CN114045306A - Luciferase complementation system plasmid and stable transfer cell strain reflecting immune synapse related signal protein and application thereof - Google Patents

Abstract

The invention provides a luciferase complementation system plasmid reflecting an immune synapse-associated signal protein, a stable transfer cell strain and application of the plasmid and the stable transfer cell strain in CAR modified cell biological activity determination. The plasmid comprises 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 stable cell line IS 293-IS-CD19, which IS obtained by transfecting the plasmid into human embryonic kidney HEK-293 cells, and stably integrates ICAM-1-Nluc, Cluc-SHP2 reporter genes and CD19 antigen. The cell strain can reflect the immunological synapse forming ability in real time, has simple determination process and accurate result, can be used for CAR modified cell biological activity assessment based on the immunological synapse forming ability, has simple assessment method, short time consumption, low detection cost and stable determination result, is beneficial to the quality control and clinical application of biological active drugs, and has high application value.

Description

Luciferase complementation system plasmid and stable transfer cell strain reflecting immune synapse related signal protein and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a luciferase complementation system plasmid reflecting immune synapse-related signaling protein, a stable transfer cell strain and application of the plasmid in CAR modified cell biological activity determination.

Background

In recent years, cell preparations are gradually maturing. 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 following small molecule chemical drugs, large molecule protein drugs, also called "live drugs". At present, cell products on the market mainly comprise traditional somatic cells, immune cells, different stem cells and the like. The detection of the in vitro biological activity of the cell product is an important index for judging the cell activity.

Chimeric Antigen Receptor (CAR) T cell immunotherapy (CAR-T therapy) in the treatment of immune cell preparations is a novel precise targeted therapy for treating tumors, and the CAR-T cells are used for repairing the intrinsic immunodeficiency of a human body, so that tumor diseases are relieved or even completely cured. CAR-T therapy is the most advanced cellular immunotherapy internationally at present, is approved for clinical treatment in the United states, China also gradually accepts clinical trial application since the beginning of 2018, and two CAR-T therapies from Xingxing medicine and Megasunon at home are reported to be on 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 method for detecting CAR modified cell mediated cytotoxicity has respective defects, such as isotope release method, time-resolved fluorescence method and reporter gene transfection method in the in vitro direct killing activity detection method, and has 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 assay targets and to establish new assay techniques in order to more rapidly assess the effectiveness of CAR-modified cells.

The immune synapse is a precise structure which is important to perform the immunological function of the T cell and is formed by the close contact between the T cell and the surface molecule of the target cell through the interaction of a receptor and a ligand. The formation of this structure helps T cells to distinguish potential antigens, increases the affinity between T cell antigen receptors and MHC-antigen peptide complexes, thereby initiating antigen recognition and activation of T cells, and studies have shown that only T cells forming an immune synapse structure can proliferate. CAR-T, as a population of T cells genetically modified to confer an immune cell-specific antigen receptor, can also form functional immune synapses. Recent related studies show that the quality of formation of immune synapses of CAR-T can be used as an index for detecting the killing activity of CAR-T, and can only be qualitatively analyzed by means of immunofluorescence techniques at present by evaluating F-actin of effector cells, tumor antigen aggregation, polarization of lytic particles, and mean fluorescence intensity of key signal molecule distribution in immune synapse structures. This method can only be studied at the level of individual CAR-modified cells, has the drawback of low throughput of sample analysis, and may lead to large variability in results due to different immunofluorescence signal reference standards.

In the traditional T cell receptor-mediated immune synapse structure, the peripheral supramolecular activation cluster region formed by binding of lymphocyte function-associated antigen-1 (LFA-1) of effector cells to intracellular adhesion receptor 1(ICAM-1) of target cells helps to maintain a more durable interaction of T cells with antigen presenting cells, thereby effectively killing the target cells. The use of LFA-1/ICAM-1 direct binding interactions has not been reported in CAR-T cell mediated immune synapse structures, and the effects on downstream mutual proteins have not been investigated.

The immune synapse structure is a complex formed by many proteins, involving interactions of many proteins. The luciferase complementation technique is a technique for detecting a signal of an interacting protein by fusing N-terminal and C-terminal fragments of luciferase, respectively, to the interacting protein to be detected. When protein interaction occurs, the interaction may cause the N-terminal and C-terminal fragments of the luciferase to recombine into an active luciferase, which in turn catalyzes the production of chemiluminescence by a substrate, such as luciferin. There is currently no report of the use of this technology in the determination of biological activity of CAR-modified cells.

