JP2020036549A - Immunostimulating emulsifier - Google Patents
Immunostimulating emulsifier Download PDFInfo
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- JP2020036549A JP2020036549A JP2018164847A JP2018164847A JP2020036549A JP 2020036549 A JP2020036549 A JP 2020036549A JP 2018164847 A JP2018164847 A JP 2018164847A JP 2018164847 A JP2018164847 A JP 2018164847A JP 2020036549 A JP2020036549 A JP 2020036549A
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- Prior art keywords
- protein
- immunostimulatory
- amino acid
- activity
- emulsifier
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
Description
本発明は、免疫賦活性乳化剤に関する。より具体的には、本発明は、免疫賦活性と乳化活性とを併せ持つタンパク質を含む免疫賦活性乳化剤に関する。 The present invention relates to an immunostimulatory emulsifier. More specifically, the present invention relates to an immunostimulatory emulsifier containing a protein having both immunostimulatory activity and emulsifying activity.
界面活性剤は、医薬、化粧品、食品等、多くの産業分野において利用されている。界面活性剤のほとんどは石油資源に由来するが、レシチンやサポニンといった生物資源に由来する界面活性剤も利用されている。近年、石油の大量消費による環境破壊や石油資源の枯渇といった問題が深刻化しており、石油原料に依存しない生物資源を原料とした機能性物質への変換が迫られている。 Surfactants are used in many industrial fields such as medicines, cosmetics, and foods. Most of the surfactants are derived from petroleum resources, but surfactants derived from biological resources such as lecithin and saponin are also used. 2. Description of the Related Art In recent years, problems such as environmental destruction and depletion of petroleum resources due to mass consumption of petroleum have become more serious, and there is an urgent need for conversion to a functional substance using biological resources that do not depend on petroleum raw materials.
生物資源に由来する界面活性剤の中でも、微生物によって生産されるものはバイオサーファクタントと呼ばれており、その研究が進められている。例えば、特許文献１には、赤色酵母を培養液中で培養して得られる糖タンパク質複合体を有効成分として含有してなる乳化剤が記載されている。また、特許文献１には、この乳化剤の糖量とタンパク質量とを求めたところ、糖とタンパク質とが約９対１の割合であることが記載されている。 Among surfactants derived from biological resources, those produced by microorganisms are called biosurfactants, and their research is being advanced. For example, Patent Document 1 describes an emulsifier containing, as an active ingredient, a glycoprotein complex obtained by culturing red yeast in a culture solution. In addition, Patent Document 1 describes that when the amount of sugar and the amount of protein of this emulsifier were determined, the ratio of sugar to protein was about 9: 1.
一方で、生物資源より抽出精製されるβ−グルカンが、免疫賦活作用があることが知られている。β−グルカンは、大麦・小麦等の穀物類、キノコ類、酵母類などに含まれることが知られている。非特許文献１では、酵母由来のβ−グルカンが、大麦・小麦由来のβ−グルカンより免疫増進効果があることが記載されている。 On the other hand, it is known that β-glucan extracted and purified from biological resources has an immunostimulating effect. β-glucan is known to be contained in cereals such as barley and wheat, mushrooms, yeasts and the like. Non-Patent Document 1 describes that β-glucan derived from yeast has an immunopotentiating effect more than β-glucan derived from barley and wheat.
酵母由来成分に乳化活性を有する成分があることは報告されているが、その成分が何であるかは具体的には明らかにされていない。また、別の酵母由来成分であるβ−グルカンは、その免疫賦活性を利用して栄養機能食品等に用いられているが、免疫賦活性以外の点で当該製品に有用な活性として利用されているものではない。 It has been reported that a component derived from yeast has an emulsifying activity, but the identity of the component is not specifically clarified. Further, β-glucan, another yeast-derived component, is used in nutritional functional foods and the like utilizing its immunostimulatory activity, but is used as a useful activity for the product in points other than immunostimulatory activity. It is not something.
