CN104087564B - Application of the slow slow-witted peptide in phosphatidylserine and treatment the nervous system disease is prepared - Google Patents
Application of the slow slow-witted peptide in phosphatidylserine and treatment the nervous system disease is prepared Download PDFInfo
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- CN104087564B CN104087564B CN201410316859.3A CN201410316859A CN104087564B CN 104087564 B CN104087564 B CN 104087564B CN 201410316859 A CN201410316859 A CN 201410316859A CN 104087564 B CN104087564 B CN 104087564B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1288—Transferases for other substituted phosphate groups (2.7.8)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
- C12P13/06—Alanine; Leucine; Isoleucine; Serine; Homoserine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
Application in health medicine the invention discloses slow slow-witted peptide of one kind and preparation method thereof and its application in phosphatidylserine (Phosphatidylserine, PS) is prepared and its pivot nervous system disease in the treatment including mental.The present invention describes slow slow-witted peptide and its encodes the polynucleotide sequence of the polypeptide.The preparation method of the polypeptide and polynucleotides is illustrated simultaneously；And application with regard to it in phosphatidylserine is prepared and its function in regulation nerve immunity system, the functional study such as the effect of antagonism Alzheimer disease (AD) done the statement of science.The polypeptide and polynucleotides pivot nervous system disease in the treatment are significantly specified, the huge DEVELOPMENT PROSPECT such as including the application in mental health medicine.
Description
The present invention describes slow slow-witted peptide and its encodes the polynucleotide sequence of the polypeptide.Illustrate the albumen simultaneously many
Peptide and polynucleotides answering in phosphatidylserine (Phosphatidylserine, PS) and treatment the nervous system disease is prepared
With the statement for waiting functional study to do science.Before significantly specifying the huge exploitation of the polypeptide and polynucleotides
Scape.
Technical field art of the present invention includes herein below.
1) molecular biology category.
2) protein molecular research field.
3) immunologic function research field.
4) disease treatment medical research field.
Background technology
1) molecular biology：The main body of the application is slow slow-witted peptide and its polynucleotide sequence for encoding the polypeptide.And
The protein gene is that mRNA is extracted from Daudi cells, and clipped, splicing allosteric, clone is obtained without film combination position
And signal peptide, the slow slow-witted peptide cDNA sequence of 199 amino acid of codified.
2) protein molecular research：Establishing stabilization expression simultaneously has enhanced green fluorescence protein and redgreen fluorescin
Fusion protein Chinese hamster ovary celI strain, high-volume extract albumen, detect its hydrolysis and phosphatidyl transfer activity, and carry out in vivo
The localization of medication, so as to be laid the foundation further to study the biocatalysis and its drug effect of slow slow-witted peptide.
3) biocatalytic Activity research：
Slow slow-witted peptide is also a kind of organized enzyme, and its substrate has phosphatidylinositols (phosph-ateidylinasito1), phosphatide
Acyl monoethanolamine (phosphatidylethanolamine) etc., but its main substrate is phosphatid ylcholine
(phosphatidylcholine, PC).In physiological conditions, delay slow-witted hydrolase polypeptide PC and produce PA and choline；On the other hand, PLD exists
In the presence of having a suitable alcoholic acceptor, moreover it is possible to be catalyzed the Binding Capacity of various hydroxyls to the base of phosphatide, form new
Phosphatide.This characteristic be transacylate activity, the reaction be called turn phosphatidyl reaction (transphosphat-idylation
Reaction) or Baseexchange reaction (base exchange).In the presence of short chain primary alcohol, PLD be preferentially catalyzed PC with
Short chain primary alcohol turns phosphatidyl (transphosphatidyIation) reaction, produces the phosphatidyl alcohol of stabilization
And water (phasphatidyIaleoho1).Turn the specific reaction that phosphatidyl reaction is detection PLD activity at present, many is ground
Study carefully the generation of the physiological product PA for also delaying slow-witted peptide using this response inhabitation.
4) research of anti-inflammatory and neu- roimmunomodulation function：
AD models are set up by with A β 1-40 intracerebral injections, and it is various using SABC, Nissl's staining, ELISA etc.
Laboratory facilities, we demonstrate that slow slow-witted peptide not only intracerebral injection without overt toxicity, and and as PS to Alzheimer disease
(AD) intervene effective.
Because can migrate to cortex, thalamus after slow slow-witted peptide injection intracerebral, small part stays in hippocampus, melts by with cell membrane
Conjunction is positioned at surface of cell membrane, and the mitigation effect similar with PS intervention effects is played to AD.Another slow slow-witted peptide not only can be to AD's
Behaviouristics changes and plays mitigation effect, moreover it is possible to suppress the pathological changes such as neuron loss, centrum fault rupture in the AD courses of disease, to god
Played a protective role through unit.Slow slow-witted peptide can also suppress T lymphocytes to the infiltration in AD model brain parenchyms and intracerebral TGF-β
1 secretion；And increasing for peripheral blood Th1 type cells can be suppressed；Especially our experiment also confirms that slow slow-witted peptide can first
Suppress the reduction of Treg cells in AD models, promote the effect of Foxp3 expression.
The mechanism of slow slow-witted peptide antagonism AD obtains PS to play its transacylate active catalytic, and then plays anti-AD by PS
Effect；By delaying the neuro-protective function that slow-witted peptide has in itself, the survival rate of neuron is improved；It is also possible to by suppressing
The release of the inflammatory mediators such as TGF-β 1, IFN-γ and promote the function such as increase of Treg cells, play the effect of antagonism AD.
The content of the invention
(1) application of slow-witted peptide is delayed：The main body of the application is that slow slow-witted peptide has phosphatidyl transfer activity, can be catalyzed soybean
Lecithin (PC) generates phosphatidylserine (PS) product；Spike not only can be in vivo positioned, there can also be regulation nerve immunity
The function of system, reaches the effect of antagonism Alzheimer disease (AD).
