CN108998451A - MiRNA-340 target gene binding sequence, recombinant plasmid and its application - Google Patents
MiRNA-340 target gene binding sequence, recombinant plasmid and its application Download PDFInfo
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Abstract
The present invention relates to molecular biology and gene expression regulation technical field, more particularly to a kind of miRNA-340 target gene binding sequence, recombinant plasmid and its application, which includes: nucleotide sequence shown in SEQIDNO:1, SEQIDNO:4, SEQIDNO:5, SEQIDNO:6, SEQIDNO:7 or SEQIDNO:19.MiRNA-340 target gene binding sequence of the invention can be in conjunction with miRNA-340, and the recombinant plasmid constructed can quickly, sensitively, and conveniently identify whether target microRNA combines 3 ' UTR regulation and control fragment of IL-17A to play regulating and controlling effect.
Description
Technical field
The present invention relates to molecular biology and gene expression regulation technical field, and in particular to a kind of miRNA-340 target base
Because of binding sequence, recombinant plasmid and its application.
Background technique
Microrna (microRNA, miRNA) is a kind of single-stranded microRNA for only having 18~25 nucleotide, is deposited extensively
It is in nematode, drosophila, plant and eucaryote including people, conservative, tissue specificity and timing with height
Property non-coding RNA molecule, do not have open reading frame (open reading frame).The gene of microRNA is located at heredity
In the introne of encoding gene, also some exists and in intergenic DNA sequences, is formed to play after mature microRNA and adjust
It is acted on after control transcription.It can be by holding non-coding region (3 ' UTR) complementary pairing with the 3 ' of target gene, in post-transcriptional level to target base
Because carrying out negative regulation, lead to mRNA Translational repression or degradation, in terms of embryonic development, cell differentiation, apoptosis, tumour
Play diversification regulating and controlling effect.
Existing research report, miRNA-340 play the role of in the pathogenesis of tumour it is very important, and in inhomogeneity
In the tumor research of type, carcinogenic or suppression cancer adjustment effect is played, includes p-AKT, EZH2, table by inhibiting related oncogene
Skin growth factor receptor etc., or liver cancer, lung cancer, colon cancer, gastric cancer are inhibited by targeting related gene, the performances such as breast cancer press down cancer
Disease effect.Meanwhile miRNA-340 may also adjust the differentiation and function of immunocyte.Studies have found that miRNA-340 can lead to
The gene targets such as downward IL-4, Bmi-1 are crossed to inhibit Th2 cell differentiation, are made in experimental allergic encephalomyelitis (EAE)
Th2 cell to Th1 cell subsets deviate.It further appreciates that, miRNA-340 inhibits Th2 access to can promote Th1, Th17 cell
Differentiation, but whether be directly targeted IL-17A influence Th17 cell, not yet studies have reported that.Clinically multiple sclerosis is according to disease
Cheng Tedian is divided into relapsing-remitting type, secondary Advancement Type and primary Advancement Type, studies have found that, from preceding 2 kinds of parting peripheral blood in patients
Middle isolated memory t cell height expresses miRNA-340, but expression has no significant change in primary Advancement Type patient, very
There is reduction phenomenon in activity hardening spot；Simultaneously be overexpressed miRNA-340, miRNA-27 in myelin specific T-cells,
MiRNA-128 can aggravate EAE in mice phenotype.
According to miRBase database, the miRNAs found at present is more than 700, with the application of high-flux sequence,
More new miRNAs will be had to be found.Possible nearly 90% human gene is regulated and controled by miRNAs, however, when being overexpressed
Or when inhibiting some miRNA, is found in numerous genes that modulation occurs and identify the target gene wherein to play a crucial role
Still there is sizable challenge.The common strategy of identification miRNA target gene is predicted using bioinformatics software at present, main
It will be according to the regularity to interact between certified miRNA and its target-gene sequence, it then follows several common principle designs
Software is completed, such as miRanda, TargetScan and TargetScanS, RNAhybrid, DIANA-microT, PicTar, and
FindTar etc..In addition, finding miRNA target gene using proteomic image also becomes a kind of new approach.
Since computer simulation has some limitations when predicting miRNA target gene, biological experimental method is utilized
MiRNA target gene can more intuitively be found.Presently mainly target gene is found from mRNA level in-site and protein level.From
MRNA level in-site, which is mainly led to finding target gene, after being overexpressed miRNA in the cell, utilizes the change of gene microarray analysis mRNA
Change to find out the target gene of corresponding miRNA.Translational repression process does not cause after the transcription that this method is mediated by miRNA
The change of mRNA level in-site, therefore the method that miRNA target gene is found in the variation according to mRNA level in-site exists centainly in recall rate
Problem.
Identification target gene most straightforward approach is examined respectively using quantitative fluorescent PCR and Western blot method at present
The variation for surveying mRNA level in-site and protein level in cell after transfecting or striking low miRNA, so that it is determined that miRNA is corresponding with target gene
Relationship.This method can greatly improve accuracy rate, but finally determine target gene, it is also necessary to identify the target site of miRNA.And
The most common method of the identification of the target site of miRNA is chemical-activated luciferase gene expression, the basic principle is that constructing fluorescence first
Plain expression of enzymes carrier will wish that 3 ' UTR of the miRNA target gene identified are building up in 3 ' UTR of luciferase gene, then will
Luciferase gene expression vector transfection cell and the expression for changing corresponding miRNA in cell, finally detect luciferase
Expression with analyze in 3 ' UTR of transfection whether the target site containing miRNA.
