CN102135497A - High-efficiency chemiluminescence sensor for detecting steroid hormone and polycyclic aromatic hydrocarbon - Google Patents
High-efficiency chemiluminescence sensor for detecting steroid hormone and polycyclic aromatic hydrocarbon Download PDFInfo
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- CN102135497A CN102135497A CN2010105950816A CN201010595081A CN102135497A CN 102135497 A CN102135497 A CN 102135497A CN 2010105950816 A CN2010105950816 A CN 2010105950816A CN 201010595081 A CN201010595081 A CN 201010595081A CN 102135497 A CN102135497 A CN 102135497A
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Abstract
The invention provides a high-efficiency chemiluminescence sensor for detecting steroid hormone and polycyclic aromatic hydrocarbon, belonging to the technical field of molecular biology and microbiology. The high-efficiency chemiluminescence sensor is characterized in that plasmid pTopo-3Alpha-F1 and plasmid p6 are converted into escherichia coli so as to establish a luminescent detecting system; the recombined plasmid p6 contains 3Alpha-HSD full regulation genes and 5257 base pairs in total; when substances such as oestrogens and the like exist in the environment, the protein expressing the regulation function by the regulation sequence can be induced on the 3Alpha-HSD on the plasmid p6; the protein can lead a starter on the plasmid pTopo-3Alpha-F1 to start, thus expressing luciferase; after the luciferase substrate is added, the chemiluminescence can be generated, and the luminous intensity can be strictly controlled by the inducer content at the moment. The high-efficiency chemiluminescence sensor has the benefits that the detecting luminous intensity can be higher by more than 10 times compared with the green fluorescent protein gene; and the detecting system is stable. The expression has amplification and reinforcement effect and the linear scope of the detection is further expanded. The high-efficiency chemiluminescence sensor can be applied to the detection on all steroid hormone and polycyclic aromatic hydrocarbons.
Description
Technical field
The invention belongs to molecular biology and microbiological technique field.
Background technology
Be accompanied by the fast development of worker, farming, animal husbandry, environment that steroid hormone and multiring aromatic hydrocarbon substance cause and food pollution are serious day by day, have brought significant damage for human lives's health.How to detect the concern of extremely domestic and international government department of this two pollutant and correlative study mechanism fast and efficiently.At present, steroid hormone and polycyclic aromatic hydrocarbon pollutant detection method are mainly chemistry and biological two big classes in environment, the food: the former mostly is chromatogram and mass spectroscopy, the large-scale costliness of instrument and equipment, and test sample is single, the sample pre-treatments complexity, and can't detect types of unknown pollutants; Some biological detection method such as cell increment experiment etc., loaded down with trivial details, the consuming time length of process, cost height, test sample is single, and is strict to testing environment, experimenter.
Summary of the invention
The objective of the invention is: a kind of high-performance bio chemistry luminescence sensor that detects steroid hormone and palycyclic aromatic is provided, plasmid pTopo-3 α-F1 and plasmid p6 are transformed respectively in the Escherichia coli, set up a kind of high-performance bio chemistry luminescent detection system of firefly luciferase gene 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 high-performance bio chemistry luminescent 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.Luciferase (Luciferase) is the general designation that occurring in nature can produce the enzyme of bioluminescence, and wherein most representative is that a kind of formal name used at school is the luciferase (firefly luciferase) in the light of firefly polypide of " Photinus pyrali ".Firefly luciferase is highly sensitive, is applicable to high flux screening, and therefore from 1986, firefly luciferase gene is used as the reporter gene of measuring gene expression, has obtained to use widely.
The present invention transforms plasmid pTopo-3 α-F1 and plasmid p6 respectively in the Escherichia coli, has set up a kind of high-performance bio chemistry luminescent detection system of firefly luciferase gene mark.3 α-regulating and controlling sequence (seeing sequence 2) 470 base-pairs and firefly luciferase reporter gene (seeing sequence 3) 1647 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 pTopo-3 α-F1 successively; Recombinant plasmid p6 contains the whole controlling genes of 3 α-HSD (seeing sequence 4), totally 5257 base-pairs.When having materials 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 pTopo-3 α-F1 start and then give expression to luciferase, after adding the luciferase substrate, can produce chemiluminescence, this moment, the luminous intensity strictness was subjected to the control of inducer content.
