CN107475292B - The preparation method of PD-1 gene defection type T lymphocyte preparations - Google Patents
The preparation method of PD-1 gene defection type T lymphocyte preparations 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 1 gene defection type T lymphocyte preparations of PD.The structure of 1 knockout carriers of PD, T separation of lymphocytes and activation, electrotransfection T lymphocytes and T7E1 digestions identification, flow cytometer screening and sequencing analysis.On the one hand the production of the gene defection type immunocyte preparation of the present invention simplifies the production routine of cell, 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, has the advantages of high specificity, significant in efficacy, side effect is low, in blood cancer
It is significant in efficacy.But cellular immunotherapy is with the presence of its shortcoming, basic reason is the PD-1 genes in T lymphocytes, PD-1
(Programmed Death 1) programmed death receptor -1：It is a kind of important immunosuppression molecule.Cancer cell is escaped immune
A kind of mechanism of killing is to generate a kind of referred to as Programmed death ligand-1 (PD-L1) 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 out 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 to shift cutaneous melanoma in first late period that is used for listed in the U.S.
(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 inhibiting tumour, greatly improve the time-to-live of patient and survival rate (60% patient was survived more than 2 years).
PD-1 antibody drugs treatment tumor efficiency is notable.However the country there is no PD-1 antibody, PD-1 Antybody therapy medicines at present
Object is still among research and development, not yet into clinical test.In addition, PD-1 Antibody preparations and purification process are complicated, the period is long, is manufactured into
This height causes antibody drug expensive, and 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 period 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 editings such as Therapeutics, Editas Medicine and CRISPR Therapeutics company, exploitation
With reference to the CAR-T therapies of gene editing technology particularly CRISPR technologies.
Invention content
The object of the present invention is to provide a kind of preparation methods of PD-1 gene defection types T lymphocyte preparations, simplify 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 knockout carrier of the band there are 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 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, identified using T7E1 digestions；
(4) flow cytometer screening and sequencing analysis
The cell collected after electrotransfection is detached 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%；
It is counted using the total volume of no 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 to mix no calcium and magnesium PBS with serum free medium RPMI 1640, then adds in EDTA
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 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 is identified through sequencing confirms that (control experiment list gRNA carrier knockout rates are for 93% for effective frameshift mutation rate
64.7%), has 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 clone sequence into pU6gRNA-Cas9-GFP expression vectors, and building can successfully 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, such cell typically contains three classes hypotype：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.In the present invention, through single gRNA expression vectors with double gRNA expression vectors to PD-1 gene knockouts reality
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%.
Usually in experiment, single gRNA knock out after in order to obtain effective knockout, need to screen effective frameshift mutation homozygote,
The process is screened since unicellular, is bred, and waiting cell proliferations, with duration, cost is big to can be used for treatment disease levels.
And the present invention knocks out PD-1 Gene Experiments using double gRNA and confirms, the effective mutation rate of cell mass after double gRNA are knocked out reaches
93%, it is even higher, 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 the two gRNA target sequences that can be realized and efficiently knock out PD-1 genes first, 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
On the one hand the production of type immunocyte preparation simplifies the production routine of cell, on the other hand improve the speed of preparation acquisition.
Description of the drawings
Fig. 1 is list gRNA knockout carrier schematic diagrames.
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 screens GFP positive cells (single gRNA expression vectors) for flow cytometer, 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 screens GFP positive cells (double gRNA expression vectors) for flow cytometer, 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.
Specific embodiment
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 editions, Science Press, 2005) or by reagent manufacturer
Proposed condition carries 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, selects two suitable target sequences (each 20bp length), then by Shanghai life work life
Two sections of complementary target sequences of object Engineering Co., Ltd synthesis (increasing the cohesive end after 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 recycling with target1 sequences
Double-strand connects, 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 Target2 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 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 2ml/ holes is added in 6 orifice plates, then adds in CD3+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
1st, by arteria brachialis extract Healthy People new blood 1mL, at once add in 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.
