CN109097401B - Preparation method of recombinant vector CAR-CD 244-anti-SMA - Google Patents
Preparation method of recombinant vector CAR-CD 244-anti-SMA Download PDFInfo
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Abstract
The invention relates to a preparation method of a recombinant vector CAR-CD244-antiPSMA, which comprises the following steps: step 1, carrying out PCR amplification on a specific antibody encoding gene by using a primer with a BfuAI restriction enzyme recognition site at the tail end; the specific antibody coding gene sequence is a partial sequence of the PSMA antibody and is a sequence 3 in a sequence table; step 2, respectively purifying the PCR products obtained in the step 1; step 3, performing single enzyme digestion on the PCR product purified in the step 2 at a BfuAI restriction enzyme recognition site, and simultaneously performing single enzyme digestion on the cloning vector containing the modified CD244 skeleton at the BfuAI restriction enzyme recognition site; step 4, purifying the product after enzyme digestion; step 5, mixing the purified product with a cloning vector containing a modified CD244 framework according to the molar ratio of 3:1 of the antibody fragment, and then using T4 ligase to perform connection, transformation, plate coating, and picking single clone to extract plasmid sequencing; the correctly ligated plasmid was designated: the recombinant vector CAR-CD 244-antiPSMA.
Description
The application is a divisional application, the application number of the original application is 201710209997.5, the application date is 3 months and 31 days in 2017, and the invention name is 'CAR-NK cell and a preparation method and application thereof'.
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of a recombinant vector CAR-CD 244-antiPSMA.
Background
Cell biotherapy is a novel cancer treatment method using the autoimmune system, and is a treatment mode with significant efficacy. It uses molecular biology technology and cell engineering technology, uses biological agent to culture and expand immune cells collected from patient in vitro, then returns to patient, so as to excite and enhance body's own immune function, and achieve the purpose of monitoring and killing pathological cells or repairing damaged cells.
Natural killer cells (NK) belong to granular lymphocytes and are important immune cells of the body. NK cells are generally considered to be derived from bone marrow lymphoid stem cells, and the differentiation and development of the NK cells depend on bone marrow or thymus microenvironment, are mainly distributed in peripheral blood and spleen, and are also present in small amounts in lymph nodes and other tissues. NK cells do not express specific antigen recognition receptors and are a third class of lymphocytes distinct from T, B lymphocytes. Compared with other lymphocytes, NK cell killing activity is not MHC-restricted, does not require antigen pre-priming, and is therefore referred to as natural killing activity. NK cell cytoplasm is rich and contains large azurophilic granules, and the content of the azurophilic granules is in positive correlation with the killing activity of NK cells, so that the killing effect of the NK cells after acting on target cells appears early, and the killing effect can be seen in 1 hour in vitro and 4 hours in vivo. The target cells of the NK cells mainly comprise certain tumor cells (including partial cell lines), virus-infected cells and the like, so that the NK cells can directly kill certain tumor cells and virus-infected cells. This means that NK cells play an important role in the body's anti-tumor, anti-viral infection and immune regulation.
CD244 is a human protein encoded by the CD244 gene, also known as NK cell receptor 2B4, an important surface receptor for NK cells. NK cells activate cytotoxic functions via the CD244 receptor.
The CAR means a Chimeric Antigen Receptor (Chimeric Antigen Receptor), and when the CAR structure and the tumor-associated Antigen antibody are fused and expressed on the cell membrane surface of the NK cell, the CAR structure and the tumor-associated Antigen antibody can endow the NK cell with the ability of specifically recognizing the corresponding tumor cell, and the NK cell is the CAR-NK cell. An intact CAR structure typically comprises an upper membrane signal region, an antibody region (typically derived from an scFV fragment corresponding to a monoclonal antibody), an extracellular proofreading region, a transmembrane region, and an intracellular signal region. The genetically modified NK cell (CAR-NK) can generate a more specific cell killing effect on cells with the antigen to which the cell killing effect is directed, so that the effect of treating tumors and the like can be achieved.
