CN113832190A

CN113832190A - Preparation method and application of trophoblast modified by genetic engineering

Info

Publication number: CN113832190A
Application number: CN202111121995.3A
Authority: CN
Inventors: 夏玉龙; 吴金芸; 叶华衍
Original assignee: Zhonghaixia Fujian Cell Biotechnology Co ltd
Current assignee: Zhonghaixia Fujian Cell Biotechnology Co ltd
Priority date: 2021-09-24
Filing date: 2021-09-24
Publication date: 2021-12-24

Abstract

The invention discloses a preparation method and application of a trophoblast modified by genetic engineering, and mainly discusses whether a K562 cell can stably express IL-21, OX40L and CD48 molecules at the same time and enhance the proliferation and tumor killing capacity of an NK cell to be researched, wherein the K562 genetically engineered cell simultaneously expressed by the three molecules is used as a trophoblast to culture the NK, so that the amplification multiple, the purity and the killing function of the NK are improved; the obtained preparation method can prepare and utilize K562 cells to simultaneously stably express membrane-bound interleukin-21 (mbIL-21), OX40L molecules and CD48 molecules to prepare an engineering cell strain named as K562-ZHX trophoblast, and then the engineering cell strain is irradiated by cobalt-60 to obtain trophoblast expanded NK cells.

Description

Preparation method and application of trophoblast modified by genetic engineering

Technical Field

The invention relates to the technical field of NK cell amplification, in particular to a preparation method and application of a trophoblast modified by genetic engineering.

Background

Cancer remains the first killer of serious threat to human health, and the incidence of cancer is increasing year by year from a global perspective. At present, the traditional cancer therapy cannot achieve good effect and cannot meet the requirement of patients on the health recovery of families of the patients. Immunotherapy of cancer is currently the most promising approach for tumor therapy recognized in the medical community. Natural killer cells (NK) are important innate immune cells in the body that, in contrast to T cells or other immune cells, rapidly release inflammatory cytokines and, without prior stimulation, lead to MHC (major histocompatibility complex) restricted killing of tumor cells. NK cells are used for tumor immunotherapy and are widely available, such as Peripheral Blood Mononuclear Cells (PBMCs), umbilical Cord Blood (CBs), Bone Marrow (BM), embryonic stem cells (MSCs), or induced pluripotent stem cells (ips).

Early studies showed that expansion of NK cells in vivo by cytokine injection did not bring any therapeutic effect to tumor patients. In recent years, a series of literature reports and clinical studies show that the NK cells are activated and amplified in vitro and then returned by veins to show good anti-tumor application prospects. NK cells account for only 5-20% of mononuclear cells in peripheral blood, and are distributed at a level inferior to that of peripheral blood in other tissues and organs. NK cells have a limited life span after reinfusion in vivo after in vitro expansion and are unable to proliferate. However, cell therapy usually requires the preparation of large numbers of cells, and the number of NK cells returned to the body directly determines the therapeutic effect. Therefore, how to efficiently expand the number of NK cells in vitro and enhance the tumor killing capability of the NK cells is the key of the NK cells for tumor immunotherapy.

Currently, various methods have been developed to expand NK cells in vitro in sufficient numbers and purity. For example, NK cells are stimulated to expand by cytokines such as IL-2, IL-15, IL-18, and IL-21. However, this method is first to search for the concentration, addition time, and the like of various cytokines. This method is time-consuming, labor-intensive, expensive, efficient, and of purity prohibitive. Another common method is to stimulate NK cell activation and expansion by trophoblasts. At present, the most commonly used trophoblasts in foreign clinical tests are irradiated K562 genetic engineering cells which simultaneously express membrane-bound IL-21 and 4-1BBL (also called Clone 9 cells), and the mode of expressing cell factors and co-stimulatory molecule ligands by the genetically modified cells is effective and can well control the production cost. However, the NK cell amplification efficiency obtained by the method is still unsatisfactory, the space for improving the amplification efficiency is greatly increased, and the defects of poor amplification multiple repeatability among PBMCs from different individual sources and the like exist. Therefore, there is an urgent need for a means for efficiently expanding high-purity NK cells.

Disclosure of Invention

The present invention aims at providing a preparation method and an application of trophoblasts modified by genetic engineering to solve the problems in the background technology.

In order to solve the technical problems, the invention provides the following technical scheme:

a preparation method of trophoblast modified by genetic engineering comprises the following steps:

(1) constructing pLV-mbIL-21-OX40L-CD48 lentiviral vector plasmid;

(2) preparing recombinant lentivirus by adopting pLV-mbIL-21-OX40L-CD48 lentivirus vector plasmid;

(3) extracting recombinant lentivirus, infecting K562 cells, selecting, subcloning, and sterilizing to obtain K562-ZHX nourishing cells.

The optimized scheme comprises the following specific steps of (1):

s1: obtaining gene coding sequences of human IL-21, human OX40L and human CD 48;

s2: sequentially connecting a human IL-21 gene coding sequence, a PGK promoter coding sequence, a human OX40L gene coding sequence, a T2A self-splicing peptide coding sequence and a CD48 gene coding sequence in series to obtain an mbiL-21-PGK-OX40L-T2A-CD48 gene sequence; wherein the sequence of the mbiL-21-PGK-OX40L-T2A-CD48 gene is shown in SEQ ID NO. 6;

s3: the gene sequence of the mbiL-21-PGK-OX40L-T2A-CD48 is constructed between BamH I and Xma I of a plasmid vector pLV-EF1a-IRES-Puro (purchased by Addgene company) to obtain a pLV-mbiL-21-OX40L-CD48 lentiviral vector plasmid.

