CN102168138A - Method for detecting androgen in environment - Google Patents
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- CN102168138A CN102168138A CN2011100432162A CN201110043216A CN102168138A CN 102168138 A CN102168138 A CN 102168138A CN 2011100432162 A CN2011100432162 A CN 2011100432162A CN 201110043216 A CN201110043216 A CN 201110043216A CN 102168138 A CN102168138 A CN 102168138A
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- 230000000694 effects Effects 0.000 claims abstract description 14
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- 238000012360 testing method Methods 0.000 claims description 18
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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
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 influence the chemical substance of human endocrine function as hormone that exists in the environment.The environment sex steroid hormone can the normal hormone physiological activity of havoc organism because but enrichment in the nature food chain is difficult to characteristics such as degraded and inactivation.The environment male sex hormone can combine with androgen receptor (AR), thus the simulation androgenic activity, the performance androgenic effect.The environment sex steroid hormone has caused people's extensive concern to human reproduction's health affected, has set up a cover fast, effectively, the method for screening test environment sex steroid hormone has become the task of top priority accurately.
The existing research of at present relevant environment male sex hormone pollution Monitoring, the androgenic method of test and appraisal environment have Hershberger test, male mouse test in pubescence etc., androgen receptor in conjunction with test, male sex hormone dependent cells propagation shaker test, male sex hormone rely on genetic transcription test etc. (Zhang Guojun. environment androgen antagonist detection method. foreign medical science. the hygiology fascicle; Foreign Medical Sciences-hygine, 2003 02 phases).The domestic scholar of having uses 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, particularly based on androgen receptor-part bonded method can not the accurate response hormone biologic activity, be difficult to reliably male sex hormone in the environment be detected.
Summary of the invention
The objective 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 specific promoter is raised by male sex hormone in the swamp eel brain aromatizing enzyme gene hypophysis, sets up a kind of by detecting the activation situation of male sex hormone to above-mentioned promotor, the method that realization detects the environment male sex hormone.
Androgenic detection method in a kind of environment, be 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), make up reporter plasmid, with reporter plasmid, swamp eel expression of androgen receptor plasmid and confidential reference items plasmid co-transfection LbetaT2 cell (mouse pituitary gonadotropic hormone cell), stimulate LbetaT2 cell after the transfection with testing sample, come male sex hormone in the test sample by the activity that detects luciferase.
Described swamp eel brain aromatizing enzyme gene hypophysis specific promoter sequence is shown in SEQ ID NO:1.
Described luciferase reporter gene carrier is pGL3-Basic.
Described confidential reference items plasmid is pRL-TK(renilla luciferase contrast reporter plasmid), as the internal reference of transfection experiment, can avoid because of the influence of the difference of transfection efficiency to the result.
Described cotransfection LbetaT2 cell agents useful for same is that this reagent transfection efficiency of Lipofectamine 2000(is higher), and in the sugared DMEM substratum of no phenol red height, cultivate after 4-6 hour after the transfection, the substratum that more renews was cultivated 22-26 hour again.
Describedly stimulating LbetaT2 cell after the transfection with testing sample, come male sex hormone in the test sample by the activity that detects luciferase, is to detect uciferase activity after sample stimulus in 22-26 hour again.
Male sex hormone in the described sample is a dihydrotestosterone, and other androgenic mechanism of action are identical with dihydrotestosterone, and method of the present invention also can be used to detect the male sex hormone of other kinds.
PGL3-Basic Photinus pyralis LUC reporter gene carrier is a kind of luciferase detection system that those skilled in the art often use, the pGL3-Basic carrier does not contain promotor and enhanser, possible promoter sequence is cloned into the upstream of luciferase gene, just can measures it and whether have 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 with swamp eel brain aromatizing enzyme gene hypophysis specific promoter sequence subclone to pGL3-Basic carrier firefly luciferase gene upstream, thereby make up a reporter plasmid.Because contain androgen response element (ARE, androgen response element nucleotide sequence is shown in SEQ ID NO:2) on the swamp eel brain aromatizing enzyme gene hypophysis specific promoter sequence, therefore can stimulate male sex hormone specifically to produce reaction.
With the above-mentioned reporter plasmid that builds, swamp eel expression of androgen receptor plasmid and confidential reference items plasmid (pRL-TK) cotransfection LbetaT2 cell, at the sugared DMEM (Gibco of no phenol red height, contain 10% no hormone foetal calf serum) cultivate in the substratum after 4-6 hour, after the substratum that more renews is cultivated 22-26 hour again, stimulate with the LbetaT2 cell of testing sample after, stimulate the back to detect uciferase activity in 22-26 hour transfection.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 is by detecting male sex hormone to the activation situation of this promotor and the existence of testing environment sexual hormoue.The technical scheme that adopts is based on detecting androgenic biologic activity, reliable results, and good reproducibility, highly sensitive, and simple to operation.
Description of drawings
Fig. 1 be among the embodiment 1 swamp eel brain aromatizing enzyme hypophysis specific promoter activity to the dose response curve of male sex hormone dihydrotestosterone DHT.EBAPII is a 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 description further, but specific embodiment is not done any qualification to the present invention.
Swamp eel brain aromatizing enzyme gene hypophysis specific promoter, its nucleotide sequence for applicant oneself clone, are announced shown in SEQ ID NO:1 on the net.
Present embodiment adopts the pGL3-Basic Photinus pyralis LUC reporter gene carrier of Promega company.
