CN104611370A - Method for rejecting B2M (beta 2-microglobulin) gene segment - Google Patents
Method for rejecting B2M (beta 2-microglobulin) gene segment Download PDFInfo
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Abstract
The invention discloses a method for rejecting a B2M (beta 2-microglobulin) gene segment. The method comprises steps as follows: (1) constructing a CRISPR/Cas9 vector system; (2) introducing a target gene into a recipient cell by the aid of the constructed CRISPR/Cas9 vector system, and rejecting the B2M gene segment; (3) detecting and identifying the transfected recipient cell. The method is a new genetic engineering method for rejecting a human cell gene in a targeted manner. According to the method, a baculovirus vector is used as a transfer system, a specific CRISPR/Cas9 component with a B2M gene rejected is efficiently expressed in a human cell, B2M gene expression is effectively blocked, and expression of a cell surface type-I HLA (human leukocyte antigen) molecule is reduced, so that a low-immunogenicity human cell is produced.
Description
Technical field
The present invention relates to field of transgenic technology, particularly the genetic engineering method of a rejecting B2M gene fragment.
Background technology
B2M (B2M) is an integral part of Human leucocyte antigen-Ⅰ quasi-molecule (HLA-I) complex body, with HLA-I molecule the expression of cell surface and cellular immunization source property closely related.The human leucocyte antigen system (HLA) being positioned at the 6th pair of the short arm of a chromosome is the genetic system of most polymorphism known today.They encode the first type and the cell surface molecule with Second-Type.HLA first type molecule has expression on all karyocyte surfaces, for presenting the peptide of originating in cell, regulates congenital immunity and acquired immunity.HLA first type molecule depends on whether suitably can be combined into a complex body with B2M (B2M) in the expression of cell surface.People B2M molecular mass is 11.6kDa, containing 99 amino acid.People B2M gene is made up of 3 exons.
By rejecting B2M gene, changing cell surface HLA first type developed by molecule, producing mouse and people's cell tested mistake (Zijlstra et al., 1990,2010 in the past of low immunogenicity; Matin et al., 2004; Zafarana et al., 2009; Riolobos et al., 2013; Torikai et al., 2013; Lu et al., 2013; Figueiredo et al., 2014).In the eighties, have employed the B2M gene in classical homologous recombination method rejecting mouse embryo stem cell, produce Chi-meric mice with these stem cells subsequently, then produce B2M negative mouse (Koller et al, 1989 by hybridization; Zijlstra et al., 1989,1990,2010).B2M-/-mouse does not express any B2M albumen measuring level, the first type MHC antigen of the almost normal work in deletion cells surface completely, but they are healthy.The efficiency that classical homologous recombination method carries out gene targeting is very low, now few use.In human embryo stem cell, the existing test of suppression (Matinet al., 2004 that RNA interference (RNAi) translates B2M; Zafarana et al., 2009).But RNAi has some shortcomings, as rejected not exclusively, and potential non-specific.A nearest research has demonstrated AAV carrier and has introduced in an end codon to B2M gene and can terminate B2M genetic expression (Riolobos et al., 2013) in advance.In human embryo stem cell, Zinc finger nuclease (ZFN) mRNA is got involved for disturbing HLA first type developed by molecule (Torikai et al., 2013) by electroporation method.Activating transcription factor sample effector nuclease (TALEN) also for disturbing B2M genetic expression (Lu et al., 2013) in human embryo stem cell.
ZFN and TALEN is the method that two kinds of early stage use customization DNA endonucleases carry out genome editor, and comparison is loaded down with trivial details, uses more expensive.Particularly TALEN molecular ratio ZFN is much bigger, is therefore difficult to efficient transfered cell, such as human embryo stem cell.The new force using endonuclease to carry out genome editor is regular intervals short palindrome tumor-necrosis factor glycoproteins (CRISPR/Cas) system of so-called cluster.This system brings a lot of benefit: simple, flexibly, cheap, be easy to programming and very efficient (Hsu et al., 2014).CRISPR/Cas9 system aims at one section of specific DNA sequence dna with RNA Guidance means.This technology uses one section of codeless mediate rna (gRNA), gRNA mediates Cas9 nuclease to specific genome target by target specific CRISPR RNA (crRNA) and a support trans-activation crRNA (tracrRNA), causes DNA double splitting of chain (DSB).Follow-up non-homologous end joining (NHEJ) activation or homologous recombination repair make the Gene interfere in target location, and correction and insertion become possibility.
