CN103484453A - MiR-222 target gene combination sequence - Google Patents

Abstract

The invention belongs to the technical filed of biological medicines, and specifically relates to a miR-222 target gene combination sequence, recombinant vectors, transformants and applications of the transformants. The miR-222 target gene combination sequence has a nucleic acid sequence shown as SEQ ID NO. 1, and thus efficient high-throughput screening evaluating platform and methods are provided for development of inhibitor medicaments of miR-222 in future, and applications of miRNA to prevention, diagnosis and treatment on tumors are substantially promoted.

Description

The target gene binding sequence of a kind of miR-222

Technical field

The invention belongs to the biological medicine technology field, be specifically related to target gene binding sequence, recombinant vectors, transformant of a kind of miR-222 and uses thereof.

Background technology

Since Lee in 1993 etc. find that in beautiful new rhabditis axei miRNA encoding gene lin-4, Reinhart in 2000 etc. find let-7 in the developmental regulation to nematode first, people find and identify hundreds of kind miRNA encoding gene in succession in tobacco, fruit bat, nematode, zebra fish, Mammals and human body.

MiRNAs is a kind of molecule that gene is finely tuned of extensive existence, and the total gene that accounts for human genome is counted 2-3%.From a structural point, miRNAs (microRNAs) is the very short non-coding regulation and control strand microRNA of a class length, about 18-24bp, on post-transcriptional level, the expression of gene is carried out to negative regulation by 3 end non-coding regions (3 ' UTR) complementary pairing with its said target mrna molecule, cause degraded or the translation of mRNA to suppress, thereby further regulate and control the various biological behavior, as process, differentiation of stem cells, apoptosis, disease and the tumour of growing occurs.

At first miRNA is expressed to the discussion changed and come from the research to B cell chronic lymphatic leukemia (CLL), thereafter, the expression that numerous research groups detect miRNA in multiple human tumor changes, therefore be considered to occur the common trait of canceration, be whom who be that fruit does not still also have clear and definite experimental evidence because of but the formation of the tumour of cancer and miRNA express variation actually.About 50% miRNA that obtains explaining is positioned the fragile site (fragile site) with Tumor-assaciated on genome, and this explanation miRNA plays vital effect in the tumour generating process.In addition, adopting biochip technology, to the miRNA from the kinds of tumors tissue samples, expression detects discovery, some miRNA down-regulated expressions, and other miRNA up-regulateds, these miRNA roles are similar to the function of cancer suppressor gene and oncogene, the researchist are arranged by miRNA called after " oncomirs ".

Research shows, miR-222(Homo sapiens miR-222) up-regulated in liver cancer, prostate cancer tissue and cancer of the stomach, glioma cell ［ Wong QW, Ching AK, Chan AW, et al.[J] .ClinCancer Res, 2010,16 (3): 867-875; Chun-Zhi Z, Lei H, An-Ling Z, et al.[J] .BMC Cancer, 2010,10:367; Galardi S, Mercatelli N, Farace MG, et al.[J] .Nucleic Acids Res, 2011,39 (9): 3892-3902 ］

Therefore, the target gene binding sequence of clear and definite miR-222, significant to its inhibitor of Future Development, by the application that greatly promotes miRNA in tumor prevention, diagnosis and treatment.

Summary of the invention

Technical problem to be solved by this invention is to provide the target gene binding sequence of miR-222, for its inhibitor medicaments of Future Development provides efficiently, screening and evaluation platform and the means of large flux.

For this reason, the invention discloses a kind of target gene binding sequence containing the miR-222 just like the described nucleotide sequence of SEQ ID NO.1.

In certain embodiments, described target gene binding sequence is as nucleotide sequence as described in SEQ ID NO.2.

In certain embodiments, described target gene binding sequence is for being DKK2 gene 3 ' UTR nucleotide sequence as described in SEQ ID NO.3.

