CN109735558A - A kind of recombinant C AR19-IL24 gene, slow virus carrier, CAR19-IL24-T cell and application - Google Patents
A kind of recombinant C AR19-IL24 gene, slow virus carrier, CAR19-IL24-T cell and application Download PDFInfo
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Abstract
The invention discloses a kind of recombinant C AR19-IL24 gene, slow virus carrier, CAR19-IL24-T cell and applications.
Description
Technical field
The invention belongs to tumour cell immunological technique fields, and in particular to a kind of recombinant C AR19-IL24 gene, slow virus
Carrier, CAR19-IL24-T cell and application.
Background technique
Chimeric antigen receptor T cell immunotherapy (Chimeric Antigen Receptor T-Cell
Immunotherapy it) is also known as CAR-T cellular immunotherapy, is a kind of to carry out immune control using Chimeric antigen receptor T cell
The novel therapies for the treatment of.Chimeric antigen receptor T cell (CAR-T) is i.e. by modification transformation in surface expression Chimeric antigen receptor
(CAR) T cell of molecule." instructing again " will be carried out to T cell by expressing the CAR molecule on T cell film, assign it to expression
The specific recognition and killing ability of the target cell of CAR molecular target, so that specificity removes the tumour target cell of patient's body.
Chimeric antigen receptor (CAR), which generally comprises, can identify that the antigen-binding portion of the antibody of certain tumour antigen (is typically derived from antibody
The scFv section of antigen binding regions), an extracellular hinge area, a transmembrane region and an intracellular signal transduction area are (usually altogether
Stimulus signal structural domain and CD3 ζ intracellular region), these components are coupled together i.e. group in certain sequence becomes a chimeric antigen
Receptor (Chimeric Antigen Receptor, CAR).
Israel scientist Zelig Eshhar is put forward for the first time CAR-T concept within 1993；Pennsylvania, USA in 2011
University Carl H.June teaches team and applies CAR-T therapy in the treatment of 1 chronic lymphocytic leukemia (CLL) patient
It is broken through.CAR-T cell therapy acute lymphatic leukemia (ALL) patient is applied successfully in the team within 2012
Emily does not recur yet so far.2013 " Science " by CAR-T cellular immunotherapy be chosen as global ten big technological breakthroughs it
It is first.Existing two CAR-T product in 2017 is approved by the FDA in the United States into clinical application, and also there is corresponding CAR-T product in the country
Clinical approval stage.
CAR-T cellular immunotherapy has become the breakthrough method for the treatment of people's specific tumors so far.CAR-T cellular immunity
The therapeutic effect of therapy and the design of recombinant C AR molecule are closely related, and CAR molecule determines the tumour that CAR-T cell is adapted to
The effect of type and CAR-T cell therapy.Report that more CAR is the second generation or third generation design, their effect at present
Effect is still to be improved, how to advanced optimize or this improvement CAR design is the key that improve CART therapeutic effect.It is existing at present
The therapeutic effect of CAR-T can be enhanced in research report, the associational cells factor IL12, IL15, IL18 and IL33, prompts other white
Interleukin may also enhance the therapeutic effect of CART.It there is no the CAR-T cell products of autocrine IL-24 at present.
Summary of the invention
The present invention is directed to overcome the deficiencies of the prior art and provide a kind of recombinant C AR19-IL24 gene, slow virus carrier,
CAR19-IL24-T cell and application.
In order to achieve the above object, technical solution provided by the invention are as follows:
The sequence of the recombinant C AR19-IL24 gene is as shown in SEQ ID NO.1.
The slow virus carrier carries above-mentioned recombinant C AR19-IL24 gene, slow virus carrier sequence such as SEQ ID NO.2
It is shown.
It is described expression recombinant C AR19-IL24 gene T cell transduceed by above-mentioned slow virus carrier prepared into T cell and
At.
The T cell co-expresses CAR and IL24.
Preferably, the T cell is to be implemented in same load for after the connection of the expressing gene of the expressing gene of CAR and IL24
In the same expression cassette of body, then carrier transduction to T cell is prepared.
