A kind of biologically active polypeptide PNIMVIQH and its preparation method and applicationTechnical field
The present invention relates to albumen fields, more particularly, to a kind of biologically active polypeptide PNIMVIQH and preparation method thereof and answerWith.
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, concurrentlyA series of biochemical reactions have been given birth to, so that protein is become polypeptide or free amino acid, is digested or passes throughThe absorption and transport of intestinal epithelial cell is directly entered the blood circulation of human body.There is also the eggs of some itself synthesis for lactic acid bacteria thallineWhite matter polypeptide fragment is utilized for bacterial growth.In these polypeptides, some is referred to as " raw with special physiological functionObject active peptide ".
It is particularly important that safe biologically active peptide is found in natural food source.In recent years, it has been found that some foodsThe polypeptides matter in object source has good bioactivity, such as corn small peptide, soybean peptide, cow's milk polypeptide.These polypeptides canTo be obtained by number of ways such as microbial fermentation, digestion enzymolysis, and biologically active polypeptide is by 2~20 mostlyAmino acid residue forms, and 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 one kind biology of its physiological activity is living from breast for the first time after opioid peptides discoveryProperty polypeptide.Jolles in 1981 et al. has found for the first time, using trypsin hydrolysis people lactoprotein, can obtain an amino acid sequenceIt is classified as the hexapeptide of Val-Glu-Pro-Ile-Pro-Tyr, experiment in vitro proves that the peptide can enhance Turnover of Mouse Peritoneal Macrophages pairThe phagocytosis of sheep red blood cell (SRBC).Migliore-Samour et al. has found the hexapeptide Thr-Thr-Met-Pro- from caseinLeu-Trp can stimulate sheep erythrocyte to the phagocytosis of mouse peritoneal macrophages and enhancing for kerekou pneumonia primaryThe resistance of bacterium.Newborn source immunomodulatory peptides (PGPIPN) the feeding rat of Li Su duckweeds et al. synthesis finds rat peritoneal macrophagesPhagocytosis and the relevant immunoloregulation function of red blood cell have significant enhancing.
Studies have shown that immune-active peptides can not only enhance immunity of organisms, the proliferation of body lymphocyte, enhancing are stimulatedThe phagocytic function of macrophage promotes the release of cell factor, improves the ability that body resists extraneous pathogenic infection, reduces machineBody incidence, and the immunological rejection of body will not be caused.
Aging is a natural phenomena, and process is often accompanied by the variation of antioxidant levels, organ-tissue, immune factor,Complicated variation occurs for middle cell factor, such as the trend that proinflammatory cytokine IL-6, IL-4, TNF-α presentation increase, IL-6It is all considered to play an important role in the generating process of geriatric disease with TNF-a.With science of heredity and molecular biologyDevelopment, the research of biological decay mechanism achieve gratifying progress.Researcher by using some model organisms, as mouse,The term single gene mutating experiment of drosophila and caenorhabditis elegan etc. finds that some genes can dramatically increase service life of these organisms and reachAs many as 6 times.
Anti-aging peptide as a kind of emerging antidotal agent, in terms of physiological function have amino acid cannot compare it is excellentGesture can generate promotion or inhibiting effect to the enzyme in organism, improve the absorption to minerals and other nutrients and profitWith removing interior free yl enhances the resistance to oxidation of body itself, to slow down aging.Therefore, the nutrition and health care of biologically active peptideEffect has become the emphasis of domestic and foreign scholars' subject study.Qiu Juan et al. pass through experimental studies have found that, milk-derived bioactive micro peptideLife span of drosophila melanogaster can effectively be extended, delay its aging, and also there is preferable antioxidation, thus it is speculated that may be wherein to be rich in coloured glazeBase peptides.SOD vigor in serum, reduces its lipid in discovery bovine colostrum extract energy conspicuousness raising the elderly's body such as the brightness in weekPeroxide and enhancing body resistance to oxidation, have certain anti-senescence function.
Have much about the research of biologically active polypeptide at present, for example Chinese patent CN105254738A discloses one kind and comesDerived from the milk-derived biologically active polypeptide DELQDKIH of beta-casein, Chinese patent CN105254739A discloses one kind and derives fromMilk-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.
Invention content
The purpose of the present invention is to provide a kind of biologically active polypeptide PNIMVIQH and its preparation method and application.
The purpose of the present invention can be achieved through the following technical solutions：
First aspect present invention provides a kind of biologically active polypeptide PNIMVIQH, amino acid sequence Pro-Asn-Ile-Met-Val-Ile-Gln-His, such as SEQ ID NO：Shown in 1.
Preferably, the biologically active polypeptide derives from Lactobacillus helveticus mycoprotein.It is derive specifically from LBH_1471 |m.1370 LBH_1471|g.1370 ORF LBH_1471|g.1370 LBH_1471|m.1370 type:complete len:327(+)LBH_1471:1-981 (+) albumen, and the amino acid residue of the 187th~194, albumen thus.LBH_1471|m.1370 LBH_1471|g.1370 ORF LBH_1471|g.1370 LBH_1471|m.1370 type:complete len:327(+)LBH_1471:1-981 (+) protein amino acid sequence such as SEQ ID NO：Shown in 3.
