CN106916206A - Hard clam polypeptide and preparation method and application - Google Patents
Hard clam polypeptide and preparation method and application Download PDFInfo
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- CN106916206A CN106916206A CN201511009515.9A CN201511009515A CN106916206A CN 106916206 A CN106916206 A CN 106916206A CN 201511009515 A CN201511009515 A CN 201511009515A CN 106916206 A CN106916206 A CN 106916206A
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- Prior art keywords
- mmp13
- polypeptide
- hard clam
- clam polypeptide
- hard
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 92
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 86
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 81
- 238000002360 preparation method Methods 0.000 title claims abstract description 21
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 21
- 230000003712 anti-aging effect Effects 0.000 claims abstract description 21
- 239000003963 antioxidant agent Substances 0.000 claims abstract description 15
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 15
- 235000006708 antioxidants Nutrition 0.000 claims abstract description 15
- 230000036541 health Effects 0.000 claims abstract description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 10
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 9
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 235000019253 formic acid Nutrition 0.000 claims description 9
- 238000005227 gel permeation chromatography Methods 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 7
- 238000001819 mass spectrum Methods 0.000 claims description 7
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- 238000004458 analytical method Methods 0.000 claims description 6
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
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- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
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- 230000009946 DNA mutation Effects 0.000 description 1
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- 108010024636 Glutathione Proteins 0.000 description 1
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- 101000777550 Homo sapiens CCN family member 2 Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000961006 Johnius belangerii Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241001123258 Meretrix meretrix Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010029350 Neurotoxicity Diseases 0.000 description 1
- BPQQTUXANYXVAA-UHFFFAOYSA-N Orthosilicate Chemical compound [O-][Si]([O-])([O-])[O-] BPQQTUXANYXVAA-UHFFFAOYSA-N 0.000 description 1
- 230000010718 Oxidation Activity Effects 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
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- UELITFHSCLAHKR-UHFFFAOYSA-N acibenzolar-S-methyl Chemical compound CSC(=O)C1=CC=CC2=C1SN=N2 UELITFHSCLAHKR-UHFFFAOYSA-N 0.000 description 1
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- 229930013930 alkaloid Natural products 0.000 description 1
- HSFWRNGVRCDJHI-UHFFFAOYSA-N alpha-acetylene Natural products C#C HSFWRNGVRCDJHI-UHFFFAOYSA-N 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 230000006933 amyloid-beta aggregation Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000004900 autophagic degradation Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical compound [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 1
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- FPAFDBFIGPHWGO-UHFFFAOYSA-N dioxosilane;oxomagnesium;hydrate Chemical compound O.[Mg]=O.[Mg]=O.[Mg]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O FPAFDBFIGPHWGO-UHFFFAOYSA-N 0.000 description 1
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- 238000004519 manufacturing process Methods 0.000 description 1
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- 239000003182 parenteral nutrition solution Substances 0.000 description 1
- 230000003617 peroxidasic effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
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- 229920000728 polyester Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
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- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
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- 230000001737 promoting effect Effects 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
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- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43509—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from crustaceans
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Insects & Arthropods (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to field of marine biotechnology, and in particular to hard clam polypeptide and preparation method and application.Hard clam polypeptide of the present invention its there is SEQ ID NO:Amino acid sequence shown in 1.Test result indicate that hard clam polypeptide MmP13 of the present invention and the like is respectively provided with good anti-oxidant, anti-aging effects.The preparation method that the present invention is provided, can make full use of clam affluent resources to obtain with strong anti-oxidation, anti-aging effects hard clam polypeptide MmP13 and the like.Hard clam polypeptide MmP13 of the present invention and the like can be used for being prepared into anti-oxidant, antiaging agent or anti-oxidant, antisenescence health product, have wide range of applications, with good economy and social effect.
Description
Technical field
The invention belongs to field of marine biotechnology, and in particular to hard clam polypeptide and preparation method and application.