The LacZ promoter gene of taking advantage of a situation among plasmid pTopo-3 α-F1 can act on regulating and controlling sequence and the promoter gene of the 3 α-HSD in itself plasmid, promote luciferase great expression thereafter, after adding the luciferase substrate, it is luminous to produce extensive chemical, and its luminous signal is had amplification, humidification.According to the luminous intensity size of this high-performance bio chemistry luminescence sensor, can calculate the relative content of specificity substance by the reference standard curve.
Construction method of the present invention:
1, plasmid pTopo-3 α-F1 is transformed among the Escherichia coli HB101, obtains a strain recombination bacillus coli HB101-F1 through two resistance screenings:
(1) get the Escherichia coli HB101 competent cell of prepared fresh, cell is evenly suspended to be placed on ice.
(2) add plasmid pTopo-3 α-F1, ice bath.
(3) 42 ℃ of water-bath heat shocks 90 seconds were placed rapidly 3 minutes on ice.
(4) add the LB nutrient culture media of 37 ℃ of preheatings, 37 ℃, 120 changeed the per minute shaken cultivation more than 60 minutes;
(5) bacterium is coated on the LB agar plate that contains kanamycins (kannmycin) and the two resistances of ampicillin (ampicillin).
(6) flat board is placed more than 1 hour at 37 ℃ of following forwards, after Liquid Absorption to be coated is advanced agar, flat board is inverted, and cultivates more than 16 hours.
(7) picking list bacterium colony in liquid LB medium culture, extracts plasmid and identifies that screening positive clone promptly obtains recombination bacillus coli HB101-F1.
2, plasmid p6 is transformed among the Escherichia coli HB101, obtains a strain recombination bacillus coli HB101-p6 through resistance screening:
(1) get the Escherichia coli HB101 competent cell of prepared fresh, cell is evenly suspended to be placed on ice.
(2) add plasmid p6, ice bath.
(3) 42 ℃ of water-bath heat shocks 90 seconds were placed rapidly 3 minutes on ice.
(4) add the LB nutrient culture media of 37 ℃ of preheatings, 37 ℃, 120 changeed the per minute shaken cultivation more than 60 minutes;
(5) bacterium is coated on the LB agar plate that contains ampicillin (ampicillin) resistance.
(6) dull and stereotyped 37 ℃ of following forwards placements 1 hour, after Liquid Absorption to be coated is advanced agar,, cultivate more than 16 hours with the flat board inversion.
(7) picking list bacterium colony in liquid LB medium culture, extracts plasmid and identifies that screening positive clone promptly obtains recombination bacillus coli HB101-p6.
3, preparation high-performance bio chemistry luminescence sensor reagent
(1) 1: 50 by volume ratio of recombination bacillus coli HB101-F1 is inoculated in the LB nutrient culture media, 37 ℃, 180～200 change shaken cultivation in the per minute constant incubator, to OD
600=2.0～4.0.
(2) taking out 4000 changeed per minute centrifugal 20～30 minutes, removed supernatant, added sterilized water with the thalline mixing, and 4000 changeed per minute centrifugal 20～30 minutes, removed supernatant, repeated twice.
(3) sterilized water and the final concentration that adds former thalline volume 1/10 is 100 μ g/ml lysozymes, abundant mixing, and-20 ℃ of multigelations three times take out, and 10000 changeed per minutes centrifugal 20～30 minutes, drew the HB101-F1 cell free system cytoplasm that supernatant is preparation.
(4) 1: 50 by volume ratio of recombination bacillus coli HB101-p6 is inoculated in the LB nutrient culture media, 37 ℃, 180～200 change shaken cultivation in the per minute constant incubator, to OD
600=2.0～4.0.
(5) taking out 4000 changeed per minute centrifugal 20～30 minutes, removed supernatant, added sterilized water with the thalline mixing, and 4000 changeed per minute centrifugal 20～30 minutes, removed supernatant, repeated twice.
(6) sterilized water and the final concentration that adds former thalline volume 1/10 is 100 μ g/ml lysozymes, abundant mixing, and-20 ℃ of multigelations three times take out, and 10000 changeed per minutes centrifugal 20～30 minutes, drew the HB101-p6 cell free system cytoplasm that supernatant is preparation.