LBH_1471|m.1370 LBH_1471|g.1370 ORF LBH_1471|g.1370 LBH_1471|m.1370type:complete len:327(+)LBH_1471:The amino acid sequence and corresponding nucleotide sequence of 1-981 (+) albumenFor existing technology, the bioactivity for encoding the nucleotide fragments energy encoding mature of this 187th~194 amino acids residue of albumen is morePeptide PNIMVIQH.
Preferably, the biologically active polypeptide has immunoloregulation function and anti-senescence function.
Second aspect of the present invention provides the nucleotide fragments for encoding the biologically active polypeptide PNIMVIQH, sequenceFor：5 '-tcc taa tat tat ggt gat tca aca-3 ', such as SEQ ID NO：Shown in 2.
Third aspect present invention provides the preparation method of the biologically active polypeptide PNIMVIQH, can pass through geneThe method of engineering is artificial synthesized, can be directly obtained from Lactobacillus helveticus thalline by the method that clasmatosis isolates and purifies, canDirectly to be prepared by chemical synthesis.
Fourth aspect present invention provides the biologically active polypeptide PNIMVIQH and is preparing with immunoloregulation functionApplication in food, health products, drug or cosmetics.
Fifth aspect present invention provides the biologically active polypeptide PNIMVIQH and is preparing the food with anti-senescence functionApplication in product, health products or drug.
Sixth aspect present invention provides the biologically active polypeptide PNIMVIQH and is preparing while having immunological regulation work(It can be with the application in the food, health products or drug of anti-senescence function.
Specifically, the biologically active polypeptide PNIMVIQH of the present invention, which can be used for preparing, reduces free radical to skin damageCosmetics, prepare the drug with immunological regulation and/or anti-aging；And due to the biologically active polypeptide of the present inventionProduct after PNIMVIQH is degraded by gastrointestinal tract still has bioactivity, thus can be also used for preparing the food such as Yoghourt,Adjust the health products of immunity, and the oral drug being used to prepare with immunological regulation and/or anti-aging.
Seventh aspect present invention, provides a kind of immunological regulation product, including the biologically active polypeptide PNIMVIQH orThe derivative of the biologically active polypeptide PNIMVIQH；The immunological regulation product includes immunological regulation food, immunological regulationHealth products, immunoregulation medicament or immunological regulation cosmetics；The derivative of the biologically active polypeptide PNIMVIQH, refers in lifeOn the amino acid side groups of object active peptides PNIMVIQH, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation,Methylate, acetylation, phosphorylation, the modifications such as esterification or glycosylation, obtained polypeptide derivative.
Eighth aspect present invention provides a kind of anti-aging product, including the biologically active polypeptide PNIMVIQH or instituteState the derivative of biologically active polypeptide PNIMVIQH；The anti-aging product include antisenility cistanche food, antisenescence health product orAntiaging agent；The derivative of the biologically active polypeptide PNIMVIQH refers to the amino in biologically active polypeptide PNIMVIQHOn sour side-chain radical, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterificationOr the modifications such as glycosylation, obtained polypeptide derivative.
Ninth aspect present invention provides product that is a kind of while having immunoloregulation function and anti-senescence function, includingThe derivative of the biologically active polypeptide PNIMVIQH or described biologically active polypeptides PNIMVIQH；With immunoloregulation function andThe product of anti-senescence function includes food, health products or drug；The derivative of the biologically active polypeptide PNIMVIQH, refer toOn the amino acid side groups of biologically active polypeptide PNIMVIQH, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylChange, methylate, acetylation, phosphorylation, the modifications such as esterification or glycosylation, obtained polypeptide derivative.
Biologically active polypeptide PNIMVIQH's of the present invention has the beneficial effect that：The biologically active polypeptide PNIMVIQH tools of the present inventionThere are preferable adjusting immunity of organisms activity and activity of fighting against senium；On the one hand, biologically active polypeptide PNIMVIQH energy of the inventionThe in-vitro multiplication ability for enough enhancing lymphocyte and macrophage improves the ability that body resists extraneous pathogenic infection, reducesBody incidence；On the other hand, the vigor of internal anti-peroxidation enzyme system can be improved, enhancing body resists the work(of external source sexual stimulusCan, to reduce organism aging process, aging and sick probability, made to developing the breast with immunoloregulation function and anti-senescence functionProduct and health products have a very important significance.
Description of the drawings
Fig. 1：Mass chromatography extraction figure (m/z=969.5253)；
Fig. 2：The second order ms figure for the segment that mass-to-charge ratio is 969.5253；
Fig. 3：Polypeptide az, by crack conditions that mass-to-charge ratio is 969.5253；
Fig. 4：The macrophages in vitro proliferative capacity of biologically active polypeptide PNIMVIQH is tested；
Fig. 5：Each group experimental animal mouse spleen situation of change；
A) it is low dosage gavage group mouse spleen organization chart；(b) it is high dose gavage group mouse spleen organization chart；(c) it isNaive mice spleen tissue；(d) it is animal model group mouse spleen organization chart；
Fig. 6：Each group mice serum IL-6 changes table；
Fig. 7：Each group mice serum TNF-α changes table.
