CN107365785A - The expression vector of NF kB activities and its regulation and control methods and applications in a kind of regulating cell - Google Patents
The expression vector of NF kB activities and its regulation and control methods and applications in a kind of regulating cell Download PDFInfo
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Abstract
The invention discloses a kind of expression vector of NF kB activities in regulating cell and its regulation and control methods and applications, the carrier includes two sequential elements, the promoter sequence and promoter downstream miRNA coded sequences of controlling gene expression；The promoter sequence inveigles subsequence and minimal promoter sequence to form by one section of NF κ B；The miRNA coded sequences are the coded sequence for the miRNA that one section of codified targets NF κ B mRNA.The transfection of the expression vector gently can be effectively reduced the intracellular NF kB activities of NF kB activity overactivities, and the vigor of normal cell is had no significant effect, so as to avoid side effect.The regulation and control method of the expression vector of the present invention is simple, quick, effective；The expression vector is used for the NF kB activities regulation and control reagent or drug molecule for preparing the closely related disease of gene therapy NF κ B overactivities, as a kind of new gene therapy agents.
Description
Technical field
The invention belongs to biological technical field, and in particular to the gene of NF- κ B (kappaB) activity in a kind of regulating cell
Expression vector and its regulation and control methods and applications.
Background technology
Nuclear factor kappaB (NF- κ B) is a kind of important control transcription factor, in many physiology and pathologic process
Play a crucial role, such as immune, cell propagation, apoptosis, inflammation, especially tumour occur.Some researchs show many diseases and NF-
The abnormal activation of κ B and its signal path is relevant.For example, NF- κ B abnormal activations in many human cancers, anti-thin by raising
Born of the same parents' apoptogene come promote existence and malignant tumour.It has been found that NF- κ B core aggregation and high NF- κ B target gene features cause
The NF- κ B paths enrichment of most of multiple myeloma cell lines, and it is sensitive to Apoptosis.Therefore, many drugmakers and
Scientist is directed to NF- kB inhibitor of the exploitation for treatment of cancer always.
Chemicals is the key areas for developing NF- kB activity inhibitor.Such as.A kind of new synthesis of Kim et al.
Compound MCAP handles Primary mouse microglia and BV2 microglias.Then induction incubation is carried out with LPS, passes through RT-
Inducible nitric oxide synthase (iNOS) in PCR, Western blotting and elisa assay cell, COX-2, pro-inflammatory cytokine,
The expression of NF- κ B and p38MAPK signaling molecules, and using difference fluorescence microscope microglia morphological change and
NF- κ B core transposition.As a result show, MCAP can suppress the iNOS and COX-2 of LPS inductions expression, illustrate that MCAP passes through suppression
NF- κ B signal paths processed, p38MAPK signal paths and proinflammatory reaction play antiinflammatory action.But regrettably these chemical drugs
Thing it is specific low.
Nowadays, gene therapy turns into preferable disease treatment strategy, because the most ailments of the mankind are by gene
Caused by structure variation or abnormal expression, so optimal disease treatment strategy is exactly to be corrected from gene level.
Substrategy and RNA jamming exposure areas is inveigled to belong to using the method for gene therapy to suppress intracellular NF- κ B activity.Inveigle sub- plan
Slightly refer to synthesize a kind of double chain DNA sequence consistent with NF- κ B cis-regulating element sequence, be conducted into cell
Afterwards, this DNA sequence dna can and the specific combinations of NF- κ B after activation, can disturb the NF- κ B after activation to enter with this
Nucleus, its specific binding with the NF- κ B binding sites on target gene is reduced, suppress the regulation and control of NF- kB activities so as to reach
The purpose of expression of target gene.Report finds to inveigle subsequence to adjust NF- κ B correlation target genes by suppressing NF- κ B activity
Expression, causes osteoclast apoptosis.Can be thin to suppress fibrosarcoma by directly suppressing NF- kB activities using trick of synthesis
Intracellular growth.Once the sub synovial cell for being introduced into rheumatoid patient, cell factor and adhesion factor are inveigled in targeting NF- κ B synthesis
Expression all reduce, and the propagation of synovial cell is also suppressed.N- kappa B decoys can regulate and control cis by identical simultaneously
The many genes of regulating element control.Meanwhile inveigle specific and relatively simple synthesis of the son with height.However, NF- κ B
Inveigle son easily by some enzymes degraded in body, it is necessary to repeat and high dose administration is to maintain NF- κ B and its signal path
Activity suppression.
RNA interference (RNAi) is the another kind strategy of gene therapy, and it refers to using double stranded rna molecule in mRNA level in-site
The sequence specific post transcriptional gene silencing process of mediation.It is small interference to be widely used in and knock out the current RNA that endogenous gene is expressed
RNA (siRNA), it has two kinds of action modes.A kind of is the double-stranded RNA (dsRNA) of transfection in vitro chemical synthesis, by cell
The silencing complex (RISC) of RNA inductions is processed into the siRNA of maturation.Another kind is that transfection can be by rna plymerase iii
Children purpura nephritis (shRNA) expression vector of (Pol III) promoter transcription, and maturation is produced by dicer in cell and RISC
siRNA.SiRNA molecules can specifically bind the mRNA of target gene so as to suppress its expression.Research finds to be suppressed with siRNA
After NF- kB activities, the induction of inflammatory cytokine is inhibited by.In addition, siRNA also causes TNF (TNF-α) place
The apoptosis of the primary synovial cell of people of reason substantially rises.Research have shown that with RNA perturbation techniques treatment rheumatoid arthritis obtain compared with
Good curative effect.Also studies have found that the NF- κ B p65 specific siRNAs of synthesis are imported in the mouse induced with IL-1 β and TNF-α
After cartilage cell, COX-2, NOS-2 and MMP-9 expression are suppressed, and show that NF- κ B p65 specific siRNAs are likely to become
The prevention and treatment medicine of osteoarthritis.However, the siRNA of chemical synthesis deliverings in vivo and its lasts be it
Stumbling-block in clinical practice.
MicroRNA (miRNA) is a kind of natural tiny RNA (~22nt), and it can also be by blocking the translation of said target mrna
Or target mrna degradation is induced to carry out regulatory gene expression as siRNA.MiRNA expression is by rna plymerase ii (Pol II)
Regulation and control, and Pol II also are responsible for mRNA expression, different from siRNA expression by RNA Polymerase III (Pol III)
Regulation and control.MiRNA is initially transcribed into long primary miRNA (pri-miRNA) precursor by Pol II, then by Drosha and Dicer cores
Sour enzyme processing, and the short hairpin RNA (shRNA) that siRNA is transcribed into extensively under Pol III promoters (such as U6) regulation and control.Cause
This, miRNA expression is also adjusted as mRNA by the element limited by transcription factor.In addition, driven with rna plymerase ii
Dynamic promoter is compared, and the regulation expression of Pol III promoters is generally more difficult.Moreover, each Pol III promoters only table
Up to single shRNA, therefore the suppression of multiple genes needs multiple promoters or carrier.Therefore, by using the Pol II of standard
Promoter (such as the main early promoter (MIEP) immediately of human cytomegalovirus (CMV)) new expression vector of exploitation, effective table
Up to targeting mRNA miRNA.
