CN114045306B - Luciferase complementary system plasmid reflecting immune synapse-related signal protein, stable transgenic cell strain and application thereof - Google Patents

Abstract

The invention provides a luciferase complementary system plasmid reflecting an immune synapse-related signal protein, a stable transgenic cell strain and application thereof in the biological activity measurement of a CAR modified cell. The plasmids include a plasmid containing ICAM-1 gene sequence and luciferase sequence Nluc on pGL3-Control plasmid, a plasmid containing SHP2 gene sequence and luciferase sequence Cluc on pGL3-Control plasmid, and a plasmid containing CD19 gene sequence. The stable transgenic cell line IS 293-IS-CD19, which IS obtained by transfecting the plasmid into human embryonic kidney cells HEK-293 cells, and stably integrates ICAM-1-Nluc and Cluc-SHP2 reporter genes and CD19 antigen. The cell strain can reflect the immune synapse forming ability in real time, has simple measurement process and accurate result, can be used for evaluating the biological activity of the CAR modified cells based on the immune synapse forming ability, has simple evaluation method, short time consumption, low detection cost and stability of measurement results, can be beneficial to quality control and clinical application of biological active drugs, and has higher application value.

Description

Luciferase complementary system plasmid reflecting immune synapse-related signal protein, stable transgenic cell strain and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a luciferase complementary system plasmid reflecting immune synapse related signal proteins, a stable transgenic cell strain and application thereof in CAR modified cell biological activity measurement.

Background

In recent years, cell preparations are gradually evolving into maturity. Cell preparations are a collective term for preparations, drugs or products for the treatment of diseases based on different cells, and are third generation drugs, also called "living drugs", following small molecule chemical drugs, macromolecular protein drugs. The cell products on the market are mainly traditional somatic cells, immune cells, different stem cells and the like. The in vitro biological activity detection of cell preparations is an important indicator for evaluating cell activity.

Chimeric antigen receptor (chimeric antigen receptor, CAR) T cell immunotherapy (CAR-T therapy) in immune cell preparation treatment is a novel accurate targeted therapy for treating tumors, and human internal immunodeficiency is repaired by CAR-T cells, so that tumor diseases are relieved and even thoroughly cured. As the most advanced cellular immunotherapy internationally at present, CAR-T therapy has been approved for clinical treatment in the united states, and china has gradually accepted clinical trial applications since 2018, and two domestic CAR-T therapies from the combination of medicine and medicine Ming-juno are reported to the market before and after 2021. Different types of CARs expressed in different effector cells show the effectiveness of different CAR-modified cells, which must be accurately assessed prior to clinical trials. The traditional methods for detecting the cytotoxicity mediated by the CAR modified cells have respective defects, such as an isotope release method, a time-resolved fluorescence method and a reporter gene transfection method in an in-vitro direct killing activity detection method, and have the defects of experimental environment limitation, low signal-to-noise ratio, long period and the like. The indirect killing activity detection mainly comprises a cytokine release method and a cell activity detection method, but also has the defects of poor specificity and the like. There is therefore a need to select new detection targets, and to establish new detection techniques in order to more rapidly evaluate the effectiveness of CAR modified cells.

Immune synapses are close contacts between T cells and target cell surface molecules through receptor-ligand interactions, forming a precise structure important for performing T cell immunological functions. The formation of this structure helps T cells to resolve potential antigens, increasing the affinity between T cell antigen receptors and MHC-antigen peptide complexes, thus initiating antigen recognition and activation of T cells, and studies have shown that proliferation can only occur in T cells that form immune synaptosomes. CAR-T is used as a T cell population which is endowed with immune cell specific antigen receptor through genetic modification, and can also form functional immune synapse. Recent studies have shown that the quality of the formation of the immune synapses of CAR-T can be used as an indicator for detecting the killing activity of CAR-T, but can only be qualitatively analyzed by means of immunofluorescence techniques at present by evaluating F-actin of effector cells, aggregation of tumor antigens, polarization of dissolved particles and average fluorescence intensity of key signal molecular distribution in the immune synapse structure. This approach can only be studied at the single CAR modified cellular level, suffers from low sample analysis throughput, and can lead to significant variability in results due to different immunofluorescence signal reference criteria.

