CN113005151A - Preparation method and application of KDR-CAR-NK cell - Google Patents
Preparation method and application of KDR-CAR-NK cell Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of KDR-CAR-NK cells. The preparation method for constructing the KDR-CAR-NK cell mainly comprises the processes of constructing a KDR-CAR sequence, constructing a KDR-CAR sequence vector, constructing the KDR-CAR-NK cell and the like. The KDR-CAR-NK cell constructed by the invention can express a KDR targeting chimeric antigen receptor, improves the targeting property, the killing property and the activity of the NK cell, enables the NK cell to break the immune suppression of the microenvironment of tumor focus positions and relieve the immune tolerance of a host, and realizes a new breakthrough in adoptive immunotherapy of allogeneic cells.
Description
Technical Field
The invention belongs to the field of biotechnology. In particular to a preparation method and application of KDR-CAR-NK cells, belonging to the technical field of tumor cell immunotherapy.
Background
Malignant tumors are diseases with multiple gene abnormalities, multiple step occurrences, and heterogeneous evolutions that seriously threaten human life and health, and the human immune system plays a crucial role in preventing the formation and progression of cancer. The occurrence and development of tumors in vivo are related to the immune dysfunction of the body, and simultaneously, tumor cells can also cause the immune suppression of the body by secreting cell factors. In recent years, tumor immunotherapy, which is a main therapeutic means for activating the human autoimmune system against malignant tumors, has shown strong antitumor activity in the treatment of various tumors such as malignant tumors of the blood system, melanoma, non-small cell lung cancer, kidney cancer, and prostate cancer. Because of its excellent curative effect and innovativeness, tumor immunotherapy has gradually become the fourth category of tumor treatment methods following surgery, radiotherapy and chemotherapy.
Among them, the use of Chimeric Antigen Receptors (CAR) modification to significantly enhance the anti-tumor activity of immune effector cells has become a preferred scheme for adoptive immune cell therapy of tumors. A CAR is a fusion molecule that comprises a tumor antigen binding domain that can specifically recognize a surface receptor of a tumor cell. CARs consist primarily of an extracellular antigen-binding portion, a hinge, a transmembrane segment, and an intracellular signaling segment. The transmembrane segment can be CD4, CD8, CD28 and CD3 zeta, and can also be single-chain or dimer of CD4, CD8, CD28, CD3 zeta and Fc epsilon RI gamma. With the development of recombinant DNA technology and the intensive research on signaling pathways, the signaling domain of CAR molecules has also evolved from the first single targeting signal to the second and third generations of multiple signaling regions containing costimulatory molecules such as CD28, CD137(4-1BB), CD134(OX40), and ICOS.
The first generation of CARs was predominantly CD3 ζ, with a short antitumor time. The second generation introduces a costimulatory molecule (CD28, 4-1BB, etc.), and clinical tests show better in vivo amplification capability and anti-tumor capability. The CAR of the third generation is added with two or more than two co-stimulatory molecules, and clinical tests show that the anti-tumor effect of the CAR is greatly increased, but the toxic and side effects are also obviously enhanced. Therefore, the second generation CAR has obvious advantages by combining curative effect and side effect, and the CAR modified T lymphocyte has made remarkable breakthrough progress in adoptive cellular immunotherapy of tumor. However, due to the limitations of the major histocompatibility complex I (MHC-1) molecule, CAR-T requires autologous cell transplantation, the process of collecting patient T cells and modifying genes is time consuming and inefficient, cell sources are limited, and it has been found clinically that not all patients can obtain large numbers of functional T cells to meet the treatment conditions. Thus, there is a need for a new source of tumor-killing immune cells as a more direct immunotherapeutic approach.
