CN110144348A - SiRNA, recombinant vector and its application expressed for striking low DEAH box unwindase 16 - Google Patents

SiRNA, recombinant vector and its application expressed for striking low DEAH box unwindase 16 Download PDF

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CN110144348A
CN110144348A CN201910325016.2A CN201910325016A CN110144348A CN 110144348 A CN110144348 A CN 110144348A CN 201910325016 A CN201910325016 A CN 201910325016A CN 110144348 A CN110144348 A CN 110144348A
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dhx16
sirna
shrna
dna
recombinant vector
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张虎
周颖婷
吴隽松
杨留才
胡明
杜欣娜
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Jiangsu Vocational College of Medicine
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Abstract

The invention belongs to molecular biology and gene engineering technology field, it is related to siRNA, recombinant vector and its application expressed for striking low DEAH box unwindase 16.The siRNA has the nucleotide sequence complementary with the mRNA of DEAH box unwindase 16.Cell proliferation experiment significantly inhibited the proliferation of breast cancer cell MCF7, mouse-borne tumor experimental result is shown, DHX16 low expression group breast cancer weight is substantially less than control group the results show that strike the expression of low DHX16 gene with the 5th day on day 4.Therefore, DEAH box unwindase 16 is new breast cancer treatment target, is had broad application prospects.

Description

SiRNA, recombinant vector and its application expressed for striking low DEAH box unwindase 16
Technical field
The invention belongs to molecular biology and gene engineering technology field, more particularly, to one kind for striking low DEAH The corresponding shRNA of the siRNA that box unwindase 16 is expressed, the siRNA, encodes the DNA of the shRNA, the recombination including the DNA Carrier, the recombinant slow virus including the recombinant vector, including the siRNA, shRNA, encode the DNA of shRNA, recombinant vector and The host cell of recombinant slow virus and their application.
Background technique
Breast cancer is one of the malignant tumour that women is common in world wide, and new hair rate and the death rate occupy all kinds of cancers It is the first.Although China's breast cancer incidence is relatively low, large population base, overall incidence number is more, and disease incidence has on year by year The trend risen, and age of onset is in rejuvenation trend.Breast cancer is in origin of cell, Histological Study, disease classification, clinical table Existing, therapeutic response and metastatic potential etc. all show great complexity and heterogeneity, limit existing breast cancer and control The validity and popularity for the treatment of method.Clinically still lack the target spot of breast cancer treatment.
DEAH box unwindase 16 (DHX16) is DEAD box protein family member.DEAD box protein is a kind of comprising asparagus fern ammonia Acid-glutamate-alanine-aspartic acid conserved motifs RNA helicase.The albuminoid participates in the more of RNA secondary structural change Kind cell processes, the assembling including RNA montage and ribosomes and spliceosome in translation initiation, nucleus and mitochondria.Base In the distribution characteristics of this albuminoid, some family members participate in embryo's generation, Sperm specific enzyme and cell growth and division.DEAD Box protein encoding gene is located at chromosome 6p21.3, i.e. major histocompatibility complex code area, this region and it is a variety of it is pernicious, Heredity and autoimmune disease are related.DHX16 is one of DEAD box protein family member, has ATP combination rna helicase Enzymatic activity participates in Pre-mRNA montage process.Importantly, mutation DHX16 can inhibit normal transhipment and expression, and mRNA Montage related gene DHX16 mononucleotide polymorphism site rs115420460 is related to lung cancer morbidity risk.However, DHX16 exists Effect during growth of breast cancers has no specific research.
Summary of the invention
It is described the object of the present invention is to provide the siRNA for being directed to new breast cancer treatment target DEAH box unwindase 16 The corresponding shRNA of siRNA, encodes the DNA of the shRNA, the recombinant vector including the DNA, the recombination including the recombinant vector Slow virus including the siRNA, shRNA, encodes the host cell of the DNA of shRNA, recombinant vector and recombinant slow virus, and Their application.
To achieve the goals above, the first aspect of the present invention provides a kind of siRNA, which has and DEAH box solution Revolve the nucleotide sequence of the mRNA complementation of enzyme 16.