(7) the recombination bacillus coli HB101-F1 cell free system cytoplasm of preparation and the recombination bacillus coli HB101-p6 cell free system cytoplasm of preparation are measured protein content respectively with the Coomassie brilliant blue method.With sterilized water two kinds of cell free system cytoplasms are diluted to 0.02mg/ml respectively, and 1: 1 by volume ratio mixes, be the high-performance bio chemistry luminescence sensor reagent of preparation.
4, set up high-performance bio chemistry emission standards detection curve
(1) with the standard items anhydrous alcohol solution of test sample, compound concentration is the standard detection liquid of 1fM/L, 1pM/L, 1nM/L, 1 μ M/L, 1mM/L respectively.
(2) get high-performance bio chemistry luminescence sensor reagent and add standard detection liquid respectively, blank adds absolute ethyl alcohol, leaves standstill, and adds the luciferase substrate again, detects its values of chemiluminescence 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:
1, the reporter gene firefly luciferase gene among plasmid pTopo-3 α-F1 (firefly luciferase:F1) utilizes reporter gene green fluorescence protein gene (GFP) to compare with this laboratory before, detect luminous intensity and exceed more than ten times, and detection system is stable.
2, beta-galactosidase gene (LacZ) promoter of taking advantage of a situation among plasmid pTopo-3 α-F1 has amplification, enhancement effect to the expression of firefly luciferase gene, has further enlarged the range of linearity (10 that detects
-15To 10
-3Every liter of mole), compare with the sensor of primitive green fluorescence protein gene (GFP) mark, the detection range of linearity of the standard detection liquid of different steroid hormones, palycyclic aromatic can exceed 3 to 9 orders of magnitude.
3, the sensor time of setting up with the cell free system detectable short, only needed 15 minutes can finish detection, detection volume is little, has stable, characteristics fast and efficiently.
4, detection method in the past must be under the prerequisite of known detection material can detection material content, and high-performance bio chemistry luminescence sensor can testing environment in the content of unknown steroid hormone and palycyclic aromatic.
5, can be applicable to the detection of all steroid hormones and palycyclic aromatic.
Description of drawings
Fig. 1, plasmid pTopo-3 α-F1 synoptic diagram;
Fig. 2, plasmid p6 synoptic diagram;
Fig. 3, stosterone high-performance bio chemistry emission standards detection curve;
Fig. 4, estradiol high-performance bio chemistry emission standards detection curve;
Fig. 5, benzopyrene high-performance bio chemistry emission standards detection curve;
Embodiment:
1, the structure of recombination bacillus coli HB101-F1
(1) get 100 μ L Escherichia coli HB101 competent cells of prepared fresh, cell is evenly suspended to be placed on ice.
(2) add plasmid pTopo-3 α-F1 3 μ L, placed on ice 30 minutes.
(3) 42 ℃ of water-bath heat shocks 90 seconds were placed rapidly 3 minutes on ice.
(4) add the LB nutrient culture media of 37 ℃ of preheatings of 600 μ L, 37 ℃, 120 changeed the per minute shaken cultivation 60 minutes;
(5) 200 μ L bacteriums are coated on the LB agar plate that contains kanamycins (kannmycin) and the two resistances of ampicillin (ampicillin).
(6) dull and stereotyped 37 ℃ of following forwards placements 1 hour, after Liquid Absorption to be coated is advanced agar, flat board is inverted incubated overnight 16 hours.
(7) picking list bacterium colony in liquid LB medium culture, extracts plasmid and identifies that screening positive clone promptly obtains recombination bacillus coli HB101-F1.
2, the structure of recombination bacillus coli HB101-p6
(1) get 100 μ L Escherichia coli HB101 competent cells of prepared fresh, cell is evenly suspended to be placed on ice.
(2) add plasmid p6 3 μ L, placed on ice 30 minutes.
(3) 42 ℃ of water-bath heat shocks 90 seconds were placed rapidly 3 minutes on ice.
