CN102643860A - Recombinant lentiviral vector aiming at hUHRF1 gene RNA (Ribonucleic Acid) interference and preparation thereof - Google Patents
Recombinant lentiviral vector aiming at hUHRF1 gene RNA (Ribonucleic Acid) interference and preparation thereof Download PDFInfo
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Abstract
The invention relates to a recombinant lentiviral vector aiming at hUHRF1 gene RNA (Ribonucleic Acid) interference and preparation thereof. The lentiviral vector aiming at ShRNA of the hUHRF1 gene is experimentally constructed; a synthetic DNA (Deoxyribonucleic Acid) segment aiming at the ShRNA is mediated through the lentiviral vector, and is in cotransfection 293 T cell culture with two vectors of pHelper 1.0 and pHelper 2.0; and after the recombinant lentiviral vector is obtained, a target cell is cotransfected, so that RNA interference aiming at the hUHRF1 gene is realized. The adopted inactive third-generation lentiviral vector (SIN) has the advantages of safety, reliability, capability of infecting nondividing cells, long-duration expression of integrating target genes into target cell gene groups, small immune reaction and the like, and is an ideal vector. According to the lentiviral vector, the interference effect on human breast cancer cells MCF-7 reaches 70-85 percent, so that the recombinant lentiviral vector lays a good experimental foundation for further research of relevant hUHRF1 genes, and can be widely used for in vivo gene treatment and gene function research.
Description
Technical field
The invention belongs to molecular biology; Biological medicine and gene engineering technology field relate generally to RNA interference recombinant lentivirus vector (LV-sh-hUHRF1) and preparation thereof to hUHRF1 (human ubiquitin-like protein containing PHD and RING domain 1) gene.
Background technology
The generation of tumour, development are multifactor causing, the process of the rapid multistage development of multistep, and the change (aneuploid) on genomic unstable or the karyomit(e) quantity, chromosome translocation, gene amplification or the like are the key characters of cancerous tumor cell.Up-to-date discovers, nearly 350 genes are oncogene in about 25,000 genes of human genome.Wherein only have the function of a few part to possess some special knowledge, and the unknown gene of most function remain further to be studied.
The hUHRF1 gene is positioned on the human chromosomal 19p13.3, is made up of 2,327 bp; Contain 18 exons, the cDNA of a 2.4kb size, 793 amino acid of encoding; Its structure comprises ubiquitin-like (ubiquitin-like) pattern of N end; A leucine zipper pattern (leucine zipper motif), the zinc finger print formula of PHD type (Zinc finger motif of PHD finger type), the ring-type zinc finger print formula (Zinc finger motif of RING finger type) of SRA-YDG structural domain and C end; In two zinc finger print formulas, contain sequence; I.e. ATP/GTP binding site, a cyclinA/E-CDK2 phosphoric acid site, two RB combining sites.HUHRF1 is at first cloned the relevant nucleoprotein gene of conduct and cell growth, and possibly have close relevant with the cell cycle circulation.
At present; The research of relevant UHRF family member function mainly concentrates on mUHRF1; Existing research report shows: the mUHRF1 gene has the cell cycle regulating of participation, DNA repairs, keeps functions such as genome stability, discovers that recently the mUHRF1 gene possibly be the new gene relevant with radiosensitivity; Because reduce the expression of mUHRF1 among the embryonic cell HEK293, cell is obviously increased x ray and ultraviolet radiosensitivity.There is 73.7% homology in the protein sequence of hUHRF1 and mUHRF1, and prompting hUHRF1 gene possibly have the effect of regulating the embryonic cell radiosensitivity too.
(RNAinterference, RNAi) technology is the conventional means of inhibition of gene expression, is gene silencing again in the RNA interference.RNA interferential principle is that this mRNA degraded takes place and causes the reticent phenomenon of genetic expression when importing with endogenous mRNA coding region homologous double-stranded RNA in the cell.
