CN108642071A - The preparation method of the dual-gene deficiency T lymphocyte preparations of PD-1 and CTLA4 - Google Patents
The preparation method of the dual-gene deficiency T lymphocyte preparations of PD-1 and CTLA4 Download PDFInfo
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Abstract
The invention belongs to technical field of molecular biology, and in particular to a kind of preparation method of the dual-gene deficiency T lymphocyte preparations of PD 1 and CTLA4.The structure of the dual-gene knockout carriers of PD 1 and CTLA4, T separation of lymphocytes and activation, electrotransfection T lymphocytes and T7E1 digestions identification, flow cytometer screening and sequencing analysis.The production of the gene defection type immunocyte preparation of the present invention, on the one hand simplifies the production routine of cell, on the other hand improves the ability that T lymphocytes kill tumour cell, and can enhance long-term tumour immunity.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to a kind of dual-gene deficiency T lymphs of PD-1 and CTLA4
The preparation method of cell preparation.
Background technology
T cell mainly identifies the Antigenic Peptide offered by cell surface major histocompatibility complex (MHC).Most of feelings
Under condition, the T cell that t cell surface antigen receptor (TCR) signal transduction can be activated with active antigen, however, relying solely on TCR letters
Number transduction pathway is difficult to trigger T cell immune response.Therefore, the effective activation of T cell and function mediation need dual signal
Synergistic effect：First, Antigenic Peptide-MHC compounds, are identified by T cell antigen receptor (TCR)；Second is that costimulatory signal, by resisting
The costimulatory molecules ligand and receptor on former presenting cell (APC) and T cell surface are to providing.Wherein, PD-1/PD-L1 is public
The most basic costimulatory signal recognized, by expressing the PD-1 in T cell and expressing the PD-L1 on antigen presenting cell surface
It interacts and generates with PD-L2.PD-1 (programmed death-1), also known as PDCDl, CD279, are CD28 immune globulins
A member in white superfamily, it finds and names when apoptosis occurs for inducing T cell hybridoma, and main expression is followed in periphery
Ring T cell, natural killer cells (NKT), in B cell and monocyte.There are two ligand PD-L1 for PD-1 molecules (also known as
CD274) and PD-L2 (also known as CD273), they are B7 family members.PD-1 is combined and can exempt to body with its ligand PD-L1
Epidemic disease system plays negativity adjustment effect.Tumor immune response early stage, circulation immunity cell T cell can identify, infiltrate and then remove
Certain cancer cells, however, the cell that some cancer cells can escape from immunosurveillance and immune-mediated by certain modes is dead
It dies.Studies have shown that costimulatory molecules PD-1, can reduce the intensity of immune response with the PD-L1 interactions of its ligand and continue
Time, this provides advantage for the immunologic escape of cancer cell.In vitro study shows to block PD-1/PD-L1 approach that can increase
Strong T cell immunizing potency, adjusts antitumor immunity of organism response.It is now recognized that costimulatory molecules and its adjusting approach are in cancer cell
Extremely important effect has been played during antineoplastic immune.Block PD-1/PD-L1 accesses that can effectively facilitate tumour-specific
Infiltration of the CD8+ T cells to tumor tissues secretes massive tumor killer factor, to inhibit the growth of tumour.
Cytotoxic t lymphocyte-associated antigen 4 (CTLA-4) is also known as CD125 molecules, is a kind of immune modulatory molecules, is
B7 molecule ligands, the inhibition molecule of contactin participate in the negative tune of immune response in conjunction with rear inducing T cell
Section, i.e., inducing T cell is reactionless, makes the cell cycle arrest of T cell in Gl phases etc., inhibits the proliferation and function of T lymphocytes.
Studies have shown that the activation of T lymphocytes can be dramatically increased with antibody blocking CTLA4 functions.Antitumor T lymphocytes are in body pair
It plays a crucial role in the immunosurveillance of malignant tumour.Experiment display, injecting anti-CTLA 4 antibody to the mouse for having transplanted tumour can support
It makes a plurality of types of tumours and antineoplastic immune can be kept for a long time.Clinical research shows to block CTLA4 that can make tumor regression.These
Data all shows that CTLA4 plays an important role in the occurrence and development of tumour.