(4) add the LB nutrient culture media of 37 ℃ of preheatings of 600 μ L, 37 ℃, 120 changeed the per minute shaken cultivation 60 minutes;
(5) 200 μ L bacteriums are coated on the LB agar plate that contains ampicillin (ampicillin) resistance.
(6) dull and stereotyped 37 ℃ of following forwards placements 1 hour, after Liquid Absorption to be coated is advanced agar, flat board is inverted incubated overnight 16 hours.
(7) picking list bacterium colony in liquid LB medium culture, extracts plasmid and identifies that screening positive clone promptly obtains recombination bacillus coli HB101-p6.
3, preparation high-performance bio chemistry luminescence sensor reagent.
(1) 1: 50 by volume ratio of recombination bacillus coli HB101-F1 is inoculated in the LB nutrient culture media, 37 ℃, 180 change shaken cultivation in the per minute constant incubator, to OD
600=3.0.
(2) taking out 4000 changeed per minute centrifugal 20 minutes, removed supernatant, added sterilized water with the thalline mixing, and 4000 changeed per minute centrifugal 20 minutes, removed supernatant, repeated twice.
(3) sterilized water and the final concentration of the former thalline volume 1/10 of adding are 100 μ g/ml lysozymes, abundant mixing ,-20 ℃ of multigelations three times take out, 10000 changeed per minute centrifugal 20 minutes, drew the recombination bacillus coli HB101-F1 cell free system cytoplasm that supernatant is preparation.
(4) 1: 50 by volume ratio of recombination bacillus coli HB101-p6 is inoculated in the LB nutrient culture media, 37 ℃, 180 change shaken cultivation in the per minute constant incubator, to OD
600=3.0.
(5) taking out 4000 changeed per minute centrifugal 20 minutes, removed supernatant, added sterilized water with the thalline mixing, and 4000 changeed per minute centrifugal 20 minutes, removed supernatant, repeated twice.
(6) sterilized water and the final concentration of the former thalline volume 1/10 of adding are 100 μ g/ml lysozymes, abundant mixing ,-20 ℃ of multigelations three times take out, 10000 changeed per minute centrifugal 20 minutes, drew the recombination bacillus coli HB101-p6 cell free system cytoplasm that supernatant is preparation.
(7) the recombination bacillus coli HB101-F1 cell free system cytoplasm of preparation and the recombination bacillus coli HB101-p6 cell free system cytoplasm of preparation are measured protein content respectively with the Coomassie brilliant blue method.With sterilized water two kinds of cell free system cytoplasms are diluted to 0.02mg/ml respectively, and 1: 1 by volume ratio mixes, be the high-performance bio chemistry luminescence sensor reagent of preparation.
4, set up stosterone standard items high-performance bio chemistry emission standards detection curve
(1) with stosterone standard items anhydrous alcohol solution, compound concentration is the standard detection liquid of 1fM/L, 1pM/L, 1nM/L, 1 μ M/L, 1mM/L respectively.
(2) get the stosterone standard detection liquid that per 100 μ L high-performance bios chemistry luminescence sensor reagent adds 5 concentration of 2 μ L respectively, the blank group adds 2 μ L absolute ethyl alcohols, totally six samples, place after 10 minutes for 30 ℃, each sample all adds the luciferase substrate of 5 μ L, detect its values of chemiluminescence with fluorescence microplate reader, and set up typical curve.
Table 1. stosterone chemiluminescence detection result
Stosterone standard items sensing range is 10
-15To 10
-6Every liter of mole detects chemiluminescence intensity and concentration and meets cubic polynomial curve: y=-16434x3+149201x2-321112x+408121, R
2=0.9973
1, the structure of recombination bacillus coli HB101-F1
(1) get 100 μ L Escherichia coli HB101 competent cells of prepared fresh, cell is evenly suspended to be placed on ice.
(2) add plasmid pTopo-3 α-F1 3 μ L, placed on ice 30 minutes.
(3) 42 ℃ of water-bath heat shocks 90 seconds were placed rapidly 3 minutes on ice.
(4) add the LB nutrient culture media of 37 ℃ of preheatings of 600 μ L, 37 ℃, 120 changeed the per minute shaken cultivation 60 minutes;
(5) 200 μ L bacteriums are coated on the LB agar plate that contains kanamycins (kannmycin) and the two resistances of ampicillin (ampicillin).