(2) feature of invention
1st, the feature of slow-witted peptide is delayed：
1) it is a kind of tasteless white or flaxen powder that this research department has purified dry slow slow-witted peptide protein product.Compared with
Soluble in water, its solubility is at normal temperatures 88-100%.Slow slow-witted peptide molecular weight of albumen is 48KD.
The transacylate activity that fusion protein delays slow-witted peptide is：20-60U/mg.Its BA effective range is about in 0.5ng/
ml—150mg/ml.Optimum range：3.0ng/ml—80μg/ml,2mg/ml-10mg/ml.
2) its coding gene sequence is without film combination position and signal peptide, the slow slow-witted peptide of 199 amino acid of codified
CDNA sequence, total length 605bp.Refer to SEQ ID NO:1.
3) amino acid polypeptide sequence of slow-witted peptide, 199 amino acid of total length are delayed.Refer to SEQ ID NO:2.
2nd, the application characteristic of slow-witted peptide is delayed
1) the present inventor has recombinantly expressed the slow slow-witted peptide with greater activity by gene expressing.Now it is verified by experiments the thing
Matter has hydrolysing activity, and with phosphatidyl transfer activity.Using slow slow-witted peptide catalysis soybean lecithin and Serine
Prepare phosphatidylserine (Phosphatidylserine, PS).
2) the present inventor is shown by lot of experimental data：Slow slow-witted peptide is respectively provided with antagonism Alzheimer disease as PS
(AD) effect.
The purpose of the present invention：
1. separate new slow slow-witted galanin peptide is provided, and it is included：Polypeptide with SEQ ID N0.2 amino acid sequences or
Its examples of conservative variations, bioactive fragment, analog or derivative.
2. the polynucleotides for encoding the polypeptide are provided.It includes a kind of nucleotide sequence or its variant being selected from the group：
The polynucleotides of polypeptide of (a) coding with SEQ ID N0.2 amino acid sequences；
(b) polynucleotides complementary with polynucleotides (a)；
C () has the polynucleotides of at least 88% homogeny with the polynucleotide sequence of (a) or (b).
I.e. the invention provides separate nucleic acid (polynucleotides), there is SEQ ID N0.2 amino acid sequences by coding substantially
The polynucleotides composition of the polypeptide of row.Polynucleotide sequence of the invention includes the nucleotide sequence of SEQ ID N0.1.
3. polynucleotides of the invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA
Or artificial synthesized DNA.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.Encoding mature
The coding region sequence of polypeptide can variant identical with the coding region sequence shown in SEQ ID N0.1 or degeneracy.Coding
The polynucleotides of the mature polypeptide of SEQ ID N0.2 include：The only coded sequence of mature polypeptide；The coded sequence of mature polypeptide
With various additional coding sequences；The coded sequence (and optional additional coding sequence) and non-coding sequence of mature polypeptide.
4., the invention further relates to the variant of foregoing description polynucleotides, its coding has identical amino acid sequence with the present invention
The polypeptide of row or the segment of polypeptide, analogs and derivatives.The variant of this polynucleotides can be that the natural equipotential for occurring becomes
The variant that allosome or non-natural occur.These nucleotide variants include substitution variants, Deletion variants and insertion variation
Body.As known in the art, allelic variant is an alternative forms for polynucleotides, and it is probably one or more nucleotides
Substitution, missing or insert, but will not from substantially change its coding polypeptide function.
5. slow-witted peptide is delayed one by one the invention provides a kind of new polypeptide, and it is substantially the ammonia as shown in SEQ ID N0.2
Base acid sequence composition.Polypeptide of the invention can be native purified product, or chemical synthesis product, or use restructuring
Technology is produced from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell).According to weight
Host used by group production decision, polypeptide of the invention can be glycosylated, or can be nonglycosylated.It is of the invention many
Peptide may also include or not include the methionine residues of starting.
6. the recombinant vector of the polynucleotides containing the slow slow-witted peptide of coding is provided.Particularly expression vector；In the present invention, coding
The polynucleotide sequence of slow slow-witted peptide can be plugged into carrier, to constitute the recombinant vector containing polynucleotides of the present invention.Bag
Include bacterial plasmid well known in the art, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus,
Retrovirus or other carriers.
Persons skilled in the art are aware that how to select appropriate carrier/transcriptional regulatory element (such as promoter, enhancing
Son etc.) and selected marker.
7. the genetically engineered host cell of the polynucleotides containing the slow slow-witted peptide of coding is provided.In the present invention, delay slow-witted peptide
Polynucleotides or the recombinant vector containing the polynucleotides can be transformed or transduced into host cell, and the polynucleotides are contained to constitute
Or the genetically engineered host cell of recombinant vector.Term " host cell " refers to prokaryotic, such as bacterial cell；Or it is low true
Nucleus, such as yeast cells；Or higher eucaryotic cells, such as mammalian cell.Representative example has：Escherichia coli, strepto-
Pseudomonas；Bacterial cell such as salmonella typhimurium；Fungal cell's such as yeast；Plant cell；Insect cell such as fruit bat S2 or Sf9；
Zooblast such as CHO, COS or Bowes melanoma cells etc..
8. the method that the slow slow-witted peptide of production is provided.With DNA sequence dna of the present invention or the restructuring containing the DNA sequence dna carry
Body conversion host cell can be carried out with routine techniques well known to those skilled in the art.When host is prokaryotes such as Escherichia coli
When, the competent cell that can absorb DNA can be harvested after exponential phase of growth, use CaCl2Method treatment, step used is in this area
It is known that.Alternative is to use MgCl2.If desired, conversion can also be carried out with the method for electroporation.When host is eucaryon
Biology, can select following DNA transfection methods：Calcium phosphate precipitation, or conventional mechanical methods such as microinjection, electricity is worn
Hole, liposome packaging etc..