Of the invention focuses on providing new breast cancer treatment target, i.e. DEAH box unwindase 16, and is not limited to specific SiRNA sequence, can according to the various methods of this field routine design be directed to the target siRNA.
The siRNA usually has the nucleotide sequence of 19~27bp, and specifically, the nucleotides sequence of the siRNA is classified as Following at least one set:
(1)DHX16-siRNA1
DHX16-si-1a:5 '-AGCCUCCAGAAGAAACGUAAA-3 ' (SEQ ID NO:1)；
DHX16-si-1b:5 '-UUUACGUUUCUUCUGGAGGCU-3, (SEQ ID NO:2)；
(2)DHX16-siRNA2
DHX16-si-2a:5 '-CGAUCUUAUAGGUUACUGGAA-3 ' (SEQ ID NO:3)；
DHX16-si-2b:5 '-UUCCAGUAACCUAUAAGAUCG-3 ' (SEQ ID NO:4)；
(3)DHX16-siRNA3
DHX16-si-3a:5 '-UGGGAUCUAGAGAAAGUGAAA-3 ' (SEQ ID NO:5)；
DHX16-si-3b:5 '-UUUCACUUUCUCUAGAUCCCA-3 ' (SEQ ID NO:6).
Second aspect of the present invention protects a kind of shRNA, is the single stranded RNA with loop-stem structure, the nucleosides of the shRNA Acid sequence is following at least one set:
(1)DHX16-shRNA1
DHX16-sh-1a:5 '-CCGGAGCCUCCAGAAGAAACGUAAACUCGAGUUUACGUUUCUUCUGGAGGCUU UUUUG-3 ' (SEQ ID NO:7)；
DHX16-sh-1b:5 '-AAUUCAAAAAAGCCUCCAGAAGAAACGUAAACUCGAGUUUACGUUUCUUCUGG AGGCU-3 ' (SEQ ID NO:8)；
(2)DHX16-shRNA2
DHX16-sh-2a:5 '-CCGGCGAUCUUAUAGGUUACUGGAACUCGAGUUCCAGUAACCUAUAAGAUCGU UUUUG-3 ' (SEQ ID NO:9)；
DHX16-sh-2b:5 '-AAUUCAAAAACGAUCUUAUAGGUUACUGGAACUCGAGUUCCAGUAACCUAUAA GAUCG-3 ' (SEQ ID NO:10)；
(3)DHX16-shRNA3
DHX16-sh-3a:5 '-CCGGUGGGAUCUAGAGAAAGUGAAACUCGAGUUUCACUUUCUCUAGAUCCCAU UUUUG-3 ' (SEQ ID NO:11)；
DHX16-sh-3b:5 '-AAUUCAAAAAUGGGAUCUAGAGAAAGUGAAACUCGAGUUUCACUUUCUCUAGA UCCCA-3 ' (SEQ ID NO:12).
Third aspect present invention provides a kind of DNA for encoding above-mentioned shRNA, the nucleotides sequence of the DNA be classified as it is following extremely It is one group few:
(1)DHX16-shDNA1
DHX16-d-1a:5 '-CCGGAGCCTCCAGAAGAAACGTAAACTCGAGTTTACGTTTCTTCTGGAGGCTTT TTTG-3 ' (SEQ ID NO:13)；
DHX16-d-1b:5 '-AATTCAAAAAAGCCTCCAGAAGAAACGTAAACTCGAGTTTACGTTTCTTCTGGA GGCT-3 ' (SEQ ID NO:14)；
(2)DHX16-shDNA2
DHX16-d-2a:5 '-CCGGCGATCTTATAGGTTACTGGAACTCGAGTTCCAGTAACCTATAAGATCGTT TTTG-3 ' (SEQ ID NO:15)；
DHX16-d-2b:5 '-AATTCAAAAACGATCTTATAGGTTACTGGAACTCGAGTTCCAGTAACCTATAAG ATCG-3 ' (SEQ ID NO:16)；
(3)DHX16-shDNA3
DHX16-d-3a:5 '-CCGGTGGGATCTAGAGAAAGTGAAACTCGAGTTTCACTTTCTCTAGATCCCATT TTTG-3 ' (SEQ ID NO:17)；
DHX16-d-3b:5 '-AATTCAAAAATGGGATCTAGAGAAAGTGAAACTCGAGTTTCACTTTCTCTAGAT CCCA-3 ' (SEQ ID NO:18).
