CN107226857A - A kind of biologically active polypeptide TIASGEPT and its preparation method and application - Google Patents
A kind of biologically active polypeptide TIASGEPT and its preparation method and application Download PDFInfo
- Publication number
- CN107226857A CN107226857A CN201710546799.8A CN201710546799A CN107226857A CN 107226857 A CN107226857 A CN 107226857A CN 201710546799 A CN201710546799 A CN 201710546799A CN 107226857 A CN107226857 A CN 107226857A
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- China
- Prior art keywords
- tiasgept
- biologically active
- active polypeptide
- polypeptide
- derivative
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Classifications
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- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4732—Casein
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Birds (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Toxicology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Dermatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to albumen field, and in particular to its amino acid sequence of a kind of biologically active polypeptide TIASGEPT and its preparation method and application, biologically active polypeptide TIASGEPT is Thr Ile Ala Ser Gly Glu Pro Thr.Tested by antioxidation in vitro, ion vitro immunization function promotes experiment, demonstrating peptide T IASGEPT has preferable antioxidation biology activity and immunoloregulation function, on the one hand there is preferable antioxidation activity, free radical that can be in removing machine body improves the quality of life；On the other hand, the biologically active polypeptide TIASGEPT of the present invention can strengthen the in-vitro multiplication ability of lymphocyte and macrophage, promote the increase of the macrophage nitric oxide amount of inducing, improve the ability that body resists extraneous pathogenic infection, the body incidence of disease is reduced, exploitation is of great significance with immunoloregulation function, the food of anti-oxidation function, health products and medicine tool.
Description
Technical field
The present invention relates to albumen field, more particularly, to a kind of biologically active polypeptide TIASGEPT and preparation method thereof and should
With.
Background technology
During cow's milk is through lactobacillus-fermented, a part of protein in cow's milk is metabolized by lactic acid bacteria to be utilized, concurrently
A series of biochemical reactions have been given birth to, protein is changed into polypeptide or free amino acid, is digested or passes through
The absorption and transport of intestinal epithelial cell is directly entered the blood circulation of human body.In these polypeptides, some has special
Physiological function, is referred to as " biologically active peptide ".
Oxidation reaction and oxidative metabolism are all vital for food and human body, and free radical and active oxygen cause
A series of oxidation reaction.When excessive free radical is formed, they can exceed protective enzyme such as superoxide dismutase, peroxide
Change the protective effect of hydrogen enzyme, so as to cause a series of side effect such as lipid oxidation, Apoptosis to produce.The oxidation of this class is anti-
Should, the shelf-life of the food containing fat is not only influenceed, also the health to human body causes certain harm, such as rheumatic arthritis, sugar
Urinate disease, artery sclerosis etc..In addition, Collins et al. researchs in 2005 find that oxidative damage of the generation of cancer also with DNA has
Close.
Some artificial synthesized antioxidant such as butylated hydroxy anisoles (BHA), 2,6- di-t-butyl -4- methylbenzenes in early days
Phenol (BHT) is applied in food, as the antioxidant of lipid, but these artificial synthesized additives have latent for human body
Risk.Therefore, safe antioxidant is found in natural food source particularly important.In the last few years, it has been found that one
The polypeptides matter of a little food sources has good antioxidation, such as corn small peptide, Soybean Peptide, cow's milk polypeptide.These
Polypeptide can be obtained by number of ways such as microbial fermentation, digestion enzymolysis, and the polypeptide with antioxidation activity is mostly
It is made up of 2~20 amino acid residues, molecular weight is less than 6000Da, contains a certain amount of hydrophobic amino acid, aromatic amino acid.
Immune-active peptides are to obtain and prove that the class biology of its physiologically active is living from breast first after opioid peptides is found
Property polypeptide.Jolles in 1981 et al. has found first, using trypsin hydrolysis people lactoprotein, can obtain an amino acid sequence
Val-Glu-Pro-Ile-Pro-Tyr hexapeptide is classified as, experiment in vitro proves that the peptide can strengthen Turnover of Mouse Peritoneal Macrophages pair
The phagocytosis of sheep red blood cell (SRBC).Migliore-Samour et al. has found the hexapeptide Thr-Thr-Met-Pro- from casein
Leu-Trp can stimulate sheep erythrocyte to the phagocytosis of mouse peritoneal macrophages and strengthen for kerekou pneumonia primary
The resistance of bacterium.Li Su duckweeds et al. find rat peritoneal macrophages with newborn source immunomodulatory peptides (PGPIPN) the feeding rat of synthesis
The phagocytosis immunoloregulation function related to red blood cell have significant enhancing.
Research shows that immune-active peptides can not only strengthen immunity of organisms, stimulates the propagation of body lymphocyte, enhancing
The phagocytic function of macrophage, promotes the release of cell factor, improves the ability that body resists extraneous pathogenic infection, reduce machine
The body incidence of disease, and the immunological rejection of body will not be caused.
The research on biologically active polypeptide has much at present, such as Chinese patent CN105254738A discloses a kind of next
The milk-derived biologically active polypeptide DELQDKIH of beta-casein is come from, Chinese patent CN105254739A discloses one kind and derived from
Milk-derived the biologically active polypeptide GTQYTD, Chinese patent CN105254740A of α s1- caseins disclose a kind of from α s2-
The milk-derived biologically active polypeptide NQFYQKF of casein.