In an optimized scheme, in step S1, the gene coding sequence of human IL-21 is shown in SEQ ID NO.1, the gene coding sequence of human OX40L is shown in SEQ ID NO.2, and the gene coding sequence of human CD48 is shown in SEQ ID NO. 3. The gene coding sequence of the PGK promoter coding sequence is shown as SEQ ID NO. 4; the gene coding sequence of the T2A self-cutting peptide coding sequence is shown in SEQ ID NO. 5.

In an optimized scheme, step S3 specifically includes:

s31: carrying out BamH I and Xmal I double enzyme digestion on the gene sequence of mbiL-21-PGK-OX40L-T2A-CD48, and carrying out electrophoretic recovery;

s32: carrying out BamH I and Xmal I double enzyme digestion on lentivirus transfer plasmid pLV-EF1a-IRES-Puro, and recovering a linearized skeleton plasmid through electrophoresis;

s33: the enzyme-digested mbl-21-PGK-OX 40L-T2A-CD48 gene is connected to the enzyme-digested pLV-EF1a-IRES-Puro skeleton plasmid through Ligation High Ver2 ligase, the plasmid is transformed to competent E.coli (DH5 alpha) cells, and purification is carried out to prepare pLV-mbl-21-OX 40L-CD48 lentiviral vector plasmid.

The optimized scheme is that the step (2) specifically comprises the following steps:

s4: preparation of recombinant lentivirus:

s41: 293T cells in logarithmic growth phase were transferred into 6-well plates containing 0.6X 10 cells per well⁶Mixing the cells with 2ml DMEM complete culture solution, and placing the mixture in a container 37The cells were cultured overnight in an incubator at room temperature, and 20ml of DMEM-complete culture solution was prepared and placed under the same conditions for overnight culture. When the confluence degree of 293FT cells is observed to reach 50% -60% under a microscope, discarding the culture solution in the 6-well plate, and then adding preheated DMEM complete culture solution into the 6-well plate, wherein the addition amount is 2 ml/well, so as to remove the cells which are not attached to the wall for later use; obtaining a culture plate containing 293T cells;

s42: the next day, mixing pLV-mbIL-21-OX40L-CD48 lentiviral vector plasmid, psPAX-1 plasmid and pMD2.G plasmid, and diluting the Opti-MEM culture medium to 1 μ g/μ L to obtain a diluent;

adding a serum-free Opti-MEM culture medium and a transfection reagent into the tube A, and uniformly mixing for 10s by vortex to obtain a solution A;

taking a tube B, adding a serum-free Opti-MEM culture medium, a diluent and a P3000 reagent into the tube B, and performing vortex for 10s to obtain a solution B;

mixing solution A and solution B, incubating at room temperature for 15min, adding into 293T cell-containing culture plate, standing at 37 deg.C and 5% CO₂Incubating for 6-8 hr, removing culture medium from the culture plate, adding DMEM complete culture medium, and incubating at 37 deg.C under 5% CO₂Continuing incubation under the condition;

s43: after 24 hours of culture, collecting the first cell supernatant;

adding DMEM complete culture solution at 37 deg.C and 5% CO₂Continuing incubation under the condition;

s44: after culturing for 52 hours, collecting the second cell supernatant, mixing with the first cell supernatant, centrifuging, collecting and transferring the supernatant;

s5: and concentrating the supernatant to obtain the recombinant lentivirus.

In an optimized scheme, step S5 specifically includes: filtering the collected supernatant, adding a Lenti-XConcentrator, incubating at 4 ℃ for 12h, centrifuging at 1500r/m for 45min, removing the supernatant, diluting, resuspending, and packaging to obtain recombinant lentivirus;

in step S5, performing titer detection after concentration, wherein the titer detection specifically comprises the steps of: infecting the recombinant lentivirus into k562 cells, and detecting the titer of the recombinant lentivirus by adopting a flow cytometry detection method.

The method comprises the following specific steps: k562 cells were added to the counting well of a Countstar counter in an amount of 1X 10⁵And is ready for use; diluting the recombinant lentivirus by 10-100 times by using RPMI-1640 complete culture solution to form diluted virus solution, then respectively adding the diluted virus solution into counting holes of a countstar counter with k562 cells, wherein the addition amount of the diluted virus solution is 1ul, 3ul, 10ul and 30ul respectively, and finally placing the countstar counter in a 5% carbon dioxide incubator at 37 ℃ for incubation for 48 hours;

20ul (i.e. containing 1X 10) was added to a 1ml centrifuge tube⁶Individual cells), 200ul PBS buffer solution, resuspension, adding 1ul anti-human CD48-FITC antibody under ice bath condition, incubating for 15 minutes, adding PBS to rotate at 1500rpm, centrifuging for 5 minutes, and washing cells twice; and adding 200ul pbs buffer solution, carrying out heavy suspension, carrying out flow cytometry detection, infecting k562 cells by the concentrated recombinant lentivirus, and determining that the number of infected positive k562 cells is equal to the number of virus particles when the infection efficiency of the k562 cells is less than 10%.