The RNA interfering process mainly contains two steps: one, double-stranded RNA is cut into the short dsrna of 21-23 base pair by the specific nucleicacidase of cell source double-stranded RNA, promptly siRNA (smallinterference RNA, siRNA); Two, the antisense strand of siRNA and nucleicacidase have formed reticent mixture (RNA-induced silencing complex, RISC), this complex body have had the mRNA that identification combines to have with siRNA homologous sequence, and at specific site this mRNA is cut off.The RNA perturbation technique has obtained using widely at present in gene therapy and research, and those proof effective siRNA/shRNA in the target experiment itself can be developed further into the medicine into RNAi simultaneously.ShRNA (short hairpin RNA) is that short hairpin RNA comprises two short inverted repeats, and one of them and goal gene are complementary, and middle loop sequence is separated and formed hairpin structure.ShRNA is processed to the siRNA goal gene of effectively degrading and suppresses its expression in vivo.But to this technology be applied to clinical treatment, need to solve the RNA interference fragment and express major issues such as continuing to reach expression efficiency.
Vectors in Gene Therapy mainly contains non-virus carrier and virus vector at present, yet non-virus carrier can't satisfy long expression, and this defective is filled up by virus vector undoubtedly.The RNA perturbation technique of slow virus mediation has in recent years been obtained good start under study for action.(lentivirus-based vector is retroviral a kind of LV) to lentiviral vectors, has retroviral substruction, but different retroviral component characteristics are arranged, and this virus not only can the transfection somatoblast but also can the transfection Unseparated Cell.Viral genome can be integrated among the host, makes the expression that the gene length time is stable, and slow virus has lower immunogenicity, and this makes slow virus become RNA interferential optimum carrier, is widely used in fields such as gene expression regulation, gene therapy at present.It is suicide property (self-inactivating that the present invention selects third generation replication defect type lentiviral vectors for use; SIN) virus; In vivo can longer expression and safe, can solve the problem such as target property, security, integration efficiency of RNA perturbation technique gene therapy.Therefore RNA interference effect long-term existence in target cell of lentiviral vectors mediation can be better performance interference effect and creates conditions.
Summary of the invention
An object of the present invention is to provide a kind of to hUHRF1 gene RNA interferential recombined lentivirus vector; This carrier is the third generation lentiviral vectors SIN of self inactivation; It is characterized in that; Described carrier S IN contains the pGCSIL-GFP/U6-ShhUHRF1 recombinant vectors, and described pGCSIL-GFP/U6-ShhUHRF1 recombinant vectors is in the MCS of pGCSIL-GFP carrier, to have connected double chain DNA fragment; The sequence of described double chain DNA fragment is a kind of (wherein S represents positive-sense strand, and AS represents antisense strand) in the following sequence:
(1)hUHRF1-homo-704：
hUHRF1-homo-704-S：
5’-GATCCAGGAGGACGTCATTTACCATTCAAGAGATGGTAAATGACGTCCTCCTTTTTTTG-3’
hUHRF1-homo-704-AS：
5’-AATTCAAAAAAAGGAGGACGTCATTTACCATCTCTTGAATGGTAAATGACGTCCTCCTG-3’
(2)hUHRF1-homo-1766：
hUHRF1-homo-1766-S：
5’-GATCCGCGCAATGTCAAGGGTGGCTTCAAGAGAGCCACCCTTGACATTGCGCTTTTTTG-3’
hUHRF1-homo-1766-AS：
5’-AATTCAAAAAAGCGCAATGTCAAGGGTGGCTCTCTTGAAGCCACCCTTGACATTGCGCG-3’
(3)hUHRF1-homo-1984：
hUHRF1-homo-1984-S：
5’-GATCCCAGTATCCAGAAGGCTACCTTCAAGAGAGGTAGCCTTCTGGATACTGTTTTTTG-3’
hUHRF1-homo-1984-AS：
5’-AATTCAAAAAACAGTATCCAGAAGGCTACCTCTCTTGAAGGTAGCCTTCTGGATACTGG-3’
Another object of the present invention provides described preparation method to hUHRF1 gene RNA interferential recombined lentivirus vector; It is characterized in that; According to the hUHRF1mRNA sequence, double chain DNA fragment has been synthesized in design, and described double chain DNA fragment is a kind of in the following sequence:
(1)hUHRF1-homo-704：
hUHRF1-homo-704-S：
5’-GATCCAGGAGGACGTCATTTACCATTCAAGAGATGGTAAATGACGTCCTCCTTTTTTTG-3’
hUHRF1-homo-704-AS：
5’-AATTCAAAAAAAGGAGGACGTCATTTACCATCTCTTGAATGGTAAATGACGTCCTCCTG-3’
(2)hUHRF1-homo-1766：
hUHRF1-homo-1766-S：
5-GATCCGCGCAATGTCAAGGGTGGCTTCAAGAGAGCCACCCTTGACATTGCGCTTTTTTG-3’
h?UHRF1-homo-1766-AS：
5’-AATTCAAAAAAGCGCAATGTCAAGGGTGGCTCTCTTGAAGCCACCCTTGACATTGCGCG-3’
(3)hUHRF1-homo-1984：
hUHRF1-homo-1984-S：
5’-GATCCCAGTATCCAGAAGGCTACCTTCAAGAGAGGTAGCCTTCTGGATACTGTTTTTTG-3’
hUHRF1-homo-1984-AS：
5’-AATTCAAAAAACAGTATCCAGAAGGCTACCTCTCTTGAAGGTAGCCTTCTGGATACTGG-3’
Then described dna fragmentation is connected in the MCS of pGCSIL-GFP carrier and is built into pGCSIL-GFP/U6-Sh hUHRF1 recombinant vectors; With pGCSIL-GFP/U6-Sh hUHRF1 recombinant vectors, pHelper1.0,2.0 3 kinds of carrier cotransfections of pHelper 293T cell cultures, obtain described recombined lentivirus vector again.