The content of the invention
It is an object of the invention to provide a kind of biologically active polypeptide TIASGEPT and its preparation method and application.
The purpose of the present invention can be achieved through the following technical solutions：
First aspect present invention is there is provided a kind of biologically active polypeptide TIASGEPT, and its amino acid sequence is Thr-Ile-
Ala-Ser-Gly-Glu-Pro-Thr, such as SEQ ID NO：Shown in 1.
Preferably, the biologically active polypeptide is milk-derived.κ-casein is derive specifically from, and is κ-ss-casein variants
The amino acid residue that A is the 145th~152.κ-ss-casein variants A amino acid sequences such as SEQ ID NO：Shown in 3.
The amino acid sequence of κ-casein and corresponding nucleotides sequence are classified as existing technology, encode κ-ss-casein variants A
The biologically active polypeptide TIASGEPT of the nucleotide fragments energy encoding mature of 145th~152 amino acids residue.
Preferably, the biologically active polypeptide has anti-oxidation function and immunoloregulation function.
There is provided the nucleotide fragments for encoding the biologically active polypeptide TIASGEPT, its sequence for second aspect of the present invention
For：5 '-acc att gct agt ggc gag cct aca-3 ', such as SEQ ID NO：Shown in 2.
Third aspect present invention can pass through gene there is provided the preparation method of the biologically active polypeptide TIASGEPT
The method of engineering is artificial synthesized, can be directly obtained from dairy products by the method isolated and purified, directly can pass through chemistry
It is synthetically prepared.
Fourth aspect present invention is preparing the food with anti-oxidation function there is provided the biologically active polypeptide TIASGEPT
Application in product, health products, medicine or cosmetics.
Fifth aspect present invention is being prepared with immunoloregulation function there is provided the biologically active polypeptide TIASGEPT
Application in food, health products or medicine.
Sixth aspect present invention is being prepared while having anti-oxidation function there is provided the biologically active polypeptide TIASGEPT
With the application in the food, health products or medicine of immunoloregulation function.
Specifically, biologically active polypeptide TIASGEPT of the invention, which can be used for preparing, reduces free radical to skin damage
Cosmetics, prepare with it is anti-oxidant and/or regulation immunity of organisms medicine；And due to the biologically active polypeptide of the present invention
Product after TIASGEPT is degraded by intestines and stomach still has a bioactivity, thus can be also used for preparing the food such as Yoghourt,
The health products of immunity are adjusted, and the oral preparation that is used for has anti-oxidant and/or regulation immunity of organisms medicine.
There is provided a kind of oxidation resistant product, including the biologically active polypeptide TIASGEPT or institute for seventh aspect present invention
State biologically active polypeptide TIASGEPT derivative；Described oxidation resistant product includes antioxidant food, antioxidant health-care product, resisted
Oxidative drug or antioxidation cosmetic product；The derivative of the biologically active polypeptide TIASGEPT, refers in biologically active polypeptide
On TIASGEPT amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetyl
The modifications, obtained polypeptide derivative such as change, phosphorylation, esterification or glycosylation.
Eighth aspect present invention there is provided a kind of immunological regulation product, including the biologically active polypeptide TIASGEPT or
The derivative of the biologically active polypeptide TIASGEPT；Described immunological regulation product includes immunological regulation food, immunological regulation
Health products or immunoregulation medicament；The derivative of the biologically active polypeptide TIASGEPT, refers in biologically active polypeptide
On TIASGEPT amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetyl
The modifications, obtained polypeptide derivative such as change, phosphorylation, esterification or glycosylation.
Ninth aspect present invention there is provided a kind of while have the product of anti-oxidation function and immunoloregulation function, including
The derivative of the biologically active polypeptide TIASGEPT or described biologically active polypeptides TIASGEPT；With anti-oxidation function and exempting from
The product of epidemic disease regulatory function includes food, health products or medicine；The derivative of the biologically active polypeptide TIASGEPT, refers to
On biologically active polypeptide TIASGEPT amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonyl
Change, methylate, acetylation, phosphorylation, esterification or glycosylation etc. modification, obtained polypeptide derivative.
Biologically active polypeptide TIASGEPT's of the present invention has the beneficial effect that：The milk-derived biologically active polypeptide of the present invention
TIASGEPT has preferable antioxidation activity and regulation immunity of organisms activity；On the one hand freedom that can be in removing machine body
Base, reduces injury of the free radical to human body；On the other hand, biologically active polypeptide TIASGEPT of the invention can also adjust body
Immunity, strengthens the multiplication capacity of lymphocyte, improves the ability that body resists extraneous pathogenic infection, reduction body morbidity
Rate, and the immunological rejection of body will not be caused, the dairy products to developing with anti-oxidation function and adjusting immunologic function
It is of great significance with health products tool.
Brief description of the drawings
Fig. 1：Mass chromatography extracts figure (m/z=775.38)；
Fig. 2：Mass-to-charge ratio is the first mass spectrometric figure of 775.38 fragment；
Fig. 3：Mass-to-charge ratio is 775.38 polypeptide az, by crack conditions；
Fig. 4：[DPPH] methanol standard curve；
Fig. 5：FeSO4Standard curve.
Embodiment
Before specific embodiments of the present invention are further described, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, the protection domain being not intended to be limiting of the invention.
