CN104894223A - Use and related drugs of human COPB2 gene - Google Patents
Use and related drugs of human COPB2 gene Download PDFInfo
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- CN104894223A CN104894223A CN201410081471.XA CN201410081471A CN104894223A CN 104894223 A CN104894223 A CN 104894223A CN 201410081471 A CN201410081471 A CN 201410081471A CN 104894223 A CN104894223 A CN 104894223A
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Abstract
The invention discloses a use and related drugs of a human COPB2 gene. The invention discloses the use of the human COPB2 gene in tumor therapy, tumor diagnosis and drug preparation. The invention further builds a human COPB2 gene small-molecular interfering RNA, a human COPB2 gene interfering nucleic acid building body and a human COPB2 gene interfering lentivirus and uses thereof. The siRNA or the siRNA sequence-containing nucleic acid building body and lentivirus can specifically inhibit expression of the human COPB2 gene, and especially the lentivirus can efficiently infect target cells, efficiently inhibit expression of the COPB2 gene in the target cells, thereby inhibiting the growth of tumor cells, promoting tumor cell apoptosis, and having important significance in tumor therapy.
Description
Technical field
The present invention relates to biological technical field, relate more specifically to purposes and the related drugs thereof of people COPB2 gene.
Background technology
The RNA interference (RNA interference, RNAi) phenomenon refer to when with double-stranded RNA (dsRNA) transfered cell of endogenous mRNA coding region section sequence homology after, there is selective degradation in this mRNA, and causes the silence of this genetic expression.Research shows; length is that the double-stranded RNA of 21-23nt specificly with post-transcriptional level can cause RNAi(Tuschl T transcribing; Zamore PD; Sharp PA, Bartel DP.RNAi:double-stranded RNA directs the ATP-dependent cleavage of mRNA at21to23nucleotide intervals.Cell2000; 101:25-33.).Tumour is the principal disease threatening human health.Though tumour patient is through chemotherapy, radiotherapy and complex therapy, five year survival rate is still very low, if intervene with the relevant gene of progress tumor invasion, new way is opened up in the treatment that can be tumour.In recent years, RNAi has become the available strategy of the gene therapy of tumour.Utilize RNAi technology can suppress proto-oncogene, generation and development (Uprichard, Susan L.The therapeutic potential of RNA interference.FEBS Letters2005 that the expression of cancer suppressor gene, Cell cycle-related genes, anti-apoptotic genes involved etc. of sudden change carrys out Tumor suppression; 579:5996-6007.).
COPB2 (coatomer protein complex, subunit beta2) be a kind of capside protein complex body, subunit β 2 (β originally), it is a kind of protein transporters, mainly be distributed in that endoplasmic reticulum, gorky are stacking, film, COPI film bubble clothing, participate in intracellular protein transport, endoplasmic reticulum gorky film bubble mediated transport, regression film bubble mediated transport, gorky's endoplasmic reticulum, interior gorky's film bubble mediated transport etc.
Studies have found that, COPB2 presents high expression level at kinds of tumors tissue.The people such as Erdogan find in adenocarcinoma of lung, COPB2 gene presents high expression level, in invasion of lung cancer process, played certain effect, Meta-analyzes and finds there is close relationship with the expression of PKCiota in the sample of mammary cancer, cervical cancer, prostate cancer and carcinoma of the pancreas and brain glue knurl.（Erdogan E,Klee EW,Thompson EA,Fields AP.，Meta-analysis of oncogenic protein kinase Ciota signaling in lung adenocarcinoma.Clin Cancer Res.2009Mar1;15(5):1527-33）。The people such as Sudo find, in mesothelioma cell, after striking the expression subtracting Copb2 gene, energy is antiproliferative effect obviously, has certain Anti-G value.(Sudo H; Tsuji AB; Sugyo A, Kohda M, Sogawa C; Yoshida C; Harada YN, Hino O, Saga T.Knockdown of COPA; identified by loss-of-function screen, induces apoptosis and suppresses tumor growth in mesothelioma mouse model.Genomics.2010Apr; 95 (4): 210-6.), based on above research, infer that it may have certain contacting with the generation of tumour development.
Summary of the invention
The object of the invention is to open and people COPB2 (coatomer protein complex, subunit beta2) methods for the treatment of of gene-correlation and medicine, with RNA interference (RNAi) for the effect of means research COPB2 gene in the survival and apoptotic process of tumour cell.
First aspect present invention, with RNA interference for means, have studied COPB2 gene to occur and developing effect in tumour, disclose a kind of method suppressing or reduce growth of tumour cell, propagation, differentiation and/or survival, the method comprises: use to tumour cell and a kind ofly specificity can suppress the transcribing or translating of COPB2 gene, or specificity can suppress the expression of eIF5B albumen or the molecule of activity, come the growth of inhibition tumor cell, propagation, differentiation and/or survival with this.
Described tumour cell is selected from it and grows and the expression of COPB2 albumen or active relevant tumour cell.Preferably, described tumour cell be selected from lung cancer, colorectal carcinoma arbitrary.
Described suppression or reduce in the method for growth of tumour cell, propagation, differentiation and/or survival, the amount of application of described molecule is enough reduce transcribing or translating of COPB2 gene, or the enough expression of reduction COPB2 albumen or the dosage of activity.Further, the expression of described COPB2 gene is at least lowered 50%, 80%, 90%, 95% or 99%.
Described molecule can be selected from but be not limited to: nucleic acid molecule, carbohydrate, lipid, small-molecule chemical medicine, antibody medicine, polypeptide, albumen or interference slow virus.