As preferably, at terminal BamH I and the EcoR I restriction enzyme site introduced of described double chain DNA fragment.
As preferably, with BamH I and EcoRI enzyme pGCSIL-GFP carrier enzyme is cut, reclaim after the big fragment it is connected back transformed competence colibacillus bacterium, picking recombinant clone with said double chain DNA fragment.
Can specificity reduce the hUHRF1 expression of gene behind this carrier transfectional cell, thereby possibly be applied to gene therapy or gene functional research.LV-sh-hUHRF1 most important characteristics provided by the invention is to provide target property hUHRF1 gene inhibition effect, and with recombined lentivirus vector as carrier, make interference effect obtain the persistence effect.Whole process of preparation is all used plasmid, avoids the traditional method adenovirus to pollute.The present invention is through the most effective hUHRF1 interference sequence of screening, and synthetic its double-stranded DNA is connected in the slow virus skeleton plasmid carrier, with helper plasmid cotransfection instrument cell 293T cell, prepares hUHRF1 interference recombinant lentivirus vector: LV-sh-hUHRF1.
Usefulness of the present invention is:
1, the pGCSIL-GFP carrier that adopts of the present invention contains the U6 promotor, can be in host cell continuous expression the little RNA of interference effect is arranged.This plasmid can be expressed the GFP GFP by the CMV promoters driven simultaneously, transfection efficiency when convenient virus is packed, and the detection of the efficiency of infection of host cells infected.The gag gene that contains HIV virus in pHelper 1.0 plasmids, the structural protein that coding virus is main; The pol gene, the enzyme of coding virus-specific; The rev gene, the regulatory factor of coding and regulating gag and pol genetic expression.The VSVg gene that contains the hsv source in pHelper 2.0 plasmids provides the virus packing needed capsid protein.Above three kinds of carrier corotation are gone into the third generation lentiviral vectors (SIN) that the 293T cell can efficiently be assembled self inactivation; Increased by two security features: one of which has made up the lentiviral vectors of self inactivation (SIN); Promptly deleted the 3 ' LTR in U3 district; Make carrier lose HIV-1 enhanser and promoter sequence, even exist all viral proteins can not transcribe out RNA.Second characteristic is to have removed the tat gene to replace the allogeneic promoter sequence, primary HIV gene like this, and 9 genes in the group have only kept 3 (gag, pol and rev) in the HIV lentiviral vectors that makes up.Therefore third generation HIV slow virus carrier system is safer.
2, the present invention is directed to the hUHRF1 target gene and design four effective interference sequences, through the slow virus interference carrier system constructing packing acquisition LV-sh-hUHRF1 of reorganization, through detecting hUHRF1 mrna expression in the target cell according to online principle; Filter out the most effectively interference fragment; Not only overcome the low transfection efficiency of non-virus carrier, the immunogenicity of also having avoided recombinant adenovirus to produce, expression time is than shortcomings such as weak points; Can be incorporated in host's the genome and stably express; Can not cause inserting inactivation, make interference effect more lasting, have and to infect Unseparated Cell, goal gene and be integrated into advantages such as target cell gene group leader time-histories is expressed, immunoreation is little; For good experiment basis is established in the further research of relevant hUHRF1 gene, and can be widely used in vivo gene treatment and gene functional research.
