CN106754725A - A kind of Chimeric antigen receptor T cell and preparation method and application - Google Patents
A kind of Chimeric antigen receptor T cell and preparation method and application Download PDFInfo
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Abstract
A kind of Chimeric antigen receptor T cell and preparation method and application, the T cell surface expression Chimeric antigen receptor CD19 CAR genes, its base sequence such as SEQ ID NO:Shown in 3.Preparation method and application present invention additionally comprises Chimeric antigen receptor T cell.The present invention adds two kinds of costimulatory signals in the structure of 3 generation CD19 CAR carriers, constructs slow virus carrier, and the effect of three generations CD19 CART is observed in cell and zoopery, it was confirmed that three generations CD19 CART can effectively kill tumour cell.
Description
Technical field
The present invention relates to a kind of Chimeric antigen receptor T cell and preparation method and application.
Background technology
Chimeric antigen receptor T cell (Chimeric Antigen Receptor T cells, CART) is repaiied by gene
The means of decorations, enable the single-stranded variable region of the monoclonal antibody of specific recognition target antigen
(scFv) expression is on T cell surface, while scFv is increased by transmembrane region with the activation of the T cell intracellular of engineer
Signal domain is grown mutually to couple.So, monoclonal antibody is combined to the specific recognition of target antigen with the function phase of T cell, is produced special
The lethal effect of the opposite sex, and CART can kill target cell in the restricted modes of non-MHC.Existing CAR-T lymphocyte technologies
The third generation is had developed to.1989, scientist Zelig Eshhar proposed the conception of CART, were sent out in subsequent CART technologies
Zhan Zhong, mainly have passed through the evolution in 3 generations, first generation CAR by identification TSA single-chain antibody (scFv) antigen binding
Portion and ITAM are constituted, extracellular to recognize tumor-marker molecule by single-chain antibody (scFv), such as CD19,
Intracellular transmits signal using CD3 molecule ζ chains, and due to no costimulatory signal, T cell generation time is short, cytokine secretion
Low, in clinical test, effect is undesirable.2011, Carl professors June treated 3 chronic pouring using two generation CART technologies
Bar cell leukemia achieves extraordinary effect, and 2 complete incidence graphs, 1 part is alleviated；Two generation CART technologies are according to selection
Costimulatory molecules difference be divided into two classes, one kind is costimulatory molecules CD28+CD3 ζ, and one kind is costimulatory molecules 4-
1BB+CD3 ζ, that is, introduce the signal sequence of costimulation, can improve cell levisticum, proliferative and time-to-live, the rush of T cell
Enter the release of cell factor, this two classes CART is achieved successfully in clinical treatment acute lymphatic leukemia.CD28
Costimulatory signal promotes T cell rapid amplifying, and 4-1BB signals promote T cell to expand, survive, the continued survival time is longer, knot
CART, referred to as three generations CART of two molecules of CD28 and 4-1BB on a CAR molecular structure are closed, i.e., simultaneously in cytoplasmic domain string
Connection is arranged CD28,4-1BB costimulatory molecules and CD3 ζ molecules, simulates what is be subject to when T cell normally plays a role substantially
Stimulus signal, compares with two generation carriers, and the signal sequence for introducing double costimulations will be with more preferable effect.
CART is the technology of the treatment tumour for obtaining remarkable break-throughs in nearly 2 years, and the technology is first in treatment with CD19 as target spot
Acute lymphatic leukemia and lymthoma on achieve successfully, according to Novartis Co., Ltd report clinical data, utilize
CD19CART treats children's B cell acute lymphatic leukemia, can reach 93% complete remission rate.At present, facing
In, the CART carrier structures for treating leukaemia and lymthoma design the structure for being all two generation carriers to bed.Although
When in clinical treatment in particular for treatment leukaemia achieve extraordinary effect, but these patients enter group condition more
It is harsh, can not still enter a group treatment for the very serious patient of the state of an illness；In addition, for CD19 CART treat lymthoma when it is complete
Direct release rate is about 30%-47%, still suffers from continuing to improve the space improved.It would therefore be highly desirable to further increase CAR molecular structures
The quantity of costimulatory molecules in cytoplasmic domain, can more preferably simulate the mechanism that T cell is activated naturally under physiology and pathologic condition,
Cell is promoted to expand, killing tumor cell, improves CD19CART therapeutic effects at the continued survival time in extension body.