The lentivirus vector is a virus vector derived from human immunodeficiency virus-1 (HIV-l), can effectively integrate exogenous genes onto a host chromosome so as to achieve the effect of persistently expressing a target sequence, and is one of common vector forms of the exogenous genes. The basic process is that the slow virus vector carrying exogenous gene is virus packed into infectious virus particle with the help of slow virus packing plasmid and cell line, and the exogenous gene is expressed in cell or living tissue through infecting cell or living tissue. The lentivirus vector can effectively infect various cells such as neuron cells, tumor cells, stem cells, myocardial cells and the like in the aspect of infection capacity. The slow virus vector can conveniently and quickly realize the long-term and stable expression of the target gene.
At present, an effective CAR-NK cell preparation method and an application precedent in tumor immunotherapy are not established.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a CAR-NK cell and a preparation method and application thereof.
The purpose of the invention and the technical problem to be solved are: the sequence of the human CD244 is modified, so that the specific protein can be expressed on the surface of cells such as NK (natural killer), and the specific protein can identify specific tumor cells and activate the NK cells, so that the specific tumor cells can be killed. The lentivirus vector containing the modified CD244 sequence can coat lentivirus, and the lentivirus can enable NK cells to express the modified CD244 protein sequence after the NK cells are infected by the lentivirus.
In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:
the invention provides a preparation method of CAR-NK cells, which comprises the following steps:
sl, linking a gene (comprising a light chain part of the specific antibody, a connecting part of the heavy chain and the light chain and a heavy chain part) encoding the specific antibody (such as a specific antibody CD19 antibody or a PSMA antibody of a target antigen of a tumor cell) to a cloning site region of the modified CAR skeleton to obtain the CAR gene,
the engineered CAR scaffold comprises sequentially linked a CD244 upper membrane signal region, a cloning site region, a CD244 extracellular hinge region, a CD244 transmembrane region, and a CD244 intracellular region,
s2, infecting the CAR gene into NK cells through lentivirus to obtain the NK cells (such as NK92MI cells) expressing the CAR protein (namely the expression protein of the CAR gene, such as the CAR-CD244-anti CD19 protein structure and the CAR-CD244-anti PSMA protein structure in the example 4), namely the CAR-NK cells.
In the CAR-NK cell preparation method, the DNA sequence of the membrane signal region on CD244 is shown as 1 st to 63 rd in sequence 1 of the sequence table;
and/or the DNA sequence of the cloning site region is shown as 64 th to 95 th in the sequence 1 of the sequence table;
and/or the DNA sequences of the CD244 extracellular hinge region, the CD244 transmembrane region and the CD244 intracellular region are shown as 96 th to 560 th in the sequence 1 of the sequence table.
In the preparation method of the CAR-NK cell, the DNA sequence of the modified CAR skeleton is shown as sequence 1 in a sequence table.
In the above method for preparing CAR-NK cells, the step S2 is performed according to a method comprising the steps of:
s21, connecting the CAR gene to a cloning site of a lentiviral vector to obtain a recombinant lentiviral vector A, and transfecting a target cell (such as 293T cell) with the recombinant lentiviral vector A to culture to obtain lentivirus;
s22, infecting the NK cells with lentiviruses to obtain the CAR-NK cells.
In the above CAR-NK cell production method, in step S21, the lentiviral vector is a Plenti lentiviral vector;
the transfection is performed using a lentiviral packaging plasmid;
the slow virus packaging plasmid is specifically psPAX2 and PMD2.G, and when in transfection, the slow virus vector, the psPAX2 and the PMD2.G are transfected into 3 xI 0 by the mass ratio of 5ug to 3.2ug to l.8ug to 7.5ug to 4.8ug to 2.7ug6(ii) individual cells of interest;
in step S22, the infecting the NK cells with the lentivirus includes a step of adding the concentrated lentivirus to a complete medium and mixing with the NK cells.
In the above CAR-NK cell production method, in step S22, the step of infecting NK cells with lentivirus further comprises a step of adding polybrene; the final concentration of the polybrene is 4 ug/mL;
the concentrated lentivirus is obtained by culturing target cells transfected by the recombinant lentivirus vector A, centrifuging the cultured supernatant for 1 hour under the condition of 20000g, and suspending the lentivirus by using a DPBS buffer solution.
