CN113265423B

CN113265423B - Construction and application of cell strain containing luciferase reporter gene of human GSTP1ARE

Info

Publication number: CN113265423B
Application number: CN202110569980.7A
Authority: CN
Inventors: 陈�田; 殷庆飞
Original assignee: Shanghai Jahwa United Co Ltd
Current assignee: Shanghai Jahwa United Co Ltd
Priority date: 2021-05-25
Filing date: 2021-05-25
Publication date: 2022-08-23
Anticipated expiration: 2041-05-25
Also published as: CN113265423A

Abstract

The invention provides an overexpression vector of a luciferase reporter gene containing human GSTP1ARE, which comprises a nucleotide sequence shown in SEQ ID NO. 2. The invention also provides a cell strain for stably expressing the luciferase reporter gene containing the human GSTP1 ARE. The invention also relates to a method for evaluating the skin sensitization risk of a reagent or a product and application of a 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

Construction and application of cell strain containing luciferase reporter gene of human GSTP1ARE

Technical Field

The invention relates to the field of cosmetic safety evaluation, in particular to an engineering cell strain which can be used for predicting and identifying skin sensitizers, in particular to an engineering cell strain containing a luciferase reporter gene of human GSTP1ARE and can be used for predicting whether cosmetic raw materials have skin sensitization risks.

Background

Cosmetics are one of the chemicals commonly used by people, and are various, such as hair cosmetics, skin care cosmetics, color cosmetics, sun protection cosmetics, freckle removal cosmetics and the like. Many adverse reactions to the skin can be caused by the quality problems or improper use of the product. In cosmetic-induced skin diseases, cosmetic contact dermatitis accounts for 70-90%. Cutaneous sensitization or Allergic Contact Dermatitis (ACD) is an immunogenic skin reaction of the skin to foreign substances, i.e. an immune-mediated skin reaction of the body after repeated exposure of the skin to a substance. This response in humans may be characterized by itching, erythema, papules, edema, blisters, fusional blisters. Allergens in cosmetics are commonly found in perfume essences, preservatives, as well as antioxidants, emulsifiers, carriers, certain sunscreens, and the like. Therefore, in order to maintain the health of cosmetic consumers and reduce the adverse reactions of cosmetics to skin, the detection of skin sensitization of cosmetic raw materials and products is very important.

Currently, methods for detecting substance sensitization include human testing, animal testing, and in vitro substitution methods. Ethical and cost cycle issues due to human trials, and cost and welfare issues for animal trialsScientists and industry researchers have been working on finding alternative methods for skin sensitization in vitro. Skin sensitization is a very complex biological process, so Economic collaboration and Development Organization (OECD) released alternative experimental guideline documents in 2012, and established a harmful Outcome Pathway (AOP) -based skin sensitization strategy by integrating several different in vitro approaches. The AOP concept is used to describe the sequence of events that occur from a molecular initiation event to the process of generation of a systematic effect. For the skin sensitization process, AOP includes four key events: the molecular initiation event (KE1) is the covalent binding of an electrophilic substance to a nucleophilic center in a skin protein, and is expressed at the molecular level, represented by Direct Peptide binding Assay (DPRA) (OECD document No. 442C); the second key event (KE2) is the activation of keratinocytes, expressed at the cellular level, by keratinocytes ARE-Nrf2[ Antioxidant response Element (Antioxidant reactive Element), Nuclear Factor NF-E2-related Factor (Nuclear Factor Erythroid2-related Factor 2, Nrf2)]Luciferase assay KeratinoSens ^TM And LuSens as representative (OECD file number 442D); the third key event (KE3) is the Activation of Dendritic Cells (DCs), which are expressed at the cellular level by Human Cell Line Activation Test (h-CLAT), Human tissue lymphoma Cell Activation Test (U937 Skin sensitivity Test, U-SENS) ^TM ) And interleukin-8 reporter assay (IL-8Luc) (OECD document No. 442E); the fourth key event (KE4) is T cell activation and proliferation, and is systemic expression at the tissue-organ level, represented by the mouse Local Lymph Node Assay (LLNA) (OECD document No. 429).

In the second key event, the principle of the test for keratinocyte activation is based on the fact that sensitizers can alter gene expression in keratinocytes. OECD certified KeratinoSens ^TM And the LuSens assay is based primarily on Keap1-Nrf2-ARE pathway activation in keratinocytes with upregulation of gene expression downstream of the ARE element. Therefore, luciferase gene was introduced by genetic engineering technique(Luc2) under the control of a promoter containing an ARE element allows assessment of the risk of potential skin sensitization of the test substance by detecting changes in luciferase activity. Such as KeratinoSens ^TM The cell model utilizes the ARE element on the human AKR1C2 gene promoter, and the LuSens cell model utilizes the ARE element on the rat NQO1 gene promoter. Due to the differences in the ARE elements from different genes, different cell models will differ in their sensitivity, specificity and accuracy for sensitizer prediction. For example, KeratinoSens compares known human body sensitization data ^TM Sensitivity, specificity and accuracy of 82%, 84% and 82% (102 species), respectively, and sensitivity, specificity and accuracy of LuSens of 78%, 79% and 79% (60 species), respectively (ref: Urbishch D, Mehling A, Guth K, Ramirez T, Honarrvar N, Kolle S, Landsiedel R et al (2015) assessment skin sensitivity in mice and men using non-animal test methods Regul toxin Pharmacol 71(2): 337-351).

At present, the Keap1-Nrf2-ARE pathway is a main regulation pathway of cell protection response to electrophilic and oxidative stress, can regulate and control the expression of detoxification, antioxidation and stress reaction enzymes and proteins, is involved in skin allergy process, and can be activated by skin sensitizers. Glutathione S-transferase 1(glutathione S-transferase P1, GSTP1) is one of glutathione mercaptotransferases (GSTs) family members that play an important role in the detoxification process by catalyzing the binding of a number of hydrophobic and electrophilic compounds to reducing glutathione. The GSTP1 gene contains ARE elements and is regulated by a Keap1-Nrf2-ARE pathway. Therefore, based on the GSTP1ARE element and luciferase, the invention unexpectedly discovers a cell model which can be used for sensitizer prediction, namely a human skin sensitizer prediction cell strain HaCaT-hGSTP1ARE-SV40p-Luc2 (also called Kerasens cell strain), and verifies that the cell model has the capability of recognizing a very strong sensitizer to a weak sensitizer and can recognize and distinguish substances without skin sensitization.

From the perspective of safe use of consumers of cosmetics, the invention constructs an engineering cell strain containing a luciferase reporter gene of a human GSTP1ARE element, namely a human skin sensitizer prediction cell strain HaCaT-hGSTP1ARE-SV40p-Luc2 (also called Kerasens cell strain), on the basis of regulation and control of the gene of a keratinocyte by an sensitizer, and the cell strain can be used for predicting whether cosmetic raw materials have skin sensitization risks or not, reducing adverse reactions of the cosmetics and having higher application value.

Disclosure of Invention

In one aspect, the invention provides an overexpression vector of a luciferase reporter gene containing human GSTP1ARE, comprising the nucleotide sequence shown in SEQ ID NO. 2 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO. 2.

On the other hand, the invention provides a cell strain for stably expressing a luciferase reporter gene containing human GSTP1ARE, the name of the cell strain is human skin sensitizer prediction cell strain HaCaT-hGSTP1ARE-SV40p-Luc2, the cell strain is preserved in China center for type culture Collection in Wuhan, China, and the preservation number is CCTCC NO: c2021113, preservation date 2021, 5 months and 14 days.

In a further aspect, the present invention provides a method of assessing the risk of skin sensitization of an agent or product, the method comprising the steps of:

(a) constructing an overexpression vector pGL4.17-hGSTP1ARE-SV40 p-Luc2(SEQ ID NO: 2);

(b) introducing an overexpression vector pGL4.17-hGSTP1ARE-SV40 p-Luc2(SEQ ID NO:2) into HaCaT cells to construct a cell strain containing a luciferase reporter gene of human GSTP1 ARE;

(c) the cell strain is adopted to detect the skin sensitization risk of the reagent or the product.

In a preferred embodiment, said step (b) comprises screening with neomycin to obtain a cell line stably expressing a luciferase reporter gene containing human GSTP1 ARE. In a preferred embodiment, the method further comprises screening cell lines after step (b) for sensitivity to the risk of skin sensitization of the agent or product. In a preferred embodiment, the agent or product to be detected is selected from: cosmetics or cosmetic raw materials. In a more preferred embodiment, the agent or product is selected from: cinnamaldehyde, 2, 4-dinitrochlorobenzene, bromothalonil, 2-mercaptobenzothiazole, cinnamyl alcohol, salicylic acid, N-dimethyl-1, 3-diaminopropane, or a combination thereof.

In still another aspect, the invention provides application of a human skin sensitizer predicted cell strain HaCaT-hGSTP1ARE-SV40p-Luc2 (preserved in China center for type culture Collection in Wuhan, China with the preservation number of CCTCC NO: C2021113 and the preservation date: 2021, 05 and 14 days) in evaluating skin sensitization risk of reagents or products. In a preferred embodiment, the reagent or product to be detected is selected from: cosmetics or cosmetic raw materials. In a more preferred embodiment, the agent or product is selected from: cinnamaldehyde, 2, 4-dinitrochlorobenzene, bromothalonil, 2-mercaptobenzothiazole, cinnamyl alcohol, salicylic acid, N-dimethyl-1, 3-diaminopropane, or a combination thereof.

Drawings

FIG. 1 shows a map of a skin sensitization prediction-associated reporter vector pGL4.17-hGSTP1ARE-SV40p-Luc 2.

FIG. 2 shows the comparison of background expression levels of Luc2mRNA of single cell clones related to the prediction of skin sensitization of multiple strains.

FIG. 3 shows a comparison of luciferase activity in single cell clones relevant to multiple strains of skin sensitization predictions under 32 μ M cinnamaldehyde-induced conditions.

FIG. 4 shows a comparison of sensitivity of relevant single-cell clones to cinnamaldehyde for multiple strains of skin sensitization predictions.

FIG. 5 shows the results of the Kerasens cell line testing for the very strong sensitizer 2, 4-dinitrochlorobenzene (luciferase activity induction and cell viability).

FIG. 6 shows the results of the Kerasens cell line testing for the strong sensitizer bromothalonil (luciferase activity induction and cell viability).

FIG. 7 shows the results of sensitizer 2-mercaptobenzothiazole (luciferase activity induction and cell viability) in the Kerasens cell line assay.

FIG. 8 shows the results of the Kerasens cell line testing for the weak sensitizer cinnamyl alcohol (luciferase activity induction and cell viability).

