CN104910255B - Polypeptide as M-ChR M1 subtype regulators and its production and use - Google Patents
Polypeptide as M-ChR M1 subtype regulators and its production and use Download PDFInfo
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Abstract
The present invention relates to polypeptide as M-ChR M1 subtype regulators and its production and use, the amino acid sequence of the polypeptide is as shown in SEQ ID NO.1, or the polypeptide is that polypeptide of the amino acid sequence as shown in SEQ ID NO.1 obtains by the substitution of one or several amino acid residues, missing or addition and with the polypeptide of M-ChR M1 hypotypes regulation activity.The present invention polypeptide effectively can be combined with M1 acceptors and its activity is adjusted, to M-ChR M1 hypotypes mediation illness, be particularly glaucoma treatment when complicated cataract, chronic obstructive pneumonia, peptic ulcer and alzheimer disease there is potential therapeutic value.
Description
Technical field
The present invention relates to the new polypeptide to M-ChR M1 hypotypes with regulation activity.The present invention also relates to described more
Peptide complicated cataract, chronic obstructive in the disease, especially glaucoma treatment that treatment is mediated by M-ChR M1 hypotypes
Purposes in pneumonia, peptic ulcer and alzheimer disease.Moreover, it relates to the preparation method of the polypeptide.
Background technology
Choline receptor is typically combined with endogenous neural conduction transmitter acetylcholine (ACh), thus active cell downstream is believed
Number Signal Transduction Pathways, produce bioactivity.Choline receptor in mammal peripheral and central nervous system is according to muscarine and cigarette
The agonist activity of alkali, M-ChR (mAChR) and nicotine receptor (nAChR) can be divided into.Wherein, mAChR presses pharmacology
Parting can be divided into M1, M2, M3, tetra- kinds of hypotypes of M4；It can be divided into m1, m2, m3, five kinds of hypotypes of m4, m5 by Molecular strain typing.M
Acceptor causes different Physiological and Biochemical effects, the difference to work may by being combined from different G-proteins at each position of whole body
The different relevant of a variety of hypotypes and Germ distribution be present from it.
M1 acceptors are distributed more widely in whole body, and more with the distribution of eye, air flue, heart, intestines and stomach and maincenter, and it is in these portions
Position has mediated some important physiological actions.In clinical practice, M1 receptor blocking pharmacons can reduce anticholinesterase and be used for green grass or young crops
The generation of cataract, can also be improved pulmonary ventilation function when light eye is treated, and can reduce gastric acid secretion, such as combine other medicines
Thing is then used for the treatment of peptic ulcer；M1 receptor stimulating agents can then improve the learning and memory function of Alzheimer patient and prolong
Delay the development of its state of an illness.Selectively acting is of great value in clinical practice in the medicine of M1 acceptors.
Current M1 receptor activity modulators are essentially all micromolecular compound, including as baogongteng A, horse
The natural products such as money alkali, also synthesize compound including oxotremorine, AC-42 etc..But choosing of most conditioning agents to M1 acceptors
Selecting property is not high, easily acts on other non-target tissues, produces toxic side effect.Therefore, new selective M1 receptor modulators are ground
It is imperative to study carefully, and improving the selectivity of M1 receptor modulators contributes to when treating relevant disease by adjusting M1 receptor actives,
Decrease or even eliminate the side effects such as xerostomia, constipation.
The content of the invention
The problem of existing for prior art, it is an object of the invention to provide a kind of alternatively property M1 receptor modulators
Polypeptide and its production and use.
The present inventor utilizes meter according to the polypeptide toxin MT7 of energy selective control M1 receptor actives special three-dimensional structure
The method of calculation machine Blast search, construct the 3-D solid structure model of M1 acceptors.Based on the spy between MT7 toxin and M1 acceptors
The opposite sex combines, the docking of integrated use protein-protein, molecular dynamics simulation, Conjugated free energy calculating, amino acid residue energy
Amount is decomposed, normal mode vibration analysis equimolecular analogy method, the interaction model between MT7 toxin and M1 acceptors has been carried out entirely
Face parses.
