CN107893078A - Target siRNA, expression vector and virion and its pharmacy application of synaptotagmin 11 - Google Patents
Target siRNA, expression vector and virion and its pharmacy application of synaptotagmin 11 Download PDFInfo
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- CN107893078A CN107893078A CN201711218449.5A CN201711218449A CN107893078A CN 107893078 A CN107893078 A CN 107893078A CN 201711218449 A CN201711218449 A CN 201711218449A CN 107893078 A CN107893078 A CN 107893078A
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- C12N2740/10011—Retroviridae
- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
- C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Abstract
The invention provides a kind of siRNA, expression vector and virion for targetting synaptotagmin 11 and its pharmacy application.Belong to biomedicine technical field, more particularly to suppress the siRNA of cynapse adaptor protein Syt11 gene expressions, contain the siRNA as active component pharmaceutical composition, to express the recombinant vector of the siRNA viral with expression, and their applications in Parkinson's and the Other diseases related to Syt11 expression.The present invention designs for the nucleotide sequence of Synaptotagmin 11 (Syt11) gene have been synthesized small molecules interference RNA, has built the shRNA expression vectors and expression virus for being directed to target sequence, the siRNA specifically targets Syt11 genes in mRNA level in-site, Syt11 expression can effectively be suppressed, for treating parkinsonism and the Other diseases related to Syt11 expression.The siRNA be transferred to after substantia nigra of midbrain area neuron can effective reticence Syt11 expression, reverse the pathogenesis of parkinsonism.
Description
Technical field
The invention belongs to biomedicine technical field, and in particular to suppress cynapse adaptor protein Syt11 gene expressions
SiRNA, contain the siRNA as active component pharmaceutical composition, express the recombinant vector of the siRNA and expression virus, with
And their applications in Parkinson's and the Other diseases related to Syt11 expression.
Background technology
Parkinson's (Parkinson diseases, PD) belong to the most important nerve of mankind nowadays with senile dementia and moved back
Row disease, for the PD incidences of disease of over-65s crowd more than 2%, the incidence of disease of more than 85 years old crowd may be up to 5%.PD is facing
Symptom complex on bed is mainly shown as bradykinesia, static tremor, the movement defects such as rigid and attitude disorders of taking action, mostly
A series of serious non-fortune such as depression, dementia, sleep deprivation, dysosmia, pain, the urinary incontinence, constipation also be present in number patient PD
Dynamic sexual dysfunction.PD pathological characters be mainly shown as substantia nigra of midbrain compact part (substantianigra pars compacta,
SNpc) the selectivity death of dopaminergic neuron and its projection area (corpus straitum, prefrontal lobe, amygdaloid nucleus, hippocampus etc.) interior DOPA
The diacrisis of amine (dopamine, DA).
Great demand of the China in terms of aging, particularly aging related neurodegenerative disease, PD diagnosis and treatment
Scientific and effective, the safe target treatment product of an urgent demand.The pathology machine of PD pathological changes at present, especially PD early stages
System, it is unclear, and PD medicine also because technology content is limited, function factor is not clear or lack high level, systematicness and
Scientific basic research, cause validity of products, security to exist and query, therefore there is presently no effective drug therapy side
Case.
Small molecules interference RNA (small interfering RNA, siRNA) has strict sequence-specific, high efficiency
With biological heredity, can specifically, efficiently promote degradation of homologous mRNA, block the expression of internal homologous gene to reach, lure into
Cells show goes out the purpose of specific gene type missing phenotype.Therefore, siRNA can be based on RNA interference (RNA interference,
RNAi) this mechanism, suppressed in a manner of sequence-specific or block any target gene interested (such as to trigger such as cancer
Etc. the gene of disease) expression, so as to reach treatment disease purpose.
Synaptotagmin Synaptotagmin-11 (Syt11) is the important member in Syt protein families.Syt11 in
Cloned by sudhof groups and identified first within 1997 Lai Syt11 is probably parkin substrate, while in recessive
Syt11 abnormal aggregation is found in the Lewy corpusculums of young patient's PD brain tissue, implies that Syt11 may be in PD related parkin
Pathologic process in play an important roll.Meanwhile the result of whole-genome association shows that Syt11 itself and PD has closely
Genetic correlation, it may be possible to Disease-causing gene (2011, Lancet) important PD, still, Syt11 whether directly participate in PD pathology
There is not been reported for process and its mechanism of causing a disease.
The content of the invention
It is an object of the invention to provide a kind of siRNA for targetting synaptotagmin Synaptotagmin-11, expression
Carrier and virion and its pharmacy application.Described siRNA specifically targets Syt11 genes, Neng Gouyou in mRNA level in-site
Effect suppresses Syt11 expression, for treating parkinsonism and the Other diseases related to Syt11 expression.
The present invention is to be achieved through the following technical solutions：
On the one hand, the invention discloses suppress synaptotagmin Synaptotagmin-11 gene expressions using siRNA
Method, target sequence corresponding to siRNA is the mRNA of Synaptotagmin-11 gene coding regions, and with Synaptotagmin-
19~21 nucleotides that 11 gene coding regions are chosen are as siRNA action target spots.
Preferably, the length of the siRNA is 15~30bp.
Preferably, target sequence corresponding to the siRNA is as follows：
Or target sequence corresponding to the siRNA is adjacent or proximate region and the energy for the target sequence that above table is listed
The target sequence for the suppression synaptotagmin Synaptotagmin-11 gene expressions effect played, described adjacent or proximate region
Scope be in people Synaptotagmin-11 mRNA from the-ATTCGGAAGAGGCGGAGTC-3 ' of sequence 5 ' to sequence 5 '-
Region within ATTCACATTACATAGGCAA-3 ' scopes.
