CN105567737A - Construction and application of miRNA-34a overexpression recombinant vector - Google Patents
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Abstract
The invention relates to construction and application of a miRNA-34a overexpression recombinant vector. Through constructing miR-34a overexpression adeno-associated virus recombinant vector and over-expressing miR-34a in hippocampus of mouse, the influence of miR-34a on anxiety disorder behavior can be determined. The research results show that the recombinant vector is successfully and efficiently expressed in overall animal level, the overexpression of miR-34a can cause anxiety disorder behavior, and thus miR-34a can be taken as the important candidate target for treating anxiety disorder behavior. The invention provides a novel technical means for treating diseases, which are caused by abnormal expression of miR-34A, such as anxiety disorder of Alzheimer's disease.
Description
Technical field
The present invention relates to the construction and application of miRNA-34a process LAN recombinant vectors, the application especially in the medicine of development alzheimer's disease.
Background technology
MicroRNA is the strand short data records tiny RNA that a class length is about 22 ~ 25 Nucleotide, it is coded protein not, but the 3 ' UTR (3 ' non-translate district) of homologous mRNA can be incorporated in the mode of incomplete base pairing with its 5 ' end 2nd ~ 8 Nucleotide, by suppressing the expression of mRNA or inducing it to degrade, negative regulate is carried out to mRNA, affects the generation of associated protein.MicroRNA-34a (miR-34a) is the one of microRNA, it is wide expression in each site tissue of whole body such as cerebral tissue, cardiac muscle, lung, liver, prostate gland, the precursor sequence of micRNA34a is as shown in sequence 1, ripe micRNA34a sequence is as shown in sequence 2, first form the precursor sequence of micRNA34a in vivo, then in body, be processed to form ripe micRNA34a.Its encoding gene is positioned No. 1 chromosome long arm 3 district 6 and is with (1p36), the disappearance at karyomit(e) (1p36) position can betide in kinds of tumor cells, as neuroblastoma, liver cancer, colorectal cancer etc., therefore, miR-34a is often used to research role in tumour occurs; In addition, miR-34a can with the mRNA non-specific binding of several genes, as MYCN, BCL2, SIRT1, NOTCH1, JAG1, CCND1, CDK6 and E2F3 etc., these genes participate in various bioprocess such as tumour in body and occur and pernicious transfer, neural generation and differentiation, vasculogenesis, apoptosis and necrosis etc., and therefore miR-34a is also widely used in research role in tumour.
But in alzheimer's disease or anxiety disorder, in role prior art, there is not been reported about miR-34a.
Summary of the invention
The present invention is by building miR-34a (miRNA-34a) process LAN recombinant expression vector, and in the efficient overexpression of whole animal level success, find that miR-34a overexpression can promote that mouse anxiety sample study of behaviour obstacle is as alzheimer's disease anxiety sample obstacle, show that miR-34a can become the important candidate targets of this type of disease for the treatment of.
The object of this invention is to provide miR-34a process LAN recombinant expression vector, it contains the sequence shown in sequence 1.
Described carrier is correlated virus recombinant vectors.
Another object of the present invention is to provide the method building this miR-34a process LAN recombinant expression vector, and the method comprises: (A) designs primer, pcr amplification miR-34a gene; (B) gene of amplification and expression vector enzyme are cut, connect goal gene and expression vector; (C) product conversion intestinal bacteria will be connected, cultivate; (D) extract recombinant plasmid after qualification and pack.
Described primer is 5 '-CGGGATCCGCAGCCTCTCCATCTTC-3 '; 5 '-GGAATTCGGCTAGGAGGATCAACACAC-3 '.
Invention additionally provides a kind of method evaluating anxiety disorder medicine or screening anxiety disorder medicine, the method comprises use miR-34a process LAN recombinant expression vector and builds anxiety disorder animal model, the improvement situation of the anxiety-like behavior obstacle before and after comparison therapy.
