CN101469315B - Two-hybrid yeast for detecting estrogen-like compound in environment and biological test method - Google Patents
Two-hybrid yeast for detecting estrogen-like compound in environment and biological test method Download PDFInfo
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Abstract
The invention provides a two-hybrid yeast for detecting estrogen-like compounds in environmental samples and a preparation method thereof, wherein the yeast contains pGBKT7-ER yeast expression plasmids and pGAD424-GRIP1 yeast expression plasmids, wherein the pGBKT7-ER yeast expression plasmids contain estrogen receptor genes, and the pGAD424-GRIP1 yeast expression plasmids contain estrogen receptor coactivated factor genetic fragments with the sequence of SEQ ID No.2. The invention also provides a bioassay method for detecting the estrogen-like compounds in the environment, which comprises: co-culturing two-hybrid yeast cells and a sample to be detected, adding a reaction liquid of o-nitrobenzene-beta-D-galactopyranoside for reaction, and calculating the concentration of the estrogen-like compounds according to the detected absorbance value of supernatant at 420 nanometers after the reaction stops. The invention adopts the two-hybrid yeast of recombinant estrogen receptor genes for test, and is more close to the actual action conditions of an endocrine system of a mammal; constructed yeast cell genes have stable character and are easy to culture and screen; the screening process of the whole estrogen-like effect is simple to operate; and the required quantity of the sample is small, and the cost is low.
Description
Technical field
The present invention relates to a kind of double cross yeast and biological test method that is used for testing environment class estrogen compound, relate to a kind of double cross yeast that comprises the female hormone receptor gene of recombinating that utilizes yeast-two hybrid technique to obtain specifically, and utilize this yeast to come the biological test method of class estrogen compound in the testing environment.
Background technology
Endocrine disrupter in the environment, also be called as environmental hormone, be meant and human producing, be discharged in the life process some toxic chemical substance in the environment that they and human body and the intravital hormone of animal have similar chemical structure, and can produce similar functions of hormones.They enter in the body, have upset the normal secretion of hormone, and the physiological process of humans and animals is got muddled, and cause function generation obstacles such as reproduction, immunity.The interference effect of the endocrine disrupter in the environment comprises: oestrogenic hormon interference effect, male sex hormone interference effect, Triiodothyronine interference effect etc., particularly oestrogenic hormon interference effect can cause biology and human reproduction system and have a strong impact on.Although it is minimum to have compound concentration in environment of oestrogenic hormon interferon activity, but in a single day it enter in human body and the animal body, can combine with specific estrogen receptor and form mixture, this mixture is further combined with the estrogen response element in nucleus (ERE), the expression of startup/inhibition downstream gene, the normal function of interference endocrine system.Further found the co-activation factor (coactor) of estrogen receptor to the estrogen receptor Its Mechanisms nineties, and set up new receptor acting theory (referring to, Hong, H., Kohlit, K., Trivedit, A., Deborah, L., Johnson, D.L. and Stallcup, M.R., 1996, Natl.Acad.Sci.USA.93:4948-4952).This theory thinks, oestrogenic hormon (perhaps class estrogen substance in the environment) with cause conformational change after the ligand binding domain (LBD) of corresponding estrogen receptor combines, further combined with the co-activation factor, promote to transcribe.
Oestrogenic hormon interference effect in the environment is studied, adopted chemical analysis and bioassay technology usually.Chemical analysis needs high resolution chromatogram/high resolution mass spectrum, except the analysis cost height, plant and instrument condition and peopleware is had quite strict requirement.Another extensively adopts be utilize reorganization estrogen receptor yeast (Yeast Estrogen Screen YES) tests, this test utilization be the single crosses yeast that reorganization estrogen receptor and receptor response element make up.Single crosses yeast method also reckons without the effect of the estrogen receptor co-activation factor.And domestic used restructuring yeast strains does not have domestic laboratory to carry out the structure work of bacterial strain nearly all from external laboratory basically at present.Therefore this reorganization bacterial classification needs long-term frozen to preserve, in case bacterial classification generation reverse mutation, proterties just is difficult to reply.Utilize double cross yeast technology that female hormone receptor gene or gene fragment and co-activation factor gene fragment are imported yeast cell simultaneously and then can overcome single crosses zymic shortcoming, and consistent with new receptor acting theory.Evidence suggests based on the theoretical nuclear receptor double cross Yeast system of setting up of new receptor acting more near the true effect situation of Mammals endocrine system (referring to, Nishihara, T., Nishikawa, J., 2000, Journal of Health Science.46 (4): 282-298).And the chamber directly makes up yeast strain by experiment, can effectively control the influence of reverse mutation, can access better quality control when measuring the oestrogenic hormon interference effect of environmental compound.
At present, adopt usually the method indication external interference of reporter gene, as the influence to genetic transcription such as compound exposure.Wherein, LacZ is exactly a kind of reporter gene of coding beta-galactosidase commonly used, and its product beta-galactosidase enzymes has strong enzymatic activity, can generate new colored compound with the specific substrates reaction, is easy to detect.Therefore, by the activity of simple mensuration beta-galactosidase enzymes, just can characterize influence to genetic transcription.
Summary of the invention
When the objective of the invention is to overcome prior art and using reorganization female hormone receptor gene single crosses yeast that class oestrogenic hormon pollutent is measured owing to lack the acceptor co-activation factor, thereby can not simulates real karyocyte truth, be easy to generate the false negative or the defective of false positive results, thereby make up and to express the double cross yeast of the estrogen receptor and the acceptor co-activation factor simultaneously, and set up the biological test method of a kind of evaluation environmental sample of economical and efficient on this basis the estrogen receptor interferon activity.This detection method is fast and convenient, and is with low cost, time saving and energy saving, be widely used, required sample size is few, and is highly sensitive, the inductive effect that can reflect estrogen receptor in the environmental sample, and calculate the toxic equivalent value of class oestrogenic hormon pollutent in the sample by typical curve.
