CN110128503A - The synthesis polypeptide and its synthetic method of a kind of anti-A β 1-42 albumen aggregation, using with the gene that encodes the synthesis polypeptide - Google Patents
The synthesis polypeptide and its synthetic method of a kind of anti-A β 1-42 albumen aggregation, using with the gene that encodes the synthesis polypeptide Download PDFInfo
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- CN110128503A CN110128503A CN201910390488.6A CN201910390488A CN110128503A CN 110128503 A CN110128503 A CN 110128503A CN 201910390488 A CN201910390488 A CN 201910390488A CN 110128503 A CN110128503 A CN 110128503A
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
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Abstract
The invention discloses a kind of synthesis polypeptide of anti-A β 1-42 albumen aggregation and its synthetic method, using with the gene that encodes the synthesis polypeptide.Synthesis polypeptide provided by the invention has the function of anti-A β 1-42 albumen aggregation activity and alleviates cognition dysfunction, can effectively prevent or treat Alzheimer disease (AD) and other neurodegenerative diseases.Therefore, synthesis polypeptide provided by the invention can be widely applied to the research and development of anti-A β 1-42 albumen aggregation, the food or drug that prevent and treat AD, the medical conditions of neurodegenerative disease can be improved to a certain extent, there is great social value and economic benefit.
Description
Technical field
The present invention relates to technical field of polypeptide, in particular to a kind of the synthesis polypeptide and its synthesis of anti-A β 1-42 albumen aggregation
Method, using with encode the synthesis polypeptide gene.
Background technique
Alzheimer disease (Alzheimer ' s disease, AD) is also known as senile dementia, is one kind with cognitive function
Neurodegenerative disease based on obstacle, core symptom include failure of memory, learning ability decline, self-care ability reduce,
Comprehension and judgment variation, behavior emotionality to things etc..AD is based on gene, environment and behavior collective effect
As a result, as aging of population process is aggravated and is effectively predicted, is treated the shortages of AD means, the quantity of global AD patient by
Year is incremented by, this disease has become after heart disease, cancer, apoplexy, leads to the fourth-largest cause of disease of old man's death, therefore develop to AD
Effective prevention measure and therapeutic agent have become the social concern to receive much attention.The typical pathological characters of AD mainly have following three
Point: one, amyloid beta (A β) abnormal deposition occurred outside brain neuron and its senile plaque (SP) being further formed;Two, refreshing
Tau abnormal protein through intracellular hyperphosphorylation assembles the neurofibrillary tangles to be formed, and neuronal cell, which tangles, will lead to carefully
Born of the same parents' apoptosis;Three, neuron loss is along with glial cells hyperplasia.The pathogenic mechanism of AD is extremely complex, and scientists propose more
Kind hypothesis, including A β cascade hypothesis, Tua albumen hypothesis, choline function hypothesis etc., wherein A β cascade hypothesis receives most wide
One of theory, currently, the research about AD focuses primarily upon A β cascade hypothesis, this hypothesis is also research prevention or treatment AD phase
Close the main flow direction of food, drug.
Β amyloid protein (A β) is that cross-film amyloid precusor protein (APP) is thin in endoplasmic reticulum, golgiosome, lysosome etc.
The micromolecule polypeptide that born of the same parents' device is generated through the proteolysis of β, gamma secretase.A β cascade hypothesis thinks that the intracorporal A β of people is in
Equilibrium state, when the A β of generation cannot by timely degradation, remove in extracellular a large amount of depositions, neurotoxin generates therewith,
And then cause AD.Deiter's cells is the main source of intracerebral A β, and usually said A β refers to A β 1-40 or A β 1-42 more, wherein
A β 1-40 tends to form typical long fibre, primarily serves and prolongs long stapled effect, and A β 1-42 is more likely to form spherical shape
Patch sample deposition, be easier to aggregation and it is not degradable, toxicity is bigger, be constitute AD senile plaque core component.According to the theory,
Reduce A β 1-42 albumen generation, improve A β 1-42 albumen degradation mechanism and inhibit A β 1-42 albumen aggregation be AD prevention,
The major target class for the treatment of and detection.