The present inventor continues using M1 acceptors as target spot, the specific interaction model based on MT7 toxin Yu M1 acceptors, should
With above-mentioned molecule simulation method, Binding peptide synthesis, radiative aglycone combination Activity determination, cell calcium current activation experiment detection side
Method, through Rational drug design, synthesize and filter out a series of polypeptides that can adjust M1 receptor actives.Specifically based on knot before this
Free energy calculating and Energy Decomposition analysis are closed, determines the Key residues of interaction in MT7 toxin with M1 acceptors be present, with reference to
The three-dimensional structure of MT7 toxin, the peptide sequence with specific three dimensional structure and surface charge properties is designed, utilizes these polypeptides
Carry out its regulation activity research with the binding activity research of M1 acceptors and to M1 acceptors, test result indicates that, these polypeptide energy
Effectively combined with M1 acceptors and its activity be adjusted, to M-ChR M1 hypotypes mediation illness, particularly green light
Complicated cataract, chronic obstructive pneumonia, peptic ulcer and alzheimer disease have potential therapeutic value during eye treatment,
Thus it is accomplished the present invention.
The present invention provides a kind of polypeptide, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO.1, or the polypeptide
It is substitution, missing or the addition that polypeptide of the amino acid sequence as shown in SEQ ID NO.1 passes through one or several amino acid residues
Polypeptide obtain and that activity is adjusted with M-ChR M1 hypotypes.
The polypeptide of the present invention can effectively be combined with M1 acceptors and its activity is adjusted, to M-ChR M1 hypotypes
The illness of mediation, particularly glaucoma treatment when complicated cataract, chronic obstructive pneumonia, peptic ulcer and alzheimer
Disease has potential therapeutic value.
It is preferred that the amino acid sequence of the polypeptide is as shown in SEQ ID NO.1~16.
The present invention also provides a kind of nucleic acid for encoding the polypeptide described in claim 1 or 2.
It is preferred that the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO.17~32.
The present invention also provides a kind of recombinant vector containing above-mentioned nucleic acid.
The present invention also provides a kind of host cell for being converted or being transfected with above-mentioned recombinant vector.
The present invention also provides a kind of preparation method of aforementioned polypeptides, including with above-mentioned host cell expression aforementioned polypeptides, so
After separate aforementioned polypeptides.
The present invention also provides a kind of pharmaceutical composition, and it includes aforementioned polypeptides and pharmaceutically acceptable auxiliary material or load
Body.
In described pharmaceutical composition, the polypeptide is the first active component, and described pharmaceutical composition also includes cholinesterase
At least one of depressant, bronchodilators, gastric acid secretion depressant and cereboactive drug are used as the second active component.
Preferably, described pharmaceutical composition includes aforementioned polypeptides and is mutually conjugated or mixes and can keep with the polypeptide
The stable preparation of the polypeptide active.
It is preferred that the preparation is nano material, liposome or oiliness compound.
The present invention also provides a kind of aforementioned polypeptides in the medicine of illness for the treatment of M-ChR M1 hypotype mediations is prepared
Purposes.
It is preferred that complicated cataract when the illness is glaucoma treatment, chronic obstructive pneumonia, peptic ulcer and Ah
The silent disease in Wurz sea.
Brief description of the drawings
Fig. 1 SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.4 and SEQ ID NO.5 polypeptides are put with M1 acceptors
Penetrate aglucon experimental result；
Fig. 2 SEQ ID NO.3-5 polypeptides stimulate carbachol the regulation and control of M1 acceptors high expressing cell release calcium current signal
As a result；
Fig. 3 SEQ ID NO.6-8 polypeptides stimulate carbachol the regulation and control of M1 acceptors high expressing cell release calcium current signal
As a result.
Embodiment
The present invention is further illustrated below in conjunction with accompanying drawing and following embodiments, it should be appreciated that accompanying drawing and following embodiments
The present invention is merely to illustrate, is not intended to limit the present invention.
The polypeptide that can adjust M-ChR M1 hypotypes activity of the present invention, its amino acid sequence is SEQ ID NO.1 institutes
Show disulfide bond be present between two of which cysteine.In addition, the polypeptide of the present invention can also be that its amino acid sequence is SEQ
Polypeptide shown in ID NO.1 is by the substitution of one or several amino acid residues, missing or addition gained, the Guang ammonia of two of which half
Disulfide bond between acid be present, arginine is required conserved amino acid, and the polypeptide is for example as shown in SEQ ID NO.2~16.
