JP4527235B2 - Novel human gene containing transcriptional regulatory region of human high affinity IgE receptor (FcεRI) α chain - Google Patents

Description

【図面の簡単な説明】
【図１】ヒト高親和性IgE受容体（FcεRI）α鎖遺伝子構造を示す図。
【符号の説明】
B; BamHI, E; EcoRI, H; HindIII, S; Sau3AI認識部位
【図２】ヒト高親和性IgE受容体（FcεRI）α鎖細胞特異的転写活性化領域の検索を示す図。
【符号の説明】
N. T.;無試験
【図３】ヒト高親和性IgE受容体（FcεRI）α鎖細胞特異的転写活性化領域の特定図。
【符号の説明】
Ｎ. T.; 無試験
【図４】ヒト高親和性IgE受容体（FcεRI）α鎖転写活性化配列の特定図。
【図５】ゲルシフトアッセイによるα鎖転写活性化配列に結合する核タンパク質の検出を示す図。
【符号の説明】
レーン1；プローブのみ、レーン2〜6；各抽出画分を含む。レーン2；competitorなし、レーン3、4；プローブと同配列のcompetitorを含む、レーン5、6；プローブ中に図4のM2と同変異を導入したcompetitorを含む。competitorはプローブの30倍分子数（レーン3、5）、60倍分子数（レーン2、4）。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a novel gene of human high affinity IgE receptor (FcεRI) α chain. Further, the present invention relates to utilization of a newly specified region having transcription activation ability. Specifically, the present invention relates to a search system for an α chain expression inhibitor using a transcriptional regulatory region, and a substance production system in animal cells.
[0002]
[Prior art]
The high-affinity IgE receptor (FcεRI) is a receptor that specifically recognizes IgE antibodies expressed on the surface of specific cells such as mast cells and basophils, and is the key to transmitting antigen-specific allergic reactions into cells. It is known that Of the three types of α-, β-, and γ-chain molecules that make up FcεRI, α-chain is expressed specifically in FcεRI and directly binds to the Fc region of IgE. It is done. In fact, anaphylactic shock does not occur in knockout mice in which the gene of FcεRIα chain is disrupted, and it has been confirmed that FcεRIα chain is essential for allergic reactions (Dombrowicz, D. et al: Cell, 75 , 969 (1993)). .
[0003]
The nucleotide sequence of the human FcεRIα chain gene has already been reported as consisting of 5 exons (Pang, J. et al: J. Immunology, 151 , 6166 (1993)). However, this gene fragment does not include a part of the 5 ′ end of the nucleotide sequence reported as human FcεRI α-chain cDNA (Kochan, JP et al: Nucleic. Acids. Res., 16 , 3584 (1988). )), It was expected that there will still be exons upstream of this first exon. Therefore, as a result of cloning and sequencing of a DNA of about 20 kb upstream of the first exon, we found two new exons (Japanese Patent Laid-Open No. 10-099081).
[0004]
[Problems to be solved by the invention]
As described above, controlling the expression of the high affinity IgE receptor (FcεRI) α chain, which is thought to play an important role in antigen-specific allergic reactions, is expected as a means for suppressing allergic reactions. However, there is only one report of the analysis we performed on the gene fragment reported by Pang et al. In 1993 (Nishiyama, C et al: J. Immunology, 163). 623 (1999)). Since the newly identified gene region exceeding 20 kb had a low activity as a site identified as a promoter, it was expected that an unknown region responsible for the transcriptional activation of this promoter was present (Japanese Patent Laid-Open No. 10-1999). -099081)).
[0005]
Therefore, an object of the present invention is to identify a region that activates transcription within a newly discovered approximately 20 kb gene region, and to construct a search system and a substance production system for an α chain expression inhibitor using the sequence. It is in.
[0006]
[Means for Solving the Problems]
In order to solve the above problems, from newly discovered human α chain gene (Japanese Patent Laid-Open No. 10-099081), fragments of various lengths and their base substitutions were prepared and linked to a reporter gene. The present invention was completed by specifying a region having transcriptional activation in the sex expression system.
