WO2016017039A1 - 炎症レポーターシステム - Google Patents
炎症レポーターシステム Download PDFInfo
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Definitions
- the present invention relates to a method for detecting an inflammatory reaction.
- the inflammatory response is one of the biological reactions that are closely related to the symptoms of many diseases, and is an important research subject in understanding their pathology and devising treatment strategies. Therefore, the development of technology that can detect the actual state of inflammation is indispensable. It has been elucidated that a general inflammatory reaction occurs by the following mechanism. That is, when an infectious source such as bacteria or virus enters the body, the infectious source is sensed by a receptor on the cell surface and cytokine secretion occurs. Using the cytokine as an index, immune cells such as macrophages migrate to the site of infection and repel the source of infection. It is said that redness, fever, pain and swelling peculiar to inflammation occur due to the active action by immune cells at the time of remission.
- Interleukin-1 beta (IL-1 ⁇ ) is known as a cytokine that is greatly involved in this inflammatory reaction and attracts attention as an inflammatory marker.
- IL-1 ⁇ is hardly secreted in the absence of inflammatory stimulus, but is known to be produced and secreted at very high levels in each tissue by inflammatory stimulus (FIG. 1).
- IL-1 ⁇ has also been found to be tightly regulated by the following characteristic two-step control.
- IL-1 ⁇ gene expression is activated by the transcription factor NF- ⁇ B induced by an inflammatory reaction, and production of proIL-1 ⁇ , which is a precursor type, is promoted with the activation of IL-1 ⁇ gene expression. Thereafter, proIL-1 ⁇ is cleaved by caspases activated by inflammasome and converted into secretable mature IL-1 ⁇ (FIG. 2).
- Non-patent Documents 1 and 2 There have already been reports on the monitoring of IL-1 ⁇ gene expression using luciferase or red fluorescent protein as a reporter molecule (Non-patent Documents 1 and 2). These reports show that a transgenic mouse into which a reporter molecule has been introduced is prepared, and that reporter signals can be detected in the inflammation model and can be used for in vivo imaging analysis. However, this method relies solely on transcriptional control, which is one factor in the cascade of inflammatory reactions, and is considered insufficient for monitoring physiological inflammatory reactions. On the other hand, a reporter system controlled by inflammasome has also been reported (Non-patent Document 3).
- An object of the present invention is to provide a technique that can detect and measure a local inflammatory reaction in a minute region in a living body with high efficiency and high sensitivity. Also provided is a transgenic mouse into which the gene vector of the present reporter system has been introduced so that the present detection method can be used easily, and further, the reporter system gene vector can be used for in vivo research and development. For the purpose.
- the inventor of the present invention monitors a physiological inflammatory reaction by utilizing a mechanism of an inflammatory reaction that is controlled in two steps by an IL-1 ⁇ gene and an inflammasome.
- the present invention was completed by constructing a reporter system capable of carrying out a high efficiency and high sensitivity, and constructing the method, a gene vector equipped with the method, and a transgenic mouse. That is, the present invention is as follows.
- a vector comprising a promoter for a gene encoding an inflammatory cytokine, a gene encoding a reporter protein, a gene encoding the inflammatory cytokine, and a gene encoding a proteolytic signal sequence.
- the reporter protein is luciferase.
- a transformant comprising the vector according to any one of (1) to (4).
- the transformant or transgenic non-human animal by inflammatory stimulation A method for detecting an inflammatory reaction, comprising detecting an inflammatory reaction induced in a human animal.
- the present invention provides a reporter system capable of monitoring an inflammatory reaction with high efficiency and high sensitivity.
- the system of the present invention can visualize inflammatory reactions and is extremely sensitive and efficient.
- LPS lipopolysaccharide
- LPS Used LPS: Sigma # L2654, used concentration: 3-4 ⁇ g / g body weight It is a figure which shows the reporter activity in RAW264 which introduce
- the vector used in the present invention comprises a fusion gene in which a plurality of genes are linked, and under the control of a gene encoding an inflammatory cytokine, a reporter molecule, a caspase recognition sequence, and proteolysis It expresses a fusion protein of a signal sequence.