Present invention also offers a kind of method evaluating therapeutic agent for alzheimer's disease or screening Alzheimer disease drugs, the method comprises use miR-34a process LAN recombinant expression vector and builds alzheimer's disease animal model, the improvement situation of the anxiety-like behavior obstacle before and after comparison therapy.
Therefore, miR-34a process LAN recombinant expression vector of the present invention can be used for the purposes of screening therapeutic agent for alzheimer's disease or anxiety disorder medicine.
Advantage of the present invention
The present invention by build miR-34a process LAN adeno-associated virus recombinant vectors and in hippocampus of mice process LAN, determine that miR-34a is on the ethological impact of anxiety disorder.Result of study shows, and this recombinant vectors is at whole animal level success high expression, and miR-34a process LAN can cause the behavior of anxiety disorder sample, shows that miR-34a can become the important candidate targets of this type of disease for the treatment of.The present invention provides new technique means for research miR-34a expresses dysfunction (as alzheimer's disease anxiety sample obstacle).
Accompanying drawing explanation
Fig. 1 is pcr amplification miR-34a gene map;
M:DNAmarker(2000bp)
1：SampleDNA
Fig. 2 is bacterium colony PCR qualification result figure;
M：DNAmarker(2000bp)
1 ~ 2: recombinant plasmid PAAV-miR-34a mono-bacterium colony; 3:pAAV-ires-GFP
The sequencing figure of gene fragment for the purpose of Fig. 3;
Fig. 4 is the picture of miR-34a stably transfected cell line under light microscopic and fluorescent microscope.
(A) picture of miR-34a stably transfected cell line under light microscopic, the scale in figure represents 100 μm.(B) be the picture under fluorescent microscope, from the visual field identical with (A).The miR-34a expression vector that the present invention builds contains GFP structural domain, and can express GFP while expression miR-34a, therefore positive cell can fluoresced green.Scale in figure represents 100 μm.
Fig. 5 is the expression of miR-34a over-express vector in hippocampus of mice CA3 district.
Fig. 6 is WT Mice brain tissues CA3 district miR-34a expression figure.
Fig. 7 is that miR-34a process LAN tests ethological effect diagram to the spacious field of WT mouse.
(A) the central area time; (B) total distance.Result is expressed as mean value ± standard error, often organizes 8-10 mouse; With t inspection, statistical analysis is carried out to result, think there is statistics difference with p<0.05, * * * p<0.001vsWT.
Fig. 8 is that miR-34a process LAN tests ethological effect diagram to WT mouse elevated plus-maze.
(A) open arms number of times is entered; (B) enter and close arm number of times; (C) the open arms time; (D) the arm time is closed; (E) enter open arms number of times to account for and enter the ratio that open arms closes arm total degree; (F) total distance.Result is expressed as mean value ± standard error, often organizes 8-10 mouse; With t inspection, statistical analysis is carried out to result, think there is statistics difference with p<0.05, * p<0.05, * * * p<0.001vsWT.
Embodiment
Below by way of embodiment, the present invention is described.
One, miR-34a over-express vector builds
1, sequence obtains and design of primers
Mouse miR-34a gene order (AccessionNo:EF606691) is found from the Nucleotide database Genbank of NCBI; then the partial sequence utilizing Oligo7 software to choose each CDS two ends is respectively analyzed; determine upstream and downstream primer; before 5 ' end of upstream primer, add protection base and BamHI restriction enzyme site sequence (CGGGATCC) again, 5 ' end of downstream primer adds protection base and EcoRI restriction enzyme site sequence (CGGAATTC).Primer entrusts the Shanghai biological company limited of raw work to synthesize, and its sequence is shown in Table 1:
Table 1.miR-34a gene amplification primer
2, the acquisition of template DNA
Cultivate Ni3T3 cell, extract STb gene with Blood & CellCultureDNAMiniKit (QIAGEN), concrete grammar reference reagent box specification sheets.