Therefore, the PD-1 genes in T lymphocytes are knocked out using CRISPR/Cas9 technologies, to activated T lymphocytes pair
The ability of tumour cell attack, realizes the immunization therapy of tumour, knocks out CTLA4 and is more advantageous to the activation of T lymphocytes, and can make
Cell obtains long-term antineoplastic immune, and the T lymphocytes of double dcc genes are with more the ability for killing tumour cell.
Invention content
The object of the present invention is to provide a kind of preparation method of the dual-gene deficiency T lymphocyte preparations of PD-1 and CTLA4,
The ability for simplifying the production routine of cell, improving T lymphocyte attacks tumour cells, and long-term tumour immunity can be enhanced.
The preparation method of the dual-gene deficiency T lymphocyte preparations of PD-1 and CTLA4 of the present invention, steps are as follows：
(1) structure of the dual-gene knockout carriers of PD-1 and CTLA4
GRNA target sequences are separately designed according to PD-1 and CTLA4 complete genome sequences (exon 1), then by two
GRNA target sequences are cloned into expression vector, and obtaining band, there are two the knockout carriers of different target sequences gRNA；
Two gRNA target sequences are respectively 5 '-GGAGTGGATAGGCCACGGCG-3 ' and 5 '-
ACACCGCTCCCATAAAGCCA-3’；
(2) T separation of lymphocytes and activation
T separation of lymphocytes is cultivated, and is activated, and is bred, and is collected；
(3) electrotransfection T lymphocytes and T7E1 digestions identification
The knockout carrier that electricity turns liquid and step (1) obtains is added in T lymphocytes and carries out electrotransfection, after electrotransfection,
Continue to cultivate, collect cell extraction genome, is identified using T7E1 digestions；
(4) flow cytometer screening and sequencing analysis
The cell collected after electrotransfection carries out isolated monoclonal cell, monoclonal cell using flow cytometer
Expand culture to get the dual-gene deficiency T lymphocyte preparations of PD-1 and CTLA4；Expand the monoclonal cell extraction after culture
Genome carries out sequencing analysis.
Expression vector described in step (1) is p2U6gRNA-Cas9-GFP expression vectors.
Electricity described in step (3) turn liquid composition it is as follows, with volume percentage：
Without calcium and magnesium PBS 90%
Serum free medium RPMI 1,640 10%；
It is in terms of 100% by the total volume of no calcium and magnesium PBS and serum free medium RPMI 1640, EDTA additions are 30g/
L, sucrose addition are 200mmol/L.
The preparation method that electricity turns liquid is to mix no calcium and magnesium PBS with serum free medium RPMI 1640, and EDTA is then added
And sucrose, refilter degerming after dissolving.
Continuation incubation time described in step (3) is 48h.
After flow cytometer screening technique is cell transfecting 48h in step (4), cell is cleaned twice with PBS, it is thin through streaming
Born of the same parents' instrument screens GFP positive monoclonal T lymphocytes, and genome is extracted in then expanding propagation culture, and connection T is carried after carrying out PCR
Body, conversion E. coli competent carry out sequencing analysis, and the T lymphs that frameshift mutation occurs for screening PD-1 and CTLA4 genes are thin
Born of the same parents (are bred) by monoclonal cell, that is, obtain novel immune cell preparation.
The present invention builds special, efficient dual-gene knockout carrier, efficient electrotransfection method, flow cytometer screening
After monoclonal cell, dual-gene deficiency T lymphocytes are screened, expand the immunocyte that numerous acquisition can be used for oncotherapy and prevention
Preparation.Two gRNA belong to independent expression unit, and carrier also includes Cas9 genes and green fluorescent protein (GFP) gene.
Dual-gene knockout carrier is thin using flow cytometer screening GFP positive monoclonals after electrotransfection T lymphocytes
Born of the same parents finally obtain dual-gene deficiency T lymphocytes, have the immunocyte condition for tumor prevention and treatment.
PD-1 the and CTLA4 gene defection type T lymphocytes of flow cytometer screening are an immunocyte preparation, be can be used for
Human body, which is fed back, prevents or treats tumour.
