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Method for obtaining II-type innate lymphocytes from mice in vivo and carrier used by same

A technology of lymphocytes and mice, applied in the field of obtaining type II innate lymphocytes

Active Publication Date: 2017-05-17
SHENZHEN INST OF ADVANCED TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although major breakthroughs have been made in the field of ILC2-related research, the research on ILC2 still faces major challenges because the number of ILC2 in normal mice is very small, and conventional immunological or biochemical research methods require a large number of cells. technical challenge

Method used

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  • Method for obtaining II-type innate lymphocytes from mice in vivo and carrier used by same
  • Method for obtaining II-type innate lymphocytes from mice in vivo and carrier used by same
  • Method for obtaining II-type innate lymphocytes from mice in vivo and carrier used by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1, Construction of pcDNA3.1-mIgκ-mIL33 recombinant vector

[0033] Through genetic recombination technology, the coding sequence of the κ chain signal peptide with mouse IgG (mIgK signal peptide nucleotide sequence and amino acid sequence are respectively referred to as SEQ ID No.: 1 and SEQ ID No.: 2, figure 1 ) and murine IL-33 active region coding sequence (see SEQ ID No.: 3 and SEQ ID No.: 4 for the nucleotide sequence and amino acid sequence of the IL-33 active region, respectively, figure 2 ) was connected between HindIII and BamHI of the pcDNA3.1 (+) eukaryotic expression vector (Invitrogen), and the pcDNA3.1-mIgκ-mIL33 recombinant plasmid was constructed.

Embodiment 2

[0034] Example 2. Isolation and purification of liver type II innate lymphocytes

[0035] 1. Tail vein high pressure injection of pcDNA3.1-mIgκ-mIL33 recombinant plasmid

[0036]In this example, within 6 to 8 seconds, a volume corresponding to about 10% of the mouse body weight (v / w) containing 20 micrograms of pcDNA3.1-mIgκ-mIL33 recombinant plasmid in normal saline or PBS solution was passed through a small Rat tail vein injection into mice.

[0037] 2. Isolation and purification of mouse liver type II innate lymphocytes

[0038] (1) At least 3 days after the high-pressure injection of the tail vein in the previous step, the accumulation of type II innate lymphocytes in the liver has been considerable (see Figure 5 , the data in the figure are the representative results of repeated experiments), killing rats: remove the eyes and let the blood out, and put them as clean as possible. (The following experiments require aseptic operation.)

[0039] (2) Liver tissue was remo...

Embodiment 3

[0055] Three days after injecting normal adult mice with different amounts of pcDNA3.1-mIgκ-mIL-33, type II innate lymphocytes were obtained from liver tissue, and the rest of the specific operations were the same as in Example 2. The number of type II innate lymphocytes that can be obtained in the liver in this example Figure 7 . It shows that the method of the present invention can induce, expand, isolate and purify high-purity and highly active type II innate lymphocytes from mice.

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Abstract

The invention provides a method for obtaining II-type innate lymphocytes from mice in vivo and a carrier used by the same. The carrier is a recombinant expression carrier which is obtained through connecting a coding sequence of k-chain signal peptide of mice IgG and a coding sequence of an active area of a mice source IL-33 to a pcDNA3.1 eukaryotic expression vector. The II-type innate lymphocytes are induced and proliferated from a liver through introduction of the recombinant expression carrier into an animal and over-expression of the mice source IL-33 in the animal, liver tissue lymphocytes are obtained through separation and are marked by using an II-type innate lymphocytes specific antibody, and the II-type innate lymphocytes are obtained through sorting by using a flow cytometry. According to the method, the high-activity II-type innate lymphocytes can be induced, greatly proliferated, separated and purified from C57BL / 6 mice.

Description

technical field [0001] The present invention relates to a method for obtaining type II innate lymphocytes from mice and the carrier used, specifically, the present invention relates to a recombinant expression vector capable of inducing and expanding a large number of type II innate lymphocytes in the liver, And a method for obtaining a large amount of type II innate lymphocytes from a mouse body by using the carrier. Background technique [0002] In recent years, with the in-depth study of innate immune cells, a group of new cell subpopulations have been discovered. This subpopulation does not express antigen-specific recognition receptors (TCR or BCR), belongs to the lymphocyte lineage in development and morphology, and is called together with natural killer (NK) cells and lymphoid tissue-induced (LTi) cells discovered in the past. Innate lymphocytes (ILCs). According to the classification method of helper T (Th) cells, the ILC family can be divided into three categories...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10
CPCC07K14/54C07K2319/02C12N5/0651C12N15/85C12N2510/00C12N2800/107
Inventor 万晓春毕嘉成崔璐璐
Owner SHENZHEN INST OF ADVANCED TECH
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