Preferably, the siRNA is unmodified, or comprising at least one nucleotides or nucleotide analog through modification；
Wherein, the decorating site of the nucleotides through modification or nucleotide analog is on the glycosyl, main chain or base of ribonucleotide；
Nucleotide analog is ribonucleotide of the main chain through modification, inosine or the tritylation of phosphorothioate group
Base.
Preferably, the siRNA comprising nucleotides of at least one through modification or nucleotide analog is targeting coding Syt11
SiNA, siRNA, dsRNA, miRNA or shRNA of nucleotide sequence.
Second aspect, the invention discloses a kind of suppression synaptotagmin Synaptotagmin-11 gene expressions
SiRNA, target sequence corresponding to the siRNA are the mRNA of Synaptotagmin-11 gene coding regions, and with
The 19-21 nucleotides that Synaptotagmin-11 gene coding regions are chosen is as siRNA action target spots.
Preferably, described siRNA length is 16~30bp.It is further preferred that siRNA length be 18~
25bp。
Preferably, target sequence corresponding to the siRNA is as follows：
。
The siRNA can relate to targeting coding unmodified or through chemical modification as the pharmaceutical composition of active component
Short interfering nucleic acid (siNA), short interfering rna (siRNA), the double-stranded RNA of Syt11 nucleotide sequences (such as mRNA sequence)
(dsRNA), Microrna (miRNA) or short hairpin RNA (shRNA).
Preferably, the siRNA is unmodified, or comprising at least one nucleotides or nucleotide analog through modification；
Wherein, decorating site is on the glycosyl, main chain or base of ribonucleotide；Nucleotide analog is the master of phosphorothioate group
Ribonucleotide, inosine or the tritylated bases of chain warp modification.
The third aspect, the invention also discloses a kind of Synaptotagmin-11 shRNA expression vectors, shRNA expression
Carrier contains above-mentioned siRNA as active component and pharmaceutically acceptable carrier.According to the present invention, described expression vector
Can be the conventional carrier of pharmaceutically acceptable this area, the carrier can express the siRNA fragment or with the siRNA
Fragment is the shRNA of active component or the various combination of these fragments.
Fourth aspect, the invention also discloses efficient silence synaptotagmin Synaptotagmin-11 shRNA tables
Da virus, shRNA expression viruses are made by following methods：
The double-stranded DNA oligo of the siRNA described in claim 1 is synthesized, it is more that double-stranded DNA oligo is inserted into expression vector
Cloning site area builds shRNA expression plasmids；By shRNA expression plasmids and helper plasmid cotransfection cells, virion is produced,
It is made after recovery concentration.
Preferably, expression virus is recombinant virus, including but not limited to slow virus (lenti virus), adenovirus
(adenovirus), other similar viruses that can realize above-mentioned function such as adeno-associated virus (AAV).
5th aspect, discloses pharmacy application, including：
The invention discloses applications of the above-mentioned siRNA in the medicine for preparing prevention and/or treatment parkinson's syndrome.
The invention discloses above-mentioned shRNA expression vectors in the medicine for preparing prevention and/or treatment parkinson's syndrome
Application.
The invention discloses above-mentioned shRNA expression virus in the medicine for preparing prevention and/or treatment parkinson's syndrome
Application.
Preferably, described medicine, the pharmaceutical composition with siRNA or using siRNA as active component are delivered to place
Chief cell, after conversion or transfection host cell, siRNA is transcribed out, degrade Syt11mRNA, and then suppresses in host cell
Synaptotagmin-11 expression.
6th aspect, the invention also discloses Syt11 in the neuron for suppressing Parkinsonian and Syt11 abnormal expressions
The method of gene expression, this method include being used as the medicine of active component by siRNA as described in relation to the first aspect or using the siRNA
Syt11 expression as described in expression vector described in composition and/or second aspect and/or the importing of the recombinant virus as described in the third aspect
Abnormal neuron and/or other relevant cells.
Compared with prior art, the present invention has technique effect beneficial below：
The present invention has synthesized small molecule interference for the nucleotide sequence design of Synaptotagmin-11 (Syt11) gene
RNA, structure specifically target for the shRNA expression vectors and expression virus, the siRNA of target sequence in mRNA level in-site
Syt11 genes, it can effectively suppress Syt11 expression, for treating parkinsonism and the other diseases related to Syt11 expression
Disease.The siRNA be transferred to after substantia nigra of midbrain area neuron can effective reticence Syt11 expression, reverse the disease of parkinsonism
Reason process.
Can by the siRNA of the present invention and/or pharmaceutical composition and/or shRNA expression plasmids and/or shRNA expression virus
Almost striatal dopamine diacrisis, limbs is reversed to transport completely in parkinsonian mouse model by the mechanism of RNA interference
A series of important Parkinson's pathogenesis such as dynamic balancing imbalance and the dopamine neuron apoptosis in later stage.This will be first
There is the drug therapy example close to complete reversing effect to parkinsonism.
Brief description of the drawings
Fig. 1 is plasmid pRNAT-H1.1/Shuttle-RFP double digestions.
Fig. 2 is Syt11 shRNA expression vector collection of illustrative plates.
Fig. 3 be Syt11 shRNA in Human Embryonic Kidney HEK 293 to Syt11 silence efficiency.
Fig. 4 is that Syt11 shRNA expression vectors transfect hippocampal neuron (Scale bar:50μm).
Fig. 5 is that Syt11 shRNA expression vectors transfect dorsal root ganglion neurons (Scale bar:20μm).