Preferably, which is characterized in that the T cell is that the expressing gene of CAR and IL24 are implemented in identical carrier not
It is prepared in expression cassette, then by carrier transduction to T cell.
The invention will be further described below:
Recombinant C AR19-IL24 gene association of the present invention interleukin-22 4 (IL-24) and embedding and antigen receptor
(CAR), the nucleic acid sequence comprising the following part of coding: the antigen-binding portion of the antibody of the specific recognition tumour antigen of extracellular region
Point, CD28 endochylema functional areas, 41BB endochylema functional areas and the CD3zeta endochylema functional areas of transmembrane segment and intracellular region, these born of the same parents
Inner region can be connected with random order, and can be with the IL-24 of autocrine.Using the recombinant C AR19-IL24 gene and contain
The efficiency of the carrier transfecting T cells of recombinant C AR19-IL24 gene is higher, and the positive T cell after transfecting has preferably
Proliferative capacity.In addition, by taking the CAR19.IL-24 of specific recognition CD19 molecule as an example, recombinant C AR19.IL of the present invention
The specificity of 24 gene pairs B cell tumour antigen CD19 molecules is high, kills CD19 with promoting CAR19.IL24-T cell-specific
+ B cell tumour.
Previous experiments result of the present invention and analysis learn that the expression of IL-24 can enhance CART treatment potential.One side
The proliferation and activation of T cell can be enhanced in face, IL24.T cell proliferation and activating potential are to influence CART product therapeutic effect
One of key factor, the T cell with stronger proliferation and activating potential, it is easier to after cultivating in vitro and feeding back to patient's body
Massive amplification, and be more advantageous to and maintain anti-tumor activity for a long time in vivo.On the other hand, IL24 shows kinds of tumor cells
Antitumor action out, such as causes tumor cell specific apoptosis, and Antineoplastic angiogenesis inhibits tumor cell invasion and transfer
Deng.In addition, IL24 molecule is smaller, it is easier to add IL24 expression cassette into CAR sequence.Based on above-mentioned experiment and analysis knot
Fruit, present invention firstly provides enhanced CAR19 (CAR19-IL24) the structure designs of fusion IL24, and construct and carry the sequence
Slow virus carrier LV-CAR19-IL24 is then prepared for the CAR-T cell of expression recombinant C AR19-IL24 gene.Its purpose exists
In the validity and safety that confirm CAR19-IL24 design, lay the foundation for the clinical application of CAR19-IL24-T cell.
By taking the CAR19.IL-24 of specific recognition CD19 molecule as an example:
1) basis has been reported the present invention first and bioinformatics mode finds specific recognition CD19 molecule
CAR19, T2A, IL24CDS sequence pass through gene chemical synthesis CAR sequence, CAR19-IL24 sequence.
2) CAR19 sequence and CAR19-IL24 sequence are connected by slow virus carrier Addgene:# by molecule clone technology
12252.Therebetween pass through T2A connection (above each segment DNA sequence temporarily non-annex).
3) acquisition normal human volunteers' peripheral blood density gradient centrifugation separation mononuclearcell (PBMC), then negative sense sorts
Total T cell (PanT cell) is obtained, stimulated using CD3/28 magnetic bead and adds IL-2 progress amplification in vitro.
4) packaging carries the slow virus of recombinant C AR19-IL24 gene, and transduction stimulates 24 hours PanT through CD3/28 magnetic bead
Cell, the enhanced CART cell of preparation expression recombinant C AR19-IL24 gene.
Compared with prior art, the invention has the benefit that
1) confirm the slow virus carrier sequential element built without mutation by being sequenced.
2) have using the CAR19.IL-24-T cell of the IL24 enhancing of slow virus carrier preparation compared to CAR19-T cell
Higher anti-tumor activity.T cell by being identified by PCR, determining and successfully incorporate CAR sequence after lentiviruses transduction,
By flow cytometry, Western Blot, Q-PCR, which is detected, determines successful expression CAR molecule.
