CN106676134B - Method for obtaining type II intrinsic lymphocytes from mice and carrier used in method - Google Patents
Method for obtaining type II intrinsic lymphocytes from mice and carrier used in method Download PDFInfo
- Publication number
- CN106676134B CN106676134B CN201611263314.6A CN201611263314A CN106676134B CN 106676134 B CN106676134 B CN 106676134B CN 201611263314 A CN201611263314 A CN 201611263314A CN 106676134 B CN106676134 B CN 106676134B
- Authority
- CN
- China
- Prior art keywords
- type
- expression vector
- liver
- lymphocyte
- murine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- 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
-
- 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
- C07K14/54—Interleukins [IL]
-
- 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
- C12N5/0651—Lymph nodes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- 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
- C12N2510/00—Genetically modified cells
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for obtaining type II inherent lymphocyte from a mouse body and a carrier used by the method, wherein the carrier is a recombinant expression carrier obtained by connecting a coding sequence of kappa chain signal peptide of mouse IgG and a coding sequence of murine IL-33 active region to a pcDNA3.1 eukaryotic expression carrier through a gene recombination technology. The recombinant expression vector is introduced into an animal body, so that murine IL-33 is overexpressed in the animal body, and the II type inherent lymphocyte is induced and amplified in the liver; then separating to obtain liver tissue lymphocyte, marking the obtained liver tissue lymphocyte with type II inherent lymphocyte specific antibody, and sorting by cell flow technology to obtain type II inherent lymphocyte. The technology of the invention can induce and largely amplify, separate and purify high-activity type II inherent lymphocytes from C57BL/6 mice.
Description
Technical Field
The present invention relates to a method for obtaining type II innate lymphocyte from mouse and a carrier used in the method, and more particularly, to a recombinant expression vector capable of inducing and amplifying a large amount of type II innate lymphocyte in liver, and a method for obtaining a large amount of type II innate lymphocyte from mouse using the vector.
Background
In recent years, as a result of intensive studies on innate immune cells, a new cell subset has been discovered. This subset does not express antigen-specific recognition receptors (TCR or BCR), is developmentally and morphologically of the lymphocyte lineage, and is known as Innate Lymphocyte (ILC) along with Natural Killer (NK) cells and Lymphoid Tissue Inducing (LTi) cells that have been found in the past. The ILC family can be further divided into three major classes according to the classification of helper T (Th) cells, based on differences in cytokine secretion and transcription factor expression: ILC1, ILC2, and ILC 3.
Type II intrinsic lymphocytes (ILC 2) are one of the newly discovered intrinsic lymphocyte populations, are mainly distributed in lung, intestinal tract, skin and other mucosal tissues, and can produce Th2 cytokines such as IL-5 and IL-13 under the stimulation of IL-25 and IL-33. ILC2 plays an important role in respiratory anti-infectious immunity and allergic diseases, and is therefore of great interest. Research has shown that ILC2 is an important source of Th2 type cell factors in the early stage of respiratory allergic diseases, and can regulate adaptive immune response; while ILC2 also plays an important role in combating parasites, viral infections, and tissue repair. Therefore, biological studies of ILC2 are of great practical significance. Despite the major breakthrough currently made in the field related to ILC2, the study of ILC2 still faces major technical challenges, since ILC2 is present in very small numbers in normal mice, whereas conventional immunological or biochemical research approaches require large numbers of cells to perform. Therefore, how to obtain high-purity ILC2 in large quantities in a short time is a major technical problem facing further research.
Disclosure of Invention
An object of the present invention is to provide a method for obtaining a large amount of type II intrinsic lymphocytes from a mouse.
Another object of the present invention is to provide a recombinant expression vector capable of inducing and amplifying a large amount of type II resident lymphocytes in the liver, which can be used for obtaining a large amount of type II resident lymphocytes from a mouse.
In one aspect, the invention provides a recombinant expression vector obtained by linking the coding sequence of the kappa chain signal peptide of mouse IgG and the coding sequence of the active region of murine IL-33 to a eukaryotic expression vector.
