CN107287164A - Target CD19 Chimeric antigen receptor T cell, preparation method and application - Google Patents
Target CD19 Chimeric antigen receptor T cell, preparation method and application Download PDFInfo
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Abstract
The invention provides targeting CD19 Chimeric antigen receptor T cell, preparation method and application, it is related to immunocyte field, it is possible to increase T cell activation ability in the treatment of CAR T cells, overcomes the problem of transfection efficiency is not enough, the killing ability to CD19 is strong.Targeting CD19 Chimeric antigen receptor T cell, surface expression targets CD19 Chimeric antigen receptor gene, the Chimeric antigen receptor gene of the targeting CD19 by CD19 single-chain antibody CD19ScFv, CD8 hinge area and transmembrane region, CD28 intracellular signal structure, 4 1BB intracellular signal structure and CD3 ζ intracellular signal domain it is in series, its base sequence such as SEQ ID NO:Shown in 2.
Description
Technical field
The present invention relates to immunocyte field, more particularly to a kind of targeting CD19 Chimeric antigen receptor T cell, preparation side
Method and application.
Background technology
B cell malignant tumour is one group of pernicious different substantiality disease of hematological system, including the white blood of acute and chronic bone-marrow-derived lymphocyte
Disease and some lymthoma hypotypes, are clinical more refractory Malignancies more because of its high recurrence rate, poor prognosis.
CD19 is not only expressed in normal pre B cell and mature B cell surface as the special cell surface differentiation antigen of B cell system,
Wide expression is on a variety of B cell malignant cell surfaces, and in candidate stem cell, thick liquid cell and other normal tissue cells
Do not express, also do not detected the presence of CD19 soluble proteins in blood.In addition, distribution of the CD19 molecules on film is more sudden and more violent
Dew, is easily combined with monoclonal antibody, and after combination without notable internalization, come off and antigenic modulation generation, therefore it is considered as
It is a promising target for treating B cell tumour.
Chimeric antigen receptor (chimeric antigen receptor), is in artificial synthesized φt cell receptor, structure
It is activation signal domain (hinge area, transmembrane region, intracellular signal transduction area) group by extracellular targeting bonding pad and T cell
Into.Chimeric antigen receptor T cell is one of tumour cell immunization therapy mode of most prospect.By the way that the T cell of patient is " heavy
Coding ", makes it to be recognized the signal transmission of activationa and proliferation T cell with specific recognition tumor associated antigen target spot after combining
To intracellular, cause t cell activation and propagation, so that effectively killing tumor cell.Can effectively it be controlled by this targeted therapy
Treat the effect that tumour is even up to healing.Compared to traditional treatment method, CAR-T treatments show big advantage, such as right
The specific killing of tumour, as long as expressing its targeted target spot can just kill, kills knurl scope extensively, and to metastatic and recurrence
Property tumour is all effective.
Although CAR-T treatments clinically achieve good curative effect, the CAR-T treatments for especially targetting CD19 are even more to make
Lymphocytic leukemia patient positive CD19 reaches 90% remission rate, but involved in the preparation process of CAR-T cells
To the step of and details it is a lot, such as activation of T cell, transfection and amplification etc., the not enough of any link is all limitation CAR-T
The principal element that cell is carried out and applied extensively, not yet compares the report of the second generation and third generation CAR, two generations in vivo at present
Difference between CAR may come from signal transduction domain incessantly, extracellular antigen binding domain (scFv), the transfection for recombinating T cell
Method, the feedback mode for recombinating T cell etc. may influence the final antitumous effect of CAR-T cells.CN106754725A is public
A kind of Chimeric antigen receptor T cell and preparation method and application is opened, preparation method does not optimize, the killing rate of target cell
It is only not strong in the activation capacity of 75% or so, CAR-T cells.Therefore, this area needs exploitation and optimization CAR-T structures and preparation badly
Method, promotes cell to expand, and improves tumor cytotoxicity efficiency.
The content of the invention
It is an object of the invention to provide a kind of targeting CD19 Chimeric antigen receptor T cell, preparation method and application, carry
T cell activation ability in high CAR-T cell therapies, overcomes the problem of transfection efficiency is not enough, the killing ability to CD19 is strong.