By conventional recombinant DNA technology, can be used to express or produce restructuring using polynucleotide sequence of the invention
Slow slow-witted peptide.In general there are following steps：
(1) with the polynucleotides (or variant) of the slow slow-witted peptide of coding of the invention, or with the restructuring containing the polynucleotides
Expression vector is converted or suitable host cell of transduceing；(2) host cell is cultivated in suitable culture medium；(3) from culture medium
Or in cell separate, protein purification.
In step (2), according to host cell used, culture medium used may be selected from various cellar cultures in culture
Base.Cultivated under conditions of host cell growth is suitable to.After host cell growth is to appropriate cell density, with suitable
Method (such as temperature transition or chemical induction) induce the promoter of selection, cell is further cultured for a period of time.
In step (3), recombinant polypeptide can be coated in intracellular or express or be secreted into extracellular on cell membrane.Such as
Fruit need, can utilize its physics, chemistry and other characteristics be separated by various separation methods and purification of Recombinant albumen.This
A little methods are well-known to those skilled in the art.These methods are included but is not limited to：Conventional renaturation process, albumen precipitation
Agent treatment (salting-out method), centrifugation, infiltration broken bacterium, ultrasonication, ultracentrifugation, sieve chromatography (gel filtration), adsorption layer
Analysis, ion-exchange chromatography, the combination of high performance liquid chroma- tography (HPLc) and other various LC technologies and these methods.
9. the special polynucleotide sequence of the slow slow-witted peptide of coding of the invention can be obtained with various methods.For example, using ability
Hybridization technique known to domain separates polynucleotides.These technologies are included but is not limited to：1) with probe and genome or cDNA texts
Storehouse hybridization detecting homologous polynucleotide sequence, and 2) antibody screening of expression library detecting with structural features
The polynucleotide passage of clone.
10. the antibody for slow slow-witted peptide of the invention is provided, and is related to a kind of to be combined with polypeptid specificity of the present invention
Antibody.
11. application the present invention relates to polypeptide of the invention or polynucleotides in phosphatidylserine is prepared.That is this hair
It is bright to digest technological process and technology that soybean lecithin produces phosphatidylserine with slow slow-witted peptide there is provided a kind of.
12. can by polypeptide of the invention, polynucleotides and its analogies, activator, antagonist ＆ inhibitor with it is suitable
Pharmaceutical carrier combination after use.These carriers can be water, glucose, ethanol, salt, buffer solution, glycerine and they
Combination.Polypeptide of the composition comprising safe and effective amount or antagonist and the carrier and excipient of effect of drugs are not influenceed.These
Composition can be used for central nervous system disease (Alzheimer disease as medicine：AD preventing and treating), including mental health care
Deng.
Other side of the invention, due to the disclosure of the technology of this paper, is to those skilled in the art apparent
's.
Brief description of the drawings
Drawings below is used to illustrate specific embodiments of the present invention, rather than limits what is be defined by the claims
The scope of the invention.
Fig. 1 is the PCR primer gel electrophoresis figure of the slow slow-witted peptide of the present invention.
Fig. 2 is the Western blot detection figures of slow slow-witted peptide albumen.The molecular weight of protein is 48kDa.
Fig. 3 is that the present invention delays slow-witted peptide and the amino acid sequence homology of human phosphatidase compares figure.FROM NET are slow slow-witted peptides.
The slow slow-witted peptides of Fig. 4 are catalyzed the thin-layer chromatogram of soybean lecithin (PC) generation phosphatidylserine (PS)
Swimming lane 1,2 is standard PS, and PS spots can clearly show before and after exposure
Swimming lane 3,4 is soybean lecithin (PC), and PC spots show clearly
Swimming lane 5,6,7 is to be catalyzed the reaction product after soybean lecithin (PC) and Serine with slow slow-witted peptide, with
Visible clearly spot in the same level of standard PS sites, illustrates to contain PS in reaction product.
Specific embodiment
Case study on implementation
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part such as sanlbrook et al., molecular cloning：Laboratory manual (New York：cold Spring Harbor Laboratory
Pres s, 1989) described in condition, or according to the condition proposed by manufacturer.
Embodiment 1:The clone of slow slow-witted peptide
Clone's series of slow slow-witted peptide：The need for research and application, we construct a series of slow slow-witted peptide clone.
The known cDNA sequence announced according to GenBank carries out design of primers with synthesis.Contain HKD1 structural genes
PLD2The upstream and downstream primer of cDNA is：
Clone 1:1F 5'-CG AAG CTT GT GTC CGT GTG TCT ATT CTG-3'(SEQ ID N0.3)；1R
5'-GC GAT ATC TCC AAG GTC AGT CAG TCG G-3'(SEQ ID N0.4)；Expected amplified fragments 248bp long.
Clone 2:Upstream and downstream primer containing HKD2 structural genes cDNA is：2F：5'-CG GAT ATC TCA ATC CTG
CAT CGC CT-3'(SEQ ID N0.5)；2R：5'-CG GGT ACC GGC CAG CTC ACT GTC CCG-3'(SEQ ID
N0.6)；Expected amplified fragments 219bp long.
Clone 3:Upstream and downstream primer containing CT structural genes cDNA is 3F：5'-CG GGT ACC TTG GCT CGG
TCT GAG CTC-3'(SEQ ID N0.7)；3R：5'-CG GGA TCC CTA TGT CCA CAC TTC TAG GG-3'
(SEQ ID N0.8)；Expected amplified fragments 132bp long.