The invention also provides a DNA molecule which is any one of the following (1) or (2):
(1) comprises a CD244 upper membrane signal area, a cloning site area, a CD244 extracellular hinge area, a CD244 transmembrane part area and a CD244 intracellular part area which are connected in sequence;
(2) is a DNA molecule formed by inserting a gene encoding a specific antibody (e.g., a specific antibody against a target antigen of a tumor cell) into the cloning site region of the DNA molecule of (1).
In the DNA molecule, the DNA sequence of the membrane signal region on the CD244 is shown as 1 st to 63 rd in the sequence 1 in the sequence table;
and/or the DNA sequence of the cloning site region is shown as 64 th to 95 th in the sequence 1 of the sequence table;
and/or the DNA sequences of the CD244 extracellular hinge region, the CD244 transmembrane region and the CD244 intracellular region are shown as 96 th to 560 th in the sequence 1 of the sequence table;
the sequence of the DNA molecule in (1) can be specifically a sequence shown in a sequence 1 in a sequence table.
The invention protects recombinant vectors (such as recombinant expression vectors, recombinant lentiviral vectors), recombinant cells (such as immune cells NK cells or T cells, or 293T cells) or recombinant cell lines containing the DNA molecules.
The invention protects the DNA molecule, the recombinant vector, the recombinant cell or the recombinant cell line, and the application of the CAR-NK cell prepared by any one of the methods in preparing a medicament (such as a tumor immunotherapy cell), a reagent or a kit for tumor therapy (such as tumor growth inhibition or tumor cell killing).
The prepared tumor treatment medicine, reagent or kit contains the following liquid: with a concentration of 5X 106The CAR-NK cells/100 ul normal saline.
The invention has the following beneficial effects:
the cell material for constructing the novel tumor immunotherapy cell is a natural killer cell (NK cell), the NK cell has low immunogenicity, variant reinfusion can be performed, and the cell source problem is solved compared with a T cell;
the tumor vaccine prepared by the invention can induce the target cell with antigen specificity to be cracked;
the engineered CAR scaffolds of the invention are equally applicable to other immune cells; the NK cell modified by the preparation method can generate a cell killing effect with stronger specificity on cells with the targeted antigen, such as tumor cells, so that the treatment effects of tumors and the like can be achieved.
The tumor immunotherapy cell prepared by the invention can be applied to cancer patients and also can be applied to foreign body reinfusion. Therefore, compared with the existing CAR-T, the tumor immunotherapy cell prepared by the invention has wider application range and lower preparation cost.
Drawings
The invention has the following drawings:
FIG. 1 is a schematic representation of an engineered CD244 scaffold linked to specific antibody encoding genes.
FIG. 2 is a schematic diagram of the recombinant lentiviral vector Plenti-CAR-CD244-anti CD 19.
FIG. 3 is an expression diagram of CD244-anti CD19 and CD244-anti SMA in NK92MI cells.
FIG. 4 shows the results of detecting the expression of CAR structure in NK cells by flow cytometry, wherein the ordinate represents the number of counts and the abscissa represents the green fluorescence intensity (log value) of CD19-FITC (GRN-Hlog).
FIG. 5 shows the killing experiment of NK92MI-CD244-anti CD19 on Daudi tumor cells.
FIG. 6 shows the killing experiment of NK92MI-CD244-antipSMA on PC3 tumor cells.
FIG. 7 shows IFN γ interferon levels measured after 20h incubation of Daudi tumor cells with NK92MI-CD244-anti CD 19.
FIG. 8 shows the IFN gamma interferon level detection after 20h incubation of the PC3 tumor cells with NK92MI-CD 244-antipSMA.
