CN109097392A - A kind of Her2-CAR-T system constituting method based on PiggyBac carrier - Google Patents

A kind of Her2-CAR-T system constituting method based on PiggyBac carrier Download PDF

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CN109097392A
CN109097392A CN201810926458.8A CN201810926458A CN109097392A CN 109097392 A CN109097392 A CN 109097392A CN 201810926458 A CN201810926458 A CN 201810926458A CN 109097392 A CN109097392 A CN 109097392A
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her2
car
piggybac
carrier
genetic fragment
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马晓冬
刘明
林展雯
李浔
黄行许
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to Her2-CAR-T system constructing technologies, more particularly to a kind of Her2-CAR-T system constituting method based on PiggyBac carrier, it is to synthesize Her2-CAR genetic fragment by design, then Her2-CAR genetic fragment is connected on Piggybac carrier and obtains Piggybac-CAR-Her2, Piggybac-CAR-Her2 is transferred in Escherichia coli again and is expanded, then plasmid is extracted from Escherichia coli, and it is added in the lymphocyte separated in human serum and is extended culture, using Nucleofector cell transfecting system transfections cotransfection plasmid Piggybac-CAR-Her2 and Piggybac Transp Osase obtains the T lymphocyte with Her2-CAR genetic fragment to T lymphocyte.The present invention uses non-viral vector PiggyBac system, avoids using security risk existing for viral vectors, has higher safety and validity, be more applicable for clinical treatment；The Nucleofector cell nuclear technology of use, foreign gene can be importing directly into nucleus, overcome liposome transfection and common electroporation, it is necessary to which dependent cells are possible to the defect for making foreign gene enter nuclear expression when dividing, cell has higher survival rate and proliferation efficiency.

Description

A kind of Her2-CAR-T system constituting method based on PiggyBac carrier
Technical field
The present invention relates to Her2-CAR-T system constructing technology, more particularly to a kind of based on PiggyBac carrier Her2-CAR-T system constituting method.
Background technique
CAR-T(Chimeric Antigen Receptor T-Cell Immunotherapy, Chimeric antigen receptor T is thin Born of the same parents' immunotherapy) it is new adoptive immunity cell therapy means developed in recent years, it is thin to modify T by genetic engineering means Born of the same parents, make it express Chimeric antigen receptor, and Chimeric antigen receptor can be identified after combining with specific recognition tumor associated antigen target spot The signal of activationa and proliferation T cell is transferred to intracellular, causes t cell activation and proliferation, thus effective killing tumor cell.CAR Molecule is broadly divided into 5 generations differentiation: I generation, specific T cell activation；In II generation, increases costimulating factor, improves cytotoxicity； III generation, while having there are two costimulating factor, improve T cell proliferative capacity and killing toxicity；In IV generation, integrates suicide gene, essence Really regulation, cytokine release (such as IL-7 and IL-15) activation；V generation, universal CAR.Compared to amplification efficiency in vitro It is low, the undesirable NK cell of cytotoxic activity, without specific cytotoxicity CIK cell and be not easy to obtain in peripheral blood And the DC cell therapy that preparation is difficult, CAR-T technology have many unique advantages, as CAR can non-MHC dependence identification tumour Antigen needs not move through APC.Tumor surface protide and lipid antigen all can serve as target spot, have extensive spectrotype；Have Costimulatory molecules can effectively enhance T cell proliferation.Therefore the technology is considered as the uniqueness side for being expected to thoroughly capture human cancer Method.
