CN110129370B - Rapid construction method of CAR-T toxicity indicating cell - Google Patents
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Abstract
The invention discloses a rapid construction method of CAR-T toxicity indicating cells, which comprises the following steps: firstly, the construction sequence is SEQ NO:1, a lentiviral plasmid; secondly, extracting the lentiviral plasmid greatly, and packaging the virus to obtain the lentivirus; again, lentiviruses infect the common cell X; finally, adding one or more biotinylation processed protein A into the universal cell U, and tightly combining with avidin on the U cell membrane to obtain the CAR-T toxicity indicating cell; according to the rapid construction method of the CAR-T toxicity indicating cell, different biotinylated protein A or a combination thereof is added, so that the cell U containing the Luciferase gene segment can be rapidly converted into various indicating cells suitable for CAR-T toxicity detection, the complicated preparation steps are greatly omitted, the preparation time is saved, and the cost is reduced.
Description
Technical Field
The invention relates to the technical field of cellular immunotherapy, in particular to a rapid construction method of CAR-T toxicity indicating cells.
Background
The chimeric antigen receptor T cell therapy CAR-T has great application prospect and value in the field of tumor therapy, and the CAR-T becomes a research direction with great prospect in the field of tumor therapy with high treatment accuracy, determined clinical curative effect and good safety.
The determination of CAR-T cytotoxicity is an important problem to be inevitable in medical research and clinical applications, and CAR-T cytotoxicity is also one of the important criteria for measuring the quality of CAR-T products. The current commonly used method for detecting cytotoxicity in vitro comprises the steps of mixing CAR-T cells and target cells, carrying out a period of culture period, labeling by adopting a flow antibody, and then measuring by using a flow cytometry method. The method has the problems of long detection time, complex operation, large error and the like.
The luciferase method is a novel method for determining the toxicity of CAR-T cells, and specifically comprises the following steps: transforming an indicating cell to stably express luciferase, wherein the number of the indicating cell and the luciferase activity value have a quantitative relationship, the indicating cell expresses tumor-associated cell membrane target antigens, CAR-T cells specifically recognize and combine the tumor-associated target antigens, the killing effect is exerted, the indicating cell is lysed, luciferase is released along with the tumor-associated cell membrane target antigens, and the unlysed cells can be located in the sediment by centrifugation.
The indicating cell is the key of a luciferase method, and the current main method for constructing the indicating cell is to utilize lentivirus carrying luciferase gene to infect a target cell (expressing a tumor cell membrane target antigen) and finally obtain a cell strain (namely the indicating cell) stably expressing luciferase after screening; in addition, if a cell line expressing a tumor-specific antigen cannot be obtained, it is also a common strategy in CAR-T cytotoxicity assays to infect a common cell line with a lentivirus so that it stably expresses both luciferase and the tumor cell membrane target antigen.
However, in any method, a series of complicated steps such as preparation of lentivirus, infection of cells, screening of stable cell strains and the like must be carried out; secondly, aiming at different CAR-T cells, a series of corresponding indicator cells are necessarily required to be prepared, and the whole indicator cell construction process has the problems of long time consumption, huge workload, low success rate and the like; finally, the detection of multi-target CAR-T cytotoxicity requires that the indicator cells express multiple tumor antigens simultaneously, which also means that the difficulty of indicator cell construction escalates. Therefore, the design of a construction method of the CAR-T toxicity indicating cell which has the advantages of short time consumption, small workload, high success rate, simple and easy steps and even can simultaneously express a plurality of tumor antigens is a problem to be solved urgently.
Disclosure of Invention
In order to solve the above problems, the present invention aims to provide a rapid construction method of CAR-T toxicity indicating cells.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a rapid construction method of CAR-T toxicity indicating cells comprises the following steps:
the construction sequence is SEQ NO:1, which lentiviral plasmid carries the following foreign gene fragments: IgK leader, avidin, CD28 transmembrane region, T2A sequence and Luciferase;
big extraction of slow virus plasmid and virus packing to obtain slow virus;
③ infecting common cells X with lentivirus, obtaining stably expressed Luciferase Luciferase and B by puromycin screening
Universal cell U for avidin;
wherein the common cell X is a tool cell, a suspension cell or an adherent tumor cell; the tool cell is 293FT cell or Jurkat cell; the suspension cells are U266 cells, RPMI8226 cells, Raji cells or K562 cells; the adherent tumor cells are MCF-7 cells or MDA-MB-231 cells;
adding one or more biotinylated protein A into a universal cell U, and tightly combining with avidin on the U cell membrane to obtain CAR-T toxicity indicating cells; biotinylated protein a is the extracellular domain portion of the cell membrane target antigen of the following cells: CD19, CD20, CD38, CS1, CD30, PDL1, GPC3, or Her2 tumor cells.
