CN102128938A - Anabolic steroids and polycyclic aromatic hydrocarbon high-efficiency bioluminescence sensor and construction method thereof - Google Patents

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

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CN102128938A
CN102128938A CN2010105950591A CN201010595059A CN102128938A CN 102128938 A CN102128938 A CN 102128938A CN 2010105950591 A CN2010105950591 A CN 2010105950591A CN 201010595059 A CN201010595059 A CN 201010595059A CN 102128938 A CN102128938 A CN 102128938A
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gfp3
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CN102128938B (en
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于源华
宋禹
于化东
何秀霞
张淑华
葛淑敏
马书林
侯巍
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Changchun University of Science and Technology
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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 such as 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

Steroid hormone and palycyclic aromatic high-performance bio fluorescent optical sensor and construction method
Technical field
The invention belongs to technical field of molecular biology.
Background technology
In recent years, the steroidal class of natural, synthetic and polycyclic aromatic hydrocarbon compounds is unconfined is discharged in the physical environment, the hormonal activity of this pollutant and to the negative influence of biosome extremely people pay close attention to.Because it is widely distributed, all has hormonal activity under extremely low concentration, environment and human health are produced serious harm, demand development urgently so can detect the new technology of steroidal class and polycyclic aromatic hydrocarbon compounds sensitive, fast and efficiently.Traditional check and analysis technology mainly is gas chromatography-mass spectrum (GC-MS), liquid chromatography-mass spectrography (HC-MS) at present, and domestic and international in addition expert has been developed into multiple steroid hormone detection system based on the research of human body steroid receptor.These detection system great majority need human body or yeast cells to cultivate, and sense cycle is long, cost is high, exist in addition sensitivity low, can only detect drawback such as a kind of compound, it is extremely limited therefore these technology being applied to the environment measuring aspect.
Comamonas testosteroni (C.testosteroni, C.T) remarkable characteristic is that it has specificity degradation of steroid class and polycyclic aromatic hydrocarbon compounds enzyme gene, and existing research about its degradation of steroid class and polycyclic aromatic hydrocarbon compounds gene expression and steroid hormone and polycyclic aromatic hydrocarbon compounds acknowledgement mechanism.Whether can be signal scheme with steroidal class and polycyclic aromatic hydrocarbon compounds with C.T, make up a kind of sensitivity, fast, simple biology sensor, be used for steroidal class and polycyclic aromatic hydrocarbon compounds potpourri in the testing environment.
Summary of the invention
The objective of the invention is: a kind of steroid hormone and palycyclic aromatic high-performance bio fluorescent optical sensor and construction method are provided, plasmid pK-3 α-GFP3 and p6 are transformed respectively in the Escherichia coli, set up a kind of bioluminescence detection system of green fluorescent protein mark, detected object is steroid hormone and palycyclic aromatic two big class materials.
Technical scheme of the present invention is:
The present invention has made up based on Comamonas testosteroni (C.testosteroni, steroid hormone C.T) and palycyclic aromatic bioluminescence detection system.Comamonas testosteroni can be with steroid hormones such as stosterones as the sole carbon source and the energy, the materials such as palycyclic aromatic of the non-steroid of can also degrading, and (the 3 α-HSD) metabolism of enzyme such as grade digest this class substrate by 3 α-hydroxysteroid dehydrogenase.Green fluorescent protein (GFP) is the reporter gene that is widely used in the biological marker field in recent years, the protein that its gene produced, light at blue wavelength region excites down, green fluorescence can be sent, the relative content of specific substrate can be calculated according to its fluorescence intensity by the reference standard curve.
The present invention transforms into plasmid pK-3 α-GFP3 and p6 in the Escherichia coli respectively and is prepared into acellular system detectable respectively, has set up a kind of bioluminescence detection system of green fluorescent protein mark.3 α-regulating and controlling sequence (seeing sequence 2) 470 base-pairs and GFP reporter gene (seeing sequence 3) 717 base-pairs such as HSD promoter have been inserted in the beta galactosidase of taking advantage of a situation (LacZ) promoter gene (seeing sequence 1) downstream at recombinant plasmid pK-3 α-GFP3 successively; Recombinant plasmid p6 contains the whole controlling genes of 3 α-HSD (seeing sequence 4), totally 5257 base-pairs.When having substrates such as estrogen in the environment, can induce the albumen of the regulating and controlling sequence expression regulation effect of 3 α on the p6 plasmid-HSD, this albumen can make the promoter on plasmid pK-3 α-GFP3 start and then give expression to GFP albumen, and the expression strictness of GFP at this moment is subjected to inducing the control of amount of substrate.
The LacZ promoter gene of taking advantage of a situation among plasmid pK-3 α-GFP3 can act on regulating and controlling sequence and the promoter gene of the 3 α-HSD in itself plasmid, promotes green fluorescent protein great expression thereafter, and fluorescence signal is had amplification, humidification.According to the fluorescence intensity size of this fluorescent optical sensor, can calculate the relative content of specific substrate by the reference standard curve.
Construction method of the present invention is:
(1) plasmid pK-3 α-GFP3 is transformed in the Escherichia coli, cultivates 12 to 14 hours, will cultivate the back Escherichia coli and be inoculated in the nutrient culture media in 1: 20 by volume, enlarged culture, it is centrifugal to cultivate back bacterium liquid, abandons supernatant, add sterilized water washing thalline, the centrifugal supernatant of abandoning, repeated washing twice adds sterilized water and lysozyme, resuspended thalline,-20 ℃ to 25 ℃ multigelations three times, centrifugal, get the acellular system cytoplasm (can preserve month for 20 ℃) that supernatant is preparation.
(2) plasmid p6 is transformed in the Escherichia coli, cultivated 12 to 14 hours, will cultivate the back Escherichia coli and be inoculated in the nutrient culture media in 1: 20 by volume, enlarged culture, it is centrifugal to cultivate back bacterium liquid, abandons supernatant, add sterilized water washing thalline, the centrifugal supernatant of abandoning, repeated washing twice adds sterilized water and lysozyme, resuspended thalline,-20 ℃ to 25 ℃ multigelations three times, centrifugal, get the acellular system cytoplasm (can preserve month for 20 ℃) that supernatant is preparation.
(3) the acellular system cytoplasm of plasmid pK-3 α-GFP3 and the acellular system cytoplasm of plasmid P6 are measured the working fluid that protein content also is prepared into protein content=0.1mg/mL respectively respectively, two kinds of working fluid 1～2: 1～10 mixing by volume.Use this and mix acellular system construction high-performance bio fluorescent optical sensor.
Set up bioluminescence standard detection curve
(1) will detect substrate standard items anhydrous alcohol solution, compound concentration is 10 respectively -3M, 10 -6M, 10 -9M, 10 -12M, 10 -15The standard detection liquid of M/L.
(2) get acellular system detectable and add standard detection liquid respectively, blank adds absolute ethyl alcohol, leaves standstill, and detects its fluorescence radiation value with fluorescence microplate reader, sets up the standard detection curve.
Detected object of the present invention is:
Steroid hormone and palycyclic aromatic two class materials, the sample detection concentration range is: 10 -15To 10 -3Every liter of mole.
The invention has the beneficial effects as follows:
Instrument at existing detection system is strict, sense cycle is long, cost is high, sensitivity is low, detect drawbacks such as thing is single, the present invention detects linear wide ranges, technical operation is simple, instrument is less demanding, and steroid hormone and polycyclic aromatic hydrocarbon pollutant in testing environment, the food suit to be applied to.
Description of drawings
Fig. 1 is plasmid pK-3 α-GFP3 synoptic diagram;
Fig. 2 is a plasmid p6 synoptic diagram;
Fig. 3 is stosterone fluoroscopic examination figure as a result;
Fig. 4 is estradiol fluoroscopic examination figure as a result;
Fig. 5 is fluorescent naphthalimide testing result figure.
Embodiment
Embodiment 1 is that example is set up bioluminescence examination criteria curve with the stosterone
1, the cytoplasmic preparation of acellular system:
(1) plasmid pK-3 α-GFP3 is transformed in the Escherichia coli, 37 ℃, cultivated 12 to 14 hours in 180～200 rpms of constant-temperature shaking culture casees, be inoculated in the Escherichia coli after cultivating in the SIN nutrient culture media in 1: 20 by volume, 37 ℃, be cultured to OD595=3.0 in 180～200 rpms of constant-temperature shaking culture casees, to cultivate 4000 rpms of the thalline in back and abandon supernatant in centrifugal 20～30 minutes, add sterilized water washing thalline, 4000 rpms centrifugal 20～30 minutes, abandon supernatant, repeated washing twice, adding sterilized water and final concentration is 100 μ g/mL lysozymes, resuspended thalline,-20 ℃ to 25 ℃ multigelations three times, 10000 rpms centrifugal 20～30 minutes, get the acellular system cytoplasm (can preserve month for 20 ℃) that supernatant is preparation.