Clone 4:The slow slow-witted peptide -6His of pEGFP-C1-
Delay the sequencer address of slow-witted peptide according to recombinant plasmid pEGFP-, commission Shanghai English fine horse biotechnology services limited public affairs
Department carries out the design and synthesis of primer.6 sequences of encoding histidine, upstream and downstream primer are continually introduced at former anti-sense primer 3 ' end
Corresponding restriction enzyme site (the restriction enzyme sites of upstream Hind III are introduced respectively；Downstream BamHI restriction enzyme sites)
And protection base.Primer sequence is as follows：
Sense primer is：F 5′-CG AAG CTT GT GTC CGT GTG TCT ATT CTG-3′(SEQ ID N0.9)
Anti-sense primer is：R 5′-CG GGA TCC CTA ATG ATG ATG ATG ATG ATG TGT CCA CAC
TTC TAG GG-3′(SEQ ID N0.10)
Primer sterilizing distilled water is dissolved, every primer concentration is 10 μM, and -20 DEG C save backup.And introduce respectively
Hind III, EcoR V, KpnI, BamH I restriction endonuclease sites.
Clone 5:The slow slow-witted peptide -6His of recombinant plasmid pcDNA3.1-
Specific steps reference《Molecular cloning》.Determined dna sequence shows:The DNA sequence dna of PCR primer and SEQ ID NO.1
1-605bp it is identical.(see Fig. 1)
The primer sequence of table 1.RT-PCR
Primer1：5'-CG AAG CTT GT GTC CGT GTG TCT ATT CTG-3'(SEQ ID NO:3)
Primer2：5'-GC GAT ATC TCC AAG GTC AGT CAG TCG G-3'(SEQ ID NO:4)
Primer3：5'-CG GAT ATC TCA ATC CTG CAT CGC CT-3'(SEQ ID NO:5)
Primer4：5'-CG GGT ACC GGC CAG CTC ACT GTC CCG-3'(SEQ ID NO:6)
Primer5：5'-CG GGT ACC TTG GCT CGG TCT GAG CTC-3'(SEQ ID NO:7)
Primer6：5'-CG GGA TCC CTA TGT CCA CAC TTC TAG GG-3'(SEQ ID NO:8)
Primer7：5'-CG AAG CTT GT GTC CGT GTG TCT ATT CTG-3'(SEQ ID NO:9)
Primer8：5'-CG GGA TCC CTA ATG ATG ATG ATG ATG ATG TGT CCA CAC TTC TAG
GG-3'(SEQ ID NO:10)
Embodiment 2:The structure of slow slow-witted peptide expression vector
PCR primer is analyzed identification using digestion with restriction enzyme method in gene inner primer PCR amplifications and gene
After correct, it is cloned on carrier pUCm-Tvector/pSK or pEGFP-C1 carrier for expression of eukaryon.Be respectively adopted digestion, PCR and
Sequencing identification its correctness.Then obtain without film combination position and signal peptide, the slow slow-witted peptide of 199 amino acid of codified
CDNA sequence.
Clone 2:The slow slow-witted peptide -6His of recombinant plasmid pcDNA3.1-
Clone 3:The slow slow-witted peptide -6His of pEGFP-C1-
Embodiment 3:The identification and activity analysis of slow slow-witted peptide albumen
The detection of slow slow-witted peptide albumen
The same 3.3.3.2 of specific detection method, if 1 swimming lane adds pre-dyed albumen Marker10 μ l, is mended to 30 μ with sample-loading buffer
L, separately sets 3 swimming lanes and adds pEGFP- to delay the Chinese hamster ovary celI strain of slow-witted peptide transfection respectively, the Chinese hamster ovary celI strain that empty plasmid pEGFP-C1 is transfected,
Each 30 μ l of total protein of untransfected Chinese hamster ovary celI strain, successively with the anti-human PLD of mouse2Primary antibody and goat anti-mouse secondary antibody are incubated.
Fusion protein pEGFP- delays the measure of slow-witted peptide activity
Fusion protein pEGFP- delays the measure of slow-witted hydrolase polypeptide activity
Preparation of samples
(1) cultured cells to be measured (about 5 × 10 of 60mm Tissue Culture Dish is got out6Cell).
(2) 3mlGENMED cleaning liquids are carefully added into, cell growth surface is covered.
(3) cleaning liquid is carefully pumped.
(4) scrape de- rod using cell and softly scrape de- cell.
(5) 3mlGENMED cleaning liquids are added, cell is mixed.
(6) the 15ml conical centrifuge tubes of precooling are moved into
(7) 4 DEG C of desk centrifuges, 300g centrifugations 5min are put into.
(8) supernatant is carefully pumped.
(9) 500 μ lGENMED lysates are added, is fully mixed.
(10) it is transferred in the centrifuge tube of the 1.5ml of precooling.
(11) strength whirlpool shakes 15 seconds.
(12) it is placed in ice bank and is incubated 30 minutes.
(13) 4 DEG C of desk centrifuges, 16000g centrifugations 5min are put into.
(14) 500 μ l supernatants to the 1.5ml centrifuge tubes of new precooling carefully are pipetted.
(15) taking 20 μ l carries out protein quantification detection.
(16) -80 DEG C are put at once preserve or be placed in ice bank and continue subsequent operation.
The quantitative determination of total protein of cell
Protein concentration is determined with Braford methods, specific method sees below：
(1) protein standard substance is prepared：0.5mgBSA is added in 1mlPBS, is completely dissolved.
(2) protein standard substance is added separately to 96 hole enzymes according to 0 μ l, 1 μ l, 2 μ l, 4 μ l, 8 μ l, 12 μ l, 16 μ l, 20 μ l
In mark reaction plate, PBS to the μ l of cumulative volume 20 is added.
(3) the μ l of protein sample 10 that will be extracted are added in 96 hole elisa Plates, and 20 μ l are added to PBS.Do 2 again
Hole.
