CN110305848A - A kind of target cell U87MG-EGRL and its construction method and application for evaluating targeting EGFR vIII antigen - Google Patents

A kind of target cell U87MG-EGRL and its construction method and application for evaluating targeting EGFR vIII antigen Download PDF

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CN110305848A
CN110305848A CN201910682087.8A CN201910682087A CN110305848A CN 110305848 A CN110305848 A CN 110305848A CN 201910682087 A CN201910682087 A CN 201910682087A CN 110305848 A CN110305848 A CN 110305848A
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egrl
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焦顺昌
张嵘
宋玉洁
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Beijing Dingcheng Taiyuan Biotechnology Co Ltd
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Abstract

The target cell U87MG-EGRL and its construction method and application that the present invention provides a kind of for evaluating targeting EGFR vIII antigen, belong to cellular immunity technical field.Target cell U87MG-EGRL of the present invention is glioma cell, can express the antigen-4 fusion protein gene of CAR-T cell target antigen EGFRvIII gene, GFP and Red Luciferase.Target cell of the present invention being capable of high efficient expression EGFRvIII antigen, not only it had expressed GFP gene but also had expressed luciferase gene, the lethal effect of assessment CAR-T cell can be reacted by luciferase, therefore the target cell can be applied to external CAR-T pharmacodynamics assessment in vivo.

Description

It is a kind of for evaluating the target cell U87MG-EGRL and its structure of targeting EGFR vIII antigen Construction method and application
Technical field
The invention belongs to cellular immunity technical fields, and in particular to a kind of for evaluating the target of targeting EGFR vIII antigen Cell U87MG-EGRL and its construction method and application.
Background technique
Chimeric antigen receptor T cell immunotherapy (Chimeric Antigen Receptor T-Cell Immunotherapy, CAR-T immunotherapy) it is a kind of novel cell immunization therapy skill developed rapidly recent years Art.It is that site-specific is identified that (single-chain antibody scFv), starting immunocompetence and attack tumour are thin by technique for gene engineering The element of born of the same parents is integrated into a gene, and in patient's autologous T lymphocytes of being transduceed, and defeated time patient's body, makes patient Specific recognition tumour cell is regained, self T-cell is activated, targetedly attacks and kill identified tumour cell Ability.
Glioma is most pernicious one of tumour, development process quickly, the mean survival time (MST) of patients with gliomas according to statistics Less than 15 months, currently without particularly effective treatment means.There is 30% or more patient to have in glioma case EGFRvIII antigen presentation.EGFRvIII is EGFR gene most common mutation in tumour, it is recognized that tumour-specific tumorigenesis Mutation.EGFRvIII is mutation neoantigen, is not expressed in the normal tissue, antigentic specificity is strong.Targeting EGFR vIII antigen For CAR-T cell for treating glioma, high specificity, toxicity of missing the target is low, Small side effects, is good selection.
The complexity and particularity of cell therapy product deposit such product between non-clinical study and clinical research In biggish population and individual difference, the non-clinical study stage possibly even carries out drug effect without suitable inside and outside disease model Evaluation is learned, therefore is very necessary in early studies in man stage entry evaluation validity of products.
Need suitable appraisement system and tool thin for pharmacodynamic study external in the clinical precursor of CAR-T immunization therapy How born of the same parents have better killing-efficiency appraisement system in vitro, and how to carry out in vivo to the volume of tumour efficiently correct Assessment is all problem to be solved.The evaluation method of Cytotoxicity in vitro efficiency includes LDH method, streaming method etc. at present, still Accurate evaluation is all unable to the fragmentation effect of target cell.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of for evaluating the target cell U87MG- of targeting EGFR vIII EGRL and its construction method and application, the target cell U87MG-EGRL both efficiently express target antigen EGFRvIII, while mistake Red luciferase gene is expressed, to provide effective tool thin for the internal Pharmacodynamics in vitro functional verification of CAR-T cell Born of the same parents.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of for evaluating the target cell U87MG-EGRL of targeting EGFR vIII, the target cell U87MG-EGRL is glioma cell, can express CAR-T cell target antigen EGFRvIII gene, GFP and Red Luciferase Antigen-4 fusion protein gene.
Preferably, the EGFRvIII gene is started by CMV promoter and is expressed, and the EGFRvIII gene has such as SEQ Nucleotide sequence shown in ID NO.1.
Preferably, in the antigen-4 fusion protein gene of the GFP and Red Luciferase include sequentially connected GFP gene, Connect peptide gene and Red Luciferase gene, the nucleotide of the antigen-4 fusion protein gene of the GFP and Red Luciferase Sequence is as shown in SEQ ID NO.5.
The present invention also provides the construction methods of the target cell U87MG-EGRL, comprising the following steps: (1) to described EGFRvIII gene is modified, the EGFRvIII target antigen sequence after must modifying；EGFRvIII target antigen after the modification The nucleotide sequence of sequence is as shown in SEQ ID NO.6；
(2) that the antigen-4 fusion protein gene of the GFP and Red Luciferase is transferred to glioma with slow-virus transfection method is thin The U87MG-GRL cell of born of the same parents' acquisition expressed fusion protein；
(3) the EGFRvIII target antigen sequence after the modification that step (1) obtains is transferred to institute with slow-virus transfection method It states in U87MG-GRL cell, obtains target cell U87MG-EGRL；
Temporal restriction relation is not present in step (1) and step (2).
Preferably, step (1) modification, including 5 ' the end addition NheI restriction enzyme sites in the EGFRvIII gene With Kozak sequence, NotI restriction enzyme sites are added at 3 ' ends.
The present invention also provides the target cell U87MG-EGRL or the target cells constructed using the construction method Application of the U87MG-EGRL in target antigen detection of expression.
The present invention also provides the target cell U87MG-EGRL or the target cells constructed using the construction method U87MG-EGRL examines the application in CAR-T Cell killing efficacy in vitro.
The present invention also provides the target cell U87MG-EGRL or the target cells constructed using the construction method U87MG-EGRL in vivo tumor formation experiment in application.