Key agents currently used for the aggregation of anti-A β 1-42 albumen include Homotaurine, epiphysin etc..Homotaurine is knot
Together in a kind of glycosaminoglycan of A β 1-42 protein monomer, the polymerization of A β 1-42 albumen can be reduced, phase II clinical trials show height
Taurine can improve the cognitive function of severe AD patient, epiphysin can antagonism A β 1-42 albumen aggregation, research finds in A β 1-
Epiphysin can reduce the aggregation of senile plaque in the transgenic mice of 42 protein overexpressions.In addition, some have anti-A β 1-42 albumen
The biologically active peptide of aggregation is also just under study for action.Biologically active peptide refers to the peptides chemical combination for having good physiological function to organism
Object, it is from a wealth of sources, including the natural polypeptides by being directly separated extraction in organism, pass through chemistry or the conjunction of biological mode synthesis
At polypeptide, wherein the time spent by natural polypeptides is long, at high cost, low output, quality are uncontrollable, however synthesis polypeptide have it is pure
Spend high, at low cost, required time less, can scale mass production the features such as, mostly use solid-phase synthesis to synthesize at this stage more
Peptide.
In contrast, the inhibitor of polypeptide has good biocompatibility and quasi- ecological nature, synthesis and modification ratio
More convenient, stability is good, and smaller with the chance of immune system interaction, penetrability is strong, much the inhibitor based on peptide
Good inhibitory effect is shown, so peptide and peptide mimics are the promising primers for preventing and treating AD.
Summary of the invention
In order to overcome the shortcomings of the prior art, the object of the present invention is to provide a kind of conjunctions of anti-A β 1-42 albumen aggregation
At polypeptide and its synthetic method, using with the gene that encodes the synthesis polypeptide.
The purpose of the present invention is to provide a kind of synthesis polypeptides of anti-A β 1-42 albumen aggregation.
The object of the invention is also to provide a kind of methods of the synthesis polypeptide of synthesis anti-A β 1-42 albumen aggregation.
The object of the invention is also to provide a kind of genes of the synthesis polypeptide of coding anti-A β 1-42 albumen aggregation.
Another object of the present invention is to provide a kind of applications of the synthesis polypeptide of anti-A β 1-42 albumen aggregation.
The purpose of the present invention is realized at least through one of following technical solution.
A kind of synthesis polypeptide of anti-A β 1-42 albumen aggregation provided by the invention, entitled LGFH, amino acid sequence are
Leu-Gly-Phe-His, as shown in SEQ ID No:1;Wherein, Leu is the corresponding residue of amino acid of leucine, and Gly is sweet ammonia
The corresponding residue of amino acid of acid, Phe are the corresponding residue of amino acid of phenylalanine, and His is that the amino acid of histidine is accordingly residual
Base.
A kind of gene of synthesis polypeptide encoding the anti-A β 1-42 albumen aggregation provided by the invention, base sequence are
CUCGGCUUCUAC, as shown in SEQ ID No:2, base number is 12bp;Wherein, CUC is the codon of leucine, and GGC is sweet
The codon of propylhomoserin, UUC are the codon of phenylalanine, and UAC is the codon of histidine.
A kind of method of synthesis polypeptide synthesizing the anti-A β 1-42 albumen aggregation provided by the invention, including walk as follows
It is rapid:
It will from the C-terminal of amino acid sequence to N-terminal according to the amino acid sequence of the synthesis polypeptide of the anti-A β 1-42 albumen aggregation
Fmoc protected amino acid and resin are coupled one by one, then remove resin and protected amino acid Side chain protective group with cutting liquid,
Synthesis polypeptide crude product is obtained, the synthesis polypeptide of the anti-A β 1-42 albumen aggregation is obtained after purifying crude.
Preferably, the resin is dichloro resin.
Further, each component volume ratio of the cutting liquid are as follows: TFA (94.5%), water (2%), EDT (2.5%), TIS
(1%)。
Further, the purifying includes: high performance liquid chromatograph purifying, and uses liquid chromatography-mass spectrography/mass spectrum (LC-
MS qualitative analysis) is carried out, its amino acid sequence is measured.
