CN111172146B - Nanoscale artificial antigen presenting cell alpha CD3-Origami aAPC, preparation method and application thereof - Google Patents
Nanoscale artificial antigen presenting cell alpha CD3-Origami aAPC, preparation method and application thereof Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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Abstract
The invention discloses a nano-scale artificial antigen presenting cell alpha CD3-Origami aAPC based on nucleic acid framework structure and a preparation method thereof, which are characterized in that M13mp18 single-chain phage DNA, single-chain DNA forming the nucleic acid framework structure and biotin modified single-chain DNA are mixed, and gradient cooling is carried out at 95-25 ℃ by a gradient PCR instrument to obtain a biotinylated nucleic acid framework structure; and sequentially adding streptavidin and biotinylated anti-CD 3 protein, and performing oscillation reaction at room temperature to obtain the nano-scale artificial antigen presenting cell (alpha CD3-Origami aAPC) based on the nucleic acid framework structure. The preparation method is simple, few in synthesis steps and short in time consumption. The alpha CD3-Origami aAPC provided by the invention is used for T cell activation, has good material biocompatibility, realizes rapid and effective proliferation of T cells, and has potential application value.
Description
Technical Field
The invention belongs to the field of immunotherapy, relates to an immune preparation and a preparation method thereof, and in particular relates to a nano-scale artificial antigen presenting cell (alpha CD3-Origami aAPC) based on a nucleic acid framework structure capable of promoting T cell activation and proliferation, and a preparation method and application thereof.
Background
Adoptive immunotherapy (Adoptive Cell Transfer Therapy, ACT) refers to the isolation of immunocompetent cells from a tumor patient, in vitro amplification and functional identification, and then reinfusion into the patient, thereby achieving the purpose of directly killing the tumor or stimulating the immune response of the body to kill the tumor cells. The key to this approach is the generation of large numbers of functional T cells to improve the immune response of the patient. Therefore, it is particularly important to develop culture platforms for T cell activation and proliferation in vitro. In vivo, T cell activation is initiated by recognition of the surface homologous polypeptide histocompatibility complex (pMHC) of Antigen Presenting Cells (APCs) by the T Cell Receptor (TCR). This interaction triggers a complex intracellular signaling network, ultimately leading to cytokine production (e.g., IL-2), cell proliferation, differentiation into different subsets. The autologous APCs are used as in-vitro stimulation platforms of T cells, and are difficult to realize in practical application due to large difference of proper cell quantity and uniformity.
Artificial antigen presenting cells (aapcs) have become a more easily produced, economically viable system than natural, and a number of cell-and non-cell-based systems have been designed as alternatives to APCs. Agonistic antibodies to CD3 (the signaling component of the TCR complex) and CD28 are often mimics of the natural ligands of the T cell activation and proliferation system. Wherein anti-CD 3 monoclonal antibodies (aCD 3 mAbs) trigger efficient and stable activation of polyclonal T cells.
Nucleic acid nanotechnology is a top-down molecular self-assembly mode, nucleic acid is used as a material unit for constructing a structure, a stable structure is spontaneously formed based on physical and chemical properties of nucleic acid molecules, a base complementary pairing principle is followed, nano-scale complex precise control is realized through reasonably designing a base chain, accurate arrangement of protein molecules can be further realized, the number of objects capable of being arranged is large, and the achievable resolution is high.
Although much research on artificial APC systems has focused on the type of molecular signaling required to induce or expand a particular T cell phenotype, little has been focused on achieving precise control of surface ligand density. The density and number of TCR ligands has a large impact on the overall signal intensity of T cells.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to solve the problem that the ligand density of the artificial antigen presenting cells is difficult to accurately control, and realize the accurate control of the number and the relative position of ligand molecules on a nucleic acid framework structure; the nucleic acid framework structure with good biocompatibility is selected as a raw material, and the prepared nano-scale artificial antigen presenting cell (alpha CD 3-origin aaPC) based on the nucleic acid framework structure has good biocompatibility, and can effectively and rapidly promote the activation and proliferation of T cells.
