CN106810612A - A kind of fusion protein for NKT cell culture, encoding gene and application - Google Patents

A kind of fusion protein for NKT cell culture, encoding gene and application Download PDF

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CN106810612A
CN106810612A CN201710048484.0A CN201710048484A CN106810612A CN 106810612 A CN106810612 A CN 106810612A CN 201710048484 A CN201710048484 A CN 201710048484A CN 106810612 A CN106810612 A CN 106810612A
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肖洪宝
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Shenzhen AIKE Hill Biotechnology Co., Ltd.
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Shenzhen Zhongjian Biotechnology Co Ltd
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Abstract

The present invention relates to biotechnology and medical domain, and in particular to a kind of fusion protein for NKT cell culture, encoding gene and application, it is used to prepare high proliferation activity, the NKT cells of High Fragmentation activity.Fusion protein ICAM1 FN are in NKT cell cultivation process, preferably have stimulated the propagation of lymphocyte, CD3/CD56 double positive cells proportions are improved simultaneously, so that PMNC amplification in vitro efficiency is 2 times of conventional method, CD3/CD56 double positive cells proportion is 2 10 times of conventional method in gained cell, the tumor activity that kills of NKT cells is enhanced, NKT cells preparation method of the invention is killed tumor activity and improves 15 35% relative to conventional method.

Description

A kind of fusion protein for NKT cell culture, encoding gene and application
Technical field
The present invention relates to biotechnology and medical domain, in particular it relates to a kind of melting for NKT cell culture Hop protein, encoding gene and application, are used to prepare high proliferation activity, the NKT cells of High Fragmentation activity.
Background technology
Cancer morbidity since have record, always in the trend for rising, particularly in the past since twenty or thirty year, with annual The speed of 3%-5% increases.The newly-increased cases of cancer in the whole world has nearly half to appear in Asia, and wherein most is in China.It is national every Minute, about 6 people made a definite diagnosis cancer.
In recent years, developing rapidly with subjects such as molecular biology and immunologys, the cellular immunotherapy of tumour into It is after another new tumor treatment model after operation, radiotherapy, chemotherapy.Cellular immunotherapy is used as the new technology hand for treating tumour Section, be by separating, activation, the antineoplastic immune cell of external mass propgation patient itself, then feed back in patient's body, with trouble The tumor-killing cell of person itself removes tumors destroyed.The treatment method with safe and effective, Small side effects, improve immunity of organisms, The features such as killing minimal residual cancer cell, prevention of tumor relapse and metastasis is greatly developed in clinical practice.This para-immunity is thin The species of born of the same parents includes cytokine-induced killer cell cell (CIK cell), and NK (NK cells) is tumor-infiltrated Lymphocyte (TIL), cytotoxic lymphocyte (CTL cells) etc..It is considered that main in immune system in traditional sense The cell of facedown tumour has two kinds, i.e. NK cells and T cell, and NKT cells are the synthesis of NK cells and T cell, together When there is the non-specific killing ability of NK cells and the specific killing function of T cell.NKT cells to be mainly characterized by T thin Rapid, the high-level property that the constancy of born of the same parents' acceptor (TCR) gene expression, the restricted and cell factor of CD1d are produced.NKT is thin Born of the same parents can strengthen immune response and can suppress immune response again, so as in antitumor, anti-infective, suppression autoimmune disease and shifting Plant and played a significant role in tolerating.
Intercellular adhesion molecule-1 (intercellular cell adhesion molecule-1, ICAM1-1) is one Important cell adhesion molecule is planted, is the film surface sugar for contacting with each other and combining between mediated cell and cell or cell and epimatrix Albumen.Cellular adhesion, growth, migration, differentiation is played an important role during apoptosis, and in wound healing, immune response, It is most important during embryonic development, metastases etc. are a series of.Rothlein is equal to 1986 in research Lymphocyte Adhesion When find that ICAM1-1 promotees the effect that is adhered for the first time.The molecular weight of ICAM-1 is between 76-114kDa, wherein nucleus 55kDa.Adhesion factor is more to be played a role in the form of analgesics screening platform.LFA-1 (lymphocyte functional antigen-1, LFA-1) is the major receptors of ICAM1-1.Through literature query and sequence ratio Right, ICAM1-1N sections of the 180 of Q28-T207 amino acid are main functional sequence, in ICAM1-1 and LFA-1 molecular binding process Middle performance key effect simultaneously maintains its bioactivity.
Fibronectin (fibronectin, FN) is a kind of multifunctional macromolecule glycoprotein in extracellular matrix, point Son amount about 440KD.FN is widely present in animal tissue and tissue fluid, and conservative is very strong during evolution.The expression of FN, The exception of degraded and institutional framework is related to a series of change of physiological diseases, including cancer, diabetes and cystic fibrosis.In the multi-functional of FN In, one of its critical function is to promote cell to be adhered growth.Being adhered for cell is that housing construction is maintained, cell growth Necessary condition.Therefore FN as the matrix of cell culture, promotes the adherent of various kinds of cell.Because FN molecular weight is big, full molecule table Up to the difficulty existed in genetic engineering, therefore FN is how studied in the form of functional polypeptide.No. 5198423 middle fingers of U.S. Patent No. Go out FN and contain three functional domains of key, i.e. cell-binding domain (cell binding domain), heparin binding domain (heparin domain) and CS-1 regions.The polypeptide may participate in cell adhesion, break up value-added main Physiological Function.The polypeptide Can be combined with the integrin family member on T cell surface, while adding anti-cd 3 antibodies mutually to be tied with the antigen receptor of T cell Close, both collective effects activation EGFR-TK pp125FAK, the then propagation by Ras pathway stimulation T cells and differentiation.
In existing PBLC preparation method, the efficiency of Lymphocyte Proliferation in Vitro is very crucial.How Improve PMNC amplification in vitro efficiency and be still the subject matter that current this area waits optimization.On the other hand, Main effect is CD3/CD56 double positive cells in NKT cells, therefore on the basis of cell proliferating number amount is improved, is improved The ratio of CD3/CD56 double positive cells seems no less important.
The content of the invention
It is double for CD3/CD56 in (PBMC) the amplification in vitro efficiency of PMNC in the prior art and NKT cells The relatively low problem of positive cell proportion, the present invention provides a kind of fusion protein, encoding gene for NKT cell culture And application, it is used to improve CD3/CD56 double positive cells in human peripheral blood mononuclear cell amplification in vitro efficiency and NKT cells Proportion.
The purpose of the present invention is achieved by following technological means：A kind of fusion egg for NKT cell culture is provided In vain, it is following albumen a) or b)：
A) protein that the amino acid sequence shown in sequence in sequence table 2 is constituted；
B) amino acid sequence of sequence 2 passes through substitution and/or missing and/or adds one or several amino in sequence table Acid and with the protein as derived from a) for NKT cell culture.
The present invention also provides the encoding gene of the fusion protein.
The encoding gene that the present invention is provided is following gene 1) or 2) or 3)：
1) its nucleotide sequence is sequence 1 in sequence table；
2) DNA fragmentation for being limited with sequence 1 under strict conditions hybridizes and encodes and be used for NKT cell culture GAP-associated protein GAPs DNA molecular；
3) there is more than 90% homology with gene 1) or 2), and encodes and be used for NKT cell culture GAP-associated protein GAPs DNA molecular.
The present invention also provides the recombinant expression carrier of the encoding gene.
The present invention also provides the fusion protein, the encoding gene, the recombinant expression carrier in NKT cell culture Application.