The content of the invention
The purpose of the present invention is achieved through the following technical solutions, and a kind of Chimeric antigen receptor T cell, surface expression is embedding
Close antigen receptor CD19CAR genes, its base sequence such as SEQ ID NO:Shown in 3.
Further, the Chimeric antigen receptor CD19CAR genes are by synthesizing 3 generation CAR structure bases of ScFV genes and synthesis
Because being spliced by over-lap PCR.
Further, the synthesis ScFV genes are to obtain coding CD8 signal peptides, sequence number by PCR method
The heavy chain amino codon optimization of CAA74659.1 is nucleotides, (G4S)3Hinge area and Serial No.
The light chain amino acid codon optimization of CAA74660.1 is the ScFV genes of the CD19 single-chain antibodies of nucleotide sequence
Nucleotide fragments, its base sequence such as SEQ ID NO:Shown in 1.
Further, 3 generation CAR structural genes of the synthesis are to obtain coding CD8 hinge areas, CD28 cross-films by PCR method
The nucleotide fragments of the CAR structural genes of the nucleotide fragments of area, cytoplasmic domain, 4-1BB cytoplasmic domains and CD3 ζ cytoplasmic domains, its base
Sequence such as SEQ ID NO:Shown in 2.
Further aim of the present invention is achieved through the following technical solutions, a kind of system of Chimeric antigen receptor T cell
Preparation Method, comprises the following steps：
(1) 3 generation slow virus CAR carriers are built：Synthesis ScFV genes or 3 generation CAR structural genes of synthesis are expanded respectively first
Increase PCR, obtain synthesizing the PCR primer of the 3 generation CAR structural gene fragments of PCR primer and synthesis of ScFV genetic fragments；Then carry out
Over-lap PCR links together the PCR primer of the two genetic fragments, obtains the CD19CAR genes of 3 generation CAR structures；Finally lead to
Pre-Lenti-EF1-MCS carriers are crossed to CD19CAR gene digestions, clone is chosen in connection, conversion, and upgrading grain, sequencing obtains sequence
The slow virus carrier Pre-Lenti-EF1-CD19CAR of the correct expression CD19CAR of row；
(2) CD19CAR slow virus is packed, T cell is infected, CART is prepared.
Further, in step (1), the primer for the amplification PCR is EcoRI-up sequences such as SEQ ID NO:Shown in 4
With BamHI-down sequences such as SEQ ID NO:Shown in 5.
Further, in step (1), the primer for the over-lap PCR is Middle-R such as SEQ ID NO:Shown in 6 and
Middle-F such as SEQ ID NO:Shown in 7.
Further, in step (1), sequencing primer is Pre-up-Seq, such as SEQ ID NO:8 shown and Pre-down-
Seq, such as SEQ ID NO:Shown in 9.
Further, in step (1), in step (1), described pair of enzyme is selected from EcoRI-HF restriction enzymes and BamHI-HF
Restriction enzyme.
Further aim of the present invention is achieved through the following technical solutions, and a kind of Chimeric antigen receptor T cell is in system
Application in standby anti-leukocythemia and lymphoid tumor medicament.
The present invention adds two kinds of costimulatory signals in the structure of 3 generation CD19CAR carriers, constructs slow virus load
Body, and the effect of three generations CD19CART is observed in cell and zoopery, it was confirmed that three generations CD19CART can be killed effectively
Dead tumour cell.
Brief description of the drawings
By reading the detailed description of hereafter preferred embodiment, various other advantages and benefit is common for this area
Technical staff will be clear understanding.Accompanying drawing is only used for showing the purpose of preferred embodiment, and is not considered as to the present invention
Limitation.And in whole accompanying drawing, identical part is denoted by the same reference numerals.In the accompanying drawings：
Fig. 1 is the evolution of CART carriers design.
Fig. 2 is that CD19CAR slow virus carriers build flow chart
Fig. 3 is ScFV genes and 3 generation CAR structural gene PCR primer electrophoretograms.Wherein, the left side is ScFV genes, and centre is
Marker (1000,2000,3000,4000,5000,6000,8000,10000bp), the right is 3 generation CAR structural genes.
Fig. 4 is overlapping PCR products electrophoretogram.Wherein, the left side be Marker (1000,2000,3000,4000,5000,
6000,8000,10000bp), the right is CD19CAR genes (1728bp).
