JP2003079368A - Reporter plasmid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a reporter plasmid having a high sensitivity and a low assay background in a reporter gene assay of an environmental hormone using the reporter plasmid. SOLUTION: Disclosed is a reporter plasmid using α2u globulin promoter as a promoter in a reporter plasmid incorporated the promoter in the upper stream of the reporter gene and a multiple cloning site for inserting an enhancer in the further upper stream of the reporter gene.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a plasmid vector for measuring enhancer activity, more specifically, an enhancer activity which can be used for determining hormone-like action concentration of endocrine disrupting substances (chemical substances such as so-called environmental hormones). The present invention relates to a plasmid vector for measurement.

[0002]

2. Description of the Related Art Industrially-derived chemical substances exist in the natural environment, and some of these chemical substances have hormone-like activity. Therefore, organisms (henceforth environmental organisms) living in the natural environment It is suspected that when it is taken into the living body, it may disturb the endocrine system of environmental organisms and humans, causing reproductive disorders, etc., which is a major social problem. As a method to deal with this problem, a method of assessing the endocrine disrupting activity of chemical substances, as well as investigating the actual conditions of exposure to environmental organisms and humans regarding chemical substances suspected of having endocrine disrupting action, elucidating the mechanism of endocrine disrupting action, and Development is an urgent task.

Conventionally, there is a reporter gene assay as a method for evaluating the endocrine-like activity of a chemical substance, for example, a method for evaluating the endocrine-like activity for an estrogen receptor which is one of hormone receptors.

This method is a method simulating the in vivo action mechanism of hormones such as estrogen. That is, hormones such as estrogen move into the nuclei of cultured cells and bind to the estrogen receptor there. This conjugate recognizes a specific nucleotide sequence of a chromosomal gene and binds to a gene (enhancer) of that specific nucleotide sequence. , Causes the transcription of genes located downstream of it. As a result, the corresponding protein is synthesized.

In the reporter gene assay, an expression gene for synthesizing a hormone receptor, a specific base sequence (enhancer) to which a hormone-receptor conjugate specifically binds, and a downstream thereof The bound promoter and the reporter gene bound further downstream are injected and exposed to the chemical substance to be evaluated.

As the reporter gene, a gene such as firefly luciferase, which can measure the amount of protein synthesized with high sensitivity such as luminescence, is used.

[0007] If the chemical substance easily binds to the receptor expressed in the cell, it binds to the receptor, and the bound substance binds to the specific base sequence of the enhancer, and as a result,
Expresses reporter proteins such as luciferase.
By determining the expression level of this reporter protein by measuring the luminescence level or the like, the extent of the hormone-like action of the chemical substance can be estimated.

Conventionally, SV40, CMV, TK, etc. have been used as promoters of the reporter plasmid for measuring the enhancer activity which is used for such purpose. FIG. 6 shows an example of a conventional reporter plasmid, in which an SV40 promoter sequence is arranged upstream of a reporter gene such as luciferase or LacZ (β-galactosidase), and an enhancer sequence is inserted further upstream thereof. The multiple cloning site of is arranged.

However, when an estrogen receptor reporter assay is carried out using these reporter plasmids, the background luminescence amount is large, and the maximum activity value due to estrogen such as estradiol is about 5 times that of the control, and the difference in light amount is small, For this reason, there are large variations in measured values.

[0010]

The present inventor has _conducted various studies in order to solve the above-mentioned problems.
When the globulin gene promoter is used as a promoter of a reporter plasmid, the above problems can be solved, and particularly, 90 to 300 bases upstream from the TATA box of the α _2u globulin promoter are effective as a promoter. The background can be reduced without reducing the efficiency of activation, resulting in a maximum activity value of 10 for control.
It became clear that the present invention could be improved by up to 20 times and the present invention was completed.

[0011] Therefore, an object of the present invention is to provide a reporter plasmid capable of obtaining measured values with high sensitivity and high reproducibility.

[0012]

In order to achieve the above object, the present invention relates to [1] a reporter plasmid comprising a promoter upstream of a reporter gene and an enhancer-inserting multiple cloning site upstream thereof. Is a α _2u globulin promoter.

[2] The promoter is located at 90 to 30 upstream from the TATA box of the α _2u globulin promoter.
The reporter plasmid according to [1], which is a promoter consisting of 0 bases.

[3] The reporter plasmid according to [1] or [2], wherein an enhancer that binds to a binding substance between an estrogen and an estrogen receptor is inserted at the multiple cloning site.

[4] The reporter plasmid according to [1] or [2], wherein an enhancer that binds to a binding product of androgen and androgen receptor is inserted into the multiple cloning site.