According to a particular embodiment of the invention, the coding sequence for the kappa chain signal peptide of mouse IgG has the sequence as shown in SEQ id No.: 1 (the corresponding amino acid sequence is shown in SEQ ID No.: 2).
According to a particular embodiment of the invention, the coding sequence of the active region of murine IL-33 has the sequence shown in SEQ ID No.: 3 (the corresponding amino acid sequence is shown in SEQ ID No.: 4).
According to a specific embodiment of the present invention, there is provided a recombinant expression vector comprising a nucleic acid sequence as set forth in SEQ ID No.: 5, and (c) a sequence shown in the specification. That is, it is a polypeptide comprising an amino acid sequence as set forth in SEQ ID No.: 5 in the open reading frame.
According to a specific embodiment of the present invention, in the recombinant expression vector of the present invention, the eukaryotic expression vector is any eukaryotic expression vector using CMV promoter or derived promoter to express protein in eukaryotic cells, such as pcdna3.1 eukaryotic expression vector, pCMV eukaryotic expression vector, and the like.
In a specific embodiment of the present invention, the recombinant expression vector of the present invention has a CMV promoter or derived promoter and has in its open reading frame the coding sequence for the mouse IgG kappa chain signal peptide and the coding sequence for the murine IL-33 active region, i.e., it is obtained by ligating the coding sequence for the mouse IgG kappa chain signal peptide and the murine IL-33 active region into the open reading frame of any eukaryotic expression vector having a CMV promoter or derived promoter, such as between the HindIII and BamHI of pcdna3.1(+) eukaryotic expression vector. In the present invention, such a recombinant expression vector (a recombinant expression vector obtained by ligating the coding sequence of kappa chain signal peptide of mouse IgG and the coding sequence of murine IL-33 active region to HindIII and BamHI of pcDNA3.1(+) eukaryotic expression vector) was named pcDNA3.1-mIg kappa-mIL 33.
The recombinant expression vector provided by the invention can induce and amplify a large amount of type II intrinsic lymphocytes in liver, and thus the invention also provides application of the recombinant expression vector in preparing a preparation for inducing and amplifying type II intrinsic lymphocytes in liver.
In another aspect, the present invention provides a method for obtaining type II innate lymphocytes from an animal comprising:
the recombinant expression vector is introduced into an animal body, so that murine IL-33 is overexpressed in the animal body, and the II type inherent lymphocyte is induced and amplified in the liver;
separating to obtain liver tissue lymphocyte, labeling the obtained liver tissue lymphocyte with type II inherent lymphocyte specific antibody, and sorting by cell flow technology to obtain type II inherent lymphocyte.
According to a specific embodiment of the present invention, the animal is a mouse in the method of the present invention for obtaining type II innate lymphocytes from an animal.
According to a specific embodiment of the present invention, the method for obtaining type II intrinsic lymphocytes from an animal according to the present invention is to introduce the recombinant expression vector into the liver of the animal by means of tail vein high pressure injection.
The tail vein high pressure injection is that a large volume of naked plasmid DNA solution is rapidly injected through the tail vein of a mouse, the amount of circulating blood is rapidly increased due to a large amount of plasmid solution, the blood is accumulated in the hepatic sinus and cannot flow back after exceeding the load of the heart, the retention time of the plasmid DNA in the hepatic sinus is prolonged, and the plasmid DNA is taken by hepatic tissue cells. The target protein can be expressed in situ by the liver cells taking up the recombinant vector.
According to a specific embodiment of the present invention, the method for obtaining type II intrinsic lymphocytes from an animal according to the present invention comprises isolating liver tissue lymphocytes from the animal at least 3 days after the intravenous administration (usually from 3 days to 7 days in the present invention).
According to a specific embodiment of the present invention, in the method for obtaining type II intrinsic lymphocytes from an animal body according to the present invention, liver tissue lymphocytes may be isolated by density gradient centrifugation (e.g., Percoll method).