Preferably, concentration of the target cell U87MG-EGRL in application is (6~10) × 103A/mL imitates target ratio For (1~20): 1.
The present invention provides a kind of for evaluating the target cell U87MG-EGRL of targeting EGFR vIII, the target cell U87MG-EGRL is glioma cell, can express CAR-T cell target antigen EGFRvIII gene, GFP and Red Luciferase Antigen-4 fusion protein gene.Target cell U87MG-EGRL of the present invention can be used for examining CAR-T cell drug effect, and being can be simultaneously Express the glioma of the antigen-4 fusion protein gene of CAR-T cellular targets antigen gene EGFRvIII and GFP and Red Luciferase Cell can truly react effect of the CAR-T cell to glioma, while thin in target since target cell is glioma cell Not only target cell antigen, but also the antigen-4 fusion protein gene of expression GFP and Red Luciferase are expressed in born of the same parents, can efficiently differentiate target Cell mortality and effector cell's death rate are swift to operate sensitive；It in vivo can be more accurate by small animal imaging system The pharmacodynamics of ground reaction CAR-T cells against tumor.
Detailed description of the invention
Fig. 1 is the pZX expression vector skeleton applied in the embodiment of the present invention；
Fig. 2 is the plasmid pZX- that the antigen-4 fusion protein gene of GFP and Red Luciferase is carried in the embodiment of the present invention GFP-T2A-LUC structure chart；
Fig. 3 is the plasmid pZX-EGFRvIII that CAR-T cell target antigen EGFRvIII gene is carried in the embodiment of the present invention Structure chart；
Fig. 4 is the U87MG-GRL cell line flow cytometer detection result constructed in the embodiment of the present invention；
Fig. 5 is that U87MG-EGRL cell carries out EGFRvIII detection of expression result in the embodiment of the present invention；
Fig. 6 is that U87MG-EGRL cell carries out luciferase testing result in the embodiment of the present invention；
Fig. 7 is that U87MG-EGRL cell is detected as target cell using small animal imaging system in the embodiment of the present invention Experimental result picture；
Fig. 8 is use CAR-EGFRvIII cell as effector cell in the embodiment of the present invention, using U87MG-GRL with Killing rate change curve of the U87MG-EGRL as target cell；
Fig. 9 is use CAR-CD19-T cell as effector cell in the embodiment of the present invention, using U87MG-GRL with Killing rate change curve of the U87MG-EGRL as target cell.
Specific embodiment
The present invention provides a kind of for evaluating the target cell U87MG-EGRL of targeting EGFR vIII, the target cell U87MG-EGRL is glioma cell, can express CAR-T cell target antigen EGFRvIII gene, GFP and Red Luciferase Antigen-4 fusion protein gene.
EGFRvIII gene of the present invention, which is preferably started by CMV promoter, expresses, and the EGFRvIII gene has such as Nucleotide sequence shown in SEQ ID NO.1.In the antigen-4 fusion protein gene of GFP and Red Luciferase of the present invention preferably Including sequentially connected GFP gene, connection peptide gene and Red Luciferase gene, wherein the nucleotide of the GFP gene Sequence preferably as shown in SEQ ID NO.2, the gene order of the Red Luciferase preferably as shown in SEQ ID NO.3, The gene order of the link peptide is preferably as shown in SEQ ID NO.4.The present invention is by the GFP gene, connection peptide gene and Red After Luciferase gene is sequentially connected with, it is preferably also included in 5 ' end addition NheI restriction enzyme sites and Kozak sequence, at 3 ' ends Not I restriction enzyme site is added, to form the antigen-4 fusion protein gene with the sequence as shown in SEQ ID NO.5.It is of the present invention Antigen-4 fusion protein gene is preferably obtained by artificial synthesized mode, and in embodiments of the present invention, You Jinwei intelligence biotechnology is limited Company's synthesis.
The present invention also provides the construction methods of the target cell U87MG-EGRL, comprising the following steps: (1) to described EGFRvIII gene is modified, the EGFRvIII target antigen sequence after must modifying；EGFRvIII target antigen after the modification The nucleotide sequence of sequence is as shown in SEQ ID NO.6；
(2) that the antigen-4 fusion protein gene of the GFP and Red Luciferase is transferred to glioma with slow-virus transfection method is thin The U87MG-GRL cell of born of the same parents' acquisition expressed fusion protein；
(3) the EGFRvIII target antigen sequence after the modification that step (1) obtains is transferred to institute with slow-virus transfection method It states in U87MG-GRL cell, obtains target cell U87MG-EGRL；
Temporal restriction relation is not present in step (1) and step (2).
The present invention modifies the EGFRvIII gene, must modify when constructing the target cell U87MG-EGRL EGFRvIII target antigen sequence afterwards；The nucleotide sequence such as SEQ ID of EGFRvIII target antigen sequence after the modification Shown in NO.6.Modification of the present invention, be preferably included in the EGFRvIII gene 5 ' end addition NheI restriction enzyme sites and Kozak sequence, in 3 ' end addition NotI restriction enzyme sites, the nucleotide sequence such as SEQ of the EGFRvIII target antigen sequence after modification Shown in ID NO.6.EGFRvIII target antigen after modification of the present invention can be obtained by artificial synthesized mode, in the present invention In embodiment, the synthesis of You Jinwei intelligence Bioisystech Co., Ltd.
The antigen-4 fusion protein gene of the GFP and Red Luciferase glioma cell is transferred to slow-virus transfection method to obtain Obtain the U87MG-GRL cell of expressed fusion protein.There is no particular determinations to the slow-virus transfection method by the present invention, utilize ability The conventional slow-virus transfection method in domain.Glioma cell of the present invention is preferably derived from ATCC cell bank, product number ForHTB-14TM。
After EGFRvIII target antigen sequence and U87MG-GRL cell after must modifying, the present invention will with slow-virus transfection method EGFRvIII target antigen sequence after the modification is transferred in the U87MG-GRL cell, obtains target cell U87MG-EGRL.This To the method that slow-virus transfection hair is transferred to, there is no particular determinations for invention, utilize the conventional method of this field.
