CN101985634B - GFP membrane type expression vector and method for sorting vector-transfected positive cells - Google Patents
GFP membrane type expression vector and method for sorting vector-transfected positive cells Download PDFInfo
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Abstract
The invention discloses a green fluorescent protein (GFP) membrane type expression vector and a method for sorting vector-transfected positive cells. The GFP membrane type expression vector comprises a multiple-cloning site, wherein a ribosome entry site and a GFP-encoded sequence are connected behind the multiple-cloning site in turn to form a transcription bicistron of the multiple-cloning site and the GFP; a signal peptide-encoded sequence is inserted between the ribosome entry site and the GFP-encoded sequence; and a hydrophobic transmembrane region-encoded sequence is added in front of a termination codon of the GFP-encoded sequence. After the GFP membrane type expression vector cloned into a target gene is successfully transfected into the cell, a unique sorting label, namely membrane type GFP, is formed on the surface of the cell, and the sorting label is not formed if the transfection is not successful. By sorting magnetic bead-combined positive cells by using a magnetic field, the sorting step is simplified, so that the positive cells can be rapidly and effectively enriched, and the method is a technical means for efficiently sorting the transfected positive cells.
Description
Technical field
The invention belongs to biological technical field; Relate to the screening of external source destination gene expression positive cell; Be particularly related to a kind of GFP (green fluorescent protein) membranous type and express carrier for expression of eukaryon as sorting indicia, and the method for carrying out the transfectional cell sorting of utilizing this carrier.
Background technology
In the biotechnology, utilize plasmid or virus vector that foreign gene is imported host cell in modern times, to make exogenous gene high-efficient expressed be a very important technology thereby in host cell, increase, transcribe, translate; No matter be the original biological behaviour of importing change host cell through foreign gene, the foreign protein that still utilizes host cell expression to have BA all has very to be used widely.But this technology has the bottleneck of a key, is exactly the not high problem of transfection efficiency.
Generally speaking, different with clone and transfection reagent, transfection efficiency also has nothing in common with each other, but totally all on the low side.Because transfection efficiency is low, behind the goal gene transfection host cell, the wild-type host cell of expression alien gene does not still account for major part; Heterogeneous cell colony has like this influenced the science of experimental result greatly for the specific cells of needs research.Thereby, after the gene transfection operation is accomplished, how from the cell mass of enormous amount, to detect and sub-elect to the few goal gene transfection positive cells of number, improve transfection efficiency, just become a key setting up the eukaryotic cell expression system.
The positive cell of screening transfection at present method commonly used is in carrier for expression of eukaryon, to add one section drug resistance gene; Screen through in cell culture system, adding corresponding cytotoxic drug generation selective pressure, for example the neomycin resistance gene of widespread usage (neor).Its principle is that thereby aminoglycoside antibiotics Xin Meisu and analogue G418 thereof can be through suppressing ribosomal function and the proteinic synthetic wild-type cell that kills.The neo genes encoding aminoglycoside phosphotransferase that carries on the carrier for expression of eukaryon, its protein product can decompose Xin Meisu and G418, can make transfection the positive cell of purpose carrier obtain resistance and can in containing the selective medium of G418, grow.Principle similarly also has the selection of Zeocin resistance, the selection of Hygromycine resistance, the selection of Puromycine resistance etc. therewith.
Though above-mentioned drug resistance gene triage techniques can effectively be removed the wild-type cell that does not import plasmid, enlarged gene transfection The Application of Technology scope, but still had weak point.Particularly; For transient transfection; Although the expression of the expression of resistant gene and goal gene is synchronous; But in the short period (a couple of days) of transient expression, Cytotoxic microbiotic can't effectively kill wild-type cell, and the heterogeneous cell mass after the transfection still can not satisfy most of experimental requirements; Carry out resistance screening to obtain often can the long-term expression goal gene stable cell line; At this moment because the resistant gene of plasmid and goal gene belong to two transcripts under the different promoters control respectively; Its process that is incorporated into the host cell gene group is at random; Thereby two genes not necessarily are incorporated into genome simultaneously, even be incorporated in the genome of same cell, because the position influence that gene inserts; Two genes might not be expressed simultaneously; When therefore through the method for resistant gene selective pressure transfectional cell being screened, the final transfectional cell that obtains is the cell of resistant gene stably express in fact, and the destination gene expression level that the investigator was concerned about becomes randomized number.And; Along with the prolongation of incubation time and the increase of passage number of times; The cell load of expressing resistant gene and goal gene is simultaneously more only expressed the cell load of resistant gene and is wanted big, the ratio that in cell mass, accounts for will further descend (except goal gene can promote indivedual Special Circumstances of cell proliferation).This scheme efficient not only consuming time but also low will sub-elect the time that the cell strain of having expressed goal gene need expend one even some months usually, and the final cell strain that obtains probably remains heterogeneous cell mass.