Fig. 6 is that Syt11 shRNA express slow virus in body-sensing dye Substantia Nigra；Wherein, signal of a displays virus in body injection
Figure, b are the brain immunofluorescence dyeing picture after virus injection January.
Fig. 7 is Syt11 shRNA slow virus in body silence efficiency；Wherein, a, b are that Syt11 post-transcriptional silencing mouse are black
Parkin and Syt11 expression in matter area and offside Substantia Nigra；C, d is that comparison virus injects mouse Substantia Nigra and offside is black
Parkin and Syt11 expression in matter area.
Fig. 8 is the expression that Syt11 shRNA slow virus can lower Syt11 in Parkinson disease model；Wherein, a is shown
Schematic diagram of the virus in body injection；B is western blot representative picture；C-f is respectively parkin silences and parkin/
The statistical chart of parkin and Syt11 expressions in the coprecipitated silent mouse Substantia Nigras of Syt11 and offside Substantia Nigra.
Fig. 9 is the apoptosis experimental result that dopamine neuron in Parkinson disease model can be reversed in Syt11 shRNA slow virus
Figure；Wherein, a, b are the apoptosis situation of dopamine neuron in parkin post-transcriptional silencing mouse Substantia Nigras and offside Substantia Nigra；
C, d is the survival condition of the coprecipitated silent mouse Substantia Nigras of parkin/Syt11 and offside Substantia Nigra dopamine neuron.
Figure 10 is the asymmetry circus movement that Syt11 shRNA slow virus reverses METH inductions；Wherein, a notes for virus
After penetrating 3,4,6,8 weeks, METH is expelled to the time lag statistics for starting asymmetry motion；B is virus injection 3, after 4,6,8 weeks,
The asymmetry motion number of turns statistics of mouse in METH injections 90min；
Figure 11 is that Syt11 shRNA slow virus reverses the limb motion in Parkinson's unbalance；Wherein, a~c is parkin
Post-transcriptional silencing causes offside somatic movement unbalance；D~f is that offside body caused by Syt11 shRNA inactivate to parkin is transported
Dynamic unbalance return action.
Embodiment
With reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and
It is not to limit.
Unless stated otherwise, reagent used in following examples, culture medium are commercial goods, unreceipted specific bar
The experimental method of part, it is generally equal according to the operation such as normal condition, used nucleic acid electrophoresis, western blot, immunofluorescence
Routinely scheme is carried out.
Embodiment 1
SiRNA and its expression vector for cynapse adaptor protein Syt11 are prepared with virus：
Synaptotagmin Syt11 gene orders are transferred from Genebank, predict its siRNA action target spot, synthesize pin
To Syt11 effective siRNA sequence；Synthesize siRNA double-strand DNA oligo, insertion expression vector multiple cloning sites area structure
ShRNA expression plasmids；By shRNA viral expression plasmids and helper plasmid cotransfection human embryo kidney (HEK) 293T cells, virus is produced
Grain, efficient silence Syt11 shRNA expression viruses are produced after recovery concentration.
1st, according to Syt11 nucleotide sequences (NM_152280 the Homo sapiens, NM_ reported in Genebank
031667 Rattusnorvegicus, NM_018804 Musmusculus), choose 19-21 for Syt11 gene coding regions
Nucleotides chooses a 19nt random oligonucleotide sequences as control as siRNA action target spots.Each siRNA
1 is shown in Table to the action target spot sequence of Syt11 genes：
The siRNA target sequences of table 1
| Target spot is numbered | Gene is numbered | Target sequence | Target area | Initiation site |
| SEQ ID NO:1 | NM_152280 | 5’-CCAATATCCGACCTAGCTT-3’ | ORF | 307 |
| SEQ ID NO:2 | NM_152280 | 5’-CCCACCATACAAGTTTATT-3’ | ORF | 443 |
| SEQ ID NO:3 | NM_152280 | 5’-GCTCAAAGGCATCAGCATA-3’ | ORF | 467 |
| SEQ ID NO:4 | NM_152280 | 5’-CCTGCTAAGCCGAGACAAA-3’ | ORF | 599 |
| SEQ ID NO:5 | NM_152280 | 5’-GGATCTTGTATAGACCAAT-3’ | ORF | 639 |
| SEQ ID NO:6 | NM_152280 | 5’-GGAATATCCAGAAGTGCAT-3’ | ORF | 1144 |
| SEQ ID NO:7 | NM_152280 | 5’-CCAGGTGTCTCTGTCATAT-3’ | ORF | 1178 |
| SEQ ID NO:8 | NM_152280 | 5’-GGTCTCTCAGGTAATCCTT-3’ | ORF | 1266 |
| SEQ ID NO:9 | NM_152280 | 5’-GCAGAAAGCGCATTGCCAA-3’ | ORF | 1309 |
| SEQ ID NO:10 | NM_152280 | 5’-GCATCGAGTTCCTCGTTAT-3’ | ORF | 1420 |
| SEQ ID NO:11 | NM_152280 | GCATCAGCATATACCCAGAGA | ORF | 475 |
| SEQ ID NO:12 | NM_152280 | ATAGACCAATTACCCATCAAA | ORF | 648 |
| SEQ ID NO:13 | NM_152280 | CATCAAAGTGCGGAGAGACAA | ORF | 518 |
| SEQ ID NO:14 | NM_152280 | ATCCTTCCTGACAAACGGCAT | ORF | 897 |
| SEQ ID NO:15 | NM_152280 | GCTTCTCTCGGGATGATGTCA | ORF | 1045 |
| SEQ ID NO:16 | NM_018804 | GCATCGAGTTCCTTGTCATTG | ORF | 1399 |
| SEQ ID NO:17 | NM_018804 | GGCTGAGATCACAAATATACG | ORF | 278 |
| SEQ ID NO:18 | NM_031667 | GGAACATTCAGAAGTGCATTA | ORF | 1049 |
| SEQ ID NO:19 | NM_031667 | GGACAAAGATGGTTCTCATAG | ORF | 441 |
| SEQ ID NO:20 | NM_031667 | GCTTTGATGTGTCACCAGTAG | ORF | 230 |
2nd, DNA oligo sequent synthesis