Description of drawings
Fig. 1 is a slow virus skeleton plasmid pGCSIL-GFP carrier structure synoptic diagram.
Fig. 2 is to hUHRF1 genetic expression interference effect picture behind the LV-sh-hUHRF1 virus infection MCF-7 cell 72h.
Negative control group adds the groups of cells sample of negative control virus infection;
HUHRF1-homo-704 organizes, and adds the groups of cells sample of hUHRF1-homo-704 virus infection;
HUHRF1-homo-1615 organizes, and adds the groups of cells sample of hUHRF1-homo-1615 virus infection;
HUHRF1-homo-1776 organizes, and adds the groups of cells sample of hUHRF1-homo-1776 virus infection;
HUHRF1-homo-1984 organizes, and adds the groups of cells sample of hUHRF1-homo-1984 virus infection.
Embodiment
The present invention combines accompanying drawing and embodiment to be further described.
Embodiment 1: the lentiviral vectors to gene hUHRF1 makes up
1, the design of oligonucleotide and synthetic
Utilize the online RNAi Series Design software BLOCK-iT RNAi Designer of Invitrogen company; Design is to 4 interference target sequences (seeing table) of hUHRF1 gene mRNA sequence (NM_001048201), and synthetic corresponding double-stranded DNA (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd).Loop structure in the LV3-shRNA template has selected for use TTCAAGAGA to avoid forming termination signal.5 ' end of positive-sense strand template has added GATCC, cuts the cohesive end complementation that the back forms with the BamHI enzyme; 5 ' end of antisense strand template has added AATTC, cuts the cohesive end complementation that the back forms with the EcoRI enzyme.
| Sequence code | The sequence title | Target sequence |
| U401 | hUHRF1-homo-704 | AGGAGGACGTCATTTACCATT |
| U402 | hUHRF1-homo-1615 | GGTCGAGATCTTTCCGGCATT |
| U403 | hUHRF1-homo-1776 | GCGCAATGTCAAGGGTGGCTT |
| U404 | hUHRF1-homo-1984 | CAGTATCCAGAAGGCTACCTT |
Double-stranded DNA answer print segment information separately is following:
(1)hUHRF1-homo-704：
hUHRF1-homo-704-S：
5’-GATCCAGGAGGACGTCATTTACCATTCAAGAGATGGTAAATGACGTCCTCCTTTTTTTG-3’
hUHRF1-homo-704-AS：
5’-AATTCAAAAAAAGGAGGACGTCATTTACCATCTCTTGAATGGTAAATGACGTCCTCCTG-3’
(2)hUHRF1-homo-1615：
hUHRF1-homo-1615-S：
5’-GATCCGGTCGAGATCTTTCCGGCATTCAAGAGATGCCGGAAAGATCTCGACCTTTTTTG-3’
hUHRF1-homo-1615-AS：
5’-AATTCAAAAAAGGTCGAGATCTTTCCGGCATCTCTTGAATGCCGGAAAGATCTCGACCG-3’
(3)hUHRF1-homo-1766：
hUHRF1-homo-1766-S：
5’-GATCCGCGCAATGTCAAGGGTGGCTTCAAGAGAGCCACCCTTGACATTGCGCTTTTTTG-3’
hUHRF1-homo-1766-AS：
5’-AATTCAAAAAAGCGCAATGTCAAGGGTGGCTCTCTTGAAGCCACCCTTGACATTGCGCG-3’
(4)hUHRF1-homo-1984：
hUHRF1-homo-1984-S：
5’-GATCCCAGTATCCAGAAGGCTACCTTCAAGAGAGGTAGCCTTCTGGATACTGTTTTTTG-3’
hUHRF1-homo-1984-AS：
5’-AATTCAAAAAACAGTATCCAGAAGGCTACCTCTCTTGAAGGTAGCCTTCTGGATACTGG-3’
ShDNA with above-mentioned sequence can produce following transcripton respectively in vivo after transcribing:
(1) hUHRF1-homo-704 transcripton:
AGGAGGACGTCATTTACCATTCAAGAGATGGTAAATGACGTCCTCCTTT
(2) hUHRF1-homo-1615 transcripton:
GGTCGAGATCTTTCCGGCATTCAAGAGATGCCGGAAAGATCTCGACCTT
(3) hUHRF1-homo-1776 transcripton:
GCGCAATGTCAAGGGTGGCTTCAAGAGAGCCACCCTTGACATTGCGCTT
(4) hUHRF1-homo-1984 transcripton:
CAGTATCCAGAAGGCTACCTTCAAGAGAGGTAGCCTTCTGGATACTGTT
Above-mentioned transcripton forms the double-stranded RNA with loop structure through self pairing, through the enzyme Dicer of specific recognition double-stranded RNA, the mode that relies on ATP progressively be cut into 21～23nt the small molecules interference RNA fragment (small interfering RNAs, siRNA).
