JP5858393B2 - Novel gene expression control method using variable region of antibody - Google Patents

Description

  The present invention relates to a novel and innovative expression vector that can be used for gene expression regulation, a transformed cell, and a gene expression regulation system using them.

  In the field of molecular biology research, heterologous expression in E. coli, yeast, mammalian cells, etc. is an important strategy for protein structure / function analysis, and various gene expression regulation systems have become widely used. For example, for the regulation of gene expression in E. coli, an expression control system using a lactose repressor using isopropyl-β-D (-)-thiogalactopyranoside (IPTG) as an inducer is used. For regulation of expression, a system using a tetracycline resistant operon using tetracycline as an inducer is used. Furthermore, gene expression regulation systems are widely applied to fields such as gene therapy and environmental monitoring. Development of gene therapy vectors for the purpose of cancer treatment that controls the expression of tumor necrosis factor using a promoter that responds to radiation is also progressing. The present inventors have so far developed a gene expression control system using an estrogen receptor and an allyl hydrocarbon receptor, and succeeded in creating a genetically modified plant for detecting environmental pollution by estrogen-like compounds and dioxins. (Non-patent Document 1 and Patent Documents 1 and 2). However, the existing gene expression regulation system has been developed using a gene expression control system inherent in living organisms. In other words, substances that serve as inducers are restricted by the presence of receptors, and we cannot set them freely.

  Currently, it is possible to artificially regulate gene expression through protein interactions. Yeast Two-Hybrid System (Non-Patent Document 2), developed in 1989 for the purpose of analyzing protein interactions by Fields, S. and Song, O., is the transcription factor GAL4 protein DNA binding region and transcription. The fact that the active region is separable is used. A protein in which a DNA binding region of GAL4 and an arbitrary protein A are fused, and a fusion protein of a transcription activation region and protein B are simultaneously expressed. Therefore, only when protein A and protein B interact, both fusion proteins can associate to function as a single transcription factor and regulate gene expression. Most transcriptional regulators can bind to a specific base sequence or promote / suppress transcription without losing its function even if the DNA-binding region or transcriptional regulatory region is separated and fused to another protein. . That is, it is possible to construct a Two-Hybrid System by replacing the DNA binding region and transcription activation region of GAL4 with those of other transcription factors.

Antibodies have been tried to be applied to disease diagnostic tests, antibody drugs, environmental monitoring, food quality control, etc. by utilizing the property of binding specifically to the substance. At present, it is no exaggeration to say that methods for producing antibodies that bind to all substances including proteins have been established, and there are no substances that cannot produce antibodies. In recent years, with the development of genetic engineering, the acquisition of antibody genes has become easier, and antibody engineering has developed rapidly. Heavy chain variable regions (V _H ) that play an important role in antigen binding and light Chain variable region (V _L ) fragments were isolated and made available. This facilitated mass production of antibody fragments and production of recombinant proteins using antibodies. In particular, the _VH and _VL fragments each have the ability to bind to an antigen, and can be said to be the minimum unit polypeptide having an antibody function. It is known that when an antigen is present, _VH and _VL fragments bind to the antigen to form a three-hybrid. An open sandwich immunochemical assay (ELISA) utilizes this property (Non-patent Document 3).

However, no gene expression regulation system using a recombinant gene in which an antibody variable region is fused with a DNA binding protein or a transcriptional activator has been reported so far, and such a technique is not disclosed in the prior art literature. There is no suggestion about the system.
JP 2004-089068 A Japanese Patent Laid-Open No. 20060-129733 Kodama, S. et al. Aryl hydrocarbon receptor (AhR) -mediated reporter gene expression systems in transgenic tobacco plants. Planta (2007) Fields, S. and Song, O. A novel genetic system to detect protein-protein interactions. Nature (1989) 340, 245-246 Ueda, H. et al. Open sandwich ELISA: A novel immunoassay based on the interaction of antibody variable region.Nature Biotechnology (1996) 14, 1714-1718

  An object of the present invention is to provide an expression vector, a transformant, and a gene expression regulation system using them, which are superior to conventional systems and can be used for novel and innovative regulation of gene expression.