Natural Killer (NK) cells are another important immune effector cell with a powerful ability to kill tumor cells and virally infected cells, and NK cell activation does not require HLA matching to mediate, and transplantation of both autologous and allogeneic NK cells has proven safe in clinical trials. In the past decade, a number of experimental approaches based on NK cell immunotherapy have been undertaken, involving NK cell sources including peripheral blood, cord blood and NK-92 cell lines. In the research, it is also found that after allogeneic NK cells are transplanted, they have few toxic and side effects such as induction of Graft Versus Host Disease (GVHD), Cytokine Release Syndrome (CRS), or neurotoxicity, so CAR-modified NK cells may be a more feasible and safer and effective tumor immune cell therapy. The NK cell line applied in the current clinical research is mainly NK-92 cells. NK-92 has strong cytotoxicity to tumor cells, and has been found to have an effect against leukemia, lymphoma, melanoma, prostate cancer and breast cancer. NK-92 cells lack the expression of inhibitory KIRs, which mediate the anti-tumor effects through the activity of perforin and granzyme. In addition, NK-92 cells are the only NK cell line approved for testing by FDA (food and Drug administration) in the United states. Like CAR-T cells, NK-92 cells are genetically engineered to express specific receptors and can also target tumor cell killing. The CAR modification endows NK cells with receptors of specific antigens, allows the NK cells to attack any potential tumor in a targeted manner, has relatively high targeting property, killing property and activity, and simultaneously can break immune suppression of a microenvironment at a tumor focus part and relieve immune tolerance of a host. CAR-modified NK-92 cells are expected to be excellent effector cells for adoptive immunotherapy of allogeneic cells.
Disclosure of Invention
Aiming at the defects generally existing in the prior art, the invention creatively provides a preparation method for constructing KDR-CAR-NK cells. The targeting property, the killing property and the activity of the NK cells are improved, so that the NK cells can break the immune suppression of a microenvironment of a tumor focus part and relieve the immune tolerance of a host, and a new breakthrough in adoptive immunotherapy of allogeneic cells is realized.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for preparing KDR-CAR-NK cells comprises the following steps:
s1, construction of KDR-CAR sequence: sequentially and serially splicing gene sequences of an intracellular signaling domain of CD8 alpha, a co-stimulatory molecule CD28 and a CD3 zeta to carry out whole-gene synthesis, reserving restriction enzyme sites Xhol I and BamH I at two ends, and connecting VEGF165 genes with corresponding enzyme sites by using T4 ligase to prepare a KDR-CAR sequence;
s2, constructing a KDR-CAR sequence vector: cloning the KDR-CAR sequence prepared in the step S1 to a lentiviral vector pcDNA3.1-EGFP to obtain a recombinant plasmid KDR-CAR sequence vector KDR-CAR/pcDNA3.1-EGFP;
s3, constructing KDR-CAR-NK cells: and (3) taking Opti-MEM as a transfection reagent, performing electroporation transfection on the KDR-CAR sequence vector KDR-CAR/pcDNA3.1-EGFP obtained in the step S2 to NK cells, and screening cells positive to fluorescent protein by using fluorescence detection to obtain the fluorescent protein-containing cell.
Preferably, the sequence information of the VEGF165 gene in step S1 is shown in SEQ ID No. 1.
AACTTTCTGCTGTCTTGGGTGCATTGGAGCCTTGCCTTGCTGCTCTACCTCCACCATGCCAAGTGGTCCCAGGCTGCACCCATGGCAGAAGGAGGAGGGCAGAATCATCACGAAGTGGTGAAGTTCATGGATGTCTATCAGCGCAGCTACTGCCATCCAATCGAGACCCTGGTGGACATCTTCCAGGAGTACCCTGATGAGATCGAGTACATCTTCAAGCCATCCTGTGTGCCCCTGATGCGATGCGGGGGCTGCTGCAATGACGAGGGCCTGGAGTGTGTGCCCACTGAGGAGTCCAACATCACCATGCAGATTATGCGGATCAAACCTCACCAAGGCCAGCACATAGGAGAGATGAGCTTCCTACAGCACAACAAATGTGAATGCAGACCAAAGAAAGATAGAGCAAGACAAGAAAAAAAATCAGTTCGAGGAAAGGGAAAGGGGCAAAAACGAAAGCGCAAGAAATCCCGGTATAAGTCCTGGAGCGTATGTGACAAGCCGAGGCGG(SEQ ID NO.1)；
Preferably, the sequence of CD8 α in step S1 is shown as SEQ ID NO.2, the sequence of CD28 is shown as SEQ ID NO.3, and the sequence of CD3 ζ is shown as SEQ ID NO. 4.
ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT(SEQ ID NO.2)
TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO.3)；
AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAAGGATCCCCC(SEQ ID NO.4)；
Preferably, in step S1, a VEGF165 fragment and a KDR-CAR fragment of the target gene are amplified by using a PCR technique, a Primer for amplifying a VEGF165 sequence is Primer-VEGF165, sequence information of an upstream Primer is shown in SEQ ID No.5, and sequence information of a downstream Primer is shown in SEQ ID No. 6;
5'-ATTGCTAGCAAGAGAGCACCCATGGCAGAAGGAG-3'(SEQ ID NO.5)
5'-GTACTCGAGCCGCCTCGGCTTGTCACATTTTTA-3'(SEQ ID NO.6)；
the Primer for amplifying the KDR-CAR is Primer-KDR-CAR, the sequence information of the upstream Primer is shown as SEQ ID NO7, and the sequence information of the downstream Primer is shown as SEQ ID NO. 8.
5'-ATTGCTAGCAAGAGAGCACCCATGGCAGAAGGAG-3'(SEQ ID NO.7)；
5'-GTAGAATTCCTCTAGAGCATGCTTAGCGAGGGG-3'(SEQ ID NO.8)；
Preferably, in the step S2, the cohesive ends of the enzyme cutting sites are added to both ends of the KDR-CAR sequence and cloned into the lentiviral vector pcdna3.1-EGFP, the cohesive end at the front section is the enzyme cutting site of the Nhe I enzyme, and the cohesive end at the end is the enzyme cutting site of the BamH I enzyme.
Preferably, the NK cells transfected by electroporation in the step S3 are NK-92 cells.
Preferably, the detection process of screening fluorescent protein positive cells by fluorescence detection in step S3 is as follows:
(1) collecting the transfected NK-92 cell suspension in a 15mL centrifuge tube, centrifuging at 1200rpm for 5min, and discarding the supernatant;
(2) adding 1mL of opti-MEM buffer solution to resuspend the cells, centrifuging to remove the supernatant, and repeating for 2-3 times;
(3) after adding 50. mu.L of opti-MEM buffer to resuspend the cells, the cells were counted so that the amount of NK-92 cells was 1X 106A plurality of; then KDR-CAR sequence vector KDR-CAR/pcDNA3.1-EGFP with the concentration of 1 mug/mug is added, the amount of the vector in each hole is 1.8 mug, opti-MEM buffer solution is used for supplementing, the total volume is 100 mug, the vector is added into an electric rotating cup, and an electric rotating experiment is carried out after resistance is tested; (confirmation: 50. mu.L buffer volume)
(4) After the electrotransfer is finished, the solution in the electrotransfer cup is transferred to a 6-hole plate, 1mL of complete culture medium is supplemented, and the 6-hole plate is placed in 5% CO2And culturing for 24-72 h in a 37 ℃ cell culture box, observing the expression of EGFP of the cells under a fluorescence inverted microscope, and screening fluorescent protein positive cells to obtain the cells expressing the KDR targeting chimeric antigen receptor NK-92.
The electroporation test of the present invention is carried out by mixing 50. mu.L of cells (at least 1X 10)6Individually) were mixed with 50. mu.L of plasmid and opti-MEM buffer, and the whole wasAccumulating 100 μ L, and adding into a cuvette for electric rotation; before electric conversion, the resistance is required to be verified to be in the range of 0.45-0.6 ohm, so that the electric rotary cup can be used.
Preferably, the resistance is tested under the condition of 150.0v and 5ms before the electricity is converted in the step (3), and the resistance range is 0.040-0.060 omega.
Preferably, KDR-CAR sequence vector 3 μ L with a concentration of 600ng/μ L is added in the step (4) to observe the expression of EGFP in the cells under a fluorescence inverted microscope.
The invention also provides application of the NK cell modified by the KDR-expressing targeting antigen receptor obtained by the preparation method in the adoptive immunotherapy direction.
In the invention, the fluorescence detection is that the plasmid vector is used and carries the fluorescent marker EGFP; the plasmid vector carries a fluorescent marker, and whether the plasmid is successfully introduced can be observed through the fluorescent marker, wherein the amount of the added plasmid is 3 mu L, and the concentration of the added plasmid is 600 ng/mu L.
Compared with the prior art, the invention has the advantages that: the invention carries out genetic engineering modification on NK-92 cells, endows the NK cells with receptors of specific antigens, improves the targeting property, the killing property and the activity of the NK cells, enables the NK cells to break immune suppression of a microenvironment of a tumor focus part and relieve immune tolerance of a host, allows the NK cells to attack any potential tumor in a targeted manner, and realizes a new breakthrough in adoptive immunotherapy of allogeneic cells.