The fourth aspect of the present invention provides a kind of recombinant vector, and the recombinant vector is (to be purchased from Shang Haiji in GV493 plasmid Triumphant Gene Tech. Company Limited) multiple cloning sites AgeI and EcoRI be inserted into the recombination that the DNA of above-mentioned coding shRNA is obtained and carry Body.
The fifth aspect of the present invention provides a kind of recombinant slow virus, and the recombinant slow virus is by above-mentioned recombinant vector and virus It packs 1.0 carrier of helper plasmid pHelper and virus packaging 2.0 carrier cotransfection mammal of helper plasmid pHelper is thin Born of the same parents obtain.
The sixth aspect of the present invention provides a kind of host cell, including above-mentioned siRNA, above-mentioned shRNA, encodes shRNA's At least one of DNA, above-mentioned recombinant vector and above-mentioned recombinant slow virus.The present invention does not have the specific type of the host cell It is particularly limited to, for example, 293T cell.
The seventh aspect of the present invention provide above-mentioned siRNA, above-mentioned shRNA, encode the DNA of shRNA, above-mentioned recombinant vector and The application of at least one of above-mentioned recombinant slow virus.
Specifically, the application is the preparation that preparation inhibits DEAH box unwindase 16 to express.
Further, the application is the preparation that preparation inhibits Cells Proliferation of Human Breast Cancer and/or growth, or pre- for preparation Anti- and/or treatment breast cancer preparation.
Cell proliferation experiment significantly inhibited mammary gland with the 5th day on day 4 the results show that strike the expression of low DHX16 gene The proliferation of cancer cell MCF7, mouse-borne tumor experimental result show that DHX16 low expression group breast cancer weight is substantially less than control group. Therefore, DEAH box unwindase 16 is new breast cancer treatment target, is had broad application prospects.
Other features and advantages of the present invention will then part of the detailed description can be specified.
Detailed description of the invention
Exemplary embodiment of the invention is described in more detail in conjunction with the accompanying drawings, it is of the invention above-mentioned and its Its purpose, feature and advantage will be apparent.
Fig. 1 shows the differential expression of DHX16 in galactophore epithelial cell and breast cancer cell.Wherein, MCF-10A (writes a Chinese character in simplified form It is normal control cell for 10A), MCF7, MDA-MB-231 (being abbreviated as 231) and T-47D are breast cancer cell.
Fig. 2 shows the influences striking low DHX16 and being proliferated to breast cancer cell MCF7.Wherein, shCtrl be control group (on), ShDHX16 be experimental group (under).
Fig. 3 shows the influence for striking low DHX16 to tumor-bearing mice growth of breast cancers.Wherein, NC is control group, shDHX16 For experimental group.
Specific embodiment
The preferred embodiment of the present invention is described in more detail below.Although the following describe preferred implementations of the invention Mode, however, it is to be appreciated that may be realized in various forms the present invention without that should be limited by the embodiments set forth herein.It is real The person that is not specified actual conditions in example is applied, is all carried out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same or instrument are not Production firm person is indicated, is the conventional products that can be obtained by commercially available purchase.
The number of source of people DHX16 involved in following embodiments is Gene ID:8449.
In following embodiments, statistical analysis is carried out using 6.0 software of GraphPad Prism.All experiment in vitro counterpoises Again more than three times.Data are indicated with means standard deviation (SD).P < 0.05 is considered to have statistical significance.
Embodiment 1
The present embodiment is for illustrating that DHX16 high is expressed in breast cancer cell line.