The reaction mechanism of the method for preparing the nano-scale artificial antigen presenting cells (alpha CD3-Origami aAPC) based on the nucleic acid framework structure, which can promote the activation and proliferation of T cells, is shown in figure 1:
the method comprises the steps of taking long M13mp18 single-stranded phage DNA as a bracket, taking short single-stranded DNA as a staple chain, selecting short single-stranded DNA with a certain number and position (namely the position of the single-stranded DNA in the whole nucleic acid framework structure) for biotinylation modification through reasonable design according to the addressability of the nucleic acid framework structure, and forming a biotinylation nucleic acid framework structure with a certain number and position after PCR annealing treatment, wherein the number of modified biotin on the nucleic acid framework is determined by the number of selected short-chain DNA, and the position of the modified biotin on the nucleic acid framework corresponds to the position of a nucleic acid framework structure design diagram.
The streptavidin is bound to the position modified with biotinylated DNA through a streptavidin-biotin system, and the biotinylated anti-CD 3 protein is bound to a nucleic acid framework structure in a fixed point manner by taking the streptavidin as a medium, namely, the streptavidin is bound to the position selected by design of the biotinylated DNA, so that the nano-scale artificial antigen presenting cell (alpha CD 3-origamiaAPC) based on the nucleic acid framework structure is finally formed.
The invention utilizes the addressability of nucleic acid framework structure to realize the accurate positioning of alpha CD3 protein molecules, realizes the preparation of nanometer artificial antigen presenting cells, and the number and the interval of biotin on the product can be accurately controlled in advance. However, commercial αcd3 magnetic beads can only realize different modification densities to a certain extent by the proportion of the magnetic beads to the αcd3 protein molecules, but are inaccurate and cannot realize nano-scale controllability.
According to the principle, the invention adopts the following technical scheme:
the invention provides a preparation method of a nano-scale artificial antigen presenting cell (alpha CD3-Origami aAPC) based on a nucleic acid framework structure, which comprises the following steps:
(1) Mixing M13mp18 single-stranded phage DNA, single-stranded DNA forming a nucleic acid framework structure and biotin-modified short single-stranded DNA in a buffer system, and performing gradient cooling to obtain a biotinylated nucleic acid framework structure;
(2) And (3) after ultrafiltration purification of the biotinylated nucleic acid framework structure in the step (1), sequentially adding streptavidin and biotinylated anti-CD 3 protein, and carrying out oscillation reaction at room temperature to obtain the nano-scale artificial antigen presenting cell (alpha CD3-origami APC) based on the nucleic acid framework structure.
Wherein in the step (1),
the molar concentration ratio of the M13mp18 single-stranded DNA, the single-stranded DNA composing the nucleic acid framework structure and the biotin-modified single-stranded DNA is 1: (5-10): (5-10); preferably, it is 1:5:5.
the buffer system is 1xTAE-Mg 2+ The concentration of magnesium ions in the buffer system is 12.5mmol/L. The gradient cooling was from 95 ℃ to 25 ℃ at 0.1 ℃ per second.
The M13mp18 single-stranded DNA refers to single-stranded circular viral DNA derived from M13mp 18. M13mp18 phage in Escherichia coli ER2738 propagation, phage through polyethylene glycol precipitation purification, and then with phenol extraction of single-stranded DNA obtained, the M13mp18 single-stranded DNA has been commercialized, often as nucleic acid frame structure scaffold chain. In a specific embodiment, the M13mp18 single stranded DNA is 7249 bases long.
The short single-stranded DNA serves as a "staple chain" of the nucleic acid framework structure, and M13mp18 can be folded and assembled into a square, triangle, cross shape or the like.