When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range
Any one numerical value can select between point and two end points.Unless otherwise defined, in the present invention all technologies for using and
Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except the specific method used in embodiment, equipment,
Outside material, according to those skilled in the art to the grasp of prior art and the record of the present invention, it can also use and this
Any method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come real
The existing present invention.
Unless otherwise indicated, disclosed in this invention experimental method, detection method, preparation method using this technology lead
Domain conventional molecular biology, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of association area.These technologies existing perfect explanation in the prior art, for details, reference can be made to Sambrook etc.
MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor
Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the
Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN
STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN
ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic
Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin
Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
The active peptide TIASGEPT's of embodiment 1 is artificial synthesized
First, the synthesis of biologically active peptide
1. RINK resin 3g (substitution value 0.3mmol/g) are weighed in 150ml reactor, with 50ml dichloromethane
(DCM) soak.
2. after 2 hours, resin is washed with nitrogen-dimethylformamide (DMF) of 3 times of resin volumes, is then drained, so
It is repeated four times, resin is drained rear stand-by.
3. a certain amount of 20% piperidines (piperidines/DMF=1 is added into reactor:4,v:V), it is placed on decolorization swinging table and shakes
20min is shaken, the Fmoc blocking groups on resin are sloughed with this.Take off after protection and to have been washed four times with the DMF of 3 times of resin volumes,
Then drain.
4. take the detection of a small amount of resin ninhydrin (nine well ninhydrins) method (each two drop of inspection A, inspection B, 100 DEG C of reactions
1min), resin has color, illustrates to be deprotected successfully.
5. weighing, amino acid Thr is appropriate and the parallel triazole (HOBT) of 1- hydroxyls-benzene is appropriate in 50ml centrifuge tube, adds
20ml DMF is dissolved, and then adds 3ml N, and N DICs (DIC) vibration shakes up 1min, treats that solution is clear
It is added to after clear in reactor, then reactor is placed in 30 DEG C of shaking table and reacted.
6. after 2 hours, with a certain amount of acetic anhydride end socket (acetic anhydride:DIEA:DCM=1:1:2,v:v:V) half an hour,
Then washed four times, drained stand-by with the DMF of 3 times of resin volumes.
7. a certain amount of 20% piperidines (piperidines/DMF=1 is added into reactor:4, v:V), it is placed on decolorization swinging table and shakes
20min is shaken, the Fmoc blocking groups on resin are sloughed with this.Take off and washed after protection with DMF four times, then drained.
8. take the detection of a small amount of resin ninhydrin (nine well ninhydrins) method (each two drop of inspection A, inspection B, 100 DEG C of reactions
1min), resin has color, illustrates to be deprotected successfully.
9. weighing, second amino acid next is appropriate and HOBT is appropriate in 50ml centrifuge tube, and the DMF for adding 25ml will
It dissolves, and the DIC vibrations for then adding 2.5ml shake up 1min, are added to after after solution clarification in reactor, then by reactor
It is placed in 30 DEG C of shaking table and reacts.
10. after 1 hour, taking a small amount of resin to detect, (each two drop of inspection A, inspection B, 100 DEG C of reactions are detected with ninhydrin method
1min), if resin is colourless, illustrate that reaction is complete；If resin has color, illustrate that condensation is incomplete, continue to react.
11. after question response is complete, resin is washed with DMF four times, then drain, a certain amount of 20% is added into reactor
Piperidines (piperidines/DMF=1:4,v:V), it is placed on decolorization swinging table and rocks 20min, the Fmoc protection groups on resin is sloughed with this
Group.Take off and washed after protection with DMF four times, then drained whether detection protection sloughs.
12. amino acid Ile, Ala, Ser, Gly, Glu, Pro and Thr are connected successively according to step 9-11.
13. after last amino acid is connected, sloughing protection, washed four times, then taken out resin with methanol with DMF
It is dry.Then with 95 cutting liquid (trifluoroacetic acids:1,2 dithioglycols:3, isopropyl base silane:Water=95:2:2:1, v:v:V) by polypeptide
Cut down from resin (every gram of resin adds 10ml cutting liquids), and with ice ether (cutting liquid：Ether=1:9,v:V) it is heavy to centrifuge
Drop four times.
So far, artificial synthesized biologically active peptide TIASGEPT.
Referring in right amount described in above step 5, step 9 calculates a theoretical use according to target synthetic quantity and yield
Amount, is multiplied by a coefficient (being 1.1 in the present embodiment), the actual amount finally obtained on the basis of theoretical amount.
Because the synthetic quantity of each target peptide is different, and yield is also different, so the amount of the amino acid weighed every time
It is just different.
The synthetic quantity of such as target peptide is 10 grams, and the yield (rate of recovery) of synthetic peptide is 90%, then the theory of amino acid
The calculation formula for the amount of weighing is：
The theory amount of weighing=10 gram * amino acid moleculars amount/target peptide molecular weight/90%. of amino acid
The result so calculated is the theory amount of weighing of amino acid, in order to ensure to obtain 10 in actual building-up process
Gram polypeptide, amino acid will weigh a little more, and the amino acid amount of weighing is typically all 1.1 times of the theoretical amount of weighing during practical operation.
Similarly, the parallel triazole (HOBT) of 1- hydroxyls-benzene, as an intermediary during Peptide systhesis, is also that theory is weighed
1.1 times of amount.