The present invention also provides the target cell U87MG-EGRL or the target cells constructed using the construction method Application of the U87MG-EGRL in target antigen detection of expression.Concentration of the target cell U87MG-EGRL of the present invention in application Preferably (0.1~3) × 106A/mL, more preferably (0.5~2) × 106A/mL, most preferably 1 × 106A/mL.
The present invention also provides the target cell U87MG-EGRL or the target cells constructed using the construction method U87MG-EGRL examines the application in CAR-T Cell killing efficacy in vitro.Target cell U87MG-EGRL of the present invention is being answered The concentration of used time is preferably (6~10) × 103A/mL, more preferably (7~9) × 103A/mL, most preferably 9 × 103A/ mL.The effect target of target cell U87MG-EGRL of the present invention is than being preferably (1~20): 1, more preferably (2.5~10): 1.This Invent the target cell U87MG-EGRL for additionally providing the target cell U87MG-EGRL or constructing using the construction method Application in tumor formation experiment in vivo.Concentration of the target cell U87MG-EGRL of the present invention in application is preferably (3~6) ×104A/mL, more preferably (4~5) × 104A/mL, most preferably 5 × 104A/mL.Below with reference to embodiment to the present invention The target cell U87MG-EGRL for being used to evaluate targeting EGFR vIII and its construction method and application provided is carried out specifically It is bright, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
Construct the antigen-4 fusion protein gene expression vector of GFP and Red Luciferase
One, material
1, slow virus skeleton plasmid pZX (slow virus carrier structure is as shown in Figure 1), slow virus packaging plasmid PSPAX2 and PMD2.G, HEK293T cell；
2, nucleotide sequence (the SEQ ID of the nucleotide sequence (SEQ ID NO.1) of Human epidermal growth factor receptor vIII gene, GFP gene NO.2), the nucleotide sequence (SEQ ID NO.3) of Red Luciferase gene, link peptide nucleotide sequence (SEQ ID NO.4), the nucleotide sequence (SEQ ID NO.5) of GFP+ link peptide+Red Luciferase, for connecting carrier The nucleotide sequence (SEQ ID NO.6) of EGFRvIII target antigen；
3, DNA sequence dna shown in SEQ ID NO.5, SEQ ID NO.6 is synthesized by Jin Weizhi Bioisystech Co., Ltd, and It is saved with plasmid form；
4, toolenzyme Nhe I, Not I are purchased from Thermo company；；
5, plasmid extraction kit, Ago-Gel QIAquick Gel Extraction Kit are purchased from TIANGEN company；
6, competent cell Stbl3 is purchased from Quan Shijin biotech firm；
7, Opti-MEM, FBS, DMEM, RPMI-1640 are purchased from Gibco company；
8, lymphocyte separation medium is purchased from the ocean Tianjin Hao company；
9, Luciferase detection kit is purchased from green skies company.
Two, the construction method of recombined lentivirus vector pZX-GFP-T2A-LUC, pZX-EGFRvIII
The construction method of the recombined lentivirus vector is as follows:
It is carried by the nucleotide sequence (SEQ ID NO.5) of the GFP+ link peptide+Red Luciferase of synthesis, for connecting The nucleotide sequence (SEQ ID NO.6) of the EGFRvIII target antigen of body is cloned into slow virus skeleton plasmid pZX respectively, respectively To recombinant slow virus plasmid pZX-GFP-T2A-LUC (as shown in Figure 2), pZX-EGFRvIII (as shown in Figure 3).
(1) slow virus skeleton plasmid pZX is subjected to double digestion using Nhe I and Not I restriction enzyme, product passes through 1% agarose gel electrophoresis, and it is tapped and recovered carrier framework (6.4kb), with the Ago-Gel reclaim reagent of Tiangeng company Box recycles corresponding segment, and measures the purity and concentration of product；
(2) by DNA fragmentation pZX respectively with GFP-T2A-LUC, EGFRvIII segment with 10 μ L systems and molar ratio 1:5 Ratio is added in Eppendorf pipe, and 10 μ L of solution I ligase is added in 16 DEG C of incubation 1h after mixing and takes 10 μ L connections Product is transferred in stbl3 competence, places 30min on ice, pipe is put into heat shock 45s in 42 DEG C of thermostat water bath, quickly Pipe is transferred in ice bath, the cooling 2min of cell is made, every pipe adds 1mL LB culture solution, then pipe is transferred on 37 DEG C of shaking tables, Incubating 45min makes bacteria resuscitation, and the transformed bacteria solution of 100 μ L is taken to be coated on Amp LB agar plate, is inverted plate, trains in constant temperature It supports in case and cultivates for 37 DEG C, 16 hours.
For picking monoclonal into 1mL LB (Amp+) fluid nutrient medium, 37 DEG C of shaking tables 200 turn over night, and inspection in second day is surveyed Sequence identifies correctly clone；
(3) by corresponding 20 μ L of recombinant slow virus plasmid glycerol stock inoculation with 200mL LB (Amp+) fluid nutrient medium in, 37 DEG C of shaking tables 200 turn over night, second day harvest bacterium solution, and row plasmid extraction (TINGEN company plasmid extraction kit) detects matter Grain purity and concentration, -20 DEG C spare.
Three, recombinant slow virus is packed
1, building slow-virus transfection experiment
(1) recovery culture HEK-293T, passage.It in day before transfection application collected by trypsinisation cell and counts, takes 2* 107Cell inoculation is placed in 37 DEG C, 5%CO to 15cm culture dish2Incubator culture, culture solution are changed to Opti-MEM and train completely Support base.
(2) on the transfection same day, two 15mL centrifuge tubes are taken, plasmid and reagent is added by system described in table 1.
1 slow virus packaging system of table
TubeA is added in Tube B and mixes, and room temperature acts on 10min, discards 16mL culture medium and 7.9mL mixture (patch is added Wall addition prevents cell to be suspended).Liquid is changed after 6h.