Described nucleic acid includes but not limited to: siRNA (esiRNA) prepared by antisense oligonucleotide, double-stranded RNA (dsRNA), ribozyme, endoribonuclease III or short hairpin RNA (shRNA).
Described double-stranded RNA, ribozyme, esiRNA or shRNA contain the promoter sequence of COPB2 gene or the information sequence of COPB2 gene.
Further, described double-stranded RNA is siRNA (siRNA).Described siRNA comprises the first chain and the second chain, described first chain and the complementary common formation RNA dimer of described second chain, and the sequence of described first chain is substantially identical with 15-27 continuous print nucleotide sequence in COPB2 gene.MRNA fragment coded by described small molecules interference RNA energy specific binding target sequence, and the expression of specificity reticent people COPB2 gene.
Further, the first chain-ordering of described siRNA is substantially identical with the target sequence in COPB2 gene.Preferably, the target sequence in described COPB2 gene contains the arbitrary sequence in SEQ ID NO:1-10.
When target sequence in described COPB2 gene is the COPB2 genetic expression of described small molecules interference RNA specificity silence, the fragment in the COPB2 gene corresponding to the mRNA fragment combined with the complementation of described small molecules interference RNA.
Preferably, described COPB2 gene source is in people.
First aspect present invention also discloses a kind of people COPB2 gene of separation at preparation or screening anti-tumor medicine, or is preparing the purposes in diagnosing tumor medicine.
Further, described tumour is selected from the arbitrary of lung cancer or colorectal carcinoma.
Described by the COPB2 gene of separation for the preparation of or screening anti-tumor medicine comprise the content of two aspects: one, to be applied to COPB2 gene for the action target of tumour cell as medicine or preparation and to prepare anti-tumor medicine or preparation; Its two, COPB2 gene is applied to screening anti-tumor medicine or preparation as medicine or preparation for the action target of tumour cell.
Described being applied to as medicine or preparation for the action target of tumour cell by COPB2 gene prepares anti-tumor medicine or preparation specifically refers to: using the target of COPB2 gene as RNA interference effect, develop the medicine for tumour cell or preparation, thus the expression level of COPB2 gene in tumour cell can be reduced.
Describedly COPB2 gene is applied to screening anti-tumor medicine as medicine or preparation for the action target of tumour cell or preparation specifically refers to: COPB2 gene is as effective object, medicine or preparation are screened, can suppress or promote that the medicine of people COPB2 genetic expression is as oncotherapy drug candidate to find.Namely COPB2 gene small molecules interference RNA (siRNA) as described in the present invention obtains for effective object screening with people COPB2 gene, can be used as the medicine with inhibition tumor cell proliferation function.In addition, such as antibody drug, small-molecule drug etc. also can using COPB2 gene and albumen thereof as effective object.
Described by COPB2 gene for the preparation of diagnosing tumor medicine, refer to preparation COPB2 gene expression product being applied to diagnosing tumor medicine as a diagnosing tumor index.
Described anti-tumor medicine is specificity can suppress transcribing or translating of COPB2 gene, or specificity can suppress the expression of COPB2 albumen or the molecule of activity, thus the expression level of COPB2 gene in reduction tumour cell, reach the object of the propagation of inhibition tumor cell, growth, differentiation and/or survival.
The anti-tumor medicine that the described COPB2 gene preparation by separation or screening obtain or diagnosing tumor medicine include but not limited to: nucleic acid molecule, carbohydrate, lipid, small-molecule chemical medicine, antibody medicine, polypeptide, albumen or interference slow virus.
Described nucleic acid includes but not limited to: siRNA (esiRNA) prepared by antisense oligonucleotide, double-stranded RNA (dsRNA), ribozyme, endoribonuclease III or short hairpin RNA (shRNA).
The amount of application of described anti-tumor medicine is enough reduce transcribing or translating of people COPB2 gene, or enough reduces the expression of people COPB2 albumen or the dosage of activity.50%, 80%, 90%, 95% or 99% is at least lowered to make the expression of people COPB2 gene.
Adopt the method for forgoing neoplasms medicine treatment tumour, mainly reached the object for the treatment of by the propagation of the expression level inhibition tumor cell reducing people COPB2 gene.Concrete, during treatment, effectively can reduce the administering substances of people COPB2 gene expression dose in patient.
Second aspect present invention discloses a kind of nucleic acid molecule reducing the separation of COPB2 genetic expression in tumour cell, and described nucleic acid molecule comprises:
A) double-stranded RNA, in described double-stranded RNA containing can under stringent condition with the nucleotide sequence of COPB2 gene recombination; Or
B) shRNA, in described shRNA containing can under stringent condition with the nucleotide sequence of COPB2 gene recombination.
Further, described double-stranded RNA comprises the first chain and the second chain, described first chain and the complementary common formation RNA dimer of described second chain, and the sequence of described first chain is substantially identical with 15-27 continuous print nucleotide sequence in COPB2 gene.Preferably, the sequence of described first chain is substantially identical with 19-23 continuous print nucleotide sequence in COPB2 gene; Better, the sequence of described first chain is substantially identical with 19 continuous print nucleotide sequences in COPB2 gene.
Further, described double-stranded RNA comprises the first chain and the second chain, described first chain and the complementary common formation RNA dimer of described second chain, and the sequence of described first chain is substantially identical with the target sequence in COPB2 gene.
The length of described double-stranded RNA first chain and the second chain is 15-27 Nucleotide; Preferably, length is 19-23 Nucleotide; Best, length is 19 Nucleotide.