Especially hard clam polypeptide MmP13 and the like (MmP13-1, MmP13-2, MmP13-3,
MmP13-4、MmP13-5)。
Background technology
Aging is the inexorable law of human life's process, is also a kind of irresistible natural phenomena, and is worked as
The fact that preceding population in the world aging aggravates, forces human society to be faced with endowment and the anti-aging pressure of arduousness
Power.It is obvious broken that the free radical theory of aging thinks that internal active oxygen and its free radical have to body cell
Bad effect, can cause the extensive injuries of various biological structures, including biomembrane denaturation, chromosome shift,
DNA mutation, disorganization and aging etc., ultimately result in body aging.With the related modern reason of aging
By carrying out anti-aging process in the way of Dietotherapy health, a kind of effective anti-aging strategy of can yet be regarded as.With
The material of food-borne is material base, by suitable extraction, preparation method, find have it is anti-oxidant,
The bioactive ingredients of life-prolonging function, help to develop the related health product of deferring senility.
Traditional medicine is to promoting longevity and neuroprotection has a long research history, and abundant marine organisms
The then provided huge resources bank of resource category.China's marine resources enrich, living marine resources tool
There is the region feature of distinctness, the works that the successive dynasties are handed down is a lot of and describes detailed, is grinding for marine resources
Study carefully and develop there is provided valuable data.Since 20 century 70s, people divide from marine organisms
Separate out tens thousand of kinds of new compounds, including peptides, protein-based, polysaccharide, alkaloids, terpene,
The types such as Macrocyclic polyester class.Wherein, peptides are the most huge classes of quantity in marine biomaterial
Compound, up to as many as tens thousand of kinds, including ocean Peptide toxin and oceanic biological active peptides etc..
Bioactivity peptides have small molecular weight, non-immunogenicity, simple structure, Small side effects, effect
Significantly the advantages of.Further, since marine organisms existence specific environment, the structure of marine organisms polypeptide with
Terrestrial organism peptide (glycopeptide) is very different, mostly small molecule cyclic peptide, containing abundant D types amino acid,
Carboxylic acid, thiophene phenol, oxazole ring, what is had also contains ethylene linkage and acetylene bond, and the biology which greatly enhances peptide is steady
Qualitative and bioavilability.Research show that biologically active peptide has various pharmacological activity, can be used for prevent and
Anti-aging and the related various diseases of aging.Kim etc. cries aunt fish (Johnius belengerii) from Pi Shi
Middle isolating active peptide can significantly inhibit peroxidatic reaction of lipid；The identification such as Shoji-Kawata one kind can be lured
The polypeptide of autophagy is led, can effectively suppress various virulence factors；Martorell etc. is from cocoa (Theobroma
Cacao antioxidation active peptides 13L (DNYDNSAGKWWVT) are separated in), it is possible to reduce A Erci
The silent disease C. Elegans Automatic Screening A β depositions in sea, the potential with prevention and treatment nerve degenerative diseases.Therefore, send out
There is anti-oxidant, anti-aging, the polypeptide active component of neuroprotection in pick living marine resources
Help lend some impetus to the comprehensive exploitation of living marine resources and utilize.
Shellfish is mild-natured, sweet, salty, have functions that enriching yin and nourishing kidney, adjust in.《The new compilation of materia medica》Record
Under shellfish meat in controlled atmosphere, effect that relieving the five internal organs, treatment are quenched one's thirst.Wherein, clam (Meretrix meretrix L.)
Also known as clam, belong to the shellfish of Bivalvia curtain clam mesh Veneridae, be the important seashells resource of China,
It is its delicious meat, nutritious, with dietotherapy very high and medical value.Hard clam polypeptide is ground at present
Study carefully the utilization for being concentrated mainly on its anti-tumor aspect, such as Chinese patent of Publication No. CN102161699A
In disclose one kind through ammonium sulfate precipitation, ultrafiltration, ion-exchange chromatography, hydrophobic chromatography, inversion layer
A kind of molecular weight is obtained after analysis for 15kDa, N- end sequence are IDEIQNTGGGTNFR, have anti-swollen
The hard clam polypeptide of tumor activity, it is applied to prevent and/or treat malignant tumour in field of biological pharmacy.But
Application and report of the current clam polypeptide in terms of anti-oxidant, anti-aging are simultaneously few, therefore, open
Hair will be equal to the prevention and treatment of anti-aging and diseases associated with senescence with the clam active polypeptide for prolonging the effect of declining
It is significant, also will provides foundation for the development and application of hard clam polypeptide class product.
