CN113388042B

CN113388042B - Recombinant protein, recombinant expression vector, recombinant cell and NK cell activated magnetic bead as well as preparation method and application thereof

Info

Publication number: CN113388042B
Application number: CN202110715596.3A
Authority: CN
Inventors: 焦顺昌; 张嵘; 袁翰; 吕鹏敏
Original assignee: Beijing Dingcheng Taiyuan Biotechnology Co ltd
Current assignee: Beijing Dingcheng Taiyuan Biotechnology Co ltd
Priority date: 2021-06-28
Filing date: 2021-06-28
Publication date: 2022-02-01
Anticipated expiration: 2041-06-28
Also published as: CN113388042A

Abstract

The invention provides a recombinant protein, a recombinant expression vector, a recombinant cell, an NK cell activation magnetic bead, a preparation method and an application thereof, and relates to the technical field of NK cell activation propagation. The invention fuses the single-chain antibody and the Streptag2 label, has simple structure, ensures that the antibody expressed by the recombinant expression vector or the recombinant cell can be directly assembled with the magnetic bead without biotinylation modification, and the assembled magnetic bead can be directly used as an amplification stimulator of the NK cell, thereby effectively amplifying the NK cell. The NK cell activating magnetic bead has the binding capacity of more than 97 percent and high specificity; the killing efficiency of the NK cell line obtained by activating and culturing the NK cell activated magnetic beads is not obviously different from that of the NK cell activated and cultured by a feeder cell method.

Description

Recombinant protein, recombinant expression vector, recombinant cell and NK cell activated magnetic bead as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of NK cell activation propagation, and particularly relates to a recombinant protein, a recombinant expression vector, a recombinant cell, an NK cell activation magnetic bead, a preparation method and an application thereof.

Background

Natural killer cells (NK) are an innate lymphocyte that can spontaneously kill tumor cells without antigen stimulation or prior sensitization, playing a key role in antiviral and antitumor immunity. The immunophenotypic characteristics are mainly CD3^-CD56⁺Is a subpopulation of lymphocytes that do not express a T cell receptor nor a B cell receptor. NK cells can kill tumor cells or virally infected cells through a variety of pathways, including direct cytotoxicity (both natural and antibody-dependent cell-mediated cytotoxicity) and indirect effects (such as cytokine production and interaction with adaptive immunity). Because of its broad spectrum and high lethality, NK cells have a very promising application prospect and are expected to become a new strategy for adoptive immunotherapy for treating tumors, and therefore, it is very necessary to develop a method for efficiently and stably amplifying NK cells in vitro.

NK cells account for about 5-10% of lymphocytes in blood, and sufficient cell number and cell purity in cell therapy are important factors of the effect of NK cells in killing tumor cells, but the proliferation of NK cells is weak, so that sufficient cells cannot be provided for administration, and clinical requirements cannot be met, so that most of treatment schemes depending on NK cells are in an early development stage, which becomes the biggest problem in clinical application. At present, the method with the highest NK cell in-vitro activation efficiency is a method of adding a feeder cell for inactivating K562 feeder cells, the positive rate of NK cells after 2-3 weeks of culture can be up to more than 90%, but inactivated feeder cells are difficult to remove, and the treatment level is difficult to achieve. In recent two years, some recombinant cytokine culture schemes are also available on the market, most of the recombinant cytokine culture schemes can stably achieve about 40-50% of positive rate, but a large amount of T cells can proliferate in the culture process, influence the subsequent use effect, possibly bring the risk of acute graft-versus-host disease (GVHD), and achieve more than 80% of positive rate, or the amplification efficiency is general, or the individual difference is serious.

Disclosure of Invention

In view of the above, the present invention is directed to a recombinant protein, a recombinant expression vector, a recombinant cell, and an NK cell-activated magnetic bead, and a preparation method and an application thereof, wherein the recombinant protein or an antibody obtained from the recombinant expression vector and the recombinant cell can be directly assembled with the magnetic bead, and the assembled magnetic bead can be directly used as an NK cell amplification stimulator to efficiently amplify NK cells.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a recombinant protein, the structure of which comprises a single-chain antibody NM₃E₂And Streptag 2.

Preferably, in the single chain antibody NM₃E₂The N terminal of the (B) is connected with an IL21 signal peptide, and the C terminal is connected with IgG1 to form IL21-NM₃E₂IgG1, the IL21-NM₃E₂The encoding gene of IgG1 comprises the nucleotide sequence shown in SEQ ID NO. 4.

Preferably, the coding gene of the IL21 signal peptide comprises a nucleotide sequence shown in SEQ ID NO. 1; single chain antibody NM₃E₂The coding gene of (A) comprises a nucleotide sequence shown in SEQ ID NO. 2.

Preferably, the encoding gene of Streptag2 includes the nucleotide sequence shown in SEQ ID NO. 3.

The invention also provides a recombinant expression vector for expressing the recombinant protein, and a basic vector of the recombinant expression vector comprises a eukaryotic expression vector pFUSE-hIgG 1-Fc.