In a traditional T cell receptor mediated immune synaptic structure, the outer Zhou Chao molecule activation cluster region formed by lymphocyte function-associated antigen-1 (LFA-1) of effector cells binding to the target cell's intracellular adhesion receptor 1 (ICAM-1) helps to maintain a more durable T cell interaction with antigen presenting cells, thereby effectively killing the target cells. LFA-1/ICAM-1 direct binding was not reported in CAR-T cell mediated immune synapse structures, and the effect on downstream cross-proteins was not studied.

An immune synapse structure is a complex formation of many proteins, involving interactions of many proteins. The luciferase complementation technique is a technique for detecting an interacting protein signal by fusing N-terminal and C-terminal fragments of luciferase, respectively, with an interacting protein to be detected. When the proteins interact, the interaction may cause the N-and C-terminal fragments of the luciferase to recombine into an active luciferase, which in turn catalyzes the production of chemiluminescence from a substrate (e.g., luciferin). There is no report on the use of this technology in CAR modified cell biological activity assays.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides a luciferase complementary system plasmid reflecting an immune synapse-related signal protein, a stable transgenic cell line, a construction method thereof and application thereof in biological activity measurement of CAR modified cells based on immune synapse formation capacity, wherein the cell line can stably integrate ICAM-1-Nluc, a Cluc-SHP2 reporter gene and a CD19 antigen, can be effectively killed by CAR-T cells taking CD19 as an antigen and can activate CAR-Jurkat (CAR-J) cells to generate IL-2 (Jurkat cells have no direct killing capacity and generate IL-2 after being activated). The invention detects the activity change of luciferase expressed by the cell strain through incubation with the CAR modified cell to reflect the immune synapse forming capacity of the CAR modified cell, and evaluates the effectiveness of the CAR modified cell through the biological activity.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a luciferase complementary system plasmid reflecting an immune synapse-related signal protein, which includes a plasmid containing ICAM-1 gene sequence and luciferase sequence Nluc on pGL3-Control plasmid, a plasmid containing SHP2 gene sequence and luciferase sequence Cluc on pGL3-Control plasmid, and a plasmid containing CD19 gene sequence.

The second aspect of the present invention provides a method for preparing the above plasmid, comprising the steps of:

step one, connecting a target gene sequence of a commercialized ICAM-1 plasmid and a luciferase sequence Nluc on a pGL3-Control plasmid with a linker through a FLAG tag sequentially by utilizing a molecular cloning technology, and constructing the linked ICAM-1 plasmid on a vector;

sequentially connecting a luciferase sequence Cluc on pGL3-Control plasmid and a commercialized SHP2 plasmid target gene sequence through His tag with a connector by using a molecular cloning technology, and constructing the connection on a vector;

and thirdly, constructing a commercialized CD19 plasmid target gene sequence on a pLV-Hygro vector by utilizing a molecular cloning technology.

Further, the joints adopted in the first step and the second step are 3X flexible linker Gly4/Ser.

Further, the sequence ICAM-1-FLAG-Nluc with the sequence of SEQ ID No.1 is obtained by ligation in the first step.

Further, the sequence Cluc-His-SHP2 with the sequence SEQ ID No.2 is obtained by connection in the second step.

Further, the vector used in the first step is pcDNA3.1 vector or pLV-Neo vector.

Further, the vector adopted in the second step is pcDNA3.1 vector or pLVX-Puro vector.

In a third aspect of the present invention, there IS provided a stably transfected cell line, 293-IS-CD19, obtained by transfecting the above plasmid into human embryonic kidney cells HEK-293 cells, stably integrating ICAM-1-Nluc with a Cluc-SHP2 reporter gene and CD19 antigen.

The fourth aspect of the present invention provides a method for constructing the stable transgenic cell line, comprising the steps of:

step one, transfecting the plasmid into HEK-293 cells by using a slow virus transfection technology;

and step two, adding puromycin, geneticin and hygromycin into the cells transfected in the step two, carrying out pressurized screening, enriching positive cells, amplifying and placing in a liquid nitrogen medium for freezing.

