CN102321655B - Pseudo-attP site based integrated general-purpose expression vector and construction method and application thereof - Google Patents

Abstract

The invention discloses a pseudo-attP site based integrated general-purpose expression vector and a construction method and application thereof. The expression vector comprises an attB sequence, pUC ori and multiple cloning sites MCS, wherein a CMV (cytomegalovirus) constitutive promoter is connected with the attB sequence, and two equidirectional LoxP elements are inserted into the backwards position of the CMV constitutive promoter; and a first fluorescent indicator protein open reading frame and a terminator thereof are arranged at the backwards positions of the LoxP elements, and a second fluorescent indicator protein open reading frame and a terminator thereof and antibiotic expression elements are arranged between the two LoxP elements. The vector can be used to screen recombinant cells stably expressing fluorescently-marked proteins without antibiotic screening markers.

Description

A kind of universal expression vector and construction process and application of integrating based on false attP site

Technical field

The invention belongs to the genetically engineered field, relate to a kind of expression vector of site-specific integration, particularly a kind of universal expression vector and construction process and application of integrating based on false attP site.

Background technology

Focus in the present scientific research just of the research of transgenic animal.Pass through transgenic technology, between can breaking kind, people isolate the economic animal that obtains to have good character, can obtain various biomaterials and pharmaceutical protein by producing bio-reactor, can break immunological rejection and carry out organ transplantation, can set up animal model and the analysis and research specific gene function of various human diseasess.Yet, still there is certain drawback in existing transgenic animal production technology: the position effect that the insertion at random of foreign gene brings, be that foreign gene is inserted into the gene inside relevant with the important vital movement of cell at random and causes its unconventionality expression, perhaps foreign gene inserts the heterochromatic zone and causes the exogenous gene expression silence.

In recent years, streptomycete phage phi C31 intergrase in can catalysis streptomyces gene group the attB site and noticeable [the Kuhstoss S of homologous recombination between the phage genome attP site, Rao RN.Analysis of the integration function of the streptomycete bacteriophage φ C31.JMol Biol, 1991,222 (4): 897-908.].The integrase mediated reorganization of φ C31 has unidirectional integration, need not extraneous chemical energy source and cofactor, competent big fragment gene effectively integrate, can play a role in multiple higher plant and zooblast and foreign gene such as can efficiently express for a long time at characteristics [Calos MP.The φ C31integrase system for gene therapy.Curr Gene Ther, 2006,6 (6): 633-645.], become the strong instrument of another genetic modification after Cre recombinase and FLP recombinase, more and more be used for transgenic research.

And relevant false attP site is studies show that aspect the genome distribution rule, and false attP site multidigit is between gene or in the gene intron, integrate rare in the exon of gene [Thyagarajan B, Liu Y, Shin S, Lakshmipathy U, Scheyhing K, Xue H

C, Strehl R, Hyllner J, Rao MS, Chesnut JD.Creation of engineered human embryonic stem cell lines using φ C31integrase.Stem Cells, 2008,26 (1): 119-126.].Thisly be partial to being integrated with of transcriptionally active district and be beneficial to expression of gene, this might be the reason that integrase mediated integration is compared with random integration generally the long period high level expression [Calos MP.The φ C31integrase system for gene therapy.Curr Gene Ther, 2006,6 (6): 633-645.].And, integration site does not prefer to transcription initiation site [Chalberg TW, Portlock JL, Olivares EC, Thyagarajan B, Kirby PJ, Hillman RT, Hoelters J, Calos MP.Integration specificity of phage φ C31integrase in the human genome.J Mol Biol, 2006,357 (1): 28-48.], [Sivalingam J such as Sivalingam, Krishnan S, Ng WH, Lee S S, Phan TT, Kon OL.Biosafety assessment of site-directed transgene integration in human umbilical cord-lining cells.Mol Ther, 2010,18 (7): 1346-1356.] find that the integration site above 70% is positioned at beyond the transcription initiation site 50kb.Therefore, from this respect, the integrase mediated potential potential safety hazard of integration of φ C31 inserts more at random and virus carrier system is much smaller.

The effectively section of having that somatic cell nuclear transfer technique is produced as transgenic animal, its outstanding advantage is that transgenosis is advanceed to the somatic cell culture stage, can effectively improve natality and control production cost [the M.B.Wheeler and E.M.Walters.Transgenic technology and applications in swine.Theriogenology2001 of transgenic animal, 56 (8): 1345-1369], but the preparation of nuclear donor cell generally need be passed through drug screening, and antibiotic-screening mark residual in the final transgenic cell reaches environmental safety safely to transgenic animal and caused potential threat.

Although in the transgenic animal production process, the residual attention that causes people gradually of antibiotic-screening mark, wherein, the Cre/LoxP system is maximum is selected to cut off selection markers [Wang S, Sun X, Ding F, Zhang K, Zhao R, Li S, Li R, Tang B, Zhang L, Liu Y, Li J, Gao F, Wang H, Wang L, Dai Y, Li N.Removal of selectable marker gene from fibroblast cells in transgenic cloned cattle by transient expression of Cre recombinase and subsequent effects on recloned embryo development.Theriogenology.2009,72 (4): 535-41.].Yet in view of Cre/LoxP system dynamics, reversible reorganization characteristic, after introducing the Cre recombinase-mediated and cutting off, need a plurality of positive colonies of picking to carry out that PCR identifies and Southern blotting assesses the recombination efficiency of Cre.But these a series of conventional sense means workloads are huge, and expend the plenty of time.Given this, a kind of effective detection and indication Cre/LoxP cut off system's exigence of efficient for the transgenic animal production process.