The present invention also provides a kind of external preparation method of people NKT cells, comprises the following steps：
S100, coated cell blake bottle：With Du Shi phosphate buffered saline coating buffers, the coating buffer includes that CD3 is mono- Clonal antibody 1-10 μ g/ml, fusion protein 5-25 μ g/ml as described in claim 1；The coating buffer is added into blake bottle Middle avoid light place 8-24h；
The preparation of S200, mononuclearcell：Human peripheral blood is extracted, the isolated single core of lymphocyte separation medium is used Cell, cell suspension is obtained using the lymph nutrient solution re-suspended cell containing autologous plasma, people's CD3 monoclonal antibodies；
The induced amplification of S300, NKT cell：Coating buffer is removed, after adding the cell suspension to Tissue Culture Flask, addition Cell factor forms lasting stimulation, is placed in CO2Cultivated in saturated humidity incubator and obtain NKT cells.
In the external preparation method of people's NKT cells that the present invention is provided, in the step S100, institute in the coating buffer The concentration of fusion protein is stated for the concentration of 10-15 μ g/ml, the people CD3 monoclonal antibodies is 4-7 μ g/ml；Take coating buffer 10ml, is added to 75cm2In blake bottle, 4 DEG C of avoid light places are overnight, standby.
In the external preparation method of people's NKT cells that the present invention is provided, in the step S200, the mononuclearcell Cleaned with DPBS twice, basis of microscopic observation is counted, and uses the pouring containing 5% autologous plasma, 50ng/ml people's CD3 monoclonal antibodies Bar cell culture fluid GT-T551 re-suspended cells, adjust cell density 3 × 106Individual/ml.
In the external preparation method of people's NKT cells that the present invention is provided, the step S300 comprises the following steps：
S301, removal coating buffer, are gently rinsed once, then gently rinsed once with 10ml GT-T551 with 10ml DPBS；
S302, the addition cell suspension are subsequently adding recombinant human interferon-γ to final concentration of 500- to the blake bottle 1000IU/ml, is placed in 37 DEG C, 5%CO224h is cultivated in saturated humidity incubator；
S303, it is separately added into RhIL-2 to final concentration of 500-1000IU/ml, recombinant human interleukin-12 to end Concentration 5-50ng/ml, interleukin 15 are to final concentration 5-50ng/ml；Every 3 days afterwards in the Tissue Culture Flask to adding cell culture Liquid, the cell culture fluid added is to contain 0.5% autologous plasma, the people CD3 monoclonal antibodies of 50ng/ml, 1000IU/ml restructuring Human interleukin-2,10ng/ml recombinant human interleukin-12s, the GT-T551 culture mediums of 10ng/ml recombinant human interleukin 15s.
In the external preparation method of people's NKT cells that the present invention is provided, in the step S303, recombined human is separately added into Proleulzin is to final concentration of 1000IU/ml, recombinant human interleukin-12 to final concentration 10ng/ml, interleukin 15 to final concentration 10ng/ml。
Fusion protein ICAM1-FN of the invention is used for NKT cell culture, and people CD3 is added in cell culture overall process Monoclonal antibody, rhIL-2, interleukin 12, interleukin 15 preferably have stimulated the propagation of lymphocyte, while carrying CD3/CD56 double positive cells proportion high, so that PMNC amplification in vitro efficiency is conventional method 2 times, CD3/CD56 double positive cells proportion is 2-10 times of conventional method in gained cell, enhances killing for NKT cells Tumor activity, NKT cells preparation method of the invention is killed tumor activity and improves 15-35% relative to conventional method.
Brief description of the drawings
Fig. 1 is before and after IPTG is induced and the SDS- of the ICAM1-FN fusion proteins of the BL21 expression of various concentrations IPTG inductions PAGE electrophoretograms；A is for before purification, B is for after purification；
Fig. 2 is the Western Blot testing results of ICAM1-FN expressing fusion proteins；
Fig. 3 is NKT cell growth curves in embodiment 1；
Fig. 4 is the FCM analysis collection of illustrative plates of PI dyeing detection NKT Cell viabilities；
Fig. 5 is the phenotype FCM analysis collection of illustrative plates of the NKT cells obtained in embodiment 1 and comparative example 1；Wherein, A It is the NKT testing results obtained in comparative example 1；B is the NKT testing results that embodiment 1 is obtained；
Fig. 6 is that the NKT cells that obtain in embodiment (A) and comparative example (B) kill tumor activity testing result figure；Wherein, A is The NKT killing-efficiencies obtained in comparative example 1；B is the NKT killing-efficiencies that embodiment 1 is obtained.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, it is right below in conjunction with drawings and Examples The present invention is described in further detail.It should be appreciated that specific embodiment described herein is only used to explain the present invention, and without It is of the invention in limiting.
Adhesion molecule1 (ICAM1-1) functional domain (Q28- between people's cell is constructed in embodiment of the present invention T207) and people's fibronectin (Fibronectin) functional domain fusion protein ICAM1-FN, and enter in e. coli bl21 Row expression and purification.The restructuring ICAM1-FN albumen of optimized dosage and people source CD3 monoclonal antibody coated cell blake bottles, are used for The mononuclearcell that culture human peripheral blood is isolated.In the incubation of mononuclearcell (PBMC), in cell culture fluid Middle addition carries out lasting stimulation less than the people CD3 monoclonal antibodies of coating concentration.Induction obtains NKT cells after 10-13 days, detects And determine that indices can reach NKT cell standards.ICAM1-FN albumen is recombinated in embodiment of the present invention and follows molecule Clone's guide,
A kind of a kind of external preparation method of people NKT cells of embodiment of the present invention offer, including precursor in NKT cells is thin The coating buffer coated cell culture of the ICAM1-FN fusion proteins containing effective dose and people's CD3 monoclonal antibodies in being used before born of the same parents' culture The step of bottle, and add in cell culture fluid in the induction of the killing cell that the human cell factor is induced and culture overall process Plus people's CD3 monoclonal antibodies, rhIL-2, interleukin 12, the step of interleukin 15；Wherein described fusion protein is behaved Intercellular adhesion molecule-1 (ICAM-1) functional domain and people's fibronectin (Fibronectin) functional domain fusion egg In vain.Concentration of the people CD3 monoclonal antibodies in the cell culture fluid is less than the concentration in the coating buffer.The present invention NKT cells preparation method introduce fusion protein ICAM1-FN in the coating stage, lymphocyte is obtained in adherent growth It is effective to stimulate.The present invention and addition people's CD3 monoclonal antibodies, rhIL-2, interleukin in cell culture overall process 12, interleukin 15 is stimulated with forming lasting induction.The embodiment method optimizes PMNC amplification in vitro effect CD3/CD56 double positive cells proportions in rate and the NKT cells for obtaining.The tumor activity that kills of NKT cells is thus enhanced, It is hopeful the effect of raising cellular immunotherapy.
Preferably, in people's NKT cells in vitro preparation methods of the embodiment of the present invention, the coating buffer of the Tissue Culture Flask In, the fusion protein concentration is 5-25 μ g/ml, and preferred concentration is 10-15 μ g/ml, and the people CD3 MAb concentrations are 1-10 μ g/ml, preferred concentration is 4-7 μ g/ml, and most preferable concentrations are 5 μ g/ml.The concentration of rhIL-2 is 500- 1000IU/ml, most preferable concentrations are 1000IU/ml, and the concentration of interleukin 12 is 5-50ng/ml, and most preferable concentrations are 10ng/ Ml, the concentration of interleukin 15 is 5-50ng/ml, and most preferable concentrations are 10ng/ml.Preferably, during prepared by NKT cells in vitro, institute People CD3 MAb concentrations are 50-100ng/ml in stating cell culture fluid.
Preferably, in people's NKT cells in vitro preparation methods of the embodiment of the present invention, glued between people's cell in the fusion protein The functional domain of attached molecule -1 (ICAM1-1) and people's fibronectin functional domain (FN) pass through flexible peptide and connect, the flexibility Peptide sequence is GGGGSGGGGS.
