CN109371062A - A kind of recombinant slow virus expression vector, recombinant cell and its application in natural killer cells culture - Google Patents
A kind of recombinant slow virus expression vector, recombinant cell and its application in natural killer cells culture Download PDFInfo
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Abstract
The present invention provides a kind of recombinant slow virus expression vectors, belong to cell engineering field.The recombinant slow virus expression vector includes the 21mb gene, 41BBL gene and MICA gene being sequentially connected in series；The 21mb gene is sequentially connected in series to obtain by signal peptide gene, IL21 gene, CD8 Hinge gene and CD8 transmembrane gene.After recombinant slow virus expression vector transfection host cell provided by the invention, host cell expression MICA and 41BBL can be made, and amalgamation and expression (and non-secreting in the culture environment) IL21 on the cell membrane of host cell.After recombinant slow virus expression vector transfection host cell provided by the invention, the recombinant cell of acquisition can play during the Fiber differentiation of NK cell to be promoted NKG2D high expression, NK cell transition is avoided to activate, and extends NK cell generation time.
Description
Technical field
The invention belongs to cell engineering fields, and in particular to a kind of recombinant slow virus expression vector, recombinant cell and
Its application in natural killer cells culture.
Background technique
Natural killer cells (natural killer cell, NK) is the important immunocyte of body, is not only swollen with anti-
Tumor, viral infection resisting and immunological regulation are related, and participate in the hair of hypersensitivity and autoimmune disease in some cases
It is raw, it can identify target cell, killing medium.The exact source of natural killer cells is not also fully aware of, it is considered that directly from
Derived from marrow.In view of the extensive use of natural killer cells, a kind of natural killer cells abductive approach of efficient stable is provided
It has broad application prospects.
Application No. is the Chinese invention patent of CN201710096536.1 disclose it is a kind of using high expression memebrane protein CD19,
The K562 engineering cell of CD137L, CD86, CD64 and transmembrane protein IL-21 combine human IL-2's mutant efficient amplification NK cell
Although method, this method solve the problems, such as that NK cell-specific identifies CD19+ cell, but be unable to effective stimulus and improve NK cell
The vital NKG2D expression of receptor of tumor-killing.Application No. is the Chinese invention patents of CN201310173235.6 to disclose
A method of K562 Cell expansions, activated lymphocyte and the NK cell transfected using CD8 α -1 segment-CD137 of interleukin-22,
This method cell-stimulating level is low, limits the proliferation tendency and killing ability of cell.
Summary of the invention
The problem of in view of background technique, the purpose of the present invention is to provide one kind to induce in natural killer cells
The NK cell-stimulating level that improves is played in the process, accelerates NK cell Proliferation, delays a kind of recombined engineering of NK cells apoptosis
Cell, and recombinant slow virus expression vector and application for constructing the recombined engineering cell.
The present invention provides a kind of recombinant slow virus expression vector, the recombinant slow virus expression vector is with slow virus carrier
Based on, including the 21mb gene, 41BBL gene and MICA gene being sequentially connected in series；The 21mb gene is by signal
Peptide gene, IL21 gene, CD8 Hinge gene and CD8 transmembrane gene are sequentially connected in series to obtain.
Preferably, the nucleotide sequence of the 21mb gene is as shown in SEQ ID No.1.
Preferably, the nucleotide sequence of the 41BBL gene is as shown in SEQ ID No.2.
Preferably, the nucleotide sequence of the MICA gene is as shown in SEQ ID No.3.
Preferably, the 21mb gene and the 41BBL gene are connected by T2A gene, the 41BBL gene and
The MICA gene is connected by T2A gene, and the nucleotide sequence of the T2A gene is as shown in SEQ ID No.4.
The present invention provides a kind of recombinant cell, the recombinant cell infects host by above-mentioned recombinant slow virus expression vector
Cell obtains.
Preferably, the host cell is human leukemia cell line K562.
Preferably, the recombinant cell is screened by the resistance culture base containing Puromycin and is obtained.
The present invention also provides above-mentioned recombinant slow virus expression vectors or recombinant cell in natural killer cells culture
Application.