On the other hand, the invention discloses a kind of recombinant vectors that comprises target gene binding sequence of the present invention.

In certain embodiments, described recombinant vectors contains Photinus pyralis LUC or ocean coelenteron luciferase reporter gene.

In certain embodiments, described recombinant vectors is the pGL3-promoter carrier.

On the other hand, the invention discloses a kind of transformant that comprises recombinant vectors of the present invention.

In one embodiment, described transformant is the U87 glioma cell.

On the other hand, the invention discloses transformant of the present invention purposes in screening miR-222 inhibitor.

The invention discloses the target gene binding sequence of miR-222, for its inhibitor medicaments of Future Development provides efficiently, screening and evaluation platform and the means of large flux, by the application that greatly promotes miRNA in tumor prevention, diagnosis and treatment.

The accompanying drawing explanation

Fig. 1 pGL3-promoter plasmid enzyme restriction figure.

Fig. 2 PCR gel electrophoresis figure.

The active detection of the two luciferases of Fig. 3.

Embodiment

The present invention also comprises by the method for the target gene binding sequence (polynucleotide) of synthetic and recombinant technology production miR-222 of the present invention.By methods known in the art, can separate and purifying polynucleotide (DNA or RNA), carrier, transformant and organism.

For carrier of the present invention, can be as phage, plasmid, clay, minichromosome, virus or retroviral vector.The carrier that can be used for the clone and/or express polynucleotide of the present invention is to copy and/or to express the carrier of polynucleotide in need copy and/or express the host cell of polynucleotide.In general, polynucleotide and/or carrier can be used for any eucaryon or prokaryotic cell prokaryocyte, comprise mammalian cell (as people (as HeLa), monkey (as Cos), rabbit (as the rabbit reticulocyte), rat, hamster (as CHO, NSO and baby hamster kidney cell) or mouse cell (as the L cell)), vegetable cell, yeast cell, insect cell or bacterial cell (as intestinal bacteria).The relevant example that is applicable to the suitable carrier of broad variety host cell can be referring to for example F.Ausubel et al., Current Protocols in Molecular Biology.Greene Publishing Associates and Wiley-Interscience (1992) and Sambrook et al. (1989).Can come great expression to can be used for for example protein of medicine, diagnostic reagent, vaccine and therapeutical agent with the host cell that contains these polynucleotide.

Having developed several different methods makes polynucleotide can operate and be connected with carrier for the sticky end via complementary.For example, can add complementary same aggressiveness sequence fragment by the DNA section in wanting insertion vector DNA.Then by the hydrogen bond connection carrier between complementary homopolymeric tail and DNA section to form recombinant DNA molecules.

The synthetic linker that contains one or more restriction site provides the method for another kind of connection DNA section and carrier.Process with phage T4DNA polysaccharase or e. coli dna polymerase I the DNA section produced by the endonuclease restrictive diges-tion, described two kinds of its 3' of polysaccharase, 5 '-exonucleolytic activity is removed outstanding γ-strand end, and fills 3 '-recessed end with its polymerization activity.Therefore, combining of these activity produced flush end DNA section.Then can catalysis the enzyme that connects of flush end DNA molecular, as be incubated together with the linkers of flush end section and large molar excess under the existence of phage T4DNA ligase enzyme.Therefore, reaction product is the DNA section that end carries the polylinker sequence.Then use these DNA sections of suitable Restriction Enzyme cracking, and be connected in the expression vector of using enzymatic lysis, described enzyme can produce the end compatible with described DNA section.Can buy from a plurality of businessmans the synthetic linker that contains a plurality of restriction endonucleases site.