- inflammatory cytokine is a cytokine produced from helper T cells, monocytes, macrophages, neutrophils, dendritic cells and the like activated by antigens such as bacteria. It is a cytokine that activates immune system cells, vascular endothelial cells, and osteoclasts.
- inflammatory cytokinines examples include IL-1 ⁇ , IL-6, IL-8, IL-12, IL-13, IL-17, IL-18, and tumor necrosis factor (TNF).
- TNF tumor necrosis factor
- the gene encoding inflammatory cytokine is expressed by the transcription factor NF- ⁇ B triggered by inflammatory reaction.
- genes encoding these inflammatory cytokines or partial sequences thereof can be used. Information on the genes encoding the inflammatory cytokines and the promoters of these genes is known.
- the partial sequence is not particularly limited as long as it has inflammatory responsiveness, and the length and region can be determined using inflammatory responsive expression enhancement, inflammatory responsive processing, or the like as an index.
- IL-1 ⁇ Accession number NM_008361.3
- IL-6 Accession number NM_031168.1
- IL-8 Accession number NM_009140.2
- IL-12 Accession number NM_001159424.1
- IL-13 Accession number NM_008355.3
- -17 Accession number NM_010552.3
- IL-18 Accession number NM_008360.1
- TNF Accession number NM_0012786601.1 IL-1 ⁇ promoter: Accession number NC — 0000686.7 IL-6 promoter: Accession number NC — 000071.6 IL-8 promoter: Accession number NC — 000071.6 IL-12 promoter: Accession No.
- NC — 0000695.6 IL-13 promoter accession number NC — 0000777.6 IL-17 promoter: Accession No. NC — 006006.6 IL-18 promoter: Accession No. NC — 000007.6 TNF promoter: Accession number NC — 0000832.6
- IL-1 ⁇ will be described as an example.
- a reporter gene is ligated downstream of the promoter of IL-1 ⁇ gene, and a gene construct in which, for example, an IL-1 ⁇ partial sequence and a proteolytic signal sequence are added downstream is prepared.
- the peptide linker encoded in the IL-1 ⁇ partial sequence includes a sequence recognized by caspase (caspase recognition sequence).
- the peptide linker encoded by the IL-1 ⁇ partial sequence is a region where caspase (caspase-1) activated by a protein complex called inflammasome acts, and the peptide linker is cleaved by caspase.
- FIG. 3 illustrates IL-1 ⁇ as an inflammatory cytokine and luciferase (Luc) as a reporter molecule.
- inflammatory cytokines and reporter molecules are not limited to IL-1 ⁇ and Luc shown in FIG.
- FIG. 3 when there is no inflammatory stimulus, the IL-1 ⁇ gene promoter does not operate, so that the expression of the reporter gene is not activated. Even if the expression leaks, the inflammasome does not work because there is no inflammatory stimulus, and the fusion protein composed of the expressed reporter molecule-caspase recognition sequence-proteolytic signal sequence is the action of the proteolytic signal sequence.
- the expression of the reporter gene is activated by the activation of the promoter by the transcription factor NF- ⁇ B, and the produced reporter molecule becomes a proteolytic signal by caspase activation by the inflammasome. It is separated from the sequence, stabilized by the reporter molecule itself, and can be detected at a high level as a luminescence signal (reporter signal) of the reporter protein. This detection result is visualized and can be confirmed by an image displayed on the monitor.
- a fusion protein comprising a reporter molecule, a caspase recognition sequence (constituting a part of the amino acid sequence of IL-1 ⁇ ) and a proteolytic signal sequence under the control of the IL-1 ⁇ gene promoter.
- a gene vector having a structure to be expressed was transiently introduced into a mouse-derived macrophage-like cell RAW264 strain.
- LPS Lipopolysaccharide, lipopolysaccharide
- transgenic mice were prepared by injecting the gene vector into pronuclear fertilized eggs of the C57BL / 6 strain. Inflammatory stimulation is given by administering LPS into the abdominal cavity of this transgenic mouse, and changes in the luminescence signal of luciferase immediately after administration, 4 hours, and 24 hours later are detected with a biological imaging apparatus. Luminescence depending on the inflammatory response can be observed in whole body tissues (Example, FIG. 5).