FIG. 9 shows the results of the Kerasens cell line testing for the non-sensitizer salicylic acid (luciferase activity induction and cell viability).

FIG. 10 shows the results of the Kerasens cell line assay for N, N-dimethyl-1, 3-diaminopropane (luciferase activity induction and cell viability).

FIG. 11 shows the effect of initial inoculum size of Kerasens cells on luciferase induction.

FIG. 12 shows the effect of Kerasens cell priming on cell viability.

FIG. 13 shows the nucleic acid sequences of DNA fragment 1 and DNA fragment 2.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill and research in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. For the purposes of the present invention, the following terms are defined.

According to the present invention, the term "cosmetic" refers to a chemical or fine chemical product that is applied by painting, spraying or the like to any part of the surface of the human body, such as skin, hair, nails, lips, etc., for the purpose of cleaning, maintaining, beautifying, modifying and changing the appearance, or for the purpose of correcting the odor of the human body and maintaining a good condition.

According to the present invention, the term "cosmetic raw material" refers to raw materials used in the production process of cosmetics, including cosmetic base materials such as oily materials, surfactants, solvents, powdery materials, high molecular polymers, and other additives; cosmetic auxiliary materials such as essence, colorant, pearling agent, antiseptic, bactericide, antioxidant and complexing agent; and special purpose raw materials, such as depilatory, hair dye, permanent wave agent, astringent, antiperspirant, deodorant, speckle removing agent, sunscreen agent, etc.

The present invention is based on the following findings: the Keap1-Nrf2-ARE pathway is a main regulation pathway of cell protection reaction to electrophilic and oxidative stress, can regulate and control the expression of detoxification, antioxidation and stress reaction enzymes and proteins, participates in the skin allergy process, and can be activated by skin sensitizers. Glutathione S-transferase P1(glutathione S-transferase P1, GSTP1) is one of glutathione mercaptotransferases (GSTs) family members that play an important role in the detoxification process by catalyzing the binding of a number of hydrophobic and electrophilic compounds to reducing glutathione. The human GSTP1 gene contains ARE elements, and the expression of the ARE elements is regulated by a Keap1-Nrf2-ARE pathway. Luciferase gene (Luc2) is a common reporter gene used in scientific research, and a GSTP1ARE element and an SV40 promoter (SV40p) ARE inserted at the upstream of the luciferase gene, and the GSTP1ARE element, the SV40 promoter and the luciferase gene (Luc2) ARE stably integrated in a keratinocyte genome to form a genetically engineered cell strain, namely a human skin sensitizer predictive cell strain HaCaT-hGSTP1ARE-SV40p-Luc2 (also called Kerases cell strain). Therefore, whether the Keap1-Nrf2-ARE pathway is activated by a skin sensitizer can be monitored by detecting the expression condition of the luciferase of the Kerasens cell, and the Kerases cell can be further used for evaluating whether a detection substance has potential skin sensitization risk.

The invention is briefly carried out as follows (examples 1 to 10): firstly, synthesizing a DNA fragment containing a GSTP1ARE element, an SV40 promoter and a luciferase gene (Luc2) by a gene total synthesis method, constructing a report vector pGL4.17-hGSTP1ARE-SV40p-Luc2, introducing the report vector into keratinocytes, and screening to obtain a genetically engineered cell strain, namely a Kerasens cell strain. Secondly, the skin sensitizer predicting capability of the Kerasens cell strain is evaluated by detecting a series of skin sensitizers or non-sensitizers with different strengths, such as 2, 4-dinitrochlorobenzene, bromothalonil, 2-mercaptobenzothiazole, cinnamyl alcohol, salicylic acid, N-dimethyl-1, 3-diaminopropane and the like, and the Kerasens cell strain is proved to be used for evaluating and predicting whether skin sensitization risks exist in cosmetic raw materials.

Meanwhile, as comparative example 1, the influence of different cell initial inoculum sizes of the Kerasens cell line on luciferase induction and the influence of cell survival rate are compared, and an appropriate cell initial inoculum size is determined, so that a stable test is provided for predicting and evaluating skin sensitizersAnd (4) preparing the system. For example, in a preferred embodiment, the initial inoculum size of cells is 1.0-2.0X10 ⁴ And each cell/hole is beneficial to the stability and repeatability of the experiment.

In addition, as comparative example 2, the known skin sensitizer, N-dimethyl-1, 3-diaminopropane, was evaluated by direct polypeptide binding assay (DPRA) for correct prediction of sensitizer, N-dimethyl-1, 3-diaminopropane, by the Kerasens cell line in comparative example 7.

In conclusion, the invention provides a human skin sensitizer prediction cell strain HaCaT-hGSTP1ARE-SV40p-Luc2 (also called Kerasens cell strain) which has high application value, strong sensitivity and is based on the luciferase reporter gene of a human GSTP1ARE element, can be used for predicting whether cosmetic raw materials have skin sensitization risk, and has high application value.

The present invention will be further illustrated below with reference to specific examples and comparative examples. However, these examples and comparative examples are only illustrative of the present invention and are not to be construed as limiting the scope of the present invention. The experimental procedures of the following examples and comparative examples, in which specific conditions are not specified, are generally conducted under conventional conditions or conditions recommended by the manufacturers.

The reagents, consumables, instruments and vendor information used in the following examples and comparative examples are as follows:

1. cells, consumables and reagents

Human immortalized keratinocytes HaCaT were purchased from shanghai yan biotechnology limited; t75 cell culture flasks and 96-well plates were purchased from Corning, USA. Fetal bovine serum, cell culture medium high-glucose DMEM, antibiotic cocktail (PSN), 0.25% trypsin-EDTA were purchased from thermo fisher, usa.

2, 4-dinitrochlorobenzene, bromothalonil, 2-mercaptobenzothiazole, cinnamyl alcohol, salicylic acid, N-dimethyl-1, 3-diaminopropane were purchased from Shanghai Allantin Biotech Ltd. Trizol was a reagent for total RNA extraction from ThermoFisher, USA; reverse transcription Kit iScript cDNA Synthesis Kit and fluorescent real-time quantitative PCR reagent iTaq ^TM Universal

Green Supermix was purchased from Bio-rad, USA; the luciferase Assay reagent Britelite plus Reporter Gene Assay System was purchased from Perkin Elmer, Inc., USA.

2. Instrument

The ultramicro ultraviolet spectrophotometry NanoDrop ONE and a fluorescence microplate reader Fluoroskan Ascent FL are purchased from Thermo corporation, the gene amplification instrument C1000 Touch and a real-time quantitative PCR instrument CFX Connect are purchased from Bio-rad corporation, and the multifunctional microplate reader VICTOR Nivo is purchased from Perkin Elmer GmbH.

Example 1: vector construction, cell strain construction and single cell cloning, purifying and identifying

First, experiment principle

Synthesizing a required DNA fragment by a whole gene synthesis method, and inserting the DNA fragment into a report vector pGL4.17[ Luc2/Neo ] to obtain an overexpression vector pGL4.17-hGSTP1ARE-SV40p-Luc 2; introducing HaCaT cells by an electroporation method, screening neomycin to obtain a stable over-expression cell strain, selecting a cell monoclonal, and detecting the over-expression condition of Luc2mRNA in the cell clone by an RT-qPCR method.

Second, Experimental methods

1. Construction of luciferase reporter Gene vector pGL4.17-hGSTP1ARE-SV40p-Luc2

A DNA fragment 1(SEQ ID NO:1) is synthesized by a whole gene synthesis method, and the sequence sequentially comprises a restriction enzyme KpnI restriction site GGTACC, an ARE element of a human GSTP1 gene promoter region, a restriction enzyme BglII restriction site AGATCT, an SV40 promoter (SV40 promoter, SV40p) sequence and a restriction enzyme HindIII restriction site AAGCTT from the 5 'end to the 3' end.

The DNA fragment 1(SEQ ID NO:1) and pGL4.17[ Luc2/Neo ] vectors were digested with restriction endonucleases KpnI and HindIII, the DNA fragment 1(SEQ ID NO:1) and the linear vector pGL4.17[ Luc2/Neo ] were recovered from agarose gel, and the DNA fragment 1(SEQ ID NO:1) was ligated with T4 DNA ligase to the linear vector pGL4.17[ Luc2/Neo ] to obtain a luciferase reporter gene vector pGL4.17-GSTP 1ARE-SV40 p-Luc2(DNA fragment 2, SEQ ID NO:2) containing a human GSTP1ARE element.

2. Construction of luciferase reporter Gene cell line containing human GSTP1ARE element

The gene vector pGL4.17-hGSTP1ARE-SV40p-Luc2 is introduced into HaCaT cells by an electroporation method, and after 48 hours, the cells ARE continuously screened and cultured for 14 days by using a culture medium containing 0.5mg/mL neomycin, and finally, the luciferase reporter gene cell strain which stably expresses the human GSTP1ARE element is obtained.

3. Detecting the overexpression condition of luciferase reporter gene cell line Luc2mRNA

In order to obtain a purified luciferase reporter gene cell strain, a series of single cell-derived clones are obtained by adopting a low-density long-term culture method.

Collecting 6 cells of the monoclonal derived cell line, extracting total RNA by using a TRIzol reagent, and carrying out reverse transcription by using a reverse transcription Kit iScript cDNA Synthesis Kit to obtain cDNA.

Real-time quantitative PCR reagent iTaq using fluorescence ^TM Universal

And performing fluorescence real-time quantitative (RT-qPCR) detection on Green Supermix, and analyzing the overexpression condition of Luc2mRNA in the luciferase reporter gene cell strain.

The primer sequences used in this example were designed as follows: the sequence of the Luc2 gene upstream primer is 5'-GTGGTGTGCAGCGAGAATAG-3', and the sequence of the downstream primer is 5'-CGCTCGTTGTAGATGTCGTTAG-3'; ACTIN gene (. beta. -ACTIN) was used as an internal control, the sequence of the upstream primer was 5'-CACCATTGGCAATGAGCGGTTC-3', and the sequence of the downstream primer was 5'-AGGTCTTTGCGGATGTCCACGT-3'.

4. Testing luciferase response sensitivity of different clones with cinnamaldehyde

Different monoclonal cell line cells were seeded in opaque 96-well white plates at 1 × 10 per well ⁴ Each monoclonal cell line was inoculated into 18 wells and cultured in a 37-degree incubator for 24 hours.

Cells were then treated with media containing final concentrations of 4 μ M, 8 μ M, 16 μ M, 32 μ M and 64 μ M cinnamaldehyde for 48 hours while DMSO control wells were set for 6 different treatment conditions, 3 replicate wells for each condition.

Luciferase activity is detected by a luciferase detection kit and a multifunctional microplate reader, and fluorescence signal data is collected and analyzed.