(3) culture harvests first viral supernatants about 20mL afterwards for 24 hours, and 4 DEG C, 1500rpm is centrifuged 10min.0.45um filter membrane Filtering, 4 DEG C of storage at least 12h after adding 4mL 5x PEG-it to mix.
(4) second batch viral supernatants about 20mL is harvested after cultivating cell 52h, 4 DEG C, 1500rpm is centrifuged 10min.0.45um Membrane filtration, 4 DEG C of storage at least 12h after adding 4mL 5x PEG-it to mix.
(5) 4 DEG C of virus liquid, 1500g are centrifuged 30min, discard supernatant, and continue to be centrifuged 5min, and exhaust supernatant, using 500 μ LDMEM culture medium, which suspends, dissolves precipitating,
(6) every pipe dispenses 100 μ L, and -80 DEG C freeze.
Embodiment 2
Target cell U87MG-EFRL building and screening
Specific slow virus infected cell method the following steps are included:
(1) U87MG cell is infected using slow virus pZX-GFP-T2A-LUC
(1) culture is diluted to 1 × 10 after infection cell digestion counts5A/mL takes 2mL that 6 orifice plate cultures are added Ware shakes up and is placed on incubator culture.
(2) packaged virus is added to culture dish, shakes up after label is good and be placed in incubator culture by next day, 2-3d After can according to cell density expand cultivate.
(2) airflow classification GFP positive cell group
(1) cell in T175 culture bottle is collected, 200 × g is centrifuged 5min, is collected simultaneously non-infected cells as control.
(2) every pipe is added 1mL PBS and washes that cell is primary, and 200 × g is centrifuged 5min.
(3) every pipe is added 500 μ LPBS and cell is resuspended into 5mL Round Bottom PolystyreneTest Tube 5mL culture medium, airflow classification GFP positive cell group, cell marking name is added in (filter membrane) after sorting in cell collecting pipe Referred to as U87MG-GRL.
(3) slow-virus infection target cell U87MG-GRL
(1) culture is diluted to 1 × 10 after infection cell digestion counts5A/mL takes 2mL that 6 orifice plate cultures are added Ware shakes up and is placed on incubator culture.
(2) packaged virus is added to culture dish, shakes up after label is good and be placed in incubator culture by next day, 2-3d After can according to cell density expand cultivate.
(4) flow cytometer detection infection pZX-EGFRvIII after target cell U87MG-EGRL target antigen EGFRvIII expression
(1) cell in T175 culture bottle is collected, 200 × g is centrifuged 5min, is collected simultaneously non-infected cells as control.
(2) every pipe is added 1mL PBS and washes that cell is primary, and 200 × g is centrifuged 5min.
(3) every pipe is added 500 μ L PBS and cell is resuspended into 5mL Round Bottom PolystyreneTest Tube (filter membrane), using the expression of Flow cytometry U87MG-EGRL target cell surface antigen EGFRvIII.
(5) airflow classification monoclonal cell
(1) cell in T175 culture bottle is collected, 200 × g is centrifuged 5min, is collected simultaneously non-infected cells as control.
(2) every pipe is added 1mL PBS and washes that cell is primary, and 200 × g is centrifuged 5min.
(3) every pipe is added 500 μ L PBS and cell is resuspended into 5mL Round Bottom PolystyreneTestTube (filter membrane) 5mL culture medium, airflow classification GFP positive cell group are added after sorting, and sorts monoclonal in cell collecting pipe Into 96 orifice plates, 1, every hole cell, the entitled U87MG-EGRL of cell marking.
Flow cytometer detection is carried out to the U87MG-GRL cell line of building, as a result as shown in figure 4, showing that its target cell GFP is expressed It is positive.
(6) monoclonal cell expands culture
(1) target cell clone is expanded culture, the good monoclonal cell of amplification state is into T75 culture bottle.
(7) surface antigen and GFP expression identification that target cell is cloned after expanding
(1) cell in T175 culture bottle is collected, 200 × g is centrifuged 5min, is collected simultaneously non-infected cells as control.
(2) every pipe is added 1mL PBS and washes that cell is primary, and 200 × g is centrifuged 5min.
(3) every pipe is added 500 μ L PBS and cell is resuspended into 5mL Round Bottom PolystyreneTest Tube (filter membrane), using the expression of Flow cytometry U87MG-EGRL target cell surface antigen EGFRvIII, as a result such as Fig. 5 institute Show, EGFRvIII surface antigen expression rate is 99.2%.
(8) the luciferase expression identification that target cell is cloned after expanding
1. the luminous efficiency that microplate reader detects the luciferase of target cell
(1) target cell is seeded in 48 orifice plates, every hole 1*105A cell；
(2) it after cell is adherent, inhales and abandons culture medium, 150 μ L lysates are added in every hole,
(3) sufficiently after cracking, 10,000-15,000g is centrifuged 3-5min, takes supernatant for measuring；
(4) melt luciferase assay reagents, and reach room temperature；
(5) by instrumentation specification unlatching Chemiluminescence Apparatus or the multifunctional enzyme mark with detection chemiluminescence function Measuring interval is set as 2 seconds by instrument, and minute is set as 10 seconds；
(6) when each sample measures, sample 20-100 μ L is taken (if sample size is enough, 100 μ L please to be added；If sample Amount deficiency can suitably reduce dosage, but preferably be consistent with the usage amount of batch sample).
(7) 100 μ L luciferase assay reagents are added, beaten with rifle or measure RLU after being mixed with other appropriate ways (relative light unit).Using reporter gene cell pyrolysis liquid as blank control.Luciferase testing result such as Fig. 6 Shown, the target cell of building all has stronger luciferase expression, and control cell has no the expression of lucifarese.