The "staple chain" required for the selection of the biotin-modified single-stranded DNA is biotinylated at the 5' end. In the present invention, the number of biotin-modified single-stranded DNA may be, but not limited to, 1 to 20, and in theory, any number and position of single-stranded DNA may be selected for biotinylation modification, but since the size of the nucleic acid framework structure is limited, the number of streptavidin that can be bound due to space problems is limited by the size of the nucleic acid framework structure.
The synthesis of the nucleic acid framework structure according to the invention is based on DNA paper folding, i.e.a long single-stranded DNA molecule (scaffold strand) extracted from M13mp18 phage is folded by a staple strand and assembled into a square, triangle, five-pointed star or the like pattern.
Wherein in the step (2),
the ultrafiltration tube was 100KD.
The molar concentration ratio of the nucleic acid framework structure to the streptavidin is 1: (5-10); preferably, it is 1:5.
the molar ratio of the concentration of streptavidin modified by the nucleic acid framework structure to the concentration of biotinylated anti-CD 3 protein is 1: (5-10); preferably 1:5. wherein the concentration of streptavidin modified by the nucleic acid framework structure is obtained by multiplying the concentration of the nucleic acid framework structure by the number of streptavidin modified.
The reaction is carried out at a temperature of 4-37 ℃, preferably by shaking at room temperature.
The reaction time is 30-60 min; preferably 30min.
The ultrafiltration tube adopted by ultrafiltration purification is 100KD, the rotation speed of ultrafiltration centrifugation is 10000g,2min, and the rotation speed of reverse buckling recovery is 2000g,2min.
M13mp18 single-stranded DNA is a commercial product, the sequence of which has been disclosed. The nucleic acid framework structures of different shapes have hundreds of single-stranded DNA (namely 'staple chain') of different sequences, the specific embodiment of the invention adopts a triangle shape, the long chain is M13mp18 single-stranded DNA with 7249 bases, 208 single-stranded DNA (the single-stranded DNA in the embodiment 1 is 208 in total, the single-stranded DNA can be selected to be subjected to biotinylation modification, and the 202 single-stranded DNA in the embodiment 1 is not subjected to biotinylation modification, and the 6 single-stranded DNA is subjected to biotinylation modification, so that the total single-stranded DNA is 208), and the triangle structure has been reported in the literature. Other shapes of nucleic acid framework structures can also be used in the present invention.
Specifically, the steps include:
(1) M13mp18 single-stranded DNA, single-stranded DNA constituting the nucleic acid framework structure, biotin-modified single-stranded DNA was prepared according to the following 1:5:5, the solution system is a 1xTAE buffer system containing 12.5mmol/L magnesium ions, and the gradient PCR instrument is used for slowly reducing the temperature from 95 ℃ to 25 ℃ at a speed of 0.1 ℃ per second, so as to obtain the biotinylated nucleic acid framework structure.
(2) And (3) ultrafiltering and purifying the nucleic acid frame structure in the step (1) by adopting an ultrafiltration tube with the granularity of 100KD, wherein the rotation speed of ultrafiltration and centrifugation is 10000g, the time is 2min, then, reversely buckling the ultrafiltration tube in a receiving tube, and centrifuging at the rotation speed of 2000g and 2min to obtain the purified biochemical nucleic acid frame structure. 1, the method comprises the following steps: 5 molar concentration (nucleic acid framework: streptavidin) after addition of streptavidin, shaking on a shaker at room temperature for 30min, purification by the same ultrafiltration, 1:5 molar concentration (nucleic acid framework structure corresponds to streptavidin: biotinylated anti-CD 3 protein) to biotinylated anti-CD 3 protein monomer, and shaking at room temperature for 30min to obtain nucleic acid framework structure-based nano-scale artificial antigen presenting cells (alpha CD3-Origami aAPC).
Wherein, in the step (1), the obtained biotinylated nucleic acid framework structure is stored in a refrigerator at 4 ℃.
Wherein, in the step (2), the ultrafiltration purification times are three times.
The preparation method of the nano-scale artificial antigen presenting cell (alpha CD3-Origami aAPC) based on the nucleic acid framework structure is simple, and has good biocompatibility and stability.