(4) each hole adds 200 μ lG250 dyeing liquors, and room temperature places 5min.First hole of standard items is set to reagent pair
According to group, the absorbance at wavelength 570nm is determined with ELIASA.
(5) according to the relation between the concentration and absorbance of protein standard, standard curve is drawn, the protein sample of calculating is dense
Degree.
The measure of slow slow-witted hydrolase polypeptide activity
Delay the continuous cyclic colorimetry immue quantitative detection reagent box of slow-witted hydrolase polypeptide organized enzyme with GENMED cells and detect slow slow-witted peptide
Hydrolysing activity, its specific Cleaning Principle is：Phosphatid ylcholine in the presence of the slow slow-witted peptide of synapse nucleoprotein sensitiveness, produce by hydrolysis
After raw phosphatidic acid and choline, by flesh choline kinase, pyruvate kinase and lactate dehydrogenase system determine reduced form nicotinoyl amine gland
Purine dinucleotides is converted into the peak change of NAD, carrys out the hydrolysis that slow-witted peptide is delayed in quantitative analysis
Activity.Delaying the slow-witted continuous circular response system of peptide is：
Concrete operation step sees below：
(1) ground control is determined
1. 170 μ lGENMED buffer solutions to the centrifuge tube of new 1.5ml are pipetted.
2. 25 μ lGENMED reaction solutions are added.
3. 10 μ lGENMED negative fluids are added.
4. whirlpool shakes 5 seconds.
5. 10min is incubated at a temperature of 30 DEG C, period whirlpool shakes 3 times, every time 5 seconds.
6. boil 5 minutes, 15min coolings are stood at room temperature.
7. it is transferred in the cuvette of 250 μ l, adds 25 μ lGENMED enzymatic liquid, fully mixes.
8. in putting spectrophotometer into, zero setting.
9. cuvette is taken out, 20 μ lGENMED background color liquid are added, is fully mixed.
10. detection in spectrophotometer is put at once, this is background empty map reading (- 5 minutes readings of 0 minute reading).
(2) sample gross activity is determined
1. 170 μ lGENMED buffer solutions to the centrifuge tube of new 1.5ml are pipetted.
2. 25 μ lGENMED reaction solutions are added.
3. 10 μ lpEGFP- are added to delay the total protein of slow-witted peptide transfection cell strain.
4. whirlpool shakes 5 seconds.
5. 10min is incubated at a temperature of 30 DEG C, period whirlpool shakes 3 times, every time 5 seconds.
6. boil 5 minutes, 15min coolings are stood at room temperature.
7. it is transferred in the cuvette of 250 μ l, adds 25 μ lGENMED enzymatic liquid, fully mixes.
8. in putting spectrophotometer into, zero setting.
9. cuvette is taken out, 20 μ lGENMED background color liquid are added, is fully mixed.
10. detection in spectrophotometer is put at once, this is sample gross activity reading (- 5 minutes readings of 0 minute reading).
(3) sample non-specific activity is determined
1. 160 μ lGENMED buffer solutions to the centrifuge tube of new 1.5ml are pipetted.
2. the 10 obligate liquid of μ lGENMED are added.
3. 10 μ lpEGFP- are added to delay the total protein of slow-witted peptide transfection cell strain.
4. it is incubated 5 minutes at a temperature of 30 DEG C, adds 25 μ lGENMED reaction solutions, whirlpool to shake 5 seconds.
5. 10min is incubated at a temperature of 30 DEG C, period whirlpool shakes 3 times, every time 5 seconds.
6. boil 5 minutes, 15min coolings are stood at room temperature.
7. it is transferred in the cuvette of 250 μ l, adds 25 μ lGENMED enzymatic liquid, fully mixes.
8. in putting spectrophotometer into, zero setting.
9. cuvette is taken out, 20 μ lGENMED background color liquid are added, is fully mixed.
10. detection in spectrophotometer is put at once, this is that sample non-specific activity reading (read for -5 minutes within 0 minute by reading
Number).
(4) sample activity is calculated
1. sample gross activity and nonspecific activity
(sample readout-background reading) × 0.25 × Sample Dilution multiple/(0.01 × 6.22 × 10 × sample protein is dense
Degree)
2. sample specific activity
Sample specific activity=sample gross activity-sample nonspecific activity
The measure of slow slow-witted peptide phosphatidyl transfer activity
With absorbance of the ELIASA bioassay standard product at 570nm wavelength, concentration and absorbance according to protein standard it
Between relation, the standard curve of drafting.
With the slow slow-witted PEPD of GENMED cells2The phosphatide of the slow slow-witted peptide of phosphatide transfer activity colorimetric determination detection kit detection
Acyl group transfer activity, its concrete principle：Substrate phosphatide p-nitrophenol in emulsion system, due to synapse nucleoprotein sensitiveness
Phospholipase D2Catalytic action, transfer phosphoester groups produce phosphatide alcohol, discharge color product p-nitrophenyl on ethanol hydroxyl
Phenol, the change (405nm wavelength) of its peak value is observed with ELIASA, carrys out quantitative analysis synapse nucleoprotein sensitiveness phospholipase D2Phosphorus
Fatty acyl group transfer activity.Its reaction system sees below：
PLD2
Phosphatidyl-p-nitrophenol+ethanol==phosphatidylalcohol+ p-
nitrophenol
Concrete operation step sees below：
(1) ground control is determined
1. 350 μ lGENMED reaction solutions to the centrifuge tube of new 1.5ml are pipetted.
2. 37 DEG C of constant incubators are put into or Water Tank with Temp.-controlled is incubated 5 minutes.
3. 50 μ lGENMED negative fluids are added, 37 DEG C of constant incubators is put into or Water Tank with Temp.-controlled is incubated 10 minutes.
4. 100 μ lGENMED terminate liquids are added, is mixed.