Chemicals, inveigles substrategy, and RNA jamming exposure areas are suppress intracellular NF- κ B and its signal path activity three
Kind main method.It is obvious that their each self-application has shortcoming.Simultaneously, it is notable that extra-inhibitory NF- kB activities
Obvious toxicity may be produced.NF- κ B play a crucial role to cancer cell.However, it is also essential for normal cell
's.The NF- kB inhibitors of research and development suppress also to block NF- κ B in Normal healthy cells to adjust while the NF- kB activities in cancer cell
Control some must cell processes, so as to cause significant dose-limiting toxicity and low therapeutic index.Therefore, these are traditional
NF- kB inhibitors nevertheless suffer from the limitation of the side effect from the intracellular NF- kB activities of extra-inhibitory.
In view of the property of NF- κ B generally existings and the physiological function of multiple-effect, during using these traditional inhibitors, it is difficult to avoid
Influence while NF- κ B pathogenic activity is suppressed to NF- κ B normal physiological functions.
The overactivity of transcription factor NF-KB is the critical event of the diseases such as inflammation, tumour, with inflammation, immune response, swollen
Physiology, the pathologic processes such as knurl generation are in close relations.Therefore develop various NF- kB activities inhibitor is turned into suppressing NF- kB activities
Inflammation and an important channel of oncotherapy.Including the small molecular core acid substance that can suppress NF- kB activities, son is such as inveigled
And siRNA (siRNA) (decoy).But gene basis NF- kB activities level is necessary to the normal physiological of cell, to thin
Born of the same parents import these small molecular core acid substances, because of its uncontrollable quantity, the extra-inhibitory of NF- kB activities are often resulted in, so as to produce
Raw serious side effect.Therefore, it is necessary to which the technical tactic for suppressing NF- kB activities to current small molecular core acid substance is transformed,
To overcome its defect, new NF- kB activity of the development with clinical value suppresses nucleic acid molecules.
The content of the invention
Goal of the invention：The problem of existing for prior art, a kind of intracellular nucleic factor NF- kB activities regulation and control of the present invention
Expression vector, the carrier can perceive the activity with NF- κ B in regulating cell, gently can be effectively reduced NF- κ B work
The intracellular NF- kB activities of property overactivity, and the vigor of normal cell is had no significant effect.The present invention solves existing skill
Art small molecular NF- kB inhibitors are to the extra-inhibitories of NF- kB activities, the problem of so as to produce serious side effect.
Present invention also offers the regulation and control methods and applications of the expression vector.
Technical scheme：To achieve these goals, as described in the present invention in a kind of regulating cell NF- kB activities gene
Expression vector, the promoter sequence expressed comprising two sequential elements, controlling gene and promoter downstream miRNA (microRNA,
MiRNA is also referred to as tiny RNA) coded sequence；The promoter sequence inveigles sub (decoy) sequence and minimum startup by one section of NF- κ B
Sub (minimal promoter) sequence composition；The miRNA coded sequences are one section of targeting NF- κ B mRNA
The miRNA of (messenger RNA, mRNA) coded sequence.
Wherein, the NF- κ B inveigle subsequence to be also referred to as NF- κ B response elements (NF- κ B responsive element)
NF- κ B including various sequences inveigle son, are one section of DNA sequence dna that can be specifically bound with NF- kB proteins, and its main sequence is special
Levy as containing the different various NF- κ B combination target spots of quantity, such as GGGACTTTCC.
Wherein, the minimal promoter includes the minimal promoter sequence of various gene sources；It is preferred that herpes simplex virus
Thymidine kinase (herpes simplex virus thymidine kinase, HSV-TK) promoter minimal promoter；Its is main
Effect is combined with basal transcription factor and rna plymerase ii, forms general transcription machine, forms the basic bar of gene expression
Part.
Preferably, the NF- κ B mRNAs (mRNA) are RelA mRNA, also referred to as p65.
Wherein, the miRNA of the targeting NF- κ B mRNA refers to miRNA and combined by nucleic acid hybridization with NF- κ BmRNA, this
With reference to the degraded that can cause NF- κ B mRNA or NF- κ B mRNA are prevented to translate into NF- kB proteins.
Preferably, the miRNA of the targeting NF- κ B mRNA includes natural miRNA or engineer miRNA, preferably adopt
MiRNA, including amiR349, amiR531 or amiR533 are manually designed, its coded sequence is respectively SEQ ID NO.1：5′-
CTT CTT CAC ACA CTG GAT TCC-3 ', SEQ ID NO.2：5 '-AAG ATG GGA TGA GAA AGG ACA-3 ',
SEQ ID NO.3：5′-CAA AGA TGG GAT GAG AAA GGA-3′.
Preferably, the expression vector is one linear (linear) or ring-type (circular) nucleic acid molecules.
Further, the nucleic acid molecules are DNA (DNA) or ribonucleic acid (RNA) molecule, including double
Chain DNA (such as adenovirus DNA molecule), single stranded DNA (adeno-associated virus molecule) or single strand RNA molecule (such as slow virus RNA molecule)
Deng.
Further, the linear nucleic acid molecule includes normal linear DNA molecular (such as pcr amplified fragment, digestion piece
Section), viral DNA molecules (such as adenovirus DNA molecule, adeno-associated virus molecule) or viral RNA molecules (such as slow virus RNA molecule)
Deng；The circular nucleic acid molecules are including DNA etc..
The method of NF- kB activities in regulating cell of the present invention, concretely comprise the following steps and described gene is imported to cell
Expression vector.
Wherein, the cell introduction method includes various types of cell nucleic acid introduction methods；It is preferred that by viral vector,
The lead-in modes such as nano-carrier, liposome, electrotransfer, particle gun.
The expression vector of NF- kB activities is used for gene therapy NF- κ B mistakes in preparation in regulating cell of the present invention
Degree is activated in the NF- kB activities regulation and control reagent or drug molecule of closely related disease (such as inflammation, tumour, autoimmune disease)
Application.
