CN112592898B - Reprogrammed NK feeder cells and preparation method and application thereof - Google Patents

Reprogrammed NK feeder cells and preparation method and application thereof Download PDF

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CN112592898B
CN112592898B CN202011518988.2A CN202011518988A CN112592898B CN 112592898 B CN112592898 B CN 112592898B CN 202011518988 A CN202011518988 A CN 202011518988A CN 112592898 B CN112592898 B CN 112592898B
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CN112592898A (en
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汤朝阳
请求不公布姓名
秦乐
吴迪
冯世忠
冯嘉昆
杨乐旋
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Guangdong Zhaotai Cell Biotechnology Co ltd
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Abstract

The invention provides a reprogrammed NK feeder cell, a preparation method and application thereof, wherein the reprogrammed NK feeder cell expresses membrane proteins IL-12, CD19, CD64 and CD86. The invention adopts the combination of IL-12, CD19, CD64 and CD86 to construct feeder cells for reprogramming NK cell culture, and the obtained reprogramming NK cells have the advantages of rapid increase speed, remarkable tumor killing capability and good functional stability, and have important application prospect in the field of immunotherapy.

Description

Reprogrammed NK feeder cells and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biology, relates to a reprogrammed NK feeder cell and a preparation method and application thereof, and in particular relates to a reprogrammed NK feeder cell and a preparation method and application thereof in culturing reprogrammed NK cells.
Background
The reprogrammed NK cells (ITNK cells) are transgenic cells with double functions of T cells and NK cells, which are obtained by performing gene editing on T cells by using CRISPR/Cas9 technology. Compared with T cells or NK cells, the reprogrammed NK cells are independent of histocompatibility complex (MHC), have significantly improved tumor killing function, and show great potential in cancer treatment.
At present, a major obstacle restricting the application of reprogrammed NK cells to immunotherapy is that it is difficult to obtain a sufficient number of reprogrammed NK cells with complete functions, and the realization of large-scale expansion of reprogrammed NK cells in vitro is a critical problem to be solved urgently.
In recent years, artificial antigen presenting cells (trophoblasts) constructed using genetic engineering techniques are increasingly being applied to in vitro expansion of NK cells. However, unlike NK cells, which have dual functions of T cells and NK cells, there is currently no effective method for amplifying and activating reprogrammed NK cells in vitro.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides a reprogramming NK feeder cell, a preparation method and application thereof, wherein the reprogramming NK feeder cell expresses a plurality of ligands for activating the reprogramming NK cell, and the number and the activity of the reprogramming NK cell are obviously improved.
To achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a reprogrammed NK feeder cell which expresses the membrane proteins IL-12, CD19, CD64 and CD86.
In the invention, IL-12, CD19, CD64 and CD86 are expressed on the surface of mammalian cells to construct feeder cells, and the feeder cells are combined with receptors on the surface of the reprogrammed NK cells to activate the reprogrammed NK cells, so that the amplification quantity and tumor cytotoxicity of the reprogrammed NK cells are remarkably improved.
Preferably, the IL-12 is expressed on the surface of the reprogrammed NK feeder cell by forming a fusion protein with the CD 8. Alpha. Transmembrane region.