Further, the screening concentration of puromycin was 2. Mu.g/mL, the screening concentration of geneticin was 1mg/mL, and the screening concentration of hygromycin was 200. Mu.g/mL.

In a fifth aspect, the present invention provides a method for evaluating biological activity of a CAR-modified cell based on immune synapse-forming ability, comprising the steps of:

step one, adding CAR modified cells into an orifice plate inoculated with the stable transgenic cell strain for co-incubation;

step two, rapidly adding a chemiluminescent substrate D-luciferin to the pore plate, and immediately reading a fluorescence value in real time after shaking for a short time at room temperature;

and thirdly, evaluating the biological activity of the CAR modified cells based on the immune synapse forming capacity through signal difference.

Further, the CAR-modified cell is a CAR-J cell or a CAR-T cell.

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

(1) Compared with the traditional detection method, the detection method is simple, the time consumption is short, the detection cost is low, and stable rotation of the strain is beneficial to ensuring the stability of the detection result;

(2) The invention takes an immune synapse signal path as a research object, designs a cell strain of an interaction luciferase reporter gene capable of reacting to protein interaction change after immune synapse formation, and is capable of reacting to immune synapse formation capacity in real time, simple in measuring process and accurate in result.

In summary, the HEK-293 cell strain constructed by the invention can stably integrate the interaction luciferase reporter gene which reacts with the protein interaction change after immune synapse formation, and can influence the complementary luciferase activity change expressed by target cells after the HEK-293 cell strain forms immune synapses with CAR modified cells. The invention establishes a simpler and faster CAR modified cell in-vitro biological activity evaluation method by using the cell strain, which is beneficial to the quality control and clinical application of biological active drugs and has higher application value.

Drawings

FIG. 1 shows the results of identifying transient expressed proteins and key proteins of the immune synapse signal pathway in HEK-293 cells according to an embodiment of the invention; wherein, FIG. 1A shows the Western blot identification results of ICAM-1-FLAG-Nluc, cluc-His-SHP2 and CD19 plasmids transiently expressed on pcDNA3.1 vector by HEK-293, the left side is negative control HEK-293, the right side is HEK-293 transfected with the above three plasmids, and beta-action is a loading internal reference protein; FIG. 1B shows the effect of the LFA-1/ICAM-1 inhibitor lifitegrast on CD19 CAR-T cell killing target cell HEK-293 (transfected with the three plasmids described above); FIG. 1C shows the effect of the LFA-1/ICAM-1 inhibitor lifitegrast on IL-2 secretion by CD19 CAR-J cells contacting target cells HEK-293 (transfected with the three plasmids described above);

FIG. 2 shows the results of the transient expression signal protein interaction and activity verification and identification of HEK-293 cells in an embodiment of the present invention; wherein, FIG. 2A shows the Western blot immunoprecipitation identification results of ICAM-1-FLAG-Nluc and Cluc-His-SHP2 plasmids transiently expressed by HEK-293 on pcDNA3.1 vector; FIG. 2B shows that ICAM-1 (P2A 4) antibody (which blocks direct binding of ICAM-1 to SHP 2) effectively inhibits luciferase activity resulting from interaction of ICAM-1-FLAG-Nluc with the Cluc-His-SHP2 fusion protein;

FIG. 3 shows the results of changes in the activity of luciferase after ICAM-1 activation of stable transformant 293-IS-CD19 according to an embodiment of the present invention; wherein FIG. 3A shows that, after ICAM-1 of 293-IS-CD19 cells IS cross-linked by an antibody (mimic immune synapse LFA-1 activates ICAM-1), cluc-His-SHP2 has reduced interaction with ICAM-1-FLAG-Nluc, CD19+Cluc-SHP2 IS a negative control; FIG. 3B shows that ICAM-1 of 293-IS-CD19 cells has reduced intracellular complementary luciferase activity after antibody cross-linking, and that Control, anti-IgG F (ab ') 2, and IgG+anti-IgG F (ab') 2 are negative controls;

FIG. 4 shows the results of luciferase activity change after incubation of stable transgenic strain 293-IS-CD19 with CAR-J (FIG. 4A) and CAR-T (FIG. 4B) cells according to an embodiment of the invention.