So, in order to solve the problem of the host cell that how to obtain the high purity expression alien gene, improve the transfection efficiency except developing new transfection reagent, the increasing technology positive cell of goal gene that has been applied to how sorting transfection is arranged in recent years.Some characteristic of utilizing transfectional cell to obtain such as dyeing of transfectional cell expression specificity or GFP, is carried out sorting through flow cytometer.
Green fluorescent protein (GFP) derives from Victoria's multitube luminescent jellyfish, and the mutant GFP albumen after genetic engineering modified can send green fluorescence under the 488nm laser radiation, and its emission peak is in the green area of visible spectrum at 509nm.GFP generally is positioned at two places as a kind of selection markers in existing commercial carrier for expression of eukaryon, and the one, (MCS) locates at MCS, and goal gene forms amalgamation and expression with GFP after inserting MCS, and promptly ripe proteic N end or C end have the GFP label; Second kind of situation is after GFP is positioned at RES (IRES), to be transcribed into bicistronic mRNA, the free tenuigenin inside that is expressed in of GFP.Generally speaking; GFP can sub-elect transfection positive cells crowd through flow cytometry as selection markers; But common flow cytometer can not carry out the branch selection operation; Airflow classification depends on expensive large-scale professional plant and instrument (like the FACS Aria type of BD company), and the higher and pair cell of required expense has certain damage, it is applied be restricted.
In recent years, utilize the specific protein molecule of cell surface, come the method for certain cell type of sorting that application is more and more widely arranged in life science and clinical practice through the antigen and antibody specific association reaction.For example people's such as the Pei Xue of military medicine research institute great waves invention " contains structure and application thereof that the CD34 marker gene is used for the carrier of transfectional cell sorting " (publication number CN1712535A).Utilize the CD34 molecule as the screening sign; Can sub-elect the transfection positive cell of expressing goal gene and CD34 molecule simultaneously; But still there is the drawback that influences its widespread use in this scheme: the CD34 molecule is the native protein of mammalian cell, is expressed in the various kinds of cell surface, like hematopoietic stem; As the label protein molecule, its range of application is just limited to CD34.The CD34 molecule can interact with CD62L molecule generation specificity, and transfectional cell ectopic expression CD34 can cause interference to the part cytologic experiment.
Summary of the invention
The technical problem that the present invention solves is to provide a kind of carrier of GFP membranous type expression; And the sorting method of the positive cell of this carrier of transfection; Overcome the defective that above-mentioned transfection efficiency is low, the mdr cell false positive high, there is limitation in sorting technology; Simplify traditional transfectional cell screening process, improve the efficiency of separation of transfectional cell.
The present invention realizes through following technical scheme:
The carrier that a kind of GFP membranous type is expressed comprises MCS, is connected with the sequence of RES and encoding green fluorescent protein after the MCS in turn, constitutes the bicistronic mRNA of transcribing of MCS and green fluorescent protein; Between the sequence of RES and encoding green fluorescent protein, insert the sequence of coded signal peptide, before the sequence terminator codon of encoding green fluorescent protein, add the sequence that the coding hydrophobicity is striden the film district.
The sequence of described signal peptide is shown in SEQ.ID.NO.1; Described hydrophobicity is striden the film region sequence shown in SEQ.ID.NO.2.
The sequence of described coded signal peptide is shown in SEQ.ID.NO.3; The sequence that described coding hydrophobicity is striden the film district is shown in SEQ.ID.NO.4.