Fig. 5 is the fragmentation effect of CD19-CART.
Fig. 6 is to inject the mouse liver table of Control or CD19CART after two weeks respectively after injecting Raji lymphoma cells
It is existing.
Fig. 7 is mouse survival curve.
Specific embodiment
The illustrative embodiments of the disclosure are more fully described below with reference to accompanying drawings.Although showing this public affairs in accompanying drawing
The illustrative embodiments opened, it being understood, however, that may be realized in various forms the disclosure without the reality that should be illustrated here
The mode of applying is limited.Conversely, there is provided these implementation methods are able to be best understood from the disclosure, and can be by this public affairs
The scope opened it is complete convey to those skilled in the art.
Embodiment 1：Pre-Lenti-EF1-CD19CAR vector constructions
1st, ScFV genes are synthesized：The heavy chain ammonia of coding CD8 signal peptides, sequence number CAA74659.1 is obtained by PCR method
Base acid codons are optimized for nucleotides, (G4S)3The light chain amino acid codon optimization of hinge area and Serial No. CAA74660.1
It is the nucleotide fragments of the ScFV genes of the CD19 single-chain antibodies of nucleotide sequence, its base sequence such as SEQ ID NO:Shown in 1.
2nd, synthesize 3 generation CAR structural genes, by PCR method obtain coding CD8 hinge areas, CD28 transmembrane regions, cytoplasmic domain,
The nucleotide fragments of the CAR structural genes of the nucleotide fragments of 4-1BB cytoplasmic domains and CD3 ζ cytoplasmic domains, its base sequence such as SEQ
ID NO:Shown in 2.
3rd, synthesis ScFV genes and 3 generation CAR structural genes are spliced by over-lap PCR, are obtained CD19CAR genes,
Such as SEQ ID NO:Shown in 3.Detailed process is as follows：
(1) design of primers：CD19CAR gene orders, single endonuclease digestion site are analyzed using sequence analysis software pDRAW32
EcoRI, BamHI are suitable for gene cloning, can be matched with the MCS (MCS) of Pre-Lenti-EF1-MCS carriers,
Design two ends primer is as follows：
EcoRI-up sequences：CGGAATTC GCCACCATGGCCTTACCAGTGACCGC, such as SEQ ID NO:Shown in 4；
BamHI-down sequences：CGGGATCC TTAGCGAGGGGGCAGGGCCTG, such as SEQ ID NO:Shown in 5.For
The primer of over-lap PCR is：
Middle-R, GTCGTGGTGGGCTTCGCTGGTGAGGAGACGGTGACTGAGG, such as SEQ ID NO:Shown in 6.
Middle-F, CCTCAGTCACCGTCTCCTCACCAGCGAAGCCCACCACGAC, such as SEQ ID NO:Shown in 7.
(2) PCR overlap-extension PCRs：After receiving the gene and primer of synthesis, enter performing PCR overlap-extension PCR.
PCR system is following (using the KOD Fx enzymes of Toyobo)：
PCR response procedures are：
PCR primer electrophoresis：See Fig. 3, gel electrophoresis, glue reclaim (Biomiga, article No. are carried out to PCR primer：DC-3511-
01), after quantitative PCR fragment concentrations, over-lap PCR is carried out.
PCR system sees below：
PCR programs are：
PCR primer electrophoresis：See Fig. 4, PCR primer gel electrophoresis, glue reclaim carries out follow-up cloning experimentation.
(3) digestion：
Endonuclease reaction system is as follows：
(4) product after digestion is reclaimed：Biomiga glue reclaim kit (article No.s：DC-3511-01).
(5) coupled reaction：
According to carrier nmole numbers：Fragment nmole number=1：3, i.e.,
Prepare linked system：
The above each component is added in 1.5mL centrifuge tubes, is fully mixed；22 DEG C of water-bath 30min；
(5) convert, apply ammonia benzyl flat board：On ice by above-mentioned connection product 50 μ L competence of addition, flick, stand 20min；
42 DEG C of water-bath heat shocks 90 seconds；5min on ice；Plus 500 μ L LB, 37 DEG C of recovery 1h；4000rpm is centrifuged 5min, stays appropriate supernatant,
Sample loading gun is resuspended, applies ammonia benzyl resistant panel；37 DEG C of incubators are overnight.10th, clone is chosen, bacterium is shaken：One company of being not added with is set during coupled reaction
The negative control of tab segments, second day observes clone's number about 50 or so in the flat board of visible addition CD19CAR fragments, hence it is evident that ratio
Not plus fragment clone's number (10) it is many, therefore judge successful connection.6 clones of random picking, shake bacterium amplification.