According to a specific embodiment of the present invention, in the method for obtaining type II intrinsic lymphocytes from an animal body according to the present invention, type II intrinsic lymphocytes are purified by flow cytometry from liver mononuclear cells using four antibodies. Preferably, the antibody is FITC-anti mouse KLRG1, PE-anti mouse Linear Cocktail, PerCP. Cy5.5-anti mouse CD90.2, APC-anti mouse IL 33R. Such antibodies are commercially available, for example from Biolegend.
The specific experiment of the invention shows that the liver of the normal mouse or the mouse injected by the control vector under high pressure almost has no ILC2 cell, while the liver of the mouse injected by the recombinant expression vector pcDNA3.1-mIg kappa-mIL 33 clearly has ILC2 cell population, and the cell can be obtained in large quantity after the sorting by flow cytometry. In addition, since the sorting strategy is based entirely on the definition of type II resident lymphocytes, the purity of the sorted cells is sufficiently high, provided that the flow cytometer is sufficiently accurate.
A large number of repeated experiments prove that the invention can induce, greatly amplify, separate and purify the II type inherent lymphocyte with high purity and high activity from the C57BL/6 mouse.
Drawings
FIG. 1 shows the nucleic acid and amino acid sequences of mIg kappa.
FIG. 2 shows the nucleic acid and amino acid sequences of mIL-33.
Figure 3 shows the flow detection and sorting strategy for type II resident lymphocytes.
FIG. 4 shows the levels of type II resident lymphocytes in the liver 3 days after tail vein injection of pcDNA3.1 and pcDNA3.1-mIg kappa-mIL-33, respectively.
FIG. 5 shows the number of type II resident lymphocytes in the liver 1-3 days after pcDNA3.1-mIg kappa-mIL-33 injection.
FIG. 6 shows that type II naive lymphocytes obtained by sorting in this manner, when cultured in vitro, produce a large amount of Th2 type cytokine but not Th1 type cytokine upon mIL-33 stimulation.
FIG. 7 shows the number of type II resident lymphocytes obtained in the liver after injecting various amounts of pcDNA3.1-mIg kappa-mIL-33 for 3 days by this method.
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. The experimental methods in the examples, in which specific conditions are not noted, are conventional methods and conventional conditions well known in the art, or conditions as recommended by the manufacturer.
Example 1 construction of the recombinant vector pcDNA3.1-mIg kappa-mIL 33
A recombinant plasmid pcDNA3.1-mIg kappa-mIL 33 was constructed by gene recombination by ligating the coding sequence of kappa chain signal peptide with mouse IgG (mIgK signal peptide nucleotide sequence and amino acid sequence are shown in SEQ ID No.: 1 and SEQ ID No.: 2, FIG. 1) and the coding sequence of murine IL-33 active region (IL-33 active region nucleotide sequence and amino acid sequence are shown in SEQ ID No.: 3 and SEQ ID No.: 4, FIG. 2) into the HindIII and BamHI of pcDNA3.1(+) eukaryotic expression vector (Invitrogen).
Example 2 isolation and purification of liver type II innate lymphocytes
1. Tail vein high pressure injection of pcDNA3.1-mIg kappa-mIL 33 recombinant plasmid
In this example, a volume of physiological saline or PBS solution containing 20. mu.g of pcDNA3.1-mIg kappa-mIL 33 recombinant plasmid corresponding to about 10% of the body weight (v/w) of a mouse was injected into the mouse through the tail vein of the mouse over a period of 6 to 8 seconds.
2. Separation and purification of mouse liver type II inherent lymphocyte
(1) After at least 3 days of tail vein hyperbaric injection in the preceding steps, type II resident lymphocytes accumulated in the liver were already significant (see fig. 5, data representing representative results of multiple replicates), and were killed: removing eyes and bleeding, and cleaning as much as possible. (the following experiments all require aseptic manipulation.)
(2) Liver tissue was harvested by aseptic technique and placed in sterile PBS on ice.
(3) Liver tissue was gently ground and filtered through a 200 mesh screen.