Drawings
Fig. 1 is a schematic structural diagram of KDR-CAR in the examples;
FIG. 2 is a graph showing the result of amplification in the examples;
FIG. 3 is a vector map of KDR-CAR/pcDNA3.1-EGFP plasmid vector in the examples;
FIG. 4 is a map of KDR-CAR transfected NK-92 cells in the examples;
FIGS. 5 and 6 are graphs of apoptosis of tumor cells detected by flow method after co-incubation of tumor cells in the examples;
FIGS. 7 and 8 are graphs showing the expression of the cytokines IFN-. gamma.and Granzyme B in the flow-type assay after co-incubation with tumor cells in the examples.
Detailed Description
The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative and not intended to limit the present invention, and all technical solutions similar or equivalent to the present invention are within the protection scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
When the PCR tandem connection is carried out, the gene sequences of a CD8 alpha (hinge region + transmembrane region), a co-stimulatory molecule CD28 intracellular region and a CD3 zeta intracellular signal conduction domain are determined firstly, and are sequentially spliced in series, the gene sequences are obtained by synthesizing the whole genes of a member biology engineering (Shanghai) corporation, and restriction enzyme sites Xhol I and BamH I are reserved at two ends of the gene sequences so as to be conveniently connected with a human targeting gene segment VEGF165 stored in a laboratory and form KDR-CAR by tandem connection. And then cloning the KDR-CAR fragment to a lentiviral vector pcDNA3.1-EGFP to obtain the lentiviral vector KDR-CAR/pcDNA3.1-EGFP.
The CD8 alpha-CD 28-CD3 zeta total gene synthesis fragment is synthesized by the corporation of Kentungsheng bioengineering (Shanghai) and Limited;
the VEGF165 gene is synthesized by Venezetian Biotechnology engineering (Shanghai) GmbH;
the restriction enzyme may be purchased from precious bioengineering (Dalian) Inc.;
the ligase can be purchased from precious bioengineering (Dalian) Inc.;
the pcDNA3.1-EGFP vector can be purchased from Anhui general biology, Inc.;
the opti-MEM buffer is commercially available from Gibco;
the BSA can be purchased from Labgic, the complete culture medium formula is 1640 complete culture medium, and the BSA consists of 90% 1640 basic culture medium and 10% fetal bovine serum (PBS), and can be purchased from Gibco company, and the cargo numbers are respectively: c11875500BT (500mL), A31608-02(500 mL);
the prepared cell membrane breaking agent is prepared by adding 3 mu L of TritonX-100(TritonX-100 is purchased from Biosharp) into 1mL of 10% BSA;
the PBS is available from Gibco corporation under the trade designation A31608-02(500mL), and paraformaldehyde is available from Seville;
the untransfected NK-92 cells used in the experiments were purchased from the China center for type culture Collection, and the lung cancer cells (A549) were purchased from the cell bank of Chinese academy of sciences;
the flow cytometer was manufactured and described as Beckmann Coulter (model: Navios).