Third, experimental results

1. The luciferase reporter gene vector pGL4.17-hGSTP1ARE-SV40p-Luc2 was successfully constructed, and the vector map is shown in FIG. 1.

2. As shown in FIG. 2, Luc2mRNA was overexpressed in the different monoclonal cell lines. GSTP1-6 with the lowest expression level is taken as a control, and Luc2mRNA relative expression levels of the other 5 single cell clones GSTP1-3, GSTP1-7, GSTP1-10, GSTP1-11 and GSTP1-12 are 51.5 times, 26.8 times, 74.8 times, 192.5 times and 646.7 times of GSTP1-6 single cell strain control in sequence (see figure 2).

3. As shown in FIGS. 3-4, the clones treated with cinnamaldehyde except for the monoclonal GSTP1-3 all showed a dose-effect relationship. From the dose-response curve, a more responsive monoclonal cell line GSTP1-10 (hereinafter referred to as Kerasens cell line) was selected for further testing of skin sensitizers and non-skin sensitizers of varying strengths.

Example 2: kerasens cell strain for detecting known extremely strong skin sensitizer 2, 4-dinitrochlorobenzene I and experimental principle

The Keap1-Nrf2-ARE pathway is a main regulation pathway of cell protection reaction to electrophilic and oxidative stress, can regulate and control the expression of detoxification, antioxidation and stress reaction enzymes and proteins, participates in the skin allergy process, and can be activated by skin sensitizers. Glutathione S-transferase 1(glutathione S-transferase P1, GSTP1) is one of glutathione mercaptotransferases (GSTs) family members that play an important role in the detoxification process by catalyzing the binding of a number of hydrophobic and electrophilic compounds to reducing glutathione. The GSTP1 gene contains ARE elements and is regulated by a Keap1-Nrf2-ARE pathway. The Kerasens cell strain is a genetically engineered epidermal keratinocyte cell strain, contains a luciferase gene Luc2 regulated by a GSTP1-ARE element, can monitor the activation condition of a Keap1-Nrf2-ARE channel in cells by detecting the luciferase induction condition, and can be further used for evaluating whether a detection substance has potential skin sensitization risk.

Second, Experimental methods

Kerasens cell culture and passage

Complete medium for culture and passage: DMEM low carbohydrate (Cat:11995, Gibco) + 10% FBS + 1% Triantibody (PSN) + 500. mu.g/mL Geneticin (G-418).

Cell passage: the fusion degree is 80-90%, and the cells can be passed through the culture medium without being too dense. And (4) carrying out passage every 2-4 days, wherein the passage ratio is 1:2-1: 6.1 flask T75 was removed, rinsed 2 times with 5mL of 1 XPBS, added 1mL of 0.25% pancreatin-EDTA and digested at 37 ℃ for 5-10 minutes. Digestion was stopped with 4mL of complete medium, pipetted evenly, cells were collected into a 15mL centrifuge tube and centrifuged at 2000rpm for 4 minutes. The old medium was aspirated off, 5mL of fresh medium was added for resuspension, and the cell suspension was taken in the new flask T75 in the proportions required for passage and supplemented to 15mL with complete medium.

The culture conditions are as follows: 5% CO2, and culturing in a constant temperature incubator at 37 ℃.

Kerasens cell inoculation

Culture medium for inoculation: DMEM low carbohydrate (Cat:11995, Gibco) + 10% FBS + 1% Triantibody (PSN), 200. mu.L per well.

Cultured Kerasens cells were removed, rinsed 2 times with 5mL of 1 XPBS, added 1mL of 0.25% pancreatin-EDTA, and digested at 37 ℃ for 5-10 minutes. Digestion was stopped with 4mL of complete medium, pipetted evenly, cells were collected into a 15mL centrifuge tube and centrifuged at 2000rpm for 4 minutes. Old medium was aspirated off, 5mL of fresh medium was added and resuspended, then 10. mu.L of cell suspension was taken and counted on a hemocytometer.

96-well plates were seeded 1.5X10 per well ⁴ The KeraSens cells, 96-well white plate, were seeded in 47 wells for luciferase assay, 36 wells for sample testing (12 test concentrations, 3 replicates per concentration), 6 wells for solvent control, 5 wells for positive control (5 test concentrations, 1 replicate per concentration); in addition, a 96-well transparent plate was seeded with 23 wells, 12 wells of test sample (12 test concentrations, 1 replicate per concentration), 6 wells as solvent control, and 5 wells as positive control (12 test concentrations, 1 replicate per concentration). Calculating the total number of cells for inoculation according to the number of the inoculated cells, and thinningCell suspension density adjusted to 7.5x10 ⁴ one/mL, 0.2mL of cell suspension was inoculated per well and then placed in a cell incubator for further 24 hours.

3. Reagent configuration

Treatment medium: DMEM low carbohydrate (Cat:11995, Gibco) + 1% FBS + 1% Triantibody (PSN), 200. mu.L per well.

3.1 solvent control: 1% DMSO (CAS No.67-68-5)

A4% DMSO solution (v/v) was prepared with 1% FBS-containing medium, and 40. mu.L of DMSO was added to 960. mu.L of the medium. When loading, 50 μ L of each well (150 μ L of culture medium in the well), the number and configuration volume of the required solvent control wells need to be calculated in advance, and there are at least 6 wells of solvent control in each 96-well white plate.

3.2 Positive control: trans-cinnamaldehyde (CAS No.14371-10-9) was set at 5 concentrations, 4-64. mu.M, 2-fold gradient.

a. Preparing 64mM cinnamaldehyde mother liquor A: molecular weight 132.16, density 1.046-1.052, 2. mu.L into 248. mu.L DMSO, and mixing.

b. Preparing 6.4mM cinnamaldehyde mother liquor B: mu.L of stock A was added to 180. mu.L of DMSO and then diluted in a 1:2 gradient to give 4 additional concentrations of stock 3.2mM, 1.6mM, 0.8mM, 0.4mM, each of at least 100. mu.L.

c. Preparing cinnamaldehyde working solution with each concentration: diluting 25 times of the mother liquor B with a 1% FBS-containing culture medium, namely adding 10. mu.L of the mother liquor B into 240. mu.L of the culture medium, and uniformly mixing for later use. When the sample was loaded, 50. mu.L of the culture medium was added to each well (150. mu.L of the culture medium was added to each well). The number of positive control holes and the configuration volume are calculated in advance, and each concentration of white board positive control with at least 1 hole positive control is calculated every 96 holes.

3.3 sample 2, 4-dinitrochlorobenzene preparation:

a. preparing 200mM of sample mother liquor A: a certain mass (about 20-40mg) of 2, 4-dinitrochlorobenzene was added to a 15mL centrifuge tube or a 1.5mL EP tube, and dissolved and mixed with a solvent DMSO to a final concentration of 200 mM.

Note: volume of DMSO, V ═ 5wp/MW-w/1000

W is the mg of material weighed, p is the purity of the material (in decimal), and MW is the molecular weight.

b. Preparing mother liquor B of each concentration of the sample: the mother liquor A was used as the highest concentration, and 100. mu.L of high concentration mother liquor B was added to 100. mu.L of DMSO to perform gradient dilution to obtain another 11 concentration mother liquors B, and finally 100. mu.L of each concentration mother liquor B (the lowest concentration was 200. mu.L) was obtained.

c. Preparing working solution of samples with various concentrations: diluting 25 times of the mother liquor with a culture medium containing 1% FBS, namely adding 10 μ L of the mother liquor into 240 μ L of the culture medium, and uniformly mixing for later use. Standing at room temperature for later use.

Note: the 12 final concentration gradients were 0.98/1.95/3.9/7.8/15.6/31.25/62.5/125/250/500/1000/2000. mu.M from low to high.

4. Sample processing cells

4.1 after 24 hours of seeded cell culture, the old medium was removed and the medium containing 1% FBS for treatment was replaced with 150. mu.L of each well.

4.2 Add the prepared solvent control, positive control and sample to a 96-well white plate and a 96-well transparent plate with a line gun, 50. mu.L per well. Solvent control group 6 duplicate wells, positive control 1 well each concentration, sample group 3 duplicate wells each concentration. Placing the mixture in an incubator for culturing for 48 hours.

5. Cell viability assay

5.1 calculating the number of the holes required to carry out toxicity test, configuring the volume for CCK-8 working fluid, and setting at least 1 hole in a blank hole.

5.2. Old medium was removed from the 96-well transparent plate and CCK-8 working solution was added by a row gun at 100. mu.L per well.

5.3 placing into a 37 degree incubator, and incubating for 2 hours (0.5-4 hours can be adjusted according to the situation).

5.4 after the incubation, the transparent plate was taken out, the plate cover was removed, and the OD was measured on a microplate reader at a wavelength of 450 nm.

5.5 after the test, data were imported into excel and cell viability, IC30 and IC50 were analyzed computationally.

And (3) activity calculation:

cell viability (%) ═ a _{Sample (I)} -A _{Blank space} ]/[A _Solvent(s) -A _{Blank space} ]×100

A _{Sample (I)} : absorbance of the well with cells, CCK-8 solution and sample;

A _solvent(s) : absorbance of wells with cells, CCK-8 solution and sample with or without solvent;

A _{blank space} : absorbance of wells with medium and CCK solution without cells.

ICx＝(Cb-Ca)*[(100-x)-Va]/(Vb-Va)+Ca

_X : percent inhibition to be calculated (%, e.g. IC30 or IC50)

Ca: minimum concentration (μ M) at which inhibition is greater than x%;

cb: maximum concentration (μ M) at which inhibition is less than x%;

va: cell viability (%) at the lowest concentration at which inhibition is greater than x%;

vb: the inhibition rate was less than the cell viability (%) at the highest concentration of x%.

6. Luciferase activity assay:

6.1 Add 100. mu.L luciferase reagent per well to make up the required amount of reagent.

6.2 remove 96 hole white board, remove old medium, use the row gun to add 100 u L1 xPBS, shake slightly manually, then remove DPBS, add luciferase reagent rapidly, each 100 u L.

6.3 detection in a fluorescent microplate reader, the LUM mode being chosen.

6.4 after the test is finished, data are imported into excel, and the induction times and EC1.5 are calculated and analyzed.

Multiple of induction ═ L _{Sample (I)} -L _{Blank space} )/(L _Solvent(s) -L _{Blank space} )

L _{Sample (I)} : the LUM value of the well with cells, luciferase reagent and sample;

L _solvent(s) : the LUM values of wells with cells, luciferase reagent and sample with or without solvent;

L _{blank space} : LUM values for wells with luciferase reagent but no cells.

