CN102128938B - 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 PDFInfo
- Publication number
- CN102128938B CN102128938B CN 201010595059 CN201010595059A CN102128938B CN 102128938 B CN102128938 B CN 102128938B CN 201010595059 CN201010595059 CN 201010595059 CN 201010595059 A CN201010595059 A CN 201010595059A CN 102128938 B CN102128938 B CN 102128938B
- Authority
- CN
- China
- Prior art keywords
- plasmid
- gfp3
- detection
- centrifugal
- escherichia coli
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000010276 construction Methods 0.000 title claims description 8
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 title abstract description 11
- 230000029918 bioluminescence Effects 0.000 title description 8
- 238000005415 bioluminescence Methods 0.000 title description 8
- 239000003263 anabolic agent Substances 0.000 title 1
- 229940070021 anabolic steroids Drugs 0.000 title 1
- 239000013612 plasmid Substances 0.000 claims abstract description 38
- 238000001514 detection method Methods 0.000 claims abstract description 37
- 241000588724 Escherichia coli Species 0.000 claims abstract description 24
- 239000003270 steroid hormone Substances 0.000 claims abstract description 17
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 13
- 239000000758 substrate Substances 0.000 claims abstract description 11
- 230000014509 gene expression Effects 0.000 claims abstract description 10
- 230000001105 regulatory effect Effects 0.000 claims abstract description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 5
- 108700008625 Reporter Genes Proteins 0.000 claims abstract description 4
- 102000005936 beta-Galactosidase Human genes 0.000 claims abstract description 3
- 108010005774 beta-Galactosidase Proteins 0.000 claims abstract description 3
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 3
- 239000006228 supernatant Substances 0.000 claims description 30
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 26
- 210000000805 cytoplasm Anatomy 0.000 claims description 23
- 238000005406 washing Methods 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 15
- 239000005090 green fluorescent protein Substances 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 13
- 125000003118 aryl group Chemical group 0.000 claims description 12
- 230000003287 optical effect Effects 0.000 claims description 11
- 239000001963 growth medium Substances 0.000 claims description 10
- 235000010335 lysozyme Nutrition 0.000 claims description 10
- 235000015097 nutrients Nutrition 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- 230000001276 controlling effect Effects 0.000 claims description 7
- 239000012530 fluid Substances 0.000 claims description 7
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 6
- 102000004144 Green Fluorescent Proteins Human genes 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 6
- 230000005855 radiation Effects 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 102000016943 Muramidase Human genes 0.000 claims description 4
- 108010014251 Muramidase Proteins 0.000 claims description 4
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 4
- 238000003390 bioluminescence detection Methods 0.000 claims description 4
- 229960000274 lysozyme Drugs 0.000 claims description 4
- 239000004325 lysozyme Substances 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 2
- 230000000694 effects Effects 0.000 claims description 2
- 229940011871 estrogen Drugs 0.000 claims description 2
- 239000000262 estrogen Substances 0.000 claims description 2
- 230000001939 inductive effect Effects 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 239000003344 environmental pollutant Substances 0.000 abstract description 3
- 231100000719 pollutant Toxicity 0.000 abstract description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 102100036504 Dehydrogenase/reductase SDR family member 9 Human genes 0.000 abstract 2
- 101000928746 Homo sapiens Dehydrogenase/reductase SDR family member 9 Proteins 0.000 abstract 2
- 102000034356 gene-regulatory proteins Human genes 0.000 abstract 1
- 108091006104 gene-regulatory proteins Proteins 0.000 abstract 1
- 230000001632 homeopathic effect Effects 0.000 abstract 1
- 229910052799 carbon Inorganic materials 0.000 description 7
- 150000003431 steroids Chemical class 0.000 description 7
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 6