Adopt the gene target of homologous recombination to remain a widely used technology allowing selected DNA sequence dna be modified in the mode that determines in advance, but have the unfavorable factor of time and effort consuming.ZFN and TALEN technology can introduce DNA DSB to stimulate NHEJ or the homologous recombination repair of easily mistake, because this increasing the efficiency of gene amendment at selected genomic locus.
Summary of the invention
In order to solve the defect in above-mentioned rejecting B2M gene fragment process, the present invention discloses a kind of method rejecting B2M gene fragment, and the present invention adopts following technical scheme to solve above-mentioned technical problem:
Reject a method for B2M gene fragment, comprise the steps:
(1) CRISPR/Cas9 carrier system is built;
(2) utilize the CRISPR/Cas9 carrier system built that goal gene is imported recipient cell, reject B2M gene fragment;
(3) recipient cell after transfection detected and identify.
Preferably, a kind ofly reject in the method for B2M gene fragment above-mentioned, CRISPR/Cas9 carrier system in described step (1), adopts following steps to build:
B2M locus Exon 2 is that the mediation sequence of target and joint sequence are after Hu Fill binds, with the pX260-Cas9 plasmid vector adhesion of BbsI-nicking, between before 3 ' end band 3bp TGG, the special intervening sequence of the B2M of the contiguous motif of region sequence (PAM) introduces carrier, described mediation sequence and joint sequence as follows:
5’...GACATTGAAGTTGACTTACTGAAGAATGGAGAGAG...3’
3’...CTGTAACTTCAACTGAATGACTTCTTACCTCTCTC...5’
The U6-B2M-crRNA component produced to be inserted between EcoRI and Xhol to use GeneArt gene to generate formation pFB-U6-B2M-crRNA plasmid from plasmid by pcr amplification subsequently;
In order to prepare pFB-CBh-Cas9-H1-tracrRNA plasmid, the CBh promotor in humanized Cas9 with px260-Cas9 plasmid is become the fragment of two each 2.8kb by pcr amplification; Adopt dual adhesion method, two fragments are pFastBac1 by limiting a some subclone with BamHI, Xhol, HindIII simultaneously; And then insert the H1-tracrRNA obtained from pX260-Cas9 plasmid; Finally, original in px260-Cas9 U6-hEMXcrRNA fragment is removed by Xbal and BamHI digestion; Xbal and BamHI continues to hang on the small segment with pcr amplification process, on the plasmid having complementary Xbal and BamHI to hang;
In order to prepare donor plasmid pFB-B2M-eGFP, belong to a left homology arm of 636-bp of B2M locus and the right side of a 687-bp to be increased by U87 cell genomic dna with homology arm and get, then be inserted into pFB-PGK-Neo-EGFP-LoxP, a pFastBac1 carrier supplies right homology arm in making before by SnaBI/Sall for left homology arm and Notl/BstBI;
The baculovirus vector (BV) of restructuring, comprise BV-B2M-crRNA, BV-Cas9/tracrRNA and BV-B2M-eGFP, use above pFB-U6-B2M-crRNA,, pFB-CBh-Cas9-H1-tracrRNA and pFB-B2M-eGFP plasmid generates respectively, and to breed in Sf9 insect cell according to Invitrogen Bac-to-Bac baculovirus expression system agreement.
Preferably, a kind ofly reject in the method for B2M gene fragment above-mentioned, by flow cytometry assay, Western blotting, immunofluorescence staining the recipient cell after transfection detected in step (3) and identify.
Preferably, a kind ofly reject in the method for B2M gene fragment above-mentioned, also comprise immune response assay step, use Elipost assay method to carry out immune response chemical examination to the recipient cell after transfection.
Compared with prior art, the present invention has following technique effect:
The invention discloses a new target gene engineering method and reject B2M gene in human body cell.This method utilizes baculovirus vector as transfer system, high expression B2M gene specific CRISPR/Cas9 assembly in human body cell, block B2M genetic expression, reduce the expression of cell surface first type HLA molecule, thus produce the human body cell of low immunogenicity.