本発明者は、乳化活性を有している生物由来成分に、さらに免疫活性を高める機能が付加されれば、乳化剤を使用する幅広い分野において機能性材料として有用となる点に着目した。本発明の目的は、生物由来物質を利用した、免疫賦活性を有する乳化剤を提供することにある。 The present inventor has paid attention to the fact that if a function of enhancing immunological activity is added to a biological component having an emulsifying activity, it will be useful as a functional material in a wide range of fields using an emulsifier. An object of the present invention is to provide an emulsifier having immunostimulatory activity using a biological substance.
本発明者は鋭意検討の結果、細胞壁タンパク質を細胞壁に固定するＧＰＩアンカーの合成に関わるＧＵＰ１遺伝子を欠損させた酵母の培養液中に、免疫賦活性と乳化活性とを併せ持つタンパク質としてＧＡＳファミリータンパク質が分泌されることを見出した。さらに、ＧＵＰ１遺伝子に加え、マンナン合成に関わるＡＮＰ１遺伝子も併せて欠損させた酵母菌体を洗浄したときに細胞から遊離する物質に、免疫賦活性と乳化活性とを併せ持ち、且つＧＡＳファミリータンパク質よりも免疫賦活性が顕著に高いＦＢＡ１タンパク質を見出した。本発明は、この知見に基づいてさらに検討を重ねることにより完成したものである。 As a result of intensive studies, the present inventors have found that a GAS-family protein as a protein having both immunostimulatory activity and emulsifying activity is contained in a culture medium of a yeast lacking the GUP1 gene involved in the synthesis of a GPI anchor for fixing a cell wall protein to the cell wall. Was found to be secreted. Furthermore, in addition to the GUP1 gene, the substance released from cells when the yeast cells, which are also deficient in the ANP1 gene involved in mannan synthesis, are washed, has both immunostimulatory activity and emulsifying activity, and has a higher activity than the GAS family protein. FBA1 protein with remarkably high immunostimulatory activity was found. The present invention has been completed by further study based on this finding.
即ち、本発明は、下記に掲げる態様の発明を提供する。
項１． 真菌由来のＦＢＡ１タンパク質を含む、免疫賦活性乳化剤。
項２． 前記ＦＢＡ１タンパク質が、
（i）配列番号１に示されるアミノ酸配列、
（ii）配列番号１において、１個又は複数個のアミノ酸残基が欠失、付加、挿入若しくは置換されたアミノ酸配列を有し、且つ乳化活性と免疫賦活性とを有するタンパク質、及び
（iii）配列番号１と７０％以上の相同性を有するアミノ酸配列を有し、且つ乳化活性と免疫賦活性とを有するタンパク質
の少なくともいずれかである、項１に記載の免疫賦活性乳化剤。
項３． 前記真菌が酵母である、項１又は２に記載の免疫賦活性乳化剤。
項４． 前記酵母がサッカロマイセス・セレビシエである、項３に記載の免疫賦活性乳化剤。
項５． 項１から４のいずれかに記載の免疫賦活性乳化剤と油分と水とを含む、免疫賦活性乳化組成物。
項６． 水中油型である、項５に記載の免疫賦活性乳化組成物。
項７． （i'）配列番号１に示されるアミノ酸配列とアフィニティタグとを有するタンパク質、
（ii'）配列番号１に示されるアミノ酸配列において、１個又は複数個のアミノ酸残基が欠失、付加、挿入若しくは置換されたアミノ酸配列とアフィニティタグとを有し、且つ乳化活性と免疫賦活性とを有するタンパク質、及び
（iii'）配列番号１に示されるアミノ酸配列と７０％以上の相同性を有するアミノ酸配列とアフィニティタグとを有し、且つ乳化活性と免疫賦活性とを有するタンパク質
の少なくともいずれかのタンパク質をコードするポリヌクレオチドを含む発現ベクターによって形質転換された細胞を作成する工程、
前記形質転換された細胞の培養を行う工程、
培養された細胞の抽出液を、前記アフィニティタグに特異的な担体に接触させ、発現タンパク質を捕捉する工程、及び
前記捕捉された発現タンパク質を回収する工程、
を含む、免疫賦活性乳化剤の製造方法。
That is, the present invention provides the following aspects of the invention.
Item 1. An immunostimulatory emulsifier comprising a fungal FBA1 protein.
