CN108794602A

CN108794602A - A kind of biologically active polypeptide PNIMVIQH and its preparation method and application

Info

Publication number: CN108794602A
Application number: CN201810716109.3A
Authority: CN
Inventors: 张少辉; 占文静; 张伯宇; 林学海; 亚辛·黑马尔; 洪志骏; 陈平; 陈承余
Original assignee: Shanghai Platinum Hui Biological Technology Co Ltd; Zhejiang Peptide Life Health Science And Technology Co Ltd
Current assignee: Shanghai Platinum Hui Biological Technology Co Ltd; Zhejiang Peptide Life Health Science And Technology Co Ltd
Priority date: 2018-06-29
Filing date: 2018-06-29
Publication date: 2018-11-13
Anticipated expiration: 2038-06-29
Also published as: CN108794602B

Abstract

The present invention relates to albumen fields, and in particular to its amino acid sequence of a kind of biologically active polypeptide PNIMVIQH and its preparation method and application, biologically active polypeptide PNIMVIQH is Pro-Asn-Ile-Met-Val-Ile-Gln-His.By the experiment of ion vitro immunization function point analysis, internal Antisenility Experiment, demonstrating polypeptide PNIMVIQH has preferable immunoloregulation function and activity of fighting against senium, on the one hand, the biologically active polypeptide PNIMVIQH of the present invention can enhance the in-vitro multiplication ability of lymphocyte and macrophage, the ability that body resists extraneous pathogenic infection is improved, body incidence is reduced；On the other hand, the vigor of internal anti-peroxidation enzyme system can be improved, enhance the function that body resists external source sexual stimulus, to reduce organism aging process, aging and sick probability, there is immunoloregulation function, the food of anti-senescence function, health products and drug to have a very important significance exploitation.

Description

A kind of biologically active polypeptide PNIMVIQH and its preparation method and application

Technical field

The present invention relates to albumen fields, more particularly, to a kind of biologically active polypeptide PNIMVIQH and preparation method thereof and answer 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, so that protein is become 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.There is also the eggs of some itself synthesis for lactic acid bacteria thalline White matter polypeptide fragment is utilized for bacterial growth.In these polypeptides, some is referred to as " raw with special physiological function Object 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 foods The polypeptides matter in object source has good bioactivity, such as corn small peptide, soybean peptide, cow's milk polypeptide.These polypeptides can To be obtained by number of ways such as microbial fermentation, digestion enzymolysis, and biologically active polypeptide is by 2~20 mostly Amino 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 discovery Property polypeptide.Jolles in 1981 et al. has found for the first time, using trypsin hydrolysis people lactoprotein, can obtain an amino acid sequence It 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 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 enhancing for kerekou pneumonia primary The resistance of bacterium.Newborn source immunomodulatory peptides (PGPIPN) the feeding rat of Li Su duckweeds et al. synthesis finds rat peritoneal macrophages Phagocytosis 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 stimulated The phagocytic function of macrophage promotes the release of cell factor, improves the ability that body resists extraneous pathogenic infection, reduces machine Body 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-6 It is all considered to play an important role in the generating process of geriatric disease with TNF-a.With science of heredity and molecular biology Development, 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 reach As 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 excellent Gesture can generate promotion or inhibiting effect to the enzyme in organism, improve the absorption to minerals and other nutrients and profit With 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 peptide Effect 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 peptide Life 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 glaze Base 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 week Peroxide 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 comes Derived from the milk-derived biologically active polypeptide DELQDKIH of beta-casein, Chinese patent CN105254739A discloses one kind and derives 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.

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.1370 type:complete len:327(+)LBH_1471:The amino acid sequence and corresponding nucleotide sequence of 1-981 (+) albumen For existing technology, the bioactivity for encoding the nucleotide fragments energy encoding mature of this 187th~194 amino acids residue of albumen is more Peptide 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, sequence For：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 gene The method of engineering is artificial synthesized, can be directly obtained from Lactobacillus helveticus thalline by the method that clasmatosis isolates and purifies, can Directly to be prepared by chemical synthesis.

