CN102719465B - Inducible tissue-specific expression vector and purpose thereof - Google Patents

Abstract

The invention belongs to the field of biological technology, relating to an inducible tissue-specific expression vector and a purpose thereof. The inducible tissue-specific expression vector mainly comprises the following structures of: a promoter EF1 alpha, an EGFP fluorescent marker gene, a BGHpoly A tailing transcription termination sequence with two ends linked with same-direction Lox sequences, and an intron sequence with internal enzyme sites introduced. The vector is pEFGLPAL-MIR using a general promoter EF1alpha to drive gene expression; the EGFP fluorescent marker gene is used for indication of the gene expression; two BDHpoly A tailing transcription termination signals linked with same-direction LoxP71 and LoxP66 are inducible by enzyme Cre. The termination signals are followed by an intron sequence, with an miRNA expression precursor is inserted into the intron. The micro RNA expression vector can successfully express specific micro RNA and the micro RNA expression can be determined by various ways. The expression vector is capable of expressing uneasily-silenced micro RNA in an ES cell, so that can be successfully applied in the preparation of transgenic mice.

Description

Inducibility tissue specific expression carrier and uses thereof

Technical field

The invention belongs to biological technical field, relate to inducibility tissue specific expression carrier and uses thereof.Be specifically related to microRNA inducibility tissue specific expression carrier and purposes, especially a kind of Cre enzyme is mediation, derivable, tissue-specific miRNA expression vector pEFG LPAL-MIR, described carrier can be with expressing microRNA, and set up inducibility, organizing specific expression transgenosis experimental animal model.

Background technology

The research report, the noncoding single stranded RNA molecule of the little endogenous that microRNA is comprised of 21-25 Nucleotide, be to be given birth to after the processing of Dicer enzyme by the single stranded RNA precursor of the base size of the approximately 70-90 with hairpin structure; Described precursor is cut the microRNA two strands into about 22 Nucleotide by RNaseIII Dicer among cell, and wherein a chain is degraded, and other one is processed to ripe microRNA.In the zooblast experiment, microRNA generally holds non-coding region by the partial sequence homology in 3 '-UTR(3 ' of target gene mRNA) combination, cause translation to stop, in the situation that seldom, cause the degraded of mRNA.The discovery of microRNA has disclosed a kind of new generegulation mechanism, aspect growth and cytodifferentiation, is rising and is having important function and significance.Research discloses, and miRNAs participates in a series of important process in vital process, as, early development, cell proliferation, apoptosis, necrocytosis, metabolism of fat and cytodifferentiation etc.Studies have found that the very large relation that has of microRNA and numerous disease, such as tumour, heart trouble etc., can further instruct the diagnosis of disease.Due to popularity and the diversity of microRNA existence, and much the expression of miRNA has the specificity of tissue and etap expression, therefore points out microRNA that more extensively various biological function may be arranged.

The at present research of diseases related need inducibility, organizing specific expression transgenosis experimental animal model, but up to now, there is not yet to be specifically designed to and build the especially miRNA expression vector of transgenosis test mice of transgenosis experimental animal model, common CMV promotor is easy to be silenced in the ES cell in addition, and these are all that the research of microRNA or the preparation of transgenosis experimental animal model have caused very large difficulty.

Summary of the invention

The objective of the invention is to overcome the deficiencies in the prior art, a kind of inducibility tissue specific expression carrier is provided, be specifically related to derivable, tissue-specific miRNA expression vector, especially a kind of Cre enzyme is mediation, derivable, tissue-specific miRNA expression vector pEFG LPAL-MIR.Described carrier can be used for expressing microRNA, and set up inducibility, organizing specific expression transgenosis experimental animal model.

Further purpose of the present invention is to provide described expression vector for expressing microRNA, and sets up purposes inducibility, in organizing specific expression transgenosis experimental animal model.

The present invention expresses miRNA in the intron mode, and described carrier, when not affecting marker gene EGFP expression, can also be realized the effective expression of miRNA; This carrier can be used for experiment in vitro and transgenic mice, and described Cre enzyme has good inducibility to the expression of this carrier.

In expression vector of the present invention, on the basis of pEGFP plasmid, added EF1 alpha promotor.

Particularly, inducibility tissue specific expression carrier of the present invention, it is characterized in that, mainly comprise following structure: EF1 α promotor, EGFP fluorescent mark gene, two ends are connected with BGH polyA tailing transcription termination sequence and the inner intron sequences of introducing restriction enzyme site of LoxP sequence in the same way; Described carrier is pEFG LPAL-MIR, has the structure of sequence 1.