The present invention is achieved through the following technical solutions:
The invention provides a kind of double cross yeast that is used for testing environment class estrogen compound, this yeast comprises pGBKT7-ER expression plasmid of yeast and pGAD424-GRIP1 expression plasmid of yeast, wherein said pGBKT7-ER expression plasmid of yeast comprises female hormone receptor gene, and described pGAD424-GRIP1 expression plasmid of yeast comprises the estrogen receptor co-activation factor gene fragment with sequence shown in SEQ ID No.2.Particularly, the preferred human estrogen acceptor gene of above-mentioned female hormone receptor gene or have the human estrogen acceptor gene fragment of sequence shown in SEQ ID No.1.Described class estrogen compound is selected from natural estrogen compound, phenols and can forms in the compound, phthalate compound, organochlorine pesticide of phenols one or more through the metabolism conversion.
Above-mentioned double cross yeast provided by the invention is specially a kind of yeast saccharomyces cerevisiae (Saccharomycescerevisiae) ER-GRIP1, its on December 27th, 2007 at the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms, Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101) carried out the biomaterial preservation, deposit number is CGMCCNo.2307.
The present invention also provides a kind of double cross zymic preparation method who is used for testing environment class estrogen compound, comprising following steps:
(1) makes up the pGBKT7-ER expression plasmid of yeast that comprises female hormone receptor gene;
(2) purifying comprises the segmental pGAD424-GRIP1 expression plasmid of yeast of estrogen receptor co-activation factor gene with sequence shown in SEQ ID No.2;
(3) adopt above-mentioned two kinds of plasmids to make up double cross yeast ER-GRIP1;
(4) screening double cross yeast ER-GRIP1.
The preferred human estrogen acceptor gene of female hormone receptor gene described in the aforesaid method or have the human estrogen acceptor gene fragment of sequence shown in SEQ ID No.1.Described class estrogen compound is selected from natural estrogen compound, phenols and can forms in the compound, phthalate compound, organochlorine pesticide of phenols one or more through the metabolism conversion.Described double cross yeast ER-GRIP 1 is specially yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ER-GRIP1, and its deposit number is CGMCC No.2307.
In above-mentioned steps (1), comprise the PCR primer of design estrogen receptor ligands in conjunction with territory ER LBD, introduce EcoRI and the single restriction enzyme site of BamHI respectively at 5 of upstream and downstream primer ' end, and upstream primer P1:5 '-TCGCCGGAATTCGCTGGAGACATGAGA-3 '; Downstream primer P2:5 '-TAACGCGGATCCTCAGACTGTGGCAGG-3 '; In above-mentioned steps (3), transform with pGBKT7-ER plasmid and pGAD424-GRIP1 plasmid simultaneously and enter brewing yeast cell Y187 (Saccharomyces cerevisiae); In above-mentioned steps (4), the method screening double cross yeast that adopts auxotroph screening and the analysis of colony lift filter membrane to combine.Wherein, the preferred colony lift filter membrane that adds estrogenic X-gal damping fluid that adopts is analyzed, and described oestrogenic hormon is 17 beta estradiols for example; Described X-gal damping fluid is 20mgmL
-1The dimethyl formamide solution of 5-bromo-4-chloro-3-indoles-β-D-galactoside.The substratum that adopts in the described auxotroph screening is SD/-Trp/-Leu, wherein comprises 20mgL
-1The VITAMIN B4 Hemisulphate, 20mgL
-1Arginine hydrochloride, 20mgL
-1One hydration histidine monohydrochloride, 30mgL
-1Isoleucine, 30mgL
-1Lysine hydrochloride, 20mgL
-1Methionine(Met), 50mgL
-1Phenylalanine, 200mgL
-1Threonine, 30mgL
-1Tyrosine, 20mgL
-1Uridylic, 150mgL
-1Xie Ansuan, 6.7gL
-1No amino acid yeast nitrogen.
Particularly, in aforesaid method, when making up the pGBKT7-ER expression plasmid of yeast, at first design the PCR primer of human estrogen acceptor ligand binding domain (hER LBD), and introduce EcoRI and the single restriction enzyme site of BamH I respectively at 5 of upstream and downstream primer ' end; (be so kind as to give with the pASV3 plasmid that contains hER LBD (sequence shown in the SEQ ID No.1) by professor Heery of Britain Nottingham university.Referring to, Pierrat, B., Heery, D.M., 1992, Gene.119 (2): 237-245) carry out pcr amplification as template; The PCR product is connected in the T-carrier with the T4 dna ligase, the T-hER LBD plasmid of structure; PGBKT7 (available from Clontech company) and T-hER LBD plasmid are used EcoRI and BamHI double digestion respectively, enzyme is cut product and is cut glue after by the agarose gel electrophoresis analysis and reclaim, pGBKT7 inserts segmental recovery product with hER LBD and adopts the T4 dna ligase to be connected, and makes up the pGBKT7-hER expression plasmid of yeast.
Particularly, in aforesaid method, during purifying pGAD424-GRIP1 expression plasmid of yeast, (professor Stallcup of California, USA university is so kind as to give with pGAD424-GRIP1.Referring to, Li, H.W., Kim, J.H., Koh, S.S., Stallcup, M.R.2003.J.Biol.Chem.Biol.279 (6): 4212-4220.) transformed competence colibacillus intestinal bacteria, screening positive clone in containing the substratum of penbritin extracts plasmid and purifying.
Particularly; in aforesaid method; when making up double cross yeast ER-GRIP1; add the pGBKT7-hER plasmid simultaneously and the pGAD424-GRIP1 plasmid mixes; and add competence brewing yeast cell Y187 (Saccharomyces cerevisiae) and hatch, obtain double cross yeast hER-GRIP1.
Particularly, in aforesaid method, during screening double cross yeast ER-GRIP1, the method screening double cross yeast that adopts auxotroph screening and the analysis of colony lift filter membrane to combine, the double cross yeast goes out positive bacterium colony by auxotroph substratum (SD/-Trp/-Leu) scalping, further uses colony lift filter membrane analytical procedure, with bacterial colony photographic reprinting to the dry filter membrane of aseptic 1#, the liquid nitrogen multigelation, smudge cells; The 1# filter membrane is placed on the 2# filter membrane that contains estrogenic X-gal damping fluid, hatch to the obvious blue bacterium colony occurring for 30 ℃, the bacterium colony that will develop the color continues to cultivate standby.