2nd, PBS the or 0.9%NaCl dilute bloods or blood plasma of 3mL is added in.Dilute blood can reduce the cohesion of red blood cell, carry
High lymphocyte harvest yield.
3rd, 4mL lymphocyte separation mediums is taken to add in centrifuge tube tube bottom, and are 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 storing more than 2h should
Centrifuge 30min.
6th, 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, containing mononuclearcell (including lymphocyte and monokaryon granulocyte).List is directly inserted into suction pipe
A nucleus layer simultaneously draws the layer, is put into another test tube.
7th, plus the lymphocyte of 10ml Hank ' s liquid dilution separation, 250 × g centrifugation 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 plate, it is placed in CO2Incubator culture 24 hours, then adding in proleulzin (final concentration 1000U/ml) stimulates 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 for adding in optimization turns 400 μ l of liquid (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-
10×106Then cell/ml adds in (1) middle PD-1 gene knockouts plasmid built to final concentration of 40 μ g/ml.0.4cm shocks by electricity
Cup, 280V voltages, shock by electricity 20ms.The full culture mediums of RPMI-1640 that preheating is added in after electrotransfection 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 ') carry out PCR amplification, PCR productions
Object using T7E1 digestions identify, as a result there is the DNA fragmentation of 550bp and 300bp, illustrate genome PD-1 genes have occurred it is prominent
Become, see 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
Middle that GFP positive cells are detached 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’)
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 8 and Figure 9：In single gRNA knocks out experiment, effective frameshift mutation has occurred in 64.7% cell
Lead to PD-1 protein inactivations, and double gRNA knockouts make effective frameshift mutation rate reach more than 93%, 15 connection product sequencings
As a result, it has been found that only there are one connection that frameshift mutation does not occur, it is seen that double gRNA knockouts can obtain sufficient amount of PD-1 genes
Inactivation type T lymphocyte populations, the cell mass can obtain the immune work(identical with PD-1 gene defect T lymphocyte homozygotes
Effect.And double gRNA knock out that the scheme T lymphocyte populations production times are short, and cost is few, and it is identical to reach homozygote T lymphocytes
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 prevention of knurl patient.
Claims (2)
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）The 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 knockout carrier of the band there are 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 is cultivated, and is activated, and is bred, and is collected；
（3）Electrotransfection T lymphocytes and T7E1 digestions identification
Electricity is added in T lymphocytes and turns liquid and step（1）Obtained knockout carrier carries out electrotransfection, after electrotransfection, continues
Cell extraction genome is collected in culture, is identified using T7E1 digestions；
Step（3）Described in electricity 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 total volume of no calcium and magnesium PBS and serum free medium RPMI 1640 as 100%, EDTA additions be 30g/L, sugarcane
Sugared addition is 200mmol/L；
（4）Flow cytometer screens and sequencing analysis
The cell collected after electrotransfection is detached using flow cytometer, and Isolated cells expand culture to get PD-
1 gene defection type T lymphocyte preparations；Expand the cell extraction genome after culture and carry out sequencing analysis；
Step（4）Obtain the herd immunity cell preparation of high effectively frameshift mutation.
2. the preparation method of PD-1 gene defection types T lymphocyte preparations according to claim 1, it is characterised in that step
（3）Described in continuation incubation time be 48h.
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Effective date of registration: 20200807 Address after: Room 302, building 1, No.36 Doukou Road, Guangdong Macao cooperative traditional Chinese medicine science and Technology Industrial Park, Hengqin new area, Zhuhai City, Guangdong Province 519000 Patentee after: Baifu Life Science Research (Zhuhai Hengqin) Co., Ltd Address before: 255000, C, 326, hi tech Pioneer Park, Zibo hi tech Zone, Shandong Co-patentee before: Wang Qinglu Patentee before: SHANDONG BAIFU GENE TECHNOLOGY Co.,Ltd. |