Preferably, using including the following steps:
(1) IL-2 of 8~12% FBS and 800~1200IU/ml are added in RPMI-1640 culture medium, is used for
The induced medium of peripheral blood mononuclear cells；
(2) upper recombinant cell and peripheral blood mononuclear cells are added to step (1) institute according to the ratio of 1.5~2.5:1
It states in induced medium, culture obtains natural killer cells.
The utility model has the advantages that the present invention provides a kind of recombinant slow virus expression vector, the recombinant slow virus expression vector with
Based lentiviral vector, including the 21mb gene, 41BBL gene and MICA gene being sequentially connected in series；The 21mb gene by
Signal peptide gene, IL21 gene, CD8 Hinge gene and CD8 transmembrane gene are sequentially connected in series to obtain.
After recombinant slow virus expression vector transfection host cell provided by the invention, host cell expression MICA can be made, to make host
Cell plays specific activation NKG2D gene during the Fiber differentiation of natural killer cells, further increases NKG2D expression
The effect of amount；Simultaneously as 21mb gene has the function of film anchoring, recombinant slow virus expression vector provided by the invention transfects place
After chief cell, can on the cell membrane of the MICA positive amalgamation and expression (and non-secreting in culture environment) IL21, so as to from
Specific is provided for it during the Fiber differentiation of Natural killer cell to support, avoids caused NK after MICA combination NKG2D
Cell transition activation；In addition, recombinant slow virus expression vector provided by the invention also has the function of expressing 41BBL gene, into
And host cell can be made to play the role of extending NK cell generation time, avoid NK Apoptosis.
The present invention also provides above-mentioned recombinant slow virus expression vectors or recombinant cell in natural killer cells culture
Application.Using application method provided by the invention, both can excessive dependence to avoid peripheral blood mononuclear cells to interleukin,
It is able to achieve efficient induction of the peripheral blood mononuclear cells to natural killer cells again.
Detailed description of the invention
Fig. 1 is the structure chart of recombinant slow virus expression vector of the present invention；
Fig. 2 is the structure chart of 21mb gene in recombinant slow virus expression vector of the present invention；
Fig. 3 is the protein expression result in flow cytometry identification of M LG trophocyte described in the embodiment of the present invention 1；
Fig. 4 is the NK cell purity shadow that difference MLG trophocyte additional proportion described in the embodiment of the present invention 1 obtains culture
Ring result；
Fig. 5 is MLG trophocyte treated NK cell described in the embodiment of the present invention 1 in vitro in cell killing experiment
Performance results；
Fig. 6 is the result data figure of MLG trophocyte culture NK cell described in the embodiment of the present invention 1.
Specific embodiment
The present invention provides a kind of recombinant slow virus expression vector, the recombinant slow virus expression vector is with slow virus carrier
Based on, including the 21mb gene, 41BBL gene and MICA gene being sequentially connected in series；The 21mb gene is by signal
Peptide gene, IL21 gene, CD8 Hinge gene and CD8 transmembrane gene are sequentially connected in series to obtain.
Recombinant slow virus expression vector of the present invention is with based lentiviral vector, including the 21mb base being sequentially connected in series
Cause, 41BBL gene and MICA gene.The present invention is not specially limited the type of the slow virus carrier, this field routine city
Sell product.In an embodiment of the present invention, the slow virus carrier is the Lenti-MSCV-Puro of ABM company production.
21mb gene of the present invention is by signal peptide gene, IL21 gene, CD8 Hinge gene and CD8
Transmembrane gene is sequentially connected in series to obtain.The structure can make the expression of IL21 gene have film Anchoring Effect, make IL21
The amalgamation and expression on the cell membrane of host cell.In the present invention, the nucleotide sequence of the signalpeptide gene is preferred
As shown in SEQ ID No.5, the amino acid sequence of the albumen after expression is as shown in SEQ ID No.9；The IL21 gene
Nucleotide sequence is preferably as shown in SEQ ID No.6, and the amino acid sequence of the albumen after expression is as shown in SEQ ID No.10；
The nucleotide sequence of the CD8 Hinge gene is preferably as shown in SEQ ID No.7, the amino acid sequence of the albumen after expression
As shown in SEQ ID No.11；The nucleotide sequence of the CD8 transmembrane gene is preferably such as SEQ ID No.8 institute
Show, the amino acid sequence of the albumen after expression is as shown in SEQ ID No.12.The nucleotide sequence of the 21mb gene is preferred
As shown in SEQ ID No.1, the amino acid sequence of the albumen after expression is as shown in SEQ ID No.13.
