CN102168138B - Method for detecting androgen in environment - Google Patents
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- CN102168138B CN102168138B CN 201110043216 CN201110043216A CN102168138B CN 102168138 B CN102168138 B CN 102168138B CN 201110043216 CN201110043216 CN 201110043216 CN 201110043216 A CN201110043216 A CN 201110043216A CN 102168138 B CN102168138 B CN 102168138B
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Abstract
The invention discloses a method for detecting androgen in the environment, which comprises the following steps of: connecting a luciferase reporter gene vector with a ricefield eel aromatase gene pituitary specific promoter, constructing a reporter plasmid, co-transfecting an LbetaT2 cell to the reporter plasmid, a ricefield eel androgen acceptor expression plasmid and an actin plasmid, stimulating the transfected LbetaT2 cell by using a sample to be tested, and detecting androgen in the sample by detecting the activity of luciferase. The detection method provided by the invention has good repeatability, high sensitivity and reliable detection result, is simple and convenient to operate and can be widely applied to androgen detection in the environment.
Description
Technical field
[0001] the present invention relates to the Environmental Biotechnology field, be specifically related to androgenic detection method in a kind of environment.
Background technology
Environmental hormone is the general name that can as hormone, affect the chemical substance of human endocrine function existed in environment.But the environment sex steroid hormone is due to enrichment in the nature food chain, and the characteristics such as hard degradation and inactivation, can the normal hormone physiological activity of havoc organism.The environment male sex hormone can with androgen receptor (AR) combination, thereby the simulation androgenic activity, the performance androgenic effect.The environment sex steroid hormone has caused people's extensive concern on the impact of human reproduction's health, set up a set of fast, effectively, the method for screening test environment sex steroid hormone has become the task of top priority accurately.
The existing research of monitoring that at present relevant environment male sex hormone pollutes, the androgenic method of test and appraisal environment have Hershberger test, male mouse test in pubescence etc., androgen receptor in conjunction with test, Androgen-dependent cell proliferation shaker test, Androgen-dependent genetic transcription test etc. (Zhang Guojun. the Environmental antiandrogen detection method. foreign medical science. the hygiology fascicle; Foreign Medical Sciences-hygine, 02 phase in 2003).The domestic scholar of having applies male sex hormone ligand-receptor binding characteristic, has developed the androgenic ELISA quantitative detecting method of a kind of environmental classes (patent number 200910191914.X).But these methods are all more loaded down with trivial details, the biologic activity that particularly method based on androgen receptor-ligand binding can not the accurate response hormone, be difficult to reliably male sex hormone in environment be detected.
Summary of the invention
The object of the invention is to according to deficiency of the prior art, androgenic detection method in a kind of environment is provided.
The present invention is achieved through the following technical solutions above-mentioned purpose:
Technical scheme of the present invention is to utilize the characteristics that in swamp eel brain aromatizing enzyme gene hypophysis, specific promoter is raised by male sex hormone, sets up a kind of by detecting the activation situation of male sex hormone to above-mentioned promotor, the method that realization is detected the environment male sex hormone.
Androgenic detection method in a kind of environment, to connect the luciferase reporter gene carrier with swamp eel brain aromatizing enzyme gene hypophysis specific promoter (this promotor only works in swamp eel hypophysis), build reporter plasmid, by reporter plasmid, swamp eel expression of androgen receptor plasmid and internal reference plasmid co-transfection LbetaT2 cell (Mouse Pituitary gonadotroph), LbetaT2 cell with after testing sample stimulation transfection, detect the male sex hormone in sample by the activity that detects luciferase.
Described swamp eel brain aromatizing enzyme gene hypophysis specific promoter sequence is as shown in SEQ ID NO:1.
Described luciferase reporter gene carrier is pGL3-Basic.
Described internal reference plasmid is pRL-TK(renilla luciferase contrast reporter plasmid), as the internal reference of transfection experiment, can avoid because of the impact on result of the difference of transfection efficiency.
Described cotransfection LbetaT2 cell agents useful for same is that this reagent transfection efficiency of Lipofectamine 2000(is higher), and in without phenol red DMEM in high glucose substratum, cultivate after 4-6 hour after transfection, the substratum more renewed is cultivated 22-26 hour again.
Describedly with testing sample, stimulating the LbetaT2 cell after transfection, by the activity that detects luciferase, detect male sex hormone in sample, is within 22-26 hour after sample stimulus, to detect uciferase activity again.
Male sex hormone in described sample is dihydrotestosterone, and other androgenic mechanism of action are identical with dihydrotestosterone, and method of the present invention also can be for detection of the male sex hormone of other kinds.
PGL3-Basic Photinus pyralis LUC Reporter gene vector is a kind of luciferase detection system that those skilled in the art often use, the pGL3-Basic carrier is not containing promotor and enhanser, possible promoter sequence is cloned into to the upstream of luciferase gene, just can measures it and whether there is the activity that starts luciferase gene expression.
Swamp eel expression of androgen receptor plasmid is the acceptor of swamp eel itself, is more suitable in the analysis of swamp eel gene promoter activity.
The present invention by swamp eel brain aromatizing enzyme gene hypophysis specific promoter sequence subclone to pGL3-Basic carrier firefly luciferase gene upstream, thereby build a reporter plasmid.Because containing androgen response element (ARE, the androgen response element nucleotide sequence is as shown in SEQ ID NO:2), therefore can stimulate and produce reaction male sex hormone specifically on swamp eel brain aromatizing enzyme gene hypophysis specific promoter sequence.