In the effective stage, siRNA is double-stranded combine with the ribozyme mixture formation RNA induce reticent mixture (RNA-induced silencing complex, RISC).RISC activates with ATP dependency mode, siRNA sex change among the RISC, and two strands is untied; Unload positive-sense strand, antisense strand still is combined on the mixture, and guiding RISC combines with homologous target RNA; Under the effect of endonuclease, said target mrna is cut off, thereby reach the effect that blocking gene is expressed.
2, the annealing of LV3-shDNA template
The synthetic double chain DNA fragment is used TE (pH8.0) dissolving respectively, and concentration is 100uM.Get corresponding positive-sense strand and antisense strand oligomer solution, according to following proportioning configuration annealing reaction system.
| Component | Volume (ul) |
| 10 * shDNA annealing buffer (1M NaCl, 0.5M Hepes, pH7.4) | 5 |
| Positive-sense strand (100uM) | 5 |
| Antisense strand (100uM) | 5 |
| Distilled water | 35 |
| Total amount | 50 |
On the PCR appearance, carry out anneal according to following program: 95 ℃ 5 minutes; 85 ℃ 5 minutes; 75 ℃ 5 minutes; 70 ℃ 5 minutes; 4 ℃ of preservations.Obtaining concentration after the anneal is the shDNA template of 10 μ M.With 50 times of gained template solution dilutions, final concentration is 200nM, is used for ligation.
3, the linearizing of LV3 carrier
Get 5ug LV3 carrier; Utilize BamHI and EcoRI restriction enzyme and the conventional double digestion system of NEB company to cut 1 hour in 37 ℃ of enzymes; Agarose electrophoresis; Use agarose gel DNA purification kit (TaKaRa company) to reclaim linearizing carrier segments, the electrophoresis detection estimated concentration, weaker concn is to 50ng/ul.
4, the structure of LV3-shRNA carrier
1) carry out the ligation of carrier according to the T4DNA ligase enzyme system of NEB company, condition is roughly following:
| Component | Volume (ul) |
| 10 * T4 connects damping fluid | 2 |
| LV3 carrier (BamHI+EcoRI double digestion) | 1 |
| ShDNA template (100nM) | 1 |
| The |
1 |
| Distilled water | 15 |
| Total amount | 20 |
22 ℃ connect 1hr, are converted into JM 109 competent cells.
2) 5 bacterium colonies of each ligation picking; Being inoculated into the LB that contains the 50ug/ml penbritin cultivates concentrated; The bacterium liquid that shakes out is chosen 2 at random and is sent to order-checking; The bacterial strain that checks order correct adopts amount extraction agent box (Shanghai Sangon Biological Engineering Technology And Service Co., Ltd) extracting in the high purity plasmid, and the gained plasmid can be used for conventional molecular biology experiment and cytologic experiment.If cytotoxicity is bigger when being used for cell transfecting, can be converted into again in the bacillus coli DH 5 alpha, prepare more high purity plasmid with test kit or CsCl ultracentrifugation then.
Encapsulating of embodiment 2:hUHRF1 gene RNA interference recombinant lentivirus
Draw high purity and do not have the recombinant virus plasmid pGCSIL-sh-hUHRF1 (20 μ g) that the intracellular toxin extracting prepares; Helper plasmid pHelper 1.0 (15 μ g) and pHelper 2.0 (10 μ g) carry out cotransfection 293T cell by Invitrogen company Lipofectamine 2000 operation instructions.