- the reporter protein reporter molecule
- luciferase for example, luciferase, GFP (green fluorescent protein), DsRed (red fluorescent protein), LacZ ( ⁇ -galactosidase) and the like can be used.
- the present invention is not limited to this.
- proteolytic signal sequence means a sequence that is positively polyubiquitinated by E3 ligase and consequently easily digested by the proteasome.
- proteolytic signal sequence used in the present invention a CL1 sequence, a PEST sequence, etc. Is mentioned. Genes encoding these proteolytic signal sequences are known and can be obtained from domestic and foreign bioreagent manufacturers.
- the transformant of the present invention can be obtained by introducing a gene vector into a host.
- the host into which the gene vector is introduced is not particularly limited, and may be a unicellular organism such as a prokaryote (E. coli, lactic acid bacteria, etc.) or a eukaryotic cell (yeast), or a human-derived cell (Hela, HEK293, etc.) Cell culture cells of mouse-derived cells (NIH3T3 etc.) or other animal cells can also be used. Methods for linking the above genes directly under the promoter are well known (Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Laboratory Press (2012)).
- Introduction of a gene vector into a host can be performed by widely known methods such as electroporation (lipoporation), a lipofection method using a commercially available lipofection reagent, and a method using a viral vector (for example, Molecular Cloning as described above). See
- Transgenic non-human animals into which this reporter gene vector has been introduced can be produced in mice, rats, dogs, monkeys, goats, etc., but are not limited to these non-human animals.
- Transgenic non-human animals can be prepared by injecting gene vector DNA into fertilized eggs of each animal using a microinjector, or embryonic stem cells (ES cells) or artificial pluripotency by homologous recombination.
- Transgenic animals can also be produced after establishing sex stem cells (iPS cells).
- the microinjection method and the like are all known methods that can be easily performed by those skilled in the art (see the above-mentioned Molecular Cloning and the like).
- the transgenic non-human animals used in the present invention are not limited to individuals, and biomaterials derived from the transgenic non-human animals such as cells, organs, tissues, embryos, and the like can also be used.
- a test substance (candidate substance) that is a candidate for an anti-inflammatory substance is not particularly limited, and examples thereof include peptides, proteins, DNA, non-peptide compounds, synthetic compounds, fermentation products, Examples thereof include cell extracts and plant extracts. These compounds may be novel compounds or known compounds.
- These test substances may form a salt.
- a salt with a physiologically acceptable acid for example, an inorganic acid
- a base for example, an organic acid
- Physiologically acceptable acid addition salts are preferred.
- the test substance may be tested on a single substance independently or on a mixture (including a library, etc.).
- Examples of the library containing a plurality of test substances include a synthetic compound library (such as a combinatorial library) and a peptide library (such as a combinatorial library).
- an inflammatory substance is administered to a transgenic non-human animal (provided with an inflammatory stimulus) to cause an inflammatory reaction, and a test substance is contacted with the animal to examine the inhibitory effect on the inflammatory reaction.
- a test substance is administered after bringing a test substance into contact with a transgenic non-human animal to induce an inflammatory reaction, and the effect of suppressing the inflammatory reaction in this animal is examined.
- the test substance that has obtained an inhibitory effect on the inflammatory reaction can be selected as a therapeutic or prophylactic agent for inflammatory diseases (for example, infection, rheumatism, allergy), that is, an anti-inflammatory agent.
- test animal transgenic non-human animal
- control animal used as the object of test substance administration
- animals of the same sex and the same age are used.
- an inflammatory substance is contacted with the transformant to examine an inhibitory effect on an inflammatory reaction
- transformation There is an embodiment in which an inflammatory substance is brought into contact with the body to induce an inflammatory reaction, and a test substance is brought into contact with the transformant to examine an inhibitory effect on the inflammatory reaction. Whether or not to suppress the inflammatory reaction is determined using whether or not the reporter protein is detected as a luminescent signal by inflammatory stimulation, and an anti-inflammatory substance is selected using the obtained detection result as an index.
- Contacting includes a mode in which a test substance is administered to a non-human animal, a mode in which the test substance is added to a transformant or a biomaterial, and a mode in which culture is performed in the presence of the test substance.