According to having selected target sequence to design shRNA interference sequences, held in normal chain 3 ' and add TTTTTT termination signals, and anti-chain
5 ' end addition termination signal complementary series, and suitable restriction endonuclease sites are added to complete vector construction, DNA at both ends
Oligo sequences are synthesized by Beijing AudioCodes biotechnology Co., Ltd.The single strain oligonucleotide sequence of synthesis is dissolved in
In annealing buffer, annealed in PCR amplification instrument, form the double-stranded DNA with cohesive end, its sequence is as follows：
SEQ ID NO:1
SEQ ID NO:2
SEQ ID NO:3
SEQ ID NO:4
SEQ ID NO:5
SEQ ID NO:6
SEQ ID NO:7
SEQ ID NO:8
SEQ ID NO:9
SEQ ID NO:10
SEQ ID NO:11
SEQ ID NO:12
SEQ ID NO:13
SEQ ID NO:14
SEQ ID NO:15
SEQ ID NO:16
SEQ ID NO:17
SEQ ID NO:18
SEQ ID NO:19
SEQ ID NO:20
SEQ ID NO:21
SEQ ID NO:22
3rd, rna interference vector is built
By taking pRNAT-H1.1/Shuttle-RFP carriers (being purchased from Genscript companies) as an example, prepared according to NEB specifications
50 μ l reaction systems, make its linearisation using BamH I and the double digestion pRNAT-H1.1/Shuttle-RFP carriers of Hind III.Reaction
System such as table 2 below：
The reaction system of table 2
After above-mentioned reaction system is well mixed, 37 DEG C of (optimum temperature) digestion 3-4h, agarose gel electrophoresis carries out digestion
Identification, referring to Fig. 1, using glue reclaim kit gel extraction purpose fragment.In Fig. 1, swimming lane：M,1kb Marker；1,
Vector；2,Vector digested by BamHⅠand HindⅢ.
ShRNAoligo after annealing is connected structure recombinant vector with the carrier linearized, coupled reaction system is as follows：
Table 3
After above-mentioned reaction system is well mixed, 16 DEG C (optimum temperature) connection 6-9h.Connection product is converted into Escherichia coli
Competent cell, in Amp+Screening positive clone on culture medium, after being expanded and being identified, shRNA recombinant expression carriers are produced,
Referring to Fig. 2.
4th, the packaging of Syt11 shRNA expression virus
Syt11 shRNA Lentiviral (L309) and helper plasmid (pRSV-REV, RRE, REV) will be carried
Respectively by 4 μ g, 2 μ g, 2 μ g, 2 μ g per 25cm2The dosage transfection HEK293T cells of culture area, transfection reagent are
Polyethylenimine " Max " (Mw 40 000), transfection method are carried out with reference to reagent teachings.After transfecting 48h, collect
Nutrient solution simultaneously centrifuges 5min in 4 DEG C with 3 000g, and supernatant is centrifuged into 120min with 50 000g again, with 1 ‰ ice-cold culture liquid
Long-pending PBS is resuspended and precipitates to obtain concentrating virus.
Embodiment 2
Syt11 shRNA are to Syt11 silence efficiencies
1st, liposome transfection people kidney embryo HEK293 cells
People's kidney embryo HEK293A cells are cultivated in DMEM (Gibco) culture medium containing 10% hyclone (FBS).
Turning for HEK293A cells is carried out using transfection reagent VigoFect (Vigorous Biotechnology Beijing Co.)
Dye, when verifying Syt 11 silence efficiency, its expression plasmid 5 shRNA expression plasmid cotransfections with randomly selecting respectively
293A cells, it is used for real time PCR detections after transfecting 72h.
2nd, mRNA extraction
HEK cells are taken, using TRIZOL lysis at room temperature cells 5min.Often add 0.2ml chloroforms using 1ml TRIZOL,
Vortex acutely shakes 15s, centrifuging and taking upper strata RNA aqueous phases after fully mixing, adds isometric isopropanol precipitating RNA, 4 DEG C, and 16
000g centrifuges 10min, removes supernatant and washs precipitation with 75% ethanol, uses ddH2O dissolving precipitations.Gained mRNA solution is through DNase
After I digestion, the absolute ethyl alcohol of 2.5 times of volumes and 0.1 times of volume 3M pH 5.2 sodium acetate are added into reaction system, in -80 DEG C
RNA is precipitated, 75% ethanol washing RNA, is precipitated with Amoxcillin deionized water dissolving, RT-PCR analyses is carried out at once after surveying concentration.
3、real time PCR
CDNA is synthesized using EasyScript First-Strand cDNA Synthesis SuperMixcDNA kits
First chain, synthetic reaction are carried out by kit explanation.Using kitGreen
QPCRSuperMix-UDG carries out real time PCR amplifications to Syt11, and reaction system is prepared to specifications, and PCR is anti-
It should be carried out on Applied Biosystems 7300real-time PCR System, reaction condition is：96 DEG C of pre-degenerations
5min, loop parameter：95 DEG C of denaturation 30s, 60 DEG C of annealing 30s, 72 DEG C of extension 45s；40 amplification cycles altogether, most after 72 DEG C
Extend 5min.The transcriptional level of Syt11mRNA in each sample is calculated according to Ct values.As a result show, shRNA in 5 randomly selected
Silence efficiency post-transcriptional silencing Syt11 that can be higher expression (>90%, Fig. 3).