[0007]
Therefore, the first of the present invention is a method for identifying a gene containing a region that performs transcriptional regulation of the human high affinity IgE receptor (FcεRI) α-chain gene and the identified gene. This DNA includes the base sequence of Sequence Listing 1. The second of the present invention is a plasmid vector containing the DNA upstream of a heterologous gene. A third aspect of the present invention is a method for using DNA for expressing a heterologous gene using the DNA.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
As described above, fragments of various lengths from newly discovered human α chain genes (JP-A-10-099081) using restriction enzyme recognition sites, polymerase chain reaction (PCR), site-specific mutagenesis, etc. And those obtained by linking the base substitution product to a reporter gene. These were subjected to a transient expression system to try to identify a transcriptional activation region that functions specifically in α-chain expressing cells.
[0009]
(1) Human high-affinity IgE receptor (FcεRI) α-chain gene transcription activation region of the human FcεRI α-chain gene covering a specific total length of 20 kbp or more, a novel first exon disclosed in JP-A-10-09081 (Ex 1A) 5 ′ of a gene fragment (Pang, J. et al .; J. Immunology, 151 , 6166 (1993)) reported in 1993 by Pang et al. From the 3′-end of a base sequence of about 1.6 kbp upstream. -The site | part which has transcription activation ability was searched about the unknown area | region to the terminal. The gene sequence of this region is shown in SEQ ID NO: 1.
[0010]
For the preparation of DNA fragments of various lengths including the first exon indicated as Ex.1A in FIG. 1, for example, a restriction enzyme site present on the chromosomal DNA can be used. Alternatively, only the target region is amplified by polymerase chain reaction (PCR), or a restriction enzyme recognition sequence is introduced into the target site on the chromosomal DNA using PCR or site-specific mutagenesis, and then the restriction enzyme is used. A method of cutting out can also be used. There are no particular limitations on the PCR method and site-directed mutagenesis method, and various kits can be used depending on the purpose. Using these methods, for example, gene fragments of various lengths as shown in FIG. 2 were linked upstream of the reporter gene, but the gene region used is not limited to the types shown in the figure. Moreover, plasmid DNA encoding luciferase is commercially available from each manufacturer as a reporter gene, but the type of plasmid is not specified. Furthermore, as the reporter gene, those that can directly quantify the amount of the protein such as CAT, β-galactosidase, growth hormone, or indirectly by using the activity as an index can be used. Moreover, depending on the PCR method and the site-specific mutation method used, unintended mutations may be introduced, so it is necessary to confirm the DNA base sequence. The method for determining the base sequence is not limited, and for example, a known method such as a DNA sequencer sold by each company can be used.
[0011]
(2) As a result of searching for a region having a promoter activation ability of about 3.8 kb around the first exon (Ex.1A) using the method of determining the transcriptional activation sequence in human FcεRI α chain gene, α chain expressing cells It was suggested that a region that specifically activates the novel promoter exists within 50 bp at a position of about 400 bp downstream of Ex.1A (FIGS. 2 and 3).
In order to identify the activation sequence present in this region, the reporter gene expression level in the mutant plasmid as shown in FIG. 4 was measured and compared. At this time, a known site-directed mutagenesis method can be used for the production of the mutant plasmid, and a kit commercially available from each company can also be used. From this experiment, it was possible to limit the sequences essential for transcriptional activation. The sequence required for transcriptional activation is shown in SEQ ID NO: 2.
[0012]
(3) Nucleoprotein that recognizes transcription activation sequence The presence of a nucleoprotein that binds to the sequence specified by the above-mentioned method can be confirmed by gel shift assay.
Isotope is generally used for labeling DNA used in the gel shift assay, but substances that can be detected with commercially available instruments such as fluorescent substances such as rhodamine can be used. In addition to the method of using the synthesized double-stranded oligo DNA after annealing, a method using PCR or the like is also possible.