(6) plasmid extraction：Biomiga endotoxin-free plasmid extraction kit (article No.s：PD1220-02).
(7) it is sequenced：Concentration plasmid order-checking higher is chosen, primer is：
Pre-up-Seq：GGAGCCTACCTAGACTCAGC, such as SEQ ID NO:Shown in 8,
Pre-down-Seq：CAACCAGGATTTATACAAGG, such as SEQ ID NO:Shown in 9,
Sequencing result is completely correct through comparing, and has obtained expressing the slow virus carrier Pre-Lenti- of CD19CAR genes
EF1-CD19CAR, structure is shown in Fig. 2.
Embodiment 2：Cell killing is tested
1st, slow virus packaging
(1) 293T cell culture is in 37 DEG C, 5%CO2In incubator, culture medium is DMEM/10%FBS.
(2) packaging virus the previous day, pancreatin digestion 293T cells, 1 × 107Cells/well plants 10cm culture dishes.
(3) during transfectional cell, in addition to Pre-Lenti-EF1-MCS-CD19CAR plasmids, every kind of plasmid will also and be packed
Plasmid psPAX2, pMD2.0G cotransfection.Wherein Pre-Lenti-EF1-MCS-CD19CAR uses 3.75 μ using 5 μ g, psPAX2
G, pMD2.0G use 1.25 μ g.During transfection, the mixture of three of the above plasmid is added in 500 μ l opti-MEM culture mediums,
The reagents of 25 μ l Lipofectamine 2000 are added in 500 μ l opti-MEM culture mediums in another micro centrifugal pipe,
Then the transfection reagent of dilution is added dropwise over the plasmid top of dilution, is mixed, centrifugation is stored at room temperature 20min, finally by plasmid
Mixture with transfection reagent is added in 10cm culture dishes, light to shake, mix, and is put into incubator.
(4) cell transfecting can harvest virus after 3 days, and 10ml bases containing Virus culture are transferred in 50ml centrifuge tubes, 4 DEG C,
Then 1250rpm, 5min, the dead 293T cells of removal floating will contain the filtering of Virus culture base, concentration, packing, -80 DEG C of jellies
Deposit, and remaining portions virus determines titre.
2nd, separation of lymphocytes
(1) draw blood：The venous blood 10ml of human body is extracted under sterile conditions.
(2) dilute：The 1640 culture medium 10ml dilutions of the blood same volume that will be obtained.
(3) 10ml human lymphocytes separating liquid (being biological Engineering Co., Ltd up to section) is added in centrifuge tube.
(4) it is slowly added to blood is adherent in centrifuge tube with electronic liquid-transfering gun.
(5) centrifuge tube is put into centrifuge, with 700g, 22 DEG C of centrifugation 25min.
(6) after centrifugation terminates, in tunica albuginea layer of the lymphocyte between upper plasma layer and separating liquid.
(7) try one's best to be drawn onto tunica albuginea layer in another centrifuge tube with 84 droppers and (add the culture mediums of 30ml 1640), note not
It is drawn onto separating liquid.
(8) 250g, 22 DEG C, 10 minutes.
(9) supernatant liquid is discarded, it is resuspended with 1640 culture mediums containing IL2, inactivated serum, count.
3rd, T cell purifying
(1) LC, takes 1x107Individual lymphocyte is in micro centrifugal pipe.
(2) 250g, 22 DEG C, 10 minutes.
(3) supernatant liquid is discarded, 80 μ L Beads enrichment buffer solutions re-suspended cells precipitation adds 20 μ l's
CD3MicroBeads (U.S. day Ni).
Placed 1 hour in (4) 4 DEG C of refrigerators, it is ensured that fully combine.
After (5) 1 hours, the Beads enrichment buffer solution of addition 1mL in micro centrifugal pipe, 250g, 5 DEG C are centrifuged 10 minutes.