Specific implementation mode
Before further describing specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to downState specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific specific in order to describeEmbodiment, the protection domain being not intended to be limiting of the invention.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical rangeAny one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using andScientific terminology is identical as the normally understood meaning of those skilled in the art of the present technique.Except used in embodiment specific method, equipment,Outside material, the record according to those skilled in the art to the grasp of the prior art and the present invention can also use and thisAny method, equipment and the material of the similar or equivalent prior art of method, equipment described in inventive embodiments, material come realThe existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neckMolecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology andThe routine techniques of related field.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 HarborLaboratory Press, 1989 and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS INMOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987 and periodic updates；theSeries METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS INENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), AcademicPress, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, ChromatinProtocols (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.
1 active peptide PNIMVIQH's of embodiment is artificial synthesized
One, the synthesis of biologically active peptide
1. RINK resin 3g (degree of substitution 0.3mmol/g) are weighed in the reactor of 150ml, with the dichloromethane of 50ml(DCM) it impregnates.
After 2.2 hours, resin is washed with nitrogen-dimethylformamide (DMF) of 3 times of resin volumes, is then drained, so weightIt is four times multiple, it is for use after resin is drained.
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 shakes20min is shaken, the Fmoc blocking groups on resin are sloughed with this.It is washed four times with the DMF of 3 times of resin volumes after having taken off protection,Then it drains.
4. taking 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 reactions1min), resin has color, illustrates to be deprotected successfully.
5. it weighs amino acid Pro in right amount and 1- hydroxyls-benzene a pair of horses going side by side triazole (HOBT) is in right amount in the centrifuge tube of 50ml, additionThe DMF of 20ml is dissolved, and the N of 3ml is then added, and N diisopropylcarbodiimide (DIC) oscillation shakes up 1min, waits for that solution is clearIt is added in reactor after clear, then reactor is placed in 30 DEG C of shaking table and is reacted.
After 6.2 hours, with a certain amount of acetic anhydride end socket (acetic anhydride:DIEA:DCM=1:1:2,v:v:V) half an hour, soIt is washed four times, is drained for use with the DMF of 3 times of resin volumes afterwards.
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 shakes20min is shaken, the Fmoc blocking groups on resin are sloughed with this.It is washed four times with DMF after having taken off protection, is then drained.
8. taking 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 reactions1min), resin has color, illustrates to be deprotected successfully.
9. weighing second amino acid next in right amount and HOBT being in right amount in the centrifuge tube of 50ml, the DMF generals of 25ml are addedIt is dissolved, and the DIC oscillations that 2.5ml is then added shake up 1min, are added in reactor after solution clarification, then by reactorIt is placed in 30 DEG C of shaking table and reacts.
After 10.1 hours, a small amount of resin is taken to detect, with ninhydrin method detection (each two drop of inspection A, inspection B, 100 DEG C of reactions1min), if resin is colourless, illustrate that the reaction was complete；If resin has color, illustrate that condensation is incomplete, the reaction was continued.
11. after complete reaction, washing resin four times with DMF, then drains, a certain amount of 20% is added into reactorPiperidines (piperidines/DMF=1:4,v:V), it is placed on decolorization swinging table and rocks 20min, the Fmoc protecting groups on resin are sloughed with thisGroup.It is washed four times with DMF after having taken off protection, then drains whether detection protection sloughs.
12. connecting amino acid Asn, Ile, Met, Val, Ile, Gln and His successively according to step 9-11.
13. after connecting the last one amino acid, protection is sloughed, is washed four times with DMF, is then taken out resin with methanolIt 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(every gram of resin adds 10ml cutting liquids) is cut down from resin, and ice ether (cutting liquid is used in combination：Ether=1:9,v:V) centrifugation is heavyDrop four times.
So far, artificial synthesized biologically active peptide PNIMVIQH.
Two, the confirmation of biologically active peptide
1) UPLC is analyzed
UPLC conditions are as follows：
Instrument：Waters ACQUITY UPLC ultra high efficiencies liquid phase-electron spray-level four bars-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 the above analysis method, using ultra high efficiency liquid phase-electron spray-level four bars-flight time mass spectrum, to bioactivityPeptide PNIMVIQH carries out chromatography and mass spectral analysis, and mass chromatography extraction figure is as shown in Figure 1, extract the second order ms at this peakFigure and az, by crack conditions are as shown in Figures 2 and 3, and the polypeptide mass-to-charge ratio that can obtain this peak is 969.5253Da, and retention time is44.8min。
3) result
From the figure 3, it may be seen that the case where being broken according to az, by, analyzes by Mascot softwares and calculates, obtain mass-to-charge ratioThe fragment sequence of 969.5253Da is Pro-Asn-Ile-Met-Val-Ile-Gln-His (PNIMVIQH), is denoted as SEQ IDNO：1.The segment and LBH_1471 | m.1370 LBH_1471 | g.1370 ORF LBH_1471 | g.1370 LBH_1471 |m.1370 type:complete len:327(+)LBH_1471:The residue sequence phase of the 187th~194,1-981 (+) albumenCorresponding, sequence is shown in SEQ ID NO：3.