Due to amino acid used in biologically active peptide building-up process and the theoretical amount of the parallel triazole (HOBT) of 1- hydroxyls-benzene
It is that those skilled in the art can calculate according to the demand of target synthetic peptide and obtain, therefore, during practical operation, in theory use
A coefficient is multiplied by the basis of amount can just synthesize target peptide, in the synthesis step and synthesis technique with reference to the present embodiment
On conditioned basic, the biologically active peptide obtained in the present embodiment can be just synthesized according to the Conventional wisdom of those skilled in the art.
2nd, the confirmation of biologically active peptide
1) UPLC is analyzed
UPLC conditions are as follows：
Instrument：Waters ACQUITY UPLC ultra high efficiency liquid phase-electron spray-level Four bar-time of-flight mass spectrometer
Chromatographic column specification：BEH C18 chromatographic columns
Flow velocity：0.4mL/min
Temperature：50℃
Ultraviolet detection wavelength：210nm
Sample size：2μL
Gradient condition：A liquid：Water containing 0.1% formic acid (v/v), B liquid：Acetonitrile containing 0.1% formic acid (v/v)
2) mass spectral analysis
Mass Spectrometry Conditions are as follows：
Ionic means：ES+
Mass range (m/z)：100-1000
Capillary voltage (Capillary) (kV)：3.0
Sampling spiroid (V)：35.0
Ion source temperature (DEG C)：115
Remove solvent temperature (DEG C)：350
Go solvent stream (L/hr)：700.0
Collision energy (eV)：4.0
Sweep time (sec)：0.25
Interior sweep time (sec)：0.02
According to above analysis method, using ultra high efficiency liquid phase-electron spray-level Four bar-flight time mass spectrum, to bioactivity
Peptide TIASGEPT carries out chromatography and mass spectral analysis, and its mass chromatography extraction figure is as shown in figure 1, extract the one-level and two at this peak
As shown in Figures 2 and 3, the polypeptide mass-to-charge ratio that can obtain this peak is 775.38Da to level mass spectrogram, and retention time is 7.89min.
3) result
From the figure 3, it may be seen that situation about being broken according to az, by, calculates by Progenesis QI software analysis, obtains matter lotus
Fragment sequence than 775.38Da is Thr-Ile-Ala-Ser-Gly-Glu-Pro-Thr (TIASGEPT), is designated as SEQ ID
NO：1.The fragment is corresponding with κ-ss-casein variants A the 142nd~152 residue sequence, κ-casamino acid sequence
GenBank numberings are AAA30433.1, and sequence is shown in SEQ ID NO：3.
The antioxidation activity experiment of the biologically active peptide of embodiment 2
Using removing free radical method (DPPH methods) and TAC method (Ferric Reducing Ability
Power FRAP methods), the biologically active polypeptide TIASGEPT obtained to embodiment 1 antioxidation activity is tested.
1st, [DPPH] method determines biologically active peptide TIASGEPT antioxidation activity in vitro
1) experiment reagent and instrument
Reagent：1,1- diphenyl -2- trinitrophenyl-hydrazines (1,1-Diphenyl-2-picrylhydrazyl [DPPH]),
Japanese Wako companies production；Methanol, Shanghai traditional Chinese medicines company provides；The milk-derived biologically active polypeptide that embodiment 1 is obtained
TIASGEPT。
Key instrument：Sunrise ELIASAs, Austrian Tecan Products；96 porocyte culture plates, the U.S.
Millipore companies manufacture；Assay balance, Meitelei-tolido Products.
2) experimental method
(1) 1mmol/L [DPPH] methanol solution
0.349mg [DPPH] is weighed with assay balance to be dissolved in 1mL methanol solutions, prepares obtained 1mmol/L
[DPPH] methanol solution, tinfoil is kept in dark place, i.e., with i.e. use.
(2) measure of [DPPH] methanol standard curve
100 μ L [DPPH] methanol standard curve samples are separately added into by table 1 in 96 orifice plates, 90min is stored at room temperature, used
ELIASA detects light absorption value at 517nm.
[DPPH] methanol of table 1 calibration curve solution is prepared
According to experimental result, using Excel matched curves and regression equation is calculated, as a result see Fig. 4 (regression equations：Y=-
0.192x+0.2271, R2=0.9991).The linear relationship of [DPPH] methanol standard curve is good, and coefficient correlation is 0.999,
Show that [DPPH] methanol standard curve preci-sion and accuracy meets detection and required.In terms of result, absorbance with
[DPPH] content is in inverse relation, and [DPPH] content is fewer, and light absorption value is higher, i.e. the ability of sample removing free radical is got over
By force.
(3) [DPPH] method determines biologically active peptide TIASGEPT antioxidation activity
1) sample sets：80 μ L concentration are added in 96 orifice plates for 1mmol/L [DPPH] methanol solution, by table 2 respectively to add
Enter the testing sample (TIASGEPT), positive control 1 (2.5mg/mL Trolox), positive control 2 of 20 μ L various concentrations
(0.025mg/mL Trolox), and negative control (phytic acid)；
2) blank group：On same 96 orifice plate, to add 80 μ L concentration as 1mmol/L [DPPH] methanol solutions and 20 μ L
The sample of deionized water does blank control.