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-associated signaling protein, a stable transfer cell strain, a construction method thereof and application thereof in CAR modified cell biological activity determination based on immune synapse forming capacity, wherein the cell strain can stably integrate ICAM-1-Nluc and Cluc-SHP2 reporter genes and CD19 antigen, can be effectively killed by CAR-T cells taking CD19 as antigen and can activate CAR-Jurkat (CAR-J) cells to generate IL-2 (the 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 to reflect the immunological synapse forming capability of CAR modified cells by incubating with the CAR modified cells, and evaluates the effectiveness of the CAR modified cells through the biological activity.

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

the first aspect of the present invention provides a luciferase complementation system plasmid reflecting an immune synapse-associated signaling protein, which comprises 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 second aspect of the present invention provides a method for preparing the above plasmid, comprising the steps of:

firstly, a commercial ICAM-1 plasmid target gene sequence and a luciferase sequence Nluc on pGL3-Control plasmid are connected with a joint through FLAG tag in sequence by utilizing a molecular cloning technology and are constructed on a vector;

connecting a luciferase sequence Cluc on pGL3-Control plasmid and a target gene sequence of commercial SHP2 plasmid with a joint through His tag in sequence by using a molecular cloning technology, and constructing the luciferase sequence Cluc on a vector;

and step three, constructing a target gene sequence of the commercial CD19 plasmid on a pLV-Hygro vector by utilizing a molecular cloning technology.

Further, the linker used in step one and step two was 3 XFlexible linker Gly 4/Ser.

Further, the sequence ICAM-1-FLAG-Nluc with the sequence SEQ ID No.1 is obtained by connection in the step one.

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

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

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

The third aspect of the invention provides a stable transgenic cell line, 293-IS-CD19, which IS obtained by transfecting the plasmid into human embryonic kidney cell HEK-293 cells, and stably integrates ICAM-1-Nluc and Cluc-SHP2 reporter genes and CD19 antigen.

The fourth aspect of the present invention provides a method for constructing the above-described stably transfected cell line, comprising the steps of:

transfecting the plasmid into HEK-293 cells by using a lentivirus transfection technology;

and step two, adding puromycin, geneticin and hygromycin into the cells transfected in the step one, enriching positive cells through pressure screening, amplifying, and placing in a liquid nitrogen medium for freezing storage.

Furthermore, the selection concentration of puromycin is 2 mug/mL, the selection concentration of geneticin is 1mg/mL, and the selection concentration of hygromycin is 200 mug/mL.

The fifth aspect of the invention provides a CAR modified cell biological activity assessment method based on immune synapse forming ability, comprising the following steps:

adding CAR modified cells into a pore plate inoculated with the stably transformed cell strain for co-incubation;

rapidly adding a chemiluminescent substrate D-luciferin into the pore plate, oscillating at room temperature for a short time, and immediately reading a fluorescence value in real time;

and step three, evaluating the biological activity of the CAR modified cells based on the immunological synapse forming ability through signal difference.

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

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

(1) compared with the traditional detection method, the detection method is simple, short in time consumption and low in detection cost, and stable transformants are favorable for ensuring the stability of the detection result;

(2) the invention takes an immune synapse signal passage as a research object, designs a cell strain of an interactive luciferase reporter gene capable of reflecting the protein interaction change after the immune synapse is formed, can reflect the forming capability of the immune synapse in real time, and has simple measuring process and accurate result.

In conclusion, the HEK-293 cell line capable of stably integrating the interactive luciferase reporter gene capable of stably responding to the protein interaction change after the formation of the immune synapse, which is constructed by the invention, can influence the complementary luciferase activity change expressed by the target cell after forming the immune synapse with the CAR modified cell. The invention establishes a simpler and faster CAR modified cell in vitro biological activity evaluation method by using the cell strain, can be beneficial to quality control and clinical application of biologically active drugs, and has higher application value.

Drawings

FIG. 1 shows the results of identifying proteins transiently expressed in HEK-293 cells and key proteins in the immune synaptic signaling pathway according to an embodiment of the present invention; wherein, FIG. 1A is the result of Western blot identification of ICAM-1-FLAG-Nluc, Cluc-His-SHP2 and CD19 plasmids which are 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-Actin is loading internal reference protein; FIG. 1B shows the effect of LFA-1/ICAM-1 inhibitor lifitegrast on CD19 CAR-T cell killing target cell HEK-293 (transfected with the three plasmids above); FIG. 1C shows the effect of LFA-1/ICAM-1 inhibitor lifitegrast on IL-2 secretion by CD19 CAR-J cells in contact with target cell HEK-293 (transfected with the three plasmids above);