In the present invention, described carrier is used EF1 alpha general starting mover to drive genetic expression; EGFP fluorescent mark gene is used to indicate the expression of gene; Then be two sections and be connected with the BDH polyA tailing transcription termination signal of LoxP71 and LoxP66 in the same way, can be by the Cre enzyme induction; In described termination signal thereafter be one section intron sequences, express the miRNA precursor and insert in this intron;

In the present invention, EF1 α promotor has higher level and expresses and be not easy to be silenced;

In the present invention, EGFP fluorescent mark gene is convenient to evaluation and the genetically modified expression of real-time monitored of transgenic mice in the future;

In the present invention, the Cre enzyme can effectively mediate these two ends and be connected with the excision of the BGH polyA tailing transcription termination sequence of LoxP sequence in the same way, thereby effectively controls whether transcribing of 3 ' sequence;

In the present invention, to including subsequence, transform, introduce restriction enzyme site, thereby the miRNA precursor can be inserted to this site, realize effective shearing of miRNA precursor, do not disturb the EGFP fluorescence gene expression of its front.

In embodiments of the invention, adopt EF1 α, to realize the high expression level of miR424 locus, and to be silenced at the gene that turn in transgenic animal future in order preventing; Introduced EGFP fluorescent mark albumen, be convenient to identify transgenic mice and monitor in real time genetically modified expression; C) at the two ends of miRNA precursor, added respectively intron donor and receptor sequence (can using the miRNA precursor from transcript as intron excision, and be processed to form ripe body), make the tandem expression of miRNA not affect the expression of EGFP; Inserted one section PolyA tailing transcription termination signal between EGFP gene and miR424 precursor, and respectively add one section LoxP sequence at the tailing signal two ends, by the mouse with myocardium specifically expressing Cre, hybridize, can be by the special cyclisation excision in the myocardial cell of this PolyA tailing transcription termination signal, realization F1 for mouse cardiac muscle in the expression of the specific miRNA of special rise, realize the tissue-specific up-regulated of miRNA, and prevented that expression from causing the death of first-generation transgenic mice.

MicroRNA inducibility tissue specific expression carrier of the present invention shows that through experiment in vitro Cre has good inducibility to this miRNA expression vector.

In the present invention, added two LoxP71 and LoxP66 sites in the same way after EGFP, these two LoxP have the site of sudden change, guarantee that on LoxP residual after recombinating with cre and karyomit(e), other LoxP recombinates.The polyA sequence that has added BDH in the middle of these two LoxP sites, make microRNA before not recombinating with the cre enzyme not express, and avoided occurring in the transgenic mice preparation that F1 generation is because microRNA crosses the embryonic death of expressing and may causing.

Carrier of the present invention has following characteristics:

1) can solve the difficulty that some lethality miRNA transgenic animal is set up: the up-regulated of some miRNA has serious illness, for example miR-1 crosses the rhythm abnormality that causes of expression, so the up-regulated of these miRNA may be lethality for animal, so this class miRNA is not easy to obtain the high expression level transgenic mice, and this carrier transgenic mice is not before hybridizing with the Cre mouse, not express miRNA, so solved the difficult problem of setting up of lethality miRNA transgenic mice;

2) can realize easily sense of organization specifically expressing and the expression of etap property of specific miRNA: the foundation that routine is set up the tissue specific expression transgenic mice is by the using-system specificity promoter, this mode has its limitation, once transgenic animal are set up, the expression that realizes its hetero-organization that can not be comparatively motor-driven.And this carrier is set up transgenic mice, can be by selecting tissue specificity and etap specific C re mouse, motor-driven raises purpose miRNA at different tissues and different steps.

3) EF1 α promotor is selected and intron is expressed the mode of miRNA, is also the assurance that realizes the miRNA effective expression, and the expression that guarantees miRNA can not disturbed the expression of series connection mark;

The EGFP gene of 4) connecting with miRNA can be simplified transgenic animal screening and Real-Time Monitoring.

The present invention has determined the expression of microRNA by the carrier transfected HEK 293 of acquisition by Fluirescence observation and RT-PCR method.

The expression vector transfected E14 cell of the present invention to building, determined the expression of microRNA in mouse ES cells by Fluirescence observation and RT-PCR method.

The present invention prepares transgenic mice with the expression vector obtained, and at F1, the mouse with green fluorescence of survival is arranged in generation.

The microRNA expression vector built in the present invention can be successful the special microRNA of expression, and can determine by several different methods the expression of microRNA.Expression vector in the present invention is in the ES cell, and microRNA expresses and is not easy to be silenced, the preparation that is applied to transgenic mice that can be successful.