Intracellular NF- kB activities regulation and control method of the invention, is a kind of new NF- kB activity control techniques, the technology can
As a kind of gene therapy skill with the closely related disease of NF- κ B overactivities (such as inflammation, tumour, autoimmune disease)
Art.
The present invention is on the basis of the NF- κ B of current trick of research and siRNA suppress the advantages of tactful and limitation, structure
Built can under the regulation and control of NF- κ B specificity promoters expression targeting NF- κ B artificial mi RNA (artificial miRNA,
AmiRNA transgene carrier), the specificity promoter inveigle sub (decoy) and a minimal promoter by NF- κ B
(minimal promoter) is formed, and expression vector of the invention is named as DMP-amiRNA, i.e., " inveigles sub- minimum to open
Mover-artificial tiny RNA " (decoy minimal promoter-artificial miRNA).The invention demonstrates that the carrier conduct
New NF- kB inhibitors, the activity with NF- κ B in regulating cell can be perceived.The carrier is perfect anti-by forming one
Road is fed back to realize the self regulating and control of intracellular NF- kB activities.In the cell for transfecting the carrier, NF- kB activities are higher, and DMP turns
Record activity is higher, and miRNA expression is more.Thus the carrier can play its NF- κ B as the sensor of intracellular NF- kB activities
Suppress function.The system combines the advantages of trick and miRNA interference.
The new NF- kB inhibitors of the present invention suppress shown in the mechanism such as principle schematic (Fig. 1) of NF- kB activities.Fig. 1 is anti-
Reflect the new NF- kB inhibitors (DMP-amiRNA) of the invention developed and its suppress the principle of intracellular NF- kB activities.The figure
Top half show that traditional small molecular core acid substance at present suppresses the strategy of NF- kB activities, that is, inveigle sub (decoy)
(the short double-stranded DNA containing NF- κ B binding sites) and siRNA (produce or imported cell by shRNA vivo transcriptions, processing
The dsRNA processing of chemical synthesis produces).Wherein siRNA transcription is completed by rna plymerase iii.The latter half of the figure is shown
New NF- κ B inhibition of gene expression carriers of the invention designed, the carrier combines trick and siRNA strategies, but assigns
Sub- New function is inveigled, i.e. NF- κ B specific enhancer functions, enhancing is merged with minimal promoter, and it is special to turn into a kind of NF- κ B
Property promoter.The specificity promoter can control the expression of downstream gene --- targeting NF- κ B artificial mi RNA ---.
Expressed miRNA can suppress NF- kB proteins p65's (the NF- κ B family members with transcriptional activation function) in translation skill
Produce, so as to reduce intracellular NF- κ B level.The carrier can perceive intracellular NF- κ B activity, determine that miRNA's is defeated
Go out.NF- kB activities are higher in cell, and combinations of the NF- κ B to DMP is more, and DMP transcriptional activities are higher, and miRNA expression is more.Instead
It, intracellular NF- kB activities are low, then miRNA expression is few, and extra-inhibitory to intracellular NF- kB activities is avoided with this, in order to avoid align
The NF- kB activities of normal cell cause to suppress, so as to avoid its side effect.It is pointed out that trick in the carrier
(decoy) in addition to playing NF- κ B specific enhancer functions, also still play and inveigle son effect, i.e., with reference to intracellular NF-
κB.But this trick being in expression vector (double-stranded cyclic DNA), inveigles son compared to conventional linear, can more support
The degraded of imperial intracellular nucleic acid enzyme, it inveigles son and enhancer difunctional more longlasting.
Beneficial effect：Compared with prior art, the invention has the advantages that：
The present invention devises a kind of NF- κ B specificity promoters (DMP), and design filters out 3 kinds of artificial mi RNAs
(amiR349, amiR531 and amiR533) molecule.By by designed NF- κ B specificity promoters and miRNA expression vector
Merged, construct a kind of miR-96 gene expression vector (pDMP- that its expression can be controlled by intracellular NF- kB activities
mCherry-amiR349、pDMP-mCherry-amiR531、pDMP-mCherry-amiR533).Tested by cell transfecting,
It was found that the transfection of the expression vector gently can be effectively reduced the intracellular NF- κ B work of NF- kB activity overactivities
Property, and the cell physiological phenotype for causing apoptosis of tumor cells after NF- kB activities suppress is produced, illustrate the validity of the carrier,
Thus a kind of new NF- kB inhibitors are demonstrated, has developed a kind of new NF- kB activities and has suppressed strategy.The new NF- of the present invention
κ B suppress carrier, and the intracellular NF- kB activities of NF- kB activity overactivities can be reduced with moderate inhibition, and to normal cell
Vigor has no significant effect, the shortcomings that overcoming current small molecule NF- kB inhibitors, avoids the excessive suppression to intracellular NF- kB activities
System, in order to avoid the NF- kB activities of normal cell are caused to suppress, so as to avoid its side effect, while in the expression vector
Inveigle son to compare and inveigle son with conventional linear, can more resist the degraded of intracellular nucleic acid enzyme, it inveigles son and enhancer difunctional
It is more longlasting.
The regulation and control method of the expression vector of the present invention is simple, quick, effective；And the expression vector can be used
In reagent or drug molecule is regulated and controled for the NF- kB activities of the closely related disease of gene therapy NF- κ B overactivities in preparation,
As a kind of new gene therapy agents, the treatment for inflammation, tumour is explored.
Brief description of the drawings
The carrier DMP-amiRNA that Fig. 1 is the present invention suppresses principle schematic；Wherein upper drawing shows inveigle son and siRNA
NF- κ B suppress approach (tradition suppress)；Suppressing approach bottom panel show DMP-amiRNA NF- κ B, (DMP-amiRNA presses down
System)；
Fig. 2 is plasmid vector collection of illustrative plates；Show the DNA element collection of illustrative plates of the 6 kinds of master plasmids carriers used in the present invention.