The invention also provides a construction method of the recombinant expression vector, which comprises the following steps: (1) adding a coding gene sequence of Streptag2 between an Fc gene and a stop codon TGA of a eukaryotic expression vector pFUSE-hIgG1-Fc to obtain a vector pFUSE-hIgG1-Fc-Streptag 2;

(2) IL21-NM₃E₂The coding gene of IgG1 is inserted between the Nco I and BgII enzyme cutting sites of the vector pFUSE-hIgG1-Fc-Streptag2 to obtain the recombinant expression vector; the IL21-NM₃E₂The encoding gene of IgG1 comprises the nucleotide sequence shown in SEQ ID NO. 4.

The invention also provides a recombinant engineering cell for expressing the recombinant protein, and the basic cell of the recombinant engineering cell comprises an expiCHO cell.

The invention also provides a preparation method of the NK cell activated magnetic bead, which comprises the following steps: and inducing the recombinant engineering cells to generate recombinant proteins, purifying and reacting with activated magnetic beads to obtain NK cell activated magnetic beads.

Preferably, the ratio of the number of activated magnetic beads to the purified recombinant protein per 150. mu.L of the reaction system is 1X 10⁷The method comprises the following steps: 15 μ g.

The invention also provides the NK cell activated magnetic bead prepared by the preparation method.

The invention also provides application of the NK cell activation magnetic bead in-vitro culture of NK cells.

The invention also provides a method for culturing NK cells in vitro, which comprises the following steps: mixing human peripheral blood mononuclear cells and the NK cell activated magnetic beads according to the number ratio of 1:1, and then culturing;

the culture medium for culture takes Human Lymphocytes Serum Free Media culture medium as a basic culture medium and also comprises: 1000IU/mL of cell culture factor IL-2, 10ng/mL of cell culture factor IL-15 and 10 percent of fetal bovine serum FBS.

Preferably, the culture conditions comprise: 37 ℃ and 5% CO₂And (4) concentration.

The invention also provides the NK cell line cultured by the method.

The invention also provides application of the NK cell line in preparation of a medicine for killing tumor cells or virus-infected cells.

The invention provides a recombinant protein, which is characterized in that a single-chain antibody and a Streptag2 label are fused, the structure is simple, the recombinant expression vector or the antibody expressed by recombinant cells can be directly assembled with magnetic beads without biotinylation modification, and the assembled magnetic beads can be directly used as an amplification stimulator of NK cells, so that the NK cells can be effectively amplified. In the embodiment of the invention, the binding condition of the magnetic beads and the recombinant protein is detected by adopting flow cytometry, the binding capacity is up to more than 97%, the specificity of the magnetic beads and the recombinant protein is high, and the magnetic beads and the recombinant protein can be used for in-vitro activation experiments of NK cells. The embodiment of the invention utilizes peripheral blood mononuclear cells (PBMC cells) and the assembled magnetic beads to carry out in-vitro activation and amplification on NK cells, pure NK cells are amplified 7 times after in-vitro activation culture for 14 days, and other cells (such as T cells) are also amplified in the activation culture process because cells used for activation initiation are PBMC cells. Meanwhile, the detection result of the flow cytometry shows that the classical NK cell (namely CD 3) is cultured for 14 days^-CD56⁺Cell population) of 63.6%, the proportion of pure NK cells (CD 3)^-CD16⁺CD56⁺Cell population) was 73.1%. The embodiment of the invention also performs an in-vitro killing experiment on the obtained NK cell line, the cell culture lasts for 7 days, and after killing for 4 hours, the effective-to-target ratio is 2: at 1, the killing efficiency of the experimental group is 77.04%; the effective target ratio is 1: the killing efficiency of the experimental group was 46.95% at 1. Therefore, the NK cell line obtained by the invention has obvious killing activity on K-562-luc2 cells. In conclusion, the scheme of the invention can obtain a large amount of NK cells with remarkable killing activity, and can be used for clinically killing tumor cells or viral infectionThe cells promote the clinical application and development of NK cells.

Drawings

FIG. 1 is a structural diagram of NK cell activated magnetic beads obtained by the present invention;

FIG. 2 is an SDS-PAGE electrophoresis of recombinant proteins;

FIG. 3 is a diagram showing the flow analysis of the binding effect of magnetic beads and antibodies;

FIG. 4 shows the cell counts of PBMC cells cultured and sampled on

days

1, 2, 3, 6, 10 and 14;

FIG. 5 shows the growth of PBMC cells after culture, under a microscope on

days

1, 2, 3, 6, 10 and 14, wherein A: cell culture 1 day picture; b: cell culture 2 days; c: cell culture 3 days; d: cell culture 6 days; e: cell culture 10 days; f: cell culture 14 days;

FIG. 6 shows the results of the flow cytometry for detecting cell phenotype;

FIG. 7 is a graph showing the results of the in vitro cell killing experiment on day 7;

FIG. 8 shows the results of the GraphPadprism software analysis of comparative example 1;

FIG. 9 shows the results of the flow cytometry test of comparative example 2;

FIG. 10 is a graph showing the results of the in vitro cell killing experiments in comparative example 3 and example 2.

Detailed Description

The invention provides a recombinant protein, which comprises a single-chain antibody NM₃E₂And Streptag 2.

