CN108314740B - Cell culture container modified by protein immobilization - Google Patents

Cell culture container modified by protein immobilization Download PDF

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CN108314740B
CN108314740B CN201810186457.4A CN201810186457A CN108314740B CN 108314740 B CN108314740 B CN 108314740B CN 201810186457 A CN201810186457 A CN 201810186457A CN 108314740 B CN108314740 B CN 108314740B
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culture container
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CN108314740A (en
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周亚凤
王绪德
申兆兴
刘雪宾
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Shanghai Gene Optimal Biotechnology Co ltd
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Shanghai Gene Optimal Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5406—IL-4
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    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/10—Petri dish
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2304—Interleukin-4 (IL-4)
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells
    • C12N2506/115—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells from monocytes, from macrophages

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Abstract

The invention discloses a cell culture container with protein directionally immobilized and modified. The invention firstly constructs a fusion protein of GMCSF and IL4 and a cell culture container with protein coated on the inner wall. The preparation method of the cell culture container comprises the following steps: his tag the protein; silanization treatment is carried out on the inner wall of the cell culture container; performing AB-NTA incubation modification on the inner wall of the cell culture container; adding NiCl 2 Coupling AB-NTA with Ni by the solution; his-tagged proteins were added and allowed to bind to nickel ions in AB-NTA, and immobilized on the inner wall of the cell culture vessel. In the process of culturing cells by using the cell culture container, the coated protein is not lost when the culture solution is replaced, so that repeated addition is not needed, the consistency of the quantity of the protein is ensured, uncontrollable factors in the whole culture process are reduced, the effect of cell culture and differentiation is ensured, and the cell culture container has the advantages of reversibility and repeated utilization.