ErbB-2 (human epidermal growth factor receptor-2, Her2) Gene, i.e. ERBB2 gene are the transmembrane glycoproteins with tyrosine kinase activity, participate in developmental embryo and adult tissue Growth, differentiation and the proliferation of cell.Research finds the overexpression of Her2 gene product and the occurrence and development of tumour and tumour Invasion is positively correlated.Researches show that Her2 in a variety of cancers such as breast cancer, colon cancer, bladder cancer, oophoroma, cervical carcinoma, cancer of the esophagus It is over-expressed in disease.Currently, CAR-T immunization therapy obtains huge progress in many clinical trials.Wherein, for people The advanced stage of skins growth factor acceptor 2 (Her2) positive can not cut off, the solid tumor of metastatic or recurrent, such as cholangiocarcinoma (biliary tract cancer, BTC), cancer of pancreas (pancreatic cancer, PC), Brain Glioma in Children (pediatric glioblastoma) and adult glioma (adult glioblastoma), CAR-T-Her2 T cell is exempted from Epidemic disease treatment is significant in efficacy, safety tolerance and no apparent dose-limiting toxicity.Have report display CAR-T-Her2 to exist Treat may be implemented in late recurrent and intractable cholangiocarcinoma and cancer of pancreas about 55% disease control rate (Disease Control Rate, DCR), the case including complete incidence graph, part alleviation and stable disease；In the meat for treating 19 recurrents In addition to realizing 42% disease in tumor (osteosarcoma, Ewing sarcoma, primary nervous ectoderm tumour, Hypertrophic small round cell neoplasm) Control rate has 1 neoplasm necrosis stove to be up to wherein 3 patients obtain complete incidence graph (Complete remission, CR) 90%, 4 patients maintain 12 weeks to 14 months stable diseases (Stable Disease, SD), wherein 3 are being extractd distal end turn It still in 6,12 later and is eased for 16 months after the tumour of shifting.Some patients have also obtained persistence after CAR-T is treated Alleviate, can maintain low concentration level up to 1 year in vivo after injecting CAR-T cell.
Nevertheless, the treatment still needs to improve there are many link, can such as find better receptor, better carrier, Better costimulatory molecules etc., wherein the safety of transfection carrier and Usefulness Pair are particularly critical in clinical treatment.Now, The main transfection mode of CAR includes the modes such as slow virus, retrovirus, electrotransfection, and viral vectors is although have preferable turn Efficiency is contaminated, but is existed because genome conformity causes the risk of insertion mutation, therefore, turns technology using non-virus carrier combination electricity, Do not need virus intervention, can get with the quite even higher transfection results of virus infection efficiency, avoid viral nucleic acid with Machine Insertion Into Host Cell influences cell normal growth and subsequent possible lesion, is the safety for promoting CAR-T cell therapy Important link.
PiggyBac (PB) system is to carry DNA fragmentation using distinctive " cut and paste " mechanism of PB transposons The transfer of " freedom " between body and genome, to effectively mediate integration of the exogenous dna fragment to genome.When swivel base, swivel base Enzyme effectively identifies special transposon sequence (ITRs), and of short duration hairpin structure is formed in conjunction with transposon ends, " shearing " After be detached from, the site TTAA of " stickup " to genome.As a kind of new transgenic approach, PiggyBac system has many excellent Gesture: (1) have the advantages that, with viral vectors compared with highly-safe, easy to operate, at low cost；(2), integration efficiency is high and higher Destination gene expression probability；(3), load capacity is big, and the insertion of bigger target fragment is, it can be achieved that polygenic coexpression； (4), transgenosis can be expressed steadily in the long term, and the wild effects such as chromosomal rearrangement are not caused after swivel base；(5), more " accurate " The insertion of copy number；(6), Insert Fragment can be freely removed after swivel base again, realization is accurately cut off；(7), non-damage can be used Witness marking replaces the conventional methods such as PCR to track transgenosis in the living body；(8), host range is wide, and transposition efficiency is high, and basic Independent of host factor.Therefore, PB transposon system can be used as mammal and cultivate the transgenosis tool of cell, and A kind of ideal non-viral genoid therapy vector.Based on above-mentioned advantage, PiggyBac vector construction Her2- is utilized CAR-T is imperative.
Summary of the invention
The object of the present invention is to provide a kind of Her2-CAR-T system constituting methods based on PiggyBac carrier；The present invention Another purpose be to provide it is a kind of based on the Her2-CAR-T system of PiggyBac vector construction in preparation for treating cancer Application in drug.