The invention is in the single-chain antibody NM₃E₂The N end of the polypeptide is preferably also connected with an IL21 signal peptide, and the coding gene of the IL21 signal peptide comprises a nucleotide sequence shown in SEQ ID NO. 1; in the single-chain antibody NM₃E₂Preferably, the C-terminus of (A) is further linked to IgG1 to form IL21-NM₃E₂IgG 1. In the present invention, the IL21 signal peptide is preferably derived from human, and can promote the secretory expression of the recombinant protein in mammalian cells. In the present invention, the recombinant protein is also referred to as recombinant NM₃E₂Streptag2 protein. Single chain antibody NM of the invention₃E₂The coding gene of (1) preferably comprises the nucleotide sequence shown in SEQ ID NO. 2. In order to make IL21-NM₃E₂IgG can be coupled directly to magnetic beads using Streptag2 with IL21-NM₃E₂IgG, the encoding gene of Streptag2 preferably comprises the nucleotide sequence shown in SEQ ID NO. 3. The gene sequence of IL21 is not particularly limited in the invention, and is preferably shown in SEQ ID NO. 1. The invention is directed to said IL21-NM₃E₂The gene encoding IgG1 is not particularly limited, and is preferably synthesized by Kingzhi Biotech Co., Ltd. In the present invention, the nucleotide sequences are all 5 'to 3' unless otherwise specified.

The eukaryotic expression vector pFUSE-hIgG1-Fc is a transient expression vector of mammalian cell protein, and the source of the basic vector is not particularly limited in the invention, and is preferably purchased from Invivogen company. The invention names the recombinant expression vector NM₃E₂Streptag2 eukaryotic expression vector.

According to the invention, a coding gene sequence of Streptag2 is added between an Fc gene of a eukaryotic expression vector pFUSE-hIgG1-Fc and a stop codon TGA to obtain a vector pFUSE-hIgG1-Fc-Streptag 2. The invention preferably entrusts Jinwei Zhi Biotech Co Ltd to modify an expression vector pFUSE-hIgG1-Fc, adds Streptag2 nucleotide sequence between Fc gene and termination codon TGA, and obtains the vector pFUSE-hIgG1-Fc-Streptag 2.

After obtaining the carrier pFUSE-hIgG1-Fc-Streptag2, the invention adds IL21-NM₃E₂The coding gene of IgG1 is inserted between the Nco I and BgII enzyme cutting sites of the vector pFUSE-hIgG1-Fc-Streptag2 to obtain the recombinant expression vector.

The present invention preferably uses the above IL21-NM₃E₂The coding gene of IgG1 is synthesized and directionally cloned to pFUSE-hIgG1-Fc-Streptag2 expression vector after NcoI and BgII linearization treatment, and the recombinant expression vector is obtained.

The invention uses the recombinant expression vector to transform DH5 alpha, and then obtains the recombinant plasmid NM after the plasmid is extracted₃E₂Streptag2 inpFUSE-hIgGle3-Fc。

The invention preferably utilizes a transfection method to construct the recombinant engineering cell, and specifically comprises the following steps: and transforming the recombinant expression vector into escherichia coli DH5 alpha, and selecting a plurality of clones for sequencing identification. The correctly sequenced recombinants (recombinant plasmids) were then placed in plasmids and used for transfection of expichcho cells (purchased from Thermo Fisher Scientific).

The transfection according to the invention preferably comprises electroporation, more preferably by means of electroporation with reference to the instructions of the electrotransformation apparatus Celetrix-CTX-1500A.

The invention also provides a method for expressing recombinant protein by using the recombinant engineering cell, which preferably comprises the following steps: after the electrotransfer is finished, the cell liquid in the electric shock tube is transferred to a pre-added 3mL solution

CHO CD04 Medium complete Medium (C.) (II)

CHO CD04 Medium basal Medium + 1% GlutaMAX from QuaCell Corp.) in six-well platesPlacing the six-hole plate at 37 deg.C and 5% CO₂The cell culture chamber (purchased from Thermo Fisher Scientific Co., Ltd.) was used to culture the cells after electroporation at a shaker rotation speed of 105rpm, and after 5 days, the culture supernatant was collected and examined for the expression of the target protein. After obtaining the recombinant protein, the invention preferably further comprises protein purification, and more preferably, the protein purification is carried out by using a ProteinA affinity chromatography column.

The induction according to the present invention is preferably the same as the above-described method for expressing a recombinant protein, and will not be described herein. In the present invention, it is preferable that the protein is purified by the protein affinity column and then the purified protein obtained is reacted with activated magnetic beads. The ratio of the number of activated magnetic beads to the purified recombinant protein in the reaction system of 150. mu.L is preferably 1X 10⁷The method comprises the following steps: 15 μ g.

In the present invention, the method for activating magnetic beads preferably includes: the stock solution of magnetic beads (Strep-Tactin mag. Microbeads, available from IBA) was shaken on a vortex shaker for 30s to obtain a solution containing about 1X 10 beads⁷And (3) placing the stock solution of the magnetic beads in a 1.5mLEP tube, adding 1mLBinding Buffer, blowing, uniformly mixing, carefully abandoning the supernatant on a magnetic frame, and repeatedly using the Bindingbuffer for cleaning once.

The reaction time of the present invention is preferably 1 hour, and the working concentration of the recombinant protein is at least 100. mu.g/mL when the reaction is carried out. After the reaction is finished, the method preferably further comprises washing once with a Binding Buffer, resuspending antibody-coupled magnetic beads (or called NK cell activated magnetic beads) in 1mL of 1 × PBS solution, counting and calculating the concentration of the antibody-coupled magnetic beads, and storing in a refrigerator at 4 ℃ for later use. The NK cell activation magnetic bead has the binding capacity of more than 97 percent and high specificity through flow cytometry detection, and can be used for in vitro culture and amplification of NK cells.