Fourth aspect present invention provides the biologically active polypeptide PNIMVIQH and is preparing with immunoloregulation function Application 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 function Application 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 damage Cosmetics, prepare the drug with immunological regulation and/or anti-aging；And due to the biologically active polypeptide of the present invention Product 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 or The derivative of the biologically active polypeptide PNIMVIQH；The immunological regulation product includes immunological regulation food, immunological regulation Health products, immunoregulation medicament or immunological regulation cosmetics；The derivative of the biologically active polypeptide PNIMVIQH, refers in life On 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 institute State the derivative of biologically active polypeptide PNIMVIQH；The anti-aging product include antisenility cistanche food, antisenescence health product or Antiaging agent；The derivative of the biologically active polypeptide PNIMVIQH refers to the amino in biologically active polypeptide PNIMVIQH On sour side-chain radical, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification Or 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, including The derivative of the biologically active polypeptide PNIMVIQH or described biologically active polypeptides PNIMVIQH；With immunoloregulation function and The product of anti-senescence function includes food, health products or drug；The derivative of the biologically active polypeptide PNIMVIQH, refer to On the amino acid side groups of biologically active polypeptide PNIMVIQH, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonyl Change, 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 invention There are preferable adjusting immunity of organisms activity and activity of fighting against senium；On the one hand, biologically active polypeptide PNIMVIQH energy of the invention The in-vitro multiplication ability for enough enhancing lymphocyte and macrophage improves the ability that body resists extraneous pathogenic infection, reduces Body 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 stimulus Can, to reduce organism aging process, aging and sick probability, made to developing the breast with immunoloregulation function and anti-senescence function Product 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 is Naive 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 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 numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, in the present invention all technologies for using and Scientific 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 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 stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and The 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 Harbor Laboratory Press, 1989 and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987 and 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.

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 weight It 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 shakes 20min 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 reactions 1min), 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, addition The 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 clear It 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, so It 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 shakes 20min 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 reactions 1min), 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 added It is dissolved, and the DIC oscillations that 2.5ml is then added shake up 1min, are added in reactor after solution clarification, then by reactor It 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 reactions 1min), 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 reactor Piperidines (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 this Group.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 methanol 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 (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 heavy Drop 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 bioactivity Peptide 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 peak Figure 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 is 44.8min。

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 ratio The fragment sequence of 969.5253Da is Pro-Asn-Ile-Met-Val-Ile-Gln-His (PNIMVIQH), is denoted as SEQ ID NO：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 (+) albumen Corresponding, 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 reality Test center；The biologically active polypeptide PNIMVIQH that embodiment 1 obtains；3- (4,5- dimethylthiazole -2) -2,5- diphenyl, four nitrogen Azoles 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 150 CO₂Incubator Heraeus companies；Dragon Wellscan MK3 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 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 After 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 solutions It 10%FBS) washes twice, cell viability examination is done in the dyeing of 0.2% trypan blue solution, confirms collected vibrant macrophage Account 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 volumes₂ 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 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 bioactivity 200 holes μ l/ of RPMI1640 complete culture solutions (10%FBS) of peptide (100,500,1000 μ g/mL), continuous culture 48 hours are scorching LPS 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 hours Three 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 570nm The 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 stimulated The proliferative conditions of cell.It is compared with blank control group, adds the polypeptide PNIMVIQH experimental groups of various concentration with experimental concentration Increase, 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 reality Test center；The biologically active polypeptide PNIMVIQH that embodiment 1 obtains；LPS is purchased from Sigma companies；Neutral red staining solution, green cloud Its biotechnology research institute produces.

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.；150 CO2 incubator Heraeus companies of Hera cell；Dragon Wellscan MK3 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 peptide 200 holes μ l/ of RPMI1640 complete culture solutions (10%FBS) of PNIMVIQH (1mg/ml) are experimental group, and addition is free of active peptide 200 holes μ l/ of RPMI1640 complete culture solutions (10%FBS) cultivated be set as blank group；And experimental group and blank group LPS 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 hole 37 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 hole L 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 PNIMVIQH Macrophage 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 generation Significant 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 chemistry Reagent Co., Ltd；Paraformaldehyde, Sinopharm Chemical Reagent Co., Ltd.；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagent Department；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 Millipore MILLEX GP0.22 μm filter membranes, Millipore Corp. of the U.S.；GL-22M high speed freezing centrifuges, Shanghai Lu Xiang instrument centrifuge instruments Co., 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 daily D-gal is subcutaneously injected with the dosage nape part of 500mg/kg, and with the dosage gavage biologically active polypeptide in 1mg/ day PNIMVIQH；Group 2 is high dose gavage group, and D-gal is subcutaneously injected with the dosage nape part of 500mg/kg daily in mouse, and The day dosage gavage biologically active polypeptide PNIMVIQH of 3mg/ only；Group 3 is blank group, mouse normal growth；Group 4 is animal mould D-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, mouse Water；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 water Supply.The every five days weight for weighing a mouse prepare D-gal injections according to the weight of mouse, and D-gal injections pass through 0.22 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, and The 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 dirty Device 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 real All operations tested during animal follow Ministry of Science and Technology's publication in 2006《Directiveness meaning about kind treatment experimental animal See》.The mouse spleen won directly is soaked in advance prepared 4% paraformaldehyde solution, to fix in the form of it.Poly Formaldehyde powder more indissoluble can be added micro sodium bicarbonate and pH value is adjusted to alkalinity, with hydrotropy.Paraformaldehyde solution It 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 group The 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 seen It examines, can be found from Fig. 5, compare the spleen section of each group mouse, for naive mice, the spleen of animal model mouse Dirty 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 occur Disorder causes anti-oxidant enzyme activity to reduce, peroxide accumulation, and then may cause the aging and atrophy of spleen.And gavage is more Then 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 pulp It is more clearly demarcated.This result illustrates, in the injection cycle of entire D-gal, experiment animal sustained is constantly by cause senescence-factor Stimulation, 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 experiment Active peptides PNIMVIQH has certain guarantor to spleen aging of the animal caused by the stimulation by the bad factor with atrophy Shield 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 chemistry Reagent Co., Ltd；Paraformaldehyde, Sinopharm Chemical Reagent Co., Ltd.；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagent Department；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 built At bio tech ltd.