Preferably, the IL-12 includes the amino acid sequence shown in SEQ ID NO. 1;
SEQ ID NO:1：
MWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAGGGGSMCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYY。
preferably, the CD 8. Alpha. Transmembrane region comprises the amino acid sequence shown in SEQ ID NO. 2;
SEQ ID NO:2：IYIWAPLAGTCGVLLLSLVITLYC。
preferably, the CD19 comprises the amino acid sequence shown in SEQ ID NO. 3;
SEQ ID NO:3：
MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHL。
preferably, the CD64 comprises the amino acid sequence shown in SEQ ID NO. 4;
SEQ ID NO:4：
MWFLTTLLLWPVDGQVDTTKAVITLQPPWVSVFQEETVTLHCEVLHLPGSSSTQWFLNGTATQTSTPSYRITSASVNDSGEYRCQRGLSGRSDPIQLEIHRGWLLLQVSSRVFTEGEPLALRCHAWVDKLVYNVLYYRNGKAFKFFHWNSNLTILKTNISHNGTYHCSGMGKHRYTSAGISVTVKELFPAPVLNTSVTSPLLEGNLVTLSCETKLLLQRPGLQLYFSFYMGSKTLRGRNTSSEYQILTARREDSGLYWCEAATEDGNVLKRSPELELQVLGLQLPTPVWFHVLFYLAVG。
preferably, the CD86 comprises the amino acid sequence shown in SEQ ID NO. 5;
SEQ ID NO:5：
MGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF。
in a second aspect, the invention provides an expression vector comprising IL-12 and CD 8. Alpha. Transmembrane region fusion protein encoding genes, CD19 encoding genes, CD64 encoding genes, and CD86 encoding genes.
Preferably, the expression vector comprises a viral vector.
Preferably, the viral vector comprises any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector, preferably a lentiviral vector.
In a third aspect, the invention provides a recombinant lentivirus prepared by co-transfecting mammalian cells with the expression vector of the second aspect and a packaging helper plasmid.
Preferably, the mammalian cells comprise any one or a combination of at least two of 293 cells, 293T cells or 293F cells.
In a fourth aspect, the present invention provides a method for preparing a reprogrammed NK feeder cell of the first aspect, said method comprising the step of introducing the recombinant lentivirus of the third aspect into K562 cells, and screening positive clones using antibiotics and a flow cytometer.
In a fifth aspect, the invention provides a medium comprising reprogrammed NK feeder cells of the first aspect.
Preferably, the medium further comprises IL-2 and/or serum.
Preferably, the culture medium comprises any one or a combination of at least two of Eagle culture medium, RPMI-1640 culture medium or Ham's F-10.
In a sixth aspect, the present invention provides a method of culturing reprogrammed NK cells, said method comprising the step of culturing reprogrammed NK cells using the medium of the fifth aspect.
Preferably, the culturing method further comprises the step of irradiating the reprogrammed NK cells.
Preferably, the dose of irradiation is 50-500 Gy.
Preferably, the preparation method of the reprogrammed NK cells is prepared by electrotransferring CRISPR/Cas9 plasmid targeting Bcl11b gene into activated T cells.
In a seventh aspect, the present invention provides a reprogrammed NK cell cultivated by the cultivation method of the sixth aspect.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention adopts the combination of IL-12, CD19, CD64 and CD86 to construct feeder cells for reprogramming NK cell culture, the feeder cells have obvious proliferation promoting and activating effects on the reprogramming NK cells, and after 2 weeks of culture, the number of the reprogramming NK cells is increased by about 120 times;
(2) Compared with T cells and NK cells, the reprogrammed NK cells have strong tumor killing capability and stable killing function, and have application prospects in the field of immunotherapy.
Drawings
FIG. 1 in vitro expansion capacity of reprogrammed NK cells after co-culturing with reprogrammed NK feeder cells;
FIG. 2 tumor cytotoxicity after co-culturing reprogrammed NK cells with reprogrammed NK feeder cells;
FIG. 3 stability of killing function after co-culture of reprogrammed NK cells with reprogrammed NK feeder cells.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
EXAMPLE 1 construction of reprogrammed NK cells
(1) The sgRNA (SEQ ID NO:6: gaccatgaactgctcacttg) was designed according to the Bcl11b gene, and restriction enzyme sites EcoRI and SalI were added at the 5 'and 3' ends of the sequence after artificial synthesis;
the synthesized sequence fragment is connected to PX458-gBCL11b carrier containing T7 promoter after EcoRI and SalI are digested, and sequencing verifies that the recombinant plasmid is constructed successfully.