Fig. 3 is interference effects of the NF- κ B miRNA to reporter gene expression；From left to right：Light field, green channel are red
Passage and fused images, enlargement ratio：200×；
Fig. 4 is the assessment schematic diagram of NF- κ B specificity promoters；Wherein A and C represents to use pDMP-EGFP and pCMV-EGFP
The 293T cell images of transfection, enlargement ratio：200 ×, in A and C：Upper is light field；It is green fluorescence channel down；B and D represents thin
The flow cytometry graph of a relation of born of the same parents' fluorescence intensity；C is control in each figure；DE is 293T cell transfectings pDMP-EGFP；BDE is
The 293T cells for being pre-processed with BAY11-7082 and being transfected with pDMP-EGFP；CE is 293T cell transfectings pCMV-EGFP；BCE is
293T cells (*, the p for being pre-processed with BAY11-7082 and being transfected with pCMV-EGFP<0.05；*, p<0.01)；
Fig. 5 is the disturbing effect schematic diagram of NF- kB activity automatic control type plasmids；Wherein A represents the 293T cell images of transfection,
Enlargement ratio：200×；B represents the flow cytometry graph of a relation of transfectional cell；A is from top to bottom：Light field, green fluorescence channel,
Red fluorescence channel and green and red fusion image；C is control in each figure；R is pCMV-EGFP-RelA；D is pDMP-
MCherry-amiR533 and pCMV-EGFP-RelA；CR is pCMV-mCherry-amiR533 and pCMV-EGFP-RelA (*, p<
0.05；*, p<0.01)；
Fig. 6 is that pDMP-mCherry-amiR533 adjusts RelA and its target gene graph of a relation；Gene table is detected by qPCR
Reach, wherein C is control；T is TNF-α；DT is that pDMP-mCherry-amiR533 adds TNF-α；CT is pCMV-mCherry-
AmiR533 adds TNF-α (*, p<0.05；**,p<0.01)；
Fig. 7 is influence graphs of a relation of the pDMP-mCherry-amiR533 to cell viability；Flow cytometry cell withers
Die；Wherein A is 93T and HepG2 cells；B is HL7702 and HepG2 cells；Control：Turn by the use of elution buffer as blank control
Contaminate cell；BAY 11-7082：Cell is handled 1 hour with 50 μM of BAY 11-7082；PDMP-mCherry-amiR533 and
pCMV-mCherry-amiR533：It is thin with pDMP-mCherry-amiR533 and pCMV-mCherry-amiR533 transfections respectively
Born of the same parents.
Embodiment
The invention will be further described with accompanying drawing with reference to embodiments.
Embodiment 1
Target NF- κ B miRNA Effect Evaluations
Method：
Material and reagent：Carrier pIRES2-EGFP is purchased from Clontech companies, pcDNATM6.2-GW/EmGFP-miR-Neg
Purchased from Invitrogen companies, pEGFP-C1 Promega companies are purchased from purchased from Clontech companies, pGL4.10-MP.Plasmid p-
MCherry, p-RelA are provided by Southeast China University's bioscience and seminar of Wang Jin sections of engineering in medicine institute laboratory structure, wherein
Plasmid p-mCherry, p-RelA are merely provided for mCherry and RelA albumen coded sequences, and its sequence is the same as public gene data
Sequence in storehouse；Restriction enzyme NheI, EcoRI, NotI, BamHI, KpnI, HindIII, NcoI, XbaI are purchased from Takara
Company； HS(Premix)、rTaq、T4Polynucleotide Kinase、T-Vector pMD19
(Simple), DNA Markers are purchased from Takara companies；Restriction enzyme A seI and NheI, two type restriction endonuclease BsmBI,
T4DNA Ligase are purchased from NEB companies；LipofectamineTM2000, Opti-MEM culture medium are purchased from Invitrogen companies；
BAY 11-7082 (NF- kB inhibitors) are purchased from green skies company；Trizol reagents are purchased from Invitrogen companies；Coke acid two
Ethyl ester (diethylpyrocarbonate, DEPC) is purchased from Sigma companies；CDNA reverse transcription reagent box is purchased from Takara companies；
Fast Green Master Mix are purchased from ABI companies；The conventional chemical such as chloroform, isopropanol reagent is purchased from traditional Chinese medicines collection
Chemical reagent Co., Ltd of group；AnnexinV-FITC/PI cell apoptosis detection kits are purchased from Kai Ji companies；293T、HepG2
Cell is purchased from Shanghai life science institute of the Chinese Academy of Sciences.
The structure of NF- κ B miRNA expression vectors：Clone to obtain mCherry fragments using carrier p-mCherry, and at this
Section Sequences upstream introduces NheI, and downstream introduces EcoRI, mCherry fragments are connected on carrier pIRES2-EGFP, forms weight
Group carrier pCMV-mCherry.Utilize carrier pcDNATM6.2-GW/EmGFP-miR-Neg clone to obtain skeleton containing miR-155 and
TK polyadenylation signal fragment, amiR is named as, and EcoRI, downstream are introduced in this section of Sequences upstream
NotI is introduced, while two type endonuclease recognized site BsmBI linker is introduced in miR155 skeletons, amiR fragments are connected into
Into carrier pCMV-mCherry, general miRNA interference carriers pCMV-mCherry-amiR is formed.Simultaneously directly from carrier
pcDNATMAmplification obtains miR-Neg fragments on 6.2-GW/EmGFP-miR-Neg, and EcoRI is introduced in upstream, and downstream introduces
NotI, miR-Neg fragments are connected into carrier pCMV-mCherry, form control vector pCMV-mCherry-miR-Neg.
RelA/p65mRNA (NCBI BC014095) complete sequence is uploaded into BLOCK-iT TM RNAi Photographing On-lines
Software (https://rnaidesigner.thermofisher.com/rnaiexpress/) design miR-96 gene sequence, and
Homology analysis is carried out through BLAST sequence alignment programs, position and its suppression efficiency according to miRNA targetings, it is determined that and synthesizing
It is used for the single stranded DNA (ssDNA) for preparing targeting RelA miRNA expression vector for 3 couple of RelA genes, in table 1
Shown in SEQ ID NO.4-9.By 3 pairs of single stranded DNAs of base pair complementarity (amiR349-F and amiR349-R, amiR531-F with
AmiR531-R, amiR533-F and amiR533-R) it is denatured respectively, then anneal to form double-stranded DNA (dsDNA).Then will
DsDNA is connected with the linear pCMV-mCherry-amiR carriers cut with BsmBI.The three of the targeting RelA/p65 genes of structure
Individual miRNA expression vector is respectively designated as pCMV-mCherry-amiR349, pCMV-mCherry-amiR531, pCMV-
mCherry-amiR533.Verified by PCR augmentation detections and DNA sequencing.
NF- κ B RNAi report carriers are built：Clone to obtain RelA full length fragments using carrier p-RelA, and in this section of sequence
Arrange upstream and introduce EcoRI, downstream introduces BamHI, RelA fragments are connected on carrier pEGFP-C1, forms recombinant vector
PCMV-EGFP-RelA (Fig. 2).Pass through DNA sequencing checking carrier.
