenase enzyme reaction generates, is measured with microplate reader or ultra-violet and visible spectrophotometer
Absorbance under 490nm wavelength.
Experimental result：
Experimental result is as shown in Figure 1, the results showed that, the growth of the present invention to heparin-agarose affinity chromatography HUVEC
Inhibited, this inhibiting effect is incrementally increased with concentration raising of the present invention is applied.This explanation, the present invention
The proliferation of the polypeptide energy strong inhibition umbilical vein endothelial cell HUVEC, and polypeptide of the present invention is thin to the tumour
The inhibited proliferation of born of the same parents is concentration dependant.
Two, the confirmatory experiment of apoptosis occurs for polypeptid induction umbilical vein endothelial cell HUVEC of the present invention
Experimental method：
Step 1: collecting logarithmic phase HUVEC cells, adjustment concentration of cell suspension is 106A cells/ml, takes 6 orifice plates,
1mL is added per hole；
Step 2: 5%CO2, cultivating 24 hours for 37 DEG C keeps cell adherent, and the target polypeptides of 0.5 μ g/mL are added；
Step 3: 5%CO2, 37 DEG C are cultivated 48 hours, are cleaned twice with cold sterile PBS, and pancreatin digestion is then added,
Suspension is made in cell and is counted；
Step 4: taking the cell that 1-5 ten thousand is resuspended, 1000G centrifuges 5min, discards supernatant the Annexin V- that 195 μ L are added
FITC combination buffers, it is slight to blow and beat, suspension is made in cell；
Step 5: into above-mentioned cell suspension, the fluorescein isothiocynate (fluorescein of 5 μ L is added
Isothiocyanate) the Annexin V and 10 μ L propidium iodides (PI), slight mixing being coupled are protected from light incubation at room temperature
15min；
Step 6: after being incubated, then 400 μ L combination buffer solutions are added into centrifuge tube, then deliver binary channels stream
Formula cytometer carry out analysis of accounts, select a length of 488nm and 535nm of excitation light wave, detection wavelength of transmitted light be 525nm and
615nm。
Annexin V-FITC, PI and various other buffer solutions used are purchased from green skies biological reagent Co., Ltd.
Experimental result：
Experimental result as shown in Fig. 2, flow cytometry the result shows that, polypeptide of the present invention mainly induces HUVEC
Early apoptosis and late apoptic occur for cell, wherein again based on late apoptic.Under peptide concentration of the present invention, occur
The cell of early apoptosis accounts for about the 8.00% of total number of cells, and the cell that late apoptic occurs accounts for about the 68.08% of total number of cells.This
Illustrate, polypeptide of the present invention can induce umbilical vein endothelial cell HUVEC and apoptosis occurs, and based on late apoptic.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and embodiment shown and described herein.
Claims (6)
1. the polypeptide extracted in a kind of nematoblast venom from fire medusa is in preparing the drug of angiogenesis of antitumor induction
Using, which is characterized in that the amino acid sequence of the polypeptide is as shown in SEQ ID No.1.
2. a kind of extracting method of the polypeptide extracted from fire medusa nematoblast venom as described in claim 1, feature exist
In including the following steps：
According to volume ratio it is 1 with 0-4 DEG C of deionized water Step 1: take tactile wrist from fire medusa living:1-1.5 is mixed, stirring,
After standing, precipitation is taken to repeat 2-4 times, centrifuged for the first time, collects sediment；
Step 2: the sediment that step 1 is obtained and the phosphoric acid hydrogen that total phosphate a concentration of 0.025mol/L, pH are 5.5-6.5
Disodium-sodium dihydrogen phosphate buffer is according to mass volume ratio 1g:40-60mL is mixed, ultrasonic disruption, and second of centrifugation is received
Collect supernatant, obtains venom protein；
Step 3: step 2 is taken to obtain venom protein, the phosphoric acid hydrogen for being 5.5-6.5 with total phosphate concentration 0.025mol/L, pH
Disodium-sodium dihydrogen phosphate buffer dilutes 3-5 times, is allowed to slowly flow through Histrap SP strong cat ion exchange columns, according to 4-6
It is that the first phosphate buffer solution that 0.085-0.090mol/L, pH are 5.5-6.5 carries out that sodium chloride concentration, which is added, in times column volume
Then it is 5.5-6.5's that elution for the first time is 0.090-0.100mol/L, pH according to 4-6 times of column volume addition sodium chloride concentration
Second phosphate buffer solution carries out second and elutes, and collects leacheate and obtains the second leacheate；
Step 4: taking step 3 to obtain the second leacheate carries out buffer solution displacement, change buffer solution is total phosphate concentration
Disodium hydrogen phosphate-the sodium dihydrogen phosphate buffer for being 7.5-8.5 for 0.025mol/L, pH is allowed to slowly flow through Histrap Q
Strong anion exchange column, it is 7.5-8.5's that according to 4-6 times of column volume, sodium chloride concentration is added, which is 0.080-0.090mol/L, pH,
Third phosphate buffer solution carries out third time elution, and it is 0.090- that sodium chloride concentration then, which is added, according to 4-6 times of column volume
0.100mol/L, pH are that the tetraphosphate buffer solution of 7.5-8.5 carries out the 4th elution, collect leacheate and obtain the 4th leaching
Washing lotion；
Step 5: the 4th leacheate that step 4 obtains is taken to carry out third time centrifugal ultrafiltration concentration, supernatant, decompression freezing are collected
Drying is to get the polypeptide.