17 type helper T lymphocytes (Th17) are the differentiation hypotypes of CD4+T cell, in the morbidity of auto-immune related disease
Important role is occupied in mechanism.Interleukin-17 A (IL-17A) is the major cytokine of Th17 secretion, in inflammatory reaction
Migration and activation to leucocyte etc. play a significant role.Psoriasis is to influence most commonly used 3 big autoimmunity disease
One of, symptom shows as pachyderma and popularity damage, can cause itch, the scales of skin that peel off and pain, and seriously affect the life of patient
Bioplasm amount, mental health and social relationships.In addition, illness is susceptible to suffer from complication compared with severe one, such as arthritis, heart disease and glycosuria
Disease has mortality risk.Currently, Leslie van der Fits et al. has confirmed autoimmune psoriasis mainly by IL-
23/IL-17A inflammation axis mediates, and pathologic process is main are as follows: pathogenic factor (such as heredity, environment, infection and physical damnification)
Cause the proinflammatory such as congenital immunity cell (such as horn cell, natural killer cells) TNF secretion-α, IL-1 β and IL-6 because
Son, the innate immune cells such as activation Dendritic Cells.The Dendritic Cells Migration of activation into immune organs such as skin lymph nodes,
Offer antigen and secrete the proinflammatory cytokines such as IL-23, naive CD4+T cell is promoted to break up to Th17.The autoreactivity of differentiation
Property Th17 migrated out from the capillary of skin, infiltrate inflammation part into skin.Obtaining stimulating again for autoantigen
Afterwards, a variety of inflammatory factors such as continue to multiply and secrete IL-17A.The factors such as IL-17A can activate horn cell, stimulate its proliferation,
Form silver bits symptom.On the other hand, the horn cell of activation can secrete again antibacterial peptide (such as LL-37 antibacterial peptide and beta-alexin),
Proinflammatory cytokines (TNF-α, IL-1 β and IL-6), chemotactic factor (CF) (CXCL8-11, CCL20) and S100 albumen etc., these factors
Congenital immunity cell can be activated again, leads to inflammation vicious circle, to maintain and aggravate psoriasis development.As it can be seen that IL-17A
Important bridge linking effect is play in the inflammation circuit of psoriasis congenital immunity and adaptive immunity.Certainly, IL-23/IL-
17A inflammation axis is only a certain important link during psoriasis pathology.Recent studies have found that gamma delta T cells and macrophage
Cell can also secrete IL-17A and promote the occurrence and development of psoriasis, and Th22 cell can secrete IL-22 and aggravate the hair of psoriasis
Exhibition, remaining pathology link need to be found.
Studies have found that IL-17 expression increases in patient with rheumatoid arthritis synovium of joint liquid and peripheral blood, IL-17
Secretion keratinocyte growth factor, hepatocyte growth factor and heparin-binding epidermal can be expressed by stimulation synovial cell
The Angiogenesis such as growth factor promote the formation of pannus, and destruction is played early stage rheumatoid arthritis；Pass through
Synovial cell and chondrocytes expressed metalloproteinases are induced, proteoglycan molecular breakdown is made, destroys cartilaginous tissue；It stimulates osteoclastic
The generation of cell plays an important role the activation of osteoclast and the absorption aspects of sclerotin in destruction of bone reaction.
In addition, finding IL-17 table in the research of the immune related diseases such as multiple sclerosis, systemic loupus erythematosus
Refer to up to exception, and using the correlation of IL-17mRNA or expressing quantity and disease light and heavy degree as the monitoring of diagnosing and treating
Mark.
Currently, glucocorticoid, immunosuppressor etc. that the treatment of autoimmune disease generallys use, although having one
Constant current modulation effect, but long-time service can all generate serious adverse reaction, and can only all slow down the development of the state of an illness, can not eradicate disease
Disease.Nearly more than 10 year, mab treatment autoimmune disease have become research hotspot, major positive active development of pharmaceuticals
Monoclonal antibody medicine with IL-23, IL-17 or IL-17 receptor etc. for target spot is used to treat psoriasis, and wherein Novartis Co., Ltd researches and develops
Secukinumab obtains European Union's approval at 2015 beginning of the years, is the first IL-17 monoclonal antibody in the whole world.For security consideration, mild or moderate is suffered from
Person cannot enjoy these monoclonal antibody medicines in a short time.
Therefore, 3 ' UTR negative regulation segment of IL-17A is specified, and constructs a kind of recombinant vector, microRNA- can be verified
The binding site sequence of 340-5p, the agonist for being directed to miR-340 for Future Development are of great significance, and will promote miR-
340 application in the diagnosis, treatment of autoimmune disease.
Summary of the invention
It is an object of the invention in view of the above drawbacks of the prior art, provide a kind of miRNA-340 target gene combination sequence
Column, recombinant plasmid and its application.
The purpose of the present invention can be realized by technical measures below:
The present invention provides a kind of miRNA-340 target gene binding sequences, which includes: SEQIDNO:1, SEQIDNO:
4, nucleotide sequence shown in SEQIDNO:5, SEQIDNO:6, SEQIDNO:7 or SEQIDNO:19.
The present invention also provides a kind of recombinant plasmid, which includes above-mentioned miRNA-340 target gene combination sequence
Column.
Preferably, the recombinant plasmid from 5 ' end to 3 ' end successively include following element: promoter sequence, reporter gene,
The miRNA-340 target gene binding sequence and termination sequence.
Preferably, which includes luciferase reporter plasmid pmir-GLO and the miRNA-340 target base
Because of binding sequence, the miRNA-340 target gene binding sequence is located at the NheI digestion of the luciferase reporter plasmid
Between site and XhoI restriction enzyme site.
The present invention also provides a kind of host cell, which contains above-mentioned recombinant plasmid.
The present invention also provides a kind of screening or the methods of identification IL-17 negative regulation preparation, and this method comprises the following steps:
Construct above-mentioned recombinant plasmid；
Make test agent and the recombinant plasmid cotransfection eukaryocyte；
Eukaryocyte after culture transfection, the uciferase activity of the eukaryocyte secretion after detection transfection；
Wherein, if uciferase activity reduces, the test agent is accredited as IL-17 negative regulation preparation.