FIG. 9 shows the results of tumor growth after in vivo fluorescence imaging of mice.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
Example 1 preparation of recombinant vector CAR-CD244-anti CD19 and recombinant vector CAR-CD244-anti SMA
1. Carrying out PCR amplification on the specific antibody encoding gene by using a primer with a BfuAI restriction enzyme recognition site at the tail end;
the specific antibody coding gene sequences used in this example are a partial sequence (sequence 2 in the sequence table) of a CD19 antibody (anti CD19) and a partial sequence (sequence 3 in the sequence table) of a PSMA antibody (anti sma);
2. respectively purifying the PCR products obtained in the step 1;
3. performing single enzyme digestion (BfuAI) on the PCR product purified in the step 2, and simultaneously performing single enzyme digestion (BfuA I) on the cloning vector containing the modified CD244 skeleton;
4. purifying the product after enzyme digestion;
5. mixing the purified product with a cloning vector containing a modified CD244 framework according to the molar ratio of the antibody fragment to 3:1, and then performing connection, transformation, plate coating, monoclonal extraction and plasmid sequencing by using T4 ligase; the correctly ligated plasmids (i.e., anti CD19 or anti SMA ligated into the cloning site region of the engineered CD244 scaffold) were designated: recombinant vector CAR-CD244-anti CD19 and recombinant vector CAR-CD244-anti SMA.
The modified CD244 framework sequence is shown as sequence 1 in a sequence table, wherein the sequences shown from 1 st to 63 rd in the sequence 1 in the sequence table are DNA sequences of a membrane signal region on CD 244; the sequence shown from the 64 th site to the 95 th site in the sequence 1 in the sequence table is a DNA sequence of a cloning site region; the DNA sequences shown from the 96 th site to the 560 th site in the sequence 1 in the sequence table are a CD244 extracellular hinge region, a CD244 transmembrane region and a CD244 intracellular region (the extracellular hinge region is contained in the transmembrane region and the intracellular region); sequences from 561 th to 575 th in a sequence 1 in a sequence table are marked by DDDDDDK-tag; the sequence shown from 576 th to 578 th in the sequence 1 of the sequence table is a stop codon.
A schematic representation of the engineered CD244 scaffold linked to specific antibody encoding genes is shown in FIG. 1: in FIG. 1, CD244leader represents the membrane signal region, V, on CD244LRepresents the light chain part of the specific antibody, GSlinker represents the connection part of the heavy chain and the light chain of the specific antibody, VHRepresents the heavy chain portion of a specific antibody, CD244 represents the extracellular hinge region, transmembrane region and intracellular partition of CD244, and DDDDK represents a DDDDK-tag label.
Example 2 preparation of recombinant Lentiviral vectors
1. Respectively using the recombinant vector CAR-CD244-anti CD19 and the recombinant vector CAR-CD244-anti SMA prepared in the example 1 as templates, and carrying out PCR amplification by using primers with BamHI and XbaI restriction enzyme cutting site sequences;
2. respectively purifying, enzyme cutting (BamHI and Xba I) and purifying the PCR product;
3. the products of step 2 were separately ligated into the Plenti lentiviral vector (named pLentiCMV/TO eGFP Puro (w159-1) between BamHI and XbaI sites, respectively, from addge, website: http:// www.addgene.org/17481/) using T4 ligase;
4. transforming, plating, picking single clone, extracting plasmid, sequencing, and respectively naming the recombinant lentivirus vector with correct sequencing as a recombinant lentivirus vector Plenti-CAR-CD244-anti CD19 and a recombinant lentivirus vector Plenti-CAR-CD244-anti SMA.
A schematic diagram of the recombinant lentiviral vector Plenti-CAR-CD244-anti CD19 is shown in FIG. 2, wherein CMV-promoter represents CMV promoter, CD244peptide is membrane signal region on CD244, anti CD19 is CD19 antibody, CD244 is CD244 extracellular hinge region, transmembrane region and intracellular region, DDDDK is DDDDK-tag marker, and WPRE is a region with function similar to 3' UTR of polyA.
Example 3 preparation and concentration of lentivirus
1. Preparation of lentiviruses
293T cells (approx. 3X 10. sup. rd.) were transfected with the recombinant lentiviral vector prepared in example 2, Plunti-CAR-CD 244-anti CD19, and psPAX2 (purchased from addge, website https:// www.addgene.org/12260/), pMD2.G (purchased from addge, website https:// www.addgene.org/12259/), at a mass ratio of 5ug:3.2ug:1.8ug6293T cells were plated on a 10cm dish), and after 8 hours, the cells were replaced with a fresh medium (DMEM medium supplemented with 10% fetal calf serum), and then the supernatant was collected every 24 hours and added with a fresh medium (DMEM medium supplemented with 10% fetal calf serum), and collected 3 times in total to obtain a lentivirus supernatant A.