In order to solve the above-mentioned problems, the present inventors have conducted intensive research and prepared a recombinant protein in which a DNA binding region or a transcriptional activation region is bound to each of the _VH and _VL fragments of an antibody, thereby producing an antigen (induction). The substance is present in the presence of V _H and V _L , and the recombinant protein exhibits a function as one transcription factor, thereby obtaining a gene expression regulation system that promotes or suppresses gene transcription, The present invention has been completed.

Accordingly, the present invention includes the following.
Item 1. A recombinant protein gene in which a heavy chain variable region of an antibody is fused with either a DNA-binding protein or a transcriptional regulatory factor; and a light chain variable region of the antibody and a heavy chain of a DNA-binding protein or a transcriptional regulatory factor An expression vector comprising a recombinant protein gene fused with a variable region and a different one,
A vector characterized by regulating the expression of a target gene in a cell, tissue or individual into which the vector has been introduced.
Item 2. A transformant into which the expression vector according to Item 1 has been introduced.
Item 3. A gene expression regulation method comprising:
The expression vector according to Item 1 is introduced into a cell, tissue, or individual having a target gene downstream of a promoter that responds to a DNA binding protein,
A method for regulating gene expression, which comprises regulating the expression of a target gene by adding an antigen specific to the fused antibody to the cell, tissue or individual.
Item 4. Item 4. The method according to Item 3, wherein the promoter and target gene that respond to a DNA-binding protein possessed by a cell, tissue, or individual are foreign genes.
Item 5. Using the antigen specific to the fused antibody as a test substance, the expression vector according to Item 1, a promoter and a target gene responsive to a DNA-binding protein, and a target gene are added to the target gene to express the target gene A method for screening a test substance, wherein the expression level is measured using as a parameter.
Item 6. A gene expression regulation system using an antigen of a specific antibody as an inducer,
A recombinant protein gene in which the heavy chain variable region of the antibody is fused with either a DNA binding protein or a transcriptional regulatory factor, and the light chain variable region of the antibody and the heavy chain of the DNA binding protein or the transcriptional regulatory factor. Simultaneous expression of a recombinant protein gene fused with another one fused with a chain variable region, a promoter responsive to a DNA binding protein, and a target gene downstream thereof in a cell, tissue or individual A gene expression regulation system characterized by the above.

  The following three points can be cited as breakthrough features of the gene expression regulation method using the antibody molecule according to the present invention. (1) Any substance (protein, organic chemical substance, inorganic chemical substance, etc.) can be set as an inducer as long as an antibody that binds to the target inducer can be obtained. (2) The target gene sequence can be freely changed by replacing the DNA binding protein with that of a transcription factor known so far. (3) Gene expression can be promoted or inhibited by using a transcriptional regulatory factor as a promoter or suppressor.

  Based on the above features, specific application examples of the gene expression regulation method of the present invention include, for example, the following three points. (1) Bioremediation field by controlling the expression of growth control genes in pollutant-degrading microorganisms with this system constructed using antibodies that specifically bind to environmental pollutants, especially dioxins and endocrine disrupting chemicals This can contribute to the prevention of secondary contamination caused by microorganisms. (2) By using an antibody against a marker protein that is specifically expressed in a disease marker, it can be applied to a gene therapy vector that controls the expression of a therapeutic gene according to the increase or decrease of the marker protein. (3) Application as an experimental technique capable of monitoring the localization of chemical substances and proteins in living bodies is also possible.

( Expression vector )
The expression vector of the present invention comprises: 1. a recombinant protein gene in which a heavy chain variable region of an antibody is fused with either a DNA-binding protein or a transcriptional regulatory factor; Includes two recombinant protein genes, a recombinant protein gene in which the light chain variable region of the antibody is fused with a DNA binding protein or transcriptional regulator that is different from the one fused with the heavy chain variable region .

  For example, these consist of a recombinant protein gene fused with an antibody heavy chain variable region and a transcriptional regulatory factor, and a recombinant protein gene fused with a light chain variable region of the antibody and a DNA binding protein. Alternatively, it may be composed of a recombinant protein gene fused with a heavy chain variable region of an antibody and a DNA binding protein, and a recombinant protein gene fused with a light chain variable region of the antibody and a transcriptional regulatory factor. May be.

  The action of the vector of the present invention is that a DNA-binding protein is produced in the presence of an antigen specific for an antibody in a cell, tissue or individual into which the vector has been introduced (hereinafter sometimes referred to as “subject”). Is to regulate the expression of a target gene downstream of a promoter that responds to.