Accompanying drawing explanation
Fig. 1 CRISPR/Cas9 is in the efficiency guiding the specific site of HEK293 cell B2M locus to disturb
The B2M locus interference that Fig. 2 BV-B2M-CRISPR guides in U87 cell
The B2M that in Fig. 3 U87 cell, BV-B2M-CRISPR causes rejects
The B2M locus target of Fig. 4 hESC (hESC)
Fig. 5 hESC after B2M rejects to the maintenance of versatility and differentiation capability
Fig. 6 human immune cells is to the reaction of B2M interference cell
Embodiment
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
The present invention discloses a kind of method rejecting B2M gene fragment, comprises the steps:
(1) CRISPR/Cas9 carrier system is built;
(2) utilize the CRISPR/Cas9 carrier system built that goal gene is imported recipient cell, reject B2M gene fragment;
(3) recipient cell after transfection detected and identify.
As shown in Figure 1, in above-mentioned steps (1), CRISPR/Cas9 carrier system builds as follows:
Manufacture the carrier of transcribing for crRNA, as Suo Shi Fig. 1 (A), a pile is containing one section of 20 Nucleotide, the mediation sequence being target with B2M locus Exon 2 and joint sequence annealed, with the adhesion of BbsI-digestedpX260-Cas9 plasmid vector, with will before 3 ' end band 3bp TGG between the special intervening sequence of B2M of the contiguous motif of region sequence (PAM) introduce carrier.The U6-B2M-crRNA cartridge clip produced inserts pFastBac1 from plasmid by pcr amplification subsequently
tM(Invitrogen) pFB-U6-B2M-crRNA plasmid is formed to use GeneArt gene to generate between EcoRI and Xhol.
When manufacturing pFB-CBh-Cas9-H1-tracrRNA plasmid, by PCR, the CBh promotor in Cas9 with the px260-Cas9 plasmid of humanization is increased into the fragment of two each 2.8kb.Adopt dual adhesion method, two fragments are pFastBac1 by limiting a some subclone with BamHI, Xhol, HindIII simultaneously.And then the H1-tracrRNA that the flourishing pX260-Cas9 plasmid of insertion EcoRI and HindIII obtains.Finally, original in px260-Cas9 U6-hEMXcrRNA fragment is removed by Xbal and BamHI digestion.Xbal and BamHI continues to hang on the small segment with pcr amplification process, on the plasmid having complementary Xbal and BamHI to hang.
When manufacturing donor plasmid pFB-B2M-eGFP, belong to a left homology arm of 636-bp of B2M locus and the right side of a 687-bp to be increased by U87 cell genomic dna with homology arm and get, then be inserted into pFB-PGK-Neo-EGFP-LoxP, a pFastBac1 carrier supplies right homology arm in making before by SnaBI/Sall for left homology arm and Notl/BstBI.In this carrier, eGFP reporter gene expression is controlled by the initial son of EF1 α.
The BV of restructuring, comprises BV-B2M-crRNA, BV-Cas9/tracrRNA and BV-B2M-eGFP, with pFB-U6-B2M-crRNA, pFB-CBh-Cas9-H1-tracrRNA and pFB-B2M-eGFP generate respectively.And breed in Sf9 insect cell according to Invitrogen Bac-to-Bac baculovirus expression system agreement.
Embodiment 1: the rejecting of B2M in people HEK293 cell
The present invention devises the mediation sequence of 20 Nucleotide, with a predeterminated position of base substitution mankind B2M gene extron 2, manufactures the B2M target CRISPR/Cas 9 carrier (Figure 1A) needed.When there being this mediation sequence, the cutting efficiency that CRISPR/Cas 9 system is expressed with plasmid vector or baculovirus vector measures chemical examination testing evaluation with T7E1 in human foetus's kidney (HEK) 293 cell.Cell is by the common transfection of plasmid vector of two expression Cas9/tracrRNA and B2M crRNA or by BV-Cas9/tracrRNA and BV-B2M-crRNA (BV-B2M-CRISPR) co-transduction.Just as shown in Figure 1B, compare with single PCR band of the about 0.46kb of control group, in the amplicon after isolated genomic DNA amplification from by the HEK293 cell of plasmid B2M target CRISPR/Cas9 system or BV-B2M-CRISPR transfection, may detect the small segment of 0.18kb and 0.28kb.These results demonstrate B2M locus DSB in HEK293 cellular genome and are induced.By the sequence of the HEK293 cell B2M locus that BV-B2M-CRISPR transduces, measure the sudden change of some insertion/deletions.In 15 parts of samples analyzed, 2 parts have and insert a pair adenosine base pair in same position, and 1 part has lacked 8 pairs of base pairs (Fig. 1 C) with native sequence ratio.Because inserting of being caused by artificial nucleic acid enzyme lacks and can occur defect by mobile reading frame by the expression of target gene, the B2M protein expression level in these cells processed is verified.Flow cytometry showed cell is after by plasmid or the process of BV-B2M-CRISPR system, and expression level lowers about 28% and 30% (Fig. 1 D) respectively.Therefore, CRISPR/Cas9 system of the present invention is proved to be the downward of B2M Gene interfere and the B2M expression that effectively can cause a specific site.
