CN104926932B - 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
- Publication number
- CN104926932B CN104926932B CN201510329507.6A CN201510329507A CN104926932B CN 104926932 B CN104926932 B CN 104926932B CN 201510329507 A CN201510329507 A CN 201510329507A CN 104926932 B CN104926932 B CN 104926932B
- Authority
- CN
- China
- Prior art keywords
- concentration
- venom
- buffer solution
- phosphate buffer
- polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
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 the drug for preparing anti-nasopharyngeal carcinoma.The polypeptide of the present invention can inhibit nasopharyngeal carcinoma tumor cell strain CNE 1, CNE 2 to grow, 2 apoptosis of induction nasopharyngeal carcinoma tumor cell strain CNE.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.
Nasopharyngeal carcinoma is the malignant tumour betided at the top of nasopharyngeal cavity with side wall.It is one of China's malignant tumour occurred frequently, morbidity
Rate is first of ear,nose & throat malignant tumour.Nasopharyngeal carcinoma radiotherapy multipair greatly has Medium sensitivity, and radiotherapy is nasopharyngeal carcinoma
Preferred therapy.But to compared with well-differentiated carcinoma, the course of disease is later and the case of recurrence after radiotherapy, operation excision and chemical drugs
Object treatment also belongs to indispensable means.Therefore, the technical issues of being urgent need to resolve there is an urgent need for a kind of conservative therapy.
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
Nasopharyngeal carcinoma tumor cell strain CNE-1, CNE-2 growth, induces nasopharyngeal carcinoma tumor cell strain CNE-2 apoptosis.
It is a still further object of the present invention to provide a kind of extracting methods of the polypeptide, and easy to operate, separating step is few, fit
It is suitable for 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 a concentration of 0.025mol/L of total phosphate, pH are the phosphorus of 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;
It is the phosphorus of 5.5-6.5 with total phosphate concentration 0.025mol/L, pH Step 3: step 2 is taken to obtain venom protein
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 0.035-0.045mol/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 0.045-0.060mol/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 0.020-0.040mol/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
0.040-0.050mol/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 0.025mol/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
It is that the first phosphate buffer solution that 0.040mol/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 0.055mol/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 0.030mol/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 0.045mol/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.
A kind of application of the polypeptide extracted in the nematoblast venom from fire medusa in the drug for preparing anti-nasopharyngeal carcinoma.
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 anti-nasopharyngeal carcinoma
The new albumen of tumor promotion, 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 0.035-0.045mol/L are 0.045-0.060mol/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, strong anion exchange column adsorbs negatively charged albumen, uses successively
The third phosphate buffer solution and sodium chloride concentration that sodium chloride concentration is 0.020-0.040mol/L are 0.040-0.050mol/
The tetraphosphate buffer solution of L elutes, and obtains target protein;
5th, the polypeptide that the present invention obtains inhibits nasopharyngeal carcinoma tumor cell it was proved that with anti-nasopharyngeal carcinoma activity
Strain CNE-1, CNE-2 growth, induces nasopharyngeal carcinoma tumor cell strain CNE-2 apoptosis, method using the present invention that can in high volume locate
Fire medusa nematoblast venom is managed, the anti-tumor active protein wherein contained is isolated.Easy to operate, separating step is few, is suitable for
Quick separating;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 polypeptide of the present invention to nasopharyngeal carcinoma tumor cell strain CNE-1, the design sketch of CNE-2 effects.
Fig. 2 is the design sketch that apoptosis occurs for polypeptid induction nasopharyngeal carcinoma tumor cell strain CNE-2 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 0.035mol/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 0.045mol/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 0.020mol/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 0.040mol/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 0.045mol/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 0.060mol/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 0.040mol/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 0.050mol/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 0.040mol/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 0.055mol/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 0.030mol/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 0.045mol/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, effect of the polypeptide of the present invention to human nasopharyngeal carcinoma tumor cell line CNE-1, CNE-2 is detected using mtt assay
Effect
Experimental method:
It is 10 with culture medium adjustment concentration of cell suspension Step 1: collecting logarithmic phase CNE-1 and CNE-2 cell5It is a thin
Born of the same parents/milliliter take 96 orifice plates, 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 L MTT 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, two kinds of KB cell CNE-1 of polypeptide pair of the present invention and
The growth of CNE-2 is inhibited, this inhibiting effect with the concentration for applying polypeptide of the present invention improve and gradually
Increase.The half lethal concentration of two kinds of cells of polypeptide pair of the present invention is respectively:0.0081 μ g/mL and 0.0073 μ g/mL.This
Illustrate, the proliferation of polypeptide energy strong inhibition nasopharyngeal carcinoma tumor cell strain CNE-1, CNE-2 of the present invention, and of the present invention
Polypeptide be concentration dependant to the inhibited proliferation of the tumour cell.
Two, the confirmatory experiment of apoptosis occurs for polypeptid induction nasopharyngeal carcinoma tumor cell strain CNE-2 of the present invention
Experimental method:
Step 1: collecting logarithmic phase CNE-2 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 0.1 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 CNE-2
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 10.89% of total number of cells, and the cell that late apoptic occurs accounts for about the 79.58% of total number of cells.