EC1.5＝(Cb-Ca)*[(1.5-Ia)/(Ib-Ia)]+Ca

Ca: lowest concentration of sample (μ M) at a fold induction greater than 1.5;

cb: maximum concentration of sample (μ M) at a fold induction of less than 1.5;

la: the induction multiple corresponding to the lowest concentration of the sample when the induction multiple is more than 1.5;

lb: the induction times are less than the induction times corresponding to the highest concentration of the sample when the induction times are less than 1.5.

7. Result judgment

7.1 the determination of any one result is performed in at least two separate experiments, at least three replicates of each experimental sample set, and if the results of the two experiments are not identical, a third experiment should be performed.

7.2 determination of Positive results: and if the results of the two or three experiments meet the following four conditions, the result is predicted to be a positive result, otherwise, the result is negative.

a. The maximum induction multiple Imax of the sample is more than or equal to 1.5, and the sample has a significant difference (t-test) compared with a control;

the survival rate of the cells under the EC1.5 concentration is more than 70 percent;

EC1.5 concentration less than 1000 μ M;

d. there was a significant dose-dependent increase in luciferase induction.

Third, experimental results

As shown in FIG. 5, the maximum induction times of Kerasens cells treated with different concentrations of 2, 4-dinitrochlorobenzene were 7.57 times as compared with the solvent control group; EC1.5 was calculated to be 2.59. mu.M, less than 1000. mu.M; EC1.5 corresponds to a cell viability of > 70%; a dose-dependent luciferase activity curve can be generated;

in conclusion, the Kerasens cell strain is used for successfully predicting the superstrong skin sensitizer 2, 4-dinitrochlorobenzene, and the Kerasens cell is proved to have the capability of evaluating whether the detected substance has potential skin sensitization risk.

Example 3: detection of known strong skin sensitizer bromothalonil by Kerasens cell line

First, principle of experiment

The Keap1-Nrf2-ARE pathway is the main regulation pathway of the cytoprotective response to electrophilic and oxidative stress, which can regulate the expression of detoxification, antioxidation and stress reaction enzymes and proteins, and the pathway participates in the skin allergy process and can be activated by skin sensitizers. Glutathione S-transferase 1(glutathione S-transferase P1, GSTP1) is one of glutathione mercaptotransferases (GSTs) family members that play an important role in the detoxification process by catalyzing the binding of a number of hydrophobic and electrophilic compounds to reducing glutathione. The GSTP1 gene contains ARE elements and is regulated by a Keap1-Nrf2-ARE pathway. The Kerasens cell strain is a genetically engineered epidermal keratinocyte cell strain, contains a luciferase gene Luc2 regulated by a GSTP1-ARE element, can monitor the activation condition of a Keap1-Nrf2-ARE channel in cells by detecting the luciferase induction condition, and can be further used for evaluating whether a detection substance has potential skin sensitization risk.

Second, Experimental methods

Kerasens cell culture and passage

Cell passage: the fusion degree is 80-90%, and the cells can be passed without being too dense. The generation is carried out every 2 to 4 days, and the generation ratio is 1:2 to 1: 6.1 flask T75 was removed, rinsed 2 times with 5mL of 1 XPBS, added 1mL of 0.25% trypsin-EDTA, and digested at 37 ℃ for 5-10 minutes. Digestion was stopped with 4mL of complete medium, pipetted evenly, cells were collected into a 15mL centrifuge tube and centrifuged at 2000rpm for 4 minutes. The old medium was aspirated off, 5mL of fresh medium was added for resuspension, and the cell suspension was taken in the new flask T75 in the proportions required for passage and supplemented to 15mL with complete medium.

Kerasens cell inoculation

Cultured Kerasens cells were removed, rinsed 2 times with 5mL of 1 XPBS, added 1mL of 0.25% pancreatin-EDTA, and digested at 37 ℃ for 5-10 minutes. Digestion was stopped with 4mL of complete medium, pipetted evenly, cells were collected into a 15mL centrifuge tube and centrifuged at 2000rpm for 4 minutes. Old medium was aspirated off, 5mL of fresh medium was added for resuspension, and 10. mu.L of cell suspension was taken and counted on a hemocytometer.

96-well plates were seeded at 1.5X10 per well ⁴ The KeraSens cells, 96-well white plate, were seeded in 47 wells for luciferase assay, 36 wells for sample testing (12 test concentrations, 3 replicates per concentration), 6 wells for solvent control, 5 wells for positive control (5 test concentrations, 1 replicate per concentration); in addition, a 96-well transparent plate was seeded with 23-well, 12-well test samples (12 test concentrations, 1 replicate per concentration), 6-well as a solvent control, and 5-well as a positive control (12 test concentrations, 1 replicate per concentration). The total number of cells for inoculation was calculated based on the number of wells for inoculated cells, and the cell suspension density was adjusted to 7.5X10 ⁴ cells/mL, 0.2mL of cell suspension per well was inoculated and then placed in a cell incubator for further 24 hours.

3. Reagent configuration

3.1 solvent control: 1% DMSO (CAS No.67-68-5)

3.2 Positive control: trans-cinnamaldehyde (CAS No.14371-10-9) was applied at 5 concentrations, 4-64. mu.M, 2-fold gradient.

a. Preparing 64mM cinnamaldehyde mother liquor A: molecular weight 132.16, density 1.046-1.052, 2. mu.L in 248. mu.L DMSO, and mixing.

3.3 detection sample bromothalonil configuration:

a. preparing 200mM of sample mother liquor A: bromothalonil in a certain mass (about 20-40mg) was added to a 15mL centrifuge tube or a 1.5mL EP tube, and dissolved and mixed with a solvent DMSO to a final concentration of 200 mM.

Note: volume V of DMSO is 5wp/MW-w/1000

W is the mg of material weighed, p is the material purity (in decimal units) and MW is the molecular weight.

c. Preparing working solution of samples with various concentrations: diluting 25 times of the mother liquor with a culture medium containing 1% FBS, namely adding 10 mu L of the mother liquor into 240 mu L of the culture medium, and uniformly mixing for later use. Standing at room temperature for later use.

4. Sample processing cells

Treatment medium: DMEM low-glycemic (Cat:11995, Gibco) + 1% FBS + 1% triple antibody (PSN), 200. mu.L per well.

4.2 Add the prepared solvent control, positive control and sample to a 96-well white plate and a 96-well transparent plate with a line gun, 50. mu.L per well. Solvent control group 6 replicate wells, positive control 1 well each concentration, sample group 3 replicate wells each concentration. Placing the mixture in an incubator for further culture for 48 hours.

5. Cell viability assay

5.4 after incubation, the transparent plate was removed, the plate cover was removed, and the OD was measured on a microplate reader at a wavelength of 450 nm.

And (3) activity calculation:

cell viability (%) ═ a _Sample(s) -A _{Blank space} ]/[A _Solvent(s) -A _{Blank space} ]×100

A _{Sample (I)} : absorbance of the well with cells, CCK-8 solution and sample;

ICx＝(Cb-Ca)*[(100-x)-Va]/(Vb-Va)+Ca

_X : percent inhibition to be calculated (%, e.g. IC30 or IC50)

Ca: minimum concentration (μ M) at which inhibition is greater than x%;

cb: maximum concentration (μ M) at which inhibition is less than x%;

6. Luciferase activity assay:

6.3 detection in a fluorescent microplate reader, the LUM mode being chosen.

L _Sample(s) : the LUM value of the well with cells, luciferase reagent and sample;

L _{blank space} : LUM values for wells with luciferase reagent without cells.

EC1.5＝(Cb-Ca)*[(1.5-Ia)/(Ib-Ia)]+Ca

Ca: lowest concentration of sample (μ M) at a fold induction greater than 1.5;

cb: highest concentration of sample (μ M) at a fold induction of less than 1.5;

7. Result judgment

7.2 determination of Positive result: and if the results of the two or three experiments meet the following four results, the result is predicted to be a positive result, otherwise, the result is predicted to be negative.

EC1.5 concentration less than 1000 μ M;

d. there was a clear dose-dependent increase in luciferase induction.

Third, experimental results

As shown in fig. 6, after the KeraSens cells were treated with bromothalonil at different concentrations, the maximum induction times were 2.49 times compared with the solvent control group; EC1.5 was calculated to be 17.56. mu.M, less than 1000. mu.M; EC1.5 corresponds to a cell viability of > 70%; a dose-dependent luciferase activity curve can be generated;

in conclusion, the Kerasens cell strain is used for successfully predicting the strong skin sensitizer bromothalonil, and the Kerasens cell is proved to have the capability of evaluating whether the detected substance has potential skin sensitization risk.

Example 4: kerasens cell line detection of known skin sensitizer 2-mercaptobenzothiazole I and experimental principle

The Keap1-Nrf2-ARE pathway is a main regulation pathway of cell protection reaction to electrophilic and oxidative stress, can regulate and control the expression of detoxification, antioxidation and stress reaction enzymes and proteins, participates in the skin allergy process, and can be activated by skin sensitizers. Glutathione S-transferase 1(glutathione S-transferase P1, GSTP1) is one of the glutathione mercaptotransferases (GSTs) family members that play an important role in the detoxification process by catalyzing the binding of a number of hydrophobic and electrophilic compounds to reducing glutathione. The GSTP1 gene contains ARE elements and is regulated by a Keap1-Nrf2-ARE pathway. The Kerasens cell strain is a genetically engineered epidermal keratinocyte cell strain, contains a luciferase gene Luc2 regulated and controlled by a GSTP1-ARE element, can monitor the activation condition of a Keap1-Nrf2-ARE pathway in cells by detecting the luciferase induction condition, and can be further used for evaluating whether a detection substance has potential skin sensitization risk.

Second, Experimental methods

Kerasens cell culture and passage

Cell passage: the fusion degree is 80-90%, and the cells can be passed through the culture medium without being too dense. The generation is carried out every 2 to 4 days, and the generation ratio is 1:2 to 1: 6.1 flask T75 was removed, rinsed 2 times with 5mL of 1 XPBS, added 1mL of 0.25% pancreatin-EDTA and digested at 37 ℃ for 5-10 minutes. Digestion was stopped with 4mL of complete medium, pipetted evenly, cells were collected into a 15mL centrifuge tube and centrifuged at 2000rpm for 4 minutes. The old medium was aspirated off, 5mL of fresh medium was added for resuspension, and the cell suspension was taken in the new flask T75 in the proportions required for passage and supplemented to 15mL with complete medium.

Kerasens cell inoculation

Culture medium for inoculation: DMEM low-glycemic (Cat:11995, Gibco) + 10% FBS + 1% triple antibody (PSN), 200. mu.L per well.