The adjusting immunity of organisms activity experiment of 2 biologically active peptide of embodiment
One, mtt assay measures the macrophages in vitro proliferative capacity experiment of biologically active polypeptide PNIMVIQH
1. experiment reagent and instrument：
Reagent：Experimental animal balb/c mouse (male 6-8 week old) Shanghai Communications University's agricultural and biological institute animal realityTest center；The biologically active polypeptide PNIMVIQH that embodiment 1 obtains；3- (4,5- dimethylthiazole -2) -2,5- diphenyl, four nitrogenAzoles bromide (MTT) Amresco companies；LPS (lipopolysaccharides) Sigma companies；Bovine serum albumin(BSA) (Bovine SerumAlbumin, BSA) Genebase companies；Three lysates, the water containing 10%SDS, 5% isobutanol and 0.012mol/L HClSolution.
Instrument and equipment：LRH-250F biochemical cultivation cases Shanghai perseverance Science and Technology Ltd.；On GL-22M high speed freezing centrifugesHai Luxiang instrument centrifuges Instrument Ltd.；Hera cell 150 CO2Incubator Heraeus companies；Dragon WellscanMK3 microplate reader Labsystems companies.
2. experimental method：
Balb/c mouse peritoneals inject 2% (w/w) the sterilizing starch solutions of 2ml, and continuous injection three days, last time is injectedThe 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, fromAfter heart pipe collects flushing liquor, supernatant is abandoned in centrifugation (1000rpm, 4 DEG C) after ten minutes, (is contained with 4 DEG C of RPMI1640 complete culture solutionsIt 10%FBS) washes twice, cell viability examination is done in the dyeing of 0.2% trypan blue solution, confirms collected vibrant macrophageAccount for 95% or more.After cell counting board reading, adjustment cell concentration to suitable concentration.
It will blow and beat to the cell suspension to suspend completely and 96 porocyte culture plates, 37 DEG C, 5%CO be added with suitable volumes2After 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 solutionsBottom washes away not adherent cell and cell fragment, obtains adherent peritoneal macrophage after purification.0.2ml is added per holeRPMI1640 complete mediums, experiment small peptide sample and LPS are added after being dissolved in culture medium in advance, start cell culture.
It is 2 × 10 that number of cells, which is added,5100 holes μ l/ of cell suspension of/ml, adherent addition after purification are more containing bioactivity200 holes μ l/ of RPMI1640 complete culture solutions (10%FBS) of peptide (100,500,1000 μ g/mL), continuous culture 48 hours are scorchingLPS to final concentration 100ng/ml was added at 24 hours for disease group.20 holes μ l/ 5%MTT are added at 44 hours, after reaching 48 hoursThree lysates in 100 holes μ l/ are added to terminate culture, after dissolving overnight, survey the suction in each hole with microplate reader at wavelength 570nmThe calculation formula of shading value (OD570), growth index (Growth Indices) is as follows：
Wherein, blank group is not apply small peptide and the cell processing group of BSA, and BSA groups are negative control.
3. experimental result and analysis：
Experimental result is shown in Fig. 4, and the addition concentration of biologically active polypeptide (PNIMVIQH) is respectively 1000 in experimental group,500,100 μ g/mL, blank group are added the PBS of corresponding amount as blank control, indicate the macrophage in the case where no LPS is stimulatedThe proliferative conditions of cell.It is compared with blank control group, adds the polypeptide PNIMVIQH experimental groups of various concentration with experimental concentrationIncrease, the proliferative capacity of macrophage is gradually increasing, in a concentration of 1000,500 μ g/mL, have significant difference (P<0.05).Illustrate that biologically active polypeptide PNIMVIQH has the ability for promoting macrophage proliferation.
Two, the rush macrophage of biologically active polypeptide PNIMVIQH swallows dimethyl diaminophenazine chloride capacity experimental
1. experiment reagent and instrument：
Reagent：Experimental animal balb/c mouse (male 6-8 week old) Shanghai Communications University's agricultural and biological institute animal realityTest center；The biologically active polypeptide PNIMVIQH that embodiment 1 obtains；LPS is purchased from Sigma companies；Neutral red staining solution, green cloudIts biotechnology research institute produces.
Instrument and equipment：LRH-250F biochemical cultivation cases Shanghai perseverance Science and Technology Ltd.；On GL-22M high speed freezing centrifugesHai Luxiang instrument centrifuges Instrument Ltd.；150 CO2 incubator Heraeus companies of Hera cell；Dragon WellscanMK3 microplate reader Labsystems companies.