2. luciferase luminous efficiency detects in animal body
(1) 1*10 is taken6A target cell inoculates to Mice Body, does 3 mouse；
(2) long to 100mm to tumour3When, the detection of luciferase luminous efficiency is carried out using small animal imaging system.It is real Result is tested as shown in fig. 7, the target cell of building is subcutaneously injected in Mice Body, small animal imaging all has as the result is shown The expression of luciferase, imaging results are good.
Embodiment 3
Target cell U87MG-EFRL cell killing Efficiency testing
(1) target cell U87MG-EFRL and U87MG-FRL are collected, is seeded in 96 orifice plates, it is thin that every hole is inoculated with 5000 targets Born of the same parents；
(2) effector cell (the CAR-T cell of targeting EGFR vIII antigen) is collected, after PBS washes twice, uses 10%FBS- 1640 are resuspended cell；
(3) effect target ratio sets 20:1, and 10: 1,5: 1,2.5: 1；
(4) after inoculation for 24 hours, by corresponding effect target than CAR-T cell is added；
(5) values of chemiluminescence is detected after killing 4h.
(6) killing-efficiency is calculated according to values of chemiluminescence.
Use CAR-EGFRvIII cell as effector cell, uses U87MG-GRL and U87MG-EGRL as target cell Killing rate change curve as shown in figure 8, CAR-EGFRvIII cell has stronger killing to make target cell U87MG-EGRL With weaker to the lethal effect for the U87MG-GRL cell for not expressing antigen；
Use CAR-CD19-T cell as effector cell, uses U87MG-GRL and U87MG-EGRL as target cell Killing rate change curve as shown in figure 9, control group CAR-CD19-T cell to target cell U87MG-GRL and U87MG-EGRL It is showed no more apparent lethal effect.
The target cell U87MG-EGRL and its construction method that the present invention provides a kind of for evaluating targeting EGFR vIII and Using, building target cell can high efficient expression EGFRvIII antigen, the target cell of targeting EGFR vIII antigen can be used as；Structure The target cell built not only expressed GFP gene but also express luciferase gene, can in vitro using GFP label to target cell into Row tracer, while the lethal effect for assessing CAR-T cell is reacted by luciferase, it is detected using Red luciferase It is high-efficient；Since the luminous efficiency of the Red luciferase of expression is higher, it is possible in vivo to the growing state of tumour It is monitored and tracks, it is equally effective for carrying out the highly difficult tracer such as tumor formation in situ.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.
Sequence table
<110>Jiao Shunchang
Beijing source Ding Chengtai Bioisystech Co., Ltd
<120>a kind of target cell U87MG-EGRL and its construction method and application for evaluating III antigen of targeting EGFR v
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2832
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atgcgaccct ccgggacggc cggggcagcg ctcctggcgc tgctggctgc gctctgcccg 60
gcgagtcggg ctctggagga aaagaaaggt aattatgtgg tgacagatca cggctcgtgc 120
gtccgagcct gtggggccga cagctatgag atggaggaag acggcgtccg caagtgtaag 180
aagtgcgaag ggccttgccg caaagtgtgt aacggaatag gtattggtga atttaaagac 240
tcactctcca taaatgctac gaatattaaa cacttcaaaa actgcacctc catcagtggc 300
gatctccaca tcctgccggt ggcatttagg ggtgactcct tcacacatac tcctcctctg 360
gatccacagg aactggatat tctgaaaacc gtaaaggaaa tcacagggtt tttgctgatt 420
caggcttggc ctgaaaacag gacggacctc catgcctttg agaacctaga aatcatacgc 480
ggcaggacca agcaacatgg tcagttttct cttgcagtcg tcagcctgaa cataacatcc 540
ttgggattac gctccctcaa ggagataagt gatggagatg tgataatttc aggaaacaaa 600
aatttgtgct atgcaaatac aataaactgg aaaaaactgt ttgggacctc cggtcagaaa 660
accaaaatta taagcaacag aggtgaaaac agctgcaagg ccacaggcca ggtctgccat 720
gccttgtgct cccccgaggg ctgctggggc ccggagccca gggactgcgt ctcttgccgg 780
aatgtcagcc gaggcaggga atgcgtggac aagtgcaacc ttctggaggg tgagccaagg 840
gagtttgtgg agaactctga gtgcatacag tgccacccag agtgcctgcc tcaggccatg 900
aacatcacct gcacaggacg gggaccagac aactgtatcc agtgtgccca ctacattgac 960
ggcccccact gcgtcaagac ctgcccggca ggagtcatgg gagaaaacaa caccctggtc 1020
tggaagtacg cagacgccgg ccatgtgtgc cacctgtgcc atccaaactg cacctacgga 1080
tgcactgggc caggtcttga aggctgtcca acgaatgggc ctaagatccc gtccatcgcc 1140
actgggatgg tgggggccct cctcttgctg ctggtggtgg ccctggggat cggcctcttc 1200
atgcgaaggc gccacatcgt tcggaagcgc acgctgcgga ggctgctgca ggagagggag 1260
cttgtggagc ctcttacacc cagtggagaa gctcccaacc aagctctctt gaggatcttg 1320
aaggaaactg aattcaaaaa gatcaaagtg ctgggctccg gtgcgttcgg cacggtgtat 1380
aagggactct ggatcccaga aggtgagaaa gttaaaattc ccgtcgctat caaggaatta 1440
agagaagcaa catctccgaa agccaacaag gaaatcctcg atgaagccta cgtgatggcc 1500