The present invention also provides the biological test method of class estrogen compound in a kind of testing environment, comprising following steps: above-mentioned double cross yeast cell and testing sample are cultivated altogether, the reaction solution that adds ortho-nitrophenyl-β-D-galactopyranoside reacts, the concentration of coming the compute classes estrogen compound according to the absorbance of detected supernatant liquor at the 420nm place after the termination reaction.
Aforesaid method specifically comprises the steps:
(1) cultivates double cross yeast ER-GRIP1 cell;
(2) typical curve is induced in drafting: at first above-mentioned double cross yeast cell is cultivated altogether with known series concentration oestrogenic hormon respectively, adding ortho-nitrophenyl-β-D-galactopyranoside reaction solution reacts, detect the absorbance of supernatant liquor after the termination reaction at the 420nm place, and, draw the betagalactosidase activity inductive dose-effect relationship typical curve of the relative double cross yeast of serial estrogen concentrations ER-GRIP1 cell with this parameter as the expression betagalactosidase activity;
(3) test sample: above-mentioned double cross yeast cell and testing sample are cultivated altogether, the reaction solution that adds ortho-nitrophenyl-β-D-galactopyranoside reacts, detect the absorbance of supernatant liquor at the 420nm place after the termination reaction, the typical curve that contrast step (2) is formulated calculates the content of class estrogen compound in the sample.
For above-mentioned biological test method, in step (1), the absorbance of regulating bacterium liquid 600nm place during with the ER-GRIP1 cell inoculation is that the absorbance at adjustment bacterium liquid 600nm place behind 0.1～0.2,30 ℃ of cultivation 24h is that 0.7～0.8 preparation is tested; Described testing sample is an organic solvent extraction liquid, and wherein oestrogenic hormon equivalent concentration is 10
-11～10
-10MolL
-1Described class estrogen compound is selected from natural estrogen compound, phenols and can forms in phenolic compound, phthalate compound, the organochlorine pesticide one or more through the metabolism conversion.Described double cross yeast ER-GRIP1 is specially yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ER-GRIP1, and its deposit number is CGMCC No.2307.
Particularly, in above-mentioned testing environment in the biological test method of class estrogen compound, the cultural method of double cross yeast ER-GRIP1 cell is: with the cell inoculation of above-mentioned recombinant gene yeast hER-GRIP1 in the SD/-Trp/-Leu liquid nutrient medium, the absorbance that detects bacterium liquid 600nm place is that the absorbance at adjustment yeast liquid 600nm place behind 0.1～0.2,30 ℃ of cultivation 24h is 0.7～0.8.
Drafting induces the typical curve concrete steps to be: 17 beta estradiols are dissolved in the dimethyl sulfoxide (DMSO) (DMSO), prepare a series of 17 beta estradiol normal concentration reaction solution (concentration ranges 2 * 10
-12～2 * 10
-6Mol/L), add 17 beta estradiol normal concentration reaction solutions in the hER-GRIP1 yeast liquid according to 0.5% ratio, preparation exposes liquid; To expose liquid and be seeded to (200 μ L/ hole) in 96 orifice plates; Cultivate the absorbance that detects the 600nm place behind the 2h for 30 ℃; The reaction solution that will contain excessive ortho-nitrophenyl-β-D-galactopyranoside again joins in 96 orifice plates; After 30 ℃ of abundant down reactions, add 1molL
-1The sodium carbonate solution termination reaction; Detect the absorbance of end product o-nitrophenol sodium with spectrophotometer at the 420nm place, and, draw the betagalactosidase activity inductive dose-effect relationship typical curve of E2 (being estradiol) to recombinant gene yeast hER-GRIP1 cell with this parameter as the expression beta-galactosidase enzymes.
The calculation formula of betagalactosidase activity u is as follows: u=(A
S-A
B)/tVDOD
S, in the formula, u is a betagalactosidase activity; T is reaction times (min); V is test volume (mL); D is a dilution factor; OD
SBe the absorbance of specimen at the 595nm place; A
SBe the absorbance OD of specimen at the 420nm place
420A
BBe the absorbancy of blank at the 420nm place.
The concrete steps of above-mentioned test sample are: the ratio according to 0.5% is added dimethyl sulfoxide (DMSO) (DMSO) dissolved testing sample in the hER-GRIP1 yeast liquid, and preparation exposes liquid; Expose liquid and be seeded to (200 μ L/ hole) in 96 orifice plates; Cultivate the absorbance that detects the 600nm place behind the 2h for 30 ℃; The reaction solution that will contain excessive ortho-nitrophenyl-β-D-galactopyranoside again joins in 96 orifice plates; After 30 ℃ of abundant down reactions, add 1molL
-1The sodium carbonate solution termination reaction; Detect the absorbance of end product o-nitrophenol sodium with spectrophotometer at the 420nm place, detect testing compound and whether have the estrogen receptor induced activity, result who is about to record and above-mentioned typical curve are relatively, and then can calculate the content of class estrogen compound in the environmental sample, thereby estimate the toxic effect of class estrogen compound in the environment.
