CN105457042A - miR-34a silent expression recombinant vector and application thereof - Google Patents
miR-34a silent expression recombinant vector and application thereof Download PDFInfo
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- CN105457042A CN105457042A CN201510979708.0A CN201510979708A CN105457042A CN 105457042 A CN105457042 A CN 105457042A CN 201510979708 A CN201510979708 A CN 201510979708A CN 105457042 A CN105457042 A CN 105457042A
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Abstract
The invention relates to the use of a miR-34a gene in the preparation of a drug for treating alzheimer disease neuro-psychic disturbance or anxiety. The miR-34a gene is shown as a sequence 1. The invention also relates to tud-mir34a for inhibiting miR-34a expression and a miR-34a silent expression recombinant vector containing the tud-mir34a. The tud-mir34a is used to construct the miR-34a silent expression recombinant vector and inhibit the great expression of miR-34a in an AD mouse hippocampal brain tissue, and miR-34a inhibition is determined to be capable of remarkably relieving AD genetically modified mouse anxiety disorder praxiology obstacle.
Description
Technical field
The present invention relates to miR-34a silence and express recombinant vector and application thereof.
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, ripe micRNA34a sequence is as shown in sequence 1, 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 chromosome (1p36) position can betide in kinds of tumor cells, as neuroblastoma, hepatocarcinoma, colorectal cancer etc., therefore, miR-34a is often used to research role in tumor 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 tumor in body and occur and pernicious transfer, neural generation and differentiation, angiogenesis, apoptosis and necrosis etc., and therefore miR-34a is also widely used in research role in tumor.
But in Alzheimer or anxiety neurosis, in role prior art, there is not been reported about miR-34a.
Summary of the invention
The present invention is directed to miR-34a to design the tud-mir34a suppressing miR-34a to express, utilize tud-mir34a build the reticent recombinant vector of miR-34a and suppress the expression that miR-34a is large in AD hippocampus of mice cerebral tissue, determine that miR-34a suppresses significantly to improve AD transgenic mice anxiety disorder sample behavioristics obstacle.Result of study shows, and this recombinant vector at whole animal level success high expression, and significantly improves AD transgenic mice anxiety sample obstacle.This result of study not only provides new technological means for the function furtheing investigate miR-34a, provides new candidate policy for AD neuropsychiatric disorders such as anxiety sample obstacle target gene therapy simultaneously.
Therefore, an object of the present invention is to provide the purposes of miR-34a gene in the medicine preparing treatment Alzheimer neuropsychiatric disorders or anxiety neurosis, described miR-34a gene is as shown in sequence 1.
Another object of the present invention is to provide a kind of tud-mir34a suppressing miR-34a to express, and its gene order is as shown in sequence 2.
Another object of the present invention is to provide miR-34a silence and expresses recombinant vector, and it contains the gene order shown in sequence 2.
Present invention also offers and build the method that miR-34a silence expresses recombinant vector, the method comprises: (A) synthesizes tud-mir34a, and its sequence is as shown in sequence in sequence table 2; (B) tud-mir34a of synthesis and carrier are carried out enzyme action, the tud-mir34a of enzyme action is connected with carrier; (C) transformation of E. coli, extracts recombiant plasmid after qualification and packs.
Invention further provides the tud-mir34a of base sequence as shown in the sequence 2 or miR-34a containing tud-mir34a silence and express the purposes of recombinant vector in the medicine preparing treatment Alzheimer neuropsychiatric disorders or anxiety neurosis.
Advantage of the present invention
The present invention devises the tud-mir34a suppressing miR-34a to express, and utilizes it effectively to build miR-34a silent carrier, and in cerebral tissue, successfully suppresses the expression of miR-34a; Determine that the suppression of miR-34a significantly can improve AD transgenic mice anxiety sample obstacle simultaneously.
Accompanying drawing explanation
Fig. 1 is enzyme action qualification result figure.
M:DNAmarker(5000bp)
1 ~ 2: recombiant plasmid pAAV-tudmiRNA-34A
3:pAAV-ZsGreen-ShRNA empty carrier order-checking qualification
The sequencing figure of genetic fragment for the purpose of Fig. 2.