  Here, as a heavy chain variable region and a light chain variable region of an antibody, those obtained by isolating a heavy chain variable region fragment and a light chain variable region fragment of an antibody derived from the same antibody can be used. There is no restriction | limiting in particular in the antibody used in this invention, What is necessary is just to select suitably according to the objective what was manufactured by genetic engineering.

  The DNA binding protein used in the present invention is not particularly limited as long as it has a gene promoter binding ability, and can be appropriately determined according to the promoter upstream of the target gene. Specific examples include DNA binding regions such as E. coli repressor (LexA), GAL4, lactose repressor, tetracycline repressor, estrogen receptor, glucocorticoid receptor, and allyl hydrocarbon receptor.

  The transcriptional regulatory factor used in the present invention is not particularly limited as long as it is a factor that promotes or suppresses the transcriptional activity of the target gene, and various transcriptional regulatory factors can be used. For example, transcription activation region of herpes simplex virus transcriptional regulatory factor (VP16), transcription activation region such as estrogen receptor, glucocorticoid receptor, allyl hydrocarbon receptor, transcription repression region such as lactose repressor, tetracycline repressor Etc., and preferably, the transcriptional activation region of VP16 can be used.

  A recombinant protein gene fused with an antibody variable region and a DNA-binding protein or a transcriptional regulatory factor can be prepared using a known genetic engineering method. More specifically, for example, mRNA is extracted from antibody-producing cells, cDNA is amplified by 5′-RACE (Rapid amplification of cDNA ends) PCR method, etc., and variable region fragments are further obtained using antibody-specific primers. It can be obtained by cloning. It can also be obtained by determining the base sequence by a sequence reaction and preparing a recombinant protein gene by a restriction enzyme treatment or the like.

  The synthetic DNA of the above recombinant protein gene can be commissioned and synthesized, for example, at Sigma Aldrich Co., Ltd. The fusion process can be performed, for example, according to an experiment document (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 3rd edition).

In addition, the expression vector of the present invention may be provided with a promoter, a marker gene for transformation, a restriction enzyme cleavage site, a terminator and the like that are usually provided in the vector. These promoters, transformation marker genes, restriction enzyme cleavage sites, terminators, and the like can be used without limitation as long as they function in the recipient into which the expression vector is introduced.
( Gene expression regulation method )
In the gene expression regulation method of the present invention, the expression vector is introduced into a cell, tissue or individual (introduced body) having a target gene downstream of a promoter responsive to a DNA binding protein, and fused to the introduced body. A method for regulating gene expression, characterized in that the expression of a target gene is regulated by adding an antigen specific to the antibody made to occur.

  Here, the promoter responsive to the DNA binding protein is not particularly limited as long as it is a promoter responsive to the DNA binding protein of the expression vector of the present invention, and any promoter may be used. Specific examples include DNA sequences to which DNA binding proteins such as LexA, GAL4, lactose repressor, tetracycline repressor, estrogen receptor, glucocorticoid receptor, and allyl hydrocarbon receptor can bind. The promoter may be endogenous or exogenous to the target substance to be introduced.

  Further, the target gene downstream of the promoter may be any gene as long as it can be translated so as to produce mRNA and optionally a protein in the recipient having the promoter. Specifically, the target to be introduced may be endogenous or exogenous. Further, it may be a gene present on the chromosome or an extrachromosomal one. Examples of exogenous substances include those derived from animals, plants, viruses, bacteria, fungi, or protozoa that can infect the recipient. The function may be known or unknown, and the function may be known in a cell of another organism, but the function may be unknown in the introduced body.

  The target to which the expression vector is introduced may be any target as long as the target gene can be transcribed into RNA or translated into protein in the cell. Specifically, the introducer used in the present invention means a cell, tissue, or individual.

  Examples of the cells used in the present invention include germline cells, somatic cells, differentiated totipotent cells, multipotent cells, divided cells, non-divided cells, parenchymal tissue cells, epithelial cells, immortalized cells, and transformed cells. It may be. Tissues include single-cell embryos or constitutive cells, multi-cell embryos, fetal tissues and the like. As specific examples of such cells, stem cells, fibroblasts, hepatocytes, blood cells, nerve cells, vascular endothelial cells, keratinocytes, melanocytes, liver cancer cells, cervical cancer cells, callus and the like are preferably used.