Preferably, in people's NKT cells in vitro preparation methods of the embodiment of the present invention, between the people's cell containing flexible peptide The DNA sequence dna of the fusion protein of Adhesion molecule1 functional domain and people's fibronectin functional domain is sequence 2 in sequence table. Particular sequence is as follows, and wherein underscore and enlarged portion are the cDNA sequence of flexible peptide GGGGSGGGGS：
ATGGCTCCCAGCAGCCCCCGGCCCGCGCTGCCCGCACTCCTGGTCCTGCTCGGGGCTCTGTTCCCAGGACCTGGCAA TGCCCAGACATCTGTGTCCCCCTCAAAAGTCATCCTGCCCCGGGGAGGCTCCGTGCTGGTGACATGCAGCACCTCCT GTGACCAGCCCAAGTTGTTGGGCATAGAGACCCCGTTGCCTAAAAAGGAGTTGCTCCTGCCTGGGAACAACCGGAAG GTGTATGAACTGAGCAATGTGCAAGAAGATAGCCAACCAATGTGCTATTCAAACTGCCCTGATGGGCAGTCAACAGC TAAAACCTTCCTCACCGTGTACTGGACTCCAGAACGGGTGGAACTGGCACCCCTCCCCTCTTGGCAGCCAGTGGGCA AGAACCTTACCCTACGCTGCCAGGTGGAGGGTGGGGCACCCCGGGCCAACCTCACCGTGGTGCTGCTCCGTGGGGAG AAGGAGCTGAAACGGGAGCCAGCTGTGGGGGAGCCCGCTGAGGTCACGACCACGGTGCTGGTGAGGAGAGATCACCA TGGAGCCAATTTCTCGTGCCGCACTGAACTGGACCTGCGGCCCCAAGGGCTGGAGCTGTTTGAGAACACCTCGGCCC CCTACCTTAGGGGTCCGGGGCCCGGGCTGCTGC TGCTGGCCGTCCTGTGCCTGGGGACAGCGGTGCCCTCCACGGGAGCCTCGAAGAGCAAGAGGCAGGCTCAGCAAATG GTTCAGCCCCAGTCCCCGGTGGCTGTCAGTCAAAGCAAGCCCGGTTGTTATGACAATGGAAAACACTATCAGATAAA TCAACAGTGGGAGCGGACCTACCTAGGCAATGCGTTGGTTTGTACTTGTTATGGAGGAAGCCGAGGTTTTAACTGCG AGAGTAAACCTGAAGCTGAAGAGACTTGCTTTGACAAGTACACTGGGAACACTTACCGAGTGGGTGACACTTATGAG CGTCCTAAAGACTCCATGATCTGGGACTGTACCTGCATCGGGGCTGGGCGAGGGAGAATAAGCTGTACCATCGCAAA CCGCTGCCATGAAGGGGGTCAGTCCTACAAGATTGGTGACACCTGGAGGAGACCACATGAGACTGGTGGTTACATGT TAGAGTGTGTGTGTCTTGGTAATGGAAAAGGAGAATGGACCTGCAAGCCCATAGCTGAGAAGTGTTTTGATCATGCT GCTGGGACTTCCTATGTGGTCGGAGAAACGTGGGAGAAGCCCTACCAAGGCTGGATGATGGTAGATTGTACTTGCCT GGGAGAAGGCAGCGGACGCATCACTTGCACTTCTAGAAATAGATGCAACGATCAGGACACAAGGACATCCTATAGAA TTGGAGACACCTGGAGCAAGAAGGATAATCGAGGAAACCTGCTCCAGTGCATCTGCACAGGCAACGGCCGAGGAGAG TGGAAGTGTGAGAGGCACACCTCTGTGCAGACCACATCGAGCGGATCTGGCCCCTTCACCGATGTTCGTGCAGCTGT TTACCAACCGCAGCCTCACCCCCAGCCTCCTCCCTATGGCCACTGTGTCACAGACAGTGGTGTGGTCTACTCTGTGG GGATGCAGTGGCTGAAGACACAAGGAAATAAGCAAATGCTTTGCACGTGCCTGGGCAACGGAGTCAGCTGCCAAGAG ACAGCTGTAACCCAGACTTACGGTGGCAACTCAAATGGAGAGCCATGTGTCTTACCATTCACCTACAATGGCAGGAC GTTCTACTCCTGCACCACAGAAGGGCGACAGGACGGACATCTTTGGTGCAGCACAACTTCGAATTATGAGCAGGACC AGAAATACTCTTTCTGCACAGACCACACTGTTTTGGTTCAGACTCGAGGAGGAAATTCCAATGGTGCCTTGTGCCAC TTCCCCTTCCTATACAACAACCACAATTACACTGATTGCACTTCTGAGGGCAGAAGAGACAACATGAAGTGGTGTGG GACCACACAGAACTATGATGCCGACCAGAAGTTTGGGTTCTGCCCCATGGCTGCCCACGAGGAAATCTGCACAACCA ATGAAGGGGTCATGTACCGCATTGGAGATCAGTGGGATAAGCAGCATGACATGGGTCACATGATGAGGTGCACGTGT GTTGGGAATGGTCGTGGGGAATGGACATGCATTGCCTACTCGCAGCTTCGAGATCAGTGCATTGTTGATGACATCAC TTACAATGTGAACGACACATTCCACAAGCGTCATGAAGAGGGGCACATGCTGAACTGTACATGCTTCGGTCAGGGTC GGGGCAGGTGGAAGTGTGATCCCGTCGACCAATGCCAGGATTCAGAGACTGGGACGTTTTATCAAATTGGAGATTCA TGGGAGAAGTATGTGCATGGTGTCAGATACCAGTGCTACTGCTATGGCCGTGGCATTGGGGAGTGGCATTGCCAACC TTTACAGACCTATCCAAGCTCAAGTGGTCCTGTCGAAGTATTTATCACTGAGACTCCGAGTCAGCCCAACTCCCACC CCATCCAGTGGAATGCACCACAGCCATCTCACATTTCCAAGTACATTCTCAGGTGGAGACCTGTGAGTATCCCACCC AGAAACCTTGGATAC
Preferably, in the building process of the fusion protein, Adhesion molecule1 and people's fibronectin between the people's cell The objective gene sequence being spliced together by PCR methods is sequence 1 in sequence table.Particular sequence is as follows：
MAPSSPRPALPALLVLLGALFPGPGNAQTSVSPSKVILPRGGSVLVTCSTSCDQPKLLGIETPLPKKEL LLPGNNRKVYELSNVQEDSQPMCYSNCPDGQSTAKTFLTVYWTPERVELAPLPSWQPVGKNLTLRCQVEGGAPRANL TVVLLRGEKELKREPAVGEPAEVTTTVLVRRDHHGANFSCRTELDLRPQGLELFENTSAPYGGGGSGGGGSLRGPGP GLLLLAVLCLGTAVPSTGASKSKRQAQQMVQPQSPVAVSQSKPGCYDNGKHYQINQQWERTYLGNALVCTCYGGSRG FNCESKPEAEETCFDKYTGNTYRVGDTYERPKDSMIWDCTCIGAGRGRISCTIANRCHEGGQSYKIGDTWRRPHETG GYMLECVCLGNGKGEWTCKPIAEKCFDHAAGTSYVVGETWEKPYQGWMMVDCTCLGEGSGRITCTSRNRCNDQDTRT SYRIGDTWSKKDNRGNLLQCICTGNGRGEWKCERHTSVQTTSSGSGPFTDVRAAVYQPQPHPQPPPYGHCVTDSGVV YSVGMQWLKTQGNKQMLCTCLGNGVSCQETAVTQTYGGNSNGEPCVLPFTYNGRTFYSCTTEGRQDGHLWCSTTSNY EQDQKYSFCTDHTVLVQTRGGNSNGALCHFPFLYNNHNYTDCTSEGRRDNMKWCGTTQNYDADQKFGFCPMAAHEEI CTTNEGVMYRIGDQWDKQHDMGHMMRCTCVGNGRGEWTCIAYSQLRDQCIVDDITYNVNDTFHKRHEEGHMLNCTCF GQGRGRWKCDPVDQCQDSETGTFYQIGDSWEKYVHGVRYQCYCYGRGIGEWHCQPLQTYPSSSGPVEVFITETPSQP NSHPIQWNAPQPSHISKYILRWRPVSIPPRNLGY
Amino acid is represented using trigram, then particular sequence is as follows：