96-well plates were seeded 1.5X10 per well ⁴ The KeraSens cells, 96-well white plate, were seeded in 47 wells for luciferase assay, 36 wells for sample testing (12 test concentrations, 3 replicates per concentration), 6 wells for solvent control, 5 wells for positive control (5 test concentrations, 1 replicate per concentration); in addition, a 96-well transparent plate was seeded with 23 wells, 12 wells of test sample (12 test concentrations, 1 replicate per concentration), 6 wells as solvent control, and 5 wells as positive control (12 test concentrations, 1 replicate per concentration). The total number of cells for inoculation is calculated according to the number of the inoculated cells, and the cell suspension density is adjusted to 7.5x10 ⁴ one/mL, 0.2mL of cell suspension was inoculated per well and then placed in a cell incubator for further 24 hours.

3. Reagent configuration

3.1 solvent control: 1% DMSO (CAS No.67-68-5)

c. Preparing cinnamaldehyde working solution with each concentration: diluting 25 times of the mother liquor B with a 1% FBS-containing culture medium, namely adding 10. mu.L of the mother liquor B into 240. mu.L of the culture medium, and uniformly mixing for later use. When the sample was loaded, 50. mu.L of the culture medium was added to each well (150. mu.L of the culture medium was added to each well). The number of the required positive control holes and the configuration volume need to be calculated in advance, and each concentration of the white board positive control of every 96 holes has at least 1 hole positive control.

3.3 detection sample 2-mercaptobenzothiazole configuration:

a. preparing 200mM of sample mother liquor A: a certain amount (about 20-40mg) of 2-mercaptobenzothiazole was added to a 15mL centrifuge tube or a 1.5mL EP tube, and dissolved and mixed with a solvent DMSO to a final concentration of 200 mM.

Note: volume V of DMSO is 5wp/MW-w/1000

4. Sample processing cells

4.1 after 24 hours of seeded cell culture, the old medium was removed and the medium containing 1% FBS for treatment was replaced by 150. mu.L per well.

4.2 Add the prepared solvent control, positive control and sample to a 96-well white plate and a 96-well transparent plate with a line gun, 50. mu.L per well. Solvent control group 6 duplicate wells, positive control 1 well each concentration, sample group 3 duplicate wells each concentration. Placing the mixture in an incubator for further culture for 48 hours.

5. Cell viability assay

And (3) activity calculation:

A _{Sample (I)} : absorbance of the well with cells, CCK-8 solution and sample;

ICx＝(Cb-Ca)*[(100-x)-Va]/(Vb-Va)+Ca

_X : percent inhibition (%, e.g. IC30 or IC50) to be calculated)

Ca: the lowest concentration (μ M) at which the inhibition rate is greater than x%;

cb: maximum concentration (μ M) at which inhibition is less than x%;

va: cell viability (%) at the lowest concentration at which the inhibition rate is greater than x%;

6. Luciferase activity assay:

6.3 detection in a fluorescent microplate reader, the LUM mode being chosen.

Multiple of induction ═ L _Sample(s) -L _{Blank space} )/(L _Solvent(s) -L _{Blank space} )

L _{blank space} : LUM values for wells with luciferase reagent but no cells.

EC1.5＝(Cb-Ca)*[(1.5-Ia)/(Ib-Ia)]+Ca

Ca: lowest concentration of sample (μ M) at a fold induction greater than 1.5;

7. Result judgment

EC1.5 concentration less than 1000 μ M;

d. there was a significant dose-dependent increase in luciferase induction.

Third, experimental results

As shown in FIG. 7, the maximum induction times of Kerasens cells treated with different concentrations of 2-mercaptobenzothiazole were 4.05 times compared with the solvent control group; EC1.5 was calculated to be 39.35. mu.M, less than 1000. mu.M; EC1.5 corresponds to a cell viability of > 70%; a dose-dependent luciferase activity curve can be generated;

in conclusion, the Kerasens cell line is used for successfully predicting the 2-mercaptobenzothiazole serving as a neutral skin sensitizer, and the Kerasens cell is proved to have the capability of evaluating whether the detection substance has potential skin sensitization risk.

Example 5: detection of cinnamyl alcohol, a known weak skin sensitizer, by Kerasens cell line

First, principle of experiment

Second, Experimental methods

Kerasens cell culture and passage

Cell passage: the fusion degree is 80-90%, and the cells can be passed without being too dense. And (4) carrying out passage every 2-4 days, wherein the passage ratio is 1:2-1: 6.1 flask T75 was removed, rinsed 2 times with 5mL of 1 XPBS, added 1mL of 0.25% pancreatin-EDTA and digested at 37 ℃ for 5-10 minutes. Digestion was stopped with 4mL of complete medium, pipetted evenly, cells were collected into a 15mL centrifuge tube and centrifuged at 2000rpm for 4 minutes. The old medium was aspirated off, 5mL of fresh medium was added for resuspension, and the cell suspension was taken in the new flask T75 in the proportions required for passage and supplemented to 15mL with complete medium.

Kerasens cell inoculation

96-well plates were seeded 1.5X10 per well ⁴ The KeraSens cells, 96-well white plate, were seeded in 47 wells for luciferase assay, 36 wells for sample testing (12 test concentrations, 3 replicates per concentration), 6 wells for solvent control, 5 wells for positive control (5 test concentrations, 1 replicate per concentration); in addition, a 96-well transparent plate was seeded with 23-well, 12-well test samples (12 test concentrations per one)1 replicate at each concentration), 6 wells are solvent controls, and 5 wells are positive controls (12 test concentrations, 1 replicate at each concentration). The total number of cells for inoculation was calculated based on the number of wells for inoculated cells, and the cell suspension density was adjusted to 7.5X10 ⁴ cells/mL, 0.2mL of cell suspension per well was inoculated and then placed in a cell incubator for further 24 hours.

3. Reagent configuration

3.1 solvent control: 1% DMSO (CAS No.67-68-5)

3.3 detection sample cinnamyl alcohol configuration:

a. preparing 200mM of sample mother liquor A: a certain amount (about 20-40mg) of cinnamyl alcohol is added into a 15mL centrifuge tube or a 1.5mL EP tube, and dissolved and mixed uniformly by a solvent DMSO to obtain a final concentration of 200 mM.

Note: volume V of DMSO is 5wp/MW-w/1000

4. Sample processing cells

4.2 Add the prepared solvent control, positive control and sample to a 96-well white plate and 96-well transparent plate with a line gun, 50. mu.L per well. Solvent control group 6 duplicate wells, positive control 1 well each concentration, sample group 3 duplicate wells each concentration. Placing the mixture in an incubator for further culture for 48 hours.

5. Cell viability assay

And (3) activity calculation:

A _{Sample (I)} : absorbance of the well with cells, CCK-8 solution and sample;

A _solvent(s) : absorbance of wells with cells, CCK-8 solution and sample with solvent;

ICx＝(Cb-Ca)*[(100-x)-Va]/(Vb-Va)+Ca

_X : percent inhibition to be calculated (%, e.g. IC30 or IC50)

cb: maximum concentration (μ M) at which inhibition is less than x%;

6. Luciferase activity assay:

6.2 remove 96 hole white board, remove old culture medium, add 100. mu.L 1 xPBS with the rifle, shake slightly manually, then remove DPBS, add luciferase reagent rapidly, 100. mu.L per hole.

6.3 detection in a fluorescent microplate reader, the LUM mode being chosen.

L _solvent(s) : wells with cells, luciferase reagents and sample with and without solventThe LUM value of (D);

L _{blank space} : LUM values for wells with luciferase reagent without cells.

EC1.5＝(Cb-Ca)*[(1.5-Ia)/(Ib-Ia)]+Ca

Ca: lowest concentration of sample (μ M) at a fold induction greater than 1.5;

lb: the fold induction is less than that corresponding to the highest concentration of the sample at which the fold induction is 1.5.

7. Result judgment

7.2 determination of Positive results: and if the results of the two or three experiments meet the following four results, the result is predicted to be a positive result, otherwise, the result is predicted to be negative.

EC1.5 concentration less than 1000 μ M;

d. there was a significant dose-dependent increase in luciferase induction.

Third, experimental results

As shown in fig. 8, after the KeraSens cells were treated with cinnamyl alcohol at different concentrations, the maximum induction times were 21.4 times compared with the solvent control group; EC1.5 was calculated to be 49.9. mu.M, less than 1000. mu.M; EC1.5 corresponds to cell viability > 70%; a dose-dependent luciferase activity curve can be generated;

in conclusion, the Kerasens cell strain is used for successfully predicting the cinnamyl alcohol serving as the weak skin sensitizer, and the Kerasens cell strain is proved to have the capability of evaluating whether a detection substance has potential skin sensitization risk.

Example 6: detection of known non-skin sensitizers salicylic acid by Kerasens cell line

First, experiment principle

Second, Experimental methods

Kerasens cell culture and passage

Cell passage: the fusion degree is 80-90%, and the cells can be passed through the culture medium without being too dense. The generation is carried out every 2 to 4 days, and the generation ratio is 1:2 to 1: 6.1 flask T75 was removed, rinsed 2 times with 5mL of 1 XPBS, added 1mL of 0.25% trypsin-EDTA, and digested at 37 ℃ for 5-10 minutes. Digestion was stopped with 4mL of complete medium, pipetted evenly, cells were collected into a 15mL centrifuge tube and centrifuged at 2000rpm for 4 minutes. The old medium was aspirated off, 5mL of fresh medium was added for resuspension, and the cell suspension was removed to 15mL in a new flask T75 at the desired ratio for passaging and supplemented with complete medium.

Kerasens cell inoculation

96-well plates were seeded 1.5X10 per well ⁴ The KeraSens cells, 96-well white plate, were seeded in 47 wells for luciferase assay, 36 wells for sample testing (12 test concentrations, 3 replicates per concentration), 6 wells for solvent control, 5 wells for positive control (5 test concentrations, 1 replicate per concentration); in addition, a 96-well transparent plate was seeded with 23-well, 12-well test samples (12 test concentrations, 1 replicate per concentration), 6-well as a solvent control, and 5-well as a positive control (12 test concentrations, 1 replicate per concentration). The total number of cells for inoculation was calculated based on the number of wells for inoculated cells, and the cell suspension density was adjusted to 7.5X10 ⁴ one/mL, 0.2mL of cell suspension was inoculated per well and then placed in a cell incubator for further 24 hours.