(2) Will be 1X 10 7 Resuspension of individual T cells in 3mL T cell medium, seeding in one well of a 6-well plate, adding a combination of 100ng/mL anti-human CD3 antibody and 100ng/mL anti-human CD28 antibody for activation;
after 3 days, the suspended cells were removed for counting, centrifuged at 300 Xg for 10min, the pellet was resuspended in 10mL Opti-MEM, centrifuged at 300 Xg for 10min, the pellet was resuspended in 100. Mu.L Opti-MEM, CRISPR/Cas9 plasmid at 40. Mu.M was added, mixed well and transferred to an electric shock cup, which was placed in a Lonza 4D-NucleofectorTM X Unit (Single electric shock cup module), and subjected to electric transfer at 800V for 4ms;
culturing the cells after electrotransformation by adopting a T551-H3 complete culture medium containing 1ng/mL IL-12, 1ng/mL IL-15 and 1ng/mL IL-18 for 72 hours, centrifuging the cells at a low speed, and replacing fresh T551-H3 complete culture medium for continuous culture for 12 hours to obtain the reprogrammed NK cells.
EXAMPLE 2 construction of reprogrammed NK feeder cells
(1) Artificially synthesizing T2A-linked nucleic acid molecules containing IL-12 and CD8 alpha transmembrane region, and coding genes of CD19, CD64 and CD86, and respectively adding EcoRI and BamHI restriction sites and protective bases thereof at two ends;
double digestion of the nucleic acid molecules with restriction enzymes EcoRI and BamHI, incubation in a37℃water bath for 30min, and recovery of the digested product containing the sticky ends using 1.5% agarose gel electrophoresis;
the digested products were ligated into EcoRI and BamHI digested linearized pLVX-EF1-MCS plasmid (containing cohesive ends) with the ligation system shown in Table 1 to give lentiviral vectors.
TABLE 1
Component (A) Dosage (mu L)
pLVX-EF1-MCS plasmid 2(50ng)
IL-12-CD19-CD64-CD86 nucleic acid molecules 10(150ng)
T4 DNA ligation buffer 2
T4 DNA ligase (NEB) 1
ddH 2 O 5
(2) Mixing helper plasmids gag/pol, rev and VSV-G with lentiviral vector in proportion, adding into a certain volume of serum-free DMEM, mixing well and standing for 15min; adding the above mixture into a cell culture flask paved with 293T cells, gently mixing, and heating at 37deg.C with 5% CO 2 Culturing in a cell incubator for 6 hours; after 6 hours, changing fresh culture medium, continuously culturing, and adding 10mM sodium butyrate solution; and collecting the culture supernatant of the lentivirus after 72 hours for purification detection to obtain the recombinant lentivirus.
(3) Wild-type K562 cells were resuspended in 10mL Opti-MEM, centrifuged at 300 Xg for 10min, the cell pellet was resuspended in 100. Mu.L buffer, recombinant lentivirus was added, and 8. Mu.g/mL polybrene and 300IU/mL IL-2 were added, and the mixture was placed at 37℃and 5% CO 2 Culturing in an incubator; after 24h, 300g was centrifuged for 5min, the supernatant was removed and fresh with 300IU/mL IL-2And re-suspending the fine sediment by the culture medium to obtain the IL-12-CD19-CD64-CD86 reprogramming NK feeder cells.
This example simultaneously constructs K562 cells expressing only any one of IL-12, CD19, CD64 or CD86, and K562 cells transfected with pLVX-EF1-MCS empty plasmid as control.
EXAMPLE 3 in vitro expansion of reprogrammed NK cells
In this example, reprogrammed NK cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, and after culturing for 24 hours, cell counting was performed to adjust the cell concentration to 2X 10 6 Equal proportions of reprogrammed NK feeder cells were added to the culture medium on day 0 and day 7, respectively, and IL-2 at a concentration of 50U/mL, 37℃and 5% CO were added 2 Co-culturing in an incubator under 500Gy irradiation conditions.