5. 100 μ lGENMED neutralizers are added, is mixed.
6. 400 μ lGENMED extracts, whirlpool are added to shake 5 seconds.
7. 4 DEG C of desk centrifuges, 16000g centrifugations 10min are put into.
8. 200 μ l upper phases are pipetted in new EP pipes.
9. 800 μ lGENMED cleaning liquids are added, is mixed.
10. the absorbance at 200 μ l ELIASA Detection wavelengths 405nm is drawn at once, and this is background blank control reading.
(2) sample gross activity is determined
1. 350 μ lGENMED reaction solutions to the centrifuge tube of new 1.5ml are pipetted.
2. 37 DEG C of constant incubators are put into or Water Tank with Temp.-controlled is incubated 5 minutes.
3. the slow slow-witted peptide fusion proteins of 50 μ lpEGFP- are added, 37 DEG C of constant incubators is put into or Water Tank with Temp.-controlled is incubated 10 points
Clock.
4. 100 μ lGENMED terminate liquids are added, is mixed.
5. 100 μ lGENMED neutralizers are added, is mixed.
6. 400 μ lGENMED extracts, whirlpool are added to shake 5 seconds.
7. 4 DEG C of desk centrifuges, 16000g centrifugations 10min are put into.
8. 200 μ l upper phases are pipetted in new EP pipes.
9. 800 μ lGENMED cleaning liquids are added, is mixed.
10. the absorbance at 200 μ l ELIASA Detection wavelengths 405nm is drawn at once, and this is sample activity reading.
(3) sample non-specific activity is determined
1. before detection starts, 50 μ lpEGFP- are pipetted and delay the total protein of slow-witted peptide transfection cell strain in the centrifuge tube of 1.5ml,
The 10 obligate liquid of μ lGENMED are added, is mixed, be incubated 5 minutes at room temperature.
2. 350 μ lGENMED reaction solutions to the centrifuge tube of new 1.5ml are pipetted, 37 DEG C of constant incubators or thermostatted water is put into
Groove is incubated 5 minutes.
3. add 60 μ l 1. solution in 2. solution, putting 37 DEG C of constant incubators into or Water Tank with Temp.-controlled is incubated 10 minutes.
4. 100 μ lGENMED terminate liquids are added, is mixed.
5. 100 μ lGENMED neutralizers are added, is mixed.
6. 400 μ lGENMED extracts, whirlpool are added to shake 5 seconds.
7. 4 DEG C of desk centrifuges are put into, 16000g is centrifuged 10 minutes.
8. 200 μ l upper phases are pipetted in new EP pipes.
9. 800 μ lGENMED cleaning liquids are added, is mixed.
10. the absorbance at 200 μ l ELIASA Detection wavelengths 405nm is drawn at once,.
(3) sample activity is calculated
1. sample gross activity and nonspecific activity
(sample readout-background reading) × 1 × 5 × Sample Dilution multiple/(0.6 × 0.01 × 18.75 × 10 × sample egg
White concentration) 2. sample specific activity
Sample specific activity=sample gross activity-sample nonspecific activity
Embodiment 4:Slow slow-witted application of the peptide in phosphatidylserine is prepared
The slow slow-witted Gly-His-Lys 3mg of freeze-drying is made enzyme liquid with 10mlPBS.
1. ultrafiltration：30KD-100KD milipore filters, filtrate is collected after nitrogen pressurization ultrafiltration.Activity to enzyme before and after ultrafiltration is surveyed
It is fixed.
2. slow slow-witted peptide (0.3mg/ml albumen) is dissolved in the Tris buffer solutions (pH6.5) that 10ml contains 0.04g aluminum oxide
In.Be stirred vigorously at 25 DEG C 30 minutes with magnetic stirring apparatus, and to stirring enzyme solutions in nonionic surface active agent is added dropwise
(selection：Sucrose ester；Sapn；Tween).It is divided to two groups, organizes one and use certain condition sonicated (10kHz -30kHz frequencies) 10min,
Group two is not processed, and is then stirred 8 hours at 25 DEG C.The sediment for obtaining is collected by the way that (12000rmp, 4 DEG C) is centrifuged, then
In -20 DEG C of freeze overnights and carry out freeze-drying.Lucifuge sealing refrigeration.
3. 2.5g Serines are placed in the reactor of the 0.02mmol/L sodium-acetate buffers equipped with 25ml, allow silk
Propylhomoserin is completely dissolved.
4. the soybean lecithin (containing more than 70% PC) of 1.5g is dissolved in 50ml ether.It is mixed with 3, mixture is placed in 28
DEG C incubator with magnetic bar stir 0.5-2 hours.
5., to 15mg enzyme preparations are added in reaction medium, reactant mixture is closed to be placed in 28 DEG C, and 4-12 is small for 200rmp stirrings
When, then stop stirring until enzyme preparation is deposited to the bottom of reactor.
6. after stratification, organic phase mixes with Serine again, and adds the enzyme preparation of recovery, repeat step 6, stands
After layering, organic phase is used to extract PS.Water obtains excessive serine after being mutually evaporated crystallization, can be used to recycle.
7. the enzyme preparation of precipitation is reclaimed, and is directly added into for second extraction after filtering.(will be to the available number of times of immobilised enzymes
Estimated.Calculating is measured with HPLC, reaction result is contrasted after counterweight is recycled.)
The purifying of 8.PS.Washed with acetate buffer solution first, stratification removes excessive serine.(use multilayer solvent
Method removes other phospholipid compositions：Alkaline aqueous solution is added, stirred, freeze, thawed, filter precipitation, remove remaining phosphatide, then
Supernatant is adjusted into pH=3~6, ether is evaporated off and is obtained raw phospholipid acyl serine.Raw phospholipid acyl serine dry powder is dissolved in n-hexane again
In, acetic acid sodium ethoxide solution is added, stirring, stratification remove the lower floor containing lipositol, and n-hexane decompression in top is steamed
It is dry to obtain high-purity phospholipid acyl serine powder) final -20 degrees Celsius of the lucifuge of preparation sealing for obtaining is stored refrigerated.To before purification,
PS after purification crosses chromatogram column analysis purity.