Provided by the inventionly be used for the double cross yeast of testing environment sample class estrogen compound and the advantage of biological detecting method is: 1) ER-GRIP1 double cross yeast cell can be expressed estrogen receptor, estrogen receptor co-activation factor protein simultaneously, support up-to-date receptor acting theory, can simulate the mechanism of action of mammalian cell inner estrogen acceptor, and the enzymic activity of the beta-galactosidase enzymes that expressing gene produces is easy to detect, and is well suited for the experimental study as toxicity of compound screening, environmental sample class estrogen effect evaluation aspect; 2) the yeast cell test is quick, easy can carry out the screening in early stage to the endocrine disrupter effect of a large amount of environmental samples or chemical substance, and for live test provides the basic data support, it is long to have remedied the live test test period, the deficiency that expense is high; 3) add 17 beta estradiols (1 * 10
-10MolL
-1) the application of X-gal damping fluid in the colony lift filter membrane is analyzed, shortened the operating time greatly, alleviated false positive results interferential possibility on the certain degree; 4) utilize SD/-Trp/-Leu auxotroph substratum to carry out yeast culture, can effectively prevent the reverse mutation of recombinant yeast cell, alleviate the situation of plasmid loss in the process of going down to posterity, make that the protein expression of recombinant yeast cell is stable, the collimation between the experimental test result is better; When 5) utilizing reorganization female hormone receptor gene double cross yeast to carry out the screening of class oestrogenic hormon pollutent and toxicity toxicological study, easy and simple to handle, time saving and energy saving, required sample size is few, and is with low cost; 6) the present invention has cloned people's female hormone receptor gene, can reflect that the class estrogen compound in the environmental sample may be to the influence of human body generation; 7) differently with the estrogen receptor of Nishihara bibliographical information clone mouse be, the female hormone receptor gene that the present invention directly clones people or gene fragment make up the double cross yeast, the risk that therefore can direct external early warning compound when measuring the oestrogenic hormon interference effect of environmental compound may exist the human endocrine system.Adopt more advanced pGBKT7 expression plasmid of yeast and Wine brewing yeast strain Y187 (Saccharomyces cerevisiae) technically, made the ER-GRIP1 bacterial strain that makes up have higher sensitivity, its EC
50Value is 7.3 * 10
-11MolL
-1, and Nishihara is reported as＞3 * 10
-10MolL
-1
Description of drawings
Below, describe the present invention in conjunction with the accompanying drawings in detail, wherein:
Fig. 1 is that 17 beta estradiols are to recombinant Saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) enzymic activity inductive dose-effect relationship typical curve.Wherein X-coordinate is 17 beta estradiol concentration, and ordinate zou is 17 beta estradiol inductive betagalactosidase activities;
Fig. 2 is for utilizing the result of chemical analysis, single crosses reorganization female hormone receptor gene yeast and three kinds of method test environment samples of double cross recombinant Saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) class estrogen effect; Wherein X-coordinate is a chemical analysis results, and ordinate zou is the test result of single crosses reorganization female hormone receptor gene yeast and double cross recombinant Saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) method; Wherein ◆ expression double cross yeast cell strain (yeast saccharomyces cerevisiae ER-GRIP1 (CGMCCNo.2307)); ■ represents the strain of single crosses yeast cell; EEQ represents 17 beta estradiol equivalent concentration (pgL
-1); YES represents the yeast detection method.
Embodiment
Yeast saccharomyces cerevisiae provided by the invention (Saccharomyces cerevisiae) ER-GRIP1 on December 27th, 2007 at the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms, Datun Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, postcode 100101) carried out the biomaterial preservation, deposit number is CGMCC No.2307.Specify the preparation of yeast saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) and the application in the biological test method of class estrogen compound in testing environment below in conjunction with embodiment.
Embodiment 1: the preparation of yeast saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) cell
1. make up the pGBKT7-hER expression plasmid of yeast that comprises female hormone receptor gene fragment (being sequence shown in the SEQ ID No.1).
At first design the PCR primer of estrogen receptor ligands, and introduce EcoRI and the single restriction enzyme site of BamHI, i.e. upstream primer P1:5 '-TCGCCG respectively at 5 of upstream and downstream primer ' end in conjunction with territory (hER LBD)
GAATTCGCTGGAGACATGAGA-3 ' (underscore partly is the restriction enzyme site of EcoRI); Downstream primer P2:5 '-TAACGC
GGATCCTCAGACTGTGGCAGG-3 ' (underscore partly is the restriction enzyme site of BamHI); Carry out pcr amplification with pASV3 plasmid (being so kind as to give) 2.5ng that contains hER LBD (sequence shown in the SEQ ID No.1) as template by professor Heery of Britain Nottingham university; PCR product (0.3pmol) is connected in the T-carrier with T4 dna ligase (1 μ L), the T-hER LBD plasmid of structure; With pGBKT7 (1 μ g, available from Clontech company) and T-hER LBD (1 μ g) plasmid use EcoRI (1 μ L) and BamHI (1 μ L) double digestion respectively, enzyme is cut product and is cut glue after by 0.8% agarose gel electrophoresis analysis and reclaim, pGBKT7 (0.03pmol) adopts T4 dna ligase (1 μ L) to be connected with the recovery product that hER LBD inserts fragment (0.3pmol), makes up the pGBKT7-hER expression plasmid of yeast.Plasmid DNA is carried out sequencing, and sequencing primer is: 5 '-TTTTCGTTTTAAAACCTAAGAGTC-3 '.
2. purifying comprises the pGAD424-GRIP1 expression plasmid of yeast of estrogen receptor co-activation factor gene fragment (being sequence shown in the SEQ ID No.2).
Transform 100 μ L competence bacillus coli DH 5 alphas with 10 μ L target plasmid pGAD424-GRIP1 (professor Stallcup of California, USA university is so kind as to give), screening positive clone in containing the substratum of penbritin extracts plasmid and purifying.0.8% agarose gel electrophoresis is identified the plasmid DNA size, simultaneously plasmid DNA is carried out sequencing, and sequencing primer is pGAD424 universal primer (examining order is finished by Sinogenomax Co., Ltd.).The described substratum that contains penbritin comprises 100mgL
-1Penbritin, 10gL
-1Tryptones, 5gL
-1Yeast extract, 10gL
-1NaCl, 15gL
-1Agarose.
3. make up the yeast saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) of double cross.
Add 0.1 μ gpGBKT7-hER plasmid simultaneously and 0.1 μ gpGAD424-GRIP1 plasmid mixes, and add the competence brewing yeast cell Y187 (Saccharomycescerevisiae) of 0.1mL prepared fresh, 200rpm is hatched 30min for 30 ℃.Add after 70 μ L dimethyl sulfoxide (DMSO) (DMSO) mix, 15min are placed in 42 ℃ of water-baths; Cell is transferred to ice bath rapidly, leaves standstill 1-2min, makes up the yeast saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) of double cross.
4. screen the yeast saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) of double cross.