Further, described double-stranded RNA is siRNA (siRNA).Further, the sequence of described siRNA first chain, as shown in SEQ ID NO:22, is specially 5 '-gcAGAUUAGAGUGUUCAAUUA-3 '.
SiRNA shown in SEQ ID NO:22 for the sequence shown in SEQ ID NO:5 be RNA disturb target sequence design, for a chain of the siRNA of people COPB2 gene, another chain i.e. sequence of the second chain and the complementation of the first chain-ordering, this siRNA can play the effect of endogenous COPB2 genetic expression in the reticent tumour cell of specificity.
Further, described shRNA comprises positive-sense strand fragment and antisense strand fragment, and connect the loop-stem structure of described positive-sense strand fragment and antisense strand fragment, the complementary of described positive-sense strand fragment and described antisense strand fragment, and the sequence of described positive-sense strand fragment is substantially identical with 15-27 continuous print nucleotide sequence in COPB2 gene.Described shRNA can become siRNA (siRNA) and then play the effect of endogenous COPB2 genetic expression in the reticent tumour cell of specificity after processing.
Further, described shRNA comprises positive-sense strand fragment and antisense strand fragment, and connect the loop-stem structure of described positive-sense strand fragment and antisense strand fragment, the complementary of described positive-sense strand fragment and described antisense strand fragment, and the sequence of described positive-sense strand fragment is substantially identical with the target sequence in COPB2 gene.
Preferably, described positive-sense strand fragment is substantially identical with 19-23 continuous print nucleotide sequence in COPB2 gene; Better, described positive-sense strand fragment is substantially identical with 19 continuous print nucleotide sequences in COPB2 gene.
Further, the sequence of the loop-stem structure of described shRNA can be selected from following arbitrary: UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU and CCACACC.
Further, the sequence of described shRNA, as shown in SEQ ID NO:23, is specially: 5 '-gcAGAUUAGAGUGUUCAAUUAUUCAAGAGAUAAUUGAACACUCUAAUCUgc-3 '.
ShRNA cuts after processing can become siRNA through enzyme, and then plays the effect of endogenous people COPB2 genetic expression in the reticent tumour cell of specificity.
The interference lentiviral vectors of gene fragment of shRNA of the present invention of encoding contains arbitrary sequence in SEQ ID NO:1-10 and complementary sequence thereof.
First chain of described double-stranded RNA or the positive-sense strand fragment of described shRNA substantially identical with the target sequence in COPB2 gene, when the target sequence of described COPB2 gene is siRNA for the COPB2 genetic expression of specificity silence, identified the fragment in the COPB2 gene corresponding to mRNA fragment of also silence by described siRNA.
Preferably, the target sequence in described COPB2 gene contains arbitrary sequence of SEQ ID NO:1-10.
Further, described COPB2 gene source is in people.
Third aspect present invention, discloses a kind of COPB2 Gene interfere nucleic acid construct, and the gene fragment of the shRNA in the nucleic acid molecule containing coding separation of the present invention, can express described shRNA.
Described people COPB2 Gene interfere nucleic acid construct can be the gene fragment clone of the aforementioned people COPB2 gene shRNA of coding is entered known carrier obtain.Further, described COPB2 Gene interfere nucleic acid construct is COPB2 Gene interfere lentiviral vectors.
COPB2 Gene interfere lentiviral vectors of the present invention the DNA fragmentation of the aforementioned COPB2 gene shRNA of coding is cloned into known carrier obtain, described known carrier mostly is lentiviral vectors, described COPB2 Gene interfere lentiviral vectors is after virus packaging becomes infectious virion, infected tumor's cell, and then transcribe out shRNA of the present invention, the steps such as processing are cut, the described siRNA of final acquisition, for the expression of the reticent COPB2 gene of specificity by enzyme.
Further, the nucleotide sequence of described COPB2 Gene interfere lentiviral vectors also containing the marker that can be detected in promoter sequence and/or codes for tumor cell; Preferably, the described marker be detected is as green fluorescent protein (GFP).
Further, described lentiviral vectors can be selected from: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti6-GW/U6-laminshrna, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, arbitrary in pGCSIL-GFP or pLenti6.2/N-Lumio/V5-GW/lacZ.
It is the people COPB2 Gene interfere lentiviral vectors of vector construction that the embodiment of the present invention specifically lists with pGCSIL-GFP, called after pGCSIL-GFP-COPB2-siRNA.
The nucleic acid molecule that the present invention is separated can be used for the medicine preparing prevention or treatment tumour, and described tumour is lung cancer or colorectal carcinoma.
COPB2 gene siRNA of the present invention can be used for the propagation of inhibition tumor cell, can be used as medicine or the preparation for the treatment of tumour further.COPB2 Gene interfere lentiviral vectors then can be used for preparing described COPB2 gene siRNA.When being used as medicine or the preparation for the treatment of tumour, be that the described nucleic acid molecule of safe and effective amount is applied to Mammals.Concrete dosage also should consider the factor such as route of administration, patient health situation, and these are all within skilled practitioners skill.
Fourth aspect present invention, discloses a kind of COPB2 Gene interfere slow virus, by aforementioned COPB2 Gene interfere lentiviral vectors slow virus packaging plasmid, clone auxiliary under, form through virus packaging.This slow virus can infected tumor's cell producing for the small molecules interference RNA of COPB2 gene, thus suppresses the propagation of lung cancer or colon cancer cell.This COPB2 Gene interfere slow virus can be used for the medicine preparing prevention or treatment tumour.