(2) plasmid p6 is transformed in the Escherichia coli, 37 ℃, cultivated 12 to 14 hours in 180～200 rpms of constant-temperature shaking culture casees, be inoculated in the Escherichia coli after cultivating in the SIN nutrient culture media in 1: 20 by volume, 37 ℃, be cultured to OD in 180～200 rpms of constant-temperature shaking culture casees 595=3.0, to cultivate 4000 rpms of the thalline in back and abandon supernatant in centrifugal 20～30 minutes, add sterilized water washing thalline, 4000 rpms centrifugal 20～30 minutes, abandon supernatant, twice of repeated washing, adding sterilized water and final concentration is 100 μ g/mL lysozymes, resuspended thalline ,-20 ℃ to 25 ℃ multigelations three times, 10000 rpms centrifugal 20～30 minutes, get the acellular system cytoplasm (can preserve month for 20 ℃) that supernatant is preparation.
(3) the acellular system cytoplasm of plasmid pK-3 α-GFP3 and the acellular system cytoplasm of plasmid P6 are measured the working fluid that protein content also is prepared into protein content=0.1mg/mL respectively respectively, and mixed by 1: 1 by volume.
2, set up bioluminescence standard detection curve
(1) with stosterone standard items anhydrous alcohol solution, compound concentration is the standard detection liquid of 1mM/L, 1 μ M/L, 1nM/L, 1pM/L, 1fM/L respectively.
(2) get 100 μ L mixing acellular system cytoplasms and add 2 μ L standard detection liquid respectively, blank adds 2 μ L absolute ethyl alcohols, 30 ℃ leave standstill 10 minutes after, detect its fluorescence radiation value with fluorescence microplate reader, set up the standard detection curve.
Fluoroscopic examination result such as following table:
Table 1. stosterone fluoroscopic examination result
Figure BSA00000390468400031
The stosterone detectability is respectively 0-1 μ M/L, and fluorescence intensity and concentration meet cubic polynomial curve: y=-284.24x 3+ 2225.9x 2-3172.7x+38422, R 2=0.994.
Embodiment 2 is that example is set up bioluminescence standard detection curve with the estradiol
1, the cytoplasmic preparation of acellular system:
(1) plasmid pK-3 α-GFP3 is transformed in the Escherichia coli, 37 ℃, cultivated 12 to 14 hours in 180～200 rpms of constant-temperature shaking culture casees, be inoculated in the Escherichia coli after cultivating in the SIN nutrient culture media in 1: 20 by volume, 37 ℃, be cultured to OD in 180～200 rpms of constant-temperature shaking culture casees 595=3.0, to cultivate 4000 rpms of the thalline in back and abandon supernatant in centrifugal 20～30 minutes, add sterilized water washing thalline, 4000 rpms centrifugal 20～30 minutes, abandon supernatant, twice of repeated washing, adding sterilized water and final concentration is 100 μ g/mL lysozymes, resuspended thalline ,-20 ℃ to 25 ℃ multigelations three times, 10000 rpms centrifugal 20～30 minutes, get the acellular system cytoplasm (can preserve month for 20 ℃) that supernatant is preparation.
(2) plasmid p6 is transformed in the Escherichia coli, 37 ℃, cultivated 12 to 14 hours in 180～200 rpms of constant-temperature shaking culture casees, be inoculated in the Escherichia coli after cultivating in the SIN nutrient culture media in 1: 20 by volume, 37 ℃, be cultured to OD in 180～200 rpms of constant-temperature shaking culture casees 595=3.0, to cultivate 4000 rpms of the thalline in back and abandon supernatant in centrifugal 20～30 minutes, add sterilized water washing thalline, 4000 rpms centrifugal 20～30 minutes, abandon supernatant, twice of repeated washing, adding sterilized water and final concentration is 100 μ g/mL lysozymes, resuspended thalline ,-20 ℃ to 25 ℃ multigelations three times, 10000 rpms centrifugal 20～30 minutes, get the acellular system cytoplasm (can preserve month for 20 ℃) that supernatant is preparation.