[0013]
Probe DNA is mixed with the nuclear fraction prepared from the cells, reacted in a solution containing an appropriate substance that inhibits non-specific binding such as poly (dI-dC), and then subjected to acrylamide gel. Protein binding to the probe can be confirmed by detection of a low mobility band.
Using this method, it was confirmed that a protein capable of binding to the above sequence was present in the α chain positive cell nucleus (FIG. 5).
[0014]
(4) Search for α-chain expression inhibitor using transcription activation sequence It is possible to search for an α-chain expression inhibitor using the specified sequence.
Add a suitable amount of test sample when culturing a cell line in which a DNA with an appropriate length containing the above sequence is linked upstream of the reporter gene, for example, wild type PB0.50 in Fig. 3 is introduced into α-chain expressing cells Then, by measuring the amount of the reporter gene product, it is possible to search for a substance exhibiting a promoter activity inhibitory effect.
[0015]
(5) Substance production in animal cells using transcription activating sequences It is also possible to use the specified sequence for substance production in animal cells.
[0016]
Increase the amount of substance production in this production system by preparing an animal expression plasmid containing one or more of this sequence in the promoter upstream or in the promoter and introducing it into an animal cell line that can activate this sequence. It is possible.
[0017]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to this.
[0018]
(1) Human high-affinity IgE receptor (FcεRI) α-chain gene transcription activation region of the human FcεRI α-chain gene covering a specific total length of 20 kbp or more, a novel first exon disclosed in JP-A-10-09081 (Ex 1A) 5 ′ of a gene fragment (Pang, J. et al .; J. Immunology, 151 , 6166 (1993)) reported in 1993 by Pang et al. From the 3′-end of a base sequence of about 1.6 kbp upstream. -The site | part which has transcription activation ability was searched about the unknown area | region to the terminal. The gene sequence of this region is shown in SEQ ID NO: 1.
[0019]
First, a DNA fragment as shown in FIG. 2 was prepared and inserted into pGV-B2 (Toyo Ink) for the region containing the first exon designated Ex.1A in FIG. Restriction enzyme sites were mainly used for excision of DNA fragments. For preparation of EB3.3, a kilosequence deletion kit (Takara Shuzo) was used, and for PH0.3, a Hin dIII site newly introduced by PCR was used. Each plasmid DNA was purified using a plasmid midi kit (Qiagen) and used for introduction of a cell line. Introduction into the cell line is as described previously (Nishiyama, C. et al: J. Immunology, 163 , 623 (1999)).
[0020]
That is, 5 μg of each plasmid DNA was added to 1 × 10 ⁷ human basophil RBL-2H3 and mouse mast cytoma PT18 together with 25 mg of the internal standard plasmid pRL-CMV (Promega). And then ingested by electroporation (950μF, 300V), culture for 24 hours, collect cells, and measure the activity of luciferase expressed in the cells with a luminometer Lumat LB9501 (Belfold) did. When measuring the activity, a Dual Luciferase assay kit (Toyo Ink or Promega) was used as a substrate, and the method followed the protocol.
[0021]
As shown in FIG. 2, the results are shown in FIG. 2.In RBL-2H3 and PT18, which are α chain positive cells, the DNA fragments indicated by EB3.3, EB3.8, PB2.5, PPv2.2, and PNd0.9 have transcription activation ability. Admitted. EN1.6 (+) shown here is a site whose sequence was shown in the previous patent specification (Japanese Patent Laid-Open No. 10-099081) as a new promoter region. Although EN1.6 (+) has a stronger transcriptional activation ability than NE1.6 (-) in the reverse direction, its value is small compared to PNd0.9 and the like described above. In addition, the activation ability of this region is not observed in Jurkat, which is an α chain non-expressing cell line. That is, it was suggested that a region that up-regulates the promoter activity upstream of Ex.1A exists in intron A ′ (FIG. 1).
