CN109608534B - A kind of cortical actin mutant 8KQ and application - Google Patents
A kind of cortical actin mutant 8KQ and application Download PDFInfo
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Abstract
The present invention provides a kind of cortical actin mutant 8KQ and its applications, belong to protein and peptide technical field, and the cortical actin mutant 8KQ has the amino acid sequence as shown in SEQ ID No:1.The cortical actin mutant 8KQ is that 8 lysine sites K107, K152, K171, K181, K193, K235, K309 and K314 of cortical actin (NP_031829.2) are sported glutamine, the charge characteristic under simulated albumin matter Acetylation status.Cortical actin mutant 8KQ of the present invention can culture cell in successful expression, can reduce and F-actin binding ability；It can be used for improving nerve synapse form；The repetition stereotypic behavior of self-closing disease disease model mouse can be improved, rapid-action, effect is obvious.
Description
Technical field
The invention belongs to protein and peptide technical field more particularly to a kind of cortical actin mutant 8KQ and its answer
With.
Background technique
Autism spectrum disorder (autism spectrum disorder, ASD) is a kind of neurodevelopmental disorder, can be led to
Cross clinical manifestation diagnosis.Newest " mental disease diagnostic & statistical manual " the 5th edition concludes the diagnostic criteria of ASD are as follows: society
Meeting communication disorder repeats mechanical movement or interest, as: seldom being exchanged with people, hiding interlocutor's sight, like repetition rotation
Toy etc..Other than above-mentioned core symptom, ASD patient is also possible to show other atypical accessory symptoms, such as different journeys
Dysnoesia, insomnia, anxiety, more dynamic, epilepsies of degree etc..It is about 1~2 ‰ in the disease incidence of ASD in 2000.But as diagnosis is marked
Quasi- and diagnostic mode improvement, developing country's children's ASD disease incidence has reached 1.5% within 2017, and clinic is mostly with row at present
Based on intervening, the drug for developing ASD treatment potential is particularly important.
The pathogenic factor of ASD is indefinite, and research thinks related with gene genetic and environmental factor at present.Hereditary variation is presented
Height heterogeneity limits ASD study of incident mechanism and clinical medicine research and development.Clinical case observation and disease animal model research
Showing ASD, there are serious nerve synapse dysfunctions.Many ASD candidate tumor susceptibility genes concentrate on excitatory synapse site, packet
Include the encoding genes such as cynapse scaffolding protein, receptor, cell adhesion molecule and cytoskeleton regulatory protein and their upstream tune
Gene is saved, these Genetic Variations directly affect dendritic spines form and structure, to change excitatory synapse number and intensity, most
Lead to synaptic function disorder eventually.SHANK3 (SH3 and multiple ankyrin repeat domains 3) gene mutation
It is first most gene associated with congenital ASD that are reported and are reported, it encodes postsynaptic scaffolding protein, leads to
It crosses and forms albumen composition with postsynaptic densification albumen 95 (PSD95), Homer, anchoring glutamate receptor is maintained in postsynaptic membrane
The stabilization of synaptic function.SHANK3 and cortical actin interact under physiological condition, maintain excitatory synapse form and knot
The stabilization of structure.It is acetylated form in the cortical actin of cynapse distribution, when its Acetylation Level reduces, cuts down cynapse and pass
The amplitude passed hinders information transmitting between neuron.The prior art focuses mostly in gene water to the research of ASD candidate's tumor susceptibility gene
It is flat.
Summary of the invention
In view of this, it is an object of the invention to a kind of cortical actin mutant 8KQ and its applications；The cortex flesh
Filamentous actin mutant 8KQ is effectively improved excitatory synapse morphosis and function by the cortical actin of simulation acetylation,
Play the role of neuroprotection.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical schemes:
A kind of cortical actin mutant 8KQ, the cortical actin mutant 8KQ have such as SEQ ID No:1
Shown in amino acid sequence.
The present invention provides the genes for encoding the cortical actin mutant 8KQ, have as shown in SEQ ID No:2
Nucleotide sequence.
The present invention provides a kind of recombinant viruses, gene and disease including encoding the cortical actin mutant 8KQ
Poisonous carrier.
Preferably, the viral vectors is adeno-associated virus or slow virus.
Preferably, the adeno-associated virus is 9 type of adenovirus.
The present invention provides the cortical actin mutant 8KQ to prepare the application in nerve protection medicine.
Preferably, the neuroprotection shows as supplement cortical actin acetylation, maintains excitatory synapse form knot
Structure and function-stable.
The present invention provides the cortical actin mutant 8KQ to treat the application in self-closing disease drug in preparation.
Preferably, the applicable object of the self-closing disease drug is self-closing disease disease model mouse.
Preferably, the self-closing disease drug is ejection preparation.
Beneficial effects of the present invention: cortical actin mutant 8KQ provided by the invention has such as SEQ ID No:1
Shown in amino acid sequence；The cortical actin mutant 8KQ is by 8 of cortical actin (NP_031829.2)
Lysine sites K107, K152, K171, K181, K193, K235, K309 and K314 sport glutamine, simulated albumin matter second
Charge characteristic under acylated state.Cortical actin mutant 8KQ of the present invention can culture cell in successful expression,
It can reduce and F-actin binding ability；The cortical actin mutant 8KQ is constructed in slow virus carrier
On can harvest virion, obtain and largely continue acetylation cortical actin mutant, for improving nerve synapse form；It will
In gland relevant viral vector, intracerebral injection is effectively improved self-closing disease disease model for the cortical actin mutant 8KQ building
The repetition stereotypic behavior of mouse, rapid-action, effect is obvious.