Description

Cell culture container modified by protein immobilization
Technical Field
The invention relates to the technical field of cell culture, in particular to a cell culture container modified by protein immobilization.
Background
Cell culture, also known as cell cloning, is an essential process in cell biology research and production of related biologicals. The cell culture can achieve the purposes of researching the life process of the cell, researching the disease and the drug mechanism, directionally differentiating the cell into other cells and the like by carrying out different treatments such as gene editing, drug treatment, induced differentiation and the like, and the cell large-scale culture can also be used for preparing biological protein drugs such as cytokines, antibodies and the like. Cell culture is an indispensable technology in the biological field.
When a certain protein is required to be added in the cell culture process, researchers often need to express and purify the protein, purified protein needs to be added every time the culture medium is replaced, the cost is relatively high in economy, and the experimental operation is relatively complicated. In addition, the stability of the product after protein expression and purification, the split charging and the preservation time of the protein and other problems can influence the cell culture and differentiation effects.
Dendritic cells, which are antigen presenting cells specific to the immune system, activate T cell immunity to specific antigens. The nobel physiology or medicine prize in 2011 was awarded to the canadian immunologist Ralph m.steinman to bring out his findings of dendritic cells and their functions in the acquired immunity. More and more researches show that the dendritic cells play an important role in the treatment of autoimmune diseases, cancers, inflammations and other diseases, and the dendritic cells have good curative effects in treating the diseases. However, since human dendritic cells are fewer and account for 1% or less of the whole blood system cells, which makes treatment of dendritic cells difficult, monocytes occupy a large proportion in the blood system and can be induced to differentiate into dendritic cells, and development of relevant stable techniques will greatly promote application of dendritic cells in disease treatment. Recent studies have shown that peripheral blood derived monocytes, GMCSF and IL4, are capable of becoming dendritic precursor cells that become mature dendritic cells induced by pro-inflammatory factors such as TNF- α, IL-1β, IL6, etc. In the culture process of dendritic cells, the cell replacement is required to be carried out for many times and the GMCSF and IL4 are added, the product stability of the GMCSF and IL4 proteins is critical for the directed differentiation of monocytes into dendritic precursor cells, and the addition of only the GMCSF without IL4 can lead the monocytes to differentiate into macrophages, so that the dendritic precursor cells cannot be cultured.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a culture container for directionally fixing fusion proteins of GMCSF and IL4 on the inner wall of the culture container, so that the whole process of inducing monocytes to differentiate into dendritic precursor cells is greatly simplified.
It is a first object of the present invention to provide a fusion protein.
It is a second object of the present invention to provide a gene.
A third object of the present invention is to provide a recombinant vector.
It is a fourth object of the present invention to provide a recombinant strain.
It is a fifth object of the present invention to provide the use of said fusion protein, said gene, said recombinant vector, said strain for inducing differentiation of monocytes into dendritic precursor cells.
It is a sixth object of the present invention to provide a cell culture vessel modified by protein-directed immobilization.
A seventh object of the present invention is to provide a method for preparing the cell culture vessel.
An eighth object of the present invention is to provide the use of the cell culture vessel in cell culture.
It is a ninth object of the present invention to provide the use of a cell culture vessel for inducing differentiation of monocytes into dendritic precursor cells.
In order to achieve the above object, the present invention is realized by the following technical scheme:
a fusion protein is a fusion protein of protein GMCSF and protein IL4, the amino acid sequence of the protein GMCSF is shown as SEQ ID NO. 1, the amino acid sequence of the protein IL4 is shown as SEQ ID NO. 2, and the amino acid sequence of the fusion protein is shown as SEQ ID NO. 3 or SEQ ID NO. 4.
A gene encoding the fusion protein of claim 1, which has a nucleotide sequence shown in SEQ ID No. 5 or SEQ ID No. 6, and falls within the scope of the present invention.
A recombinant vector contains a gene with a nucleotide sequence shown as SEQ ID NO. 5 or SEQ ID NO. 6, and also belongs to the protection scope of the invention.
A recombinant strain containing the recombinant vector also belongs to the protection scope of the invention.
The use of any of said fusion protein, said gene, said recombinant vector or said recombinant strain to induce differentiation of monocytes into dendritic cells is also within the scope of the present invention.
A cell culture container with protein directional immobilization modification, wherein the inner wall of the cell culture container is coated with protein.
Preferably, the inner wall of the cell culture container is made of glass, polystyrene, polyethylene and other materials, and the material can be used for coating protein molecules.
The preparation method of the cell culture container comprises the following steps when the inner wall of the culture container is glass:
s1, adding His tag to protein;
s2, silanization treatment is carried out on the inner wall of the cell culture container;
s3, performing AB-NTA incubation modification on the inner wall of the cell culture container;
s4, adding NiCl 2 Coupling NTA with Ni ions by the solution to finish nickel ion coating;
s5, adding protein containing His tag, and carrying out affinity with the coated nickel ion to fix the protein on the inner wall of the cell culture container.
AB-NTA refers to agarose gel beads (AB) to which NTA is covalently coupled.
Preferably, the protein is a fusion protein with an amino acid sequence shown as SEQ ID NO. 3 or SEQ ID NO. 4.
The application of the cell culture container in cell culture also belongs to the protection scope of the invention.