Detailed Description

The invention researches the change relation between the activation of target cell ICAM-1 and the combination of protein tyrosine phosphatase-2 (SHP 2) of downstream combined protein SH2 in the cell immune synapse structure of the CAR, and constructs an ICAM-1/SHP2 interaction signaling probe by utilizing a luciferase complementation technology. And the probe is used for researching the dynamic change of ICAM-1/SHP2 protein interaction in target cells in the formation process of the immune synapse of the CAR modified cells, so as to evaluate the effectiveness of the CAR modified cells. Based on the above, the invention provides a luciferase complementary system plasmid reflecting the immune synapse-related signal protein, a stable transgenic cell strain and application thereof in the biological activity measurement of CAR modified cells.

The present invention will be described in detail and specifically by way of the following specific examples and drawings to provide a better understanding of the present invention, but the following examples do not limit the scope of the present invention.

The methods described in the examples are carried out using conventional methods, if not specified, and the reagents used are, if not specified, conventional commercially available reagents or reagents formulated by conventional methods.

Example 1

The embodiment provides a luciferase complementary system plasmid reflecting an immune synapse-related signal protein and verifies the plasmid, and the construction and verification process of the plasmid comprises the following steps:

(1) Constructing a plasmid;

1) The sequence ICAM-1-FLAG-Nluc (the target gene sequence of the commercial ICAM-1 plasmid and the luciferase sequence (Nluc, aa 2-416) on pGL3-Control plasmid are sequentially connected with 3X flexible linker Gly4/Ser by FLAG tag, and SEQ ID No. 1) is connected to pcDNA3.1 vector by enzyme cleavage sites Kpn I and Xba I to obtain plasmid pc3.1-ICAM-1-FLAG-Nluc.

2) The sequence Cluc-His-SHP2 (luciferase sequence (Cluc, aa 398-550) on pGL3-Control plasmid and the target gene sequence of commercialized SHP2 plasmid are connected with 3X flexible linker Gly4/Ser by His tag in sequence by using molecular cloning technology, and SEQ ID No. 2) is connected to pcDNA3.1 vector by enzyme cleavage sites Kpn I and Xba I respectively to obtain plasmid pc3.1-Cluc-His-SHP2.

3) All plasmids were sequenced to verify correct.

(2) Cell transfection;

HEK-293 cell plates were allowed to stand overnight. And (3) respectively transfecting HEK-293 cells with human CD19 expression plasmids on pc3.1-ICAM-1-FLAG-Nluc, pc3.1-Cluc-His-SHP2 and a purchased pcDNA3.1 vector by using a Lipofectamine 3000 reagent, placing the HEK-293 cells in a carbon dioxide incubator for culturing for 6 hours, changing liquid, collecting partial cells after 48 hours to prepare Western blot protein samples, and carrying out a CAR modified cell killing activity test on the rest cells.

(3) Identifying the transient expression of the cell fusion protein;

cell lysate RIPA HEK-293 cells transfected with the three plasmids are treated to prepare a Westernblot protein loading buffer solution. The fusion protein expression was detected by using the original protein antibody and the tag protein antibody, respectively, by using a chemiluminescence method, and the result is shown in FIG. 1A, and HEK-293 cells normally express the target protein.

(4) Identifying the CAR-T cell immune synapse key signal protein;

the CAR-T cells were tested for their ability to kill target cells using the Eu-TDA release assay. After incubating fluorescent dyes in HEK-293 target cells transfected with the three plasmids, the cells were seeded in a 96-well plate with a U-shaped bottom at a proper density, CAR-T cells were pre-incubated with 1. Mu.M lifitegrast (LFA-1/ICAM-1 inhibitor) for one hour, added to the target cells at different densities, the supernatant was collected three hours later, substrates were added, signal values were read using a time-resolved fluoroimmunoassay analyzer, and the cell dye release rate was analyzed. The results are shown in FIG. 1B, wherein the lifitegrast effectively inhibits the killing ability of CAR-T to HEK-293 cells transfected with three plasmids, indicating the importance of LFA-1/ICAM-1 binding in the immune synapse structure of CAR-T.