2. experimental method：
It is 2 × 10 that number of cells, which is added,6100 holes μ l/ of cell suspension of/ml, adherent be added after purification contain active peptide200 holes μ l/ of RPMI1640 complete culture solutions (10%FBS) of PNIMVIQH (1mg/ml) are experimental group, and addition is free of active peptide200 holes μ l/ of RPMI1640 complete culture solutions (10%FBS) cultivated be set as blank group；And experimental group and blank groupLPS to 10 μ g/ml of final concentration is added when culture is arrived for 24 hours；After continuing culture to 48h, cell culture fluid is abandoned in suction.PBS cleans hole37 DEG C of 80 holes μ l/ of dimethyl diaminophenazine chloride dye liquor are added behind bottom, inhales abandon dye liquor after ten minutes, after being cleaned twice with PBS, 150 μ are added per holeL cell pyrolysis liquid (glacial acetic acid：Absolute ethyl alcohol=1:1, v/v).After 4 DEG C of dissolvings overnight, absorbance value is measured at wavelength 540nm(OD540)。
3. experimental result and analysis：
1 biologically active polypeptide PNIMVIQH of table promotees the measurement of macrophage phagocytosis dimethyl diaminophenazine chloride ability
| Experiment packet | Inflammation group absorbance value (OD540) |
| Blank group | 0.1102±0.0728 |
| Experimental group | 0.1462±0.0362** |
Note：*, compared with negative control, significant difference (P < 0.05)
*, compared with negative control group, significant difference (P < 0.01)
Experimental result is shown in Table 1, compared with cell blank, the inflammation group of addition 1mg/ml biologically active polypeptides PNIMVIQHMacrophage phagocytosis dimethyl diaminophenazine chloride ability obviously increases, and compared with cell blank group, has significant difference (P < 0.01).Illustrate that biologically active polypeptide PNIMVIQH has macrophages in vitro phagocytosis dimethyl diaminophenazine chloride ability in the case where there is inflammation generationSignificant facilitation.
The activity of fighting against senium of 3 biologically active peptide of embodiment is tested
One, experiments of the biologically active polypeptide PNIMVIQH to internal spleen tissue structure function
1. experiment reagent and instrument：
Reagent：Experimental animal ICR mouse (male 5 week old), Shanghai City Experimental Animal Center；D-gal, Chinese medicines group chemistryReagent Co., Ltd；Paraformaldehyde, Sinopharm Chemical Reagent Co., Ltd.；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagentDepartment；The biologically active polypeptide PNIMVIQH that embodiment 1 obtains.
Instrument and equipment：The ultra-clean water of CM-230 types mole, Shanghai Moller scientific instrument Co., Ltd；Mi Libo MilliporeMILLEX GP0.22 μm filter membranes, Millipore Corp. of the U.S.；GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument centrifuge instrumentsCo., Ltd.
2. experimental method：
(1) Animal Aging model modeling
After a week by the raising of ICR mouse adaptability, it is divided into 4 groups, every group 6.Group 1 is low dosage gavage group, and mouse is dailyD-gal is subcutaneously injected with the dosage nape part of 500mg/kg, and with the dosage gavage biologically active polypeptide in 1mg/ dayPNIMVIQH；Group 2 is high dose gavage group, and D-gal is subcutaneously injected with the dosage nape part of 500mg/kg daily in mouse, andThe day dosage gavage biologically active polypeptide PNIMVIQH of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is animal mouldD-gal, and the physiology salt of gavage a concentration of 0.9% is subcutaneously injected with the dosage nape part of 500mg/kg daily in type group, mouseWater；The injection cycle of D-gal and the gavage period of polypeptide are 42 days.It replaces bedding and padding within every 3 days, and ensures feed and distilled waterSupply.The every five days weight for weighing a mouse prepare D-gal injections according to the weight of mouse, and D-gal injections pass through0.22 μm of needle cylinder type filter membrane filtering, it is sterile to ensure.
(2) animal viscera is obtained
After the completion of experimental period, to pluck the blood that eyeball blood taking method obtains mouse, disconnected neck puts to death mouse after obtaining blood, andThe body of mouse is placed on low temperature ice box afterwards, and wins brain, spleen, liver and the kidney of mouse rapidly, what is obtained is dirtyDevice is placed in the 1.5mL centrifuge tubes to sterilize in advance, and all organ samples are maintained in -80 DEG C of refrigerators standby inspection.Disposition is realAll operations tested during animal follow Ministry of Science and Technology's publication in 2006《Directiveness meaning about kind treatment experimental animalSee》.The mouse spleen won directly is soaked in advance prepared 4% paraformaldehyde solution, to fix in the form of it.PolyFormaldehyde powder more indissoluble can be added micro sodium bicarbonate and pH value is adjusted to alkalinity, with hydrotropy.Paraformaldehyde solutionIt prepares and needs to complete in draught cupboard.
(3) sample detection
The making of histotomy：Mouse spleen sample need at least fix 24 hours in 4% paraformaldehyde solution.Spleen groupThe wax stone knitted makes, slice is completed with the HE dyeing commission Shanghai bio tech ltd Wei Ao.