The present invention obtains specificity, efficient identification target site using bioinformatics technique and effectively cuts off target site
GRNA target sequences, then clone sequence into pU6gRNA-CMV-Cas9-GFP expression vectors, and building successfully can be simultaneously
Two expression vectors for carrying different target sequences gRNA of expression：p2U6gRNA-Cas9-GFP.
Expression vector is transferred to work by the present invention on the basis of building gRNA with Cas9 expression vectors using electrotransfection technology
The T lymphocytes of change, electrotransfection is using the electrotransfection buffer solution of optimization, and transformation efficiency is high, and cell mortality is low, more effective to obtain
The dual-gene deficiency T lymphocytes of PD-1 and CTLA4.
Beneficial effects of the present invention are as follows：
The present invention determines the two gRNA target sequences that can be realized and efficiently knock out PD-1 and CTLA4 genes first,
Then optimize electrotransfection buffer solution, realize the efficient electrotransfection of T lymphocytes and ensure the survival rate of cell after electrotransfection, finally
GFP positive cells are screened using flow cytometer, the homozygote T lymphocytes of the dual-gene frameshift mutation of separation screening obtain PD-
The dual-gene deficient cells of 1 and CTLA4.The production of the gene defection type immunocyte preparation of the present invention, on the one hand simplifies cell
Production routine, on the other hand improve the ability that T lymphocytes kill tumour cell, and long-term tumour immunity can be enhanced.
Description of the drawings
Fig. 1 is list PD-1 knockout carrier schematic diagrames.
Fig. 2 is the intermediate carrier used in the double knockout carriers of structure.
Fig. 3 is the dual-gene knockout carrier schematic diagrames of PD-1 and CTLA4.
Fig. 4 is the T lymphocytes of activation.
Fig. 5 is T7E1 digestion qualification result of the electrotransfection after 48 hours.
Fig. 6 is PD-1 gene sequencing results.
Fig. 7 is CTLA4 gene sequencing results.
Fig. 8 is the western blotting figures of the dual-gene deficient cells strains of PD-1 and CTLA4.
Specific implementation mode
The present invention is described further with reference to embodiments.
Test method without specific conditions in following embodiment, usually according to normal condition such as《Molecular Cloning: A Laboratory
Guide》Condition described in reference books commonly used in the art such as (third edition, Science Presses, 2005), or press reagent manufacturer
Proposed condition carries out.
Embodiment 1
(1) structure of the dual-gene knockout carriers of PD-1 and CTLA4
1. the design of gRNA target sequences
PD-1 complete genome sequences (EF064716.1) and CTLA4 complete genome sequences (NG_011502.1), base are obtained from NCBI
In CRISPR-DO website design gRNA target sequences, then according to the parameter of setting, select two genes suitable
Target sequences (each 20bp length) are then synthesized two sections of complementary target sequences by Shanghai Sheng Gong bioengineering Co., Ltd
(increasing the cohesive end after BbsI digestions).
Such as：PD-1 oligo：F 5’-ATAG GGAGTGGATAGGCCACGGCG-3’
R 5’-AAAT CGCCGTGGCCTATCCACTCC-3’
CTLA4 oligo：F 5’-ATAG ACACCGCTCCCATAAAGCCA-3’
R 5’-AAAT TGGCTTTATGGGAGCGGTGT-3’
2. knockout carrier is built
With BbsI digestion pU6gRNA-CMV-Cas9-GFP expression vectors, formed with PD-1oligo sequences after recycling
Oligo double-strands connect, and structure is containing there are one the PD-1 gene knockout carriers (Fig. 1) of gRNA.
Using pU6gRNA-CMV-Cas9-GFP expression vectors as template, with primer gRNA-R：5’-
ACAGAATTCGTCAATAATCAATGTCATTAATTAAGG-3 ' and pU6gRNA-CMV-Cas9-GFP-F：5’-
ACAAAGCTTTAGTTATTAATAGTAATCAATTACGGGG-3 ' linearizes expression vector, and breaking point is located at gRNA and CMV
Between promoter sequence, and HindIII and EcoRI restriction enzyme sites are added at two sections.