A kind of synthesis polypeptide of anti-A β 1-42 albumen aggregation provided by the invention can be applied to prepare anti-A β 1-42 albumen
The food or drug of aggregation, while can also be applied to the research and development of food, drug for preventing or treating AD.
Compared with prior art, the invention has the advantages that and the utility model has the advantages that
The present invention provides the synthesis polypeptide of anti-A β 1-42 albumen aggregation, has effects that significant anti-A β 1-42 albumen aggregation, can
Improve memory, alzheimer's disease delayed to fall ill, can be widely applied to prepare anti-A β 1-42 albumen aggregation, prevention or treatment Ah
The research and development of the food or drug of the silent disease in Wurz sea.Thus, synthesis polypeptide provided by the invention can be to AD and other nervus retrogressions
Disease is effectively prevented or is treated, and improves the medical conditions of neurodegenerative disease to a certain extent, has certain society
It can be worth and economic benefit.
Detailed description of the invention
Fig. 1 a is the high-efficient liquid phase chromatogram for the polypeptide LGFH that embodiment 1 synthesizes;
Fig. 1 b is liquid chromatography-mass spectrography/mass spectrum (LC-MS) figure for the polypeptide LGFH that embodiment 1 synthesizes;
Fig. 2 a is negative control group (Control group), model group (Model group) in embodiment 2 and is separately added into
Synthesis polypeptide (24 h of preincubate) that concentration is 0.05 mM, the synthesis polypeptide (24 h of preincubate) that concentration is 0.1 mM, concentration are
The MTT cell survival rate column of the synthesis polypeptide (preincubate is for 24 hours) of 0.5 mM, the synthesis polypeptide (24 h of preincubate) that concentration is 1 mM
Shape figure, wherein " * " indicates there is significant difference between the group and control group;
The polypeptide low dose group of 0.1 mM when Fig. 2 b is negative control group, model group, synthesis polypeptide 24 h of preincubate in embodiment 2
With the cell branch office streaming figure of the polypeptide high dose group of 0.5 mM.
Fig. 2 c be negative control group in embodiment 2, model group, synthesis polypeptide (24 h of preincubate) concentration be 0.1 mM it is more
The A β 1-42 albumen aggregation rate for the polypeptide high dose group that peptide low dose group and synthesis polypeptide (24 h of preincubate) concentration are 0.5 mM
Column diagram, wherein " * " indicates there is significant difference between the group and control group;
Fig. 3 a is negative control group (Control group), model group (Model group) in embodiment 3 and is separately added into
Synthesis polypeptide (48 h of preincubate) that concentration is 0.05 mM, the synthesis polypeptide (48 h of preincubate) that concentration is 0.1 mM, concentration are
Synthesis polypeptide (48 h of the preincubate) concentration of 0.5 mM is the MTT cell survival rate column of the synthesis polypeptide (48 h of preincubate) of 1 mM
Shape figure, wherein " * " indicates there is significant difference between the group and control group;
Fig. 3 b be negative control group in embodiment 3, model group, synthesis polypeptide (48 h of preincubate) concentration be 0.1 mM polypeptide it is low
The cell branch office streaming figure for the polypeptide high dose group that dosage group and synthesis polypeptide (48 h of preincubate) concentration are 0.5 mM.
Fig. 3 c be negative control group in embodiment 3, model group, synthesis polypeptide (48 h of preincubate) concentration be 0.1 mM it is more
The A β 1-42 albumen aggregation rate for the polypeptide high dose group that peptide low dose group and synthesis polypeptide (48 h of preincubate) concentration are 0.5 mM
Column diagram, wherein " * " indicates there is significant difference between the group and control group.
Specific embodiment
Specific implementation of the invention is described further below in conjunction with attached drawing and example, but implementation and protection of the invention
It is without being limited thereto.If it is existing to be that those skilled in the art can refer to it is noted that there is the not special process of detailed description below
Technology realize or understand.Reagents or instruments used without specified manufacturer, be considered as can by it is commercially available be commercially available it is normal
Advise product.
