CN107475292A - The preparation method of the gene defection type T lymphocyte preparations of PD 1 - Google Patents
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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 gene defection type T lymphocyte preparations of PD 1.The structure of the knockout carriers of PD 1, 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, the production routine of cell is on the one hand simplified, on the other hand improve the speed of preparation acquisition.
Description
Technical field
The invention belongs to technical field of molecular biology, and in particular to a kind of PD-1 gene defection types T lymphocyte preparations
Preparation method.
Background technology
CAR-T technologies (T lymphocytes are transformed using gene engineering method) in cellular immunotherapy are to treat at present
Most significant tumour cell therapy is imitated, forth generation has been developed to by the intracellular signal molecule amount for being continuously increased CAR elements, with
Nonspecific DC cell therapies of tradition etc., which are compared, possesses the advantages of high specificity, evident in efficacy, side effect is low, in blood cancer
It is evident in efficacy.But cellular immunotherapy is with the presence of its shortcoming, its basic reason is the PD-1 genes in T lymphocytes, PD-1
(Programmed Death 1) programmed death acceptor -1：It is a kind of important immunosuppression molecule.Cancer cell is escaped immune
A kind of mechanism of killing, it is that a kind of referred to as Programmed death ligand-1 (PD-L1) is produced by its surface, as this PD-L1
Being connected on the PD-1 albumen of a kind of immunocyte T cell causes T cell to inactivate.T cell cannot find tumour to exempting from
Epidemic disease system sends the signal of attack tumour.
And the method that relative maturity solves T cell inactivation at present is antibody technique.Such as：2014 Nian Shiguibao companies develop
Opdivo (PD-1 inhibitor Nivolumab) successively Japan and the U.S. listing, and MSD Corp. develop Keytruda
(PD-1 inhibitor Pemboolizamab) is then first and is used for late period transfer cutaneous melanoma in U.S.'s listing
(Melanoma) inhibitor, the PD-1 inhibitor is in the clinical trial of these cutaneous melanoma late period transporting patients, quilt
It was found that routinely suppressing tumour, greatly improve time-to-live and the survival rate of patient (60% patient was survived more than 2 years).
PD-1 antibody drugs treatment tumor efficiency is notable.But the country there is no PD-1 antibody, PD-1 Antybody therapy medicines at present
Thing is still among research and development, not yet into clinical test.In addition, PD-1 Antibody preparations and purge process are complicated, cycle length, it is manufactured into
This height, cause antibody drug expensive, in fact ordinary people is difficult to bear this high medical expense.
Therefore, the PD-1 genes in T lymphocytes are knocked out using CRISPR/Cas9 technologies, so as to activated T lymphocytes pair
The ability of tumour cell attack, realizes the immunization therapy of tumour.Program cell preparation process is relatively easy, the cycle is short, cost
It is low, it is more suitable for China's national situation and ordinary people patient.At present, almost all of CAR-T therapies company all with gene editing company
Carry out cooperation, 6 CAR-T therapies enterprises and Intellia including Novartis, Juno and Cellectis
The combination among the strong ones of the gene editing such as Therapeutics, Editas Medicine and CRISPR Therapeutics company, exploitation
With reference to the CAR-T therapies of gene editing technology particularly CRISPR technologies.
The content of the invention
It is an object of the invention to provide a kind of preparation method of PD-1 gene defection types T lymphocyte preparations, simplifies thin
The production routine of born of the same parents, the speed for improving preparation acquisition.
The preparation method of PD-1 gene defection types T lymphocyte preparations of the present invention, step are as follows：
(1) structure of PD-1 knockout carriers
Two different gRNA target sequences are designed according to PD-1 complete genome sequences, then by two different gRNA
Target sequences are cloned into expression vector, obtain the knockout carrier with two different target sequences gRNA；
Two different gRNA target sequences be respectively 5 '-TGTGAGGAGTGGATAGGCCA-3 ' and 5 '-
AGGGCCCGGCGCAATGACAG-3’；
(2) T separation of lymphocytes and activation
T separation of lymphocytes, cultivate, activate, breed, collect；
(3) electrotransfection T lymphocytes and T7E1 digestions identification
The knockout carrier progress electrotransfection that electricity turns liquid and step (1) obtains is added in T lymphocytes, after electrotransfection terminates,
Continue to cultivate, collect cell extraction genome, identified using T7E1 digestions；
(4) flow cytometer screening and sequencing analysis
The cell that electrotransfection is collected after terminating is separated using flow cytometer, and Isolated cells expand culture, i.e.,
Obtain PD-1 gene defection type T lymphocyte preparations；Expand the cell extraction genome after culture and carry out sequencing analysis.