Summary of the invention

The problem that the present invention solves is to provide a kind of universal expression vector and construction process and application of integrating based on false attP site, overcome the defective that existing gene integration and antibiotic-screening aspect exist, for body-cell neucleus transplanting makes up the nuclear donor cell that the transgene clone embryo provides the antibiotic-free selection markers, improve the security that transgenic animal produce.

The present invention is achieved through the following technical solutions:

A kind of universal expression vector of integrating based on false attP site, comprise attB sequence, pUC ori and multiple clone site MCS, the CMV constitutive promoter is connected with the attB sequence, two LoxP elements are in the same way inserted in CMV constitutive promoter downstream, be provided with first fluorescence indicator protein open reading frame and the terminator thereof in LoxP element downstream, between two LoxP elements, be provided with the second fluorescence indicator protein open reading frame and terminator thereof and antibiotic-screening element.

Described attB sequence is shown in SEQ.ID.NO.1, and described CMV constitutive promoter is Pcmv IE promotor.

Described multiple clone site MCS comprises following restriction enzyme enzyme recognition site:

MluI, BamHI, PacI, PspOMI, SacI, FseI, AclI, XhoI, PvuI, KpnI, HindIII, BglII and EcoRI.

Described two LoxP element and interior outside enzymes thereof are in the same way cut the sequence of recognition site shown in SEQ.ID.NO.2.

The described first fluorescence indicator protein open reading frame is green fluorescent protein eGFP open reading frame, and the second fluorescence indicator protein open reading frame is red fluorescent protein DsRed open reading frame, and its terminator is the Sv40 terminator.

Described antibiotic-screening element is the KanR/neoR Expression element, stops the CMV constitutive promoter the first fluorescence indicator protein to be leaked the Sv40 terminator of expressing also to be provided with one between two LoxP elements.

A kind of construction process of the universal expression vector of integrating based on false attP site may further comprise the steps:

1) by the SOE-PCR method, the attB sequence of AseI restriction enzyme site and protection base is contained at the two ends of clone's acquisition shown in SEQ.ID.NO.1;

2) by the SOE-PCR method, the clone obtains shown in SEQ.ID.NO.2 two LoxP element and the interior outside enzyme LoxP2 sequences of cutting recognition site thereof in the same way;

3) by the SOE-PCR method,, the clone obtains the MCS sequence that comprises a plurality of cloning sites shown in SEQ.ID.NO.3;

4) with BamHI/BglII double digestion pEGFP-C1 carrier, gel obtains the pEGFP-B2 carrier after reclaiming carrier framework fragment and connection;

By the AseI restriction enzyme site attB sequence clone is gone into the pEGFP-B2 carrier and obtain the pAttB-eGFP carrier;

By the NheI/AgeI restriction enzyme site LoxP2 sequence clone is gone into the pAttB-eGFP carrier and obtain the pAttB-Loxp2-eGFP carrier;

5) by the SpeI/AflII restriction enzyme site, to comprise the prokaryotic promoter of KanR/neoR gene open reading frame, expressing K anR gene, the SV40 early promoter of expressing the neoR gene and the KanR/neoR Expression element of HSV TK polyA terminator, and be cloned into and obtain the pAL2G-neo carrier in the pAttB-Loxp2-eGFP carrier;

And then by the AflII/NotI restriction enzyme site Sv40pA terminator is cloned into the pAL2G-neo carrier, obtain the pAL2G-neopA carrier;

6) with MluI/EcoO109I difference double digestion MCS sequence and pAL2G-neopA carrier, gel reclaims the fragment after enzyme is cut, and obtain the pANG-MCS carrier after the fragment that will reclaim connects then;

7) by the AscI/SpeI restriction enzyme site DsRed1 open reading frame and terminator sequence clone thereof are gone into the pANG-MCS carrier, obtain the universal expression vector pARNG that integrates based on false attP site.

The described universal expression vector of integrating based on false attP site is in the application of the reconstitution cell of the antibiotic-free selection markers of screening stably express fluorescent mark albumen.

The described universal expression vector of integrating based on false attP site carries out drug screening after carrying out cell transfecting, obtain the reconstitution cell of expression vector stable transfection; Then it is carried out the processing that the LoxP sequence is sheared in the Cre reorganization, the reconstitution cell of screening stably express first fluorescin, the reconstitution cell of the antibiotic-free selection markers of fluorescent mark albumen is expressed in acquisition.

Being treated to of LoxP sequence sheared in described Cre reorganization:

Reconstitution cell after the drug screening is comprised the transfection of the expression vector of Cre element, perhaps it is hatched in containing the environment that is connected with the Cre recombinase of wearing the film peptide.

Compared with prior art, the present invention has following beneficial technical effects:

1, the universal expression vector of integrating based on false attP site provided by the invention, after carrying out cell transfecting, integrase mediated down at streptomycete phage phi C31, can be incorporated into false attP site in the gene of eucaryote cell group locus specificity, its distribution be partial between gene or gene intron in the zone of transcriptionally active, reticent and other potential safety hazards of the genetic expression of having avoided plasmid to insert at random causing, thus make that transgenosis continues to efficiently express.

2, the universal expression vector of integrating based on false attP site provided by the invention, because LoxP-second fluorescent mark-microbiotic circuit elements design, can carry out drug screening by G418, obtain not only to have the positive cell clone of G418 resistance but also stably express red fluorescent protein, got rid of on the one hand because indivedual wild-type cell is cloned the false positive of the insensitive formation of G418, the continuing to efficiently express and can reflect that also integration site is positioned at karyomit(e) transcriptionally active district of red fluorescent protein on the other hand, screening is suitable for the recombinant chou that transgenosis efficiently expresses.At this moment, because two effects of terminator between the LoxP in the same way, the first fluorescent mark green fluorescent protein is not expressed.