In specific embodiment, a kind of synthesis polypeptide of anti-A β 1-42 aggregation provided by the invention, entitled LGFH, amino acid
Sequence are as follows: Leu-Gly-Phe-His, as shown in sequence table SEQ ID No:1;
Wherein, Leu is the corresponding residue of amino acid of leucine, and Gly is the corresponding residue of amino acid of glycine, and Phe is phenylpropyl alcohol ammonia
The corresponding residue of amino acid of acid, His are the corresponding residue of amino acid of histidine.
The synthesis polypeptide of anti-A β 1-42 albumen aggregation of the invention can pass through polypeptide solid-state reaction method or technique for gene engineering
Synthesis;
Wherein, when synthesizing by polypeptide solid-state reaction method, using standard Fmoc scheme, dichloro resin is selected;It is protected using Fmoc
Amino acid N end, each protected amino acid are Fmoc- L-Leu-OH, Fmoc-Gly-OH, Fmoc-L-Phe-OH, Fmoc-L-
His -OH.Protected amino acid and resin, which carry out coupling one by one, can be used coupling reagent and the activation examination of synthesis in solid state field routine
Agent, including using 4-dimethylaminopyridine (DMAP) and dicyclohexylcarbodiimide (DCC) complete first protected amino acid with
The coupling of resin carries out the coupling between remaining protected amino acid using I-hydroxybenzotriazole (HOBt).It is arrived according to peptide C end
Protected amino acid and resin are coupled by the amino acid sequence of N-terminal one by one, then lysate removing resin and protected amino acid
Side chain protective group obtains synthesis polypeptide crude product, and after synthesis polypeptide purifying crude, the synthesis for obtaining anti-A β 1-42 albumen aggregation is more
Peptide.
In specific embodiment, the present invention is based on A β to cascade hypothesis, using E22G-A β 42-mCherry HEK-293 transgenosis
Cell model prevents synthesis polypeptide LGFH to carry out the in vitro study of AD senile plaque aggregation.
Experiment in vitro is carried out using tetracycline induction E22G-mcherry Hek-293 cell strain.Tetracycline can induce A β
Albumen is assembled in the cell and generates toxicity, the pathological development process of analog AD patient's body neuronal cell senile plaque.Four
The induction of ring element makes aβ protein form red fluorescent protein in the cell and issues red fluorescence, using CytoFlexS fluidic cell
Instrument positioned to mcherry red fluorescence, quantitative detection.
Embodiment 1
Solid-phase synthesis synthesis polypeptide LGFH
1, synthetic route process flow
Using dichloro resin as carrier, first resin swelling, then by the active site on the C-terminal carboxyl of first amino acid and resin
Chlorine reaction carries out dehydrating condensation and connects second amino acid, take off again after the completion of condensation after first amino acid connects on resin
Fmoc protection.According to the amino acid sequence repetitive operation as shown in SEQ ID No:1, remaining ammonia has successively been connect from C-terminal to N-terminal
Base acid, finally cuts down polypeptide from resin with cutting reagent, and the volume ratio of each component is TFA in the cutting reagent
(94.5%), water (2%), EDT (2.5%), TIS (1%).
2, synthesis process
Using dichloro resin as carrier, first dichloro resin swelling, 1.28 g dichloro resins is weighed, is put into reaction column, then exists
20 mL DCM reagents are added in reaction column, vibrate 30 min, activation is stand-by.First amino acid is connected, is leached out by husky core
The Fmoc-L-His-OH of 1.05 times of dichloro resin moles is added in DCM solvent, adds 10 times of dichloro resin moles
DIEA(diisopropylethylamine), it is eventually adding 10 mL DMF dissolution, vibrates 1 h.With DMF and DCM alternately cleaning 6 after having reacted
Time.DMF solution of 20 mL containing volume fraction for 20% piperidines is added, removes DMF solution after 5 min.20 mL are added containing volume
Score is the DMF solution of 20% piperidines, and 15 min of oscillation are deprotected.Then, piperidine solution is removed, 15 grainy resin (dichloros are taken
Resin), it is washed three times with ethyl alcohol, ninhydrin, pyridine and each drop of phenol is added and becomes then in 105 DEG C of -110 DEG C of 5 min of heating
Navy blue is positive reaction, can be feminine gender if non-discolouring, need to be deprotected again after the next amino acid of continued access.It connects
Get off and cleaned for the first time, successively with 15 mL DMF(N, dinethylformamide), 15 mL methanol and 15 mL DMF cleaning,
The number of cleaning is twice.It is added the Fmoc-L-Phe-OH of 3 times of resins (dichloro resin) mole, 3 times of resin moles
HBTU is dissolved with the DMF solution of 10 mL DMF, and the DIEA of 10 times of resin moles is added, and 30 min of reaction are condensed.