Recombinant slow virus expression vector of the present invention includes the 21mb gene, 41BBL gene and MICA base being sequentially connected in series
Cause.In the present invention, the nucleotide sequence of the 41BBL gene is preferably as shown in SEQ ID No.2, the albumen after expression
Amino acid sequence is as shown in SEQ ID No.14.The nucleotide sequence of the MICA gene preferably as shown in SEQ ID No.3,
The amino acid sequence of albumen after expression is as shown in SEQ ID No.15.In the present invention, the 21mb gene and the 41BBL
Gene preferably passes through T2A gene and connects, and the nucleotide sequence of the T2A gene is preferably as shown in SEQ ID No.4, table
The amino acid sequence of albumen after reaching is as shown in SEQ ID No.16.In the present invention, the 41BBL gene and the MICA base
Because it is also preferred that being connected by T2A gene.
The present invention provides a kind of recombinant cell, the recombinant cell infects host by above-mentioned recombinant slow virus expression vector
Cell obtains.In the present invention, the host cell preferably includes human leukemia cell line K562.The present invention is to the infection
Method is not particularly limited, method of this field conventionally used for slow virus carrier infection host cell.Utilize the recombination
After Lentiviral infects host cell, metainfective host cell is preferably placed in containing Puromycin's by the present invention
In resistance culture base, using recombinant slow virus expression vector to the resistance of Puromycin, screening obtains having high ability to express
Recombinant cell.
The present invention also provides above-mentioned recombinant slow virus expression vectors or recombinant cell in natural killer cells culture
Application.
In the present invention, the application preferably includes following steps:
(1) IL-2 of 8~12% FBS and 800~1200IU/ml are added in RPMI-1640 culture medium, is used for
The induced medium of peripheral blood mononuclear cells；
(2) above-mentioned recombinant cell and peripheral blood mononuclear cells are added to step (1) according to the ratio of 1.5~2.5:1
In the induced medium, culture obtains natural killer cells.
FBS and IL-2 is first added in the present invention in RPMI-1640 culture medium, prepares induced medium.In the present invention, institute
RPMI-1640 culture medium is stated preferably to buy from Gibco company, article No. 11875.In the present invention, the additive amount of the FBS is excellent
It is selected as 8~12%, more preferably 10%；The additive amount of the IL-2 is preferably 800~1200IU/ml, more preferably 1000IU/
ml.The present invention is not particularly limited the source of the FBS and the IL-2, this field conventional commercial product.In this hair
In bright embodiment, the FBS is bought from Gibco company, article No. 10437.The certainly double aigret medicine companies of IL-2 purchase, commodity
Name: Xin Jier；Specification: 1,000,000 units every.
After obtaining induced medium, above-mentioned recombinant cell and peripheral blood mononuclear cells are added to step by the present invention simultaneously
(1) in the induced medium, culture obtains natural killer cells.In the present invention, the recombinant cell and the peripheral blood
The quantity of mononuclearcell is than being preferably 1.5~2.5:1, more preferably 2:1.
Below with reference to embodiment to a kind of recombinant slow virus expression vector provided by the invention, recombinant cell and its in nature
Application in killing cell culture is described in detail, but they cannot be interpreted as the limit to the scope of the present invention
It is fixed.