3) it is detected by ability of cell proliferation and determines that CAR19.IL-24-T cell has stronger proliferative capacity.
4) it is detected by co culture system in vitro cytotoxicity and determines that CAR19.IL-24-T cell has stronger tumor-killing to imitate
Fruit.
In addition, thinking according to the present invention, can choose different CAR extracellular region-intracellular domain and combines with IL24, with
Adapt to different tumor cell types.It can choose different connection original parts to construct IL-24 and CAR molecule in a different order
In the same expression cassette.It can be co-expressed in T cell in different expression cassette by IL-24 and CAR are separated.
In short, the present invention combines interleukin-22 4 and Chimeric antigen receptor to obtain preferably in immunotherapy of tumors is applied
Therapeutic effect can effectively prepare CAR19-IL24-T cell using the present invention, express the chimeric antigen of target tumor antigen
Receptor and interleukin-22 4.Compared with CD19-CART cell, CAR19-IL24-T cell has stronger tumor function of killing to increase with better
Grow ability.And confirm that CAR19-IL24-T cell is a kind of safe, special, effective CART cell, there is important clinic to answer
With value.
Detailed description of the invention
Fig. 1 is CAR19 and CAR19-IL24 schematic diagram；
Fig. 2 is that the protein expression level figure of CAR19 and CAR19-IL24 in the cell is detected using Western Blot；
Fig. 3 is that CAR19-IL24-T cell GFP expression figure is observed using inverted fluorescence microscope；
Fig. 4 is the CAR19 molecule that the surface CAR19-T and CAR19-IL24-T is detected using Flow cytometry；
Fig. 5 is the Function detection result figure of CAR19.IL-24-T cell；In figure, E: effectiveness cell, difference LV-CAR19-
The CAR19-IL24-T cell of IL24 transduction, the CAR19-T cell of LV-CAR19 transduction, and transduce LV-GFP's with batch
T cell；T: target cell is the Raji cell of the CD19 positive；CD19 positive cell (i.e. Raji is detected using Annexin-V APC+
Cell) apoptosis situation, ordinate indicate target cell apoptosis ratio；
Fig. 6 is to be schemed using PCR from the plasmid amplification IL-24 of the existing sequence containing IL-24；
Fig. 7 is that bacterium solution PCR identifies information drawing.
Specific embodiment
The building of 1 CAR19 expression cassette and slow virus carrier
(1) basis has been reported and bioinformatics mode finds the FMC63scFv sequence of specific recognition CD19 molecule
Column, design transmembrane region and intracellular signal transduction region sequence and gene chemical synthesis sequence (CAR19), and the design of CAR19 sequence both ends has
Bbs1 restriction enzyme site, the sequence are loaded into pUC57 plasmid (pUC57-CAR19).
(2) Bbs1 digestion pUC57-CAR19 will hold respectively in CAR19 sequence 5 ' and 3 ' and generate BamH1 and Age1 viscosity end
End.Two generation of BamH1 and Age1 double digestion slow virus carrier plasmid pHQ-009 is used simultaneously.(attached digestion system)
(3) glue recycles pHQ-009 skeleton 7379bp and CAR19 sequence 1534bp.It is constructed using T4DNA Ligase connection
Plasmid pHQ-010, (25 DEG C) of Connection Time room temperature are no more than 10min or 16 DEG C of water-bath and stay overnight.(attached linked system)
(4) 5 μ l of connection product is taken, is converted using Stbl3.(attached Stbl3 transformation system)
Picking monoclonal carries out bacterium solution PCR identification after (5) 14 hours.(attached bacterium solution PCR system and condition)
(6) bacterium solution PCR positive colony is taken to carry out Sanger sequencing identification.
(7) take Sanger sequencing identification sequence and the consistent clone of theoretical sequence to carry out plasmid and mention pHQ-010 greatly, for containing
There are two generation slow virus carrier plasmids of CAR19 sequence.