As shown in FIG. 1, the number of cells increased significantly after the co-culture of reprogrammed NK cells with IL-12-CD19-CD64-CD86 reprogrammed NK feeder cells, and the number of reprogrammed NK cells increased by about 120-fold after 2 weeks of culture, significantly above that of the control group.
EXAMPLE 4 in vitro killing of reprogrammed NK cells
The cultured reprogrammed NK cells, T cells and NK cells of example 3 were individually associated with 5X 10 3 Co-culturing melanoma cells A375 in a U-shaped 96-well plate, wherein the ratio of effector cells to target cells (E: T) is 1:1, and repeating each group of experiments for 3 times;
after 18 hours of co-culture, 100 μl/well of Luciferase substrate (1×) was added to the 96-well plate, the cells were resuspended and immediately assayed by a multifunctional microplate reader for 1 second for RLU (relative light unit), and the killing efficiency evaluation method was performed by Luciferase (Luciferase) quantification, and the killing effect of different reprogrammed NK cells on a375 was compared in vitro as follows:
100% × (control well reading-experimental well reading)/control well reading (blank reading without cells can be ignored)
As a result, as shown in FIG. 2, the killing efficiency of the reprogrammed NK cells was higher than that of the T cells and NK cells, whereas the killing efficiency of the reprogrammed NK cells obtained by co-culture with IL-12-CD19-CD64-CD86-K562 cells was higher.
EXAMPLE 5 killing stability of reprogrammed NK cells
The reprogrammed NK cells were further co-cultured with IL-12-CD19-CD64-CD86-K562 feeder cells for 1 month, and cells were collected every 5 days to examine their killing effect on A375 cells.
As a result, as shown in FIG. 3, the reprogrammed NK cells were maintained at a stable killing ability substantially within a month, and after 20 days of co-culture with feeder cells, the cells exhibited a degree of depletion, which was manifested by a reduction in cell volume and apoptosis of a part of the cells, probably due to an excessive promotion of the proliferation of reprogrammed NK cells by feeder cells, and therefore, after about 14 days of culture of reprogrammed NK cells with feeder cells, feeder cells were removed and cultured using basal medium.
In conclusion, the invention adopts the feeder cells expressing the membrane immobilized IL-12, CD19, CD64 and CD86 to co-culture with the reprogramming NK cells, and the obtained reprogramming NK cells retain the inherent properties of T cells and NK cells, have enhanced tumor killing capacity and stable tumor killing effect, and have important significance in the field of cellular immunotherapy.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
SEQUENCE LISTING
<110> Guangdong Zhaotai in vivo biomedical technology Co., ltd
<120> a reprogrammed NK feeder cell, method for producing the same and use thereof
<130> 202014
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 557
<212> PRT
<213> artificial sequence
<400> 1
Met Trp Pro Pro Gly Ser Ala Ser Gln Pro Pro Pro Ser Pro Ala Ala
1 5 10 15
Ala Thr Gly Leu His Pro Ala Ala Arg Pro Val Ser Leu Gln Cys Arg
20 25 30
Leu Ser Met Cys Pro Ala Arg Ser Leu Leu Leu Val Ala Thr Leu Val
35 40 45
Leu Leu Asp His Leu Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro
50 55 60
Asp Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg
65 70 75 80
Ala Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr
85 90 95
Pro Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys
100 105 110
Thr Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu
115 120 125
Ser Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys
130 135 140
Leu Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser
145 150 155 160
Ile Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn
165 170 175
Ala Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn
180 185 190
Met Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser
195 200 205
Glu Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys
210 215 220
Thr Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala
225 230 235 240
Gly Gly Gly Gly Ser Met Cys His Gln Gln Leu Val Ile Ser Trp Phe
245 250 255
Ser Leu Val Phe Leu Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys
260 265 270
Lys Asp Val Tyr Val Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly
275 280 285