(5) Identifying the CAR-J cell immune synapse key signal protein;

the CAR-J cells indirectly reflect the ability to kill target cells using IL-2 release to detect effector cell activation. HEK-293 target cells transfected with the three plasmids were seeded in a 96-well plate with a U-shaped bottom at an appropriate density, CAR-J cells were pre-incubated for 1. Mu.M lifitegrast for one hour and then added to the target cells at a 10-fold cell density, and supernatants were collected three hours and six hours later, respectively, and the supernatant content was measured using an IL-2ELISA kit to analyze the activation of effector cells. The results are shown in FIG. 1C, which shows the importance of LFA-1/ICAM-1 binding in the immune synapse structure of CAR-J, in that the Lifitegrast effectively inhibits the IL-2 secretion capacity of CAR-J on HEK-293 cells transfected with three plasmids.

In summary, the killing potential of CAR modified cells can be effectively inhibited by inhibiting LFA-1/ICAM-1 binding in the immune synaptic structure, demonstrating the importance of ICAM-1 activation of target cells in the immune synaptic structure.

Example 2

In this example, the immune synapse key signal protein plasmid constructed in example 1 was subjected to intracellular activity verification, and specific operation steps and results are as follows:

(1) Westernblot immunoprecipitation assay

The collected cells were lysed on ice using Co-IP lysate (20 mM Tris-HCl, pH 7.5, 150mM NaCl,1mM EDTA and 1% TRITON X-100, containing protease and phosphatase inhibitor) for 30min, centrifuged at 12000rpm for 10min at 4℃and the supernatant incubated with FLAG-M2 beads overnight at 4 ℃. The beads were then rinsed three times with TBS, loaded with loading buffer and boiled at 97℃for 5min. And then carrying out subsequent experiments according to the Western blot step.

As a result, as shown in FIG. 2A, the fusion protein ICAM-1-FLAG-Nluc and Cluc-His-SHP2 directly interacted with each other in HEK-293 cells.

(2) Fusion protein complementation luciferase activity verification

HEK-293 cells transfected with the above three plasmids were preincubated with 100. Mu.g/mL IgG or ICAM-1 (P2A 4) antibody, followed by 1X 10 ⁵ Per mL,100 μl/well was added to 96 well plates and the final concentration of 3mM D-luciferin substrate was added immediately and the change in fluorescence signal value was read immediately in real time.

As shown in FIG. 2B, ICAM-1 (P2A 4) antibodies effectively inhibited the fluorescent signal values generated by ICAM-1/SHP2 interactions, indicating that the intracellular fluorescent signal was derived from the fusion protein interactions. The specificity of the fusion plasmid expression probe protein is proved, and the fusion plasmid expression probe protein has biological activity value.

In summary, the specificity of the fluorescent probe was demonstrated by the luciferase activity that can be produced by direct interaction of ICAM-1-FLAG-Nluc with the Cluc-His-SHP2 fusion protein in HEK-293 cells.

Example 3

The embodiment provides a stable transgenic strain 293-IS-CD19, and the specific construction method comprises the following steps:

(1) Determination of optimal screening concentration of puromycin, geneticin and hygromycin;

HEK-293 cells are inoculated into a 24-hole cell culture plate, puromycin, geneticin or hygromycin with different screening concentrations are added, and the concentration range is that: puromycin (0.1. Mu.g/mL-10. Mu.g/mL for 2-3 days), geneticin (0.1 mg/mL-2 mg/mL for 7-14 days), hygromycin (10. Mu.g/mL-500. Mu.g/mL for 5-7 days) were observed for cell growth morphology, and it was confirmed that puromycin screening concentration was 2. Mu.g/mL, geneticin screening concentration was 1mg/mL, and hygromycin screening concentration was 200. Mu.g/mL.

(2) Plasmid construction

1) The sequence ICAM-1-FLAG-Nluc was ligated to the pLV-Neo vector via the cleavage sites EcoR I, xba I, respectively, to give the plasmid pLV-ICAM-1-FLAG-Nluc-Neo.

