CN102827873A - Cre fusion protein functional test cell and modified Cre fusion protein - Google Patents

Abstract

The invention discloses a Cre fusion protein functional test cell and a modified Cre fusion protein. The Cre fusion protein functional test cell takes an HEK (human embryonic kidney) 293 cell as a host cell, a pAcmy (African cassava mosaic virus)-stop-EGFP (enhanced green fluorescent protein) carrier and phi C31 integrase are used for co-transfection of the host cell. Through the action of the phi C31 integrase, the carrier which carries an LoxP (locus of X-over P1) element having a Cre recombinase detection function is integrated to a pseudo attP (attachment P) site of an HEK 293 cell line in a single copying manner, so a stable and reliable cell model is provided for researching the Cre fusion protein membrane penetrating efficiency and biological activity. The Cre fusion protein HNTC (hemagglutinin-neuraminidase cytotoxic T) which is modified by TAT (transactivator) and NLS (nuclear localization signal) polypeptides has high membrane penetrating efficiency and good biological activity, can be effectively used as a tool for genetic manipulation at a mammalian cell chromosome level.

Description

Cre fusion rotein after a kind of Cre fusion rotein Function detection cell and the modification

Technical field

The invention belongs to recombination function and detect the cell technology field, relate to a kind of Cre fusion rotein Function detection cell and modify after the Cre fusion rotein.

Background technology

Cre belongs to recombinase family; It is to derive from the P1 phage; Can act on mammalian cell and cause dna sequence dna specificity recombinant protein (Nagy A:Cre recombinase:the universal reagent for genome tailoring.Genesis 2000; 26:99-109), it is to be the albumen of 38kD by the phage-coded size of P1, can discern the loxp site of 34bp size.Recombinase is not strict to the configuration requirement of substrate, can be super coiled DNA, can be linear DNA yet.Under the effect of Cre enzyme, two in the same way the dna fragmentation between the loxp site will be deleted; DNA between two reverse loxp sites (the Hoess R H that will overturn; Abremski K.Mechanism of strand cleavage and exchange in the Cre lox site specific recombination system [J] .MolBiol; 1985,181 (3): 351-362.) genetic recombination of Cre enzyme mediation can fix a point, regularly, the specificity control of fixed tissue.This system was found in 1981 first; 1993 by (Gu H such as Gu; Zou Y R; Rajewsky K.Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-LoxP-mediated gene targeting [J] .Cell; 1993,73 (6): be widely used in research field such as gene clone, gene trap, gene integration, gene elmination since 1155-1164.) researchist formally uses as a kind of genetic manipulation means and embodied huge advantage.

The Cre recombinase is through accurately cutting DNA and being connected again; Can mediate target DNA comprises inversion, transposition, cuts off or the rearrangement of form such as integration; Thereby make and the eukaryotic gene group is carried out genetic modification, and transgenic animal and plant transfer expression of gene effectively controlled become possibility in the karyomit(e) level.This system has become the strong instrument that the aspects such as site-directed integration, gene trap and chromosome engineering of evaluation, the foreign gene of deletion at specific gene, gene function are studied at present, and is being widely used aspect genetically modified yeast, plant, insect, the DNA reorganization of mammiferous inside and outside.

Therefore, be necessary to make up a kind of mammalian cell and detect clone.The element of the detection cre/loxp system efficiency that is used at present to make up; Mostly be with blocking with the placed in-line transcription terminator of a plurality of cis in the middle of a constitutive promoter and a kind of reporter gene; There are two LoxP in the same way the terminator both sides; Through modes such as cell transfecting or virus transductions, random integration (or additive method) or fixed point are practiced shooting on the mammalian cell genome then.But this random integration (or additive method) possibly produce following situation: insert sudden change 1.: the integration of virus vector mediation causes inserting sudden change easily and causes that it is failure that cell is built; 2. gene silencing: and the random integration of non-virus carrier also is easily integrated into the heterochromatic zone, causes transgene silencing, makes that newly-established clone can't the normal expression reporter gene, and can not normally indicate the cre cutting efficiency; 3. non-single copy is integrated: under the prerequisite of the preceding two kinds of situation of eliminating; Suppose that non-virus carrier random integration site all is positioned at the transcriptionally active district; Integrate if non-single copy has taken place, except possibly influencing the cell normal growth, unnecessary LoxP sequence also can have influence on the checking of Cre recombinase function.