Example 1A method for preparing KDR-CAR-NK cells
The preparation method of the KDR-CAR-NK cell comprises the following steps:
s1, construction of KDR-CAR sequence: sequentially and serially splicing gene sequences of an intracellular signaling domain of CD8 alpha, a co-stimulatory molecule CD28 and a CD3 zeta to carry out whole-gene synthesis, reserving restriction enzyme sites Xhol I and BamH I at two ends, and connecting VEGF165 genes with corresponding enzyme sites by using T4 ligase to prepare a KDR-CAR sequence; FIG. 2 is a diagram showing the detection results of amplification, wherein lane 1 is the VEGF165 gene PCR amplification product, lane 2 is the marker lane, and lane 3 is the whole gene synthesis CD8 α -CD28-CD3 ζ fragment amplification product;
the sequence information of the VEGF165 gene is shown in SEQ ID NO. 1; the sequence of the CD8 alpha is shown as SEQ ID NO.2, the sequence of the CD28 is shown as SEQ ID NO.3, and the sequence of the CD3 zeta is shown as SEQ ID NO. 4;
the PCR technology is utilized to amplify target gene VEGF165 fragments and KDR-CAR fragments, a Primer for amplifying VEGF165 sequences is Primer-VEGF165, the sequence information of an upstream Primer is shown as SEQ ID NO.5, and the sequence information of a downstream Primer is shown as SEQ ID NO. 6; the Primer for amplifying the KDR-CAR is Primer-KDR-CAR, the sequence information of the upstream Primer is shown as SEQ ID NO7, and the sequence information of the downstream Primer is shown as SEQ ID NO. 8; the PCR reaction is described in table 1 below:
TABLE 1 PCR reaction System
| Reagent | Amount of the composition used | Final concentration |
| PrimerSTAR Max Premix(2ⅹ) | |
1× |
| Upstream primer (SEQ ID NO.5) | 15pmol | 0.3μM |
| Downstream primer (SEQ ID NO.6) | 15pmol | 0.3μM |
| pUC57-CAR | 100ng | |
| Sterilized water | Up to 50μL |
The PCR reaction procedure was as follows:
s2, constructing a KDR-CAR sequence vector: cloning the KDR-CAR sequence prepared in the step S1 to a lentiviral vector pcDNA3.1-EGFP to obtain a recombinant plasmid KDR-CAR sequence vector KDR-CAR/pcDNA3.1-EGFP;
adding cohesive ends of enzyme cutting sites at two ends of a KDR-CAR sequence, cloning the cohesive ends into a lentiviral vector pcDNA3.1-EGFP, wherein the cohesive end at the front section is the enzyme cutting site of Nhe I enzyme, and the cohesive end at the tail end is the enzyme cutting site of BamH I enzyme;
s3, constructing KDR-CAR-NK cells: performing electroporation transfection on the KDR-CAR sequence vector KDR-CAR/pcDNA3.1-EGFP obtained in the step S2 to NK cells by using Opti-MEM as a transfection reagent, and screening cells positive to fluorescent protein by using fluorescence detection to obtain the fluorescent protein-containing cell;
the electroporation transfected NK cells are NK-92 cells;
the detection process for screening the cells positive to the fluorescent protein by using the fluorescence detection comprises the following steps:
(1) collecting the transfected NK-92 cell suspension in a 15mL centrifuge tube, centrifuging at 1200rpm for 5min, and discarding the supernatant;
(2) adding 1mL of opti-MEM buffer solution to resuspend the cells, centrifuging to remove the supernatant, and repeating for 2-3 times;
(3) after adding 50. mu.L of opti-MEM buffer to resuspend the cells, the cells were counted so that the amount of NK-92 cells was 1X 106A plurality of; then KDR-CAR sequence vector KDR-CAR/pcDNA3.1-EGFP with the concentration of 1 mug/mug is added, the amount of the vector in each hole is 1.8 mug, opti-MEM buffer solution is used for supplementing, the total volume is 100 mug, the vector is added into an electric rotating cup, and an electric rotating experiment is carried out after resistance is tested;
(4) after the electrotransfer is finished, the solution in the electrotransfer cup is transferred to a 6-hole plate, 1mL of complete culture medium is supplemented, and the 6-hole plate is placed in 5% CO2And culturing for 24-72 h in a 37 ℃ cell culture box, observing the expression of EGFP of the cells under a fluorescence inverted microscope, and screening fluorescent protein positive cells to obtain the cells expressing the KDR targeting chimeric antigen receptor NK-92.
And (3) testing the resistance under the condition of 150.0v and 5ms before the electricity is converted, wherein the resistance range is 0.040-0.060 omega.
And (3) adding KDR-CAR sequence vector 3 muL with the concentration of 600 ng/muL into the step (4), observing the expression of EGFP of the cell under a fluorescence inverted microscope, and screening cells with positive fluorescent protein by using fluorescence detection.