The use of the cell culture vessel in inducing differentiation of monocytes into dendritic cells is also within the scope of the present invention.
Compared with the prior art, the invention has the following beneficial effects:
the cell culture container prepared by the invention can avoid the repeated addition of two proteins of GMCSF and IL4 in the cell culture process by only coating the fusion protein once, and is economical and practical; meanwhile, the instability of the protein quality in the preparation process for many times is avoided, so that uncontrollable factors in the whole culture process are reduced, and the experimental operation is simplified. In addition, the protein with His tag can be directionally fixed on the glass cell culture dish coated with nickel, and after the solution containing imidazole is added, the protein can be eluted and the fresh protein is re-fixed, so that the method has the advantages of reversibility and multiple use.
Drawings
FIG. 1 shows a schematic representation of seamless cloning of GMCSF and IL4 fusion proteins.
FIG. 2 shows the results of PAGE gel electrophoresis after preliminary purification of GMCSF-IL4 and IL4-GMCSF nickel column.
FIG. 3 is a schematic diagram of a glass cell culture dish coated with a GMCSF-IL4 and IL4-GMCSF fusion protein.
Detailed Description
The invention will be further elaborated in connection with the drawings and the specific embodiments described below, which are intended to illustrate the invention only and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
EXAMPLE 1 construction of pET28 a-hGGMCSF-IL 4, pET28a-hIL4-GMCSF plasmids
The pET28 a-hGGMCSF-IL 4 plasmid and pET28a-hIL4-GMCSF plasmid are constructed by adopting a seamless cloning method, related primers are designed according to the requirements of a seamless cloning experimental method, plasmids pET50 b-hGGMCSF and pET50b-hIL4 (constructed by Shanghai Jinuo Biotechnology Co., ltd.) respectively containing GMCSF genes are used as DNA templates, and after PCR and glue recovery, seamless cloning is carried out. The nucleotide sequence of the GMCSF is shown as SEQ ID NO. 7, and the amino acid sequence is shown as SEQ ID NO. 1; the nucleotide sequence of IL4 is shown as SEQ ID NO. 8, and the amino acid sequence is shown as SEQ ID NO. 2.
1. hGGMCSF-linker, linker-IL4, hIL4-linker and linker-GMCSF fragments were amplified, respectively.
(1) PCR amplification of hGGMCSF-lingker, linker-GMCSF fragment pET50 b-hGGMCSF was used as DNA template, and the primers were as follows:
GMCSF-linker F：
GMCSF-linker R：
Linker-GMCSF F：
Linker-GMCSF R：
italics are with pET28a overlap region, underlined are BamHI and XhoI cleavage sites, and bolded are linker region.
PCR amplification system:
PCR amplification conditions
After the PCR amplification is completed, 1% agarose gel electrophoresis is carried out, then gel cutting is carried out, gel recovery is carried out by using a radicular gel recovery kit, and the concentration is measured by an ultraviolet spectrophotometer.
(2) PCR amplification of hIL4-linker, linker-IL4 fragment uses pET50b-hIL4 as DNA template, and the primers are:
hIL4-linker F：
hIL4-linker R：
linker-IL4F：
linker-IL4R：
the italic portion is with pET28a overlap region, the underlined portion is XhoI and BamHI cleavage sites, and the bolded portion is linker region. The PCR amplification system of the linker-IL4 fragment and the amplification conditions are the same as those of the hGGMCSF-linker fragment, and the concentration is recovered by cutting gel after the PCR amplification is completed.
2. Seamless cloning constructs pET28 a-hGGMCSF-IL 4, pET28a-hIL4-GMCSF plasmids.
Use of a seamless cloning kit Trelief from the company Optimum of the Prime biotechnology TM SoSoo Cloning Kit to make seamless cloning experiments, the specific operation is as follows:
(1) The pET28a plasmid was double digested with BamHI, xhoI from NEB.
Double enzyme digestion system:
and (3) performing enzyme digestion at 37 ℃ for 2 hours, performing 1% agarose gel electrophoresis, recovering large-fragment target DNA by using the radicular gelatin recovery kit gel, and determining the concentration of a gelatin recovery product by using a spectrophotometer.
(2) Sample addition according to the instructions of the seamless cloning kit:
reacting at 50deg.C for 40min, mixing with 30 mu lBL, standing on ice for 30min, heat-shocking at 42deg.C for 90s, standing on ice for 3min, adding 500. Mu.l LB without antibiotics, shaking at 37deg.C and 200rpm for 1h, centrifuging at low speed, removing part of supernatant, leaving 100. Mu.l liquid, blowing thallus to mix, tu Kana antibiotic plate, culturing overnight at 37deg.C incubator, picking monoclonal colony, culturing with Carna antibiotic LB, and delivering bacterial liquid to the Propioneer biosciences, and sequencing.
3. Results
Sequencing results show that the nucleotide for encoding the fusion protein GMCSF-IL4 is shown as SEQ ID NO. 5, and the amino acid sequence is shown as SEQ ID NO. 3; the nucleotide for coding IL4-GMCSF is shown as SEQ ID NO. 6, and the amino acid sequence is shown as SEQ ID NO. 4. Obtaining positive recombinant strain.
Example 2 cell culture Container with glass inner wall
1. Expression and purification of hGGMCSF-IL 4, hIL4-GMCSF fusion protein
Positive clones were selected for correct sequencing in example 1, and cultured overnight at 37℃and 200rpm with 5ml of LB containing a carbazate antibiotic. The following day, the overnight culture broth was transferred at 1:100 into 50mL of liquid LB containing the kana antibiotic, at 37 ℃,200rpm, and incubated for 3 hours (bulk culture to about od600=0.6 to 0.8). Transferring to 1L LB culture medium containing kana antibiotic, culturing at 37deg.C and 200rpm for 5-6 hr, cooling to 16deg.C, culturing, adding 0.5mmol/L IPTG, and culturing overnight. The overnight expressed cells were collected by centrifugation at 4000rpm for 10min, 40ml of equilibration (20 mM Tris-Cl, pH8.0, 0.5MNaCl, 40mM imidazole) resuspended cells, sonicated on 40% power ice, sonicated for 10S, stopped for 10S. After the completion of the sonication, the supernatant was collected by high-speed centrifugation at 12000rpm for 10min, and subjected to preliminary purification by a nickel column.
The treated 1ml column volume of nickel column was subjected to protein loading, followed by washing with 5ml of equilibration solution 20mM Tris-Cl, 0.5M NaCl, 40mM imidazole, followed by washing with 5ml of washing solution (20 mM Tris-Cl, 0.5M NaCl, 100mM imidazole), and finally eluting with 3ml of eluent (20 mM Tris-Cl, 0.5M NaCl, 300mM imidazole), 500. Mu.l of eluted protein solution was collected per centrifuge tube at the time of elution. hGGMCSF-IL 4 and hIL4-GMCSF fusion proteins are subjected to ion exchange purification, and concentration is carried out to determine the protein concentration.