The invention also provides the nano-scale artificial antigen presenting cell alpha CD3-Origami aAPC based on the nucleic acid framework structure obtained by the preparation method. The number and the interval of the alpha CD3 protein molecules modified by the alpha CD3-Origami aAPC are controllable, and are determined by the number and the position of the biotinylated single-stranded DNA selected during the design of the nucleic acid structure.
In one embodiment, the product is a triangular framework structure with sides of about 120nm, each side incorporating two αcd3D protein molecules with a 40nm spacing.
The invention also provides application of the nano-scale artificial antigen presenting cell (alpha CD3-Origami aAPC) based on the nucleic acid framework structure in preparation of medicaments for activation and proliferation of T cells, such as mouse T cells.
In the present invention, the nucleic acid framework-based nano-sized artificial antigen presenting cells (αcd 3-origamaapcs) can be used for activation and proliferation of mouse T cells. The method has the following characteristics: (1) the number and relative position of anti-CD 3 proteins on nucleic acid framework-based nanoscale artificial antigen presenting cells (αCD3-Origami aAPCs) can be precisely controlled; (2) the nucleic acid framework structure has good biocompatibility and can not produce a killing effect on cells; (3) the nano-scale artificial antigen presenting cell (alpha CD3-Origami aAPC) based on nucleic acid framework structure can realize the rapid activation and effective proliferation of T cells.
In the present invention, the nucleic acid framework-based nanoscale artificial antigen presenting cells (αcd 3-origamaapcs) include, but are not limited to, loading with only one anti-CD 3 protein molecule.
In the present invention, the biotin-modified single-stranded DNA can achieve the regulation of the number and spacing on the αCD3-origin aAPC of the antigen presenting cell.
In the present invention, the number of biotin sites designed in the nucleic acid framework-based nano-sized artificial antigen presenting cell (αcd 3-origamaapc) may be 1 to 20, for example, 6.
In the present invention, the biotin site-to-site distance designed in the nucleic acid framework-based nano-sized artificial antigen presenting cell (αcd 3-origamaapc) may be 10nm to 100nm, for example, may be 40nm.
In the present invention, the nucleic acid framework-based nanoscale artificial antigen presenting cells (αcd 3-origamaapcs) include, but are not limited to, those used for mouse T cell activation and proliferation.
The preparation method of the nano-scale artificial antigen presenting cell (alpha CD3-Origami aAPC) based on the nucleic acid framework structure is simple, and can realize the accurate control of the number and the spacing of protein molecules. The nanometer artificial antigen presenting cell (alpha CD3-Origami aAPC) provided by the invention has good biocompatibility, and can realize effective activation and proliferation of mouse T cells. The nanometer artificial antigen presenting cell (alpha CD3-Origami aAPC) provides an effective platform for proliferation of T cells, and has potential application value in adoptive immunotherapy.
Drawings
FIG. 1 is a diagram showing the reaction mechanism of the present invention for preparing nanoscaled artificial antigen presenting cells (αCD3-Origami aAPCs) based on nucleic acid framework structure for activation and proliferation of T cells.
FIG. 2 is an atomic force characterization diagram of biotinylated nucleic acid structures and nano-sized artificial antigen presenting cells based on nucleic acid framework structures (αCD3-Origami aAPCs) prepared in example 1; wherein the ordinate of the small graph in the upper right-hand graph in fig. 2 is height/nm and the ordinate of the small graph in the lower right-hand graph is height/nm.
FIG. 3 shows cytotoxicity experiments in a control group and an experimental group (biotinylated nucleic acid structure, streptavidin-linked nucleic acid structure prepared in example 1).
FIG. 4 shows the expression of early signal CD69 protein on the surface of T cells at 16 hours in a control group and an experimental group (. Alpha. CD3-Origami aAPC prepared in example 1).
FIG. 5 is a comparison of interferon-gamma release concentrations in a control group and an experimental group (. Alpha. CD3-Origami aAPC prepared in example 1).