The building of 2 CAR19.IL-24 expression cassettes and slow virus carrier
(1) plasmid amplification IL-24 of the PCR from the existing sequence containing IL-24 is used, is introduced respectively using upstream and downstream primer
Xma1 and HindIII restriction enzyme site.(attached plasmid PCR system and condition are shown in Table 1, using PCR from the existing sequence containing IL-24
Plasmid amplification IL-24 figure is shown in Fig. 6)
Table 1
(2) PCR product is subjected to glue recovery purifying 723bp.PGEM-T-easy carrier is connected into using T4DNA Ligase.
According to the amount being added in OD value and skeleton and fragment length estimation linked system, with Insert Fragment: mole of skeleton
Amount ratio prepares system (being shown in Table 2) between 3:1~10:1:
Table 2
(3) 5 μ l of connection product is taken, is converted using DH5 α, " Lan Bai " screening is carried out.
It takes DH5 α competence to be placed in ice bath and dissolves 5min.
5 μ l connection products/100 μ l competence are added thereto.
30min is stood in ice water.
42 DEG C of water-bath 90S.It not shake
Cooling 3min is quickly transferred in ice water.
Add the 900 μ L of culture medium without Amp resistance, is placed in 37 DEG C of shaking tables-revolving speed 150rpm*45min.
Prepare the plate (the ammonia benzyl resistant panel containing IPTG/X-Gal) of blue and white screening: by the 100mM IPTG of 10 μ l
It is coated on the surface of ammonia benzyl resistant panel with 40 μ L 20mg/ml X-gal, and is protected from light just to set 30 minutes in room temperature and is allowed to absorb
It is spare afterwards.
Take it is appropriate f) in bacterium solution be applied to plate in g), be placed in 37 DEG C of incubators, be inverted culture 14 hours.
(4) picking white colonies carry out Sanger sequence verification.Take Sanger sequencing identification sequence consistent with theoretical sequence
Clone carry out plasmid mention pHQ-040 greatly.
(5) Xma1 and HindIII double digestion pHQ-040 is used；Use Age1 and HindIII double digestion pUC57-
CAR19。
According to the form below prepares pHQ-040 reaction system (being shown in Table 3):
Table 3
According to the form below prepares pUC57-CAR19 reaction system (table 4):
Table 4
(6) the IL-24 sequence 723bp and pUC57-CAR19 skeleton 4290bp in glue recycling pHQ-040.Use T4DNA
Ligase connection constructs plasmid pHQ-036, and (25 DEG C) of Connection Time room temperature are no more than 10min or 16 DEG C of water-bath and stay overnight.It is (attached
Junctor system)
According to the amount being added in OD value and skeleton and fragment length estimation linked system, with Insert Fragment: mole of skeleton
Amount ratio prepares system (table 5) between 3:1~10:1:
Table 5
(7) 5 μ l of connection product is taken, is converted using DH5 α.(attached DH5 α transformation system)
A) it takes DH5 α competence to be placed in ice bath and dissolves 5min.
B) 5 μ l connection products/100 μ l competence are added thereto.
C) 30min is stood in ice water.
D) 42 DEG C of water-bath 90S.It not shake
E) cooling 3min is quickly transferred in ice water.
F) add the 900 μ L of culture medium without Amp resistance, be placed in 37 DEG C of shaking tables-revolving speed 150rpm*45min.
G) take it is appropriate f) in bacterium solution be applied to Amp resistant panel, be placed in 37 DEG C of incubators, be inverted culture 14 hours.
Picking monoclonal carries out bacterium solution PCR identification after (8) 14 hours.(attached bacterium solution PCR system and condition, PCR information drawing are shown in
Fig. 7)
Monoclonal sterile small Tip picking to 300 μ l LB is trained into base, after being placed in 37 DEG C of shaking tables-revolving speed 150rpm*45min
For following PCR (table 6):
Table 6
(9) bacterium solution PCR positive colony is taken to carry out Sanger sequencing identification.