The sorting method of the positive transfectional cell behind the GFP membranous type expression vector transfectional cell,, may further comprise the steps:
1) MCS at GFP membranous type expression vector inserts the external source goal gene, constitutes the bicistronic mRNA of transcribing of external source goal gene and green fluorescent protein; Then behind GFP membranous type expression vector transfection host cell, collect and treat that isolated cells processes single-cell suspension liquid;
2) will resist GFP monoclonal antibody and two diamagnetic pearls to mix, and make anti-GFP monoclonal antibody be attached to the surface of two diamagnetic pearls;
3) add the two diamagnetic pearls that combined anti-GFP monoclonal antibody at the single-cell suspension liquid of processing, fully mixing makes the two diamagnetic pearls that combined anti-GFP monoclonal antibody combine with the positive cells transfected that the GFP membranous type is expressed;
4) under the action of a magnetic field, with positive cells transfected that has combined two diamagnetic pearls and the cellular segregation that does not combine two diamagnetic pearls; Collection has combined the positive cells transfected of two diamagnetic pearls, and magnetic bead is separated with positive cells transfected, obtains the positive cells transfected of purifying.
Described two diamagnetic pearls are that magnetic bead is connected with the antibody that can discern anti-GFP monoclonal antibody through connecting DNA.
Described anti-GFP monoclonal antibody is the mouse endogenous antibody, and two diamagnetic pearls are that magnetic bead is connected with the monoclonal antibody of the anti-mouse of people through connecting DNA.
Compared with prior art, the present invention has following beneficial technical effects:
1, the carrier of GFP membranous type expression provided by the invention; Under the guiding of signal peptide, pass cytolemma behind the egfp expression; And stride in hydrophobicity under the effect in film district and be bonded to surface of cell membrane, become the new screening label of positive transfectional cell---membranous type GFP (mGFP); And simultaneously because goal gene will be transcribed bicistronic mRNA with green fluorescence protein gene formation after inserting MCS; Realize the coexpression of goal gene and membranous type GFP, membranous type GFP just becomes the surface molecular mark of the positive transfectional cell that goal gene expressed like this.Also promptly, be cloned into after the GFP membranous type expression vector transfectional cell success of goal gene, can form unique screening label---membranous type GFP, do not have then can not forming of transfection success at cell surface.
And through specificity combination, immunomagnetic beads absorption carrying out sorting cells to the GFP monoclonal antibody; And because the specificity of expressing and the specificity of antigen-antibody recognition reaction, the consistence that reconstitution cell goal gene that can guarantee like this to be screened and GFP screen the label positive expression; Especially membranous type GFP is the antigen-antibody identification of cell surface in screening as the cell surface molecule mark, can simplify the screening step, realizes the fast enriching of positive cell.
2, eukaryotic cell commonly used does not have endogenous GFP gene, only if the external source transfection, otherwise do not express membranous type GFP molecule, membranous type GFP can not cause false positive results as evaluation or selective marker; And, do not exist on the eukaryotic cell can with GFP high-affinity bonded native ligand protein molecular, can not influence the biological behaviour of expressing the GFP cell, reduced the interference of experimental implementation to the result.
3, the present invention utilizes gene engineering method to make GFP be fixed in surface of cell membrane through striding the film region sequence; This sequence is that 29 hydrophobic amino acids constitute; The cytolemma lipid is double-deck to be fixed in the cytolemma outside surface with the GFP molecule thereby insert; Do not contain the cytoplasmic region part, influence the biological behaviour of cell so can not transduce outer signals.
4, the external source goal gene inserts behind the MCS to transcribe with mGFP becomes a bicistronic mRNA; The aminoterminal of green fluorescent protein is connected with signal peptide sequence; What carboxyl terminal was connected with hydrophobic amino acid strides the film region sequence; GFP albumen can be on film, expressed behind this expression vector of cell transfecting, the external source goal gene can also be expressed simultaneously.Under same promotor, express the ORF of two fragment genes respectively; Make the expression of the new sign of expression and cell surface of external source goal gene have good dependency, and the cell of the expression film sign mGFP that this dependency has guaranteed to sub-elect also is simultaneously the cell of expressing goal gene.
5, the cell sorting method of GFP-transfected membranous type expression vector provided by the invention; Through the flow process that the GFP antigen and antibody specific combines, immunomagnetic beads adsorbs, the magnetic field sorting combines the positive cell of magnetic bead; Simplified the screening step, can be fast effective enrichment positive cell, and avoided the defective of the long-term screening of microbiotic; Can also improve the screening ratio of transfection positive cell, be a kind of technique means that can efficiently sub-elect the transfection positive cell.