The extraction of RNA and reverse transcription quantitative PCR (RT-qPCR)
1, Total RNAs extraction: the present embodiment carries out at low temperature.Cell culture carries out in 6 orifice plates, removes culture medium, PBS 1000 μ l of Trizol, shaking table 10min is added in rinsing 3 times, every hole；It is collected into 1.5ml centrifuge tube, 200 μ l chlorine are added in every pipe It is imitative, 30sec is acutely mixed, 15min is stood, 4 DEG C of 12000rpm are centrifuged 15min；400 μ l of gentle aspiration supernatant liquid is to another In new centrifuge tube, isometric isopropanol is added, is gently mixed by inversion, 4 DEG C of 12000rpm are centrifuged 10min；Supernatant is abandoned, 1ml is added 75% ethanol wash sediment, 4 DEG C of 12000rpm are centrifuged 10min；Supernatant is discarded as far as possible, dries 10min, every pipe at room temperature 10 μ l are added without RNase water, dissolve, spectrophotometer is quantitative.
2, reverse transcription: every 25 μ l reverse transcription system includes 100pmol random primer, 2 μ g total serum IgEs, 1 μ of M-MLV reverse transcriptase 0.625 1.25 μ l, 5 × M-MLV buffer of μ l, dNTPs (10mM) of l, RNase inhibitor, 5 μ l, remaining uses ddH2O polishing is to 25 μ l.Reaction condition are as follows: 37 DEG C of 1h, 95 DEG C of 5min.
3, quantitative PCR: every 20 μ l reaction system includes 2 × PCR Mix (ABI) 10 μ l, each 0.4 μ l of upstream and downstream primer, CDNA 1 μ l, ddH2O 8.2μl.Reaction condition are as follows: 94 DEG C of 2min, 94 DEG C of 15s, 60 DEG C of 40s, 40 circulations.It is used in experiment To primer sequence be shown in Table 1.
1 fluorescence quantitative RT-PCR primer sequence of table
Such as Fig. 1, RT-qPCR as the result is shown in MDA-MB-231, MCF7 and T47D cell DHX16 expression compared with control group MCF- 10A cell significantly increases (P < 0.001, P < 0.001, P < 0.001).
Embodiment 2
The present embodiment is for illustrating that the expression for striking low DHX16 can inhibit breast cancer to be proliferated and/or grow.
One, the preparation of RNAi slow virus clone
1, shot design
3 RNAi target sequences, DHX16 gene 3 are designed according to RNAi sequence design principle for DHX16 gene order A RNA interfered target sequence is respectively as follows: 5'-TGGGATCTAGAGAAAGTGAAA-3'(SEQ ID NO:23), 5'- CGATCTTATAGGTTACTGGAA-3'(SEQ ID NO:24) and 5'-AGCCTCCAGAAGAAACGTAAA-3'(SEQ ID NO:25).Negative control (NC) RNA interfered target sequence are as follows: 5'-TTCTCCGAACGTGTCACGT-3'(SEQ ID NO:26).
Based on above-mentioned RNA disturbance target point design siRNA it is as shown in table 2, siRNA used in embodiment be DHX16-si-1a, DHX16-si-1b, DHX16-si-2a, DHX16-si-2b, DHX16-si-3a, DHX16-si-3b, NC-si-a and NC-si-b For the sequence designed for negative control group:
Table 2
Title SiRNA sequence 5'-3'
DHX16-si-1a 5 '-AGCCUCCAGAAGAAACGUAAA-3 ', SEQ ID NO:1
DHX16-si-1b 5 '-UUUACGUUUCUUCUGGAGGCU-3 ', SEQ ID NO:2
DHX16-si-2a 5 '-CGAUCUUAUAGGUUACUGGAA-3 ', SEQ ID NO:3
DHX16-si-2b 5 '-UUCCAGUAACCUAUAAGAUCG-3 ', SEQ ID NO:4
DHX16-si-3a 5 '-UGGGAUCUAGAGAAAGUGAAA-3 ', SEQ ID NO:5
DHX16-si-3b 5 '-UUUCACUUUCUCUAGAUCCCA-3 ', SEQ ID NO:6
NC-si-a 5 '-UUCUCCGAACGUGUCACGU-3 ', SEQ ID NO:27
NC-si-b 5 '-ACGUGACACGUUCGGAGAA-3 ', SEQ ID NO:28
Table 3 shows the nucleotide sequence of shRNA used, and shRNA used in embodiment is DHX16-sh-1a, DHX16-sh- 1b, DHX16-sh-2a, DHX16-sh-2b, DHX16-sh-3a, DHX16-sh-3b, NC-sh-a and NC-sh-b are control group sequence Column:
Table 3
Table 4 shows the nucleotide sequence of the DNA of code used shRNA, and the DNA of the code used shRNA of embodiment is DHX16-d-1a, DHX16-d-1b, DHX16-d-2a, DHX16-d-2b, DHX16-d-3a, DHX16-d-3b, NC-d-a and NC- D-b is control group sequence:
Table 4
2, carrier digestion
50 μ l digestion systems are prepared according to table 5.Various reagents are sequentially added by tab sequential, are gently blown and beaten with pipettor mixed Even, brief centrifugation is placed in 37 DEG C of reaction 3h.Agarose gel electrophoresis is carried out to carrier digestion products, recycles purpose band.