Fig. 3 is the picture of TudmiR-34a stably transfected cell line under light microscopic and fluorescence microscope.
(A) picture of TudmiR-34a stably transfected cell line under light microscopic, the scale in figure represents 100 μm; (B) this is the picture under fluorescence microscope, from the visual field identical with (A).The TudmiR-34a expression vector that we build contains GFP domain, and can express GFP while expression miR-34a, therefore positive cell can fluoresced green.Scale in figure represents 100 μm.
Fig. 4 is the expression of the reticent expression vector of TudmiR-34a in hippocampus of mice CA3 district.
Fig. 5 is APP
swe/ PS1
m146V/ Tau
p301Ltransgenic mouse brain tissue CA3 district TudmiR-34a expression.
Fig. 6 is that the expression of miR-34a silence tests ethological impact to the spacious field of Tg mice.
(A) the central area time; (B) total distance.Result is expressed as meansigma methods ± standard error, often organizes 8-10 mice; With t inspection, statistical analysis is carried out to result, think there is statistics difference with p<0.05, * * p<0.01vsTg.
Fig. 7 is that the expression of miR-34a silence tests ethological effect diagram to Tg 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 meansigma methods ± standard error, often organizes 8-10 mice; With t inspection, statistical analysis is carried out to result, think there is statistics difference with p<0.05, * p<0.05, * * p<0.01vsTg.
Detailed description of the invention
Below by way of detailed description of the invention, the present invention is described.
One, the reticent expression vector establishment of miR-34a
1, sequence obtains and design of primers
From the Nucleotide data base of NCBI, Genbank finds mice miR-34a gene order (AccessionNo:EF606691), the long-acting suppression TudmiR-34a sequence special according to the structural design miR-34a of TudRNA, adds BamHI, HindIII restriction enzyme site at aim sequence two ends.Sequence is delivered to the raw work synthesis in Shanghai.
TudmiR-34a：
GGATCCGGTGGCGCTAGGATCATCAACACCGTCACAGAAATCTTCGACCAACACAAGTATTCTGGTCACAGAATACAACACCGTCACAGAAATCTTCGACCAACACAAGATGATCCTAGCGCCACCTTTTTTAAGCTT
2, the double digestion of genes of interest and expression vector
(1) TudmiR-34a gene enzyme action system (50ul):
Genes of interest: 30ul
Reaction buffer:5ul
Water: 10ul
HindIII:2.5ul
BanHI:2.5ul
(2) pAAV-ZsGreen-ShRNA carrier enzyme action system (50ul):
Carrier: 20ul
Buffer：5ul
Water: 20ul
HindIII：2.5ul
BamHI：2.5ul
Above-mentioned reaction system is placed in 37 DEG C, spends the night or enzyme action 5 hours.Electrophoresis detection digestion products, and cut glue recovery.
3, genes of interest and pAAV-ZsGreen-ShRNA is connected
It (is a kind of gland relevant viral vector that the genes of interest miR-34a of above-mentioned enzyme action is connected to enzyme action carrier pAAV-ZsGreen-ShRNA, purchased from Shanghai Sheng Gong biological engineering company limited) upper formation recombiant plasmid pAAV-TudmiR-34a, pAAV-ZsGreen-ShRNA and the sterilized water of getting enzyme action are negative control simultaneously, prepare the linked system of 10ul by table 1:
Table 1.10 μ lmiR-34a linked system
| pAAV-ZsGreen-ShRNA | 1ul |
| TudmiR-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.
4, 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 culture medium, are placed in shaking table 200 rotating speed 1 hour.Bacterium liquid is coated on ampicillin plate, is labeled as pAAV-TudmiR-34a, pAAV (-) respectively.Cultivate 14h, recombiant plasmid pAAV-TudmiR-34a flat board for 37 DEG C and grow single bacterium colony, and pAAV (-) flat board does not have single bacterium colony.
5, enzyme action qualification and order-checking
(1) enzyme action qualification
Random picking 2 single bacterium colonies, shake bacterium picking plasmid, use HindIII and BglII double digestion recombinant vector and pAAV-ZsGreen-ShRNA empty carrier, the results are shown in Figure 1.
