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Purpose of Rap1b gene

A gene and drug technology, applied in genetic engineering, microbial determination/inspection, and other methods of inserting foreign genetic materials, etc., can solve the problems of long treatment course, high cost, and insignificant effect.

Inactive Publication Date: 2019-02-01
KUNMING UNIV OF SCI & TECH +1
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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; because it is particularly urgent to find a high-efficiency and low-cost inhibitor that can target the treatment of type I hypersensitivity

Method used

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  • Purpose of Rap1b gene
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  • Purpose of Rap1b gene

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1: Constructing a plasmid capable of editing the target gene and transfecting it into the cells so that it can function in the cells, and then screening the Rap1b knockout cell line for subsequent experiments;

[0049] (1) Plasmid construction

[0050] A. Design and synthesis of gRNA

[0051] To design a pair of gDNA of about 20bp, we can design it through the following online tool: CRISPRDesign from MIT: http: / / crispr.mit.edu / ; Design the gRNA sequence targeting the Rap1b coding gene, as shown in the following table:

[0052] Table 1: gRNA-Rap1b nucleic acid sequence

[0053]

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

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

[0056] ① Dissolve each gRNA to a final concentration of 100 μM, and phosphorylate and anneal the sgRNA according to the following formula;

[0057] Element

Dosage (μL)

sgRNA forward sequence (100μM)

1

sgRNA r...

Embodiment 2

[0095] The proliferation rate of mast cells has a certain influence on the overall degranulation level of cells, and the mast cell sensitizer C48 / 80 can induce mast cell activation and degranulation, and the degree of degranulation is related to the ability of mast cells to accept C48 / 80 sensitization signals certain correlation. In order to verify the effect of Rap1b on the proliferation of mast cells and the acceptance of C48 / 80 signals, we used CCK-8 assay to detect the proliferation rate of P815WT and Rap1b KO cells and the inhibition rate of cell proliferation induced by C48 / 80 (10 μg / mL) ,Specific steps are as follows:

[0096] (1) Culture and passage of mast cell lines P815WT and Rap1b KO cells

[0097] A. Cultivation of P815WT and Rap1b 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;

[0098] B. Passage of P815WT and Rap1b KO cells: When the cell density ...

Embodiment 3

[0110] The degree of degranulation of mast cells directly reflects the state of allergic reactions in the body. The pre-synthesized granule medium in the cells contains substances such as tryptase and β-hexosaminidase, which can be used as markers of mast cell degranulation. In order to verify the effect of Rap1b on mast cell activation and degranulation, toluidine blue staining, ELISA and chromogenic methods were used in this experiment. The specific steps are as follows:

[0111] (1) Culture and passage of mast cell lines P815WT and Rap1b KO cells

[0112] A. Cultivation of P815WT and Rap1b 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;

[0113] B. Passage of P815WT and Rap1b KO cells: When the cell density in step A reaches 80%, it needs to be passaged. Collect the cell culture solution in the culture bottle into a centrifuge tube, rinse with preheated phosphat...

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Abstract

The invention discloses a purpose of a Rap1b gene, which is characterized in that the Rap1b gene is used as a drug target in screening of anti-type I hypersensitivity drugs; by establishing an in-vitro allergy model of P815 mast cells induced by a sensitizer C48 / 80, the knockout of Rap1b has the function of promoting the activation of mast cells, which is characterized in that Rap1b can alleviatea degranulation reaction of mast cells under a sensitized state, inhibits the release of tryptase and beta-hexosaminidase, inhibits the synthesis and secretion of inflammatory factors, and inhibits the expression of inflammatory genes at a mRNA level; knockout of Rap1b activates a MAPK signal pathway and promotes phosphorylation of AKT, which demonstrates that Rap1b inhibits MAPK and AKT signal pathways in intracellular signal pathways, provides a theoretical basis for clinical treatment of type I hypersensitivity reactions, and screens the novel drug targets.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to the screening of type I hypersensitivity target therapy inhibitors caused by mast cell activation, that is, the application of Rap1b gene as a drug target in screening anti-type I hypersensitivity drugs. Background technique [0002] Allergic diseases (allergic diseases) include allergic rhinitis, asthma, conjunctivitis, eczema, food allergy, etc., which are common clinical diseases and frequently-occurring diseases, and have been listed by the World Health Organization (WHO) as the focus 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 total prevale...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883G01N33/68C12N15/90C12N9/22
CPCG01N33/6893C12N9/22C12N15/907C12Q1/6883G01N2800/7095
Inventor 徐天瑞虞姣姣安输郭利群张新宇郝佩琪
Owner KUNMING UNIV OF SCI & TECH
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