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Application of signal-regulatory protein alpha (SIRPalpha) in preparation of medicines for preventing and treating allergic diseases

A technology of signal regulation protein and allergic reaction, applied in the field of medical bioengineering, can solve the problems such as the application of SIRPα that has not been reported in the literature

Inactive Publication Date: 2012-05-02
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is currently no literature reporting the application of SIRPα in the preparation of drugs for the prevention and treatment of allergic diseases
There is also no literature report on SIRPα regulating mast cell activation

Method used

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  • Application of signal-regulatory protein alpha (SIRPalpha) in preparation of medicines for preventing and treating allergic diseases
  • Application of signal-regulatory protein alpha (SIRPalpha) in preparation of medicines for preventing and treating allergic diseases
  • Application of signal-regulatory protein alpha (SIRPalpha) in preparation of medicines for preventing and treating allergic diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Gene cloning, interference sequence design and plasmid construction and identification

[0042] 1. Construction of SIRPα expression plasmid

[0043] Material:

[0044] 1) The SIRPα expression vector pcDNA3.1A was purchased from Invitrogen; the interference expression vector pSUPER was purchased from Oligoengine; the interference fragment used for transient transfection was synthesized by Invitrogen.

[0045] 2) The cloning primers are the start and end primer sequences of the mouse SIRPα mRNA open reading frame, which were synthesized by Shanghai Sangong.

[0046] 3) The cloning process uses restriction endonuclease and T4 ligase purchased from NEB Company.

[0047] 4) 293T cell line was purchased from Shanghai Institute of Cellular Sciences, Chinese Academy of Sciences.

[0048] 5) The transfection reagent PEI was purchased from Polyplus-transfection SA.

[0049]6) The mouse-derived cDNA library was constructed by reverse transcription method (see Molecu...

Embodiment 2

[0064] Example 2: Isolation, culture and identification of mast cells

[0065] Material:

[0066] 1) c57BL / 6 mice were purchased from Shanghai Bikai Animal Company.

[0067] 2) IL3 and SCF were purchased from peprotech company, and DMEM medium and antibiotics for culture were purchased from GIBCO company.

[0068] 3) FceRI and c-kit antibodies for identification were purchased from eBioscience.

[0069] method:

[0070] Femur bone marrow of c57 mice aged 6-8 weeks was flushed out with a syringe, and cultured in DMEM medium for 4-8 weeks. Add 10% FBS, 10ng / ml IL3, 20ng / ml SCF to the culture medium and add antibiotics at the same time. The expression of FceRI and c-kit on the cell surface of the cultured mast cells was detected by flow cytometry.

[0071] result:

[0072] like figure 2 As shown, the cultured cells express the mast cell marker molecule FceRI ( figure 2 A) and c-kit ( figure 2 B), the expression cell purity is above 95%.

Embodiment 3

[0073] Example 3: SIRPα regulates mast cell degranulation response and cytokine expression.

[0074] Material:

[0075] 1) Mast cell cell line, mast cell isolated and cultured in primary generation (obtained in Example 2)

[0076] 2) design synthetic interference fragments, interference plasmids and overexpression plasmids (obtained in Example 1)

[0077] 3) Cell model IgE and antigen DNP-BSA used to induce mast cell activation and degranulation were purchased from SIGMA Company.

[0078] 4) F-actin-specific fluorescent probe Alexa 488phalloidin was purchased from Invitogen

[0079] 5) Calcium ion indicator Fluo-3, AM was purchased from Invitogen.

[0080] 6) PCR detection reagents were purchased from TAKARA Company, and primers were synthesized by Shanghai Sangong.

[0081] method:

[0082] 1) Establishment of stable cell lines

[0083] RBL2H3 cells (from ATCC) were transferred to a 6-well plate, and the signal regulatory protein expression plasmid and the control pla...

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Abstract

The invention belongs to the technical field of medical bioengineering. SIRPalpha is a member of an inhibitory receptor protein family. The immunoloregulation effect of a dendritic cell (DC) has been reported, the SIRPalpha can inhibit the DC maturation induced by LPS and the IL-12 secretion of the matured DC, the SIRPalpha on the surface of the DC can bidirectionally and negatively regulate the function of a T cell and the DC through the action of the SIRPalpha with a ligand CD47 on the T cell. No application of the SIRPalpha in the preparation of medicines for preventing and treating allergic diseases is reported in literatures at present. The invention aims to provide a new purpose of the SIRPalpha. SIRPalpha gene is cloned, a plasmid is constructed, and overexpression or the SIRPalpha expression interference can be carried out in the invention. The application of the SIRPalpha in the preparation of the medicines for preventing and treating the allergic diseases is provided in the invention.

Description

technical field [0001] The invention belongs to the technical field of medical bioengineering. Specifically, the present invention relates to the application of a signal regulatory protein α (SIRPα) in the preparation of drugs for the prevention and treatment of allergic diseases. Background technique [0002] Signal-regulatory proteins α (signal-regulatory proteins, SIRPα) is a member of the inhibitory receptor protein family. Rat protein tyrosine phosphatase SIRP α was first identified in MOLECULAR AND CELL BIOLOGY in 1996 and NATURE in 1997. SIRPα, also called SHPS-1, CD172, P84, is a membrane protein mainly expressed in myeloid cells, GeneID: 19261. The structure of SIRPα consists of an outer membrane segment containing 3 immunoglobulin-like structures and an ITIM motif It consists of an intracellular segment and a transmembrane region of 24 amino acids. Due to its special cell location and structure, it determines that it transmits different signals in different cell...

Claims

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Application Information

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IPC IPC(8): A61K48/00C12N15/867C12N15/861A61P37/08
Inventor 王红阳董立巍王敏谈冶雄潘宇飞
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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