Embodiment 2: the rejecting of B2M in people U87 cell
Cloned by the human body cell that BV-B2M-CRISPR disturbs for choosing stable B2M gene, the present invention has manufactured a donor vehicle based on BV, BV-B2M-eGFP, cartridge clip for you to choose is integrated into B2M locus (Fig. 2 A) at specific site by homologous recombination.U87 glioma cell, a kind of can easily by the human cell line that BV transduces, transduceed by BV-B2M-CRISPR and select 3 weeks with G418.The cell of survival becomes the eGFP positive, and stable eGFP expresses and continues at least 3 months (Fig. 2 B).By using individual and eGFP complementary one to one, the PCR gamete in a right homology arm downstream in B2M site detects one section of 1.4kb band, and pcr gene somatotype (Fig. 2 C and D) is used to check that eGFP donor cartridge clip is integrated at the specific site of B2M locus.Other a pair and primary B2M allelic complementation, the gamete of DNA fragmentation of amplification one section of 0.9kb is used to differentiate that B2M isozygotys the U87 cell clone of rejecting.PCR extends and carries out 40 seconds only to allow the short fragment amplification of primary B2M allelotrope.By checking 56 stable clones got from single cell clone, find that 49 clones's (87.5%) show B2M specific site and integrate (Fig. 2 E).But have integration donor cartridge clip to enter in the clone of specific site at 40, neither one shows diallele integrated transgene.Gene sequencing is carried out to the stable U87 having B2M specific site donor cartridge clip the to integrate clone from 10 random chooses, finds that target area is inserted and lack sudden change (Fig. 2 E), confirm to produce B2M and isozygoty and reject the failure of U87 cell.This failure may be relevant with the cancer cell characteristics of this Human cell line.However, gRT-PCR, western blotting and flow cytometry confirm B2M mRNA and albumen clones the large downward of acceptance of the bid at all verified B2M heterozygosis knockout cells.
Embodiment 3: in human pluripotent stem cells, B2M rejects
After proving the validity of BV-B2M-CRISPR in mankind U87 cell, the present invention utilizes it to test this system to the high transduction efficiency of baculovirus vector on human pluripotent stem cell H1hESC.Complete after BV transduces 72 hours, G418 screening starts to enrich eGFP positive cell.The iPSC bacterium colony of the eGFP positive continues expansion when normal cultivation again by mechanical grading subsequently.Pure eGFP is positive, and hESC bacterium colony obtains (Fig. 4 A) after about 3 months in amplification.In these clones, the per-cent of eGFP positive cell was 18.5% at the 20th day, rose to 98.6% by the 100th day.With above-described pcr gene classifying method, present invention obtains 2 B2M isozygoty reject hESC clone.As shown in Figure 4 C, in clone 2.3, the amplification of one section of 1.4kb demonstrates the integration of donor cartridge clip at B2M locus, and the disappearance of 0.9kb fragment demonstrates diallele change.Western blot analysis confirms the disappearance (Fig. 4 D) of B2M protein expression this B2M-/-hESC clone.Flow cytometry also demonstrates the expression (Fig. 4 E) that cell surface does not almost have B2M albumen.As expectation, a heterozygosis (B2M+/-) hESC clone, clone 7.1 (Fig. 4 C), B2M protein expression lowers (Fig. 4 D, E).