One aspect of the present invention provides a kind of targeting CD19 Chimeric antigen receptor T cell, surface expression targeting CD19's
Chimeric antigen receptor gene, the Chimeric antigen receptor gene of the targeting CD19 by CD19 single-chain antibody CD19ScFv, CD8
Hinge area and transmembrane region, CD28 intracellular signal domain, 4-1BB intracellular signal domain and CD3 ζ intracellular signal knot
Structure domain is in series, its base sequence such as SEQ ID NO:Shown in 2, its amino acid sequence such as SEQ ID NO:Shown in 3.
As optimal technical scheme, CD19 single-chain antibody CD19ScFv sequences are by mouse anti human CD 19 antibody cloning FMC63
Heavy chain and light-chain variable sequence codon optimization and obtain, its base sequence such as SEQ ID NO:Shown in 1.
Another aspect of the present invention provides the preparation method of targeting CD19 Chimeric antigen receptor T cell, including following step
Suddenly：
(1) structure of CD19-CAR slow virus carriers
Restriction enzyme site is added at above-mentioned targeting CD19 Chimeric antigen receptor gene order two ends, with Lentiviral
Plv-EF1a double digestions, reclaim DNA fragmentation, connect, conversion, and picking monoclonal extracts plasmid, by digestion and sequencing identification,
Obtain CD19-CAR slow virus carriers Plv-EF1a-CD19ScFv；
(2) preparation of slow virus
Extract slow virus skeleton plasmid Plv-EF1a, slow virus expression plasmid Plv-EF1a-CD19ScFv, helper plasmid
PsPAX2 and pMD2.G, determines plasmid concentration, prepares rotaring redyeing system, transfects HEK293T/17 cells, separates to obtain slow virus concentration
Liquid；
(3) separation and culture of T cell
Healthy peripheral blood is taken, lymphocyte is separated, culture 24-48h is stimulated.
(4) infection and amplification of T cell
The T cell vaccination that above-mentioned stimulation is cultivated adds the slow virus concentrate of step (2) to culture plate, infects 8-
12h, obtains targeting CD19 Chimeric antigen receptor T cell.
As optimal technical scheme, restriction enzyme site is added at above-mentioned targeting CD19 Chimeric antigen receptor gene order two ends
BamH1 and Sal1, double digestion is carried out with Lentiviral Plv-EF1a with restriction enzyme BamH1 and Sal1.
As optimal technical scheme, the preparation rotaring redyeing system transfects HEK293T/17 cells, separates to obtain slow virus concentration
The concrete operations of liquid are：The HEK293T/17 cell culture that cell state is good is taken, by expression plasmid Plv-EF1a or Plv-
EF1a-CD19ScFv, helper plasmid psPAX2 and pMD2.G are with 9:6:3 mass ratio and LipoFilter transfection reagents and nothing
Serum DMEM culture mediums prepare rotaring redyeing system, and nutrient solution is changed after cotransfection HEK293T/17 cells, 12h after incubation；
Collect viral supernatants in 24h, 48h after liquid is changed in transfection respectively, centrifuge, filtering, the virus liquid after filtering exceed the speed limit from
The heart, discards supernatant, and precipitation is resuspended with serum-free medium, obtains slow virus concentrate.
As optimal technical scheme, the concrete operations of separation and the culture of the T cell are：Healthy peripheral blood is taken, is centrifuged
After stay autologous plasma standby, the isometric normal saline dilution of remaining haemocyte is added to the upper strata of lymphocyte separation medium, from
The heart, draws middle tunica albuginea confluent monolayer cells, adds brine, and centrifugation obtains the lymphocyte of separation；
Lymphocyte after separation adds the autologous plasma of 2% volume to be resuspended with T lymphocyte culture mediums, and it is close to adjust cell
Degree, is inoculated in the coated culture plate of CD3 and CD28 antibody that concentration is 5ug/ml, stimulates culture 24h-48h.
As optimal technical scheme, supplement final concentration of 500U/ml's in the culture plate of the autologous plasma containing 2% volume
RhIL-2,25ng/ml rhIL-7 and 50ng/ml recombinant human interleukin 15.