293T cells (about 3X 10. multidot. TM.) were transfected with recombinant lentiviral vector Plunti-CAR-CD 244-antipSMA prepared in example 2 and psPAX2, pMD2.G vector at a mass ratio of 5ug:3.2ug:1.8ug6293T cells were plated on a 10cm dish), and after 8 hours, the cells were replaced with a fresh medium (DMEM medium supplemented with 10% fetal bovine serum), and then the supernatant was collected every 24 hours and added with a fresh medium (DMEM medium supplemented with 10% fetal bovine serum), and collected 3 times in total, to obtain a lentivirus supernatant B.
2. Concentration of lentiviruses
Respectively loading the lentivirus supernatants A and B obtained in the step 1 into a high-speed centrifuge tube according to 30 ml/tube, centrifuging by using a high-speed centrifuge for 1 hour at 20000g, collecting the lentiviruses on the tube wall, suspending by using 100ul of DPBS buffer solution, respectively obtaining concentrated lentiviruses A and B, and storing at-80 ℃.
Example 4 preparation of CAR-NK cells by concentrated lentivirus-infected NK cells and detection
1. Concentrated lentivirus infected NK cells
The concentrated lentiviruses A and B prepared in example 3 were added to 1ml of complete medium, and 10 ml of complete medium, respectively5NK92MI cells (purchased from ATCC, website: https:// www.atcc.org /) were mixed (polybrene with a final concentration of 4ug/ml was added) to obtain recombinant cells NK92MI-CD244-anti CD19 and recombinant cells NK92MI-CD244-anti SMA (the recombinant cells obtained here were mixed cells, wherein CAR-NK cells refer to recombinant cells NK92MI-CD244-anti CD19 and NK92MI-CD244-anti SMA of NK92 MI).
2. Detection of expression of CAR Structure in NK cells
The recombinant cells NK92MI-CD244-anti CD19 obtained in the step 1 and the cells of the 293T transfection vector Plenti-CAR-CD244-anti CD19 obtained in the example 3 are subjected to Western blot to detect the expression condition of the surface antibody: western blot detection was performed after incubation of cells transfected with the Plenti empty vector from NK92MI and cells transfected with the Plenti empty vector from NK92MI and recombinant cells NK92MI-CD244-anti CD19, respectively, using CD19-FITC antigen as a control, and the results are shown in FIG. 3.
The results in FIG. 3 show that NK92MI-CD244-anti CD19 and 293T transfected vector Plenti-CAR-CD244-anti CD19 cells were able to specifically express CAR-CD244-anti CD19 protein structure.
Expression detection of cells of the recombinant cell NK92MI-CD244-antiPSMA and the 293T transfection vector Plenti-CAR-CD244-antiPSMA obtained in example 3 was performed according to the method, and as a result, cells of the NK92MI-CD244-antiPSMA and the 293T transfection vector Plenti-CAR-CD244-antiPSMA can specifically express a CAR-CD244-antiPSMA protein structure.
3. Flow cytometry detection of CAR-NK protein Structure expression in CAR-NK cells
The specific operation is carried out according to the instructions of the flow-type antigen marker, and specifically comprises the following steps:
taking about 10 cells to be detected (recombinant cells NK92MI-CD244-anti CD19 in step 2)5Centrifuging individual cells and removing supernatant;
suspending cells to be detected by using 200ul DPBS, adding a flow type antigen marker, gently mixing, incubating for 30 minutes in a shaking table (100 revolutions) at 37 ℃, and centrifuging to remove a supernatant;
suspending the cells by using 500ul of complete culture medium, culturing for 5 minutes in an incubator at 37 ℃, centrifuging and removing supernatant;
after resuspending the cells with 200ul DPBS, the cells were tested on the machine.
As shown in figure 4, the flow cytometry is used for detecting the expression of the CAR protein structure on the cell surface of the recombinant cell NK92MI-CD244-anti CD19, the darker solid line gray part without edges is a control without any modified NK92MI cells, the lighter solid line gray part with edges is a CD19-FITC antigen incubated by the recombinant cell NK92MI-CD244-anti CD19, and the displacement indicates that the CD19 antibody is expressed on the cell membrane surface of the recombinant cell NK92MI-CD244-anti CD 19; the borderline is the cell count corresponding to the position of the fluorescence signal on the abscissa.