Preferably, the biotinylation-treated protein A is of two or three kinds.
Preferably, the commonly used cells X are 293FT cells or K562 cells.
Preferably, the biotinylated protein a is part of the extracellular domain of the cell membrane target antigen of CD19, CD20 and CD 30.
A preferable rapid construction method of CAR-T toxicity indicating cells comprises the following steps: 1 comprising the steps of:
the full gene synthesis sequence is shown as SEQ NO:1, designing the upstream and downstream enzyme cutting sites as XhoI and BamHI respectively;
the gene fragment and pLVX-IRES-Puro plasmid are subjected to double enzyme digestion by XhoI and BamHI, and the cut gel is recovered and then connected;
thirdly, transforming the ligation product into Stbl3 competent cells, and coating LB culture medium plates containing ampicillin resistance;
fourthly, picking a single clone and culturing the single clone in an LB culture medium containing ampicillin resistance for 8-12 hours;
fifthly, carrying out double enzyme digestion on the plasmids containing the XhoI and the BamHI, verifying the gel electrophoresis, and storing after verifying the correctness of the plasmids by sequencing.
Compared with the prior art, the invention has the following advantages:
according to the rapid construction method of the CAR-T toxicity indicating cell, different biotinylated protein A (tumor target antigen ectodomain part) or a combination thereof is added, so that a cell U containing a Luciferase gene fragment can be rapidly converted into various indicating cells suitable for CAR-T toxicity detection, the fussy preparation steps are greatly omitted, the preparation time is saved, and the cost is reduced; because the indicator cell for detecting the CAR-T toxicity can simultaneously have luciferase and one or more tumor target antigens, the indicator cell can be used as an indicator cell of one antigen or an indicator cell of multiple antigens and can meet the detection of the single-target or multi-target CAR-T cytotoxicity.
FIG. 1 is a schematic diagram of the principle of the rapid method of construction of CAR-T toxicity-indicating cells;
FIG. 2 is a schematic diagram of a foreign gene fragment carried by a lentiviral plasmid;
FIG. 3 is a graph showing the relationship between the activity (luminescence intensity) of luciferase and the number of cells;
FIG. 4 is a schematic representation of the killing effect of CD19 CAR-T.
Detailed Description
The invention aims to provide a rapid construction method of CAR-T toxicity indicating cells, which is realized by the following technical scheme:
a rapid construction method of CAR-T toxicity indicating cells is shown in figure 1, and comprises the following steps:
the construction sequence is SEQ NO:1, which lentiviral plasmid carries the following foreign gene fragments: IgK leader (signal peptide of immunoglobulin K), avidin (avidin), CD28 transmembrane region, T2A sequence, and Luciferase (Luciferase), as shown in fig. 2;
big extraction of slow virus plasmid and virus packing to obtain slow virus;
③ infecting common cells X by lentivirus, and obtaining universal cells U stably expressing Luciferase and avidin through puromycin screening;
wherein the common cell X is a tool cell, a suspension cell or an adherent tumor cell; the tool cell is 293FT cell or Jurkat cell; the suspension cells are U266 cells, RPMI8226 cells, Raji cells or K562 cells; the adherent tumor cells are MCF-7 cells or MDA-MB-231 cells;
adding the biotinylated protein A into a universal cell U, and tightly combining with avidin on the U cell membrane to obtain a single antigen CAR-T toxicity indicating cell; biotinylated protein a is the extracellular domain portion of the cell membrane target antigen of the following cells: CD19, CD20, CD38, CS1, CD30, PDL1, GPC3, or Her2 tumor cells;
adding a plurality of different biotinylated protein A into a universal cell U, and tightly combining with avidin on a U cell membrane to obtain a multi-antigen CAR-T toxicity indicating cell; biotinylated protein a is the extracellular domain portion of the cell membrane target antigen of the following cells: CD19, CD20, CD38, CS1, CD30, PDL1, GPC3, or Her2 tumor cells.
Preferably, the biotinylation-treated protein A is of two or three kinds.
Preferably, the commonly used cells X are 293FT cells or K562 cells.