- the test substance may be inoculated into the animal by injection or the like.
- Examples of the mode in which the test substance is added to the transformant or the biomaterial include adding the test substance to a cell culture, adding the test substance to a tissue, an organ, or the like.
- “Culture” means any of cells, culture media, and cell extracts.
- “Culturing in the presence of a test substance” means culturing under conditions where the cell and the test substance are in contact with each other, and the contact of the test substance with the cells or the like is, for example, a cell culture medium.
- a test substance is added to various buffers (for example, HEPES buffer, phosphate buffer, phosphate buffered saline, Tris-HCl buffer, etc.), and the cells are incubated for a certain period of time. be able to.
- the concentration of the test substance added to the culture varies depending on the type of compound (solubility, toxicity, etc.), but is appropriately selected within the range of 100 ng / ml to 10 ⁇ g / ml, for example. Examples of the incubation time include 4 to 48 hours.
- the present invention is a transformant in which a promoter containing a gene encoding a proinflammatory cytokine, a gene encoding a reporter protein, a gene encoding the proinflammatory cytokine, and a gene encoding a proteolytic signal sequence is introduced.
- a kit for detecting an inflammatory reaction or screening for an anti-inflammatory substance, including a transgenic non-human animal is provided.
- the transformant or transgenic non-human animal of the present invention is used as a detection agent for inflammatory diseases or inflammatory reactions
- the transformant or transgenic non-human animal is used as another reagent, for example, distilled water, buffer reagent. , Inflammation-inducing substances, instructions for use, and the like.
- PCR kit used in any part is Prime Star (Takara)
- template DNA is mouse ES cell-derived genomic DNA
- reaction conditions are 98 ° C .; 10 seconds, 55 ° C .; 5 seconds, 72 ° C .; 35 minutes cycle for 2 minutes It is.
- the primers used vary from part to part and are as follows. 1st part Second part Third part 4th part Each portion was linked using a ClaI site, EcoT22I site, and SmaI site.
- the entire region of the mouse-derived IL-1 ⁇ gene partial (17-216aa) region was divided and cloned using the PCR method.
- the PCR kit used was Prime Star (Takara), the template DNA was a reverse transcription product derived from mouse placenta, and the reaction conditions were 98 ° C .; 10 seconds, 55 ° C .; 5 seconds, 72 ° C .; 35 cycles of 1 minute.
- the primers used were a 5-side primer; aaagggtaccatgagaatgaccgtgtttttg (SEQ ID NO: 10), 3-side primer; aaaactcgagaaaaccgtttttccattctttttc (SEQ ID NO: 11).
- the gene vector constructed as described above was transiently introduced into mouse-derived macrophage-like cells RAW264.
- Effectene Qiagen
- LPS LPS (Sigma # L2654) was added to the culture medium of transiently expressing cells at a concentration of 2 ⁇ g / mL, and the amount of luciferase luminescence 48 hours after the addition was quantitatively measured with a luminometer.
- an LPS-free group was provided.
- a vector in which GL4 was connected downstream of the promoter of IL-1 ⁇ gene was prepared, and this was similarly tested on cells transfected with this.
- the gene vector cut out and purified was injected into 200 pronuclear fertilized eggs collected from C57BL / 6 strain mice, and 71 offspring were obtained. Genotypes were analyzed using genomic DNA extracted from the body tissue of the litter, and 18 founder mice having a gene vector inserted into the genome were obtained. Four founder mice were crossed with wild type C57BL / 6 strain mice to produce F1 generation mice, and the reaction of the reporter molecule when LPS (Sigma # L2654) was administered intraperitoneally was examined. As a result, system no. In the individual of M1, the S / N ratio was most excellent, and this strain was established as an inflammatory reporter mouse. (FIG. 8).
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Abstract
Description
一般的な炎症反応は、次のメカニズムにより起こることが解明されている。すなわち、細菌やウイルスなどの感染源が体内に侵入すると、感染源は細胞表面の受容体で感知されサイトカインの分泌が起こる。そのサイトカインを指標にしてマクロファージなどの免疫細胞が感染部位に遊走され、感染源を撃退する。その撃退時の免疫細胞による活発な働きから、炎症時に特有の発赤、発熱、疼痛、腫脹が起こるとされている。
IL−1βは、以下の特徴的な二段階の制御により厳密に調節されることも分かっている。IL−1βの遺伝子発現は、炎症反応によって惹起される転写因子NF−κBにより活性化され、IL−1β遺伝子発現の活性化に伴い、前駆型であるproIL−1βの産生が促進される。その後、proIL−1βはインフラマソームで活性化されたカスパーゼによって切断を受け、分泌可能な成熟型IL−1βに変換される(図2)、というものである。
また一方、インフラマソームで制御されるレポーターシステムについても報告されている(非特許文献3)。この報告では、非炎症時には凝集構造をとることで不活性状態となっているレポーター分子が、炎症刺激によりインフラマソームが機能することで単量体化して活性を示すように設計されている。しかし、これもインフラマソームだけに頼っており、感度としては十分でなく、また、このシステムは、生体マウスにおいて機能するかどうかについての検証がなされていない。
すなわち、本発明は以下の通りである。
(2)炎症性サイトカインがインターロイキン1βである(1)に記載のベクター。
(3)レポータータンパク質がルシフェラーゼである(1)又は(2)に記載のベクター。
(4)炎症性サイトカインをコードする遺伝子は、カスパーゼにより認識されるペプチドをコードするポリヌクレオチド配列を含むものである(1)~(3)のいずれか1項に記載のベクター。
(5)前記(1)~(4)のいずれか1項に記載のベクターを含む形質転換体。
(6)前記(1)~(4)のいずれか1項に記載のベクターが導入されたトランスジェニック非ヒト動物。
(7)非ヒト動物がマウスである(6)に記載のトランスジェニック非ヒト動物。
(8)炎症性刺激によりレポータータンパク質が発光シグナルとして検出される、(5)に記載の形質転換体又は(6)若しくは(7)に記載のトランスジェニック非ヒト動物。
(9)前記(5)に記載の形質転換体又は(6)~(8)のいずれか1項に記載のトランスジェニック非ヒト動物を用いて、炎症性刺激により当該形質転換体又はトランスジェニック非ヒト動物に惹起される炎症反応を検出することを特徴とする炎症反応の検出方法。
(10)炎症性サイトカインをコードする遺伝子は、炎症反応により惹起される転写因子NF−κBにより発現されるものである、(9)に記載の方法。
(11)炎症性刺激によりレポータータンパク質を発光シグナルとして検出する、(9)又は(10)に記載の方法。
(12)前記(5)に記載の形質転換体又は(6)~(8)のいずれか1項に記載のトランスジェニック非ヒト動物に、炎症性刺激下で候補物質を接触させ、炎症反応の有無を指標として抗炎症物質を選択することを特徴とする抗炎症物質のスクリーニング方法。
(13)前記(5)に記載の形質転換体又は(6)~(8)のいずれか1項に記載のトランスジェニック非ヒト動物を含む、炎症反応検出用又は抗炎症物質のスクリーニング用キット。
1.ベクター等及び検出方法
本発明において使用されるベクターは、複数の遺伝子を連結した融合遺伝子を含むものであり、炎症性サイトカインをコードする遺伝子のプロモーター制御下で、レポーター分子、カスパーゼ認識配列及びタンパク質分解シグナル配列の融合タンパク質を発現するものである。
本発明において、「炎症性サイトカイン」とは、細菌などの抗原などによって活性化されたヘルパーT細胞、単球、マクロファージ、好中球、樹状細胞などから産生されるサイトカインであり、マクロファージやその他の免疫系細胞、血管内皮細胞、破骨細胞を活性化するサイトカインである。このような炎症性サイトカインとして、例えばIL−1β、IL−6、IL−8、IL−12、IL−13、IL−17、IL−18、腫瘍壊死因子(TNF)などが挙げられる。炎症性サイトカインをコードする遺伝子は、炎症反応により惹起される転写因子NF−κBにより発現される。