To achieve the goals above, present invention employs following technical solutions:
First aspect of the invention is to provide a kind of Her2-CAR-T system constituting method based on PiggyBac carrier, It is that Her2-CAR genetic fragment is synthesized by design, then Her2-CAR genetic fragment is connected on Piggybac carrier and is obtained Piggybac-CAR-Her2, then Piggybac-CAR-Her2 is transferred in Escherichia coli and is expanded, then from Escherichia coli Middle extraction plasmid, and be added in the lymphocyte separated in human serum and be extended culture, using Nucleofector cell Cotransfection plasmid Piggybac-CAR-Her2 and Piggybac Transposase is to T lymphocyte for transfection system transfection, described Method the following steps are included:
Step 1, Her2-CAR genetic fragment design synthesis, searches the single-chain variable fragments sequence of Her2, and root According to the characteristic of PiggyBac carrier, design synthesis include the single chain antibody fragments scFv that there is specificity to Her2 antigen, Cross-film hinge, costimulating factor CD28 and 4-1BB segment and the source intracellular delivery signal CD3 ζ in the source myc-tag, CD8 Genetic fragment, obtain Her2-CAR genetic fragment；
Step 2, the PiggyBac vector construction with Her2-CAR segment will be walked using the method for digestion, connection and conversion Rapid one obtained Her2-CAR genetic fragment is connected on PiggyBac carrier, is obtained with Her2-CAR genetic fragment Piggybac-CAR-Her2；
Step 3, Her2-CAR genetic test and screening obtain band by the method for gene sequencing come what detecting step two synthesized The Piggybac carrier of Her2-CAR genetic fragment, whether Her2-CAR gene order is correct, obtains band, Her2-CAR gene Piggybac-CAR-Her2；
Step 4, the Piggybac-CAR-Her2 amplification with Her2-CAR gene have Her2-CAR for what step 3 obtained The Piggybac carrier of gene is transferred in Escherichia coli, makes the Piggybac-CAR-Her2 with Her2-CAR gene big Massive amplification in enterobacteria；
Step 5, plasmid extracts in Escherichia coli, using Escherichia coli in the big pumping kit extraction step four of endotoxin-free plasmid Plasmid DNA；
Step 6, the T lymphocyte building with Her2-CAR gene, separates lymphocyte, and T lymph is added from human serum The Plasmid DNA that cell culture fluid, INF- γ, OKT-3, hIL-2 and step 5 obtain is extended culture, uses Nucleofector cell transfecting system transfections cotransfection plasmid Piggybac-CAR-Her2 and Piggybac Transposase Into T cell, the T lymphocyte with Her2-CAR gene is obtained.
Preferably, the Piggybac carrier is pMD 18-T simple vector.
Preferably, the Escherichia coli are E. coli competent TOP10.
Preferably, the sequence of the Her2-CAR genetic fragment is SEQ ID NO.2.
Preferably, the gene order of the Piggybac-CAR-Her2 is SEQ ID NO.1.
Another aspect of the present invention is to provide a kind of Her2-CAR-T system based on PiggyBac vector construction and is making The application being ready for use in treating cancer drug.
Preferably, the cancer includes cholangiocarcinoma, pancreas, Brain Glioma in Children and adult glioma.
Compared with prior art, beneficial effects of the present invention:
(1) non-viral vector PiggyBac system is used, avoids using security risk existing for viral vectors, has higher Safety and validity, be more applicable for clinical treatment.
(2) the Nucleofector cell nuclear technology used, can be importing directly into nucleus for foreign gene, overcome Liposome transfection and common electroporation, it is necessary to which dependent cells are possible to the defect for making foreign gene enter nuclear expression when dividing, carefully Born of the same parents have higher survival rate and proliferation efficiency.
Detailed description of the invention
Fig. 1 is the gene timing diagram after present invention building；
Fig. 2 is to select positive colony after the present invention constructs to carry out digestion qualification figure；
Fig. 3 is the proof diagram of gene order of the present invention；
Fig. 4 is the proof diagram of CAR expression after transfected Jurkat cells of the present invention.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is described in detail, but the present invention can be defined by the claims and The multitude of different ways of covering is implemented.