Embodiment 3
Syt11 shRNA expression plasmid transfected neurons
1st, liposome transfection hippocampal neuron
P0-1 days Hippocampus of Mice are taken, 0.25%trypsin37 DEG C of digestion~12min, are obtained after digestion tissue is dispelled
To cell suspension.Cell suspension is dropped in be covered with poly-L-lysine (Sigma) slide/culture dish in, with containing 2%
B27,0.5mM L-glutamine and 5 μM of cytosine arabinoside Neurobasal (Gibco) are cultivated.Training
The shRNA expression plasmids in embodiment 2 are transfected by hippocampus god using Lipofectamine 2000 (Invitrogen) after supporting 5 days
Through member, expressions of the shRNA in neuron is observed after 12-14 days.As a result it is as shown in Figure 4, it can be seen that through liposome method
After transfection, it is possible to find the hippocampal neuron of great expression green fluorescent protein, the positive cell of green fluorescent protein are success
Transfection and expression shRNA neuron.Show that Syt11 shRNA expression vectors are successfully transferred in hippocampal neuron.
2nd, electroporation transfection dorsal root ganglion neurons
60g male rats are taken, its chest section and waist section backbone is separated, chooses DRGs, with containing 1mg/ml
In Collagenase 1A and 0.3mg/ml Trypsin digestive juice, 40min is digested in 37 DEG C, section is gently dispelled, obtained
Cell suspension.Electricity is carried out using the μ l transfection system MPK10096 systems (Invitrogen) of NeonTM 100
Turn, the cell after transfection is placed in the DMEM-F12 containing 10%FBS, and cell suspension is dropped in and is covered with poly-L-lysine
(Sigma) in slide/culture dish, cultivated with the DMEM-F12 containing 10%FBS, electricity observes shRNA in nerve after turning 24h
Expression in member.As a result referring to Fig. 5, after electroporated method transfection, it is possible to find the Dorsal root of great expression green fluorescent protein
Ganglion neuronal, the positive cell of green fluorescent protein are Successful transfection and express shRNA neuron, show Syt11
ShRNA expression vectors are successfully transferred in the neuron.
Embodiment 4
Syt11 shRNA slow virus contaminates in body-sensing
1st, Syt11 shRNA slow virus brain stereotactic is injected
By taking C57 adult mices as an example, intraperitoneal injection 1.5g/kg urethanes are anaesthetized, and its body temperature is maintained using heating blanket
O is provided at~37 DEG C, while using oxygen mask2Keep mouse in good condition.Will as horizontal reference point using bregma and lambda
Mouse is fixed on stereotaxic apparatus, and an aperture is opened in mouse head injection position with cranial drill, with equipped with 32G tack pins
10 μ l micro syringes draw 2 μ l viral concentrations liquid (slow virus titres:108–109/ml；AAV2/9 titres：1012–1013/ ml),
Unilateral injection is to black substance region (- 3mm AP, 1.25mm ML, 4mm DV), and after suturing scalp, mouse is placed on 37 DEG C of heating blankets
Recovered.
2nd, Immunofluorescence test virus injection and efficiency of infection
Histotomy：Experimental mouse is anaesthetized sb. generally using urethane (1.5g/kg, i.p.), is entered with 50ml physiological saline
Row cardiac perfusion, after use 4% paraformaldehyde instead and pre-fixed.Brain tissue is taken out, 4 DEG C is put into 4% PFA and fixed
Night.Respectively serial dehydration is carried out with 10%, 20% and 30% sucrose solution.After brain tissue is repaired, wrapped using OCT glue
Bury, cut into slices on freezing microtome, for immunofluorescence dyeing.
Immunofluorescence：5min is punched with PBS brain washes piece 3 times, then with the PBS solution room temperature of the X-100 containing 0.3%Triton,
Then 1h is closed in the PBS solution containing 2%BSA.By brain piece, room temperature is incubated in the 2%BSA-PBS solution containing corresponding primary antibody
1h (or 4 DEG C of overnight incubations) is educated, is washed 3 times with the PBS containing 2%BSA, is then incubated at room temperature 1h, PBS solution with corresponding fluorescence secondary antibody
After washing 3 times, mounting is carried out with Dako mountants.IMAQ is carried out using LSM710 inverted fluorescence microscopes.Referring to Fig. 6, its
In, schematic diagram of a displays virus in body injection；B is the brain immunofluorescence dyeing picture after virus injection January, and the TH positives are more
Bar amine neuron, the positive cells for virus infection of GFP, Scale bar:500μm.TH positive cells are dopamine neuron,
Green fluorescent protein positive cell is Successful transfection and expresses shRNA neuron.As a result show, Syt11 shRNA are sick slowly
Poison is successfully injected to unilateral nigra area, and has higher efficiency of infection to the dopamine neuron of this brain area.
Embodiment 5
Syt11 shRNA slow virus is in body silence efficiency
1. protein extraction
After experimental mouse anesthesia, cardiac perfusion is carried out with~section cerebrospinal fluid ice-cold 50ml, rapid broken end taking-up brain tissue,
And the brain water flat slice of~200 μ m-thicks is cut on Leica VT 1000S slicers, the histotomy containing midbrain is collected,
Dozen viral side is cut respectively and offside Substantia Nigra brain tissue is homogenized.After homogenate, 16 4 DEG C of 000g centrifugation 15min, take
Clearly, isometric 2 Х SDS-PAGE sample buffers are added, 95-100 DEG C is boiled 5-6min, produces protein sample, for Western
Blot is analyzed.
