CN100447153C - Novel 11 peptide, preparation method and application thereof - Google Patents
Novel 11 peptide, preparation method and application thereof Download PDFInfo
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- CN100447153C CN100447153C CNB001057995A CN00105799A CN100447153C CN 100447153 C CN100447153 C CN 100447153C CN B001057995 A CNB001057995 A CN B001057995A CN 00105799 A CN00105799 A CN 00105799A CN 100447153 C CN100447153 C CN 100447153C
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- peptide
- app11
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- isoleucine
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Classifications
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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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/582—Recycling of unreacted starting or intermediate materials
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a new 11 peptide, it is composed of 11 amino acid residues, its sequence is isoleucine-aspartic acid-threonine-lysine-glutamic acid-glycine-isoleucine-leucine-glutamine-tyrosine-cysteine (IDTKEGILQYC), and its application for curing neuronal degenerative disease and having the effect of neurotrophic.
Description
The present invention relates to chemistry of peptides, particularly relate to a kind of 11 new peptides, Preparation Method And The Use.
The APP11 peptide is a kind of new polypeptide that we find first, still finds no any report that closes the APP11 peptide so far.
The mid-90 was once found the APP17 peptide; beta amyloid peptide precursor protein (β-Amyloidprecursor protein; APP) the peptide section of 319-335 position has neurotrophic effect in the peptide chain, comprising: promote axon growth, the trauma of cerebral nerve cell that increases synaptic density, can protect ischemic to cause.
It is short than the APP17 peptide to the objective of the invention is to seek a kind of peptide chain, synthetic easy than the APP17 peptide, has the polypeptide of neurotrophic effect.We have finally found a kind of 11 new peptides through in a few years research, the APP11 peptide, and it not only has neurotrophic effect, and may treat the neurone degeneration.
APP11 peptide of the present invention is a beta amyloid peptide precursor protein N end 63-73 position peptide section, be made up of 11 amino-acid residues, sequence is Isoleucine-aspartic acid-Threonine-LYS-GLU-glycine-Isoleucine-leucine-glutamine-tyrosine-halfcystine (IDTKEGILQYC).We are through studying the biological activity that APP N holds the polypeptide fragment of various different lengthss repeatedly, finally find and definite APP11 peptide has neurotrophic effect.
Our APP11 peptide of discovering has following function up to now:
1. external, human neuroblastoma strain SY5Y cell cell space area and aixs cylinder length be can increase, cell proliferation and survival ability promoted.
2. improve the expression of some key proteins in the learning and memory function of diabetic mice and the hippocampal neuron.
3. improve diabetic mice sciatic nerve conduction of velocity and some protein expressions.
Above result shows that the APP11 peptide has neurotrophic effect, and has the ability of improving experimental neurone degeneration.
APP11 peptide of the present invention adopts solid phase method synthetic; the main thought of solid-phase peptide synthetic is: earlier the amino acid whose carboxyl of the C-terminal that will synthesize peptide chain is connected with the same insoluble macromolecular compound of the structure of covalent linkage (resin); be combined in amino acid on the solid phase carrier as amino component with this then; through the deaminize protecting group and with excessive activated carboxyl component reaction, spreading peptide chain.Such step can repeatedly go on repeatedly, reaches the length of required synthetic peptide chain at last.Figure below has been represented this building-up process.
Raw material:
HMP resin (P-hydroxymethyl phenoxy methyl poly ethenoid resin)
Fmoc-AA (amino acid of 9-fluorenyl methoxy carbonyl acyl group protection)
NMP n-formyl sarcolysine base pyrrolidone
The DCM methylene dichloride
MeoH methyl alcohol
The Piperidine piperidines
The DMAP dimethyl aminopyridine
The HOBT hydroxybenzotriazole
The DCC dicyclohexylcarbodiimide
The TFA trifluoroacetic acid
The EDT 1
The thio phenyl methyl ether
Crystallization phenol
Acetonitrile
Instrument:
The polypeptide automatic DNA synthesizer DNA
Rotary Evaporators
High performance liquid chromatograph
Freeze drier
Synthetic method:
Take by weighing HMP resin 100mg, replacing equivalent is 1.0meq, promptly 0.1mmol in the reaction chamber of American AB 1431A type polypeptide automatic DNA synthesizer DNA, by synthesizer automatically with specific AA by different being linked in sequence, the coupling rate reaches 99%.React as follows:
1. amino acid whose activation (HOBt/DCC method)
The amino acid of Fmoc protection
HOBt/DCC
2. connect amino acid to resin
3. the Fmoc protecting group of deaminize acid
4. amino acid whose activation (HOBt/DCC method)
5. coupling
New link coupled peptide-resin
Repeat 3-5 until end of synthesis, obtain the peptide resin of APP11 peptide.