The Chimeric antigen receptor T cell that another aspect of the present invention provides targeting CD19 is preparing anti-B cell malignant tumour
Application in medicine.
Compared with prior art, positive and beneficial effect of the invention is：
1st, targeting CD19 of the invention Chimeric antigen receptor T cell, surface expression targets CD19 Chimeric antigen receptor
Gene, the single-chain antibody CD19ScFvCD19 sequences for targetting CD19 in CD19 Chimeric antigen receptor gene are resisted by mouse anti human CD 19
Body clone FMC63 heavy chain and the optimization of light-chain variable sequence codon are obtained, and the fragmentation effect with CAR-T cells is close
Correlation, significantly improves killing ability of the CAR-T cells to CD19.
2nd, optimization of the targeting CD19 of the invention Chimeric antigen receptor T cell preparation method Jing Guo step so that prepare
CAR-T cells activation capacity it is strong, CD3 ratio is high, and the transduction efficiency of T cell is high.
3rd, preparation method of the invention more effectively can stimulate T cell to breed, and improve the quality and quantity of gained T cell,
Ensure the good fragmentation effect of sufficient amount of CAR-T cells plays.
Brief description of the drawings
Fig. 1 illustrates for the slow virus plasmid construct of the Chimeric antigen receptor gene containing targeting CD19 of the embodiment of the present invention
Figure；
Fig. 2 is the Lentiviral Plv-EF1a-CD19ScFv of embodiment of the present invention restriction enzyme
The electroresis appraisal figure of BamH1/Sal1 double digestion fragments；
Fig. 3 for the embodiment of the present invention flow cytomery culture before and after CD3 positive T cells ratio chart；
T cell surface Fab is CAR expression figure after Fig. 4 infects for the flow cytomery of the embodiment of the present invention；
When Fig. 5 imitates target ratio for the flow cytomery difference of the embodiment of the present invention, the ratio that target cell is killed by T cell
Variation diagram；
When Fig. 6 detects different effect target ratios for the LDH cytotoxicities of the embodiment of the present invention, the ratio that target cell is killed by T cell
Example variation diagram.
Embodiment
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described implementation
Example only a part of embodiment of the invention, rather than whole embodiments.Based on the embodiment in the present invention, this area is common
The every other embodiment that technical staff is obtained under the premise of creative work is not made, belongs to the model that the present invention is protected
Enclose.
The embodiments of the invention provide a kind of targeting CD19 Chimeric antigen receptor T cell, the chimeric antigen is additionally provided
The preparation method and application of recipient T cells, are illustrated below with reference to specific embodiment.
Embodiment 1
Target the preparation of CD19 Chimeric antigen receptor
Target CD19 Chimeric antigen receptor gene by CD19 single-chain antibody CD19ScFv, CD8 hinge area and cross-film
The intracellular signal domain in area, CD28 intracellular signal structure and CD3 ζ is in series, and its structural representation is shown in Fig. 1, its alkali
Basic sequence such as SEQ ID NO:Shown in 2, length is 1584bp.
Wherein, CD19 single-chain antibody CD19ScFv sequences by mouse anti human CD 19 antibody cloning FMC63 heavy chain and light chain
Variable region sequences codon optimizes and obtained, its base sequence such as SEQ ID NO:Shown in 1, length is 726bp.
Embodiment 2
The structure of CD19-CAR slow virus carriers
The Chimeric antigen receptor gene order two ends for targetting CD19 add BamH1 and Sal1 restriction enzyme sites, complete sequence respectively
Synthesis.Sequence and Lentiviral Plv-EF1a after synthesis carry out double digestion with restriction enzyme BamH1 and Sal1.
Digestion system is as follows：
Plasmid containing composition sequence
Or Plv-EF1a empty carrier plasmids 4ug
BamH1 2ul
Sal1 2ul
10XBuffer 5ul
ddH2O is supplemented to 50ul
Digestion products enter row agarose gel electrophoresis, and the correct fragment of size is scaled off, and utilize (the purchase of glue reclaim kit
From Takara companies) reclaim DNA fragmentation.