In aforesaid method, when screening the yeast saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) of double cross, the method screening double cross yeast that adopts auxotroph screening and the analysis of colony lift filter membrane to combine, the double cross yeast goes out positive bacterium colony by auxotroph substratum (SD/-Trp/-Leu) scalping, further use colony lift filter membrane analytical procedure, with bacterial colony photographic reprinting to the dry filter membrane of aseptic 1#, liquid nitrogen multigelation 3～5 times, smudge cells; The 1# filter membrane placed contain 17 beta estradiols (1 * 10
-10MolL
-1) the 2# filter membrane of X-gal damping fluid on, 30 ℃ hatch occur up to the clone significantly blue.The bacterium colony of colour developing is positive bacterium colony, and is standby behind the positive bacterium colony continuation cultivation 24h.Described SD/-Trp/-Leu comprises 20mgL
-1The VITAMIN B4 Hemisulphate, 20mgL
-1Arginine hydrochloride, 20mgL
-1One hydration histidine monohydrochloride, 30mgL
-1Isoleucine, 30mgL
-1Lysine hydrochloride, 20mgL
-1Methionine(Met), 50mgL
-1Phenylalanine, 200mgL
-1Threonine, 30mgL
-1Tyrosine, 20mgL
-1Uridylic, 150mgL
-1Xie Ansuan, 6.7gL
-1No amino acid yeast nitrogen; The X-gal damping fluid is 20mgmL
-1The dimethyl formamide solution of 5-bromo-4-chloro-3-indoles-β-D-galactoside.
Embodiment 2: utilize the yeast saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) of double cross to measure 17 beta estradiol (E
2) effect come the drawing standard curve.
Yeast cell is cultivated: with yeast saccharomyces cerevisiae ER-GRIP1 (CGMCCNo.2307) cell inoculation of the double cross of preparation in SD/-Trp/-Leu liquid nutrient medium (composition with embodiment 1 in identical), detect the absorbance (is blank with the substratum) at bacterium liquid 600nm place, regulate absorbance and be that to cultivate the absorbance of adjusting bacterium liquid 600nm place behind the 24h in 0.1～0.2,30 ℃ of airbath shaking tables be 0.7～0.8.
Drawing standard curve: get bacteria suspension 0.995mL to corresponding Eppendorf pipe, add 5 μ LDMSO (blank) or 5 μ L DMSO dissolved E
2Each 200 μ L transfers in 96 orifice plates successively with above solution.In 130rpm, 30 ℃ in 96 orifice plate shaking table shaking culture 2h then; After cultivating end, at first detect the absorbance at 600nm place; Remove 150 μ L yeast liquid; Add 120 μ L assay buffer (adding 3.33mL concentration is 0.1% SDS solution and 270 μ L beta-mercaptoethanols in the damping fluid of every 100mL basis) and 20 μ L chloroforms, the pre-10min that cultivates on 30 ℃ constant temperature shaking table; Reaction solution (the 0.04gL that adds 40 μ L ortho-nitrophenyl-β-D-galactopyranosides again
-1), start enzyme reaction, along with further cultivation, can see gradually that nutrient solution presents more and more stronger yellow; 30 ℃ are fully reacted back (for 17 beta estradiols, such process needs 20min approximately) down, add 100 μ L sodium carbonate solution (1molL
-1) termination reaction; Get 200 μ L supernatant liquors detect end product o-nitrophenol sodium with spectrophotometer at the 420nm place absorbance, and, draw E with this parameter as the expression beta-galactosidase enzymes
2Betagalactosidase activity inductive dose-effect relationship typical curve (Fig. 1) to recombination yeast saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) cell.
The calculation formula of betagalactosidase activity u is as follows: u=(A
S-A
B)/tVDOD
S, in the formula, u is a betagalactosidase activity; T is reaction times (min); V is test volume (mL); D is a dilution factor; OD
SAbsorbance for specimen 595nm place; A
SBe the absorbance OD of specimen at the 420nm place
420A
BBe the absorbancy of blank at the 420nm place.
The above-mentioned reaction solution that contains excessive ortho-nitrophenyl-β-D-galactopyranoside comprises: 21.51gL
-1Na
2HPO
412H
2O, 6.22gL
-1NaH
2PO
42H
2O, 0.75gL
-1KCl, 0.25gL
-1MgSO
47H
2O, 0.04gL
-1Ortho-nitrophenyl-β-D-galactopyranoside.
As shown in Figure 1, E
2EC to yeast saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) the cell betagalactosidase activity inductive dose-effect relationship typical curve of double cross
50Value is 7.3 * 10
-11MolL
-1, 2 * 10
-11～2 * 10
-10MolL
-1The scope internal memory is in linear relationship; E with world report
2EC in the recombination yeast system
50Be 1 * 10
-11～3 * 10
-9MolL
-1Between the result conform to, the double cross yeast that the present invention preparation is described is for E
2Susceptibility close with the system that sets up of other method of report, show that tentatively double cross female hormone receptor gene yeast measurement system can be used as the quantitative analysis standard of the toxic effect of class estrogen compound in the environment.
Embodiment 3: the class oestrogenic hormon pollutent toxicity of utilizing yeast saccharomyces cerevisiae ER-GRIP1 (CGMCC No.2307) the biological test method evaluation environmental sample of double cross.
Gather the water inlet of the different water factories in certain city, water outlet respectively as sample.Adopt the method for Solid-Phase Extraction, add the class oestrogenic hormon pollutent in methylene dichloride, the normal hexane extraction sample, the organic solvent that extracts is dried up in high pure nitrogen, add the test sample solution that DMSO dissolves and be prepared into arbitrary concentration in the typical curve linearity range.
Utilize the class oestrogenic hormon pollutent toxicity of yeast saccharomyces cerevisiae ER-GRIP1 (CGMCCNo.2307) the biological test method evaluation environmental sample of the double cross described in the embodiment 1, the measurement result of measurement result and single crosses reorganization female hormone receptor gene yeast method, mass spectrometric analysis method compares, and sees Fig. 2.