3, the acquisition of goal gene
The primer newly synthesized is added TEbuffer to dissolve, the ultimate density of adjustment primer is 10 μMs, with above-mentioned DNA for template, adds primer, PrimeSTARMaxDNAPolymerase.
PCR reaction system (10ul):
PCR reaction conditions is as follows:
PCR circulation is 30, and the sepharose qualification of race 1% after PCR completes, the results are shown in Figure 1.
Above-mentioned reaction system is expanded 10 times, determines goal gene according to Marker, cut glue and reclaim (step reclaims test kit with reference to glue).
4, the double digestion of goal gene and expression vector
(1) miR-34a gene enzyme cuts system (50ul):
(2) pAAV-ZSGREEN-miRNA carrier enzyme cuts system (50ul):
Above-mentioned reaction system is placed in 37 DEG C, spends the night or enzyme cuts 5 hours.Electrophoresis detection digestion products, and cut glue recovery.PAAV-ZSGREEN-miRNA carrier is purchased from excellent precious biological (VT2243).
5, goal gene and pAAV-ZSGREEN-miRNA is connected
The goal gene miR-34a cut by above-mentioned enzyme is connected to enzyme and cuts formation recombinant plasmid PAAV-miR-34a on carrier pAAV-ZSGREEN-miRNA, get pAAV-ZSGREEN-miRNA that enzyme cuts and sterilized water is negative control simultaneously, prepare the linked system of 10ul by table 2:
Table 2.10 μ lmiR-34a linked system
| pAAV-ZSGREEN-miRNA | 1ul |
| miR-34a | 3ul |
| T4Ligase buffer | 1ul |
| T4Ligase | 0.5 |
| H 2O | 5.5ul |
Then be placed in 22 DEG C of 3-6 hour, or 4 DEG C are spent the night.
6, transformation of E. coli Top10 and coated plate
Get 5ul to connect product and negative control and be added to respectively in the competence Top10 of 50ul, soft mixing, 4 DEG C of ice bath 30min, then 42 DEG C of heat shock 1min, 4 DEG C of coolings, add 100ul non-resistant substratum, are placed in shaking table 200 rotating speed 1 hour.Bacterium liquid is coated on ampicillin plate, is labeled as pAAV-MIR-34A, pAAV (-) respectively.Cultivate 14h, recombinant plasmid pAAV-MIR-34A flat board for 37 DEG C and grow single bacterium colony, and pAAV (-) flat board does not have single bacterium colony.
7, bacterium colony PCR identifies and order-checking
(1) bacterium colony PCR identifies
Random picking 10 single bacterium colonies, utilize pAAV-ires-GFP for template, primer miR-34a-F, miR-34a-R carry out pcr amplification, the results are shown in Figure 2 simultaneously.
(2) order-checking qualification
1, No. 2 sample is served Hai Shenggong order-checking, the results are shown in Figure 3.
8, recombinant plasmid is extracted in a large number
After order-checking is correct, the correct bacterium liquid of order-checking is pressed 1:500 enlarged culturing, puts 37 DEG C of overnight incubation.Collect bacterium liquid, extract plasmid, method is with reference to E.Z.N.A.Endo-FreePlasmidMaxKitSpin specification sheets.
9, encapsidated adenovirus correlated virus AAV9
According to AAV virus package kit specification sheets step by recombinant plasmid pAAV-miR-34a, PAAV-GFP (contrast) respectively with AAV2/9, pHELP plasmid coinfection AAV9 cell, encapsidated adenovirus correlated virus, the state of 24h observation of cell and fluorescence intensity, the results are shown in Figure 4.48h collects viral supernatants, and ultracentrifugation purified virus, finally the virus PBS of purifying is dissolved, quantitative analysis.
10, virus titer detects
(1) get 20ul concentrating virus liquid, add 1ulRNAse-freeDNAse, mixing, 37 DEG C of water-bath 30min.