The polynucleotide inset should be operably connected on the suitable promotor compatible with the host cell of expressing polynucleotide.Promotor can be strong promoter and/or inducible promoter.The example of some promotors of enumerating comprises phage PL promotor of a specified duration, intestinal bacteria lac, trP, phoA, tac promotor, SV40 is early stage and late promoter and retrovirus LTR promotor.Other suitable promotor is well known by persons skilled in the art.Express recombinant vectors and further contain transcription initiation, termination site, and contain at transcriptional domain the ribosome bind site that is useful on translation.The encoding part of the transcript that recombinant vectors is expressed can comprise the translation initiation codon that is positioned at the starting point place and the terminator codon that suitably is positioned at the end that is translated polypeptide (UAA, UGA or UAG).

As mentioned above, expression vector can comprise at least one selective marker.Described mark comprises Tetrahydrofolate dehydrogenase, G418, glutamine synthase, neomycin resistance, Photinus pyralis LUC, the ocean coelenteron luciferase for eukaryotic cell culture; And for tsiklomitsin, kantlex or the acillin resistant gene of intestinal bacteria and other microbial culture.Suitably host's representative example includes but not limited to: bacterial cell, as intestinal bacteria, streptomycete and salmonella typhimurium cell; The fungal cell, as yeast cell (as yeast saccharomyces cerevisiae or pichia pastoris phaff); Insect cell, as fruit bat S2 and noctuid SF9 cell; Zooblast, as CHO, COS, NSO, 293 and the Bowes melanoma cells; And vegetable cell.The appropriate culture medium of above-mentioned host cell and culture condition are known in the art.

The present invention also comprises the host cell that contains nucleotide sequence of the present invention, and described nucleotide sequence can operate and be connected with one or more allos control region (as promotor and/or enhanser) through technology known in the art.Can select to regulate the expression of the gene order of inserting, or can be according to required particular form modification and the host strain of processed gene product.Under the existence of some inductor, the expression that some promotor starts can raise; Therefore, can control the expression through the polypeptide of genetic modification.In addition, the different hosts cell has the mechanism of distinctive and special translation, translation post-treatment and modification (as phosphorylation, cracking) protein.Can select suitable clone to carry out desirable modification and processing to guarantee the exogenous protein to expressing.

By the transfection of calcium phosphate transfection, the mediation of DEAE-dextran, transfection, electroporation, transduction, infection or other method of cation lipid mediation, nucleic acid of the present invention and nucleic acid recombinant vectors can be imported to host cell.Described method is described in the laboratory manual of a plurality of standards, as Davis et al., and Basic Methods In Molecular Biology (1986).

The target gene binding sequence (polynucleotide) of miR-222 of the present invention of encoding can be connected to breed in the host with the carrier that contains selective marker.In general, can be at throw out, import plasmid vector in the mixture as calcium phosphate precipitation thing or itself and charged lipids.If carrier is virus, can uses suitable package cell line in vitro it to be packed, then transduce to host cell.

Can identify by well-known technology the cell successfully transformed, contain the cell of DNA recombinant vectors of the present invention.For example, can cultivate the cell of importing expression recombinant vectors gained to produce required polypeptide.Collect and lysing cell, use as Southem (1975) J.Mol.Biol.95,503 or Berent et al (1985) Biotech.3,208 described methods, detect the existence of DNA in its DNA content.Perhaps, use the existence of protein in the antibody test supernatant liquor.

For the routine techniques used in practice of the present invention is described in further detail, the practitioner can be referring to textbook and the comment of cells involved biology, weave construction and embry standard.Comprise that Teratocarcinomas and embryonic stem cell:A practical approach[E.J.Robertson compiles, IRL publishes company limited, 1987]; The volumes such as Guide to techniques in Mouse Development[P.M.Wasserman, academic press, 1993]; Embryonic Stem Cell Differentiation in Vitro[M.V.Wiles, Meth.Enzymol.225:900,1993]; Properties and uses of Embryonic Stem Cells:Prospects for Application to Human Biology and Gene Therapy[P.D.Rathjen etc., Reprod.Fertil.Dev.10:31,1998].