The content of the invention
In view of this, it is many it is an object of the invention to provide the clam with anti-oxidant and anti-aging activity
Peptide and preparation method and application.
To realize the purpose of the present invention, the present invention is adopted the following technical scheme that.
A kind of hard clam polypeptide MmP13, it has SEQ ID NO:Amino acid sequence shown in 1.
The present invention is centrifuged after clam software is homogenized, and takes supernatant drying, and ethanol is extracted, milipore filter cuts
Stay, gel chromatography is separated, the isolated hard clam polypeptide MmP13 of reversed-phase liquid chromatography tandem mass spectrum, its tool
There are SEQ ID NO:Amino acid sequence shown in 1, sequence is specially LSDRLEETGGASS.
Present invention also offers with SEQ ID NO:The hard clam polypeptide of the amino acid sequence shown in 1
Replace, lack or add the ammonia obtained by one or more amino acid residues in the amino acid sequence of MmP13
The hard clam polypeptide MmP13 analogs of base acid sequence.Described 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, missing are added, can be with
Carried out in any site of amino acid sequence, as long as the polypeptide with improved amino acid sequence has resisting
Oxidation and anti-aging activity.Similar, the number of the amino acid for replacing, lacking or adding
It is arbitrary, as long as the polypeptide with improved amino acid sequence has anti-oxidant and anti-aging activity.
Wherein, in some embodiments, the hard clam polypeptide MmP13 analogs, it has SEQ ID
NO:Amino acid sequence shown in 2, sequence is specially QLSDRLEETGGASS, is named as MmP13-1.
In some embodiments, the hard clam polypeptide MmP13 analogs, it has SEQ ID NO:3
Shown amino acid sequence, sequence is specially IEQLSDRLEETGGASS, is named as MmP13-2.
In some embodiments, the hard clam polypeptide MmP13 analogs, it has SEQ ID NO:4
Shown amino acid sequence, sequence is specially QIEQLSDRLEETGGASS, is named as MmP13-3.
In some embodiments, the hard clam polypeptide MmP13 analogs, it has SEQ ID NO:5
Shown amino acid sequence, sequence is specially LSDRLEETGGASSIQHE, is named as MmP13-4.
In some embodiments, the hard clam polypeptide MmP13 analogs, it has SEQ ID NO:6
Shown amino acid sequence, sequence is specially QIEQLSDRLEETGGASSIQHE, is named as
MmP13-5。
Divide present invention provides the DNA for encoding hard clam polypeptide MmP13 of the present invention and the like
Son.Due to the degeneracy of codon, there may be many kinds can encode particular polypeptide of the present invention
Nucleotide sequence.For DNA points that encodes hard clam polypeptide MmP13 of the present invention and the like
Son, those skilled in the art can easily using existing known method manufacture synthesis.Such as, lead to
The codon that selection corresponds to the amino acid residue of the amino acid sequence described in constituting is crossed, can be easily true
DNA molecular of the fixed and offer corresponding to hard clam polypeptide MmP13 and the like the amino acid sequence of polypeptide.
Present invention also offers the preparation method of the hard clam polypeptide, it is centrifuged after the homogenate of clam software, is taken
Clear to dry, ethanol extracts the molten polypeptide of alcohol, is that 3kDa milipore filters are retained with molecular cut off, collects molecule
Amount<The ultrafiltration component of 3kDa, after gel tswett's chromatography methods is separated, reversed-phase liquid chromatography tandem mass spectrum is separated.
Preferably, being centrifuged in described preparation method, after the homogenate as 5000g~15000g is centrifuged
10min~50min.
Preferably, in described preparation method, the ethanol is extracted as the extraction of the ethanol of addition 60%~80%,
Extraction time is 12~24h.