Generally speaking, the transfection efficiency of common mammalian cell promptly has about 20% cell expressing goal gene in the cell mass after the transfection about 20%; And the sorting through present technique, the enrichment positive cell rises to more than 95% the positive cell ratio.
Description of drawings
Fig. 1 is the plasmid map of the membranous type GFP expression vector pIRES-mGFP that makes up of the present invention;
Fig. 2 is the schematic flow sheet of the cell sorting method of transfection membranous type GFP expression vector.
Embodiment
The present invention has made up the carrier that a kind of GFP membranous type is expressed; After MCS, be connected with the sequence of RES and encoding green fluorescent protein in turn; Constitute the bicistronic mRNA of transcribing of MCS and green fluorescent protein, make the goal gene coexpression of green fluorescent protein and insertion MCS; And before the sequence terminator codon of encoding green fluorescent protein, add the sequence that the coding hydrophobicity is striden the film district; (leading sequence, LS) shown in SEQ.ID.NO.1, (transmembrane sequence, the sequence of 29 hydrophobic amino acids TM) is shown in SEQ.ID.NO.2 as striding the film district for the signal peptide sequence of 20 hydrophobic amino acids.
Make the green fluorescent protein with the goal gene coexpression under the guiding of signal peptide, pass cytolemma like this, and stride in hydrophobicity under the effect in film district and be bonded to surface of cell membrane, become the new screening label of positive transfectional cell---membranous type GFP.
The cell sorting method of GFP-transfected membranous type expression vector provided by the invention has not only been simplified traditional loaded down with trivial details time-consuming procedure again of transfectional cell screening, and has improved the efficiency of separation of transfectional cell, will be widely used at life science.
A kind of membranous type expressing green fluorescent protein is as the carrier for expression of eukaryon of sorting indicia; The sequence of encoding green fluorescent protein is positioned at after the RES; Be built into the bicistronic mRNA of transcribing of MCS and green fluorescent protein, between the sequence of RES and encoding green fluorescent protein, insert
The carrier of expressing in the face of the GFP membranous type down elaborates:
At first, select green fluorescent protein (GFP) as the cell surface marker molecule.GFP has the advantage of following several respects as the surface of cell membrane sign: 1.GFP pair cell toxicity is extremely low, and instantaneous and stably express does not all influence the survival and the biological behaviour of cell, has been widely used in many research fields of life science at present; 2.GFP three-dimensional structure is that typical β is barrel-shaped, comprises βZhe Die and α spiral, and fluorophor is included in wherein, has good anti-cancellation structure, can tolerate the operation of long period; 3.GFP send bright green fluorescence after being stimulated, transfectional cell can pass through the detection of multiple instrument qualitative, quantitatives such as fluorescent microscope, board-like fluorescent quantitation appearance or flow cytometer; 4. eukaryotic cell commonly used does not have endogenous GFP gene, only if the external source transfection, otherwise do not express the GFP molecule, can not cause false positive results as evaluation or selective marker; 5. on eukaryotic cell, do not exist can with GFP high-affinity bonded native ligand protein molecular, can not influence the biological behaviour of expressing the GFP cell, reduced the interference of experimental implementation to the result.
Secondly; (leading sequence LS), adds before the GFP terminator codon and strides film district (transmembrane sequence through between IRES and GFP, inserting signal peptide sequence; TM) sequence; Pass and be anchored to the cytolemma outer surface after this genetically engineered operation makes GFP express, become the new screening label-membranous type GFP of transfectional cell, be called mGFP.It is double-deck that the TM sequence is only inserted the cytolemma lipid, do not contain the cytoplasmic region part, can the transfer cell external signal and influence the biological behaviour of cell.
Become a bicistronic mRNA because the effect of IRES sequence, external source goal gene insert behind the MCS to transcribe with mGFP, under same promotor, express the ORF (ORF) of two fragment genes respectively.Therefore, host cell if express the GFP label protein on the film, then is bound to express the external source goal gene after operating through transfection.The reconstruction of such genetically engineered can be so that the expression of the new sign of the expression of external source goal gene and cell surface has good dependency, and the cell of the expression film sign mGFP that this dependency has guaranteed to sub-elect also is simultaneously the cell of expression goal gene.
With the be configured to embodiment of PIRES2-AcGFP1 carrier, the Construction of eukaryotic process of green fluorescent protein as sorting indicia is described below to PIRES2-mGFP carrier as shown in Figure 1.