FIG. 6 shows proliferation of a control group and an experimental group (. Alpha. CD3-Origami aAPC prepared in example 1) on day 3.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings. The procedures, conditions, experimental methods, etc. for carrying out the present invention are common knowledge and common knowledge in the art, except for the following specific references, and the present invention is not particularly limited.
Example 1
(1) One 7249 base long M13mp18 single-stranded DNA, 202 short single-stranded DNA constituting the nucleic acid framework structure, 6 biotin-modified short single-stranded DNA were prepared according to a sequence of 1:5:5, the solution system is a 1xTAE buffer system containing 12.5mM magnesium ions, and the solution system is slowly reduced from 95 ℃ to 25 ℃ at a speed of 0.1 ℃ per second by using a gradient PCR instrument, so as to obtain the biotinylated nucleic acid framework structure.
(2) And (3) ultrafiltering and purifying the nucleic acid structure in the step (1) by adopting an ultrafiltration tube with the granularity of 100KD, wherein the rotation speed of ultrafiltration and centrifugation is 10000g, the time is 2min, then, inversely buckling the ultrafiltration tube in a receiving tube, and centrifuging the rotation speed of 2000g and 2min to obtain the purified biotinylated nucleic acid framework structure. 1, the method comprises the following steps: 5, after adding streptavidin, shaking for 30min at room temperature on a shaker, purifying by the same ultrafiltration, the molar ratio of streptavidin molar concentration to biotinylated anti-CD 3 protein molecule according to the nucleic acid framework structure is 1:5, adding biotinylated alpha CD3 protein (CD 3 molecule is an important mark of T cell, and consists of gamma, delta, epsilon, zeta and eta polypeptide chains, wherein the cytoplasmic regions of the gamma, delta, epsilon, zeta and eta polypeptide chains all contain ITAM, have the function of transmitting TCR signals, activate the T cell, and oscillate at room temperature for 30min to prepare the nano-scale artificial antigen presenting cell (alpha CD 3-origin aaPC) based on nucleic acid framework, wherein the product is a triangular framework structure with the side length of about 120nm, and two alpha CD3D protein molecules with the distance of 40nm are combined on each side.
As shown in FIG. 2, from the atomic force microscope characterization and height charts, it was demonstrated that the anti-CD 3 protein molecules were indeed bound at pre-designed sites, demonstrating that the prepared nano-sized artificial antigen presenting cells (. Alpha. CD3-Origami aAPCs) of nucleic acid framework structure did achieve precise control of the position and number of protein molecules.
Example 2
The nucleic acid construct prepared in Experimental example 1 and the streptavidin-linked nucleic acid construct were filtered with a 0.22 μm sterile filter to remove bacteria from the solution. The degerming structure is used for a mouse T cell toxicity experiment and is used as an experimental group;
(1) Filling the edge wells of the 96-well plate with sterile PBS; a lymphocyte suspension of the mouse is obtained by using a lymphocyte separation liquid, T cells are obtained by using a Ni Long Maozhu method, the cell suspension concentration is adjusted to 50000/mL, and 100 mu L of the cell suspension is added to each hole.
(2) 5% CO2, 37℃incubation, adding 10 u L nucleic acid structure and streptavidin nucleic acid structure, to a final concentration of 1nM, 10nM, 20nM, each set of 3 multiple wells.
(3) The cells were placed in an incubator and incubated at 5% CO2 for 24h at 37 ℃.
(4) mu.L of 5mg/mLMTT was added to each well and the culture was continued for 4 hours.
(5) After the culture was terminated, the cell supernatant was removed by centrifugation, 150. Mu.L of dimethyl sulfoxide was added to each well and resuspended, and the shaking table was oscillated at low speed for 10 minutes, and then the absorbance of each well at OD490 nm was measured by using a microplate reader.