IDTKEGILQYC-resin 350mg cuts peptide chain fall from resin: with TFA (trifluoroacetic acid) cutting peptide chain, use EDT, thio phenyl methyl ether, H
2O makes scavenging agent, at room temperature reacts 3.0 hours, removes cutting reagent, uses extracted with diethyl ether again, and the crude product that obtains the APP11 peptide is 100mg, and yield is 85%.
The purifying of APP11 peptide
The high performance liquid chromatography separation and purification:
Condition: chromatographic column C
810 * 100mm
The chromatographic instrument ABI 151A type U.S.
Mobile phase A-0.1%TFA (trifluoroacetic acid) H
2O
B-0.1%TFA (trifluoroacetic acid) is in 60% acetonitrile
Detect wavelength 214nm
Flow velocity 4ml/ minute
Gradient 20-60%B was in 30 minutes
HPLC (high performance liquid chromatography) analyzes
Chromatographic column: C
184.6 * 150mm
Moving phase: A-0.1%TFA (trifluoroacetic acid) H
2O
B-0.1%TFA (trifluoroacetic acid) is in acetonitrile
Detect wavelength: 214nm
Flow velocity: 1ML/min
Gradient: 0-60%B was in 30 minutes
Analytical results is seen accompanying drawing 1.
The evaluation of APP11 peptide
APP11 peptide AA proximate analysis the results are shown in accompanying drawing 2.
Sequence: IDTKEGILQYC (Isoleucine-aspartic acid-Threonine-LYS-GLU-glycine-Isoleucine-leucine-glutamine-tyrosine-halfcystine)
Do standard with Gly (glycine)
Annotate: Gln (glutamine) is destroyed in the acidolysis process to become Glu (L-glutamic acid), so Glu (L-glutamic acid) is 2, and Gln (glutamine) is 0.
AA proximate analysis result shows and conforms to the component of purpose peptide, proves that synthetic is successful.
The neurotrophic effect of APP11 peptide of the present invention and improve the neurone degeneration and prove by following experiment.
1. experimental technique: adopt internationally recognized experimental technique to carry out water maze laboratory, immunohistochemical staining and neuronal cell cultures.
1) water maze laboratory
3 all row water maze tests are trained 2 every day behind the animal Cheng Mo, and continuous 5 days, the 1st day 2 cecums, the 2nd day 3 cecums, the 3rd, 4,5 day 4 cecums, running into cecum with head is 1 mistake, writes down the time and the wrong reaction number of times of every mouse covering the race.
2) hippocampus neuron immunohistochemistry research
The capable frozen section of mouse brain after fixing carries out the immunohistochemical staining of various antibody.Sp immunohistochemical methods test kit is available from Zymed Laboratories company.By specification experimentizes.
The immunohistochemical methods quantitative analysis: each 3 of (1) every treated animals, every in corresponding hippocampus position serial section, gets 1 every 2, promptly gets 15 section countings altogether.(2) with Pentium Visilog 5 image analysis software 5-6 positive cell selected in every section carrying out the immunohistochemical methods mark at random, survey its endochylema average gray.Data information is handled: enumeration data is represented with mean ± standard deviation, the test of significance analysis of variance.
3) experiment in vitro: use human neuroblastoma strain SY5Y, cell culture condition is to add 10% foetal calf serum in the MEM substratum.The SY5Y cell is divided into control group, damage group and APP11 peptide protection group.1. MTT bromination (3-[4,5-dimethylthiazole-2-yl] phenylbenzene tetrazolium, Sigma product) metabolic rate is measured:
Is that 1 * 103.ml-1 is inoculated in 96 orifice plates (Costar product) with the SY5Y cell with density, is divided into control group, damage group and the protection of APP11 peptide group after 3 days, every group 8 hole.The 4th day every hole, inoculation back adds MTT (5mg.ml-1) 20 μ l, and 37oC was hatched 4 hours, the sucking-off nutrient solution, and every hole adds DMSO200 μ l, and jolting 10 minutes is measured 550nm place's optical density value (OD) with microplate reader (Diagnostic Pasteur LP400).
(MTT is a tetrazolium bromide, after the cultured cells picked-up, generates first by the plastosome metabolism and praises (formazane), so the plastosome vigor is vigorous more, it is many more that first is praised generation, and optical density value is also high more.By measuring the MTT metabolic rate, situation) but reacting cells is survived.