Then, using pU6gRNA-CMV-Cas9-GFP expression vectors as template, with primer gRNA-F：5-’
ACAAAGCTTAAGGTCGGGCAGGAAGAGGG-3 ' and gRNA-R：5’-
ACAGAATTCGTCAATAATCAATGTCATTAATTAAGG-3 ' clones gRNA expression units, and is connected into pUC19, then passes through
BbsI digestions are connected into CTLA4oligo sequences and obtain intermediate carrier (Fig. 2).It is linearized with HindIII and EcoRI double digestions
PU6gRNA-CMV-Cas9-GFP expression vectors and intermediate carrier, connect the two after recycling, and acquisition can express two differences
The knockout carrier p2U6gRNA-Cas9-GFP of target sequences gRNA, is shown in Fig. 3.
(2) T separation of lymphocytes and activation
①CD3+Antibody is coated with tablet
The PBS buffer solution in the holes 2ml/ is added in 6 orifice plates, CD is then added3+Antibody is placed in 37 to 10 μ g/ml of final concentration
℃CO2Incubator is incubated 2 hours, then sucks PBS, tablet is cleaned 2 times with fresh preheating PBS, then with serum-free RPMI-
1640 culture mediums clean 1 time.
2. mature T separation of lymphocytes and culture
1, by arteria brachialis extract Healthy People new blood 1mL, at once be added the 1.5mL containing 10 μ L anticoagulant heparin agent from
In heart pipe.To keep the activity of lymphocyte, detached at once after blood sampling.
2, PBS the or 0.9%NaCl dilute bloods or blood plasma of 3mL is added.Dilute blood can reduce the cohesion of red blood cell, carry
High lymphocyte harvest yield.
3, it takes 4mL lymphocyte separation mediums that centrifuge tube tube bottom is added, and is warming up to room temperature.
4, the blood sample after 4ml dilutions is drawn with Pasteur glass pipette, separation of lymphocytes is slowly taped against along tube wall
Above liquid, liquid layer interface is not upset.
5, horizontal rotor centrifugation 2000r/min or 700 × g centrifuges 20min, 20 DEG C of room temperature.The blood of storage 2h or more is answered
Centrifuge 30min.
6, tube bottom is red blood cell after centrifuging, and middle layer is separating liquid, and top layer is blood plasma.It is between plasma layer and separating liquid
The finer and close tunica albuginea of a thin layer, (including lymphocyte and monokaryon granulocyte) containing mononuclearcell.It is directly inserted into list with suction pipe
A nucleus layer simultaneously draws the layer, is put into another test tube.
7, plus the lymphocyte of 10ml Hank ' s liquid dilution separation, 250 × g centrifuge 10min, abandon supernatant.Repeated washing 2
It is secondary, remove blood platelet and anticoagulant substances.Finally 10 times of tallies being diluted with PBS and counting cell, Trypan Blue determines that cell is deposited
Motility rate>95%.
3. T lymphocyte activators and breeding
T the lymphocyte precipitates 2. full culture mediums of RPMI-1640 are resuspended in the 7th step, are transferred to (1.) CD3+Antibody
In coated 6 orifice plates, it is placed in CO2Incubator culture 24 hours, proleulzin (final concentration 1000U/ml), which is then added, to stimulate carefully
Intracellular growth, acquired results are shown in Fig. 4.
(3) electrotransfection T lymphocytes and T7E1 digestions identification
It collects T lymphocytes to be cleaned one time with PBS, the electricity that optimization is added turns 400 μ l of liquid (formulas：Without calcium and magnesium PBS 90%,
EDTA 30g/L, serum free medium RPMI 1,640 10%, sucrose 200mmol/L) be resuspended to final concentration 5-10 ×
106Then the PD-1 built in (1) and the dual-gene knockout plasmids of CTLA4 is added to final concentration of 40 μ g/ml in cell/ml.