8h is replaced by perfect medium after the transfection, in 37 ℃, 5%CO
2After continuing in the incubator to cultivate 48h, collect and be rich in slow virus particulate cell conditioned medium liquid.4 ℃, 4000g removed behind the cell debris in centrifugal 10 minutes and to obtain slow virus with 0.45 μ M filter filtering supernatant subsequent use, can satisfy general test cell line.If will obtain the slow virus liquid concentrator that the slow virus of higher concentration obtains high titre after can be to it further concentrated and purified, packing virus liquid concentrator-80 ℃ prolonged preservation is got wherein one and is carried out the viral biology titer determination according to the following steps.
1.293T when cell was cultured to the 80-90% fusion in the 6cm petridish, the nutrient solution that inclines was used twice in 3ml D-Hank ' s solution washing cell.
2. (0.05%, Gibco), behind the mixing, the careful suction removed pancreatin solution, places 3-5 minute for 37 ℃ to add 1ml Trypsin-EDTA solution.
3. add 2ml again and contain the DMEM nutrient solution of 10%FBS, piping and druming makes cell form single cell suspension.
4. blood counting chamber is counted, with cell dilution to 3 * 10
5Cell/ml.
5. by 3 * 10
4The concentration of cells/well is inoculated 96 orifice plates, behind the mixing in 37 ℃ of 5%CO
2Cultivate 24h.
6. with DMEM training liquid ten times dilution 3-5 the gradient (according to cell state, if necessary can add Polybrene that final concentration be 5ug/ml) of slow virus stoste (10-20ul) with 15%FBS.
7. inhale and go the nutrient solution in 96 orifice plates, every hole to add the viral liquid of 100 μ l dilution, utilize Lentivirus-NC virus liquid (the lucky agate in Shanghai, 1 * 10 simultaneously
8TU/ml) set up negative control group, in 37 ℃ of 5%CO
2Cultivate 24h.
8. inhale and abandon the virus dilution liquid in 96 orifice plates, every hole adds the DMEM training liquid of 150 μ l 15%FBS, and (according to cell state, can tell 1/3-1/5 if necessary) is in 37 ℃ of 5%CO
2Continue cultivation 48,72h.
9. through fluorescent microscope or FACS counting fluorocyte, under fluorescent microscope, judge transfection efficiency (efficient is about more than 80%), calculate virus titer in conjunction with extension rate through observing the GFP expression.
Embodiment 3: target cell infects test and genetic expression suppresses effect analysis
1, target cell is infected test
According to the following steps human breast cancer cell MCF-7 (available from Shanghai Inst. of Life Science, CAS cell resource center) is carried out the virus infection experiment:
When 1) the MCF-7 cell was cultured to the 80-90% fusion in the 10cm petridish, the nutrient solution that inclines was used twice in 3ml D-Hank ' s solution washing cell.
2) (0.05%, Gibco), behind the mixing, the careful suction removed pancreatin solution, places 3-5 minute for 37 ℃ to add 1ml Trypsin-EDTA solution.
3) add 2ml DMEM nutrient solution again, piping and druming makes cell form single cell suspension.
4) blood counting chamber counting is by 10 * 10
5The concentration of cells/well is inoculated 6 orifice plates, 37 ℃ of 5%CO behind the mixing
2Cultivated 24 hours.
5) with slow virus stoste 200ul, with five times of dilutions of DMEM training liquid of 10%FBS.
6) suction goes the nutrient solution in 6 orifice plates, every hole to add the viral liquid of above-mentioned 1ml dilution, utilizes Lentivirus-NC virus liquid (the lucky agate in Shanghai, 1 * 10 simultaneously
8TU/ml) set up negative control group, in 37 ℃ of 5%CO
2Cultivate 24h.
7) the virus dilution liquid in 6 orifice plates is abandoned in suction, and every hole adds the DMEM training liquid of 1.5ml 10%FBS, and (according to cell state, can tell 1/3-1/5 if necessary) is in 37 ℃ of 5%CO
2Continue to cultivate, under fluorescent microscope, judge transfection efficiency (efficient is about more than 70%) through observation GFP expression, receive appearance behind the 72h, the gained cell is used for RNA extraction and follow-up Real-time PCR detection.