And in mammalian cell; Streptomycete phage c31 intergrase is proved to be the intergrase that can react with specific sequence in the genomic dna (being false attP site); It can stably be incorporated into foreign gene on genomic those the specific sequences of mammalian cell; It is partial to be incorporated into genome transcriptionally active district with single copy, inserts the transgenation risk that causes at random thereby greatly reduced foreign gene.The cytogene group-specific site of found c31 intergrase identification has more than 100; Bioinformatic analysis to integration site shows that they have security (T.W.Chalberg preferably; Et al.Integration specificity of phiC31 integrase in the human genome.J Mol Biol.2006.357.28-48); Point out it as a useful instrument that in mammalian cell, carries out genetic manipulation and gene therapy; Be suitable for verifying that Cre wears the structure of film peptide efficient clone; I.e. mediation contains the carrier of verifying element and is incorporated into mammalian genes group transcriptionally active district with single form that copies, thereby normally exercises the function that its checking Cre fusion rotein is worn film ability and BA.

Summary of the invention

The problem that the present invention solves be a kind of Cre fusion rotein Function detection cell and modify after the Cre fusion rotein, with checking Cre fusion rotein wear membrane efficiency and biological activity, thereby optimize of the application of Cre fusion rotein in genes involved engineering field.

The present invention realizes through following technical scheme:

A kind of Cre fusion rotein Function detection carrier; Comprise Pcmv IE promotor; The upper reaches in Pcmv IE promotor are provided with the attB sequence; The downstream of Pcmv IE promotor are provided with two Loxp sequences in the same way, are provided with BGH terminator, BGH terminator and SV40A terminator successively between two Loxp sequences in the same way, and the downstream of Loxp sequence are provided with fluorescent mark gene and antibiotic-screening gene; When the Loxp sequence and between three terminators behind Cre recombinase effect down cut, Pcmv IE promotor and fluorescent mark gene splicing start the fluorescent mark expression of gene.

Described carrier is pAcmv-stop-EGFP, comprises the following element and the order of connection: attB sequence-Pcmv IE promotor-Loxp sequence-BGH terminator-BGH terminator-SV40A terminator-Loxp sequence-fluorescent mark gene-antibiotic-screening gene;

Described fluorescent mark gene is read frame for the EGFP gene, and the antibiotic-screening gene is neo, and this carrier is called carrier.

The nucleotide sequence of said pAcmv-stop-EGFP is shown in SEQ.ID.NO.1.

A kind of Cre fusion rotein Function detection cell, host cell is the HEK293 cell, with pAcmv-stop-EGFP carrier and phiC31 intergrase expression vector pCMVint cotransfection host cell; Utilize the reconstitution cell of the positive transfection of G418 resistance screening, and screen attB-CMV-stop-EGFP expression vector wherein is integrated into the false attP of host cell site with the form of single copy cell.

The application of described Cre fusion rotein Function detection cell in detecting Cre fusion rotein shear active.

Described Cre fusion rotein is the Cre fusion rotein of wearing film peptide and nuclear localization signal modification with protein transduction function.

A kind of through TAT and the peptide modified Cre fusion rotein of NLS, begin to be respectively 6 * His zone, NLS polypeptide zone, TAT polypeptide zone and Cre enzyme zone from the N end; Wherein the sequence of the amino-acid residue in NLS polypeptide zone is: Pro-Lys-Lys-Lys-Arg-Lys-Val, the sequence of the amino-acid residue in AT polypeptide zone is: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg.

The preparation method of the Cre fusion rotein that described TAT and NLS are peptide modified, concrete steps are following:

1) pcr amplification obtains having the Cre nucleotide sequence of SacI and XhoI recognition sequence; After amplified production cut through SacI and XhoI enzyme; Recovery is connected on the same prokaryotic expression carrier pET28a (+) that cuts through SacI and XhoI enzyme, obtains the pET28a-Cre recombinant plasmid;

2) produce by primer annealing and have the NLS-TAT nucleotide sequence of NdeI and SacI recognition sequence sticky end, and be connected to and pass through the pET28a-Cre recombinant plasmid that NdeI and SacI enzyme are cut, obtain the pET28a-HNTC recombinant plasmid;

3) with the pET28a-HNTC recombinant plasmid transformed in e. coli strain bl21, select 5-8 male colony inoculation in the LB of kalamycin resistance substratum, at 16～22 ℃ with 0.2mM IPTG abduction delivering Cre fusion rotein;

4) the proteic bacterium of abduction delivering is through the ultrasonic disruption thalline, discharge fusion rotein after, inhale column purification through the affine layer of Ni ion chelating, obtain the albumen behind the purifying.