Example 5 killing experiment of CAR-NK cells on tumor cells
Get 104NK92MI-CD244-anti CD19 cells and 104Individual B-lymphocytoma (Daudi) cells were incubated at 1:1, with NK92MI as a control;
5,000 NK92MI-CD244-anti CD1 cells and 104Daudi was incubated with NK92MI as control, 0.5: 1;
2,500 NK92MI-CD244-anti CD19 cells and 104Daudi was incubated with NK92MI as control and scored as 0.25: 1;
1,250 NK92MI-CD244-anti CD19 cells and 104Daudi was incubated with NK92MI as control, 0.125: 1;
NK92MI-CD244-antipSMA was incubated with prostate cancer (PC3) cells, in the proportions described above, using NK92MI as a control.
Three sets of parallel controls were set up and cell viability was measured (general procedure) using the CellTox-Green (promega G8471) kit after 20 hours incubation and charted.
The results are shown in FIGS. 5 and 6, in which the diamond-shaped points represent the degree of tumor cell lysis under the condition of unmodified NK92MI cells; the square point is the tumor lysis degree under the condition of NK92MI cells modified by the method of example 4 (NK92MI-anti CD19 represents NK92MI-CD244-anti CD19, and NK92MI-anti SMA represents NK92MI-CD244-anti SMA). The results demonstrate that recombinant CD 244-modified NK cells, i.e. CAR-NK recombinant cells, kill tumor cells better than unmodified NK cells.
Example 6 detection of IFN-. gamma.Interferon levels after 20h incubation of Daudi, PC3 cells with CAR-NK cells
Get 105Individual NK92MI-CD244-anti CD19 and 105The Daudi cells were incubated in 500ul of medium for 20h (denoted NK92MI-anti CD19+ Daudi); unmodified NK92MI cells were used as control (denoted NK92MI + Daudi);
get 105NK92MI-CD244-antipSMA cell and 105The PC3 cells are incubated in 500ul of culture medium for 20h (marked as NK92MI-anti PSMA + PC 3); unmodified NK92MI cells were used as control (denoted NK92MI + PC 3);
centrifuging the incubated cells, taking the supernatant, and detecting the IFN gamma interferon level according to the specific method according to the kit use instructions (different kit methods are slightly different).
The results are shown in fig. 7 and fig. 8, wherein NK92MI is the result of incubation of unmodified NK92MI cells alone, NK92MI-anti CD19 is the result of incubation of NK92MI-CD244-anti CD19 alone, and NK92MI-anti sma is the result of incubation of NK92MI-CD244-anti sma alone.
The results show that: the recombinant CD244 modified NK cells namely CAR-NK cell NK92MI-CD244-anti CD19 and NK92MI-CD244-anti SMA can release more IFN gamma after incubation of corresponding tumor cells, and therefore the CAR-NK activity is higher than that of unmodified common NK cells, and the killing effect on the tumor cells is higher.
Example 7 in vivo tumor model experiment in Experimental animals
A total of 9 healthy Nude mice (Nude Mouse) were randomly divided into 3 groups, 3 mice per group, and 3 groups were a placebo group, an NK cell control group, and a CAR-NK cell group. All mice were injected with 5X 10 injections near the chest axilla6One PC3 tumor cell/cell with GFP fluorescent protein marker and fed normally for 14 days. After 14 days, all groups of mice were subjected to in vivo fluorescence imaging and tumor growth in the mice was recorded. After fluorescence imaging, injection points for each group of mouse PC3 tumor cells were performed separatelyThe following administrations were carried out:
injecting 100ul of physiological saline into mice in a blank control group;
NK cell control group mice were injected with 100ul of a common NK92MI cell suspension in a total of 5X 106NK cells/100 ul physiological saline;
mice of CAR-NK cell group were injected with CAR-NK (NK92MI-CD244-AntiPSMA) cell suspension (concentration 5X 10) prepared in example 4 for PC3 tumor cells6Individual CAR-NK (NK92MI-CD244-antiPSMA) cells/100 ul saline).