[0022]
From the results in FIG. 2, it was predicted that the enhancer region was present in about 600 bp between NheI and NdeI directly under Ex.1A. Next, a plasmid in which this 600 bp was deleted stepwise was prepared and the case of FIG. Similarly, it was introduced into α-chain expressing cells, and the activity of luciferase as a reporter gene was measured. The plasmid was prepared by introducing a restriction enzyme site (HindIII or BamHI) using the PCR method.
[0023]
The results are shown in FIG. In the case of RBL-2H3, the activity observed for DNA fragments with a length of PB0.50 or more is markedly reduced at PB0.45 or less. This tendency was also confirmed in KU812, which is also an α chain positive human cell line. From this, it was predicted that the enhancer sequence that functions in the α-chain expressing cell line is present in about 50 bp that is present in PB0.50 and not included in PB0.45.
[0024]
(2) Determination of transcriptional activation sequence in human FcεRI α chain gene As described above, a region having a promoter activation ability was searched for about 3.8 kb around the first exon (Ex.1A). It was suggested that a region that activates a novel promoter exists within 50 bp at a position about 400 bp downstream of Ex.1A (FIGS. 2 and 3).
In order to identify the activation sequence present in this region, a mutant plasmid as shown in Fig. 4 is prepared, introduced into an α-chain expressing cell line, and the activity of luciferase expressed in the cell is measured and compared. did. The mutant plasmid was prepared using the site-directed mutagenesis kit (Stratagene) using PB0.50 determined to have enhancer activity in FIG. 3 as a template. Cell transformation, activity measurement, and the like were performed in the same manner as in FIGS.
[0025]
As a result, as shown in FIG. 4, a significant decrease in activity was observed only when a mutation was introduced into the 5-base TTCCA sequence in this region. The effect was common to all the α chain positive cells used, RBL-2H3, PT18, and KU812. From this experiment, the enhancer sequence was limited to one region (SEQ ID NO: 2) containing TTCCA.
[0026]
(3) Nucleoprotein recognizing transcription activation sequence The presence of a nucleoprotein that binds to the sequence specified by the above-mentioned method was confirmed by gel shift assay.
In the gel shift assay, an AGCCCTACTTTTCCATTTAGGGAGAT sequence containing a TTCCA sequence and a complementary sequence were synthesized and rhodamine-labeled at the 5 ′ end (Japanese flour) was used after annealing. Preparation of nuclear fraction, reaction of probe DNA and nuclear protein, electrophoresis on acrylamide gel, and the like followed the methods already reported (Nishiyama, C. et al: J. Immunology, 163 , 623 (1999)).
[0027]
That is, after recovering KU812 or PT18, it was washed with a phosphate buffer, and buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 μg / ml leupeptin and 1 μg / ml aproptinin) And then left at 4 ° C. for 10 minutes. Subsequently, Nonidet P-40 was added to 0.5% and allowed to stand for another 15 minutes, and then centrifuged at 6000 × g for 1 minute. The resulting precipitate fraction was suspended in an extraction buffer (20 mM HEPES, pH 7.9, 400 mM KCl, 1.5 mM MgCl ₂ , 0.2 mM EDTA, 1 mM DTT, 1 mM PMSF, 1 μg / ml leupeptin and 1 μg / ml aproptinin), Furthermore, what was left to stand for 1 hour was centrifuged at 10000 × g for 5 minutes, and the supernatant fraction was used as the nuclear extract fraction. 5 pmol of the labeled probe and 10 μg of the nuclear extract protein were reacted for 20 minutes in a 15 μl solution containing 500 ng of poly (dI-dC), 1 mM MgCl ₂ , 30 mM KCl, 10 mM HEPES, pH 7.9, 1 mM DTT, 5% glycerol. Thereafter, the gel was applied to a 4% polyacrylamide gel and subjected to electrophoresis at 120 V for 2.5 hours using 0.5 × TBE buffer (45 mM trisborate, pH 8.0, 45 mM boric acid, 1 mM EDTA). FMBIO (Takara Shuzo) was used to detect rhodamine.