2. experimental method：

(1) Animal Aging model modeling

(2) animal viscera is obtained

(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% group It knits homogenate, under the conditions of 4 DEG C after 4000g centrifugations, takes supernatant, discard precipitation, operated according to kit specification, or set It 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 contains Increase (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-dose Stimulation, the excess injection of D-gal without completely destroy Mice Body in SOD enzyme systems, illustrate in injection cycle, experiment Animal continuously by the stimulation for causing senescence-factor, can lead to the reduction of SOD contents in Different Organs, but same When 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 model Group 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 for Estimate the accumulation situation of animal body lipid peroxide, therefore it follows that animal model group mouse is causing aging model to be formed In 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 from And oxidative damage is caused, occur a large amount of lipid peroxide in liver organization, MDA is as lipid peroxide, in animal The raising of in-vivo content can reflect the reduction of Antioxidant Enzymes vigor in Mice Body from side.And polypeptide gavage group Mouse Liver The significant decrease of dirty MDA contents, illustrate the intake of polypeptide PNIMVIQH can be effectively protected vital tissue organ from it is bad because Son 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 chemistry Reagent Co., Ltd；Paraformaldehyde, Sinopharm Chemical Reagent Co., Ltd.；Sodium chloride, the limited public affairs of Chinese medicines group chemical reagent Department；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..

2. experimental method：

(1) Animal Aging model modeling

(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, and The 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 blood Clearly.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 2006 Publication《Guiding opinion about kind treatment experimental animal》.The mouse spleen won directly is soaked in prepared in advance In 4% paraformaldehyde solution, to fix in the form of it.Paraformaldehyde powder more indissoluble, micro sodium bicarbonate can be added will PH 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 dilutions At 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 mouse 100 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 diluting Ratio 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 Plate Liquid, and ELISA Plate is placed on blotting paper, it is careful to pat, remove surplus liquid.By preheated biotin antiantibody work Make 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's PBS 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 preheated ABC 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 time Impregnate 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 song Line 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. 7 167.31 ± 24.78pg/mL, 4.54 ± 0.66pg/mL, compared to the increase (P that conspicuousness is presented in normal group<0.01), therefore It is considered that due to continuously injecting cause senescence-factor, animal model group mouse is caused aging occur in cell factor level The 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 because The 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 mould Type 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 obtain Inhibition to a certain extent；From the perspective of inflammation, mouse inflammation because of caused by oxidation has obtained effective inhibition；From declining From 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 obtain Control.

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 general Principle is applied in other embodiment without having to go through creative labor.Therefore, the present invention is not limited to the above embodiments, ability Field 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's Within 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

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 1

Pro Asn Ile Met Val Ile Gln His

1 5

<210> 2

<211> 24

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<400> 2

tcctaatatt atggtgattc aaca 24

<210> 3

<211> 326

<212> PRT

<213>Artificial sequence (Artificial Sequence)