Embodiment 1
(1) Preparatory work of experiment
Lymphocyte separation medium, T lymphocyte culture solution are U.S. Invitrogen Products, Nucleofector lipofectamine is purchased from Lonza company, and INF- γ, OKT-3 and hIL-2 antibody is public purchased from PeproTech Department, Myc-tag antibody are purchased from Cell signaling company, and the big pumping kit of endotoxin-free plasmid and gel reclaims kit are equal Purchased from Zymo research company of the U.S., PCR reagent is purchased from precious bioengineering (Dalian) Co., Ltd, various restriction endonucleases and T4 Ligase is purchased from NEB company.
(2) experimentation
The construction method of Her2-CAR-T based on PiggyBac non-virus carrier: it according to the characteristic of PiggyBac carrier, looks into Single-chain antibody variable region (scFv) gene fragment order of Her2 is looked for, and designing synthesis includes from Her2 antigentic specificity Single-chain antibody (the single-chain that heavy chain of antibody and light chain variable region segment are connected to form by intermediate connection molecule Variable fragment, scFv), intermediate segment is made of the connection and transmembrane segment in the source CD8, and segment is by transmitting in film The effector molecule of signal such as CD3 ζ composition.The target gene of synthesis is connected on pMD 18-T simple vector, is used Target gene is connected on PiggyBac carrier by the method for digestion, connection and conversion, is carried out using vector-specific primers PCR amplification and be sequenced it is whether correct to detect the objective gene sequence of synthesis, connection product is then transferred to Escherichia coli sense By in state TOP10, making the massive amplification in Escherichia coli of the PiggyBac carrier containing target fragment.Using nothing after constructing successfully The big pumping kit of endotoxin plasmid extracts plasmid.Human serum is taken, lymphocyte separation medium separates lymphocyte, and T lymph is added Cell culture fluid, INF- γ, OKT-3 and hIL-2 are extended culture.It is total using Nucleofector cell transfecting system transfections Transfected plasmids Piggybac-CAR-Her2 and Piggybac Transposase are to T cell, after 48 hours, collect cell, myc- Tag fluorescence antibody label, uses the expression of flow cytomery Her2-CAR.The segment designs synthetic method are as follows: passes through Design synthesis Her2-CAR segment, the segment mainly include the single chain antibody fragments scFv for having specificity to Her2 antigen, The cross-film hinge in the source myc-tag, CD8, costimulating factor CD28 and 4-1BB segment and intracellular delivery signal CD3 ζ, then Her2-CAR is connected on Piggybac carrier.
(3) analysis of experimental results
Gene timing diagram after building is as shown in Figure 1, experiments have shown that gene order and the gene order of design synthesis are completely the same；
Fig. 2 is to select positive colony after constructing to carry out digestion qualification figure；
Fig. 3 is the partial gene sequence of verifying；
Fig. 4 is Her2-CAR in T cell surface expression, identification and sorting.
The foregoing is merely the preferred embodiments of invention, are not intended to limit the scope of the invention, all to utilize this It simply modifies or converts made by description of the invention content, be applied directly or indirectly in other relevant technical fields, same Reason is included within the scope of the present invention.