After detected sample sample-adding is finished, 90min is stored at room temperature, light absorption value is detected at 517nm with ELIASA.Under
Formula calculates free radical scavenging activity, and experimental result is shown in Table 2.
Formula：
Table 2 [DPPH] method determines the antioxidation activity result of biologically active polypeptide
From table 2 it can be seen that having under the same conditions as the 2.5mg/mL of positive control Trolox most strong clear
Except the ability of free radical, free radicals all in solution can be almost removed, are secondly 0.025mg/mL Trolox, phytic acid, work
Property polypeptide.Peptide T IASGEPT removes [DPPH] free radical rate and bell is presented down with change in concentration, and is in concentration
Peak is reached at 2.5mg/mL, is 25.85%.
2nd, FARP methods determine biologically active peptide TIASGEPT antioxidant activity in vitro
1) experiment reagent and instrument
TAC detection kit (Ferric Reducing Ability of Plasma FRAP methods), is purchased from
The green skies biotechnology company in Shanghai；FeSO4Solution (10mmol/L), watermiscible vitamin E (Trolox solution) (10mmol/
L), the milk-derived biologically active polypeptide TIASGEPT that embodiment 1 is obtained.
Key instrument：Sunrise ELIASAs, Austrian Tecan Products；96 porocyte culture plates, the U.S.
Millipore companies manufacture；Assay balance, Meitelei-tolido Products；HWS26 type electric-heated thermostatic water baths, Shanghai
One permanent Science and Technology Ltd.'s manufacture.
2) experimental method
(1) preparation of FRAP working solutions
According to TAC detection kit, TPTZ 7.5mL dilutions, the μ L solution of TPTZ 750, detection are buffered
The μ L of liquid 750 are well mixed, and are incubated in 37 DEG C of water-baths, are finished in 2 hours h.
(2)FeSO4The making of standard curve curve is determined
180 μ LFRAP working solutions are first added in 96 orifice plates, 5 μ L FeSO are added by table 34Calibration curve solution, is gently mixed
Even, 37 DEG C are incubated after 3-5min, and light absorption value is determined at 593nm with ELIASA.
The FeSO of table 34The solution of standard curve determination is prepared
FeSO4Concentration is in good proportional relation, FeSO with light absorption value4Concentration is higher, and light absorption value is higher.FeSO of the present invention4
Standard curve result is shown in Fig. 5, and the linear relationship of standard curve is good, and coefficient correlation is 0.998, FeSO4The precision of standard curve
Degree and the degree of accuracy meet detection and required, available for follow-up calculating.
(3) FRAP methods determine biologically active polypeptide TIASGEPT oxidation resistance
First added in 96 orifice plates in 180 μ L FRAP working solutions, blank control wells and add 5 μ L ddH2O, sample detection hole
5 μ L phytic acid are added in 5 μ L testing samples of interior addition, positive control, are gently mixed, 37 DEG C are incubated after 3-5min, are existed with ELIASA
Light absorption value is determined at 593nm.TAC representation is with FeSO4The concentration of standard liquid is represented.Count according to the following formula
Free radical scavenging activity is calculated, experimental result is shown in Table 4.
The FARP methods of table 4 determine biologically active polypeptide TIASGEPT TAC result
By TAC method (Ferric Reducing Ability Power FRAP methods) to polypeptide
TIASGEPT external total antioxidant activity is determined, it is found that biologically active polypeptide TIASGEPT has preferable oxygen reduction
The ability of compound matter；In the case of concentration is 4mg/mL, peptide T IASGEPT total antioxidation level reaches 0.0231mmol/g；
Illustrate biologically active polypeptide TIASGEPT TAC, higher than the plant with weak antioxidation activity under comparable sodium
Acid, with conspicuousness (p>0.05) difference.Therefore, can assert the biologically active polypeptide TIASGEPT of invention has significant antioxygen
Change ability.
The promotion immunity of organisms activity experiment of the biologically active peptide of embodiment 3
First, mtt assay determines biologically active polypeptide TIASGEPT vitro lymphocyte proliferation capacity experimental
1) experiment material and instrument：
Reagent and material：Experimental animal balb/c mouse (male 6-8 week old, Shanghai Communications University's agricultural and biological institute
Animal experimental center)；The milk-derived biologically active polypeptide TIASGEPT that embodiment 1 is obtained；Mouse lymphocyte extract solution (is purchased from
Suo Laibao companies)；RPMI1640 culture mediums (are purchased from GIBCO companies)；3- (4,5- dimethylthiazole-the 2)-nitrogen of 2,5- diphenyl four
Azoles bromide (MTT, purchased from Amresco companies)；ConA (ConA, purchased from Sigma companies)；Bovine serum albumin(BSA) (BSA,
Purchased from Genebase companies)；Pepsin (is purchased from Sigma companies)；Pancreatin (Corolase PP, purchased from AB companies).
Instrument：LRH-250F biochemical cultivation cases, Shanghai perseverance Science and Technology Ltd.；GL-22M high speed freezing centrifuges, Shanghai
Lu Xiang instrument centrifuges Instrument Ltd.；Hera cell 150CO2 incubators, Heraeus companies；Dragon Wellscan
MK3 ELIASAs, Labsystems companies；ALPHA1-2-LD vacuum freeze driers, Christ companies；Ultra performance liquid chromatography-
Quadrupole rod time of-flight mass spectrometer, waters companies.