5 carrier digestion system of table
Reagent Volume (μ l)
ddH2O 41
10 × reaction buffer 5
The GV493 Plasmid DNA (1 μ g/ μ L) of purifying 2
AgeI(10U/μl) 1
EcoRI(10U/μl) 1
Total 50
3, shDNA anneals to form double-stranded DNA
Pairs of shDNA dry powder is dissolved in annealing buffer after synthesis, 90 DEG C of water-bath 15min, cooled to room temperature.
4, carrier connects
The carrier that double digestion linearizes is connected with annealing double-stranded DNA by T4DNA ligase, 16 DEG C of connection 1-3h.
3 carrier linked system of table
Reagent Volume (μ l)
Linearized vector (100ng/ μ l) 1
Double-stranded DNA (100ng/ μ l) 1
10 × T4DNA ligase buffer solution 2
T4DNA ligase 1
ddH2O Complement to 20
5, it converts
10 μ L connection reaction products are added in 100 μ L competent cells, flicks and is mixed under tube wall number, placed on ice 30min；42 DEG C of heat shock 90s, ice bath are incubated for 2min；500 μ L LB culture mediums are added, are placed in 37 DEG C of shaking table shaken cultivation 1h；It takes suitable Amount bacterium solution is uniformly coated on the plate containing corresponding antibiotic, and culture 12-16h is inverted in constant incubator.
6, sequencing identification
The positive colony transformant identified is inoculated in the LB liquid medium containing corresponding antibiotic in right amount, 37 DEG C of trainings 12-16h is supported, appropriate bacterium solution is taken to be sequenced, identified.
7, plasmid transfection and slow virus harvest
Virus packaging is related to three plasmids altogether: carrying tool carrier plasmid (Ji Kai GV493 carrier), the virus of target sequence Pack helper plasmid (Ji Kai Helper 1.0) and virus packaging helper plasmid (Ji Kai Helper 2.0).Using three plasmid corotation Contaminate 293T cell.
Before transfection for 24 hours, it with the 293T cell of trypsin digestion logarithmic growth phase, is adjusted with the culture medium containing 10% serum Cell density about 5 × 106/ 15ml is reinoculated on 10cm Tissue Culture Dish, 37 DEG C, 5%CO2Culture in incubator；For 24 hours to thin It can be used to transfect when born of the same parents' density is up to 70%~80%；2h is changed to serum free medium before transfecting；Sterile centrifugation tube is taken, is added Each DNA solution (20 μ g of GV493 plasmid, 1.0 pHelper vector plasmid, 15 μ g, 2.0 pHelper vector plasmid, 10 μ g), with phase The triumphant transfection reagent of Ji of volume is answered to be uniformly mixed, adjustment total volume is 1ml, incubates 15min at room temperature；Mixed liquor is slowly added dropwise Into 293T cell culture fluid, mix, in 37 DEG C, 5%CO2It is cultivated in cell incubator；It is discarded after culture 6h mixed containing transfection With the culture medium of object, the PBS cleaning that 10ml is added is primary, falls after the soft transfection mixture for shaking culture dish to wash remnants It abandons；
It is slowly added to the cell culture medium 20ml containing 10% serum, in 37 DEG C, 5%CO2Continue to cultivate 48- in incubator 72h。
8, slow virus concentration and purifying and quality inspection
According to cell state, the 293T cell supernatant of 48h (transfection can be calculated as 0h) after transfection is collected；In 4 DEG C, 4000g is centrifuged 10min, removes cell fragment；With 0.45 μm of filter filtering supernatant in 40ml ultracentrifugation pipe；Trim respectively Ultracentrifugation pipe with vial supernatant is put into Beckman ultracentrifuge, 4 DEG C, 25000rpm by sample one by one, It is centrifuged 2h；It after centrifugation, discards supernatant, removal as far as possible remains in the liquid on tube wall, and virus is added and saves liquid, gently repeatedly Piping and druming is resuspended；Through after completely dissolution, after high speed centrifugation 10000rpm, 5min, supernatant being taken to dispense as required；
The key Quality Control of slow virus includes physical state detection, Sterility testing and virus titer detection.