Cytobiology technology can be at " the current scheme in cytobiology " [J.S.Bonifacino etc., Wiley & Sons] in find.Reagent related to the present invention, cloning vector and genetic manipulation test kit can obtain from commercial supplier, for example BioRad, Stratagene, Invitrogen, ClonTech and sigma-Aldrich company.

Usually in " animal cell culture: basic fundamental handbook " latest edition, (R.I.Freshney edits cell culture processes, Wiley & Sons); " cell cultures general technology " (M.A.Harrison and I.F.Rae, Cambridge University publishes); In " embryonic stem cell: method and operational provisions " (K.Turksen edits, and Humana publishes), description is arranged.Tissue culture medium (TCM) and reagent can obtain from commercial supplier, for example Gibco/BRL, Nalgene-Nunc International, Sigma Chemical Co. and ICN Biomedicals.

The type of the substratum that the present invention is used is restriction not, as long as can be used for the substratum that stem cell is cultivated.CO in this class substratum ₂concentration preferably 5%, but the invention is not restricted to this.

Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition or the condition of advising according to manufacturer.Unless otherwise defined, the same meaning that all specialties of using in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.

Instrument and reagent

Table-type high-speed refrigerated centrifuge (Eppendorf company)

The Multifunction fluorescent microplate reader

Gel imaging system

Desk centrifuge

Electronic balance

Biological electrophoresis image analysis system

50 * TAE solution

Agrose

Embodiment 1. builds the pGL3-DKK2-promoters-basic plasmid

(1) with the conventional DH5 α competent cell prepared of pGL3-promoter Plasmid Transformation, increased, alkaline lysis prepares the pGL3-promoter plasmid, the purity of electrophoresis detection plasmid and content, with XbaI, plasmid is carried out to enzyme and cut, the test kit gel reclaims the enzyme of large fragment and cuts product;

(2) clone, the enzyme of 3 ' UTR sequence of DKK2 gene are cut and purifying

Adopt database TargetScan5.2, the miR-222 binding sequence in 3 ' UTR sequence of prediction DKK2 gene is as nucleotide sequence as described in SEQ ID NO.1, then designs its primer and is

Primer 1:5 '-GATCGCCGTGTAAT tCTAGAgTTTCATTGCCCTCT-3 ';

Primer 2: 5 '-CGTCGATAACTCTAGACAAAGTCAATAGCTGC-3 ';

Take genomic dna as template, utilize conventional PCR reaction to be increased, reclaim the PCR product with the test kit gel, by the PCR product XbaI enzyme cutting reclaimed;

(3) the pGL3-promoter carrier of above-mentioned acquisition and DKK2 gene 3 ' UTR fragment are carried out to ligation;

(4) screening recon, by the direct transformed competence colibacillus intestinal bacteria of above-mentioned connection product, through selecting in the LB substratum that contains penbritin, cultivate, picking colony immediately from the flat board of transformant, extract on a small quantity plasmid with alkaline lysis after amplification cultivation standby, cut the pcr amplification result through enzyme: the purpose band appears at the 774bp left and right, the DKK2 target gene (seeing accompanying drawing 2) that confirms successfully to have increased, through order-checking, identifies as nucleotide sequence as described in SEQ ID NO.2 simultaneously.Plasmid called after pGL3-DKK2-3 ' the UTR-promoter plasmid (hereinafter to be referred as " DKK2-WT ") that sequence is correct.