Experiment-evaluation of cell killing experiment Effect
(1) Mixing untransfected NK cells, KDR-CAR-NK cells and lung cancer cells (A549) according to the proportion of effector cells to target cells of 2:1 and 5:1, culturing for 72 hours, and detecting the expression of cytokines IFN-gamma and Granzyme B by using a flow cytometer;
(2) collecting target cells (A549) in a 15mL centrifuge tube, centrifuging at 1200rpm for 5min, and discarding the supernatant;
(3)1mL of complete medium resuspend the cells at 1X 10 per well5One was inoculated in the lower chamber of a transwell cell culture plate (Corning 0.4 μm cat # 3450);
(4) respectively mixing 2 × 105Each, 5 × 105The individual KDR-CAR-NK cells were seeded in the upper chamber at 5% CO2Culturing the cells in a 37 ℃ cell culture box for 72 hours and then using the cells in subsequent experiments;
(5) collecting KDR-CAR-NK cells in co-culture, adding 1mL PBS for resuspension, discarding supernatant, and repeating twice;
(6) adding 500 μ L paraformaldehyde for fixation for 30 min;
(7) adding 500 μ L of BSA cell disrupting agent, and standing for 1 h;
(8) adding an (IFN-gamma/Granzyme B antibody, incubating in a dark place, and detecting on a computer;
(9) evaluation of cell killing Effect
When the untransfected cells were incubated with the target cells, no secretion of the corresponding cytokine was detected; however, after the target cell is incubated with the target cell, secretion of Granzyme B and interferon r is detected to be increased significantly, and meanwhile, after the tumor cell is incubated with the target cell, apoptosis of the tumor cell is detected, fig. 5 and 6 are apoptosis graphs of flow-type detection tumor cell after co-incubation of the tumor cell in the examples, and fig. 7 and 8 are expression graphs of flow-type detection cytokines IFN-gamma and Granzyme B after co-incubation of the tumor cell in the examples, which shows that the KDR-CAR-NK target cell has a killing effect on the target cell and the tumor cell.
Finally, it should be noted that the above-described embodiments are described to facilitate understanding and use of the invention by those of ordinary skill in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Sequence listing
<110> university of Guangdong department of pharmacy
<120> preparation method and application of KDR-CAR-NK cell
<130> 2021.2.24
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 510
<212> DNA
<213> VEGF165 Gene sequence (VEGF165 gene sequence)
<400> 1
aactttctgc tgtcttgggt gcattggagc cttgccttgc tgctctacct ccaccatgcc 60
aagtggtccc aggctgcacc catggcagaa ggaggagggc agaatcatca cgaagtggtg 120
aagttcatgg atgtctatca gcgcagctac tgccatccaa tcgagaccct ggtggacatc 180
ttccaggagt accctgatga gatcgagtac atcttcaagc catcctgtgt gcccctgatg 240
cgatgcgggg gctgctgcaa tgacgagggc ctggagtgtg tgcccactga ggagtccaac 300
atcaccatgc agattatgcg gatcaaacct caccaaggcc agcacatagg agagatgagc 360
ttcctacagc acaacaaatg tgaatgcaga ccaaagaaag atagagcaag acaagaaaaa 420
aaatcagttc gaggaaaggg aaaggggcaa aaacgaaagc gcaagaaatc ccggtataag 480
tcctggagcg tatgtgacaa gccgaggcgg 510
<210> 2
<211> 135
<212> DNA
<213> CD8 alpha Gene sequence (CD8 alpha gene sequence)
<400> 2
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 3
<211> 204
<212> DNA
<213> CD28 Gene sequence (CD 8. alpha. gene sequence)
<400> 3
ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60
gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 120
aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 180
cgcgacttcg cagcctatcg ctcc 204
<210> 4
<211> 351
<212> DNA
<213> CD3 ζ Gene sequence (CD3 ζ gene sequence)
<400> 4
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac 180
aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 240
cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 300
acctacgacg cccttcacat gcaggccctg ccccctcgct aaggatcccc c 351
<210> 5
<211> 34
<212> DNA
<213> Primer-VEGF165-F
<400> 5
attgctagca agagagcacc catggcagaa ggag 34
<210> 6
<211> 33
<212> DNA
<213> Primer-VEGF165-R
<400> 6
gtactcgagc cgcctcggct tgtcacattt tta 33
<210> 7
<211> 34
<212> DNA
<213> Primer-KDR-CAR-F
<400> 7
attgctagca agagagcacc catggcagaa ggag 34
<210> 8
<211> 33
<212> DNA
<213> Primer-KDR-CAR-R
<400> 8
gtagaattcc tctagagcat gcttagcgag ggg 33
Claims (10)
1. A method for preparing KDR-CAR-NK cells, which is characterized by comprising the following steps:
s1, construction of KDR-CAR sequence: sequentially and serially splicing gene sequences of an intracellular signaling domain of CD8 alpha, a co-stimulatory molecule CD28 and a CD3 zeta to carry out whole-gene synthesis, reserving restriction enzyme sites Xhol I and BamH I at two ends, and connecting VEGF165 genes with corresponding enzyme sites by using T4 ligase to prepare a KDR-CAR sequence;
s2, constructing a KDR-CAR sequence vector: cloning the KDR-CAR sequence prepared in the step S1 to a lentiviral vector pcDNA3.1-EGFP to obtain a recombinant plasmid KDR-CAR sequence vector KDR-CAR/pcDNA3.1-EGFP;
s3, constructing KDR-CAR-NK cells: and (3) taking Opti-MEM as a transfection reagent, performing electroporation transfection on the KDR-CAR sequence vector KDR-CAR/pcDNA3.1-EGFP obtained in the step S2 to NK cells, and screening cells positive to fluorescent protein by using fluorescence detection to obtain the fluorescent protein-containing cell.