The optimized scheme is that the step (3) is specifically as follows:

sequentially adding the recombinant lentivirus and polybrene according to the final MOI value of the composite recombinant lentivirus of 5, and uniformly mixing to form a recombinant lentivirus mixture;

infecting K562 cells by using a recombinant lentivirus mixture, sorting and subcloning after infecting for 72 hours to obtain a K562-ZHX engineering cell strain;

and performing cobalt-60 irradiation sterilization on the K562-ZHX engineering cell strain, wherein the dosage of cobalt in the cobalt-60 irradiation sterilization is 100gy, and preparing the K562-ZHX trophoblast. The cell density of k562-mbIL-21-OX40L-CD48 trophoblasts is 1 x 10⁷The cells are frozen at-20 ℃.

In an optimized scheme, the feeder cells are used for in-vitro amplification culture of NK cells. The method comprises the following specific steps:

s6: and (3) PBMC cell extraction: the method comprises the following specific steps:

(1) transferring 10ml of whole blood into a 50ml centrifuge tube, adding 10ml of PBS solution for dilution, and gently mixing uniformly;

(2) two 15ml centrifuge tubes were taken and 5ml of Ficoll solution was added first. Then, slightly adding the diluted blood to the Ficoll upper layers of two centrifuge tubes, wherein the two solutions are required to be mixed together, and 10ml of diluted blood is added into each centrifuge tube;

(3)2000rpm, 20min, and noting that the deceleration setting must be set to be noblack or only 1-2 braking;

(4) the cell layer of PBMC is white. The layer of cells can now be pipetted into another clean 15ml centrifuge tube; adding fresh culture medium to 10-15ml, centrifuging at 1500rpm for 5min, and removing supernatant; adding 5ml of culture medium to suspend cells, counting and then performing subsequent tests such as plating and the like;

s7: in vitro expansion of NK cells:

s71: taking K562-ZHX trophoblast cells, performing water bath resuscitation at 37 ℃, washing with PBS buffer solution, and centrifuging to obtain washed K562-ZHX trophoblast cells;

s72: adding PBMC cells into a culture flask containing NK cell complete culture medium, re-suspending, adding washed K562-ZHX trophoblasts, mixing uniformly to form a culture cell fluid I, transferring the culture cell fluid I into an incubator at 37 ℃ and 5% CO₂Culturing under the condition, centrifuging to remove supernatant when culturing to 3 days, supplementing an NK cell complete culture medium, uniformly mixing, and continuing culturing;

s73: when the cells were cultured until day 7, an NK cell complete medium was added to the flask so that the density of PBMC cells was 2X 10⁶Adding washed K562-ZHX feeder cells into the cells per ml, mixing uniformly, and culturing for 7 days to obtain NK cells.

In an optimized scheme, when NK cells are amplified in vitro, the number of PBMC cells and K562-ZHX trophoblasts is 1:1, the cell density ratio of PBMC cells to K562-ZHX feeder cells was 1:1

In an optimized scheme, the NK cell complete culture medium adopted in the steps comprises the following components of inactivated autologous plasma, gentamicin, an L500 culture solution and IL-2 interleukin; the addition amount of the IL-2 interleukin is that each milliliter of the L500 culture solution contains 200 IU and 300IU of the IL-2 interleukin; the addition amount of gentamicin is 50-100IU of gentamicin in per ml of L500 culture solution.

The preparation method of the NK cell complete medium comprises the following steps: adding 5 percent of inactivated autologous plasma into 95 percent of L500 culture solution according to the volume percentage, uniformly mixing, then adding IL-2 interleukin and gentamicin, and uniformly mixing to form the NK cell complete culture medium.

Compared with the prior art, the invention has the following beneficial effects:

OX40 is a costimulatory molecule of NK cells, and after OX40 is combined with OX40 receptor (OX40L), intracellular part of OX40 can recruit tumor necrosis factor related molecules (TRAFs), increase secretion of cytokines including IL-2, IL-4, IL-5 and IFN-gamma and further promote effective proliferation of NK cells; CD48 is a ligand of 2B4, and 2B4 is a key receptor on the surface of NK cells, and CD48 can increase the expression of cytokines including IFN-gamma so as to promote the effective proliferation of the NK cells; however, whether the K562 cells can stably express IL-21, OX40L and CD48 molecules and enhance the proliferation and tumor killing capacity of NK cells is needed to be researched, and the K562 genetically engineered cells simultaneously expressed by the three molecules are used as trophoblasts to culture NK cells, so that the amplification factor, the purity and the killing function of the NK cells are improved.

1. In the invention, IL-21 and OX40L molecules are stably expressed on the surface of K562 cell feeder cells, and CD48 molecules can be expressed at the same time. OX40L and CD48 molecules serve as natural ligands of NK cell activating receptor 2B4 and can promote activation of NK cells, and NKg2d provides main signals for activation of the NK cells and can cover inhibitory signals received by other NK receptors.

2. The invention simultaneously expresses plasmid vectors of three molecules of mbIL-21, OX40L and CD48, then packaging lentiviruses, and sorting and amplifying K562-ZHX cells of mbIL-21+ OX40L + CD48+ by a method of infecting K562 with recombinant lentiviruses, and obtaining trophoblasts required by NK activation after receiving irradiation treatment. The construction of the lentivirus vector carrying the three molecular genes and the corresponding recombinant lentivirus is avoided, so that the workload is saved, the working time is shortened, and a large amount of cost is saved. The method can effectively improve the construction power of the K562-ZHX engineering cells and reduce the construction difficulty.

3. K562 cells simultaneously expressed by the molecules of mbIL-21, OX40L and CD48 are used as trophoblasts for activating and culturing NK cells, the amplification multiple of the NK cells is as high as 300 times, the secretion capacity of tumor killing effector molecules is improved by about 5 times, and the amplification multiple repeatability among different PBMCs is good.