The structure of the NK cell-activating magnetic bead of the present invention is preferably as shown in FIG. 1.

The invention also provides a method for culturing NK cells in vitro, which comprises the following steps: mixing human peripheral blood mononuclear cells (PBMC cells) and the NK cell activated magnetic beads according to the number ratio of 1:1, and culturing;

the culture medium for culture takes Human Lymphocytes Serum Free Media culture medium (LY-08 culture medium for short) as a basal culture medium and also comprises: 1000IU/mL of cell culture factor IL-2, 10ng/mL of cell culture factor IL-15 and 10 percent of fetal bovine serum FBS.

In the present invention, the source of the PBMC cells is not particularly limited, and the PBMC cells can be extracted by themselves, but in the present embodiment, human PBMC cells are preferably extracted by collecting peripheral blood of the elbow vein of a volunteer and separating the blood by density gradient centrifugation using a Ficoll lymphocyte separation medium (purchased from TBD).

The present invention preferably uses the culture medium to resuspend the PBMC cells and adjust the cell density to 1.5X 10⁶mL, following human PBMC cells: NK cell activating magnetic beads ═ 1:1, inoculating the human PBMC cells and the NK cell activated magnetic beads into a six-hole plate, uniformly mixing, and placing the six-hole plate at the temperature of 37 ℃ and 5% CO₂The cell culture box of (2) is used for carrying out in-vitro amplification culture. The invention carries out complete liquid change after the culture for 48 hours, then carries out liquid supplement or liquid change treatment every other day, each liquid supplement or liquid change culture medium contains the cell culture factor, and the cell density is maintained at 1 x 10⁶/mL。

The invention also provides the NK cell line cultured by the method.

The invention adopts flow cytometry to detect the NK cell line, and shows that the classical NK cell (namely CD 3) is cultured for 14 days^-CD56⁺Cell population) of 63.6%, the proportion of pure NK cells (CD 3)^-CD16⁺CD56⁺Cell population) was 73.1%, and the amplification effect was good.

The NK cell line has strong killing capacity on tumor cells, and has no obvious difference from the killing efficiency of NK cells activated and cultured by a feeder cell method.

The following examples are provided to illustrate a recombinant protein, a recombinant expression vector, a recombinant cell and an NK cell-activated magnetic bead, and methods for preparing and using the same, but they should not be construed as limiting the scope of the present invention.

Example 1

One, NM₃E₂Construction of Streptag2 eukaryotic expression vector

The expression vector was modified to pFUSE-hIgG1-Fc-Streptag2 by Kingzhi Biotech, Inc. by adding Streptag2 nucleotide sequence (SEQ ID NO.3) between the Fc gene of expression vector pFUSE-hIgG1-Fc (purchased from Invivogen) and the stop codon TGA.

Synthesis of IL21-NM by Jinzhi Biotechnology Ltd₃E₂-IgG1(SEQ ID NO.4)。

Synthesizing IL21-NM from whole gene₃E₂IgG1 was directionally cloned into pFUSE-hIgG1-Fc-Streptag2 expression vector linearized with Nco I and BgIIL, transformed into E.coli DH 5. alpha. and several clones were picked for sequencing identification. The correctly sequenced recombinants (recombinant plasmids) were then placed in plasmids and used for transfection of expichcho cells (purchased from Thermo Fisher Scientific).

By sequencing, IL21-NM₃E₂The full-length gene sequence of IgG is identical to that of SEQ ID NO.4, and is consistent with the expectation.

II, recombination NM₃E₂Expression and purification of Streptag2 protein

1. Recombinant NM₃E₂Expression of Streptag2 protein

expiCHO cells were counted and 2X 10 cells were taken⁷Individual expihcho cells were used for plasmid transfection. Following an electropolymerization Buffer 103 partA:transfection Buffer was prepared at a ratio of 1:1 (available from Celetrix corporation) for electrophoresis Buffer 103part B, and recombinant plasmid NM was taken₃E₂Streptag2 inpFSUSE-hIgGle 3-Fc, adding the prepared transfection buffer solution and the prepared recombinant plasmid into the cell sediment, uniformly mixing, transferring into a 120 μ L electric shock tube (purchased from Celetrix company), operating with reference to the instruction of an electrotransfer Celetrix-CTX-1500A during electric shock, transferring the cell liquid in the electric shock tube into 3mL of the electric shock tube after the electric shock is finished

CHO CD04 Medium complete Medium (C.) (II)

CHO CD04 Medium basal Medium + 1% GlutaMAX from QuaCell corporation) were placed in a six-well plate at 37 ℃ with 5% CO₂The cell culture chamber (purchased from Thermo Fisher Scientific Co., Ltd.) was used to culture the cells after electroporation at a shaker rotation speed of 105rpm, and after 5 days, the culture supernatant was collected and examined for the expression of the target protein.

2. Recombinant NM₃E₂Purification of Streptag2 protein

Adding a buffer solution of 20mM PB and 200mM NaCl into the cell culture supernatant after transfection to adjust the pH to 7.5;

protein purification chromatography column: ProteinA affinity chromatography column (available from GE Healthcare, column volume 1.0 mL);

buffer a (buffera): PBS, pH 7.4;

buffer b (bufferb): 0.1M Glycine, pH 3.0;

buffer c (buffer c): 0.1M Glycine, pH 2.7.