Preferably, in described preparation method, the gel chromatography is separated and is specially Sephadex G-25
Gel chromatography column to MW<3kDa polypeptide fractions are separated, with the loading volume and 20 of 500 μ L
The concentration of mg/mL carries out inserting needle sample-adding, with water as mobile phase, the speed of 1mL/min eluted, it is purple
Light absorption value of the eluent of external detector detection Each point in time at 280nm, collects in 200min
Elution fraction.
Preferably, in described preparation method, reversed-phase liquid chromatography tandem mass spectrum is separated to be specially and takes receipts
Elution fraction in the 200min of collection, is dissolved in formic acid containing 0.1v/v%, the aqueous solution of 2v/v% acetonitriles
In, nanoliter liquid chromatography-mass spectrometry；Described nanoliter of liquid chromatography-mass spectrometry be nanoliter
The trapping column sample injection of liquid chromatogram, with 0.1v/v% formic acid, 2v/v% acetonitriles the aqueous solution with 2.5
The flow velocity wash-out 10min of μ L/min, is then carried out with the acetonitrile solution of 0.1v/v% formic acid, 2v/v% water
Gradient elution, liquid phase gradient be 50min in B liquid from 5~35%, analytical column is reverse-phase chromatographic column,
Carry out scanning of the mass spectrum analysis simultaneously.The analytical column can be C18 reverse-phase chromatographic columns, and specification is 75 μ ms
15cm, 3 μm,
Paraquat is an electron-like acceptor, acts on the redox reaction of cell, and activation in the cell is
Oxygen radical is the basis of toxic action, the excessive super oxide anion free radical and hydrogen peroxide for being formed
Etc. (H2O2) many tissue organ cell's membrane lipid peroxidatios can be caused, so as to cause multisystem histoorgan
Infringement.Therefore, suppressing its oxidative damage to cell can effectively alleviate neurotoxicity.The present invention will
The hard clam polypeptide MmP13 is added in the C. Elegans Automatic Screening nutrient solution of paraquat modeling, regularly counts beautiful line
The ratio of worm survival, detects the influence of the oxidative stress that the hard clam polypeptide MmP13 is induced paraquat,
Result shows that hard clam polypeptide MmP13 of the present invention has significant antioxidation.
Life-span, as most reliable, the most representational measurement index of aging, can have rated from integral level
Bion or the aging degree of colony.The present invention is by described hard clam polypeptide MmP13 and the like
(MmP13-1, MmP13-2, MmP13-3, MmP13-4, MmP13-5) is added to the adult initial stage
Wild-type C nematode in cultivate after, timing counts the survival condition of C. Elegans Automatic Screening, detects the clam
Influences of the polypeptide MmP13 and the like to the C. Elegans Automatic Screening life-span.Result shows, clam of the present invention
Polypeptide MmP13 and the like can extend the C. Elegans Automatic Screening time-to-live under field conditions (factors), with prolonging
The effect of slow senescence process.
Therefore, the invention provides described hard clam polypeptide MmP13 and the like (MmP13-1,
MmP13-2, MmP13-3, MmP13-4, MmP13-5) preparing anti-oxidant, anti-aging medicine
In application.
Those skilled in the art can be by hard clam polypeptide MmP13 of the present invention and the like
(MmP13-1, MmP13-2, MmP13-3, MmP13-4, MmP13-5) is directly or indirectly added
Prepare pharmaceutically acceptable various conventional auxiliary materials required during different dosage forms, such as filler, disintegrant,
Lubricant, adhesive etc., in traditional drug formulations method, be made common dosage forms for example tablet, capsule,
Parenteral solution, oral liquid, granule, pill, powder and pill etc..Wherein, filler such as starch,
Lactose, sucrose, glucose, mannitol and silicic acid；Disintegrant such as agar, calcium carbonate, potato starch or
Tapioca, alginic acid, some silicate and sodium carbonate, low-substituted hydroxypropyl cellulose；Lubricant is such as
Talcum powder, calcium stearate, magnesium stearate, solid polyethylene glycol, Sodium Laurylsulfate；Adhesive such as carboxylic first
Base cellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and Arabic gum.