As can be seen from Figure 2, no matter be single crosses yeast cell and the bioassay of double cross yeast cell, between the toxic equivalent of resulting result and mass spectroscopy class oestrogenic hormon pollutent good correlationship is arranged.Double cross yeast cell bioassay result is more near chemical analysis (being mass spectroscopy), and and the single crosses recombinant gene yeast cell tests method that adopts usually at present in the world good comparability is arranged.The result of Fig. 2 represents and can judge the toxic equivalent of class oestrogenic hormon pollutent in the environmental sample or the size of its potential eco-toxicity with the method for the present invention's proposition.
Sequence table
SEQ?ID?No.1
＜110〉Ecological Environment Research Center, Chinese Academy of Sciences
＜120〉be used for the double cross yeast and the biological test method of testing environment class estrogen compound
<130>?DIC07110101
<160>2
<170>PatentIn?version?3.3
<210>1
<211>942
<212>DNA
＜213〉people
<400>1
gctggagaca?tgagagctgc?caacctttgg?ccaagcccgc?tcatgatcaa?acgctctaag 60
aagaacagcc?tggccttgtc?cctgacggcc?gaccagatgg?tcagtgcctt?gttggatgct 120
gagcccccca?tactctattc?cgagtatgat?cctaccagac?ccttcagtga?agcttcgatg 180
atgggcttac?tgaccaacct?ggcagacagg?gagctggttc?acatgatcaa?ctgggcgaag 240
agggtgccag?gctttgtgga?tttgaccctc?catgatcagg?tccaccttct?agaatgtgcc 300
tggctagaga?tcctgatgat?tggtctcgtc?tggcgctcca?tggagcaccc?agggaagcta 360
ctgtttgctc?ctaacttgct?cttggacagg?aaccagggaa?aatgtgtaga?gggcatggtg 420
gagatcttcg?acatgctgct?ggctacatca?tctcggttcc?gcatgatgaa?tctgcaggga 480
gaggagtttg?tgtgcctcaa?atctattatt?ttgcttaatt?ctggagtgta?cacatttctg 540
tccagcaccc?tgaagtctct?ggaagagaag?gaccatatcc?accgagtcct?ggacaagatc 600
acagacactt?tgatccacct?gatggccaag?gcaggcctga?ccctgcagca?gcagcaccag 660
cggctggccc?agctcctcct?catcctctcc?cacatcaggc?acatgagtaa?caaaggcatg 720
gagcatctgt?acagcatgaa?gtgcaagaac?gtggtgcccc?tctatgacct?gctgctggag 780
atgctggacg?cccaccgcct?acatgcgccc?actagccgtg?gaggggcatc?cgtggaggag 840
acggaccaaa?gccacttggc?cactgcgggc?tctacttcat?cgcattcctt?gcaaaagtat 900
tacatcacgg?gggaggcaga?gggtttccct?gccacggtct?ga 942
SEQ?ID?No.2
<210>2
<211>4374
<212>DNA
＜213〉mouse
<400>2
ggagaaaaca?cctctgaccc?gtccagggca?gagaccagaa?aacgcaagga?atgtcccgac 60
cagctcggac?ccagccccaa?aaggagcact?gagaaacgga?accgcgagca?ggagaataag 120
tacatagagg?agctggccga?tctgatcttc?gcaaacttta?atgatattga?caacttcaac 180
ttcaaacctg?acaaatgtgc?catcctaaaa?gaaactgtga?agcagatccg?ccagatcaaa 240
gagcaagaga?aagcagcagc?tgccaacata?gatgaagtgc?agaagtcaga?tgtgtcgtcc 300
acggggcagg?gtgtcatcga?caaggatgca?ctggggccca?tgatgcttga?ggccctcgat 360
gggttcttct?tcgttgtgaa?cctggaaggc?agtgtggtgt?tcgtgtcaga?gaatgtgaca 420
cagtatctac?ggtataacca?agaagagctg?atgaacaaga?gtgtctacag?catcctgcat 480
gtcggggacc?acactgaatt?tgtcaagaac?ctgctgccaa?agtccatggt?gaatggagga 540
tcctggtctg?gagaacctcc?caggcggacg?agccatacct?tcaactgtcg?catgctggtg 600
aagcctttgc?cagattcaga?agaggaaggc?catgatagcc?aggaagccca?tcagaaatac 660
gaggcgatgc?agtgcttcgc?tgtgtctcag?cccaagtcca?tcaaagagga?aggcgaagat 720
ttgcagtcct?gcttgatttg?tgtggcacga?agagtcccca?tgaaggaaag?accaactctt 780
ccctcatcag?aaagctttac?cacccgccag?gacctccaag?gcaagatcac?ttcactggac 840
actagcacca?tgagagccgc?catgaagccg?ggctgggaag?atctggtaag?aagatgcatt 900
cagaagttcc?acacacagca?tgaaggggag?tctctatcat?atgccaagag?gcatcaccat 960
gaagttctga?gacaagggtt?ggcgttcagt?cagatctatc?gtttttcttt?gtctgatggc 1020
actctcgttg?ctgcacaaac?caagagcaaa?ctcatccgtt?ctcagactac?taatgagcct 1080
cagcttgtaa?tatctttaca?catgcttcac?agagagcaga?atgtatgtgt?aatgaatccg 1140
gatctgactg?gacaagcgat?ggggaagcca?ttgaatccaa?ttagctctag?cagccctgcc 1200
caccaggccc?tgtgcagtgg?gaacccaggt?caggacatga?ccctcggtag?caatataaat 1260
tttcccatga?atggcccaaa?ggaacaaatg?ggcatgccta?tgggcaggtt?tggtggttct 1320
gggggcatga?accatgtgtc?aggcatgcag?gcaaccactc?ctcagggtag?taactatgca 1380
ctcaaaatga?acagtccctc?gcaaagcagc?cccggcatga?acccggggca?agccagctcc 1440
gtgctctccc?caaggcagcg?catgagcccc?ggcgtggctg?gcagtcctcg?catcccaccc 1500
agtcagtttt?cccctgcagg?aagcttgcat?tcccctgtgg?gagtttgcag?cagcacagga 1560