And (3) purification process: the protein affinity column was pretreated with buffer A using AKTAexplorer 100 type protein purification system (available from GE Healthcare), and the culture supernatant collected above was loaded after adjusting pH to 7.5 with 20mM PB buffer and 200mM NaCl, and the effluent was collected. After the sample loading is finished, the column is equilibrated by using at least 1.5mL of Buffer A, after the equilibration, the column is respectively eluted by using Buffer B and Buffer C, the target protein eluent is collected (1% of 1M Tris is required to be added in advance to a collection tube of the eluent, the pH value of the eluent is neutralized by 8.0, and the final concentration of Tris is about 10mM), and finally, the concentrated dialysis is carried out to the Buffer PBS.

The results show that the final purified recombinant NM₃E₂The Streptag2 protein was analyzed by SDS-PAGE electrophoresis, the SDS-PAGE electrophoresis under reducing and non-reducing conditions is shown in FIG. 2, and after purification by protein affinity chromatography column, the recombinant NM protein₃E₂The purity of Streptag2 protein is as high as 95%.

Preparation of magnetic beads

The stock solution of magnetic beads (Strep-Tactin mag. Microbeads, available from IBA) was shaken on a vortex shaker for 30s to obtain a solution containing about 1X 10 beads⁷Adding 1mL Binding Buffer into the stock solution of the magnetic beads in a 1.5mL EP tube, blowing and uniformly mixing, carefully discarding supernatant on a magnetic frame, and repeatedly using the Binding Buffer to clean once; adding the recombinant NM purified by a ProteinA affinity chromatography column₃E₂Streptag2 protein into activated magnetic beads, mixing, reacting at room temperature for 1h, wherein the recombinant NM is added₃E₂The working concentration of Streptag2 protein is at least 100 μ g/mL; washing once by using Binding Buffer, resuspending the antibody-coupled magnetic beads in 1mL of 1 XPBS solution, counting and calculating the concentration of the antibody-coupled magnetic beads (the structure is shown in figure 1), and storing in a refrigerator at 4 ℃ for later use.

Taking the magnetic beads coupled with the antibody in a flow tube, and detecting the magnetic beads and NM by adopting flow cytometry₃E₂Binding of Streptag2 recombinant proteins. The flow cytometry analysis results are shown in FIG. 3, which shows that the magnetic beads Strep-Tactin mag. Microbeads and recombinant NM of IBA₃E₂The binding capacity of Streptag2 protein is up to 97%, and the specificity of both is high.

Example 2

In vitro amplification culture of NK cells

Collecting peripheral blood of elbow vein of volunteer, separating and extracting human PBMC cells by density gradient centrifugation with Ficoll lymphocyte separation medium (purchased from TBD), adding fetal bovine serum FBS (purchased from gibco) and IL-2Human lymphocyes Serum Free Media (LY-08) (available from Gilles Biotech, hereinafter abbreviated as LY-08 culture medium) for (Human recombinant interleukin-2 for injection, available from Erlu pharmaceutical Co.) and IL-15 (available from novoprotein Co.) were resuspended, followed by cell counting, and the cell density was adjusted to 1.5X 10⁶mL, following human PBMC cells: magnetic beads are 1:1, inoculating the PBMC cells and the magnetic beads into a six-hole plate, uniformly mixing, and placing the six-hole plate at the temperature of 37 ℃ and 5% CO₂The cell culture box of (2) is used for carrying out in-vitro amplification culture. Wherein the working concentration of the added cell culture factor IL-2 is 1000IU/mL, the working concentration of IL-15 is 10ng/mL, and the working concentration of the fetal bovine serum FBS is 10%. Completely changing the culture medium after 48h, and supplementing or changing the culture medium every other day, wherein the cell culture factor is contained in each supplemented or changed culture medium, and the cell density is maintained at 1 × 10⁶mL, with cell counting. Then at 0d, 7d and 14d, respectively, taking the cells, performing cell phenotype detection by using flow cytometry, and counting cell proliferation times, wherein the cell phenotype detects the classical NK cell phenotype (namely CD 3)^-CD56⁺The cell population of (a).

As shown in FIGS. 4-6, the pure NK cells were amplified 7 times after 14 days of in vitro activation culture, and other cells (such as T cells) were also amplified during the activation culture process because the cells used for the activation initiation are PBMC cells. Meanwhile, the detection result of the flow cytometry shows that the classical NK cell (namely CD 3) is cultured for 14 days^-CD56⁺Cell population) of 63.6%, the proportion of pure NK cells (CD 3)^-CD16⁺CD56⁺Cell population) was 73.1%.

Example 3

In vitro killing experiment of NK cells

NK cells cultured for 7 days in example 2 were collected and effector cell density was adjusted to 4X 10⁶Perml and 2X 10⁶Per mL; the target cells, human myeloid leukemia cell K-562-luc2 (purchased from ATCC company,

CCL-243-LUC2^TM) And adjusting the density of the target cells to 2X 10⁶mL, i.e. effective target ratio of 2: 1 and 1: 1; according to different effective target ratios, respectively adding a total of 200 mu L of effector cells and target cells with different concentrations into a round-bottom 96-well plate, and dividing the effector cells and the target cells into two groups:

target cell group: 100 μ L of target cells +100 μ L of LY-08 complete medium, 3 multiple wells per group;

experimental groups: mu.L of target cells + 100. mu.L of NK cells cultured for 7 days in example 1, 3 duplicate wells per group;

placing the two groups of mixed cells at 37 deg.C and 5% CO₂Cultured for 4h in a cell culture box and utilizes One-Lite^TMThe Luciferaaseassay system kit (from Vazyme) detected the remaining K-562-luc2 cells and the killing efficiency of NK cells was calculated according to the formula (results are expressed as mean of 3 replicates).