  Furthermore, specific examples of the individual used as the recipient in the present invention include those belonging to plants, animals, protozoa, viruses, bacteria, or fungal species. The plant may be monocotyledonous, dicotyledonous or gymnosperm, and the animal may be a vertebrate or invertebrate. Preferred microorganisms as the recipients of the present invention are those used in agriculture or industry. Fungi include organisms in both mold and yeast forms.

  As a method for introducing a vector into a recipient, when the recipient is a cell or tissue, the lithium chloride method (Ito, H. et al. Transformation of intact yeast cells treated with alkali cations. Journal of Bacteriology (1983 153 (1), 163-8), calcium chloride method (Cohen et al., Proc. Natl. Acad. Sci. USA, 69: 2110 (1972)), protoplast method (Mol. Gen. Genet., 168: 111 (1979)), competent method (J. Mol. Biol., 56: 209 (1971)), calcium phosphate method, electroporation method, lipofection method, virus infection or transformation method, and the like. Examples of the method for introduction into the embryo include microinjection, electroporation, and virus infection. When the introducer is a plant, methods such as injection or perfusion into the body cavity or stromal cells of the plant body, spraying, Agrobacterium method, electroporation method, or particle bombardment method are used. In the case of an animal individual, it is introduced systemically by oral, topical, parenteral (including subcutaneous, intramuscular and intravenous administration), vaginal, rectal, nasal, ophthalmic, intraperitoneal administration, etc. The method, electroporation method, virus infection or the like is used.

  The antigen specific to the fused antibody means an antigen specific to the antibody of the expression vector of the present invention. Specifically, the substance is not particularly limited as long as it is a substance that can specifically bind to the antibody, and can be widely selected from proteins, high molecular / low molecular organic compounds, inorganic compounds, and the like.

  In the present invention, regulation of target gene expression means promoting or suppressing (inhibiting) the transcription of the target gene.

The degree of target gene expression regulation is determined by comparing the accumulation of RNA of the target gene, or the amount of protein encoded by the target gene, with no treatment of the antigen into the introduction of the expression vector of the present invention and with the treatment. Can be measured. The amount of mRNA can be measured by a commonly used method known per se. Specifically, for example, Northern blotting method, RT (reverse transcription) -PCR method or the like can be used. In addition, the amount of protein produced can be measured by Western blotting using an antibody whose antigen is the protein encoded by the target gene, or by measuring the enzyme activity of the protein encoded by the target gene.
( Screening method )
As an application example of the gene expression regulation method of the present invention, a cell, tissue, or the like having an expression vector of the present invention, a promoter responsive to a DNA-binding protein, and a target gene using an antigen specific to the fused antibody as a test substance Examples include a method of screening a test substance by adding to an individual and measuring the expression level using the expression of the target gene as an index.

According to the screening method of the present invention, the localization of chemical substances and proteins in a living body is monitored by performing enzymatic activity of a target gene by pigment deposition, fluorescence, luminescence, etc., and observing with the naked eye, a microscope, etc. Is also possible.
( Gene expression regulation system )
The present invention further relates to a gene expression regulation system using an antigen of a specific antibody as an inducer. 1. a recombinant protein gene in which the heavy chain variable region of the antibody is fused with either a DNA-binding protein or a transcriptional regulator; 2. a recombinant protein gene in which the light chain variable region of the antibody is fused with a DNA-binding protein or a transcriptional regulator other than the one fused with the heavy chain variable region; Provided is a gene expression regulation system characterized by simultaneously expressing a promoter responsive to a DNA binding protein and a target gene downstream thereof in a cell, tissue or individual. As one embodiment of the gene expression regulation system of the present invention, there is a system as schematically shown in FIG.

  Here, the “inducing substance” means an antigen specific for the antibody possessed by the expression vector of the present invention. By adding an inducer to this system, as shown in FIG. 1, the antibody variable region fused to the transcriptional regulatory factor is linked to the other antibody variable region (ie, the antibody variable region fused to the DNA binding protein). The entire region acts as a transcription factor, and can suppress or promote the transcriptional activity of the target gene downstream of the DNA-binding protein-responsive promoter.

  That is, the gene expression regulation system of the present invention can be used for various applications by appropriately selecting the combination of the antibody to be used and the target gene.