2) The sequence Cluc-His-SHP2 is connected to a pLVX-Puro vector through enzyme cutting sites Xho I and Xba I respectively to obtain a plasmid pLVX-Cluc-His-SHP2-Puro.

3) The pcDNA3.1 vector sequence CD19 was ligated to the pLV-Hygro vector via the cleavage sites EcoRI and BamH I, respectively, to give the plasmid pLV-CD19-HA-Hygro.

4) All plasmids were sequenced to verify correct.

(3) Collecting slow viruses;

HEK-293T cell plates were allowed to stand overnight. HEK-293T cells were transfected with pLV-CD19-HA-Hygro, pLV-ICAM-1-FLAG-Nluc-Neo or pLVX-Cluc-His-SHP2-Puro plasmid, respectively, and psPAX2 and pMD2.G plasmids using Lipofectamine 3000 reagent, and cultured in a carbon dioxide incubator for 6 hours, and then the supernatant was collected after 48 hours, filtered and concentrated to obtain high titer lentiviruses.

(4) Cell transfection and expansion;

HEK-293 cell plates were allowed to stand overnight. Adding lentivirus with different titers and auxiliary infectious agent polybrene, culturing in a carbon dioxide incubator for 24 hours, changing the liquid, culturing for 48 hours, adding a screening agent, screening for 14 days, and obtaining stable transgenic cell strain 293-IS-CD19, and performing expanded culture under the condition of DMEM+10% FBS (1 mug/mL puromycin, 500 mug/mL geneticin and 100 mug/mL hygromycin).

(5) Crosslinking and activating 293-IS-CD19 stable transfer cell ICAM-1;

293-IS-CD19 cells were incubated with 15. Mu.g/mL ICAM-1 (6.5B5) antibody in 0.1% FBS medium for 1 hour at 37℃after which the cells were washed twice in 0.1% FBS medium, followed by Co-IP assay after 20min incubation with 20. Mu.g/mL anti-mouse IgG F (ab') 2 at 37℃as shown in FIG. 3A, SHP2 binding was reduced after ICAM-1 antibody cross-linking. For the activity assay, the final concentration of 3mM D-luciferin was added immediately after the addition of the above concentrations of anti-mouse IgG F (ab') 2, and the fluorescence signal value was read in real time. As a result, the complementary fluorescent signal was decreased after ICAM-1 antibody was crosslinked as shown in FIG. 3B. The potential biological activity changes are reflected by the simulation of ICAM-1 activation in immune synapses by ICAM-1 antibody cross-linking.

Example 4

The embodiment provides an application of the stable transgenic strain 293-IS-CD19 to the determination of the biological activity of CAR modified cells based on the immune synapse forming ability, and the specific operation method comprises the following steps:

the measuring step comprises the following steps: 293-IS-CD19 cells were cultured in a complete medium (heat-inactivated fetal bovine serum FBS 10mL, DMEM medium 90mL, mixed well, and stored at 4 ℃) at 37℃under 5% carbon dioxide, and cells with good growth state were taken for the experiment. Under aseptic conditions, cells were trypsinized, trypsinized in complete medium and resuspended, centrifuged at 1000rpm for 2 min, the supernatant removed, resuspended in complete medium and the cell concentration adjusted to 2X 10 ⁶ mu.L/mL, 50. Mu.L/well, was seeded in 96-well plates. Different densities of effector cell CAR-J modified cells and CAR-T cells taking CD19 as antigen are respectively regulated, and 0:1,1:1,3:1 and 10:1 (effector cells: target cell ratio) are inoculated into a 96-well plate added with target cells, and different numbers of Jurkat cells are supplemented at the same time, so that the total amount of cells in each well in the system is 1.1X10 ⁶ And each. The final concentration of 3mM D-luciferin was immediately added and the fluorescent signal value was read in real time, with the reading time set to within one hour.

The results show in fig. 4A and 4B that as the concentration of effector cells increases, the fluorescence signal decreases, there is a quantitative relationship, and the stability at 30min is improved, suggesting that the new detection model is successfully established.