Fifth aspect present invention, disclose a kind of pharmaceutical composition for preventing or treat tumour, its active substance contains the nucleic acid molecule of aforesaid separation, one or more the combination in COPB2 Gene interfere nucleic acid construct or COPB2 Gene interfere slow virus.
Further, described pharmaceutical composition contains double-stranded RNA described in 1 ~ 99wt%, shRNA, COPB2 Gene interfere nucleic acid construct or COPB2 Gene interfere slow virus, and pharmaceutically acceptable carrier, thinner or vehicle.
When preparing these compositions, usually by activeconstituents and mixed with excipients, or with vehicle dilution, or wrap in can in the carrier that exists of capsule or sachet.When vehicle plays thinner effect, it can be solid, semisolid or the fluent material medium as vehicle, carrier or activeconstituents.Therefore, composition can be tablet, pill, pulvis, solution, syrup, sterilizing injecting solution etc.The example of suitable vehicle comprises: lactose, glucose, sucrose, sorbyl alcohol, N.F,USP MANNITOL, starch, Microcrystalline Cellulose, polyvinylpyrrolidone, Mierocrystalline cellulose, water, etc.Preparation also can comprise: wetting agent, emulsifying agent, sanitas (as methyl hydroxybenzoate and propyl ester), sweeting agent etc.
The invention also discloses the application of described pharmaceutical composition in the arbitrary anti-tumor medicine preparing treatment lung cancer or colorectal carcinoma.
The treatment being applied as tumour of this pharmaceutical composition provides a kind of method, is specially a kind of method of prevention or treatment target in-vivo tumour, comprises and being applied in object by the pharmaceutical composition described in effective dose.Further, described tumour is selected from the arbitrary of lung cancer or colorectal carcinoma.
When described pharmaceutical composition is used for prevention or treatment target in-vivo tumour, need the pharmaceutical composition described in effective dose to be applied in object.Adopt the method, growth, propagation, the recurrence of described tumour and/or shift suppressed.Further, the growth of described tumour, propagation, recurrence and/or transfer at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% part suppressed.
The object of described method can be people.
Sixth aspect present invention, disclose a kind of test kit for reducing the COPB2 genetic expression in tumour cell, described test kit comprises: the nucleic acid molecule being present in the described separation in container, COPB2 Gene interfere nucleic acid construct, and/or described COPB2 Gene interfere slow virus.
In sum, the present invention devises 10 RNAi target sequences for people COPB2 gene, build corresponding COPB2RNAi carrier, wherein the RNAi carrier pGCSIL-GFP-COPB2-siRNA of encoding sequence SEQ ID NO:5 significantly can lower the expression of COPB2 gene at mRNA level in-site and protein level.Use slow virus (lentivirus, be abbreviated as Lv) carry RNAi carrier pGCSIL-GFP-COPB2-siRNA as genetic manipulation instrument the RNAi sequence for COPB2 gene can efficiently be imported lung cancer H1299 cell and colorectal carcinoma RKO cell target, reduce the expression level of COPB2 gene, significantly suppress the multiplication capacity of above-mentioned tumour cell.Therefore the COPB2 gene silencing of lentivirus mediated is the potential clinical non-operative treatment mode of malignant tumour.
SiRNA provided by the invention or comprise the nucleic acid construct of this siRNA sequence, slow virus specificity can suppress the expression of people COPB2 gene, especially slow virus, efficiently can infect target cell, suppress the expression of COPB2 gene in target cell expeditiously, and then the growth of inhibition tumor cell, promote apoptosis of tumor cells, significant in oncotherapy.
Accompanying drawing explanation
Fig. 1: pGCSIL-GFP Plasmid diagram
Fig. 2: COPB2-RNAi slow virus infected lung cancer H1299 cell and colorectal carcinoma RKO cell after 5 days, and the expression level of COPB2mRNA significantly reduces
Fig. 3: COPB2-RNAi slow virus infected lung cancer H1299 cell after 5 days, caused cell inhibitory effect
Fig. 4: COPB2-RNAi slow virus infected colorectal carcinoma RKO cell after 5 days, caused cell inhibitory effect
Embodiment
The generation development that the present invention is based on multiple eukaryotic translation initiation factor and tumour is closely related, and COPB2, as a kind of eukaryotic translation initiation factor, infers that it may may take part in generation and the development of malignant tumour.
The present invention relates to one group of small molecules interference RNA for people COPB2 gene (siRNA) sequence, rna interference vector and RNA and disturb slow virus.Choose the target site of people COPB2mRNA coding region sequence as siRNA, according to the preferred 15-27 of continuous print 10-30(, more preferably 19-23 in target site) individual base sequence design siRNA target sequence.By gene clone, the nucleic acid construct of the above-mentioned siRNA of construction expression, the slow virus of the above-mentioned siRNA of packaging expression.Cell experiment proves, above-mentioned siRNA sequence can the expression of endogenous COPB2 gene in the reticent human tumor cells of specificity.
Contriver finds, can the propagation of inhibition tumor cell effectively after the expression of COPB2 gene of transferring person under adopting RNAi method, and this achievement in research shows that COPB2 gene is proto-oncogene, can be used as the target spot of oncotherapy.Contriver synthesizes further and tests the multiple siRNA for COPB2 gene, filter out the expression that effectively can suppress COPB2 and then the siRNA suppressing lung cancer H1299 cell and colorectal carcinoma RKO Cells Cell Proliferation and growth, complete the present invention on this basis.