(4) Centrifuge at 1060g for 10min at 4 ℃ and discard the supernatant. (centrifugation was performed at 4 ℃ C. except for the step of density gradient centrifugation)
(5) Density gradient centrifugation: resuspend the cell pellet with 3ml 42% Percoll, add slowly to 3ml 70% Percoll (GE healthcare) surface, centrifuge 1260g for 30 min.
20ml 70% Percoll formulation: 12.6ml Percoll stock solution (GE Healthcare) +
1.4ml 10×PBS+6ml 1×PBS
20ml of 42% Percoll formulation: 7.56ml Percoll stock solution (GE Healthcare) +
840ul 10×PBS+11.6ml 1×PBS
(6) The intermediate layer cells were removed to a new 15 ml centrifuge tube, filled with PBS, centrifuged at 1060g for 10 minutes and the supernatant discarded.
(7) A small number of cells were removed and stained with trypan blue and counted.
(8) Cell suspension was added to rat serum to block Fc receptors and label fluorescent antibodies: FITC-anti-mouse KLRG1, PE-anti-mouse Linear Cocktail, PerCP. Cy5.5-anti-mouse CD90.2, APC-anti-mouse IL33R (antibodies all from Biolegend), incubated at 4 ℃ in the absence of light for 15 min.
(9) PBS was added thereto, and washed twice at 1060 g.times.10 min at 4 ℃.
(10) And (3) sorting the Lineage-IL33R + CD90.2+ KLRG1+ cells by using a flow cytometer, namely ILC2 cells.
See fig. 3 and 4 for flow cytometric sorting of ILC2 cells. In FIG. 3, the left panel circles single cells from a liver monocyte suspension using FSC-A and FSC-H, the middle panel circles single cells from the above single cells from Linear-ST 2+ cells under Linear negative ST2 positive conditions, and the right panel circles CD90.2 positive KLRG1 positive cells from the above Linear-ST 2+ cells, i.e., ILC2 cells.
As can be seen from the experimental results in FIG. 4, the liver of the normal mouse or the mouse injected with the control vector under high pressure has almost no ILC2 cell, and the liver of the mouse injected with the pcDNA3.1-mIg kappa-mIL 33 recombinant vector has a clear ILC2 cell population, which is then sorted by flow cytometry to obtain the cell in large quantity (FIG. 5). The cells thus selected produced significant amounts of Th2 type cytokines such as IL-5 and IL-13, but failed to produce Th1 type cytokines such as IFN-. gamma. (FIG. 6), when cultured in vitro at 50000/mL density, 2 days after stimulation with mIL-33 (e.g., 50ng/mL mIL-33), indicating that the cells had superior activity.
Example 3
After injecting normal adult mice with different amounts of pcDNA3.1-mIg kappa-mIL-33 for 3 days, type II resident lymphocytes were obtained from liver tissues, and the rest of the detailed procedures were the same as in example 2. The number of type II resident lymphocytes that can be obtained in the liver in this example is shown in FIG. 7. The method of the present invention is used in inducing, amplifying, separating and purifying great amount of high purity and high activity type II inherent lymphocyte from mouse.
Sequence listing
<110> Shenzhen advanced technology research institute
<120> method for obtaining type II innate lymphocytes from mice and carrier used in the method
<130>GAI16CN1304
<160>5
<170>PatentIn version 3.5
<210>1
<211>60
<212>DNA
<213> mice
<220>
<223> coding sequence for the
<220>
<221>CDS
<222>(1)..(60)
<400>1
atg gag aca gac aca ctc ctg cta tgg gta ctg ctg ctc tgg gtt cca 48
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
ggt tcc act ggt 60
Gly Ser Thr Gly
20
<210>2
<211>20
<212>PRT
<213> mice
<220>
<223> signal peptide of
<400>2
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly
20
<210>3
<211>477
<212>DNA
<213> mice
<220>
<223> coding sequence of IL-33 active region of mouse
<220>
<221>CDS
<222>(1)..(477)
<400>3
agc atc caa gga act tca ctt tta aca cag tct cct gcc tcc ctg agt 48
Ser Ile Gln Gly Thr Ser Leu Leu Thr Gln Ser Pro Ala Ser Leu Ser
1 5 10 15
aca tac aat gac caa tct gtt agt ttt gtt ttg gag aat gga tgt tat 96
Thr Tyr Asn Asp Gln Ser Val Ser Phe Val Leu Glu Asn Gly Cys Tyr
20 25 30
gtg atc aat gtt gac gac tct gga aaa gac caa gag caa gac cag gtg 144
Val Ile Asn Val Asp Asp Ser Gly Lys Asp Gln Glu Gln Asp Gln Val
35 40 45
cta cta cgc tac tat gag tct ccc tgt cct gca agt caa tca ggc gac 192
Leu Leu Arg Tyr Tyr Glu Ser Pro Cys Pro Ala Ser Gln Ser Gly Asp
50 55 60
ggt gtg gat ggg aag aag ctg atg gtg aac atg agt ccc atc aaa gac 240
Gly Val Asp Gly Lys Lys Leu Met Val Asn Met Ser Pro Ile Lys Asp
65 70 75 80
aca gac atc tgg ctg cat gcc aac gac aag gac tac tcc gtg gag ctt 288
Thr Asp Ile Trp Leu His Ala Asn Asp Lys Asp Tyr Ser Val Glu Leu
85 90 95
caa agg ggt gac gtc tcg cct ccg gaa cag gcc ttc ttc gtc ctt cac 336
Gln Arg Gly Asp Val Ser Pro Pro Glu Gln Ala Phe Phe Val Leu His
100 105 110
aaa aag tcc tcg gac ttt gtt tca ttt gaa tgc aag aat ctt cct ggc 384
Lys Lys Ser Ser Asp Phe Val Ser Phe Glu Cys Lys Asn Leu Pro Gly
115 120 125
act tac ata gga gta aaa gat aac cag ctg gct cta gtg gag gag aaa 432
Thr Tyr Ile Gly Val Lys Asp Asn Gln Leu Ala Leu Val Glu Glu Lys
130 135 140
gat gag agc tgc aac aat att atg ttt aag ctc tcg aaa atc taa 477
Asp Glu Ser Cys Asn Asn Ile Met Phe Lys Leu Ser Lys Ile
145 150 155
<210>4
<211>158
<212>PRT
<213> mice
<220>
<223> mouse IL-33 active region
<400>4
Ser Ile Gln Gly Thr Ser Leu Leu Thr Gln Ser Pro Ala Ser Leu Ser
1 5 10 15
Thr Tyr Asn Asp Gln Ser Val Ser Phe Val Leu Glu Asn Gly Cys Tyr
2025 30
Val Ile Asn Val Asp Asp Ser Gly Lys Asp Gln Glu Gln Asp Gln Val
35 40 45
Leu Leu Arg Tyr Tyr Glu Ser Pro Cys Pro Ala Ser Gln Ser Gly Asp
50 55 60
Gly Val Asp Gly Lys Lys Leu Met Val Asn Met Ser Pro Ile Lys Asp
65 70 75 80
Thr Asp Ile Trp Leu His Ala Asn Asp Lys Asp Tyr Ser Val Glu Leu
85 90 95
Gln Arg Gly Asp Val Ser Pro Pro Glu Gln Ala Phe Phe Val Leu His
100 105 110
Lys Lys Ser Ser Asp Phe Val Ser Phe Glu Cys Lys Asn Leu Pro Gly
115 120 125
Thr Tyr Ile Gly Val Lys Asp Asn Gln Leu Ala Leu Val Glu Glu Lys
130 135 140
Asp Glu Ser Cys Asn Asn Ile Met Phe Lys Leu Ser Lys Ile
145 150 155
<210>5
<211>537
<212>DNA
<213>Artificial Sequence
<220>
<223> open reading frame
<400>5
atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccactggt 60
agcatccaag gaacttcact tttaacacag tctcctgcct ccctgagtac atacaatgac 120
caatctgtta gttttgtttt ggagaatgga tgttatgtga tcaatgttga cgactctgga 180
aaagaccaag agcaagacca ggtgctacta cgctactatg agtctccctg tcctgcaagt 240
caatcaggcg acggtgtgga tgggaagaag ctgatggtga acatgagtcc catcaaagac 300
acagacatct ggctgcatgc caacgacaag gactactccg tggagcttca aaggggtgac 360
gtctcgcctc cggaacaggc cttcttcgtc cttcacaaaa agtcctcgga ctttgtttca 420
tttgaatgca agaatcttcc tggcacttac ataggagtaa aagataacca gctggctcta 480