Preferably, the eukaryocyte is yeast cells, insect cell, plant cell, zooblast or people's cell.
The present invention also provides a kind of construction methods of above-mentioned recombinant plasmid, and this method comprises the following steps:
Using mouse gene group DNA as template, with nucleotide sequence shown in (i) SEQ ID No.2 and SEQ ID No.3 or
(ii) nucleotide sequence shown in SEQ ID No.9 and SEQ ID No.10 or (iii) SEQ ID No.11 and SEQ ID No.12
Nucleotide sequence shown in shown nucleotide sequence or (iv) SEQ ID No.13 and SEQ ID No.14 or (v) SEQ ID No.15
It is classified as primer with nucleotides sequence shown in SEQ ID No.16, carries out PCR amplification；
Sequence obtained by PCR amplification is connected to the multiple cloning sites of luciferase reporter plasmid pmir-GLO.
The present invention also provides above-mentioned miRNA-340 target gene binding sequences or above-mentioned recombinant plasmid to detect
MiRNA-340 is to the active application of IL-17A gene regulation.
The present invention also provides above-mentioned miRNA-340 target gene binding sequences or above-mentioned recombinant plasmid to study
MiRNA-340 is to the application in candidate targets IL-17A biological function or regulatory mechanism.
The present invention also provides above-mentioned miRNA-340 target gene binding sequences or above-mentioned recombinant plasmid in target gene is
Application in the miRNA-340 functional study of IL-17A.
MiRNA-340 target gene binding sequence of the invention can be in conjunction with miRNA-340, the recombinant plasmid energy of building
It is enough that quickly, sensitively, and conveniently whether identification target microRNA combines 3 ' UTR regulation and control fragment of IL-17A to play regulating and controlling effect；
MiRNA-340 target gene binding sequence of the invention and recombinant plasmid can be applied to detection miRNA-340 to IL-17A gene
In regulating and controlling effect, research miRNA-340 can also be applied to in candidate targets IL-17A biological function or regulatory mechanism,
And applied to target gene in the miRNA-340 functional study of IL-17A.
Detailed description of the invention
Fig. 1 is pmir-GLO plasmid construct figure used in the embodiment of the present invention.
Fig. 2 is the complete segment report recombinant plasmid of 3 ' UTR and empty control plasmid of IL-17A gene in the embodiment of the present invention
Uciferase activity comparison diagram.
Fig. 3 is the fluorescein of 3 ' the UTR fragment deletions report recombinant plasmid of each IL-17A gene in the embodiment of the present invention
Enzymatic activity comparison diagram.
Fig. 4 is 3 ' UTR nucleotide alignments' analysis charts of IL-17A gene between different plant species.
Fig. 5 is 3 ' UTR negative regulation segments of difference miRNA and IL-17A gene report recombinant plasmid in the embodiment of the present invention
The uciferase activity comparison diagram of cotransfection.
Fig. 6 is that miRNA-340 is mutated from 3 ' the UTR segments or binding site of different IL-17A genes in the embodiment of the present invention
The uciferase activity comparison diagram of segment report recombinant plasmid cotransfection.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawing and specific implementation
Invention is further described in detail for example.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention,
It is not intended to limit the present invention.
The present invention is by 3 ' UTR nucleic acid sequence of Cloning of mouse IL-17A or its segment, the 3 ' UTR of IL-17A that will be cloned into
Nucleic acid sequence or its segment build up eukaryotic gene expression vector in vitro, and reporter gene imports eukaryocyte, obtain energy
It is enough to realize to 3 ' the UTR nucleic acid sequence of IL-17A of reporter gene expression negative regulation or its segment, and further specify the IL-
The negative regulation segment of 3 ' UTR nucleic acid sequence of 17A.3 ' UTR nucleic acid sequence of IL-17A or its segment are built up into eukaryon base in vitro
Because of the miRNA-340 cotransfection eukaryocyte of expression vector and overexpression, the experimental results showed that, miRNA-340 can be with IL-
3 ' UTR nucleic acid sequence of 17A or its segment combine, i.e., 3 ' UTR nucleic acid sequence of IL-17A or its segment are miRNA-340 target gene
Binding sequence, IL-17A gene are the target genes of miRNA-340, and miRNA-340 targets and inhibit the expression of IL-17A gene, right
IL-17A gene plays the role of negative regulation.
Specifically, 3 ' the UTR nucleic acid sequence of mouse IL-17A cloned in embodiments of the present invention or its segment difference
For nucleotide sequence shown in SEQIDNO:1, SEQIDNO:4, SEQIDNO:5, SEQIDNO:6 and SEQIDNO:7, further verify
The negative regulation segment of 3 ' UTR nucleic acid sequence of specific IL-17A is nucleotide sequence shown in SEQIDNO:19.Also, experiment card
It is bright, core shown in above-mentioned SEQIDNO:1, SEQIDNO:4, SEQIDNO:5, SEQIDNO:6, SEQIDNO:7 or SEQIDNO:19
Nucleotide sequence can be in conjunction with miRNA-340.
MiRNA-340 target gene binding sequence provided in an embodiment of the present invention is 3 ' UTR nucleic acid sequence of IL-17A or its piece
Section, the sequence are DNA form, which includes genomic DNA or artificial-synthetic DNA, as long as the sequence includes SEQIDNO:
Nucleotide sequence shown in 19 can achieve the object of the present invention；Further, the sequence include: SEQIDNO:1,
Nucleotide sequence shown in SEQIDNO:4, SEQIDNO:5, SEQIDNO:6 or SEQIDNO:7.