The invention provides siRNA (siRNA) sequence of a series of interference people COPB2 gene, constructing can the slow virus of the reticent COPB2 genetic expression of specificity.The present invention studies discovery, for siRNA and the RNAi slow virus of people COPB2 gene design, and stable expression of also lowering COPB2 gene specifically, and effectively suppress the propagation of human tumor cells.The present invention shows that COPB2 gene can promote growth of tumour cell, is expected to the target spot becoming early diagnosis of tumor and treatment.And, by the expression of the reticent COPB2 gene of RNAi mode, can be used as the effective means of Tumor suppression development.
Mentality of designing of the present invention is:
The present invention screens by the following method and obtains a kind of people COPB2 gene RNAi slow virus: from Genbank, transfer people COPB2 gene order; Prediction siRNA site; Synthesize the effective siRNA sequence for COPB2 gene, two ends are containing the double-stranded DNA Oligo of restriction enzyme site cohesive end; Be connected with double-stranded DNA Oligo after lentiviral vectors double digestion, the RNAi plasmid of construction expression COPB2 gene siRNA sequence; By assistant carrier (Packing Mix, Sigma-aldrich company) the cotransfection HEKC 293T that RNAi plasmid and slow virus packaging need, produce recombinant slow virus particle, the slow virus of efficient reticent COPB2 gene can be obtained.
Based on aforesaid method, the invention provides the Effective target site (specifically as shown in SEQ ID NO1-10) of 10 interference COPB2 genes, construct the slow virus of special interference people COPB2 gene.
The present invention simultaneously also discloses a kind of people COPB2 gene RNAi slow virus (COPB2-RNAi) and preparation and application thereof.
This research finds, utilizes the RNAi method of lentivirus mediated, after reducing the expression in tumour cell of COPB2 gene, and can the propagation of effective inhibition tumor cell.This research shows, COPB2 gene is a proto-oncogene, tumor cell proliferation can be promoted, occur in tumour and in development, there is important biological function, COPB2 gene can be the target of oncotherapy, and the COPB2 gene specific silence of lentivirus mediated can be used as a kind of new tool of oncotherapy.
The present invention is set forth further below in conjunction with embodiment.Should be understood that embodiment only for illustration of the present invention, but not limit the scope of the invention.In embodiment, the experimental technique of unreceipted actual conditions and the reagent of undeclared formula are conveniently condition, as works such as ［ beautiful ］ Sambrook.J; Huang Peitang etc. translate.Molecular cloning texts guide, the third edition.Beijing: the condition of the condition described in Science Press 2002 or manufacturers's suggestion is carried out or configures.
Embodiment 1 is for the preparation of people COPB2 gene RNAi slow virus
1. screening is for the effective siRNA target spot of people COPB2 gene
COPB2(NM_004766.2 is transferred from Genbank) gene information; Design the effective siRNA target spot for COPB2 gene.Table 1 lists wherein 10 effective siRNA target sequences for COPB2 gene.
Table 1 target is in the siRNA target sequence of people COPB2 gene
| SEQ ID NO. | TargetSeq | Initiation site |
| 1 | CATTAGTAAATGTCAGTTT | 2097 |
| 2 | TCAGACTATTCAGCACAAT | 1131 |
| 3 | CCACGATTCTTCAGAGTAT | 1257 |
| 4 | AGCCAAACAATACCCACTT | 2526 |
| 5 | AGATTAGAGTGTTCAATTA | 305 |
| 6 | CTAGATCTGATCGAGTTAA | 166 |
| 7 | TGCTACTGAGGAATCATTT | 1565 |
| 8 | TGACTAAACAGATCATTAT | 2871 |
| 9 | AGATTAGAGTGTTCAATTA | 367 |
| 10 | CCACGATTCTTCAGAGTAT | 1319 |
2. the preparation of lentiviral vectors
Double-stranded DNA Oligo sequence (table 2) of two ends containing Age I and EcoR I restriction enzyme site cohesive end is synthesized for siRNA target spot (for SEQ ID NO:5); Act on pGCSIL-GFP carrier (Shanghai JiKai Gene Chemical Technology Co., Ltd provides, Fig. 1) with Age I and EcoR I restriction enzyme, make its linearizing, agarose gel electrophoresis qualification endonuclease bamhi.
Table 2 two ends contain the double-stranded DNA Oligo of Age I and EcoR I restriction enzyme site cohesive end
| 5’ | Neck | Ring | Neck | 3’ | SEQ | |
| Positive-sense strand | CCGG | gcAGATTAGAGTGTTCAATTA | TTCAAGAGA | TAATTGAACACTCTAATCTgc | TTTTTG | 11 |
| Antisense strand | AATTCAAAAA | gcAGATTAGAGTGTTCAATTA | TCTCTTGAA | TAATTGAACACTCTAATCTgc | 12 |
By T4DNA ligase enzyme, by double digestion linearizing, (it is as shown in table 4 that enzyme cuts system, 37 DEG C, reaction 1h) the carrier DNA double-stranded DNA Oligo good with purifying is connected, spend the night in 16 DEG C of connections in suitable buffer system (linked system is as shown in table 5), reclaim connection product.By fresh competent escherichia coli cell (conversion operation reference: Molecular Cloning: A Laboratory guide second edition 55-56 page) prepared by connection product conversion calcium chloride.Growing bacterium clone surface at connection converted product is stained with, and be dissolved in 10 μ l LB substratum, mixing gets 1 μ l as template; The upstream and downstream of RNAi sequence, designs general PCR primer (upstream primer sequence: 5 '-CCTATTTCCCATGATTCCTTCATA-3 ' (SEQ ID NO:15) in lentiviral vectors; Downstream primer sequence: 5 '-GTAATACGGTTATCCACGCG-3 ' (SEQ ID NO:16), carries out PCR identification experiment (, as table 6-1, reaction conditions is as table 6-2 for PCR reaction system).The clone positive to PCR qualification checks order and compare of analysis, and the clone that comparison is correct is the carrier of the expressed rna i for SEQ ID NO:5 successfully constructed, called after pGCSIL-GFP-COPB2-siRNA.