2. cell counting
3. serum lactic dehydrogenase (LDH) spills rate mensuration: measure method (colorimetry) shown in the test kit according to LDH, measure 450nm place optical density value, do the LDH typical curve simultaneously.
4. morphological observation: use the medical image Computerized image processing system and measure cell axon length and cell space area.
5. statistical procedures is used SPSS software and is done the t inspection statistics.
Material: press the complete synthesis APP11 peptide of preceding method by the contriver.Laboratory animal is mouse and rat, and mouse is the Kunming kind, body weight 32-37 gram, 8 ages in week.Rat is Wistar, about 250 grams, and 16 ages in week.Animal model preparation and control: be divided into control group (C group), diabetic groups (DM group) and APP11 peptide treatment group (APP11 peptide+DM group)
Preparation of table 1. animal model and administration
The diabetic mice model
Male mouse of kunming, body weight 32-37 gram is divided into three groups: normal control group (C group), diabetes control group (DM group), APP11 peptide control group (11P+DM group) at random.11P+DM organizes behind diabetes Cheng Mo and 2 weeks injected the APP11 peptide to 4 peritheliums, and each 0.35ug/, once a day.200mg/kg body weight dosage abdominal injection STZ is pressed in the mouse fasting after 12 hours, survey non-fasting serum glucose after three days, thinks that greater than 15mM/L person diabetes model duplicates successfully.
The diabetes rat model clone method is the same.
2. experimental result
1) experiment in the body
1. diabetic mice water maze laboratory result
Table 2. has been swum the omnidistance required time of water maze
*Compare p＜0.05 with the DM group
*Compare p＜0.01 with the DM group
Table 3. water maze test errors reaction times
*Compare p＜0.05 with the DM group
*Compare p＜0.01 with the DM group
The water maze test result shows: DM group covering the race time lengthening, the wrong reaction increased frequency has been compared significant differences with the C group, has compared significant difference with DM+APP11 peptide group.The result shows that the DM group exists study, memory dysfunction, and the APP11 peptide has the improvement effect to study, the memory dysfunction of diabetic mice.
2. nervous tissue immunohistochemical staining
Three kinds of PS-1 antibody positives of three groups of mouse hippocampus of table 4. cell number relatively
## compares P＜0.01 with the C group,
*Compare P＜0.01 with the DM group
Three groups of mouse hippocampus of table 5. NF, NGF antibody positive cell number are relatively
*Compare P＜0.01 with the DM group
Three kinds of PS-1 antibody positives of three groups of mouse hippocampus of table 6. cell gray scales tristimulus values relatively
## compares P＜0.01 with the C group,
*Compare P＜0.01 with the DM group
Three groups of mouse hippocampus of table 7. NF, NGF antibody positive cell gray scales tristimulus values relatively
*Compare P＜0.01 with the DM group
Above result shows that the APP11 peptide has a significant effect to the expression of mouse hippocampus neuron NF, NT-3, PS-1.
3. the change of some composition in peripheral nerve tissue and the blood
A) the APP11 peptide can make the diabetic sciatic nerve conduction of velocity recover normal.
Table 8.APP11 peptide 3 μ g/ days of subcutaneous injection, after February to the influence of rat conduction of velocity
*Compare P＜0.05 with DM+11P and C group
B) the APP11 peptide does not influence blood sugar and blood glucose regulation hormone.
Table 9. normal rat is quiet only to push away APP11 peptide 50 μ g/, to the influence of blood sugar
Table 10. normal rat is quiet only to push away APP11 peptide 10 μ g/, to the influence of blood glucose regulation hormone
C1: quiet push away the APP11 peptide before, C2: quiet push away the APP11 peptide after.P＞0.05
C) the APP11 peptide is to the influence of renal function
3 μ g/ days of table 11. subcutaneous injection APP11 peptide, after February to the influence of renal function
*Compare P＜0.05 with the C group
# and DM group be P＜0.05 relatively
Above presentation of results, the APP11 peptide has the function of improving the diabetes rat neuronal degeneration, and does not finish by the lowering blood glucose approach.
2) experiment in vitro
1. MTT metabolic rate measurement result shows: APP11 peptide group MTT metabolic rate is higher than control group, and significant difference is arranged.
Table 12.APP 11 peptides influence SY5Y MTT metabolic rate
*Compare P＜0.05 with control group
2. cell counting result shows: APP11 peptide group cell count all was higher than control group at the 1st, 2,3 day, and significant difference is arranged.