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

Cre fusion rotein Function detection carrier provided by the invention and cells exclude: 1. insert sudden change: the integration of virus vector mediation causes inserting sudden change easily and causes that it is failure that cell is built; 2. gene silencing: and the random integration of non-virus carrier also is easily integrated into the heterochromatic zone, causes transgene silencing, makes that newly-established clone can't the normal expression reporter gene, and can not normally indicate the cre cutting efficiency; 3. non-single copy is integrated: under the prerequisite of the preceding two kinds of situation of eliminating; Suppose that non-virus carrier random integration site all is positioned at the transcriptionally active district; Integrate if non-single copy has taken place, except possibly influencing the cell normal growth, unnecessary LoxP sequence also can have influence on the checking of Cre recombinase function.

Screening provided by the invention obtain through TAT and the peptide modified Cre fusion rotein HNTC of NLS, it wears the membrane efficiency height, biological activity is good, can be effective as the instrument of genetic manipulation on the mammalian cell karyomit(e) level.

Description of drawings

Fig. 1 is that Cre recombinase functional verification clone makes up synoptic diagram and principle of work thereof;

Fig. 2 is a checking carrier pACMV-stop-EGFP function on cell levels;

Fig. 3-1 is the logarithm (log of average Ct value to the sample copy number ₂N) mapping obtains the absolute quantitation typical curve;

Fig. 3-2 is the solubility curve analytical results of specificity amplification primer TGFP;

Fig. 4-1～4-2 is to detecting clone 293-C31-L2GFP functional verification;

Fig. 5-1～5-5 is a Cre Expression of Fusion Protein purifying.Wherein Fig. 5-1 is the Cre fusion rotein synoptic diagram of the various combination of TAT and NLS modification; Fig. 5-2 cuts evaluation figure for the Cre fusion protein prokaryotic expression carrier enzyme of preparation different modifying; Fig. 5-3 is to be the protein purification outcome procedure of example with fusion rotein His-NLS-TAT-Cre (HNTC); Fig. 5-4 is the SDS-PAGE detected result of various cre fusion roteins; Fig. 5-5 is the immunoblotting result who utilizes the specificity antibody of cre recombinase.

Fig. 6-1 is that the fluorescence situation is expressed in the 293-C31-L2GFP effect for microscopic examination different modifying Cre recombinant protein pair cell;

Fig. 6-2 is the recombination efficiency of flow cytometry detection by quantitative different modifying Cre fusion rotein.

Embodiment

Below in conjunction with concrete embodiment the present invention is done further detailed description, said is to explanation of the present invention rather than qualification.

1.Cre foundation and the functional verification of recombinase functional verification clone 293-C31-L2GFP

1.1 the structure of carrier pACMV-stop-EGFP

It is following to be used to set up the carrier pACMV-stop-EGFP building process that detects cre recombinase active clone:

With carrier pcdna3.1 (+) (Invitrogen company) is template, obtains two sections BGH terminator sequences through PCR method amplification, one section fragment P1 that has AscI and SpeI restriction enzyme site wherein, and its amplimer is:

Upstream primer: TTGGCGCGCCACCCGCTGATCAG;

Downstream primer: CCGGACTAGTGGTTCTTTCCGCCTCAGAAGCC;

Another section has the fragment P2 of SpeI and AflII restriction enzyme site, and its amplimer is:

Upstream primer: GGACTAGTACCCGCTGATCAGCC;

Downstream primer: AGACTTAAGTGGTTCTTTCCGCCTC;

With carrier pEGFP-C1 is template, increases to such an extent that two ends are the SV40A terminator sequence P3 of AflII and NotI restriction enzyme site through PCR method, and its amplimer is:

Upstream primer: CCATCTTAAGTGATCATAATCAGCCATACCAC;

Downstream primer: AGACTGCGGCCGCTAAGATACATTG.