Build pGCSIL-GFP-Scr-siRNA negative control plasmids, negative control siRNA target sequence is 5 '-TTCTCCGAACGTGTCACGT-3 ' (SEQ ID NO:17).When building pGCSIL-GFP-Scr-siRNA negative control plasmids, double-stranded DNA Oligo sequence (table 3) of Age I and EcoR I restriction enzyme site cohesive end is contained, all same pGCSIL-GFP-COPB2-siRNA of all the other construction processs, authentication method and condition for Scr siRNA target spot synthesis two ends.
Table 3 two ends contain the double-stranded DNA Oligo of Age I and EcoR I restriction enzyme site cohesive end
| 5’ | Neck | Ring | Neck | 3’ | SEQ | |
| Positive-sense strand | CCGG | TTCTCCGAACGTGTCACGT | TTCAAGAGA | ACGTGACACGTTCGGAGAA | TTTTTG | 13 |
| Antisense strand | AATTCAAAAA | TTCTCCGAACGTGTCACGT | TCTCTTGAA | ACGTGACACGTTCGGAGAA | 14 |
By the carrier of T4DNA ligase enzyme by double digestion linearizing (it is as shown in table 4 that enzyme cuts system, 37 DEG C, reaction 1h)
Table 4pGCSIL-GFP plasmid enzyme restriction reaction system
| Reagent | Volume (μ l) |
| PGCSIL-GFP plasmid (1 μ g/ μ l) | 2.0 |
| 10×buffer | 5.0 |
| 100×BSA | 0.5 |
| Age I(10U/μl) | 1.0 |
| EcoR I(10U/μl) | 1.0 |
| dd H 2O | 40.5 |
| Total | 500 |
Table 5 carrier DNA and double-strand double-stranded DNA Oligo ligation system
| Reagent | Positive control (μ l) | From connecting contrast (μ l) | Connecting groups (μ l) |
| Linearizing carrier DNA (100ng/ μ l) | 1.0 | 1.0 | 1.0 |
| The double-stranded DNA Oligo (100ng/ μ l) of annealing | 1.0 | - | 1.0 |
| 10 × T4 phage DNA ligase enzyme damping fluid | 1.0 | 1.0 | 1.0 |
| T4 phage DNA ligase enzyme | 1.0 | 1.0 | 1.0 |
| dd H 2O | 16.0 | 17.0 | 16.0 |
| Total | 20.0 | 20.0 | 20.0 |
Table 6-1PCR reaction system
| Reagent | Volume (μ l) |
| 10×buffer | 2.0 |
| dNTPs(2.5mM) | 0.8 |
| Upstream primer | 0.4 |
| Downstream primer | 0.4 |
| Taq polysaccharase | 0.2 |
| Template | 1.0 |
| ddH 2O | 15.2 |
| Total | 20.0 |
Table 6-2PCR reaction system program setting
3. pack COPB2-siRNA slow virus
Extract the DNA of RNAi plasmid pGCSIL-GFP-COPB2-siRNA with the plasmid extraction test kit of Qiagen company, be mixed with 100ng/ μ l storage liquid.
24h before transfection, with the HEKC 293T cell of tryptic digestion logarithmic phase, with the DMEM perfect medium adjustment cell density containing 10% foetal calf serum for 1.5 × 10
5cell/ml, is inoculated in 6 orifice plates, 37 DEG C, 5%CO
2cultivate in incubator.Transfection is can be used for when cell density reaches 70%-80%.2h before transfection, the original substratum of sucking-off, adds the perfect medium that 1.5ml is fresh.According to the explanation of the MISSION Lentiviral Packaging Mix test kit of Sigma-aldrich company, Packing Mix(PVM is added in a sterile centrifugation tube) 20 μ l, PEI12 μ l, plasma-free DMEM medium 400 μ l, get the plasmid DNA of the above-mentioned extracting of 20 μ l, add to above-mentioned PVM/PEI/DMEM mixed solution.
Above-mentioned transfection miscellany is at room temperature hatched 15min, is transferred in the substratum of HEKC 293T cell, 37 DEG C, 5%CO
216h is cultivated in incubator.Discard the developing medium containing transfection miscellany, PBS solution is washed, and adds perfect medium 2ml, continues to cultivate 48h.Collecting cell supernatant liquor, Centricon Plus-20 centrifugal ultrafiltration unit (Millipore) purifying and concentrated slow virus, step is as follows: (1) 4 DEG C, the centrifugal 10min of 4000g, removing cell debris; (2) 0.45 μm of frit supernatant liquors are in 40ml ultracentrifugation pipe; (3) 4000g is centrifugal, 10-15min, to the viral concentration volume needed; (4), after centrifugal end, filtering cup and filtered solution collection cups are below separated, tipped upside down on by filtering cup on sample collection cup, centrifugal 2min centrifugal force is no more than 1000g; (5) Centrifuge Cup is removed from sample collection cup, in sample collection cup, be viral concentration liquid.By after the packing of viral concentration liquid in-80 degrees Celsius of preservations.The sequence of first chain of the siRNA contained in viral concentration liquid is as shown in SEQ ID NO:15.The wrapping process of contrast slow virus, with COPB2-siRNA slow virus, only replaces pGCSIL-GFP-COPB2-siRNA carrier with pGCSIL-GFP-Scr-siRNA carrier.