FIG. 2 shows the interaction and activity verification and identification results of HEK-293 cells in one embodiment of the present invention; wherein, FIG. 2A shows the Western blot immunoprecipitation identification results of ICAM-1-FLAG-Nluc and Cluc-His-SHP2 plasmid transiently expressed on pcDNA3.1 vector by HEK-293; FIG. 2B shows that ICAM-1(P2A4) antibody (which blocks direct binding of ICAM-1 to SHP2) 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 protein interaction and luciferase activity change after ICAM-1 activation of stably transfected 293-IS-CD19 in one embodiment of the present invention; wherein, FIG. 3A shows that after 293-IS-CD19 cells ICAM-1 are subjected to antibody cross-linking (simulating immune synapse LFA-1 activating ICAM-1), the interaction between Cluc-His-SHP2 and ICAM-1-FLAG-Nluc IS weakened, and CD19+ Cluc-SHP2 IS a negative control; FIG. 3B shows that ICAM-1 of 293-IS-CD19 cells was cross-linked with antibody, and the activity of intracellular complementation luciferase was decreased, and Control, anti-IgG F (ab ')2 and IgG + anti-IgG F (ab')2 were negative controls;

FIG. 4 shows the results of luciferase activity changes of stably transfected 293-IS-CD19 after incubation with CAR-J (FIG. 4A) and CAR-T (FIG. 4B) cells in one embodiment of the present invention.

Detailed Description

The invention researches the change relation between the activation of target cells ICAM-1 in a CAR modified cellular immune synapse structure and the combination of protein tyrosine phosphatase-2 (SHP2) of downstream binding protein SH2, and constructs a signaling probe for ICAM-1/SHP2 interaction by utilizing a luciferase complementation technology. And the probe is applied to research the dynamic change of ICAM-1/SHP2 protein interaction in target cells in the immune synapse forming process 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 complementation system plasmid reflecting an immune synapse-associated signaling protein, a stable transfer cell strain and application of the plasmid and the stable transfer cell strain in CAR modified cell biological activity determination.

The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.

In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.

Example 1

This example provides and verifies a luciferase complementation system plasmid reflecting an immune synapse-associated signaling protein, and the construction and verification process of the plasmid includes the following steps:

(1) constructing plasmids;

1) the sequence ICAM-1-FLAG-Nluc (obtained by connecting a commercial ICAM-1 plasmid target gene sequence and a luciferase sequence (Nluc, aa2-416) on pGL3-Control plasmid by using a molecular cloning technology through FLAG tag and 3 Xflexible linker Gly4/Ser, and SEQ ID No.1) is connected to a pcDNA3.1 vector through enzyme cutting sites Kpn I and Xba I to obtain the plasmid pc 3.1-ICAM-1-FLAG-Nluc.

2) The sequence Cluc-His-SHP2 (luciferase sequence (Cluc, aa398-550) on pGL3-Control plasmid and commercial SHP2 plasmid target gene sequence are connected by His tag and 3 Xflexible linker Gly4/Ser to obtain SEQ ID No.2) through enzyme cutting sites Kpn I and Xba I respectively to be connected to pcDNA3.1 vector to obtain the plasmid pc3.1-Cluc-His-SHP 2.

3) All plasmids were verified to be correct by sequencing.

(2) Cell transfection;

HEK-293 cell plates overnight. HEK-293 cells are transfected by human CD19 expression plasmids on pc3.1-ICAM-1-FLAG-Nluc, pc3.1-Cluc-His-SHP2 and a purchased pcDNA3.1 vector respectively by using a Lipofectamine 3000 reagent, the cells are placed in a carbon dioxide incubator for 6 hours and then changed, a part of the cells are collected after 48 hours to prepare Western blot protein samples, and the remaining cells are subjected to CAR modified cell killing activity tests.

(3) Identifying transient expression of cell fusion protein;

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

(4) Identification of CAR-T cell immune synapse key signaling protein;

CAR-T cells were tested for their ability to kill target cells using the Eu-TDA release assay. HEK-293 target cells transfected with the above three plasmids were inoculated with a fluorescent dye at an appropriate density in a U-bottom 96-well plate, and CAR-T cells were preincubated with 1. mu.M lifitegrast (LFA-1/ICAM-1 inhibitor) for one hour, then added to the target cells at different densities, after three hours, the supernatant was collected, the substrate was added, and the signal value was read using a time-resolved fluoroimmunoassay analyzer to analyze the rate of the cell dye release method. The results are shown in FIG. 1B, and lifitegrast effectively inhibits killing ability of CAR-T to HEK-293 cells transfected with three plasmids, indicating the importance of LFA-1/ICAM-1 binding in CAR-T immune synapse structure.