Then it carries out cleaning for second, successively be cleaned with 15 milliliters of DMF, 15 ml methanols and 15 milliliters of DMF, the number of cleaning is two
It is secondary.Solvent is removed, 15 dichloro resins are taken, is washed three times with ethyl alcohol, ninhydrin, pyridine and each 200mL of phenol is added, then exists
105 DEG C of -110 DEG C of 5 min of heating, colourless is positive reaction, then needs to be condensed again if blue.It repeats to grasp according to above method
Make, has successively connect remaining amino acid, completed the extension of peptide chain.It connects Deng the last one amino acid with the synthesis of posterior restoration peptide just
Completion has been reacted, with DMF washing reaction 3 times, DCM washing reaction 3 times, methanol washing reaction 3 times, has drained peptide resin finally with complete
At the last contraction phase.
3, the cutting of amino acid side chain deprotection and resin
15 mL of configuration cuts liquid, the wherein percent by volume of cutting liquid each component are as follows: TFA (94.5%), water (2%), EDT
(2.5%),TIS(1%).Resin (dichloro resin) is fitted into flask, (30 DEG C) 2 h of oscillation of constant temperature.Lysate is blown with nitrogen
It is dry, it is subsequently poured into centrifuge tube, pours into 40 mL ether.It seals and puts into a centrifuge 5 min of centrifugation, centrifugation rate is
3000rpm outwells supernatant, leaves bottom white precipitate.It is washed 6 times with ether again, then room temperature volatilizes to get crude product peptide.
4, HPLC is purified
The crude product peptide is put into vessel first, is completely dissolved with the acetonitrile solution that 30-50 mL volume fraction is 50%, is surpassed
2 min of sound.Again with 0.45 μm of membrane filtration lysate.Take 3 μ l lysate analysis level HPLC analysis crude product peptide in order to rear
Continuous preparation.Mobile phase is water and acetonitrile, 30 min of time, gradient elution, first by HPLC start gradient balance 5 min then into
Sample, start gradient volume ratio are as follows: water 95%, acetonitrile 5% terminate gradient volume ratio are as follows: water 5%, acetonitrile 95%.The sample that will have been dissolved
Do sample introduction preparation.It prepares HPLC and balances 10 min, start gradient are as follows: water 95%, acetonitrile 5% terminate gradient are as follows: water 25%, acetonitrile
75%, 40 min of gradient timetable.Collect the sample come out from detector.Above procedure is closed in 12 channel polypeptide of SYMPHONY type
Cheng Yizhong is completed, and the polypeptide (synthesis polypeptide of anti-A β 1-42 albumen aggregation) of synthesis is pure through SHIMADZU high performance liquid chromatograph
Change, purity reaches 99% or more, and carries out qualitative analysis using liquid chromatography-mass spectrography/mass spectrometric hyphenated technique (LC-MS), measures it
Amino acid sequence.
The high-efficient liquid phase chromatogram and liquid chromatography-mass spectrography/mass spectrum (LC-MS) figure of the polypeptide of synthesis are respectively such as Fig. 1 a and figure
Shown in 1b, by Fig. 1 a and Fig. 1 b analysis shows, the primary amino acid sequences of the polypeptide of synthesis be Leu-Gly-Phe-His to get
To target polypeptides, synthesis obtains the synthesis polypeptide of anti-A β 1-42 albumen aggregation.
Embodiment 2
The synthesis polypeptide of synthesis polypeptide LGFH(anti-A β 1-42 albumen aggregation) 24 h anti-A β 1-42 albumen aggregation in vitro of preincubate
Activity experiment
1, experimental method
The preparation of culture medium: take 35 mL high glucose mediums (DMEM), take 4 mL fetal calf serums (FBS), 1 mL L-Glutamine,
The Blasticidin S antibiotic of the Hygromycin B of 40 μ L and 20 μ L is configured to the complete medium of 40 mL.