The present invention will encode the genetic recombination of the cortical actin mutant 8KQ to adeno-associated virus or slow virus
In, it can be realized the great expression of cortical actin mutant 8KQ, scale industrial production is applied to improve Synaptic Morphology
With dysfunction pharmaceutical composition, it is used to prepare neuroprotection class drug.
Detailed description of the invention
Fig. 1 is that excitatory synapse expresses acetylation cortical actin, wherein (A) is the neuron of in vitro culture
(DIV21) immunofluorescence dyeing picture；(B) it is analyzed for the common location of PSD95 and cortactin, ac-cortactin；
Acetylation Level after Fig. 2 is incubated for jointly for the Ac-CoA of cortical actin and various concentration；Left figure is western
The expression of the acetylation cortical actin of blot detection, coomassie brilliant blue staining show substrate cortex Actin
Amount；Right figure is the statistical chart of acetylation cortical actin relative level in western blot result, defining contrast group acetyl
Changing cortical actin level is 100%, and data source is in independent experiment three times；
Fig. 3 is the identification of cortex Actin acetylation sites, wherein (A) is that LC-MS/MS identifies to obtain the dynamic egg of cortex flesh
8 lysines are acetylation in white repeat region；(B) the cortical actin mutant of HA label is overexpressed for HEK293 cell
8KQ and western blot detects plasmid expression；
Fig. 4 is F-actin combination sxemiquantitative experimental result picture；
Fig. 5 is the influence for being overexpressed 8KQ to neuronal excitability postsynaptic currents, wherein (A) is the nerve for being overexpressed 8KQ
For member compared with the neuronal excitability postsynaptic currents of control are whole, (B) is the Amplitude Comparison of electric current, and (C) is the frequency of electric current
Rate compares；
Fig. 6 is that the analog form of acetylation cortical actin is influenced on mouse ASD sample behaviouristics is improved；Wherein (A) is
Adeno-associated virus Naoliqing capsule injects schematic diagram；It (B) is adeno-associated virus injection and Behavior test arrangement of time schematic diagram；
(C) it repeats to compare in mechanical experiment from mao time of supervising the cooking for every group of mouse.
Specific embodiment
The present invention provides a kind of cortical actin mutant 8KQ, the cortical actin mutant 8KQ to have such as
Amino acid sequence shown in SEQ ID No:1, specific as follows:
Wherein the site with underscore is rite-directed mutagenesis site；The cortical actin mutant
8KQ is by 8 lysine sites of cortical actin (NP_031829.2, amino acid sequence is as shown in SEQ ID No:3)
K107, K152, K171, K181, K193, K235, K309 and K314 sport glutamine, under simulated albumin matter Acetylation status
Charge characteristic.Residue in lysine (K) usually occurs for the acetylation of protein, can under the catalysis of transacetylase, with
Covalent bond, i.e. charging neutrality occur for acetyl donor acetyl coenzyme A (Ac-CoA).The present invention sports the K of pI=9.74
The glutamine (Q) of pI=5.65, i.e. charging neutrality under simulation Acetylation status, obtain the acetylation shape of cortical actin
Formula 8KQ.
In the present invention, the expression plasmid including the cortical actin mutant 8KQ can be cultivated in vitro
Successful expression in HEK293 cell can reduce and F-actin binding ability；The cortical actin is mutated
Body 8KQ building can harvest virion on slow virus carrier, obtain and largely continue acetylation cortical actin mutant, use
In improvement nerve synapse form；The cortical actin mutant 8KQ is constructed in gland relevant viral vector, intracerebral injection has
Effect improves the repetition stereotypic behavior of self-closing disease disease model mouse, rapid-action, and effect is obvious.
The present invention also provides the genes for encoding the cortical actin mutant 8KQ, have such as SEQ ID No:2 institute
The nucleotide sequence shown；It is specific as follows:
The gene of the coding cortical actin mutant 8KQ is to pass through
The gene (NM_007803.5, nucleotide sequence is as shown in SEQ ID No:4) for encoding cortical actin is subjected to rite-directed mutagenesis
Obtaining the wherein site with underscore afterwards is mutational site；" C " is sported by " A ".
The present invention provides a kind of recombinant virus, gene and viral vectors including the cortical actin 8KQ.At this
In invention, the viral vectors is preferably adeno-associated virus or slow virus；The adeno-associated virus is preferably 9 type of adenovirus
AAV9, the 9 type AAV9 of adenovirus being capable of the special high expression in nervous system；The slow virus carrier is preferably GV509.This
Invention is not particularly limited the source of the adeno-associated virus and slow virus, using this field conventional commercial product.This
Invention does not have particular/special requirement to the preparation method of the recombinant virus, and the preparation method using the recombinant virus of this field routine is
It can.In specific implementation process of the present invention, the gene order of the coding cortical actin mutant 8KQ is connected to carrier
Recombinant virus is obtained between the restriction enzyme site BamHI and AfeI of GV509.In the present invention, the cortical actin will be encoded
The genetic recombination of mutant 8KQ can be realized a large amount of of cortical actin mutant 8KQ into adeno-associated virus or slow virus
Expression, scale industrial production are applied to improve Synaptic Morphology and dysfunction pharmaceutical composition, are used to prepare neuroprotection class medicine
Object.
The present invention also provides the cortical actin mutant 8KQ to prepare the application in nerve protection medicine.