0.4cm electric shock cups, 280V voltages, shock by electricity 20ms.The full culture mediums of RPMI-1640 that preheating is added after electrotransfection are placed in dioxy
Change and continue to cultivate in carbon incubator, after 48 hours, collect cell extraction genome, with identification primer PD-1 (PD1F：5’-
CCCAGCCAGCACTCTGGCCT-3’；PD1R：5 '-CTGCCCGGAGGGACTGGTAGGC-3 ') carry out PCR amplification, PCR product
Identify the DNA fragmentation of 550bp and 300bp as a result occur, it is prominent to illustrate that the PD-1 genes of genome have occurred using T7E1 digestions
Become, sees Fig. 5.
(4) flow cytometer screening and sequencing analysis
Electrotransfection T lymphocytes collect cell and clean cell 2 times with PBS, be placed in 1ml PBS buffer solution after 48 hours
It is middle that GFP positive cells are detached using flow cytometer, detach monoclonal cell.
Isolated cells expand culture, take 1-2 × 106Cell extracts genome, utilizes identification primer PD-1 (PD1F：
5’-CCCAGCCAGCACTCTGGCCT-3’；PD1R：5 '-CTGCCCGGAGGGACTGGTAGGC-3 ') and CTLA4 (CTLA4F '：
5’-CCAGAGGCAGCTTCTTTTCCGCC-3’；CTLA4R’：5 '-GGAATACAGAGCCAGCCAAGCC-3 ') PCR is carried out,
Carrier T is connected after PCR product recycling, then converts E. coli competent, picking monoclonal bacterium carries out sanger sequencing analysis,
As a result as shown in Figure 6 and Figure 7：Screening obtains dual-gene deficiency T lymphocyte homozygotes, which, which has, is better than PD-1 genes
The homozygous immune effect of defect T lymphocytes.
(5) western blotting are analyzed
After sequencing homozygous cell amplification breeding, cell is collected, total protein is extracted, carries out western blotting
The expression of PD-1 and CTLA4 genes in the cell is analyzed, internal reference albumen is done with β-actin, the results are shown in Figure 8, this is thin
The expression that can't detect two kinds of albumen in born of the same parents illustrates that PD-1 and CTLA4 genes have lacked in the T lymphocytes obtained.
In the present invention double gRNA knock out dual-gene scheme obtain T lymphocytes production time it is short, cost is few, and dual-gene
Deficiency homozygote T lymphocytes can enhance the effect of killing tumour cell, and can enhance long-term tumour immunity, so this hair
Bright constructed PD-1 and the dual-gene deficiency T lymphocyte populations of CTLA4 are a novel immune cell preparation, can be used for tumour trouble
The prevention of person.
Sequence table
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Claims (4)
1. a kind of preparation method of the dual-gene deficiency T lymphocyte preparations of PD-1 and CTLA4, it is characterised in that steps are as follows：
(1) structure of the dual-gene knockout carriers of PD-1 and CTLA4
GRNA target sequences are separately designed according to PD-1 and CTLA4 complete genome sequences, then by two gRNA target sequences
Lek is grand into expression vector, and obtaining band, there are two the knockout carriers of different target sequences gRNA；
Two gRNA target sequences are respectively 5 '-GGAGTGGATAGGCCACGGCG-3 ' and 5 '-
ACACCGCTCCCATAAAGCCA-3’；
(2) T separation of lymphocytes and activation
T separation of lymphocytes is cultivated, and is activated, and is bred, and is collected；
(3) electrotransfection T lymphocytes and T7E1 digestions identification
The knockout carrier that electricity turns liquid and step (1) obtains is added in T lymphocytes and carries out electrotransfection, after electrotransfection, continues
Cell extraction genome is collected in culture, is identified using T7E1 digestions；
(4) flow cytometer screening and sequencing analysis
The cell collected after electrotransfection carries out isolated monoclonal cell using flow cytometer, and monoclonal cell expands
Culture is to get the dual-gene deficiency T lymphocyte preparations of PD-1 and CTLA4；Expand the monoclonal cell after culture and extracts gene
Group carries out sequencing analysis.
2. the preparation method of the dual-gene deficiency T lymphocyte preparations of PD-1 according to claim 1 and CTLA4, special
Sign is that the expression vector described in step (1) is p2U6gRNA-Cas9-GFP expression vectors.