  For example, by using the gene expression regulation system of the present invention, the system constructed using antibodies that specifically bind to environmental pollutants, particularly dioxins and endocrine disrupting chemicals, allows growth control genes in pollutant-degrading microorganisms. Controlling the expression of can contribute to the avoidance of secondary contamination by microorganisms, which is a problem in the field of bioremediation. In addition, by using an antibody against a marker protein that is specifically expressed in a disease marker, it can be applied to a gene therapy vector that controls the expression of a therapeutic gene according to the increase or decrease of the marker protein.

The following are some examples of combinations of antibodies and target genes that can be used in this system.
(Specific example 1)
Antibody: Anti-glucose antibody Target gene: Insulin Insulin expression is induced in the presence of glucose. By producing enteric bacteria and mammalian cells into which this system has been introduced, it is possible to produce cells for treating diabetes that produce insulin when the blood glucose level increases.
(Specific example 2)
Antibody: Anti-tumor marker (CEA etc.) Antibody target gene: Tumor necrosis factor such as cytokinin TNFa Expression of tumor necrosis factor TNFa is induced in the presence of the tumor marker CEA. By producing mammalian cells into which this system has been introduced, cancer therapeutic cells that synthesize tumor necrosis factor when tumor markers are increasing can be produced.
(Specific example 3) Reporter such as antibody: anti-estrogen antibody target gene: GFP (Green Fluorescent Protein) GFP expression is induced in the presence of female hormone estrogen. By creating transgenic mice with this system, observing GFP fluorescence, and monitoring tissues and cells containing female hormones in real time, new knowledge in molecular biology research can be obtained.
(Specific example 4)
Antibody: Anti-oseltamivir phosphate antibody Target gene: Reporter such as GFP Expression of GFP is induced in the presence of oseltamivir phosphate, the main component of Tamiflu. By creating transgenic mice with this system, observing GFP fluorescence, and monitoring tissues and cells in which oseltamivir phosphate is present in real time, new insights into the pharmacokinetics of Tamiflu can be obtained.

EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated in detail, this invention is not limited to these.
Example 1
(Method)
Construction of plasmid for expressing recombinant protein using antibody variable region in yeast _VH and _{VL of} anti-bisphenol A antibody BBA-2187 (JP 2002-253259 A) and LexA DNA binding region or VP16 transcription activity Recombinant protein genes linked to the activated region were constructed. Specifically, PCR is performed using the pER8XEV plasmid as a template for the LexA gene and the pX6XEV plasmid as a template for the VP16 gene with specific primers (including restriction enzyme cleavage sites) at both ends of each gene. Inserted into. After confirming the DNA sequence of the amplified fragment, it was cleaved with a restriction enzyme and inserted into a yeast expression vector. An antibody gene treated with a nuclear translocation signal (NLS) or restriction enzyme was inserted into it to construct an antibody transcription factor gene. FIG. 2 shows all 32 types of recombinant protein genes constructed.

Insert a gene containing the LexA DNA binding region into the yeast expression vector pESC-TRP (Stratagene) and a gene containing the VP16 transcriptional activation region between the GAL1 promoter and CYC1 terminator of pECS-HIS (Stratagene) to construct an expression plasmid did.
Yeast Transformation Using the constructed expression plasmid, the yeast L40 strain (recombinant yeast strain carrying the lacZ gene downstream of the LexA responsive promoter) (Invitrogen) was transformed into the lithium chloride method (Ito, H. et al. Transformation of Journal of Bacteriology (1983) 153 (1), 163-8) was transformed, and recombinant yeast was selected on SD-HIS-TRP medium. A total of 128 types of yeast strains were obtained by combining all 32 types of recombinant proteins with those having a LexA DNA binding region and those having a VP16 transcriptional activation region.
lacZ activity measurement Recombinant yeast is cultured in 2mL SD-HIS-TRP (2% glucose) medium at 30 ℃ for 24 hours, then in 2mL SD-HIS-TRP (2% galactose) medium containing bisphenol A at 24 ℃. Incubate for hours.
The cells were collected from 1 mL of the culture solution by centrifugation and then suspended in 1 mL Z buffer (60 mM Na ₂ HPO ₄ , 40 mM NaH ₂ PO ₄ , 1 mM MgSO ₄ , 10 mM KCl, 35 mM s-mercaptoethanol). The cells were collected again by centrifugation, suspended in 445 μL of Z buffer, 20 μL chloroform and 35 μL 0.1% SDS were added and stirred, and this was used as the enzyme solution. 20 μL of 4 mg / ml O-Nitrophenylgalactoside (ONPG) was added to 100 μL of the enzyme solution, and the mixture was kept at 28 ° C. When colored yellow, the reaction was stopped by adding 50 μL of 1M Na ₂ CO ₃ . 200 μL of the reaction solution was transferred to a 96-well plate, and the absorbance at 415 nm was measured with a microplate reader (MTP-500, Corona). The lacZ activity was calculated by the following formula. lacZ activity (unit / mL · OD ₆₀₀ ) = OD ₄₁₅ × 1000 / OD ₆₀₀ × reaction time (min) × enzyme solution (mL)
(result)
A total of 128 recombinant yeast strains were prepared by combining all 32 types of recombinant proteins with those having LexA DNA binding regions and those having VP16 transcriptional activation regions. Among them, when bisphenol A was treated with recombinant yeast expressing XL that fused LexA DNA-binding region and _VL and HP fused with _VH and VP16 transcriptional activation region, lacZ activity was reduced compared to the case without treatment. Increased 208 times (Figure 3). From the above results, it was shown that a gene expression regulator using the antibody antigen as an inducer can be created using the antibody _VH and _VL .