Embodiment 2 real-time fluorescence quantitative RT-PCR method detects the silence efficiency of COPB2 gene
The people lung cancer H1299 cell and the colorectal carcinoma RKO cell that are in logarithmic phase carry out trysinization, and (cell count is about 5 × 10 to make cell suspension
4/ ml) be inoculated in 6 orifice plates, be cultured to cytogamy degree and reach about 30%.According to infecting plural number (MOI, H1299:1; RKO:2) value, virus prepared by the embodiment 1 adding sufficient quantity, replaced medium after cultivation 24h, after time of infection reaches 5 days, collecting cell.According to the Trizol process specifications of Invitrogen company, extracted total RNA.According to the M-MLV process specifications of Promega company, RNA reverse transcription is obtained cDNA(reverse transcription reaction system in table 7,42 DEG C of reaction 1h, then in 70 DEG C of water-baths, water-bath 10min makes reversed transcriptive enzyme inactivation).
TP800 type Real time PCR instrument (TAKARA) is adopted to carry out Real_time quantitative detection.The primer of COPB2 gene is as follows: upstream primer 5 '-GTGGGGACAAGCCATACCTC-3 ' (SEQ ID NO:18) and downstream primer 5 '-GTGCTCTCAAGCCGGTAGG-3 ' (SEQ ID NO:19).With house-keeping gene GAPDH for internal reference, primer sequence is as follows: upstream primer 5 '-TGACTTCAACAGCGACACCCA-3 ' (SEQ ID NO:20) and downstream primer 5 '-CACCCTGTTGCTGTAGCCAAA-3 ' (SEQ ID NO:21).By the proportional arrangement reaction system in table 8.
Table 7 reverse transcription reaction system
| Reagent | Volume (μ l) |
| 5×RT buffer | 4.0 |
| 10mM dNTPs | 2.0 |
| RNasin | 0.5 |
| M-MLV-RTase | 1.0 |
| DEPC H 2O | 3.5 |
| Total | 11.0 |
Table 8Real-time PCR reaction system
| Reagent | Volume (μ l) |
| SYBR premix ex taq: | 10.0 |
| Upstream primer (2.5 μMs): | 0.5 |
| Downstream primer (2.5 μMs): | 0.5 |
| cDNA | 1.0 |
| ddH 2O | 8.0 |
| Total | 20.0 |
Setting program is two-step approach Real-time PCR: denaturation 95 DEG C, 15s; Each step sex change 95 DEG C afterwards, 5s; Annealing extension 60 DEG C, 30s; Carry out 45 circulations altogether.Read light absorption value in the extension stage at every turn.After PCR terminates, 95 DEG C of sex change 1min, are then cooled to 55 DEG C, and DNA double chain is fully combined.To 95 DEG C from 55 DEG C, each walks increase by 0.5 DEG C, keeps 4s, reads light absorption value simultaneously, makes melting curve.Adopt 2-
Δ Δ Ctanalytical method calculates and has infected the gene expression abundance of COPB2mRNA.Infect the cell of contrast virus (Lv-Scr-siRNA) in contrast.Experimental result (Fig. 2) shows, in people lung cancer H1299 cell and colorectal carcinoma RKO cell, the expression level of COPB2mRNA has lowered 82.0% and 75.5%.
Embodiment 3 detects the multiplication capacity infecting the tumour cell of COPB2-siRNA slow virus
The people lung cancer H1299 cell and the colorectal carcinoma RKO cell that are in logarithmic phase carry out trysinization, and (cell count is about 5 × 10 to make cell suspension
4/ ml) be inoculated in 6 orifice plates, be cultured to cytogamy degree and reach about 30%.According to infecting plural number (MOI, H1299:1; RKO:2), add the virus of sufficient quantity, replaced medium after cultivation 24h, after time of infection reaches 5 days, collect each experimental group cell being in logarithmic phase.The resuspended one-tenth cell suspension (2 × 10 of perfect medium
4/ ml), be about 2000/hole with cell density, inoculate 96 orifice plates.Often organize 5 multiple holes, every hole 100 μ l.After completing plate, put 37 DEG C, 5%CO
2incubator is cultivated.From after bed board second day, every day was detected with Cellomics instrument (Thermo Fisher) and reads plate once, and continuous detecting reads plate 5 days.By the input parameter of adjustment Cellomics arrayscan, calculate the quantity of the cell of the band green fluorescence in each scanning orifice plate exactly, statistics is carried out to data and draws, draw cell proliferation curve (result as Figure 3-Figure 4).Result shows, slow virus infects each tumour of group at cell injuring model after 5 days, and rate of propagation significantly slows down, far below the rate of propagation of control group tumour cell.
The above; be only preferred embodiment of the present invention; not to any formal and substantial restriction of the present invention; should be understood that; for those skilled in the art; under the prerequisite not departing from the inventive method, also can make some improvement and supplement, these improve and supplement and also should be considered as protection scope of the present invention.All those skilled in the art, without departing from the spirit and scope of the present invention, a little change made when utilizing disclosed above technology contents, the equivalent variations of modifying and developing, be Equivalent embodiments of the present invention; Meanwhile, all according to substantial technological of the present invention to the change of any equivalent variations that above-described embodiment is done, modify and differentiation, all still belong in the scope of technical scheme of the present invention.