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

CAR-J cells indirectly reflect the ability to kill target cells by detecting effector cell activation using IL-2 release. HEK-293 target cells transfected with the three plasmids are planted in a U-shaped bottom 96-well plate at a proper density, CAR-J cells are preincubated for 1 mu M lifitegrast for one hour, then added into the target cells at a cell density of 10 times, supernatants are collected after three hours and six hours respectively, and IL-2ELISA kits are used for detecting the content of the supernatants and analyzing the activation condition of effector cells. The results are shown in FIG. 1C, and the significance of LFA-1/ICAM-1 binding in CAR-J immune synapse structures is shown by the fact that lifitegrast effectively inhibits the IL-2 secretion ability of CAR-J on HEK-293 cells transfected with three plasmids.

Taken together, 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 intracellular activity of the immune synapse key signal protein plasmid constructed in example 1 was verified, and the specific operation steps and results are as follows:

(1) westernblot immunoprecipitation assay

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

As shown in FIG. 2A, the fusion proteins ICAM-1-FLAG-Nluc and Cluc-His-SHP2 had direct interactions 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 of IgG or ICAM-1(P2A4) antibody, followed by 1X 10⁵100. mu.L/well of each 96-well plate was added immediately with a final concentration of 3mM D-luciferin substrate, and changes in fluorescence signal values were read immediately in real time.

The results are shown in FIG. 2B, where the ICAM-1(P2A4) antibody effectively inhibited the fluorescence signal generated by the ICAM-1/SHP2 interaction, indicating that the intracellular fluorescence signal was derived from the interaction of the fusion protein. The specificity of the fusion plasmid expression probe protein is proved, and the fusion plasmid expression probe protein has biological activity value.

In conclusion, the luciferase activity generated by the direct interaction of ICAM-1-FLAG-Nluc and the Cluc-His-SHP2 fusion protein in HEK-293 cells proves the specificity of the fluorescent probe.

Example 3

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

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

HEK-293 cells were seeded in 24-well cell culture plates and puromycin, geneticin or hygromycin was added at different screening concentrations, ranging from: puromycin (0.1-10 mug/mL for 2-3 days), geneticin (0.1-2 mg/mL for 7-14 days), hygromycin (10-500 mug/mL for 5-7 days) and cell growth morphology were observed, confirming puromycin screening concentration of 2 mug/mL, geneticin screening concentration of 1mg/mL and hygromycin screening concentration of 200 mug/mL.

(2) Plasmid construction

1) The sequence ICAM-1-FLAG-Nluc is connected to a pLV-Neo vector through enzyme cutting sites EcoR I and Xba I respectively to obtain a plasmid pLV-ICAM-1-FLAG-Nluc-Neo.

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

3) The sequence CD19 on the pcDNA3.1 vector is connected to the pLV-Hygro vector through enzyme cutting sites EcoR I and BamH I respectively to obtain a plasmid pLV-CD 19-HA-Hygro.

4) All plasmids were verified to be correct by sequencing.

(3) Collecting lentiviruses;

HEK-293T cell plates overnight. And transfecting HEK-293T cells with pLV-CD19-HA-Hygro, pLV-ICAM-1-FLAG-Nluc-Neo or pLVX-Cluc-His-SHP2-Puro plasmids, psPAX2 and pMD2.G plasmids respectively by using a Lipofectamine 3000 reagent, culturing in a carbon dioxide incubator for 6 hours, changing the culture solution, collecting the supernatant after 48 hours, filtering and concentrating to obtain the high-titer lentivirus.

(4) Cell transfection and amplification;

HEK-293 cell plates overnight. Adding lentivirus with different titers and polybrene serving as an auxiliary infection reagent, placing the mixture into a carbon dioxide incubator for 24 hours, changing the culture solution, continuing to culture for 48 hours, adding a screening agent for screening for 14 days to obtain a stable cell strain 293-IS-CD19, and carrying out amplification culture on the cell strain, wherein the culture solution conditions are DMEM + 10% FBS (1 mu g/mL puromycin, 500 mu g/mL geneticin and 100 mu g/mL hygromycin).