Cell culture：293T cells, which are used, contains 10% (v/v) FBS (HyClone), 100units/mL penicillin and 100g/
The DMEM of mL streptomysins is containing 5% (v/v) CO2Humidified incubator in 37 DEG C culture.By cell with 1 × 105Individual cell/cm2
Density be seeded in 24 orifice plates, be incubated 24 hours (degree of converging about 80%).Separated and used with EndoFree plasmid kits (CWBio)
In all carriers of transfection.Before transfection, culture medium is carefully taken out, cell is rinsed with 500 μ L PBS.Then, to each hole
500 μ LOPTI-MEM culture mediums of middle addition are simultaneously incubated 2 hours.By using the transfection reagent Lipofectamine based on lipid
Expression vector is transfected into cell by 2000 (Invitrogen).After 6 hours, mixture is removed, adds complete medium, is continued
Cell is cultivated to 24 hours.
Cell transfecting：By pCMV-mCherry-amiR349/amiR531/amiR53/ and pCMV-mCherry-miR-Neg
Plasmid of 4 kinds of plasmids respectively with report carrier pCMV-EGFP-RelA is mixed by 500ng and 250ng of mass number.Press
The operating instructions of Lipofectamine 2000, the above-mentioned plasmid mixed is distinguished into cotransfection into 293T cells.
Cell detection：Observe cultivating to 24h 293T cells to be put under inverted fluorescence microscope and take pictures, pass through fluorescence
Expression come preliminary assessment targeting RelA genes miRNA interference carriers interference effect, and filter out interference effect most
Good miRNA.
As a result：
Designed by miRNA, carrier restructuring and sequence verification, successfully construct three NF- κ B miRNA interference carriers, point
It is not pCMV-mCherry-amiR349, pCMV-mCherry-amiR531 and pCMV-mCherry-amiR533, a feminine gender
Control vector pCMV-mCherry-miR-Neg and a report carrier pCMV-EGFP-RelA (Fig. 2).In order to assess three kinds
The interference effect that miRNA is expressed RelA, respectively by three kinds of miRNA expression vectors and report carrier pCMV-EGFP-RelA corotation
Contaminate to 293T cells.Interference effect is judged by green caused by EGFP and red fluorescent protein (RFP) and red fluorescence intensity
Fruit.As a result show, red fluorescence intensity caused by three kinds of miRNA is basically identical, illustrates that three kinds of miRNA are similar in transfectional cell
Express (Fig. 3).However, green fluorescence intensity caused by three kinds of miRNA is significantly different.The green fluorescence of negative control group cell is most
By force, show that negative control miRNA does not influence reporter expression.By contrast, three kinds of targeting NF- κ B miRNA is disturbed
Reporter gene expression.Wherein, amiR533 causes most significant disturbing effect (Fig. 3).As a result show, the expression of RelA genes by
To these artificial miRNA interference, particularly amiR533.Therefore, these miRNA can be used for subsequent experiment.
Table 1 is used for the single stranded DNA (ssDNA) for preparing targeting RelA miRNA expression vector
Embodiment 2
The Effect Evaluation of NF- κ B specificity promoters
Method：
Vector construction：One section of 52-bp fragment containing NF- κ B classics binding sites on cloning vector pGL4.32 is simultaneously ordered
It is entitled to inveigle son, its sequence SEQ ID NO.10：5'-GGG AAT TTC CGG GGA CTT TCC GGG AAT TTC CGG
GGA CTT TCC GGG AAT TTC C-3', as NF- κ B specific regulatory elements.According to the sequences Design and synthesize
Two single stranded oligonucleotides, its sequence is as shown in the SEQ ID NO.11-12 of table 2.By two single strand oligonucleotide Acid denaturations and move back
Fire forms double-stranded DNA；The upstream and downstream of the double-stranded DNA introduces respectively can be with KpnI and HindIII digestion cohesive end sequences
Arrange the sequence of annealing.Then (MP, minimum open the linear pGL4.10-MP cut the double-stranded DNA and KpnI and HindIII double digestions
Mover) connect, recombinant vector is named as pGL4.10-DMP.Thus, the minimum promoter (sequence that pGL4.10-DMP contains
SEQ ID NO.13：5'-TAG AGG GTA TAT AAT GGA AGC TCG ACT TCC AG-3'), it, which is combined, inveigles sub- shape
Into NF- κ B specificity promoters.EGFP fragments are cloned by pEGFP-C1.NcoI and XbaI are introduced respectively with downstream at its upstream
Restriction enzyme site.The fragment is connected to structure NF- κ B specificity promoter carriers in pGL4.10-DMP, is named as pDMP-EGFP
(Fig. 2).Pass through PCR detections and DNA sequencing checking carrier.Carrier pEGFP-C1 is named as pCMV-EGFP (Fig. 2), so as to
PDMP-EGFP is compared.
Cell culture：293T cells, which are used, contains 10% (v/v) FBS (HyClone), 100units/mL penicillin and 100g/
The DMEM of mL streptomysins is containing 5% (v/v) CO2Humidified incubator in 37 DEG C culture.By cell with 1 × 105Individual cell/cm2
Density be seeded in 24 orifice plates, be incubated 24 hours (degree of converging about 80%).Separated and used with EndoFree plasmid kits (CWBio)
In all carriers of transfection.Before transfection, culture medium is carefully taken out, cell is rinsed with 500 μ L PBS.Then, to each hole
500 μ L OPTI-MEM culture mediums of middle addition are simultaneously incubated 2 hours.By using the transfection reagent Lipofectamine based on lipid
Expression vector is transfected into cell by 2000 (Invitrogen companies).After 6 hours, mixture is removed, adds complete medium,
Continue to cultivate cell 18h or 42h.
Cell transfecting：293T cells are handled 1 hour with 25 μM of BAY 11-7082 (BAY).Then, by pDMP-EGFP and
PCMV-EGFP is transfected into the 293T cells through processing, is cultivated 24 hours respectively.
Cell detection：The cell of transfection is imaged by fluorescence microscope, then with 10mg/mL trypsase/
EDTA solution digestions, carry out flow cytometry.
As a result：
NF- κ B trick subsequences are connected on minimal promoter so as to build NF- κ B specificity promoters, are named as
DMP promoters.Then the expression EGFP of DMP regulation and control report carrier pDMP-EGFP is constructed, the transcription for assessing DMP is lived
Property.The report carrier pCMV-EGFP that the present embodiment also constructs the expression EGFP under people CMV MIEP regulation and control is compared.Respectively
This two carriers are transfected to 293T cells.As a result show, expression of the DMP successes induction of reporter gene；However, its transcription is lived
Property is far below strong promoter MIEP (Fig. 4).