2, hUHRF1 genetic expression suppresses effect analysis
1) extraction of total RNA
Carry out the extraction of total RNA according to the following steps:
(1) nutrient solution in 6 orifice plates is abandoned in suction, and every hole adds 1ml Trizol (Invitrogen).
(2) the rifle head piping and druming of handling with DEPC makes the complete cracking of cell.
(3) lysate is transferred in the 1.5ml EP pipe of DEPC processing, room temperature was placed 10 minutes.
(4) add 200 μ l trichloromethanes (analytical pure), the concuss mixing, room temperature was placed 10 minutes.
(5) 4 ℃ of 12000g centrifugal 10 minutes, draw supernatant liquid to new centrifuge tube, add isopyknic Virahol (analytical pure), precipitation at room temperature 10 minutes.
(6) 4 ℃ of 12000g centrifugal 15 minutes, abandon supernatant.
(7) deposition with 500 μ l75% washing with alcohol once.Centrifugal 5 minutes of 4 ℃ of 12000g reclaim deposition, abandon supernatant.
(8) normal temperature is inverted and was dried 10 minutes.
(9) with 20 μ l DEPC-H
2The O dissolution precipitation is measured OD
260, OD
280, calculate RNA concentration.
(10) integrity of agarose electrophoresis inspection RNA.
2) the RNA rt obtains cDNA
Utilize the M-MLV reversed transcriptive enzyme test kit of TaKaRa company, carry out the RNA rt, generate cDNA by its description of product.
3, Real-time PCR detects
1) adopt software design Real-time PCR to detect primer, primer sequence information is following, and is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
2) according to the form below preparation reaction system
| Component | Final concentration | Volume |
| 2 * Real-time PCR Master Mix (the lucky agate in Shanghai) | 1× | 10μl |
| ?FPrimer(20uM) | 0.1μM | 0.1μl |
| ?RPrimer?(20uM) | 0.1μM | 0.1μl |
| The cDNA template | - | 2μl |
| RTaq archaeal dna polymerase (5U/ μ l) (TaKaRa) | 2.5U/μl | 0.4μl |
| Distilled water | To 20 μ l |
3) utilize Mx3000Real-time PCR appearance (Stratagen) to react reaction conditions: 95 ℃, sex change in 3 minutes; 95 ℃, 30 seconds, 62 ℃, 40 seconds, totally 40 circulations.
, as confidential reference items hUHRF1mRNA content results behind each virus infection is handled with the hGAPDH gene, and calculated the ratio of hUHRF1mRNA in itself and the negative control.The result shows (see figure 2); To hUHRF1 gene RNA interferential recombinant slow virus hUHRF1-homo-704, hUHRF1-homo-1766, hUHRF1-homo-1984 all can effectively suppress the HUHRF1 expression of gene; Suppress the about 70-85% of effect, thereby can be used for follow-up hUHRF1 gene functional research.And the inhibition effect of hUHRF1-homo-1615 is relatively poor, therefore in experiment from now on, will not adopt.
Claims (4)
1. one kind is directed against hUHRF1 gene RNA interferential recombined lentivirus vector; This carrier is the third generation lentiviral vectors SIN of self inactivation; It is characterized in that; Described carrier S IN contains pGCSIL-GFP/U6-Sh hUHRF1 recombinant vectors, and described pGCSIL-GFP/U6-ShhUHRF1 recombinant vectors is in the MCS of pGCSIL-GFP carrier, to have connected double chain DNA fragment; The sequence of described double chain DNA fragment is a kind of in the following sequence:
(1)hUHRF1-homo-704：
hUHRF1-homo-704-S：
5’-GATCCAGGAGGACGTCATTTACCATTCAAGAGATGGTAAATGACGTCCTCCTTTTTTTG-3’
hUHRF1-homo-704-AS：
5’-AATTCAAAAAAAGGAGGACGTCATTTACCATCTCTTGAATGGTAAATGACGTCCTCCTG-3’
(2)hUHRF1-homo-1766：
hUHRF1-homo-1766-S：
5’-GATCCGCGCAATGTCAAGGGTGGCTTCAAGAGAGCCACCCTTGACATTGCGCTTTTTTG-3’