(2) 1, No. 2 sample is served Hai Shenggong order-checking, the results are shown in Figure 2.
6, recombiant plasmid is extracted in a large number
After order-checking is correct, the correct bacterium liquid of order-checking is pressed 1:500 amplification culture, puts 37 DEG C of overnight incubation.Collect bacterium liquid, extract plasmid, method is with reference to E.Z.N.A.Endo-FreePlasmidMaxKitSpin description.
7, encapsidated adenovirus correlated virus AAV9
According to AAV virus package kit description step by recombiant plasmid pAAV-TudmiR-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 3.48h collects viral supernatants, and ultracentrifugation purified virus, finally the virus PBS of purification is dissolved, quantitative analysis.
8, 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 E.C. 3.4.21.64,37 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-tudmiRNA-34A：1.7x1012vg/ml
rAAV9-GFP：3.0x1012vg/ml
Detecting the titre of virus according to QPCR, is 1012vg/ml by PBS virus dilution to final titre.
Two, hippocampus of mice CA3 Naoliqing capsule injection
(1) induced anesthesia laboratory animal (APPSwe/PS1M146V/TauP301L transgenic mice)
(2) animal brain solid is fixing
The mice of anesthesia is moved to position finder region, unscrews the nose bar knob on position finder, the front tooth of mice is stuck in circular hole; Pre-pressing nose bar, regulates the longitudinal separation of whole adapter to suitable position; Ear bar is inserted mice auditory meatus, the left and right ear bar of balance adjustment, line and ear bar between mice 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 mice cranium face maintenance level, lock screw, fixing head; By padded for the health of mice.
(3) brain operation of opening cranium
Open cold light source, regulate brightness, illuminate operative site; With povidone iodine 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 TudmiR34a carrier, the motion arm being connected to position finder makes it fixing; According to mice 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 impact 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 impact point; Running fix instrument motion arm, observes liquid crystal display screen 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; Labelling is carried out immediately below needle point; Beat drill for craniotomy 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 position finder 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, mice 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 microscope slide, rear 4 DEG C of preservations of having cut into slices.
(6) section puts into the acetone of 4 DEG C of pre-coolings, 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 SABC 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 4.
Four, Q-PCR
(1) get Hippocampus CA 3 Region tissue, miRNeasyMiniKit (QIAGEN) extracts RNA, concrete grammar reference reagent box description
(2) sonde method Hairpin-itmiRNAs quantitatively and u6 calibration qRT-PCR test kit (GenePharma) carry out RealTimeRT-PCR, concrete grammar reference reagent box description, the results are shown in Figure 5.
As shown in Figure 5, utilize the method for realtimeRTPCR, detect the level of TudmiR-34a in 8 monthly age Mice brain tissues CA3 districts, result shows, and compared with the control mice at same monthly age, in injection TudmiR-34a Mice brain tissues, the level of miR-34a obviously changes.Using U6snRNA as internal reference, the Ct value of TudmiR-34a is carried out standardization.Data representation meansigma methods ± 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<0.05vsTg.
Five, anxiety behavioristics is detected
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 6.
(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 mice 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) mice is taken out (mice 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, mice is taken out (every mice accepts once to test) from mining site case; 5% alcoholic solution cleaning square chest inwall and bottom surface, in order to avoid the remaining information (as the stool, urine of animal, abnormal smells from the patient) 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.
Express the ethological impact of Tg mouse anxiety to study miR-34a silence, we construct the reticent expression vector of miR-34a and locating injection has carried out the experiment of spacious field to Tg hippocampus of mice CA3 district.Result shows, and compared with Tg mice, miR-34a silence is expressed Tg mice and occurred obvious increase (P<0.01) (Fig. 6 A) in the time of central area; The total distance there was no significant difference of motion (Fig. 6 B) of two groups of mices, illustrates the motor capacity of two groups of mices and zero difference.Prompting miR-34a silence expresses the anxiety behavior that significantly can improve Tg mice.