The present invention observes B2M rejecting further and does not have a significant effect to hESC Growth of Cells and shape.Clone 2.3 and clone 7.1 continue to express the mark of multipotential stem cell, comprise Sox2, Nanog, Oct4 and SSEA4 (Fig. 5 A, B) and embryoid body formed chemically examine in maintain that to be divided into 3 be paotoblastic ability (Fig. 5 C and D).Teratoma forms chemical examination B2M and isozygotys and reject hESC and clone 2.3 and carry out.All 10 mouse of implanting hESC grow teratoma within February.Tissue (Fig. 5 E) from all 3 germinal layers has been found to the histological inspection of the differentiated tissue in monster.Therefore, the characteristic of hESC multipotential stem cell and differentiation capability do not receive the impact of B2M eliminating system.
The first type HLA defect after embodiment 4:B2M rejects and the immunogenicity of the first type HLA deficient cells
The main purpose of this research generates the first circular type HLA deficient cells of low immunity.The present invention depicts B2M heterozygosis and rejects (+/-) U87 nucleus B2M and to isozygoty rejecting (-/-) hESC cell.B2M+/-U87 cell relatively primary U87 cell HLA I surface expression levels reduces, in clone 9, approximately reduce by 50% (Fig. 6 A).When the reaction of allogeneic ion vitro immunization with normal human subject PBMC cell cultures after with IFN γ Elispot chemical examination assessment time, primary U87 cell significantly increases in PBMC the cell producing the immune interference factor and compares dormancy and respond lymphocytic quantity (IFN γ dot frequency), but B2M+/-U87 cell does not have (Fig. 6 B).Heteroimmunity reaction for B2M+/-U87 cell is also checked.Main mice spleen cell is used to the effector cell with primary U87 or U87B2M+/-cell co-culture by the primary U87 cytositimulation of mitomycin process after one week.The cytotoxic killer certificate of analysis U87B2M+ be decomposed/-cell percentages is significantly lower than primary U87 cell (Fig. 6 C).
Undifferentiated hPSC HLA I developed by molecule level is very low, and HLA II level cannot detect.When in this research generate B2M isozygoty reject hESC analyzed time, observe cell HLA I, HLA II and costimulatory molecules CD40, CD80, CD83 and CD86 molecule are feminine gender.As proving before, the HLA I developed by molecule level of hESC cell surface breaks up the gentle rising of rear only meeting in vitro and in vivo, but then can acutely rise after cell IFN γ process.For the reaction of assessment alloimmunity, the B2M generated in above-mentioned experiment isozygotys and rejects hESC and be divided into fibroblast (Fig. 6 D), then with IFN γ process.Although the fibroblast surface HLA I developed by molecule level of breaking up from primary hESC cell is significantly promoted to 66.1% from 1.6%, the fibroblast surface HLA I expression level broken up in B2M-/-hESC is but significantly not different, only from 0.9% to 1.6% (Fig. 6 E).During assessment alloimmune originality, allergen mankind PBMC chemically examines assessment (Fig. 6 F) with Elispot after HLAI defect or primary hESC cell are cultivated when having IFN-γ process and not processing.When not having IFN γ process, the fibroblast of primary hESC and B2M-/-hESC differentiation all only have stimulated PBMC low-level secretion IFN γ.After IFN γ process, the fibroblast of primary hESC differentiation dose-dependently stimulates PBMC to secrete IFN γ.It should be noted that HLA I defect that B2M rejects significantly reduces the level that PBMC secretes IFN γ, to a similar level not having the fibroblast of IFN γ process to observe there.These results demonstrate and create and can prolong the reduced immunogenicity hESC keeping immunoglogical unresponsiveness in raw cell.
In transfection experiment, HEK293 cell is planted at 6 well culture dish with the density of 1 × 105 every well.When reaching 70% bout, the cell in each well contains the pFB-U6-B2M-crRNA of 1.2 μ g, the plasmid/liposomal mixtures transfection of 1.2 μ gpFB-CBh-Cas9-H1-tracrRNA and and 5 μ l liposomes 2000 with a kind of.In baculovirus transduction experiment, HEK293 cell, U87 cell and H1hESC planted in 6 well culture dish on 0th, and next day with baculovirus vector with the every cell transduction of each BV carrier infection multiplicity (MOI) 100 4 hours.Subsequently, cell culture medium is removed, and is changed to general substratum.Transduce within 3rd, rise G418 medicament selection transduction U87 nucleus H1hESC and carry out 3 weeks.Then single cell clone is filtered out by limiting dilution or unicellular sifting machine.
The DNeasy blood of genomic dna Qiagene is separated with organization tool case.Full RNA TRIzol reagent is separated.