Recombinant plasmid provided in an embodiment of the present invention be will it is above-mentioned include SEQIDNO:1, SEQIDNO:4, SEQIDNO:5,
The miRNA-340 target gene binding sequence of nucleotide sequence shown in SEQIDNO:6, SEQIDNO:7 or SEQIDNO:19 is in body
The eukaryotic gene expression vector being built into outside.Further, which is Reporter System, successively from 5 ' ends to 3 ' ends
Including following element: promoter sequence, reporter gene, above-mentioned miRNA-340 target gene binding sequence and termination sequence.More into
One step, which includes luciferase reporter plasmid pmir-GLO and the miRNA-340 target gene combination sequence
Column, the miRNA-340 target gene binding sequence are located at the NheI restriction enzyme site and XhoI enzyme of the luciferase reporter plasmid
Between enzyme site.
Specifically, the present invention is by by 3 ' UTR nucleic acid sequence of IL-17A or its segment (miRNA-340 target gene combination sequence
Column) to 3 ' ends of reporter gene, formation 3 ' UTR of reporter sequences-IL-17A passes through and verifies 3 ' UTR core of IL-17A for building
Whether acid sequence can carry out negative regulation to the expression of reporter gene, and then whether verify 3 ' UTR nucleic acid sequence of IL-17A can be right
The expression of IL-17A carries out negative regulation.
Above-mentioned miRNA-340 target gene binding sequence or above-mentioned recombinant plasmid can be applied to detection miRNA-340
To IL-17A gene regulation effect in, can also be applied to research miRNA-340 to candidate targets IL-17A biological function
Or in regulatory mechanism, and applied to target gene in the miRNA-340 functional study of IL-17A.
The screening of the embodiment of the present invention or the method for identifying IL-17 negative regulation preparation, following steps:
Construct above-mentioned recombinant plasmid；
Make test agent and the recombinant plasmid cotransfection eukaryocyte；
Eukaryocyte after culture transfection, the uciferase activity of the eukaryocyte secretion after detection transfection；
Wherein, if uciferase activity reduces, the test agent is accredited as IL-17 negative regulation preparation.
The recombinant plasmid of the embodiment of the present invention can be constructed according to following construction method, be included the following steps:
Using mouse gene group DNA as template, with nucleotide sequence shown in (i) SEQ ID No.2 and SEQ ID No.3 or
(ii) nucleotide sequence shown in SEQ ID No.9 and SEQ ID No.10 or (iii) SEQ ID No.11 and SEQ ID No.12
Nucleotide sequence shown in shown nucleotide sequence or (iv) SEQ ID No.13 and SEQ ID No.14 or (v) SEQ ID No.15
It is classified as primer with nucleotides sequence shown in SEQ ID No.16, carries out PCR amplification；
Sequence obtained by PCR amplification is connected to the multiple cloning sites of luciferase reporter plasmid pmir-GLO.
Wherein, (i) nucleotide sequence shown in SEQ ID No.2 and SEQ ID No.3, (ii) SEQ ID No.9 and SEQ
Nucleotide sequence shown in ID No.10, nucleotide sequence, (iv) SEQ shown in (iii) SEQ ID No.11 and SEQ ID No.12
Nucleotide sequence shown in ID No.13 and SEQ ID No.14, nucleotide shown in (v) SEQ ID No.15 and SEQ ID No.16
Sequence is followed successively by nucleotide sequence shown in SEQIDNO:1, SEQIDNO:4, SEQIDNO:5, SEQIDNO:6, SEQIDNO:7
PCR amplification primer.
In order to keep the narration of this disclosure more detailed with it is complete, below for embodiments of the present invention and specific real
It applies example and proposes illustrative description；But this not implements or uses the unique forms of the specific embodiment of the invention.Embodiment
In cover multiple specific embodiments feature and to construction with operate these specific embodiments method and step it is suitable with it
Sequence.However, can also reach identical or impartial function and sequence of steps using other specific embodiments.
It will be appreciated by those skilled in the art that following embodiment is merely to illustrate the present invention rather than limitation is of the invention
Range.Test method without specific conditions in following embodiment, usually according to normal condition, such as Sambrook et al.,
Molecular cloning: condition described in laboratory manual, or according to the normal condition proposed by manufacturer.
The clone of 1 mouse IL-17A of embodiment, 3 ' UTR nucleic acid sequence
Step S101: inquiring 3 ' UTR nucleic acid sequence of mmu-IL-17A in NCBI is the 535th base in IL-17A gene
To the 1171st base (as shown in SEQ ID No.1), design primer are as follows:
F:5 '-CGGCTAGCCGACAGAGACCCGCGGCTGACCCCTAAG-3 ' (as shown in SEQ ID No.2)；
R:5 '-CCGCTCGAGCGGTTTAAAACAGAGTAGGGAGCT-3 ' (as shown in SEQ ID No.3).
Step S102: using mouse gene group DNA as template, with nucleotides sequence shown in SEQ ID No.2 and SEQ ID No.3
It is classified as primer, carries out routine PCR reaction amplification using high fidelity enzyme, electrophoresis carries out gel recycling PCR product after running glue, uses
NheI, XhoI carry out double digestion, and by digestion products recovery purifying.
The recombination reporter plasmid of the building complete segment of 3 ' UTR of pmir-GLO-IL-17A of embodiment 2
Step S201: being expanded with the DH5 α competent cell that the conversion of pmir-GLO plasmid is routinely prepared, small to mention reagent
Box prepares pmir-GLO plasmid, electrophoresis detection plasmid purity and quality, carries out double digestion to plasmid with NheI, XhoI, gel returns
Receive the digestion products of kit recycling large fragment；Pmir-GLO plasmid map as shown in Figure 1, the present embodiment pmir-GLO plasmid
For pmirGLO Dual-Luciferase miRNA Target Expression carrier (being abbreviated as pmirGLO).
Connection, conversion and the clonal expansion of recombinant plasmid
Step S202: by 3 ' UTR PCR product of mmu-IL-17A after digestion prepared by embodiment 1 and pmir-GLO matter
Grain is attached reaction.
Step S203: connection product transformed competence colibacillus cell DH5 α, the selectivity on the LB culture plate containing ammonia benzyl mycin
Culture, picked clones bacterium colony, amplification cultivation.