CAR genetic fragments after recovery are connected with carrier segments using kit (being purchased from Takara companies) is connected
Connect, 16 DEG C of connections are stayed overnight.
Connection product 10ul is transferred in DH5a competence, overnight incubation in bacteriological incubator is placed.
Monoclonal bacterium colony is taken, expands culture in the LB nutrient solutions containing ampicillin.
By the correct clone of PCR preliminary screenings connection, mini-scale plasmid extracts kit extracts plasmid, by BamH1 and
Sal1 double digestions and sequencing identification, restriction enzyme digestion and electrophoresis figure are as shown in Fig. 2 wherein, M1 is 15000bp Marker, and M2 is 2000bp
Marker, 1 swimming lane is the Plv-EF1a-CD19ScFv of digestion, and correct plasmid is named as Plv-EF1a-CD19ScFv.Take
The correct bacterium solution of Partial Characterization is frozen in -80 DEG C of conservations.
Embodiment 3
Target the preparation of CD19 Chimeric antigen receptor T cell
(1) prepared by slow virus
Slow virus skeleton plasmid Plv-EF1a, slow disease are extracted with the big extraction reagent kit of endotoxin-free plasmid (Qiangen companies)
Malicious expression plasmid Plv-EF1a-CD19ScFv, helper plasmid psPAX2 and pMD2.G, is extracted according to operational manual, acquisition
Plasmid is through spectrophotometric determination concentration；
Take the HEK293T/17 cells that cell state is good, be inoculated in culture dish, quantity using after 24h cell fusion degree as
80% or so is advisable, with the DMEM nutrient solution cultures containing 10%FBS.
After 24h, by expression plasmid Plv-EF1a or Plv-EF1a-CD19ScFv and helper plasmid psPAX2 and pMD2.G with
9:6:3 mass ratio prepares rotaring redyeing system with LipoFilter transfection reagents and plasma-free DMEM medium, is incubated after 15min
Cotransfection HEK293T/17 cells, 12h changes fresh medium.
The 24h after liquid is changed in transfection, collects viral supernatants, 4 DEG C, 3000rpm centrifugations 15min respectively during 48h.Viral supernatants are used
4.5um filters are filtered, and the vial supernatant after filtering is transferred in ultracentrifugation pipe, 4 DEG C, 20000rpm centrifugations 2h.Discard
Clearly, precipitation presses 1 with serum-free medium:50 ratio is resuspended, you can obtains viral concentrate, is dispensed into 1.5mlEP pipes, often manages
200ul, -80 DEG C save backup.
(2) separation and culture of T cell
Stay autologous plasma standby after taking health donors peripheral blood, centrifugation with anticoagulant heparin pipe.Remaining haemocyte is with equal volume
Normal saline dilution, is then gently added to the upper strata of lymphocyte separation medium (GE companies), 400g centrifugations 30min；In absorption
Between tunica albuginea confluent monolayer cells, add physiology salt washing cell, 100g centrifugation 10min.
After the lymphocyte of acquisition is washed twice through physiology salt, with T lymphocyte culture mediums X-VIVO15 (Lonza companies)
Plus 2% volume autologous plasma be resuspended, and adjust cell density for 2 × 106/ ml, be inoculated in containing concentration for 5ug/ml CD3 and
In the culture plate of CD28 antibody, slow-virus infection is carried out after stimulating culture 24h-48h.
Final concentration of 500U/ml rhIL-2 (IL-2), 25ng/ml recombinant human interleukin are supplemented in culture medium
Element 7 (IL-7) and 50ng/ml recombinant human interleukin 15 (IL-15).
(3) infection and amplification of T cell
Cell after above-mentioned stimulation culture is pressed per hole 2 × 106Individual cell number is inoculated into RetroNectin coated 12
In well culture plate, coating buffer concentration used is 20ug/ml, each hole 500ul, and 4 DEG C are stayed overnight or 37 DEG C of 2h coatings.By in -80 DEG C
The viral concentration liquid frozen melts through room temperature, and each hole adds 200ul, and adds final concentration of 8ug/ml polybrene.