3. the extracting method of the polypeptide extracted from fire medusa nematoblast venom as claimed in claim 2, which is characterized in that also
Include the following steps：
According to volume ratio it is 1 with 2 DEG C of deionized water Step 1: take tactile wrist from fire medusa living:1.3 mixing, stirring are stood
Afterwards, it takes precipitation to be repeated 3 times, centrifuges for the first time, collect sediment；
Step 2: the phosphoric acid hydrogen two that the sediment that step 1 is obtained is 6.0 with total phosphate a concentration of 0.025mol/L, pH
Sodium-sodium dihydrogen phosphate buffer is according to mass volume ratio 1g:50mL is mixed, ultrasonic disruption, and supernatant is collected in second of centrifugation
Liquid obtains venom protein；
Step 3: step 2 is taken to obtain venom protein, the disodium hydrogen phosphate-for being 6.0 with total phosphate concentration 0.025mol/L, pH
Sodium dihydrogen phosphate buffer dilutes 4 times, is allowed to slowly flow through Histrap SP strong cat ion exchange columns, according to 5 times of column volumes
It is that the first phosphate buffer solution that 0.087mol/L, pH are 6.0 is once eluted that sodium chloride concentration, which is added, then according to 5
It is that the second phosphate buffer solution that 0.095mol/L, pH are 6.0 carries out second and elutes that sodium chloride concentration, which is added, in times column volume,
It collects leacheate and obtains the second leacheate；
Step 4: taking step 3 to obtain the second leacheate carries out buffer solution displacement, change buffer solution is total phosphate concentration
Disodium hydrogen phosphate-the sodium dihydrogen phosphate buffer for being 8.0 for 0.025mol/L, pH is allowed to slowly flow through by Histrap Q
Strong anion exchange column, it is that the third phosphate that 0.085mol/L, pH are 8.0 is slow that sodium chloride concentration, which is added, according to 5 times of column volumes
It rushes solution and carries out third time elution, it is that 0.095mol/L, pH are 8.0 that sodium chloride concentrations then, which are added, according to 5 times of column volumes
Tetraphosphate buffer solution carries out the 4th elution, collects leacheate and obtains the 4th leacheate；
Step 5: the 4th leacheate that step 4 obtains is taken to carry out third time centrifugal ultrafiltration concentration, supernatant, decompression freezing are collected
Drying is to get the polypeptide.
4. the extracting method of the polypeptide extracted from fire medusa nematoblast venom as claimed in claim 3, which is characterized in that institute
The molecular cut off for stating super filter tube used in step 5 ultrafiltration is 30K.
5. the extracting method of the polypeptide extracted from fire medusa nematoblast venom as claimed in claim 4, which is characterized in that institute
Stating the operating condition once centrifuged is：Centrifugal force 1000G, 4 DEG C, 15min；The operating condition of the secondary centrifuging is：Centrifugal force
13000G, 4 DEG C, 2h；The operating condition centrifuged three times is：Centrifugal force 5000G, 4 DEG C, 1h.
6. the extracting method of the polypeptide extracted from fire medusa nematoblast venom as claimed in claim 5, which is characterized in that institute
The first phosphate buffer solution and the second phosphate buffer solution stated are the phosphoric acid of a concentration of 0.025mol/L of total phosphate
Disodium hydrogen-sodium dihydrogen phosphate buffer；Third phosphate buffer solution and tetraphosphate buffer solution are total phosphate
Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer of a concentration of 0.025mol/L.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101181305A (en) * | 2007-11-07 | 2008-05-21 | 淮海工学院 | Extract of acaleph toxin as well as preparation method and usage thereof |
| CN103739694A (en) * | 2014-01-29 | 2014-04-23 | 中国科学院海洋研究所 | Method for extracting toxin protein from white cyanea tentacles |
| CN103819552A (en) * | 2014-02-17 | 2014-05-28 | 中国人民解放军第二军医大学 | Cyanea capillata polypeptide growth factor, preparation method and applications thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101181305A (en) * | 2007-11-07 | 2008-05-21 | 淮海工学院 | Extract of acaleph toxin as well as preparation method and usage thereof |
| CN103739694A (en) * | 2014-01-29 | 2014-04-23 | 中国科学院海洋研究所 | Method for extracting toxin protein from white cyanea tentacles |
| CN103819552A (en) * | 2014-02-17 | 2014-05-28 | 中国人民解放军第二军医大学 | Cyanea capillata polypeptide growth factor, preparation method and applications thereof |
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