The accompanying drawing explanation

Fig. 1 shows the LPAL-mir-363 by pEFG, after pEFG LPAL-mir-363 cre, pEFG LPAL-mir-424, (used transfection reagent LTX) in plasmid transfection mouse stem cells E14 after pEFG LPAL-mir-424 cre, after 24h, by observing green fluorescence, differentiates plasmid expression.

Fig. 2 shows in the present invention extracting cell RNA after cell transfecting 48h, detects the expression of miR-363 and miR-424 after tailing method RT-PCR, and 18s does internal reference, and after cre, miRNA can express.

Fig. 3 show in the present invention specific intron primer loxp71 BGH F/loxp66 BGH T PCR show the miRNA shearing after intron diminish.

Embodiment

embodiment 1 builds pEFG LPAL-MIR

1) insert EF1 alpha promotor

Primer according to the design of pEBG plasmid sequence with corresponding restriction enzyme site, by EF1 alpha promoter sequence, through the PCR-enzyme, cut-ligation is building up in the pEGFP-c1 plasmid, compare EF1 alpha promoter sequence without sudden change through order-checking, and the plasmid called after pEFG that this is built.

2) insert the LoxP-polyA-LoxP sequence

The LoxP71 of each 35bp of chemosynthesis and LoxP66.The polyA1 sequence comes from BGH polyA sequence in pcDNA3.1 (+).Method series connection by 3 sections sequences with overlapping PCR, order is LoxP71-polyA1-LoxP66, and two LoxP sites maintenance cis-position directions, and by this segment DNA, through enzyme, cut-ligation is building up in the pEFG carrier, through the sudden change of order-checking comparison nothing, called after pEFG LPAL.

3) insert the intron sequence

Intron (intron) sequence comes from the pRL-renilla plasmid, intron D and intron R are introduced to Bgl II successively by overlapping PCR method centre, EcoR V, Sall I restriction enzyme site, and before the DNA of acquisition is inserted into to pEFGFP plasmid SV40 polyA sequence, the plasmid called after pEFG LPAL-MIR built, total length is that 6287bp(has the structure as sequence 1).

the application of embodiment 2 pEFG LPAL-MIR

1) build pEFG LPAL-mir-363 and pEFG LPAL-mir-424

Mir-363 and mir-424 precursor sequence are reacted to acquisition by PCR, and be inserted in pEFG LPAL-MIR carrier, through the sudden change of order-checking nothing.

2) plasmid is external recombinates with cre enzyme (NEB)

Reaction system (20ul):

Plasmid 5-10ng

10×Buffer 2ul

Cre 0.5ul

ddH2O to 20ul

37°C 30min，72°C 10min。

Transform the TOP10 competence, 37 ° of C 12h, clone PCR is identified (primer is loxp71 BGH F/loxp66 BGH T), without obvious band be the plasmid of cyclisation excision, and through the order-checking confirmation.

3) expression of pEFG LPAL-MIR in mouse embryo stem cell

As shown in Figure 1, by pEFG LPAL-mir-363, after pEFG LPAL-mir-363 cre, pEFG LPAL-mir-424, (use transfection reagent LTX) in plasmid transfection mouse stem cells E14 after pEFG LPAL-mir-424 cre, after 24h, by observing green fluorescence, differentiate plasmid expression.

As shown in Figure 2, extracting cell RNA after cell transfecting 48h detects the expression of miR-363 and miR-424 after tailing method RT-PCR, and 18s does internal reference, and after cre, miRNA can express.

As shown in Figure 3, after specific intron primer loxp71 BGH F/loxp66 BGH T PCR shows the miRNA shearing, intron diminishes.

embodiment 3 prepares transgenic mice

(ApaL I) after pEFG LPAL-mir-363 and pEFG LPAL-mir-424 plasmid linearization proceeded to according to a conventional method to the zygote of mouse, this zygote is implanted in the female mice body, the result demonstration, F1 is accredited as transgenic mice for mouse through egfp expression.