Above three terminators that increase are cloned among the carrier pAttB-LoxP2-eGFP two corresponding MCSs between the LoxP sequence in the same way in the same way in order, obtain carrier pACMV-stop-EGFP.Its plasmid map is as shown in Figure 1; The attB sequence is positioned at the upper reaches of Pcmv promotor; The downstream of Pcmv promotor are provided with two Loxp sequences in the same way; Be provided with BGH terminator, BGH terminator and SV40A terminator successively between two Loxp sequences in the same way, the downstream of Loxp sequence are provided with fluorescent mark gene EGFP and antibiotic-screening gene neo.The element order of connection is attB sequence-Pcmv IE-Loxp sequence-BGH terminator-BGH terminator-SV40A terminator-Loxp sequence, and its nucleotide sequence is shown in SEQ.ID.NO.1.

1.2 carrier pACMV-stop-EGFP functional verification

Press table 1 grouping transfection HEK293 cell (available from Chinese Academy of Sciences typical case culture collection council cell bank), checking carrier pACMV-stop-EGFP fluorescence report function from the cell levels.

The grouping transfection of table 1 different carriers

Below respectively organize plasmid with X-tremeGENE HP DNA Transfection Reagent test kit (Roche company) transient transfection HEK293 clone, observe each experimental group luciferase expression situation down at inverted fluorescence microscope (Nikon Eclipse Ti) behind the 24h.Detected result is as shown in Figure 2, and BLK is for using H ₂O replaces plasmid to carry out transfection as negative control, and (wavelength 420～485nm) excites and do not observe fluorescence down the result at blue light; PAcmv-stop-EGFP and the independent transfection group of pCAG-Cre-IP plasmid are at the blue-light excited green fluorescence of not observing down; This stop cassette that shows that 3 * polyA terminator cis connects and composes plays and stops Pcmv IE promotor to the EGFP gene transcription, and pCAG-Cre-IP can expressing green fluorescent protein yet in addition; And pAcmv-stop-EGFP and pCAG-Cre-IP plasmid co-transfection group can be observed stronger green fluorescence signal under blue-light excited; Prompting Cre recombinase is expressed the back stop cassette between two LoxP sequences on the pAcmv-stop-EGFP carrier is sheared; Make Pcmv IE promotor and EGFP gene read the frame splicing, thus great expression green fluorescent protein (referring to shear history shown in Figure 1).This shows that the active measuring element of checking Cre among the carrier pAcmv-stop-EGFP can be used for subsequent detection.

1.3 the foundation of clone 293-C31-L2GFP

1) mensuration of G418 minimum lethal concentration: the G418 that in the nutrient solution (the DMEM cell culture fluid that contains serum) of HEK293 cell (host cell), adds the different concns gradient respectively screened 8 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 cre Function detection plasmid pACMV-stop-EGFP and phiC31 intergrase expression vector pCMVint cotransfection;

Because cre Function detection plasmid pACMV-stop-EGFP 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 in containing the DMEM cell culture fluid of serum, adds final concentration behind the transfection HEK293 clone 36h and be minimum lethal concentration (600 μ g/ml) screens; With the negative contrast of HEK293 cell of untransfected, its cell culture fluid is added with the G418 of same concentration.

Behind the transfectional cell G418 screening 8d, the cell of control group is all dead; The positive reconstitution cell of the transfection that shows as the G418 resistance reduced by half the G418 concentration of substratum continues screening at the bottom of cell covers with ware, uses the pancreas enzyme-EDTA peptic cell then, adds the not DMEM cell culture fluid enlarged culturing of added with antibiotic again.

1.4 the copy number of reconstitution cell is identified (based on the absolute quantitation method of real-time quantitative PCR technology)

1) standard substance preparation

1. real-time fluorescence quantitative PCR. adopt reaction system (the SYBR Premix Ex Taq of 20 μ l PCR ^TM, TaKaRa) press StepOne plus quantitative fluorescent PCR (ABI company) service manual setting, 95 ℃ of 10s with condition; 95 ℃ of 5s, 60 ℃ of 30s (40 circulations); 95 ℃ of 15s; 60 ℃ of 30s; 95 ℃ of 15s. reaction systems mixing and seal in 96 orifice plates (BIOplastics EU 0.2mL Thin-wall 8-tube strip) with ultra-clean light lid (BIOplastics EU Optical Wide area 8-cap strip). reaction result uses StepOne Software v2.1 to collect, analyze. and all samples are all done 3 repetitions in same block of plate, get average.