The present invention will be described in detail below.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION The reporter plasmid of the present invention comprises, as shown in FIG. 1, a promoter 4 upstream of a reporter gene 2 and a multiple cloning site 6 for inserting an enhancer upstream thereof, which are sequentially incorporated into the plasmid.

The reporter gene 2 may be any as long as it expresses a protein whose abundance can be detected with high sensitivity using a chemical substance, light or the like.
Specifically, LacZ, a luciferase that can detect luminescence
Examples include genes that express reporter proteins such as (β-galactosidase).

The promoter 4 used in the present invention is α _2u
It is a globulin promoter. As a promoter
The α _2u globulin promoter may be used as it is, but particularly, a portion of 90 to 300 bases upstream from the TATA box of the α _2u globulin promoter is preferable, and more preferably 1 to upstream from the TATA box.
It is a portion from 00 to 150 bases. By using this base moiety, as will be described later, when performing a reporter gene assay for environmental hormones using this reporter plasmid, the chemiluminescence background of the measurement system is greatly reduced without reducing the transcription activation efficiency. As a result, the maximum activation ratio with respect to the control can be improved by 10 to 20 times.

The nucleotide sequence of the α _2u globulin promoter (SEQ ID NO: 1) is shown below.

[0021]

TABLE 1 5'-GAACACCCACTGTTTTTCTTGGAAATATGCTTTGTGAAATGTATTAGTGAAAAAAATCAATCCATAGGA GATGAGATTGCCAAGTTGGAAAAGGGCAGGAACAATCCTTGGCTTCACATCAGTACATGAGAAAACATTCCA AAAAGCCTGAGGGAAGGAGGCCCATATGAGAAGGAAAAAAAAAACACTGGAACCCAGAGAGAGTATAAAGAC GAGCAAAGTGCTGAAGGTGGAGTGTGGGCATCATCAGCAGAGAGAGATTGTCCCAACAGAGAGGCAATTCTA TTCCCTACCAACATG-3 'multiple cloning site 6 is for inserting an enhancer that binds to the conjugate compound and receptor that responds to compounds of interest in the assay. Specific examples of the multi-cloning site 6 include 5'-GGTACCGAGCTCTTACTCGTGCTAGCCCGGGCTCGAGATCTGCGATCTAAGTAAGCTT-3 '(SEQ ID NO: 7).

Examples of the enhancer include receptors that bind female hormones such as estradiol and male hormones such as testosterone, and enhancers that bind to the conjugates of these hormones.

The method for producing the above reporter plasmid is as follows:
When looking at female hormone activity, after incorporating a female hormone response element (ERE) as an enhancer into the multicloning site of a commercially available luciferase reporter vector (pGL3 basic vector, manufactured by Promega, etc.), start from the start codon of the luciferase gene to ERE. It can be produced by incorporating an α _2u globulin promoter in between.

When a reporter gene assay for environmental hormones against estrogen is carried out using the above reporter plasmid, the above reporter plasmid, a chemical substance, and a plasmid for expressing a female hormone receptor are put into a cultured cell and cultured for a predetermined period of time. The amount of luciferase expression is measured as the amount of luminescence. The strength of the hormone action of the chemical substance is estimated based on the intensity of the luminescence.

The receptor expression plasmid is a commercially available mammalian cell expression vector (pCI; Promega Co., pCDNA
3.3; Invitrogen, etc.) and each hormone receptor gene can be easily prepared. Also, cells that already express hormone receptors (such as MCF-7 cells that express female hormone receptors).
Etc., the introduction of the hormone receptor expression plasmid can be omitted.

The present invention will be described in more detail with reference to the following examples.

[0027]

Examples Example 1 (Synthesis of reporter plasmid for measuring female hormone) ER
E (SEQ ID NO: 2) was synthesized. Commercial reporter plasmid (brand name pGL3-basic vector, Promega) 1
After cutting μg with restriction enzymes Kpn I and Sac I, the above ERE was incorporated to prepare an ERE / pGL3-basic vector.

Further, the plasmid (ERE / pGL3-basic vector) was cut with restriction enzymes Nco I and Xho I, and AUG100 (SEQ ID NO: 4) was inserted between the start codons of ERE and luciferase.

After transforming Escherichia coli with the ERE / pGL3-basic vector incorporating the above AUG100, colonies were cultured in a medium. Purification of plasmid (GFX ^TM Micro Plas
midPrep Kit, Amersham Pharmacia or plasmid purification kit, Qiagen) to perform ERE-AUG100
-Luc + reporter plasmid was obtained.