The pIRES2-AcGFP carrier is to the construction step of pIRES2-mGFP carrier:
1. the chemical synthesis coding signal peptide with stride the Nucleotide of film region sequence; 5 ' end adds the BstXI site; 3 ' end adds the XbaI site; In the middle of both design add BamHI, KpnI, EcoRI, four kinds of restriction enzyme sites of HindIII (promptly hold 3 ' end to be from 5 ': BstXI-LS-(BamHI, KpnI, EcoRI, HindIII)-TM-XbaI), sequence is inserted the pUC57-T-simple carrier with T-A clone mode, called after pUC-LSTM; Wherein the nucleotide sequence of coded signal peptide is shown in SEQ.ID.NO.3, and the nucleotide sequence that coding is striden the film region sequence is shown in SEQ.ID.NO.4.
2. be template with pIRES2-AcGFP carrier (Clontech company, article No. 632435), the design primer amplification goes out GFP, and adds BamHI and HindIII site at GFP fragment two ends, is connected into above-mentioned pUC-LSTM carrier after enzyme is cut, called after pUC-LGT;
3. from above-mentioned pUC-LGT carrier, cut out LS-GFP-TM with BstXI and XbaI enzyme, and enzyme is cut pIRES-AcGFP, difference glue recovery fragment and carrier;
4. connect the pIRES-AcGFP carrier of LS-GFP-TM and excision GFP, make up successful carrier pIRES2-mGFP.
Through the operation of said gene engineering, original pIRES2-AcGFP carrier changes to constitute expresses the proteic pIRES2-mGFP carrier of membranous type GFP.
Referring to Fig. 2, the cell sorting method of GFP-transfected membranous type expression vector may further comprise the steps:
1), the goal gene nucleic acid fragment of needs research is inserted into the MCS place of above-mentioned carrier, the GFP membranous type expression vector of construction expression external source goal gene through gene engineering method; Utilize liposome mediated-method or methods such as calcium phosphate mediated method or electroporation will make up successful expression vector transfection host cell, collect transfectional cell after 24～48 hours, process single-cell suspension liquid;
2) will resist GFP monoclonal antibody and two diamagnetic pearls to mix, and make anti-GFP monoclonal antibody be attached to the surface of two diamagnetic pearls;
3) add the two diamagnetic pearls that combined anti-GFP monoclonal antibody at the single-cell suspension liquid of processing, fully mixing makes two diamagnetic pearls combine with the membranous type GFP of positive cells transfected surface expression;
4) utilize external magnetic field to sub-elect the transfection positive cell of expressing membranous type GFP, utilize again and shear damping fluid (Releasing Buffer) effect, the transfection positive cell of expressing membranous type GFP is separated with immunomagnetic beads, thereby obtain the transfection positive cell of sorting purifying.
Elaborate in the face of this method down.Main commercialization reagent and equipment: anti-mouse two diamagnetic pearl: CELLection
TMPan Mouse IgG Kit test kit (Dynal company, article No. 115-31D); Magnet stand is used to provide external magnetic field, adopts Dynal MPC-S type magnet stand (Dynal company, article No. 120-20D); RPMI1640 cell culture medium (Hyclone company); NBCS (SIJIQING company); Bovine serum albumin BSA (Sigma company); Vertical mixed instrument (the Ningbo Xin Zhi HS-3 of company type).
White row reagent preparation: PBS damping fluid: 0.15mol/L, pH 7.4; Buffer1: contain mass/volume than being the PBS damping fluid of 0.1%BSA; Buffer2: the RPMI1640 cell culture medium that contains volume ratio and be 1% NBCS; Specificity combines the mouse monoclonal antibody FMU-GFP.5 of GFP.
1, the collection of transfectional cell
Utilize liposome mediated-method or methods such as calcium phosphate mediated method or electroporation will make up successful expression vector transfection host cell, collect transfectional cell after 24～48 hours, process individual cells suspension-s.
If host cell is a suspended culture cell, 1200rpm then, 5min is centrifugal, abandons supernatant, with the fresh culture re-suspended cell deposition of proper volume; If the cell of adherent culture needs to exhaust substratum, digest with 0.25% pancreatin-0.02%EDTA-PBS damping fluid; Make cell become suspended state; 1200rpm then, 5min is centrifugal, abandons supernatant; With the fresh culture re-suspended cell deposition of proper volume, process individual cells suspension-s.