2. the foundation of typical curve. according to P.Song (SONG P; CA I C Q; SKOKUTM; Et al.Qurntitative real-time PCR as a screening tool for estimating transgene copy number inWH ISKERSTM-derived transgenic maize [J] .Plant Cell Rep, 2002,20 (10): the 948-954.) method of structure standard substance; The plasmid pACMV-stop-EGFP that will contain foreign gene mixes with the HEK293 cell genomic dna; Setting contains the standard control of 0.5,1,2,4,8 and 16 transgenic copy number, and is specific as follows: suppose that the plasmid size that contains transgenic fragment is a bp, HEK293 cell genomic dna consumption is b ng; Transgenic fragment completely random ground (or the head links to each other) from beginning to end inserts on the karyomit(e), because human gene group DNA monoploid (haploid) size is 3 * 10 ⁹Bp, the transgenic positive HEK293 cell genomic dna of b ng contains the quality of transgenic copy and is so:

$\frac{a\×b\×0.5}{3\×{10}^9}\mathrm{ng}.$

Concrete is that the TGFP primer sequence is (amplified production is 129bp) with primer TGFP amplification GFP gene fragment:

Upstream primer: CGACGGCAACTACAAGAC;

Downstream primer: TAGTTGTACTCCAGCTTGTGC.

2) detected result of copy number of foreign gene

1. absolute quantitation typical curve. for the relation of confirming that Ct value and initial copy are counted N in the Real-timePCR amplification; Transgenic plasmid and wild-type HEK293 cell genomic dna is mixed; Be provided with the standard control that contains 0.5,1,2,4,8,16 copy respectively; With primer TGFP amplification exogenous genetic fragment (129bp), the equal Ct value of making even is with the logarithm (log of average Ct value to the sample copy number ₂N) mapping obtains absolute quantitation typical curve (Fig. 3-1), and quantitatively the Equation for Calculating formula is: log2N=-1.3448Ct+37.548, coefficient of determination R ²=0.9987, show that standard substance prepare successfully.For the specificity of assessing reaction and identify product, carrying out melt curve analysis analysis (Fig. 3-2) after the amplification. have and have only near about 88 ℃ one unimodal, the specificity of primer amplification is very good.

2. the evaluation of transgenic copy number. the HEK293 cell clone of getting different reorganization respectively carries out real-time quantitative PCR; Amplification GFP gene; And carrying out repeated experiments 3 times, numerical value is represented with

(mean+SD).Real-time PCR between 0.06 to 0.11, shows that the result is accurately credible to reorganization HEK293 cell clone qualification result standard deviation, and the result is: No. 2, reconstitution cell and No. 5 clones integrate for single copy; No. 1, No. 4 and No. 6 is that two copies are integrated; No. 3 the clone is the integration of 3 copies.

1.5 the half-nest type inverse PCR is identified integration site

In view of above analytical results, No. 2 clones and No. 5 clones of getting single copy integration carry out the integration site analysis.

10 μ g genomic dnas are with isocaudarner NheI and 37 ℃ of digested overnight of SpeI.After ethanol sedimentation, phenol/chloroform extracting, the genomic dna that digested is with 4 ℃ of connections of spending the night of 1000 T4-DNA of unit ligase enzymes (NEB company).After connecting product process ethanol sedimentation, phenol/chloroform extracting, be dissolved in 20 μ l TE buffer and process template.

Template to make is carried out first round PCR with primer HNIF1:CCCATAGTAACGCCAATA (upper reaches) and HNIR:CGATGTAGGTCACGGTCT (downstream), is undertaken by following condition: 94 ℃ of 5min, 1 circulation; 94 ℃, 30s, 51-61 ℃ 45s, 72 ℃ of 5min, 30 circulations; 72 ℃, 10min, 1 circulation obtains first round PCR product.

1 μ l first round PCR product as second take turns PCR template, increase with primer HNIF2:TAGCGGTTTGACTCACG (upper reaches) and HNIR:CGATGTAGGTCACGGTCT (downstream), by following reaction: 94 ℃ of 5min, a circulation; 94 ℃, 30s, 50-60 ℃ 45s, 72 ℃ of 5min, 30 circulations; 72 ℃, 10min.

Second takes turns the PCR product carries out agarose gel electrophoresis, reclaims connection pMD9-T carrier and send order-checking.Sequencing result carries out BLAST and analyzes on NCBI.

BLAST result shows, in No. 2 reconstitution cells, the integration incident occurs on No. 10 karyomit(e) (Fig. 4-1), is positioned at the INPP5A gene; And in No. 5 reconstitution cells, the integration incident occurs on No. 22 euchromosomes (Fig. 4-2), the intervening sequence between CRYBA4 gene and MN1 gene, and all distance meets false attP site characteristic greater than 300kb.