After injection and 7 days after normal feeding, all groups of mice were subjected to in vivo fluorescence imaging, and the tumor status in the mice was recorded.
The results are shown in FIG. 9, where the left, middle and right three columns in FIG. 9 are: the results of the blank control group, the NK cell control group and the CAR-NK cell group after the injection of the first behavior and the results of the second behavior after the normal feeding for 7 days show that the average tumor volume in the CAR-NK cell group mice is obviously smaller and the tumor growth is obviously inhibited compared with the blank control group and the NK cell control group in the figure 9 result.
Those not described in detail in this specification are within the skill of the art.
<110> Beijing Zhinuo medical science and technology Co Ltd
<120> preparation method of recombinant vector CAR-CD244-antiPSMA
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 578
<212> DNA
<213> Artificial sequence
<400> 1
atgctggggc aagtggtcac cctcatactc ctcctgctcc tcaaggtgta tcagggcaaa 60
ggattttgca ggtaaatttc ccgggacctg caaaacagga ctgtcagaat gcccatcagg 120
aattcagatt ttggccgttt ttggtgatca tcgtgattct aagcgcactg ttccttggca 180
cccttgcctg cttctgtgtg tggaggagaa agaggaagga gaagcagtca gagaccagtc 240
ccaaggaatt tttgacaatt tacgaagatg tcaaggatct gaaaaccagg agaaatcacg 300
agcaggagca gacttttcct ggagggggga gcaccatcta ctctatgatc cagtcccagt 360
cttctgctcc cacgtcacaa gaaccagcat atacattata ttcattaatt cagccttcca 420
ggaagtctgg atccaggaag aggaaccaca gcccttcctt caatagcact atctatgaag 480
tgattggaaa gagtcaacct aaagcccaga accctgctcg attgagccgc aaagagctgg 540
agaactttga tgtttattcc gatgacgatg acaagtga 578
<210> 2
<211> 774
<212> DNA
<213> Artificial sequence
<400> 2
gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60
acatgcactg tctcaggggt gtcattaccc gactatggtg taagctggat tcgccagcct 120
ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 180
tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 240
aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 300
tacggtggta gctatgctat ggactactgg ggtcaaggaa cctcagtcac cgtctcctca 360
ggtggtggtg gttccggtgg tggtggttcc ggtggtggtg gttccgacat ccagatgaca 420
cagactacat cctccctgtc tgcctctctg ggagacagag tcaccatcag ttgcagggca 480
agtcaggaca ttagtaaata tttaaattgg tatcagcaga aaccagatgg aactgttaaa 540
ctcctgatct accatacatc aagattacac tcaggagtcc catcaaggtt cagtggcagt 600
gggtctggaa cagattattc tctcaccatt agcaacctgg agcaagaaga tattgccact 660
tacttttgcc aacagggtaa tacgcttccg tacacgttcg gaggggggac taagttggaa 720
ataacacggg ctgatgctgc accaactgta tccatcttcc caccatccag taat 774
<210> 3
<211> 834
<212> DNA
<213> Artificial sequence
<400> 3
agtcctgccc agttcctgtt tctgttagtg ctctggattc aggaaaccaa cggtgatgtt 60
gtaatgaccc agactccact cactttgtcg gttaccattg gacaaccagc ctctatctct 120
tgcaagtcaa gtcagagcct cttatatagt aatggaaaaa cctatttgaa ttggttatta 180
cagaggccag gccagtctcc aaagcgccta atctatctgg tgtctaaact ggactctgga 240
gtccctgaca ggttcactgg cagtggatca ggaacagatt ttacactgaa aatcagcaga 300
gtggaggctg aggatttggg agtttattac tgcgtgcaag gtacacattt tcctcacacg 360
ttcggagggg ggaccaagct ggaaataaaa cggggtggct caggatcggg tggctctggc 420
tctggtggct caggatcgaa cttcgggctc agcttgattt tccttgtcct tgttttaaaa 480
ggtgtccagt gtgaagtgaa ggtggtggag tctgggggag gcttagtgaa gcctggagcg 540
tctctgaaac tctcctgtgc agcctctgga ttcactttca gtaactatgg catgtcttgg 600
gttcgccaga cttcagacaa gaggctggag tgggtcgcat ccattagtag tggtggtgat 660
agcaccttct atgcagacaa tgtaaagggc cgattcacca tctccagaga gaatgccaag 720
aacaccctgt acctgcaaat gagtagtctg aagtctgagg acacggcctt gtattactgt 780
gcaagagacg atctatttaa ctggggccaa ggcaccactc tcacagtctc atca 834