Two, slow-virus transfection
To guarantee gene jamming effectiveness, the present embodiment designs 3 RNA disturbance target points for DHX16 gene, and will carry not 3 plasmid equal proportions mixing with target spot carries out slow virus packaging, so that it is guaranteed that striking for target gene subtracts after virus infected cell Efficiency.
Cell secondary culture is into 6 orifice plates after 12-16 hours: virus liquid 0.15ml and fresh cell medium mixed, Ratio is that 0.5ml fresh medium and 0.65 μ l polybrene (final concentration 4ng/ml) are added in 0.15ml vial supernatant； Pre- mixed virus infection liquid is added in aim cell culture dish, and cell density is no more than 50% at this time.After being incubated overnight It is replaced with fresh medium.
After infection 3 days, logarithmic growth phase cell carries out cell proliferation experiment.
Three, cell proliferation experiment
Above-mentioned control group and the cell in logarithmic growth phase for striking low DHX16 expression are subjected to pancreatin digestion, are made thin Born of the same parents' suspension；Cell suspension (cell number is about 3000) is inoculated in 96 orifice plates, is counted respectively at the 1st day, 2 days, 3 days, 4 days, 5 days Cell quantity is calculated, growth curve is drawn.
As shown in Fig. 2, cell proliferation experiment is the results show that strike the expression of low DHX16 gene, it is aobvious with the 5th day on day 4 Write the proliferation (P=0.0018, P=0.016) for inhibiting breast cancer cell MCF7.
Four, human breast cancer in nude mice lotus knurl is tested
Cell suspension is respectively prepared in control group and the MCF7 breast cancer cell line for striking low DHX16, carries out nude mice fat pad Plantation.Every group of 6 mouse, every mouse inoculation 100 μ l, 2 × 106A cell.After 1 month, tumor size and volume are detected, into Row statistical analysis.
As shown in figure 3, mouse-borne tumor experimental result is shown, the gross tumor volume of DHX16 low expression group is substantially less than control group (P<0.01)。
Various embodiments of the present invention are described above, above description is exemplary, and non-exclusive, and It is not limited to disclosed each embodiment.Without departing from the scope and spirit of illustrated each embodiment, for this skill Many modifications and changes are obvious for the those of ordinary skill in art field.