- 229960005309 estradiol Drugs 0.000 description 6
- 229930182833 estradiol Natural products 0.000 description 6
- 241000589518 Comamonas testosteroni Species 0.000 description 5
- UFWIBTONFRDIAS-UHFFFAOYSA-N Naphthalene Chemical compound C1=CC=CC2=CC=CC=C21 UFWIBTONFRDIAS-UHFFFAOYSA-N 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 230000001086 cytosolic effect Effects 0.000 description 3
- 235000019441 ethanol Nutrition 0.000 description 3
- 125000005909 ethyl alcohol group Chemical group 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- XJHABGPPCLHLLV-UHFFFAOYSA-N benzo[de]isoquinoline-1,3-dione Chemical compound C1=CC(C(=O)NC2=O)=C3C2=CC=CC3=C1 XJHABGPPCLHLLV-UHFFFAOYSA-N 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000003054 hormonal effect Effects 0.000 description 2
- 101710172561 3alpha-hydroxysteroid dehydrogenase Proteins 0.000 description 1
- 102100024089 Aldo-keto reductase family 1 member C2 Human genes 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108010085012 Steroid Receptors Proteins 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 102000005969 steroid hormone receptors Human genes 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
一种甾体类激素及多环芳烃高效生物荧光传感器，属于分子生物学技术领域，其特征是：将质粒pK-3α-GFP3和p6分别转化进大肠杆菌中并分别制备成非细胞体系检测试剂，在重组质粒pK-3α-GFP3的顺势β-半乳糖苷酶(LacZ)启动子基因下游依次插入了3α-HSD启动子等调控序列468碱基对及GFP报告基因722碱基对；可以诱导p6质粒上3α-HSD的调控序列表达调控作用的蛋白，该蛋白可使质粒pK-3α-GFP3上的启动子启动进而表达出GFP蛋白，此时GFP的表达量严格受到诱导底物量的控制。有益效果是：针对现有检测系统的仪器要求严格、检测周期长、成本高、灵敏度低、检测物单一等弊端，本发明检测线性范围宽，技术操作简单，仪器要求不高，适宜推广应用于检测环境、食品中甾体类激素及多环芳烃污染物。
A high-efficiency bioluminescent sensor for steroid hormones and polycyclic aromatic hydrocarbons, which belongs to the technical field of molecular biology, is characterized in that: the plasmids pK-3α-GFP3 and p6 are respectively transformed into Escherichia coli and prepared into non-cell system detection reagents respectively , 468 base pairs of the regulatory sequence such as the 3α-HSD promoter and 722 base pairs of the GFP reporter gene were sequentially inserted downstream of the homeopathic β-galactosidase (LacZ) promoter gene of the recombinant plasmid pK-3α-GFP3; it can induce The regulatory sequence of 3α-HSD on the p6 plasmid expresses a regulatory protein, which can activate the promoter on the plasmid pK-3α-GFP3 and then express the GFP protein. At this time, the expression level of GFP is strictly controlled by the amount of the induced substrate. . The beneficial effects are: in view of the disadvantages of the existing detection system, such as strict requirements for instruments, long detection period, high cost, low sensitivity, and single detection object, the present invention has a wide detection linear range, simple technical operation, and low requirements for instruments, and is suitable for popularization and application Detect steroid hormones and polycyclic aromatic hydrocarbons pollutants in the environment and food.
Description
Technical field
The invention belongs to technical field of molecular biology.
Background technology
In recent years, natural, artificial synthetic steroid and polycyclic aromatic hydrocarbon compounds is unconfined is discharged in 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 have hormonal activity under extremely low concentration, environment and human health are produced serious harm, so can new technology sensitive, that detect fast and efficiently steroid and polycyclic aromatic hydrocarbon compounds demand development urgently.Traditional detection analytical technology is mainly gas chromatography-mass spectrum (GC-MS), liquid chromatography-mass spectrography (HC-MS) at present, and domestic and international expert has been developed into multiple steroid hormone detection system based on the research of human body steroid receptor in addition.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 the 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 with C.T take steroid and polycyclic aromatic hydrocarbon compounds as signal scheme, build a kind of sensitivity, fast, simple biology sensor, be used for steroid and polycyclic aromatic hydrocarbon compounds potpourri in 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 Escherichia coli, set up a kind of bioluminescence detection system of Green Fluorescent Protein, detected object is steroid hormone and the large class material of palycyclic aromatic two.