Period prepares filtering pillar, and Filter column is placed on magnet, and with the buffer solution rinse of 500 μ l, (buffer solution is stayed with gravity
Under).
(6) cell abandons supernatant after being centrifuged, and the buffer solution of 500 μ l is resuspended, adds pillar, and buffer solution is downward with gravity, carefully
After the outflow of born of the same parents' suspension, pillar is washed four times with the buffer solution of 500 μ l.
(7) pillar is laid down from magnet, is gone out T cell with 1mL buffer solutions, to 1ml centrifuge tubes.
(8) 250g, 5 DEG C are centrifuged 10 minutes.
(9) 1640 culture mediums containing IL2, containing inactivated serum are resuspended, count.
4th, T cell slow-virus infection
(1) purifying T is counted, and 2 × 106Individual T cell/hole (6 orifice plate) plantation, after overnight incubation, adds the control that MOI is 2
Virus liquid (empty carrier is packed, Control) and Pre-Lenti-EF1-MCS-CD19CAR virus liquids, infection is overnight.
(2) infect second day, add 1ml fresh cultures.
(3) infect the 3rd day, T cell is fully activated, molecular marker for increased proliferation, and T cell now is transferred into the blake bottles of 25cm 2.
(4) after infecting 5 days, killing experiments are carried out.
5th, killing experiments
(K562-CD19, the stabilization set up by the method for slow-virus infection is thin up to the K562 cells of CD19 molecules for count table
Born of the same parents system), 1 × 104Individual K562-CD19 cells/wells (96 orifice plate)；Count T cell (the Control virus liquids infection of virus infection
Control T cells and Pre-Lenti-EF1-MCS-CD19CAR virus liquids infection CD19CAR-T cells), according to effect target
Than 10:1,5:1,2.5:1 ratio adds K562-CD19 cells upper strata, and experimental design is shown in Table 1.
The killing experiments of table 1. design table (target cell：K562.CD19)
6th, measurement is killed：The LDH of cells and supernatant is released into by measuring dead cell to kill determining CAR-T cells
The effect of dead K562-CD19 target cells.
(1) operation is according to Promega kit specifications (the non-radioactive cell toxicity detections of CytoTox 96, article No.：
G1780)。
(2) killing experiments overnight incubation in step 5, after about 18h, maximum release aperture adds 10 × cell pyrolysis liquid.
After (3) 2 hours, 50 μ l supernatants are taken per hole, add 50 μ l LDH zymolytes, after being stored at room temperature 20 minutes, enzyme mark
Absorbance OD492 at instrument measurement 492nm.
(4) killing rate is calculated, and computing formula is
Experimental port be different effect targets than the OD492 that obtains, it is Control T cells or CD19CAR-T that effector cell is spontaneous
The OD492 of cell, target cell is spontaneous to be discharged for K562-CD19 is minimum, and target cell is the maximum releases of K562-CD19 to the maximum.Calculate
The killing rate for obtaining is shown in Fig. 5, and target cell is the cell that K562 expresses CD19, effector cell for control (Control) T cell and
CD19-CART cells, as can be seen from Figure 5, CD19-CART cells can express the K562 cells of CD19 with specific killing, show
Pre-Lenti-EF1-MCS-CD19CAR-T is successfully constructed.
Embodiment 3：Zoopery
1st, mouse species：It is NSG immunodeficient mouses strain (lacking T cell, B cell and NK cell functions).2nd, tumour
Inoculation：Using Raji lymphoma cell lines, 1x106Cell/200ul PBS tail vein injections (6 mouse).
3rd, CART injections：After Raji tumor cell inoculations 2 days, tail vein injection Control or CD19CART cell
(1x107Cell/200ul PBS, one group of 3 mouse), see Fig. 6.It will be appreciated from fig. 6 that control CART can not effectively suppress lymthoma
In the growth (white patch is the tumour that lymphoma cell is formed) of liver, CD19CART can effectively suppress lymthoma to cell.
4th, observed every 2 days, measure Mouse Weight, until CART cell infusions are after 2 weeks, control group (Control) mouse
Start hind limb paralysis occur, dead successively, anatomic observation records the death time, arranges survivorship curve, sees Fig. 7.As shown in Figure 7,
CD19CART has been obviously prolonged the mouse survival time.