Sequence table
<110>Ma Xiaodong
<120>a kind of Her2-CAR-T system constituting method based on PiggyBac carrier
<141> 2018-08-01
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7845
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actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata 60
catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa 120
agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa 180
atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa 240
tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac 300
gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa 360
ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct 420
aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa 480
gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc 540
gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc 600
aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 660
gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 720
cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg 780
gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg 840
ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg 900
tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag 960
gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata 1020
ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa 1080
aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag 1140
ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc 1200
cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg 1260
ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga 1320
cacctggggg gatacgggga aaaggcctcc acggccaagg atctgcgatc gctccggtgc 1380
ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg gaggggtcgg 1440
caattgaacg ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta 1500
ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag tagtcgccgt 1560
gaacgttctt tttcgcaacg ggtttgccgc cagaacacag ctgaagcttc gaggggctcg 1620
catctctcct tcacgcgccc gccgccctac ctgaggccgc catccacgcc ggttgagtcg 1680
cgttctgccg cctcccgcct gtggtgcctc ctgaactgcg tccgccgtct aggtaagttt 1740
aaagctcagg tcgagaccgg gcctttgtcc ggcgctccct tggagcctac ctagactcag 1800
ccggctctcc acgctttgcc tgaccctgct tgctcaactc tacgtctttg tttcgttttc 1860
tgttctgcgc cgttacagat ccaagctgtg accggcgcct actctagagc caccatggat 1920
ttccaggtgc agatattctc ctttctcctc atatcagcct ctgtgatcat gagcagagga 1980
gatatacaga tgacacaatc tccatctagt ctgtctgcct cagtcggtga tcgcgttacc 2040
atcacttgta gggcaagcca ggacgtgaat acagccgttg cctggtatca gcagaaacct 2100
ggaaaggctc ccaagctgct gatctatagc gccagtttcc tgtatagcgg agttccctcc 2160
agattcagtg gtagcaggag tggcacagat ttcactctca caatcagcag cctccagcca 2220
gaggactttg ctacttacta ttgccaacag cactatacca ctcctcccac atttggccag 2280
ggcaccaaag tcgagattaa gcgcacaggg tctacaagcg gtagcggaaa gccaggatca 2340
ggcgaaggca gcgaggtcca gctggtggaa tctggaggtg gactggtgca acccggagga 2400
tctctgcgcc tctcatgtgc cgcaagcggg ttcaacatta aggacactta cattcactgg 2460
gtcaggcagg cacctgggaa gggactcgaa tgggtggcta ggatctatcc aaccaacggc 2520
tacactcgct acgcagactc agtcaagggt cgctttacca tatcagccga tacttctaag 2580
aacaccgcct acctgcaaat gaactcactg agggctgagg acaccgcagt gtactactgc 2640
tctaggtggg gtggagatgg cttctatgct atggatgtgt gggggcaggg caccctcgtg 2700
accgtcagta gtgccgctgg gtcagagcag aaactgatct ccgaagaaga tctgttcgtc 2760
cccgtgttcc tgcctgccaa gccaacaact acccctgctc cacgaccacc tactccagca 2820
cctaccatcg caagtcagcc cctgtcactg cgacctgagg cttgccggcc agcagctgga 2880
ggagcagtgc acacccgagg cctggacttc gcatgcgata tctacatttg ggcaccactg 2940
gctggaacct gtggggtcct gctgctgagc ctggtcatca ccctgtattg taaccacaga 3000
aataggagca aacgctcccg actgctgcat tccgactaca tgaacatgac acctcggaga 3060
ccaggcccca ctagaaagca ttaccagcca tatgccccac ccagggattt cgcagcctat 3120
cggagccggt tcagcgtcgt gaaaaggggg cgcaagaaac tgctgtacat cttcaagcag 3180
ccttttatgc gcccagtgca gacaactcag gaggaagacg gatgctcttg tcggttccca 3240
gaggaggagg aaggaggctg cgagctgaga gtgaagttca gccggagcgc cgatgcacca 3300
gcatatcagc agggacagaa tcagctgtac aacgagctga atctgggcag gcgcgaggaa 3360
tatgacgtgc tggataagcg acgaggacgg gaccccgaaa tgggaggaaa acccagaagg 3420
aagaaccctc aggaggggct gtataatgaa ctgcagaaag acaagatggc tgaggcatac 3480
agcgaaattg gaatgaaagg agagcgccga cgggggaagg gacacgatgg gctgtaccag 3540
ggactgtcaa ccgccactaa agatacctac gacgcactgc acatgcaggc tctgccccca 3600
agagaattcg aaggatccgc ggccgctgag ggcagaggaa gtcttctaac atgcggtgac 3660
gtggaggaga atcccggccc ttccgggatg accgagtaca agcccacggt gcgcctcgcc 3720
acccgcgacg acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc 3780
gccacgcgcc acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa 3840
ctcttcctca cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc 3900
gcggtggcgg tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc 3960
ggcccgcgca tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc 4020
ctcctggcgc cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg 4080
cccgaccacc agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc 4140
gagcgcgccg gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac 4200
gagcggctcg gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg 4260