Technical scheme of the present invention is:
The present invention has built based on the steroid hormone of Comamonas testosteroni (C.testosteroni, C.T) and palycyclic aromatic bioluminescence detection system.Comamonas testosteroni can be with steroid hormones such as stosterones as sole carbon source and the energy, the materials such as palycyclic aromatic of the non-steroid of can also degrading, and (metabolism of the enzyme such as 3 α-HSD) digests this class substrate by 3 α-hydroxysteroid dehydrogenase.Green fluorescent protein (GFP) is the reporter gene that is widely used in recent years the biological marker field, the protein that its gene produces, under the light of blue wavelength region excites, 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 plasmid pK-3 α-GFP3 and p6 respectively in Escherichia coli and is prepared into respectively acellular system detection reagent, has set up a kind of bioluminescence detection system of Green Fluorescent Protein.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 the substrates such as estrogen in 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 of GFP strictly is subject to inducing the control of amount of substrate at this moment.
The LacZ promoter gene of taking advantage of a situation in plasmid pK-3 α-GFP3 can act on regulating and controlling sequence and the promoter gene of 3 α in itself plasmid-HSD, 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 Escherichia coli, cultivates 12 to 14 hours, and after cultivating, Escherichia coli were inoculated in nutrient culture media in 1: 20 by volume, enlarge and cultivate, after cultivating, bacterium liquid is centrifugal, 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 Escherichia coli, cultivated 12 to 14 hours, after cultivating, Escherichia coli were inoculated in nutrient culture media in 1: 20 by volume, enlarge and cultivate, after cultivating, bacterium liquid is centrifugal, 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 the acellular system cytoplasm of plasmid pK-3 α-GFP3 and plasmid P6 measured respectively protein content and be prepared into respectively the working fluid of protein content=0.1mg/mL, 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.
Set up bioluminescence standard detection curve
(1) with detection 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 detection reagent and add respectively standard detection liquid, blank adds absolute ethyl alcohol, and is standing, detects its fluorescence radiation value, Criterion detection curve with fluorescence microplate reader.
Detected object of the present invention is:
Steroid hormone and palycyclic aromatic two class materials, the sample detection concentration range is: 10
-15To 10
-3Mole every liter.
The invention has the beneficial effects as follows:
Instrument for existing detection system is strict, sense cycle is long, cost is high, sensitivity is low, detect the drawbacks such as thing is single, the present invention detects linear wide ranges, technical operation is simple, instrument is less demanding, suitablely is applied to steroid hormone and polycyclic aromatic hydrocarbon pollutant in testing environment, food.
Description of drawings
Fig. 1 is plasmid pK-3 α-GFP3 schematic diagram;
Fig. 2 is plasmid p6 schematic 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 sets up bioluminescence examination criteria curve as an example of stosterone example
1, the cytoplasmic preparation of acellular system:
(1) plasmid pK-3 α-GFP3 is transformed in Escherichia coli, 37 ℃, cultivated 12 to 14 hours in 180～200 rpms of constant-temperature shaking incubators, 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 incubators, after cultivating, 4000 rpms of thalline were abandoned 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 supernatant and be the acellular system cytoplasm (can preserve month for 20 ℃) of preparation.
(2) plasmid p6 is transformed in Escherichia coli, 37 ℃, cultivated 12 to 14 hours in 180～200 rpms of constant-temperature shaking incubators, 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 incubators
595=3.0, after cultivating, 4000 rpms of thalline were abandoned 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 the acellular system cytoplasm of plasmid pK-3 α-GFP3 and plasmid P6 measured respectively protein content and be prepared into respectively the working fluid of protein content=0.1mg/mL, and mixing 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 respectively 2 μ L standard detection liquid, blank adds 2 μ L absolute ethyl alcohols, 30 ℃ after standing 10 minutes, detects its fluorescence radiation value, Criterion detection curve with fluorescence microplate reader.