Embodiment 5:The qualitative, quantitative of PS
Thin-layer chromatography is legal
The preparation of standard sample：1mg standard PC, PS are weighed respectively, are dissolved in 1ml chloroforms or diethyl ether solution, sealing refrigeration
It is standby.The preparation of thin layer chromatography board：0.3g sodium cellulose glycolates are added in 60ml distilled water, 70 DEG C of heating water baths are complete
3 are pressed after dissolving：1 ratio adds 20g silica whites, grinding uniform application on a glass, to dry in the shade, and baking oven is put into after being completely dried
30min is activated at 110 DEG C, after taking-up naturally cools to room temperature, drying box is put into standby.
The selection of PS solvents：
Chloroform：Methyl alcohol：Acetone：Water：Ammoniacal liquor=73：27：10：2：4(V/V)
Point sample：Draw appropriate standard solution and testing sample, the point sample on lamellae respectively with capillary syring.Point sample
The chromatography cylinder equipped with solvent is put into after finishing to launch.
Coloration method：After by being placed in iodine cylinder dye after the organic solvent volatilization totally of the thin layer plate surface that solvent launches
Color.
(need reagent：Silica gel, sodium cellulose glycolate, chloroform, acetone, sodium acetate, methyl alcohol, ether, ammoniacal liquor, PC standards
Product, PS standard items)
PS is quantified
The silica gel plate being fully deployed by thin-layer chromatography, the spot of phosphorous fat above it is scraped from lamellae respectively
Carry out to be transferred in big syringe, each sample adds the ether of isodose pressure flush repeatedly, the liquid after flushing loads and first passes through in advance
By in the container of precision weighing.Placement overnight, remeasures after ether evaporates, according to front and rear mathematic interpolation each sample
In content of phospholipid.
Embodiment 6:Intervention study of the slow slow-witted peptide to Alzheimer disease (AD) animal model
Experimental technique：
The intervention of AD animal models：
After intraperitoneal injection 2% yellow Jackets (40mg/kg) anesthetized rat, rat is fixed on stereotaxic instrument, often
Rule preserved skin sterilization, cuts skin of head, reference《Rat stereotaxic atlas》, positioning bilateral hippocampus CA1 areas are (with bregma as former
Point, 3.0mm, opens 2.0mm backward by left and right, and depth is 3.5mm under skull surface).Then bore and open skull, expose endocranium, it is micro-
4 μ l are slowly contained 10 μ g A β by amount injector from the brain surface vertical inserting needle of anchor point1-40Slow-witted (low dose of peptide purification liquid slow with 0.08 μ l
Amount group), the SPSS of slow slow-witted peptide purifications liquid (middle dose group) of 0.4 μ l or the slow slow-witted refined solution of the peptides 2 (high dose group) of 0.8 μ l
Injection, injection time 10min, let the acupuncture needle remain at a certain point 10min, makes medicine fully infiltrate local organization, and the withdraw of the needle is sewed up the incision, conventinal breeding；Every
Day carries out identical injection.And the 3rd, 6,9 days PS of exclusive use corresponding dosage after final injection carry out bilateral hippocampus respectively
Injection is intervened.
Zoological specimens are gathered：
All slow slow-witted peptide injection groups and slow slow-witted peptide intervention group rat entered rower in the same day after last time determined with Morris water
This collection.Row Culling heart blood after intraperitoneal injection 2% yellow Jackets (40mg/kg) anesthetized rat, blood specimen normal temperature stands 2h, treats
3500rpm centrifugations 10min after blood clotting, takes supernatant, and -80 DEG C preserve for ELISA detections.Every group takes 6 fast quick-breaks of animal
Head, takes brain on ice, separates half brain of left and right, and left half brain is placed in fixes 48h in neutral formalin, after right half brain is weighed plus by 10% mass
Volume ratio adds the physiological saline of 4 DEG C of precoolings to be fully ground, 3000rpm centrifugation 5min, and taking -80 DEG C of homogenate supernatant and preserving is used for
ELISA is detected.Remaining rats underwent heart perfusion.It is fixed on after rat deep anaesthesia on dissection plate, cuts thorax abdomen open, exposes the heart to the open air
Dirty, left apex of the heart inserting needle cuts off right auricle of heart, and perfusion 200ml PBS to the internal organs such as heart, lung, liver take off white rear perfusion 200ml more than 4%
It is stiff that four limbs occurs in polyformaldehyde to rat.Broken end takes brain, separates half brain of left and right, and left brain is placed in neutral formalin is overnight used for paraffin afterwards
Embedding.Each group rat retains after spleen, lymph node neutral formalin are fixed is used for FFPE.
Morris determined with Morris water：
The corresponding time carries out water maze location navigation testing inspection to each group rat after surgery, lasts 3 days, respectively from 4 not
Same mark point, by rat, head is put into water towards pool wall at the radian of quadrant edge 1/2, and finding platform in record 2min is taken
Between (escape latency).If rat to enter fail to find platform in 2min after water, 15s is placed on platform and stopped, guide
Learning and memory, escape latency is recorded as 2min.Training every time is spaced more than 60s.
ELISA is detected
Operated according to kit explanation, in slow slow-witted peptide intervention group and slow slow-witted peptide injection group rat blood serum, brain homogenate
IL-17, IL-4, TGF-β 1, IL-21 and IFN-γ detected.
Experimental result shows：
1. slow-witted peptide intracerebral injection is delayed without overt toxicity.