2nd, Western hybridizes
Gained protein solution is subjected to electrophoresis, and electricity goes to pvdf membrane, is sealed with the PBS solution room temperature containing 5% skimmed milk power
1h is closed, washes film 3 times, 3-4h (or 4 DEG C of overnight incubations) is incubated at room temperature in the PBST solution containing 2%BSA containing corresponding primary antibody, is used
PBST is washed and is incubated at room temperature 1h after film 5 times with corresponding secondary antibody again, and PBST is washed after film 5 times in the double infrared laser imaging systems of Odyssey
In be scanned.As a result referring to Fig. 7, wherein, a, b are in Syt11 post-transcriptional silencing mouse Substantia Nigras and offside Substantia Nigra
Parkin and Syt11 expression；C, d be comparison virus inject in mouse Substantia Nigra and offside Substantia Nigra parkin and
Syt11 expression, the results showed that, Syt11 shRNA slow virus can succeed silence brain area Syt11 expression.
Embodiment 6
Protective effect of the Syt11 shRNA slow virus to dopamine neuron
1. the foundation of Parkinson's pathological model
Parkin (PARK2) Inactivating mutations are to cause the pathogenetic major reason of Parkinson, to establish unilateral Parkinson
Sick pathological model, parkin shRNA expression viruses are built, by its fixed-point injection to mouse unilateral nigra area, post-transcriptional silencing
Region parkin expression.Using western blot detections parkin silence efficiency, using the dye of dopamine neuron
Parkinson's pathological change caused by color and count evaluation parkinshRNA.Referring to Fig. 8, wherein, a displays virus is in body injection
Schematic diagram；B is western blot representative picture；C-f is respectively parkin silences and parkin/Syt11 coprecipitated silent small
The statistical chart of parkin and Syt11 expressions in mouse Substantia Nigra and offside Substantia Nigra.Referring to Fig. 9, a, b are after parkin is transcribed
The apoptosis situation of dopamine neuron in silence mouse Substantia Nigra and offside Substantia Nigra；C, d is parkin/Syt11 coprecipitated silent small
Mouse Substantia Nigra and the survival condition of offside Substantia Nigra dopamine neuron.As a result show, post-transcriptional silencing parkin expression can
Syt11 expression is lifted, and causes the apoptosis of later stage dopamine neuron.
2nd, post-transcriptional silencing Syt11 expresses the protective effect to dopamine neuron
By Syt11 shRNA expression virus, Unilateral injection to mouse Substantia Nigra, is adopted simultaneously with parkinshRNA expression virus
With western blot detections parkin and Syt11 expression, dyeing and count evaluation using dopamine neuron
Syt11 shRNA are to dopamine neuron apoptosis and the reversing effect of missing.As a result show, Syt11 shRNA can not only be lowered
Syt11 expression, referring to Fig. 8, and the apoptosis phenomenon of the dopamine neuron caused by parkin silences can be reversed completely,
Referring to Fig. 9.
Embodiment 7
The dyskinesia of Parkinson's can be reversed in Syt11 shRNA slow virus
1st, the asymmetric circus movement of methamphetamine induction
Carry out meth (Methamphetamine, METH) and lure within 3 weeks, 4 weeks, 6 weeks and 8 weeks after virus injection
The asymmetry circus movement experiment led.Mouse is previously positioned in experimental box 30min to adapt to experimental situation, then according to
8mg/kg dosage intraperitoneal injection METH, the time required to there is offside circus movement first in statistics, and the number of turns in 90min.
If not occurring asymmetry motion in 90min, motion time lag is designated as 90min.Referring to Figure 10, wherein, a be virus injection 3,
4th, after 6,8 weeks, METH is expelled to the time lag statistics for starting asymmetry motion；B is virus injection 3, after 4,6,8 weeks, METH notes
Penetrate the asymmetry motion number of turns statistics of mouse in 90min.As a result show, intraperitoneal injection METH can succeed silence after inducible transcription
The asymmetry circus movement of parkin mouse, and Syt11shRNA can almost reverse this dyskinesia completely.
2nd, footprint gait analysis
Sole coats red, black two kinds of non-toxic pigments respectively before and after mouse, and experiment mice is daily in the wide 10cm of long 100cm
Training is run 3 times on high 10cm mining site runway, carries out carrying out the gait marking and analysis after training for three days on end, statistics is left respectively
Step spacing and the overlapping cases (centre of the palm distance) of front and rear sole between the right crus of diaphragm palm, to assess the stabilization between mouse or so paces
Property.Referring to Figure 11, the results showed that, post-transcriptional silencing parkin can induce its offside somatic movement it is unbalance (fluctuation of Walk spacing becomes big,
Front and rear sole overlaying horizontal reduces), and Syt11 shRNA can successfully reverse this dyskinesia.
Sequence table
<110>Xi'an Communications University
<120>Target siRNA, expression vector and virion and its pharmacy application of synaptotagmin -11
<160> 22
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Claims (31)
- A kind of 1. method for suppressing synaptotagmin Synaptotagmin-11 gene expressions using siRNA, it is characterised in that Target sequence corresponding to siRNA is the mRNA of Synaptotagmin-11 gene coding regions, and with Synaptotagmin-11 genes 19~21 nucleotides that code area is chosen are as siRNA action target spots.
- 2. suppress the side of synaptotagmin Synaptotagmin-11 gene expressions using siRNA as claimed in claim 1 Method, it is characterised in that the length of the siRNA is 15~30bp.