MetAlaProSerSerProArgProAlaLeuProAlaLeuLeuValLeuLeuGlyAlaLeuPheProGlyProGlyAs nAlaGlnThrSerValSerProSerLysValIleLeuProArgGlyGlySerValLeuValThrCysSerThrSerC ysAspGlnProLysLeuLeuGlyIleGluThrProLeuProLysLysGluLeuLeuLeuProGlyAsnAsnArgLys ValTyrGluLeuSerAsnValGlnGluAspSerGlnProMetCysTyrSerAsnCysProAspGlyGlnSerThrAl aLysThrPheLeuThrValTyrTrpThrProGluArgValGluLeuAlaProLeuProSerTrpGlnProValGlyL ysAsnLeuThrLeuArgCysGlnValGluGlyGlyAlaProArgAlaAsnLeuThrValValLeuLeuArgGlyGlu LysGluLeuLysArgGluProAlaValGlyGluProAlaGluValThrThrThrValLeuValArgArgAspHisHi sGlyAlaAsnPheSerCysArgThrGluLeuAspLeuArgProGlnGlyLeuGluLeuPhe
GluAsnThrSerAlaProTyrGlyGlyGlyGlySerGlyGlyGlyGlySerLeuArgGlyProGlyProGlyLeuLe uLeuLeuAlaValLeuCysLeuGlyThrAlaValProSerThrGlyAlaSerLysSerLysArgGlnAlaGlnGlnM etValGlnProGlnSerProValAlaValSerGlnSerLysProGlyCysTyrAspAsnGlyLysHisTyrGlnIle AsnGlnGlnTrpGluArgThrTyrLeuGlyAsnAlaLeuValCysThrCysTyrGlyGlySerArgGlyPheAsnCy sGluSerLysProGluAlaGluGluThrCysPheAspLysTyrThrGlyAsnThrTyrArgValGlyAspThrTyrG luArgProLysAspSerMetIleTrpAspCysThrCysIleGlyAlaGlyArgGlyArgIleSerCysThrIleAla AsnArgCysHisGluGlyGlyGlnSerTyrLysIleGlyAspThrTrpArgArgProHisGluThrGlyGlyTyrMe tLeuGluCysValCysLeuGlyAsnGlyLysGlyGluTrpThrCysLysProIleAlaGlu
LysCysPheAspHisAlaAlaGlyThrSerTyrValValGlyGluThrTrpGluLysProTyrGlnGlyTrpMetMe tValAspCysThrCysLeuGlyGluGlySerGlyArgIleThrCysThrSerArgAsnArgCysAsnAspGlnAspT hrArgThrSerTyrArgIleGlyAspThrTrpSerLysLysAspAsnArgGlyAsnLeuLeuGlnCysIleCysThr GlyAsnGlyArgGlyGluTrpLysCysGluArgHisThrSerValGlnThrThrSerSerGlySerGlyProPheTh rAspValArgAlaAlaValTyrGlnProGlnProHisProGlnProProProTyrGlyHisCysValThrAspSerG lyValValTyrSerValGlyMetGlnTrpLeuLysThrGlnGlyAsnLysGlnMetLeuCysThrCysLeuGlyAsn GlyValSerCysGlnGluThrAlaValThrGlnThrTyrGlyGlyAsnSerAsnGlyGluProCysValLeuProPh eThrTyrAsnGlyArgThrPheTyrSerCysThrThrGluGlyArgGlnAspGlyHisLeu
TrpCysSerThrThrSerAsnTyrGluGlnAspGlnLysTyrSerPheCysThrAspHisThrValLeuValGlnTh rArgGlyGlyAsnSerAsnGlyAlaLeuCysHisPheProPheLeuTyrAsnAsnHisAsnTyrThrAspCysThrS erGluGlyArgArgAspAsnMetLysTrpCysGlyThrThrGlnAsnTyrAspAlaAspGlnLysPheGlyPheCys ProMetAlaAlaHisGluGluIleCysThrThrAsnGluGlyValMetTyrArgIleGlyAspGlnTrpAspLysGl nHisAspMetGlyHisMetMetArgCysThrCysValGlyAsnGlyArgGlyGluTrpThrCysIleAlaTyrSerG lnLeuArgAspGlnCysIleValAspAspIleThrTyrAsnValAsnAspThrPheHisLysArgHisGluGluGly HisMetLeuAsnCysThrCysPheGlyGlnGlyArgGlyArgTrpLysCysAspProValAspGlnCysGlnAspSe rGluThrGlyThrPheTyrGlnIleGlyAspSerTrpGluLysTyrValHisGlyValArg
TyrGlnCysTyrCysTyrGlyArgGlyIleGlyGluTrpHisCysGlnProLeuGlnThrTyrProSerSerSerGl yProValGluValPheIleThrGluThrProSerGlnProAsnSerHisProIleGlnTrpAsnAlaProGlnProS erHisIleSerLysTyrIleLeuArgTrpArgProValSerIleProProArgAsnLeuGlyTyr
Realization of the invention is described in detail below in conjunction with specific embodiment.
Embodiment 1
The structure of 1.ICAM1-FN plasmids
ICAM1-1 and FN gene orders according to people, design two pairs of primers：The forward primer P1 of people ICAM1-1, reversely draws Thing P2；The forward primer P3 of people FN, reverse primer P4.And objective gene sequence is obtained by two-step pcr.Primer sequence is as follows：
ICAM1-F：5′-CATCATCATCATCATCAGACATCTGTGTCCCC-3′
ICAM1-R:5′–GTAGGGGGCCGAGGTGTT- CT-3′
FN-F:5′-CTTAGGGGTCCGGGGCC CGG-GCT-3′
FN-R:5′-AGACTGCAGGTCGACGTATCCAAGGTTTCTGGGTGGGATACTC-3′
The end of ICAM1-F primers 5 ' introduces NdeI restriction enzyme sites (underscore and italicized item), and the end of ICAM1-R primers 5 ' introduces The cDNA (underscore thickened portion) of flexible peptide is encoded, the plasmid (being purchased from Origene) with the sequences of ICAM1-1cDNA containing someone is Template.The end of FN-F primers 5 ' introduces the cDNA sequence (underscore thickened portion) of flexible peptide GGGGSGGGGS, and FN-R primers 5 ' are held HindIII restriction enzyme sites (underscore and italicized item) is introduced, is template with the plasmid purchased from Origene, the work(of amplification people FN Can region.Two sections of gene orders of gained are named with ICAM1 and FN respectively.Obtained by Standard PCR..PCR reaction systems are as follows: DNA profiling 20ng, 100nM forward primer, 100nM reverse primers, the μ l of Taq polymerase 0.5, buffer solution 5 μ l, 10mM dNTP are double Steam water to 50 μ l.PCR reaction conditions are as follows：95 degree 5 minutes, (94 degree 30 seconds, 57 degree 30 seconds, 72 degree 45 seconds), 2-34 circulations, 72 Degree 10 minutes, 4 degree of preservation genes of interest.