Step S204: a small amount of plasmid of small extraction reagent kit extraction is spare, and Standard PCR verifies whether Successful amplification recombination,
Purpose band meets purpose nucleic acid sequence in 636bp or so, while through sequencing identification.The correct plasmid of sequence is named as pmir-
3 ' UTR plasmid of GLO-IL-17A.
The building of 3 ' UTR fragment deletion reporter plasmid of embodiment 3IL-17A
S301: different primers shown in design table 1, respectively with the pmir-GLO-IL-17A 3 ' of embodiment 2 built
The complete segment recombination reporter plasmid of UTR is that template carries out PCR, obtains a series of 3 ' the UTR piece of IL-17A of missing different lengths
Section: F1 (+535~+1131), F2 (+622~+1131), F3 (+759~+1131), F4 (+849~+1131), F5 (+914~+
1131), wherein F1 is the 535th base to the 1131st base (as shown in SEQ ID No.4) in IL-17A gene, F2 IL-
622nd base to the 1131st base (as shown in SEQ ID No.5) in 17A gene, F3 be IL-17A gene in the 759th base extremely
1131st base (as shown in SEQ ID No.6), F4 are the 849th base to the 1131st base (such as SEQ ID in IL-17A gene
Shown in No.7), F5 is the 914th base to the 1131st base in IL-17A gene (as shown in SEQ ID No.8).
The primer of 3 ' UTR deletion fragment of table 1IL-17A
Step S302:PCR product agarose gel electrophoresis, recycling, double digestion, connection, conversion escherichia coli DH5a.
Step S303: digestion is identified after small upgrading grain, and the recombinant plasmid for identifying correct positive colony is sequenced.
Step S304: a series of 3 ' UTR fragment deletion of the pmir-GLO-IL-17A recombination report matter of fragment deletions is obtained
Grain, successively referred to as plasmid F1, plasmid F2, plasmid F3, plasmid F4 and plasmid F5.
The negative regulation of the complete segment of 3 ' UTR of embodiment 4IL-17A gene acts on
By the complete segment report recombinant plasmid of 3 ' UTR of pmir-GLO-IL-17A prepared by embodiment 2 and blank
Pmir-GLO plasmid distinguishes transfected Jurkat cells, Jurkat cell is pressed 5 × 10 in transfection first 1 day5Density be inoculated in 48 holes
Plate contains 10% fetal calf serum culture medium, is placed in 5%CO237 DEG C of incubator are incubated overnight；Using Lipo3000 by pmir-GLO-
3 ' UTR plasmid of IL-17A, fragment deletion plasmid, sky pmir-GLO carrier distinguish transfected Jurkat cells；After transfection for 24 hours, it is sucked out
Culture medium is added PBS and washs cell, abandons the residual liquid in net each hole as far as possible.With Dual-luciferase reportor systerm reagent
Box detects each group uciferase activity.The light of firefly in each experimental group is measured by Dual-Luciferase reporter gene detection system
The fluorescent value (FLuc) of luciferase and the fluorescent value (RLuc) of renilla luciferase, taking F/R is that relative activity value is counted
According to statistics, as a result as shown in Figure 2, wherein "-" indicate blank pmir-GLO plasmid group, "+" indicate pmir-GLO-IL-17A
The complete segment of 3 ' UTR reports recombinant plasmid group.
Wherein, steps are as follows for specific luciferase assays:
A) cell is collected, 1ml PBS (500 μ l/ times) mildly blows and beats cell, moves in 1.5ml EP pipe；
B) it is centrifuged, RT, 600g, 5min, abandons supernatant, bullet, which beats test tube bottom, disperses precipitating；
C) with ddH2O dilution 5 × lysate (Passive Lysis Buffer) to 1 ×, according to sample size is collected, add suitable
Amount mixes, it is proposed that volume is as shown in table 2；
2 lysate dosage of table suggests table
| Plate types | Lysate dosage (μ l) |
| 6 orifice plates | 500 |
| 24 orifice plates | 100 |
| 48 orifice plates | 65 |
| 96 orifice plates | 20 |
D) it puts to sufficiently cracking on shaking table, 25 DEG C, 300rpm, 15min；
E) room temperature is centrifuged, and 600g, 5min take supernatant to be detected；
F) analysis software is opened, read plate region is set；
G) 20 μ l of sample to be tested is added, 50 μ l firefly luciferases are added and detect liquid LAR II, are read after mixing
Firefly RLU(Relative Light Unit)；
H) 50 μ l renilla luciferases are added and detect liquid, Renilla RLU is read after mixing；
I) reflect the activation degree of reporter gene between different samples according to Firefly/Renilla ratio.
The experimental results showed that the Jurkat of the complete segment report Transfected Recombinant Plasmid of 3 ' UTR of pmir-GLO-IL-17A is thin
The uciferase activity of intracrine is said lower than the uciferase activity that the Jurkat cell of blank pmir-GLO plasmid transfection is secreted
The complete segment of 3 ' UTR of bright IL-17A gene can be with negative regulation uciferase activity.
The determination of the negative regulation segment of 3 ' UTR of embodiment 5IL-17A gene
5 IL-17A, 3 ' UTR fragment deletion reporter plasmid prepared by embodiment 3 distinguishes transfected Jurkat cells, transfection
After for 24 hours, culture medium is sucked out, PBS is added and washs cell, abandons the residual liquid in net each hole as far as possible.With Dual-Luciferase report
It accuses system kit and detects each group uciferase activity, by Dual-Luciferase Reporter Assay System reagent
100 μ L PLB are added into every hole for box specification, and room temperature shaker shakes the abundant lytic cell of 20min.20 μ L LAR- are added in every hole
II reagent, is put on instrument, reads luciferase fluorescent value, 20 μ L Stop&Glo reagents are then added, are put on instrument, reads
Take renilla luciferase fluorescent value.Firefly in each experimental group is measured by Dual-Luciferase reporter gene detection system
The fluorescent value (FLuc) of luciferase and the fluorescent value (RLuc) of renilla luciferase, taking F/R is that relative activity value carries out data
Statistics, as a result as shown in Figure 3, wherein F1 segment report recombinant plasmid group, F2 segment report recombination matter are followed successively by under upper
Grain group, F3 segment report recombinant plasmid group, F4 segment report recombinant plasmid group and F5 segment report recombinant plasmid group.