(Preparation of cultured cells containing female hormone receptor expression gene and reporter plasmid) 60% -80%
A confluent cell culture dish was prepared [immediately before the gene transfer, the medium was removed by washing twice with 5 ml Eagle basal medium (EMEM)].

Female hormone expression plasmid (hER / pCI)
2μg and ERE-AUG100-Luc + reporter plasmid 4μ
After adding g to 300 μl of EMEM, LipofectAMINE PLUS
After adding 60 μl of a reagent (Gibco BRL) and mixing, the mixture was left for 15 minutes (solution A). LipofectAMINE in another container at the same time
(Gibco BRL) 15 μl after mixing 12 μl and 300 μl EMEM
It was left for a minute (solution B). After mixing (A solution) and (B solution), 2.4 ml of EMEM was added to prepare about 3 ml of a DNA preparation solution, which was then added to the above-mentioned cell culture dish and left for 30 minutes. Then, 3 ml of EMEM containing 20% fetal bovine serum was added, followed by culturing overnight.

(Method for detecting activity of female hormone) Cell seeding 1 × 10 5 cells / ml of the cells prepared as described above
To 100 μl / well (10000 / well).

As a chemical substance, a natural female hormone (17
β estradiol, Wako Pure Chemical Industries, Ltd.) was added to the plate at various concentrations, and the cells were cultured in a CO 2 incubator for 24 hours.

Remove the plate from the incubator,
After discarding the medium, the plate was washed twice with phosphate buffer (10 mM, pH 7.2). Then, a cell lysing agent (Cell Cultu
15 μl of re Lysis Reagent 5X, Promega Co., Ltd.), and allowed to stand for 10 minutes. Then, a luminescence substrate [luciferase assay kit, Promega Co., Ltd.] was added, and a chemiluminescence measurement device (LUMI star, BMG Lab Tecnologies Co., Ltd.) was added. Was used to measure the amount of chemiluminescence. The result is shown in FIG.

Comparative Example 1 The amount of chemiluminescence was measured in the same manner as in Example except that the commercially available reporter plasmid pGL3 (manufactured by Promega Corp.) was used. The results are shown in Fig. 2 (B).

As is apparent from FIG. 2, in the case of the reporter plasmid according to the present invention, the transcription activity is increased by 4 times as compared with the case of the conventional reporter plasmid. Figure 3 shows the measurement background.
It was shown to.

When the background of Example 1 and the background of Comparative Example 1 are compared, it is found that the background of Example 1 is about 1/4 smaller, and the dispersion of measured values is also smaller.

Example 2 In the same manner as in Example 1, the promoter upstream of TATA, AUG150 (SEQ ID NO: 5) having 150 bases, and the promoter having 200 bases were used.
A reporter plasmid with AUG200 (SEQ ID NO: 6) was prepared. Using these reporter plasmids and the reporter plasmid AUG100 (SEQ ID NO: 4) prepared in Example 1, the same operation as in Example 1 was carried out to determine the transcription activity of E2 (17β estradiol). Indicated.

From FIG. 4, it can be seen that the number of promoter bases is highest when the number of bases upstream of TATA is about 100.

Example 3 By following the same procedure as in Example 2, AUG100 (SEQ ID NO: 4) having 100 promoter nucleotides upstream of TATA, and AUG150 having 150 promoters.
After preparing a reporter plasmid of (SEQ ID NO: 5) and AUG200 having 200 bases (SEQ ID NO: 6), an androgen responsive element ARE (SEQ ID NO: 2) was used as an enhancer instead of the female hormone responsive element ERE (SEQ ID NO: 3). ) Was inserted. Using these reporter plasmids, the same procedure as in Example 2 was carried out, and DHT, which is a kind of androgen, was used.
The results of determining the transcriptional activity of (dihydrotestosterone, Wako Pure Chemical Industries) are shown in FIG.

From FIG. 5, it can be seen that, when observing the action of androgen, the transcription activity is highest when the number of promoter bases upstream of TATA is about 150.

[0042]

INDUSTRIAL APPLICABILITY In the present invention, since the reporter plasmid uses the α _2u globulin promoter as a promoter, when it is used in a reporter gene assay, the transcription activity is high and the background is low.