Fluoroscopic examination result such as following table:
Table 1. stosterone fluoroscopic examination result
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 sets up bioluminescence standard detection curve as an example of estradiol example
1, the cytoplasmic preparation of acellular system:
(1) plasmid pK-3 α-GFP3 is transformed in Escherichia coli, 37 ℃, cultivated 12 to 14 hours in 180～200 rpms of constant-temperature shaking incubators, 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 incubators
595=3.0, after cultivating, 4000 rpms of thalline were abandoned 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 Escherichia coli, 37 ℃, cultivated 12 to 14 hours in 180～200 rpms of constant-temperature shaking incubators, 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 incubators
595=3.0, after cultivating, 4000 rpms of thalline were abandoned 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 the acellular system cytoplasm of plasmid pK-3 α-GFP3 and plasmid P6 measured respectively protein content and be prepared into respectively the working fluid of protein content=0.1mg/mL, and mixing 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 respectively 2 μ L standard detection liquid, blank adds 2 μ L absolute ethyl alcohols, 30 ℃ after standing 10 minutes, detects its fluorescence radiation value, Criterion detection curve with fluorescence microplate reader.
Fluoroscopic examination result such as following table:
Table 2. estradiol fluoroscopic examination result
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 sets up bioluminescence standard detection curve as an example of naphthalene example
1, the cytoplasmic preparation of acellular system:
(1) plasmid pK-3 α-GFP3 is transformed in Escherichia coli, 37 ℃, cultivated 12 to 14 hours in 180～200 rpms of constant-temperature shaking incubators, 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 incubators
595=3.0, after cultivating, 4000 rpms of thalline were abandoned 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 Escherichia coli, 37 ℃, cultivated 12 to 14 hours in 180～200 rpms of constant-temperature shaking incubators, 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 incubators
595=3.0, after cultivating, 4000 rpms of thalline were abandoned 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 the acellular system cytoplasm of plasmid pK-3 α-GFP3 and plasmid P6 measured respectively protein content and be prepared into respectively the working fluid of protein content=0.1mg/mL, and mixing 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 respectively 2 μ L standard detection liquid, blank adds 2 μ L absolute ethyl alcohols, 30 ℃ after standing 10 minutes, detects its fluorescence radiation value, Criterion detection curve with fluorescence microplate reader.Fluoroscopic examination result such as following table:
Table 3. fluorescent naphthalimide testing result
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.
Claims (5)
1. a steroid hormone and palycyclic aromatic high-performance bio fluorescent optical sensor, it is characterized in that: plasmid pK-3 α-GFP3 and p6 are transformed respectively in Escherichia coli and be prepared into respectively acellular system detection reagent, set up a kind of bioluminescence detection system of Green Fluorescent Protein; Be that to have inserted successively 3 α-HSD promoter regulation sequence be that 470 base-pairs and the GFP albumen reporter gene of sequence 2 is 717 base-pairs of sequence 3 for sequence 1 downstream at the beta galactosidase promoter gene of taking advantage of a situation of recombinant plasmid pK-3 α-GFP3; It is sequence 4 that recombinant plasmid p6 contains the whole controlling genes of 3 α-HSD, totally 5257 base-pairs; When having the estrogen substrate in 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 this moment, GFP protein expression amount strictly was subject to inducing the control of amount of substrate.
2. the construction method of a steroid hormone and palycyclic aromatic high-performance bio fluorescent optical sensor, its construction method is:
A, plasmid pK-3 α-GFP3 are transformed in Escherichia coli, cultivate 12 to 14 hours, and after cultivating, Escherichia coli were inoculated in nutrient culture media in 1: 20 by volume, enlarge and cultivate, after cultivating, bacterium liquid is centrifugal, 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 Escherichia coli, cultivated 12 to 14 hours, after cultivating, Escherichia coli were inoculated in nutrient culture media in 1: 20 by volume, enlarge and cultivate, after cultivating, bacterium liquid is centrifugal, 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 is measured respectively protein content and be prepared into respectively the working fluid of protein content=0.1mg/mL, 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, with detection 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 the acellular system and detect reagent and add respectively standard detection liquid, blank adds absolute ethyl alcohol, and is standing, detects its fluorescence radiation value, Criterion detection curve with fluorescence microplate reader.