gtggaggaga aagatgagag ctgcaacaat attatgttta agctctcgaa aatctaa 537
Claims (8)
1. A recombinant expression vector that links the coding sequence of the k-chain signal peptide of mouse IgG and the coding sequence of the murine IL-33 active region into the open reading frame of a eukaryotic expression vector with a CMV promoter or a derivative promoter; the recombinant expression vector has a sequence shown in SEQ ID No: 5, and (b) a sequence shown in the specification; the eukaryotic expression vector is selected from pcDNA3.1 eukaryotic expression vectors.
2. The recombinant expression vector of claim 1, wherein the coding sequence of the k-chain signal peptide of mouse IgG has the amino acid sequence as set forth in SEQ ID No: 1; the coding sequence of the murine IL-33 active region has the sequence shown in SEQ ID No: 3, and (b) is shown in the specification.
3. Use of a recombinant expression vector according to any one of claims 1 to 2 for the preparation of a formulation for inducing and expanding type II resident lymphocytes in the liver.
4. A method of obtaining type II resident lymphocytes from an animal, the method comprising:
introducing the recombinant expression vector of any one of claims 1-2 into mouse liver, so that the mouse liver over-expresses murine IL-33, thereby inducing and amplifying type II innate lymphocytes in the liver;
separating to obtain liver tissue lymphocyte, labeling the obtained liver tissue lymphocyte with type II inherent lymphocyte specific antibody, and sorting by cell flow technology to obtain type II inherent lymphocyte.
5. The method of claim 4, wherein the recombinant expression vector is introduced into mouse liver by tail vein high pressure injection.
6. The method of claim 4, wherein the liver tissue lymphocytes are isolated by density gradient centrifugation.
7. The method of claim 4, wherein the type II resident lymphocytes are purified by flow cytometry sorting from liver mononuclear cells using four antibodies.
8. The method of claim 7 wherein the antibody is FITC-anti-murine KLRG1, PE-anti-murine LineagCocktail, PerCP. Cy5.5 anti-murine CD90.2, APC-anti-murine IL 33R.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611263314.6A CN106676134B (en) | 2016-12-30 | 2016-12-30 | Method for obtaining type II intrinsic lymphocytes from mice and carrier used in method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611263314.6A CN106676134B (en) | 2016-12-30 | 2016-12-30 | Method for obtaining type II intrinsic lymphocytes from mice and carrier used in method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106676134A CN106676134A (en) | 2017-05-17 |
| CN106676134B true CN106676134B (en) | 2020-03-06 |
Family
ID=58849588
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611263314.6A Active CN106676134B (en) | 2016-12-30 | 2016-12-30 | Method for obtaining type II intrinsic lymphocytes from mice and carrier used in method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106676134B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410820A (en) * | 2018-03-14 | 2018-08-17 | 南京鼓楼医院 | A method of isolating and purifying intrinsic lymphocyte from mouse intestinal mucosa |
| CN118056898A (en) * | 2022-11-18 | 2024-05-21 | 深圳先进技术研究院 | Method for separating intestinal mucosa lamina propria lymphocyte |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104001168A (en) * | 2014-06-07 | 2014-08-27 | 中国医学科学院医学生物学研究所 | Method for constructing IL-33 presentation VLP (Virus-Like Particle) vaccine used in active immunotherapy of chronic asthma |
-
2016
- 2016-12-30 CN CN201611263314.6A patent/CN106676134B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104001168A (en) * | 2014-06-07 | 2014-08-27 | 中国医学科学院医学生物学研究所 | Method for constructing IL-33 presentation VLP (Virus-Like Particle) vaccine used in active immunotherapy of chronic asthma |
Non-Patent Citations (5)
| Title |
|---|
| "Enhanced production of secretory glycoprotein VSTM1-v2 with mouse IgGk signal peptide in optimized HEK293F transient transfection";Huihui Liu et al.,;《Journal of Bioscience and Bioengineering》;20150630;第121卷(第2期);第133页摘要部分和第134页左栏第5段 * |
| "GenBank：D84070.1";GenBank;《NCBI》;20160614;第1页 * |
| "Innate-Type and Acquired-Type";Tomohiro Yoshimoto et al.,;《Allergology International》;20141231(第63期);第6页左栏第2段、附图3-第7页左栏第1段 * |
| "NCBI Reference Sequence: NM_001164724.1";GenBank;《NCBI》;20160904;第1、3页 * |
| "上皮源性细胞因子IL-33通过激活细胞ILC2参与哮喘Th2型免疫应答";李艳等;《第九届全国免疫学学术大会》;20141231;第58页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106676134A (en) | 2017-05-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DE102016115246B3 (en) | NEW T-CELL RECEPTORS AND THEIR USE IN IMMUNOTHERAPY | |
| JP6665141B2 (en) | Identification of CD8 + T cells that are CD161hi and / or IL18Rahi and have rapid drug efflux ability | |
| CN108368520A (en) | The genome project of pluripotent cell is transformed | |
| Dooms et al. | Interleukin-2 enhances CD4+ T cell memory by promoting the generation of IL-7Rα–expressing cells | |
| US10400215B2 (en) | Adoptive transfer of CD8+ T cell clones derived from central memory cells | |
| CN108472346A (en) | Chimerical receptor containing TRAF inducement structures domain and compositions related and method | |
| JP2021516996A (en) | Biologically related orthogonal cytokine / receptor pair | |
| CN110291402A (en) | The method of identification peptide epitopes, molecule and associated uses in conjunction with such epitope | |
| CN111566221A (en) | Methods for NK cell transduction | |
| JP2021524248A (en) | Chimeric antigen receptor with modified linker domain and its use | |
| WO2021142835A1 (en) | Strengthened receptor for improving immune cell function | |
| US8815543B2 (en) | Methods of producing antigen-specific B cell populations in the presence of CD40:CD40L, BAFF:BAFF receptor and Fas:FasL stimulation | |
| CN107106609A (en) | Stimulate and extend the composition and method of T cell | |
| JP2022526372A (en) | CAR for use in the treatment of HvG disease | |
| CN106676134B (en) | Method for obtaining type II intrinsic lymphocytes from mice and carrier used in method | |
| JP2022520777A (en) | An adjuvant capable of boosting the proliferation of immune cells in vivo | |
| Asami et al. | Cultivation and characterization of planarian neuronal cells isolated by fluorescence activated cell sorting (FACS) | |
| CN108251442B (en) | FLT3 chimeric antigen receptor and application thereof | |
| US20200215117A1 (en) | Mesenchymal stem cell over-expressing cxcr5, preparation method and use thereof | |
| CN112679618A (en) | TRBC 1-targeted humanized chimeric antigen receptor, T cell and application | |
| JP2012518407A (en) | Isolated monocyte population and related therapeutic applications | |
| WO2021232050A1 (en) | Natural killer cell immunotherapy for the treatment or prevention of sars-cov-2 infection | |
| JP2003529363A (en) | Production of TcR gamma delta T cells | |
| KR20220041934A (en) | Therapeutic immune cells with improved function and method for preparing the same | |
| CN111676195A (en) | UCAR immune cell for treating T cell tumor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |