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Method for determining glucocorticoid mixture based on transgenic engineering cell strain

A transgenic engineering, glucocorticoid technology, applied in genetic engineering, botanical equipment and methods, biochemical equipment and methods, etc. problems

Inactive Publication Date: 2020-08-07
INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A large number of known and unknown glucocorticoid mixtures and their metabolites may be missed or undetectable, and existing methods such as mass spectrometry and enzyme-linked immunosorbent immunoassays have an imbalance between high throughput, sensitivity, time and cost
[0005] At present, a large number of investigations and risk monitoring of glucocorticoid mixtures have not been carried out, mainly due to the high cost and long detection time

Method used

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  • Method for determining glucocorticoid mixture based on transgenic engineering cell strain
  • Method for determining glucocorticoid mixture based on transgenic engineering cell strain
  • Method for determining glucocorticoid mixture based on transgenic engineering cell strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Method for determination of glucocorticoid mixture based on GFP-GR-puro Hela cell transgenic engineering cell line

[0039] 1. Target gene synthesis

[0040] The whole GFP-GR gene (as shown in SEQ ID NO: 1) was synthesized (pTet-GFP-GR) by Nanjing GenScript Company.

[0041] 2. Plasmid extraction and sequencing

[0042] pTet-GFP-GR was transformed into Escherichia coli Trans5α competent cells by heat shock method, spread on LB (Amp) plates, and cultured at 37°C. Pick a monoclonal strain, culture it overnight in 100mL LB (Amp) liquid medium, and use the Gene star endotoxin-free plasmid extraction kit for plasmid extraction.

[0043] Plasmid concentration 857.6ng / μL, OD 260 / 280=1.89; 20uL sample was taken and sent to Sangon for sequencing and identification.

[0044] 3. Carrier Construction

[0045] Sequence the correct plasmid, use PCR to amplify the GFP-GR target fragment, and purify the PCR product; the lentiviral vector plasmid FV026 has been digested and linear...

Embodiment 2

[0060] Testing and validation of glucocorticoid mixtures

[0061] 1. Experimental method

[0062] Perform 17β-estradiol (17β-Estradiol) (negative), dexamethasone (Dexa) (positive) to the stable strain cell GFP-GR-puro Hela that embodiment 1 screens out, and several other glucocorticoid drugs In the administration experiment, the action time was 0.5h, and then the cytoplasmic nuclear migration response was detected.

[0063] In order to eliminate the influence factors in the medium and serum as much as possible, the cell medium was replaced with phenol red-free MEM (+10% activated charcoal-treated serum, 10% CD-FBS) complete medium 1 day in advance. Subsequently, the GFP-GR-puro Hela digestion was counted according to 10 4 Each well was inserted into a black 96-well plate (3603, corning). After culturing overnight, add blank control (DMSO), negative control estradiol (17β-E2), positive drug dexamethasone (Dexa) and several other glucocorticoid drugs (prednisone and methylpre...

Embodiment 3

[0069] 1. Experimental method

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Abstract

The invention discloses a transgenic engineering cell strain and a method for determining a glucocorticoid mixture based on the cell strain. According to the cell strain, a green fluorescent protein GFP and a glucocorticoid receptor GR fusion protein gene are integrated into a Hela cell chromosome in a lentiviral infection mode, puromycin puro screening is conducted, and finally the Hela cell strain stably expressing GFP-GR is obtained. Based on a Hela cell strain for stable expression of GFP-GR, by measuring a migration ratio of a GFP-GR binding hormone ligand migrated from a cytoplasm regionto a cell nucleus region, the positive detection rate of the glucocorticoid mixtures in different samples can be rapidly detected in high flux and low cost, the sensitivity can reach 10<-9> mol / L interms of dexamethasone, the content of the glucocorticoid mixture in the environment and food can be rapidly screened, the harm degree is clear, and risk prevention and control are guided.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for measuring glucocorticoid mixtures based on GFP-GR-puro Hela cell transgenic engineering cell lines. Background technique [0002] Lentivirus vector is a gene therapy vector developed on the basis of human immunodeficiency virus-1 (HIV-1). There are three types of viral vectors commonly used in genetic engineering: retroviruses, adenoviruses, and lentiviruses. Among them, retroviral vectors can only infect cells in the dividing stage, and the vector capacity is limited; after adenovirus infects cells, the target gene generally cannot be integrated into the chromosome of the host cell, and can only be expressed in transient infection; and lentivirus is different from The first two viruses not only have the ability to infect dividing and non-dividing cells, but also have the characteristics of large vectors to accommodate exogenous gene fragments and long-term expression o...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/62G01N33/74
CPCC12N5/0693C12N15/86C07K14/721C07K14/72G01N33/74C12N2510/00C12N2740/15043C07K2319/00
Inventor 李耘刘畅钱永忠邱静任亚林李金娟
Owner INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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