aatagccata?gttataccaa?cagttccctc?aatgcactgc?aagccctcag?cgagggccat 1620
ggggtctcac?tcgggtcctc?gctggcttca?ccggacctaa?aaatgggcaa?tttgcaaaac 1680
tccccagtta?atatgaatcc?tcccccactc?agcaagatgg?gaagcttgga?ctccaaagac 1740
tgttttggac?tttatgggga?gccctcagaa?ggtacaactg?gacaagcaga?ggccagctgc 1800
catcctgaag?aacaaaaggg?gcccaatgat?tccagcatgc?cccaggcggc?cagcggggac 1860
agggctgagg?gacacagccg?gctgcatgac?agcaaagggc?agaccaaact?cctgcagctg 1920
ctgaccacca?agtccgacca?gatggagcct?tcacccttgc?ccagctcctt?gtcggacaca 1980
aacaaggact?caacagggag?cttgcctggg?cctgggtcca?cgcatggcac?ctcgctcaag 2040
gagaagcata?agattttgca?cagactctta?caggacagca?gttcccctgt?ggacttggcc 2100
aagctgacag?cagaagccac?aggcaaagag?ctgagccagg?agtccagcag?cacagctcct 2160
gggtcggaag?tgactgtcaa?acaggagcca?gcgagcccca?agaagaaaga?gaatgcacta 2220
ctgcgctatt?tgctcgacaa?agatgatact?aaagatattg?gtttaccgga?aataaccccc 2280
aaactcgagc?gactggacag?taagacagat?cctgccagta?acacaaagtt?aattgctatg 2340
aaaactgtga?aggaggaggt?gagctttgag?cccagtgacc?agcctggcag?cgagctggac 2400
aacttggaag?agattttgga?tgatttgcag?aacagtcagt?taccacagct?tttcccagac 2460
acaaggccag?gagctcctac?tgggtcagtt?gacaagcaag?ccatcatcaa?tgacctcatg 2520
caactcacag?ctgacagcag?tcccgtccca?cctgccggag?cccagaaggc?agcactgcgc 2580
atgtcacaga?gcacttttaa?taacccacga?ccagggcaac?tgggcaggtt?attgccaaac 2640
cagaacttac?cacttgacat?cactttgcaa?agcccaactg?gtgctggacc?tttcccacca 2700
atcagaaaca?gtagccccta?ctcagtgata?cctcagccag?gaatgatggg?taaccaaggg 2760
atgctaggaa?gccaaggaaa?cttagggaac?aatagcacag?gaatgattgg?cagcagcact 2820
tcccggccca?gcatgccttc?tggggaatgg?gcaccacaga?gtccagctgt?gagagtcact 2880
tgtgctgcta?ccactggtgc?catgaaccga?ccagtccaag?gaggcatgat?tcggaaccca 2940
acagccagca?tccccatgcg?agccaacagc?cagcctggcc?aaagacagat?gcttcagtct 3000
caggtcatga?acataggccc?ttctgagtta?gagatgaaca?tgggaggacc?tcagtataat 3060
caacagcagg?cccctccgaa?ccaaactgcc?ccgtggcctg?agagcatcct?gcctatagac 3120
caggcatcgt?ttgccagcca?gaacaggcag?cccttcggca?gctcccctga?tgacctgctg 3180
tgtccacatc?ctgcagcaga?gtcgccaagc?gatgagggcg?ctcttcttga?ccagctgtat 3240
ctggccttgc?ggaacttcga?tggccttgag?gagattgata?gagctctggg?gataccagaa 3300
ctggtcagcc?agagccaagc?tgtggatgca?gagcagttct?caagtcagga?gtccagcata 3360
atgctggagc?agaagccccc?cgttttccca?cagcagtacg?catctcaggc?acaaatggcc 3420
cagggtggct?ataatcccat?gcaagatcca?aactttcaca?ccatgggaca?gcggccaaat 3480
tacaccacac?tccgtatgca?gccacggcca?ggcctcaggc?ccacaggcat?tgtacagaac 3540
cagccaaacc?aactgagact?tcagcttcag?caccgcctcc?aagcacagca?gaatcgccag 3600
ccgcttatga?atcagatcag?cagtgtttcc?aatgtgaacc?tgactctgag?gcctggagtg 3660
cccactcagg?ctcctattaa?tgcacagatg?ctggcccaga?ggcagaggga?aatcctcaac 3720
caacaccttc?ggcagaggca?gatgcagcag?caggtgcagc?agcggactct?gatgatgaga 3780
ggacagggct?tgaatgtgac?cccaagcatg?gtggctcccg?ctggcctacc?agcagccatg 3840
agcaatcccc?ggatccccca?ggccaatgcc?cagcagttcc?catttcctcc?gaactacgga 3900
ataagtcaac?aacctgatcc?tggctttact?ggggctacga?ctccccagag?tcctctaatg 3960
tctccccgga?tggcacatac?tcagagtccc?atgatgcagc?agtctcaagc?caacccagcc 4020
taccagccca?cctcagacat?gaatggatgg?gcacagggga?gcatgggtgg?aaacagcatg 4080
ttctcacagc?agtccccacc?acactttggg?caacaagcaa?acaccagcat?gtatagtaac 4140
aacatgaaca?tcagtgtgtc?gatggcaacc?aacacgggtg?gcttgagcag?catgaaccag 4200
atgacatgcc?agatgagcat?gacctcagtg?acctccgtgc?ctacgtcagg?actgccctcc 4260
atgggtcccg?agcaggtcaa?tgaccctgct?ctgaggggag?gcaacctttt?cccaaaccaa 4320
ctgcctggaa?tggacatgat?caagcaggag?ggagatgcat?ctcggaaata?ctgc 4374
Claims (15)
1. double cross yeast that is used for testing environment sample class estrogen compound or natural estrogen compound, it is characterized in that, contain pGBKT7-ER expression plasmid of yeast and pGAD424-GRIP1 expression plasmid of yeast in this yeast, it is the human estrogen acceptor gene fragment shown in the SEQ ID No.1 that wherein said pGBKT7-ER expression plasmid of yeast comprises sequence, it is the estrogen receptor co-activation factor gene fragment shown in the SEQ ID No.2 that the pGAD424-GRIP1 expression plasmid of yeast comprises sequence, and used yeast is yeast saccharomyces cerevisiae Y187.