2. The preparation method according to claim 1, wherein the sequence information of the VEGF165 gene in step S1 is shown in SEQ ID No. 1.
3. The method according to claim 1, wherein the sequence of CD8 α in step S1 is represented by SEQ ID NO.2, the sequence of CD28 is represented by SEQ ID NO.3, and the sequence of CD3 ζ is represented by SEQ ID NO. 4.
4. The preparation method according to claim 1, wherein in step S1, the target gene VEGF165 fragment and KDR-CAR fragment are amplified by using PCR technique, the Primer for amplifying VEGF165 sequence is Primer-VEGF165, the sequence information of upstream Primer is shown in SEQ ID No.5, and the sequence information of downstream Primer is shown in SEQ ID No. 6; the Primer for amplifying the KDR-CAR is Primer-KDR-CAR, the sequence information of the upstream Primer is shown as SEQ ID NO.7, and the sequence information of the downstream Primer is shown as SEQ ID NO. 8.
5. The method of claim 1, wherein in step S2, the cohesive ends of the restriction sites are added to both ends of the KDR-CAR sequence and cloned into the lentiviral vector pcdna3.1-EGFP, the cohesive end at the front end is the restriction site of Nhe I enzyme, and the cohesive end at the end is the restriction site of BamH I enzyme.
6. The method for preparing the recombinant human NK cells according to claim 1, wherein the NK cells transfected by electroporation in the step S3 are NK-92 cells.
7. The method according to claim 1, wherein the step S3 of screening for cells positive for fluorescent protein by fluorescence detection comprises:
(1) collecting the transfected NK-92 cell suspension in a 15mL centrifuge tube, centrifuging at 1200rpm for 5min, and discarding the supernatant;
(2) adding 1mL of opti-MEM buffer solution to resuspend the cells, centrifuging to remove the supernatant, and repeating for 2-3 times;
(3) after adding 50. mu.L of opti-MEM buffer to resuspend the cells,counting to make NK-92 cells amount to 1 × 106A plurality of; then KDR-CAR sequence vector KDR-CAR/pcDNA3.1-EGFP with the concentration of 1 mug/mug is added, the amount of the vector in each hole is 1.8 mug, opti-MEM buffer solution is used for supplementing, the total volume is 100 mug, the vector is added into an electric rotating cup, and an electric rotating experiment is carried out after resistance is tested;
(4) after the electrotransfer is finished, the solution in the electrotransfer cup is transferred to a 6-hole plate, 1mL of complete culture medium is supplemented, and the 6-hole plate is placed in 5% CO2And culturing for 24-72 h in a 37 ℃ cell culture box, observing the expression of EGFP of the cells under a fluorescence inverted microscope, and screening fluorescent protein positive cells to obtain the cells expressing the KDR targeting chimeric antigen receptor NK-92.
8. The method according to claim 7, wherein the resistance is measured at 150.0v for 5ms before the electrotransfer in step (3), and the resistance is in the range of 0.040-0.060 Ω.
9. The method according to claim 6, wherein the KDR-CAR sequence vector is added to the sample in step (4) at a concentration of 600 ng/. mu.L and 3. mu.L is subjected to fluorescent inverted microscope to observe the expression of EGFP in the cells.
10. Use of KDR-expressing targeting antigen receptor-modified NK cells obtained by the preparation method according to any one of claims 1 to 9 for adoptive immunotherapy.
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