In order to verify DMP NF- κ B specificity, transfected respectively with NF- kB inhibitors with pDMP-EGFP and pCMV-EGFP
The 293T cells of BAY11-7082 pretreatments.As a result finding BAY11-7082 processing causes in the cell of two carriers of transfection
Reporter lowers (Fig. 4).However, in transfection has pDMP-EGFP cell, EGFP expression is reduced to 70.1% (p<
0.05) cell for, transfecting pCMV-EGFP is then 45.8% (p<0.01).As a result show, compared to CMV promoter, DMP is to NF-
κ B are more specific, indicate DMP NF- κ B specificity.
The NF- κ B of table 2 inveigle subsequence
Embodiment 3
The Effect Evaluation of NF- κ B automatic control type miRNA expression vectors
Method：
Vector construction：DMP fragments are cloned by carrier pDMP-EGFP, insert AseI and NheI in upstream and downstream respectively
Restriction enzyme site.DMP fragments are connected in the recombinant vector pCMV-mCherry-amiR533 for removing CMV promoter, build NF-
κ B automatic control type miRNA expression vectors, are named as pDMP-mCherry-amiR533 (Fig. 2).Entered by PCR amplifications and gene sequencing
Row carrier is verified.Meanwhile pCMV-mCherry-amiR533 is also constructed as control (Fig. 2), verified and carried by DNA sequencing
Body.
Cell culture：293T cells with contain 10% (v/v) FBS (HyClone companies), 100units/mL penicillin with
The DMEM of 100g/mL streptomysins is containing 5% (v/v) CO2Humidified incubator in 37 DEG C culture.By cell with 1 × 105It is individual thin
Born of the same parents/cm2Density be seeded in 24 orifice plates, be incubated 24 hours (degree of converging about 80%).With EndoFree plasmid kits (CWBio)
Separate all carriers for transfection.Before transfection, culture medium is carefully taken out, cell is rinsed with 500 μ L PBS.Then, to
500 μ L OPTI-MEM culture mediums are added in each hole and are incubated 2 hours.By using the transfection reagent based on lipid
Expression vector is transfected into cell by Lipofectamine 2000 (Invitrogen).After 6 hours, mixture is removed, is added
Complete medium, continue to cultivate cell 48h.
Cell transfecting：With pCMV-EGFP-RelA and pDMP-mCherry-amiR533 or pCMV-mCherry-amiR533
Cotransfection 293T cells, and be incubated 24 hours.PCMV-EGFP-RelA and elution buffer are respectively as positive and blank control.
Cell detection：Disturbing effect is assessed by double fluorescence imagings and flow cytometry.
As a result：
In order to build NF- κ B automatic control type miRNA expression vectors, by the NF- κ B specificity promoters (DMP) of above-mentioned checking and
Targeting NF- κ B miRNA (amiR533) reassemble into new plasmid vector pDMP-mCherry-amiR533.Using the carrier,
The expection of amiR533 expressions is by intracellular NF- κ B autogenous controls.AmiR533 expressions are by depending on intracellular NF- κ B
Activity.Similar pCMV-mCherry-amiR533 is built as control vector.In order to assess NF- κ B automatic control types miRNA expression
The disturbing effect of carrier, respectively with pDMP-mCherry-amiR533, pCMV-mCherry-amiR533 and pCMV-EGFP-
RelA cotransfection 293T cells.As a result show, pCMV-mCherry-amiR533 causes amiR533 expression to be higher than pDMP-
mCherry-amiR533.Correspondingly, pCMV-mCherry-amiR533 compares pDMP- to the inhibition of reporter gene expression
MCherry-amiR533 more significant (Fig. 5 A).
Flow cytometry shows that pDMP-mCherry-amiR533 transfections only result in significantly reducing for fluorecyte quantity
(p<0.01), but the relative intensity of fluorescence of cell does not have significant decrease.By contrast, pCMV-mCherry-amiR533 is transfected
The quantity of fluorecyte and relative intensity of fluorescence is caused to significantly reduce (Fig. 5 B).As shown by data pDMP-mCherry-amiR533
Regulation activity it is much gentleer than pCMV-mCherry-amiR533, imply that pDMP-mCherry-amiR533 can be used for adjust
NF- κ B intracellular reactive.Data are also shown that the strong promoter as mankind CMV MIEP cannot be used for NF- κ B targets
Expression to miRNA because itself intrinsic high transcriptional activity will necessarily extra-inhibitory NF- kB activities, it is important so as to damage
The normal physiological function of transcription factor.Simultaneously pDMP-mCherry-amiR349, pDMP-mCherry-amiR531 result with
PCMV-mCherry-amiR533 is similar, can be used for gentle regulation NF- κ B intracellular reactive.
Embodiment 4
RelA and its target gene are regulated and controled by NF- κ B automatic control types miRNA expression vector
Method：
Cell culture：HepG2 cells with contain 10% (v/v) FBS (HyClone), 100units/mL penicillin with
The DMEM of 100g/mL streptomysins is containing 5% (v/v) CO2Humidified incubator in 37 DEG C culture.By cell with 1 × 105It is individual thin
Born of the same parents/cm2Density be seeded in 6 orifice plates, be incubated 24 hours (degree of converging about 80%).With EndoFree plasmid kits (CWBio)
Separate all carriers for transfection.Before transfection, culture medium is carefully taken out, cell is rinsed with 500 μ L PBS.Then, to
500 μ L OPTI-MEM culture mediums are added in each hole and are incubated 2 hours.By using the transfection reagent based on lipid
Expression vector is transfected into cell by Lipofectamine 2000 (Invitrogen).After 6 hours, mixture is removed, is added
Complete medium, continue to cultivate cell 48h.
Cell transfecting：Transfected respectively with 4000ng pDMP-mCherry-amiR533 or pCMV-mCherry-amiR533
To the HepG2 cells in 6 orifice plates, elution buffer is blank control.After cultivating 48h, cell stimulates 1 with TNF-α (10ng/mL)
Hour.
Gene expression detection：Cell is collected, total serum IgE, reverse transcription synthesis complementary DNA (cDNA) are extracted with Trizol.Pass through
QPCR quantitative analyses RelA and its target gene, including BCL3, NFKB1, CD54, NFKBIA, CXCL1, PTGS2, CCL2, NFKB2
With MMP9 expression.Primer up and down for qPCR refers to table 3 as shown in SEQ ID NO.14-35.