As can be seen from the above examples, the present invention constructs a cell line capable of reflecting the biological activity of the immune synapse-forming ability, which can reduce the activity of complementary luciferase when incubated with CAR modified cells, and the biological activity of a sample can be evaluated by detecting the activity of luciferase expressed by the cell line.

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. It will be apparent to those skilled in the art that any equivalent modifications and substitutions of the present invention are intended to be within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Sequence listing

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ctacattctg gagacatagc ttactgggac gaagacgaac acttcttcat cgttgaccgc 120

ctgaagtctc tgattaagta caaaggctat caggtggctc ccgctgaatt ggaatccatc 180

ttgctccaac accccaacat cttcgacgca ggtgtcgcag gtcttcccga cgatgacgcc 240

ggtgaacttc ccgccgccgt tgttgttttg gagcacggaa agacgatgac ggaaaaagag 300

atcgtggatt acgtcgccag tcaagtaaca accgcgaaaa agttgcgcgg aggagttgtg 360

tttgtggacg aagtaccgaa aggtcttacc ggaaaactcg acgcaagaaa aatcagagag 420

atcctcataa aggccaagaa gggcggaaag atcgccgtgt caggaggtgg aggttctgga 480

ggtggtggat ccggtggagg tggatcacat catcaccatc accatatgac atcgcggaga 540

tggtttcacc caaatatcac tggtgtggag gcagaaaacc tactgttgac aagaggagtt 600

gatggcagtt ttttggcaag gcctagtaaa agtaaccctg gagacttcac actttccgtt 660

agaagaaatg gagctgtcac ccacatcaag attcagaaca ctggtgatta ctatgacctg 720

tatggagggg agaaatttgc cactttggct gagttggtcc agtattacat ggaacatcac 780

gggcaattaa aagagaagaa tggagatgtc attgagctta aatatcctct gaactgtgca 840

gatcctacct ctgaaaggtg gtttcatgga catctctctg ggaaagaagc agagaaatta 900

ttaactgaaa aaggaaaaca tggtagtttt cttgtacgag agagccagag ccaccctgga 960

gattttgttc tttctgtgcg cactggtgat gacaaagggg agagcaatga cggcaagtct 1020

aaagtgaccc atgttatgat tcgctgtcag gaactgaaat acgacgttgg tggaggagaa 1080

cggtttgatt ctttgacaga tcttgtggaa cattataaga agaatcctat ggtggaaaca 1140

ttgggtacag tactacaact caagcagccc cttaacacga ctcgtataaa tgctgctgaa 1200

atagaaagca gagttcgaga actaagcaaa ttagctgaga ccacagataa agtcaaacaa 1260

ggcttttggg aagaatttga gacactacaa caacaggagt gcaaacttct ctacagccga 1320

aaagagggtc aaaggcaaga aaacaaaaac aaaaatagat ataaaaacat cctgcccttt 1380

gatcatacca gggttgtcct acacgatggt gatcccaatg agcctgtttc agattacatc 1440

aatgcaaata tcatcatgcc tgaatttgaa accaagtgca acaattcaaa gcccaaaaag 1500

agttacattg ccacacaagg ctgcctgcaa aacacggtga atgacttttg gcggatggtg 1560

ttccaagaaa actcccgagt gattgtcatg acaacgaaag aagtggagag aggaaagagt 1620

aaatgtgtca aatactggcc tgatgagtat gctctaaaag aatatggcgt catgcgtgtt 1680

aggaacgtca aagaaagcgc cgctcatgac tatacgctaa gagaacttaa actttcaaag 1740

gttggacaag ggaatacgga gagaacggtc tggcaatacc actttcggac ctggccggac 1800

cacggcgtgc ccagcgaccc tgggggcgtg ctggacttcc tggaggaggt gcaccataag 1860

caggagagca tcatggatgc agggccggtc gtggtgcact gcagtgctgg aattggccgg 1920

acagggacgt tcattgtgat tgatattctt attgacatca tcagagagaa aggtgttgac 1980

tgcgatattg acgttcccaa aaccatccag atggtgcggt ctcagaggtc agggatggtc 2040

cagacagaag cacagtaccg atttatctat atggcggtcc agcattatat tgaaacacta 2100

cagcgcagga ttgaagaaga gcagaaaagc aagaggaaag ggcacgaata tacaaatatt 2160

aagtattctc tagcggacca gacgagtgga gatcagagcc ctctcccgcc ttgtactcca 2220

acgccaccct gtgcagaaat gagagaagac agtgctagag tctatgaaaa cgtgggcctg 2280

atgcaacagc agaaaagttt cagatga 2307

Claims (10)