3, the universal expression vector of integrating based on false attP site provided by the invention, after positive reconstitution cell is obtained in screening, by Cre reorganization shearing action, remove two the antibiotic-screening mark between the LoxP, red fluorescent protein gene and terminators in the same way, make CMV promotor and eGFP gene splicing, green fluorescent protein is expressed, and prompting Cre recombinase cuts off successfully, and reconstitution cell is converted to green fluorescent protein as cue mark.

By the screening (selected by flow cytometry apoptosis) to green fluorescent protein, obtain the transgenic cell of the antibiotic-free selection markers of stably express green fluorescent protein (genetically modified organism label) then, can be used as the nuclear donor cell and produce transgenic animal by SCNT.So both pass through antibiotic pressure and selected to obtain to express stable cell, and then again it had been excised and had converted to the green fluorescence mark, got rid of the influence of antibiotic-screening mark to transgenic animal safety and environmental safety.

4, the universal expression vector of integrating based on false attP site provided by the invention, at problem such as vector integration site, transgene expression level and antibiotic-screening mark be residual, can be applicable to make up the nuclear donor cell that the transgene clone embryo provides the antibiotic-free selection markers for body-cell neucleus transplanting, thereby the safe cultivation system of the transgenic animal of setting up a kind of antibiotic-free selection markers provides valuable technology platform for carrying out the animal transgenic research.

Description of drawings

Fig. 1-a～Fig. 1-e is the detected through gel electrophoresis figure of SOE-PCR amplification attB fragment, LoxP2 fragment and MCS fragment;

Fig. 2 is construction process schema of the present invention;

Fig. 3 is carrier system pie graph of the present invention;

Fig. 4 a～Fig. 4-c cuts the evaluation of evaluation and pcr amplified fragment for the enzyme of vector construction intermediate carrier of the present invention;

Fig. 5 a～Fig. 5-b is that the enzyme of expression vector pARNG is cut evaluation;

The two fluorescently-labeled indication situation that makes up among the expression vector pARNG after Fig. 6 recombinates for fluorescent microscope detects;

The two fluorescently-labeled indication situation that makes up among the expression vector pARNG after Fig. 7 recombinates for Western Blotting detects;

Fig. 8 a～Fig. 8-f is that flow cytometer detects the two fluorescently-labeled indication situation that makes up among the back expression vector pARNG that recombinates.

Embodiment

The universal expression vector of integrating based on false attP site provided by the invention, this carrier system can be under the mediation of streptomycete phage phi C31 intergrase, be incorporated into false attP site in the gene of eucaryote cell group locus specificity, thereby make that transgenosis continues to efficiently express, the problem of resolved vector integration site, transgene expression level; Then after drug screening obtains positive cell, excision antibiotic-screening mark under the effect that the Cre reorganization is sheared, and detect by two fluorescence report system and to cut off efficient, finally go out to cut off fully the transgenic cell of selection markers through selected by flow cytometry apoptosis, be used for body-cell neucleus transplanting, produce the transgenic animal of antibiotic-free selection markers, thereby improve the transgenic animal security, provide valuable technology platform for carrying out the animal transgenic research.Below in conjunction with the structure of concrete carrier with detect that the present invention is described in further detail, the explanation of the invention is not limited.

A kind of construction process of the universal expression vector of integrating based on false attP site may further comprise the steps:

1) by the SOE-PCR method, the attB sequence of AseI restriction enzyme site and protection base is contained at the two ends of clone's acquisition shown in SEQ.ID.NO.1; Concrete operations are:

A, with four couples of primer attB1 F/attB1 R, attB2F/attB2R, attB3F/attB3R, attB4F/attB4R be by the synthetic fragment attB1 of SOE-PCR method, attB2, attB3 and attB4 (Fig. 1-a); The used template of this step SOE-PCR is primer itself, as attB1F/attB1R itself be primer also be template, because these two pairs of primers have the reverse complementary sequence of 15-20bp, complementary sequence combination during annealing, under the effect of Taq enzyme, extend the polishing fragment during PCR, form the template of next round SOE-PCR;

attB1F：ttcgacgcgt?agttattaat?gtcgacgatg?taggtcacgg?tctcgaagcc?gcgg

attB1R：cgcgcccggg?gagcccaagg?gcacgccctg?gcacccgcac?cgcggcttcg?agac

attB2F：ggctccccgg?gcgcgtactc?cacctcaccc?atctggtcca?tcatgatgaa?cggg

attB2R：gcgccgcgcg?ttcgccggga?tcaactaccg?ccacctcgac?ccgttcatca?tgat

attB3F：gcgaacgcgc?ggcgcaccgg?gaagccctcg?ccctcgaaac?cgctgggcgc?ggtg

attB3R：acccgccgac?gccgtcgcac?gtcccgtgct?caccgtgacc?accgcgccca?gcgg

attB4F：acggcgtcgg?cgggtgcgga?tacgcggggc?agcgtcagcg?ggttctcgac?ggtc

attB4R：aacgacgcgt?tactattaat?gtcgacatgc?ccgccgtgac?cgtcgagaac?ccgc

B, being template with fragment attB1 and attB2 respectively, is primer with attB1F and attB2R, by the synthetic fragment attB12 of SOE-PCR method (Fig. 1-b); Equally, be template withfragment attB 3 and attB 4, be primer with attB3F and attB4R, by the synthetic fragment attB34 of SOE-PCR method (Fig. 1-b);

C, being template with fragment attB12 and attB34 at last, is primer with attB1F and attB4R, and by the synthetic attB sequence of SOE-PCR method, its concrete sequence is (Fig. 1-c) shown in SEQ.ID.NO.1.