Hard clam polypeptide MmP13 of the present invention and the like (MmP13-1, MmP13-2, MmP13-3,
MmP13-4, MmP13-5) anti-oxidant, anti-aging health products can also be prepared into.
As shown from the above technical solution, the invention provides hard clam polypeptide and preparation method and application.This
It has SEQ ID NO to invent the hard clam polypeptide:Amino acid sequence shown in 1.Test result indicate that this hair
Bright described hard clam polypeptide MmP13 and the like is respectively provided with good anti-oxidant, anti-aging effects.This hair
The preparation method of bright offer, can make full use of clam affluent resources to obtain with strong anti-oxidation, anti-aging
Effect hard clam polypeptide MmP13 and the like.Hard clam polypeptide MmP13 of the present invention and the like can
For being prepared into anti-oxidant, antiaging agent or anti-oxidant, antisenescence health product, have a wide range of application
It is general, with good economy and social effect.
Brief description of the drawings
In order to illustrate more clearly about the embodiment of the present invention or technical scheme of the prior art, below will be to reality
The accompanying drawing to be used needed for example or description of the prior art is applied to be briefly described.
Fig. 1 shows the gel chromatography separation chromatography figure of embodiment 1；
Fig. 2 shows the high-efficient liquid phase chromatogram of hard clam polypeptide MmP13；
Fig. 3 shows the mass spectrogram of hard clam polypeptide MmP13；
Fig. 4 show the resistant to paraquat of embodiment 2 induction oxidative stress experiment in hard clam polypeptide MmP13 to beautiful line
The survivorship curve of worm survival rate；
Fig. 5 show the C. Elegans Automatic Screening life experiment of embodiment 3 in hard clam polypeptide MmP13 to C. Elegans Automatic Screening survival rate shadow
Loud survivorship curve；
Fig. 6 show the C. Elegans Automatic Screening life experiment of embodiment 3 in hard clam polypeptide MmP13-1 to C. Elegans Automatic Screening survival rate
The survivorship curve of influence；
Fig. 7 show the C. Elegans Automatic Screening life experiment of embodiment 3 in hard clam polypeptide MmP13-2 to C. Elegans Automatic Screening survival rate
The survivorship curve of influence；
Fig. 8 show the C. Elegans Automatic Screening life experiment of embodiment 3 in hard clam polypeptide MmP13-3 to C. Elegans Automatic Screening survival rate
The survivorship curve of influence；
Fig. 9 show the C. Elegans Automatic Screening life experiment of embodiment 3 in hard clam polypeptide MmP13-4 to C. Elegans Automatic Screening survival rate
The survivorship curve of influence；
Hard clam polypeptide MmP13-5 is survived to C. Elegans Automatic Screening during Figure 10 shows the C. Elegans Automatic Screening life experiment of embodiment 3
The survivorship curve of rate influence.
Specific embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is carried out clear, complete
Site preparation is described, it is clear that described embodiment is only a part of embodiment of the invention, rather than whole
Embodiment.Based on the embodiment in the present invention, those of ordinary skill in the art are not making creativeness
The every other embodiment obtained under the premise of work, belongs to the scope of protection of the invention.
For a further understanding of the present invention, the present invention is elaborated with reference to specific embodiment.
The preparation of embodiment 1, hard clam polypeptide MmP13
Fresh clam carries out treatment of shelling, and silt is washed 3~4 times with water successively cleaning, and clam is soft
Body is fully homogenized, and homogenate is centrifuged 10min through 5000g, takes supernatant vacuum freeze drying, is added
60~80% ethanol extract 24h, 10000g centrifugation 30min, and it is 3kDa to take supernatant molecular cut off
After milipore filter retention, MW is obtained<The ultrafiltration component of 3kDa.