2) combining of the monoclonal antibody of anti-GFP and two diamagnetic pearls:
Get 100 μ l, two diamagnetic pearl suspensions and (contain 4 * 10
7Individual magnetic bead), behind the adding 1ml Buffer1 thorough washing, on magnet stand, leaves standstill 1min, inhale and abandon supernatant, add 1ml Buffer1 mixing; Add 2 μ g monoclonal antibody FMU-GFP.5 again, place on the vertical mixing appearance 4 ℃ to put upside down mixing 30～60min; On magnet stand, leave standstill 1min, inhale and abandon supernatant, again with Buffer1 washing 2 times.
The monoclonal antibody of anti-GFP can be commercialization reagent or white row preparation, and commercialization reagent: the how tame biotech company that comprises Sigma all has commercial monoclonal antibody to provide.
Embodiments of the invention are selected the FMU-GFP.5 monoclonal antibody; The preparation method of this monoclonal antibody: what immunogen adopted is prokaryotic expression recombinant GFP albumen; Immunity Balb/c mouse; Extracting spleen cell and Sp2/0 myeloma cell are merged, and obtain combining the proteic monoclonal antibody of GFP through indirect elisa method screening, limiting dilution assay cloning.Concrete grammar is seen open source literature: Ran Zhuang; Yuan Zhang; Rui Zhang, Chaojun Song, Kun Yang; Angang Yang, Boquan Jin.Purification of GFP fusion proteins with high purity and yield by monoclonal antibody-coupled affinity column chromatography.Protein Expression and Purification.2008; 59 (1): 138-143, the researchist of this area can ask for the FMU-GFP.5 monoclonal antibody from the disclosed laboratory of above-mentioned document easily.The required anti-GFP specific monoclonal antibody of this step also can buy obtain from biotech company, for example green skies biotech company (product article No.: AG281).
3) add the two diamagnetic pearls that are combined with anti-GFP monoclonal antibody
The monoclonal antibody of anti-GFP with join in the individual cells suspension-s of processing after two diamagnetic pearls combine; In the liquid phase buffering system, immobilised specificity combines combination that the anti-monoclonal antibody of GFP of GFP can high-affinity to express the positive transfectional cell of membranous type GFP.
4) utilize the external magnetic field sorting to express the cell of certain films surface marker
1, using Buffer1 adjustment transfectional cell density is 1 * 10
7, get the 1ml cell suspension and be combined with the magnetic bead mixing of monoclonal antibody, put on the vertical mixing appearance 4 ℃ and put upside down mixing 20min; On magnet stand, leave standstill 2min then, inhale and abandon supernatant to remove the negative cells that does not combine magnetic bead; Add 1ml Buffer1 mixing, on magnet stand, leave standstill 1min, inhale and abandon supernatant, repeat 3 times washed cell, exhaust supernatant for the last time.
2, add 200 μ l in the Buffer2 of 37 ℃ of preheatings re-suspended cell; Adding 4 μ l DNase I solution digestion connects and is connected DNA (the connection antibody that has on the commercialization reagent immunomagnetic beads is with the DNA chain of magnetic bead or be connected DNA) between immunomagnetic beads and the anti-mouse monoclonal antibody of people; Put upside down mixing 15min under the room temperature, firmly blow and beat suspension 5～10 times so that immunomagnetic beads and positive cell dissociate with the pipettor suction nozzle.On magnet stand, leave standstill 2min, inhale and abandon supernatant, repeat 3 times washed cell.The cell that obtains is highly purified transfection positive cell.
The structure of embodiment 1:pIRES2-mGFP-CD226 carrier
The CD226 gene is inserted pIRES2-mGFP carrier as shown in Figure 1.The CD226 gene source is in the pEF-BOS-CD226 carrier; ORF ORF gene order according to pIRES2-mGFP MCS restriction enzyme site and CD226; The design primer is introduced XhoI and BamHI restriction enzyme site, and primer sequence and high-fidelity PCR reaction conditions are following:
P1:gcctcgagat ggattatcct actttact 28 (underscore is the XhoI site)
P2:ggggatcctt aaactctagt ctttggtct 29 (underscore is the BamHI site)
100 μ l reaction systems comprise: pEF-BOS-CD226 plasmid 0.1 μ l; Each 5ul of primer P1 and P2 (10 μ M), dNTP (2.5mM) 8ul, high-fidelity DNA polymerase PrimerStar (TaKaRa company) 0.5 μ l; 5 * PCR Buffer 20ul, distilled water 81.4 μ l.Reaction conditions: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 30sec; 59 ℃ of annealing 45sec, 72 ℃ are extended 90sec; 35 circulations; 72 ℃ are extended 10min.