ICAM1-FN is connected with pGEM-T carriers, and structure obtains pGEMT-ICAM1-FN plasmids.Recombinant plasmid is in E.coli Conversion incubation is carried out in DH5a competent cells (Promega), then by ampicillin Screening Treatment after blue or green containing ammonia benzyl It is incubated again in the culture medium of mycin.Enter performing PCR with ICAM-1-F and FN-R primer pair bacterium solutions to identify, PCR terminates through agarose Detected through gel electrophoresis, the bacterium solution with genes of interest band is positive bacterium solution, preserves strain, and sequencing identification is carried out to plasmid.
2. the expression of the structure and ICAM1-FN of expression vector pColdII-ICAM1-FN
ICAM-FN fusion proteins are cut off and cloned from plasmid pGEM T-ICAM1-FN with restriction endonuclease NdeI, HindIII To pColdII plasmids, for protein expression.Recombinant plasmid carries out conversion incubation in competent cell BL21 (DE).With ICAM-1-F and FN-R primer pair bacterium solutions enter performing PCR identification and sequencing identification.It is seeded to through the positive bacteria of sequencing identification and contains ammonia The IPTG of 0.1-1mM is added in the LB fluid nutrient mediums of parasiticin, when culture to OD600 reaches 0.6 carries out low temperature (15 DEG C) Induced expression.1ml is sampled when before induction with induction 3h respectively, for the detection of destination protein expression.
3. the expression and identification of destination protein
Cell precipitation is collected with the bacterium solution each 1ml, 10000g, 4 DEG C for inducing 3h, centrifugation 5min before collecting induction, thalline sinks Form sediment resuspended with 80 μ l bacterial lysates, add albumen sample-loading buffer, boiling water bath 10min to prepare protein sample in the sample.Egg 8% protein adhesive is splined in vain, destination protein molecular weight is about 83kDa. protein adhesives (SDS-PAGE) result and sees Figure 1A such as Figure 1A institutes Show, destination protein is expressed in soluble protein form.Expression quantity is different under the IPTG inductions of various concentrations.Final choice 0.5mM IPTG induces 3h, is the inductive condition of purpose protein expression.According to above-mentioned treatment albumen, western-blot (Diagnosis of Sghistosomiasis is carried out Mark) detection.Testing result is shown in Fig. 2.There is band in positive bacteria correspondence duct at molecular weight 83kD, and blank bacterium duct is without phase Answer band.
4. the purifying of ICAM1-FN is recombinated
The positive bacterium solution 10000g for being transferred to recombinant plasmid pColdII-ICAM1-FN is taken, 4 DEG C, 5min, collects thalline is centrifuged. Thalline is resuspended in bacterial lysate, and ice-bath ultrasonic cracks thalline to bacterium solution and becomes clarification.By the thalline suspension after ultrasonication 12000g, is centrifuged 10min by 4 DEG C, collects supernatant, loading, with Ni2+-NTA post affinitive layer purification destination proteins, and priority Eluted with the imidazoles of 25 and 300mmol/L.The final PB with pH 7.5 is dialysed, and 0.22 μm of filter membrane was filtered Bacterium, 4 DEG C save backup.Destination protein sample after purification is carried out into SDS-PAGE detections, testing result is shown in Figure 1B.
The preparation of 5.NKT cells
μ g/ml, the ICAM1-FN fusion proteins of the monoclonal antibodies of CD3 containing people 5 are added in Du Shi phosphate buffers (DPBS) 10 μ g/ml configure coating buffer.Coating buffer 10ml is taken, 75cm is added to2In blake bottle, 4 DEG C of avoid light places are overnight, standby.
Extract healthy volunteer peripheral blood 50ml, anticoagulant heparin, room temperature centrifugation (700g, 20 points)；Upper plasma is drawn, is put 56 DEG C, 30 points in water-bath.Then after 4 DEG C static 15 points, it is centrifuged (900g, 30 points), takes 4 DEG C of autologous plasma and save backup. Above-mentioned 700g is taken, 20 separate heart rear lower cell component, add D-PBS to 50ml, mix, be added slowly to 2 equipped with 20ml people In the 50ml centrifuge tubes of lymphocyte separation medium, room temperature centrifugation (800g, 15 points).Tunica albuginea confluent monolayer cells are drawn, is added to equipped with 5ml RPMI1640 50ml centrifuge tubes in.It is 50ml to cumulative volume to add RPMI1640, is centrifuged (600g, 10 minutes), abandons supernatant. 50ml RPMI are added again, is centrifuged (600g, 10 minutes), abandon supernatant.Finally with blood plasma, 50ng/ containing 5% above-mentioned preparation The LCF GT-T551 re-suspended cells of ml people's CD3 monoclonal antibodies, adjust cell density 3 × 106Individual/ml.
75cm overnight will be coated with2Blake bottle discards coating buffer, with 10ml DPBS and 10ml GT-T551 respectively rinsings one It is secondary.And cell suspension obtained above is added into the 75cm2Blake bottle, is subsequently adding the recombined human of final concentration of 1000IU/ml Interferon-γ (INF- γ), is put into incubator 24h.24 were as a child separately added into addition：The restructuring of final concentration of 1000IU/ml Human interleukin-2 (IL-2), the recombinant human interleukin-12 (IL-12) of final concentration 10ng/ml, the interleukin 15 of final concentration 10ng/ml (IL-15).And contain GT-T551 culture mediums, 5% autologous plasma, the people CD3 of 50ng/ml to benefit in the Tissue Culture Flask in every 3 days Monoclonal antibody, 1000IU/ml RhIL-2s (IL-2), 10ng/ml recombinant human interleukin-12s (IL-12), 10ng/ml The cell culture fluid of recombinant human interleukin 15 (IL-15).
6.NKT cells indices are detected
Cell breeds the cell sampling cultivated in 0d, 4d, 8d, 10d, 12d are to above-mentioned steps, carries out cytometer Number, draws cell growth curve as shown in Figure 3.The cell of above-mentioned culture is contaminated through fluorescent dye PI (propidium iodide) in 12d Color, the motility rate for obtaining NKT cells by FCM analysis reaches more than 96%.Testing result is as shown in Figure 4.In the 12d pairs The cell of above-mentioned culture carries out lymphocyte phenotype detection, checks CD3+/CD56+ cell proportions.BD Calibur fluidic cells The testing result that instrument is carried out is as shown in Figure 5.12d is obtained in above-mentioned steps NKT cells with by the CFSE (vinegar of Fluoresceincarboxylic acid two Hydrochlorate succinimide ester) dyeing tumour cell K562 mixed culture a period of time after, dyeed with PI, wherein CFSE and The cell of PI double labellings is the tumour cell of NKT cell killings.Testing result is as shown in Figure 6.
Comparative example 1
Step 1-4 is with embodiment 1.
Only coating buffer composition is different from embodiment in the preparation process of NKT cells.Specific method for coating is as follows：Take containing someone The DPBS coating buffer 10ml of the μ g/ml of CD3 monoclonal antibodies 5 and the μ g/ml of people FN active fragments 10, are added to 75cm2In blake bottle, 4 DEG C of avoid light places are overnight, standby.
Experimental result
(1) cell propagation detection
Cell sampling count results in embodiment 1 and comparative example 1 are as shown in table 1.Cell growth curve is drawn, such as Shown in Fig. 3.
Table 1
Embodiment 1 0.3 1.10±0.15 23.15±2.87 143.68±4.92 365.49±7.36
Comparative example 1 0.3 0.63±0.12 20.81±4.78 88.32±4.29 224.37±7.90
Being can be seen that by table 1 and Fig. 3, NKT cell average proliferation multiples prepared by the method for the present invention are 1418.3 ± 19.76；NKT cell average proliferation multiples prepared by conventional method are 747.9 ± 18.40.NKT prepared by the method for the present invention is thin Born of the same parents' proliferation activity is apparently higher than conventional method, about the 2 of conventional method times.