Using the gel chromatography column (1.5cm × 1.2m, Bio-Rad company) of Sephadex G-25 to MW
<The polypeptide fractions of 3kDa carry out gel chromatography separation.With the loading volume of 500 μ L and 20mg/mL
Concentration carries out inserting needle sample-adding, with liquid phase constant flow pump as power, one-level water as mobile phase, the speed of 1mL/min
Degree is eluted, and detects the eluent of Each point in time at 280nm by UV UV-detectors
Light absorption value.Result is shown in Fig. 1
By the visible MW of Fig. 1 results<The polypeptide fractions of 3kDa are main to obtain after being separated through gel chromatography
To 5 components, respectively F1, F2, F3, F4 and F5.
F1 components are taken to be dissolved in formic acid containing 0.1v/v%, in the aqueous solution of 2v/v% acetonitriles, carry out online
Nanoliter liquid chromatography mass combination analysis, nanoliter liquid chromatogram is the Eksigent of American AB SCIEX companies
nanoLC-UltraTMTwo-dimentional system, mass spectrum is the systems of TripleTOF 5600.In the trapping of nanoliter flow
Post (3 μm,) on sample introduction, kinetic pump with 0.1v/v% formic acid, the aqueous solution of 2v/v% acetonitriles,
Flow velocity carries out desalination 10min for 2.5 μ L/min, then with 0.1v/v% formic acid, the acetonitrile of 2v/v% water
Solution carries out gradient elution, liquid phase gradient be in 50min B liquid from 5~35%, analytical column is 75 μ
M × 15cm, C18-3 μm,Scanning of the mass spectrum analysis is carried out simultaneously, as a result such as table 1.
The hard clam polypeptide sequence of table 1
| Polypeptide title | Amino acid sequence | Sequence number |
| MmP13 | SEQ ID NO:1 | |
| MmP13-1 | SEQ ID NO:2 | |
| MmP13-2 | SEQ ID NO:3 | |
| MmP13-3 | SEQ ID NO:4 | |
| MmP13-4 | SEQ ID NO:5 | |
| MmP13-5 | SEQ ID NO:6 |
The oxidative stress experiment of embodiment 2, resistant to paraquat induction
Hard clam polypeptide MmP13 prepared by embodiment 1 is 0.5mM, 1mM, 2mM according to concentration
It is separately added into the C. Elegans Automatic Screening nutrient solution of 70mM paraquat modelings with 4mM, positive controls are added
The reduced glutathione (GSH) of 2mM makees positive tested material, at the same blank control group add it is isometric
S Medium, are placed in 20 DEG C of cultures, and a C. Elegans Automatic Screening survival rate is counted every about 12h, until all
Insect is dead.Survival rate is represented with survivorship curve, using Kaplan-meier method com-parison and analysis hard clam polypeptides
The otherness of MmP13 and the like groups and control group.Result is as shown in Figure 4.
Result shows that hard clam polypeptide MmP13 concentration prepared by embodiment 1 is 0.5mM, 1mM, 2mM
It is respectively provided with significant antioxidation during with 4mM, and antioxygen better than the GSH under comparable sodium is turned into
With.
Embodiment 3, life experiment
Hard clam polypeptide MmP13 prepared by embodiment 1 according to 1mM, 2mM and 4mM concentration,
And the analog MmP13-1 of the hard clam polypeptide MmP13 for preparing embodiment 1, MmP13-2,
MmP13-3, MmP13-4, MmP13-5 are added separately to the open country at adult initial stage with the concentration of 4mM
In raw type C. Elegans Automatic Screening, control group adds isometric S Medium, is placed in 20 DEG C and persistently cultivates, and
A survival condition for each group C. Elegans Automatic Screening was counted every 1 day under microscope, until all C. Elegans Automatic Screenings are dead
Die.The survival rate of statistical disposition each group nematode, and result is represented with survivorship curve, using Kaplan-meier
The otherness of method com-parison and analysis hard clam polypeptide MmP13 and the like and control group.Result such as Fig. 5-10
It is shown.