Because the attB sequence derives from streptomycete phage phi C31 genome originally, consider the difficulty of template preparation, the attB sequence so employing SOE-PCR increases, the sequence that contains the AseI restriction enzyme site simultaneously in its two ends design, so that be cloned into universal expression vector, thereby realize its site-specific integration.

2) by the SOE-PCR method, the clone obtains shown in SEQ.ID.NO.2 two LoxP element and the interior outside enzyme LoxP2 sequences of cutting recognition site (inboard recognition site is AscI, SpeI, AflII and NotI, and site, the outside is NheI and AgeI) thereof in the same way; Concrete operations are:

A, with primer LoxP2F1 and LoxP2R1 by the synthetic fragment LoxP1 of the method for SOE-PCR (Fig. 1-a); The used template of this step SOE-PCR is primer itself, as LoxP2F1/LoxP2R1 itself be primer also be template, because these two pairs of primers have the reverse complementary sequence of 15-20bp, complementary sequence combination during annealing, under the effect of Taq enzyme, extend the polishing fragment during PCR, form the template of next round SOE-PCR;

B, be template with fragment LoxP1, LoxP2F1 and LoxP2R2 are primer, (Fig. 1-b), specifically shown in SEQ.ID.NO.2, wherein the LoxP sequence is ataacttcgt atagcataca ttatacgaag ttat to its sequence by the synthetic fragment LoxP2 of the method for SOE-PCR;

LoxP2F1：ctagctagct?agataacttc?gtatagcata?cattatacga?agttatttgg?cgcgccttg

LoxP2R1：tagtttagcg?gccgcaaatg?ccttaagatg?gactagtcca?aggcgcgcca?aataac

LoxP2R2：gcgaccggta?gataacttcg?tataatgtat?gctatacgaa?gttatagttt?agcggccgc

The LoxP2 sequence that obtains by SOE-PCR amplification: 1. obtain two LoxP sequences in the same way, be used for realizing that the Cre reorganization cuts off; Two in the same way the LoxP sequence outside increase NheI and the AgeI restriction enzyme site makes the LoxP2 sequence can be cloned into carrier; 3. between two LoxP sequences in the same way, increase the insertion that AscI, SpeI, AflII and NotI restriction enzyme site are convenient to KanR/neoR Expression element, DsRed open reading frame and SV40 terminator.

3) by the SOE-PCR method, the clone obtains the MCS sequence that comprises a plurality of cloning sites shown in SEQ.ID.NO.3, wherein contain 13 restricted endoenzymes and cut recognition site, be successively: MluI, BamHI, PacI, PspOMI, SacI, FseI, AclI, XhoI, PvuI, KpnI, HindIII, BglII and EcoRI; What it was concrete is operating as:

A, with two couples of primer MCS1F/MCS1R and MCS2F/MCS2R by the synthetic fragment MCS1 of SOE-PCR method and MCS2 (Fig. 1-d); The used template of this step SOE-PCR is primer itself, as MCS1F/MCS1R itself be primer also be template, because these two pairs of primers have the reverse complementary sequence of 15-20bp, complementary sequence combination during annealing, under the effect of Taq enzyme, extend the polishing fragment during PCR, form the template of next round SOE-PCR.

MCS1F：tcgacgcgtc?gcggatccgt?gccttaatta?aggactgggc?ccattgcgag?ctc

MCS1R：ctcgagggtg?caacgttcgc?ttggccggcc?ttgaccgagc?tcgcaatggg?ccc

MCS2F：aacgttgcac?cctcgagcgg?atcgatcgat?tggggtaccc?cgccaagctt?gg

MCS2R：atggcagggc?ctgccggaat?tcggtgaaga?tcttctccca?agcttggcgg?gg

Being template with fragment MCS1 and MCS2 respectively, is primer with MCS1F and MCS2R, by the synthetic fragment MCS of SOE-PCR method (Fig. 1-e);

Carrying out above-mentioned steps 1)～3) amplification the time, fragment for its amplification is carried out detected through gel electrophoresis simultaneously, and its detected result is shown in Fig. 1-a～1-e: swimminglane 1～5 is respectively detected through gel electrophoresis fragment attB1 (93bp), attB2 (93bp), attB3 (93bp), attB4 (91bp) and LoxP1 (97bp) among Fig. 1-a;Swimming lane 1,2 is detected through gel electrophoresis fragment attB12 (171bp) among Fig. 1-b, andswimming lane 3,4 is fragment attB34 (169bp), and swimming lane 5,6 is fragment LoxP2 (141bp);Swimming lane 1,2 is detected through gel electrophoresis fragment attB (325bp) among Fig. 1-c;Swimming lane 1,2 is respectively gel electrophoresis electrophoresis detection fragment MCS1 (89bp) and MCS2 (89bp) among Fig. 1-d;Swimming lane 1,2 is detected through gel electrophoresis fragment MCS (161bp) among Fig. 1-e; MCS is detected primer adopts following primer:

EGFP-C?for?primer：catggtcctg?ctggagttcg?tg

pUC_ori_rev?primer：ggtctgacgc?tcagtggaac?g

Referring to vector construction schema shown in Figure 2, the structure flow process of each element of carrier in carrier is described below:

4) with BamHI/BglII double digestion pEGFP-C1 carrier (Clontech), remove the multiple clone site of carrier itself, gel reclaims the carrier framework fragment, and 4 ℃ of connections of spending the night obtain recombinant vectors pEGFP-B2;

With BamHI/BglII double digestion pDsRed1-C1 carrier (Clontech), remove the multiple clone site of carrier itself, gel reclaims the carrier framework fragment, and 4 ℃ of connections of spending the night obtain recombinant vectors pDsRed1-B2;

The AgeI/HindIII double digestion of above-mentioned two carriers is identified shown in Fig. 4-a, the wherein negative contrast of 1 swimming lane, be AgeI/HindIII double digestion plasmid pEGFP-C1, can cut out 3900bp and 800bp two bands, andswimming lane 2 and 3 is respectively AgeI/HindIII double digestion plasmid pEGFP-B2 and pDsRed1-C1, owing to removed the multiple clone site that contains HindIII, be about the single band of 4700bp so can only cut out length;

By the AseI restriction enzyme site attB sequence fragment that increases is cloned into the pEGFP-B2 carrier then and obtains the pAttB-eGFP carrier; Enzyme is cut evaluation shown in Fig. 4-b: design a pair of detection primer TaseI F and TaseI R in AseI recognition site both sides, andswimming lane 2 negative contrast pEGFP-C1, the PCR product is 135bp, andswimming lane 1 is pAttB-EGFP, and the PCR product is 432bp; As follows to the detection primer that AseI single endonuclease digestion site insertion sequence is identified:

TaseI?F：cttttgctca?catgttcttt?cc

TaseI?R：tgtaacgcgg?aactccatat?a

By the NheI/AgeI restriction enzyme site LoxP2 sequence fragment that increases is cloned into the pAttB-eGFP carrier and obtains the pAttB-Loxp2-eGFP carrier;

5) by the amplification KanR/neoR expressed intact element of PCR method from the carrier pEGFP-C1 (comprise KanR/neoR gene open reading frame, the prokaryotic promoter of expressing K anR gene, express SV40 early promoter and the HSV TK polyA terminator of neoR gene); The primer that amplification is adopted is to as follows:

Kneo?F：ggactagtgc?gtcaggtggc?acttttcg

Kneo?R：agccttaagc?cccgacgttg?gctg

Pass through the SpeI/AflII restriction enzyme site then, the KanR/neoR Expression element is cloned into obtains the pAL2G-neo carrier in the pAttB-Loxp2-eGFP carrier; Enzyme is cut evaluation shown in Fig. 4-c: obtain 5091bp and 1745bp two bands;

Sv40pA terminator on the carrier pEGFP-C1 is increased, by the AflII/NotI restriction enzyme site Sv40pA terminator is cloned into the pAL2G-neo carrier again, obtain the pAL2G-neopA carrier;

6) with MluI/EcoO109I difference double digestion MCS sequence fragment and pAL2G-neopA carrier, remove the KanR/neoR Expression element (being the KanR/neoR Expression element that carries on the skeleton carrier pEGFP-C1) in last two the LoxP sequences of the carrier pAL2G-neopA outside, gel reclaims the fragment after enzyme is cut, and will obtain the pANG-MCS carrier after the fragment connection (4 ℃ are spent the night) of reclaiming then;

7) method of PCR is from carrier pDsRed1-B2 amplification DsRed1 open reading frame and terminator thereof, and the primer that amplification is adopted is as follows:

DsRedpA?F：ataggcgcgc?caccatggtg?cg

DsRedpA?R：ccggactagt?cattgatgag?tttggacaaa?ccac

By the AscI/SpeI restriction enzyme site DsRed1 open reading frame and terminator sequence clone thereof are gone into the pANG-MCS carrier then, obtain the universal expression vector pARNG that integrates based on false attP site.

Carry out following enzyme for constructed expression vector pARNG and cut evaluation:

AscI/SpeI double digestion qualification result is shown in Fig. 5-a, wherein,swimming lane 1 is identified carrier pARNG for the AscI/SpeI double digestion, obtains about 5000bp and 900bp two bands, swimminglane 2 is identified carrier pARNG for the AscI single endonuclease digestion, and obtaining length is the single band of 5918bp.

Qualification result shown in Fig. 5-b, wherein,swimming lane 1,2 for the plasmid pARNG that is untreated as negative control, swimminglane 3 be AscI single endonuclease digestion linearized vector pARNG as positive control, obtain the single band of 5918bp; Swimming lane 4 is AseI single endonuclease digestion carrier pARNG, obtains 5621bp and 297bp two bands, proves that attB successfully is connected to the AseI site of carrier pARNG; Swimming lane 5-17 is respectively the single endonuclease digestion of MCS recognition site, all obtains the single band of 5918bp, proves that 13 restriction enzyme enzyme recognition sites all are unique in the carrier pARNG multiple clone site.