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 in plasmid pK-3 α-GFP3 can act on regulating and controlling sequence and the promoter gene of 3 α in itself plasmid-HSD, green fluorescent protein great expression after promotion plasmid pK-3 α-GFP3 has amplification, humidification to fluorescence signal.
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, 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
-3Mole every liter.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010595059 CN102128938B (en) | 2010-12-20 | 2010-12-20 | Anabolic steroids and polycyclic aromatic hydrocarbon high-efficiency bioluminescence sensor and construction method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010595059 CN102128938B (en) | 2010-12-20 | 2010-12-20 | Anabolic steroids and polycyclic aromatic hydrocarbon high-efficiency bioluminescence sensor and construction method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102128938A CN102128938A (en) | 2011-07-20 |
| CN102128938B true CN102128938B (en) | 2013-06-19 |
Family
ID=44267101
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010595059 Expired - Fee Related CN102128938B (en) | 2010-12-20 | 2010-12-20 | Anabolic steroids and polycyclic aromatic hydrocarbon high-efficiency bioluminescence sensor and construction method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102128938B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110684792B (en) * | 2019-10-14 | 2022-01-11 | 江南大学 | Glycolic acid sensor capable of being eliminated repeatedly and construction method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000049150A1 (en) * | 1999-02-18 | 2000-08-24 | National University Of Singapore | Chimeric gene constructs for generation of fluorescent transgenic ornamental fish |
| WO2000071565A2 (en) * | 1999-05-21 | 2000-11-30 | The Regents Of The University Of California | Fluorescent protein indicators |
| CN101539524A (en) * | 2009-03-06 | 2009-09-23 | 于源华 | Fluorescence detection card and fluorescence detection method for organic contaminants in food and environment |
-
2010
- 2010-12-20 CN CN 201010595059 patent/CN102128938B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000049150A1 (en) * | 1999-02-18 | 2000-08-24 | National University Of Singapore | Chimeric gene constructs for generation of fluorescent transgenic ornamental fish |
| WO2000071565A2 (en) * | 1999-05-21 | 2000-11-30 | The Regents Of The University Of California | Fluorescent protein indicators |
| CN101539524A (en) * | 2009-03-06 | 2009-09-23 | 于源华 | Fluorescence detection card and fluorescence detection method for organic contaminants in food and environment |
Non-Patent Citations (10)
| Title |
|---|
| A model on the regulation of 3α-hydroxysteroid dehydrogenase/carbonyl reductase expression in Comamonas testosteroni;Guangming Xiong等;《Chemico-Biological Interactions》;20010131;第130-132卷;第723-736页 * |
| Cis- and trans-regulatory elements of 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase as biosensor system for steroid determination in the environment;Guangming Xiong等;《Chemico-BiologicalInteractions》;20091231;第178卷;第215-220页 * |
| Guangming Xiong等.A model on the regulation of 3α-hydroxysteroid dehydrogenase/carbonyl reductase expression in Comamonas testosteroni.《Chemico-Biological Interactions》.2001,第130-132卷 |
| Guangming Xiong等.Cis- and trans-regulatory elements of 3 alpha-hydroxysteroid dehydrogenase/carbonyl reductase as biosensor system for steroid determination in the environment.《Chemico-BiologicalInteractions》.2009,第178卷 |
| 于源华等.环境中甾体激素降解茵的筛选及功能基因的克隆.《长春理工大学学报》.2010,第33卷(第1期), |
| 于源华等.生物荧光方法快速测定环境水体中的甾体类激素.《分析化学》.2010,第38卷(第7期), |