<400> 3

Met Lys Lys Asp Asn Cys Thr Ala Met Leu Val Gly Lys Asp Ala Ser

1 5 10 15

Ile Asp Gly Ser Thr Met Ile Ala Arg Asp Glu Asp Gly Tyr Gly Gly

20 25 30

Ile Asn Glu Lys Leu Phe Val Val Asn Lys Ala Arg His Tyr Asp Glu

35 40 45

Asp Tyr Val Ser Lys Tyr Asn Gly Phe Lys Met His Leu Glu Gly Asp

50 55 60

Gly Cys Lys Trp Thr Ala Ala Pro Thr Ala Asp Asp Ser Glu Gly Arg

65 70 75 80

Trp Asp Glu Gln Gly Ile Asn Glu Tyr Asn Val Ala Met Ser Ala Thr

85 90 95

Glu Thr Glu Ala Thr Asn Ala Arg Cys Leu Gly His Asp Pro Leu Val

100 105 110

Glu Asp Gly Ile Asn Glu Asp Ser Met Val Tyr Ile Thr Leu Pro Phe

115 120 125

Val Lys Thr Ala Arg Glu Gly Val Leu Arg Leu Gly Arg Leu Ile Glu

130 135 140

Lys Tyr Gly Thr Gly Glu Thr Asn Gly Ile Ala Phe Ser Asp Asn Lys

145 150 155 160

Glu Val Trp Tyr Leu Glu Thr Gly Ala Gly His Gln Trp Val Ala Ala

165 170 175

Arg Val Pro Asp Asp Ser Tyr Ala Ile Cys Pro Asn Ile Met Val Ile

180 185 190

Gln His Val Asn Phe Asp Asp Pro Asp Asn Phe Met Tyr Ser Glu Gly

195 200 205

Ile Gln Glu Phe Val Glu Lys Asn His Leu Asn Asn Ser Thr Asp Gly

210 215 220

Ser Phe Ser Phe Arg Asp Ile Phe Gly Thr Lys Asp Glu Ala Asp Ala

225 230 235 240

Phe Tyr Asn Thr Pro Arg Thr Trp Tyr Gly Gln Lys Leu Phe Asn Pro

245 250 255

Ser Ile Glu Gln Asp Pro Thr Ser Gln Glu Met Pro Phe Thr Arg Val

260 265 270

Pro Glu Lys Lys Ile Gly Val Glu Asp Val Gln Lys Phe Leu Ser Ser

275 280 285

His Tyr Asn Gly Thr Pro Tyr Asp Pro Met Gly Thr Phe Ser Ser Gly

290 295 300

Ser Glu Lys Glu Gln Lys Met Phe Arg Ser Ile Ala Leu Asp Arg Asn

305 310 315 320

Gln Glu Ser Ser Ile Leu

325

Claims

1. a kind of biologically active polypeptide PNIMVIQH, which is characterized in that its amino acid sequence is Pro-Asn-Ile-Met-Val- Ile-Gln-His。

2. a kind of biologically active polypeptide PNIMVIQH according to claim 1, which is characterized in that the biologically active polypeptide From Lactobacillus helveticus mycoprotein.

3. encoding the nucleotide fragments of biologically active polypeptide PNIMVIQH described in claim 1, which is characterized in that the nucleotide The sequence of segment such as SEQ ID NO：Shown in 2.

4. the preparation method of biologically active polypeptide PNIMVIQH as described in claim 1, which is characterized in that pass through genetic engineering Method is artificial synthesized or Lactobacillus helveticus thalline is directly obtained by the method that clasmatosis isolates and purifies, or directly passing through Be synthetically prepared.

5. the application of biologically active polypeptide PNIMVIQH as described in claim 1, which is characterized in that the biologically active polypeptide Applications of the PNIMVIQH in preparing the food with immunoloregulation function, health products, drug or cosmetics.

6. the application of biologically active polypeptide PNIMVIQH as described in claim 1, which is characterized in that the biologically active polypeptide Applications of the PNIMVIQH in preparing the food with anti-senescence function, health products or drug.

7. the application of biologically active polypeptide PNIMVIQH as described in claim 1, which is characterized in that the biologically active polypeptide Applications of the PNIMVIQH in preparing the food with immunoloregulation function and anti-senescence function, health products or drug.

8. a kind of immunological regulation product, which is characterized in that including biologically active polypeptide PNIMVIQH as described in claim 1 or institute State the derivative of biologically active polypeptide PNIMVIQH；The immunological regulation product includes immunological regulation food, immunological regulation guarantor Strong product, immunoregulation medicament or immunological regulation cosmetics；The derivative of the biologically active polypeptide PNIMVIQH, refers in biology On the amino acid side groups of active peptides PNIMVIQH, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, first Base, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.

9. a kind of anti-aging product, which is characterized in that including biologically active polypeptide PNIMVIQH or described as described in claim 1 The derivative of biologically active polypeptide PNIMVIQH；The anti-aging product includes antisenility cistanche food, antisenescence health product or anti- Senescence drug；The derivative of the biologically active polypeptide PNIMVIQH refers to the amino acid in biologically active polypeptide PNIMVIQH On side-chain radical, aminoterminal or c-terminus carry out hydroxylating, carboxylated, be carbonylated, methylate, acetylation, phosphorylation, esterification or Polypeptide derivative that is glycosylation modified, obtaining.

10. a kind of product with immunoloregulation function and anti-senescence function, which is characterized in that including as described in claim 1 The derivative of biologically active polypeptide PNIMVIQH or described biologically active polypeptides PNIMVIQH；With immunoloregulation function and anti-ageing The product of old function includes food, health products or drug；The derivative of the biologically active polypeptide PNIMVIQH, refers in biology On the amino acid side groups of active peptides PNIMVIQH, aminoterminal or c-terminus carry out hydroxylating, carboxylated, carbonylation, first Base, acetylation, phosphorylation, esterification or glycosylation modified, obtained polypeptide derivative.