Glu Met Val Val Leu Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr
290 295 300
Trp Thr Leu Asp Gln Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu
305 310 315 320
Thr Ile Gln Val Lys Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His
325 330 335
Lys Gly Gly Glu Val Leu Ser His Ser Leu Leu Leu Leu His Lys Lys
340 345 350
Glu Asp Gly Ile Trp Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro
355 360 365
Lys Asn Lys Thr Phe Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg
370 375 380
Phe Thr Cys Trp Trp Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser
385 390 395 400
Val Lys Ser Ser Arg Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly
405 410 415
Ala Ala Thr Leu Ser Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr
420 425 430
Glu Tyr Ser Val Glu Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu
435 440 445
Glu Ser Leu Pro Ile Glu Val Met Val Asp Ala Val His Lys Leu Lys
450 455 460
Tyr Glu Asn Tyr Thr Ser Ser Phe Phe Ile Arg Lys Pro Asp Pro Pro
465 470 475 480
Lys Asn Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val
485 490 495
Ser Trp Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser
500 505 510
Leu Thr Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys
515 520 525
Asp Arg Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys
530 535 540
Asn Ala Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr
545 550 555
<210> 2
<211> 24
<212> PRT
<213> artificial sequence
<400> 2
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys
20
<210> 3
<211> 313
<212> PRT
<213> artificial sequence
<400> 3
Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met
1 5 10 15
Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp
20 25 30
Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln
35 40 45
Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu
50 55 60
Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile
65 70 75 80
Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu
85 90 95
Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr
100 105 110
Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp
115 120 125
Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro
130 135 140
Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala
145 150 155 160
Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro
165 170 175
Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro
180 185 190
Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser
195 200 205
Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser
210 215 220
Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp
225 230 235 240
Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala
245 250 255
Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu
260 265 270
Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly
275 280 285
Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu
290 295 300
Cys Ser Leu Val Gly Ile Leu His Leu
305 310
<210> 4
<211> 299
<212> PRT
<213> artificial sequence
<400> 4
Met Trp Phe Leu Thr Thr Leu Leu Leu Trp Pro Val Asp Gly Gln Val
1 5 10 15
Asp Thr Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser Val
20 25 30
Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu Pro
35 40 45
Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln Thr
50 55 60
Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser Gly
65 70 75 80
Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp Pro Ile Gln
85 90 95
Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln Val Ser Ser Arg Val
100 105 110
Phe Thr Glu Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Val Asp
115 120 125
Lys Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe Lys
130 135 140
Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile Ser
145 150 155 160
His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr Thr
165 170 175
Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro Val
180 185 190
Leu Asn Thr Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val Thr
195 200 205
Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly Leu Gln Leu
210 215 220
Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu Arg Gly Arg Asn Thr
225 230 235 240
Ser Ser Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly Leu
245 250 255
Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg Ser
260 265 270
Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro Val
275 280 285
Trp Phe His Val Leu Phe Tyr Leu Ala Val Gly