Sequence table
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<213>artificial sequence (Artificial Sequence)
<400> 3
cgaucuuaua gguuacugga a 21
<210> 4
<211> 21
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 4
uuccaguaac cuauaagauc g 21
<210> 5
<211> 21
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ugggaucuag agaaagugaa a 21
<210> 6
<211> 21
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 6
uuucacuuuc ucuagauccc a 21
<210> 7
<211> 58
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ccggagccuc cagaagaaac guaaacucga guuuacguuu cuucuggagg cuuuuuug 58
<210> 8
<211> 58
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 8
aauucaaaaa agccuccaga agaaacguaa acucgaguuu acguuucuuc uggaggcu 58
<210> 9
<211> 58
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ccggcgaucu uauagguuac uggaacucga guuccaguaa ccuauaagau cguuuuug 58
<210> 10
<211> 58
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 10
aauucaaaaa cgaucuuaua gguuacugga acucgaguuc caguaaccua uaagaucg 58
<210> 11
<211> 58
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ccggugggau cuagagaaag ugaaacucga guuucacuuu cucuagaucc cauuuuug 58
<210> 12
<211> 58
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 12
aauucaaaaa ugggaucuag agaaagugaa acucgaguuu cacuuucucu agauccca 58
<210> 13
<211> 58
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
ccggagcctc cagaagaaac gtaaactcga gtttacgttt cttctggagg cttttttg 58
<210> 14
<211> 58
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
aattcaaaaa agcctccaga agaaacgtaa actcgagttt acgtttcttc tggaggct 58
<210> 15
<211> 58
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ccggcgatct tataggttac tggaactcga gttccagtaa cctataagat cgtttttg 58
<210> 16
<211> 58
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
aattcaaaaa cgatcttata ggttactgga actcgagttc cagtaaccta taagatcg 58
<210> 17
<211> 58
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ccggtgggat ctagagaaag tgaaactcga gtttcacttt ctctagatcc catttttg 58
<210> 18
<211> 58
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
aattcaaaaa tgggatctag agaaagtgaa actcgagttt cactttctct agatccca 58
<210> 19
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
ggatgaggca cacgaaagga 20
<210> 20
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
aacacagggg cgtcatcaaa 20
<210> 21
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ggcacccagc acaatgaaga 20
<210> 22
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
actcctgctt gctgatccac 20
<210> 23
<211> 21
<212> DNA
<213> Homo sapiens
<400> 23
tgggatctag agaaagtgaa a 21
<210> 24
<211> 21
<212> DNA
<213> Homo sapiens
<400> 24
cgatcttata ggttactgga a 21
<210> 25
<211> 21
<212> DNA
<213> Homo sapiens
<400> 25
agcctccaga agaaacgtaa a 21
<210> 26
<211> 19
<212> DNA
<213> Homo sapiens
<400> 26
ttctccgaac gtgtcacgt 19
<210> 27
<211> 19
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 27
uucuccgaac gugucacgu 19
<210> 28
<211> 19
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 28
acgugacacg uucggagaa 19
<210> 29
<211> 57
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 29
ccgguucucc gaacguguca cguuucaaga gaacgugaca cguucggaga auuuuug 57
<210> 30
<211> 57
<212> RNA
<213>artificial sequence (Artificial Sequence)
<400> 30
aauucaaaaa uucuccgaac gugucacguu cucuugaaac gugacacguu cggagaa 57
<210> 31
<211> 57
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
ccggttctcc gaacgtgtca cgtttcaaga gaacgtgaca cgttcggaga atttttg 57
<210> 32
<211> 57
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
aattcaaaaa ttctccgaac gtgtcacgtt ctcttgaaac gtgacacgtt cggagaa 57

Claims (10)

1. a kind of siRNA, which is characterized in that the siRNA has the nucleotides sequence complementary with the mRNA of DEAH box unwindase 16 Column.
2. siRNA according to claim 1, which is characterized in that the nucleotides sequence of the siRNA is classified as following at least one Group:
(1)DHX16-siRNA1
DHX16-si-1a:5 '-AGCCUCCAGAAGAAACGUAAA-3 '；
DHX16-si-1b:5 '-UUUACGUUUCUUCUGGAGGCU-3 '；
(2)DHX16-siRNA2
DHX16-si-2a:5 '-CGAUCUUAUAGGUUACUGGAA-3 '；
DHX16-si-2b:5 '-UUCCAGUAACCUAUAAGAUCG-3 '；
(3)DHX16-siRNA3
DHX16-si-3a:5 '-UGGGAUCUAGAGAAAGUGAAA-3 '；
DHX16-si-3b:5 '-UUUCACUUUCUCUAGAUCCCA-3 '.
