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Use of L-Plastin gene

A gene and drug technology, applied in the field of screening anti-allergic drugs and inhibitor screening for type I hypersensitivity targeted therapy, can solve the problems of ineffective effect, high cost, and long treatment course

Inactive Publication Date: 2019-01-18
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the clinical treatment of allergic diseases mainly adopts symptomatic treatment, such as the use of antihistamine drugs and hormones, and allergen-specific immunotherapy (Allergen-specific immunotherapy). Long time, high cost, and sometimes the effect is not obvious

Method used

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  • Use of L-Plastin gene
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  • Use of L-Plastin gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Constructing a plasmid capable of editing the target gene and transfecting it into cells so that it can function in the cell, and then screening out the cell line with L-Plastin knockout for subsequent experiments

[0050] (1) Plasmid construction

[0051] A. Design and synthesis of gRNA

[0052] To design a pair of gDNA of about 20bp, we can design it through the following online tools: CRISPRDesign of MIT: http: / / crispr.mit.edu / ; design the gRNA sequence targeting the gene encoding L-Plastin, as shown in the following table Show:

[0053]

[0054] Note: The lowercase part is the nucleic acid sequence connected to the plasmid;

[0055] B. Cloning sgRNA into pSpCas9(BB) plasmid

[0056] Dissolve each gRNA to a final concentration of 100 μM, and phosphorylate and anneal the sgRNA according to the following recipe;

[0057] Place the above mixture in a PCR machine, and use the following program to phosphorylate and anneal sgRNA: 37°C for 30 minutes...

Embodiment 2

[0093] Example 2: Mast cell sensitizer C48 / 80 can induce mast cell activation and degranulation, and the degree of degranulation has a certain correlation with the ability of mast cells to accept C48 / 80 sensitization signals

[0094] In order to verify the effect of L-Plastin on mast cells accepting C48 / 80 signal, we used CCK-8 assay to detect the cell proliferation inhibition rate of P815 WT and L-Plastin KO cells induced by C48 / 80 (10 μg / mL), Specific steps are as follows:

[0095] (1) Culture and passage of mast cell lines P815 WT and L-Plastin KO cells

[0096] A. Cultivation of P815 WT and L-Plastin KO cells: P815 was cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS), 1% penicillin and streptomycin at 37°C and 5% CO 2 Environment;

[0097] B. Passaging of P815 WT and L-Plastin KO cells: When the cell density reaches 80% in step A, passaging is required. Collect the cell culture solution in the culture flask into a centrifuge tube and rinse wit...

Embodiment 3

[0105] Experimental purpose and method: The degree of degranulation of mast cells directly reflects the condition of the body's allergic reaction. The pre-synthesized granule medium in the cells contains substances such as histamine and β-hexosaminidase, which can act as mast cell degranulation. Particle markers. In order to verify the effect of L-Plastin on mast cell activation and degranulation, this experiment uses toluidine blue staining method, ELISA method and chromogenic method for detection. The specific steps are as follows:

[0106] (1) Culture and passage of mast cell lines P815 WT and L-Plastin KO cells

[0107] A. The culture of P815 WT and L-Plastin KO cells: P815 was cultured in high-glucose DMEM medium, containing 10% fetal bovine serum (FBS), 1% penicillin and streptomycin, cultured at 37°C, 5% CO 2 Environment;

[0108] B. Passaging of P815 WT and L-Plastin KO cells: When the cell density reaches 80% in step A, passaging is required. Collect the cell cultur...

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Abstract

The invention discloses the use of an L-Plastin gene, which is an application of the L-Plastin gene as a drug target in screening a type I hypersensitivity targeted therapeutic inhibitor, and the invention is induced by constructing a sensitizer C48 / 80 The P815 mast cell in vitro allergy model found that L-Plastin knockdown has the effect of inhibiting mast cell activation, can effectively inhibitthe degree of degranulation of mast cells in sensitized state, and relieve the release of histamine and beta-hexosaminidase Represses the synthesis and secretion of inflammatory factors; inhibits theexpression of inflammatory genes and the expression of key receptors in the mast cell activation pathway, and significantly inhibits the RAF-MEK-ERK signaling pathway and inhibits AKT in the intracellular signaling pathway. Phosphorylation levels have been shown to have anti-allergic effects at the cellular level, providing a theoretical basis for clinical treatment of type I hypersensitivity reactions and the application of screening for new drug targets.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to screening inhibitors for type I hypersensitivity targeted therapy caused by activation of mast cells, using L-Plastin gene as a drug target to screen antiallergic drugs. Background technique [0002] Allergic diseases (allergic diseases) include allergic rhinitis, asthma, conjunctivitis, eczema, food allergies, etc., which are common clinical diseases and frequently-occurring diseases, and have been listed by the World Health Organization (WHO) as the priority of the 21st world One of the three major diseases to be prevented and controlled is a major health problem in the world. [0003] According to the statistics of the World Allergy Organization (WAO), the incidence of allergic diseases has increased by at least 4-5 times in the past 30 years, and is increasing at a rate of more than 1% per year. At present, the global prevalence rate has reached 22%. It is est...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/113
CPCC12N15/113C12Q1/6883C12N2310/10
Inventor 安输虞姣姣徐天瑞郭利群张新宇郝佩琪
Owner KUNMING UNIV OF SCI & TECH
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