In the polypeptide of the present invention, disulfide bond is respectively formed between two Cys amino acid in sequence, constrains simulating peptide topological structure
For cyclic peptide so that simulating peptide can keep stable β-pleated sheet structure, and the structure is the core of simulating peptide and M1 acceptor interactions
Part；0-2 amino acid can be retained respectively by being formed on the outside of the Cys amino acid of disulfide bond, and its inner side can retain 3-18 amino
Acid, the length depending on formed β-pleated sheet structure；Meanwhile the centre position between two Cys amino acid should be by Pro-Arg
Composition, the two amino acid are the Key residues for identifying M1 acceptors, are the necessary components of MT7 simulating peptides.
The polypeptide of the present invention can obtain by the following method.
It is homologous using computer according to the polypeptide toxin MT7 of energy selective control M1 receptor actives special three-dimensional structure
The method that mould is built, using the crystal structure of beta adrenergic as template, construct the 3-D solid structure model of M1 acceptors.Based on MT7
Specific binding between toxin and M1 acceptors, the docking of integrated use protein-protein, molecular dynamics simulation, with reference to from
By that can calculate, amino acid residue Energy Decomposition, normal mode vibration analysis equimolecular analogy method, between MT7 toxin and M1 acceptors
Interaction model carried out comprehensive analysis.Then, using M1 acceptors as target spot, the specificity based on MT7 toxin Yu M1 acceptors
Interaction model, using above-mentioned molecule simulation method, Binding peptide synthesis, radiative aglycone combination Activity determination, cell calcium current
Activation experiment detection method, through Rational drug design, synthesize and filter out a series of polypeptides that can adjust M1 receptor actives.Specifically
It is based on Conjugated free energy calculating and Energy Decomposition analysis before this, determines interaction be present with M1 acceptors in MT7 toxin
Key residues, with reference to the three-dimensional structure of MT7 toxin, design the polypeptide sequence with specific three dimensional structure and surface charge properties
Row, carry out its regulation activity research with the binding activity research of M1 acceptors and to M1 acceptors using these polypeptides.
Radiative aglycone combination Activity determination result shows, the affinity of polypeptide of the invention and M1 M-ChRs with small point
Sub- control drug carbachol (CCh) is close, or even part of polypeptide and the affinity of M1 acceptors exceed carbachol (referring to figure
1).And the polypeptide of the present invention has the function of selective depression M1 acceptors.
Cell calcium current activation experiment testing result shows that polypeptide of the invention is in the Chinese hamster ovary celI of stable expression hM1 acceptors
Energy antagonism agonist carbachol stimulates the release of calcium current (referring to Fig. 2,3).The polypeptide of the present invention is M-ChR M1 hypotypes
Negative sense other structure regulation polypeptide, the other structure regulation polypeptide of this negative sense has the ability of antagonism M-ChR M1 hypotype intrinsic activities.
Therefore, polypeptide of the invention can effectively be combined with M1 acceptors and its activity is adjusted, to M-ChR
M1 hypotypes mediation illness, particularly glaucoma treatment when complicated cataract, chronic obstructive pneumonia, peptic ulcer and A Er
Zi Haimo diseases have potential therapeutic value.
On the other hand, the present invention provides a kind of nucleic acid for separating and purifying, and it can encode any one above-mentioned polypeptide (amino
Acid sequence be SEQ ID NO.1 polypeptide, or amino acid sequence be SEQ ID NO.1 described in polypeptide pass through one or several ammonia
Substitution, missing or the polypeptide for adding gained of base acid residue), its sequence is for example as shown in SEQ ID NO.17~32.
It should be understood that：Various modifications can be carried out in the nucleotide sequence of the nucleic acid of the present invention, as long as the modification does not change
Become the amino acid sequence of the polypeptide by code area translation.And, it might even be possible in the region beyond code area is handled, draw
Enter various modifications or alterations, as long as they do not influence the expression of gene.In other words, the nucleotides of the present invention can be modified
Sequence, at least gained nucleotide sequence have identical or a bioactivity of functional equivalent, and they are also in the model of the present invention
In enclosing.