Constructed its system's pie graph of universal expression vector pARNG as shown in Figure 3, (pUC ori is the replication origin of plasmid comprising attB sequence, pUC ori, for plasmid can copy amplification in bacterium, derive from the pEGFP-C1 carrier) and multiple clone site MCS; Homologous recombination can take place with false attP site (pseudo attP) in the gene of eucaryote cell group in the attB sequence under the mediation of streptomycete phage phi C31 intergrase, thereby be integrated into the transcriptionally active district in the gene of eucaryote cell group with making carrier pARNG locus specificity, be conducive to transgenosis and continue to efficiently express; Multiple clone site MCS comprises 13 unique restriction enzyme enzyme recognition sites, is used for the clone of goal gene element;

Also be provided with sequencing primer EGFP-C for primer and pUC ori rev primer for the order-checking of MCS site insertion sequence simultaneously;

CMV constitutive promoter (deriving from the pEGFP-C1 carrier) is connected with the attB sequence, two LoxP elements are in the same way inserted in CMV constitutive promoter downstream, be provided with first fluorescence indicator protein open reading frame (eGFP) and the terminator thereof in LoxP element downstream, between two LoxP elements, be provided with the second fluorescence indicator protein open reading frame (DsRed) and terminator and KanR/neoR Expression element, also be provided with one simultaneously and stop the CMV constitutive promoter the first fluorescence indicator protein to be leaked the Sv40 terminator of expressing; Two LoxP circuit elements design are in order to carry out the screening of positive cell under medicine pressure, and after screening successfully, excise by the Cre recombinase, remove the antibiotic-screening mark, and make CMV promotor and eGFP gene splicing, be converted to green fluorescent protein as cue mark:

Expression vector is incorporated into by the attB sequence after the false attP of the gene of eucaryote cell group site, through G418 drug screening, obtains not only to have the positive cell clone of G418 resistance but also stably express red fluorescent protein; Handle positive cell clone with the Cre recombinase then, remove two the antibiotic-screening mark between the LoxP, red fluorescent protein gene and terminators in the same way, and make CMV promotor and eGFP gene splicing, green fluorescent protein is expressed, prompting Cre recombinase cuts off successfully, the transgenic cell of the antibiotic-free selection markers of screening stably express green fluorescent protein.

The following describes constructed pARNG carrier in the application of the transgenic cell of the antibiotic-free selection markers of screening stably express green fluorescent protein, specifically comprise the operation by cell cultures, transfection, fluorescent screening.

1, based on the transient transfection cell, to the detection of fluorescent marker

1) preparation of host cell

The HEK293 cell inoculation in the DMEM nutrient solution that contains 10% foetal calf serum, is put into 37 ℃, 5%CO with 6 well culture plates of inoculating cell₂In the incubator.Treat to carry out when cell grows to 70%-90% and converges rate transfection.

2) preparation of transfection composite

A. prepare 6 aseptic centrifuge tubes of 1.5mL, add 200 μ l Opti-MEM substratum respectively;

B. in centrifuge tube, add Opti-MEM (1 μ l), pARNG plasmid vector (1 μ g), pDsRed1-C1 plasmid vector (1 μ g), pEGFP-C1 plasmid vector (1 μ g), pARNG plasmid vector (1 μ g)+pCAG-Cre-IP plasmid vector (1 μ g), pCAG-Cre-IP plasmid vector (1 μ g) respectively;

PCAG-Cre-IP plasmid vector (Li P, Tong C, Mehrian-Shai R, Jia L, Wu N, Yan Y, Maxson RE, Schulze EN, Song H, Hsieh CL, Pera MF, Ying QL.Germline competent embryonic stem cells derived from rat blastocysts.Cell.2008Dec26; 135 (7): adding 1299-310.) is to shear for the reorganization that realizes the Loxp sequence of Cre;

C. of short duration soft vortex (being no more than 10s);

D. add 8 μ l X-tremeGENE HP DNA Transfection Reagent respectively;

E. of short duration soft vortex;

F. room temperature (15 ℃-25 ℃) is hatched 15-30min.

3) transfection composite is dropwise added in the cell of 6 well culture plates successively, and at 6 well culture plates difference mark HEK293, pARNG, pDsRed1-C1, pEGFP-C1, pARNG+pCAG-Cre-IP, pCAG-Cre-IP;

4) behind the incubated cell 24h, the reconstitution cell that will carry out different reorganization schemes carries out following detection:

A, reconstitution cell is observed under the inverted fluorescence microscope respectively.The result is as shown in Figure 6: negative control group HEK293 does not all observe fluorescence under green and blue excitation light; Experimental group pARNG and positive controls pDsRed1-C1 can observe red fluorescence under green exciting light, and do not observe green fluorescence under blue excitation light; This shows that first fluorescent mark this moment (green fluorescence) is not expressed, and second fluorescent mark (red fluorescence) is expressed, and second fluorescent mark (red fluorescence) can be used as the fluorescence indication of pARNG carrier reorganization;

Positive controls pEGFP-C1 does not observe red fluorescence and expresses under green exciting light, and can observe green fluorescence under blue excitation light, and control group pCAG-Cre-IP is all not luminous after being excited; Experimental group pARNG+pCAG-Cre-IP observes red fluorescence and expresses under green exciting light, under blue excitation light, observe green fluorescence, two of this explanations sequence between the Loxp element are in the same way cut off by Cre reorganization, the green fluorescence mark is activated son and activates expression, thereby observes green fluorescence.

The two fluorescence report systems of above detected result proof can be used as the indication that positive-selecting and Cre enzyme are cut Loxp-microbiotic element: red fluorescence can be used as the mark of transfection success, and green fluorescence can be used as the mark that the microbiotic element is sheared.

B, the cell of reorganization is respectively applied to Western Blotting detects.Collect and respectively organize cell in the PBS of 1mL precooling solution, centrifugal 5 minutes of 1000rpm, supernatant discarded, 60 μ l Western and IP cell pyrolysis liquid (the green skies) re-suspended cell, add cOmplete Protease Inhibitor Cocktail Tablets (Roche) and be adjusted to working concentration, cracking 30min on ice, whole protein is added sample-loading buffer, 100 ℃ of sex change 5min, 12% SDS-PAGE gel electrophoresis is transferred to sample on the pvdf membrane from the SDS-PAGE gel by semidrying then, 4 ℃ of night incubation of primary antibodie, washing, the two anti-2.5h of hatching, the exposure of eECL Western Blot Kit highly sensitive chemiluminescence detection kit is used in the washing back.