(10) it takes Sanger sequencing identification sequence and the consistent clone of theoretical sequence to carry out plasmid and mentions pHQ-036 greatly.
(11) Bbs1 digestion pHQ-036 is used, glue recycles CAR19.IL-24 sequence 2215bp；Simultaneously using BamH1 and
Age1 double digestion two generations slow virus carrier plasmid pHQ-009, glue recycle pHQ-009 skeleton 7379bp；Use T4DNA Ligase
Connection building plasmid pHQ-035, is the two generation lentivirus sequences containing CAR19.IL-24 sequence.
3 CAR 19 and CAR19.IL-24 slow virus prepare and (are shown in Table 7):
Table 7
4 CAR 19-T cells and the preparation of CAR19.IL-24-T cell
(1) acquisition normal human volunteers' peripheral blood density gradient centrifugation separation mononuclearcell (PBMC), then negative sense divides
Choosing obtains total T cell (PanT cell), is stimulated using CD3/28 magnetic bead and adds IL-2 progress amplification in vitro.
(2) CAR 19 and CAR19.IL-24 slow virus are transduceed respectively stimulates 24 hours PanT cells through CD3/28 magnetic bead,
It prepares CAR19-T cell and expresses the enhanced CART cell of recombinant C AR19.IL-24 gene.
The detection of 5 CAR19.IL-24-T cells
The detection of CAR expression
See Fig. 2, what CAR19 arrow indicated is the position of CAR19 albumen, and the instruction of IL24 arrow is IL24 protein positions.
Binding protein Marker:26616 (ThermoFisher) instruction, prompts the CAR19 of cell inner expression, CAR19-IL24 and pre-
The CAR19 albumen of phase is consistent with IL24 albumen size.
See Fig. 3, is transduceed after T cell using slow virus carrier LV-CAR19-IL24, the GFP carried will table in the cell
It reaches.Left figure is bright field image, and right figure is the fluorescent image under the same visual field.Prompt slow virus carrier LV-CAR19-IL24 transduction
T cell express GFP, indicator virus transduction success and CAR19-IL24 expression.
See Fig. 4, is transduceed after T cell using slow virus carrier LV-CAR19, LV-CAR19-IL24, the CAR19 carried
Molecule will be in T cell surface expression.The antibody test CAR19 molecule being coupled using APC, the figure area Zhong M1 are apparent positive letter
Number, after prompting CAR19 molecule to there is higher expression, left figure to prompt slow virus carrier LV-CAR19 transduction on T cell surface
There is 55.96% T cell expression CAR19 molecule, there is 63.8% T cell expression after right figure prompt LV-CAR19-IL24 transduction
CAR19 molecule.
The Function detection of 6 CAR19.IL-24-T cells
Cytotoxicity detection
Fig. 5 shows that, when the cell proportion of E:T is 5-8, CAR19-IL24-T cell and CAR19-T cell can be shown
Promote the effect of target cell apoptosis, it is often more important that, the rush apoptosis effect of CAR19-IL24-T is obviously more preferable compared with CAR19-T.