hUHRF1-homo-1766-AS：
5’-AATTCAAAAAAGCGCAATGTCAAGGGTGGCTCTCTTGAAGCCACCCTTGACATTGCGCG-3’
(3)hUHRF1-homo-1984：
hUHRF1-homo-1984-S：
5’-GATCCCAGTATCCAGAAGGCTACCTTCAAGAGAGGTAGCCTTCTGGATACTGTTTTTTG-3’
hUHRF1-homo-1984-AS：
5’-AATTCAAAAAACAGTATCCAGAAGGCTACCTCTCTTGAAGGTAGCCTTCTGGATACTGG-3’。
2. the described preparation method to hUHRF1 gene RNA interferential recombined lentivirus vector of claim 1 is characterized in that, according to the hUHRF1mRNA sequence, double chain DNA fragment has been synthesized in design, and described double chain DNA fragment is a kind of in the following sequence:
(1)hUHRF1-homo-704：
hUHRF1-homo-704-S：
5’-GATCCAGGAGGACGTCATTTACCATTCAAGAGATGGTAAATGACGTCCTCCTTTTTTTG-3’
hUHRF1-homo-704-AS：
5’-AATTCAAAAAAAGGAGGACGTCATTTACCATCTCTTGAATGGTAAATGACGTCCTCCTG-3’
(2)hUHRF1-homo-1766：
hUHRF1-homo-1766-S：
5’-GATCCGCGCAATGTCAAGGGTGGCTTCAAGAGAGCCACCCTTGACATTGCGCTTTTTTG-3’
hUHRF1-homo-1766-AS：
5’-AATTCAAAAAAGCGCAATGTCAAGGGTGGCTCTCTTGAAGCCACCCTTGACATTGCGCG-3’
(3)hUHRF1-homo-1984：
hUHRF1-homo-1984-S：
5’-GATCCCAGTATCCAGAAGGCTACCTTCAAGAGAGGTAGCCTTCTGGATACTGTTTTTTG-3’
hUHRF1-homo-1984-AS：
5 '-AATTCAAAAAACAGTATCCAGAAGGCTACCTCTCTTGAAGGTAGCCTTCTGGATAC TGG-3 ' is connected to described dna fragmentation in the MCS of pGCSIL-GFP carrier then and is built into pGCSIL-GFP/U6-Sh hUHRF1 recombinant vectors; With pGCSIL-GFP/U6-ShhUHRF1 recombinant vectors, pHelper1.0,2.0 3 kinds of carrier cotransfections of pHelper 293T cell cultures, obtain described recombined lentivirus vector again.
3. the preparation method to hUHRF1 gene RNA interferential recombined lentivirus vector according to claim 2 is characterized in that, at terminal BamH I and the EcoRI restriction enzyme site introduced of described double chain DNA fragment.
4. the preparation method to hUHRF1 gene RNA interferential recombined lentivirus vector according to claim 2; It is characterized in that; With BamH I and Eco RI enzyme pGCSIL-GFP carrier enzyme is cut; After reclaiming big fragment it is connected back transformed competence colibacillus bacterium, picking recombinant clone with said double chain DNA fragment.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110872599A (en) * | 2018-08-29 | 2020-03-10 | 徐州医科大学 | AURKA/UHRF1 double-gene co-interference lentivirus and application thereof in drugs for treating colorectal cancer |
| CN111363744A (en) * | 2020-02-26 | 2020-07-03 | 暨南大学 | Berberine-mediated UHRF1 gene inhibition and application thereof in preparation of medicines for treating multiple myeloma |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101575615A (en) * | 2009-04-30 | 2009-11-11 | 中南大学 | Construction of recombined lentivirus vector aiming at PKC gamma gene RNA interference and application thereof |
-
2012
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101575615A (en) * | 2009-04-30 | 2009-11-11 | 中南大学 | Construction of recombined lentivirus vector aiming at PKC gamma gene RNA interference and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 李新莉: "人UHRF1基因对乳腺肿瘤细胞BT-549生长的影响", 《苏州大学学报（医学版）》 * |
| 李新莉: "人类UHRF1基因肿瘤生物学功能的研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110872599A (en) * | 2018-08-29 | 2020-03-10 | 徐州医科大学 | AURKA/UHRF1 double-gene co-interference lentivirus and application thereof in drugs for treating colorectal cancer |
| CN111363744A (en) * | 2020-02-26 | 2020-07-03 | 暨南大学 | Berberine-mediated UHRF1 gene inhibition and application thereof in preparation of medicines for treating multiple myeloma |
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