(2) cell mortality detection
Cell sampling in embodiment 1 is dyeed through fluorescent dye PI (propidium iodide), and NKT is obtained by FCM analysis The motility rate of cell reaches more than 96%, as shown in Figure 4.
(3) cell phenotype detection
The flow cytomery result of cell phenotype is shown in Fig. 5.
The method of the present invention prepare NKT cell phenotype results be：CD3+/CD56+T cell proportions are 70-80% (Fig. 5 The right side, Data002), CD3+/CD56+T cell proportions are 30-40% in NKT prepared by conventional method (Fig. 5 is left, Data001).
(4) NKT cells kill tumor activity
With BDCalibur flow cytomeries, by the method for the present invention (embodiment 1) and conventional method, (contrast is real respectively Applying example 1) NKT for preparing kills tumor activity, and testing result is shown in Fig. 6.Compare 1 in effect target：Under conditions of 1, prepared by the method for the present invention NKT cells kill ratio of outflow for 30-40%, the NKT cells of conventional method kill ratio of outflow for 10-20%.
It is thin that the preparation method of the cytokine induced kill cell that the present invention is provided significantly increases the single core of peripheral blood CD3/CD56 double positive cells proportions in the outer amplification efficiency of cell space and NKT cells, while also improving CD3/ in NKT cells The ratio of cd8 cell, and enhance NKT cells kill tumor activity.
The foregoing is only the preferred embodiments of the present invention, be not intended to limit the invention, it is all it is of the invention spirit and Any modification, equivalent and improvement for being made within principle etc., should be included within the scope of the present invention.
Sequence table
<110>Shenzhen Zhong Jian Bioisystech Co., Ltd
<120>A kind of fusion protein for NKT cell culture, encoding gene and application
<130>
<160> 2
<210> 1
<211> 2619
<212> DNA
<213>Artificial sequence
<400> 1
ATGGCTCCCA GCAGCCCCCG GCCCGCGCTG CCCGCACTCC TGGTCCTGCT CGGGGCTCTG 60
TTCCCAGGAC CTGGCAATGC CCAGACATCT GTGTCCCCCT CAAAAGTCAT CCTGCCCCGG 120
GGAGGCTCCG TGCTGGTGAC ATGCAGCACC TCCTGTGACC AGCCCAAGTT GTTGGGCATA 180
GAGACCCCGT TGCCTAAAAA GGAGTTGCTC CTGCCTGGGA ACAACCGGAA GGTGTATGAA 240
CTGAGCAATG TGCAAGAAGA TAGCCAACCA ATGTGCTATT CAAACTGCCC TGATGGGCAG 300
TCAACAGCTA AAACCTTCCT CACCGTGTAC TGGACTCCAG AACGGGTGGA ACTGGCACCC 360
CTCCCCTCTT GGCAGCCAGT GGGCAAGAAC CTTACCCTAC GCTGCCAGGT GGAGGGTGGG 420
GCACCCCGGG CCAACCTCAC CGTGGTGCTG CTCCGTGGGG AGAAGGAGCT GAAACGGGAG 480
CCAGCTGTGG GGGAGCCCGC TGAGGTCACG ACCACGGTGC TGGTGAGGAG AGATCACCAT 540
GGAGCCAATT TCTCGTGCCG CACTGAACTG GACCTGCGGC CCCAAGGGCT GGAGCTGTTT 600
GAGAACACCT CGGCCCCCTA CGGCGGTGGC GGTAGCGGTG GTGGTGGTTC TCTTAGGGGT 660
CCGGGGCCCG GGCTGCTGCT GCTGGCCGTC CTGTGCCTGG GGACAGCGGT GCCCTCCACG 720
GGAGCCTCGA AGAGCAAGAG GCAGGCTCAG CAAATGGTTC AGCCCCAGTC CCCGGTGGCT 780
GTCAGTCAAA GCAAGCCCGG TTGTTATGAC AATGGAAAAC ACTATCAGAT AAATCAACAG 840
TGGGAGCGGA CCTACCTAGG CAATGCGTTG GTTTGTACTT GTTATGGAGG AAGCCGAGGT 900
TTTAACTGCG AGAGTAAACC TGAAGCTGAA GAGACTTGCT TTGACAAGTA CACTGGGAAC 960
ACTTACCGAG TGGGTGACAC TTATGAGCGT CCTAAAGACT CCATGATCTG GGACTGTACC 1020
TGCATCGGGG CTGGGCGAGG GAGAATAAGC TGTACCATCG CAAACCGCTG CCATGAAGGG 1080
GGTCAGTCCT ACAAGATTGG TGACACCTGG AGGAGACCAC ATGAGACTGG TGGTTACATG 1140
TTAGAGTGTG TGTGTCTTGG TAATGGAAAA GGAGAATGGA CCTGCAAGCC CATAGCTGAG 1200
AAGTGTTTTG ATCATGCTGC TGGGACTTCC TATGTGGTCG GAGAAACGTG GGAGAAGCCC 1260
TACCAAGGCT GGATGATGGT AGATTGTACT TGCCTGGGAG AAGGCAGCGG ACGCATCACT 1320
TGCACTTCTA GAAATAGATG CAACGATCAG GACACAAGGA CATCCTATAG AATTGGAGAC 1380
ACCTGGAGCA AGAAGGATAA TCGAGGAAAC CTGCTCCAGT GCATCTGCAC AGGCAACGGC 1440
CGAGGAGAGT GGAAGTGTGA GAGGCACACC TCTGTGCAGA CCACATCGAG CGGATCTGGC 1500
CCCTTCACCG ATGTTCGTGC AGCTGTTTAC CAACCGCAGC CTCACCCCCA GCCTCCTCCC 1560
TATGGCCACT GTGTCACAGA CAGTGGTGTG GTCTACTCTG TGGGGATGCA GTGGCTGAAG 1620
ACACAAGGAA ATAAGCAAAT GCTTTGCACG TGCCTGGGCA ACGGAGTCAG CTGCCAAGAG 1680
ACAGCTGTAA CCCAGACTTA CGGTGGCAAC TCAAATGGAG AGCCATGTGT CTTACCATTC 1740
ACCTACAATG GCAGGACGTT CTACTCCTGC ACCACAGAAG GGCGACAGGA CGGACATCTT 1800
TGGTGCAGCA CAACTTCGAA TTATGAGCAG GACCAGAAAT ACTCTTTCTG CACAGACCAC 1860
ACTGTTTTGG TTCAGACTCG AGGAGGAAAT TCCAATGGTG CCTTGTGCCA CTTCCCCTTC 1920
CTATACAACA ACCACAATTA CACTGATTGC ACTTCTGAGG GCAGAAGAGA CAACATGAAG 1980
TGGTGTGGGA CCACACAGAA CTATGATGCC GACCAGAAGT TTGGGTTCTG CCCCATGGCT 2040
GCCCACGAGG AAATCTGCAC AACCAATGAA GGGGTCATGT ACCGCATTGG AGATCAGTGG 2100
GATAAGCAGC ATGACATGGG TCACATGATG AGGTGCACGT GTGTTGGGAA TGGTCGTGGG 2160
GAATGGACAT GCATTGCCTA CTCGCAGCTT CGAGATCAGT GCATTGTTGA TGACATCACT 2220
TACAATGTGA ACGACACATT CCACAAGCGT CATGAAGAGG GGCACATGCT GAACTGTACA 2280