SEQUENCE LISTING
<110>Central South University
<120>a kind of recombinant C AR19-IL24 gene, slow virus carrier, CAR19-IL24-T cell and application
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 2947
<212> DNA
<213>artificial synthesized
<400> 1
gatcgccacc atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc 60
attcctcctg atcccagaca tccagatgac acagactaca tcctccctgt ctgcctctct 120
gggagacaga gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg 180
gtatcagcag aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca 240
ctcaggagtc ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat 300
tagcaacctg gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc 360
gtacacgttc ggagggggga ctaagttgga aataacaggc tccacctctg gatccggcaa 420
gcccggatct ggcgagggat ccaccaaggg cgaggtgaaa ctgcaggagt caggacctgg 480
cctggtggcg ccctcacaga gcctgtccgt cacatgcact gtctcagggg tctcattacc 540
cgactatggt gtaagctgga ttcgccagcc tccacgaaag ggtctggagt ggctgggagt 600
aatatggggt agtgaaacca catactataa ttcagctctc aaatccagac tgaccatcat 660
caaggacaac tccaagagcc aagttttctt aaaaatgaac agtctgcaaa ctgatgacac 720
agccatttac tactgtgcca aacattatta ctacggtggt agctatgcta tggactactg 780
gggtcaagga acctcagtca ccgtctcctc agcggccgca attgaagtta tgtatcctcc 840
tccttaccta gacaatgaga agagcaatgg aaccattatc catgtgaaag ggaaacacct 900
ttgtccaagt cccctatttc ccggaccttc taagcccttt tgggtgctgg tggtggttgg 960
gggagtcctg gcttgctata gcttgctagt aacagtggcc tttattattt tctgggtgag 1020
gagtaagagg agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg 1080
gcccacccgc aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc 1140
cagagtgaag ttcagcagga gcgcagacgc ccccgcgtac cagcagggcc agaaccagct 1200
ctataacgag ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg 1260
ccgggaccct gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa 1320
tgaactgcag aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg 1380
ccggaggggc aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac 1440
ctacgacgcc cttcacatgc aggccctgcc ccctcgctat gagggcagag gaagtctgct 1500
aacatgcggt gacgtcgagg agaatcctgg cccaccgggt atgaattttc aacagaggct 1560
gcaaagcctg tggactttag ccagcagacc cttctgccct cctttgctgg cgacagcctc 1620
tcaaatgcag atggttgtgc tcccttgcct gggttttacc ctgcttctct ggagccaggt 1680
atcaggggcc cagggccaag aattccactt tgggccctgc caagtgaagg gggttgttcc 1740
ccagaaactg tgggaagcct tctgggctgt gaaagacact atgcaagctc aggataacat 1800
cacgagtgcc cggctgctgc agcaggaggt tctgcagaac gtctcggatg ctgagagctg 1860
ttaccttgtc cacaccctgc tggagttcta cttgaaaact gttttcaaaa actaccacaa 1920
tagaacagtt gaagtcagga ctctgaagtc attctctact ctggccaaca actttgttct 1980
catcgtgtca caactgcaac ccagtcaaga aaatgagatg ttttccatca gagacagtgc 2040
acacaggcgg tttctgctat tccggagagc attcaaacag ttggacgtag aagcagctct 2100
gaccaaagcc cttggggaag tggacattct tctgacctgg atgcagaaat tctacaagct 2160
cgagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccaccggt 2220
cgccaccatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga 2280
gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc 2340
cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg 2400
gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca 2460
catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac 2520
catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga 2580
caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct 2640
ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca 2700
gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca 2760
gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga 2820
caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca 2880
catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta 2940
caagtaa 2947
<210> 2
<211> 9594
<212> DNA
<213>artificial synthesized
<400> 2
agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag 60
caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg 120
tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact 180
gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct 240
ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 300
aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 360
tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc 420
gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc 480
ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa 540
ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg 600
ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata 660
aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc 720
tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga 780
caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc 840
aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca 900
aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg 960
agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga 1020
gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata 1080
ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcctcaatg 1140
acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg 1200
ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag 1260
ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt 1320
tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt 1380
aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt 1440
aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag 1500
aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata 1560
acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta 1620
agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta 1680
tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa 1740
gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat 1800
cggttaactt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta 1860
gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa 1920
aattttatcg atcacgagac tagcctcgag aagcttgata tcgaattcca cggggttggg 1980
gttgcgcctt ttccaaggca gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg 2040
ttccgggaaa cgcagcggcg ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag 2100
cgtcacccgg atcttcgccg ctacccttgt gggccccccg gcgacgcttc ctgctccgcc 2160
cctaagtcgg gaaggttcct tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc 2220
acgtctcact agtaccctcg cagacggaca gcgccaggga gcaatggcag cgcgccgacc 2280
gcgatgggct gtggccaata gcggctgctc agcagggcgc gccgagagca gcggccggga 2340
aggggcggtg cgggaggcgg ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg 2400
gtgttccgca ttctgcaagc ctccggagcg cacgtcggca gtcggctccc tcgttgaccg 2460
aatcaccgac ctctctcccc agggggatcg ccaccatgct tctcctggtg acaagccttc 2520
tgctctgtga gttaccacac ccagcattcc tcctgatccc agacatccag atgacacaga 2580
ctacatcctc cctgtctgcc tctctgggag acagagtcac catcagttgc agggcaagtc 2640
aggacattag taaatattta aattggtatc agcagaaacc agatggaact gttaaactcc 2700
tgatctacca tacatcaaga ttacactcag gagtcccatc aaggttcagt ggcagtgggt 2760
ctggaacaga ttattctctc accattagca acctggagca agaagatatt gccacttact 2820
tttgccaaca gggtaatacg cttccgtaca cgttcggagg ggggactaag ttggaaataa 2880
caggctccac ctctggatcc ggcaagcccg gatctggcga gggatccacc aagggcgagg 2940
tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg tccgtcacat 3000
gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc cagcctccac 3060
gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac tataattcag 3120
ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt ttcttaaaaa 3180
tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat tattactacg 3240
gtggtagcta tgctatggac tactggggtc aaggaacctc agtcaccgtc tcctcagcgg 3300
ccgcaattga agttatgtat cctcctcctt acctagacaa tgagaagagc aatggaacca 3360
ttatccatgt gaaagggaaa cacctttgtc caagtcccct atttcccgga ccttctaagc 3420
ccttttgggt gctggtggtg gttgggggag tcctggcttg ctatagcttg ctagtaacag 3480
tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac agtgactaca 3540
tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc tatgccccac 3600
cacgcgactt cgcagcctat cgctccagag tgaagttcag caggagcgca gacgcccccg 3660
cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga agagaggagt 3720
acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag ccgagaagga 3780
agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg gaggcctaca 3840
gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc ctttaccagg 3900
gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc ctgccccctc 3960
gctatgaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccac 4020
cgggtatgaa ttttcaacag aggctgcaaa gcctgtggac tttagccagc agacccttct 4080
gccctccttt gctggcgaca gcctctcaaa tgcagatggt tgtgctccct tgcctgggtt 4140
ttaccctgct tctctggagc caggtatcag gggcccaggg ccaagaattc cactttgggc 4200
cctgccaagt gaagggggtt gttccccaga aactgtggga agccttctgg gctgtgaaag 4260
acactatgca agctcaggat aacatcacga gtgcccggct gctgcagcag gaggttctgc 4320
agaacgtctc ggatgctgag agctgttacc ttgtccacac cctgctggag ttctacttga 4380
aaactgtttt caaaaactac cacaatagaa cagttgaagt caggactctg aagtcattct 4440
ctactctggc caacaacttt gttctcatcg tgtcacaact gcaacccagt caagaaaatg 4500
agatgttttc catcagagac agtgcacaca ggcggtttct gctattccgg agagcattca 4560
aacagttgga cgtagaagca gctctgacca aagcccttgg ggaagtggac attcttctga 4620
cctggatgca gaaattctac aagctcgagg gcagaggaag tctgctaaca tgcggtgacg 4680
tcgaggagaa tcctggccca ccggtcgcca ccatggtgag caagggcgag gagctgttca 4740
ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg 4800
tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca 4860
ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc 4920
agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc 4980
ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc 5040
gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg 5100
acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca 5160
acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc 5220
acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg 5280
gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca 5340
aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga 5400
tcactctcgg catggacgag ctgtacaagt aaagcggccg cgtcgacaat caacctctgg 5460
attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat 5520
gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt 5580
tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca 5640
ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg 5700
ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg 5760
aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca 5820
attccgtggt gttgtcgggg aagctgacgt cctttccatg gctgctcgcc tgtgttgcca 5880
cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc 5940
ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc 6000
agacgagtcg gatctccctt tgggccgcct ccccgcctgg aattcgagct cggtaccttt 6060
aagaccaatg acttacaagg cagctgtaga tcttagccac tttttaaaag aaaagggggg 6120
actggaaggg ctaattcact cccaacgaag acaagatctg ctttttgctt gtactgggtc 6180
tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct 6240
taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga 6300
ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtag 6360
tagttcatgt catcttatta ttcagtattt ataacttgca aagaaatgaa tatcagagag 6420
tgagaggaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 6480
tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 6540