The present invention also provides expression vector, and it is SEQ ID NO.1 that it, which includes the encoding amino acid sequence of at least one copy,
The nucleotide sequence of shown peptide.The present invention also provides another expression vector, and it includes the coded amino acid sequence of at least one copy
It is classified as polypeptide of the polypeptide described in SEQ ID NO.1 by the substitution of one or several amino acid residues, missing or addition gained
The nucleotide sequence of (such as polypeptide as shown in SEQ ID NO.2~16).
The present invention also provides protokaryon or eukaryotic host cell, the host cell contain above-mentioned expression vector.
The present invention also provides the Pharmaceutical composition of regulation M1 receptor actives, including the medicinal effective dose as active material
Amino acid sequence is that the polypeptide described in SEQ ID NO.1 polypeptide or SEQ ID NO.1 passes through one or several amino acid residues
Substitution, the polypeptide of missing or addition gained, and pharmaceutical carrier, diluent and/or adjuvant.
Pharmaceutically acceptable carrier species available for the present invention includes, physiological saline, sterilized water, Ringer's solution, slow
Rush salt solution, glucose solution, maltodextrin solution, glycerine, ethanol, liposome.They can be used alone or are applied in combination.Root
According to demand, composition can also be further comprising other usually used additives of such as antioxidant, buffer solution.In addition, root
According to mode of administration, composition can be configured to together with diluent, dispersant, surfactant, bonding agent and lubricant
Injection dosage form, such as aqueous solution, suspension, emulsion, or be configured to oral dosage form, as pill, capsule, particle or
Tablet.After being conjugated with the carrier, target organ, tissue specific antibodies or part, active component can be guided to described
Organ or tissue.Generally carrier, excipient or additive known in the art can be used in the present invention, and can be used
Carrier, the species of excipient or additive be not limited to the example.According to purpose or demand, led according to affiliated technology
Usual method, method of administration, therapeutically effective amount used in domain can be by composition as described above or preparations suitably
Give individual.Method of administration can for example have oral, non-bowel, subcutaneous, intraperitoneal, intrapulmonary and intranasal administration.
Preferably, polypeptide of the invention can be with keeping its activity stabilized preparation to be mutually conjugated or mix.The preparation example
Nano material, liposome or oiliness compound in this way.The polypeptide and nano material, liposome Polymer material of the present invention is conjugated
When, the compound generated after conjugated can make the polypeptide of the present invention more stably be transported to target cell in body.The present invention
Polypeptide and the mixture of oiliness compound or a variety of oiliness compounds when mixing, resulting mixture can also make this hair
Bright polypeptide is more stably transported to target cell in body.
The polypeptide of the present invention can be used for preparing the disease mediated medicine for the treatment of M1 acceptors.The disease includes but unlimited
In：Complicated cataract, chronic obstructive pneumonia, peptic ulcer and alzheimer disease during glaucoma treatment.
The polypeptide of the present invention not only can adjust M1 receptor actives when being used alone, and treat the disease that M1 acceptors are mediated,
And can be with clinically conventional corresponding disease therapeuticing medicine such as cholinesterase inhibitor, bronchodilators, gastric acid secretion
Depressant and cereboactive drug etc. share, and can show synergistic therapeutic action to disease.
The polypeptide of the present invention can be obtained by biological method, such as with above-mentioned host cell expression polypeptide, and it is entered
Row separation.The polypeptide of the present invention can also use the method (such as solid-phase synthesis) of chemical synthesis to be prepared, and purity is high, point
Small, the high specificity of son amount, non-immunogenicity, securely and reliably.
The invention will now be further described with reference to specific embodiments, advantages of the present invention and feature will be with description and
It is apparent.But these embodiments are only exemplary, do not form any restrictions to the scope of the present invention.People in the art
Member to the details and form of technical solution of the present invention it should be understood that can enter without departing from the spirit and scope of the invention
Row modifications or substitutions, but these modifications and replacement are each fallen within protection scope of the present invention.