agcgtggaca acccccacgt gtgccgcctg ctgggcatct gcctcacctc caccgtgcag 1560
ctcatcacgc agctcatgcc cttcggctgc ctcctggact atgtccggga acacaaagac 1620
aatattggct cccagtacct gctcaactgg tgtgtgcaga tcgcaaaggg catgaactac 1680
ttggaggacc gtcgcttggt gcaccgcgac ctggcagcca ggaacgtact ggtgaaaaca 1740
ccgcagcatg tcaagatcac agattttggg ctggccaaac tgctgggtgc ggaagagaaa 1800
gaataccatg cagaaggagg caaagtgcct atcaagtgga tggcattgga atcaatttta 1860
cacagaatct atacccacca gagtgatgtc tggagctacg gggtgactgt ttgggagttg 1920
atgacctttg gatccaagcc atatgacgga atccctgcca gcgagatctc ctccatcctg 1980
gagaaaggag aacgcctccc tcagccaccc atatgtacca tcgatgtcta catgatcatg 2040
gtcaagtgct ggatgataga cgcagatagt cgcccaaagt tccgtgagtt gatcatcgaa 2100
ttctccaaaa tggcccgaga cccccagcgc taccttgtca ttcaggggga tgaaagaatg 2160
catttgccaa gtcctacaga ctccaacttc taccgtgccc tgatggatga agaagacatg 2220
gacgacgtgg tggatgccga cgagtacctc atcccacagc agggcttctt cagcagcccc 2280
tccacgtcac ggactcccct cctgagctct ctgagtgcaa ccagcaacaa ttccaccgtg 2340
gcttgcattg atagaaatgg gctgcaaagc tgtcccatca aggaagacag cttcttgcag 2400
cgatacagct cagaccccac aggcgccttg actgaggaca gcatagacga caccttcctc 2460
ccagtgcctg aatacataaa ccagtccgtt cccaaaaggc ccgctggctc tgtgcagaat 2520
cctgtctatc acaatcagcc tctgaacccc gcgcccagca gagacccaca ctaccaggac 2580
ccccacagca ctgcagtggg caaccccgag tatctcaaca ctgtccagcc cacctgtgtc 2640
aacagcacat tcgacagccc tgcccactgg gcccagaaag gcagccacca aattagcctg 2700
gacaaccctg actaccagca ggacttcttt cccaaggaag ccaagccaaa tggcatcttt 2760
aagggctcca cagctgaaaa tgcagaatac ctaagggtcg cgccacaaag cagtgaattt 2820
attggagcat ga 2832
<210> 2
<211> 717
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atgagtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60
gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120
aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 180
gtcactactt tcggttatgg tgttcaatgc tttgcgagat acccagatca tatgaaacag 240
catgactttt tcaagagtgc catgcctgaa ggttatgtac aggaaagaac tatatttttc 300
aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt 360
aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa 420
ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga 480
atcaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540
cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600
ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 660
cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caaataa 717
<210> 3
<211> 1647
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
atggaaacag aaagagaaga aaacgttgtc tacggcccac tgccattcta cccgatcgag 60
gagggctctg ccggcatcca attgcacaag tacatgcaac aatacgccaa gctcggcgcc 120
atcgccttca gtaacgccct gacaggcgtc gacatcagct accagcagta cttcgacatc 180
acgtgcagac tcgccgaggc tatgaagaac tacggcatga agccagaagg acacatcgct 240
ctctgtagcg agaactgcga agagttcttc attcctgttc tggctggtct ttacatcgga 300
gttacagtcg cgccaactaa cgaaatttat acacttagag agctgaacca cagtctgggg 360
atagcccaac ctactatcgt attctctagc aggaagggcc tgcccaaagt gcttgaggtg 420
cagaagaccg tgacttgcat caaaaccatt gtcatcctgg acagtaaggt caacttcggc 480
ggttatgact gcgtagagac cttcattaag aaacacgtcg agctgggctt tcctgccacc 540
tcatttgtgc ccatcgacgt caaagaccgg aagcaccaca ttgctctgct tatgaactct 600
tccggttcca cagggctgcc caaaggagta gagatcactc acgaggccct ggtcacgaga 660
ttctctcacg ctaaggaccc tatatacggc aatcaggtgg ccccaggtac cgctatcctg 720
actgtcgtgc ctttccacca cggcttcgga atgttcacta ctttgggcta ctttgcctgc 780
ggttaccgga ttgtcatgct tactaagttc gacgaggagc ttttcctgcg cacacttcag 840
gattacaagt gcactacagt aatcctggtg ccgacactgt tcgcaattct taataggtct 900
gagctccttg ataagtttga cctctctaac ctgactgaaa tagccagcgg tggtgctcca 960
cttgccaagg agatcggcga ggctgttgca agaagattca acctcccagg cgtccggcag 1020
ggatatggac tcaccgagac taccagtgcc tttatcatca ctcctaaggg cgacgacaag 1080
ccgggagcca gcggcaaggt cgtgcctctg ttcaaggtga agattattga cctcgatacc 1140
aagaaaacgt tgggtgtcaa cagacgggga gaaatctgcg tgaaaggacc atctcttatg 1200
ttgggataca cgaacaatcc tgaagccacc agagaaacta ttgacgagga aggctggctg 1260
cacacgggtg acatcgggta ctacgacgag gatgagcact tctttatagt cgaccgcctg 1320
aaatctctca ttaagtataa aggataccaa gtgccaccag ctgaactgga gtctgtgctc 1380
ctgcaacacc ctaacattag agatgctggt gtggccgggg ttcccgacag cgaggcaggc 1440
gagctgcctg gagccgtcgt tgtgatggaa aagggaaaga caatgactga gaaagaaatc 1500
gtagactatg taaactccca ggtggtcaac cacaagcggc tgaggggcgg cgtgcggttc 1560
gtagatgaag tccccaaggg gctcacagga aagatcgacg cgaaagttat cagggagata 1620
ctcaagaaac ctcaagcagg tgggtag 1647
<210> 4
<211> 54
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54
<210> 5
<211> 2449
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
gctagcgcgg ccgcgccgcc accatgagta aaggagaaga acttttcact ggagttgtcc 60
caattcttgt tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg 120