3. Reagent configuration

3.1 solvent control: 1% DMSO (CAS No.67-68-5)

A4% DMSO solution (v/v) was prepared in a medium containing 1% FBS, and 40. mu.L DMSO was added to 960. mu.L of the medium. When loading, 50 μ L of each well (150 μ L of culture medium in the well), the number and configuration volume of the required solvent control wells need to be calculated in advance, and there are at least 6 wells of solvent control in each 96-well white plate.

c. Preparing cinnamaldehyde working solution with each concentration: diluting 25 times of the mother liquor B with a culture medium containing 1% FBS, namely adding 10 mu L of the culture medium into 240 mu L of the mother liquor B, and uniformly mixing for later use. When the sample was loaded, 50. mu.L of each well (150. mu.L of medium in each well) was added. The number of positive control holes and the configuration volume are calculated in advance, and each concentration of white board positive control with at least 1 hole positive control is calculated every 96 holes.

3.3 preparation of salicylic acid for detection samples:

a. preparing 200mM of sample mother liquor A: a certain mass (about 20-40mg) of salicylic acid is added into a 15mL centrifuge tube or a 1.5mL EP tube, dissolved and mixed uniformly by a solvent DMSO, and the final concentration is 200 mM.

Note: volume V of DMSO is 5wp/MW-w/1000

4. Sample processing cells

5. Cell viability assay

5.1 calculating the number of the holes required to carry out the toxicity test, configuring the volume used by the CCK-8 working fluid, and enabling the blank hole to have at least 1 hole.

And (3) activity calculation:

A _Sample(s) : absorbance of the well with cells, CCK-8 solution and sample;

ICx＝(Cb-Ca)*[(100-x)-Va]/(Vb-Va)+Ca

_X : percent inhibition to be calculated (%, e.g. IC30 or IC50)

Ca: minimum concentration (μ M) at which inhibition is greater than x%;

cb: maximum concentration (μ M) at which inhibition is less than x%;

6. Luciferase activity assay:

6.3 detection in a fluorescent microplate reader, the LUM mode being chosen.

L _{blank space} : LUM values for wells with luciferase reagent but no cells.

EC1.5＝(Cb-Ca)*[(1.5-Ia)/(Ib-Ia)]+Ca

Ca: lowest concentration of sample (μ M) at a fold induction greater than 1.5;

7. Result judgment

EC1.5 concentration less than 1000 μ M;

d. there was a significant dose-dependent increase in luciferase induction.

Third, experimental results

As shown in FIG. 9, the Kerasens cells treated with different concentrations of salicylic acid had a maximum induction factor of 1.35 times and less than 1.5 times, and had no skin sensitization ability, compared with the solvent control group.

In conclusion, salicylic acid was successfully predicted to be a non-skin sensitizer by using the Kerasens cell line, and the Kerasens cell line was proved to have the ability to distinguish the non-skin sensitizer.

Example 7: detection of known skin sensitizers N, N-dimethyl-1, 3-diaminopropane by Kerasens cell line

First, principle of experiment

The Keap1-Nrf2-ARE pathway is the main regulation pathway of the cytoprotective response to electrophilic and oxidative stress, which can regulate the expression of detoxification, antioxidation and stress reaction enzymes and proteins, and the pathway participates in the skin allergy process and can be activated by skin sensitizers. Glutathione S-transferase 1(glutathione S-transferase P1, GSTP1) is one of glutathione mercaptotransferases (GSTs) family members that play an important role in the detoxification process by catalyzing the binding of a number of hydrophobic and electrophilic compounds to reducing glutathione. The GSTP1 gene contains ARE elements and is regulated by a Keap1-Nrf2-ARE pathway. The Kerasens cell strain is a genetically engineered epidermal keratinocyte cell strain, contains a luciferase gene Luc2 regulated and controlled by a GSTP1-ARE element, can monitor the activation condition of a Keap1-Nrf2-ARE pathway in cells by detecting the luciferase induction condition, and can be further used for evaluating whether a detection substance has potential skin sensitization risk.

Second, Experimental methods

Kerasens cell culture and passage

Kerasens cell inoculation

3. Reagent configuration

3.1 solvent control: 1% DMSO (CAS No.67-68-5)

b. Preparing 6.4mM cinnamaldehyde mother liquor B: mu.L of stock A was added to 180. mu.L of DMSO and then diluted in a 1:2 gradient to give 4 additional concentrations of stock, 3.2mM, 1.6mM, 0.8mM, 0.4mM, each of at least 100. mu.L.

3.3 test sample N, N-dimethyl-1, 3-diaminopropane configuration:

a. preparing 200mM of sample mother liquor A: a certain amount (about 20-40mg) of N, N-dimethyl-1, 3-diaminopropane was added to a 15mL centrifuge tube or a 1.5mL EP tube, and dissolved in DMSO solvent to mix well to a final concentration of 200 mM.

Note: volume V of DMSO is 5wp/MW-w/1000

b. Preparing mother liquor B of each concentration of the sample: using the mother liquor A as the highest concentration, 100. mu.L of the high concentration mother liquor B was added to 100. mu.L of DMSO to carry out gradient dilution to obtain 11 additional concentration mother liquors B, and finally 100. mu.L of each concentration mother liquor B (200. mu.L of the lowest concentration) was obtained.

4. Sample processing cells

5. Cell viability assay

And (3) activity calculation:

A _Sample(s) : absorbance of the well with cells, CCK-8 solution and sample;

ICx＝(Cb-Ca)*[(100-x)-Va]/(Vb-Va)+Ca

_X : percent inhibition to be calculated (%, e.g. IC30 or IC50)

Ca: minimum concentration (μ M) at which inhibition is greater than x%;

cb: maximum concentration (μ M) at which inhibition is less than x%;

6. Luciferase activity assay:

6.3 detection in a Fluoromicroplate reader, LUM mode was chosen.

Fold induction ═ L _Sample(s) -L _{Blank space} )/(L _Solvent(s) -L _{Blank space} )

L _{blank space} : LUM values for wells with luciferase reagent but no cells.

EC1.5＝(Cb-Ca)*[(1.5-Ia)/(Ib-Ia)]+Ca

Ca: lowest concentration of sample (μ M) at a fold induction greater than 1.5;

7. Result judgment

EC1.5 concentration less than 1000 μ M;

d. there was a clear dose-dependent increase in luciferase induction.

Third, experimental results

As shown in fig. 10, after the KeraSens cells were treated with different concentrations of N, N-dimethyl-1, 3-diaminopropane, the maximum induction factor was 3.41 times compared to the solvent control group; EC1.5 was calculated to be 65.87 μ M, less than 1000 μ M; EC1.5 corresponds to a cell viability of > 70%; a dose-dependent luciferase activity curve can be generated;

in conclusion, the Kerasens cell strain is used for successfully predicting the skin sensitizer N, N-dimethyl-1, 3-diaminopropane, and the Kerasens cell has the capability of evaluating whether the detected substance has potential skin sensitization risk.

Comparative example 1: detection of known skin sensitizers by Kerasens cell line

First, principle of experiment

The Keap1-Nrf2-ARE pathway is a main regulation pathway of cell protection reaction to electrophilic and oxidative stress, can regulate and control the expression of detoxification, antioxidation and stress reaction enzymes and proteins, participates in the skin allergy process, and can be activated by skin sensitizers. Glutathione S-transferase 1(glutathione S-transferase P1, GSTP1) is one of glutathione mercaptotransferases (GSTs) family members that play an important role in the detoxification process by catalyzing the binding of a number of hydrophobic and electrophilic compounds to reducing glutathione. The GSTP1 gene contains ARE elements and is regulated by a Keap1-Nrf2-ARE pathway. The Kerasens cell strain is a genetically engineered epidermal keratinocyte cell strain, contains a luciferase gene Luc2 regulated and controlled by a GSTP1-ARE element, can monitor the activation condition of a Keap1-Nrf2-ARE pathway in cells by detecting the luciferase induction condition, and can be further used for evaluating whether a detection substance has potential skin sensitization risk.

In this comparative example, Kerasens cells of different initial inoculum sizes were treated with the known sensitizer cinnamaldehyde and the appropriate initial inoculum size of cells was selected in terms of both luciferase activity and cell viability.

Second, Experimental methods

Kerasens cell culture and passage

Kerasens cell inoculation

The 96-well plate was inoculated with 6 densities, respectivelyIs 0.5x10 ⁴ 1.0x10 per well ⁴ 1.5x10 pieces/hole ⁴ 2.0x10 pieces/hole ⁴ 2.5x10 pieces/hole ⁴ 3.0x10 pieces/hole ⁴ Per well. One 96-well plate was inoculated with 6 wells per density, and 3 96-well white plates were prepared for luciferase activity measurement, and 3 96-well transparent plates were used for cell viability measurement.

Suspensions of varying cell densities were prepared, seeded with 0.2mL of cell suspension per well, and placed in a cell incubator for further 24 hours.

3. Reagent configuration

3.1 solvent control: 1% DMSO (CAS No.67-68-5)

c. Preparing cinnamaldehyde working solution with each concentration: diluting 25 times of mother liquor B with 1% FBS-containing culture medium, namely adding 1440 μ L of culture medium into 60 μ L, and mixing uniformly for later use. When the sample was loaded, 50. mu.L of each well (150. mu.L of medium in each well) was added.

4. Sample processing cells

4.2 prepared solvent control, cinnamaldehyde samples of different concentrations were added to a 96-well white plate and a 96-well transparent plate using a line gun, 50. mu.L per well. Solvent control 6 replicate wells per 96 well plate, 1 well each for different cinnamaldehyde concentrations corresponding to different cell densities. Placing the mixture in an incubator for further culture for 48 hours.

5. Cell viability assay

5.1 calculating the number of the holes required to carry out toxicity test, configuring the volume for CCK-8 working fluid, and setting at least 6 holes in blank holes.

5.5 after the test is finished, data are imported into excel, and cell viability is calculated and analyzed.

And (3) activity calculation:

A _{Sample (I)} : absorbance of the well with cells, CCK-8 solution and sample;

ICx＝(Cb-Ca)*[(100-x)-Va]/(Vb-Va)+Ca

6. Luciferase activity assay:

6.3 detection in a fluorescent microplate reader, the LUM mode being chosen.

6.4 after the test is finished, data are imported into excel, and the induction times are calculated and analyzed.

L _{blank space} : LUM values for wells with luciferase reagent but no cells.

Third, experimental results

As shown in FIG. 11, different initial inoculum sizes of Kerasens cells had a greater effect on luciferase activity at 1.0X10 ⁴ At one/well, the maximum induction fold was generated and decreased with increasing initial inoculum size. 0.5 × 10 in different starting inoculum sizes under 32 μ M cinnamaldehyde treatment conditions ⁴ At counts/well, the maximum induction value was generated. Although the initial inoculum size had a significant effect on luciferase activity in the cells, treatment with cinnamaldehyde produced a dose-dependent luciferase activity curve at different initial inoculum sizes of cells.