(6) At the same time, zeroing wells (medium, MTT, dimethyl sulfoxide) and control wells (cells, nucleic acid structures of the same concentration, medium, MTT, dimethyl sulfoxide) were set.
(7) Cell viability (cell viability) was calculated:
cell viability = (nucleic acid structure group a value-zeroed well a value)/(control well a value-zeroed well a value) ×100%
As shown in fig. 3, cytotoxicity experiments were performed with T cells. The experimental results show that the survival rate of the cells under different concentrations of nucleic acid structures is close to 100%. This indicates that the nucleic acid structure has little toxicity to T cells, i.e., that the nucleic acid structure has good biocompatibility.
Example 3
The nucleic acid framework-based nano-sized artificial antigen presenting cells (. Alpha.CD3-Origami aAPC) prepared in Experimental example 1 were used for T cell activation and proliferation as experimental groups.
The experiment adopts a lymphocyte separation liquid and a Ni Long Maozhu method to obtain T cells, adopts a fluorescent antibody flow cytometry to detect the expression of CD69 on the surface of the T cells, adopts an ELISA method to detect the cytokine release amount of the T cells, and adopts a CFSE method to detect the proliferation condition of the T cells.
(1) T cell harvesting
After 6-8 weeks of mice cervical dislocation are killed, spleen is ground to obtain single cell suspension, and then lymphocyte suspension is obtained by separating lymph separating liquid. T cells were obtained by the ni Long Maozhu method and resuspended in RPMI1640 complete medium for use.
(2) CD69 protein expression assay
In a 96-well plate, 10 were added per well 5 The freshly isolated T cells were incubated with 200. Mu.L of RPMI1640 complete medium containing 10nM of nucleic acid framework-based nanoscopic artificial antigen presenting cells (. Alpha.CD 3-origamiaAPC) for 16h, 2. Mu.g/mL of anti-CD 28 was added to the medium to give a control group of 200. Mu.L of complete medium. Three sets of duplicate wells per set. The collected cells were washed with PBS and stained with αCD69-PE in FACS buffer (PBS/0.1% heat-inactivated-FBS/0.1% sodium azide) for 30min at 4 ℃. After resuspension of cells with FACS buffer, upflow cytometryThe PE was excited using a 488nm laser.
(3) Cytokine detection
In 96-well plates, 105 freshly isolated T cells were added to each well and incubated for 16h with 200. Mu.L of RPMI1640 complete medium containing 10nM of αCD 3-origamiaAPC, 2. Mu.g/mL of anti-CD 28 was added to the medium, and 200. Mu.L of complete medium was added as a control. Interferon-gamma the concentration of interferon-gamma in the supernatant was determined using a mouse interferon-gamma enzyme-linked immunosorbent assay kit.
(4) CFSE proliferation
Fluorescent dye 5- (-6) -carboxyfluorescein diacetate succinimidyl ester CFSE was stored in 10mM stock solution in dimethyl sulfoxide. The concentration of T cells just separated is regulated to be 0.5-10x10 6 Per mL, the fluorescent dye was diluted with PBS. Cells were incubated in 1mL complete medium containing 5. Mu.M fluorescent dye at room temperature in the dark for 5min. The staining of the markers was stopped by adding 10 volumes of 20℃PBS containing 5% heat-inactivated-FBS, and then centrifuged at 1500g for 5min. Cells were washed with PBS containing 5% heat-inactivated-FBS at 20 ℃. In 96-well plates, 5X10 was added per well 4 Cells were incubated with 200. Mu.L of RPMI1640 complete medium containing 10nM of aCD 3-Origami aAPC for 3 days, 2. Mu.g/mL of anti-CD 28 was added to the medium to give a control group with 200. Mu.L of complete medium. After cells were collected, washed with PBS, stained with dead/live dye, detected by an up-flow cytometer, PE was excited with a 488nm laser, and the dead/live dye was excited with a 405nm laser. The collected data were further analyzed for proliferation using FlowJo.