IL−6:アクセッション番号NM_031168.1
IL−8:アクセッション番号NM_009140.2
IL−12:アクセッション番号NM_001159424.1
IL−13:アクセッション番号NM_008355.3
IL−17:アクセッション番号NM_010552.3
IL−18:アクセッション番号NM_008360.1
TNF:アクセッション番号NM_001278601.1
IL−1βプロモーター:アクセッション番号NC_000068.7
IL−6プロモーター:アクセッション番号NC_000071.6
IL−8プロモーター:アクセッション番号NC_000071.6
IL−12プロモーター:アクセッション番号NC_000069.6
IL−13プロモーター:アクセッション番号NC_000077.6
IL−17プロモーター:アクセッション番号NC_000067.6
IL−18プロモーター:アクセッション番号NC_000075.6
TNFプロモーター:アクセッション番号NC_000083.6
IL−1β遺伝子のプロモーター下流にレポーター遺伝子を連結し、その下流に例えばIL−1β部分配列と蛋白質分解シグナル配列を付加した遺伝子コンストラクトを作製する。IL−1β部分配列においてコードされるペプチドリンカーにはカスパーゼにより認識される配列(カスパーゼ認識配列)を含む。そして、当該IL−1β部分配列によりコードされるペプチドリンカーは、インフラマソームと呼ばれるタンパク質複合体により活性化されたカスパーゼ(カスパーゼ−1)が作用する領域であり、カスパーゼにより当該ペプチドリンカーが切断される。
図3において、炎症刺激がない場合には、IL−1β遺伝子プロモーターが作動しないので、レポーター遺伝子の発現は活性化されることはない。仮に、発現がリークしたとしても、やはり炎症刺激がないためインフラマソームが働かず、発現したレポーター分子−カスパ−ゼ認識配列−蛋白質分解シグナル配列からなる融合蛋白質は、その蛋白質分解シグナル配列の作用により、積極的にユビキチン−プロテアソーム系で分解されることになる。
他方、炎症刺激がある場合、転写因子であるNF−κBによりプロモーターが作動することでレポーター遺伝子の発現は活性化され、産生されたレポーター分子は、インフラマソームによるカスパーゼの活性化により蛋白質分解シグナル配列から切り離され、レポーター分子それ単独で安定化し、レポータータンパク質の発光シグナル(レポーターシグナル)として高レベルで検出することができる。この検出結果は可視化され、モニターに表示された画像により確認することができる。
また、当該遺伝子ベクターをC57BL/6系統の前核期受精卵にインジェクションすることにより、トランスジェニックマウスを作製した。このトランスジェニックマウスの腹腔内にLPSを投与することで炎症刺激を与え、投与直後と、4時間後、24時間後のルシフェラーゼの発光シグナルの変化を生体イメージング装置で検出する。全身の組織において、炎症反応に依存する発光が観察することができる(実施例、図5)。
レポータータンパク質(レポーター分子)としては、例えば、ルシフェラーゼ、GFP(緑色蛍光蛋白質)、DsRed(赤色蛍光蛋白質)、LacZ(β−ガラクトシダーゼ)などを利用することができ、また、遺伝子ベクターもプラスミドDNA、ウイルスベクターなどの形態をとることができるが、本発明はこれに限定されるものではない。
「タンパク質分解シグナル配列」とは、E3リガーゼにより積極的にポリユビキチン化され、結果としてプロテアソームにより消化されやすい配列を意味し、本発明において使用されるタンパク質分解シグナル配列として、CL1配列やPEST配列などが挙げられる。これらのタンパク質分解シグナル配列をコードする遺伝子は公知であり、国内外のバイオ試薬メーカーなどから入手することができる。