The influence that table 13. cell counting observation APP11 peptide is grown to SY5Y (x ± s)
*Compare with control group: P＜0.05,
*Compare with control group: P＜0.01
3. LDH spills the demonstration of rate measurement result: after the cell count markization, APP11 peptide group LDH compares with control group all highly significant difference.
Table 14.APP11 peptide spills the influence of rate to SY5Y LDH
*Compare with control group: P＜0.01
4. morphological observation: the APP11 Toplink promotes neuron axon growth and cell space increase.
Aixs cylinder length: C group 42.22 ± 22.07APP11 peptide group 89.67 ± 39.38
Cell space area: C group 749.69 ± 207.65APP11 peptide group 1101.66 ± 355.31
We think that the possible mechanism of APP11 peptide treatment neuron regression is: diabetes animal model has the performance of neurodegeneration, expression decreased such as neurotrophic factor NT-3, BDNF, NGF, prompting may be owing to support the nerve survival factor of neuronal function to reduce, conducted signal intensity is lower than threshold value after making the acceptor of multiple survival factors, and the neurone genetic transcription can not be activated and cause neuronal degeneration.After giving exogenous APP11 peptide, can activate neuronal function, the treatment neuronal degeneration by talking with the signal conduction that strengthens the nerve survival factor in the signal conductive process mutually.Because the APP11 peptide does not influence blood sugar and regulates hormone, its effect is irrelevant with Regular Insulin.The neurotrophic effect of APP11 peptide may pass through g protein coupled receptor, activates IRS-1, finally plays the effect of control diabetes nerve unit regression.
The APP11 peptide will be in the world first with the treatment nerve degenerative diseases medicine.The inside and outside studies show that the APP11 peptide has the effect that strengthens neuronic survival ability and reduce injurious factor, so can expect that the treatment spectrum of APP11 peptide can comprise at least:
1. early, mid-term alzheimer disease
2. diabetic neuropathy
3. alleviating cerebral apoplexy damages neurone
Climacteric syndromes
5. brain and nerve injury
6. Parkinson's disease (remain experiment confirm)
But the APP11 peptide also has very big potentiality.We find that it has therapeutic action to diabetic nephropathy recently, the experimental lung arterial hypertension there is hypotensive effect, therefore the result of treatment that small vessel disease is become if can repeat then understanding and the treatment small vessel disease becomes, provides the new target position of drug effect-improve peripheral nerve degeneration, and this will become the control small vessel disease and become new ideas as hypertension, diabetes etc.
APP11 peptide of the present invention can use separately, also can make suitable agent shape with carrier, the vehicle of various acceptance.
Annex 1: the main reference document of research method
1.Fuller?SJ，Storey?E，et?al.Intracellular?production?of?βA4?Amyloid?ofAlzheimer’s?Disease：Modulation?by?phosphoralytion?and?lack?of?coupling?tothe?secretion?of?the?Amyloid?precursor?protein.1995，34，8091-8098，Biochemistry
Produce the β A4 4 amyloid of senile dementia in the cell: regulate and shortage 4 amyloid precursor protein excretory coupling journal of biological chemistry by phosphorylation
2.Ostrerova?N，Petrucelli?L，et?al.α-Synuclein?shares?physical?and?functionalhomology?with?14-3-3?proteins.The?Journal?of?Neurosci.1999，19(14)：5782-5791
α-common nucleoprotein and 14-3-3 albumen have the homology Journal of Neuroscience on physiological function
3.Faircloth GT, Stewart D, Clement JJ.A simple screening procedure for thequantitative measurement of cytotoxicity to resting primary lymphocytecultures.Journal of Tissue Culture Methods.1988,11 (4): cytotoxicity was quantitative during 201-205 lymphocyte of former generation was cultivated, the simple examination step tissue culture method magazine of mensuration
4. Zhao Yong plum, Zhao Zhiwei, Ji Zhijuan etc.The APP17 peptide is to the influence China geriatrics magazine 18 (5): 3061999 of diabetic mice micro-tubular structure and Protein tau phosphorylation related enzymes
Qian Yuying Zhao Yong plum etc.The APP17 peptide is to the influence China geriatrics magazine 18 (6): 1999 of diabetic mice study, memory function and hippocampus NT-3, choline acetylase neuron expression
Claims (2)
1. sequence is 11 peptides of Isoleucine-aspartic acid-Threonine-LYS-GLU-glycine-Isoleucine-leucine-glutamine-tyrosine-halfcystine.
2. contain 11 peptides of significant quantity claim 1 and the pharmaceutical composition that acceptable assistant agent has neurotrophic effect and treatment neurone retrograde affection.
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