(2) 4 DEG C, 12000rpm, centrifugal 10min, get 10ul supernatant in another aseptic 1.5mlEP pipe.
(3) 90ulDilutionBuffer (1mMTris-HCl, pH8.0,0.1mMEDTA, 150mMNaCl) is added, mixing, 100 DEG C of metal bath reaction 10min.
(4) naturally cool to room temperature, add 1ul Proteinase K, 37 DEG C of water-bath 1h.
(5) 100 DEG C of metal bath reaction 10min, naturally cool to room temperature.
(6) Q-PCR template is used as by after above-mentioned diluted sample, quantitative analysis of virus titre.
rAAV9-mir-34a：1.1x10
12vg/ml
rAAV9-GFP：2.7x10
12vg/ml
Detecting the titre of virus according to QPCR, is 10 by PBS virus dilution to final titre
12vg/m
Two, hippocampus of mice CA3 Naoliqing capsule injection
(1) induced anesthesia laboratory animal (APP
swe/ PS1
m146V/ Tau
p301Ltransgenic mice)
(2) animal brain solid is fixing
The mouse of anesthesia is moved to orientator region, unscrews the nose bar knob on orientator, the front tooth of mouse is stuck in circular hole; Pre-pressing nose bar, regulates the longitudinal separation of whole adapter to suitable position; Ear bar is inserted mouse duct, the left and right ear bar of balance adjustment, line and ear bar between mouse two ear is made on the same line, to ensure that the position scale of left and right ear bar is identical, compress ear bar, then nose bar is compressed, up-down adjustment front tooth folder height simultaneously, and front and back regulate adapter position, make mouse cranium face maintenance level, lock screw, fixing head; By padded for the health of mouse.
(3) brain operation of opening cranium
Open cold light source, regulate brightness, illuminate operative site; With Iodophor and cotton ball soaked in alcohol sterilization skin; Cut off scalp, remove periosteum; With dry cotton ball, skull surface is cleaned out.
(4) location drilling
Syringe is sucked miR34a over-express vector, the motion arm being connected to orientator makes it fixing; According to mouse skull sutura, find bregma point (Bregma); Reference point (zero point) using this as three-dimensional system of coordinate; According to brain map, determine to inject the coordinate figure of object point hippocampus CA3 relative to bregma point (Bregma), i.e. ML value (X-axis), AP value (Y-axis), DV value (Z axis); This experiment with X, Y, Z (2.3mm, 2.18mm, 2.10mm) for object point; Running fix instrument motion arm, observes liquid crystal display numerical value simultaneously; Needle tip is made to arrive the top X of target, Y (2.3mm, 2.18mm); Then operate Z axis, make most advanced and sophisticated decline, near skull surface; Mark is carried out immediately below needle point; Beat craniotomy drill power supply, and be adjusted to suitable rotating speed and hole.