gtgaaggtga tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac 180
tacctgttcc atggccaaca cttgtcacta ctttcggtta tggtgttcaa tgctttgcga 240
gatacccaga tcatatgaaa cagcatgact ttttcaagag tgccatgcct gaaggttatg 300
tacaggaaag aactatattt ttcaaagatg acgggaacta caagacacgt gctgaagtca 360
agtttgaagg tgataccctt gttaatagaa tcgagttaaa aggtattgat tttaaagaag 420
atggaaacat tcttggacac aaattggaat acaactataa ctcacacaat gtatacatca 480
tggcagacaa acaaaagaat ggaatcaaag ttaacttcaa aattagacac aacattgaag 540
atggaagcgt tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg 600
tccttttacc agacaaccat tacctgtcca cacaatctgc cctttcgaaa gatcccaacg 660
aaaagagaga ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca 720
tggatgaact atacaaagag ggcagaggaa gtcttctaac atgcggtgac gtggaggaga 780
atcccggccc tatggaaaca gaaagagaag aaaacgttgt ctacggccca ctgccattct 840
acccgatcga ggagggctct gccggcatcc aattgcacaa gtacatgcaa caatacgcca 900
agctcggcgc catcgccttc agtaacgccc tgacaggcgt cgacatcagc taccagcagt 960
acttcgacat cacgtgcaga ctcgccgagg ctatgaagaa ctacggcatg aagccagaag 1020
gacacatcgc tctctgtagc gagaactgcg aagagttctt cattcctgtt ctggctggtc 1080
tttacatcgg agttacagtc gcgccaacta acgaaattta tacacttaga gagctgaacc 1140
acagtctggg gatagcccaa cctactatcg tattctctag caggaagggc ctgcccaaag 1200
tgcttgaggt gcagaagacc gtgacttgca tcaaaaccat tgtcatcctg gacagtaagg 1260
tcaacttcgg cggttatgac tgcgtagaga ccttcattaa gaaacacgtc gagctgggct 1320
ttcctgccac ctcatttgtg cccatcgacg tcaaagaccg gaagcaccac attgctctgc 1380
ttatgaactc ttccggttcc acagggctgc ccaaaggagt agagatcact cacgaggccc 1440
tggtcacgag attctctcac gctaaggacc ctatatacgg caatcaggtg gccccaggta 1500
ccgctatcct gactgtcgtg cctttccacc acggcttcgg aatgttcact actttgggct 1560
actttgcctg cggttaccgg attgtcatgc ttactaagtt cgacgaggag cttttcctgc 1620
gcacacttca ggattacaag tgcactacag taatcctggt gccgacactg ttcgcaattc 1680
ttaataggtc tgagctcctt gataagtttg acctctctaa cctgactgaa atagccagcg 1740
gtggtgctcc acttgccaag gagatcggcg aggctgttgc aagaagattc aacctcccag 1800
gcgtccggca gggatatgga ctcaccgaga ctaccagtgc ctttatcatc actcctaagg 1860
gcgacgacaa gccgggagcc agcggcaagg tcgtgcctct gttcaaggtg aagattattg 1920
acctcgatac caagaaaacg ttgggtgtca acagacgggg agaaatctgc gtgaaaggac 1980
catctcttat gttgggatac acgaacaatc ctgaagccac cagagaaact attgacgagg 2040
aaggctggct gcacacgggt gacatcgggt actacgacga ggatgagcac ttctttatag 2100
tcgaccgcct gaaatctctc attaagtata aaggatacca agtgccacca gctgaactgg 2160
agtctgtgct cctgcaacac cctaacatta gagatgctgg tgtggccggg gttcccgaca 2220
gcgaggcagg cgagctgcct ggagccgtcg ttgtgatgga aaagggaaag acaatgactg 2280
agaaagaaat cgtagactat gtaaactccc aggtggtcaa ccacaagcgg ctgaggggcg 2340
gcgtgcggtt cgtagatgaa gtccccaagg ggctcacagg aaagatcgac gcgaaagtta 2400
tcagggagat actcaagaaa cctcaagcag gtgggtagga attcggatc 2449
<210> 6
<211> 2855
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
gctagcgccg ccaccatgcg accctccggg acggccgggg cagcgctcct ggcgctgctg 60
gctgcgctct gcccggcgag tcgggctctg gaggaaaaga aaggtaatta tgtggtgaca 120
gatcacggct cgtgcgtccg agcctgtggg gccgacagct atgagatgga ggaagacggc 180
gtccgcaagt gtaagaagtg cgaagggcct tgccgcaaag tgtgtaacgg aataggtatt 240
ggtgaattta aagactcact ctccataaat gctacgaata ttaaacactt caaaaactgc 300
acctccatca gtggcgatct ccacatcctg ccggtggcat ttaggggtga ctccttcaca 360
catactcctc ctctggatcc acaggaactg gatattctga aaaccgtaaa ggaaatcaca 420
gggtttttgc tgattcaggc ttggcctgaa aacaggacgg acctccatgc ctttgagaac 480
ctagaaatca tacgcggcag gaccaagcaa catggtcagt tttctcttgc agtcgtcagc 540
ctgaacataa catccttggg attacgctcc ctcaaggaga taagtgatgg agatgtgata 600
atttcaggaa acaaaaattt gtgctatgca aatacaataa actggaaaaa actgtttggg 660
acctccggtc agaaaaccaa aattataagc aacagaggtg aaaacagctg caaggccaca 720
ggccaggtct gccatgcctt gtgctccccc gagggctgct ggggcccgga gcccagggac 780
tgcgtctctt gccggaatgt cagccgaggc agggaatgcg tggacaagtg caaccttctg 840
gagggtgagc caagggagtt tgtggagaac tctgagtgca tacagtgcca cccagagtgc 900
ctgcctcagg ccatgaacat cacctgcaca ggacggggac cagacaactg tatccagtgt 960
gcccactaca ttgacggccc ccactgcgtc aagacctgcc cggcaggagt catgggagaa 1020
aacaacaccc tggtctggaa gtacgcagac gccggccatg tgtgccacct gtgccatcca 1080
aactgcacct acggatgcac tgggccaggt cttgaaggct gtccaacgaa tgggcctaag 1140
atcccgtcca tcgccactgg gatggtgggg gccctcctct tgctgctggt ggtggccctg 1200
gggatcggcc tcttcatgcg aaggcgccac atcgttcgga agcgcacgct gcggaggctg 1260
ctgcaggaga gggagcttgt ggagcctctt acacccagtg gagaagctcc caaccaagct 1320
ctcttgagga tcttgaagga aactgaattc aaaaagatca aagtgctggg ctccggtgcg 1380
ttcggcacgg tgtataaggg actctggatc ccagaaggtg agaaagttaa aattcccgtc 1440
gctatcaagg aattaagaga agcaacatct ccgaaagcca acaaggaaat cctcgatgaa 1500
gcctacgtga tggccagcgt ggacaacccc cacgtgtgcc gcctgctggg catctgcctc 1560
acctccaccg tgcagctcat cacgcagctc atgcccttcg gctgcctcct ggactatgtc 1620