遺伝子ベクターを導入する宿主も特に限定はされず、原核生物(大腸菌、乳酸菌等)や真核細胞(酵母)等の単細胞生物であってもよく、また、ヒト由来細胞(Hela,HEK293等)やマウス由来細胞(NIH3T3等)の株化培養細胞、あるいはその他の動物細胞も使用できる。プロモーター直下に上記遺伝子を連結する方法は周知である(Molecular Cloning:A Laboratory Manual(4th Edition)、Cold Spring Harbor Laboratory Press(2012))。遺伝子ベクターの宿主への導入は、電気穿孔法(エレクトロポレーション)、市販のリポフェクション試薬を用いたリポフェクション法、ウイルスベクターによる方法など、広く公知の方法によって実施することができる(上記Molecular Cloning等を参照のこと)。
本発明において使用されるトランスジェニック非ヒト動物は、個体に限定されるものではなく、当該トランスジェニック非ヒト動物由来の生体材料、例えば細胞、器官、組織、胚などを使用することもできる。
本発明において、抗炎症物質の候補となる被検物質(候補物質)は、特に限定されるものではなく、例えば、ペプチド、タンパク質、DNA、非ペプチド性化合物、合成化合物、発酵生産物、細胞抽出液、植物抽出液などが挙げられ、これら化合物は新規化合物であってもよいし、公知化合物であってもよい。これら被検物質は塩を形成していてもよく、被検物質の塩としては、生理学的に許容される酸(例えば無機酸など)や塩基(例えば有機酸など)などとの塩が用いられ、生理学的に許容される酸付加塩が好ましい。被検物質は、単一の物質を独立に試験しても、混合物(ライブラリーなどを含む)について試験をしてもよい。複数の被検物質を含むライブラリーとしては、合成化合物ライブラリー(コンビナトリアルライブラリーなど)、ペプチドライブラリー(コンビナトリアルライブラリーなど)などが挙げられる。
本発明において、形質転換体を使用する場合は、形質転換体に被検物質を接触させた後、この形質転換体に炎症性物質を接触させて炎症反応の抑制効果を調べる態様、及び形質転換体に炎症性物質を接触させて炎症反応を惹起し、この形質転換体に被検物質を接触させて炎症反応の抑制効果を調べる態様がある。
炎症反応を抑制するかどうかは、炎症性刺激によりレポータータンパク質が発光シグナルとして検出されるか否かを指標とし、得られる検出結果を指標として抗炎症物質を選択する。
培養物中に添加される被検物質の濃度は化合物の種類(溶解度、毒性等)により異なるが、例えば、100ng/ml~10μg/mlの範囲で適宜選択される。インキュベート時間としては、例えば、4~48時間が挙げられる。
本発明は、炎症性サイトカインをコードする遺伝子のプロモーター、レポータータンパク質をコードする遺伝子、前記炎症性サイトカインをコードする遺伝子、及びタンパク質分解シグナル配列をコードする遺伝子を含むベクターが導入された形質転換体又はトランスジェニック非ヒト動物を含む、炎症反応検出用又は抗炎症物質のスクリーニング用キットを提供する。
本発明の形質転換体又はトランスジェニック非ヒト動物を炎症性疾患や炎症反応の検出薬として用いる場合には、上記形質転換体又はトランスジェニック非ヒト動物を他の試薬、例えば、蒸留水、緩衝試薬、炎症惹起物質、使用説明書などを含めることができる。
マウス由来IL−1β遺伝子の上流約5kbpの領域を、マウス由来細胞から抽出したゲノムDNAを材料としてクローニングした。クローニング領域の直下にHSV由来のTK遺伝子プロモーターを融合し、この融合プロモーターの下流にPhotinus pyralis由来 改変型ルシフェラーゼ(GL4,約1.7kbp)、さらにその下流にマウス由来IL−1βの部分配列(17−216aa)をコードする塩基配列、さらにその下流にCL1(Saccharomyces cerevisiae由来)−PEST(マウス由来)配列、SV40由来ポリA配列を接続したベクター(図7)(配列番号1)を構築した。
IL−1β遺伝子のクローニングは以下の通り行った。
全領域を4部分に分割して、それぞれPCR法を用いてクローニングした。いずれの部分においても使用したPCRキットはPrime Star(Takara)、鋳型DNAはマウスES細胞由来ゲノムDNA、反応条件は98℃;10秒、55℃;5秒、72℃;2分の35回サイクルである。使用プライマーは部分ごとに異なり、以下の通りである。
第1部分
第2部分
第3部分
第4部分
なお、各部分の連結はClaIサイト、EcoT22Iサイト、SmaIサイトを用いて行った。