To pGL3-Basic carrier firefly luciferase gene upstream, ordinary method makes up a reporter plasmid, called after EBAPI with above-mentioned swamp eel brain aromatizing enzyme gene hypophysis specific promoter sequence subclone in the present invention.
LbetaT2 cell (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), adopt the Lipofectamine 2000(1 μ L/ hole of Invitrogen then) reporter plasmid (380 ng), swamp eel expression of androgen receptor plasmid (50 ng, oneself makes up, swamp eel androgen receptor cDNA coding region is cloned in the pcDNA3.0 expression vector of Invitrogen company), and the confidential reference items plasmid pRL-TK(20 ng of Promega company) cotransfection is in the LbetaT2 cell, at the sugared DMEM (Gibco of no phenol red height, contain 10% no hormone foetal calf serum) cultivate in the substratum after 4 hours, after the substratum that more renews is cultivated 24 hours again, with testing sample (is environmental water sample in the present embodiment, handle through degerming) transfectional cell is stimulated, stimulate back 24 hours lysing cell, adopt two luciferase reporter 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
＜110〉Zhongshan University
＜120〉androgenic detection method in a kind of environment
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Claims (7)
1. androgenic detection method in the environment, be to connect the luciferase reporter gene carrier with swamp eel brain aromatizing enzyme gene hypophysis specific promoter, make up reporter plasmid, with reporter plasmid, swamp eel expression of androgen receptor plasmid and confidential reference items plasmid co-transfection LbetaT2 cell, stimulate LbetaT2 cell after the transfection with testing sample, come male sex hormone in the test sample by the activity that detects luciferase.
2. according to androgenic detection method in the described environment of claim 1, it is characterized in that described swamp eel brain aromatizing enzyme gene hypophysis specific promoter sequence is shown in SEQ ID NO:1.
3. according to androgenic detection method in the described environment of claim 1, it is characterized in that described luciferase reporter gene carrier is pGL3-Basic.
4. according to androgenic detection method in the described environment of claim 1, it is characterized in that described confidential reference items plasmid is pRL-TK.
5. according to androgenic detection method in the described environment of claim 1, it is characterized in that described cotransfection LbetaT2 cell agents useful for same is Lipofectamine 2000, and cultivate after 4-6 hour in the sugared DMEM substratum of no phenol red height after the transfection, the substratum that more renews was cultivated 22-26 hour again.
6. according to androgenic detection method in the described environment of claim 1, it is characterized in that described with the LbetaT2 cell after the testing sample stimulation transfection, coming male sex hormone in the test sample by the activity that detects luciferase, is to detect uciferase activity after sample stimulus in 22-26 hour again.
7. according to androgenic detection method in the described environment of claim 1, it is characterized in that described male sex hormone is a dihydrotestosterone.
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| CN 201110043216 CN102168138B (en) | 2011-02-23 | 2011-02-23 | Method for detecting androgen in environment |
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| CN 201110043216 CN102168138B (en) | 2011-02-23 | 2011-02-23 | Method for detecting androgen in environment |
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| CN102168138B CN102168138B (en) | 2013-06-12 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103965348A (en) * | 2014-04-30 | 2014-08-06 | 中国水产科学研究院淡水渔业研究中心 | Monopterus albus estrogen receptor alpha gene, encoding protein and enzyme-linked immunosorbent assay method |
| CZ306626B6 (en) * | 2015-11-05 | 2017-04-05 | Masarykova Univerzita | A set for determining analytes, its preparation and the method of determining analytes |
| CN106872433A (en) * | 2017-03-31 | 2017-06-20 | 中国农业大学 | Beta cyclodextrin functionalized carbon point material and its method for being applied to detection testosterone |
| CN109061201A (en) * | 2018-09-05 | 2018-12-21 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of detection method of antiandrogen effect |
| CN114106142A (en) * | 2021-11-03 | 2022-03-01 | 中山大学 | Ricefield eel 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 |
-
2011
- 2011-02-23 CN CN 201110043216 patent/CN102168138B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| YANG ZHANG ET AL: "Two cytochrome P450 aromatase genes in the hermaphrodite ricefield eel Monopterus albus: mRNA expression during ovarian development and sex change.", 《JOURNAL OF ENDOCRINOLOGY》 * |
| 周芳 等: "黄鳝AR基因的克隆与表达分析", 《湖北省遗传学会、江西省遗传学会2006年学术年会暨学术讨论会论文摘要集》 * |
| 张扬: "黄鳝一种芳香化酶CYP19a1b基因黄鳝一种芳香化酶CYP19a1b基因，表达及转录调控机制的研究", 《中山大学博士学位论文》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103965348A (en) * | 2014-04-30 | 2014-08-06 | 中国水产科学研究院淡水渔业研究中心 | Monopterus albus estrogen receptor alpha gene, encoding protein and enzyme-linked immunosorbent assay method |
| CZ306626B6 (en) * | 2015-11-05 | 2017-04-05 | Masarykova Univerzita | A set for determining analytes, its preparation and the method of determining analytes |
| CN106872433A (en) * | 2017-03-31 | 2017-06-20 | 中国农业大学 | Beta cyclodextrin functionalized carbon point material and its method for being applied to detection testosterone |
| 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 |
| CN109061201A (en) * | 2018-09-05 | 2018-12-21 | 中国农业科学院农业质量标准与检测技术研究所 | A kind of detection method of antiandrogen effect |
| 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 |
| CN114106142A (en) * | 2021-11-03 | 2022-03-01 | 中山大学 | Ricefield eel growth prolactin antiserum and preparation method and application thereof |
| 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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