[0043]

[Sequence list]                                   SEQUENCE LISTING <110> Chemicals Evaluation and Research Institute, Japan <120> alpha 2u globulin promoter <160> 7 <210> 1 <211> 300 <212> DNA <213> Artificial Sequence <400> 1 GAACACCCAC TGTTTTTCTT GGAAATATGC TTTGTGAAAT GTAT TAGTGA 50 AAAAAATCAA TCCATAGGAG ATGAGATTGC CAAGTTGGAA AAGG GCAGGA 100 ACAATCCTTG GCTTCACATC AGTACATGAG AAAACATTCC AAAA AGCCTG 150 AGGGAAGGAG GCCCATATGA GAAGGAAAAA AAAAACACTG GAAC CCAGAG 200 AGAGTATAAA GACGAGCAAA GTGCTGAAGG TGGAGTGTGG GCAT CATCAG 250 CAGAGAGAGA TTGTCCCAAC AGAGAGGCAA TTCTATTCCC TACC AACATG 300 <120> ARE <210> 2 <211> 81 <212> DNA <213> Artificial Sequence <400> 2 GGTACCAAGC TAGAACCTGT TCTGATCAAG CTAGAACAGC ATGT TCTGAT 50 CAAGCTAGAA CAGCATGTTC TGATCGAGCT C <120> ERE <210> 3 <211> 90 <212> DNA <213> Artificial Sequence <400> 3 GGTACCAAAG TCAGGTCACA GTGACCTGAT CAAAAGTCAG GTCA CAGTGA 50 CCTGATCAAA AGTCAGGTCA CAGTGACCTG ATCAGAGCTC <120> AUG100 <210> 4 <211> 151 <212> DNA <213> Artificial Sequence <400> 4 CTCGAGAGGG AAGGAGGCCC ATATGAGAAG GAAAAAAAAA CACT CGAACC 50 CAAAGAGAGT ATAAAGATGA GCAACGTGCT TGGAGGTGGA GTGT GGGCAC 100 CATCAGCAAA GAGATTGTCC CGACAGAGAG GCAATTCTAT TCCC TACCAC 150 C <120> AUG150 <210> 5 <211> 201 <212> DNA <213> Artificial Sequence <400> 5 CTCGAGACAA TCCTTGGCTT CACATCAGTA CATGAGAAAA CATT CCAAAA 100 AGCCTGAGGG AAGGAGGCCC ATATGAGAAG GAAAAAAAAA ACAC TCAAAC 150 CCAGAGAGAG TATAAAGACG AGCAAAGTGC TGGAGGTGGA GTGT GGGCAC 200 CATCCACAGA GAGATTGTCC CGACAGAGAG GCAATTCTAT TCCC TACCAC 250 C <120> AUG200 <210> 6 <211> 250 <212> DNA <213> Artificial Sequence <400> 6 CTCGAGAAAA AATCAATCCA TAGGAGATGA GATTGCCAAG TTGG AAAGGG 50 CAGGAACAAT CCTTGGCTTC ACATCAGTAC ATGAGAAAAC ATTC CAAAAA 100 GCCTGAGGGA AGGAGGCCCA TATGAGAAGG AAAAAAAAAA CACT GGAACC 150 CAGAGAGAGT ATAAAGACGA GCAAAGTGCT GAAGGTGGAG TGTG GGCATC 200 ATCAGCAGAG AGATTGTCCC AACAGAGAGG CAATTCTATT CCCT ACCACC 250 <120> Multiple cloning site <210> 7 <211> 58 <212> DNA <213> Artificial Sequence <400> 7 GGTACCGAGC TCTTACTCGT GCTAGCCCGG GCTCGAGATC TGCGATCTAA 50 GTAAGCTT

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing an example of the reporter plasmid of the present invention.

2 (A) is a graph showing the relationship between the transcription activation rate and estrogen concentration obtained in Example 1 and FIG. 2 (B).

FIG. 3 is a graph showing background chemiluminescence intensities of Example 1 and Comparative Example 1.

FIG. 4 is a graph showing the relationship between the number of promoter bases and the transcription activity when the action of female hormone is detected in the plasmid of the present invention.

FIG. 5 is a graph showing the relationship between the number of promoter bases and the transcription activity when the action of androgen is detected in the plasmid of the present invention.

FIG. 6 is an explanatory view showing a conventional reporter plasmid for measuring enhancer activity.

[Explanation of symbols]

2 Reporter gene 4 Promoter 6 multiple cloning sites

Claims (4)

[Claims] 1. A reporter plasmid comprising a promoter upstream of a reporter gene and an enhancer insertion multiple cloning site upstream thereof, wherein the promoter is an α _2u globulin promoter. 2. The reporter plasmid according to claim 1, wherein the promoter is a promoter consisting of 90 to 300 bases upstream from the TATA box of the α _2u globulin promoter. 3. The reporter plasmid according to claim 1 or 2, wherein an enhancer that binds to a conjugate of estrogen and an estrogen receptor is inserted at the multiple cloning site. 4. The reporter plasmid according to claim 1 or 2, wherein an enhancer that binds to a conjugate of androgen and an androgen receptor is inserted into the multiple cloning site.