cgggaacaca aagacaatat tggctcccag tacctgctca actggtgtgt gcagatcgca 1680
aagggcatga actacttgga ggaccgtcgc ttggtgcacc gcgacctggc agccaggaac 1740
gtactggtga aaacaccgca gcatgtcaag atcacagatt ttgggctggc caaactgctg 1800
ggtgcggaag agaaagaata ccatgcagaa ggaggcaaag tgcctatcaa gtggatggca 1860
ttggaatcaa ttttacacag aatctatacc caccagagtg atgtctggag ctacggggtg 1920
actgtttggg agttgatgac ctttggatcc aagccatatg acggaatccc tgccagcgag 1980
atctcctcca tcctggagaa aggagaacgc ctccctcagc cacccatatg taccatcgat 2040
gtctacatga tcatggtcaa gtgctggatg atagacgcag atagtcgccc aaagttccgt 2100
gagttgatca tcgaattctc caaaatggcc cgagaccccc agcgctacct tgtcattcag 2160
ggggatgaaa gaatgcattt gccaagtcct acagactcca acttctaccg tgccctgatg 2220
gatgaagaag acatggacga cgtggtggat gccgacgagt acctcatccc acagcagggc 2280
ttcttcagca gcccctccac gtcacggact cccctcctga gctctctgag tgcaaccagc 2340
aacaattcca ccgtggcttg cattgataga aatgggctgc aaagctgtcc catcaaggaa 2400
gacagcttct tgcagcgata cagctcagac cccacaggcg ccttgactga ggacagcata 2460
gacgacacct tcctcccagt gcctgaatac ataaaccagt ccgttcccaa aaggcccgct 2520
ggctctgtgc agaatcctgt ctatcacaat cagcctctga accccgcgcc cagcagagac 2580
ccacactacc aggaccccca cagcactgca gtgggcaacc ccgagtatct caacactgtc 2640
cagcccacct gtgtcaacag cacattcgac agccctgccc actgggccca gaaaggcagc 2700
caccaaatta gcctggacaa ccctgactac cagcaggact tctttcccaa ggaagccaag 2760
ccaaatggca tctttaaggg ctccacagct gaaaatgcag aatacctaag ggtcgcgcca 2820
caaagcagtg aatttattgg agcatgagcg gccgc 2855

Claims (9)

1. a kind of for evaluating the target cell U87MG-EGRL of targeting EGFR vIII antigen, which is characterized in that the target cell U87MG-EGRL is glioma cell, can express CAR-T cell target antigen EGFRvIII gene, GFP and Red Luciferase Antigen-4 fusion protein gene.
2. target cell U87MG-EGRL according to claim 1, which is characterized in that the EGFRvIII gene is started by CMV Son starting expression, the EGFRvIII gene have the nucleotide sequence as shown in SEQ ID NO.1.
3. target cell U87MG-EGRL according to claim 1, which is characterized in that GFP the and Red Luciferase's Include sequentially connected GFP gene, connection peptide gene and Red Luciferase gene in antigen-4 fusion protein gene, the GFP and The nucleotide sequence of the antigen-4 fusion protein gene of Red Luciferase is as shown in SEQ ID NO.5.
4. the construction method of any one of claims 1 to 3 target cell U87MG-EGRL, which is characterized in that including following step It is rapid: (1) the EGFRvIII gene to be modified, the EGFRvIII target antigen sequence after must modifying；After the modification The nucleotide sequence of EGFRvIII target antigen sequence is as shown in SEQ ID NO.6；
(2) antigen-4 fusion protein gene of the GFP and Red Luciferase glioma cell is transferred to slow-virus transfection method to obtain Obtain the U87MG-GRL cell of expressed fusion protein；
(3) the EGFRvIII target antigen sequence after the modification that step (1) obtains is transferred to slow-virus transfection method described In U87MG-GRL cell, target cell U87MG-EGRL is obtained；
Temporal restriction relation is not present in step (1) and step (2).
5. construction method according to claim 4, which is characterized in that step (1) described modification is included in the EGFRvIII 5 ' the end addition NheI restriction enzyme sites and Kozak sequence of gene, in 3 ' end addition NotI restriction enzyme sites.
6. any one of claims 1 to 3 target cell U87MG-EGRL utilizes the construction method structure of claim 4 or 5 Application of the target cell U87MG-EGRL built in target antigen detection of expression.
7. any one of claims 1 to 3 target cell U87MG-EGRL utilizes the construction method structure of claim 4 or 5 The target cell U87MG-EGRL built examines the application in CAR-T Cell killing efficacy in vitro.
8. any one of claims 1 to 3 target cell U87MG-EGRL utilizes the construction method structure of claim 4 or 5 The target cell U87MG-EGRL built in vivo tumor formation experiment in application.
9. according to the application of any one of claim 6~8, which is characterized in that the target cell U87MG-EGRL is in application Concentration be (6~10) × 103A/mL, effect target ratio are (1~20): 1.
CN201910682087.8A 2019-07-26 2019-07-26 A kind of target cell U87MG-EGRL and its construction method and application for evaluating targeting EGFR vIII antigen Pending CN110305848A (en)

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CN113943751A (en) * 2021-10-20 2022-01-18 扬州大学 Target cell for H7N9 subtype avian influenza serum killing effect determination and identification method

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