By the above-mentioned reporter plasmid built, swamp eel expression of androgen receptor plasmid and internal reference plasmid (pRL-TK) cotransfection LbetaT2 cell, without phenol red DMEM in high glucose (Gibco, containing 10% without the hormone foetal calf serum) cultivate 4-6 hour in substratum after, after the substratum more renewed is cultivated 22-26 hour again, LbetaT2 cell with testing sample after to transfection is stimulated, and within after stimulating 22-26 hour, detects uciferase activity.According to whether obviously the raise existence of the androgenic activity material that can detect environmental sample to be measured of uciferase activity.
Compared with prior art, the present invention has following beneficial effect:
The singularity that the present invention utilizes swamp eel brain aromatizing enzyme gene hypophysis specific promoter raised by male sex hormone, the existence of testing environment sexual hormoue by detecting male sex hormone to the activation situation of this promotor.The technical scheme adopted is based on detecting androgenic biologic activity, and reliable results is reproducible, highly sensitive, and simple to operation.
The accompanying drawing explanation
Fig. 1 is the dose response curve of swamp eel brain aromatizing enzyme hypophysis specific promoter activity to male sex hormone dihydrotestosterone DHT in embodiment 1.EBAPII is swamp eel brain aromatizing enzyme hypophysis specific promoter reporter plasmid; The positive control plasmid of AREtk.
Embodiment
Below in conjunction with specific embodiment, the present invention is done further and describes, but specific embodiment is not done any restriction to the present invention.
Swamp eel brain aromatizing enzyme gene hypophysis specific promoter, its nucleotide sequence, as shown in SEQ ID NO:1, for applicant oneself clone, is announced on the net.
The present embodiment adopts the pGL3-Basic Photinus pyralis LUC Reporter gene vector of Promega company.
The present invention is by above-mentioned swamp eel brain aromatizing enzyme gene hypophysis specific promoter sequence subclone to pGL3-Basic carrier firefly luciferase gene upstream, and ordinary method builds a reporter plasmid, called after EBAPI.
LbetaT2 cell (the present embodiment is granted by Pamela doctor Mellon of Univ California-San Diego USA, can buy on the market) is transferred to 24 orifice plates (every hole 10
5cell), then adopt the Lipofectamine 2000(1 μ L/ hole of Invitrogen) reporter plasmid (380 ng), swamp eel expression of androgen receptor plasmid (50 ng, oneself builds, swamp eel androgen receptor cDNA coding region is cloned in the pcDNA3.0 expression vector of Invitrogen company), and the internal reference plasmid pRL-TK(20 ng of Promega company) cotransfection is in the LbetaT2 cell, without phenol red DMEM in high glucose (Gibco, containing 10% without the hormone foetal calf serum) cultivate 4 hours in substratum after, after the substratum more renewed is cultivated 24 hours again, with testing sample, (in the present embodiment, be environmental water sample, through degerming, process) transfectional cell is stimulated, stimulate latter 24 hours lysing cell, adopt two luciferase report gene detection systems (Promega) to measure uciferase activity.By whether the raise existence of the androgenic activity material that just can detect environmental sample to be measured of uciferase activity.
SEQUENCE LISTING
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<120 > androgenic detection method in a kind of environment
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Claims (3)
1. androgenic detection method in an environment, to connect the luciferase reporter gene carrier with swamp eel brain aromatizing enzyme gene hypophysis specific promoter, build reporter plasmid, by reporter plasmid, swamp eel expression of androgen receptor plasmid and internal reference plasmid co-transfection LbetaT2 cell, with the LbetaT2 cell after testing sample stimulation transfection, detect the male sex hormone in sample by the activity that detects luciferase, wherein, described swamp eel brain aromatizing enzyme gene hypophysis specific promoter sequence is as shown in SEQ ID NO:1, described luciferase reporter gene carrier is pGL3-Basic, described internal reference plasmid is pRL-TK, described male sex hormone is dihydrotestosterone.
2. androgenic detection method in environment according to claim 1, it is characterized in that described cotransfection LbetaT2 cell agents useful for same is Lipofectamine 2000, and after transfection, in without phenol red DMEM in high glucose substratum, cultivate after 4-6 hour, the substratum more renewed is cultivated 22-26 hour again.
3. androgenic detection method in environment according to claim 1, it is characterized in that described with the LbetaT2 cell after testing sample stimulation transfection, detecting male sex hormone in sample by the activity that detects luciferase, is within 22-26 hour after sample stimulus, to detect uciferase activity again.
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| CN103965348B (en) * | 2014-04-30 | 2016-06-15 | 中国水产科学研究院淡水渔业研究中心 | Monopterus albus (Zuiew) estrogen receptorαgene, encoding proteins and enzyme-linked immune detection method |
| CZ2015787A3 (en) * | 2015-11-05 | 2017-04-05 | Masarykova Univerzita | A set for determining analytes, its preparation and the method of determining analytes |
| CN106872433B (en) * | 2017-03-31 | 2019-03-08 | 中国农业大学 | Beta-cyclodextrin functionalization carbon dots material and its applied to detection testosterone method |
| CN109061201B (en) * | 2018-09-05 | 2022-03-01 | 中国农业科学院农业质量标准与检测技术研究所 | Method for detecting antiandrogen effect |
| CN114106142B (en) * | 2021-11-03 | 2024-05-14 | 中山大学 | Monopteri albi growth prolactin antiserum and preparation method and application thereof |
| CN115181748A (en) * | 2022-08-08 | 2022-10-14 | 湖州师范学院 | Biological detection method of male hormone |
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