32 DEG C, 1800rpm centrifuges 12 orifice plate 1.5h.After be placed in 37 DEG C, saturated humidity is 5% CO28- is infected in incubator
Fresh X-VIVO15 nutrient solutions are changed after 12h, and supply autologous plasma and the factor IL-2, IL-7, IL-15.Change every three days
Fresh medium, so as to obtain targeting CD19 Chimeric antigen receptor T cell.
Embodiment 4
The ratio and transduction efficiency of flow cytometer detection T lymphocytes
Fraction is taken respectively after the isolated uncultivated lymphocyte of peripheral blood and slow-virus infection 7 days thin
Nutrient solution is discarded after born of the same parents, centrifugation, cell is washed with PBS, cell is resuspended with 100ulPBS afterwards and APC anti-human are added
Streaming Guan Zhongyong flow cytomeries are transferred to after CD3 (being purchased from Biolegend companies) streaming antibody labeled cells, mark,
As a result Fig. 3 is seen.Fig. 3 result shows that CD3 ratio is significantly raised after amplification compared to before amplification, and ratio is up to more than 90%
The cell of 7d empty carrier Plv-EF1a groups and experiment Plv-EF1a-CD19ScFv groups after infection is taken respectively, is used
PerCP anti-mouse IgG, F (ab ') 2 (being purchased from Jackson companies) antibody labeling T cell surface C AR expression efficiency.
As a result see in Fig. 4, Fig. 4 and show the expression of empty vector control group cell nonreactive CD19 Chimeric antigen receptors, experimental group cell has
The expression of anti-CD19 Chimeric antigen receptors.
Embodiment 5
Lethal effect of the CAR-T cells to CD19 positive cells
Flow cytometer detection：Take two groups (control group and experimental group) infect after the T lymphocytes of 7-10 days, with the CD19 positives
Nalm-6 cells are co-cultured, and set the quantity ratio respectively 2 of T lymphocytes and Nalm-6 cells:1 and 10：1, by two groups of T cells
Co-cultured respectively with tumour cell after 24h, collect cell, with PE anti-human CD19 streaming antibody labeled cells, carried out
The ratio change of flow cytometer detection CD19 positive cells, is as a result shown in Fig. 5.As shown in Figure 5, the CAR-T cells containing CAR genes can
Specific recognition CD19 tumour antigens, the Nalm-6 cell positive to CD19, with stronger killing-efficiency, reach 95% with
On, huge potentiality are shown in treatment tumour, compared to targeting CD19 of the prior art Chimeric antigen receptor T cell
Tumor cytotoxicity efficiency significantly improve.
LDH cytotoxicities are detected：Two groups of T cells are collected, the ratio for setting effector cell and target cell is respectively 2:1、5:1
With 10:1, plant in 96 orifice plates, three multiple holes of every kind of setting, after 24h co-cultivation, centrifuge cell culture plate, in absorption
Clearly, 150ulLDH release reagents are added and are mixed, continues to cultivate 1h, centrifuges 96 orifice plates, take supernatant 120ul, add 96 new holes
In plate, and add 60ulLDH detection working solutions.Lucifuge is incubated 30min, and absorbance is determined at 490nm.The absorbance of survey is subtracted
Ground control hole absorbance, cytotoxicity or apoptosis rate (%)=(processing sample absorbance-sample controls hole absorbance)/(thin
The absorbance of born of the same parents' maximum enzyme activity-sample controls hole absorbance) × 100.The apoptosis rate of difference group cell is shown in Fig. 6, such as Fig. 6
Shown, the CAR-T cells containing CAR genes have specific killing activity to the cell for expressing CD19, compared with control treatment
Significant difference.It is identical with above-mentioned flow cytometer detection result, compared with prior art with High Fragmentation rate.