The preparation of synthesis polypeptide (LGFH) solution: the polypeptide LGFH of 4.543 mg, the complete medium described in 10 mL are weighed
Dissolution, after crossing 0.22 μm of filter, mother liquid concentration is 1 mM, then mother liquor is diluted to above-mentioned experiment with the complete medium
Required concentration.
2 mg/mL tetracyclines are prepared: being weighed the tetracycline of 10 mg, prepared with the 1 í PBS buffer solution of 5 mL, mistake
After 0.22 μm of filter, -20 DEG C be kept in dark place it is spare.
It is cultivated and is tested using E22G-mcherry Hek-293 cell.Experimental group: negative control group
(Control group, E22G-mcherry Hek-293 cell is added without tetracycline induction);Model control group (Model
Group, E22G-mcherry Hek-293 cell, are induced using tetracycline);Polypeptide LGFH low dose group (0.1 mM) and polypeptide
LGFH high dose group (0.5 mM), every group sets three in parallel.
Plating cells are carried out using 6 orifice plates, the number of cells in every hole is 20000, after 24 h of cell adherent growth, according to reality
It tests grouping and is separately added into the complete medium and synthesis polypeptide solution (LGFH polypeptide solution).After cultivating 24 h, to each group into
Row changes liquid, is separately added into the complete medium and synthesis polypeptide solution (LGFH polypeptide solution) according to experimental group, wherein mould
Type control group, polypeptide LGFH low dose group (0.1 mM) and polypeptide LGFH high dose group (0.5 mM) need to separately be added tetracycline, and four
Mass fraction of the ring element in complete medium is 10 μ g/mL, continues 72 h of Fiber differentiation.Using CytoFlexS fluidic cell
Instrument detect to mcherry red fluorescence, using the channel ECD under 561 exciters.Wherein, the calculating of A beta-aggregation rate
It is as follows:
A beta-aggregation rate=processing group average fluorescent strength/model group intensity * 100%.
Embodiment 3
The synthesis polypeptide of synthesis polypeptide LGFH(anti-A β 1-42 albumen aggregation) 48 h anti-A β 1-42 albumen aggregation in vitro of preincubate
Activity experiment
1, experimental method
The preparation of culture medium: take 35 mL high glucose mediums (DMEM), take 4 mL fetal calf serums (FBS), 1 mL L-Glutamine,
The Blasticidin S antibiotic of the Hygromycin B of 40 μ L and 20 μ L is configured to the complete medium of 40 mL.
The preparation of synthesis polypeptide (LGFH) solution: the polypeptide LGFH of 4.543 mg, the complete medium described in 10 mL are weighed
Dissolution, after crossing 0.22 μm of filter, mother liquid concentration is 1 mM, then mother liquor is diluted to above-mentioned experiment with the complete medium
Required concentration.
2 mg/mL tetracyclines are prepared: being weighed the tetracycline of 10 mg, prepared with the 1 í PBS buffer solution of 5 mL, mistake
After 0.22 μm of filter, -20 DEG C be kept in dark place it is spare.
It is cultivated and is tested using E22G-mcherry Hek-293 cell.Experimental group: negative control group
(Control group, E22G-mcherry Hek-293 cell is added without tetracycline induction);Model group (Model group,
E22G-mcherry Hek-293 cell, is induced using tetracycline);Polypeptide LGFH low dose group (0.1 mM) and polypeptide LGFH high
Dosage group (0.5 mM), every group sets three in parallel.
Plating cells are carried out using 6 orifice plates, the number of cells in every hole is 20000, after 24 h of cell adherent growth, according to reality
It tests grouping and is separately added into the complete medium and synthesis polypeptide solution (LGFH polypeptide solution).After cultivating 48 h, to each group into
Row changes liquid, is separately added into the complete medium and synthesis polypeptide solution (LGFH polypeptide solution) according to experimental group, wherein mould
Type control group, polypeptide LGFH low dose group (0.1 mM) and polypeptide LGFH high dose group (0.5 mM) need to separately be added tetracycline, and four
Mass fraction of the ring element in complete medium is 10 μ g/mL, continues 72 h of Fiber differentiation.Using CytoFlexS fluidic cell
Instrument detect to mcherry red fluorescence, using the channel ECD under 561 exciters.Wherein, the calculating of A beta-aggregation rate
It is as follows:
A beta-aggregation rate=processing group average fluorescent strength/model group intensity * 100%.