tgcatgaccc gcaagcccgg tgcctgaatc taggtcgaca atcaacctct ggattacaaa 4320
atttgtgaaa gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac 4380
gctgctttaa tgcctttgta tcatgcgtta actaaacttg tttattgcag cttataatgg 4440
ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc 4500
tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggaatt gactcaaatg 4560
atgtcaatta gtctatcaga agctcatctg gtctcccttc cgggggacaa gacatccctg 4620
tttaatattt aaacagcagt gttcccaaac tgggttctta tatcccttgc tctggtcaac 4680
caggttgcag ggtttcctgt cctcacagga acgaagtccc taaagaaaca gtggcagcca 4740
ggtttagccc cggaattgac tggattcctt ttttagggcc cattggtatg gctttttccc 4800
cgtatccccc caggtgtctg caggctcaaa gagcagcgag aagcgttcag aggaaagcga 4860
tcccgtgcca ccttccccgt gcccgggctg tccccgcacg ctgccggctc ggggatgcgg 4920
ggggagcgcc ggaccggagc ggagccccgg gcggctcgct gctgccccct agcgggggag 4980
ggacgtaatt acatccctgg gggctttggg ggggggctgt ccctgatatc tataacaaga 5040
aaatatatat ataataagtt atcacgtaag tagaacatga aataacaata taattatcgt 5100
atgagttaaa tcttaaaagt cacgtaaaag ataatcatgc gtcattttga ctcacgcggt 5160
cgttatagtt caaaatcagt gacacttacc gcattgacaa gcacgcctca cgggagctcc 5220
aagcggcgac tgagatgtcc taaatgcaca gcgacggatt cgcgctattt agaaagagag 5280
agcaatattt caagaatgca tgcgtcaatt ttacgcagac tatctttcta gggttaatct 5340
agctgcatca ggatcatatc gtcgggtctt ttttccggct cagtcatcgc ccaagctggc 5400
gctatctggg catcggggag gaagaagccc gtgccttttc ccgcgaggtt gaagcggcat 5460
ggaaagagtt tgccgaggat gactgctgct gcattgacgt tgagcgaaaa cgcacgttta 5520
ccatgatgat tcgggaaggt gtggccatgc acgcctttaa cggtgaactg ttcgttcagg 5580
ccacctggga taccagttcg tcgcggcttt tccggacaca gttccggatg gtcagcccga 5640
agcgcatcag caacccgaac aataccggcg acagccggaa ctgccgtgcc ggtgtgcaga 5700
ttaatgacag cggtgcggcg ctgggatatt acgtcagcga ggacgggtat cctggctgga 5760
tgccgcagaa atggacatgg ataccccgtg agttacccgg cgggcgcgct tggcgtaatc 5820
atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 5880
agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 5940
tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 6000
aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 6060
cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 6120
ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 6180
ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 6240
cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 6300
actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 6360
cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 6420
tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 6480
gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 6540
caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 6600
agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 6660
tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 6720
tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 6780
gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 6840
gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 6900
aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 6960
atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 7020
gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 7080
acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 7140
ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 7200
tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 7260
ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 7320
ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 7380
atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 7440
taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 7500
catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 7560
atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 7620
acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 7680
aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 7740
ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 7800
cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcat 7845
<210> 2
<211> 408
<212> DNA
<213> Artificial sequence
<400> 2
tctagagcca ccatggattt ccaggtgcag atattctcct ttctcctcat atcagcctct 60
gtgatcatga gcagaggaga tatacagatg acacaatctc catctagtct gtctgcctca 120
gtcggtgatc gcgttaccat cacttgtagg gcaagccagg acgtgaatac agccgttgcc 180
tggtatcagc agaaacctgg aaaggctccc aagctgctga tctatagcgc cagtttcctg 240
tatagcggag ttccctccag attcagtggt agcaggagtg gcacagattt cactctcaca 300
atcagcagcc tccagccaga ggactttgct acttactatt gccaacagca ctataccact 360
cctcccacat ttggccaggg caccaaagtc gagattaagc gcacaggg 408

Claims (8)

1. a kind of Her2-CAR-T system constituting method based on PiggyBac carrier, which is characterized in that it is closed by design At Her2-CAR genetic fragment, then Her2-CAR genetic fragment is connected on Piggybac carrier and obtains Piggybac- CAR-Her2, then Piggybac-CAR-Her2 is transferred in Escherichia coli and is expanded, matter is then extracted from Escherichia coli Grain, and be added in the lymphocyte separated in human serum and be extended culture, using Nucleofector cell transfecting system Cotransfection plasmid Piggybac-CAR-Her2 and Piggybac Transposase is transfected to T lymphocyte, is had The T lymphocyte of Her2-CAR genetic fragment.