Preferably, the biotinylated protein a is part of the extracellular domain of the cell membrane target antigen of CD19, CD20 and CD30 cells.
A preferable rapid construction method of CAR-T toxicity indicating cells comprises the following steps: 1 comprising the steps of:
the full gene synthesis sequence is shown as SEQ NO:1, designing the upstream and downstream enzyme cutting sites as XhoI and BamHI respectively;
the gene fragment and pLVX-IRES-Puro plasmid are subjected to double enzyme digestion by XhoI and BamHI, and the cut gel is recovered and then connected;
thirdly, transforming the ligation product into Stbl3 competent cells, and coating LB culture medium plates containing ampicillin resistance;
fourthly, picking a single clone and culturing the single clone in an LB culture medium containing ampicillin resistance for 8-12 hours;
fifthly, carrying out double enzyme digestion on the plasmids containing the XhoI and the BamHI, verifying the gel electrophoresis, and storing after verifying the correctness of the plasmids by sequencing.
The kit used for the plasmid macroextraction in the embodiment of the invention is a Tiangen plasmid macroextraction kit. Biotinylated CD19, CD20, and CD30 extracellular domain proteins are all provided by ACROBiosystems;
the invention is further described with reference to specific examples.
Example 1
Construction of Lentiviral plasmids
1. The gene fragment shown in figure 1 is synthesized from the whole gene, and the sequence is SED NO:1, the fragment contains IgK leader, avidin, CD28 transmembrane region, T2A sequence and Firefly-derived Luciferase, and the upstream and downstream enzyme cutting sites are respectively XhoI and BamHI;
2. the gene fragment and pLVX-IRES-Puro plasmid are cut by XhoI and BamHI; cutting and recovering the gel, and connecting by a conventional molecular biological method;
3. the ligation products were transformed into Stbl3 competent cells, plated on LB medium plate (ampicillin resistance)
4. Selecting the monoclonal antibody to an LB culture medium for culture for 8-12 hours;
5. extracting plasmids, carrying out XhoI and BamHI double enzyme digestion, verifying whether the size of a strip is correct by combining gel electrophoresis, and further carrying out plasmid sequencing by using a universal primer to verify the correctness; the universal primers are provided by Biotechnology engineering (Shanghai) GmbH;
6. the plasmid was stored at-20 ℃ and designated pLVX-avidin-Luc-IRES-Puro.
Second, preparation of lentivirus
1. Plasmid major grape
The plasmid pLVX-avidin-Luc-IRES-Puro is transformed into stbl3 competence conventionally, a single clone is picked up to 500ml conical flask (containing 100ml LB culture medium), cultured overnight, the plasmid is extracted by using plasmid extraction box, ddH2Eluting the plasmid with Nanadrop, wherein the concentration of the plasmid is more than 1 mug/mul;
2. virus package
(1) Preparing a solution A: adding 1.2ml of opti-MEM into a 1.5ml EP tube, adding the following plasmids, carrying out vortex oscillation for 5-10 seconds, uniformly mixing, and adding the plasmids in a mass ratio of:
psPAX2 7.5μg
pMD2.G 2.5μg
pLVX-avidin-Luc-IRES-Puro 10μg
(2) preparing a solution B: taking a 15ml centrifuge tube, adding 1.5ml of opti-MEM and 200060 mu l of lipo, and carrying out vortex oscillation for 5-10 seconds;
(3) adding the solution A into the solution B, performing vortex oscillation for 5-10 seconds, and standing at 20-30 ℃ for 20 minutes;
(4) preparation of 293T cells: laying 293T cells in a 10cm plate until the fusion degree of the 293T cells reaches 80%;
(5) uniformly dripping the A, B mixed solution into the 10cm plate, standing for 3-5 minutes, shaking up lightly, and culturing for 60 hours in an incubator at 37 ℃;
(6) centrifuging at 2000rpm for 10 min, removing cell debris, and filtering with 0.45 μm filter membrane;
(7) collecting supernatant, subpackaging and storing at-80 ℃.