4. The preparation method of the invention can lay a practical foundation for obtaining the NK cells with high purity, high amplification times and high killing activity.

The invention provides a method for rapidly and conveniently preparing trophoblasts for NK cell amplification, which comprises the steps of preparing an engineering cell strain named as K562-ZHX trophoblasts by simultaneously stably expressing a membrane-bound interleukin-21 (mbIL-21), an OX40L molecule and a CD48 molecule by using K562 cells, and irradiating the engineering cell strain by using cobalt-60 to obtain the trophoblasts for amplifying NK cells, wherein the activity of the NK cells prepared by the method is improved by 3 times, the cell amplification energy is improved by 300 times, and a large amount of time and cost can be saved.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:

FIG. 1 is a schematic diagram of the tandem connection of the gene sequence of mbiL-21-PGK-OX40L-T2A-CD48 disclosed in the invention;

FIG. 2 is a pLV-EF1a-IRES-Puro vector plasmid map disclosed in the present invention;

FIG. 3 is a plasmid map of pLV-mbIL-21-OX40L-CD48 lentiviral vector disclosed in the present invention;

FIG. 4 is a schematic representation of the NK cell proliferation curve in the assay of the invention;

FIG. 5 is a schematic diagram showing the detection of the positive rate of NK cells in the detection assay of the present invention;

FIG. 6 is a schematic diagram showing the detection of IFN-. gamma.secretion ability before and after amplification of NK cells in the assay of the present invention;

FIG. 7 shows the expression of IL-21 (anti-human IL-21 polyclonal antibody) measured by Western Blot assay in the assay of the present invention;

FIG. 8 shows the expression of OX40L and CD48 proteins (anti-humanOX40L rabbit polyclonal antibody) detected by Western Blot assay in the assay of the invention;

FIG. 9 shows the expression of OX40L and CD48 proteins (anti-human CD48 polyclonal antibody (Thermo Fisher, Cat # PA5-114716)) as measured by the Western Blot assay in the assay of the invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the following examples of the present application, the raw materials used are as follows:

DMEM complete medium was purchased from Hyclone; the PEI kit is purchased from Thermo company; plasmid purification kits were purchased from Qiagen; the whole gene synthesis is completed by a PCR method by Jinzhi company; the ligase for gene and vector was purchased from TOYOBO; anti-human CD48-FITC, anti-human IL-21-PE antibody from Thermo company, anti-human OX40 antibody from Abcam company; the enzyme digestion kit is purchased from NEB company; the seamless cloning kit is purchased from Suzhou Yuhengshi, Inc.; l500 medium was purchased from Takara.

The NK cell complete culture medium comprises inactivated autologous plasma, gentamicin, an L500 culture solution, IL-2 interleukin and the addition amount of the IL-2 interleukin, wherein each ml of the L500 culture solution contains 200-300IU of IL-2 interleukin, and each ml of the L500 culture solution contains 50-100IU of gentamicin;

Example 1:

(1) construction of pLV-mbIL-21-OX40L-CD48 lentiviral vector plasmid:

s1: obtaining gene coding sequences of human IL-21, human OX40L and human CD 48; wherein the gene coding sequence of the human IL-21 is shown as SEQ ID NO.1, the gene coding sequence of the human OX40L is shown as SEQ ID NO.2, and the gene coding sequence of the human CD48 is shown as SEQ ID NO. 3. The gene coding sequence of the PGK promoter coding sequence is shown as SEQ ID NO. 4; the gene coding sequence of the T2A self-splicing peptide coding sequence is shown as SEQ ID NO. 5.

S2: sequentially connecting a human IL-21 gene coding sequence, a PGK promoter coding sequence, a human OX40L gene coding sequence, a T2A self-splicing peptide coding sequence and a CD48 gene coding sequence in series to obtain an mbiL-21-PGK-OX40L-T2A-CD48 gene sequence; wherein the sequence of the mbiL-21-PGK-OX40L-T2A-CD48 gene is shown in SEQ ID NO. 6; specifically, it can be shown in FIG. 1;

s3: the gene sequence of the mbiL-21-PGK-OX40L-T2A-CD48 is constructed between BamH I and Xma I of a plasmid vector pLV-EF1a-IRES-Puro (purchased by Addgene company) to obtain a pLV-mbiL-21-OX40L-CD48 lentiviral vector plasmid. The plasmid map of pLV-mbIL-21-OX40L-CD48 lentiviral vector can be specifically shown in FIG. 3;

s33: the enzyme-digested mbl-21-PGK-OX 40L-T2A-CD48 gene is connected to the enzyme-digested pLV-EF1a-IRES-Puro skeleton plasmid through Ligation High Ver2 ligase, the plasmid is transformed to competent E.coli (DH5 alpha) cells, and the plasmid vector is purified to prepare pLV-mbl-21-OX 40L-CD48 lentivirus transfer plasmid vector.

(2) Recombinant lentiviruses were prepared using pLV-mbIL-21-OX40L-CD48 lentiviral vector plasmids:

s4: preparation of recombinant lentivirus:

s41: 293T cells in logarithmic growth phase were transferred into 6-well plates containing 0.6X 10 cells per well⁶The cells and 2ml of DMEM complete medium were mixed well and incubated overnight in an incubator at 37 ℃ while 20ml of DMEM complete medium was prepared and incubated overnight under the same conditions for further use.