Two is anti-: HRP-labeled Goat Anti-Mouse IgG (H+L) (the green skies), HRP-labeled Goat Anti-Rabbit IgG (H+L) (the green skies).

Western Blotting detected result is as shown in Figure 7: with Living

DsRed Polyclonal Antibody (Clontech) carries out Western blotting as primary antibodie and detects, sample pARNG, pDsRed1-C1 and pARNG+pCAG-Cre-IP detect the specific band that size is about 28KD, prompting RFP (red fluorescent protein) is expressed, and sample HEK293, pEGFP-C1 and pCAG-Cre-IP do not detect specific band, and RFP is not expressed in prompting;

With Living

GFP Monoclonal Antibody (Clontech) carries out Western blotting as primary antibodie and detects, sample pEGFP-C 1 and pARNG+pCAG-Cre-IP detect the specific band that size is about 30KD, prompting GFP is expressed, and other samples all do not detect specific band, and GFP is not expressed in prompting; GAPDH is as the standardized confidential reference items of Western blot protein.

Above Western blotting detected result is consistent with the fluorescent microscope observed result, and two fluorescence report system can be used as the indication that positive-selecting and Cre enzyme are cut Loxp-microbiotic element.

C, the cell of reorganization is used for fluidic cell detects, prepare single cell suspension with trypsin digestion, cell counting is used for BD FACSAria flow cytometry analysis.

The result shows: Fig. 8-a sample HEK293 detects less than any fluorescent signal; Fig. 8-b sample pARNG only detects the red fluorescence signal; Fig. 8-c sample pDsRed1-C1 only detects the red fluorescence signal; Fig. 8-d sample pEGFP-C1 only detects the green fluorescence signal; Fig. 8-e sample pARNG+pCAG-Cre-IP can detect a small amount of red fluorescence signal and a large amount of green fluorescence signal; Fig. 8-f sample pCAG-Cre-IP detects less than any fluorescent signal.The flow cytometry conclusion is consistent with fluorescent microscope observed result and Western Blotting detected result.

The two fluorescence report systems of above detected result proof can be used as the indication of positive-selecting and Cre excision Loxp-microbiotic element.

2, the screening of the transgenic cell of the antibiotic-free selection markers of stably express green fluorescent protein:

1) mensuration of G418 minimum lethal concentration: the G418 that adds the different concns gradient in the nutrient solution (the DMEM cell culture fluid that contains serum) of bovine fetal fibroblast (host cell) respectively screened 12 days, measured Normocellular G418 minimum lethal concentration.When the concentration of G418 in the cell culture fluid 〉=600 μ g/ml, all dead at the microscopically visible cell, so the minimum lethal concentration of G418 is 600 μ g/ml.

2) cell transfecting and screening:

Treat that host cell grows to 70%-90% and converges rate, use X-tremeGENE HP DNA Transfection Reagent transfection reagent, host cell is carried out the transfection of pARNG expression vector;

Because universal expression vector pARNG comprises G418 resistant gene neoR, under the positive cell that is incorporated into the neoR gene on the genome can be survived in the nutrient solution that contains finite concentration G418, and the normal cell of untransfected can be killed by this medicine.

The G418 that adds final concentration behind the universal expression vector pARNG transfection bovine fetal fibroblast 24h and be minimum lethal concentration (600 μ g/ml) in containing the DMEM cell culture fluid of serum screens; With the negative contrast of the bovine fetal fibroblast of untransfected, its cell culture fluid is added with the G418 of same concentration.

Behind the transfectional cell G418 screening 12d, the cell of control group is all dead; The positive reconstitution cell of the transfection that shows as the G418 resistance is observed under fluorescent microscope, observed the second fluorescence indicator protein RFP and whether express.To not only having the positive cell clone of G418 resistance but also stably express red fluorescent protein, the G418 concentration of substratum is reduced by half lasting screening at the bottom of cell covers with ware, use the pancreas enzyme-EDTA peptic cell then, add the not DMEM cell culture fluid enlarged culturing of added with antibiotic again.

3) the Cre recombinase is handled:

The positive cell that not only has G418 resistance but also stably express red fluorescent protein is inoculated in the DMEM nutrient solution that contains 10% foetal calf serum, 6 well culture plates of inoculating cell are put into 37 ℃, 5%CO₂In the incubator.Treating to carry out when cell grows to 70%-90% and converges rate the Cre recombinase handles.

3.1Cre plasmid transfection sieve method:

A. prepare the aseptic centrifuge tube of 1.5mL, add 200 μ l Opti-MEM substratum;

B. in centrifuge tube, add pCAG-Cre-IP plasmid vector (2 μ g);

C. of short duration soft vortex (being no more than 10s);

D. add 8 μ l X-tremeGENE HP DNA Transfection Reagent respectively;

E. of short duration soft vortex;

F. room temperature (15 ℃-25 ℃) is hatched 15-30min.

G. transfection composite is dropwise added in the cell of 6 well culture plates;

H. behind the incubated cell 24h, the cell of the antibiotic-free mark that obtains recombinating.

I. will observe the reconstitution cell that the screening green fluorescence is expressed under the reconstitution cell inverted fluorescence microscope;

J. the cell with reorganization is used for Western Blotting detection, the reconstitution cell that the screening green fluorescence is expressed;

K. the cell with reorganization is used for the reconstitution cell that fluidic cell sorting green fluorescence is expressed, and finally obtains the positive cell of antibiotic-free selection markers.