The pGL3-promoter carrier that embodiment 2. builds containing DKK2 gene 3 ' UTR fragment mutant nucleotide sequence

Change the TAG base in the TGTAGCA in DKK23 ' UTR into the CGA base, design its primer according to pGL3-DKK2-3 ' UTR-promoter plasmid sequence and be

Primer 3:5 '-ACAGAAAGCTTTGCGACAGAATATATTT-3 ';

Primer 4:5 '-CAAATATATTCTGTCGCAAAGCTTTCTGT-3 ';

Prepared pGL3-DKK2-3 ' the UTR-promoter plasmid of the embodiment 1 of take is template, adopt TaKaRa MutanBEST Kit, according to test kit specification sheets step operation, with reference to embodiment 1(4) step filter out the pGL3-promoter carrier (hereinafter to be referred as " DKK2-Mut ") containing DKK2 gene 3 ' UTR fragment mutant nucleotide sequence

Embodiment 3. luciferases are active to be detected

Use the U87 glioma cell: with containing 10% tire calf serum, obtaining nutrient solution and be made into the individual cells suspension, with every hole 1000-10000 cell, be inoculated into 96 orifice plates, every pore volume 200ul, 12-24 hour adherent rear cell carries out transfection while reaching 50% left and right fusion rate; According to the routine techniques method, DKK2-WT plasmid, DKK2-Mut plasmid, empty pGL3-promoter carrier are combined respectively to renilla luciferase pRL-SV40 plasmid co-transfection U87 glioma cell, form DKK2-WT group, DKK2-Mut group and DKK2-Ctrl group, added respectively miR-222 mimics, miR-222 inhibitor or equal-volume nutrient solution (as blank) simultaneously; The Dual-Luciferase detection is carried out in transfection after 24 hours

Dual-Luciferase test experience step:

1. lysing cell: the reporter gene cell pyrolysis liquid is fully mixed, add as follows the reporter gene cell pyrolysis liquid, fully lysing cell.

A. for attached cell: after exhausting cell culture fluid, can directly add the reporter gene cell pyrolysis liquid; For suspension cell: centrifugal going adds reporter gene thin after supernatant

Cellular lysate liquid.

B. fully after cracking, 10,000-15, the centrifugal 3-5 minute of 000g, get supernatant for measuring.

Annotate: can measure immediately luciferase after lysis, also can be first frozen, after treating, measure again.Frozen sample need melt, and is measured after reaching room temperature again.

Vessel type 96 orifice plate 48 orifice plate 24 orifice plate 12 orifice plate 6 orifice plates

Reporter gene cell pyrolysis liquid (microlitre/hole) 100 150 200 300 500

2. melt Photinus pyralis LUC detection reagent and Renilla luciferase and detect damping fluid, and reach room temperature.Renilla luciferase detection substrate (100X) is placed in ice bath

Or standby on ice chest.

3. need the amount of 100 microlitres according to each sample, get appropriate Renilla luciferase and detect damping fluid, according to 1:100, add Renilla luciferase detection substrate (100X) to join

Make Renilla luciferase testing liquid.For example, 1 milliliter of Renilla luciferase detects in damping fluid and adds 10 microlitre Renilla luciferase detection substrates

(100X) can prepare into about 1 milliliter of Renilla luciferase testing liquid after fully mixing.

4. press the instrumentation specification sheets and open fluor tester, measuring interval is made as to 2 seconds, minute is made as 10 seconds.

5. during each sample determination, sample thief 20-100 microlitre (if sample size is enough, please add 100 microlitres, but the usage quantity of each sample will be consistent), add 100 microlitres

The Photinus pyralis LUC detection reagent, beat or mix rear mensuration RLU (relative light unit) with other appropriate ways with rifle.Take the reporter gene cell pyrolysis liquid as blank

Contrast.

6. after completing said determination Photinus pyralis LUC step, add 100 microlitre Renilla luciferase testing liquid, with rifle, beat or mix rear survey with other appropriate ways

Determine RLU (relative light unit).

7. in the situation that take the Renilla luciferase as internal reference, the RLU value that the RLU value obtained by Photinus pyralis LUC mensuration obtains divided by the Renilla luciferase assay.According to

The ratio obtained carrys out the activation degree of more different sample room purpose reporter genes.