The experimental results showed that 3 ' UTR fragment deletion F1 segment of pmir-GLO-IL-17A reports recombinant plasmid group, F2 segment
Report that recombinant plasmid group, F3 segment report recombinant plasmid group, the Jurkat cell of F4 segment report recombinant plasmid group transfection are secreted
Uciferase activity lower than F5 segment report recombinant plasmid group transfection Jurkat cell secretion uciferase activity, explanation
F1 segment (as shown in SEQ ID No.4), F2 segment (as shown in SEQ ID No.5), F3 segment (as shown in SEQ ID No.6)
It is that IL-17A segment (as shown in SEQ ID No.7) can be with negative regulation uciferase activity, F5 segment (such as SEQ ID with F4
Shown in No.8) there is no negative regulation effect to uciferase activity, therefore, the negative regulation segment of 3 ' UTR of IL-17A gene includes
In F1 segment (as shown in SEQ ID No.4), F2 segment (as shown in SEQ ID No.5), F3 segment is (such as SEQ ID No.6 institute
Show) and F4 be IL-17A segment (as shown in SEQ ID No.7) within, meanwhile, the negative regulation segment of 3 ' UTR of IL-17A gene
It is not included within F5 segment (as shown in SEQ ID No.8), then, the negative regulation segment of 3 ' UTR of IL-17A gene is IL-
849th base to the 914th base in 17A gene (as shown in SEQ ID No.19).
The potential miRNA binding site of the negative regulation segment of 3 ' UTR of IL-17A gene
According to bioinformatics software miRanda, Targetscan (http://www.targetscan.org/),
RNAhybrid(http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/)、RNA22(http://
Cbcsrv.watson.ibm.com/rna22.html), the negative regulation segment of on-line prediction miRNA and IL17A-3'UTR may
The target site be combineding with each other.3 ' the UTR nucleic acid sequence of IL-17A of mouse, rat, the mankind are compared respectively, and identified
Segment deletion-primers sequence location and candidate's microRNA quasi-step matrix site out, as a result as shown in figure 4, being especially located at conserved region
The microRNA binding site in domain, using comprehensive analysis, obtain it is larger may be by the candidate of 3 ' UTR negative regulation IL-17A
MicroRNA:miR-340, miR-21, miR-466I.
The negative regulation segment that embodiment 6 constructs 3 ' UTR of IL-17A gene reports recombinant plasmid
According to the negative regulation segment report recombination matter of 3 ' UTR of the plasmid construction method building IL-17A gene of embodiment 3
Grain.
Regulating and controlling effect of the 7 difference candidate miRNA of embodiment to IL-17A gene
MiR-340, miR-21, miR-466I analog mimics are ordered from Ji Ma biotech firm, it, will according to operation instruction
Three kinds of analogs are prepared with embodiment 6 thin containing 3 ' UTR negative regulation segment reporter plasmid cotransfection 293T of IL-17A respectively
Born of the same parents, experimental result play negative regulation effect in combination with IL-17A3 ' UTR as shown in figure 5, filtering out miR-340, wherein in Fig. 5
In, 293T cell, corresponding missense are transfected with miR-340 (A), miR-21 (B), miR-466I (C) analogies (mimics) respectively
Oligonucleotides (Negative control, NC) compares, and Dual-luciferase reportor systerm kit detects each group luciferase
Activity.
Regulating and controlling effect of the embodiment 8miRNA-340 to IL-17A gene
The building of the microRNA binding site mutant reporter plasmid of prediction: respectively to potential microRNA binding site
Rite-directed mutagenesis and deletion mutation are carried out, rite-directed mutagenesis is carried out to specific site using over-lap PCR (overlapping PCR) method
And in addition deletion mutation devises a pair of comprising mutational site weight in addition to using pair of primers used in amplification wild-type fragment
Folded extension primer (A-F and A-R).First round PCR uses wild type F and A-R amplified fragments A first；Simultaneously with A-F and wild type R
Amplified fragments B, two sections of PCR products (A and B) respectively by agarose gel electrophoresis separation, recycling and after purification, mixed in equal amounts make
For the template of second of PCR, second of PCR then is carried out with wild primers again, amplification products therefrom is specific mutation
Nucleic acid sequence.PCR product is inserted into respective carrier respectively after digestion, obtaining recombinant plasmid need to confirm that building is correct through digestion,
And it is mismatched through sequencing identification without base.
Recycle mutant reporter plasmid and miR-340 cotransfection 293T cell, Dual-luciferase reportor systerm kit
Detect each group uciferase activity, verifying miR-340 whether negative regulation IL-17A.