SEQUENCE LISTING
<110>Coordinate magnificent medical diagnostic techniqu Co., Ltd in Qingdao
<120>Target CD19 Chimeric antigen receptor T cell, preparation method and application
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 726
<212> DNA
<213>It is artificial synthesized
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gacatacaga tgacccagac tactagctct ctgagtgcaa gtctcgggga cagggttaca 60
atttcatgcc gggcctccca ggacatctcc aaatacctta attggtatca gcagaaacct 120
gatggaaccg tgaagctctt gatctatcac acatcccggc tgcatagcgg agttccttcc 180
aggttcagtg gtagtggcag cggaacagac tatagcctca ccatcagcaa cctggaacaa 240
gaagatatcg caacatactt ctgtcagcag gggaatacac ttccttacac ttttgggggc 300
ggcaccaaac tggagatcac cggaggcgga ggctccggag gagggggaag cgggggaggt 360
gggtctgaag taaagctgca ggagagcggg ccaggcctcg tggctccgag tcagagtttg 420
agcgttacat gtacagtgtc cggcgtaagc ttgcccgatt acggcgtctc atggattcgc 480
caacctccaa ggaagggcct ggagtggctc ggggttattt ggggctctga gactacatat 540
tataatagcg cactcaagtc taggcttact atcatcaagg ataattcaaa gagccaggtc 600
tttcttaaga tgaattcact gcagactgac gacacggcca tttactactg cgcgaaacat 660
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agctca 726
<210> 2
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<213>It is artificial synthesized
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ccggacatac agatgaccca gactactagc tctctgagtg caagtctcgg ggacagggtt 120
acaatttcat gccgggcctc ccaggacatc tccaaatacc ttaattggta tcagcagaaa 180
cctgatggaa ccgtgaagct cttgatctat cacacatccc ggctgcatag cggagttcct 240
tccaggttca gtggtagtgg cagcggaaca gactatagcc tcaccatcag caacctggaa 300
caagaagata tcgcaacata cttctgtcag caggggaata cacttcctta cacttttggg 360
ggcggcacca aactggagat caccggaggc ggaggctccg gaggaggggg aagcggggga 420
ggtgggtctg aagtaaagct gcaggagagc gggccaggcc tcgtggctcc gagtcagagt 480
ttgagcgtta catgtacagt gtccggcgta agcttgcccg attacggcgt ctcatggatt 540
cgccaacctc caaggaaggg cctggagtgg ctcggggtta tttggggctc tgagactaca 600
tattataata gcgcactcaa gtctaggctt actatcatca aggataattc aaagagccag 660
gtctttctta agatgaattc actgcagact gacgacacgg ccatttacta ctgcgcgaaa 720
cattactatt acggtggcag ctacgcaatg gactactggg gccagggtac atccgtcacc 780
gtgagctcaa caactacacc agcgccccgc ccccctaccc cggcacccac aatcgccagt 840
cagccactct ccctgagacc agaagcctgc agacccgccg ccggcggagc tgtccacacc 900
cgaggcctcg atttcgcatg cgacatctat atttgggctc ctctggcagg cacctgtggg 960
gtacttctgc tctcactggt tatcaccctt tactgccggt ccaagcgcag tcggctgctt 1020
cactccgact atatgaacat gaccccaagg aggcccggcc ccactagaaa gcactatcag 1080
ccttacgctc ccccgagaga cttcgccgcc tacagatcca aacggggcag aaagaaactc 1140
ctgtatatat tcaaacaacc atttatgaga ccagtacaaa ctactcaaga ggaagatggc 1200
tgtagctgcc gatttccaga agaagaagaa ggaggatgtg aactgagagt gaagttcagc 1260
aggagcgcag acgcccccgc gtaccagcag ggccagaacc agctctataa cgagctcaat 1320
ctaggacgaa gagaggagta cgatgttttg gacaagagac gtggccggga ccctgagatg 1380
gggggaaagc cgagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1440
aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1500
cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1560
atgcaggccc tgccccctcg ctaa 1584
Claims (8)
1. targetting CD19 Chimeric antigen receptor T cell, surface expression targets CD19 Chimeric antigen receptor gene, and its feature exists
In,
The Chimeric antigen receptor gene of the targeting CD19 by CD19 single-chain antibody CD19ScFv, CD8 hinge area and cross-film
Area, CD28 intracellular signal domain, 4-1BB intracellular signal domain and CD3 ζ intracellular signal domain series connection structure
Into its base sequence such as SEQ ID NO:Shown in 2.
2. targeting CD19 according to claim 1 Chimeric antigen receptor T cell, it is characterised in that
CD19 single-chain antibody CD19ScFv sequences by mouse anti human CD 19 antibody cloning FMC63 heavy chain and light-chain variable sequence
Codon optimizes and obtained, its base sequence such as SEQ ID NO:Shown in 1.
3. the preparation method of the Chimeric antigen receptor T cell of the targeting CD19 described in claim 1 or 2, it is characterised in that including
Following steps：
(1) structure of CD19-CAR slow virus carriers
Restriction enzyme site is added at above-mentioned targeting CD19 Chimeric antigen receptor gene order two ends, with Lentiviral Plv-
EF1a double digestions, reclaim DNA fragmentation, connect, conversion, and picking monoclonal extracts plasmid, by digestion and sequencing identification, obtained
CD19-CAR slow virus carriers Plv-EF1a-CD19ScFv；
(2) preparation of slow virus
Extract slow virus skeleton plasmid Plv-EF1a, slow virus expression plasmid Plv-EF1a-CD19ScFv, helper plasmid psPAX2
And pMD2.G, plasmid concentration is determined, rotaring redyeing system is prepared, HEK293T/17 cells is transfected, separates to obtain slow virus concentrate；
(3) separation and culture of T cell
Healthy peripheral blood is taken, lymphocyte is separated, culture 24-48h is stimulated.
(4) infection and amplification of T cell
The T cell vaccination that above-mentioned stimulation is cultivated adds the slow virus concentrate of step (2) to culture plate, infects 8-12h, obtains
CD19 Chimeric antigen receptor T cell must be targetted.
4. the preparation method of targeting CD19 according to claim 3 Chimeric antigen receptor T cell, it is characterised in that
Restriction enzyme site BamH1 and Sal1 are added at above-mentioned targeting CD19 Chimeric antigen receptor gene order two ends, with slow virus
Expression vector Plv-EF1a carries out double digestion with restriction enzyme BamH1 and Sal1.
5. the preparation method of targeting CD19 according to claim 3 Chimeric antigen receptor T cell, it is characterised in that
The preparation rotaring redyeing system, transfects HEK293T/17 cells, separate the concrete operations of slow virus concentrate are：
The HEK293T/17 cell culture that cell state is good is taken, by expression plasmid Plv-EF1a or Plv-EF1a-CD19ScFv,
Helper plasmid psPAX2 and pMD2.G are with 9:6:3 mass ratio and LipoFilter transfection reagents and plasma-free DMEM medium
Rotaring redyeing system is prepared, nutrient solution is changed after cotransfection HEK293T/17 cells, 12h after incubation；
Viral supernatants are collected in 24h, 48h after liquid is changed in transfection respectively, are centrifuged, filtering, the virus liquid ultracentrifugation after filtering is abandoned
Fall supernatant, precipitation is resuspended with serum-free medium, obtain slow virus concentrate.
6. the preparation method of targeting CD19 according to claim 3 Chimeric antigen receptor T cell, it is characterised in that
The concrete operations of separation and the culture of the T cell are：
Stay autologous plasma standby after taking healthy peripheral blood, centrifugation, the isometric normal saline dilution of remaining haemocyte is added to pouring
Middle tunica albuginea confluent monolayer cells are drawn in the upper strata of bar cell separation liquid, centrifugation, add brine, and centrifugation obtains the lymph of separation
Cell；
Lymphocyte after separation adds the autologous plasma of 2% volume to be resuspended with T lymphocyte culture mediums, and adjusts cell density,
It is inoculated in the coated culture plate of CD3 and CD28 antibody that concentration is 5ug/ml, stimulates culture 24h-48h.
7. the preparation method of targeting CD19 according to claim 6 Chimeric antigen receptor T cell, it is characterised in that
Final concentration of 500U/ml rhIL-2,25ng/ml are supplemented in the culture plate of autologous plasma containing 2% volume
RhIL-7 and 50ng/ml recombinant human interleukin 15.
8. the Chimeric antigen receptor T cell of the targeting CD19 described in claim 1 or 2 is preparing anti-B cell malignant tumor medicine
In application.
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