290 295
<210> 5
<211> 323
<212> PRT
<213> artificial sequence
<400> 5
Met Gly Leu Ser Asn Ile Leu Phe Val Met Ala Phe Leu Leu Ser Gly
1 5 10 15
Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe Asn Glu Thr Ala Asp Leu
20 25 30
Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln Ser Leu Ser Glu Leu Val
35 40 45
Val Phe Trp Gln Asp Gln Glu Asn Leu Val Leu Asn Glu Val Tyr Leu
50 55 60
Gly Lys Glu Lys Phe Asp Ser Val His Ser Lys Tyr Met Gly Arg Thr
65 70 75 80
Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg Leu His Asn Leu Gln Ile
85 90 95
Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile His His Lys Lys Pro Thr
100 105 110
Gly Met Ile Arg Ile His Gln Met Asn Ser Glu Leu Ser Val Leu Ala
115 120 125
Asn Phe Ser Gln Pro Glu Ile Val Pro Ile Ser Asn Ile Thr Glu Asn
130 135 140
Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile His Gly Tyr Pro Glu Pro
145 150 155 160
Lys Lys Met Ser Val Leu Leu Arg Thr Lys Asn Ser Thr Ile Glu Tyr
165 170 175
Asp Gly Ile Met Gln Lys Ser Gln Asp Asn Val Thr Glu Leu Tyr Asp
180 185 190
Val Ser Ile Ser Leu Ser Val Ser Phe Pro Asp Val Thr Ser Asn Met
195 200 205
Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys Thr Arg Leu Leu Ser Ser
210 215 220
Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln Pro Pro Pro Asp His Ile
225 230 235 240
Pro Trp Ile Thr Ala Val Leu Pro Thr Val Ile Ile Cys Val Met Val
245 250 255
Phe Cys Leu Ile Leu Trp Lys Trp Lys Lys Lys Lys Arg Pro Arg Asn
260 265 270
Ser Tyr Lys Cys Gly Thr Asn Thr Met Glu Arg Glu Glu Ser Glu Gln
275 280 285
Thr Lys Lys Arg Glu Lys Ile His Ile Pro Glu Arg Ser Asp Glu Ala
290 295 300
Gln Arg Val Phe Lys Ser Ser Lys Thr Ser Ser Cys Asp Lys Ser Asp
305 310 315 320
Thr Cys Phe
<210> 6
<211> 20
<212> DNA
<213> artificial sequence
<400> 6
gaccatgaac tgctcacttg 20

Claims (7)

1. A reprogrammed NK feeder cell, wherein said reprogrammed NK feeder cell expresses the membrane proteins IL-12, CD19, CD64 and CD86;
the IL-12 and CD8 alpha transmembrane region are fused and expressed on the surface of the reprogrammed NK feeder cell;
the amino acid sequence of the IL-12 is shown as SEQ ID NO. 1;
the amino acid sequence of the CD8 alpha transmembrane region is shown as SEQ ID NO. 2;
the amino acid sequence of the CD19 is shown as SEQ ID NO. 3;
the amino acid sequence of the CD64 is shown as SEQ ID NO. 4;
the amino acid sequence of the CD86 is shown as SEQ ID NO. 5.
2. An expression vector comprising an IL-12 encoding gene, a gene encoding a CD8 a transmembrane region, a CD19 encoding gene, a CD64 encoding gene, and a CD86 encoding gene;
the amino acid sequence of the IL-12 is shown as SEQ ID NO. 1;
the amino acid sequence of the CD8 alpha transmembrane region is shown as SEQ ID NO. 2;
the amino acid sequence of the CD19 is shown as SEQ ID NO. 3;
the amino acid sequence of the CD64 is shown as SEQ ID NO. 4;
the amino acid sequence of the CD86 is shown as SEQ ID NO. 5;
the expression vector is a lentiviral vector.
3. The expression vector of claim 2, wherein the expression vector comprises a viral vector;
the viral vector includes any one of a lentiviral vector, a retroviral vector, or an adeno-associated viral vector.
4. A recombinant lentivirus prepared by co-transfecting a mammalian cell with the expression vector of any one of claims 2-3 and a packaging helper plasmid;
the mammalian cells include 293T cells.
5. A method of preparing a reprogrammed NK feeder cell of claim 1, comprising the step of introducing the recombinant lentivirus of claim 4 into a K562 cell, and screening positive clones using antibiotics and flow cytometry.
6. A culture medium comprising the reprogrammed NK feeder cell of claim 1;
the medium also includes IL-2 and/or serum;
the culture solution of the culture medium comprises any one or a combination of at least two of Eagle culture solution, RPMI-1640 culture medium or Ham's F-10.
7. A method of culturing reprogrammed NK cells, said method comprising the step of culturing reprogrammed NK cells using the medium of claim 6;
the culture method further comprises the step of irradiating the reprogrammed NK cells;
the irradiation dose is 50-500 Gy;
the preparation method of the reprogramming NK cells is to prepare the reprogramming NK cells by electrotransferring CRISPR/Cas9 plasmids targeting the Bcl11b genes into activated T cells.
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