3. experimental result and analysis：
In this experiment, 4 groups of mouse are shared, the mouse normal growth of wherein blank group is not affected by any environmental stimuli,3 groups of mouse of remaininging receive the long term injections of D-gal.Using light microscope, the spleen section of separate groups of mice is seenIt examines, can be found from Fig. 5, compare the spleen section of each group mouse, for naive mice, the spleen of animal model mouseDirty red pulp is fuzzy with white pulp boundary, and atrophy occurs in white pulp, shows that long-term D-gal injections make the glycometabolism approach of mouse occurDisorder causes anti-oxidant enzyme activity to reduce, peroxide accumulation, and then may cause the aging and atrophy of spleen.And gavage is moreThen white pulp atrophy degree is lighter relative to animal model group mouse for the spleen tissue of the mouse of peptide group, and the boundary of red pulp and white pulpIt is more clearly demarcated.This result illustrates, in the injection cycle of entire D-gal, experiment animal sustained is constantly by cause senescence-factorStimulation, lead to the aging and atrophy of spleen.Therefore from the changes in microstructure the case where from the point of view of, the biology invented in this experimentActive peptides PNIMVIQH has certain guarantor to spleen aging of the animal caused by the stimulation by the bad factor with atrophyShield acts on.
Two, the experiment that biologically active polypeptide PNIMVIQH acts on intracorporeal organ antioxidant levels
1. experiment reagent and instrument：
Reagent：Experimental animal ICR mouse (male 5 week old), Shanghai City Experimental Animal Center；D-gal, Chinese medicines group chemistryReagent Co., Ltd；Paraformaldehyde, Sinopharm Chemical Reagent Co., Ltd.；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagentDepartment；The biologically active polypeptide PNIMVIQH that embodiment 1 obtains；BCA protein reagent boxes, Science and Technology Ltd. of Nanjing Keygen Biotech；MDA lipid peroxide kits, Science and Technology Ltd. of Nanjing Keygen Biotech；SOD superoxide dismutase kits, Nanjing is builtAt bio tech ltd.
Instrument and equipment：The ultra-clean water of CM-230 types mole, Shanghai Moller scientific instrument Co., Ltd；Mi Libo MilliporeMILLEX GP0.22 μm filter membranes, Millipore Corp. of the U.S.；GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument centrifuge instrumentsCo., Ltd.
2. experimental method：
(1) Animal Aging model modeling
After a week by the raising of ICR mouse adaptability, it is divided into 4 groups, every group 6.Group 1 is low dosage gavage group, and mouse is dailyD-gal is subcutaneously injected with the dosage nape part of 500mg/kg, and with the dosage gavage biologically active polypeptide in 1mg/ dayPNIMVIQH；Group 2 is high dose gavage group, and D-gal is subcutaneously injected with the dosage nape part of 500mg/kg daily in mouse, andThe day dosage gavage biologically active polypeptide PNIMVIQH of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is animal mouldD-gal, and the physiology salt of gavage a concentration of 0.9% is subcutaneously injected with the dosage nape part of 500mg/kg daily in type group, mouseWater；The injection cycle of D-gal and the gavage period of polypeptide are 42 days.It replaces bedding and padding within every 3 days, and ensures feed and distilled waterSupply.The every five days weight for weighing a mouse prepare D-gal injections according to the weight of mouse, and D-gal injections pass through0.22 μm of needle cylinder type filter membrane filtering, it is sterile to ensure.
(2) animal viscera is obtained
After the completion of experimental period, to pluck the blood that eyeball blood taking method obtains mouse, disconnected neck puts to death mouse after obtaining blood, andThe body of mouse is placed on low temperature ice box afterwards, and wins brain, spleen, liver and the kidney of mouse rapidly, what is obtained is dirtyDevice is placed in the 1.5mL centrifuge tubes to sterilize in advance, and all organ samples are maintained in -80 DEG C of refrigerators standby inspection.Disposition is realAll operations tested during animal follow Ministry of Science and Technology's publication in 2006《Directiveness meaning about kind treatment experimental animalSee》.The mouse spleen won directly is soaked in advance prepared 4% paraformaldehyde solution, to fix in the form of it.PolyFormaldehyde powder more indissoluble can be added micro sodium bicarbonate and pH value is adjusted to alkalinity, with hydrotropy.Paraformaldehyde solutionIt prepares and needs to complete in draught cupboard.
(3) sample detection
All internal organs that need to be detected, grind at low ambient temperatures, and 4 DEG C of sterile PBS solutions is used in combination to be diluted to 10% groupIt knits homogenate, under the conditions of 4 DEG C after 4000g centrifugations, takes supernatant, discard precipitation, operated according to kit specification, or setIt is to be measured in -80 DEG C of refrigerators.
3. experimental result and analysis：
The variation of SOD contents in 2 each group experimental animal mouse Different Organs of table
Note：* mark is compared with model group, significant difference (P<0.05)；* marks are compared with model group,Significant difference (P<0.01), similarly hereinafter.
As can be known from Table 2, relative to animal model group mouse, the SOD in the liver and kidney of polypeptide gavage group mouse containsIncrease (the P of conspicuousness is presented in amount<0.01).Although meaning the mouse of polypeptide gavage group by the D-gal of long-term, high-doseStimulation, the excess injection of D-gal without completely destroy Mice Body in SOD enzyme systems, illustrate in injection cycle, experimentAnimal continuously by the stimulation for causing senescence-factor, can lead to the reduction of SOD contents in Different Organs, but sameWhen take in a certain amount of polypeptide PNIMVIQH to the oxidative damage in Mice Body have certain protective role.