Embodiment 1
The building of 1, MLG (Membrane Ligand GrowthBooster transmembrane ligand basal growth support) trophocyte:
Human leukemia cell line K562 (American Type Culture Collection；Rockville, MD) pass through
RPMI-1640 culture containing 10%FBS.By by " film anchoring " IL21 (IL-21 is merged with CD8), 41BBL and the common structure of MICA
It is built in slow virus carrier, carries out viral packaging.Viral packaging process include: by suspension 293T expression system, with liposome or
Polymer transfection reagent (such as PEI 25K, Polyscience；Transdux, SBI etc.) transiently transfect pLenti-MLG slow virus
Skeleton plasmid and slow virus pack helper plasmid, then express slow virus to cell and secrete in cells and supernatant.Concentration
Viral supernatants are harvested, infectious titer is obtained, then by supplier guidance concentration addition Transdux (SBI), infection K562 is thin
Born of the same parents, so that building obtains K562-mbl21-41BBL-MICA trophocyte.Carrier is resistant, by containing Puromycin
(Gibco) resistance culture base screens high expressing cell.
To screen high expressing cell, as a result as shown in Figure 3 the present invention is sorted with flow cytometry.Fig. 3 the result shows that:
It is identified through flow cytometry, three kinds of albumen are expressed in MLG cell surface height.
The present invention carries out unicellular separation to highly expressed stable transfected cells using flow cytometer, establishes and stablizes expression
The trophocyte monoclonal cell system of above-mentioned memebrane protein element.
The building of 2, MLG trophocytes is for Activation In Vitro and amplification peripheral blood mononuclear cells
Trophocyte is collected by centrifugation and washed once with PBS.Take 2 × 107A trophocyte is suspended in 2ml RPMI-
(serum is free of) in 1640 culture mediums.Mitomycin C (being purchased from Sigma, the use of concentration is 50ug/ml) is added, is incubated in 37 DEG C
30min is to stop the division of cell.With complete medium centrifuge washing cell precipitation twice, i.e., culture peripheral blood list operable for activation
A nucleus uses.
Fresh separated or the peripheral blood mononuclear cells frozen and trophocyte are contained according to the ratio addition of 2:1
The RPMI-1640 culture medium of 1000IU/ml IL-2 and 10%FBS (Gibco) is cultivated.Every two days one subcultures of replacement
(old culture medium being discarded after centrifugation, be changed to the fresh culture containing IL-2).Fresh culture containing IL-2, and adjust thin
Born of the same parents' density is to 0.5~1 × 106/ml.After culture 21 days, harvest induces successful NK cell.
3. the present invention carries out 5 day short time culture of peripheral blood PBMC, knot by the way that the MLG trophocyte of different proportion is added
Fruit is as shown in Figure 4.
As shown in Figure 4, the NK cell purity that the additional proportion of different trophocytes obtains culture has an impact.When MLG is thin
When born of the same parents' additional proportion is 0.5 times of PBMC, induced NK cell effect is best, is 37.3, much larger than the induction mode of other ratios.
4, cell in vitro killing experiments (function that verifying generates NK):
By CFSE dye marker K562 cell, K562 target cell 2 × 10 is inoculated in the every hole of 6 orifice plates5It is a, and according to
The NK cell that culture obtains is added in effect target ratio (Effector:Target, E:T) 10:1.Killing experiments use identical with amplification
Culture medium carries out (IL-2 containing 1000U/ml).The whole cells of collection in 4 hours, are added after washing after the NK cell that culture is added
PI is dyed 30 minutes, carries out dead cell detection to CFSE positive group.As a result as shown in Figure 5.
Fig. 5 is the results show that the NK cell ratio turned out by MLG has stronger killing vigor using 41BBL trophocyte.
Cause target cell death rate (i.e. PI stained positive ratio) higher after MLG-NK killing.
5. the excellent results of the induction of natural killer cells compared with the existing technology:
Common Human peripheral blood NK cells culture is often used the K562 trophocyte for being overexpressed 41BBL gene.The present invention exists
Further MLG trophocyte is constructed by IL21 the and NKG2D ligand MIC-A of combined use cross-film expression on the basis of this to go forward side by side
The NK cell culture experiments of derived from peripheral blood are gone.As a result as shown in Figure 6.
Fig. 6 the result shows that: after carrying out cell culture in 21 days using MLG trophocyte, CD56 is positive, and the NK of CD3 feminine gender is thin
Born of the same parents' ratio is significantly increased, and ratio (98.1%) is much higher than traditional 41BBL-K562 cell.