(3) the acellular system cytoplasm of plasmid pK-3 α-GFP3 and the acellular system cytoplasm of plasmid P6 are measured the working fluid that protein content also is prepared into protein content=0.1mg/mL respectively respectively, and mixed by 1: 1 by volume.
2, set up bioluminescence standard detection curve
(1) with estradiol standard items anhydrous alcohol solution, compound concentration is the standard detection liquid of 1mM/L, 1 μ M/L, 1nM/L, 1pM/L, 1fM/L respectively.
(2) get 100 μ L mixing acellular system cytoplasms and add 2 μ L standard detection liquid respectively, blank adds 2 μ L absolute ethyl alcohols, 30 ℃ leave standstill 10 minutes after, detect its fluorescence radiation value with fluorescence microplate reader, set up the standard detection curve.
Fluoroscopic examination result such as following table:
Table 2. estradiol fluoroscopic examination result
Figure BSA00000390468400041
The estradiol detectability is respectively 0-1 μ M/L, and fluorescence intensity and concentration meet cubic polynomial curve: y=3.1852x 3-344.84x 2+ 3185.1x+33801, R 2=0.9969.
Embodiment 3 is that example is set up bioluminescence standard detection curve with the naphthalene
1, the cytoplasmic preparation of acellular system:
(1) plasmid pK-3 α-GFP3 is transformed in the Escherichia coli, 37 ℃, cultivated 12 to 14 hours in 180～200 rpms of constant-temperature shaking culture casees, be inoculated in the Escherichia coli after cultivating in the SIN nutrient culture media in 1: 20 by volume, 37 ℃, be cultured to OD in 180～200 rpms of constant-temperature shaking culture casees 595=3.0, to cultivate 4000 rpms of the thalline in back and abandon supernatant in centrifugal 20～30 minutes, add sterilized water washing thalline, 4000 rpms centrifugal 20～30 minutes, abandon supernatant, twice of repeated washing, adding sterilized water and final concentration is 100 μ g/mL lysozymes, resuspended thalline ,-20 ℃ to 25 ℃ multigelations three times, 10000 rpms centrifugal 20～30 minutes, get the acellular system cytoplasm (can preserve month for 20 ℃) that supernatant is preparation.
(2) plasmid p6 is transformed in the Escherichia coli, 37 ℃, cultivated 12 to 14 hours in 180～200 rpms of constant-temperature shaking culture casees, be inoculated in the Escherichia coli after cultivating in the SIN nutrient culture media in 1: 20 by volume, 37 ℃, be cultured to OD in 180～200 rpms of constant-temperature shaking culture casees 595=3.0, to cultivate 4000 rpms of the thalline in back and abandon supernatant in centrifugal 20～30 minutes, add sterilized water washing thalline, 4000 rpms centrifugal 20～30 minutes, abandon supernatant, twice of repeated washing, adding sterilized water and final concentration is 100 μ g/mL lysozymes, resuspended thalline ,-20 ℃ to 25 ℃ multigelations three times, 10000 rpms centrifugal 20～30 minutes, get the acellular system cytoplasm (can preserve month for 20 ℃) that supernatant is preparation.
(3) the acellular system cytoplasm of plasmid pK-3 α-GFP3 and the acellular system cytoplasm of plasmid P6 are measured the working fluid that protein content also is prepared into protein content=0.1mg/mL respectively respectively, and mixed by 1: 1 by volume.
2, set up bioluminescence standard detection curve
(1) with naphthalene standard items anhydrous alcohol solution, compound concentration is the standard detection liquid of 1mM/L, 1 μ M/L, 1nM/L, 1pM/L, 1fM/L respectively.
(2) get 100 μ L mixing acellular system cytoplasms and add 2 μ L standard detection liquid respectively, blank adds 2 μ L absolute ethyl alcohols, 30 ℃ leave standstill 10 minutes after, detect its fluorescence radiation value with fluorescence microplate reader, set up the standard detection curve.Fluoroscopic examination result such as following table:
Table 3. fluorescent naphthalimide testing result
Figure BSA00000390468400051
The estradiol detectability is respectively 0-1 μ M/L, and fluorescence intensity and concentration meet cubic polynomial curve: y=12.815x 3-125.23x 2+ 1444.4x+36335, R 2=0.9298.