3. a kind of shRNA, which is characterized in that the nucleotides sequence of the shRNA is classified as following at least one set:
(1)DHX16-shRNA1
DHX16-sh-1a:5 '-CCGGAGCCUCCAGAAGAAACGUAAACUCGAGUUUACGUUUCUUCUGGAGGCUUUUU UG-3'；
DHX16-sh-1b:5 '-AAUUCAAAAAAGCCUCCAGAAGAAACGUAAACUCGAGUUUACGUUUCUUCUGGAGG CU-3'；
(2)DHX16-shRNA2
DHX16-sh-2a:5 '-CCGGCGAUCUUAUAGGUUACUGGAACUCGAGUUCCAGUAACCUAUAAGAUCGUUUU UG-3'；
DHX16-sh-2b:5 '-AAUUCAAAAACGAUCUUAUAGGUUACUGGAACUCGAGUUCCAGUAACCUAUAAGAU CG-3'；
(3)DHX16-shRNA3
DHX16-sh-3a:5 '-CCGGUGGGAUCUAGAGAAAGUGAAACUCGAGUUUCACUUUCUCUAGAUCCCAUUUU UG-3'；
DHX16-sh-3b:5 '-AAUUCAAAAAUGGGAUCUAGAGAAAGUGAAACUCGAGUUUCACUUUCUCUAGAUCC CA-3’。
4. a kind of DNA for encoding shRNA as claimed in claim 3, which is characterized in that the nucleotides sequence of the DNA is classified as following It is at least one set of:
(1)DHX16-shDNA1
DHX16-d-1a:5 '-CCGGAGCCTCCAGAAGAAACGTAAACTCGAGTTTACGTTTCTTCTGGAGGCTTTTT TG- 3'；
DHX16-d-1b:5 '-AATTCAAAAAAGCCTCCAGAAGAAACGTAAACTCGAGTTTACGTTTCTTCTGGAGG CT- 3'；
(2)DHX16-shDNA2
DHX16-d-2a:5 '-CCGGCGATCTTATAGGTTACTGGAACTCGAGTTCCAGTAACCTATAAGATCGTTTT TG- 3'；
DHX16-d-2b:5 '-AATTCAAAAACGATCTTATAGGTTACTGGAACTCGAGTTCCAGTAACCTATAAGAT CG- 3'；
(3)DHX16-shDNA3
DHX16-d-3a:5 '-CCGGTGGGATCTAGAGAAAGTGAAACTCGAGTTTCACTTTCTCTAGATCCCATTTT TG- 3'；
DHX16-d-3b:5 '-AATTCAAAAATGGGATCTAGAGAAAGTGAAACTCGAGTTTCACTTTCTCTAGATCC CA- 3’。
5. a kind of recombinant vector, which is characterized in that the recombinant vector be GV493 plasmid multiple cloning sites AgeI and EcoRI insertion such as encodes the recombinant vector that the DNA of coding shRNA as claimed in claim 4 is obtained.
6. a kind of recombinant slow virus, which is characterized in that recombinant slow virus recombinant vector as described in claim 5 and virus Pack helper plasmid pHelper1.0 carrier and virus packaging helper plasmid pHelper2.0 carrier cotransfection mammalian cell It obtains.
7. a kind of host cell, which is characterized in that including siRNA of any of claims 1 or 2, as claimed in claim 3 ShRNA, the coding DNA of shRNA as claimed in claim 4, recombinant vector described in claim 5 and as claimed in claim 6 At least one of recombinant slow virus.
8. siRNA of any of claims 1 or 2, shRNA as claimed in claim 3, coding shRNA's as claimed in claim 4 The application of at least one of recombinant vector described in DNA, claim 5 and recombinant slow virus as claimed in claim 6.
9. application according to claim 8, wherein the application is the system that preparation inhibits DEAH box unwindase 16 to express Agent.
10. application according to claim 8, wherein the application is that preparation inhibits Cells Proliferation of Human Breast Cancer and/or growth Preparation, or be preparation prevention and/or treatment breast cancer preparation.
CN201910325016.2A 2019-04-22 2019-04-22 SiRNA, recombinant vector and its application expressed for striking low DEAH box unwindase 16 Withdrawn CN110144348A (en)

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Publication number Priority date Publication date Assignee Title
US20190054122A1 (en) * 2015-07-31 2019-02-21 Regents Of The University Of Minnesota Intracellular genomic transplant and methods of therapy
WO2017074788A1 (en) * 2015-10-27 2017-05-04 The Broad Institute Inc. Compositions and methods for targeting cancer-specific sequence variations

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刘静: "《分子生物学实验指导》", 31 December 2015, 中南大学出版社 *
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