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
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<210> 6
<211> 465
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
atggagagga ttgtcatctg tctgatggtc atcttcttgg ggacactggt ccacaaatca 60
agctcccaag gtcaagatcg ccacatgatt agaatgcgtc aacttataga tattgttgat 120
cagctgaaaa attatgtgaa tgacttggtc cctgaatttc tgccagctcc agaagatgta 180
gagacaaact gtgagtggtc agctttttcc tgctttcaga aggcccaact aaagtcagca 240
aatacaggaa acaatgaaag gataatcaat gtatcaatta aaaagctgaa gaggaaacca 300
ccttccacaa atgcagggag aagacagaaa cacagactaa catgcccttc atgtgattct 360
tatgagaaaa aaccacccaa agaattccta gaaagattca aatcacttct ccaaaagatg 420
attcatcagc atctgtcctc tagaacacac ggaagtgaag attcc 465
<210> 7
<211> 135
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 8
<211> 63
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60
acc 63
<210> 9
<211> 21
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 9
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 10
<211> 155
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 10
Met Glu Arg Ile Val Ile Cys Leu Met Val Ile Phe Leu Gly Thr Leu
1 5 10 15
Val His Lys Ser Ser Ser Gln Gly Gln Asp Arg His Met Ile Arg Met
20 25 30
Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn Tyr Val Asn Asp
35 40 45
Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp Val Glu Thr Asn Cys
50 55 60
Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala Gln Leu Lys Ser Ala
65 70 75 80
Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val Ser Ile Lys Lys Leu
85 90 95
Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg Arg Gln Lys His Arg
100 105 110
Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys Lys Pro Pro Lys Glu
115 120 125
Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys Met Ile His Gln His
130 135 140
Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser
145 150 155
<210> 11
<211> 45
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 11
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 12
<211> 21
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 12
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
20
<210> 13
<211> 242
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 13
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Met Glu Arg Ile Val Ile Cys Leu Met Val Ile
20 25 30
Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln Asp Arg
35 40 45
His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys
50 55 60
Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp
65 70 75 80
Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys Ala
85 90 95
Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile Asn Val
100 105 110
Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala Gly Arg
115 120 125
Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr Glu Lys
130 135 140
Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu Gln Lys
145 150 155 160
Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu Asp Ser
165 170 175
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
180 185 190
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
195 200 205
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
210 215 220
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
225 230 235 240
Ile Thr
<210> 14
<211> 254
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 14
Met Glu Tyr Ala Ser Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro
1 5 10 15
Pro Ala Pro Arg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val
20 25 30
Ala Gly Leu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe
35 40 45
Leu Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser
50 55 60
Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp
65 70 75 80
Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val
85 90 95
Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp
100 105 110
Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu
115 120 125
Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe
130 135 140
Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser
145 150 155 160
Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala
165 170 175
Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala
180 185 190
Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala
195 200 205
Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His
210 215 220
Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val
225 230 235 240
Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu
245 250
<210> 15