| 液相色谱一串联质谱同时测定动物组织中22种同化激素;陈慧华等;《分析化学》;20090228;第37卷(第2期);第181-186页 * |
| 环境中甾体激素降解茵的筛选及功能基因的克隆;于源华等;《长春理工大学学报》;20100331;第33卷(第1期);第108-111页 * |
| 生物荧光方法快速测定环境水体中的甾体类激素;于源华等;《分析化学》;20100731;第38卷(第7期);第1048-1051页 * |
| 陈慧华等.液相色谱一串联质谱同时测定动物组织中22种同化激素.《分析化学》.2009,第37卷(第2期), |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102128938A (en) | 2011-07-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Line et al. | Physiological levels of nitrate support anoxic growth by denitrification of Pseudomonas aeruginosa at growth rates reported in cystic fibrosis lungs and sputum | |
| Junne et al. | A two‐compartment bioreactor system made of commercial parts for bioprocess scale‐down studies: impact of oscillations on Bacillus subtilis fed‐batch cultivations | |
| Patterson et al. | Codon optimization of bacterial luciferase (lux) for expression in mammalian cells | |
| Werlen et al. | Measurement of biologically available naphthalene in gas and aqueous phases by use of a Pseudomonas putida biosensor | |
| Tescione et al. | Construction and evaluation of a metal ion biosensor | |
| Crespo et al. | Pseudomonas aeruginosa exhibits deficient biofilm formation in the absence of class II and III ribonucleotide reductases due to hindered anaerobic growth | |
| Downie et al. | Transparent soil microcosms allow 3D spatial quantification of soil microbiological processes in vivo | |
| CN103981263B (en) | The quantitative detecting method of antibiotics resistance gene in soil | |
| Wecker et al. | Design of a new biosensor for algal H2 production based on the H2-sensing system of Rhodobacter capsulatus | |
| Rajoka et al. | Kinetics and thermodynamics of ethanol production by a thermotolerant mutant of Saccharomyces cerevisiae in a microprocessor‐controlled bioreactor | |
| Bueno et al. | Regulation of the emissions of the greenhouse gas nitrous oxide by the soybean endosymbiont Bradyrhizobium diazoefficiens | |
| CN102128938B (en) | Anabolic steroids and polycyclic aromatic hydrocarbon high-efficiency bioluminescence sensor and construction method thereof | |
| Altermann et al. | Tailored nanoparticles with the potential to reduce ruminant methane emissions | |
| CN102147364B (en) | Efficient microbial fluorescent sensor for detecting steroids hormone and polycyclic aromatic hydrocarbon | |
| Ovcharova et al. | C-type natriuretic peptide acts as a microorganism-activated regulator of the skin commensals Staphylococcus epidermidis and Cutibacterium acnes in dual-species biofilms | |
| Zhao et al. | Optimization of photosynthetic bacteria wastewater treatment and study of microbial species diversity | |
| Zhou et al. | Study on the effect of key genes ME2 and adhE during Luzhou-flavor baijiu brewing | |
| Pooley et al. | Continuous culture of photobacterium | |
| Alessi et al. | A rapid, sensitive, low-cost assay for detecting hydrogenotrophic methanogens in anaerobic digesters using loop-mediated isothermal amplification | |
| Nie et al. | Enhanced intracellular glutathione synthesis and excretion capability of Candida utilis by using a low pH‐stress strategy | |
| MD3625G2 (en) | Heptaazotate of hexa-(µ-?-alanine -(O,O'))-µ3-oxo-tri(aqua)triferrum(III) 3,5-hydrate and process for Spirulina platensis biomass obtaining | |
| Darusman et al. | Spontaneous expression of the gene of KI67 and P53 in cynomolgus monkeys infected with papillomavirus | |
| CN101372713B (en) | Method for detecting Francisella tularensis using multiple PCR technology | |
| CN207958369U (en) | A kind of microbial detection device | |
| CN102818801A (en) | Method for determining ATP |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130619 Termination date: 20131220 |