The design of 1 polypeptide of the present invention of embodiment
The Interactions Mode combined according to MT7 polypeptides with M1 receptor-specifics, corresponding to the binding domain of MT7 polypeptides, sheet
Inventor designs a plurality of MT7 simulating peptides that can regulate and control M1 acceptors, and the sequence of which part polypeptide is as follows：
It is the amino acid sequence shown in SEQ ID NO.1；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.2；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.3；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.4；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.5；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.6；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.7；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.8；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.9；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.10；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.11；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.12；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.13；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.14；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.15；Particular sequence is described as：
It is the amino acid sequence shown in SEQ ID NO.16；Particular sequence is described as：
Aforementioned polypeptides are the simulating peptide of MT7 toxin, and disulfide bond is respectively formed between two Cys amino acid in its sequence, are constrained
Simulating peptide topological structure is cyclic peptide so that simulating peptide can keep stable β-pleated sheet structure, and the structure is simulating peptide and M1 acceptors
The core of interaction；0-2 amino acid can be retained respectively by being formed on the outside of the Cys amino acid of disulfide bond, can be with the inside of it
Retain 3-18 amino acid, the length depending on formed β-pleated sheet structure；Meanwhile the interposition between two Cys amino acid
Putting should be made up of Pro-Arg, and the two amino acid are the Key residues for identifying M1 acceptors, be the necessary composition portion of MT7 simulating peptides
Point.
Embodiment 2：The synthesis of polypeptide of the present invention
1) laboratory apparatus and material：
Dimethylformamide (DMF), piperidines, resin, dichloromethane a heatable brick bed (DCM), Kaiser kits, I-hydroxybenzotriazole
(HOBt), tetramethylurea hexafluorophosphate (HBTU), diisopropylethylamine (DIEA), methanol, various amino acid, Solid-phase Polypeptide close
Cheng Guan；
2) experimental procedure：
Weigh resin and put into Solid-phase synthesis peptides pipe (hereinafter referred to as reactor), add appropriate DMF swellings 10
Minute.Take out DMF and carry out Fmoc deprotection reactions, i.e., plus in right amount contain the DMF solution of 20% piperidines, be sufficiently stirred, taken out after 5 minutes
Fall solution, the DMF solution for rejoining 20% piperidines deprotects 7 minutes.Deprotection liquid is taken out, resin is washed 4-5 times with DMF,
DMF is taken out, is washed 1-2 times with DCM, takes a small amount of resin (about 5~10mg) to be washed 2 times with ethanol in test tube from reactor,
Kaiser methods detect and record color, prepare to feed intake, and are reacted into amino acid condensation.Respectively according to SEQ ID NO.1-SEQ ID
The amino acid sequence order of NO.16 peptides takes corresponding amino acid, HOBt to be dissolved in appropriate container with DMF, adds after dissolving completely
Enter DIEA to activate 5 minutes, add load weighted HBTU, dissolve activation 5 minutes under ice-water bath, put into reactor, stirring is anti-
Should.After 90 minutes, a small amount of resin is taken from reactor in test tube, is washed 2 times with ethanol, the detection of Kaiser methods.Take out reaction
Liquid in device, washed 2 times with DMF, take out DMF, obtain the peptide resin after first amino acid condensation.To gained peptide resin weight
It is multiple to carry out " Fmoc deprotection --- amino acid condensation " reactions steps above, finished to last amino acid reaction.React
Bi Hou, DCM washing resin 2-3 times, takes out solvent, adds methanol stirring and shrinks (5min+5min), takes out methanol, continue to drain
15-20min.The peptide resin synthesized is taken out in reactor, round-bottomed flask is transferred to, is drained in drier, split at room temperature
Solution two hours.Solution after resin filter is concentrated under vacuum lyophilized.Crude product peptide 5.0mg is dissolved in 0.5mL ultra-pure waters, with full
It is 7 to adjust pH with sodium bicarbonate aqueous solution, adds 0.01mL hydrogen peroxide, shaken at room temperature, and RP-HPLC monitors reaction process,
20min reaches reaction end, with vinegar acid for adjusting pH value to 2~3 terminating reactions so that crude product polypeptide aoxidizes to be formed with two
The polypeptide of the present invention of sulfide linkage.Thick peptide is further reverse HPLC-purified using preparative, ensures HPLC detection purity ＞ 98%, obtains
Serial No. SEQ ID NO.1-SEQ ID NO.16 peptide.Resulting pure peptide is reflected using mass spectrum (MS, electrospray)
Fixed, the relevant physicochemical character of SEQ ID NO.1-SEQ ID NO.16 peptides is as shown in table 1.