TGCTTCGGTC AGGGTCGGGG CAGGTGGAAG TGTGATCCCG TCGACCAATG CCAGGATTCA 2340
GAGACTGGGA CGTTTTATCA AATTGGAGAT TCATGGGAGA AGTATGTGCA TGGTGTCAGA 2400
TACCAGTGCT ACTGCTATGG CCGTGGCATT GGGGAGTGGC ATTGCCAACC TTTACAGACC 2460
TATCCAAGCT CAAGTGGTCC TGTCGAAGTA TTTATCACTG AGACTCCGAG TCAGCCCAAC 2520
TCCCACCCCA TCCAGTGGAA TGCACCACAG CCATCTCACA TTTCCAAGTA CATTCTCAGG 2580
TGGAGACCTG TGAGTATCCC ACCCAGAAAC CTTGGATAC 2619
<210> 2
<211> 873
<212> PRT
<213>Artificial sequence
<400> 2
Met Ala Pro Ser Ser Pro Arg Pro Ala Leu 10
Pro Ala Leu Leu Val Leu Leu Gly Ala Leu 20
Phe Pro Gly Pro Gly Asn Ala Gln Thr Ser 30
Val Ser Pro Ser Lys Val Ile Leu Pro Arg 40
Gly Gly Ser Val Leu Val Thr Cys Ser Thr 50
Ser Cys Asp Gln Pro Lys Leu Leu Gly Ile 60
Glu Thr Pro Leu Pro Lys Lys Glu Leu Leu 70
Leu Pro Gly Asn Asn Arg Lys Val Tyr Glu 80
Leu Ser Asn Val Gln Glu Asp Ser Gln Pro 90
Met Cys Tyr Ser Asn Cys Pro Asp Gly Gln 100
Ser Thr Ala Lys Thr Phe Leu Thr Val Tyr 110
Trp Thr Pro Glu Arg Val Glu Leu Ala Pro 120
Leu Pro Ser Trp Gln Pro Val Gly Lys Asn 130
Leu Thr Leu Arg Cys Gln Val Glu Gly Gly 140
Ala Pro Arg Ala Asn Leu Thr Val Val Leu 150
Leu Arg Gly Glu Lys Glu Leu Lys Arg Glu 160
Pro Ala Val Gly Glu Pro Ala Glu Val Thr 170
Thr Thr Val Leu Val Arg Arg Asp His His 180
Gly Ala Asn Phe Ser Cys Arg Thr Glu Leu 190
Asp Leu Arg Pro Gln Gly Leu Glu Leu Phe 200
Glu Asn Thr Ser Ala Pro Tyr Gly Gly Gly 210
Gly Ser Gly Gly Gly Gly Ser Leu Arg Gly 220
Pro Gly Pro Gly Leu Leu Leu Leu Ala Val 230
Leu Cys Leu Gly Thr Ala Val Pro Ser Thr 240
Gly Ala Ser Lys Ser Lys Arg Gln Ala Gln 250
Gln Met Val Gln Pro Gln Ser Pro Val Ala 260
Val Ser Gln Ser Lys Pro Gly Cys Tyr Asp 270
Asn Gly Lys His Tyr Gln Ile Asn Gln Gln 280
Trp Glu Arg Thr Tyr Leu Gly Asn Ala Leu 290
Val Cys Thr Cys Tyr Gly Gly Ser Arg Gly 300
Phe Asn Cys Glu Ser Lys Pro Glu Ala Glu 310
Glu Thr Cys Phe Asp Lys Tyr Thr Gly Asn 320
Thr Tyr Arg Val Gly Asp Thr Tyr Glu Arg 330
Pro Lys Asp Ser Met Ile Trp Asp Cys Thr 340
Cys Ile Gly Ala Gly Arg Gly Arg Ile Ser 350
Cys Thr Ile Ala Asn Arg Cys His Glu Gly 360
Gly Gln Ser Tyr Lys Ile Gly Asp Thr Trp 370
Arg Arg Pro His Glu Thr Gly Gly Tyr Met 380
Leu Glu Cys Val Cys Leu Gly Asn Gly Lys 390
Gly Glu Trp Thr Cys Lys Pro Ile Ala Glu 400
Lys Cys Phe Asp His Ala Ala Gly Thr Ser 410
Tyr Val Val Gly Glu Thr Trp Glu Lys Pro 420
Tyr Gln Gly Trp Met Met Val Asp Cys Thr 430
Cys Leu Gly Glu Gly Ser Gly Arg Ile Thr 440
Cys Thr Ser Arg Asn Arg Cys Asn Asp Gln 450
Asp Thr Arg Thr Ser Tyr Arg Ile Gly Asp 460
Thr Trp Ser Lys Lys Asp Asn Arg Gly Asn 470
Leu Leu Gln Cys Ile Cys Thr Gly Asn Gly 480
Arg Gly Glu Trp Lys Cys Glu Arg His Thr 490
Ser Val Gln Thr Thr Ser Ser Gly Ser Gly 500
Pro Phe Thr Asp Val Arg Ala Ala Val Tyr 510
Gln Pro Gln Pro His Pro Gln Pro Pro Pro 520
Tyr Gly His Cys Val Thr Asp Ser Gly Val 530
Val Tyr Ser Val Gly Met Gln Trp Leu Lys 540
Thr Gln Gly Asn Lys Gln Met Leu Cys Thr 550
Cys Leu Gly Asn Gly Val Ser Cys Gln Glu 560
Thr Ala Val Thr Gln Thr Tyr Gly Gly Asn 570
Ser Asn Gly Glu Pro Cys Val Leu Pro Phe 580
Thr Tyr Asn Gly Arg Thr Phe Tyr Ser Cys 590
Thr Thr Glu Gly Arg Gln Asp Gly His Leu 600
Trp Cys Ser Thr Thr Ser Asn Tyr Glu Gln 610
Asp Gln Lys Tyr Ser Phe Cys Thr Asp His 620
Thr Val Leu Val Gln Thr Arg Gly Gly Asn 630
Ser Asn Gly Ala Leu Cys His Phe Pro Phe 640
Leu Tyr Asn Asn His Asn Tyr Thr Asp Cys 650
Thr Ser Glu Gly Arg Arg Asp Asn Met Lys 660
Trp Cys Gly Thr Thr Gln Asn Tyr Asp Ala 670
Asp Gln Lys Phe Gly Phe Cys Pro Met Ala 680
Ala His Glu Glu Ile Cys Thr Thr Asn Glu 690
Gly Val Met Tyr Arg Ile Gly Asp Gln Trp 700
Asp Lys Gln His Asp Met Gly His Met Met 710
Arg Cys Thr Cys Val Gly Asn Gly Arg Gly 720
Glu Trp Thr Cys Ile Ala Tyr Ser Gln Leu 730
Arg Asp Gln Cys Ile Val Asp Asp Ile Thr 740
Tyr Asn Val Asn Asp Thr Phe His Lys Arg 750
His Glu Glu Gly His Met Leu Asn Cys Thr 760
Cys Phe Gly Gln Gly Arg Gly Arg Trp Lys 770
Cys Asp Pro Val Asp Gln Cys Gln Asp Ser 780
Glu Thr Gly Thr Phe Tyr Gln Ile Gly Asp 790
Ser Trp Glu Lys Tyr Val His Gly Val Arg 800
Tyr Gln Cys Tyr Cys Tyr Gly Arg Gly Ile 810
Gly Glu Trp His Cys Gln Pro Leu Gln Thr 820
Tyr Pro Ser Ser Ser Gly Pro Val Glu Val 830
Phe Ile Thr Glu Thr Pro Ser Gln Pro Asn 840
Ser His Pro Ile Gln Trp Asn Ala Pro Gln 850
Pro Ser His Ile Ser Lys Tyr Ile Leu Arg 860
Trp Arg Pro Val Ser Ile Pro Pro Arg Asn 870
Leu Gly Tyr 873

Claims (10)

1. a kind of fusion protein for NKT cell culture, is following albumen a) or b)：
A) protein that the amino acid sequence shown in sequence in sequence table 2 is constituted；
B) in sequence table sequence 2 amino acid sequence by substitution and/or missing and/or add one or several amino acid and With the protein as derived from a) for NKT cell culture.