In the present invention, the cortical actin mutant 8KQ being capable of charge under the Acetylation status of simulated cortical actin
Feature can be effectively improved excitatory synapse morphosis and function, play the role of neuroprotection.In the present invention, the mind
It is protected and preferably shows as supplement cortical actin acetylation, maintain excitatory synapse morphosis and function-stable.
The present invention also provides the cortical actin mutant 8KQ to treat answering in self-closing disease drug in preparation
With.The present invention is not particularly limited the applicable object of the self-closing disease drug, in embodiments of the present invention preferably with self-closing disease
Disease model mouse is object.The present invention is not particularly limited the dosage form of the self-closing disease drug, normal using this field
Dosage form is advised, in embodiments of the present invention preferably ejection preparation.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood
For limiting the scope of the present invention.
Embodiment 1
Excitatory synapse expresses acetylation cortical actin
In advance with poly-D-lysine (PLL) coating culture dish or glass slide, room temperature coating is stayed overnight or 37 DEG C of coating 2h.Recycling
PLL, rinsed with sterile water 3 times, culture dish or glass slide after coating are placed in 4 DEG C and can be reserved for 1 week.HBSS buffer is pre-chilled, and infuses
Operation keeps cryogenic sterile before meaning digestion.Birth same day suckling mouse ice is swooned, 75% alcohol disinfecting takes rapidly brain.It is shelled in HBSS
From hippocampus/cortical tissue, gently it is transferred in the EP pipe equipped with fresh HBSS.It is cut into small pieces, 37 DEG C of pancreatin (0.125%) digestion
During which 30min tissue block is softly resuspended every 10min.With containing 10%FBS F12 culture medium terminate digestion, 800rpm from
Heart 1min, removes supernatant.F12+10%FBS culture medium is added to be resuspended, 37 DEG C of standing 5min.Cell mass is softly blown and beaten with pipettor
Block stands 2min, and supernatant is neuron suspension.Cell count, bed board.Culture for 24 hours afterwards changes culture medium into
Neurobasal (Gibco#10888022) culture medium, and add 2%B27 (Gibco#17504044) and 1%Glutamax
(Gibco#35050061), and every 2 days, half amount changes liquid.
In vitro culture neuron, the temperature to be aspired for stability of condition of culture (37 DEG C), stable CO2It is horizontal (5%), higher
Relative saturation humidity (95%) expresses cynapse marker protein PSD95, referred to as PSD95 until being within 21 days (DIV21) mature neuron
Aggregation.Postsynaptic densification albumen 95 (PSD95) is excitatory synapse marker.Using immunofluorescence dyeing, cortex is marked respectively
Actin (cortactin) and acetylation cortical actin (ac-cortactin) are in postsynaptic expression position.
Immunofluorescence dyeing concrete operations are as follows: the neuron PBS buffer solution of apposition growth on the cover slip is rinsed 3
It is secondary, 5min/ times.The fixed 10min of PFA (4%) room temperature of pre-cooling, PBS rinsing 3 times, 5min/ times.(PBS matches 0.2%Triton
System) room temperature permeabilization 10min, PBS rinsing 3 times, 5min/ times.5%BSA (PBST preparation) room temperature closes 30min, primary antibody (confining liquid
Prepare) 4 DEG C be incubated overnight.PBST is rinsed 3 times, 10min/ times.Secondary antibody (confining liquid preparation) is incubated at room temperature 1.5h.PBST rinsing 3
It is secondary, 10min/ times, mounting.Laser co-focusing (Zeiss LSM 780) obtains immunofluorescence results such as Figure 1A, and ImageJ can be analyzed
The common location situation of cortical actin and acetylation cortical actin and PSD95, by common location coefficient (overlap
Coefficient it) indicates.Cortical actin (cortactin) and acetylation cortical actin (ac-cortactin) exist
Postsynaptic expression has a common location with PSD95, immunofluorescence dyeing as the result is shown acetylation cortical actin in postsynaptic content
It is higher.
Embodiment 2
Research shows that the acetylation of cortical actin is adjusted by transacetylase (PCAF) and deacetylase (HDAC6)
Section, is likely present non-enzymatic acetylation.Acetyl coenzyme A (Ac-CoA) is the unique next of acetyl group needed for histone acetylation
Source, the acetylation modification that intracellular Ac-CoA concentration influences protein are horizontal.To inquire into this possibility and its biological significance,
External acetylation experiment is carried out.Reaction buffer: Tris-HCl (50mM, pH8.0), EDTA (0.1mM), DTT (1mM) are sweet
Oily (10%), sodium butyrate (10mM) use preceding addition protease inhibitors.Reaction substrate: cortical actin, acetyl donor
Ac-CoA.By reaction system in 30 DEG C of incubation 1h, obtained reaction product is used for Western blot antibody test, used
Antibody is general acetylation antibody acetylated-lysine；The additional amount of substrate cortical actin is by coomassie brilliant blue staining
It indicates.In vitro in acetylization reaction, found after the Ac-CoA of cortical actin and various concentration is incubated for jointly, with
Raised trend is also presented in the raising of Ac-CoA concentration, cortical actin Acetylation Level, and there is the two significant be positively correlated (to scheme
2), illustrate that cortical actin acetylation and Ac-CoA level are closely related.