This explanation, polypeptide of the present invention can induce nasopharyngeal carcinoma tumor cell strain CNE-2 and notable apoptosis occur.
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. a kind of application of the polypeptide extracted in nematoblast venom from fire medusa in the drug for preparing anti-nasopharyngeal carcinoma, feature exist
In 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 HistrapSP strong cat ion exchange columns, according to 4-6
It is that the first phosphate buffer solution that 0.035-0.045mol/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 0.045-0.060mol/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 0.020-0.040mol/L, pH,
Third phosphate buffer solution carries out third time elution, and it is 0.040- that sodium chloride concentration then, which is added, according to 4-6 times of column volume
0.050mol/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 packet
Include 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 sediment that step 1 is obtained and a concentration of 0.025mol/L of total phosphate, the phosphoric acid hydrogen two that pH is 6.0
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, and with total phosphate concentration 0.025mol/L, the disodium hydrogen phosphate-that pH is 6.0
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 0.040mol/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 0.055mol/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
For 0.025mol/L, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer that pH is 8.0 is allowed to slowly flow through by Histrap Q
Strong anion exchange column, it is that the third phosphate that 0.030mol/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 0.045mol/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.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510329507.6A CN104926932B (en) | 2015-03-31 | 2015-06-15 | The polypeptide extracted in a kind of nematoblast venom from fire medusa and its application |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN2015101483259 | 2015-03-31 | ||
CN201510148325 | 2015-03-31 | ||
CN201510329507.6A CN104926932B (en) | 2015-03-31 | 2015-06-15 | The polypeptide extracted in a kind of nematoblast venom from fire medusa and its application |
Publications (2)
Publication Number | Publication Date |
---|---|
CN104926932A CN104926932A (en) | 2015-09-23 |
CN104926932B true CN104926932B (en) | 2018-08-03 |
Family
ID=54114375
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201510329507.6A Expired - Fee Related CN104926932B (en) | 2015-03-31 | 2015-06-15 | The polypeptide extracted in a kind of nematoblast venom from fire medusa and its application |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN104926932B (en) |
Citations (3)
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 |
-
2015
- 2015-06-15 CN CN201510329507.6A patent/CN104926932B/en not_active Expired - Fee Related
Patent Citations (3)
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 |
Also Published As
Publication number | Publication date |
---|---|
CN104926932A (en) | 2015-09-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107441501A (en) | Drug-loaded liposome of antibacterial peptide modification and its production and use | |
CN105586379B (en) | A kind of preparation method with the collagen active peptide for inhibiting cancer cell multiplication effect | |
CN104630318B (en) | A kind of preparation method of small water turtle antineoplastic polypeptide | |
CN104277134B (en) | A kind of preparation method and applications of the dictyophora fungus polysaccharide-chelates of zinc with anti-tumor activity | |
CN100475840C (en) | Wasp antibiotic peptide and preparing method and use thereof | |
CN101037468A (en) | Preparation method of oyster active peptides | |
CN105801690B (en) | A kind of trypsin inhibitor and its preparation method and application | |
CN107751697A (en) | Chlorella extract phycocyanin drink with function preparation method | |
CN101020715B (en) | Process of extracting and preparing deer nerve growth factor (DEER NGF) | |
CN109608557A (en) | Polysaccharides extracts Isolation and purification method, Lycium chinense glycopeptide and preparation method | |
CN104311645B (en) | Spirulina polypeptide P1 and its application with bacteriostatic activity | |
CN106729601A (en) | Placental lipo-glucosaminoglycan, polypeptide bigeminy immunopotentiator and preparation method thereof | |
CN104306399B (en) | A kind of deer spleen extract extracting method and the application in immunity medicine is improved | |
CN104356218B (en) | The preparation of Scolopendra subspinipes analgesia peptide precursor protein Ssm A and its product Ssm A1 and Ssm A2 and application | |
CN104926932B (en) | The polypeptide extracted in a kind of nematoblast venom from fire medusa and its application | |
CN102516382B (en) | Antimicrobial peptide Hainanenin-5 of Amolops hainanensis, gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis, and application of gene of antimicrobial peptide Hainanenin-5 of Amolops hainanensis | |
CN104877022B (en) | The polypeptide extracted in a kind of nematoblast venom from fire medusa and its application | |
CN103172721B (en) | Amolops hainanensis antimicrobial peptide Hainanenin-1, and gene, separation purification, chemical synthesis and application thereof | |
CN101709083B (en) | Fibrinolytic protein from scorpion tails, preparation method and application thereof | |
CN109400669B (en) | Extraction method and application of micromolecular protein of walnut kernel peel | |
CN103805663A (en) | Method for separating, purifying and extracting bioactive peptide from marine product | |
CN103755417A (en) | Production method of selenium-rich hericium erinaceus mycelia through liquid fermentation | |
CN104877023B (en) | The polypeptide extracted in a kind of nematoblast venom from fire medusa and its application | |
CN113813291A (en) | Preparation method of animal medicinal material freeze-dried powder | |
CN110218717A (en) | A kind of preparation method of ganoderma lucidum polysaccharide and its protective effect to the dry inactivation of superoxide dismutase |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180803 Termination date: 20190615 |
|
CF01 | Termination of patent right due to non-payment of annual fee |