2. Immobilization of hGGMCSF-IL 4, hIL4-GMCSF fusion protein
And coating the glass cell culture dish with nickel, namely sequentially performing the steps of silanization, incubation of AB-NTA and NiCl to provide Ni ions for coating the nickel ions, and completing the nickel ion coating process.
The specific operation is as follows: adding 2% gamma-glycidoxypropyl trimethoxysilane (Glymo) toluene steam into a glass cell culture dish, standing for 12 hours at room temperature, and washing with ultra-pure water for 3 times; after the addition of Na containing 2% (wt/vol) AB-NTA (nitrilotriacetic acid-agarose gel beads) 2 CO 3 The solution (0.01M, pH 10) was left at 60℃for 16h and washed 3 times with ultrapure water; finally adding 10mM NiCl 2 And 5mM glycine (pH 8.0) buffer, washed 3 times with ultrapure water at room temperature for 2 h.
After coating nickel ions on a glass cell culture dish, adding the hGGMCSF-IL 4 and hIL4-GMCSF purified proteins prepared in the example 2 into the glass culture dish, and placing the mixture in a cell culture box at 37 ℃ for 1-2 hours to finish fixation of fusion proteins. After fixation, the culture medium can be used for the induction culture of dendritic cells.
EXAMPLE 3 culture of monocytes to dendritic cells
The fusion protein can be directly used for cell culture after being fixed on a glass cell culture dish.
(1) Collecting human peripheral blood mononuclear cells by using a blood cell separator;
(2) Further purifying mononuclear cells (PBMCs) by lymphocyte separation medium density gradient centrifugation;
(3) Washing with serum-free culture solution for 2 times to obtain PBMC with purity of more than 90%.
The prepared peripheral blood mononuclear cells are cultured by using the glass cell culture dish with the fusion protein fixed, which is prepared in the embodiment 2, wherein the cell culture medium is 10% FCS+serum-free medium, the culture condition of a cell culture box is 37 ℃ and the concentration of 5% carbon dioxide, the cell culture medium is changed every other day, the cells are cultured for 6 to 7 days, the generation of pseudo feet of the cells can be observed by a microscope, and the successful induction of dendritic precursor cells is proved.
Sequence listing
<110> Shanghai Jinuo Biotech Co., ltd
<120> a cell culture vessel modified by protein immobilization
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Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln
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Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His Val
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Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys Gln
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Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met Met
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Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser Cys
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Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp
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Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu Gly
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Arg Phe Leu Lys Arg Leu Asp Arg Asn Leu Trp Gly Leu Ala Gly Leu
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Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp
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Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu
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gcacccgccc gctcgcccag ccccagcacg cagccctggg agcatgtgaa tgccatccag 60
gaggcccggc gtctcctgaa cctgagtaga gacactgctg ctgagatgaa tgaaacagta 120
gaagtcatct cagaaatgtt tgacctccag gagccgacct gcctacagac ccgcctggag 180
ctgtacaagc agggcctgcg gggcagcctc accaagctca agggcccctt gaccatgatg 240
gccagccact acaagcagca ctgccctcca accccggaaa cttcctgtgc aacccagatt 300
atcacctttg aaagtttcaa agagaacctg aaggactttc tgcttgtcat cccctttgac 360
tgctgggagc cagtccagga gggaggtggc gggagtggag gtggcgggag tggaggtggc 420
gggagtcaca agtgcgatat caccttacag gagatcatca aaactttgaa cagcctcaca 480
gagcagaaga ctctgtgcac cgagttgacc gtaacagaca tctttgctgc ctccaagaac 540
acaactgaga aggaaacctt ctgcagggct gcgactgtgc tccggcagtt ctacagccac 600
catgagaagg acactcgctg cctgggtgcg actgcacagc agttccacag gcacaagcag 660
ctgatccgat tcctgaaacg gctcgacagg aacctctggg gcctggcggg cttgaattcc 720
tgtcctgtga aggaagccaa ccagagtacg ttggaaaact tcttggaaag gctaaagacg 780
atcatgagag agaaatattc aaagtgttcg agctag 816
<210> 6
<211> 816
<212> DNA
<213> person (Homo sapiens)
<400> 6
cacaagtgcg atatcacctt acaggagatc atcaaaactt tgaacagcct cacagagcag 60
aagactctgt gcaccgagtt gaccgtaaca gacatctttg ctgcctccaa gaacacaact 120
gagaaggaaa ccttctgcag ggctgcgact gtgctccggc agttctacag ccaccatgag 180
aaggacactc gctgcctggg tgcgactgca cagcagttcc acaggcacaa gcagctgatc 240
cgattcctga aacggctcga caggaacctc tggggcctgg cgggcttgaa ttcctgtcct 300
gtgaaggaag ccaaccagag tacgttggaa aacttcttgg aaaggctaaa gacgatcatg 360
agagagaaat attcaaagtg ttcgagcgga ggtggcggga gtggaggtgg cgggagtgga 420
ggtggcggga gtgcacccgc ccgctcgccc agccccagca cgcagccctg ggagcatgtg 480
aatgccatcc aggaggcccg gcgtctcctg aacctgagta gagacactgc tgctgagatg 540
aatgaaacag tagaagtcat ctcagaaatg tttgacctcc aggagccgac ctgcctacag 600
acccgcctgg agctgtacaa gcagggcctg cggggcagcc tcaccaagct caagggcccc 660
ttgaccatga tggccagcca ctacaagcag cactgccctc caaccccgga aacttcctgt 720
gcaacccaga ttatcacctt tgaaagtttc aaagagaacc tgaaggactt tctgcttgtc 780
atcccctttg actgctggga gccagtccag gagtag 816
<210> 7
<211> 381
<212> DNA
<213> person (Homo sapiens)
<400> 7
gcacccgccc gctcgcccag ccccagcacg cagccctggg agcatgtgaa tgccatccag 60
gaggcccggc gtctcctgaa cctgagtaga gacactgctg ctgagatgaa tgaaacagta 120
gaagtcatct cagaaatgtt tgacctccag gagccgacct gcctacagac ccgcctggag 180
ctgtacaagc agggcctgcg gggcagcctc accaagctca agggcccctt gaccatgatg 240
gccagccact acaagcagca ctgccctcca accccggaaa cttcctgtgc aacccagatt 300
atcacctttg aaagtttcaa agagaacctg aaggactttc tgcttgtcat cccctttgac 360
tgctgggagc cagtccagga g 381
<210> 8
<211> 390
<212> DNA
<213> person (Homo sapiens)
<400> 8
cacaagtgcg atatcacctt acaggagatc atcaaaactt tgaacagcct cacagagcag 60
aagactctgt gcaccgagtt gaccgtaaca gacatctttg ctgcctccaa gaacacaact 120
gagaaggaaa ccttctgcag ggctgcgact gtgctccggc agttctacag ccaccatgag 180
aaggacactc gctgcctggg tgcgactgca cagcagttcc acaggcacaa gcagctgatc 240
cgattcctga aacggctcga caggaacctc tggggcctgg cgggcttgaa ttcctgtcct 300
gtgaaggaag ccaaccagag tacgttggaa aacttcttgg aaaggctaaa gacgatcatg 360
agagagaaat attcaaagtg ttcgagctag 390