(6) dull and stereotyped 37 ℃ of following forwards placements 1 hour, after Liquid Absorption to be coated is advanced agar, flat board is inverted incubated overnight 16 hours.
(7) picking list bacterium colony in liquid LB medium culture, extracts plasmid and identifies that screening positive clone promptly obtains recombination bacillus coli HB101-F1.
2, the structure of recombination bacillus coli HB101-p6
(1) get 100 μ L Escherichia coli HB101 competent cells of prepared fresh, cell is evenly suspended to be placed on ice.
(2) add plasmid p6 3 μ L, placed on ice 30 minutes.
(3) 42 ℃ of water-bath heat shocks 90 seconds were placed rapidly 3 minutes on ice.
(4) add the LB nutrient culture media of 37 ℃ of preheatings of 600 μ L, 37 ℃, 120 changeed the per minute shaken cultivation 60 minutes;
(5) 200 μ L bacteriums are coated on the LB agar plate that contains ampicillin (ampicillin) resistance.
(6) dull and stereotyped 37 ℃ of following forwards placements 1 hour, after Liquid Absorption to be coated is advanced agar, flat board is inverted incubated overnight 16 hours.
(7) picking list bacterium colony in liquid LB medium culture, extracts plasmid and identifies that screening positive clone promptly obtains recombination bacillus coli HB101-p6.
3, preparation high-performance bio chemistry luminescence sensor reagent.
(1) 1: 50 by volume ratio of recombination bacillus coli HB101-F1 is inoculated in the LB nutrient culture media, 37 ℃, 180 change shaken cultivation in the per minute constant incubator, to OD
600=3.0.
(2) taking out 4000 changeed per minute centrifugal 20 minutes, removed supernatant, added sterilized water with the thalline mixing, and 4000 changeed per minute centrifugal 20 minutes, removed supernatant, repeated twice.
(3) sterilized water and the final concentration of the former thalline volume 1/10 of adding are 100 μ g/ml lysozymes, abundant mixing ,-20 ℃ of multigelations three times take out, 10000 changeed per minute centrifugal 20 minutes, drew the recombination bacillus coli HB101-F1 cell free system cytoplasm that supernatant is preparation.
(4) 1: 50 by volume ratio of recombination bacillus coli HB101-p6 is inoculated in the LB nutrient culture media, 37 ℃, 180 change shaken cultivation in the per minute constant incubator, to OD
600=3.0.
(5) taking out 4000 changeed per minute centrifugal 20 minutes, removed supernatant, added sterilized water with the thalline mixing, and 4000 changeed per minute centrifugal 20 minutes, removed supernatant, repeated twice.
(6) sterilized water and the final concentration of the former thalline volume 1/10 of adding are 100 μ g/ml lysozymes, abundant mixing ,-20 ℃ of multigelations three times take out, 10000 changeed per minute centrifugal 20 minutes, drew the recombination bacillus coli HB101-p6 cell free system cytoplasm that supernatant is preparation.
(7) the recombination bacillus coli HB101-F1 cell free system cytoplasm of preparation and the recombination bacillus coli HB101-p6 cell free system cytoplasm of preparation are measured protein content respectively with the Coomassie brilliant blue method.With sterilized water two kinds of cell free system cytoplasms are diluted to 0.02mg/ml respectively, and 1: 1 by volume ratio mixes, be the high-performance bio chemistry luminescence sensor reagent of preparation.
4, set up estradiol standard items high-performance bio chemistry emission standards detection curve
(1) with estradiol standard items anhydrous alcohol solution, compound concentration is the standard detection liquid of 1fM/L, 1pM/L, 1nM/L, 1 μ M/L, 1mM/L respectively.
(2) get the estradiol standard detection liquid that per 100 μ L high-performance bios chemistry luminescence sensor reagent adds 5 concentration of 2 μ L respectively, the blank group adds 2 μ L absolute ethyl alcohols, totally six samples, place after 10 minutes for 30 ℃, each sample all adds the luciferase substrate of 5 μ L, detect its values of chemiluminescence with fluorescence microplate reader, and set up typical curve.
Table 2. estradiol chemiluminescence detection result
Estradiol standard items sensing range is every liter of 10-15 to a 10-6 mole, detects chemiluminescence intensity and concentration and meets cubic polynomial curve: y=-15615x3+147277x2-340838x+436881, R
2=0.9844
1, the structure of recombination bacillus coli HB101-F1
(1) get 100 μ L Escherichia coli HB101 competent cells of prepared fresh, cell is evenly suspended to be placed on ice.
(2) add plasmid pTopo-3 α-F1 3 μ L, placed on ice 30 minutes.
(3) 42 ℃ of water-bath heat shocks 90 seconds were placed rapidly 3 minutes on ice.
(4) add the LB nutrient culture media of 37 ℃ of preheatings of 600 μ L, 37 ℃, 120 changeed the per minute shaken cultivation 60 minutes;
(5) 200 μ L bacteriums are coated on the LB agar plate that contains kanamycins (kannmycin) and the two resistances of ampicillin (ampicillin).
(6) dull and stereotyped 37 ℃ of following forwards placements 1 hour, after Liquid Absorption to be coated is advanced agar, flat board is inverted incubated overnight 16 hours.
(7) picking list bacterium colony in liquid LB medium culture, extracts plasmid and identifies that screening positive clone promptly obtains recombination bacillus coli HB101-F1.
2, the structure of recombination bacillus coli HB101-p6
(1) get 100 μ L Escherichia coli HB101 competent cells of prepared fresh, cell is evenly suspended to be placed on ice.
(2) add plasmid p6 3 μ L, placed on ice 30 minutes.
(3) 42 ℃ of water-bath heat shocks 90 seconds were placed rapidly 3 minutes on ice.
(4) add the LB nutrient culture media of 37 ℃ of preheatings of 600 μ L, 37 ℃, 120 changeed the per minute shaken cultivation 60 minutes;
(5) 200 μ L bacteriums are coated on the LB agar plate that contains ampicillin (ampicillin) resistance.
(6) dull and stereotyped 37 ℃ of following forwards placements 1 hour, after Liquid Absorption to be coated is advanced agar, flat board is inverted incubated overnight 16 hours.
(7) picking list bacterium colony in liquid LB medium culture, extracts plasmid and identifies that screening positive clone promptly obtains recombination bacillus coli HB101-p6.
3, preparation high-performance bio chemistry luminescence sensor reagent.
(1) 1: 50 by volume ratio of recombination bacillus coli HB101-F1 is inoculated in the LB nutrient culture media, 37 ℃, 180 change shaken cultivation in the per minute constant incubator, to OD
600=3.0.
(2) taking out 4000 changeed per minute centrifugal 20 minutes, removed supernatant, added sterilized water with the thalline mixing, and 4000 changeed per minute centrifugal 20 minutes, removed supernatant, repeated twice.
(3) sterilized water and the final concentration of the former thalline volume 1/10 of adding are 100 μ g/ml lysozymes, abundant mixing ,-20 ℃ of multigelations three times take out, 10000 changeed per minute centrifugal 20 minutes, drew the recombination bacillus coli HB101-F1 cell free system cytoplasm that supernatant is preparation.
(4) 1: 50 by volume ratio of recombination bacillus coli HB101-p6 is inoculated in the LB nutrient culture media, 37 ℃, 180 change shaken cultivation in the per minute constant incubator, to OD
600=3.0.
(5) taking out 4000 changeed per minute centrifugal 20 minutes, removed supernatant, added sterilized water with the thalline mixing, and 4000 changeed per minute centrifugal 20 minutes, removed supernatant, repeated twice.
(6) sterilized water and the final concentration of the former thalline volume 1/10 of adding are 100 μ g/ml lysozymes, abundant mixing ,-20 ℃ of multigelations three times take out, 10000 changeed per minute centrifugal 20 minutes, drew the recombination bacillus coli HB101-p6 cell free system cytoplasm that supernatant is preparation.
(7) the recombination bacillus coli HB101-F1 cell free system cytoplasm of preparation and the recombination bacillus coli HB101-p6 cell free system cytoplasm of preparation are measured protein content respectively with the Coomassie brilliant blue method.With sterilized water two kinds of cell free system cytoplasms are diluted to 0.02mg/ml respectively, and 1: 1 by volume ratio mixes, be the high-performance bio chemistry luminescence sensor reagent of preparation.