The PCR product adopts XhoI and BamHI to carry out double digestion after the fast purifying test kit is purified, and carries out fast purifying once more; Get pIRES2-mGFP simultaneously and carry out double digestion, use the fast purifying test kit to purify with XhoI and BamHI.PCR product after enzyme is cut is connected under the effect of T4DNA ligase enzyme with linear carrier, and condition is: 16 ℃, and reaction overnight.Transformed into escherichia coli E.coli, Kan resistance LB is dull and stereotyped in the shop, and the picking clone identifies after 37 ℃ of incubated overnight, obtains plasmid pIRES2-mGFP-CD226.
Embodiment 2:pIRES2-mGFP-CD226 carrier transfection CHO cell
DMEM cell culture medium with containing 10% NBCS is cultivated Chinese hamster ovary celI in the 75cm2 culturing bottle, converge rate to 80%, exhausts substratum, digests with 0.25% pancreatin-0.02%EDTA-PBS damping fluid; Make cell become suspended state, cell is changed in the 15ml centrifuge tube, 1200rpm, 5min is centrifugal; Abandon supernatant, with the electroporation damping fluid re-suspended cell deposition of proper volume, 1200rpm then; The centrifugal supernatant of abandoning of 5min, washed cell 2 times is processed individual cells suspension-s; Counting cells is suspended in cell in the electroporation damping fluid again, and the adjustment final concentration of cells is 1 * 10
7Individual cell/ml.Get the 1ml cell suspension and put into electric revolving cup, add 50 μ g recombinant vectors pIRES2-mGFP-CD226, ice bath 10min, the electroporation transfection CHO cell, the electroporation apparatus parameter is provided with: voltage 300V, electric weight 250 μ F.With an amount of substratum diluting cells, the G418 selectivity is cultivated 48h, presses the preceding method harvested cell after the transfection.
Embodiment 3: the immunological magnetic bead sorting of transfection positive cell
Get 100ul two diamagnetic pearl suspensions and (contain 4 * 10
7Individual magnetic bead), add 1ml Buffer1 thorough washing, on magnet stand, leave standstill 1min, inhale and abandon supernatant, add 1ml Buffer1 mixing, add 2 μ g monoclonal antibody FMU-GFP.5, place on the vertical mixing appearance and put upside down mixing, 4 ℃, 30～60min.On magnet stand, leave standstill 1min, inhale and abandon supernatant, again with Buffer1 washing 2 times;
Chinese hamster ovary celI is conventional behind the electroporation transfection cultivated 48 hours, made foreign gene be able to express.Exhaust substratum, digest, make cell become suspended state with 0.25% pancreatin-0.02%EDTA-PBS damping fluid; Cell is changed in the 15ml centrifuge tube, 1200rpm, 5min is centrifugal; Abandon supernatant, with proper volume Buffer1 washed cell 2 times, and the adjustment cell density is 1 * 10
7Get 1ml cell suspension and the magnetic bead mixing that is combined with monoclonal antibody, put on the vertical mixing appearance and put upside down mixing, 4 ℃, 20min; On magnet stand, leave standstill 2min then, inhale and abandon supernatant to remove the negative cells that does not combine magnetic bead.Add 1ml Buffer1 mixing, on magnet stand, leave standstill 1min, inhale and abandon supernatant, repeat 3 times washed cell, exhaust supernatant for the last time.
Add 200ul in the Buffer2 of 37 ℃ of preheatings re-suspended cell; 8 add the digestion of 4ul DNase I solution connects DNA; To cut off being connected of magnetic bead and positive cell, put upside down mixing 15min under the room temperature, firmly blow and beat suspension 5-10 time so that cell separates with magnetic bead with the pipettor suction nozzle.On magnet stand, leave standstill 2min, inhale and abandon supernatant, repeat 3 times washed cell.The cell that obtains is highly purified transfection positive cell, can directly carry out next step analysis or add the substratum continued cultivating.