Embodiment 3
The identification of cortical actin acetylation sites
In order to further inquire into effect of the cortical actin acetylation in post-synaptic structures and function point analysis, utilize
LC-MS/MS method identifies the acetylation sites of cortical actin in external acetylation experiment (Fig. 2).Coomassie brilliant blue is contaminated
The SDS adhesive tape for the cortical actin position that color is shown is cut, and is completed by Hangzhou Jing Jie Biotechnology Co., Ltd subsequent
LC-MS/MS process.Detailed process is as follows: adhesive tape being shredded, 50% acetonitrile of ammonium hydrogen carbonate containing 50mM (NH4HCO3) is used
(acetonitrile) blob of viscose is decolourized.5min is incubated for using 100% acetonitrile to be dehydrated blob of viscose, removes liquid phase in system later,
Dithiothreitol (DTT) (dithiothreitol) solution of final concentration of 10mM, 37 DEG C of incubation 60min are added.Reuse 100%
Acetonitrile is incubated for dehydration, removes the iodo-acetamide (iodoacetamide) that final concentration of 55mM is added after liquid phase, room temperature, which is protected from light, incubates
Educate 45min.It is cleaned later using the ammonium hydrogen carbonate of final concentration of 50mM, reuses 100% acetonitrile and be incubated for dehydration.Finally use
Blob of viscose is resuspended in 50mM ammonium hydrogen carbonate containing 10ng/ μ l trypsase (trypsin), is incubated for 1h on ice.It is extra in sample to remove
Solution after, by blob of viscose 37 DEG C enzymatic hydrolysis overnight.Peptide fragment after enzymatic hydrolysis successively uses 50% acetonitrile/5% formic acid and 100% acetonitrile
Blob of viscose is extracted, the freezing of peptide fragment solution is spin-dried for rear spare.After peptide fragment is dissolved with mobile phase A, through 1000 ultra high efficiency liquid of EASY-nLC
Phase system is separated.Mobile phase A is the aqueous solution containing 0.1% formic acid and 2% acetonitrile, Mobile phase B be containing 0.1% formic acid and
The aqueous solution of 90% acetonitrile.Liquid phase gradient setting: 0~22min4%~35%B phase；22~27min35%~80%B phase；27
~30min80%B phase, flow velocity maintain 400nL/min.Peptide fragment is injected NSI ion after separating via ultra high efficiency liquid phase systems
It carries out ionizing in source and then be analyzed into Thermo ScientificTM Q ExactiveTM Plus mass spectrum.Ion source electricity
Pressure is set as 2.0kV, and peptide fragment parent ion and its secondary fragment are all detected and analyzed using high-resolution Orbitrap.Level-one
Scanning of the mass spectrum range is set as 350-1800m/z, and scanning resolution is set as 70,000；Orbitrap scanning resolution is set as
17,500.Data acquisition scheme scans (DDA) program using data dependence type, i.e., the selection signal intensity highest after level-one scanning
Preceding 20 peptide fragment parent ions sequentially enter HCD collision cell using 28% crushing energy carry out fragmentation, equally successively carry out two
Grade mass spectral analysis.In order to improve mass spectrographic effective rate of utilization, automatic growth control (AGC) is set as 5E4, and signal threshold value is set as
5000ions/s, maximum injection length are set as 200ms, and the dynamic exclusion time of tandem mass spectrum scanning is set as 15s to reduce
The multiple scanning of parent ion.Second order ms data are retrieved using Proteome Discoverer 1.3.Retrieval parameter setting
As follows: database is set as cortactin protein sequence；Digestion mode is set as Trypsin/P；Leakage enzyme site number is set as 4；
Level-one parent ion quality error tolerance is set as 10ppm；The quality error tolerance of secondary fragment ions is set as 0.02Da；
Fixed modification is set as cysteine alkylation；It is variable to modify the acetylation for being set as lysine, the oxidation of methionine and egg
The acetylation of white N-terminal；The marking of peptide fragment ion requires to be higher than 20, and qualification result peptide confidence is set as High.
As shown in figure 3, share 8 sites be detected with acetylation modification (K107, K152, K171, K181, K193,
K235, K309 and K314).These sites are all in the repeat region structural domain of cortical actin.Utilize change charge
Strategy, the cortical actin mutant 8KQ of design construction simulation acetylation cortical actin form, i.e., 8 second
Acylated residue lysine (K, pI=9.74) is sported negatively charged glutamine (Q, pI=5.65), simulates Acetylation status
Under charging neutrality；Similarly, 8 acetylation residue K are sported arginine similar in isoelectric point (R, pI=10.76), is constructed
Obtain the simulation non-acetylated form cortactin-8KR of cortactin.Mutation process is by site-directed mutagenesis kit
(QuikChange Site-directed Mutagenesis kit) is completed.Plasmid cortactin-8KQ and 8KR are existed
PEI transfection is carried out in HEK293 cell, is extracted cell holoprotein after 48h and is carried out westernblot detection, HA antibody test proves
8KQ and 8KR successful expression (Fig. 3).
Embodiment 4
The biological action of acetylation cortical actin
Cortical actin in conjunction with F-actin and can adjust the nucleation process of actin, influence cytoskeleton activity
Property.This function of cortical actin is influenced by its acetylation modification is horizontal, and when cortical actin acetylation is hindered
Its factor in conjunction with F-actin.When acetylation cortex flesh, which moves egg level, to be reduced, cortex flesh moves egg energy in conjunction with F-actin
Power enhances (Fig. 4).
The operation of F-actin vitro binding assay is as follows: G-actin buffer (G-buffer) and F- needed for preparing experiment
Actin buffer (F-buffer), i.e. G-buffer:Tris-HCl (5mM, pH8.0), CaCl2(0.2mM)、ATP(0.2mM)、
DTT(0.5mM)；F-buffer:Tris-HCl (2mM, pH8.0), CaCl2(0.2mM)、ATP(1mM)、DTT(0.2mM)、KCl
(50mM)、MgCl2(2mM).By the actin protein dissolution of purchase in G-buffer, -80 DEG C of storages.
In vitro culture HEK293 cell transfects 8KQ and 8KR respectively, reject cell culture medium after 48h, is floated with the PBS of pre-cooling
It washes twice, the G-buffer of appropriate volume pre-cooling is added, is incubated for 15min on ice.Neuron is scraped off into culture dish with cell scraper,
Suspension is placed in the glass homogenizer of pre-cooling, is homogenized on ice about 30 times.10000g, is centrifuged 10min, takes supernatant, can make by 4 DEG C
The expression of endogenous cortactin is detected for input.Cell protein solution concentration tune is neat, by 1mg protein solution and 30 μ g
Actin albumen mixes the ice bath 1h in F-buffer.10% (W/V) sucrose solution of 1/4 total volume is added, and (F-buffer matches
System), 100000g, is centrifuged 1h by 4 DEG C.The supernatant (S) obtained at this time is cannot be in conjunction with cortactin in cell protein solution
G-actin is precipitated as the compound of cortactin combination F-actin.To the G-buffer that proper volume is added in precipitating (P),
4 DEG C of incubation 2h, make F-actin be changed into G-actin.The S of acquisition and P are respectively used to Western blot detection, due to reality
It is consistent to test total actin, total cortactin amount in system, so need to only compare what different experiments group be combined with each other with F-actin
The amount of cortactin.
External F-actin combination sxemiquantitative experimental result is shown, compared with wild type cortical actin (WT),
Cortactin-8KQ simulates the cortical actin of acetylation, declines with F-actin binding ability；Conversely, cortactin-
8KR and F-actin binding ability rise (Fig. 4).This is the results show that cortactin-8KQ, 8KR of building can simulate acetyl
Change the function with non-acetylation cortactin.
Embodiment 5
The expression of cortical actin mutant 8KQ does not influence normal neuronal meta function
As method in vitro culture neuron infected slow virus LV-cortactin 8KQ- to the 10th day in embodiment 1
EGFP, Technique of unicell patch clamp records neuron electrophysiological function situation at the 14th day.Using EPC-10/2 amplifier
(HEKA, Germany) carries out Whole-cell recording with voltage-clamp mode.Recording electrode is by borosilicate glass capillary tube
(Warner Instruments) self-control, resistance are 3~5M Ω.Only the full cell patch of series resistance < 15M Ω is for remembering
Record.Film potential is maintained at -70mV, solution composition in electrode: 130mM K-IAO, 1mM EGTA, 5mM Na2ATP, 2mM
MgATP, 0.3mM Na2GTP, 5mM QX-314 and 10mM HEPES (pH 7.3).Bath foam composition: 25mM HEPES (pH
7.3), 128mM NaCl, 30mM glucose, 5mM KCl, 5mM CaCl2,1mM MgCl2,50 μM of D-AP5,20 μM of pockets are male
Red alkali.D-AP5, Bic and QX-314 come from TOCRIS Bioscience, and other chemicals come from Sigma-
Aldrich.When recording mEPSC, 1 μM of tetraodotoxin (TOCRIS Bioscience) is added in bath foam.It uses
PATCHMASTER software (HEKA, Germany) acquisition data, and use MiniAnalysis software (Synaptosoft),
Clampfit (Molecular Devices) and Igor (Wavemetrics) are analyzed.
Slow virus LV-cortactin 8KQ-EGFP can mediate 8KQ to express, and be overexpressed in wild (WT) neuron
8KQ does not have an impact neuronal excitability excitatory postsynaptic currents (Fig. 5 A), only expresses compareing for EGFP with infecting
(control) it compares, the amplitude (Fig. 5 B) and frequency (Fig. 5 C) for being overexpressed the neuronal excitability postsynaptic currents of 8KQ are showed no
Significant changes.Illustrate that the overexpression of acetylation cortical actin 8KQ has no effect on the function of wild neuron.
Embodiment 6
Intracerebral injection simulation acetylation cortical actin can improve mouse self-closing disease sample behavior
By Naoliqing capsule injection technique, adeno-associated virus AAV9-CamKII-GFP- is injected to mouse brain cortical region
8KQ (structure such as Fig. 6 A).1% yellow Jackets are injected intraperitoneally according to the ratio of 0.1ml/g, after mouse is anesthetized, are fixed on brain
On stereotaxic instrument, it is ensured that head steady.Erythromycin ointment is applied to eyeball of mouse, protection cornea is because long-time is exposed and lamp
Light irradiation causes to damage.75% alcohol disinfecting mouse head coat and skin, it is careful to remove along sagittal seaming and cutting~1cm long notch
Skin, with 2% hydrogen peroxide clean the surface fascia, exposure skull.The position mouse bregma (Bregma) is marked and records, it is vertical using brain
Body position indicator coordinate, mobile micro syringe position, label is following everywhere respectively: (1) anteroposterior (AP),
2.0mm；Mediolateral (ML), 0.5mm；Dorsoventral (DV), 1.3mm；(2) AP, -1.0mm；ML, 2.0mm；DV,
0.8mm.Dental drill (0.5mm) carefully drills, and is considered to be worth doing with cotton swab cleaning surrounding bone.Micro syringe (Hamilton) is fixed on electricity
On sub- micro-injection pump, 2min is stopped after knit stitch to designated position, injection speed selects 0.2 μ l/min, stops after injection
2min, slow needle.After all position injections, sew up the incision.