When the confluence degree of 293FT cells is observed to reach 50% -60% under a microscope, discarding the culture solution in the 6-well plate, and then adding preheated DMEM complete culture solution with the addition of 2 ml/well on the 6-well plate to remove the cells which are not attached to the wall for later use;

s42: according to Invitrogen^TM Lipofectamine^TM3000 instructions for the operation of the transfection reagent(s),

the following day, 1. mu.g of pLV-mbIL-21-OX40L-CD48 lentiviral vector plasmid, 0.6ug of PAX-1 plasmid and 0.4ug of pMD2G plasmid were mixed, and the mixture was diluted to 1. mu.g/. mu.L in a serum-free Opti-MEM medium to obtain a diluted solution;

adding 50 μ L of serum-free Opti-MEM medium and 7 μ L of Lipofectamine 3000 (transfection reagent) into tube A, and mixing by vortexing for 10s to obtain solution A;

adding 250 mu L of serum-free Opti-MEM medium, 4 mu g of diluent and 6 mu L P3000 reagent into the tube B, and vortexing for 10s to obtain solution B;

mixing solution A and solution B, incubating at room temperature for 15min, removing 1mL of culture medium from each well of 293T cell-containing culture plate to make each well have a volume of 1mL, adding 500 μ L of solution A-solution B into each well, and adding liquid against the wall of the well to prevent cell damage; the plate was gently stirred to distribute it evenly. Standing at 37 deg.C for 5% CO₂Incubating for 6 hours under the conditions, aspirating the culture medium containing the liposome-DNA complex from each well, treating the aspirated culture medium with 10% bleaching solution, treating it again, adding DMEM complete medium, and incubating at 37 deg.C and 5% CO₂Continuing incubation under the condition;

s43: after 24 hours of culture, collecting the first cell supernatant, collecting 2mL of cell supernatant from each well, placing into a 15mL conical tube and storing at 4 ℃;

s44: after culturing for 52 hours, the second cell supernatant was collected, and 2mL of the cell supernatant was collected from each well, mixed with the first cell supernatant, and centrifuged to make the total volume of the collected supernatant 4 mL. Centrifuging the supernatant at 2000 r/m for 5min at room temperature to remove cell debris; the supernatant was collected and transferred, and the supernatant was filtered through a 0.45 μm filter to obtain a filtrate, and the cell pellet was discarded.

S5: concentrating the supernatant to obtain the recombinant lentivirus:

transferring the collected supernatant to a sterile centrifuge tube, adding a Lenti-XConcentator, and incubating at 4 ℃ for 12h to concentrate the virus, wherein the volume ratio of the Lenti-X Co centator to the supernatant is 1: and 3, centrifuging at the rotating speed of 1500r/m for 45min, removing the supernatant, diluting according to the dilution multiple of 10-100 times, adding a DMEM basic culture medium to resuspend virus precipitates, and subpackaging the virus at 50 mu L/tube for later use to obtain the recombinant lentivirus.

S5, concentrating, and performing titer detection, wherein the titer detection comprises the following specific steps: infecting the recombinant lentivirus into k562 cells, and detecting the titer of the recombinant lentivirus by adopting a flow cytometry detection method.

The method comprises the following specific steps: adding k562 cells into a counting hole on a Countstar counter, wherein the adding amount of the k562 cells is 1105 for later use; diluting the recombinant lentivirus by 10-100 times by using RPMI-1640 complete culture solution to form diluted virus solution, then respectively adding the diluted virus solution into counting holes of a countstar counter with k562 cells, wherein the addition amount of the diluted virus solution is 1ul, 3ul, 10ul and 30ul respectively, and finally placing the countstar counter in a 5% carbon dioxide incubator at 37 ℃ for incubation for 48 hours;

20ul (i.e. containing 1X 10) was added to a 1ml centrifuge tube⁶Individual cells), 200ul pbs buffer, resuspension, 1ul anti-human CD48-FITC antibody added under ice bath conditions, and incubation for 15minAdding PBS to rotate at 1500rpm, centrifuging for 5min, and washing the cells twice; and adding 200ul pbs buffer solution, carrying out heavy suspension, carrying out flow cytometry detection, infecting k562 cells by the concentrated recombinant lentivirus, and determining that the number of infected positive k562 cells is equal to the number of virus particles when the infection efficiency of the k562 cells is less than 10%.

Sequentially adding the recombinant lentivirus and 6ug/ml polybrene according to the final MOI value of the composite recombinant lentivirus of 5, and uniformly mixing to form a recombinant lentivirus mixture;

infecting K562 cells by using a recombinant lentivirus mixture, sorting IL-21+ OX40L + CD48+ K562 cells by flow cytometry 72 hours after infection, and obtaining K562-mIL-21-OX40L-CD48 by subcloning to obtain a K562-ZHX engineering cell strain;

and performing cobalt-60 irradiation sterilization on the K562-ZHX engineering cell strain to obtain the K562-ZHX trophoblast. Wherein the dosage of the cobalt in the cobalt-60 irradiation sterilization is 100 Gy; the cell density of K562-ZHX feeder cells is 1X 10⁷One per ml. Freezing and storing at-20 deg.C.

Example 2:

taking the NK cells prepared in the example 1, and carrying out in-vitro amplification culture, wherein the specific steps are as follows:

s6: and (3) PBMC cell extraction:

the method comprises the following specific steps:

s7: in vitro expansion of NK cells:

s73: when the cells were cultured until day 7, an NK cell complete medium was added to the flask so that the density of PBMC cells was 2X 10⁶Adding washed K562-ZHX trophoblasts into the cells per ml, mixing the cells uniformly, and culturing the cells for 7 days to obtain NK cells. When NK cells are amplified in vitro, the number of PBMC cells and K562-ZHX trophoblasts is 1:1, the cell density ratio of PBMC cells to K562-ZHX feeder cells was 1: 1.

control group: k562 cells were taken as feeder cells.