2) experimental method：
Mouse spleen is taken under aseptic condition, mouse lymphocyte is extracted with lymphocyte extract solution, Yuan Dynasty's culture is carried out.With
Cell density is adjusted to 2.5 × 10 by complete RPMI1640 nutrient solutions6Individual/mL.Sequentially added in 96 porocyte culture plates：
100 μ L mouse lymphocyte suspensions, 100 μ L RPMI1640 complete culture solutions, 20 μ L ConAs, 100 μ L samples.In addition,
Blank control group (pH7.2~7.4,3mol/L PBS) and negative control group (500 μ g/mL BSA) are set, and research shows that its is right
Do not influenceed in vitro lymphocyte proliferation.Every group of 3 parallel laboratory test samples.In 5%CO2Cultivated in 37 DEG C of incubators after 68h,
20 μ L MTT are added under aseptic condition per hole, continue to cultivate 4h, careful abandoning supernatant adds 100 μ L dimethyl sulfoxide (DMSO)s per hole,
37 DEG C of biochemical cultivation cases hatch 10min, shake up, light absorption value is determined at 570nm with ELIASA.
Vitro lymphocyte proliferation ability represents that computational methods are as follows with stimulus index：
In formula：A1For blank control at the 570nm under light absorption value；A2For negative control group at the 570nm under extinction
Value, A3For experimental group at the 570nm under light absorption value.
3) experimental result and analysis
Experimental result is shown in Table 5.As shown in Table 5, the bar for being 100 μ g/mL in biologically active peptide TIASGEPT mass concentration
Under part, milk-derived biologically active peptide TIASGEPT stimulus index is more than BSA, illustrates that TIASGEPT can stimulate body to a certain extent
The propagation of outer mouse lymphocyte.And TIASGEPT stimulus index has reached 1.159, and negative control group has significance difference
Different (P<0.05).It therefore, it can assert that active peptides TIASGEPT has the ability for remarkably promoting mouse lymphocyte propagation,
Can be edible as a kind of health products or additive, it is possible to increase the immunity of animal and human body.
Influences of the biologically active polypeptide TIASGEPT of table 5 to vitro lymphocyte proliferation
| Experiment packet | Stimulus index SI |
| Negative control group | 1 |
| TIASGEPT | 1.159±0.035* |
Note：* labelled notation has significant difference (P ＜ 0.05) to be compared with negative control.
2nd, mtt assay determines biologically active polypeptide TIASGEPT macrophages in vitro multiplication capacity experiment
1) experiment reagent and instrument
Reagent：Experimental animal balb/c mouse (male 6-8 week old) Shanghai Communications University agricultural and biological institute animal reality
Test center；The milk-derived biologically active polypeptide TIASGEPT that embodiment 1 is obtained；3- (4,5- dimethylthiazole -2) -2,5- hexichol
Base tetrazole bromide (MTT) Amresco companies；LPS (lipopolysaccharides) Sigma companies；Bovine serum albumin(BSA) (Bovine Serum
Albumin, BSA) Genebase companies；Three lysates, the water containing 10%SDS, 5% isobutanol and 0.012mol/L HCl
Solution.
Instrument and equipment：LRH-250F biochemical cultivation cases Shanghai perseverance Science and Technology Ltd.；On GL-22M high speed freezing centrifuges
Hai Luxiang instrument centrifuges Instrument Ltd.；Hera cell 150CO2Incubator Heraeus companies；Dragon Wellscan
MK3 ELIASA Labsystems companies.
2) test method：
Balb/c mouse peritoneal injections 2ml 2% (w/w) sterilizing starch solutions, continuous injection three days, last time is injected
The neck that breaks after 24 hours is put to death.Skin of abdomen is peelled off, 4 DEG C of phosphate buffers (PBS) is drawn with syringe and rinses abdominal cavity repeatedly, from
Heart pipe is collected after flushing liquor, and centrifugation (1000rpm, 4 DEG C) abandons supernatant after 10 minutes, (is contained with 4 DEG C of RPMI1640 complete culture solutions
10%FBS) wash twice, cell viability examination is done in the dyeing of 0.2% trypan blue solution, confirm the vibrant macrophage collected
Account for more than 95%.After cell counting count board reading, adjustment cell concentration to suitable concn.
It will blow and beat to the cell suspension suspended completely with suitable volumes 96 porocyte culture plates of addition, 37 DEG C, 5%CO2
After being cultivated 4 hours under environment, liquid in hole is abandoned in suction, and cell culture plate well is carefully cleaned with 37 DEG C of RPMI1640 complete culture solutions
Bottom, washes away not adherent cell and cell fragment, obtains adherent peritoneal macrophage after purification.0.2ml is added per hole
RPMI1640 complete mediums, experiment is dissolved in after culture medium and added in advance with small peptide sample and LPS, starts cell culture.