<211> 383
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 15
Met Gly Leu Gly Pro Val Phe Leu Leu Leu Ala Gly Ile Phe Pro Phe
1 5 10 15
Ala Pro Pro Gly Ala Ala Ala Glu Pro His Ser Leu Arg Tyr Asn Leu
20 25 30
Thr Val Leu Ser Trp Asp Gly Ser Val Gln Ser Gly Phe Leu Thr Glu
35 40 45
Val His Leu Asp Gly Gln Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys
50 55 60
Arg Ala Lys Pro Gln Gly Gln Trp Ala Glu Asp Val Leu Gly Asn Lys
65 70 75 80
Thr Trp Asp Arg Glu Thr Arg Asp Leu Thr Gly Asn Gly Lys Asp Leu
85 90 95
Arg Met Thr Leu Ala His Ile Lys Asp Gln Lys Glu Gly Leu His Ser
100 105 110
Leu Gln Glu Ile Arg Val Cys Glu Ile His Glu Asp Asn Ser Thr Arg
115 120 125
Ser Ser Gln His Phe Tyr Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn
130 135 140
Leu Glu Thr Lys Glu Trp Thr Met Pro Gln Ser Ser Arg Ala Gln Thr
145 150 155 160
Leu Ala Met Asn Val Arg Asn Phe Leu Lys Glu Asp Ala Met Lys Thr
165 170 175
Lys Thr His Tyr His Ala Met His Ala Asp Cys Leu Gln Glu Leu Arg
180 185 190
Arg Tyr Leu Lys Ser Gly Val Val Leu Arg Arg Thr Val Pro Pro Met
195 200 205
Val Asn Val Thr Arg Ser Glu Ala Ser Glu Gly Asn Ile Thr Val Thr
210 215 220
Cys Arg Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg
225 230 235 240
Gln Asp Gly Val Ser Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val
245 250 255
Leu Pro Asp Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile
260 265 270
Cys Gln Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly
275 280 285
Asn His Ser Thr His Pro Val Pro Ser Gly Lys Val Leu Val Leu Gln
290 295 300
Ser His Trp Gln Thr Phe His Val Ser Ala Val Ala Ala Ala Ala Ile
305 310 315 320
Phe Val Ile Ile Ile Phe Tyr Val Arg Cys Cys Lys Lys Lys Thr Ser
325 330 335
Ala Ala Glu Gly Pro Glu Leu Val Ser Leu Gln Val Leu Asp Gln His
340 345 350
Pro Val Gly Thr Ser Asp His Arg Asp Ala Thr Gln Leu Gly Phe Gln
355 360 365
Pro Leu Met Ser Asp Leu Gly Ser Thr Gly Ser Thr Glu Gly Ala
370 375 380
<210> 16
<211> 18
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 16
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
1 5 10 15
Gly Pro
Claims (10)
1. a kind of recombinant slow virus expression vector, which is characterized in that the recombinant slow virus expression vector is with slow virus carrier
Basis, including the 21mb gene, 41BBL gene and MICA gene being sequentially connected in series；The 21mb gene is by signal peptide
Gene, IL21 gene, CD8 Hinge gene and CD8 transmembrane gene are sequentially connected in series to obtain.
2. recombinant slow virus expression vector according to claim 1, which is characterized in that the nucleotides sequence of the 21mb gene
Column are as shown in SEQ ID No.1.
3. recombinant slow virus expression vector according to claim 1, which is characterized in that the nucleotide of the 41BBL gene
Sequence is as shown in SEQ ID No.2.
4. recombinant slow virus expression vector according to claim 1, which is characterized in that the nucleotides sequence of the MICA gene
Column are as shown in SEQ ID No.3.
5. recombinant slow virus expression vector according to claim 1, which is characterized in that the 21mb gene and described
41BBL gene is connected by T2A gene, and the 41BBL gene and the MICA gene are connected by T2A gene,
The nucleotide sequence of the T2A gene is as shown in SEQ ID No.4.
6. a kind of recombinant cell, which is characterized in that the recombinant cell disease of the recombinant lentiviral as described in Claims 1 to 5 any one
Malicious expression vector infection host cell obtains.
7. recombinant cell according to claim 6, which is characterized in that the host cell is human leukemia cell line
K562。
8. recombinant cell according to claim 7, which is characterized in that the recombinant cell is by containing Puromycin's
The screening of resistance culture base obtains.
9. described in recombinant slow virus expression vector or claim 6~8 any one described in Claims 1 to 5 any one
Application of the recombinant cell in natural killer cells culture.
10. application according to claim 9, which is characterized in that the application includes the following steps:
(1) IL-2 of 8~12% FBS and 800~1200IU/ml are added in RPMI-1640 culture medium, obtains for periphery
The induced medium of blood mononuclear cell；
(2) by recombinant cell described in claim 6~8 any one and peripheral blood mononuclear cells according to the ratio of 1.5~2.5:1
Example is added in step (1) described induced medium, and culture obtains natural killer cells.