And (3) detection test:

1. K562-ZHX engineered cell lines were prepared according to the method disclosed in example 1, and expression of mbiL-21, OX40L and CD48 in K562-ZHX cells was detected by Western Blot as follows:

(1) harvesting of k562-ZHX cells 1X 10⁶Adding 100ul RIPA lysate (containing 1% PMSF), mixing by vortex for 30s, and standing on ice for 30 min; centrifuging the sample at 4 ℃ and 12000rpm for 30min, and transferring the supernatant into a new centrifuge tube;

(2) adding 6 × protein loading to working concentration (1 ×), heating at 100 deg.C for 6min to denature protein, and directly subjecting the sample to SDS electrophoresis or storing in refrigerator at-20 deg.C for use.

(3) Respectively taking 40 mu L of the cell lysate, loading the cell lysate to 10% SDS-PAGE at 120V for 120min to finish electrophoresis;

(4) transferring the protein in SDS-PAGE to a PVDF membrane by a constant current of 350mA for 120 minutes;

(5) placing the PVDF membrane after membrane conversion in a sealing solution containing 5% of skimmed milk, and sealing overnight at 4 ℃;

(6) the PVDF membrane after blocking is respectively put into 10ml primary antibodies containing rabbit anti-human IL-21(Thermo Fisher, Cat # PA5-115407), rabbit anti-human OX40L (Thermo Fisher, # PA 5-2452) and rabbit anti-human CD48(Thermo Fisher, # PA 5-2452) with the concentration of 0.5ug/ml, and is incubated for 1h at room temperature;

(7) the PVDF membrane incubated with the primary antibody was washed 5 times with TBST for 5 minutes each time;

(8) putting the washed PVDF membrane into 10ml of goat anti-rabbit IgG secondary antibody (abcam, ab97051) which is diluted according to the proportion of 1:100000, and incubating for 1h at room temperature; TBST washing for 5 times, each time for 5 min;

(9) after addition of ECL hypersensitivity developing solution, photographs were taken with a solar imaging system (Shanghai Tiannen technologies Co., Ltd. # Tanon-4600 SF).

And (4) conclusion: by the method, the expression of IL-21, OX40L and CD48 proteins can be detected by the prepared K562-ZHX engineering cells, and the K562-ZHX cell strains can be successfully constructed as shown in figures 7-9.

2. Taking K562 cells (K562/NK) and K562-ZHX (K562-ZHX/NK) as NK cells of the feeder cells for 14 days, and specifically operating according to the scheme disclosed in example 2; NK cells cultured for 14 days were counted to prepare an NK cell proliferation graph, and as shown in FIG. 4, it was found that NK cells proliferated by 300 times. Compared with the method that the K562 cell is taken as a trophoblast, the amplification efficiency of the NK cell is improved by 10 times.

3. And (3) detecting the positive rate of NK cell increase:

taking K562 cells (K562/NK) and K562-ZHX (K562-ZHX/NK) as NK cells of feeder cells for 14 days, respectively, specifically, according to the scheme disclosed in example 2, taking the NK cells cultured for 14 days, diluting the cell density to 1.0 × 106/ml, staining with CD3-FITC and CD56CD16-PE antibodies, and detecting by a flow cytometer, wherein the positive rate of the NK cells is 92.2% and is far higher than that of the NK cells induced and activated by K562; according to the invention, the NK cells are cultured by adopting the k562-ZHX feeder cells, so that the positive rate of the NK cells is greatly improved.

The specific detection schematic diagram is shown in figure 5.

4. Detection of cytokine secretion ability of NK cells:

taking K562 cells (K562/NK) and K562-ZHX (K562-ZHX/NK) as NK cells of the feeder cells for 14 days, and specifically operating according to the scheme disclosed in example 2; the expression level of IFN-effector cytokines was determined by ELISA. Detection shows that the IFN-level of NK cell expression induced by K562-ZHX is obviously higher than that of NK cell induced by K562 serving as a trophoblast by more than 6 times. The invention can obtain NK cells with stronger immune response capability.

The specific detection schematic diagram is shown in fig. 6.

And (4) conclusion: the invention provides a method for rapidly and conveniently preparing trophoblasts for NK cell amplification, which comprises the steps of simultaneously stably expressing membrane-bound interleukin-21 (mbIL-21), OX40L molecules and CD48 molecules by using K562 cells to prepare an engineering cell strain named as K562-ZHX trophoblasts, and irradiating the engineering cell strain by using cobalt-60 to obtain the trophoblasts for NK cell amplification, wherein the activity of the NK cells prepared by the method is improved by 3 times, the cell amplification energy is improved by more than 300 times, and a large amount of time and cost can be saved.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Mediterranean gorges (Fujian) cell Biotech Co., Ltd

<120> preparation method and application of trophoblast modified by genetic engineering