4, set up benzopyrene standard items high-performance bio chemistry emission standards detection curve
(1) with benzopyrene standard items anhydrous alcohol solution, compound concentration is the standard detection liquid of 1fM/L, 1pM/L, 1nM/L, 1 μ M/L, 1mM/L respectively.
(2) get the benzopyrene standard detection liquid that per 100 μ L high-performance bios chemistry luminescence sensor reagent adds 5 concentration of 2 μ L respectively, the blank group adds 2 μ L absolute ethyl alcohols, totally six samples, place after 10 minutes for 30 ℃, each sample all adds the luciferase substrate of 5 μ L, detect its values of chemiluminescence with fluorescence microplate reader, and set up typical curve.
Table 3. benzopyrene chemiluminescence detection result
Benzopyrene standard items sensing range is 10
-15To 10
-3Every liter of mole detects chemiluminescence intensity and concentration and meets cubic polynomial curve: y=-7930.5x3+77226x2-145810x+286787, R
2=0.9814.
Claims (5)
1. high-performance bio chemistry luminescence sensor that detects steroid hormone and palycyclic aromatic, it is characterized in that: plasmid pTopo-3 α-F1 and plasmid p6 are transformed respectively in the Escherichia coli, set up a kind of high-performance bio chemistry luminescent detection system of firefly luciferase gene mark, inserted 3 α-regulating and controlling sequence 470 base-pairs and firefly luciferase reporter gene 1647 base-pairs such as HSD promoter successively in the beta galactosidase promoter gene downstream of taking advantage of a situation of recombinant plasmid pTopo-3 α-F1; Recombinant plasmid p6 contains the whole controlling genes of 3 α-HSD, totally 5257 base-pairs; When having materials 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 pTopo-3 α-F1 start and then give expression to luciferase, after adding the luciferase substrate, can produce chemiluminescence, this moment, the luminous intensity strictness was subjected to the control of inducer content.
2. a kind of high-performance bio chemistry luminescence sensor that detects steroid hormone and palycyclic aromatic according to claim 1, it is characterized in that: the LacZ promoter gene of taking advantage of a situation among plasmid pTopo-3 α-F1 can act on regulating and controlling sequence and the promoter gene of the 3 α-HSD in itself plasmid, promote luciferase great expression thereafter, after adding the luciferase substrate, it is luminous to produce extensive chemical, and its luminous signal is had amplification, humidification.
3. a kind of high-performance bio chemistry luminescence sensor that detects steroid hormone and palycyclic aromatic according to claim 1, it is characterized in that:, can calculate the relative content of specificity substance by the reference standard curve according to the luminous intensity size of this high-performance bio chemistry luminescence sensor.
4. a kind of high-performance bio chemistry luminescence sensor that detects steroid hormone and palycyclic aromatic according to claim 1, it 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.
5. a kind of high-performance bio chemistry luminescence sensor that detects steroid hormone and palycyclic aromatic according to claim 1, its construction method is:
A, plasmid pTopo-3 α-F1 is transformed among the Escherichia coli HB101, obtain a strain recombination bacillus coli HB101-F1 through two resistance screenings;
(1) get the Escherichia coli HB101 competent cell of prepared fresh, cell is evenly suspended to be placed on ice;
(2) add plasmid pTopo-3 α-F1, ice bath;
(3) 42 ℃ of water-bath heat shocks 90 seconds were placed rapidly 3 minutes on ice;
(4) add the LB nutrient culture media of 37 ℃ of preheatings, 37 ℃, 120 changeed the per minute shaken cultivation more than 60 minutes;
(5) bacterium is coated on the LB agar plate that contains kanamycins (kannmycin) and the two resistances of ampicillin (ampicillin);
(6) flat board is placed more than 1 hour at 37 ℃ of following forwards, after Liquid Absorption to be coated is advanced agar, flat board is inverted, and cultivates more than 16 hours;
(7) picking list bacterium colony in liquid LB medium culture, extracts plasmid and identifies that screening positive clone promptly obtains recombination bacillus coli HB101-F1;