2. the Her2-CAR-T system constituting method according to claim 1 based on PiggyBac carrier, which is characterized in that It the described method comprises the following steps:
Step 1, Her2-CAR genetic fragment design synthesis, searches the single-chain variable fragments sequence of Her2, and root According to the characteristic of PiggyBac carrier, design synthesis include the single chain antibody fragments scFv that there is specificity to Her2 antigen, Cross-film hinge, costimulating factor CD28 and 4-1BB segment and the source intracellular delivery signal CD3 ζ in the source myc-tag, CD8 Genetic fragment, obtain Her2-CAR genetic fragment；
Step 2, the PiggyBac vector construction with Her2-CAR segment will be walked using the method for digestion, connection and conversion Rapid one obtained Her2-CAR genetic fragment is connected on PiggyBac carrier, is obtained with Her2-CAR genetic fragment Piggybac-CAR-Her2；
Step 3, Her2-CAR genetic test and screening obtain band by the method for gene sequencing come what detecting step two synthesized The Piggybac carrier of Her2-CAR genetic fragment, whether Her2-CAR gene order is correct, obtains band, Her2-CAR gene Piggybac-CAR-Her2；
Step 4, the Piggybac-CAR-Her2 amplification with Her2-CAR gene have Her2-CAR for what step 3 obtained The Piggybac carrier of gene is transferred in Escherichia coli, makes the Piggybac-CAR-Her2 with Her2-CAR gene big Massive amplification in enterobacteria；
Step 5, plasmid extracts in Escherichia coli, using Escherichia coli in the big pumping kit extraction step four of endotoxin-free plasmid Plasmid DNA；
Step 6, the T lymphocyte building with Her2-CAR gene, separates lymphocyte, and T lymph is added from human serum The Plasmid DNA that cell culture fluid, INF- γ, OKT-3, hIL-2 and step 5 obtain is extended culture, uses Nucleofector cell transfecting system transfections cotransfection plasmid Piggybac-CAR-Her2 and Piggybac Transposase Into T cell, the T lymphocyte with Her2-CAR gene is obtained.
3. the Her2-CAR-T system constituting method according to claim 1 or 2 based on PiggyBac carrier, feature exist In the Piggybac carrier is pMD 18-T simple vector.
4. the Her2-CAR-T system constituting method according to claim 1 or 2 based on PiggyBac carrier, feature exist In the Escherichia coli are E. coli competent TOP10.
5. the Her2-CAR-T system constituting method according to claim 1 or 2 based on PiggyBac carrier, feature exist In the sequence of the Her2-CAR genetic fragment is SEQ ID NO.2.
6. the Her2-CAR-T system constituting method according to claim 1 or 2 based on PiggyBac carrier, feature exist In the gene order of the Piggybac-CAR-Her2 is SEQ ID NO.1.
7. a kind of application being used in treating cancer drug based on the Her2-CAR-T system of PiggyBac vector construction in preparation.
8. application according to claim 7, which is characterized in that the cancer includes cholangiocarcinoma, pancreas, Brain Glioma in Children With adult glioma.
CN201810926458.8A 2018-08-15 2018-08-15 A kind of Her2-CAR-T system constituting method based on PiggyBac carrier Pending CN109097392A (en)

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CN112458116A (en) * 2020-10-19 2021-03-09 广州重磅生物科技有限公司 Construction method of PD-1 knockout CD19CAR-T cell
CN112795593A (en) * 2019-11-13 2021-05-14 路春光 Construction method and clinical application of novel non-viral vector TSCM gene therapy

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CN112795593A (en) * 2019-11-13 2021-05-14 路春光 Construction method and clinical application of novel non-viral vector TSCM gene therapy
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Application publication date: 20181228