tgtatcttat catgtctggc tctagctatc ccgcccctaa ctccgcccag ttccgcccat 6600
tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc 6660
tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcgtcgag 6720
acgtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac 6780
aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc 6840
ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 6900
gcagcctgaa tggcgaatgg cgcgacgcgc cctgtagcgg cgcattaagc gcggcgggtg 6960
tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg 7020
ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg 7080
ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt 7140
agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt 7200
tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta 7260
tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa 7320
atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt 7380
cccaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat 7440
acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg 7500
aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc 7560
attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga 7620
tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga 7680
gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg 7740
cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc 7800
tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac 7860
agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact 7920
tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca 7980
tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg 8040
tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact 8100
acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg 8160
accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg 8220
tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat 8280
cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc 8340
tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat 8400
actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt 8460
tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc 8520
cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt 8580
gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac 8640
tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt 8700
gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct 8760
gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga 8820
ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac 8880
acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg 8940
agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt 9000
cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc 9060
tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg 9120
gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc 9180
ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc 9240
ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag 9300
cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca 9360
ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat 9420
taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg 9480
tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga 9540
ttacgccaag cgcgcaatta accctcacta aagggaacaa aagctggagc tgca 9594
Claims (6)
1. a kind of recombinant C AR19-IL24 gene, which is characterized in that the sequence of the recombinant C AR19-IL24 gene such as SEQ ID
Shown in NO.1.
2. a kind of slow virus carrier, which is characterized in that the slow virus carrier carries recombinant C AR19- described in claim 1
IL24 gene, the slow virus carrier sequence is as shown in SEQ ID NO.2.
3. a kind of T cell for expressing recombinant C AR19-IL24 gene, which is characterized in that the expression recombinant C AR19-IL24 gene
T cell transduceed by slow virus carrier as claimed in claim 2 and be prepared into T cell.
4. a kind of T cell, which is characterized in that the T cell co-expresses CAR and IL24.
5. T cell as claimed in claim 4, which is characterized in that the T cell is the table by the expressing gene of CAR and IL24
Up to after gene connection, it is implemented in the same expression cassette of identical carrier, then carrier transduction to T cell is prepared.
6. T cell as claimed in claim 4, which is characterized in that the T cell is to construct the expressing gene of CAR and IL24
It is prepared in the different expression cassettes of identical carrier, then by carrier transduction to T cell.
Priority Applications (2)
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| CN201811517078.5A CN109735558B (en) | 2018-12-12 | 2018-12-12 | Recombinant CAR19-IL24 gene, lentiviral vector, CAR19-IL24-T cell and application |
| CN202210613661.6A CN116479022A (en) | 2018-12-12 | 2018-12-12 | Recombinant CAR19-IL24 gene, lentiviral vector, CAR19-IL24-T cell and application |
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| CN201811517078.5A CN109735558B (en) | 2018-12-12 | 2018-12-12 | Recombinant CAR19-IL24 gene, lentiviral vector, CAR19-IL24-T cell and application |
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| CN201811517078.5A Active CN109735558B (en) | 2018-12-12 | 2018-12-12 | Recombinant CAR19-IL24 gene, lentiviral vector, CAR19-IL24-T cell and application |
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| CN111423517B (en) * | 2020-04-15 | 2023-05-12 | 赛德特生物制药有限公司 | Tumor cell stem-restricted CAR and application thereof |
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| CN116479022A (en) | 2023-07-25 |
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