Genomic dna extracts after transfection or transduction from by the cell of B2M CRISPR/Cas9 system process on the 3rd.One section of 0.5kb, utilizes B2M point to disturb introduction amplification containing the fragment of CRISPR cut point in B2M locus from genomic dna, middle use PCR SuperMix High Fidelity.PCR primer, through purifying, deactivates, again overdoes and use the T7 restriction endonuclease I not mating sensitivity to digest.Fragment is finally separated with 2.0% agarose gel.
When carrying out pcr gene somatotype, use the model genomic dna of 200ng, HiFi polysaccharase, each introduction of 0.5 μm of ol/l, the magnesium chloride of dNTP and 2mmol/l of 0.25mmol/l.The temperature that overdoes is 55 degrees Celsius or 65 degrees Celsius, increases in a thermocycler, to carry out 35 circulations.The a bit of extension time (40 seconds) is used to only increase primary type allelotrope.
For RT-PCR, RNA SuperScript III first chain generation system reverse transcription.Pcr amplification carries out 15 seconds at 94 degrees Celsius, and 55 degrees Celsius are carried out 20s, then carry out 45 seconds at 72 degrees Celsius, carries out 35 circulations altogether.The product increased is analyzed on 1.5% agarose gel.SYBR Green PCR master mix is used to real-time quantitative PCR, and (95 degrees Celsius lower 60 seconds, 1 circulation; 95 degrees Celsius lower 15 seconds, and 60 degrees Celsius lower 1 minute, 40 circulations).Result is normalized according to beta-actin mRNA level in-site subsequently.
The existence of B2M or cell surface first type HLA molecule uses Prep-cy5.5 to mark anti-B2M or APC with flow cytometry assay and marks anti-HLA (abc) antibody (BD Biosciences) and detect in on-fixed cell.Flow cytometry is also used to detect H1hESCs Regular Insulin-EDTA and the expression of eGFP in hESC after B2M target again after rinsing.FACS Calibur stream type cell analyzer C6 is used.
In western blot analysis, cell rinses with cold PBS and decomposes in containing proteinase inhibitor, RIPA damping fluid.At 4 deg. celsius with 10,000g after centrifugal 20 minutes, a supernatant liquor collected then protein concn BCA Protein Assay tool kit determines.After 12%SDS-PAGE glue is separated, protein is transferred on pvdf membrane and carries out immunoblotting.The anti-B2M antibody of trace detects.After rinsing with PBS at four times, horseradish peroxidase-labeled secondary antibody is added into.Ag-Ab combination enhanced chemiluminescence manifests.
In immunofluorescence dyeing reaction, cell 4% trioxymethylene is fixed, and rinses, and increases penetrance, blocks.Sample and following antibody overnight incubation (4 degrees Celsius): OCT3/4, SOX2, Nanog and SSEA4.Immune response antigen marks against murine IgG (1:500) or Alexa Fluor@594 through Alexa Fluor@594 and marks anti-rabbit IgG (1:500) and dye visual.Nuclear targeting is by the DAPI (PBS of 1:1000; 5 minutes) realize.The image of fluorescence labeled cell is obtained by a reverse fluorescent microscope of Zeiss.
Elipsot chemical examination is the method for the cell of the independent secrete cytokines of a kind of highstrung detection.This method is used to test the alloreactivity of normal blood donor by the post-stimulatory peripheral blood lymphocytes of allosome B2M knockout cell.PBMC (2 × 105) responsively cell and irritation cell (2x 104U87 or 4x 104FLC) at a 96-well, prior covering anti-human gamma disturb because of) Elipsot culture dish jointly at one 37 degrees Celsius, incubated overnight in the humidification incubator of 5% carbonic acid gas.The explanation of IFN γ Elispot work box manufacturer is followed in the operation of culture dish.Spot by an Elispot culture dish reader Auto-counting for scanning analysis.
The heteroimmunity of the U87 of B2M interference is also tested with main mice spleen cell.Spleen takes out from primary female C57Bl/6 mouse (6-8 week), is divided into fritter.Add Liberase and reach 0.6mg.ml to ultimate density.Suspension is cultivated 15 minutes under 37 degrees Celsius, adds EDTA and reaches 10mM to ultimate density.Splenocyte is rinsed by a MESH film subsequently.Single Spleen cell suspensions is used and the U87 cell co-culture of ametycin process stimulates.After 1 week, the effector cell that U87 stimulated with as primary U87 or the U87B2M+/-cell of target cell in triplicate mode, in 37 degrees Celsius of lower co-cultivation 4 hours under different effector cells and target cell ratio.Cytotoxicity activity is with the chemical examination of CytoTox 96 non-radioactive cell toxicity and according to manufacturer's protocol measure.