B, plasmid p6 is transformed among the Escherichia coli HB101, obtain a strain recombination bacillus coli HB101-p6 through resistance screening;
(1) get the Escherichia coli HB101 competent cell of prepared fresh, cell is evenly suspended to be placed on ice;
(2) add plasmid p6, ice bath;
(3) 42 ℃ of water-bath heat shocks 90 seconds were placed rapidly 3 minutes on ice;
(4) add the LB nutrient culture media of 37 ℃ of preheatings, 37 ℃, 120 changeed the per minute shaken cultivation more than 60 minutes;
(5) bacterium is coated on the LB agar plate that contains ampicillin (ampicillin) resistance;
(6) dull and stereotyped 37 ℃ of following forwards placements 1 hour, after Liquid Absorption to be coated is advanced agar,, cultivate more than 16 hours with the flat board inversion;
(7) picking list bacterium colony in liquid LB medium culture, extracts plasmid and identifies that screening positive clone promptly obtains recombination bacillus coli HB101-p6;
C, preparation high-performance bio chemistry luminescence sensor reagent
(1) 1: 50 by volume ratio of recombination bacillus coli HB101-F1 is inoculated in the LB nutrient culture media, 37 ℃, 180～200 change shaken cultivation in the per minute constant incubator, to OD
600=2.0～4.0;
(2) taking out 4000 changeed per minute centrifugal 20～30 minutes, removed supernatant, added sterilized water with the thalline mixing, and 4000 changeed per minute centrifugal 20～30 minutes, removed supernatant, repeated twice;
(3) sterilized water and the final concentration that adds former thalline volume 1/10 is 100 μ g/ml lysozymes, abundant mixing, and-20 ℃ of multigelations three times take out, and 10000 changeed per minutes centrifugal 20～30 minutes, drew the HB101-F1 cell free system cytoplasm that supernatant is preparation;
(4) 1: 50 by volume ratio of recombination bacillus coli HB101-p6 is inoculated in the LB nutrient culture media, 37 ℃, 180～200 change shaken cultivation in the per minute constant incubator, to OD
600=2.0～4.0;
(5) taking out 4000 changeed per minute centrifugal 20～30 minutes, removed supernatant, added sterilized water with the thalline mixing, and 4000 changeed per minute centrifugal 20～30 minutes, removed supernatant, repeated twice;
(6) sterilized water and the final concentration that adds former thalline volume 1/10 is 100 μ g/ml lysozymes, abundant mixing, and-20 ℃ of multigelations three times take out, and 10000 changeed per minutes centrifugal 20～30 minutes, drew the HB101-p6 cell free system cytoplasm that supernatant is preparation;
(7) the recombination bacillus coli HB101-F1 cell free system cytoplasm of preparation and the recombination bacillus coli HB101-p6 cell free system cytoplasm of preparation are measured protein content respectively with the Coomassie brilliant blue method.With sterilized water two kinds of cell free system cytoplasms are diluted to 0.02mg/ml respectively, and 1: 1 by volume ratio mixes, be the high-performance bio chemistry luminescence sensor reagent of preparation;
D, set up high-performance bio chemistry emission standards detection curve
(1) with the standard items anhydrous alcohol solution of test sample, compound concentration is the standard detection liquid of 1fM/L, 1pM/L, 1nM/L, 1 μ M/L, 1mM/L respectively;
(2) get high-performance bio chemistry luminescence sensor reagent and add standard detection liquid respectively, blank adds absolute ethyl alcohol, leaves standstill, and adds the luciferase substrate again, detects its values of chemiluminescence with fluorescence microplate reader, sets up the standard detection curve.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645109A (en) * | 2016-12-29 | 2017-05-10 | Tcl集团股份有限公司 | Method, device and system for detecting hormones |
| CN106995805A (en) * | 2017-03-26 | 2017-08-01 | 海南大学 | A kind of engineering bacteriophage quick detection microorganism of lysozyme mark |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106645109A (en) * | 2016-12-29 | 2017-05-10 | Tcl集团股份有限公司 | Method, device and system for detecting hormones |
| CN106995805A (en) * | 2017-03-26 | 2017-08-01 | 海南大学 | A kind of engineering bacteriophage quick detection microorganism of lysozyme mark |
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| CN102135497B (en) | 2012-09-05 |
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