The killing efficiency calculation formula is as follows: killing efficiency ═ 100% for (1-experimental/target cell).

After the cells are cultured for 7 days, the results of in vitro killing experiments are shown in figure 7, and after 4 hours of killing, the effective-to-target ratio is 2: at 1, the killing efficiency of the experimental group is 77.04%; the effective target ratio is 1:1, the killing efficiency of the experimental group is 46.95%, and the NK cells activated and cultured by the invention have obvious killing activity on K-562-luc2 cells.

Comparative example 1

Antibody-coupled magnetic beads and recombinant NM₃E₂Effect of Streptag2 protein formulation

1) Recombinant NM₃E₂Dilution of Streptag2 protein

Recombination of NM₃E₂Streptag2 protein was diluted using a gradient method according to 1: 2 was diluted down with 1 x PBS solution for 10 concentrations, the highest concentration being the original concentration, i.e.: 284. mu.g/mL, stored at 4 ℃ in a refrigerator protected from light for later use.

2) Flow cytometry analysis

Taking equivalent magnetic bead Strep-Tactinmag. Microbeads, and respectively adding the recombinant NM with different concentrations₃E₂The Streptag2 protein was coupled, i.e.: mixing magnetic bead stock solution (Strep-Tactin mag. micro)beads) was oscillated on a vortex oscillator for 30s, which was taken to include about 1X 10⁷Adding 1mLBinding Buffer into a 1.5mL EP tube to blow and mix uniformly, carefully abandoning supernatant on a magnetic frame, and repeatedly using the Binding Buffer to clean once; adding the above different concentrations of recombinant NM₃E₂Mixing Streptag2 protein into activated magnetic beads, and reacting at room temperature for 1 h; washing once by using Binding Buffer, adding a flow-type secondary antibody PE anti-human IgG Fc (purchased from BioLegend), and incubating for 20min at room temperature in a dark place; taking antibody-coupled magnetic beads in a flow tube, and detecting the magnetic beads and NM (nanometer) with different concentrations by adopting flow cytometry₃E₂Binding of Streptag2 recombinant proteins.

3) Data processing and analysis

Data analysis was performed using FlowJo V10 software and GraphPad Prism software.

Analysis of the data by FlowJo V10 software (table 1) and GraphPad Prism software (fig. 8) after detection by flow cytometry showed that recombinant NM was present at different concentrations₃E₂Microbeads of the Streptag2 protein, the highest binding ability and sensitivity to the magnetic beads Strep-Tactin mag of IBA, was the original concentration of recombinant NM₃E₂Streptag2 protein, i.e. added recombinant NM₃E₂The working concentration of Streptag2 protein was 284 μ g/mL, followed by a concentration of 1: 2 equal dilution of the first two gradients, the three concentrations of beads and recombinant protein were all more specific, therefore, in antibody recombination NM₃E₂Recombinant NM added when Streptag2 protein was coupled to magnetic beads₃E₂The working concentration of Streptag2 protein was at least 100. mu.g/mL.

TABLE 1FlowJo V10 software analysis results

Concentration (μ g/mL)	Geometric mean (PE-A)
		284	13876
142	13060
		71	15046
35.5	12871
		17.75	8166
8.875	2847
		4.4375	387
2.21875	59.8
		1.109375	34.7
0.5546875	31.4
		0.27734375	6036
0.138671875	6552

Comparative example 2

The problem of the proportion of magnetic beads to cells during the activation of NK cells

1) Preparation of magnetic beads

Shaking the stock solution of magnetic beads (Strep-Tactin mag. Microbeads) on a vortex oscillator for 30s to obtain a solution containing about 1 × 10⁷Adding 1mL Binding Buffer into the stock solution of the magnetic beads in a 1.5mL EP tube, blowing and uniformly mixing, carefully discarding supernatant on a magnetic frame, and repeatedly using the Binding Buffer to clean once; addition of recombinant NM₃E₂Streptag2 protein into activated magnetic beads, mixing, reacting at room temperature for 1h, wherein the recombinant NM is added₃E₂The working concentration of Streptag2 protein is at least 100 μ g/mL; washing once by using Binding Buffer, resuspending the antibody-coupled magnetic beads in 1mL of 1 × PBS solution, counting and calculating the concentration of the antibody-coupled magnetic beads, and storing in a refrigerator at 4 ℃ for later use.

2) Extraction of human PBMC cells

Collecting peripheral blood of elbow vein of volunteer, separating and extracting human PBMC cell by density gradient centrifugation with Ficoll lymphocyte separation solution, suspending with LY-08 culture medium containing fetal bovine serum FBS, IL-2 and IL-15, counting cells, and adjusting cell density to 1.5 × 10⁶and/mL. Wherein the working concentration of the added cell culture factor IL-2 is 1000IU/mL, the working concentration of IL-15 is 10ng/mL, and the working concentration of the fetal bovine serum FBS is 10%.