- 3. suppress the side of synaptotagmin Synaptotagmin-11 gene expressions using siRNA as claimed in claim 1 Method, it is characterised in that target sequence corresponding to the siRNA is as follows：Or target sequence corresponding to the siRNA is the adjacent or proximate region for the target sequence that above table is listed and can played Suppression synaptotagmin Synaptotagmin-11 gene expressions effect target sequence, described adjacent or proximate region model Enclose be in people Synaptotagmin-11mRNA from the-ATTCGGAAGAGGCGGAGTC-3 ' of sequence 5 ' to sequence 5 '- Region within ATTCACATTACATAGGCAA-3 ' scopes.
- 4. suppress the side of synaptotagmin Synaptotagmin-11 gene expressions using siRNA as claimed in claim 1 Method, it is characterised in that the siRNA is unmodified, or comprising at least one nucleotides or nucleotide analog through modification； Wherein, the decorating site of the nucleotides through modification or nucleotide analog is on the glycosyl, main chain or base of ribonucleotide；Nucleotide analog is ribonucleotide of the main chain through modification, inosine or the trityl choline of phosphorothioate group Base.
- 5. suppress the side of synaptotagmin Synaptotagmin-11 gene expressions using siRNA as claimed in claim 4 Method, the siRNA comprising nucleotides of at least one through modification or nucleotide analog are targeting coding Syt11 nucleotide sequences SiNA, siRNA, dsRNA, miRNA or shRNA.
- A kind of 6. siRNA for suppressing synaptotagmin Synaptotagmin-11 gene expressions, it is characterised in that the siRNA Using be Synaptotagmin-11 gene coding regions mRNA as corresponding to target sequence, and with Synaptotagmin-11 genes compile 19~21 nucleotides that code area chooses are as siRNA action target spots, so as to suppress synaptotagmin Synaptotagmin- 11 gene expressions.
- 7. suppress the siRNA of synaptotagmin Synaptotagmin-11 gene expressions, its feature as claimed in claim 6 It is, the length of the siRNA is 15~30bp.
- 8. suppress the siRNA of synaptotagmin Synaptotagmin-11 gene expressions, its feature as claimed in claim 6 It is, the siRNA is unmodified, or comprising at least one nucleotides or nucleotide analog through modification；Wherein, repaiied The nucleotides of decorations or the decorating site of nucleotide analog can be on the glycosyls, main chain or base of ribonucleotide；Nucleotide analog is ribonucleotide of the main chain through modification, inosine or the trityl choline of phosphorothioate group Base.
- 9. suppress the siRNA of synaptotagmin Synaptotagmin-11 gene expressions, its feature as claimed in claim 8 It is, wherein the siRNA comprising nucleotides of at least one through modification or nucleotide analog, or targeting coding Syt11 nucleotides SiNA, dsRNA, miRNA or shRNA of sequence.
- 10. suppressing the siRNA of synaptotagmin Synaptotagmin-11 gene expressions as claimed in claim 6, it is special Sign is that target sequence corresponding to the siRNA is as follows：Or target sequence corresponding to the siRNA be above table list the adjacent or proximate region of sequence and can play Suppression synaptotagmin Synaptotagmin-11 gene expressions effect target sequence；Described adjacent or proximate region model Enclose is from-the ATTCGGAAGAGGCGGAGTC-3 ' of sequence 5 ' to-ATTCACATTACATAGGCAA-3 ' scopes of sequence 5 ' in mRNA Within region.
- A kind of 11. method for the medicine for preparing prevention and/or treatment parkinson's syndrome, it is characterised in that utilize claim SiRNA in 6-10 described in any one prepares the medicine of prevention and/or treatment parkinson's syndrome.
- 12. a kind of medicine for preventing and/or treating parkinson's syndrome, it is characterised in that it is utilized in claim 6-10 SiRNA described in any one prepares the medicine of prevention and/or treatment parkinson's syndrome.
- 13. a kind of medicine for preventing and/or treating parkinson's syndrome, it is characterised in that by any one in claim 1-4 It is described using siRNA suppress synaptotagmin Synaptotagmin-11 gene expressions method apply prepare prevention and/ Or the medicine prepared in the medicine for the treatment of parkinson's syndrome.
- A kind of 14. medicine for preventing and/or treating parkinson's syndrome, it is characterised in that：Described medicine is unmodified SiRNA fragment or the siRNA fragment through modification, or the group of unmodified siRNA fragment and the siRNA fragment through modification Close, or the combination of the different siRNA fragments through modification；Or the medicine is with unmodified siRNA fragment or through repairing The siRNA fragment of decorations, or the combination of unmodified siRNA fragment and the siRNA fragment through modification, or it is different Medicine of the combination of siRNA fragment through modification as active component；SiRNA is delivered to host cell by the medicine, after conversion or transfection host cell, transcribes out siRNA, is degraded Syt11mRNA, and then suppress the expression of Synaptotagmin-11 in host cell.
- 15. medicine as claimed in claim 14, it is characterised in that it is using according to any one in claim 1-5 Described method suppresses the medicine that synaptotagmin Synaptotagmin-11 gene expressions obtain.
- 16. medicine as claimed in claim 14, it is characterised in that：Using any one of claim 6-10 siRNA。
- A kind of 17. carrier, it is characterised in that siRNA of the expression vector using in claim 6~10 described in any one as Active component.
- 18. carrier as claimed in claim 17, it is characterised in that it is Synaptotagmin-11shRNA expression vectors.
- 19. a kind of carrier, it is characterised in that it utilizes method any one of claim 1-5, suppresses synaptotagmin Synaptotagmin-11 gene expressions.