3.2Cre protein transduction method:

In the Cre treatment step, express the plasmid vector random integration of Cre recombinase in the positive cell genome, bring unnecessary risk, can use and be connected with in the Cre recombinase adding DMEM cell culture fluid of wearing the film peptide, hatch 24h, carry out observation detection and the fluidic cell sorting of green fluorescent protein then, finally obtain the positive cell of antibiotic-free selection markers.

Claims (10)

1. universal expression vector of integrating based on false attP site, it is characterized in that, comprise attB sequence, pUC ori and multiple clone site MCS, the CMV constitutive promoter is connected with the attB sequence, two LoxP elements are in the same way inserted in CMV constitutive promoter downstream, be provided with first fluorescence indicator protein open reading frame and the terminator thereof in LoxP element downstream, between two LoxP elements, be provided with the second fluorescence indicator protein open reading frame and terminator thereof and antibiotic-screening element;

Described attB sequence is shown in SEQ.ID.NO.1;

The ordering of said structure fragment as shown in Figure 3.

2. the universal expression vector of integrating based on false attP site as claimed in claim 1 is characterized in that described CMV constitutive promoter is Pcmv IE promotor.

3. the universal expression vector of integrating based on false attP site as claimed in claim 1 is characterized in that described multiple clone site MCS comprises following restriction enzyme enzyme recognition site:

MluI, BamHI, PacI, PspOMI, SacI, FseI, AclI, XhoI, PvuI, KpnI, HindIII, BglII and EcoRI.

4. the universal expression vector of integrating based on false attP site as claimed in claim 1 is characterized in that described two LoxP element and interior outside enzymes thereof are in the same way cut the sequence of recognition site shown in SEQ.ID.NO.2.

5. the universal expression vector of integrating based on false attP site as claimed in claim 1, it is characterized in that, the described first fluorescence indicator protein open reading frame is green fluorescent protein eGFP open reading frame, the second fluorescence indicator protein open reading frame is red fluorescent protein DsRed open reading frame, and its terminator is the Sv40 terminator.

6. the universal expression vector of integrating based on false attP site as claimed in claim 1, it is characterized in that, described antibiotic-screening element is the KanR/neoR Expression element, also is provided with one between two LoxP elements and stops the CMV constitutive promoter the first fluorescence indicator protein to be leaked the Sv40 terminator of expressing.

7. the construction process based on the universal expression vector of false attP site integration is characterized in that, may further comprise the steps:

1) by the SOE-PCR method, the attB sequence of AseI restriction enzyme site and protection base is contained at the two ends of clone's acquisition shown in SEQ.ID.NO.1;

2) by the SOE-PCR method, the clone obtains shown in SEQ.ID.NO.2 two LoxP element and the interior outside enzyme LoxP2 sequences of cutting recognition site thereof in the same way;

3) by the SOE-PCR method, the clone obtains the MCS sequence that comprises a plurality of cloning sites shown in SEQ.ID.NO.3;

4) with BamHI/BglII double digestion pEGFP-C1 carrier, gel obtains the pEGFP-B2 carrier after reclaiming carrier framework fragment and connection;

By the AseI restriction enzyme site attB sequence clone is gone into the pEGFP-B2 carrier and obtain the pAttB-eGFP carrier;

By the NheI/AgeI restriction enzyme site LoxP2 sequence clone is gone into the pAttB-eGFP carrier and obtain the pAttB-Loxp2-eGFP carrier;

5) by the SpeI/AflII restriction enzyme site, to comprise the prokaryotic promoter of KanR/neoR gene open reading frame, expressing K anR gene, the SV40 early promoter of expressing the neoR gene and the KanR/neoR Expression element of HSV TK polyA terminator, and be cloned into and obtain the pAL2G-neo carrier in the pAttB-Loxp2-eGFP carrier;

And then by the AflII/NotI restriction enzyme site Sv40pA terminator is cloned into the pAL2G-neo carrier, obtain the pAL2G-neopA carrier;

6) with MluI/EcoO109I difference double digestion MCS sequence and pAL2G-neopA carrier, gel reclaims the fragment after enzyme is cut, and obtain the pANG-MCS carrier after the fragment that will reclaim connects then;

7) by the AscI/SpeI restriction enzyme site DsRed1 open reading frame and terminator sequence clone thereof are gone into the pANG-MCS carrier, obtain the universal expression vector pARNG that integrates based on false attP site.

8. the described universal expression vector of integrating based on false attP site of claim 1 is in the application of the reconstitution cell of the antibiotic-free selection markers of screening stably express fluorescent mark albumen.

9. application as claimed in claim 8 is characterized in that, the universal expression vector of integrating based on false attP site carries out drug screening after carrying out cell transfecting, obtain the reconstitution cell of expression vector stable transfection; Then it is carried out the processing that the LoxP sequence is sheared in the Cre reorganization, the reconstitution cell of screening stably express first fluorescin, the reconstitution cell of the antibiotic-free selection markers of fluorescent mark albumen is expressed in acquisition.

10. application as claimed in claim 9 is characterized in that, being treated to of LoxP sequence sheared in described Cre reorganization:

Reconstitution cell after the drug screening is comprised the transfection of the expression vector of Cre element, perhaps it is hatched in containing the environment that is connected with the Cre recombinase of wearing the film peptide.

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