Experimental result (Fig. 3), cross and express 3 ' the UTR fluorescence activity that miR-222 can lower DKK2, and after miR-222 is suppressed, 3 ' the UTR fluorescence activity of DKK2 raises, and the change of miR-222 simultaneously can not cause the fluorescence activity of blank group and Seed Sequences sudden change group., this shows that the TGTAGCA in DKK23 ' UTR is the miR-222 binding sequence.

Scope of the present invention is not subject to the restriction of described specific embodiments, and described embodiment, only as the single example of illustrating all respects of the present invention, also comprises method and the component of functional equivalent in the scope of the invention.In fact, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to description and accompanying drawing above.Within described improvement also falls into the scope of appended claims.Every piece of reference mentioned above is listed this paper in as a reference all in full.

Sequence table

<110 > Xinhua Hospital Attached to Medical School, Shanghai Jiaotong Univ.

<120 > the target gene binding sequence of a kind of miR-222

<130 > specification sheets sequence table

<160>3

<170>PatentIn version 3.3

<210>1

<211>7

<212>DNA

<213>Homo sapiens

<400>1

tgtagca 7

<210>2

<211>756

<212>DNA

<213>Homo sapiens

<400>2

gtttcattgc cctctataag cttctgacta gccaatggca tcatccaatt ttcttcccaa 60

acctctgcag catctgcttt attgccaaag ggctagtttc ggttttctgc agccattgcg 120

gttaaaaaat ataagtagga taacttgtaa aacctgcata ttgctaatct atagacacca 180

cagtttctaa attctttgaa accactttac tacttttttt aaacttaact cagttctaaa 240

tactttgtct ggagcacaaa acaataaaag gttatcttat agtcgtgact ttaaactttt 300

gtagaccaca attcactttt tagttttctt ttacttaaat cccatctgca gtctcaaatt 360

taagttctcc cagtagagat tgagtttgag cctgtatatc tattaaaaat ttcaacttcc 420

cacatatatt tactaagatg attaagactt acattttctg cacaggtctg caaaaacaaa 480

aattataaac tagtccatcc aagaaccaaa gtttgtataa acaggttgct ataagcttgg 540

tgaaatgaaa atggaacatt tcaatcaaac atttcctata taacaattat tatatttaca 600

atttggtttc tgcaatattt ttcttatgtc caccctttta aaaattatta tttgaagtaa 660

tttatttaca ggaaatgtta atgagatgta ttttcttata gagatatttc ttacagaaag 720

ctttgtagca gaatatattt gcagctattg actttg 756

<210>3

<211>2174

<212>DNA

<213>Homo sapiens

<400>3

tcaccattga ggaacatcat caattgcaga ctgtgaagtt gtgtatttaa tgcattatag 60

catggtggaa aataaggttc agatgcagaa gaatggctaa aataagaaac gtgataagaa 120

tatagatgat cacaaaaagg gagaaagaaa acatgaactg aatagattag aatgggtgac 180

aaatgcagtg cagccagtgt ttccattatg caacttgtct atgtaaataa tgtacacatt 240

tgtggaaaat gctattatta agagaacaag cacacagtgg aaattactga tgagtagcat 300

gtgactttcc aagagtttag gttgtgctgg aggagaggtt tccttcagat tgctgattgc 360

ttatacaaat aacctacatg ccagatttct attcaacgtt agagtttaac aaaatactcc 420

tagaataact tgttatacaa taggttctaa aaataaaatt gctaaacaag aaatgaaaac 480

atggagcatt gttaatttac aacagaaaat taccttttga tttgtaacac tacttctgct 540

gttcaatcaa gagtcttggt agataagaaa aaaatcagtc aatatttcca aataattgca 600

aaataatggc cagttgttta ggaaggcctt taggaagaca aataaataac aaacaaacag 660

ccacaaatac ttttttttca aaattttagt tttacctgta attaataaga actgatacaa 720

gacaaaaaca gttccttcag attctacgga atgacagtat atctctcttt atcctatgtg 780