Result shows that hard clam polypeptide MmP13 prepared by embodiment 1 is 1mM, 2mM and 4mM in concentration
When, the C. Elegans Automatic Screening mean survival time under field conditions (factors) can be extended, and presented in concentration range
Significant concentration dependent, and with the effect of lengthening the life of concentration 4mM be optimal；Simultaneously, it has been found that with most
Good concentration also can for MmP13-1, MmP13-2, MmP13-3, MmP13-4, MmP13-5 of 4mM
In enough significantly extension C. Elegans Automatic Screenings life-span under field conditions (factors), show hard clam polypeptide MmP13 of the present invention
And the like the effect with anti-aging process.
Claims (10)
1. a kind of hard clam polypeptide, it is characterised in that it has SEQ ID NO:Amino acid sequence shown in 1.
2. have and replace, lack or add by the amino acid sequence of hard clam polypeptide described in claim 1
Plus the hard clam polypeptide of the amino acid sequence obtained by one or more amino acid residues.
3. hard clam polypeptide according to claim 2, it is characterised in that with SEQ ID NO:2~6 it
Amino acid sequence shown in one.
4. the preparation method of hard clam polypeptide described in claim 1, it is characterised in that after the homogenate of clam software
Centrifugation, takes supernatant drying, and ethanol extracts the molten polypeptide of alcohol, is that 3kDa milipore filters are retained with molecular cut off,
Collect molecular weight<The ultrafiltration component of 3kDa, after gel tswett's chromatography methods is separated, reversed-phase liquid chromatography series connection matter
Spectrum is separated.
5. preparation method according to claim 4, it is characterised in that centrifugation is 5000 after the homogenate
G~15000g is centrifuged 10min~50min.
6. preparation method according to claim 4, it is characterised in that the ethanol is extracted as adding
60%~80% ethanol is extracted.
7. preparation method according to claim 4, it is characterised in that the gel chromatography is separated
Specially the gel chromatography column of Sephadex G-25 is to MW<3kDa hard clam polypeptide components are separated,
Inserting needle sample-adding is carried out with the concentration of the loading volume of 500 μ L and 20mg/mL, with water as mobile phase, 1
The speed of mL/min is eluted, and the eluent of UV-detector detection Each point in time is at 280nm
Light absorption value, collect 200min in elution fraction.
8. preparation method according to claim 4, it is characterised in that reversed-phase liquid chromatography is connected matter
Spectrum separates the elution fraction for being specially and taking in the 200min of collection, is dissolved in formic acid containing 0.1v/v%, 2
In the aqueous solution of v/v% acetonitriles, nanoliter liquid chromatography-mass spectrometry；Described nanoliter of liquid chromatogram-matter
Spectrum combination analysis are the trapping column sample injection in nanoliter liquid chromatogram, with 0.1v/v% formic acid, 2v/v% second
The aqueous solution of nitrile elutes 10min with the flow velocity of 2.5 μ L/min, then with 0.1v/v% formic acid, 2v/v%
The acetonitrile solution of water carries out gradient elution, and liquid phase gradient is that B liquid, from 5~35%, divides in 50min
Analysis post is reverse-phase chromatographic column, while carrying out scanning of the mass spectrum analysis.
9. hard clam polypeptide described in claims 1 to 3 any one is in anti-oxidant, anti-aging medicine is prepared
Application.
10. hard clam polypeptide described in claims 1 to 3 any one is preparing anti-oxidant, anti-aging health care
Application in product.
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| CN201511009515.9A CN106916206A (en) | 2015-12-25 | 2015-12-25 | Hard clam polypeptide and preparation method and application |
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| CN108295018A (en) * | 2018-02-28 | 2018-07-20 | 磐安县派普特生物科技有限公司 | The preparation method of facial mask is retained containing clam active polypeptide |
| CN110639228A (en) * | 2019-09-27 | 2020-01-03 | 泗县瑞星精密机械有限公司 | Biomaterial extraction system based on homogenate device |
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| CN108295018A (en) * | 2018-02-28 | 2018-07-20 | 磐安县派普特生物科技有限公司 | The preparation method of facial mask is retained containing clam active polypeptide |
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