  The LexA DNA binding region and the VP16 transcriptional activation region do not contain NLS, and it was expected that NLS was added to the recombinant protein for nuclear translocation. Was not essential. Since a substance smaller than 40 kDa can pass through the nuclear pore and enter the nucleus by free diffusion, it can be presumed that the recombinant protein of less than 26 kDa can enter the nucleus by free diffusion.

The following three points can be cited as breakthrough features of the gene expression regulation system using antibody molecules of the present invention. (1) Any substance (protein, organic chemical substance, inorganic chemical substance, etc.) can be set as an inducer as long as an antibody that binds to the target inducer can be obtained. (2) The target DNA sequence can be freely changed by replacing the DNA binding region with that of a transcription factor known so far. (3) It is also possible to suppress gene expression by replacing the transcription activation region with a repression region.
Example 2
<Method>
Construction of plasmid for expressing recombinant protein using antibody variable region in yeast Produces anti-PCB80 (3,3 ', 5,5'-Tetrachlorobiphenyl) antibody Mab-4444 (JP-A-2005-247822) V _H and V _L were cloned from the hybridoma cells, respectively, and their DNA sequences and deduced amino acid sequences were determined (FIG. 4). Next, a recombinant protein gene was constructed in which LexA DNA binding region or VP16 transcriptional activation region was bound to _VH and _VL . Specifically, using the pER8XEV plasmid as a template for the LexA gene and the pX6XEV plasmid as a template for the VP16 gene, PCR is performed with specific primers (including restriction enzyme cleavage sites) at both ends of each gene, and the amplified fragment is inserted into the vector. did. After confirming the DNA sequence of the amplified fragment, it was cleaved with a restriction enzyme and inserted into a yeast expression vector. An antibody gene treated with NLS or restriction enzyme was inserted into the antibody transcription factor gene XNH / NPL. Insert a gene containing the LexA DNA binding region into the yeast expression vector pESC-TRP (Stratagene) and a gene containing the VP16 transcriptional activation region between the GAL1 promoter and CYC1 terminator of pECS-HIS (Stratagene) to construct an expression plasmid did.

Yeast Transformation Using the constructed expression plasmid, the yeast L40 strain (recombinant yeast strain carrying the lacZ gene downstream of the LexA responsive promoter) (Invitrogen) was transformed into the lithium chloride method (Ito, H. et al. Transformation of Journal of Bacteriology (1983) 153 (1), 163-8) was transformed, and recombinant yeast was selected on SD-HIS-TRP medium.