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 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 mice 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) mice is taken out in cage, mice 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, mice is taken out (every mice accepts once to test) from mining site case; 5% alcoholic solution cleaning square chest inwall and bottom surface, in order to avoid the remaining information (as the stool, urine of animal, abnormal smells from the patient) 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.
Elevated plus-maze test, by mice in the face of open arms is placed on the middle section of Elevated plus-maze, arrives by record mice the anxiety behavior that time of each arm and number of times etc. weigh mice.Compared with Tg mice, miR-34a silence expresses the number of times that Tg mice enters open arms significantly increases (P<0.01) (Fig. 7 A), and enters the number of times closing arm and significantly reduce (P<0.05) (Fig. 7 B).Simultaneously, compared with Tg mice, miR-34a silence expresses the time that Tg mice enters open arms significantly increases (P<0.05) (Fig. 7 C), and enters the time of closing arm and significantly reduce (P<0.05) (Fig. 7 D).Compared with Tg mice, miR-34a silence expression Tg mice enters open arms number of times and accounts for the percentage ratio entering open arms number of times He close arm number of times, significant increase (P<0.01) (Fig. 7 E), it has the trend of rising to the Preference of open arms.The total distance there was no significant difference of motion (Fig. 7 F) of two groups of mices, illustrates the motor capacity of two groups of mices and zero difference.To sum up, all results show, compared with Tg mice, miR-34a silence expresses the anxiety behavior that significantly can improve Tg mice.
Claims (5)
- The purposes of 1.miR-34a gene in the medicine preparing treatment Alzheimer neuropsychiatric disorders or anxiety neurosis, described miR-34a gene is as shown in sequence 1.
- 2. suppress the tud-mir34a that miR-34a expresses, its gene order is as shown in sequence 2.
- 3.miR-34a silence expresses recombinant vector, and it contains the gene order shown in sequence 2.
- 4. build the method that miR-34a silence expresses recombinant vector, the method comprises: (A) synthesizes tud-mir34a, and its sequence is as shown in sequence in sequence table 2; (B) tud-mir34a of synthesis and carrier are carried out enzyme action, the tud-mir34a of enzyme action is connected with carrier; (C) transformation of E. coli, extracts recombiant plasmid after qualification and packs.
- 5.tud-mir34a or the silence of the miR-34a containing tud-mir34a express the purposes of recombinant vector in the medicine preparing treatment Alzheimer neuropsychiatric disorders or anxiety neurosis, and the base sequence of described tud-mir34a is as shown in sequence 2.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105567737A (en) * | 2015-12-30 | 2016-05-11 | 广州凯拓生物科技开发有限公司 | Construction and application of miRNA-34a overexpression recombinant vector |
| WO2018165929A1 (en) * | 2017-03-15 | 2018-09-20 | 深圳市博奥康生物科技有限公司 | Dual mirna inhibitory expression vector, construction method and application thereof |
| WO2018170759A1 (en) * | 2017-03-22 | 2018-09-27 | 深圳市博奥康生物科技有限公司 | Recombinant ad-140-148a-185-tud adenovirus, and construction and application thereof |
-
2015
- 2015-12-23 CN CN201510979708.0A patent/CN105457042B/en active Active
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| BRIAN GEORGE DIAS等: "Amygdala-Dependent Fear Memory Consolidation via miR-34a and Notch Signaling", 《NEURON》 * |
| 伍开明: "下调miR-21抑制实验性肝纤维化及其机制研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567737A (en) * | 2015-12-30 | 2016-05-11 | 广州凯拓生物科技开发有限公司 | Construction and application of miRNA-34a overexpression recombinant vector |
| WO2018165929A1 (en) * | 2017-03-15 | 2018-09-20 | 深圳市博奥康生物科技有限公司 | Dual mirna inhibitory expression vector, construction method and application thereof |
| WO2018170759A1 (en) * | 2017-03-22 | 2018-09-27 | 深圳市博奥康生物科技有限公司 | Recombinant ad-140-148a-185-tud adenovirus, and construction and application thereof |
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| CN105457042B (en) | 2019-01-04 |
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