A group: miR-340mimics respectively with without containing (W/O ,+914~+1131) or contain (W/ ,+535~+1131)
3 ' the UTR reporter plasmid cotransfection 293T cell of IL-17A of miR-340 binding site；B group: miR-340mimics is respectively and just
The report of normal 3 ' UTR reporter plasmid of IL-17A (WT ,+535~+1131) and miR-340 binding site mutation (Mutated)
Plasmid co-transfection 293T cell, Dual-luciferase reportor systerm kit detect each group uciferase activity, experimental result such as Fig. 6
It is shown, illustrate that miRNA-340 has negative regulation effect to IL-17A gene.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>
<120>miRNA-340 target gene binding sequence, recombinant plasmid and its application
<160> 19
<170> PatentIn version 3.5
<210> 1
<211> 637
<212> DNA
<213>mouse
<400> 1
acagagaccc gcggctgacc cctaagaaac ccccacgttt ctcagcaaac ttacttgcat 60
ttttaaaaca gttcgtgcta ttgattttca gcaaggaatg tggattcaga ggcagattca 120
gaattgtctg ccctccacaa tgaaaagaag gtgtaaaggg gtcccaaact gcttcgtgtt 180
tgtttttctg tggactttaa attatttgtg tatttacaat atcccaagat agctttgaag 240
cgtaacttat tttaatgaag tatctacatt attattatgt ttctttctga agaagacaaa 300
attcaagact cagaaatttt attatttaaa aggtaaagcc tatatttata tgagctattt 360
atgaatctat ttatttttct tcagtatttg aagtattaag aacatgattt tcagatctac 420
ctagggaagt cctaagtaag attaaatatt aatggaaatt tcagctttac tatttgttta 480
tttaaggttc tctcctctga atggggtgaa aaccaaactt agttttatgt ttaataactt 540
tttaaattat tgaagattca aaaaattgga taatttagct ccctactctg ttttaaaaaa 600
aaattgtaac aatatcactg taataataaa gttttgg 637
<210> 2
<211> 36
<212> DNA
<213>artificial sequence
<400> 2
cggctagccg acagagaccc gcggctgacc cctaag 36
<210> 3
<211> 33
<212> DNA
<213>artificial sequence
<400> 3
ccgctcgagc ggtttaaaac agagtaggga gct 33
<210> 4
<211> 597
<212> DNA
<213>mouse
<400> 4
acagagaccc gcggctgacc cctaagaaac ccccacgttt ctcagcaaac ttacttgcat 60
ttttaaaaca gttcgtgcta ttgattttca gcaaggaatg tggattcaga ggcagattca 120
gaattgtctg ccctccacaa tgaaaagaag gtgtaaaggg gtcccaaact gcttcgtgtt 180
tgtttttctg tggactttaa attatttgtg tatttacaat atcccaagat agctttgaag 240
cgtaacttat tttaatgaag tatctacatt attattatgt ttctttctga agaagacaaa 300
attcaagact cagaaatttt attatttaaa aggtaaagcc tatatttata tgagctattt 360
atgaatctat ttatttttct tcagtatttg aagtattaag aacatgattt tcagatctac 420
ctagggaagt cctaagtaag attaaatatt aatggaaatt tcagctttac tatttgttta 480
tttaaggttc tctcctctga atggggtgaa aaccaaactt agttttatgt ttaataactt 540
tttaaattat tgaagattca aaaaattgga taatttagct ccctactctg ttttaaa 597
<210> 5
<211> 520
<212> DNA
<213>mouse
<400> 5
ctattgattt tcagcaagga atgtggattc agaggcagat tcagaattgt ctgccctcca 60
caatgaaaag aaggtgtaaa ggggtcccaa actgcttcgt gtttgttttt ctgtggactt 120
taaattattt gtgtatttac aatatcccaa gatagctttg aagcgtaact tattttaatg 180
aagtatctac attattatta tgtttctttc tgaagaagac aaaattcaag actcagaaat 240
tttattattt aaaaggtaaa gcctatattt atatgagcta tttatgaatc tatttatttt 300
tcttcagtat ttgaagtatt aagaacatga ttttcagatc tacctaggga agtcctaagt 360
aagattaaat attaatggaa atttcagctt tactatttgt ttatttaagg ttctctcctc 420
tgaatggggt gaaaaccaaa cttagtttta tgtttaataa ctttttaaat tattgaagat 480
tcaaaaaatt ggataattta gctccctact ctgttttaaa 520
<210> 6
<211> 373
<212> DNA
<213>mouse
<400> 6
caagatagct ttgaagcgta acttatttta atgaagtatc tacattatta ttatgtttct 60
ttctgaagaa gacaaaattc aagactcaga aattttatta tttaaaaggt aaagcctata 120
tttatatgag ctatttatga atctatttat ttttcttcag tatttgaagt attaagaaca 180
tgattttcag atctacctag ggaagtccta agtaagatta aatattaatg gaaatttcag 240
ctttactatt tgtttattta aggttctctc ctctgaatgg ggtgaaaacc aaacttagtt 300
ttatgtttaa taacttttta aattattgaa gattcaaaaa attggataat ttagctccct 360
actctgtttt aaa 373
<210> 7
<211> 283
<212> DNA
<213>mouse
<400> 7
aattttatta tttaaaaggt aaagcctata tttatatgag ctatttatga atctatttat 60
ttttcttcag tatttgaagt attaagaaca tgattttcag atctacctag ggaagtccta 120
agtaagatta aatattaatg gaaatttcag ctttactatt tgtttattta aggttctctc 180
ctctgaatgg ggtgaaaacc aaacttagtt ttatgtttaa taacttttta aattattgaa 240
gattcaaaaa attggataat ttagctccct actctgtttt aaa 283
<210> 8
<211> 218
<212> DNA
<213>mouse
<400> 8
ttcagtattt gaagtattaa gaacatgatt ttcagatcta cctagggaag tcctaagtaa 60
gattaaatat taatggaaat ttcagcttta ctatttgttt atttaaggtt ctctcctctg 120
aatggggtga aaaccaaact tagttttatg tttaataact ttttaaatta ttgaagattc 180
aaaaaattgg ataatttagc tccctactct gttttaaa 218
<210> 9
<211> 36
<212> DNA
<213>artificial sequence
<400> 9
cggctagccg acagagaccc gcggctgacc cctaag 36
<210> 10
<211> 33
<212> DNA
<213>artificial sequence
<400> 10
ccgctcgagc ggtttaaaac agagtaggga gct 33
<210> 11
<211> 32
<212> DNA
<213>artificial sequence
<400> 11
cggctagccg tcagcaagga atgtggattc ag 32
<210> 12
<211> 33
<212> DNA
<213>artificial sequence
<400> 12
ccgctcgagc ggtttaaaac agagtaggga gct 33
<210> 13
<211> 32
<212> DNA
<213>artificial sequence
<400> 13
cggctagccg caagatagct ttgaagcgta ac 32
<210> 14
<211> 33
<212> DNA
<213>artificial sequence
<400> 14
ccgctcgagc ggtttaaaac agagtaggga gct 33
<210> 15
<211> 29
<212> DNA
<213>artificial sequence
<400> 15
cggctagccg aattttatta tttaaaagg 29
<210> 16
<211> 33
<212> DNA
<213>artificial sequence
<400> 16
ccgctcgagc ggtttaaaac agagtaggga gct 33
<210> 17
<211> 33
<212> DNA
<213>artificial sequence
<400> 17
cggctagccg tcagtatttg aagtattaag aac 33
<210> 18
<211> 33
<212> DNA
<213>artificial sequence
<400> 18
ccgctcgagc ggtttaaaac agagtaggga gct 33
<210> 19
<211> 66
<212> DNA
<213>mouse
<400> 19
aattttatta tttaaaaggt aaagcctata tttatatgag ctatttatga atctatttat 60
ttttct 66
Claims (11)
1. a kind of miRNA-340 target gene binding sequence, which is characterized in that the sequence include: SEQIDNO:1, SEQIDNO:4,
Nucleotide sequence shown in SEQIDNO:5, SEQIDNO:6, SEQIDNO:7 or SEQIDNO:19.