lacZ activity measurement Recombinant yeast is cultured in 2mL SD-HIS-TRP (2% glucose) medium at 30 ° C for 24 hours, then in 2mL SD-HIS-TRP (2% galactose) medium containing PCB80 for 24 hours. Cultured. The cells were collected from 1 mL of the culture solution by centrifugation and then suspended in 1 mL Z buffer (60 mM Na ₂ HPO ₄ , 40 mM NaH ₂ PO ₄ , 1 mM MgSO ₄ , 10 mM KCl, 35 mM β-mercaptoethanol). The cells were collected again by centrifugation, suspended in 445 μL of Z buffer, 20 μL chloroform and 35 μL 0.1% SDS were added and stirred, and this was used as the enzyme solution. 20 μL of 4 mg / ml O-Nitrophenylgalactoside (ONPG) was added to 100 μL of the enzyme solution, and the mixture was kept at 28 ° C. When colored yellow, the reaction was stopped by adding 50 μL of 1M Na ₂ CO ₃ . 200 μL of the reaction solution was transferred to a 96-well plate, and the absorbance at 415 nm was measured with a microplate reader (MTP-500, Corona). The lacZ activity was calculated by the following formula.
lacZ activity (unit / mL · OD ₆₀₀ ) = OD ₄₁₅ × 1000 / OD ₆₀₀ × reaction time (min) × enzyme solution (mL)
<Result>
Recombinant yeast strains having LexA-NLS-V _H and NLS-VP16- _VL were prepared. When this recombinant yeast strain was treated with 100 ng / mL PCB80, the lacZ activity was significantly increased about 1.7 times compared with the case where it was not treated (DMSO, PCB80 solvent) (FIG. 5). Furthermore, when the concentration of PCB80 to be treated was increased to 1000 ng / mL, LacZ activity increased in a concentration-dependent manner (FIG. 6). Furthermore, LacZ activity significantly increased after 4 hours of PCB80 treatment, and then increased linearly until 16 hours (FIG. 7). This recombinant yeast strain showed slight cross-reactivity to PCB169, which has high cross-reactivity with monoclonal antibodies in addition to PCB80, and increased LacZ activity (FIG. 8). From the above results, it is possible to create a gene expression regulatory factor that uses the antigen of the antibody as an inducer in the same way when using not only the anti-bisphenol A antibody but also the anti-PCB80 antibody _VH and _VL. Indicated.

FIG. 1 is a schematic diagram showing one embodiment of the gene expression regulation system of the present invention. FIG. 2 is a recombinant protein actually constructed in the examples. FIG. 3 is a graph showing that the activity in the recombinant yeast is increased. FIG. 4 shows the DNA sequence of the VL (A) and VH (B) regions of the anti-PCB80 antibody Mab-4444 and the deduced amino acid sequence. FIG. 5 shows LacZ activity in recombinant yeast XNH / NPL treated with PCB80. FIG. 6 shows concentration-dependent LacZ activity in recombinant yeast XNH / NPL treated with PCB80. FIG. 7 shows LacZ activity with PCB80 treatment time in recombinant yeast XNH / NPL. FIG. 8 shows LacZ activity in recombinant yeast XNH / NPL treated with various PCB congeners.

Claims (5)

A recombinant protein gene in which a heavy chain variable region of an antibody is fused with either a DNA-binding protein or a transcriptional regulatory factor; and a light chain variable region of the antibody and a heavy chain of a DNA-binding protein or a transcriptional regulatory factor An expression vector comprising a recombinant protein gene fused with a variable region and a different one,
An expression vector characterized by regulating the expression of a target gene in a cell, tissue or individual into which the vector has been introduced. A transformant obtained by introducing the expression vector according to claim 1 ,
A transformant for regulating the expression of a target gene in the cell, tissue or non-human individual . A gene expression regulation method comprising:
Introducing the expression vector of claim 1 into a cell, tissue or non-human individual having a target gene downstream of a promoter responsive to a DNA binding protein;
A gene expression regulation method for regulating expression of a target gene by adding an antigen specific to a fused antibody to the cell, tissue or non-human individual.   The method according to claim 3, wherein the promoter and target gene that respond to a DNA-binding protein possessed by a cell, tissue or non-human individual are foreign genes. A gene expression regulation system using an antigen of a specific antibody as an inducer,
A recombinant protein gene in which the heavy chain variable region of the antibody is fused with either a DNA binding protein or a transcriptional regulatory factor, and the light chain variable region of the antibody and the heavy chain of the DNA binding protein or the transcriptional regulatory factor. Recombinant protein gene fused with the one that is fused with the variable chain region, and a promoter that responds to a DNA binding protein and a target gene downstream thereof are simultaneously expressed in cells, tissues, or non-human individuals A gene expression regulation system characterized by comprising: