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Applications of novel human latent leukocyte differentiation antigen TMIGD2 discovered by utilization of immunomic technology

A protein and antibody technology, applied in the field of use and preparation of polynucleotides and polypeptides, can solve problems such as non-functional reports

Inactive Publication Date: 2013-12-04
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to search for candidate leukocyte differentiation antigens that function in the immune system, Peking University Human Disease Gene Research Center cooperates with the National Human Genome Northern Research Center, using the strategy of immune genomics, with the whole genome as the background, according to the expression and structural characteristics of immune cell membrane molecules , and formulated the following four selection criteria: (1) Highly expressed in immune cells; (2) Characteristic expression profile, that is, specific expression only on certain types of immune cells; (3) Preference for single Transmembrane type I, II, V cell membrane protein; (4) There is no functional report yet

Method used

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  • Applications of novel human latent leukocyte differentiation antigen TMIGD2 discovered by utilization of immunomic technology
  • Applications of novel human latent leukocyte differentiation antigen TMIGD2 discovered by utilization of immunomic technology
  • Applications of novel human latent leukocyte differentiation antigen TMIGD2 discovered by utilization of immunomic technology

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1. Selection of Human Potential Leukocyte Differentiation Antigen TMIGD2

[0039] In order to find candidate immune cell membrane molecules that function in the immune system, Peking University Human Disease Gene Research Center cooperates with the National Human Genome North Research Center, using the strategy of immune genomics, with the whole genome as the background, according to the expression and structural characteristics of immune cell membrane molecules , and formulated the following four selection criteria: (1) Highly expressed in immune cells; (2) Characteristic expression profile, that is, specific expression only on certain types of immune cells; (3) Preference for single Transmembrane type I and type II cell membrane protein; (4) There is no function report yet. To this end, the inventors selected 721_B_lymphoblasts, CD4+T-cells, CD8+T-cells, PB-BDCA4+Dentritic_Cells, PB-CD14+Monocytes, PB-CD19+Bcells, PB-CD56+NKCells, BM-CD33+Myeloid , BM-CD34+, ...

Embodiment 2

[0041] Example 2. Carry out cell expression analysis of human TMIGD2 by RT-PCR method

[0042] First, total cellular RNA was extracted. Harvest 2~10×10 6 For different cells, add appropriate amount of TRIZOL reagent. Incubate at room temperature for 10 minutes to completely separate the nucleoprotein fraction. Add 0.2ml chloroform per ml TRIZOL, vortex for 15 seconds, and place at room temperature for 2-3 minutes. Centrifuge at 12000g for 15 minutes at 4°C. After centrifugation, take the upper aqueous phase and place it in a new EP tube, add 0.5 ml of isopropanol to each ml of TRIZOL, and let it stand at room temperature for 10 minutes. Centrifuge at 12000g for 10 minutes at 4°C. Discard the supernatant and wash with 1 ml of 75% ethanol per ml of TRIZOL. Centrifuge at 7500g for 5 minutes at 4°C. Discard the supernatant and let the precipitated RNA dry naturally at room temperature. Add appropriate amount of DEPC water to dissolve and mix well. Perform RNA quantificati...

Embodiment 3

[0046] Embodiment 3 Human TMIGD2 recombinant expression

[0047] Firstly, the extracellular domain fragment of TMIGD2 was amplified by PCR. Use the primers described in SEQ No.13 and 14 to carry out PCR amplification with pcDB-TMIGD2-myc-his as a template, then use Nco I and Xho I to double-enzyme digest the PCR product and prokaryotic expression vectors pGEX4T-1 and pET -32a-c(+), the TMIGD2 ectoregion fragment after enzyme digestion was connected to two kinds of vectors at 16°C for 12 hours, transformed into E. coli DH5α, the transformant was grown on LA plate medium, the clone was picked, and the plasmid was extracted. PCR identification, selection of positive clones, and sequencing, pGEX4T-1-TMIGD2 is the expression plasmid of the extracellular segment of TMIGD2 with a GST tag at the N-terminus and the signal peptide is removed, and pET-32a-c(+)-TMIGD2 is an expression plasmid with a GST tag at the N-terminus. The expression plasmid of TMIGD2 extracellular segment with Tr...

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Abstract

The invention describes a novel human latent leukocyte differentiation antigen TMIGD2 (Transmembrane and immunoglobulin domain-containing protein 2, TMIGD2). The invention selects the novel human latent leukocyte differentiation antigen TMIGD2 by utilization of an immunomic strategy. The invention discloses a TMIGD2 polypeptide, and applications of the polynucleotide coding sequence of the TMIGD2 polypeptide. The invention proves that the TMIGDW refers to an immune cell-membrane molecule highly-expressed in the human immune system. A function, of the protein molecule, that the protein molecule can inhibit secretion of cytokines and killing molecules of NK cells and can inhibit the killing activity of the NK cells, and that the protein molecule may be a novel immune cell inhibitory receptor are disclosed by the invention. The invention also discloses a cell which is likely to contain a TMIGD2 ligand.

Description

technical field [0001] The present invention relates to a novel human potential leukocyte differentiation antigen TMIGD2. The present invention also relates to the use and preparation of such polynucleotides and polypeptides. TMIGD2 is a novel human potential leukocyte differentiation antigen selected by immunomics strategy. TMIGD2 is highly expressed in the human immune system, expressed in human resting NK cells and up-regulated with the activation of IL-2. Antibody cross-linking can inhibit the secretion of cytokines and killing factors by IL-2-activated NK cells, and inhibit the killing activity of IL-2-activated NK cells on K562 cells. Its ligand may exist on the Hela cell membrane or in its culture supernatant. Background technique [0002] Leukocyte Differentiation Antigen (LDA) mainly refers to the cell surface molecules that appear or disappear during the differentiation and maturation of hematopoietic stem cells into different lineages, different stages of diffe...

Claims

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Application Information

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IPC IPC(8): C07K14/705C12N15/12C12N15/63C12N1/21C12P21/02C07K16/28G01N33/68
Inventor 韩文玲马大龙李婷刘凯王平章
Owner PEKING UNIV
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