The adeno-associated virus (AAV9-CamKII-GFP-8KQ) has excitor nerve member specific expression promoter CamKII,
It is consistent with the highly expressed neuron type of DIP2A to express position.Dip2a knock-out mice passes through CRISPR Cas9 by this laboratory
Technology preparation finds there is self-closing disease sample behaviouristics phenotype using Behaviors survey screening, including repetition stereotyped action is (excessively autonomous
Manage hair) and slight human communication disorders, as mouse self-closing disease model in subsequent experimental.Virus is 4 weeks after mouse is born
Injection was spread by 4 weeks virus, Behavior test progress (Fig. 6 B) in 8 weeks after mouse is born.
Mouse is placed in the mouse cage for being covered with clean padding, adapts to environment 10min.It records in following 10min, mouse repeats
The frequency and the time of hair are uprightly explored and manage in stereotyped action-.Adeno-associated virus infects the 8KQ expression of mediation, can remedy Dip2a
KO repeats mechanical autonomous grooming (Fig. 6 C).The KO mouse that virus is compareed with injection compares, injection 8KQ adeno-associated virus
The time of mouse from hair of supervising the cooking is substantially reduced.Therefore, 8KQ has part therapeutic effect to ASD.
As can be seen from the above embodiments, cortical actin mutant 8KQ provided by the invention passes through rite-directed mutagenesis, Neng Goumo
The acetylation state of charge of quasi- cortical actin；The cortical actin mutant 8KQ can improve mouse self-closing disease sample
Behavior.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>Northeast Normal University
<120>a kind of cortical actin mutant 8KQ and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
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Met Trp Lys Ala Ser Ala Gly His Ala Val Ser Ile Thr Gln Asp Asp
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Gly Gly Ala Asp Asp Trp Glu Thr Asp Pro Asp Phe Val Asn Asp Val
20 25 30
Ser Glu Lys Glu Gln Arg Trp Gly Ala Lys Thr Val Gln Gly Ser Gly
35 40 45
His Gln Glu His Ile Asn Ile His Lys Leu Arg Glu Asn Val Phe Gln
50 55 60
Glu His Gln Thr Leu Lys Glu Lys Glu Leu Glu Thr Gly Pro Lys Ala
65 70 75 80
Ser His Gly Tyr Gly Gly Lys Phe Gly Val Glu Gln Asp Arg Met Asp
85 90 95
Arg Ser Ala Val Gly His Glu Tyr Gln Ser Gln Leu Ser Lys His Cys
100 105 110
Ser Gln Val Asp Ser Val Arg Gly Phe Gly Gly Lys Phe Gly Val Gln
115 120 125
Met Asp Arg Val Asp Gln Ser Ala Val Gly Phe Glu Tyr Gln Gly Lys
130 135 140
Thr Glu Lys His Ala Ser Gln Gln Asp Tyr Ser Ser Gly Phe Gly Gly
145 150 155 160
Lys Tyr Gly Val Gln Ala Asp Arg Val Asp Gln Ser Ala Val Gly Phe
165 170 175
Asp Tyr Gln Gly Gln Thr Glu Lys His Glu Ser Gln Lys Asp Tyr Ser
180 185 190
Gln Gly Phe Gly Gly Lys Tyr Gly Ile Asp Lys Asp Lys Val Asp Lys
195 200 205
Ser Ala Val Gly Phe Glu Tyr Gln Gly Lys Thr Glu Lys His Glu Ser
210 215 220
Gln Lys Asp Tyr Val Lys Gly Phe Gly Gly Gln Phe Gly Val Gln Thr
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Asp Arg Gln Asp Lys Cys Ala Leu Gly Trp Asp His Gln Glu Lys Leu
245 250 255
Gln Leu His Glu Ser Gln Lys Asp Tyr Lys Thr Gly Phe Gly Gly Lys
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Phe Gly Val Gln Ser Glu Arg Gln Asp Ser Ser Ala Val Gly Phe Asp
275 280 285
Tyr Lys Glu Arg Leu Ala Lys His Glu Ser Gln Gln Asp Tyr Ala Lys
290 295 300
Gly Phe Gly Gly Gln Tyr Gly Val Gln Gln Asp Arg Met Asp Lys Asn
305 310 315 320
Ala Ser Thr Phe Glu Glu Val Val Gln Val Pro Ser Ala Tyr Gln Lys
325 330 335
Thr Val Pro Ile Glu Ala Val Thr Ser Lys Thr Ser Asn Ile Arg Ala
340 345 350
Asn Phe Glu Asn Leu Ala Lys Glu Arg Glu Gln Glu Asp Arg Arg Lys
355 360 365
Ala Glu Ala Glu Arg Ala Gln Arg Met Ala Lys Glu Arg Gln Glu Gln
370 375 380
Glu Glu Ala Arg Arg Lys Leu Glu Glu Gln Ala Arg Ala Lys Lys Gln
385 390 395 400
Thr Pro Pro Ala Ser Pro Ser Pro Gln Pro Ile Glu Asp Arg Pro Pro
405 410 415
Ser Ser Pro Ile Tyr Glu Asp Ala Ala Pro Phe Lys Ala Glu Pro Ser
420 425 430
Tyr Arg Gly Ser Glu Pro Glu Pro Glu Tyr Ser Ile Glu Ala Ala Gly
435 440 445
Ile Pro Glu Ala Gly Ser Gln Gln Gly Leu Thr Tyr Thr Ser Glu Pro