The relevant physicochemical character of table 1.SEQ ID NO.1-SEQ ID NO.16 peptides
Embodiment 3：Radioligand bind assay
1) laboratory apparatus and material：
CO2gas incubator, laboratory ultrapure water system, Biohazard Safety Equipment, low-temperature and high-speed centrifuge, Ultrasound Instrument, analysis
Balance, 96 orifice plate nutsch filters, liquid scintillation instrument, constant water bath box, [3H] NMS radioligands, carbachol, strychnia, atropine,
PEI, glass fiber filter；
2) experimental procedure：
The culture of CHO-M1 cells：Every bottle of cell 5mL nutrient solution, 1:7 passages (105Cells/mL), 48h is covered with.Discard
After nutrient solution, 2mL PBS cells, 0.5mL pancreatin is added after discarding.After about digesting 1min, it is seen that cellular layer crushes, and adds
3mL fresh mediums.Blow even rear passage or kind ware, every bottle of biography 0.5mL cell suspension.If 10cm culture dishes are planted, in culture dish
Middle addition 9mL fresh mediums, and 1mL cell suspensions are added, blow even.48~72h of cell in culture dish is grown to 80%,
Cell membrane can be collected；
The preparation of memebrane protein：Cell is washed twice with ice-cold KHB buffer solutions, is added KHB buffer solution 2mL, is gently scraped cell,
Cell is collected in 15mL centrifuge tubes, 3000rpm centrifuges 5min at 4 DEG C, and cell is resuspended.Ice-water bath ultrasonication, ultrasound condition
For：20% peak power, suspend 3 seconds within 5 seconds per ultrasound, 12000rpm centrifugations 20min, is resuspended in suitable at continuous ultrasound 5 times, 4 DEG C
In amount KHB buffer solutions (in terms of every μ L of ware 200), memebrane protein is obtained, often the μ L of pipe 600 are dispensed, and -80 DEG C freeze.Protein quantification uses
BCA methods；
Radioligand saturation experiment：The memebrane protein frozen is taken out, with ice-cold KHB buffer solutions by 1 after melting:8
Dilution, pressed in the orifice plate of U-shaped bottom 96 and memebrane protein dilution (equivalent to the μ L of memebrane protein stoste 10) is added per the μ L of hole 80, then added successively
Entering the KHB buffer solutions of different volumes and [3H] NMS radioligands makes constant total volume be 200 μ L.In saturation experiment, each
Hole sequentially adds the μ L of KHB buffer solutions 110,100,80,60,40,20,0, sequentially adds 10,20,40,60,80,100,120 μ L
[3H] NMS radioligand working solutions supply 200 μ L volumes, [3H] NMS radioligand working solutions concentration of addition is 100pCi/
μ L, about 1.17nM ([3H] NMS radioligands original liquid concentration is 1 μ Ci/ μ L, 85.5Ci/mmol, 11.7 μM).Set [3H]
NMS radioligand concentration is incremented by the range of 0.0585~0.702nM；In non-specific binding experiment, each hole adds film
The μ L of diluted protein solution 80,100 μM of μ L of Atro solution 20 (10 μM of final concentration), then sequentially add in each hole KHB buffer solutions 80,
60th, 40,20,0 μ L, 20,40,60,80,100 μ L [3H] NMS radioligand working solutions is sequentially added and supply 200 μ L volumes, altogether
5 non-specific binding concentration gradients are set, and [3H] NMS radioligand concentration is incremented by the range of 0.12~0.58nM.Instead
After answering mixture to be incubated 30min in 37 DEG C of water-baths, it is placed in 96 orifice plate Vacuum filtration devices after cleaning and filters removal supernatant
Liquid, memebrane protein and ligand trapses in connection are made (to be tied in advance with 0.3%PEI is dipped to reduce non-specificity to 96 orifice plate films
Close) on, get filter membrane express developed with ultra-pure water and remove free ligand three times, remove filter membrane and be placed in 100 DEG C of oven for baking 4min, it is complete
Full drying filter membrane takes out filter membrane after becoming fragile, and adds 1~2mL scintillation solutions, sealer, cpm numbers is counted on liquid scintillation counter；
Radioligand competion experiment：In competion experiment, the dilution of 80 μ L memebrane proteins is added per hole in 96 orifice plates
Liquid, μ L of [3H] NMS radioligands working solution 80, the μ L of KHB buffer solutions 20 and the μ L of polypeptide gradient solution of the present invention 20 to be measured, cumulative volume
Keep 200 μ L.When determining total binding value, 20 μ L polypeptide solutions of the present invention are substituted for 20 μ L KHB buffer solutions, remaining volume is not
Become；When determining non-specific binding, 20 μ L polypeptide solutions of the present invention are substituted for 100 μM of 20 μ L atropine solution.As sun
Property control carbachol (CCh gradient is arranged to 1 μM~100mM, totally 6 concentration；The concentration of polypeptide of the present invention is 100nM
~10mM, totally 6 gradient concentrations.It is incubated and post processing is consistent with Binding experiment；
Data processing：Data analysis passes through GraphPad Prism (ver.5.01；San Diego, CA) software completion.