Claims (5)
1. the carrier that the GFP membranous type is expressed is characterized in that, comprises MCS, is connected with the sequence of RES and encoding green fluorescent protein after the MCS in turn, constitutes the bicistronic mRNA of transcribing of MCS and green fluorescent protein; Between the sequence of RES and encoding green fluorescent protein, insert the sequence of coded signal peptide, before the sequence terminator codon of encoding green fluorescent protein, add the sequence that the coding hydrophobicity is striden the film district;
The sequence of described signal peptide is shown in SEQ.ID.NO.1; Described hydrophobicity is striden the film region sequence shown in SEQ.ID.NO.2.
2. the carrier that GFP membranous type as claimed in claim 1 is expressed is characterized in that the sequence of described coded signal peptide is shown in SEQ.ID.NO.3; The sequence that described coding hydrophobicity is striden the film district is shown in SEQ.ID.NO.4.
3. the sorting method of the positive transfectional cell behind the described GFP membranous type of the claim 1 expression vector transfectional cell is characterized in that, may further comprise the steps:
1) MCS at GFP membranous type expression vector inserts the external source goal gene, constitutes the bicistronic mRNA of transcribing of external source goal gene and green fluorescent protein; Then behind GFP membranous type expression vector transfection host cell, collect and treat that isolated cells processes single-cell suspension liquid;
2) will resist GFP monoclonal antibody and two diamagnetic pearls to mix, and make anti-GFP monoclonal antibody be attached to the surface of two diamagnetic pearls;
3) add the two diamagnetic pearls that combined anti-GFP monoclonal antibody at the single-cell suspension liquid of processing, fully mixing makes the two diamagnetic pearls that combined anti-GFP monoclonal antibody combine with the positive cells transfected that the GFP membranous type is expressed;
4) under the action of a magnetic field, with positive cells transfected that has combined two diamagnetic pearls and the cellular segregation that does not combine two diamagnetic pearls; Collection has combined the positive cells transfected of two diamagnetic pearls, and magnetic bead is separated with positive cells transfected, obtains the positive cells transfected of purifying.
4. the sorting method of the positive transfectional cell behind the GFP membranous type expression vector transfectional cell as claimed in claim 3 is characterized in that, described two diamagnetic pearls are that magnetic bead is connected with the antibody that can discern anti-GFP monoclonal antibody through connecting DNA.
5. the sorting method of the positive transfectional cell behind the GFP membranous type expression vector transfectional cell as claimed in claim 3; It is characterized in that; Described anti-GFP monoclonal antibody is the mouse endogenous antibody, and two diamagnetic pearls are that magnetic bead is connected with the monoclonal antibody of the anti-mouse of people through connecting DNA.
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| CN103388001B (en) * | 2013-07-16 | 2016-01-20 | 中国人民解放军第四军医大学 | A kind of method containing thrombin cleavage site GST membranous type expression vector and transfection positive cell |
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|---|---|---|---|---|
| US6316181B1 (en) * | 1998-04-24 | 2001-11-13 | Virginia Commonwealth University | Establishment of cell lines with persistent expression of a green fluorescent protein (GFP) using a pIRES/EGFP DNA vector construct |
| CN101463362A (en) * | 2009-01-15 | 2009-06-24 | 中国农业大学 | Expression vector for fusion expression of green fluorescent protein, construction method and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6316181B1 (en) * | 1998-04-24 | 2001-11-13 | Virginia Commonwealth University | Establishment of cell lines with persistent expression of a green fluorescent protein (GFP) using a pIRES/EGFP DNA vector construct |
| CN101463362A (en) * | 2009-01-15 | 2009-06-24 | 中国农业大学 | Expression vector for fusion expression of green fluorescent protein, construction method and use thereof |
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| Title |
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| RAN ZHUANG, ET AL.Purification of GFP fusion proteins with high purity and yield.《PROTEIN EXPRESSION AND PURIFICATION》.2008,第59卷138-143. * |
| 曲笑霞,等.一个用于转染细胞分选的双顺反子载体的构建及其应用.《生物化学与生物物理进展》.2005,第32卷(第9期),889-894. * |
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| US11554141B2 (en) | 2014-04-01 | 2023-01-17 | Rubius Therapeutics, Inc. | Methods and compositions for immunomodulation |
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