Claims (1)
1. A preparation method of a recombinant vector CAR-CD244-antipSMA is characterized by comprising the following steps:
step 1, carrying out PCR amplification on a specific antibody encoding gene by using a primer with a BfuAI restriction enzyme recognition site at the tail end;
the specific antibody coding gene sequence is sequence 3 in the sequence table;
step 2, purifying the PCR product obtained in the step 1;
step 3, performing single enzyme digestion on the PCR product purified in the step 2 at a BfuAI restriction enzyme recognition site, and simultaneously performing single enzyme digestion on the cloning vector containing the modified CD244 skeleton at the BfuAI restriction enzyme recognition site;
step 4, purifying the product after enzyme digestion;
step 5, mixing the purified product with a cloning vector containing a modified CD244 framework according to the molar ratio of 3:1 of the antibody fragment, and then using T4 ligase to perform connection, transformation, plate coating, and picking single clone to extract plasmid sequencing; the cloning site region of the modified CD244 skeleton is connected with antiPSMA, and the obtained plasmid is named as: the recombinant vector CAR-CD244-anti PSMA;
the modified CD244 framework sequence is shown as sequence 1 in a sequence table, wherein the sequences shown from 1 st to 63 rd in the sequence 1 in the sequence table are DNA sequences of a membrane signal region on CD 244; the sequence shown from the 64 th site to the 95 th site in the sequence 1 in the sequence table is a DNA sequence of a cloning site region; the DNA sequences shown from the 96 th site to the 560 th site in the sequence 1 in the sequence table are a CD244 extracellular hinge region, a CD244 transmembrane region and a CD244 intracellular region; sequences from 561 th to 575 th in a sequence 1 in a sequence table are marked by DDDDDDK-tag; the sequence shown from 576 th to 578 th in the sequence 1 of the sequence table is a stop codon.
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| CN107267463A (en) * | 2017-08-23 | 2017-10-20 | 安徽惠恩生物科技股份有限公司 | A kind of Car NK cell preparation methods for breast cancer |
| CN107759701B (en) * | 2017-10-27 | 2021-07-02 | 杭州优善生物科技有限公司 | Chimeric antigen receptor, NK cell modified by chimeric antigen receptor, coding DNA, mRNA, expression vector, preparation method and application |
| CN109810995B (en) | 2017-12-06 | 2020-10-02 | 阿思科力(苏州)生物科技有限公司 | Nucleotide sequence for coding CAR, ROBO1CAR-NK cell for expressing CAR and preparation and application thereof |
| CN108220294A (en) | 2018-02-11 | 2018-06-29 | 南京医科大学 | CRISPR/Cas9 carriers combine and its application in gene knockout |
| CN109136192A (en) * | 2018-09-20 | 2019-01-04 | 北京呈诺医学科技有限公司 | A kind of preparation method of iCAR-NK cell |
| CN109762844A (en) * | 2019-01-31 | 2019-05-17 | 北京呈诺医学科技有限公司 | A kind of CAR-NK cell preparation method of target mesothelin |
| CN109825528A (en) * | 2019-02-21 | 2019-05-31 | 北京呈诺医学科技有限公司 | A kind of CAR-NK cell preparation method for Huppert's disease |
| CN113461830B (en) * | 2021-07-22 | 2022-04-26 | 徐州医科大学 | Umbilical cord blood-derived CD 19-targeted CAR-NK cell and preparation method thereof |
| CN114835820B (en) * | 2022-04-14 | 2023-01-06 | 呈诺再生医学科技(珠海横琴新区)有限公司 | Chimeric Fc receptor for genetically modified pluripotent stem cells and natural killer cells |
| CN117384858B (en) * | 2023-12-12 | 2024-03-08 | 山东丽山生物科技有限公司 | Preparation method and application of chimeric antigen receptor T cells |
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