More than comprehensive, will carry out follow-up test with No. 5 clones of the reconstitution cell called after 293-C31-L2GFP that single copy form is incorporated into the false attP of HEK293 cellular genome site.

1.6 detect clone 293-C31-L2GFP functional verification:

1) preparation of host cell

The HEK293 cell line cell is inoculated in respectively in the DMEM nutrient solution that contains 10% foetal calf serum, the 35mm petridish of inoculating cell is put into 37 ℃, 5%CO ₂In the incubator.Treat to carry out when cell grows to 70%-90% and converges rate transfection.

2) preparation of transfection composite

A. prepare 2 aseptic centrifuge tubes of 1.5mL, add 200 μ l Opti-MEM substratum respectively;

B. the adding that in centrifuge tube, adds Opti-MEM (1 μ l), pCAG-Cre-IP plasmid vector (1 μ g) pCAG-Cre-IP plasmid vector respectively is in order to realize that Cre shears the reorganization of Loxp sequence;

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 HEK293 cell of 35mm petridish successively, and on petridish, distinguish mark " HEK293 " and " HEK293+CrePL ".

4) same method pair cell is that 293-C31-L2GFP carries out transient transfection, corresponding Tissue Culture Dish marked " 293-C31-L2GFP " and " 293-C31-L2GFP+CrePL ".

5) behind the incubated cell 36h, will carry out the reconstitution cell of different reorganization schemes and under inverted fluorescence microscope, observe, the result is shown in Fig. 4-2.

The fluorescent microscope observed result shows: a.293-C31-L2GFP cell does not originally show any fluorescence under inverted fluorescence microscope is blue-light excited; And after process is expressed Cre recombinase plasmid pCAG-Cre-IP transient transfection; The Cre recombinase-mediated excises two Stop Cassette between the LoxP in the same way; Make CMV promotor and EGFP read the frame splicing; Expressing green fluorescent protein, therefore under fluorescent microscope through observing green fluorescence under blue-light excited.Above result proves that clone 293-C31-L2GFP can be used for verifying the function of Cre recombinase active.

2. Cre expressing fusion protein, purifying and the functional verification of modifying based on TAT and NLS

The polypeptide (TAT, aminoacid sequence are YGRKKRRQRRR) that the proteic 47-57 amino acids of HIV-1 virus TAT is formed can form the protein product that merges with goal gene, directly shifts the protein transduction technology that gets into cell.

Nuclear localization signal (nuclear localization signal; NLS) be a kind ofly to be present in the cell in the numerous protein and aminoacid sequence that mediating protein is transported in nucleus by tenuigenin; Wherein be present in the large T antigen of simian virus 40 (SV40); Be one and comprise 7 amino acid whose small peptides (NLS, aminoacid sequence are PKKKRKV).

TAT or NLS can be used for constituting cre and wear the film peptide, and its effect is respectively that mediation cre fusion rotein passes cytolemma effectively and is delivered to nucleus getting into cytoplasmic cre fusion rotein, thereby function takes place the LoxP target sequence on the identification karyomit(e).This protein transduction technology because shift an instantaneous existence, get into degradable activated protein form in the cell, thereby greatly reduce the unfavorable aspect of other transgenosis approach.And fusion rotein function conformation senior with it is closely related; And only be difficult to by rule of thumb from protein prediction not the cre of isomorphic map wear the film peptide and mammalian cell, wear membrane efficiency and biological activity; So be necessary the function of the Cre fusion rotein that more various TAT, NLS modify, thereby filter out a kind of most effective, active best cre fusion rotein.

2.1 various Cre expressing fusion protein and purifying based on TAT and NLS modification

Consider the needs of various cre Expression of Fusion Protein purifying, adopt essentially identical affinity chromatography method, so the label of 6 Histidines is set.Designed its different modification protocols according to TAT, permutation and combination that NLS is different with Cre, specifically shown in Fig. 5-1, wherein: H is 6 * His, and N is NLS, and T is TAT, and C is the Cre enzyme; Shearing splicing through each Expression element after clear and definite its modification protocols can be accomplished modification.The splicing that mainly wherein a kind of below HNTC (its aminoacid sequence is shown in SEQ.ID.NO.2) is modified to its element of example explanation prepares scheme.