1. A luciferase complementary system plasmid reflecting an immune synapse-related signal protein is characterized by comprising a plasmid containing an ICAM-1 gene sequence and a luciferase sequence Nluc on a pGL3-Control plasmid, a plasmid containing an SHP2 gene sequence and a luciferase sequence Cluc on a pGL3-Control plasmid, and a plasmid containing a CD19 gene sequence;

the plasmid containing ICAM-1 gene sequence and luciferase sequence Nluc on pGL3-Control plasmid is obtained by ligating the sequence shown as SEQ ID No.1 to pcDNA3.1 vector through cleavage sites Kpn I, xba I;

the plasmid containing the SHP2 gene sequence and the luciferase sequence Cluc on the pGL3-Control plasmid is obtained by connecting a sequence shown as SEQ ID No.2 to a pcDNA3.1 vector through cleavage sites Kpn I and Xba I;

the CD19 gene is human CD19 gene

The plasmid containing the CD19 gene sequence was obtained by constructing the CD19 gene sequence on a pLV-Hygro vector.

2. A method for preparing a luciferase complementary system plasmid according to claim 1, comprising the steps of:

step one, connecting an ICAM-1 gene sequence and a luciferase sequence Nluc on pGL3-Control plasmid with a linker through a FLAG tag sequentially by utilizing a molecular cloning technology, and constructing the linked adaptor on a vector;

sequentially connecting a luciferase sequence Cluc and an SHP2 gene sequence on pGL3-Control plasmid with a connector through His tag by using a molecular cloning technology, and constructing the luciferase sequence Cluc and the SHP2 gene sequence on a vector;

and thirdly, constructing the CD19 gene sequence on a pLV-Hygro vector by utilizing a molecular cloning technology.

3. The method according to claim 2, wherein the linker used in the first and second steps is 3X flexible linker Gly ₄ Ser。

4. The method according to claim 2, wherein the vector used in the first step is pcDNA3.1 or pLV-Neo.

5. The method according to claim 2, wherein the vector used in the second step is pcDNA3.1 or pLVX-Puro vector.

6. A stably transfected cell line, characterized by being a 293-IS-CD19 cell line, obtained by transfecting the luciferase complementation system plasmid of claim 1 into human embryonic kidney cells HEK-293 cells, stably integrating ICAM-1-Nluc with a Cluc-SHP2 reporter gene and CD19 antigen.

7. The method for constructing a stable transgenic cell line according to claim 6, comprising the steps of:

step one, transfecting the luciferase complementary system plasmid according to claim 1 into HEK-293 cells by using a lentiviral transfection technique;

and step two, adding puromycin, geneticin and hygromycin into the cells transfected in the step two, carrying out pressurized screening, enriching positive cells, amplifying and placing in a liquid nitrogen medium for freezing.

8. The method according to claim 7, wherein the puromycin is selected at a concentration of 2 μg/mL, the geneticin is selected at a concentration of 1mg/mL, and the hygromycin is selected at a concentration of 200 μg/mL.

9. A method for evaluating the in vitro biological activity of CAR modified cells based on the ability of immune synapse formation, comprising the steps of:

step one, adding the CAR modified cells to an aperture plate inoculated with the stably transformed cell strain of claim 6 for co-incubation;

step two, rapidly adding a chemiluminescent substrate D-luciferin to the pore plate, and immediately reading a fluorescence value in real time after shaking for a short time at room temperature;

and thirdly, evaluating the biological activity of the CAR modified cells based on the immune synapse forming capacity through signal difference.

10. The assessment method according to claim 9, wherein the CAR-modified cells are CAR-J cells or CAR-T cells.