全領域を一割してPCR法を用いてクローニングした。使用したPCRキットはPrime Star(Takara)、鋳型DNAはマウス胎盤由来の逆転写産物、反応条件は98℃;10秒、55℃;5秒、72℃;1分の35回サイクルである。
使用プライマーは5側プライマー;aaaggtaccgatgagaatgacctgttctttg(配列番号10)、3側プライマー;aaactcgagaaaccgtttttccatcttcttc(配列番号11)である。
上記の通り構築した遺伝子ベクターを、マウス由来マクロファージ様細胞RAW264株に一過的に導入した。
トランスフェクションには、Effectene(Qiagen)を用い、トランスフェクション後24時間の細胞を回収して実験に供試した。一過的な発現細胞の培養液に、2μg/mLの濃度でLPS(Sigma #L2654)を添加し、添加後48時間のルシフェラーゼ発光量を、ルミノメーターで定量測定した。対照として、LPS無添加群を設けた。また、従来型のIL−1β遺伝子発現のみを検出するレポーターシステムとして、IL−1β遺伝子のプロモーター下流にGL4を接続したベクターを作製し、これをトランスフェクション処理した細胞で同様に試験を行った。
切り出し精製した遺伝子ベクターを、C57BL/6系統のマウスから採取した前核期受精卵200個にインジェクションし、71匹の産子を得た。産子の体組織から抽出したゲノムDNAを用いて遺伝子型を解析し、ゲノムに遺伝子ベクターが挿入されているファウンダーマウス18匹を得た。ファウンダーマウス4匹それぞれを野生型C57BL/6系統マウスと交配させ、F1世代マウスを作出し、LPS(Sigma #L2654)を腹腔内に投与したときのレポーター分子の反応を調べた。
その結果、系統番号No.M1の個体において、最もS/N比に優れており、この系統を炎症レポーターマウスとして確立した。(図8)。
確立した炎症レポーターマウスの腹腔内に、3mg/kg wtの濃度でLPS(Sigma #L2654)を投与した。投与後0h、4h、24時間に生体イメージング装置(IVIS)を用いて、ルシフェラーゼ発光を観察した。その結果、全身の組織から、ルシフェラーゼの発光シグナルを捉えることができた(図5)。
Claims (13)
- 炎症性サイトカインをコードする遺伝子のプロモーター、レポータータンパク質をコードする遺伝子、前記炎症性サイトカインをコードする遺伝子、及びタンパク質分解シグナル配列をコードする遺伝子を含むベクター。
- 炎症性サイトカインがインターロイキン1βである請求項1に記載のベクター。
- レポータータンパク質がルシフェラーゼである請求項1又は2に記載のベクター。
- 炎症性サイトカインをコードする遺伝子は、カスパーゼにより認識されるペプチドをコードするポリヌクレオチド配列を含むものである請求項1~3のいずれか1項に記載のベクター。
- 請求項1~4のいずれか1項に記載のベクターを含む形質転換体。
- 請求項1~4のいずれか1項に記載のベクターが導入されたトランスジェニック非ヒト動物。
- 非ヒト動物がマウスである請求項6に記載のトランスジェニック非ヒト動物。
- 炎症性刺激によりレポータータンパク質が発光シグナルとして検出される、請求項5に記載の形質転換体又は請求項6若しくは7に記載のトランスジェニック非ヒト動物。
- 請求項5に記載の形質転換体又は請求項6~8のいずれか1項に記載のトランスジェニック非ヒト動物を用いて、炎症性刺激により当該形質転換体又はトランスジェニック非ヒト動物に惹起される炎症反応を検出することを特徴とする炎症反応の検出方法。
- 炎症性サイトカインをコードする遺伝子は、炎症反応により惹起される転写因子NF−κBにより発現されるものである、請求項9に記載の方法。
- 炎症性刺激によりレポータータンパク質を発光シグナルとして検出する、請求項9又は10に記載の方法。
- 請求項5に記載の形質転換体又は請求項6~8のいずれか1項に記載のトランスジェニック非ヒト動物に、炎症性刺激下で候補物質を接触させ、炎症反応の有無を指標として抗炎症物質を選択することを特徴とする抗炎症物質のスクリーニング方法。
- 請求項5に記載の形質転換体又は請求項6~8のいずれか1項に記載のトランスジェニック非ヒト動物を含む、炎症反応検出用又は抗炎症物質のスクリーニング用キット。
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