Figure ISA00000390468600011
Figure ISA00000390468600021
Figure ISA00000390468600031
Figure ISA00000390468600041

Claims (5)

1. steroid hormone and palycyclic aromatic high-performance bio fluorescent optical sensor, it is characterized in that: transform into plasmid pK-3 α-GFP3 and p6 in the Escherichia coli respectively and be prepared into acellular system detectable respectively, set up a kind of bioluminescence detection system of green fluorescent protein mark; 3 α-regulating and controlling sequence 470 base-pairs and GFP reporter gene 717 base-pairs such as HSD promoter have been inserted in the beta galactosidase promoter gene downstream of taking advantage of a situation at recombinant plasmid pK-3 α-GFP3 successively; Recombinant plasmid p6 contains the whole controlling genes of 3 α-HSD, totally 5257 base-pairs; When having substrates such as estrogen in the environment, can induce the albumen of the regulating and controlling sequence expression regulation effect of 3 α on the p6 plasmid-HSD, this albumen can make the promoter on plasmid pK-3 α-GFP3 start and then give expression to GFP albumen, and the expression strictness of GFP at this moment is subjected to inducing the control of amount of substrate.
2. the construction method of steroid hormone and palycyclic aromatic high-performance bio fluorescent optical sensor, its construction method is:
A, plasmid pK-3 α-GFP3 are transformed in the Escherichia coli, cultivate 12 to 14 hours, will cultivate the back Escherichia coli and be inoculated in the nutrient culture media in 1: 20 by volume, enlarged culture, it is centrifugal to cultivate back bacterium liquid, abandons supernatant, add sterilized water washing thalline, the centrifugal supernatant of abandoning, repeated washing twice adds sterilized water and lysozyme, resuspended thalline,-20 ℃ to 25 ℃ multigelations three times, centrifugal, get the acellular system cytoplasm that supernatant is preparation;
B, plasmid p6 is transformed in the Escherichia coli, cultivated 12 to 14 hours, will cultivate the back Escherichia coli and be inoculated in the nutrient culture media in 1: 20 by volume, enlarged culture, it is centrifugal to cultivate back bacterium liquid, abandons supernatant, add sterilized water washing thalline, the centrifugal supernatant of abandoning, repeated washing twice adds sterilized water and lysozyme, resuspended thalline,-20 ℃ to 25 ℃ multigelations three times, centrifugal, get the acellular system cytoplasm that supernatant is preparation;
C, the acellular system cytoplasm of the acellular system cytoplasm of plasmid pK-3 α-GFP3 and plasmid P6 measured protein content respectively and be prepared into the working fluid of protein content=0.1mg/mL respectively, two kinds of working fluids mix by volume at 1～2: 1～10; Use this and mix acellular system construction high-performance bio fluorescent optical sensor;
D, will detect substrate standard items anhydrous alcohol solution, compound concentration is 10 respectively -3M, 10 -6M, 10 -9M, 10 -12M, 10 -15The standard detection liquid of M/L;
F, get acellular system detectable and add standard detection liquid respectively, blank adds absolute ethyl alcohol, leaves standstill, and detects its fluorescence radiation value with fluorescence microplate reader, sets up the standard detection curve.
3. a kind of steroid hormone according to claim 1 and palycyclic aromatic high-performance bio fluorescent optical sensor, it is characterized in that: the LacZ promoter gene of taking advantage of a situation among plasmid pK-3 α-GFP3 can act on regulating and controlling sequence and the promoter gene of the 3 α-HSD in itself plasmid, promote green fluorescent protein great expression thereafter, fluorescence signal is had amplification, humidification.
4. a kind of steroid hormone according to claim 1 and palycyclic aromatic high-performance bio fluorescent optical sensor is characterized in that: according to the fluorescence intensity size of this fluorescent optical sensor, calculate the relative content of specific substrate by the reference standard opisometer.
5. a kind of steroid hormone according to claim 1 and palycyclic aromatic high-performance bio fluorescent optical sensor is characterized in that: detected object is steroid hormone and palycyclic aromatic two class materials, and the sample detection concentration range is 10 -15To 10 -3Every liter of mole.
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