(5) brain medicine micro-injection
Mobile Z axis, makes needle point pointed end move, and needle point continues to enter brain tissue downwards through skull surface, when the Z axis display of orientator digital display module is to (2.1mm), illustrate that syringe needle has arrived injection target area X, Y, Z (2.30mm, 2.18mm, 2.10mm); Injection rate is 1ul/min, and volume injected is 1ul/ side, starts injection; Let the acupuncture needle remain at a certain point 5min after injection, allows medicine fully absorb;
(6) skin closure and animal revive
With suture by brain skin closure, sew up 3-4 pin; After stitching, coat appropriate penicillin at suture; Withdraw from ear bar, unclamp nose bar, mouse is taken out from instrument; After reviving, rearging cage can be put back to and carry out other experiments follow-up;
Three, frozen section
(1) draw materials: mouse brain tissue took out after 10 days by vector injection
(2) fixing: tissue to be put into 4% paraformaldehyde, 4 DEG C of fixing more than 24h (comprising 24h);
(3) dewater: the tissue after fixing is put into 30% sucrose and dewaters, (be generally 24h) when tissue is deposited in after bottom solution, can carry out embedding and cutting into slices;
(4) embed: freezing microtome is opened by 2h in advance, and temperature reaches-20 DEG C and can use.Refrigerator tray adds the freezing embedding medium of OCT, makes rapidly embedding medium OCT solidify at fast precooling place;
(5) cut into slices: repairing the sheet stage, tissue is achieved the goal region; At dicing phase, thickness is set in 15 μm and cuts into slices, section is transferred on slide glass, rear 4 DEG C of preservations of having cut into slices;
(6) section puts into the acetone of 4 DEG C of precoolings, at 4 DEG C of fixing 10min; PBS embathes, and shaking table slowly shakes 3 times, each 5min;
(7) blot section moisture around, around tissue, draw the circle of a suitable size with immunohistochemical methods pen;
(8) DAPI dyeing: each tissue dropping 50 μ lDAPI dye liquor, room temperature lucifuge hatches 3min, and PBST embathes, and shaking table slowly shakes 3 times, each 3min;
(9) mounting: use filter paper to blot and organize surrounding liquid, the anti-fluorescence quenching dripping 15 μ about l carries out mounting;
(10) observe: use laser confocal microscope to observe; The results are shown in Figure 5.
Four, Q-PCR
(1) get Hippocampus CA 3 Region tissue, miRNeasyMiniKit (QIAGEN) extracts RNA, concrete grammar reference reagent box specification sheets
(2) probe method Hairpin-itmiRNAs quantitatively and u6 calibration qRT-PCR test kit (GenePharma) carry out RealTimeRT-PCR, concrete grammar reference reagent box specification sheets, the results are shown in Figure 6.
As shown in Figure 6, utilize the method for realtimeRTPCR, detect the level of miR-34a in 8 monthly age Mice brain tissues CA3 districts, result shows, and compared with the WT control mice at same monthly age, in injection miR-34a Mice brain tissues, the level of miR-34a obviously changes.Using U6snRNA as internal reference, the Ct value of miR-34a is carried out stdn.Data representation mean value ± standard error shown in figure.With t inspection, statistical analysis (n=3) is carried out to result, think there is statistics difference with p<0.05, * * * p<0.001vsWT.
Five, anxiety study of behaviour detects
In order to study miR-34a process LAN to the ethological impact of WT mouse anxiety, we construct miR-34a over-express vector and locating injection has carried out the experiment of spacious field to WT hippocampus of mice CA3 district.
1, spacious field experiment
Experimental technique reference literature
behavBrainRes.2014; 275:219-24, specific experiment step is as follows, the results are shown in Figure 7.
(1) experiment is carried out under quiet environment; Before experiment, animal was transferred to Behaviors survey room in 2 hours in advance, independent for every mouse is placed on rearging cage;
(2) in Xeye Behavior surveillance system, carry out the design of experiment porch, spacious field areas is split into 16 lattice regions, and wherein 12 is outer region, and 4 is middle section;
(3) mouse is taken out (mouse is experimenter dorsad) in cage, be positioned over spacious center court gently, allow it freely explore environment 5min and record;
(4) stop shooting after observing certain hour, mouse is taken out (every mouse accepts once to test) from mining site case; 5% spirituous solution cleaning square chest inwall and bottom surface, in order to avoid the remaining information (as the stool, urine of animal, smell) of animal last time affects next test result; Change animal, continue experiment;
(5), after experiment terminates, derive in data subordinate act Hygienic monitoring on hands of childhood system, and carry out the analysis of data.
Result shows, and compared with WT mouse, miR-34a process LAN WT mouse has occurred obvious downtrending (P<0.001) (Fig. 7 A) in the time of central area; The total distance there was no significant difference of motion (Fig. 7 B) of two groups of mouse, illustrates the motor capacity of two groups of mouse and indifference.The anxiety behavior of prompting miR-34a process LAN on WT mouse makes a significant impact.