As shown in fig. 12, different initial amounts of KeraSens cells had a greater effect on cell viability. Under the condition of lower-concentration cinnamic aldehyde (less than or equal to 16 mu M) treatment, the difference is not great; but at higher concentrations of cinnamaldehyde (32 μ M and 64 μ M) treatment, the low initial inoculum cell viability was low, even approaching 0; with increasing initial inoculum size, cell survival rate gradually increased at 3.0 × 10 ⁴ Cell viability reached 186% per well.

Considering luciferase activity and cell survival rate, the initial inoculation amount of the cells is 1.0-2.0x10 ⁴ Preferably one/well. This is advantageous for the stability and reproducibility of the experiment.

Comparative example 2: DPRA method for detecting known skin sensitizers N, N-dimethyl-1, 3-diaminopropane

First, principle of experiment

The direct polypeptide binding test (DPRA) is one of the skin sensitization in-vitro substitution test methods of cosmetic raw materials, and can be combined with other skin sensitization substitution tests to comprehensively evaluate the skin sensitization reaction. The principle of the DRPA test method is that a chemical method is used for simulating the combination of a sensitizing substance and synthetic cysteine polypeptide and lysine polypeptide, the consumption condition of the two polypeptides is measured by using high performance liquid chromatography, and the skin sensitization capacity of a test object is predicted according to the consumption rate of the polypeptides.

In this comparative example, known skin sensitizers, N-dimethyl-1, 3-diaminopropane, were evaluated by the DPRA method to compare the predictions of sensitizers by Kerasens cells in example 7.

Second, Experimental methods

The specific operation of the DPRA test refers to group standard of skin sensitization test of cosmetic raw materials-direct polypeptide binding test (DPRA) published by the Association of daily chemicals industries of Shanghai in 2018, and standard number T/SHRH 013-2018.

1. Instrument and reagent consumable

1.1 reagents

1.1.1 polypeptide fragments

Cysteine polypeptide (Ac-RFAACAA-COOH, molecular weight 751.9, purity > 90%)

Lysine polypeptide (Ac-RFAAKAA-COOHA, molecular weight 776.2, purity > 90%)

1.1.2 acetonitrile (chromatographic grade)

1.1.3 water, primary water specified in GB/T6682

1.1.4 trifluoroacetic acid (analytically pure)

1.1.5 sodium dihydrogen phosphate dihydrate (analytically pure)

1.1.6 disodium hydrogen phosphate dodecahydrate (analytically pure)

1.1.7 ammonium acetate (analytical grade)

1.1.8 Ammonia (analytical purity)

1.1.9 cinnamic aldehyde (analytically pure)

1.2 instruments

1.2.1 high performance liquid chromatograph

1.2.2 chromatographic columns

C18 column (2.1mm X100 mm X3.5 micron) or equivalent chromatographic column

2. Test method

2.1 preparation of polypeptide stock solutions

2.1.1 cysteine polypeptide stock solutions: weighing appropriate amount of cysteine polypeptide, and preparing into 0.667 mmol.L with phosphate buffer solution of pH7.5 ^-1 The solution of (1).

ph7.5 phosphate buffer: 18mL of 0.1 mmol. multidot.L ^-1 Sodium dihydrogen phosphate solution and 82 mL0.1mmol. L ^-1 The disodium hydrogen phosphate solution was mixed and the pH adjusted to 7.5.

2.1.2 lysine polypeptide stock solutions: weighing appropriate amount of lysine polypeptide, and preparing into 0.667 mmol.L with ammonium acetate buffer solution of pH10.2 ^-1 The solution of (1).

ph10.2 ammonium acetate buffer: 1.542g of ammonium acetate was taken, dissolved in 200mL of water, and the pH was adjusted to 10.2 with aqueous ammonia.

2.2 preparation of test substance and Positive control solution

On the test day, a test substance and a positive control solution were prepared at a concentration of 100 mmol.L ^-1 Volume 3mL, positive control cinnamaldehyde. The solvent is preferably acetonitrile, and if not, it can be dissolved in water, water/acetonitrile (1:1), isopropanol, acetone, and acetone/acetonitrile (1:1), or dissolved in 300. mu.L dimethyl sulfoxide and diluted with 2700. mu.L acetonitrile, or dissolved in 1500. mu.L dimethyl sulfoxide and diluted with 1500. mu.L acetonitrile.

2.3 preparation of polypeptide Standard solution

8mL of phosphate buffer and 8mL of ammonium acetate buffer were mixed with 2mL of acetonitrile, respectively, to prepare dilution buffers. Take 1600 μ L0.667 mmol.L ^-1 Polypeptide stock solution (0.667 mmol. L) ^-1 ) Adding into 400 μ L acetonitrile to obtain polypeptide standard mother liquor (0.534 mmol. L) ^-1 ). Diluting and mixing the polypeptide standard mother liquor and the dilution buffer solution in equal proportion to prepare the polypeptide standard mother liquor with the concentration of 0.534 mmol.L ^-1 、0.267mmol·L ^-1 、0.1335mmol·L ^-1 、0.0667mmol·L ^-1 、0.0334mmol·L ^-1 、0.0167mmol·L ^-1 、0mmol·L ^-1 The polypeptide standard solution of (4).

2.4 preparation of high Performance liquid chromatography test solutions

A1 mL sample injection bottle is taken, the reagents are added according to the table 1 and mixed evenly, and 3 parts of each sample and each reference are prepared in parallel. And placing the sampling bottle at 25 +/-2.5 ℃ and incubating for 24 +/-2 hours in the dark, detecting by using high performance liquid chromatography respectively, and completing the whole detection within 30 hours. Before and after the reaction, the sample injection bottle needs to be observed and whether precipitation occurs or not is recorded. When precipitation occurs before incubation, the consumption of the polypeptide cannot be calculated, a positive result is available, and a negative result is uncertain; if only the precipitate appears after incubation, the low-speed centrifugation of 100-400x g is adopted to lead the precipitate to be gathered to the bottom of the sample injection bottle for further sample injection.

Table 1: blank control, co-elution control and sample preparation table

2.5 high Performance liquid chromatography detection

2.5.1 Mobile phase

Mobile phase A: 1000. mu.L of trifluoroacetic acid was added to 1LHPLC grade water.

Mobile phase B: to 1L of acetonitrile was added 850. mu.L of trifluoroacetic acid.

2.5.2 high Performance liquid chromatography conditions

C18 chromatographic column, with a column temperature of 25 ℃, a sample injection amount of 3-10 μ L, and detecting the peak area of the polypeptide at 220 nm. The chromatographic elution conditions are shown in Table 2 (adjusted as appropriate).

Table 2: gradient elution condition of high performance liquid chromatography

Time/min	Flow rate/mL min ^-1	Mobile phase A/%)	Mobile phase B/%)
				0	0.35	90	10
10	0.35	75	25
				11	0.35	10	90
13	0.35	10	90
				13.5	0.35	90	10
20	0.35	90	10

2.5.3 high performance liquid chromatography sample injection sequence

Table 3: high performance liquid chromatography sample injection sequence

2.5.4 calculation of the consumption Rate of the polypeptide

Consumption rate ═ 1- (peak area of polypeptide reaction/polypeptide peak area of blank) ] × 100%

3. Conditions for test establishment

The following conditions are met, and the test conditions are met:

3.1 Standard Curve: r is ² ＞0.990。

3.2 blank control A average polypeptide concentration should be 0.5 + -0.05 mmol.L-1

3.3 average positive control polypeptide consumption rate: the cysteine polypeptide should be between 60.8% and 100%, and the consumption rate SD is less than 14.9%. Lysine polypeptide should be between 40.2% and 69.4%, consumption rate SD is less than 11.6%.

3.4 blank B, C Peak area CV < 15.0% (n ═ 9)

The following conditions are met, and the test sample results are valid:

3.5 sample cysteine polypeptide consumption rate SD < 14.9%, sample lysine polypeptide consumption rate SD < 11.6%.

3.6 solvent blank C should have an average polypeptide concentration of 0.5. + -. 0.05 mmol.L-1.

4DPRA prediction model

4.1 Using a 1: 10 cysteine polypeptide and 1: the 50 lysine polypeptide model was used to identify the test substances (see Table 4). Table 4 shows 1: 10 cysteine polypeptide/1: 50 lysine polypeptide model.

TABLE 4

4.2 when the test substance co-eluted with the lysine polypeptide, the judgment was made only by using the cysteine polypeptide model (see Table 5).

Table 5 shows 1: 10 cysteine polypeptide model.

TABLE 5

Rate of cysteine polypeptide consumption	Extent of reaction	DPRA prediction results
			The consumption rate is less than or equal to 13.89 percent	No or slight reaction	Negative of
13.89％<The consumption rate is less than or equal to 23.09 percent	Weak reaction	Positive for
			23.09％<The consumption rate is less than or equal to 98.24 percent	Moderate reaction	Positive for
98.24％<The consumption rate is less than or equal to 100 percent	Strong reaction	Positive for

4.3 when the test substance is co-eluted with the cysteine polypeptide and the lysine polypeptide at the same time, the conclusion is judged to be 'uncertain'.

Third, experimental results

Table 6 shows the results of testing N, N-dimethyl-1, 3-diaminopropane by the DPRA method. As shown in the following Table, the results of the DPRA test showed that N, N-dimethyl-1, 3-diaminopropane had a cysteine polypeptide consumption rate of 0, a lysine polypeptide consumption rate of 0, and an average elimination rate of 0, and N, N-dimethyl-1, 3-diaminopropane was predicted to be a non-sensitizer.

TABLE 6

Through the implementation of comparative example 2 and comparison example 7, the Kerasens cell strain is shown to be capable of making up the deficiency of the DPRA test to a certain extent in predicting some skin sensitizers, such as N, N-dimethyl-1, 3-diaminopropane and the like, and improving the sensitivity, specificity and accuracy of prediction in the '2-out-of-3' integrated test strategy.