Third, slow virus infection K562 cell
1. Take 2X 105Adding 0.5ml of IMDM complete culture medium (containing 10% FBS) into each K562 cell for resuspension, placing the cells in a 12-well plate, adding polybrene to 6 mu g/ml and 0.5ml of virus supernatant, fully mixing the cells, and centrifuging the cells at 3000rpm for 4 hours at room temperature;
2. adding an equal volume of fresh medium after 4 hours to dilute polybrene; continuously culturing for 3-4 days, and changing the liquid every other day;
3. carrying out multiple passages according to a conventional puromycin screening method to obtain a puromycin resistant cell K562-avidin-Luc;
4. extracting puromycin resistant cells K562-avidin-Luc in the step 3 by a conventional method, detecting the expression condition of the gene SEQ NO:1 by using a conventional fluorescent quantitative PCR method, and detecting the activity of the Luciferase by using a Firefoy Luciferase detection kit to prove that avidin and the Luciferase are stably expressed in the K562-avidin-Luc cells.
Fourth, 293FT cell infected by lentivirus
1. Take 5X 105293FT cells, to which 0.5ml IM was addedDM complete medium (containing 10% FBS) heavy suspension, placed in 12 hole plate, adding polybrene to 4 μ g/ml and 0.5ml virus supernatant, fully mixing, 3000rpm room temperature centrifugation for 4 hours;
2. adding an equal volume of fresh medium after 4 hours to dilute polybrene; continuously culturing for 3-4 days, and changing the liquid every other day;
3. carrying out multiple passages according to a conventional puromycin screening method to obtain a puromycin resistant cell 293 FT-avidin-Luc;
4. extracting the puromycin resistant cell 293FT-avidin-Luc in the step 3 by a conventional method, detecting the expression condition of the gene SEQ NO:1 by using a conventional fluorescent quantitative PCR method, and detecting the activity of the Luciferase by using a Firefley Luciferase detection kit to prove that avidin and the Luciferase are stably expressed in the 293FT-avidin-Luc cell.
The K562 cells and 293FT cells in step three and step four can be replaced by other commonly used cell lines, including but not limited to, Jurkat and other tool cells and other tumor cell lines, such as U266, RPMI8226, Raji and other suspension cells and MCF-7, MDA-MB-231 and other adherent tumor cell lines.
Example 2
Construction of K562 cells carrying the CD19 antigen
1. Culturing the K562-avidin-Luc cells constructed in the third step in a 12-well plate to enable the cells to be in a logarithmic growth phase;
2. the CD19 antigen protein extracellular domain amino acid sequence is shown in SEQ NO. 2, and the sequence can be externally expressed by entrusted companies, and histidine tags are added at the tail ends of the sequence for easy purification. After purification of the protein, the company was requested to carry out biotinylation. Alternatively, biotinylated CD19 extracellular domain protein is commercially available and is commercially available, such as from ACROBiosystems.
3. Take 1X 106Adding the biotinylated CD19 antigen ectodomain protein obtained in the K562-avidin-Luc cell to a final concentration of 10. mu.g/ml, and incubating at 4 ℃ for 30 minutes;
4. the supernatant was removed by centrifugation, resuspended in IMDM complete medium (containing 10% FBS), and the procedure was repeated 1 time to obtain K562 cells carrying the CD19 antigen.
The obtained K562 cells carrying the CD19 antigen are combined with the extracellular domain of the CD19 antigen on the basis of stably expressing Luciferase and can be used as indicator cells for detecting CD19CAR-T cytotoxicity. By using the method of the Firefoy luciferase assay kit, the luciferase activity (luminescence intensity) and the cell number are analyzed to present a linear relation within a certain cell number range, as shown in FIG. 3.
Example 3 construction of K562 cells carrying CD19 and CD30 antigens
1. Culturing the K562-avidin-Luc cells constructed in the third step in a 12-well plate to enable the cells to be in a logarithmic growth phase;
2. preparing biotinylated CD19 and CD30 extracellular domain proteins;
3. take 1X 106Adding the biotinylated CD19 and CD30 ectodomain protein into K562-avidin-Luc cells at the same time to a final concentration of 10 μ g/ml, and incubating at 4 ℃ for 60 minutes;
4. the supernatant was removed by centrifugation, resuspended in IMDM complete medium (containing 10% FBS), and the procedure was repeated 1 time to obtain K562 cells bearing the CD19 and CD30 antigens.
The obtained K562 cells carrying CD19 and CD30 antigens combine with CD19 and CD30 antigen extracellular domains on the basis of stable expression of Luciferase, and can be used as indicator cells for detecting CAR-T cytotoxicity of targeting CD19 and CD 30.