[0028]
The results are as shown in FIG. By adding a nuclear extract to the probe DNA, multiple bands were detected for both PT18 and KU812 (lane 2). Competitors 1 and 2 were used as unlabeled competitors that inhibit the binding of probe DNA and nucleoprotein. The competitor 1 is the same sequence as the labeled probe, and the competitor 2 is the labeled probe sequence in which mutations are introduced into the TTCCA sequence identified in the experiment of FIG. The comparison between lanes 3 and 4 and lanes 5 and 6 in FIG. 5 suggested the presence of a protein whose binding was inhibited in the wild type but not in the mutant type. That is, it was confirmed that a protein capable of binding to the above TTCCA sequence was present in the α chain positive cell nucleus.
[0029]
(4) Search for α chain expression inhibitor using transcription activation sequence Using the identified sequence, a search system for α chain expression inhibitor was constructed.
[0030]
After introducing wild type PB0.50 in Fig. 3 into α-chain expressing cell lines such as RBL-2H3 and PT18, dispense into 96-well plates. Thereafter, an appropriate amount of a test sample was added and further cultured overnight, and then the luciferase activity in each well was measured. As test samples, actinomycete cultures and compound libraries were used.
[0031]
【The invention's effect】
The human FcεRI α chain-derived DNA base sequence shown in the present invention and the α chain expression inhibitor obtained from a search system using the same can provide a new allergy treatment method and antiallergic agent. In addition, by introducing this base sequence into a vector used for gene recombination, the productivity of the target substance in the recombinant can be improved.
[0032]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the structure of a human high affinity IgE receptor (FcεRI) α chain gene.
[Explanation of symbols]
B; BamHI, E; EcoRI, H; HindIII, S; Sau3AI recognition site FIG. 2 is a diagram showing a search for a human high affinity IgE receptor (FcεRI) α chain cell specific transcription activation region.
[Explanation of symbols]
NT; untested FIG. 3 is a specific view of the human high affinity IgE receptor (FcεRI) α chain cell specific transcription activation region.
[Explanation of symbols]
N. T .; no test FIG. 4 is a specific view of the human high affinity IgE receptor (FcεRI) α chain transcription activation sequence.
FIG. 5 shows detection of nucleoprotein that binds to α-chain transcription activation sequence by gel shift assay.
[Explanation of symbols]
Lane 1; probe only, lanes 2 to 6; each extract fraction is included. Lane 2; no competitor, lanes 3 and 4; containing a competitor having the same sequence as the probe, lanes 5 and 6; containing a competitor having the same mutation introduced in FIG. 4 as M2. The competitor is 30 times the number of probes (lanes 3 and 5) and 60 times the number of molecules (lanes 2 and 4).

Claims (7)

A gene that activates the expression of a human high-affinity IgE receptor (FcεRI) α-chain gene comprising the nucleotide sequence set forth in SEQ ID NO: 2. In the base sequence described in SEQ ID NO: 2, the base sequence excluding the 11th to 15th base sequences (TTCCA sequence) is a base sequence in which 1 to 5 bases are substituted and / or inserted and / or deleted. A gene containing A vector comprising the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence contained in the gene of claim 2. 4. The vector of claim 3, selected from the group consisting of a plasmid vector, a viral vector, and a liposome vector. A host cell transformed with the vector according to claim 3 or 4. The host cell according to claim 5, wherein the host cell is selected from the group consisting of Escherichia coli, yeast, insect cells, COS cells, mink lung epithelial cells, blood cells, fibroblasts, and tumor cells. When culturing a cell line using the base sequence of SEQ ID NO: 2 or the base sequence contained in the gene of claim 2 and introducing a plasmid in which a DNA containing this base sequence is linked upstream of the reporter gene into an α-chain expressing cell A method of searching for an expression inhibitor of human high affinity IgE receptor (FcεRI) α chain, comprising adding a test sample and measuring the amount of a reporter gene product.

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