Expression vector described in step (1) is pU6gRNA-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%；
Counted using the cumulative volume without calcium and magnesium PBS and serum free medium RPMI 1640 as 100%, EDTA additions are 30g/
L, sucrose addition are 200mmol/L.
The preparation method that electricity turns liquid is will to be mixed without calcium and magnesium PBS with serum free medium RPMI 1640, then add EDTA
And sucrose, refiltered after dissolving degerming.
Continuation incubation time described in step (3) is 48h.
It is, thin through streaming after flow cytometer screening technique is cell transfecting 48h in step (4) with PBS cell twice
Born of the same parents' instrument screens GFP positive t lymphocytes, that is, obtains the herd immunity cell preparation of high effectively frameshift mutation.
The present invention builds special, efficient double gRNA knockout carriers, efficient electrotransfection method, flow cytometer screening
The immunocyte preparation that can be used for oncotherapy and prevention is obtained afterwards.Two gRNA belong to independent expression unit, and carrier also wraps
Gene containing Cas9 and green fluorescent protein (GFP) gene.
Double gRNA knockout carriers screen GFP positive cells after electrotransfection T lymphocytes, using flow cytometer, positive
Cellular genome confirms that (control experiment list gRNA carrier knockout rates are for 93% for effective frameshift mutation rate through sequencing identification
64.7%), possesses the immunocyte condition for tumor prevention and treatment.
The PD-1 gene defection type T lymphocyte populations of flow cytometer screening are an immunocyte preparation, available for human body
Feed back prevention or treatment tumour.
The present invention obtains specificity, efficient identification target site using bioinformatics technique and effectively cuts off target site
GRNAtarget sequences, then sequence is cloned into pU6gRNA-Cas9-GFP expression vectors, successfully construct to express simultaneously
Two expression vectors for carrying different target sequences gRNA.
Expression vector is transferred to work by the present invention on the basis of structure gRNA with Cas9 expression vectors using electrotransfection technology
The T lymphocytes of change, for electrotransfection using the electrotransfection buffer solution of optimization, transformation efficiency is high, and cell mortality is low, more effective to obtain
PD-1 gene defection type T lymphocyte populations.
The present invention screens GFP positive cells by flow cytometer, and such cell typically contains three class hypotypes：1. point is prominent
Become；2. monoallelic missing (i.e. a PD-1 allele normal expression, and another PD-1 allele is knocked)；③
PD-1 genes are knocked completely.It is real to PD-1 gene knockouts with double gRNA expression vectors through single gRNA expression vectors in the present invention
Test it was found that, in single gRNA experiments the 3. kind situation account for 64.7% (i.e. effective frameshift mutation) of the whole circumstances, it is and double
GRNA knock out it is experimentally confirmed that the 3. kind situation account for the whole circumstances and reach more than 93%.
Generally in experiment, in order to obtain effective knockout, it is necessary to screen effective frameshift mutation homozygote after single gRNA knockouts,
The process is screened since unicellular, bred, and waiting cell proliferation, it uses duration, and cost is big to can be used for treating disease levels.
And the present invention knocks out PD-1 Gene Experiments using double gRNA and confirmed, the effective mutation rate of cell mass after double gRNA are knocked out reaches
93%, even more high, single celled effect can be competent at completely, and double gRNA are knocked out and are obtained cell mass, cell radix is big, breeding
Time to quantity available is short, and cost is low, so the knockout scheme obtains a kind of new herd immunity cell preparation.
Beneficial effects of the present invention are as follows：
The present invention determines first can realize two gRNA target sequences for efficiently knocking out PD-1 genes, then excellent
Change electrotransfection buffer solution, realize the efficient electrotransfection of T lymphocytes and ensure the survival rate of cell after electrotransfection, finally utilize stream
Formula cell instrument screens GFP positive cell groups, obtains gene editing type immunocyte preparation.Its PD-1 gene defect cell obtained
Group, PD-1 genes inactivation rate are suitable with the cell mass that traditional unicellular homozygote is screened and obtained.The gene defect of the present invention
The production of type immunocyte preparation, the production routine of cell is on the one hand simplified, on the other hand improve the speed of preparation acquisition.
Brief description of the drawings
Fig. 1 is single gRNA knockout carriers schematic diagram.
Fig. 2 is the intermediate carrier used in the double gRNA knockout carriers of structure.
Fig. 3 is double gRNA knockout carrier schematic diagrames.
Fig. 4 is the T lymphocytes of activation.
Fig. 5 is that (T7E1 is not added in+representative addition T7E1 enzymes ,-representative to T7E1 digestions qualification result of the electrotransfection after 48 hours
The negative control of enzyme).
Fig. 6 is that flow cytometer screens GFP positive cells (single gRNA expression vectors), and the small figure in the upper left corner is cellular morphology ginseng
Number, the small figure in the upper right corner is cell viability parameter, and the small figure in the lower left corner is unicellular parameter, and the small figure in the lower right corner is GFP positive parameters.