With plasmid pCAG-Cre-IP (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.2008 Dec 26; 135 (7): 1299-310) be template, the condition of the pcr amplified reaction of amplification Cre: 95 ℃, 30 seconds; 55 ℃, 30 seconds; 72 ℃, 1 minute 30 seconds; Primer sequence is following:

Cresx?F：TTC? ATGTCCAATTTACTGACCG

Cresx?R：CCG?

CTAATCGCCATCTTC

5 ' end contains SacI and XhoI restriction enzyme site respectively in the primer, is respectively goal gene cre recombinase 5 thereafter and ' holds and 3 ' terminal sequence contains 50mmol/L KCL in the reaction system of 50 μ l; 10mmol/L Tris-CL, (pH8.5), 1; 5mmol/L MgCl2; 200 μ mol/L dntp, 10pmol primer, the LATaq archaeal dna polymerase of 1U (TaKaRa Company products).On Veriti 96-well Thermal Cycler (ABI company) PCR appearance, press 30 circulations of following conditioned response: 95 ℃, 30 seconds; 55 ℃, 30 seconds; 72 ℃, 1 minute 30 seconds.

The DNA glue that amplified production is given birth to worker company with Shanghai reclaims the test kit purifying; After respectively amplification PCR products and plasmid pET-28a (+) (Novagen Company products) being carried out double digestion digestion with restriction enzyme SacI and XhoI, reclaim dna fragmentation respectively, be connected to expression vector plasmid pET-28a (+) with the dna ligation kit of TaKaRa company; 16 ℃ of connections are after 16 hours; Connect product and use transformed into escherichia coli DH5a, after the flat board of the LB substratum of kantlex (final concentration is 50 μ g/mL) spends the night, the positive colony enzyme that screens cut evaluation after; And check order, obtain the pET28a-Cre recombinant plasmid.

The NLS-TAT nucleotide sequence that has NdeI and SacI recognition sequence sticky end is produced by primer annealing, is directly connected to the pET28a-Cre recombinant plasmid that process NdeI and SacI enzyme are cut, and obtains the pET28a-HNTC recombinant plasmid.The dna sequence analysis result shows that aminoacid sequence and the predefined modification protocols after the translation of PCR product representative is identical.Then with recombinant plasmid transformed e. coli bl21 (DE3) (Novagen company).In the LB liquid nutrient medium that contains kantlex (final concentration 50 μ g/mL), host bacterium BL21 (pET28a-HNTC) is cultured to logarithmic phase at 37 ℃, and adding IPTG is 0.2mM to final concentration, and 18 ℃ are continued to cultivate 30 hours.Centrifugal collection thalline destroys thalline with the broken bacterium appearance of UW, and centrifugal collection supernatant is with can (6 * HIS-Tag) bonded affinity columns (Novagen Company products) have obtained the cre fusion rotein of purifying with the Histidine polypeptide.Through the SDS-PAGE electrophoresis, obtain single band (Fig. 5-3) at about 38kDa place.Collect the purified proteins sample, utilize the ultrafiltration post to concentrate the cre fusion rotein, be stored in-70 ℃ of refrigerators to 2mg/mL.

Use method likewise, the splicing order that changes element just can prepare the recombinant plasmid pET28a-HNTC that expresses HTNC, HTCN, HNCT, HCNT, HCTN, HTC, HNC, HCN, HCT, WTCre fusion rotein, pET28a-HTNC pET28a-HTCN; PET28a-HNCT, pET28a-HCNT, pET28a-HCTN; PET28a-HTC, pET28a-HNC, pET28a-HCN; PET28a-HCT, pET28a-WTCre; Expression respectively then, purifying and the concentrated various fusion roteins that obtain.

Fig. 5-2 cuts evaluation figure for the Cre fusion protein prokaryotic expression carrier enzyme of preparation different modifying; Fig. 5-3 is to be the protein purification outcome procedure of example with fusion rotein His-NLS-TAT-Cre (HNTC); Fig. 5-4 is the SDS-PAGE detected result of various cre fusion roteins; The immunoblotting result of specificity antibody who utilizes the cre recombinase is shown in Fig. 5-5.

