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Construction of cell strain containing luciferase reporter gene of human GSTP1 ARE and application thereof

A technology of luciferase and reporter gene, applied to genetically modified cells, cells modified by introducing foreign genetic material, applications, etc., can solve problems such as differences

Active Publication Date: 2021-08-17
SHANGHAI JAHWA UNITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to differences in ARE elements from different gene sources, different cell models will have different predictions (sensitivity, specificity and accuracy) of allergens

Method used

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  • Construction of cell strain containing luciferase reporter gene of human GSTP1 ARE and application thereof
  • Construction of cell strain containing luciferase reporter gene of human GSTP1 ARE and application thereof
  • Construction of cell strain containing luciferase reporter gene of human GSTP1 ARE and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Vector construction, cell line construction and single cell clone purification and identification

[0044] 1. Experimental principle

[0045] The required DNA fragment was synthesized by the whole gene synthesis method and inserted into the reporter vector pGL4.17[luc2 / Neo] to obtain the overexpression vector pGL4.17-hGSTP1 ARE-SV40p-Luc2; it was introduced into HaCaT cells by electroporation, and Neomycin screening was performed to obtain stable overexpression cell lines, single cell clones were selected, and RT-qPCR method was used to detect the overexpression of Luc2 mRNA in the cell clones.

[0046] 2. Experimental method

[0047] 1. Construction of luciferase reporter gene vector pGL4.17-hGSTP1 ARE-SV40p-Luc2

[0048] DNA fragment 1 (SEQ ID NO: 1) was synthesized by the whole gene synthesis method, the sequence from the 5' end to the 3' end is the restriction endonuclease KpnI restriction site GGTACC, the human GSTP1 gene promoter region ARE element, ...

Embodiment 2

[0065] Example 2: Detection of known extremely strong skin sensitizer 2,4-dinitrochlorobenzene by KeraSens cell line 1. Experimental principle

[0066] The Keap1-Nrf2-ARE pathway is the main regulatory pathway of cytoprotective responses to electrophilic and oxidative stress, which can regulate the expression of detoxification, antioxidant and stress response enzymes and proteins. This pathway is involved in the process of skin allergy and can be induced by skin Sensitive substances are activated. Glutathione S-transferase 1 (glutathione S-transferase P1, GSTP1) is a member of the glutathione sulfhydryl transferase (GSTs) family, which catalyzes the interaction of many hydrophobic and electrophilic compounds with reducing gluten Glutathione binding plays an important role in the detoxification process. The GSTP1 gene contains ARE elements, which are regulated by the Keap1-Nrf2-ARE pathway. The KeraSens cell line is a genetically engineered epidermal keratinocyte cell line, w...

Embodiment 3

[0137] Example 3: Detection of known strong skin sensitizer bromothalonil by KeraSens cell line

[0138] 1. Experimental principle

[0139] The Keap1-Nrf2-ARE pathway is the main regulatory pathway of cytoprotective responses to electrophilic and oxidative stress, which can regulate the expression of detoxification, antioxidant and stress response enzymes and proteins. This pathway is involved in the process of skin allergy and can be induced by skin Sensitive substances are activated. Glutathione S-transferase 1 (glutathione S-transferase P1, GSTP1) is a member of the glutathione sulfhydryl transferase (GSTs) family, which catalyzes the interaction of many hydrophobic and electrophilic compounds with reducing gluten Glutathione binding plays an important role in the detoxification process. The GSTP1 gene contains ARE elements, which are regulated by the Keap1-Nrf2-ARE pathway. The KeraSens cell line is a genetically engineered epidermal keratinocyte cell line, which contai...

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Abstract

The invention provides an overexpression vector of a luciferase reporter gene containing human GSTP1ARE. The overexpression vector comprises a nucleotide sequence as shown in SEQ ID NO: 2. The invention also provides a cell strain capable of stably expressing the luciferase reporter gene containing the human GSTP1ARE. The invention further relates to a method for evaluating the skin sensitization risk of a reagent or a product and an application of the cell strain stably expressing the luciferase reporter gene containing the human GSTP1ARE in evaluating the skin sensitization risk of the reagent or the product.

Description

technical field [0001] The invention relates to the field of safety evaluation of cosmetics, and is actually an engineered cell line that can be used to predict and identify skin allergens, specifically an engineered cell line containing a luciferase reporter gene of human GSTP1 ARE, which can be used to predict cosmetics Whether the raw material has a skin sensitization risk. Background technique [0002] Cosmetics are one of the chemicals commonly used by people, and there are many types, such as hair care, skin care, make-up, sunscreen, and freckle removal. Due to product quality problems or improper use, many skin adverse reactions will be caused. Cosmetic contact dermatitis accounts for 70-90% of skin diseases caused by cosmetics. Skin sensitization or allergic contact dermatitis (Allergic Contact Dermatitis, ACD) is an immunogenic skin reaction of the skin to foreign substances, that is, an immune-mediated skin reaction produced by the body after the skin is repeated...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/65C12N15/54C12N5/10C12Q1/02
CPCC12N15/85C12N15/65C12N9/1088C12N5/0629G01N33/5044C12Y205/01018C12N2800/107C12N2510/04
Inventor 陈田殷庆飞
Owner SHANGHAI JAHWA UNITED
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