Example 4
Construction of K562 cells carrying CD19, CD20 and CD30 antigens simultaneously
1. Culturing the K562-avidin-Luc cells constructed in the third step in a 12-well plate to enable the cells to be in a logarithmic growth phase;
2. preparing biotinylated CD19, CD20, and CD30 extracellular domain proteins;
3. take 1X 106Adding the biotinylated CD19, CD20 and CD30 extracellular domain proteins into the K562-avidin-Luc cells at the same time to a final concentration of 10 μ g/ml, and incubating for 60 minutes at 4 ℃;
4. the supernatant was removed by centrifugation, resuspended in IMDM complete medium (containing 10% FBS), and the procedure was repeated 1 time to obtain K562 cells bearing the CD19, CD20, and CD30 antigens.
The obtained K562 cells carrying CD19, CD20 and CD30 antigens combine with CD19, CD20 and CD30 antigen extracellular domains on the basis of stable expression of Luciferase, and can be used as indicator cells for detecting CAR-T cytotoxicity of targeting CD19, CD20 and CD30 at the same time.
Example 5
Application of K562 cells carrying CD19 antigen as CD19CAR-T cytotoxicity indicator cells
1. The K562 cells carrying the CD19 antigen constructed in step five were suspended to 1X 10 using IMDM complete medium (containing 10% FBS)7Taking 10 mul/well and adding into a 96-well plate;
2. the CD19CAR-T cell preparation process is shown in the literature (Onco Targets ther.2018,11: 1767-1776);
3. CD19CAR-T cells were suspended to 1X 10 with IMDM complete Medium (10% FBS)7(ii)/ml, adding corresponding volumes to the wells such that the ratio of the number of CD19CAR-T cells to the number of indicator cells is 0:1 (control), 1:1 (experimental), 2:1 (experimental), 5:1 (experimental), 10:1 (experimental), and each ratio is at least 3 replicate wells;
3. make up 150 μ l per well with IMDM complete medium (containing 10% FBS);
4、CO2culturing for 24h in an incubator;
5. centrifuging at 300g for 5min to remove culture medium supernatant;
6. adding lysis solution into the cell sediment according to the method of a Firefly Luciferase assay kit (Promega E1501), and determining the Luciferase activity in the lysate;
7. calculating the Killing Effect of the CD19CAR-T, Kiling Effect (%) (control group bioluminescence intensity-experimental group bioluminescence intensity)/control group bioluminescence intensity 100; as shown in FIG. 4, the CD19CAR-T is calculated to have obvious cytotoxicity, and K562 cells carrying CD19 antigen obtained by the preparation method can be used as CD19CAR-T cytotoxicity indicator cells.
Sequence listing
<110> Shandong Oncuo Biotech Co., Ltd
<120> method for quickly constructing CAR-T toxicity indicating cells
<141> 2019-05-06
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2505
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gcggaagcgg gtatcaccgg cacgtggtac aaccagtctg gttctacctt caccgttacc 180
gcgggtgcgg acggtaacct gaccggtcag tacgaaaacc gtgcgcaggg cactggttgc 240
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ggtggtgcgg aagcgcgtat caacacccag tggaacctga cctacgaagg tggttctggt 420
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aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 780
acccgcaagc atgaaggtag aggtagtctc ctcacatgtg gcgatgtaga agagaaccct 840
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gacgggaccg ctggcgagca gctgcataaa gccatgaagc gctacgccct ggtgcccggc 960
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ggctacggcc tgacagaaac aaccagcgcc attctgatca cccccgaagg ggacgacaag 1920
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agcggctacg ttaacaaccc cgaggctaca aacgctctca tcgacaagga cggctggctg 2100
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ctgcaacacc ccaacatctt cgacgccggg gtcgccggcc tgcccgacga cgatgccggc 2280
gagctgcccg ccgcagtcgt cgtgctggaa cacggtaaaa ccatgaccga gaaggagatc 2340
gtggactatg tggccagcca ggttacaacc gccaagaagc tgcgcggtgg tgttgtgttc 2400
gtggacgagg tgcctaaagg actgaccggc aagttggacg cccgcaagat ccgcgagatt 2460
ctcattaagg ccaagaaggg cggcaagatc gccgtctagg gatcc 2505
<210> 2
<211> 538
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp Asn Ala Val
1 5 10 15
Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln Gln Leu Thr
20 25 30
Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu Ser Leu Gly
35 40 45
Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile Trp Leu Phe
50 55 60
Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu Cys Gln Pro
65 70 75 80
Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr Val Asn Val
85 90 95
Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp Leu Gly Gly
100 105 110
Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro Ser Ser Pro
115 120 125
Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala Lys Asp Arg
130 135 140
Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro Arg Asp Ser
145 150 155 160
Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro Gly Ser Thr
165 170 175
Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser Arg Gly Pro
180 185 190
Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser Leu Leu Ser
195 200 205
Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp Val Met Glu
210 215 220
Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala Gly Lys Tyr
225 230 235 240
Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu Glu Ile Thr
245 250 255
Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly Gly Trp Lys
260 265 270
Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu Cys Ser Leu
275 280 285
Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg Arg Lys Arg
290 295 300
Lys Arg Met Thr Asp Pro Thr Arg Arg Phe Phe Lys Val Thr Pro Pro
305 310 315 320
Pro Gly Ser Gly Pro Gln Asn Gln Tyr Gly Asn Val Leu Ser Leu Pro
325 330 335
Thr Pro Thr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala Ala Gly Leu
340 345 350
Gly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp Val Gln Ala
355 360 365
Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly Pro Glu Glu
370 375 380
Glu Glu Gly Glu Gly Tyr Glu Glu Pro Asp Ser Glu Glu Asp Ser Glu
385 390 395 400
Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu Ser Gln Asp
405 410 415
Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro Leu Gly Pro Glu Asp
420 425 430
Glu Asp Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu Asp Glu Glu
435 440 445
Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser Pro His Gly
450 455 460
Ser Ala Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Ala Gly Ser Gln
465 470 475 480
Ser Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro Gln Leu Arg
485 490 495
Ser Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp Ala Asp Ser
500 505 510
Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp Pro Ala Trp Gly Gly
515 520 525
Gly Gly Arg Met Gly Thr Trp Ser Thr Arg
530 535
Claims (1)
1. A rapid construction method of CAR-T toxicity indicating cells, which is characterized in that: the method comprises the following steps:
the construction sequence is SEQ NO:1, which lentiviral plasmid carries the following foreign gene fragments: IgK leader, avidin, CD28 transmembrane region, T2A sequence and Luciferase;
wherein the construction sequence is SEQ NO:1 comprising the steps of:
the full gene synthesis sequence is shown as SEQ NO:1, designing the upstream and downstream enzyme cutting sites as XhoI and BamHI respectively;
the gene fragment and pLVX-IRES-Puro plasmid are subjected to double enzyme digestion by XhoI and BamHI, and the cut gel is recovered and then connected;
thirdly, transforming the ligation product into Stbl3 competent cells, and coating LB culture medium plates containing ampicillin resistance;
fourthly, picking a single clone and culturing the single clone in an LB culture medium containing ampicillin resistance for 8-12 hours;
carrying out double enzyme digestion of XhoI and BamHI and carrying out gel electrophoresis verification on the plasmids, carrying out plasmid sequencing and verifying the correctness, and then storing;
big extraction of slow virus plasmid and virus packing to obtain slow virus;
③ infecting common cells X by lentivirus, and obtaining universal cells U stably expressing Luciferase and avidin through puromycin screening;
wherein the common cell X is 293FT cell or K562 cell;
adding two or three biotinylated protein A into a universal cell U, and tightly combining with avidin on the U cell membrane to obtain CAR-T toxicity indicating cells; biotinylated protein a is the extracellular domain portion of the cell membrane target antigen of the following cells: CD19, CD20, and CD 30.
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| CN108474002A (en) * | 2015-10-22 | 2018-08-31 | 朱诺治疗学有限公司 | For the method for transduction, reactant box, reactant and equipment |
| CN108753728A (en) * | 2018-06-14 | 2018-11-06 | 焦顺昌 | A kind of target cell and its preparation method and application for examining CAR-T Cell killing efficacies |
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| WO1997005266A1 (en) * | 1995-07-25 | 1997-02-13 | Introgene B.V. | Methods and means for targeted gene delivery |
| CN106282237A (en) * | 2015-05-27 | 2017-01-04 | 北京大学 | A kind of biotin-avidin-Lentiviral and CAR-T cell preparation method and visualization scheme |
| CN108474002A (en) * | 2015-10-22 | 2018-08-31 | 朱诺治疗学有限公司 | For the method for transduction, reactant box, reactant and equipment |
| CN106086078A (en) * | 2016-07-08 | 2016-11-09 | 宜明细胞生物科技有限公司 | A kind of CAR T cell toxicity instruction carrier |
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