Saturation experiment result is first using unit point band Hill Coefficient Fittings, then be total to using unit point total binding with non-specific binding
Fitting, calculate Hill coefficients (Hill), Bmax (Bmax) and equilibrium dissociation constant (Kd) when acceptor combines；It is competing
Strive Binding experiment result and only calculate the radiative aglycone combination rate added after polypeptide of the present invention, draw response curve and be compared；
In the analysis, adding relatively low radiative aglycone combination rate after polypeptide of the present invention represents test polypeptide to M1 acceptors
With compared with high binding affinity.Have found, the polypeptide of the present invention tested in the analysis in the analysis with M1 M-ChRs
Affinity it is close with small molecule control drug carbachol (CCh).More generally, part of polypeptide and the affinity of M1 acceptors are found
Power exceedes carbachol, SEQ ID NO.1, SEQ ID NO.10, SEQ ID NO.4 and SEQ ID NO.5 and carbachol parent
Comparing result is as shown in Figure 1 with joint efforts.The knot similar to SEQ ID NO.1 polypeptides is obtained with other polypeptides of the present invention
Fruit.
Embodiment 4：The calcium current release regulation and control analysis of M1 acceptors
1) laboratory apparatus and material：
CO2gas incubator, laboratory ultrapure water system, Biohazard Safety Equipment, carbachol, strychnia, atropine,
Multi-function microplate reader, the quick dyestuff of calcium (Calcium 4), probenecid；
2) experimental procedure：
Cell culture and bed board：Cell is taken out from CO2 incubators, is observed under the microscope, cell is bright, adherent equal
It is even, during density suitable (80% degree of converging), it is adapted to bed board.The culture medium in blake bottle is discarded, adds the washing of 5mL PBSs
Cell, ensure that each place touches PBS, discard PBS.1mL 0.05%Trypsin-EDTA are added,
About 1min is placed, micro- Microscopic observation cell, after most of change is circular, adds 3mL MEM full nutrient solutions, is blown and beaten to single thin
Born of the same parents.Cell count is carried out using cell counter, appropriate MEM full nutrient solutions is added, cell density is adjusted to 8 × 105cells/
mL.The cell of above-mentioned density is layered in the porocyte culture plates of black 96,75 or 100 μ L/well, is transferred to the training of CO2 incubators
Support 12~24h；
Polypeptide active measure of the present invention：The volume of the quick dyestuff of calcium of needs is calculated by the gauge in 75 or 100 μ L/ holes, is used
HBSS buffer solutions dissolve the dyestuffs of Calcium 4, and adding probenecid storing solution (500mM, 1N NaOH dissolvings are prepared) makes its concentration
For 5mM.In activator screening experiment, the quick dyestuff of the calcium prepared is added to (100 μ L in the cell of 96 orifice plates by 100 μ L/ holes
Cells/ holes).Plank is transferred to CO2 incubators, 30min is incubated, is then incubated at room temperature 15~30min again, is utilized
The ligand solution of gradient concentration is added 96 orifice plate kinds and carries out intracellular calcium current inspection by Flexstation III multi-function microplate readers
Survey.In other structure adjusts screening experiment, the calcium ion dyestuff prepared is added to (75 μ L in the cell of 96 orifice plates by 75 μ L/ holes
Cells/ holes), then the polypeptide solution of the present invention of fixed concentration is added in the cell of 96 orifice plates (by 50 μ L/ holes).By plank
CO2 incubators are transferred to, 30min is incubated, is then incubated at room temperature 15~30min again, it is using Flexstation III that gradient is dense
The CCh solution of degree, which is added in 96 orifice plates, carries out intracellular calcium current detection, and excitation wavelength is 485nm during detection, and Detection wavelength is
525nm, 2~3min of METHOD FOR CONTINUOUS DETERMINATION, one group of data can be obtained per 1.52s；
Data processing：Intracellular calcium current regulation and control result is mapped, EC50 values are obtained after non-linear S curve fitting, and
CCh concentration dependant response curve is normalized CHO-M1 cells after polypeptide of the present invention is acted on, and passes through fitting
EC50 offsets characterize other structure adjustment effect of the polypeptide of the present invention to M1 acceptors；
In the analysis, relatively low EC50 skews multiple represents that test polypeptide has other structure agonist activity to M1 acceptors, compared with
High EC50 skew multiples represent that test polypeptide has other structure antagonistic activity to M1 acceptors.It is expected that polypeptide of the present invention is expressed stable
Energy antagonism agonist carbachol stimulates the release of calcium current in the Chinese hamster ovary celI of hM1 acceptors, and SEQ ID NO.3-8 peptides are to M1 acceptors
Regulation and control result as shown in Fig. 2-3 and table 2.