2, elevated plus-maze test
Experimental technique reference literature
behavBrainRes.2015; 288:39-49, specific experiment step is as follows, the results are shown in Figure 8.
(1) experiment is carried out under quiet environment; Before experiment, animal was transferred to Behaviors survey room in 2 hours in advance, independent for every mouse is placed on rearging cage;
(2) in Xeye Behavior surveillance system, carry out the design of experiment porch, superior cross labyrinth closes arm by two open arms and two and a middle section forms;
(3) mouse is taken out in cage, mouse is placed on overhead central area gently towards open arms, allow it freely explore environment 5min and record;
(4) stop shooting after observing certain hour, mouse is taken out (every mouse accepts once to test) from mining site case; 5% spirituous solution cleaning square chest inwall and bottom surface, in order to avoid the remaining information (as the stool, urine of animal, smell) of animal last time affects next test result; Change animal, continue experiment;
(5), after experiment terminates, derive in data subordinate act Hygienic monitoring on hands of childhood system, and carry out the analysis of data.
As shown in Figure 8, elevated plus-maze test, by mouse in the face of open arms is placed on the middle section of Elevated plus-maze, arrives by record mouse the anxiety behavior that time of each arm and number of times etc. weigh mouse to result.Compared with WT mouse, the number of times that miR-34a process LAN WT mouse enters open arms significantly reduces (P<0.001) (Fig. 8 A), and enters the number of times closing arm and significantly increase (P<0.001) (Fig. 8 B).Simultaneously, compared with WT mouse, the time that miR-34a process LAN WT mouse enters open arms significantly reduces (P<0.05) (Fig. 8 C), and enters the time of closing arm and significantly increase (P<0.05) (Fig. 8 D).Compared with WT mouse, miR-34a process LAN WT mouse enters open arms number of times and accounts for the per-cent entering open arms number of times He close arm number of times, significant minimizing (P<0.001) (Fig. 8 E), it has the trend of rising to the Preference closing arm.The total distance there was no significant difference of motion (Fig. 8 F) of two groups of mouse, illustrates the motor capacity of two groups of mouse and indifference.To sum up, all results show, compared with WT mouse, the anxiety behavior of miR-34a process LAN WT mouse significantly increases.Judgement of proof miR-34a over-express vector can be used for curative effect or the screening Alzheimer disease drugs of evaluating therapeutic agent for alzheimer's disease.
Claims (6)
1. a miR-34a process LAN recombinant expression vector, it contains the sequence shown in sequence 1.
2. miR-34a process LAN recombinant expression vector according to claim 1, wherein said carrier is correlated virus recombinant vectors.
3. build the method for the miR-34a process LAN recombinant expression vector described in claim 1 or 2, the method comprises: (A) designs primer, pcr amplification miR-34a gene; (B) gene of amplification and expression vector enzyme are cut, connect goal gene and expression vector; (C) product conversion intestinal bacteria will be connected, cultivate; (D) extract recombinant plasmid after qualification and pack.
4. method according to claim 3, wherein said primer is 5 '-CGGGATCCGCAGCCTCTCCATCTTC-3 '; 5 '-GGAATTCGGCTAGGAGGATCAACACAC-3 '.
5. evaluate a method for anxiety disorder medicine or screening anxiety disorder medicine, the method comprises use miR-34a process LAN recombinant expression vector and builds anxiety disorder animal model, the improvement situation of the anxiety-like behavior obstacle before and after comparison therapy.
6. evaluate the method for therapeutic agent for alzheimer's disease or screening Alzheimer disease drugs for one kind, the method comprises use miR-34a process LAN recombinant expression vector and builds alzheimer's disease animal model, the improvement situation of the anxiety-like behavior obstacle before and after comparison therapy.
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| EP3983549A4 (en) * | 2019-06-17 | 2023-06-28 | Biorchestra Co., Ltd. | Compositions and methods for preparing an alzheimer's disease animal model using microrna |
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