SEQUENCE LISTING

<110 > Fudan University

<120 > inducibility tissue specific expression carrier and uses thereof

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 6287

<212> DNA

<213> Artificial

<400> 1

tagttattat cgacattgat tattgactag ttattaatcc tcggcctctg agctattcca 60

gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagcttt gcaaagatgg 120

ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga 180

gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag 240

aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac 300

tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat 360

ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag 420

gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg 480

ccttgaatta cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt 540

tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg 600

agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc 660

ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac 720

gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc 780

ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag 840

gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc 900

tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc 960

ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag 1020

ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa 1080

aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc 1140

caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg 1200

gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg 1260

gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct 1320

caagcctcag acagtggttc aaagtttttt tcttccattt caggtgtcgt gaggaattcc 1380

tcgactagcg ctaccggtcg ccaccatggt gagcaagggc gaggagctgt tcaccggggt 1440

ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg 1500

cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg 1560

caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt 1620

cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg 1680

ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga 1740

ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa 1800

ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta 1860

tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat 1920

cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg 1980

ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc 2040

caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct 2100

cggcatggac gagctgtaca agtccggata gaatagtacc gttcgtatag catacattat 2160

acgaagttat catcatcacc atcaccattg agtttaaacc cgctgatcag cctcgactgt 2220

gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga 2280

aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag 2340

taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga 2400

agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac 2460

cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg 2520

tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt 2580

cgctttcttc ataacttcgt atagcataca ttatacgaac ggtatccgga ctcagatcct 2640

agataacttc tgacacaaca gtctcgaact taagctgcag aagttggtcg tgaggcactg 2700

ggcaggtaag tatcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcttgt 2760

cgagacagag aagactcttg cgtttctgat aggcaccaag tagatctgat atcgtcgacg 2820

cactattggt cttactgaca tccactttgc ctttctctcc acaggtgtcc actcccagtt 2880

caattacagc tcttaaggct agagtactta atacgactca ctatagctcg acggtaccgc 2940

gggcccgatc caccggatct agataactga tcataatcag ccataccaca tttgtagagg 3000

ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg 3060

caattgttgt tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca 3120

tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac 3180

tcatcaatgt atcttaaggc gtaaattgta agcgttaata ttttgttaaa attcgcgtta 3240

aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 3300

aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca 3360

ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 3420

ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta 3480

aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg 3540

gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg 3600

gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca 3660

ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 3720

tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 3780

aggaagagtc ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa 3840

agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa 3900

ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca 3960

attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 4020

gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 4080

ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 4140

tttgcaaaga tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga 4200

ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa 4260

cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt 4320

ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg 4380

ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa 4440

gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 4500

cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt 4560

gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact 4620

cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg 4680

ccagccgaac tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg 4740

acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc 4800

atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 4860

gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc 4920

gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg 4980

ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg 5040

attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct 5100

ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc 5160

taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa 5220

gacagaataa aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg 5280

gctggcactc tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt 5340

ccttttcccc accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg 5400

gggcggcagg ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac 5460

ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa 5520

tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat 5580

cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc 5640

taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg 5700

gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag ttaggccacc 5760

acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg 5820

ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg 5880

ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa 5940

cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg 6000

aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga 6060

gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct 6120

gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca 6180

gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc 6240

ctgcgttatc ccctgattct gtggataacc gtattaccgc catgcat 6287

Claims (5)

1. inducibility tissue specific expression carrier, is characterized in that, mainly comprises following structure: EF1 α promotor, EGFP fluorescent mark gene, BGH polyA tailing transcription termination sequence and intron sequences; Described carrier is pEFGLPAL-MIR, has the structure of sequence 1.

2. by inducibility tissue specific expression carrier claimed in claim 1, it is characterized in that, described BGHpolyA tailing transcription termination sequence is that two ends are connected with the transcription termination sequence of LoxP sequence in the same way.

3. by inducibility tissue specific expression carrier claimed in claim 1, it is characterized in that its inner restriction enzyme site of introducing of described intron sequences.

4. the construction process of the inducibility tissue specific expression carrier of claim 1, is characterized in that, it comprises step:

1) insert EF1 alpha promotor

The EF1alpha promoter sequence is inserted in the pEGFP-c1 plasmid, obtains plasmid pEFG;

2) insert the LoxP-polyA-LoxP sequence

Method series connection by LoxP71 and LoxP66 and BGH polyA sequence with overlapping PCR, obtaining order is the DNA section of LoxP71-polyA1-LoxP66, is inserted in the pEFG carrier, obtains plasmid pEFG LPAL;

3) insert the intron sequence

Intron sequences intron D and intron R are by introducing successively Bgl II in the middle of overlapping PCR method, EcoR V, Sall I restriction enzyme site, and before the DNA obtained is inserted to pEFG LPAL plasmid SV40polyA sequence, build to obtain pEFG LPAL-MIR, total length is 6287bp, has the structure as sequence 1.

5. by the method for claim 4, it is characterized in that, the LoxP71 of described step 2 and LoxP66 are chemosynthesis; Described two LoxP sites are the cis-position direction.

Images