Slow slow-witted peptide toxicity test result shows that rat does not occur weak, vomiting, poor appetite, troubled, erects after surgery
Hair, expiratory dyspnea, the spasm even poisoning symptom such as death.Time gradient experimental result shows each number of days rat after slow slow-witted peptide injection
Hippocampus is showed no there is pyramidal tract fault-layer-phenomenon, and closely in order, cell is full, only at 0 day for each number of days Hippocampal cell arrangement
And 2 Tian Zu rat cerebral tissues there is slight oedema, gap is slightly broadening, and visible more amount activated microglia, illustrate
Slow to stay first 0-2 days that peptide is injected into intracerebral, there is irritability inflammation, oedema in intracerebral, but over time, inflammation gradually disappears
Lose.And slow slow-witted peptide injection group HE dye piece results further show, the slow slow-witted multiple intracerebral injection of peptide will not cause cones tomography,
The toxicity such as neuron loss change.Shown in Nissl neuron statistics, delay slow-witted peptide injection Zu CA1 areas neuron number
Slightly reduce, but still be significantly higher than 14 days groups of AD models, infer that reason is that multiple injection is led because CA1 is slow slow-witted peptide injection site
Mechanism is caused to damage, so as to cause neuron number irritability to reduce.And the neuron number at other positions is compared to physiology salt
Water group is then without substantially change.Morris determined with Morris water result confirms that individually injection will not cause rat memory occur to slow slow-witted peptide
The behaviouristics such as decline change, and the slow slow-witted peptide intracerebral injection of ELISA results prompting is to IL-17, IL-4, TGF-β 1, IFN-γ and IL-
21 expression has no significant effect, and points out slow slow-witted peptide to inject normal rat intracerebral to Th17, Th1/Th2, Tfh and B cell
Expression without influence.The infiltration of CD3, CD79a, Foxp3 positive cell, the sun of TGF-β 1 are not found in slow slow-witted peptide injection each group intracerebral
Property 14 days groups of cell number and physiological saline close to and considerably less than 14 days groups of AD models, these results are then further characterized by slow slow-witted
Peptide intracerebral injection has no overt toxicity.
2. it is effective that slow-witted peptide intervention AD is delayed.
Migrated to cortex, thalamus after slow slow-witted peptide injection intracerebral, small part stays in hippocampus, is positioned by with cell membrane fusion
In surface of cell membrane, the mitigation effect similar with PS intervention effects is played to AD.Slow slow-witted peptide can not only change to the behaviouristics of AD
Mitigation effect is played in change, moreover it is possible to suppress the pathological changes such as neuron loss, centrum fault rupture in the AD courses of disease, neuron is played
Protective effect.Slow slow-witted peptide can also suppress T lymphocytes to the infiltration and the secretion of intracerebral TGF-β 1 in AD model brain parenchyms；
And increasing for peripheral blood Th1 type cells can be suppressed；This experiment also confirms that slow slow-witted peptide can suppress Treg in AD models first
The reduction of cell；Another slow slow-witted peptide also reaches as PS not by the level of increase or suppression Th2, Th17, Tfh and B cell
Arrive intervention effect.The mechanism of slow slow-witted peptide antagonism AD obtains PS to play its transacylate active catalytic, and then is sent out by PS
Wave the effect of anti-AD；By delaying the neuro-protective function that slow-witted peptide has in itself, the survival rate of neuron is improved；It is also possible to lead to
Cross the release that suppresses the inflammatory mediator such as TGF-β 1, IFN-γ and promote the function such as increase of Treg cells, play antagonism AD's
Effect.
Claims (9)
1. a kind of slow slow-witted peptide, is the polypeptide of the amino acid sequence shown in SEQ ID N0.2.
2. a kind of polynucleotide sequence for encoding slow slow-witted peptide, is the polynucleotides shown in SEQ ID N0.1.
3. polynucleotides of polypeptide of the encoding amino acid sequence as shown in SEQ ID N0.2.
4. with the polynucleotides of the polynucleotides complementation described in claim 3.
5., containing the recombinant vector of the polynucleotides described in Claims 2 or 3 or 4, the recombinant vector is expression vector.
6. slow slow-witted application of the peptide in phosphatidylserine is prepared described in claim 1,
It is characterized in that：The transacylate activity of slow slow-witted peptide is： 20-60U/mg.
7. the polynucleotides described in the slow slow-witted peptide or claim any one of 2-4 described in claim 1 are preparing treatment A Erci
Application in the silent medicine in sea.
8. application according to claim 7, it is characterised in that：Medicine is polypeptide, polynucleotides are combined with pharmaceutical carrier；Institute
It is water, glucose, ethanol, salt, buffer solution, glycerine or combinations thereof to state carrier.
9. application according to claim 7, it is characterised in that：By slow slow-witted peptide or the difference of slow slow-witted peptide and phosphatidylserine
The mixture of ratio, adds pharmaceutic adjuvant.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1904041A (en) * | 2005-07-25 | 2007-01-31 | 福建医科大学 | Human recombination phospholipase D2, its preparation method and application in preparation medicine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1904041A (en) * | 2005-07-25 | 2007-01-31 | 福建医科大学 | Human recombination phospholipase D2, its preparation method and application in preparation medicine |
Non-Patent Citations (3)
| Title |
|---|
| Expresson of the N-terminus truncated phosphotase D in Escherichia coli and characterization of its anti-inflammatory activity;ZHU Ling et al.;《Chinese Journal of Zoonoses》;20081231;991-998 * |
| pEGFP-rhPLD2融合基因在CHO细胞株中的稳定表达;陈秋雁等;《中国人兽共患病学报》;20091231;775－778 * |
| 磷脂酶D2及其在神经退行性疾病中的作用;张洁莹;《国际神经病学神经外科学杂志》;20061231;492－495 * |
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