3) Inoculation of

Respectively taking 6 × 10⁶2, 3 x 10⁶And 1.2X 10⁷Antibody-coupled magnetic beads and 6X 10⁶Inoculation of human PBMC cells was performed according to magnetic bead: human PBMC cells ═ 1: 1. 1: 2 and 2: 1, inoculating the PBMC cells and the magnetic beads coupled with the antibody into a six-hole plate, uniformly mixing, and placing the six-hole plate at the temperature of 37 ℃ and 5% CO₂The cell culture box of (2) is used for carrying out in-vitro amplification culture. Completely changing the culture medium after 48h, and supplementing or changing the culture medium every other day, wherein the cell culture factor is contained in each supplemented or changed culture medium, and the cell density is maintained at 1 × 10⁶/mL。

4) Flow cytometry detection

Are respectively at the firstAt 0d and 7d, taking cells, performing cell phenotype detection by flow cytometry, and counting cell proliferation times, wherein the cell phenotype detects the classical NK cell phenotype (namely CD 3)^-CD56⁺The cell population of (a).

The results of cell culture for 7 days and cell phenotype detection by flow cytometry are shown in FIG. 9, when the magnetic beads: human PBMC cells ═ 1:1 hour, can guarantee the stable activation effect of NK cell under the quantity of certain magnetic bead and antibody, consequently when carrying out the activation experiment of NK cell in vitro, the proportion of magnetic bead and human PBMC cell is 1: the comprehensive effect is best when 1.

Comparative example 3

Comparison of the trophoblast method for NK cell activation with the method described in example 2 (trophoblast-free method)

1) NK cell activation method (trophoblast method):

collecting peripheral blood of elbow vein of volunteer, separating and extracting human PBMC cell by density gradient centrifugation with Ficoll lymphocyte separation solution, suspending with LY-08 culture medium containing fetal bovine serum FBS, IL-2 and IL-15, counting cells, and adjusting cell density to 1 × 10⁶and/mL. Wherein the working concentration of the added cell culture factor IL-2 is 1000IU/mL, the working concentration of IL-15 is 10ng/mL, and the working concentration of the fetal bovine serum FBS is 10%.

Following human PBMC cells: trophoblast K562/IL21[ human Chronic myelogenous leukemia cell (interleukin 21 gene modification) from national laboratory cell resource sharing platform, resource No. 3111C0001CCC000457]2: 1 ratio of human PBMC and feeder cells K562/IL21 were inoculated into a T25 cell culture flask (purchased from Corning Corp.), mixed well and then the T25 cell culture flask was placed at 37 ℃ with 5% CO₂The cell culture box of (2) is used for carrying out in-vitro amplification culture. 7d was supplemented with feeder cells K562/IL21 in an amount of one half of the number of NK cells at that time. Absolutely static state is ensured in the first 48h of cell culture, complete liquid change is carried out after 48h, liquid supplement or liquid change treatment is carried out every other day, the cell culture factors are contained in each liquid supplement or liquid change culture medium, and the cell density is maintained at 1 × 10⁶/mL。

2) NK cell activation method (non-feeder cell method):

the specific implementation procedure is the same as in example 2.

3) In vitro killing experiment of NK cells:

taking effector cells, namely NK cells cultured for 7 days by a feeder cell method and a non-feeder cell method, and adjusting the density of various effector cells to 4 × 10⁶Perml and 2X 10⁶Per mL; taking target cells, i.e. human myeloid leukemia cell K-562-luc2, and adjusting the target cell density to 2X 10⁶mL, i.e. effective target ratio of 2: 1 and 1: 1; according to different effective target ratios, respectively adding a total of 200 mu L of effector cells and target cells with different concentrations into a round-bottom 96-well plate, and totally dividing into three groups:

experimental groups: 100 mu L of target cells and 100 mu L of NK cells cultured for 7 days by a non-feeder cell method, and each group is provided with 3 multiple wells;

control group: culturing 7-day NK cells by a target cell + feeder cell method in 100 mu L, and making 3 multiple holes in each group;

placing the three groups of mixed cells at 37 deg.C and 5% CO₂The cell culture box of (1) was cultured for 4 hours, the remaining K-562-luc2 cells were detected using the One-Lit (TM) Luciferase Assay system kit, and the killing efficiency of NK cells was calculated according to the formula (the results are expressed as the mean of 3 replicate wells).

After killing for 4h, substituting into a formula to calculate the killing efficiency as shown in table 2 and fig. 10, wherein the effective target ratio is 2: at 1, the killing efficiency of the examples and the comparative examples is 77.04% and 76.60%, respectively; the effective target ratio is 1: at 1, the killing efficiencies of the examples and comparative examples were 46.95% and 37.95%, respectively. Therefore, the killing efficiency of NK cells activated and cultured by the non-feeder cell method of the invention is not obviously different from that of NK cells activated and cultured by the feeder cell method in the comparative example.