(results)
As shown in FIG. 4, after co-culturing. Alpha. CD3-Origami aAPC with T cells for 16 hours, about 30% of the cell surface of the experimental group expressed the CD69 protein, the early T cell signaling molecule, with a significant increase over the control group. It was demonstrated that the prepared nucleic acid framework-structured nanoscale artificial antigen presenting cells (. Alpha.CD3-origamiaAPC) did achieve efficient early activation of T cells.
As shown in fig. 5, by comparing the released interferon- γ concentrations of the experimental group and the control group, it was found that the released interferon- γ concentration of the experimental group was significantly higher than that of the control group. It was demonstrated that the prepared nucleic acid framework-structured nanoscale artificial antigen presenting cells (. Alpha.CD3-origamiaAPC) did achieve efficient early activation of T cells.
As shown in FIG. 6, by cell proliferation analysis of the experimental group and the control group, it was found that about 70% of the cells in the experimental group were significantly proliferated, while the cells in the control group were not significantly proliferated. It was demonstrated that the prepared nucleic acid framework structured nano-sized artificial antigen presenting cells (. Alpha.CD3-origamiaAPC) did achieve efficient proliferation of T cells.
The foregoing description is only of a few specific embodiments of the present invention and is not intended to limit the present invention. It is within the scope of the present invention that the use of nucleic acid framework-based nano-sized artificial antigen presenting cells (. Alpha.CD3-Origami aAPC) for T cell activation and proliferation, obtained by the same or similar methods, is within the spirit and principles of the present invention.
Claims (8)
1. A method for preparing a nucleic acid framework-based nano-sized artificial antigen presenting cell αcd 3-origamaapcs, comprising the steps of:
(1) Mixing M13mp18 single-stranded phage DNA, single-stranded DNA forming a nucleic acid framework structure and biotin-modified short single-stranded DNA in a buffer system, and performing gradient cooling to obtain a biotinylated nucleic acid framework structure; the molar concentration ratio of the M13mp18 single-stranded phage DNA, the single-stranded DNA composing the nucleic acid framework structure and the biotin-modified short single-stranded DNA is 1: (5-10): (5-10);
(2) Adding streptavidin and biotinylated anti-CD 3 protein to react after ultrafiltration purification of the biotinylated nucleic acid framework structure in the step (1) to obtain the nano-scale artificial antigen presenting cell alpha CD3-Origami APC based on the nucleic acid framework structure; the molar concentration ratio of the nucleic acid framework structure to the streptavidin is 1: (5-10); the molar ratio of the nucleic acid framework structure to the streptavidin to biotinylated αcd3 protein molecule is 1: (5-10).
2. The method of claim 1, wherein the biotin-modified short single stranded DNA is spaced 10 to 100nm apart on an αcd3-Origami APC.
3. The method of claim 1, wherein in step (1), the buffer system is 1x TAE-Mg 2+ Wherein the magnesium ion concentration is 12.5mmol/L.
4. The method of claim 1, wherein in step (1), the gradient of cooling is from 95 ℃ to 25 ℃ at 0.1 ℃ per second.
5. The method of claim 1, wherein in the step (2), the ultrafiltration tube used for ultrafiltration purification is 100KD, the rotational speed of ultrafiltration centrifugation is 10000g,2min, and the rotational speed during back-off recovery is 2000g,2min.
6. The method of claim 1, wherein the nucleic acid framework-based nano-sized artificial antigen presenting cell αCD3-origamaaAPC is obtained.
7. The nucleic acid framework-based nano-sized artificial antigen presenting cell αcd3-Origami aAPC of claim 6, wherein the number and spacing of αcd3 protein molecules modified by the αcd3-Origami aAPC is controllable, and is determined by the number and position of biotinylated single-stranded DNA selected in designing the nucleic acid structure.
8. Use of a nucleic acid framework-based nano-sized artificial antigen presenting cell αcd3-Origami aapcs according to claim 6 or 7 for the preparation of a medicament for T cell activation and proliferation.
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