The above, the only present invention preferably specific embodiment, but protection scope of the present invention is not limited thereto,
Any one skilled in the art the invention discloses technical scope in, the change or replacement that can be readily occurred in,
Should all be included within the scope of the present invention.Therefore, protection scope of the present invention should be with the protection model of the claim
Enclose and be defined.
SEQUENCE LISTING
<110>Beijing Puri gold Science and Technology Ltd.
<120>A kind of Chimeric antigen receptor T cell and preparation method and application
<130> 2016
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 789
<212> DNA
<213>ScFV genes
<400> 1
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120
accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180
ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240
tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300
caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360
ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420
ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480
ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540
cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600
tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660
gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720
cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780
gtctcctca 789
<210> 2
<211> 939
<212> DNA
<213>CAR structural genes
<400> 2
ccagcgaagc ccaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 60
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 120
acgagggggc tggacttcgc cccacgcaaa attgaagtta tgtatcctcc tccttaccta 180
gacaatgaga agagcaatgg aaccattatc catgtgaaag ggaaacacct ttgtccaagt 240
cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg tggagtcctg 300
gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag gagtaagagg 360
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 420
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc caaacggggc 480
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 540
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga 600
gtgaagttca gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat 660
aacgagctca atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg 720
gaccctgaga tggggggaaa gccgcagaga aggaagaacc ctcaggaagg cctgtacaat 780
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 840
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 900
tacgacgccc ttcacatgca ggccctgccc cctcgctaa 939
<210> 3
<211> 1728
<212> DNA
<213>CD19 CAR genes
<400> 3
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120
accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180
ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240
tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300
caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360
ggggggacca agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc 420
ggcggatctg aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc 480
ctgtccgtca catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt 540
cgccagcctc cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca 600
tactataatt cagctctcaa atccagactg accatcatca aggacaactc caagagccaa 660
gttttcttaa aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa 720
cattattact acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc 780
gtctcctcac cagcgaagcc caccacgacg ccagcgccgc gaccaccaac accggcgccc 840
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900
gcagtgcaca cgagggggct ggacttcgcc ccacgcaaaa ttgaagttat gtatcctcct 960
ccttacctag acaatgagaa gagcaatgga accattatcc atgtgaaagg gaaacacctt 1020
tgtccaagtc ccctatttcc cggaccttct aagccctttt gggtgctggt ggtggttggt 1080
ggagtcctgg cttgctatag cttgctagta acagtggcct ttattatttt ctgggtgagg 1140
agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 1200
cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 1260
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 1320
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 1380
gaactgagag tgaagttcag caggagcgca gacgcccccg cgtaccagca gggccagaac 1440
cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 1500
cgtggccggg accctgagat ggggggaaag ccgcagagaa ggaagaaccc tcaggaaggc 1560
ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1620
ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1680
aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgctaa 1728
<210> 4
<211> 34
<212> DNA
<213>It is artificial synthesized
<400> 4
cggaattcgc caccatggcc ttaccagtga ccgc 34
<210> 5
<211> 29
<212> DNA
<213>It is artificial synthesized
<400> 5
cgggatcctt agcgaggggg cagggcctg 29
<210> 6
<211> 40
<212> DNA
<213>It is artificial synthesized
<400> 6
gtcgtggtgg gcttcgctgg tgaggagacg gtgactgagg 40
<210> 7
<211> 40
<212> DNA
<213>It is artificial synthesized
<400> 7
cctcagtcac cgtctcctca ccagcgaagc ccaccacgac 40
<210> 8
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 8
ggagcctacc tagactcagc 20
<210> 9
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 9
caaccaggat ttatacaagg 20
Claims (10)
1. a kind of Chimeric antigen receptor T cell, it is characterised in that the T cell surface expression Chimeric antigen receptor CD19CAR bases
Cause, its base sequence such as SEQ ID NO:Shown in 3.
2. Chimeric antigen receptor T cell according to claim 1, it is characterised in that the Chimeric antigen receptor CD19CAR bases
Because being spliced by over-lap PCR by synthesizing ScFV genes and 3 generation CAR structural genes of synthesis.
3. Chimeric antigen receptor T cell according to claim 2, it is characterised in that the synthesis ScFV genes are to pass through
PCR method obtains coding CD8 signal peptides, the heavy chain amino codon optimization of sequence number CAA74659.1 is nucleotides,
(G4S)3The light chain amino acid codon optimization of hinge area and Serial No. CAA74660.1 is single-stranded for the CD19 of nucleotide sequence
The nucleotide fragments of the ScFV genes of antibody, its base sequence such as SEQ ID NO:Shown in 1.