2. the encoding gene of fusion protein described in claim 1.
3. encoding gene according to claim 2, it is characterised in that：The encoding gene is following base 1) or 2) or 3) Cause：
1) its nucleotide sequence is sequence 1 in sequence table；
2) DNA fragmentation for being limited with sequence 1 under strict conditions hybridizes and encodes and be used for NKT cell culture GAP-associated protein GAPs DNA molecular；
3) there is more than 90% homology, and coding and the DNA for NKT cell culture GAP-associated protein GAPs with gene 1) or 2) Molecule.
4. the recombinant expression carrier of encoding gene described in Claims 2 or 3 is contained.
5. recombinantly expressed described in encoding gene, claim 4 described in fusion protein, Claims 2 or 3 described in claim 1 and carried Application of the body in NKT cell culture.
6. a kind of external preparation method of people NKT cells, it is characterised in that comprise the following steps：
S100, coated cell blake bottle：With Du Shi phosphate buffered saline coating buffers, the coating buffer includes CD3 monoclonals Antibody 1-10 μ g/ml, fusion protein 5-25 μ g/ml as described in claim 1；Kept away during the coating buffer is added into blake bottle Light places 8-24h；
The preparation of S200, mononuclearcell：Human peripheral blood is extracted, it is thin using the isolated single core of lymphocyte separation medium Born of the same parents, cell suspension is obtained using the lymph nutrient solution re-suspended cell containing autologous plasma, people's CD3 monoclonal antibodies；
The induced amplification of S300, NKT cell：Coating buffer is removed, after adding the cell suspension to Tissue Culture Flask, cell is added The factor forms lasting stimulation, is placed in CO2Cultivated in saturated humidity incubator and obtain NKT cells.
7. the external preparation method of people NKT cells according to claim 6, it is characterised in that in the step S100, institute The concentration of fusion protein described in coating buffer is stated for the concentration of 10-15 μ g/ml, the people CD3 monoclonal antibodies is 4-7 μ g/ml； Coating buffer 10ml is taken, 75cm is added to2In blake bottle, 4 DEG C of avoid light places are overnight, standby.
8. the external preparation method of people NKT cells according to claim 6, it is characterised in that in the step S200, institute State mononuclearcell to be cleaned with DPBS twice, basis of microscopic observation is counted, using mono- containing 5% autologous plasma, 50ng/ml people CD3 The LCF GT-T551 re-suspended cells of clonal antibody, adjust cell density 3 × 106Individual/ml.
9. the external preparation method of people NKT cells according to claim 6, it is characterised in that the step S300 includes Following steps：
S301, removal coating buffer, are gently rinsed once, then gently rinsed once with 10ml GT-T551 with 10ml DPBS；
S302, the addition cell suspension are subsequently adding recombinant human interferon-γ to final concentration of 500- to the blake bottle 1000IU/ml, is placed in 37 DEG C, 5%CO224h is cultivated in saturated humidity incubator；
S303, it is separately added into RhIL-2 to final concentration of 500-1000IU/ml, recombinant human interleukin-12 to final concentration 5-50ng/ml, interleukin 15 are to final concentration 5-50ng/ml；Every 3 days afterwards to adding cell culture fluid in the Tissue Culture Flask, The cell culture fluid added is to contain 0.5% autologous plasma, the people CD3 monoclonal antibodies of 50ng/ml, 1000IU/ml recombined humans Proleulzin, 10ng/ml recombinant human interleukin-12s, the GT-T551 culture mediums of 10ng/ml recombinant human interleukin 15s.
10. the external preparation method of people NKT cells according to claim 9, it is characterised in that in the step S303, It is separately added into RhIL-2 to final concentration of 1000IU/ml, recombinant human interleukin-12 to final concentration 10ng/ml, Bai Jie Plain 15 to final concentration 10ng/ml.
CN201710048484.0A 2017-01-20 2017-01-20 A kind of fusion protein for NKT cell culture, encoding gene and application Pending CN106810612A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107475194A (en) * 2017-10-09 2017-12-15 天津长和生物技术有限公司 NKT cell culture mediums, cultural method and the application of the two
CN113699107A (en) * 2021-10-27 2021-11-26 东莞再立健生物科技有限公司 Peripheral blood NKT cell culture solution and culture method

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102352342A (en) * 2011-09-30 2012-02-15 上海柯莱逊生物技术有限公司 Method for amplifying cytokine induced kill cells (CIK) and CIK cell preparation
CN102676453A (en) * 2011-03-17 2012-09-19 中国医学科学院肿瘤研究所 Method for culturing natural killer (NK) and/or natural killer T (NKT) cells
CN102732481A (en) * 2012-06-13 2012-10-17 郑意端 Preparation method of human cytokine-induced killer cells
CN103849599A (en) * 2014-03-18 2014-06-11 加思葆（北京）医药科技有限公司 Culture medium efficiently amplifying autologous NK cells and cultural method
CN105087483A (en) * 2015-07-24 2015-11-25 广州赛莱拉干细胞科技股份有限公司 Immune cell culture medium and culture method, as well as application of ginsenoside
CN105219712A (en) * 2015-11-16 2016-01-06 广州赛莱拉干细胞科技股份有限公司 A kind of NKT cell culture medium and NKT cell culture processes
CN106434556A (en) * 2016-11-22 2017-02-22 上海市公共卫生临床中心 Method for in-vitro inducing amplification of type-I NKT (natural killer T) cells

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102676453A (en) * 2011-03-17 2012-09-19 中国医学科学院肿瘤研究所 Method for culturing natural killer (NK) and/or natural killer T (NKT) cells
CN102352342A (en) * 2011-09-30 2012-02-15 上海柯莱逊生物技术有限公司 Method for amplifying cytokine induced kill cells (CIK) and CIK cell preparation
CN102732481A (en) * 2012-06-13 2012-10-17 郑意端 Preparation method of human cytokine-induced killer cells
CN103483454A (en) * 2012-06-13 2014-01-01 深圳市阳溪生物科技有限公司 Fusion protein used for preparing human cytokine induced killer cells
CN103849599A (en) * 2014-03-18 2014-06-11 加思葆（北京）医药科技有限公司 Culture medium efficiently amplifying autologous NK cells and cultural method
CN105087483A (en) * 2015-07-24 2015-11-25 广州赛莱拉干细胞科技股份有限公司 Immune cell culture medium and culture method, as well as application of ginsenoside
CN105219712A (en) * 2015-11-16 2016-01-06 广州赛莱拉干细胞科技股份有限公司 A kind of NKT cell culture medium and NKT cell culture processes
CN106434556A (en) * 2016-11-22 2017-02-22 上海市公共卫生临床中心 Method for in-vitro inducing amplification of type-I NKT (natural killer T) cells

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JOHANSSON S ET AL.: "Fibronectin-integrin interactions", 《FRONT BIOSCI》 *
VENTER,J.C. ET AL: "fibronectin 1, isoform CRA_d [Homo sapiens]", 《GENBANK: EAW70533.1》 *
王惠明 等: "Prostein31-39特异的、HLA-A*2.1限制性NKT细胞表型及功能初步研究", 《第三军医大学学报》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107475194A (en) * 2017-10-09 2017-12-15 天津长和生物技术有限公司 NKT cell culture mediums, cultural method and the application of the two
CN107475194B (en) * 2017-10-09 2018-07-24 天津长和生物技术有限公司 The application of NKT cell culture mediums, cultural method and the two
CN113699107A (en) * 2021-10-27 2021-11-26 东莞再立健生物科技有限公司 Peripheral blood NKT cell culture solution and culture method
CN113699107B (en) * 2021-10-27 2022-02-01 东莞再立健生物科技有限公司 Peripheral blood NKT cell culture solution and culture method

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