(5)293-IS-CD19 stable cell ICAM-1 cross-linking activation;

293-IS-CD19 cells were incubated with 15. mu.g/mL ICAM-1(6.5B5) antibody in 0.1% FBS medium at 37 ℃ for 1 hour, after which the cells were washed twice in 0.1% FBS medium, after which they were incubated with 20. mu.g/mL anti-mouse IgG F (ab')2 at 37 ℃ for 20min before performing the Co-IP assay, with the results shown in FIG. 3A, and 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 anti-mouse IgG F (ab')2 at the above concentration, and the fluorescence signal was read in real time. As a result, as shown in FIG. 3B, the complementary fluorescent signal was decreased after ICAM-1 antibody was crosslinked. The stimulation of ICAM-1 activation in immune synapses by ICAM-1 antibody cross-linking was mimicked, reflecting potential biological activity changes.

Example 4

The present embodiment provides an application of the above-mentioned stable transformant 293-IS-CD19 in determining biological activity of CAR-modified cells based on immune synapse-forming ability, and the specific operation method comprises the following steps:

the determination step comprises: 293-IS-CD19 cells were cultured in complete medium (heat-inactivated fetal bovine serum FBS 10mL, DMEM 90mL, mixed, stored at 4 ℃) at 37 ℃ and 5% carbon dioxide, and cells in good growth state were used for the assay. Under aseptic conditions, cells were trypsinized, pancreatin was neutralized with complete medium and cells were resuspended, centrifuged at 1000rpm for 2 minutes, the supernatant was discarded, resuspended with complete medium and the cell concentration was adjusted to 2X 10⁶Each well was seeded at 50. mu.L/mL in 96-well plates. The effector cell CAR-modified cell CAR-J and CAR-T cell with CD19 as antigen were adjusted to different densities, and inoculated into 96-well plate with target cells added therein at 0:1, 1:1, 3:1, 10:1 (effector cell: target cell ratio), and supplemented with Jurkat cells in different amounts, so that the total amount of cells per well in the system was 1.1 × 10⁶And (4) respectively. The final concentration of 3mM D-luciferin was immediately added and the fluorescence signal values were read in real time with the reading time set to within one hour.

The result is shown in fig. 4A and 4B, as the concentration of the effector cells increases, the fluorescence signal decreases, and a dose-effect relationship exists, and the fluorescence signal tends to be stable at 30min, which indicates that the establishment of a new detection model is successful.

According to the embodiments, the cell strain capable of reflecting the biological activity of the immunological synapse forming ability is constructed, the cell strain can reduce the activity of the complementary luciferase under the condition of being incubated with the CAR modified cell, and the biological activity of the sample can be evaluated by detecting the activity of the luciferase expressed by the cell strain.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. It will be appreciated by those skilled in the art that any equivalent modifications and substitutions are within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

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 complementation system plasmid reflecting immune synapse-associated signaling 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.

2. The method for preparing the plasmid of claim 1, comprising the steps of:

firstly, a commercial ICAM-1 plasmid target gene sequence and a luciferase sequence Nluc on pGL3-Control plasmid are connected with a joint through FLAG tag in sequence by utilizing a molecular cloning technology and are constructed on a vector;

connecting a luciferase sequence Cluc on pGL3-Control plasmid and a target gene sequence of commercial SHP2 plasmid with a joint through His tag in sequence by using a molecular cloning technology, and constructing the luciferase sequence Cluc on a vector;

and step three, constructing a target gene sequence of the commercial CD19 plasmid 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 3 XFlexible linker Gly 4/Ser.

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

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

6. A stable transgenic cell line 293-IS-CD19 cell line, which IS obtained by transfecting the plasmid of claim 1 into HEK-293 cells of human embryonic kidney cells, and stably integrates ICAM-1-Nluc and Cluc-SHP2 reporter genes and CD19 antigen.

7. The method of claim 6, comprising the steps of:

step one, transfecting the plasmid of claim 1 into HEK-293 cells by using a lentivirus transfection technology;

and step two, adding puromycin, geneticin and hygromycin into the cells transfected in the step one, enriching positive cells through pressure screening, amplifying, and placing in a liquid nitrogen medium for freezing storage.

8. The method according to claim 7, wherein the puromycin screening concentration is 2 μ g/mL, the geneticin screening concentration is 1mg/mL, and the hygromycin screening concentration is 200 μ g/mL.

9. A method for evaluating biological activity of CAR modified cells based on immunological synapse-forming ability, comprising the steps of:

step one, adding CAR modified cells into a pore plate inoculated with the stable transfused cell strain of claim 6 for co-incubation;

rapidly adding a chemiluminescent substrate D-luciferin into the pore plate, oscillating at room temperature for a short time, and immediately reading a fluorescence value in real time;

and step three, evaluating the biological activity of the CAR modified cells based on the immunological synapse forming ability through signal difference.

10. The assessment method of claim 9, wherein said CAR-modified cell is a CAR-J cell or a CAR-T cell.

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