4. Chimeric antigen receptor T cell according to claim 2, it is characterised in that the synthesis 3 generation CAR structural genes
It is the core that coding CD8 hinge areas, CD28 transmembrane regions, cytoplasmic domain, 4-1BB cytoplasmic domains and CD3 ζ cytoplasmic domains are obtained by PCR method
The nucleotide fragments of the CAR structural genes of acid fragments, its base sequence such as SEQ ID NO:Shown in 2.
5. the preparation method of a kind of Chimeric antigen receptor T cell as described in one of claim 1-4, it is characterised in that including
Following steps：
(1) 3 generation slow virus CAR carriers are built：Synthesis ScFV genes or 3 generation CAR structural genes of synthesis are expanded respectively first
PCR, obtains synthesizing the PCR primer of the 3 generation CAR structural gene fragments of PCR primer and synthesis of ScFV genetic fragments；Then weight is carried out
Folded PCR links together the PCR primer of the two genetic fragments, obtains the CD19CAR genes of 3 generation CAR structures；Finally will
Clone is chosen in Pre-Lenti-EF1-MCS carriers and CD19CAR gene double digestions, connection, conversion, and upgrading grain, sequencing obtains sequence
The slow virus carrier Pre-Lenti-EF1-CD19CAR of the correct expression CD19CAR of row；
(2) CD19CAR slow virus is packed, T cell is infected, CART is prepared.
6. the preparation method of Chimeric antigen receptor T cell according to claim 5, it is characterised in that in step (1), uses
In the amplification PCR primer be EcoRI-up sequences such as SEQ ID NO:With BamHI-down sequences such as SEQ ID shown in 4
NO:Shown in 5.
7. the preparation method of Chimeric antigen receptor T cell according to claim 5, it is characterised in that in step (1), uses
In the over-lap PCR primer be Middle-R such as SEQ ID NO:With Middle-F such as SEQ ID NO shown in 6:Shown in 7.
8. the preparation method of Chimeric antigen receptor T cell according to claim 5, it is characterised in that in step (1), institute
Sequencing primer is stated for Pre-up-Seq, such as SEQ ID NO:8 shown and Pre-down-Seq, such as SEQ ID NO:Shown in 9.
9. the preparation method of Chimeric antigen receptor T cell according to claim 5, it is characterised in that in step (1), institute
State double enzymes and be selected from EcoRI-HF restriction enzymes and BamHI-HF restriction enzymes.
10. according to one of claim 1-4 described Chimeric antigen receptor T cell in anti-leukocythemia and lymphoid tumor medicament is prepared
Application.
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| CN107603995A (en) * | 2017-07-31 | 2018-01-19 | 北京普瑞金科技有限公司 | A kind of CD19 CART and its application |
| CN107937428A (en) * | 2017-11-28 | 2018-04-20 | 马晓冬 | A kind of carrier construction method of integration microRNA and CAR functions |
| CN108017716A (en) * | 2018-01-02 | 2018-05-11 | 赖沛龙 | Chimeric antigen receptor, slow virus carrier, expression cell and medicine comprising C5aR intracellular domain |
| CN109468282A (en) * | 2018-11-22 | 2019-03-15 | 青岛协和华美医学诊断技术有限公司 | Preparation method and application of chimeric antigen receptor T cells targeting CD19 |
| CN111566219A (en) * | 2017-12-27 | 2020-08-21 | 环球生命科技咨询美国有限责任公司 | Methods and kits for viral vector isolation |
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| CB02 | Change of applicant information |
Address after: Zi street Jinhui road Shenzhen city Guangdong province 518122 Pingshan District pit No. 14 Shenzhen Biomedical Innovation Industrial Park, building 1, 402 Applicant after: Shenzhen Purim biopharmaceutical Co., Ltd. Address before: 100161 Beijing city Fengtai District Wanfeng Road No. 308 Shunhe business office building room 8422 Applicant before: BEIJING PREGENE BIOTECHNOLOGY COMPANY,LTD. |
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| CB02 | Change of applicant information | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170531 |
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| RJ01 | Rejection of invention patent application after publication |