2, experimental result
Negative control group and blank control group (Control group) are the cell that tetracycline induction is not added, model group
(Model group) is then the cell that tetracycline induction is added, and LGFH is the experimental group that various concentration synthesis polypeptide LGFH is added.
The synthesis polypeptide LGFH of negative control group and various concentration precoat educate 24 h MTT cell survival rate figure it is as shown in Figure 2 a;It is negative
Control, model control group, the polypeptide high dose group of the polypeptide low dose group of 0.1 mM and 0.5 mM, which are precoated, educates branch office's stream of 24 h
Formula figure and A β 1-42 albumen aggregation rate column diagram are respectively as shown in Fig. 2 b, 2c;
It by embodiment 2 it is found that when polypeptide 24 h of preincubate, can be seen that from Fig. 2 a, the survival rate of cell is in certain concentration dependant
Property, there is high concentration suppression.Have when peptide concentration is 0.1 mM and promotees cell Proliferation phenomenon but be not present with control group existing
Significant difference, when peptide concentration is 0.5 mM cytotoxicity is not present between 80%-100% in cell survival rate, therefore can
0.1,0.5 mM, two kinds of concentration are selected to carry out subsequent experimental;By Fig. 2 b branch office streaming figure it is found that the mCherry of model control group is glimmering
Luminous intensity is higher than negative control group, and the mCherry fluorescence intensity of polypeptide administration group decreases compared to model control group.With
MCherry average fluorescent strength shows A β 1-42 albumen aggregation rate, by Fig. 2 c A β 1-42 albumen aggregation column diagram it is found that phase
Compared with negative control group, the polypeptide A β 1-42 albumen aggregation rate conspicuousness of model control group increases, and illustrates modeling success.Compared to
The A β 1-42 albumen aggregation rate of model control group, the polypeptide administration group of high (0.5 mM), low (0.1 mM) dosage is in conspicuousness
It reduces, wherein the A β 1-42 albumen aggregation rate decline of polypeptide low dose group is more, illustrates that polypeptide LGFH can reduce A β 1-42 albumen
Aggregation rate, and the anti-A β 1-42 albumen congregational rate of low dose group is better than high dose group.
The synthesis polypeptide LGFH of negative control group and various concentration, which precoats, educates MTT cell survival rate figure such as Fig. 3 a institute of 48 h
Show;Negative control, model control group, the polypeptide high dose group of the polypeptide low dose group of 0.1 mM and 0.5 mM precoat and educate 48 h's
Branch office's streaming figure and A β 1-42 albumen aggregation rate column diagram respectively as shown in Fig. 3 b, 3c.
By embodiment 3 it is found that when polypeptide 48 h of preincubate, it can be seen that from Fig. 3 a, there are concentration dependants for the survival rate of cell
Property, there is high concentration suppression.There is proliferation when peptide concentration is 0.1 mM but there is no existing with control group
Significant difference, cytotoxicity is not present between 80%-100% in cell survival rate when peptide concentration is 0.5 mM, therefore optional
It selects 0.1,0.5 mM, two kinds of concentration and carries out subsequent experimental;By Fig. 3 b branch office streaming figure it is found that the mCherry fluorescence of model control group
Intensity is higher than negative control group, and the mCherry fluorescence intensity of polypeptide administration group decreases compared to model control group.Using
MCherry average fluorescent strength shows A β 1-42 albumen aggregation rate, figure is assembled by Fig. 3 c A β 1-42 albumen it is found that compared to
The polypeptide A β 1-42 albumen aggregation rate conspicuousness of negative control group, model control group increases, and illustrates modeling success.Compared to model
Control group, the A β 1-42 albumen aggregation rate of the polypeptide administration group of high (0.5 mM), low (0.1 mM) dosage are in conspicuousness reduction,
Wherein the A β 1-42 albumen aggregation rate decline of polypeptide low dose group is more, illustrates that polypeptide LGFH can reduce the aggregation of A β 1-42 albumen
Rate, and the anti-A β 1-42 albumen congregational rate of low dose group is better than polypeptide high dose group.