2. polypeptide of the present invention of table is to CCh excitement M1 acceptors EC50The regulation and control of value
| Sample | Concentration | EC50(nM) | EC50Deviation ratio |
| Control | 100μM | 151 | 1 |
| Strychnia | 100μM | 51.3 | 0.340 |
| SEQ ID NO.3 | 100μM | 233 | 1.542 |
| SEQ ID NO.4 | 100μM | 230 | 1.520 |
| SEQ ID NO.5 | 100μM | 246 | 1.628 |
| SEQ ID NO.6 | 100μM | 231 | 1.530 |
| SEQ ID NO.7 | 100μM | 191 | 1.263 |
| SEQ ID NO.8 | 100μM | 290 | 1.920 |
The polypeptide of the present invention can effectively be combined with M1 acceptors and its activity is adjusted, to M-ChR M1 hypotypes
The illness of mediation, particularly glaucoma treatment when complicated cataract, chronic obstructive pneumonia, peptic ulcer and alzheimer
Disease has potential therapeutic value.
Claims (11)
- A kind of 1. polypeptide, it is characterised in that the amino acid sequence of the polypeptide such as SEQ ID NO. 5 or SEQ ID NO. 6 institutes Show.
- A kind of 2. nucleic acid for encoding the polypeptide described in claim 1.
- A kind of 3. recombinant vector of the nucleic acid containing described in claim 2.
- 4. a kind of recombinant vector with described in claim 3 converts or the host cell of transfection.
- 5. the preparation method of the polypeptide described in a kind of claim 1, it is characterised in that including with the host described in claim 4 Polypeptide described in cell expression claim 1, then separates the polypeptide described in claim 1.
- 6. a kind of pharmaceutical composition, it is characterised in that including the polypeptide described in claim 1 and pharmaceutically acceptable auxiliary Material or carrier.
- 7. pharmaceutical composition according to claim 6, it is characterised in that the carrier is mutually conjugated or mixed with the polypeptide Close and the stable preparation of the polypeptide active can be kept.
- 8. pharmaceutical composition according to claim 7, it is characterised in that the preparation is nano material, liposome or oil Property compound.
- 9. pharmaceutical composition according to claim 6, it is characterised in that the polypeptide is the first active component, the medicine Compositions also include at least one of cholinesterase inhibitor, bronchodilators and gastric acid secretion depressant and are used as the Two active components.
- 10. the polypeptide described in a kind of claim 1 is preparing the illness for the treatment of M-ChR M1 hypotypes abnormal activation mediation Purposes in medicine.
- 11. purposes according to claim 10, it is characterised in that the complicated cataract when illness is glaucoma treatment, Chronic obstructive pneumonia and peptic ulcer.
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| CN101745097A (en) * | 2008-12-12 | 2010-06-23 | 海南大学 | Alpha-conotoxins from Hainan province for specific blockage of acetylcholine receptor and application thereof |
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| CN101745097A (en) * | 2008-12-12 | 2010-06-23 | 海南大学 | Alpha-conotoxins from Hainan province for specific blockage of acetylcholine receptor and application thereof |
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