Obtain after adherent peritoneal macrophage after purification, experimental group adds per hole is dissolved with biologically active polypeptide LPLP
The μ l/ holes of RPMI1640 complete culture solutions (10%FBS) 200 of (1mg/ml), continuously cultivate 48h；Negative control group is per hole solubilization
Solution has BSA (500 μ g/mL) the μ l/ holes of RPMI1640 complete culture solutions (10%FBS) 200；Blank group addition RPMI1640 is complete
The μ l/ holes of nutrient solution (10%FBS) 200, continuously cultivate 48h.Also, experimental group, negative control group and blank group are set just respectively again
Often organize and inflammation group；Inflammation group adds LPS to final concentration of 100ng/ml when 24h is arrived in culture；Normal group is not added with LPS；And
Normal group and inflammation group add the μ l/ holes of 5%MTT 20 in 44h；Cell culture reaches that 100 μ l/ holes are added after 48h three is molten
Liquid is solved to terminate culture, after dissolving overnight, the absorbance (OD570) in each hole is surveyed with ELIASA under wavelength 570nm, growth refers to
The calculation formula of number (Growth Indices) is as follows：
Wherein, blank nutrient solution is the RPMI1640 complete culture solutions containing 10%FBS.
3) experimental result and analysis
Experimental result is shown in Table 6, as shown in Table 6, under conditions of addition 1mg/ml biologically active polypeptides TIASGEPT, normally
The macrophage of group and inflammation group has propagation.And compared with negative control group, there is significant difference (P ＜ 0.01).Say
Gelatine/biological activity peptide T IASGEPT has significant proliferation function to macrophages in vitro.
The influence that the biologically active polypeptide TIASGEPT of table 6 breeds to macrophages in vitro
| Experiment packet | Normal group GI | Inflammation group GI |
| Negative control group | 1 | 1 |
| TIASGEPT(1mg/ml) | 1.103±0.0520** | 1.159±0.0385** |
Note：* represent to be compared with negative control, there is significant difference (P ＜ 0.05)；* represents to be compared with negative control group,
There is significant difference (P ＜ 0.01)
3rd, the measure (Griess methods) of the biologically active polypeptide TIASGEPT rush macrophage nitric oxide amount of inducing
1) experiment reagent and instrument
Reagent：Experimental animal balb/c mouse (male 6-8 week old) Shanghai Communications University agricultural and biological institute animal reality
Test center；The milk-derived biologically active polypeptide TIASGEPT that embodiment 1 is obtained；LPS, purchased from Sigma companies；Neutral red staining
Liquid, green skies biotechnology research institute production.
Instrument and equipment：LRH-250F biochemical cultivation cases Shanghai perseverance Science and Technology Ltd.；On GL-22M high speed freezing centrifuges
Hai Luxiang instrument centrifuges Instrument Ltd.；Hera cell 150CO2 incubator Heraeus companies；Dragon Wellscan
MK3 ELIASA Labsystems companies.
2) test method：
It is 2 × 10 to add number of cells6/ ml μ l/ the holes of cell suspension 100, it is adherent to add after purification containing peptide
The μ l/ holes of RPMI1640 complete culture solutions (10%FBS) 200, inflammation group adds LPS to the μ g/ml of final concentration 10 in 24h, continuously
Cultivate after 48h, collect the μ l/ holes of nutrient solution supernatant 50, add Griess reagents 1 and Griess reagents successively in nutrient solution supernatant
2 each 50 μ l/ holes, after reacting at room temperature 10 minutes, determine absorbance (OD540) under 540nm wavelength.
3) experimental result and analysis：
The biologically active polypeptide TIASGEPT of table 7 promotees the measure of the macrophage nitric oxide amount of inducing
| Experiment packet | Normal group | Inflammation group |
| Cell blank | 0.0592±0.00525 | 0.3241±0.0381 |
| TIASGEPT 1mg/ml | 0.1289±0.0257** | 0.4899±0.0241** |
| TIASGEPT 0.5mg/ml | 0.1232±0.0130** | 0.3356±0.0305** |
| TIASGEPT 0.1mg/ml | 0.2474±0.0193** |
Note：*, compared with negative control, there is significant difference (P ＜ 0.05)；
*, is compared with negative control group, there is significant difference (P ＜ 0.01)
Experimental result is shown in Table 7, as shown in Table 7, and biologically active polypeptide TIASGEPT is added in experimental group, and concentration is respectively
1mg/mL and 0.5mg/mL, an oxygen of the promotion macrophage grown under inflammatory conditions is made for growing under normal circumstances with LPS
Changing the nitrogen amount of inducing has facilitation.Compared with cell blank group, with significant difference (P<0.05).Work as biologically active polypeptide
TIASGEPT addition concentration is 0.1mg/mL, is compared in the case where LPS makes inflammatory conditions, also macrophage nitric oxide can be promoted to lure
The increase of raw amount, and with significant difference (P<0.05).But compared with the cell blank group grown under normal circumstances, do not have
Significant difference.Illustrating that biologically active polypeptide TIASGEPT has under the conditions of finite concentration promotes macrophage nitric oxide to lure
The raw increased ability of amount.
The above-mentioned description to embodiment is understood that for ease of those skilled in the art and using invention.
Person skilled in the art obviously can easily make various modifications to these embodiments, and described herein general
Principle is applied in other embodiment without passing through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability
Field technique personnel are according to the announcement of the present invention, and not departing from improvement and modification that scope made all should be the present invention's
Within protection domain.
<110>Zhejiang Hui Tai life and healths Science and Technology Ltd.