<160> 6

<170> SIPOSequenceListing 1.0

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<211> 462

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgagatcca gtcctggcaa catggagagg attgtcatct gtctgatggt catcttcttg 60

gggacactgg tccacaaatc aagctcccaa ggtcaagatc gccacatgat tagaatgcgt 120

caacttatag atattgttga tcagctgaaa aattatgtga atgacttggt ccctgaattt 180

ctgccagctc cagaagatgt agagacaaac tgtgagtggt cagctttttc ctgctttcag 240

aaggcccaac taaagtcagc aaatacagga aacaatgaaa ggataatcaa tgtatcaatt 300

aaaaagctga agaggaaacc accttccaca aatgcaggga gaagacagaa acacagacta 360

acatgccctt catgtgattc ttatgagaaa aaaccaccca aagaattcct agaaagattc 420

aaatcacttc tccaaaaggt atctacctta agtttcattt ga 462

<210> 2

<211> 552

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggaaaggg tccaacccct ggaagagaat gtgggaaatg cagccaggcc aagattcgag 60

aggaacaagc tattgctggt ggcctctgta attcagggac tggggctgct cctgtgcttc 120

acctacatct gcctgcactt ctctgctctt caggtatcac atcggtatcc tcgaattcaa 180

agtatcaaag tacaatttac cgaatataag aaggagaaag gtttcatcct cacttcccaa 240

aaggaggatg aaatcatgaa ggtgcagaac aactcagtca tcatcaactg tgatgggttt 300

tatctcatct ccctgaaggg ctacttctcc caggaagtca acattagcct tcattaccag 360

aaggatgagg agcccctctt ccaactgaag aaggtcaggt ctgtcaactc cttgatggtg 420

gcctctctga cttacaaaga caaagtctac ttgaatgtga ccactgacaa tacctccctg 480

gatgacttcc atgtgaatgg cggagaactg attcttatcc atcaaaatcc tggtgaattc 540

tgtgtccttt ga 552

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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atgtgctcca gaggttggga ttcgtgtctg gctctggaat tgctactgct gcctctgtca 60

ctcctggtga ccagcattca aggtcacttg gtacatatga ccgtggtctc cggcagcaac 120

gtgactctga acatctctga gagcctgcct gagaactaca aacaactaac ctggttttat 180

actttcgacc agaagattgt agaatgggat tccagaaaat ctaagtactt tgaatccaaa 240

tttaaaggca gggtcagact tgatcctcag agtggcgcac tgtacatctc taaggtccag 300

aaagaggaca acagcaccta catcatgagg gtgttgaaaa agactgggaa tgagcaagaa 360

tggaagatca agctgcaagt gcttgaccct gtacccaagc ctgtcatcaa aattgagaag 420

atagaagaca tggatgacaa ctgttatctg aaactgtcat gtgtgatacc tggcgagtct 480

gtaaactaca cctggtatgg ggacaaaagg cccctcccaa aggagctcca gaacagtgtg 540

cttgaaacca cccttatgcc acataattac tccaggtgtt atacttgcca agtcagcaat 600

tctgtgagca gcaagaatgg cacggtctgc ctcagtccac cctgtaccct ggcccggtcc 660

tttggagtag aatggattgc aagttggcta gtggtcacgg tgcccaccat tcttggcctg 720

ttacttactt aa 732

<210> 4

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg 60

gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc 120

gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca 180

ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac 240

gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt 300

ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg 360

gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct 420

ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt 480

cctcatctcc gggcctttcg 500

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

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gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54

<210> 6

<211> 2399

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggatccgcca ccatgagatc cagtcctggc aacatggaga ggattgtcat ctgtctgatg 60