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| CN201811520275.2A CN109371062A (en) | 2018-12-12 | 2018-12-12 | A kind of recombinant slow virus expression vector, recombinant cell and its application in natural killer cells culture |
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| CN109810197A (en) * | 2019-02-25 | 2019-05-28 | 上海尚泰生物技术有限公司 | Artificial antigen presenting cell and its construction method for efficient amplification NK |
| CN110396131A (en) * | 2019-08-23 | 2019-11-01 | 北京鼎成肽源生物技术有限公司 | A kind of Chimeric antigen receptor, recombinant vector, recombinant cell and the application of ErbB2 single-chain antibody, targeting people ErbB2 |
| CN110592014A (en) * | 2019-08-14 | 2019-12-20 | 广东美赛尔细胞生物科技有限公司 | Method for continuously removing feeder cells in vitro and in vivo without irradiation in NK cell therapy |
| CN110904052A (en) * | 2019-12-30 | 2020-03-24 | 北京鼎成肽源生物技术有限公司 | Culture method of SNK cells |
| CN110951694A (en) * | 2019-12-30 | 2020-04-03 | 北京鼎成肽源生物技术有限公司 | Preparation method of autologous trophoblast and culture method of SNK cells |
| CN111041048A (en) * | 2019-12-30 | 2020-04-21 | 北京鼎成肽源生物技术有限公司 | Preparation method of limited-generation trophoblast and culture method of SNK cells |
| CN111073856A (en) * | 2019-12-28 | 2020-04-28 | 郑州大学第一附属医院 | Trophoblast, preparation method thereof and application thereof in NK cell amplification |
| CN112592898A (en) * | 2020-12-21 | 2021-04-02 | 广东昭泰体内生物医药科技有限公司 | Reprogrammed NK feeder cell and preparation method and application thereof |
| CN112608901A (en) * | 2020-12-21 | 2021-04-06 | 广东昭泰体内生物医药科技有限公司 | Artificial antigen presenting cell and preparation method and application thereof |
| CN112626028A (en) * | 2020-12-21 | 2021-04-09 | 广东昭泰体内生物医药科技有限公司 | Engineered cell for activating NK-like cell and preparation method and application thereof |
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| CN109810197A (en) * | 2019-02-25 | 2019-05-28 | 上海尚泰生物技术有限公司 | Artificial antigen presenting cell and its construction method for efficient amplification NK |
| CN110592014A (en) * | 2019-08-14 | 2019-12-20 | 广东美赛尔细胞生物科技有限公司 | Method for continuously removing feeder cells in vitro and in vivo without irradiation in NK cell therapy |
| CN110396131A (en) * | 2019-08-23 | 2019-11-01 | 北京鼎成肽源生物技术有限公司 | A kind of Chimeric antigen receptor, recombinant vector, recombinant cell and the application of ErbB2 single-chain antibody, targeting people ErbB2 |
| CN111073856A (en) * | 2019-12-28 | 2020-04-28 | 郑州大学第一附属医院 | Trophoblast, preparation method thereof and application thereof in NK cell amplification |
| CN111041048B (en) * | 2019-12-30 | 2021-07-06 | 北京鼎成肽源生物技术有限公司 | Preparation method of limited-generation trophoblast and culture method of SNK cells |
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| CN112626028A (en) * | 2020-12-21 | 2021-04-09 | 广东昭泰体内生物医药科技有限公司 | Engineered cell for activating NK-like cell and preparation method and application thereof |
| CN112592898A (en) * | 2020-12-21 | 2021-04-02 | 广东昭泰体内生物医药科技有限公司 | Reprogrammed NK feeder cell and preparation method and application thereof |
| CN112608901B (en) * | 2020-12-21 | 2023-11-21 | 广东昭泰细胞生物科技有限公司 | Artificial antigen presenting cell, preparation method and application thereof |
| CN112626028B (en) * | 2020-12-21 | 2023-11-21 | 广东昭泰细胞生物科技有限公司 | Engineering cell for activating NK-like cells and preparation method and application thereof |
| CN112592898B (en) * | 2020-12-21 | 2024-02-13 | 广东昭泰细胞生物科技有限公司 | Reprogrammed NK feeder cells and preparation method and application thereof |
| CN117645669A (en) * | 2022-08-29 | 2024-03-05 | 星奕昂(上海)生物科技有限公司 | Immune cell with membrane-bound IL-21 and preparation method and application thereof |
| WO2024046072A1 (en) * | 2022-08-29 | 2024-03-07 | 星奕昂(上海)生物科技有限公司 | Immune cell having membrane-bound il-21, and preparation method therefor and use thereof |
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