450 455 460
Val Tyr Glu Thr Thr Glu Ala Pro Gly His Tyr Gln Ala Glu Asp Asp
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Thr Tyr Asp Gly Tyr Glu Ser Asp Leu Gly Ile Thr Ala Ile Ala Leu
485 490 495
Tyr Asp Tyr Gln Ala Ala Gly Asp Asp Glu Ile Ser Phe Asp Pro Asp
500 505 510
Asp Ile Ile Thr Asn Ile Glu Met Ile Asp Asp Gly Trp Trp Arg Gly
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<213>artificial sequence (Artificial Sequence)
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atgtggaaag cctctgcagg ccatgctgtg tccatcacgc aggatgatgg aggagctgat 60
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gctaaaaccg tgcagggatc ggggcaccag gaacacatca acattcacaa gcttcgagag 180
aatgtcttcc aagaacacca gacgctcaag gagaaggagc tggaaacggg acccaaggct 240
tcccacggct atggcgggaa gttcggtgtg gagcaggata ggatggacag atcagccgtg 300
ggccatgagt accagtcgca gctttccaag cactgctcac aagtggactc ggtccggggc 360
ttcggaggca agttcggtgt ccagatggac agggtggatc agtctgctgt aggctttgaa 420
taccagggga agactgagaa gcatgcctcc cagcaagact actctagtgg cttcggtggc 480
aaatacggtg tgcaagctga ccgtgtagac cagagtgccg tgggctttga ctaccagggc 540
cagacggaga agcatgagtc tcagaaagat tactcccaag gttttggtgg caaatatggg 600
attgacaagg acaaggtgga taaaagtgct gtgggctttg agtatcaagg caagacagag 660
aagcacgaat cccagaaaga ctatgtaaaa ggctttggag gacagtttgg tgtgcagaca 720
gacagacagg acaagtgtgc ccttggctgg gaccatcagg agaagctgca gctgcatgaa 780
tcccaaaaag actataagac tggtttcgga ggcaaatttg gtgttcagtc cgagaggcag 840
gactcctccg ctgtggggtt tgattacaag gagagattgg ccaagcacga gtcccagcaa 900
gactatgcca aaggattcgg cgggcagtat ggggtgcagc aggatcggat ggacaagaat 960
gcatccacct ttgaagaagt ggtccaggtg ccatctgcct atcagaagac tgtccccatt 1020
gaggccgtaa ccagcaaaac cagtaatatc cgtgctaact ttgaaaacct ggcaaaggag 1080
agagagcagg aggacaggcg gaaggcagaa gccgagagag ctcagcggat ggccaaagaa 1140
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acgccccctg catcccctag tcctcaacca attgaagaca gaccaccctc cagccccatc 1260
tatgaggatg cagctccgtt caaggccgag ccgagctacc gaggtagcga acctgagcct 1320
gagtacagca tcgaggccgc aggcattcct gaggctggca gccagcaagg cctgacctat 1380
acatcagagc ccgtgtacga gactacagag gctcctggcc actatcaagc agaggatgac 1440
acctacgatg ggtatgagag tgacctgggc atcacagcca tcgccctgta tgactaccag 1500
gctgctggcg atgatgagat ctcctttgac cctgatgaca tcatcaccaa catagaaatg 1560
attgacgatg gctggtggcg tggggtgtgc aagggcagat acgggctctt cccagccaac 1620
tatgtggagc tgcggcagta g 1641
<210> 3
<211> 546
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Met Trp Lys Ala Ser Ala Gly His Ala Val Ser Ile Thr Gln Asp Asp
1 5 10 15
Gly Gly Ala Asp Asp Trp Glu Thr Asp Pro Asp Phe Val Asn Asp Val
20 25 30
Ser Glu Lys Glu Gln Arg Trp Gly Ala Lys Thr Val Gln Gly Ser Gly
35 40 45
His Gln Glu His Ile Asn Ile His Lys Leu Arg Glu Asn Val Phe Gln
50 55 60
Glu His Gln Thr Leu Lys Glu Lys Glu Leu Glu Thr Gly Pro Lys Ala
65 70 75 80
Ser His Gly Tyr Gly Gly Lys Phe Gly Val Glu Gln Asp Arg Met Asp
85 90 95
Arg Ser Ala Val Gly His Glu Tyr Gln Ser Lys Leu Ser Lys His Cys
100 105 110
Ser Gln Val Asp Ser Val Arg Gly Phe Gly Gly Lys Phe Gly Val Gln
115 120 125
Met Asp Arg Val Asp Gln Ser Ala Val Gly Phe Glu Tyr Gln Gly Lys
130 135 140
Thr Glu Lys His Ala Ser Gln Lys Asp Tyr Ser Ser Gly Phe Gly Gly
145 150 155 160
Lys Tyr Gly Val Gln Ala Asp Arg Val Asp Lys Ser Ala Val Gly Phe
165 170 175
Asp Tyr Gln Gly Lys Thr Glu Lys His Glu Ser Gln Lys Asp Tyr Ser
180 185 190
Lys Gly Phe Gly Gly Lys Tyr Gly Ile Asp Lys Asp Lys Val Asp Lys
195 200 205
Ser Ala Val Gly Phe Glu Tyr Gln Gly Lys Thr Glu Lys His Glu Ser
210 215 220
Gln Lys Asp Tyr Val Lys Gly Phe Gly Gly Lys Phe Gly Val Gln Thr
225 230 235 240
Asp Arg Gln Asp Lys Cys Ala Leu Gly Trp Asp His Gln Glu Lys Leu
245 250 255
Gln Leu His Glu Ser Gln Lys Asp Tyr Lys Thr Gly Phe Gly Gly Lys
260 265 270
Phe Gly Val Gln Ser Glu Arg Gln Asp Ser Ser Ala Val Gly Phe Asp
275 280 285
Tyr Lys Glu Arg Leu Ala Lys His Glu Ser Gln Gln Asp Tyr Ala Lys
290 295 300
Gly Phe Gly Gly Lys Tyr Gly Val Gln Lys Asp Arg Met Asp Lys Asn
305 310 315 320
Ala Ser Thr Phe Glu Glu Val Val Gln Val Pro Ser Ala Tyr Gln Lys
325 330 335
Thr Val Pro Ile Glu Ala Val Thr Ser Lys Thr Ser Asn Ile Arg Ala