To those skilled in the art, obviously the invention is not restricted to the details of above-mentioned one exemplary embodiment, and when not deviating from spirit of the present invention or essential characteristic, the present invention can be realized in other specific forms.Therefore, no matter from which point, all should embodiment be regarded as exemplary, and be nonrestrictive, scope of the present invention is limited by claims instead of above-mentioned explanation, and all changes be therefore intended in the implication of the equivalency by dropping on claim and scope are included in the present invention.Any Reference numeral in claim should be considered as the claim involved by limiting.
In addition, be to be understood that, although this specification sheets is described according to embodiment, but not each embodiment only comprises an independently technical scheme, this narrating mode of specification sheets is only for clarity sake, those skilled in the art should by specification sheets integrally, and the technical scheme in each embodiment also through appropriately combined, can form other embodiments that it will be appreciated by those skilled in the art that.
Claims (4)
1. reject a method for B2M gene fragment, it is characterized in that, comprise the steps:
(1) CRISPR/Cas9 carrier system is built;
(2) utilize the CRISPR/Cas9 carrier system built that goal gene is imported recipient cell, reject B2M gene fragment;
(3) recipient cell after transfection detected and identify.
2. a kind of method rejecting B2M gene fragment according to claim 1, is characterized in that, CRISPR/Cas9 carrier system in described step (1), adopts following steps to build:
B2M locus Exon 2 is that the mediation sequence of target and joint sequence are after Hu Fill binds, with the pX260-Cas9 plasmid vector adhesion of BbsI-nicking, between before 3 ' end band 3bp TGG, the special intervening sequence of the B2M of the contiguous motif of region sequence (PAM) introduces carrier, described mediation sequence and joint sequence as follows:
5’...G A C A T T G A A G T T G A C T T A C T G A A G A A T G G A G A G A G...3’
3’...C T G T A A C T T C A A C T G A A T G A C T T C T T A C C T C T C T C...5’
The U6-B2M-crRNA component produced to be inserted between EcoRI and Xhol to use GeneArt gene to generate formation pFB-U6-B2M-crRNA plasmid from plasmid by pcr amplification subsequently;
In order to prepare pFB-CBh-Cas9-H1-tracrRNA plasmid, the CBh promotor in humanized Cas9 with px260-Cas9 plasmid is become the fragment of two each 2.8kb by pcr amplification; Adopt dual adhesion method, two fragments are pFastBac1 by limiting a some subclone with BamHI, Xhol, HindIII simultaneously; And then insert the H1-tracrRNA obtained from pX260-Cas9 plasmid; Finally, original in px260-Cas9 U6-hEMXcrRNA fragment is removed by Xbal and BamHI digestion; Xbal and BamHI continues to hang on the small segment with pcr amplification process, on the plasmid having complementary Xbal and BamHI to hang;
In order to prepare donor plasmid pFB-B2M-eGFP, belong to a left homology arm of 636-bp of B2M locus and the right side of a 687-bp to be increased by U87 cell genomic dna with homology arm and get, then be inserted into pFB-PGK-Neo-EGFP-LoxP, a pFastBac1 carrier supplies right homology arm in making before by SnaBI/Sall for left homology arm and Notl/BstBI;
The baculovirus vector (BV) of restructuring, comprise BV-B2M-crRNA, BV-Cas9/tracrRNA and BV-B2M-eGFP, use above pFB-U6-B2M-crRNA,, pFB-CBh-Cas9-H1-tracrRNA and pFB-B2M-eGFP plasmid generates respectively, and to breed in Sf9 insect cell according to Invitrogen Bac-to-Bac baculovirus expression system agreement.
3. a kind of method rejecting B2M gene fragment according to claim 1, it is characterized in that, by flow cytometry assay, Western blotting, immunofluorescence staining the recipient cell after transfection detected in step (3) and identify.
4. a kind of method rejecting B2M gene fragment according to claim 1, is characterized in that, also comprise immune response assay step, uses Elipost assay method to carry out immune response chemical examination to the recipient cell after transfection.
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