- 20. carrier as claimed in claim 19, it is characterised in that it is Synaptotagmin-11shRNA expression vectors.
- A kind of 21. method for the medicine for preparing prevention and/or treatment parkinson's syndrome, it is characterised in that utilize claim Carrier described in any one of 17-20 prepares medicine.
- 22. a kind of medicine for preventing and/or treating parkinson's syndrome, it is characterised in that utilize any one of claim 17-20 Described carrier prepares said medicine.
- A kind of 23. shRNA expression viruses of silence synaptotagmin Synaptotagmin-11, it is characterised in that the shRNA Expression virus is made by following methods：The double-stranded DNA oligo of the siRNA in claim 6~10 described in any one is synthesized, double-stranded DNA oligo is inserted into table ShRNA expression plasmids are built up to vector multiple cloning site area；By shRNA expression plasmids and helper plasmid cotransfection cells, produce Virion, it is made after recovery concentration.
- 24. silence synaptotagmin Synaptotagmin-11 as claimed in claim 23 shRNA expression viruses, it is special Sign is that the virus is the one or more in slow virus, adenovirus, adeno-associated virus.
- 25. a kind of method for the shRNA expression viruses for preparing silence synaptotagmin Synaptotagmin-11, its feature exist In shRNA expression viruses are made by following methods：The double-stranded DNA oligo of the siRNA in claim 6~10 described in any one is synthesized, double-stranded DNA oligo is inserted Expression vector multiple cloning sites area builds shRNA expression plasmids；By shRNA expression plasmids and helper plasmid cotransfection cells, production Raw virion, it is made after recovery concentration.
- A kind of 26. method for the medicine for preparing prevention and/or treatment parkinson's syndrome, it is characterised in that utilize claim Virus in 23 or 24 prepares the medicine of prevention and/or treatment parkinson's syndrome.
- A kind of 27. method for the medicine for preparing prevention and/or treatment parkinson's syndrome, it is characterised in that utilize claim Virus prepared by the method in 25 prepares the medicine of prevention and/or treatment parkinson's syndrome.
- 28. a kind of medicine for preventing and/or treating parkinson's syndrome, it is characterised in that it is to utilize claim 23 or 24 In virus prepare prevention and/or treatment parkinson's syndrome medicine.
- 29. a kind of medicine for preventing and/or treating parkinson's syndrome, it is characterised in that it is to utilize to make in claim 25 Prevention and/or the medicine for the treatment of parkinson's syndrome prepared by standby viral preparation method.
- 30. a kind of medicine for preventing and/or treating parkinson's syndrome, it is characterised in that the medicine is by claim 6-10 SiRNA described in any one claim is used as activity using the siRNA described in any one of claim 6-10 claim The medicine of composition imports the neuron of the Syt11 abnormal expressions and/or other relevant cells.
- A kind of 31. carrier, it is characterised in that the carrier by the siRNA described in any one of claim 6-10 claim or The Syt11 tables are imported using the siRNA described in any one of claim 6-10 claim as the medicine of active component Neuron and/or other relevant cells up to exception.
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| CN108959843A (en) * | 2018-06-06 | 2018-12-07 | 北京大学 | The chemical small molecule drug computational screening method of targeted rna |
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| CN112867495A (en) * | 2018-10-19 | 2021-05-28 | 韩国生命工学研究院 | Gastric cancer therapeutic composition comprising SYT11 inhibitor as active ingredient |
| WO2022253196A1 (en) * | 2021-06-01 | 2022-12-08 | 苏州百脉得生物科技有限公司 | Synaptotagmin-11 mrna as marker of parkinson's disease and use thereof |
| WO2022253195A1 (en) * | 2021-06-01 | 2022-12-08 | 苏州百脉得生物科技有限公司 | Parkinson's syndrome marker and use thereof |
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| CN108959843A (en) * | 2018-06-06 | 2018-12-07 | 北京大学 | The chemical small molecule drug computational screening method of targeted rna |
| CN108959843B (en) * | 2018-06-06 | 2021-07-06 | 北京箭牧科技有限公司 | Computer screening method of chemical small molecule drug of target RNA |
| CN112867495A (en) * | 2018-10-19 | 2021-05-28 | 韩国生命工学研究院 | Gastric cancer therapeutic composition comprising SYT11 inhibitor as active ingredient |
| US20210361694A1 (en) * | 2018-10-19 | 2021-11-25 | Korea Research Institute Of Bioscience And Biotechnology | Method for preventing or treating cancer using syt11 inhibitor |
| EP3868385A4 (en) * | 2018-10-19 | 2021-12-22 | Korea Research Institute of Bioscience and Biotechnology | Gastric cancer treatment composition comprising syt11 inhibitor as active ingredient |
| CN109706173A (en) * | 2019-01-31 | 2019-05-03 | 齐齐哈尔大学 | A kind of carrier pZSW-1 reducing lung carcinoma cell multidrug resistance by RNAi silencing Egr1 gene |
| CN112704567A (en) * | 2019-10-25 | 2021-04-27 | 复旦大学附属华山医院 | Experimental method for determining anatomical functional boundary of lateral Subnucleus (SLD) of animal pons dorsum |
| WO2022253196A1 (en) * | 2021-06-01 | 2022-12-08 | 苏州百脉得生物科技有限公司 | Synaptotagmin-11 mrna as marker of parkinson's disease and use thereof |
| WO2022253195A1 (en) * | 2021-06-01 | 2022-12-08 | 苏州百脉得生物科技有限公司 | Parkinson's syndrome marker and use thereof |
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