TABLE 2 killing effect of in vitro cell experiment

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Beijing ancient cooking peptide source Biotechnology Ltd

<120> recombinant protein, recombinant expression vector, recombinant cell and NK cell activated magnetic bead, and preparation method and application thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 60

<212> DNA

<213> Homo sapiens

<400> 1

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60

<210> 2

<211> 1395

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 2

atggaggtgc agctggtgga gagcggaggc ggagtggtga ggcccggcgg atctctgagg 60

ctgagctgcg ctgcctccgg cttcaccttc gacgactacg gcatgagctg ggtgagacaa 120

gcccccggca agggactgga gtgggtgagc ggaatcaact ggaacggagg ctccaccggc 180

tacgctgaca gcgtgaaggg aagatttacc atctctagag acaacgccaa gaactctctg 240

tatctgcaga tgaacagcct cagagccgag gatacagccg tctactactg tgccagagga 300

aggtctctgc tgtttgacta ctggggccaa ggcacactgg tcacagtgag cagaggaggc 360

ggcggaagcg gaggaggagg atccggcggc ggaggctcca gcgagctcac acaagacccc 420

gccgtgagcg tggctctggg ccagaccgtg agaatcacat gccaaggcga ttctctgagg 480

agctactacg ccagctggta ccagcagaag cccggccaag cccccgtgct ggtcatctac 540

ggcaagaaca atagaccttc cggcattccc gacagattca gcggcagctc cagcggcaac 600

acagcctctc tgacaatcac cggcgcccaa gccgaagacg aggccgacta ctactgcaac 660

tctagagaca gctccggcaa ccacgtggtg tttggaggcg gcaccaagct caccgtgctg 720

agatctgtgg agtgcccacc ttgcccagca ccacctgtgg caggaccttc agtcttcctc 780

ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 840

gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 900

gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 960

gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 1020

gtctccaaca aaggcctccc atcctccatc gagaaaacca tctccaaagc caaagggcag 1080

ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag 1140

gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1200

agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1260

tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1320

ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1380

ctgtctccgg gtaaa 1395

<210> 3

<211> 87

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 3

tggtcccatc cgcaatttga gaaggggggc ggatctggag ggggcagtgg aggttccgct 60

tggagccacc cccagttcga aaagtga 87

<210> 4

<211> 1542

<212> DNA

<213> Artificial sequence (artificial sequence)

<400> 4

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60

atggaggtgc agctggtgga gagcggaggc ggagtggtga ggcccggcgg atctctgagg 120

ctgagctgcg ctgcctccgg cttcaccttc gacgactacg gcatgagctg ggtgagacaa 180

gcccccggca agggactgga gtgggtgagc ggaatcaact ggaacggagg ctccaccggc 240

tacgctgaca gcgtgaaggg aagatttacc atctctagag acaacgccaa gaactctctg 300

tatctgcaga tgaacagcct cagagccgag gatacagccg tctactactg tgccagagga 360

aggtctctgc tgtttgacta ctggggccaa ggcacactgg tcacagtgag cagaggaggc 420

ggcggaagcg gaggaggagg atccggcggc ggaggctcca gcgagctcac acaagacccc 480

gccgtgagcg tggctctggg ccagaccgtg agaatcacat gccaaggcga ttctctgagg 540

agctactacg ccagctggta ccagcagaag cccggccaag cccccgtgct ggtcatctac 600

ggcaagaaca atagaccttc cggcattccc gacagattca gcggcagctc cagcggcaac 660

acagcctctc tgacaatcac cggcgcccaa gccgaagacg aggccgacta ctactgcaac 720

tctagagaca gctccggcaa ccacgtggtg tttggaggcg gcaccaagct caccgtgctg 780

agatctgtgg agtgcccacc ttgcccagca ccacctgtgg caggaccttc agtcttcctc 840

ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 900

gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 960

gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 1020

gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 1080

gtctccaaca aaggcctccc atcctccatc gagaaaacca tctccaaagc caaagggcag 1140

ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag 1200

gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1260

agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1320

tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1380

ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1440

ctgtctccgg gtaaatggtc ccatccgcaa tttgagaagg ggggcggatc tggagggggc 1500

agtggaggtt ccgcttggag ccacccccag ttcgaaaagt ga 1542

Claims

1. A preparation method of NK cell activated magnetic beads is characterized by comprising the following steps: inducing the recombinant engineering cells to generate recombinant proteins, purifying and reacting with activated magnetic beads to obtain NK cell activated magnetic beads; the magnetic beads are Strep-Tactin mag.

The basic cells of the recombinant engineering cells comprise expiCHO cells, the genome of the recombinant engineering cells comprises a recombinant expression vector, and the construction method of the recombinant expression vector comprises the following steps:

(1) adding a coding gene sequence of Streptag2 between an Fc gene and a stop codon TGA of a eukaryotic expression vector pFUSE-hIgG1-Fc to obtain a vector pFUSE-hIgG1-Fc-Streptag 2;

(2) IL21-NM₃E₂The coding gene of IgG1 is inserted between the Nco I and BgII enzyme cutting sites of the vector pFUSE-hIgG1-Fc-Streptag2 to obtain the recombinant expression vector, and the IL21-NM₃E₂The encoding gene of the-IgG 1 is a nucleotide sequence shown in SEQ ID NO. 4.

2. NK cell-activated magnetic beads prepared by the method according to claim 1.

3. Use of the NK cell activating magnetic bead according to claim 2 for in vitro culturing NK cells.

4. A method for culturing NK cells in vitro, comprising the steps of: mixing human peripheral blood mononuclear cells and the NK cell activated magnetic beads of claim 2in a number ratio of 1:1, and culturing;