The above results show polypeptide preincubation time either 24 h or 48 h of same concentrations, there is anti-A β 1-
The effect of 42 albumen aggregation;When polypeptide preincubation time is identical, it is poly- that the peptide concentration of high and low dose all has anti-A β 1-42 albumen
The effect of collection, moreover, the anti-A β 1-42 albumen congregational rate of 0.1 mM polypeptide low dose group is better than polypeptide high dose group.Therefore,
The preferable Development process for improving memory, inhibiting AD is all had when 0.1 mM synthesis polypeptide LGFH 24 h and 48 h of preincubate, it can
Applied to preparing anti-A β 1-42 albumen aggregation and preventing, treating the food or drug of AD, to the nerve including AD disease
Degenerative disease is effectively prevented and treated.
Above embodiments are only preferrred embodiment of the present invention, for explaining only the invention, are not intended to limit the present invention, this
Field technical staff should belong to guarantor of the invention without departing from change made under spirit of the invention, replacement, modification etc.
Protect range.
Sequence table
<110>South China Science & Engineering University
<120>synthesis polypeptide and its synthetic method of a kind of anti-A β 1-42 albumen aggregation, using with the base that encodes the synthesis polypeptide
Cause
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4
<212> PRT
<213>artificial synthesized (artificial sequence)
<400> 1
Leu Gly Phe His
1
<210> 2
<211> 12
<212> DNA/RNA
<213>artificial synthesized (artificial sequence)
<400> 2
cucggcuucu ac 12
Claims (7)
1. a kind of synthesis polypeptide of anti-A β 1-42 albumen aggregation, which is characterized in that entitled LGFH, amino acid sequence Leu-
Gly-Phe-His, as shown in SEQ ID No:1;Wherein, Leu is the corresponding residue of amino acid of leucine, and Gly is glycine
The corresponding residue of amino acid, Phe are the corresponding residue of amino acid of phenylalanine, and His is the corresponding residue of amino acid of histidine.
2. a kind of gene for the synthesis polypeptide for encoding anti-A β 1-42 albumen aggregation described in claim 1, which is characterized in that base
Sequence is CUCGGCUUCUAC, and as shown in SEQ ID No:2, base number is 12 bp;Wherein, CUC is the codon of leucine,
GGC is the codon of glycine, and UUC is the codon of phenylalanine, and UAC is the codon of histidine.
3. a kind of method of the synthesis polypeptide of anti-A β 1-42 albumen aggregation described in synthesis claim 1, which is characterized in that including such as
Lower step:
It will from the C-terminal of amino acid sequence to N-terminal according to the amino acid sequence of the synthesis polypeptide of the anti-A β 1-42 albumen aggregation
Fmoc protected amino acid is coupled one by one from the first amino acid of C-terminal and resin, is then carried out one by one with solid-phase synthesis
Dehydrating condensation between amino acid forms peptide chain, removes resin and protecting group with cutting liquid, obtains synthesis polypeptide crude product, will synthesize
The synthesis polypeptide of the anti-A β 1-42 albumen aggregation is obtained after polypeptide purifying crude.
4. synthetic method according to claim 3, which is characterized in that the resin is dichloro resin.
5. synthetic method according to claim 3, which is characterized in that the cutting liquid presses volume percentage, composition
Ingredient include 94.5% TFA, 2% water, 2.5% EDT and 1% TIS.
6. synthetic method according to claim 3, which is characterized in that the purifying is pure by high performance liquid chromatograph
Change, and qualitative analysis is carried out using liquid chromatography-mass spectrography/mass spectrometric hyphenated technique, measures its amino acid sequence.
7. a kind of synthesis polypeptide of anti-A β 1-42 albumen aggregation described in claim 1 is in the food for preparing anti-A β 1-42 albumen aggregation
Application in product or drug.
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