3. the preparation method of the dual-gene deficiency T lymphocyte preparations of PD-1 according to claim 1 and CTLA4, special
It is as follows that the electricity that sign is described in step (3) turns liquid composition, with volume percentage：
Without calcium and magnesium PBS 90%
Serum free medium RPMI 1,640 10%；
It is in terms of 100% by the total volume of no calcium and magnesium PBS and serum free medium RPMI 1640, EDTA additions are 30g/L, sugarcane
Sugared addition is 200mmol/L.
4. the preparation method of the dual-gene deficiency T lymphocyte preparations of PD-1 according to claim 1 and CTLA4, special
Sign is that the continuation incubation time described in step (3) is 48h.
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| CN107384959A (en) * | 2017-08-02 | 2017-11-24 | 山东百福基因科技有限公司 | The preparation method of the dual-gene deficiency T lymphocyte preparations of PD 1 and CTLA4 |
| CN108531457A (en) * | 2018-04-10 | 2018-09-14 | 杭州荣泽生物科技有限公司 | A kind of method that Cas9/RNP knocks out T cell PD-1 and LAG3 gene and prepares CAR-T cells |
| CN109136190A (en) * | 2018-04-11 | 2019-01-04 | 北京中诚华科生物科技有限公司 | A kind of BTLA for resisting tumour immunity and inhibiting environment-/-The preparation method and application of T lymphocyte |
| JP7338894B2 (en) | 2018-09-11 | 2023-09-05 | ボード オブ リージェンツ，ザ ユニバーシティ オブ テキサス システム | A genetic mouse model of autoimmune adverse events and immune checkpoint blockade therapy |
| CN111440799B (en) * | 2020-04-17 | 2021-03-02 | 皖南医学院第一附属医院（皖南医学院弋矶山医院） | Kit for realizing multiple immune checkpoint gene editing immune cells through CRISPR-Cpf1 mediation and application thereof |
| CN117169518B (en) * | 2023-11-03 | 2024-01-19 | 赛德特(北京)生物工程有限公司 | Method and kit for detecting CD3 antibody residues in T lymphocyte preparation |
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| CN107384959A (en) * | 2017-08-02 | 2017-11-24 | 山东百福基因科技有限公司 | The preparation method of the dual-gene deficiency T lymphocyte preparations of PD 1 and CTLA4 |
| CN107475292A (en) * | 2017-08-02 | 2017-12-15 | 山东百福基因科技有限公司 | The preparation method of the gene defection type T lymphocyte preparations of PD 1 |
| WO2018041121A1 (en) * | 2016-08-31 | 2018-03-08 | Beijing Biocytogen Co., Ltd | Genetically modified non-human animal with human or chimeric ctla-4 |
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| US20170020922A1 (en) * | 2015-07-16 | 2017-01-26 | Batu Biologics Inc. | Gene editing for immunological destruction of neoplasia |
| US20170152506A1 (en) * | 2015-08-28 | 2017-06-01 | Batu Biologics, Inc. | Inactivation of lymphocyte immunological checkpoints by gene editing |
| CN106480097A (en) * | 2016-10-13 | 2017-03-08 | 南京凯地生物科技有限公司 | Knocking out that people PD 1 is gene constructed using CRISPR/Cas9 technology can the method for targeting MSLN novel C AR T cell and its application |
| CN106480027A (en) * | 2016-09-30 | 2017-03-08 | 重庆高圣生物医药有限责任公司 | CRISPR/Cas9 targeting knock out people PD 1 gene and its specificity gRNA |
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2017
- 2017-08-02 CN CN201710649973.1A patent/CN107384959A/en active Pending
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2018
- 2018-05-18 CN CN201810482548.2A patent/CN108642071A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018041121A1 (en) * | 2016-08-31 | 2018-03-08 | Beijing Biocytogen Co., Ltd | Genetically modified non-human animal with human or chimeric ctla-4 |
| CN107384959A (en) * | 2017-08-02 | 2017-11-24 | 山东百福基因科技有限公司 | The preparation method of the dual-gene deficiency T lymphocyte preparations of PD 1 and CTLA4 |
| CN107475292A (en) * | 2017-08-02 | 2017-12-15 | 山东百福基因科技有限公司 | The preparation method of the gene defection type T lymphocyte preparations of PD 1 |
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