Claims (14)
1. the purposes of the people COPB2 gene be separated in preparation or screening anti-tumor medicine.
2. purposes as claimed in claim 1, is characterized in that, described tumour be selected from lung cancer, colorectal carcinoma arbitrary.
3. reduce a nucleic acid molecule for the separation of COPB2 genetic expression in tumour cell, described nucleic acid molecule comprises:
A) double-stranded RNA, in described double-stranded RNA containing can under stringent condition with the nucleotide sequence of COPB2 gene recombination; Or
B) shRNA, in described shRNA containing can under stringent condition with the nucleotide sequence of COPB2 gene recombination.
4. the nucleic acid molecule be separated as claimed in claim 3, it is characterized in that, described double-stranded RNA comprises the first chain and the second chain, described first chain and the complementary common formation RNA dimer of described second chain, and the sequence of described first chain is substantially identical with the target sequence in COPB2 gene; Described shRNA comprises positive-sense strand fragment and antisense strand fragment, and connect the loop-stem structure of described positive-sense strand fragment and antisense strand fragment, the complementary of described positive-sense strand fragment and described antisense strand fragment, and the sequence of described positive-sense strand fragment is substantially identical with the target sequence in COPB2 gene.
5. the nucleic acid molecule be separated as described in claim 3 or 4, it is characterized in that, the target sequence of described COPB2 gene contains arbitrary sequence in SEQ ID NO:1-10.
6. the nucleic acid molecule be separated as described in claim 3 or 4, it is characterized in that, described double-stranded RNA is siRNA, and the sequence of this siRNA first chain is as shown in SEQ ID NO:22.
7. the nucleic acid molecule be separated as described in claim 3 or 4, it is characterized in that, the sequence of described shRNA is as shown in SEQ ID NO:23.
8. a COPB2 Gene interfere nucleic acid construct, the gene fragment containing the shRNA in the nucleic acid molecule be separated described in the arbitrary claim of coding claim 3-7, can express described shRNA.
9. COPB2 Gene interfere nucleic acid construct as claimed in claim 8, is characterized in that, described COPB2 Gene interfere nucleic acid construct is interference lentiviral vectors.
10. COPB2 Gene interfere nucleic acid construct as claimed in claim 9, is characterized in that, described interference lentiviral vectors obtains after the gene fragment clone of the described shRNA of coding is entered lentiviral vectors, and described lentiviral vectors is selected from:
pLKO.1-puro、pLKO.1-CMV-tGFP、pLKO.1-puro-CMV-tGFP、pLKO.1-CMV-Neo、pLKO.1-Neo、pLKO.1-Neo-CMV-tGFP、pLKO.1-puro-CMV-TagCFP、pLKO.1-puro-CMV-TagYFP、
PLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti6-GW/U6-laminshrna, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or
Arbitrary in pLenti6.2/N-Lumio/V5-GW/lacZ.
11. 1 kinds of COPB2 Gene interfere slow viruss, by disturb described in the arbitrary claim of claim 9-10 lentiviral vectors slow virus packaging plasmid, clone auxiliary under, form through virus packaging.
12. 1 kinds for preventing or treat the pharmaceutical composition of tumour, its active substance contains the nucleic acid molecule of the separation described in the arbitrary claim of claim 3-7, COPB2 Gene interfere nucleic acid construct described in the arbitrary claim of claim 8-10, and/or COPB2 Gene interfere slow virus according to claim 11.
The application of pharmaceutical composition described in 13. claims 12 in the anti-tumor medicine of preparation treatment cancer of the stomach, lung cancer, colorectal carcinoma or glioma.
14. 1 kinds of test kits for reducing COPB2 genetic expression in tumour cell, it is characterized in that, described test kit comprises: be present in container, the nucleic acid molecule of the separation described in the arbitrary claim of claim 3-7, COPB2 Gene interfere nucleic acid construct described in the arbitrary claim of claim 8-10, and/or COPB2 Gene interfere slow virus according to claim 11.
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| CN107519491A (en) * | 2017-09-04 | 2017-12-29 | 无锡市人民医院 | The purposes of COPB2 inhibitor |
| CN107779453A (en) * | 2016-08-30 | 2018-03-09 | 上海吉凯基因化学技术有限公司 | The purposes and its related drugs of people's TRIP13 genes |
| CN111304327A (en) * | 2020-02-24 | 2020-06-19 | 宋程 | Application of human GRPEL2 gene and related product |
| CN111304327B (en) * | 2020-02-24 | 2024-04-19 | 宋程 | Application of human GRPEL gene and related products |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107779453A (en) * | 2016-08-30 | 2018-03-09 | 上海吉凯基因化学技术有限公司 | The purposes and its related drugs of people's TRIP13 genes |
| CN107519491A (en) * | 2017-09-04 | 2017-12-29 | 无锡市人民医院 | The purposes of COPB2 inhibitor |
| CN107519491B (en) * | 2017-09-04 | 2020-08-25 | 无锡市人民医院 | Use of COPB2 inhibitor |
| CN111304327A (en) * | 2020-02-24 | 2020-06-19 | 宋程 | Application of human GRPEL2 gene and related product |
| CN111304327B (en) * | 2020-02-24 | 2024-04-19 | 宋程 | Application of human GRPEL gene and related products |
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