Fig. 7 is that flow cytometer screens GFP positive cells (double gRNA expression vectors), and the small figure in the upper left corner is cellular morphology ginseng
Number, the small figure in the upper right corner is cell viability parameter, and the small figure in the lower left corner is unicellular parameter, and the small figure in the lower right corner is GFP positive parameters.
Fig. 8 is the sequencing analysis (single gRNA expression vectors) to PD-1 catastrophes in GFP positive cells.
Fig. 9 is the sequencing analysis (double gRNA expression vectors) to PD-1 catastrophes in GFP positive cells.
Embodiment
The present invention is described further with reference to embodiments.
The experimental method of unreceipted actual conditions in following examples, generally according to normal condition such as《Molecular Cloning: A Laboratory
Guide》Condition described in reference books commonly used in the art such as (third editions, Science Press, 2005), or by reagent manufacturer
Proposed condition is carried out.
Embodiment 1
(1) structure of PD-1 knockout carriers
1. the design of gRNA target sequences
PD-1 complete genome sequences (EF064716.1) are obtained from NCBI, based on CRISPR-DO website design gRNA target
Sequence, then according to the parameter of setting, two suitable target sequences (each 20bp length) are selected, then by Shanghai life work life
Two sections of complementary target sequences of thing Engineering Co., Ltd synthesis (cohesive end after increase BbsI digestions).
Such as：Target1：F 5’-ATAGTGTGAGGAGTGGATAGGCCA-3’
R 5’-AAATTGGCCTATCCACTCCTCACA-3’
Target2：F 5’-ATAGAGGGCCCGGCGCAATGACAG-3’
R 5’-AAATCTGTCATTGCGCCGGGCCCT-3’
2. knockout carrier is built
With BbsI digestion pU6gRNA-CMV-Cas9-GFP expression vectors, the oligo formed after recovery with target1 sequences
Double-strand connects, and builds the PD-1 gene knockout carriers (Fig. 1) containing a 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 breakaway poing 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 Target2 sequences and obtain intermediate carrier (Fig. 2).Linearized with HindIII and EcoRI double digestions
PU6gRNA-CMV-Cas9-GFP expression vectors and intermediate carrier, connect the two after recovery, and acquisition can express two differences
Target sequences gRNA knockout carrier, is shown in Fig. 3.
(2) T separation of lymphocytes and activation
①CD3+Antibody is coated with flat board
The PBS in 2ml/ holes is added in 6 orifice plates, then adds CD3+Antibody is placed in 37 to the μ g/ml of final concentration 10
℃CO2Incubator is incubated 2 hours, then sucks PBS, with fresh preheating PBS flat board 2 times, then with serum-free RPMI-
1640 culture mediums clean 1 time.
2. mature T separation of lymphocytes and culture
1st, by arteria brachialis extract Healthy People new blood 1mL, at once add the 1.5mL containing 10 μ L anticoagulant heparin agent from
In heart pipe.To keep the activity of lymphocyte, separated at once after blood sampling.
2nd, 3mL PBS or 0.9%NaCl dilute bloods or blood plasma is added.Dilute blood can reduce the cohesion of red blood cell, carry
High lymphocyte harvest yield.
3rd, take 4mL lymphocyte separation mediums to add centrifuge tube ttom of pipe, and be warming up to room temperature.
4th, 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.
5th, horizontal rotor centrifugation 2000r/min or 700 × g centrifugations 20min, 20 DEG C of room temperature.The blood for depositing more than 2h should
Centrifuge 30min.
6th, ttom of pipe is red blood cell after centrifuging, and intermediate layer is separating liquid, and the superiors are blood plasma.It is between plasma layer and separating liquid
The finer and close tunica albuginea of a thin layer, containing mononuclearcell (including lymphocyte and monokaryon granulocyte).List is directly inserted into suction pipe
Individual nucleus layer simultaneously draws the layer, is put into another test tube.
7th, plus 10ml Hank ' s liquid dilutes the lymphocyte of separation, 250 × g centrifugation 10min, abandons 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 plate, CO is placed in2Incubator culture 24 hours, then adding proleulzin (final concentration 1000U/ml) stimulates carefully
Intracellular growth, acquired results are shown in Fig. 4.