Sequence listing

<110> Shanghai's family Joint sharps Ltd

<120> construction and application of cell strain containing luciferase reporter gene of human GSTP1ARE

<130> 212870

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 276

<212> DNA

<213> Artificial sequence

<400> 1

ggtacccgcg agcggccggc gccgtgactc agcactgggg cggagcgggg cgggaccacc 60

cagatctgcg atctgcatct caattagtca gcaaccatag tcccgcccct aactccgccc 120

atcccgcccc taactccgcc cagttccgcc cattctccgc cccatcgctg actaattttt 180

tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga 240

ggcttttttg gaggcctagg cttttgcaaa aagctt 276

<210> 2

<211> 5818

<212> DNA

<213> Artificial sequence

<400> 2

ggcctaactg gccggtaccc gcgagcggcc ggcgccgtga ctcagcactg gggcggagcg 60

gggcgggacc acccagatct gcgatctgca tctcaattag tcagcaacca tagtcccgcc 120

cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatcg 180

ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca 240

gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagcttg gcaatccggt 300

actgttggta aagccaccat ggaagatgcc aaaaacatta agaagggccc agcgccattc 360

tacccactcg aagacgggac cgccggcgag cagctgcaca aagccatgaa gcgctacgcc 420

ctggtgcccg gcaccatcgc ctttaccgac gcacatatcg aggtggacat tacctacgcc 480

gagtacttcg agatgagcgt tcggctggca gaagctatga agcgctatgg gctgaataca 540

aaccatcgga tcgtggtgtg cagcgagaat agcttgcagt tcttcatgcc cgtgttgggt 600

gccctgttca tcggtgtggc tgtggcccca gctaacgaca tctacaacga gcgcgagctg 660

ctgaacagca tgggcatcag ccagcccacc gtcgtattcg tgagcaagaa agggctgcaa 720

aagatcctca acgtgcaaaa gaagctaccg atcatacaaa agatcatcat catggatagc 780

aagaccgact accagggctt ccaaagcatg tacaccttcg tgacttccca tttgccaccc 840

ggcttcaacg agtacgactt cgtgcccgag agcttcgacc gggacaaaac catcgccctg 900

atcatgaaca gtagtggcag taccggattg cccaagggcg tagccctacc gcaccgcacc 960

gcttgtgtcc gattcagtca tgcccgcgac cccatcttcg gcaaccagat catccccgac 1020

accgctatcc tcagcgtggt gccatttcac cacggcttcg gcatgttcac cacgctgggc 1080

tacttgatct gcggctttcg ggtcgtgctc atgtaccgct tcgaggagga gctattcttg 1140

cgcagcttgc aagactataa gattcaatct gccctgctgg tgcccacact atttagcttc 1200

ttcgctaaga gcactctcat cgacaagtac gacctaagca acttgcacga gatcgccagc 1260

ggcggggcgc cgctcagcaa ggaggtaggt gaggccgtgg ccaaacgctt ccacctacca 1320

ggcatccgcc agggctacgg cctgacagaa acaaccagcg ccattctgat cacccccgaa 1380

ggggacgaca agcctggcgc agtaggcaag gtggtgccct tcttcgaggc taaggtggtg 1440

gacttggaca ccggtaagac actgggtgtg aaccagcgcg gcgagctgtg cgtccgtggc 1500

cccatgatca tgagcggcta cgttaacaac cccgaggcta caaacgctct catcgacaag 1560

gacggctggc tgcacagcgg cgacatcgcc tactgggacg aggacgagca cttcttcatc 1620

gtggaccggc tgaagagcct gatcaaatac aagggctacc aggtagcccc agccgaactg 1680

gagagcatcc tgctgcaaca ccccaacatc ttcgacgccg gggtcgccgg cctgcccgac 1740

gacgatgccg gcgagctgcc cgccgcagtc gtcgtgctgg aacacggtaa aaccatgacc 1800

gagaaggaga tcgtggacta tgtggccagc caggttacaa ccgccaagaa gctgcgcggt 1860

ggtgttgtgt tcgtggacga ggtgcctaaa ggactgaccg gcaagttgga cgcccgcaag 1920

atccgcgaga ttctcattaa ggccaagaag ggcggcaaga tcgccgtgta ataattctag 1980

agtcggggcg gccggccgct tcgagcagac atgataagat acattgatga gtttggacaa 2040

accacaacta gaatgcagtg aaaaaaatgc tttatttgtg aaatttgtga tgctattgct 2100

ttatttgtaa ccattataag ctgcaataaa caagttaaca acaacaattg cattcatttt 2160

atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gcaagtaaaa cctctacaaa 2220

tgtggtaaaa tcgataagga tccgtttgcg tattgggcgc tcttccgctg atctgcgcag 2280

caccatggcc tgaaataacc tctgaaagag gaacttggtt agctaccttc tgaggcggaa 2340

agaaccagct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag 2400

gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag 2460

gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc 2520

cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc 2580

atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat 2640

tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcgattctt 2700

ctgacactag cgccaccatg atcgaacaag acggcctcca tgctggcagt cccgcagctt 2760

gggtcgaacg cttgttcggg tacgactggg cccagcagac catcggatgt agcgatgcgg 2820

ccgtgttccg tctaagcgct caaggccggc ccgtgctgtt cgtgaagacc gacctgagcg 2880

gcgccctgaa cgagcttcaa gacgaggctg cccgcctgag ctggctggcc accaccggcg 2940

taccctgcgc cgctgtgttg gatgttgtga ccgaagccgg ccgggactgg ctgctgctgg 3000

gcgaggtccc tggccaggat ctgctgagca gccaccttgc ccccgctgag aaggtttcta 3060

tcatggccga tgcaatgcgg cgcctgcaca ccctggaccc cgctacctgc cccttcgacc 3120

accaggctaa gcatcggatc gagcgtgctc ggacccgcat ggaggccggc ctggtggacc 3180

aggacgacct ggacgaggag catcagggcc tggcccccgc tgaactgttc gcccgactga 3240

aagcccgcat gccggacggt gaggacctgg ttgtcacaca cggagatgcc tgcctcccta 3300

acatcatggt cgagaatggc cgcttctccg gcttcatcga ctgcggtcgc ctaggagttg 3360

ccgaccgcta ccaggacatc gccctggcca cccgcgacat cgctgaggag cttggcggcg 3420

agtgggccga ccgcttctta gtcttgtacg gcatcgcagc tcccgacagc cagcgcatcg 3480

ccttctaccg cttgctcgac gagttctttt aatgatctag aaccggtcat ggccgcaata 3540

aaatatcttt attttcatta catctgtgtg ttggtttttt gtgtgttcga actagatgct 3600

gtcgaccgat gcccttgaga gccttcaacc cagtcagctc cttccggtgg gcgcggggca 3660

tgactatcgt cgccgcactt atgactgtct tctttatcat gcaactcgta ggacaggtgc 3720

cggcagcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 3780

cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 3840

gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 3900

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 3960

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 4020

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 4080

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 4140

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 4200

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 4260

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 4320

aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 4380

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 4440

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 4500

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 4560

gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 4620

tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag cggccgcaaa 4680

tgctaaacca ctgcagtggt taccagtgct tgatcagtga ggcaccgatc tcagcgatct 4740

gcctatttcg ttcgtccata gtggcctgac tccccgtcgt gtagatcact acgattcgtg 4800

agggcttacc atcaggcccc agcgcagcaa tgatgccgcg agagccgcgt tcaccggccc 4860

ccgatttgtc agcaatgaac cagccagcag ggagggccga gcgaagaagt ggtcctgcta 4920

ctttgtccgc ctccatccag tctatgagct gctgtcgtga tgctagagta agaagttcgc 4980

cagtgagtag tttccgaaga gttgtggcca ttgctactgg catcgtggta tcacgctcgt 5040

cgttcggtat ggcttcgttc aactctggtt cccagcggtc aagccgggtc acatgatcac 5100

ccatattatg aagaaatgca gtcagctcct tagggcctcc gatcgttgtc agaagtaagt 5160

tggccgcggt gttgtcgctc atggtaatgg cagcactaca caattctctt accgtcatgc 5220

catccgtaag atgcttttcc gtgaccggcg agtactcaac caagtcgttt tgtgagtagt 5280

gtatacggcg accaagctgc tcttgcccgg cgtctatacg ggacaacacc gcgccacata 5340

gcagtacttt gaaagtgctc atcatcggga atcgttcttc ggggcggaaa gactcaagga 5400

tcttgccgct attgagatcc agttcgatat agcccactct tgcacccagt tgatcttcag 5460

catcttttac tttcaccagc gtttcggggt gtgcaaaaac aggcaagcaa aatgccgcaa 5520

agaagggaat gagtgcgaca cgaaaatgtt ggatgctcat actcgtcctt tttcaatatt 5580

attgaagcat ttatcagggt tactagtacg tctctcaagg ataagtaagt aatattaagg 5640

tacgggaggt attggacagg ccgcaataaa atatctttat tttcattaca tctgtgtgtt 5700

ggttttttgt gtgaatcgat agtactaaca tacgctctcc atcaaaacaa aacgaaacaa 5760

aacaaactag caaaataggc tgtccccagt gcaagtgcag gtgccagaac atttctct 5818

Claims

1. An overexpression vector of luciferase reporter gene containing human GSTP1ARE, which is a nucleotide sequence shown in SEQ ID NO. 2.

2. A cell strain for stably expressing a luciferase reporter gene containing human GSTP1ARE is characterized in that the name of the cell strain is human skin sensitizer predicted cell strain HaCaT-hGSTP1ARE-SV40p-Luc2, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2021113.

3. a method of assessing the risk of skin sensitization of an agent or product for non-diagnostic, non-therapeutic purposes, the method comprising the steps of:

(a) constructing an overexpression vector pGL4.17-hGSTP1ARE-SV40p-Luc2, wherein the nucleotide sequence of the overexpression vector is SEQ ID NO. 2;

(b) introducing an overexpression vector pGL4.17-hGSTP1ARE-SV40p-Luc2 into HaCaT cells to construct a cell strain containing a luciferase reporter gene of human GSTP1 ARE;

4. The method of claim 3, wherein the step (b) comprises screening with neomycin to obtain a cell line stably expressing the luciferase reporter gene containing human GSTP1 ARE.

5. The method of claim 3, wherein the method further comprises screening cell lines after step (b) for susceptibility to a risk of skin sensitization of the agent or product.

6. A method according to claim 3, wherein the reagent or product to be detected is selected from: cosmetics or cosmetic raw materials.

7. The method of claim 5 or 6, wherein the agent or product is selected from the group consisting of: cinnamaldehyde, 2, 4-dinitrochlorobenzene, bromothalonil, 2-mercaptobenzothiazole, cinnamyl alcohol, salicylic acid, N-dimethyl-1, 3-diaminopropane, or a combination thereof.

8. Use of the cell strain of claim 2 for the evaluation of the risk of skin sensitization of a reagent or product for non-diagnostic, non-therapeutic purposes.

9. Use according to claim 8, wherein the reagent or product to be detected is selected from: cosmetic or cosmetic raw material.

10. Use according to claim 9, wherein the agent or product is selected from: cinnamaldehyde, 2, 4-dinitrochlorobenzene, bromothalonil, 2-mercaptobenzothiazole, cinnamyl alcohol, salicylic acid, N-dimethyl-1, 3-diaminopropane, or a combination thereof.