The situation of change of MDA contents in 3 each group experimental animal mouse Different Organs of table
As can be known from Table 3, the liver MDA content of animal model group mouse is 27.86 ± 7.34nmol/L, with animal modelGroup compares, and significant difference (P is presented in the MDA contents in two groups of mouse livers of polypeptide gavage<0.01).Since MDA can be used forEstimate the accumulation situation of animal body lipid peroxide, therefore it follows that animal model group mouse is causing aging model to be formedIn the process, due to the long term injections of excessive D-gal, the glycometabolism approach of mouse is made to get muddled, generate a large amount of free radicals fromAnd oxidative damage is caused, occur a large amount of lipid peroxide in liver organization, MDA is as lipid peroxide, in animalThe raising of in-vivo content can reflect the reduction of Antioxidant Enzymes vigor in Mice Body from side.And polypeptide gavage group Mouse LiverThe significant decrease of dirty MDA contents, illustrate the intake of polypeptide PNIMVIQH can be effectively protected vital tissue organ from it is bad becauseSon stimulation generates a large amount of lipid peroxide.
Three, the experiment that immune cell factor in serum is acted on of biologically active polypeptide PNIMVIQH
1. experiment reagent and instrument：
Reagent：Experimental animal ICR mouse (male 5 week old), Shanghai City Experimental Animal Center；D-gal, Chinese medicines group chemistryReagent Co., Ltd；Paraformaldehyde, Sinopharm Chemical Reagent Co., Ltd.；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagentDepartment；The biologically active polypeptide PNIMVIQH that embodiment 1 obtains；BCA protein reagent boxes, Science and Technology Ltd. of Nanjing Keygen Biotech；ELISA cell factors Quick kit (TNF-α and IL-6), Wuhan Boster Biological Technology Co., Ltd..
Instrument and equipment：The ultra-clean water of CM-230 types mole, Shanghai Moller scientific instrument Co., Ltd；Mi Libo MilliporeMILLEX GP0.22 μm filter membranes, Millipore Corp. of the U.S.；GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument centrifuge instrumentsCo., Ltd.
2. experimental method：
(1) Animal Aging model modeling
After a week by the raising of ICR mouse adaptability, it is divided into 4 groups, every group 6.Group 1 is low dosage gavage group, and mouse is dailyD-gal is subcutaneously injected with the dosage nape part of 500mg/kg, and with the dosage gavage biologically active polypeptide in 1mg/ dayPNIMVIQH；Group 2 is high dose gavage group, and D-gal is subcutaneously injected with the dosage nape part of 500mg/kg daily in mouse, andThe day dosage gavage biologically active polypeptide PNIMVIQH of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is animal mouldD-gal, and the physiology salt of gavage a concentration of 0.9% is subcutaneously injected with the dosage nape part of 500mg/kg daily in type group, mouseWater；The injection cycle of D-gal and the gavage period of polypeptide are 42 days.It replaces bedding and padding within every 3 days, and ensures feed and distilled waterSupply.The every five days weight for weighing a mouse prepare D-gal injections according to the weight of mouse, and D-gal injections pass through0.22 μm of needle cylinder type filter membrane filtering, it is sterile to ensure.
(2) animal viscera and serum are obtained
After the completion of experimental period, to pluck the blood that eyeball blood taking method obtains mouse, disconnected neck puts to death mouse after obtaining blood, andThe body of mouse is placed on low temperature ice box afterwards, after mouse blood is stored at room temperature 1 hour, 15min is centrifuged with 3000g, detaches bloodClearly.Serum keeping is to be checked in -80 DEG C of refrigerators.All operations during disposition experimental animal follow the Ministry of Science and Technology in 2006Publication《Guiding opinion about kind treatment experimental animal》.The mouse spleen won directly is soaked in prepared in advanceIn 4% paraformaldehyde solution, to fix in the form of it.Paraformaldehyde powder more indissoluble, micro sodium bicarbonate can be added willPH value is adjusted to alkalinity, with hydrotropy.The preparation needs of paraformaldehyde solution are completed in draught cupboard.