2. a kind of recombinant plasmid, which is characterized in that the recombinant plasmid includes miRNA-340 target gene as described in claim 1
Binding sequence.
3. recombinant plasmid according to claim 2, which is characterized in that the recombinant plasmid, which is held from 5 ' ends to 3 ', successively includes
Following element: promoter sequence, reporter gene, miRNA-340 target gene binding sequence as described in claim 1 and termination sequence
Column.
4. recombinant plasmid according to claim 3, which is characterized in that the recombinant plasmid includes luciferase reporter gene matter
Grain pmir-GLO and the miRNA-340 target gene binding sequence, the miRNA-340 target gene binding sequence are located at described glimmering
Between the NheI restriction enzyme site and XhoI restriction enzyme site of light element enzyme reporter plasmid.
5. a kind of host cell, which is characterized in that the host cell contains such as the described in any item recombination matter of claim 2-4
Grain.
6. a kind of method of screening or identification IL-17 negative regulation preparation, which is characterized in that this method comprises the following steps:
Construct recombinant plasmid as claimed in claim 4；
Make test agent and the recombinant plasmid cotransfection eukaryocyte；
Eukaryocyte after culture transfection, the uciferase activity of the eukaryocyte secretion after detection transfection；
Wherein, if uciferase activity reduces, the test agent is accredited as IL-17 negative regulation preparation.
7. the method for screening according to claim 6 or identification IL-17 negative regulation preparation, which is characterized in that the eukaryon
Cell is yeast cells, insect cell, plant cell, zooblast or people's cell.
8. a kind of construction method of recombinant plasmid as claimed in claim 4, which is characterized in that this method comprises the following steps:
Using mouse gene group DNA as template, with nucleotide sequence or (ii) shown in (i) SEQ ID No.2 and SEQ ID No.3
Shown in nucleotide sequence shown in SEQ ID No.9 and SEQ ID No.10 or (iii) SEQ ID No.11 and SEQ ID No.12
Nucleotide sequence shown in nucleotide sequence or (iv) SEQ ID No.13 and SEQ ID No.14 or (v) SEQ ID No.15 and
Nucleotides sequence shown in SEQ ID No.16 is classified as primer, carries out PCR amplification；
Sequence obtained by PCR amplification is connected to the multiple cloning sites of luciferase reporter plasmid pmir-GLO.
9. miRNA-340 target gene binding sequence described in claim 1 or the described in any item recombination matter of claim 2 to 4
Grain is in detection miRNA-340 to the active application of IL-17A gene regulation.
10. miRNA-340 target gene binding sequence described in claim 1 or the described in any item recombination matter of claim 2 to 4
Grain is in research miRNA-340 to the application in candidate targets IL-17A biological function or regulatory mechanism.
11. miRNA-340 target gene binding sequence described in claim 1 or the described in any item recombination matter of claim 2 to 4
Application of the grain in the miRNA-340 functional study that target gene is IL-17A.
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Citations (2)
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|---|---|---|---|---|
| JP2016026486A (en) * | 2014-06-24 | 2016-02-18 | 美智代 津留 | Ossification of spine ligament model non-human animal, diabetes model non-human animal, ossification of spine ligament model cell, diabetes model cell, screening method, detection method of ossification of spine ligament lesion cell, ossification of spine ligament diagnostic kit and ossification of spine ligament therapeutic agent |
| WO2016172008A1 (en) * | 2015-04-24 | 2016-10-27 | University Of Massachusetts | Modified aav constructions and uses thereof |
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2018
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|---|---|---|---|---|
| JP2016026486A (en) * | 2014-06-24 | 2016-02-18 | 美智代 津留 | Ossification of spine ligament model non-human animal, diabetes model non-human animal, ossification of spine ligament model cell, diabetes model cell, screening method, detection method of ossification of spine ligament lesion cell, ossification of spine ligament diagnostic kit and ossification of spine ligament therapeutic agent |
| WO2016172008A1 (en) * | 2015-04-24 | 2016-10-27 | University Of Massachusetts | Modified aav constructions and uses thereof |
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| BANERJEE A ET AL.: "NM_010552.3", 《GENBANK》 * |
| 李阳: "MiR-340靶向EZH2逆转miRNAs异常表达抑制三阴性乳腺癌进展的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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