2. double cross yeast according to claim 1 is characterized in that, described class estrogen compound is selected from one or more in phenols class estrogen compound, phthalate compound, the organochlorine pesticide.
3. double cross yeast according to claim 1 is characterized in that, described double cross yeast is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) ER-GRIP1, and its deposit number is CGMCCNo.2307.
4. one kind according to each described double cross zymic preparation method in the claim 1 to 3, it is characterized in that this method may further comprise the steps:
(1) makes up the pGBKT7-ER expression plasmid of yeast that comprises described female hormone receptor gene;
(2) purifying comprises the pGAD424-GRIP1 expression plasmid of yeast of described estrogen receptor co-activation factor gene;
(3) adopt above-mentioned two kinds of plasmids to make up double cross yeast ER-GRIP1;
(4) screening double cross yeast ER-GRIP1.
5. preparation method according to claim 4, it is characterized in that, step (1) comprises the PCR primer of design estrogen receptor ligands in conjunction with territory ER LBD, wherein introduce EcoR I and the single restriction enzyme site of BamH I respectively at 5 of upstream and downstream primer ' end, and upstream primer P1:5 '-TCGCCGGAATTCGCTGGAGACATGAGA-3 '; Downstream primer P2:5 '-TAACGCGGATCC TCAGACTGTGGCAGG-3 '.
6. preparation method according to claim 5 is characterized in that, transforms with pGBKT7-ER plasmid and pGAD424-GRIP1 plasmid simultaneously in step (3) and enters brewing yeast cell Y187 (Saccharomyces cerevisiae).
7. preparation method according to claim 5 is characterized in that, the method that adopts auxotroph screening and the analysis of colony lift filter membrane to combine in step (4) is screened described double cross yeast.
8. preparation method according to claim 7 is characterized in that, adopts the colony lift filter membrane that adds estrogenic X-gal damping fluid to analyze in step (4).
9. preparation method according to claim 8 is characterized in that, described oestrogenic hormon is 17 beta estradiols.
10. preparation method according to claim 8 is characterized in that, described X-gal damping fluid is 20mgmL
-1The dimethyl formamide solution of 5-bromo-4-chloro-3-indoles-β-D-galactoside.
11. preparation method according to claim 7 is characterized in that, the substratum that adopts in the described auxotroph screening is not for adding tryptophane and leucic SD substratum, and this substratum is by 20mgL
-1The VITAMIN B4 Hemisulphate, 20mgL
-1Arginine hydrochloride, 20mgL
-1One hydration histidine monohydrochloride, 30mgL
-1Isoleucine, 30mgL
-1Lysine hydrochloride, 20mgL
-1Methionine(Met), 50mgL
-1Phenylalanine, 200mgL
-1Threonine, 30mgL
-1Tyrosine, 20mgL
-1Uridylic, 150mgL
-1Xie Ansuan, 6.7gL
-1No amino acid yeast nitrogen is formed.
12. the biological test method of class estrogen compound in the testing environment, it is characterized in that, this method comprises the steps: each described double cross yeast cell and testing sample in the claim 1 to 4 are cultivated altogether, the reaction solution that adds ortho-nitrophenyl-β-D-galactopyranoside reacts, the concentration of coming the compute classes estrogen compound according to the absorbance of detected supernatant liquor at the 420nm place after the termination reaction.
13. biological test method according to claim 12 is characterized in that, described method comprises the steps:
(1) cultivates double cross yeast ER-GRIP1 cell;
(2) typical curve is induced in drafting: at first described double cross yeast cell is cultivated altogether with known series concentration oestrogenic hormon respectively, adding ortho-nitrophenyl-β-D-galactopyranoside reaction solution reacts, detect the absorbance of supernatant liquor after the termination reaction at the 420nm place, and, draw the betagalactosidase activity inductive dose-effect relationship typical curve of the described relatively double cross yeast of serial estrogen concentrations ER-GRIP1 cell with this parameter as the expression betagalactosidase activity;
(3) test sample: described double cross yeast cell and testing sample are cultivated altogether, the reaction solution that adds ortho-nitrophenyl-β-D-galactopyranoside reacts, the detection reaction supernatant liquor is at the absorbance at 420nm place after the termination reaction, and the typical curve that contrast step (2) is formulated calculates the content of class estrogen compound in the sample.
14. biological test method according to claim 13, it is characterized in that, in step (1), the absorbance of regulating bacterium liquid 600nm place during with the ER-GRIP1 cell inoculation is that the absorbance at adjustment bacterium liquid 600nm place behind 0.1～0.2,30 ℃ of cultivation 24h is that 0.7～0.8 preparation is tested.
15., it is characterized in that described testing sample is an organic solvent extraction liquid according to each described biological test method in the claim 12 to 14, wherein oestrogenic hormon equivalent concentration is 10
-11～10
-10MolL
-1
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| CN103555601A (en) * | 2013-11-13 | 2014-02-05 | 华南师范大学 | Recombinant yeast for detecting environmental estrogen and constructing method and use of recombinant yeast |
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| CN104498376A (en) * | 2014-12-19 | 2015-04-08 | 中国科学院生态环境研究中心 | Recombinant estrogen receptor fluorescent two-hybrid yeast as well as preparation method and application |
| CN105866441B (en) * | 2015-01-19 | 2017-07-28 | 中国科学院生态环境研究中心 | A kind of method for detecting estrogen or oestrogen-like hormone compound concentration |
| CN104711204A (en) * | 2015-03-05 | 2015-06-17 | 中国科学院生态环境研究中心 | Double-cross yeast gER beta 1-GRIP1 for reorganizing estrogen receptor of gobiocypris rarus and application of double-cross yeast gER beta 1-GRIP1 |
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| CN110564753A (en) * | 2019-08-15 | 2019-12-13 | 中国海洋大学 | Yeast recombination system and method for detecting environmental estrogen interferents |
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