(3) sample detection
It is indicated according to kit specification, draws standard curve first, standard items powder is prepared with standard dilutionsAt the solution of 1000pg/mL, then serial dilution is 500pg/mL, 250pg/mL, 125pg/mL, 62.5pg/mL, 31.3pg/mL,15.6pg/mL waiting various concentrations.Each concentration gradient solution pipettes 100 μ L in the ELISA Plate of coated antibody.Draw mouse100 μ L of blood serum sample, be added in same ELISA Plate (need to press when detecting calculating again if blood serum sample is inadequate, after can suitably dilutingRatio is converted).ELISA Plate is covered, places it under 37 DEG C of environment and is incubated 90min.After completion of the reaction, it carefully gets rid of in ELISA PlateLiquid, and ELISA Plate is placed on blotting paper, it is careful to pat, remove surplus liquid.By preheated biotin antiantibody workMake liquid to sequentially add in each hole of ELISA Plate by 100 μ L of every hole, at 37 DEG C, reacts 60min.After completion of the reaction, utilize 0.01M'sPBS solution is washed 3 times, and the PBS of 100 μ L is added in every hole every time, impregnates 1min hypsokinesis and removes solution, 3 times repeatedly.It will be preheatedABC working solutions are sequentially added by every hole 0.1ml, 37 DEG C of reaction 30min.After completion of the reaction, 5 times are washed with 0.01M PBS, every timeImpregnate 1min or so.The TMB developing solutions for balancing 30min at 37 DEG C are sequentially added by 90 μ L of every hole, 37 DEG C are protected from light 8-12min.TMB terminate liquids are sequentially added by every hole 0.1ml, blue is vertical at this time turns yellow, and OD values are measured in 450nm with microplate reader.Known concentration is done by the standard protein of cell factor to be serially diluted, standard curve is drawn out after measuring OD values, according to standard songLine can extrapolate the content of cell factor in sample.
3. experimental result and analysis：
The situation of change of cell factor in 4 each group mice serum of table
IL-6 and TNF-α content are respectively out of model group Mice Body that can be found that in this experiment in table 4, Fig. 6, Fig. 7167.31 ± 24.78pg/mL, 4.54 ± 0.66pg/mL, compared to the increase (P that conspicuousness is presented in normal group<0.01), thereforeIt is considered that due to continuously injecting cause senescence-factor, animal model group mouse is caused aging occur in cell factor levelThe symptom of property inflammation, and the IL-6 of the mice serum of polypeptide gavage group is effectively controlled with TNF-α content.According to cell becauseThe experimental result of son, the serum levels of inflammatory cytokines IL-6 of polypeptide gavage group mouse, the secretion level of TNF-α are below animal mouldType group, from the point of view of oxidative damage angle, mouse oxidative damage caused by due to free radical is attacked, Peroxidation Product is accumulated may obtainInhibition to a certain extent；From the perspective of inflammation, mouse inflammation because of caused by oxidation has obtained effective inhibition；From decliningFrom the point of view of old angle, a series of geriatric diseases caused by mouse aging caused by long term injections D-gal are possible to obtainControl.
The above description of the embodiments is intended to facilitate ordinary skill in the art to understand and use the invention.Person skilled in the art obviously easily can make various modifications to these embodiments, and described herein generalPrinciple is applied in other embodiment without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, abilityField technique personnel announcement according to the present invention, improvement and modification made without departing from the scope of the present invention all should be the present invention'sWithin protection domain.
Sequence table
<110>Zhejiang Hui Tai life and healths Science and Technology Ltd.；The Shanghai bio tech ltd Bo Hui
<120>A kind of biologically active polypeptide PNIMVIQH and its preparation method and application
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Ile Asn Glu Lys Leu Phe Val Val Asn Lys Ala Arg His Tyr Asp Glu
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Asp Tyr Val Ser Lys Tyr Asn Gly Phe Lys Met His Leu Glu Gly Asp
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Gly Cys Lys Trp Thr Ala Ala Pro Thr Ala Asp Asp Ser Glu Gly Arg
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Trp Asp Glu Gln Gly Ile Asn Glu Tyr Asn Val Ala Met Ser Ala Thr
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Glu Asp Gly Ile Asn Glu Asp Ser Met Val Tyr Ile Thr Leu Pro Phe
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Val Lys Thr Ala Arg Glu Gly Val Leu Arg Leu Gly Arg Leu Ile Glu
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Lys Tyr Gly Thr Gly Glu Thr Asn Gly Ile Ala Phe Ser Asp Asn Lys
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Glu Val Trp Tyr Leu Glu Thr Gly Ala Gly His Gln Trp Val Ala Ala
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Arg Val Pro Asp Asp Ser Tyr Ala Ile Cys Pro Asn Ile Met Val Ile
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Gln His Val Asn Phe Asp Asp Pro Asp Asn Phe Met Tyr Ser Glu Gly
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Ile Gln Glu Phe Val Glu Lys Asn His Leu Asn Asn Ser Thr Asp Gly
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Ser Phe Ser Phe Arg Asp Ile Phe Gly Thr Lys Asp Glu Ala Asp Ala
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Phe Tyr Asn Thr Pro Arg Thr Trp Tyr Gly Gln Lys Leu Phe Asn Pro
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Ser Ile Glu Gln Asp Pro Thr Ser Gln Glu Met Pro Phe Thr Arg Val
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Pro Glu Lys Lys Ile Gly Val Glu Asp Val Gln Lys Phe Leu Ser Ser
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His Tyr Asn Gly Thr Pro Tyr Asp Pro Met Gly Thr Phe Ser Ser Gly
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Ser Glu Lys Glu Gln Lys Met Phe Arg Ser Ile Ala Leu Asp Arg Asn
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Gln Glu Ser Ser Ile Leu
325