Claims (1)

1. The application of a protein-oriented immobilized modified cell culture container in inducing monocytes to differentiate into dendritic precursor cells is characterized in that the inner wall of the cell culture container is coated with protein, wherein the protein is fusion protein of protein GMCSF and protein IL4, the amino acid sequence of the protein GMCSF is shown as SEQ ID NO. 1, the amino acid sequence of the protein IL4 is shown as SEQ ID NO. 2, the amino acid sequence of the fusion protein is shown as SEQ ID NO. 3 or SEQ ID NO. 4, and the cell container is used for inducing monocytes to differentiate into dendritic precursor cells; when the inner wall of the culture container is glass, the preparation method of the cell culture container comprises the following steps:
s1, adding His tag to protein;
s2, silanization treatment is carried out on the inner wall of the cell culture container;
s3, performing AB-NTA incubation modification on the inner wall of the cell culture container;
s4, adding NiCl2 solution to couple NTA with Ni to finish nickel ion coating;
s5, adding protein containing His tag, and carrying out affinity with the coated nickel ion to fix the protein on the inner wall of the cell culture container.
CN201810186457.4A 2018-03-07 2018-03-07 Cell culture container modified by protein immobilization Active CN108314740B (en)

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CN107208064A (en) * 2015-01-29 2017-09-26 国立大学法人东京大学 The cultural method of cell, the agglutination body of cell, cell agglutination controlling agent and culture medium
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