(3) electrotransfection T lymphocytes and T7E1 digestions identification
T lymphocytes PBS one time is collected, the electricity for adding optimization turns the μ l of liquid 400 (formulas：Without calcium and magnesium PBS 90%
(V/V), EDTA 30g/L, serum free medium RPMI 1,640 10% (V/V), sucrose 200mmol/L) it is resuspended to final concentration 5-
10×106Cell/ml, (1) middle PD-1 gene knockouts plasmid built is then added to final concentration of 40 μ g/ml.0.4cm shocks by electricity
Cup, 280V voltages, shock by electricity 20ms.Electrotransfection the adds preheating full culture mediums of RPMI-1640 after terminating are placed in carbon dioxide culture
Continue to cultivate in case, after 48 hours, collect cell extraction genome, with identification primer Target1 (PD1F：5’-
GACTGGGCACAGGAGTGGGAGG-3’；PD1R：5 '-GCCTGACTCAGGCCAGGATCC-3 ') enter performing PCR amplification, PCR productions
Thing identifies 550bp and 300bp DNA fragmentation as a result occur, illustrate that the PD-1 genes of genome are dashed forward using T7E1 digestions
Become, see Fig. 5.
(4) flow cytometer screening and sequencing analysis
Electrotransfection T lymphocytes are after 48 hours, collect cell PBS cell 2 times, are placed in 1ml PBSs
Middle that GFP positive cells are separated using flow cytometer, the T separation of lymphocytes processes that single gRNA is knocked out are shown in Fig. 6, double
The T separation of lymphocytes processes that gRNA is knocked out are shown in Fig. 7.
Isolated cells expand culture, take 1-2 × 106Cell extracts genome, using identifying primer Target1
(PD1F：5’-GACTGGGCACAGGAGTGGGAGG-3’；PD1R：5 '-GCCTGACTCAGGCCAGGATCC-3 ') and Target2
(PD1F’：5-’GGCTTTGTGGGGCCACCCAGCCCCT-3’；PD1R’：5’-CCTCGTGCGGCCCGGGAGCAGATG-3’)
Enter performing PCR, carrier T is connected after PCR primer recovery, then convert E. coli competent, picking monoclonal bacterium carries out sanger
Sequencing analysis, as a result as shown in Figure 8 and Figure 9：In single gRNA knocks out experiment, there occurs effective frameshift mutation for 64.7% cell
Cause PD-1 protein inactivations, and double gRNA knockouts make effective frameshift mutation rate reach more than 93%, 15 connection products sequencings
As a result find that only a connection is without generation frameshift mutation, it is seen that double gRNA, which are knocked out, can obtain sufficient amount of PD-1 genes
Inactivation type T lymphocyte populations, the cell mass can obtain is immunized work(with PD-1 gene defect T lymphocyte homozygotes identical
Effect.And double gRNA knock out that the scheme T lymphocyte populations production times are short, and cost is few, and can reach homozygote T lymphocyte identicals
Effect, so the PD-1 gene defection type T lymphocyte populations constructed by the present invention are a novel immune cell preparation, available for swelling
The preventing and treating of knurl patient.
Claims (4)
1. a kind of preparation method of PD-1 gene defection types T lymphocyte preparations, it is characterised in that step is as follows：
(1) structure of PD-1 knockout carriers
Two different gRNA target sequences are designed according to PD-1 complete genome sequences, then by two different gRNA
Target sequences are cloned into expression vector, obtain the knockout carrier with two different target sequences gRNA；
Two different gRNA target sequences be respectively 5 '-TGTGAGGAGTGGATAGGCCA-3 ' and 5 '-
AGGGCCCGGCGCAATGACAG-3’；
(2) T separation of lymphocytes and activation
T separation of lymphocytes, cultivate, activate, breed, collect；
(3) electrotransfection T lymphocytes and T7E1 digestions identification
The knockout carrier progress electrotransfection that electricity turns liquid and step (1) obtains is added in T lymphocytes, after electrotransfection terminates, is continued
Culture, cell extraction genome is collected, identified using T7E1 digestions；
(4) flow cytometer screening and sequencing analysis
The cell that electrotransfection is collected after terminating is separated using flow cytometer, and Isolated cells expand culture, produce PD-
1 gene defection type T lymphocyte preparations；Expand the cell extraction genome after culture and carry out sequencing analysis.
2. the preparation method of PD-1 gene defection types T lymphocyte preparations according to claim 1, it is characterised in that step
(1) expression vector described in is pU6gRNA-Cas9-GFP expression vectors.
3. the preparation method of PD-1 gene defection types T lymphocyte preparations according to claim 1, it is characterised in that step
(3) electricity described in turn liquid composition it is as follows, with volume percentage：
Without calcium and magnesium PBS 90%
Serum free medium RPMI 1,640 10%；
Counted using the cumulative volume without calcium and magnesium PBS and serum free medium RPMI 1640 as 100%, EDTA additions are 30g/L, sugarcane
Sugared addition is 200mmol/L.
4. the preparation method of PD-1 gene defection types T lymphocyte preparations according to claim 1, it is characterised in that step
(3) the continuation incubation time described in is 48h.
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| CN117169518A (en) * | 2023-11-03 | 2023-12-05 | 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 |
| 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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