As a result：
, will in order to verify whether pDMP-mCherry-amiR533 can adjust the expression of endogenous NF- κ B and its target gene
PDMP-mCherry-amiR533, pCMV-mCherry-amiR533 distinguish transfection HepG 2 cell.It is intracellular in order to change
NF- kB activities, with TNF-α, a kind of known NF- κ B stimulants, stimulate cell.Pass through qPCR quantitative analyses RelA and some
The expression of typical target gene.As a result show, expression (Fig. 6) of the TNF-α success induction of RelA and its target gene.Two kinds of amiRNA
Expression vector inhibits the induced expression of RelA and its target gene (Fig. 6) to varying degrees.However, and pDMP-mCherry-
AmiR533 (Fig. 6) is compared, in the cell of pCMV-mCherry-amiR533 transfections, the induced expression of RelA and its target gene by
To more significant suppress, show pDMP-mCherry-amiR533 to RelA and its regulation milder of target gene.PDMP- simultaneously
mCherry-amiR349、
PDMP-mCherry-amiR531 result is similar with pCMV-mCherry-amiR533, to RelA and its target gene
Regulation it is also gentle.
Table 3 detects the qPCR primers of NF- κ B and its expression of target gene
Embodiment 5
Influence of the automatic control type expression vector to cell viability
Method：
Cell culture：293T, HepG2 and HL7702 cell, which are used, contains 10% (v/v) FBS (HyClone companies),
The DMEM of 100units/mL penicillin and 100g/mL streptomysins is containing 5% (v/v) CO2Humidified incubator in 37 DEG C training
Support.By cell with 1 × 105Individual cell/cm2Density be seeded in 24 orifice plates or 6 orifice plates, be incubated 24 hours (degree of converging about 80%).
All carriers for transfection are separated with EndoFree plasmid kits (CWBio).Before transfection, culture medium is carefully taken out,
Cell is rinsed with 500 μ L PBS.Then, 500 μ L OPTI-MEM culture mediums are added into each hole and are incubated 2 hours.By making
Expression vector is transfected into cell with the transfection reagent Lipofectamine 2000 (Invitrogen) based on lipid.6 is small
Shi Hou, mixture is removed, add complete medium, continue to cultivate cell 18h or 42h.
Cell transfecting：Transfected respectively with 800ng pDMP-mCherry-amiR533 or pCMV-mCherry-amiR533
293T, HepG2 and HL7702 cell.Positive control is used as by the use of 50 μM of BAY processing cells within 1 hour.
Cell detection：By all cell dissociations, collection, pass through fluidic cell (ACEA NovoCyte companies) AnnexinV-
FITC/PI binary channels is detected.
As a result：
NF- κ B play Anti-G value frequently by the target gene for suppressing Apoptosis.Therefore, joined by fluidic cell film
Albumen V-FITC/PI binary channels have evaluated influences of the pDMP-mCherry-amiR533 to Apoptosis.At BAY 11-7082
The cell of reason makees positive control.As a result show, pDMP-mCherry-amiR533 causes the early stage (right lower quadrant) of two cells
The total cell apoptosis rate represented with late period (right upper quadrant) apoptotic cell is minimum (Fig. 7 A).Carrier pCMV-mCherry-amiR533
Higher apoptosis rate is result in respect to pDMP-mCherry-amiR533.BAY 11-7082 are common as suppression NF- kB activities
Reagent, cause highest apoptosis rate, significantly larger than two miR533 expression vectors (Fig. 7 A).It is obvious that small-molecule chemical suppresses
Agent causes high apoptosis by strong extra-inhibitory NF- kB activities.Apoptosis caused by NF- κ B miRNA interference is much smaller than small point
Sub- chemical inhibitor.The NF- κ B miRNA interference of strong promoter (MIEP) regulation and control causes than by NF- κ B specificity promoters
(DMP) the higher apoptosis of the interference of regulation and control.PDMP-amiR533 NF- κ B miRNA disturb the influence to cell viability minimum.
Apoptosis assay also shows that miRNA interference causes 293T Apoptosis to be far below HepG2 cells.Reason is that 293T is that gene changes
Good Human embryonic kidney cells, and HepG2 is human liver cancer cell, the former has the NF- kB activity lower than the latter.
Finally, pDMP-mCherry-amiR533 is further have evaluated to normal person's hepatic cell line HL7702 Apoptosis
Influence.Negative controls (38-40) of the HL7702 as NF- κ B overactivities, is contrasted with human hepatocarcinoma cells HepG2.Together
Sample, two kinds of cell lines are handled with pCMV-mCherry-amiR533, pDMP-mCherry-amiR533 and BAY 11-7082,
Apoptosis is analyzed by Annexin V-FITC/PI two-channel flow cytometrics.As a result show, pDMP-mCherry-
AmiR533 does not induce HL7702 Apoptosis (Fig. 7 B).However, pCMV-mCherry-amiR533 induction HL7702 cells are notable
Apoptosis.Importantly, BAY 11-7082 are also induction of the notable apoptosis (Fig. 7 B) of HL7702 cells.These as shown by data,
PDMP-mCherry-amiR533 tool NF- κ B specificity, and chemically NF- kB inhibitors BAY 11-7082 and strong promoter
MIEP is not that complete NF- κ B are specific, it is thus possible to causes the side effect in applying.PDMP-mCherry-amiR533 is aligned
Ordinary person liver cell HL7702 activity means that this new NF- kB inhibitors can eliminate NF- κ B excessive activations without influence
NF- κ B overactivity in cell (such as cancer or inflammatory cell), but not to normal affecting cells.PDMP- simultaneously
MCherry-amiR349/amiR531 result is similar with pCMV-mCherry-amiR533, can eliminate NF- κ B excessive activations
Cell in NF- κ B overactivity, but not to normal affecting cells.