2.2 above various Cre fusion rotein functions are verified based on clone 293-C31-L2GFP

Inoculation detects clone 293-C31-L2GFP in 12 porocyte culture plates of the DMEM cell culture fluid that contains 10% foetal calf serum, 37 ℃, cultivates in the 5%CO2 cell culture incubator.When cell density reaches 50-70%, add respectively with hatching altogether 3 hours in the filtering various Cre fusion roteins adding cell culture fluids of 0.22 μ m filter, PBS changes the normal cell nutrient solution after cleaning, 36 observations as a child.The DNA recombination function (Fig. 6-1) of Cre fusion rotein transduction in mammalian cell that under inverted fluorescence microscope, has the then qualitative explanation purifying of green fluorescence cell to obtain.Use the digestion of trypsinase-EDTA to be individual cells, detect the cell number that green fluorescence is arranged through flow cytometer, detection by quantitative goes out the activity and the recombination efficiency (Fig. 6-2) of cre fusion rotein, and the result shows that HNTC's is active best.

Claims (8)

1. Cre fusion rotein Function detection carrier; It is characterized in that; Comprise Pcmv IE promotor, be provided with the attB sequence at the upper reaches of Pcmv IE promotor, the downstream of Pcmv IE promotor are provided with two Loxp sequences in the same way; Be provided with BGH terminator, BGH terminator and SV40A terminator successively between two Loxp sequences in the same way, the downstream of Loxp sequence are provided with fluorescent mark gene and antibiotic-screening gene; When the Loxp sequence and between three terminators behind Cre recombinase effect down cut, Pcmv IE promotor and fluorescent mark gene splicing start the fluorescent mark expression of gene.

2. Cre fusion rotein Function detection carrier as claimed in claim 1; It is characterized in that; Described carrier is pAcmv-stop-EGFP, comprises the following element and the order of connection: attB sequence-Pcmv IE promotor-Loxp sequence-BGH terminator-BGH terminator-SV40A terminator-Loxp sequence-fluorescent mark gene-antibiotic-screening gene;

Described fluorescent mark gene is read frame for the EGFP gene, and the antibiotic-screening gene is neo, and this carrier is called carrier.

3. Cre fusion rotein Function detection carrier as claimed in claim 1 is characterized in that the nucleotide sequence of said pAcmv-stop-EGFP is shown in SEQ.ID.NO.1.

4. a Cre fusion rotein Function detection cell is characterized in that host cell is the HEK293 cell, with pAcmv-stop-EGFP carrier and phiC31 intergrase expression vector pCMVint cotransfection host cell; Utilize the reconstitution cell of the positive transfection of G418 resistance screening, and screen attB-CMV-stop-EGFP expression vector wherein is integrated into the false attP of host cell site with the form of single copy cell.

5. the application of the described Cre fusion rotein of claim 4 Function detection cell in detecting Cre fusion rotein shear active.

6. application as claimed in claim 5 is characterized in that, described Cre fusion rotein is the Cre fusion rotein of wearing film peptide and nuclear localization signal modification with protein transduction function.

7. one kind through TAT and the peptide modified Cre fusion rotein of NLS, it is characterized in that, begins to be respectively 6 * His zone, NLS polypeptide zone, TAT polypeptide zone and Cre enzyme zone from the N end; Wherein the sequence of the amino-acid residue in NLS polypeptide zone is: Pro-Lys-Lys-Lys-Arg-Lys-Val, the sequence of the amino-acid residue in AT polypeptide zone is: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg.

8. the preparation method of the peptide modified Cre fusion rotein of described TAT of claim 7 and NLS is characterized in that concrete steps are following:

1) pcr amplification obtains having the Cre nucleotide sequence of SacI and XhoI recognition sequence; After amplified production cut through SacI and XhoI enzyme; Recovery is connected on the same prokaryotic expression carrier pET28a (+) that cuts through SacI and XhoI enzyme, obtains the pET28a-Cre recombinant plasmid;

2) produce by primer annealing and have the NLS-TAT nucleotide sequence of NdeI and SacI recognition sequence sticky end, and be connected to and pass through the pET28a-Cre recombinant plasmid that NdeI and SacI enzyme are cut, obtain the pET28a-HNTC recombinant plasmid;

3) with the pET28a-HNTC recombinant plasmid transformed in e. coli strain bl21, select 5-8 male colony inoculation in the LB of kalamycin resistance substratum, at 16～22 ℃ with 0.2mM IPTG abduction delivering Cre fusion rotein;

4) the proteic bacterium of abduction delivering is through the ultrasonic disruption thalline, discharge fusion rotein after, inhale column purification through the affine layer of Ni ion chelating, obtain the albumen behind the purifying.

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