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Anabolic steroids and polycyclic aromatic hydrocarbon high-efficiency bioluminescence sensor and construction method thereof

A fluorescent sensor and polycyclic aromatic hydrocarbon technology, applied in the field of molecular biology, can solve the problems of long detection period, high cost, low sensitivity, etc., and achieve the effect of low instrument requirements, wide detection linear range, and simple technical operation

Inactive Publication Date: 2011-07-20
CHANGCHUN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of these detection systems require human or yeast cell culture, the detection period is long, the cost is high, and there are disadvantages such as low sensitivity and only one compound can be detected, so the application of these technologies to environmental detection is extremely limited

Method used

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  • Anabolic steroids and polycyclic aromatic hydrocarbon high-efficiency bioluminescence sensor and construction method thereof
  • Anabolic steroids and polycyclic aromatic hydrocarbon high-efficiency bioluminescence sensor and construction method thereof
  • Anabolic steroids and polycyclic aromatic hydrocarbon high-efficiency bioluminescence sensor and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Taking testosterone as an example to establish a bioluminescence detection standard curve

[0026] 1. Preparation of cell plasma in acellular system:

[0027] (1) Transform the plasmid pK-3α-GFP3 into Escherichia coli, and cultivate it in a constant temperature shaking incubator at 37°C at 180-200 rpm for 12 to 14 hours, and inoculate the cultured Escherichia coli at a volume ratio of 1:20 Cultivate in SIN medium at 37°C, 180-200 rpm in a constant temperature shaking incubator until OD595=3.0, centrifuge the cultured bacteria at 4000 rpm for 20-30 minutes, discard the supernatant, add sterile water to wash the bacteria , centrifuge at 4000 rpm for 20-30 minutes, discard the supernatant, repeat washing twice, add sterile water and a final concentration of 100 μg / mL lysozyme, resuspend the bacteria, freeze and thaw three times at -20°C to 25°C, Centrifuge at 10,000 rpm for 20-30 minutes, and take the supernatant as the prepared acellular system cytoplasm (can b...

Embodiment 2

[0037] Example 2 Taking estradiol as an example to establish a bioluminescence standard detection curve

[0038] 1. Preparation of cell plasma in acellular system:

[0039] (1) Transform the plasmid pK-3α-GFP3 into Escherichia coli, and cultivate it in a constant temperature shaking incubator at 37°C at 180-200 rpm for 12 to 14 hours, and inoculate the cultured Escherichia coli at a volume ratio of 1:20 In SIN medium, cultivate to OD in 37°C, 180-200rpm constant temperature shaking incubator 595= 3.0, centrifuge the cultured cells at 4000 rpm for 20-30 minutes, discard the supernatant, add sterile water to wash the cells, centrifuge at 4000 rpm for 20-30 minutes, discard the supernatant, repeat washing twice, add sterile Bacterial water and a final concentration of 100 μg / mL lysozyme, resuspended bacteria, repeated freezing and thawing three times at -20°C to 25°C, centrifuged at 10,000 rpm for 20 to 30 minutes, and the supernatant was the prepared acellular system cytoplasm ...

Embodiment 3

[0049] Embodiment 3 takes naphthalene as an example to establish a bioluminescence standard detection curve

[0050] 1. Preparation of cell plasma in acellular system:

[0051] (1) Transform the plasmid pK-3α-GFP3 into Escherichia coli, and cultivate it in a constant temperature shaking incubator at 37°C at 180-200 rpm for 12 to 14 hours, and inoculate the cultured Escherichia coli at a volume ratio of 1:20 In SIN medium, cultivate to OD in 37°C, 180-200rpm constant temperature shaking incubator 595 = 3.0, centrifuge the cultured cells at 4000 rpm for 20-30 minutes, discard the supernatant, add sterile water to wash the cells, centrifuge at 4000 rpm for 20-30 minutes, discard the supernatant, repeat washing twice, add sterile Bacterial water and a final concentration of 100 μg / mL lysozyme, resuspended bacteria, repeated freezing and thawing three times at -20°C to 25°C, centrifuged at 10,000 rpm for 20 to 30 minutes, and the supernatant was the prepared acellular system cytop...

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Abstract

The invention relates to an anabolic steroids and polycyclic aromatic hydrocarbon high-efficiency bioluminescence sensor, belonging to the technical field of molecular biology; the bioluminescence sensor is characterized in that plasmids pK-3[alpha]-GFP3 and p6 are respectively transformed into colibacillus and respectively prepared into cell-free system testing agent; 3[alpha]-HSD promoters suchas regulatory sequence 486 base pair and GFP reporter gene 722 base pair are inserted at the down stream of the cis-[belta]-galactosidase (LacZ) promoter gene of recombinant plasmid pK-3[alpha]-GFP3 in sequence; the regulatory sequence of 3[alpha]-HSD on p6 plasmid can be induced to express protein with regulatory function; the protein can promote the promoter on the plasmids pK-3[alpha]-GFP3 so as to express the GFP protein; at the moment, the expression quantity of the GFP is controlled by the quantity of induction substrate strictly. The bioluminescence sensor and the construction method thereof have the following benefits: aiming at the shortcomings of strict instrument requirement, long detection cycle, high cost, low sensitivity and single test piece in the existing testing system, the bioluminescence sensor and the construction method thereof have wide test linearity width, simple technical operation and low instrument requirement, and are applicable to popularize and apply to detect anabolic steroids and polycyclic aromatic hydrocarbon pollutant in the environment and food.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. Background technique [0002] In recent years, natural and synthetic steroids and polycyclic aromatic hydrocarbons have been discharged into the natural environment without restriction. The hormone activity and negative impact on organisms of these pollutants have attracted much attention. Due to its wide distribution and hormonal activity at extremely low concentrations, it poses serious hazards to the environment and human health. Therefore, new technologies that can detect steroids and polycyclic aromatic hydrocarbons sensitively, quickly and efficiently are urgently needed to be developed. At present, the traditional detection and analysis techniques are mainly gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (HC-MS). In addition, domestic and foreign experts have developed a variety of steroids based on the research on human steroid hormone r...

Claims

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Application Information

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IPC IPC(8): G01N33/74G01N21/64C12N15/70C12N15/65
Inventor 于源华宋禹于化东何秀霞张淑华葛淑敏马书林侯巍
Owner CHANGCHUN UNIV OF SCI & TECH
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