SEQUENCE LISTING
<110>Southeast China University
<120>The expression vector of NF- kB activities and its regulation and control methods and applications in a kind of regulating cell
<130> 2017
<160> 35
<170> PatentIn version 3.3
<210> 1
<211> 21
<212> DNA
<213>It is artificial synthesized
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<400> 3
caaagatggg atgagaaagg a 21
<210> 4
<211> 64
<212> DNA
<213>It is artificial synthesized
<400> 4
tgctgcttct tcacacactg gattccgttt tggccactga ctgacggaat ccagtgtgaa 60
gaag 64
<210> 5
<211> 64
<212> DNA
<213>It is artificial synthesized
<400> 5
cctgcttctt cacactggat tccgtcagtc agtggccaaa acggaatcca gtgtgtgaag 60
aagc 64
<210> 6
<211> 64
<212> DNA
<213>It is artificial synthesized
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tgctgaagat gggatgagaa aggacagttt tggccactga ctgactgtcc tttcatccca 60
tctt 64
<210> 7
<211> 64
<212> DNA
<213>It is artificial synthesized
<400> 7
cctgaagatg ggatgaaagg acagtcagtc agtggccaaa actgtccttt ctcatcccat 60
cttc 64
<210> 8
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<212> DNA
<213>It is artificial synthesized
<400> 8
tgctgcaaag atgggatgag aaaggagttt tggccactga ctgactcctt tcttcccatc 60
tttg 64
<210> 9
<211> 64
<212> DNA
<213>It is artificial synthesized
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cctgcaaaga tgggaagaaa ggagtcagtc agtggccaaa actcctttct catcccatct 60
ttgc 64
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<212> DNA
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gggaatttcc ggggactttc cgggaatttc cggggacttt ccgggaattt cc 52
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cgggaatttc cggggacttt ccgggaattt ccggggactt tccgggaatt tcca 54
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<213>It is artificial synthesized
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agcttggaaa ttcccggaaa gtccccggaa attcccggaa agtccccgga aattcccggt 60
ac 62
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<211> 32
<212> DNA
<213>It is artificial synthesized
<400> 13
tagagggtat ataatggaag ctcgacttcc ag 32
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<213>It is artificial synthesized
<400> 14
cctggagcag gctatcagtc 20
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<211> 20
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<213>It is artificial synthesized
<400> 15
atgggatgag aaaggacagg 20
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<212> DNA
<213>It is artificial synthesized
<400> 16
ttcctctggt gaacctgcct ac 22
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<212> DNA
<213>It is artificial synthesized
<400> 17
cgtgtctccg tcctcatctg c 21
<210> 18
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 18
gtgcagagga aacgtcagaa 20
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<211> 20
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<213>It is artificial synthesized
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gtgggaagct ataccctgga 20
<210> 20
<211> 17
<212> DNA
<213>It is artificial synthesized
<400> 20
aggtgacgct gaatggg 17
<210> 21
<211> 16
<212> DNA
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<400> 21
gctcgggcaa tgggtt 16
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<400> 22
ctccgagact ttcgaggaaa tac 23
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<213>It is artificial synthesized
<400> 23
gccattgtag ttggtagcct tca 23
<210> 24
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<213>It is artificial synthesized
<400> 24
cactgctgct cctgctcct 19
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<213>It is artificial synthesized
<400> 25
ggctatgact tcggtttgg 19
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<213>It is artificial synthesized
<400> 26
cgaggtgtat gtatgagtgt 20
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agtgggtaag tatgtagtgc 20
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<400> 29
acacttgctg ctggtgattc t 21
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<400> 30
gccgaaagac ctatccca 18
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<400> 31
agccgctgcc tctgaagt 18
<210> 32
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<213>It is artificial synthesized
<400> 32
aggacggcaa tgctgatg 18
<210> 33
<211> 18
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<213>It is artificial synthesized
<400> 33
tcgtagttgg cggtggtg 18
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<213>It is artificial synthesized
<400> 34
atttggtcgt attgggcg 18
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ctcgctcctg gaagatgg 18
Claims (12)
1. the expression vector of NF- kB activities in a kind of regulating cell, it is characterised in that include two sequential elements, regulation and control
The promoter sequence and promoter downstream miRNA coded sequences of gene expression；The promoter sequence inveigles son by one section of NF- κ B
Sequence and minimal promoter sequence composition；The miRNA coded sequences are the miRNA that one section of codified targets NF- κ B mRNAs
Coded sequence.
2. expression vector according to claim 1, it is characterised in that it is various that the NF- κ B inveigle subsequence to include
The NF- κ B of sequence inveigle son, are one section of DNA sequence dna that can be specifically bound with NF- kB proteins, its main sequence be characterized as containing
The different various NF- κ B combination target spots of quantity.
3. expression vector according to claim 1, it is characterised in that the minimal promoter comes including various genes
The minimal promoter sequence in source.
4. expression vector according to claim 1, it is characterised in that the NF- κ B mRNAs are RelAmRNA.
5. expression vector according to claim 1, it is characterised in that the miRNA of the targeting NF- κ B mRNA is
Hybridized by nucleic acid and combined with NF- κ B mRNA, the miRNA that NF- κ B mRNA degrade or translation prevents can be caused.
6. expression vector according to claim 1, it is characterised in that the miRNA of the targeting NF- κ B mRNAs,
Including natural miRNA and engineer miRNA；The engineer miRNA includes amiR349, amiR531 or amiR533, its
Coded sequence is respectively SEQ ID NO.1：5 '-CTT CTT CAC ACA CTG GAT TCC-3 ', SEQ ID NO.2：5′-
AAG ATG GGA TGA GAA AGG ACA-3 ', SEQ ID NO.3：5′-CAA AGA TGG GAT GAG AAA GGA-3′.
7. expression vector according to claim 1, it is characterised in that the expression vector is a linear or ring
Shape nucleic acid molecules.
8. expression vector according to claim 7, it is characterised in that the nucleic acid molecules be DNA or
Ribonucleic acid molecule, including double-stranded DNA, single stranded DNA or single strand RNA molecule.
9. expression vector according to claim 7, it is characterised in that the linear nucleic acid molecule includes normal linear
DNA molecular, viral DNA molecules or viral RNA molecules；The circular nucleic acid molecules include DNA.
10. a kind of method of NF- kB activities in regulating cell, it is characterised in that import the gene described in claim 1 to cell
Expression vector.
11. according to the method for claim 10, it is characterised in that the cell introduction method includes various types of nucleic acid
Cell introduction method.
12. the expression vector of NF- kB activities is close for gene therapy NF- κ B overactivities in preparation in a kind of regulating cell
Application in the NF- kB activities regulation and control reagent or drug molecule of cut related disorders.
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| CN108220336A (en) * | 2017-12-14 | 2018-06-29 | 东南大学 | Based on intracellular NF- kB activities activation effect gene in NF- κ B overactivities intracellular gene expression and application |
| CN108410893A (en) * | 2018-02-27 | 2018-08-17 | 东南大学 | A kind of tumour cell specific response expression vector started by NF- κ B and its expression product and application |
| WO2019047962A1 (en) * | 2017-09-11 | 2019-03-14 | 东南大学 | GENE EXPRESSION CARRIER FOR REGULATING NF-κB ACTIVITY IN CELL AND REGULATION METHOD THEREFOR AND APPLICATION THEREOF |
| CN111249477A (en) * | 2020-03-12 | 2020-06-09 | 东南大学 | Composition for killing cancer cells based on gene interference vector and iron nanoparticles and application thereof |
| CN111304249A (en) * | 2020-02-17 | 2020-06-19 | 东南大学 | Novel CRISPR Cas13a-gRNA expression vector and application thereof |
| CN115161289A (en) * | 2022-03-14 | 2022-10-11 | 东南大学 | Recombinant adeno-associated virus for treating inflammatory diseases and construction method and application thereof |
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| CN107365785B (en) | 2020-02-18 |
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