<120>A kind of biologically active polypeptide TIASGEPT and its preparation method and application
<160>3
<170> PatentIn version 3.3
<210> 1
<211> 8
<212> PRT
<213>Artificial sequence
<220>
<223>Biologically active polypeptide
<400> 1
Thr Ile Ala Ser Gly Glu Pro Thr
5
<210> 2
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<223>Biologically active polypeptide coded sequence
<400> 2
accattgcta gtggcgagcc taca 24
<210> 3
<211> 190
<212> PRT
<213>Artificial sequence
<220>
<223>κ-ss-casein variants A amino acid sequences
<400> 3
<400> 3
Met Met Lys Ser Phe Phe Leu Val Val Thr Ile Leu Ala Leu Thr
5 10 15
Leu Pro Phe Leu Gly Ala Gln Glu Gln Asn Gln Glu Gln Pro Ile
20 25 30
Arg Cys Glu Lys Asp Glu Arg Phe Phe Ser Asp Lys Ile Ala Lys
35 40 45
Tyr Ile Pro Ile Gln Tyr Val Leu Ser Arg Tyr Pro Ser Tyr Gly
50 55 60
Leu Asn Tyr Tyr Gln Gln Lys Pro Val Ala Leu Ile Asn Asn Gln
65 70 75
Phe Leu Pro Tyr Pro Tyr Tyr Ala Lys Pro Ala Ala Val Arg Ser
80 85 90
Pro Ala Gln Ile Leu Gln Trp Gln Val Leu Ser Asn Thr Val Pro
95 100 105
Ala Lys Ser Cys Gln Ala Gln Pro Thr Thr Met Ala Arg His Pro
110 115 120
His Pro His Leu Ser Phe Met Ala Ile Pro Pro Lys Lys Asn Gln
125 130 135
Asp Lys Thr Glu Ile Pro Thr Ile Asn Thr Ile Ala Ser Gly Glu
140 145 150
Pro Thr Ser Thr Pro Thr Thr Glu Ala Val Glu Ser Thr Val Ala
155 160 165
Thr Leu Glu Asp Ser Pro Glu Val Ile Glu Ser Pro Pro Glu Ile
170 175 180
Asn Thr Val Gln Val Thr Ser Thr Ala Val
185 190
Claims (10)
1. a kind of biologically active polypeptide TIASGEPT, it is characterised in that its amino acid sequence is Thr-Ile-Ala-Ser-Gly-
Glu-Pro-Thr。
2. a kind of biologically active polypeptide TIASGEPT according to claim 1, it is characterised in that the biologically active polypeptide
For milk-derived.
3. encode the nucleotide fragments of biologically active polypeptide TIASGEPT described in claim 1, it is characterised in that the nucleotides
The sequence of fragment such as SEQ ID NO：Shown in 2.
4. biologically active polypeptide TIASGEPT as claimed in claim 1 preparation method, it is characterised in that pass through genetic engineering
Method is artificial synthesized, or is directly obtained from dairy products by the method isolated and purified, or is directly prepared by chemical synthesis.
5. biologically active polypeptide TIASGEPT as claimed in claim 1 application, it is characterised in that the biologically active polypeptide
Applications of the TIASGEPT in food, health products, medicine or cosmetics with anti-oxidation function are prepared.
6. biologically active polypeptide TIASGEPT as claimed in claim 1 application, it is characterised in that the biologically active polypeptide
Applications of the TIASGEPT in food, health products or medicine with immunoloregulation function is prepared.
7. biologically active polypeptide TIASGEPT as claimed in claim 1 application, it is characterised in that the biologically active polypeptide
Applications of the TIASGEPT in food, health products or medicine with anti-oxidation function and immunoloregulation function is prepared.
8. a kind of oxidation resistant product, it is characterised in that including biologically active polypeptide TIASGEPT or described as claimed in claim 1
Biologically active polypeptide TIASGEPT derivative；Described oxidation resistant product includes antioxidant food, antioxidant health-care product, antioxygen
Chemical drug thing or antioxidation cosmetic product；The derivative of the biologically active polypeptide TIASGEPT, refers in biologically active polypeptide
On TIASGEPT amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetyl
Change, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
9. a kind of immunological regulation product, it is characterised in that including biologically active polypeptide TIASGEPT as claimed in claim 1 or institute
State biologically active polypeptide TIASGEPT derivative；
Described immunological regulation product includes immunological regulation food, immunological regulation health products or immunoregulation medicament；
The derivative of the biologically active polypeptide TIASGEPT, refers to the amino acid side chain base in biologically active polypeptide TIASGEPT
In group, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification or glycosylating
Modification, obtained polypeptide derivative.
10. a kind of product with anti-oxidation function and immunoloregulation function, it is characterised in that including as claimed in claim 1
Biologically active polypeptide TIASGEPT or described biologically active polypeptides TIASGEPT derivative；With anti-oxidation function and immune tune
Saving the product of function includes food, health products or medicine；The derivative of the biologically active polypeptide TIASGEPT, refers in biology
On active peptides TIASGEPT amino acid side groups, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, first
Base, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.
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Denomination of invention: A bioactive peptide tiasgept and its preparation method and Application Effective date of registration: 20210630 Granted publication date: 20200414 Pledgee: Zhejiang Cangnan rural commercial bank Limited by Share Ltd. Pledgor: ZHEJIANG HUITAI LIFE HEALTH TECHNOLOGY Co.,Ltd. Registration number: Y2021330000661 |
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