gtcatcttct tggggacact ggtccacaaa tcaagctccc aaggtcaaga tcgccacatg 120

attagaatgc gtcaacttat agatattgtt gatcagctga aaaattatgt gaatgacttg 180

gtccctgaat ttctgccagc tccagaagat gtagagacaa actgtgagtg gtcagctttt 240

tcctgctttc agaaggccca actaaagtca gcaaatacag gaaacaatga aaggataatc 300

aatgtatcaa ttaaaaagct gaagaggaaa ccaccttcca caaatgcagg gagaagacag 360

aaacacagac taacatgccc ttcatgtgat tcttatgaga aaaaaccacc caaagaattc 420

ctagaaagat tcaaatcact tctccaaaag gtatctacct taagtttcat tttttgggtg 480

ctggtggtgg ttggtggagt cctggcttgc tatagcttgc tagtaacagt ggcctttatt 540

attttctggg tgtaggggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt 600

tagcagcccc gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca 660

catccaccgg taggcgccaa ccggctccgt tctttggtgg ccccttcgcg ccaccttcta 720

ctcctcccct agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac 780

aaatggaagt agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga 840

agcgggtagg cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc 900

agaggctggg aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg 960

gcgcccgaag gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg 1020

cgctgttctc ctcttcctca tctccgggcc tttcggccac catggaaagg gtccaacccc 1080

tggaagagaa tgtgggaaat gcagccaggc caagattcga gaggaacaag ctattgctgg 1140

tggcctctgt aattcaggga ctggggctgc tcctgtgctt cacctacatc tgcctgcact 1200

tctctgctct tcaggtatca catcggtatc ctcgaattca aagtatcaaa gtacaattta 1260

ccgaatataa gaaggagaaa ggtttcatcc tcacttccca aaaggaggat gaaatcatga 1320

aggtgcagaa caactcagtc atcatcaact gtgatgggtt ttatctcatc tccctgaagg 1380

gctacttctc ccaggaagtc aacattagcc ttcattacca gaaggatgag gagcccctct 1440

tccaactgaa gaaggtcagg tctgtcaact ccttgatggt ggcctctctg acttacaaag 1500

acaaagtcta cttgaatgtg accactgaca atacctccct ggatgacttc catgtgaatg 1560

gcggagaact gattcttatc catcaaaatc ctggtgaatt ctgtgtcctt gagggcagag 1620

gaagtcttct aacatgcggt gacgtggagg agaatcccgg cccttgctcc agaggttggg 1680

attcgtgtct ggctctggaa ttgctactgc tgcctctgtc actcctggtg accagcattc 1740

aaggtcactt ggtacatatg accgtggtct ccggcagcaa cgtgactctg aacatctctg 1800

agagcctgcc tgagaactac aaacaactaa cctggtttta tactttcgac cagaagattg 1860

tagaatggga ttccagaaaa tctaagtact ttgaatccaa atttaaaggc agggtcagac 1920

ttgatcctca gagtggcgca ctgtacatct ctaaggtcca gaaagaggac aacagcacct 1980

acatcatgag ggtgttgaaa aagactggga atgagcaaga atggaagatc aagctgcaag 2040

tgcttgaccc tgtacccaag cctgtcatca aaattgagaa gatagaagac atggatgaca 2100

actgttatct gaaactgtca tgtgtgatac ctggcgagtc tgtaaactac acctggtatg 2160

gggacaaaag gcccctccca aaggagctcc agaacagtgt gcttgaaacc acccttatgc 2220

cacataatta ctccaggtgt tatacttgcc aagtcagcaa ttctgtgagc agcaagaatg 2280

gcacggtctg cctcagtcca ccctgtaccc tggcccggtc ctttggagta gaatggattg 2340

caagttggct agtggtcacg gtgcccacca ttcttggcct gttacttact taacccggg 2399

Claims

1. A method for preparing trophoblast modified by genetic engineering is characterized by comprising the following steps: the method comprises the following steps:

(1) constructing pLV-mbIL-21-OX40L-CD48 lentiviral vector plasmid;

(3) and extracting recombinant lentivirus, infecting K562 cells, performing subcloning after sorting, and sterilizing to obtain K562-ZHX trophoblast cells.

2. The method for preparing trophoblast cells modified by genetic engineering according to claim 1, wherein the method comprises the following steps: the step (1) is specifically as follows:

s3: the gene sequence of the mbiL-21-PGK-OX40L-T2A-CD48 is constructed into a plasmid vector to obtain a pLV-mbiL-21-OX40L-CD48 lentiviral vector plasmid.

3. The method for preparing trophoblast cells modified by genetic engineering according to claim 2, wherein: in step S1, the gene coding sequence of human IL-21 is shown in SEQ ID NO.1, the gene coding sequence of human OX40L is shown in SEQ ID NO.2, and the gene coding sequence of human CD48 is shown in SEQ ID NO. 3.

4. The method for preparing trophoblast cells modified by genetic engineering according to claim 2, wherein: step S3 specifically includes:

s33: the enzyme-digested mbl-21-PGK-OX 40L-T2A-CD48 gene is connected to the enzyme-digested pLV-EF1a-IRES-Puro skeleton plasmid through Ligation High Ver2 ligase, the plasmid is transformed to competent E.coli (DH5 alpha) cells, and the plasmid is purified to prepare pLV-mbl-21-OX 40L-CD48 lentiviral vector plasmid.

5. The method for preparing trophoblast cells modified by genetic engineering according to claim 1, wherein the method comprises the following steps: the step (2) is specifically as follows:

s4: preparation of recombinant lentivirus:

s41: 293T cells were seeded in a culture plate containing DMEM complete medium at 37 ℃ with 5% CO₂Incubating under the condition to obtain a culture plate containing 293T cells;

s43: after 24 hours of culture, collecting the first cell supernatant;

adding DMEM complete culture solution at 37 deg.C and 5% CO₂Continue under the conditionIncubation;

s5: and concentrating the supernatant to obtain the recombinant lentivirus.

6. The method for preparing trophoblast cells modified by genetic engineering according to claim 1, wherein the method comprises the following steps: the step (3) is specifically as follows:

and performing cobalt-60 irradiation sterilization on the K562-ZHX engineering cell strain to obtain the K562-ZHX trophoblast.

7. The method for preparing trophoblast cells modified by genetic engineering and the use thereof according to claim 5, wherein the method comprises the following steps: step S5 specifically includes: filtering the collected supernatant, adding a Lenti-XConcentrator, incubating at 4 ℃ for 12h, centrifuging at 1500r/m for 45min, removing the supernatant, diluting, resuspending, and packaging to obtain recombinant lentivirus;

8. Use of the trophoblast prepared according to the preparation method of any one of claims 1 to 7, wherein: the feeder cells are used for in vitro amplification culture of NK cells.

9. Use according to claim 8, characterized in that: the method comprises the following specific steps:

s6: extracting PBMC cells;

s7: in vitro expansion of NK cells:

s72: adding PBMC cells into a culture flask containing NK cell complete culture medium, re-suspending, adding washed K562-ZHX trophoblasts, mixing uniformly to form a culture cell fluid I, transferring the culture cell fluid I into an incubator at 37 ℃ and 5% CO₂Culturing under the condition, centrifuging to remove supernatant when culturing to 3 days, supplementing an NK cell complete culture medium, and continuing culturing after uniformly mixing;

s73: when the cells were cultured until day 7, the NK cell complete medium was added to the flask so that the cell density of PBMC cells was 2X 10⁶Adding washed K562-ZHX feeder cells into the cells per ml, mixing uniformly, and culturing for 7 days to obtain NK cells.

10. Use according to claim 9, characterized in that: when NK cells are amplified in vitro, the number of PBMC cells and K562-ZHX trophoblasts is 1:1, the cell density ratio of PBMC cells to K562-ZHX feeder cells was 1: 1.