attcctgctc tgaatgcatt atattttcca aactataccc ataaattgtg actagtaaaa 840

tacttacaca gagcagaatt ttcacagatg gcaaaaaaat ttaaagatgt ccaatatatg 900

tgggaaaaga gctaacagag agatcattat ttcttaaaga ttggccataa cctgtatttt 960

gatagaatta gattggtaaa tacatgtatt catacatact ctgtggtaat agagacttga 1020

gctggatctg tactgcactg gagtaagcaa gaaaattggg aaaacttttt cgtttgttca 1080

ggttttggca acacatagat catatgtctg aggcacaagt tggctgttca tctttgaaac 1140

caggggatgc acagtctaaa tgaatatctg catgggattt gctatcataa tatttactat 1200

gcagatgaat tcagtgtgag gtcctgtgtc cgtactatcc tcaaattatt tattttatag 1260

tgctgagatc ctcaaataat ctcaatttca ggaggtttca caaaatggac tcctgaagta 1320

gacagagtag tgaggtttca ttgccctcta taagcttctg actagccaat ggcatcatcc 1380

aattttcttc ccaaacctct gcagcatctg ctttattgcc aaagggctag tttcggtttt 1440

ctgcagccat tgcggttaaa aaatataagt aggataactt gtaaaacctg catattgcta 1500

atctatagac accacagttt ctaaattctt tgaaaccact ttactacttt ttttaaactt 1560

aactcagttc taaatacttt gtctggagca caaaacaata aaaggttatc ttatagtcgt 1620

gactttaaac ttttgtagac cacaattcac tttttagttt tcttttactt aaatcccatc 1680

tgcagtctca aatttaagtt ctcccagtag agattgagtt tgagcctgta tatctattaa 1740

aaatttcaac ttcccacata tatttactaa gatgattaag acttacattt tctgcacagg 1800

tctgcaaaaa caaaaattat aaactagtcc atccaagaac caaagtttgt ataaacaggt 1860

tgctataagc ttggtgaaat gaaaatggaa catttcaatc aaacatttcc tatataacaa 1920

ttattatatt tacaatttgg tttctgcaat atttttctta tgtccaccct tttaaaaatt 1980

attatttgaa gtaatttatt tacaggaaat gttaatgaga tgtattttct tatagagata 2040

tttcttacag aaagctttgt agcagaatat atttgcagct attgactttg taatttagga 2100

aaaatgtata ataagataaa atctattaaa tttttctcct ctaaaaactg aaaaaaaaaa 2160

aaaaaaaaaa aaaa 2174

Claims (10)

1. one kind containing the target gene binding sequence just like the miR-222 of the described nucleotide sequence of SEQ ID NO.1.

2. the target gene binding sequence of miR-222 according to claim 1, is characterized in that described target gene binding sequence is for nucleotide sequence as described in SEQ ID NO.2.

3. the target gene binding sequence of miR-222 according to claim 1, is characterized in that described target gene binding sequence is for DKK 2 gene 3 ' UTR nucleotide sequences as described in SEQ ID NO.3.

4. the recombinant vectors of a target gene binding sequence that comprises the described miR-222 of claim 1.

5. recombinant vectors according to claim 4, is characterized in that described target gene binding sequence is for nucleotide sequence as described in SEQ ID NO.2.

6. recombinant vectors according to claim 4, is characterized in that described target gene binding sequence is for DKK2 gene 3 ' UTR nucleotide sequence as described in SEQ ID NO.3.

7. recombinant vectors according to claim 6, is characterized in that described recombinant vectors contains Photinus pyralis LUC or ocean coelenteron luciferase reporter gene.

8. recombinant vectors according to claim 7, is characterized in that described recombinant vectors is the pGL3-promoter carrier.

9. a transformant that comprises the described recombinant vectors of any one in claim 4-8.

10. the purposes of the described transformant of claim 9 in screening miR-222 inhibitor.

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