340 345 350
Asn Phe Glu Asn Leu Ala Lys Glu Arg Glu Gln Glu Asp Arg Arg Lys
355 360 365
Ala Glu Ala Glu Arg Ala Gln Arg Met Ala Lys Glu Arg Gln Glu Gln
370 375 380
Glu Glu Ala Arg Arg Lys Leu Glu Glu Gln Ala Arg Ala Lys Lys Gln
385 390 395 400
Thr Pro Pro Ala Ser Pro Ser Pro Gln Pro Ile Glu Asp Arg Pro Pro
405 410 415
Ser Ser Pro Ile Tyr Glu Asp Ala Ala Pro Phe Lys Ala Glu Pro Ser
420 425 430
Tyr Arg Gly Ser Glu Pro Glu Pro Glu Tyr Ser Ile Glu Ala Ala Gly
435 440 445
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450 455 460
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485 490 495
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500 505 510
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<213>artificial sequence (Artificial Sequence)
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gctaaaaccg tgcagggatc ggggcaccag gaacacatca acattcacaa gcttcgagag 180
aatgtcttcc aagaacacca gacgctcaag gagaaggagc tggaaacggg acccaaggct 240
tcccacggct atggcgggaa gttcggtgtg gagcaggata ggatggacag atcagccgtg 300
ggccatgagt accagtcgaa gctttccaag cactgctcac aagtggactc ggtccggggc 360
ttcggaggca agttcggtgt ccagatggac agggtggatc agtctgctgt aggctttgaa 420
taccagggga agactgagaa gcatgcctcc cagaaagact actctagtgg cttcggtggc 480
aaatacggtg tgcaagctga ccgtgtagac aagagtgccg tgggctttga ctaccagggc 540
aagacggaga agcatgagtc tcagaaagat tactccaaag gttttggtgg caaatatggg 600
attgacaagg acaaggtgga taaaagtgct gtgggctttg agtatcaagg caagacagag 660
aagcacgaat cccagaaaga ctatgtaaaa ggctttggag gaaagtttgg tgtgcagaca 720
gacagacagg acaagtgtgc ccttggctgg gaccatcagg agaagctgca gctgcatgaa 780
tcccaaaaag actataagac tggtttcgga ggcaaatttg gtgttcagtc cgagaggcag 840
gactcctccg ctgtggggtt tgattacaag gagagattgg ccaagcacga gtcccagcaa 900
gactatgcca aaggattcgg cgggaagtat ggggtgcaga aggatcggat ggacaagaat 960
gcatccacct ttgaagaagt ggtccaggtg ccatctgcct atcagaagac tgtccccatt 1020
gaggccgtaa ccagcaaaac cagtaatatc cgtgctaact ttgaaaacct ggcaaaggag 1080
agagagcagg aggacaggcg gaaggcagaa gccgagagag ctcagcggat ggccaaagaa 1140
agacaggagc aggaggaggc gcgcaggaag ctggaagagc aagccagagc caagaagcag 1200
acgccccctg catcccctag tcctcaacca attgaagaca gaccaccctc cagccccatc 1260
tatgaggatg cagctccgtt caaggccgag ccgagctacc gaggtagcga acctgagcct 1320
gagtacagca tcgaggccgc aggcattcct gaggctggca gccagcaagg cctgacctat 1380
acatcagagc ccgtgtacga gactacagag gctcctggcc actatcaagc agaggatgac 1440
acctacgatg ggtatgagag tgacctgggc atcacagcca tcgccctgta tgactaccag 1500
gctgctggcg atgatgagat ctcctttgac cctgatgaca tcatcaccaa catagaaatg 1560
attgacgatg gctggtggcg tggggtgtgc aagggcagat acgggctctt cccagccaac 1620
tatgtggagc tgcggcagta g 1641
Claims (10)
1. a kind of cortical actin mutant 8KQ, which is characterized in that the cortical actin mutant 8KQ has such as SEQ
Amino acid sequence shown in ID No:1.
2. encoding the gene of cortical actin mutant 8KQ described in claim 1, which is characterized in that have such as SEQ ID
Nucleotide sequence shown in No:2.
3. a kind of recombinant virus, which is characterized in that including encoding the cortical actin mutant 8KQ's in claim 2
Gene and viral vectors.
4. recombinant virus according to claim 3, which is characterized in that the viral vectors is adeno-associated virus or slow disease
Poison.
5. recombinant virus according to claim 4, which is characterized in that the adeno-associated virus is 9 type of adenovirus.
6. cortical actin mutant 8KQ described in claim 1 is preparing the application in nerve protection medicine.
7. application according to claim 6, which is characterized in that the neuroprotection shows as supplement cortical actin second
It is acylated, maintain excitatory synapse morphosis and function-stable.
8. cortical actin mutant 8KQ described in claim 1 treats the application in self-closing disease drug in preparation.
9. application according to claim 8, which is characterized in that the applicable object of the self-closing disease drug is self-closing disease
Model mice.
10. application according to claim 8, which is characterized in that the self-closing disease drug is ejection preparation.
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