Item 2. The FBA1 protein is
(I) an amino acid sequence represented by SEQ ID NO: 1,
(Ii) a protein having an amino acid sequence in which one or more amino acid residues are deleted, added, inserted or substituted in SEQ ID NO: 1, and having an emulsifying activity and an immunostimulatory activity, and (iii) Item 2. The immunostimulatory emulsifier according to Item 1, which has an amino acid sequence having 70% or more homology with SEQ ID NO: 1 and is at least one of a protein having emulsifying activity and immunostimulating activity.
Item 3. Item 3. The immunostimulatory emulsifier according to Item 1 or 2, wherein the fungus is yeast.
Item 4. Item 4. The immunostimulatory emulsifier according to Item 3, wherein the yeast is Saccharomyces cerevisiae.
Item 5. Item 5. An immunostimulatory emulsified composition comprising the immunostimulatory emulsifier according to any one of Items 1 to 4, an oil component and water.
Item 6. Item 6. The immunostimulatory emulsified composition according to Item 5, which is an oil-in-water type.
Item 7. (I ′) a protein having the amino acid sequence represented by SEQ ID NO: 1 and an affinity tag,
(Ii ′) the amino acid sequence represented by SEQ ID NO: 1, which has an amino acid sequence in which one or more amino acid residues have been deleted, added, inserted, or substituted, and an affinity tag, and has emulsifying activity and immunostimulation. And (iii ′) a protein having an amino acid sequence having at least 70% homology with the amino acid sequence shown in SEQ ID NO: 1 and an affinity tag, and having an emulsifying activity and an immunostimulatory activity. Producing a cell transformed by an expression vector comprising a polynucleotide encoding at least one of the proteins,
Culturing the transformed cells,
Contacting the extract of the cultured cells with a carrier specific to the affinity tag, and capturing the expressed protein; and recovering the captured expressed protein,
A method for producing an immunostimulatory emulsifier, comprising:
本発明によれば、免疫賦活性と乳化活性とを併せ持つ真菌由来のタンパク質を用いることで、生物由来物質を利用した、免疫賦活性を有する乳化剤が提供されるため、乳化剤を用いる幅広い分野において機能性材料として適用することが可能となる。 According to the present invention, by using a fungal protein having both immunostimulatory activity and emulsifying activity, an emulsifier having immunostimulatory activity utilizing a biological substance is provided. It can be applied as a conductive material.
［１．免疫賦活性乳化剤］
本発明の免疫賦活性乳化剤は、真菌由来のＦＢＡ１タンパク質を含む。ＦＢＡ１タンパク質は、一種類のタンパク質が乳化活性と免疫賦活性とを併せ持つ。
[1. Immunostimulating emulsifier]
The immunostimulatory emulsifier of the present invention contains a fungal-derived FBA1 protein. One type of FBA1 protein has both emulsifying activity and immunostimulatory activity.
［１−１．乳化活性及び免疫賦活性］
本発明のＦＢＡ１タンパク質が兼備する乳化活性及び免疫賦活性は、当該タンパク質が有する生理活性のうちの一部であるが、本発明のＦＢＡ１タンパク質の範囲を定める際に指標となる生理活性である。
[1-1. Emulsifying activity and immunostimulatory activity]
The emulsifying activity and immunostimulatory activity of the FBA1 protein of the present invention, which are a part of the physiological activities of the protein, are used as an index when defining the range of the FBA1 protein of the present invention.
本発明の免疫賦活性乳化剤によって発揮される乳化活性は、ＦＢＡ１タンパク質を水中に含む水性液体に油を加えて混合することで、乳化物を得ることで確認することができる。乳化物は、ミセルの形成により確認することができる。本発明の免疫賦活性乳化剤は、例えば水中油滴型（Ｏ／Ｗ）のミセルを形成しやすい。なお、乳化活性の大小は、乳化相の体積によって判断することができる。ＦＢＡ１タンパク質を水中に含む水性液体の量と油の量との割合によっては、混合直後に生じる乳化相が単相となる場合と、水性液体相及び／又は油相との複相となる場合とがある。混合直後に単相となる場合は、混合後の所定時間放置後に水性液体相及び／又は油相と分離される場合は、分離した乳化相の体積が大きいほど、乳化活性が高いと判断することができる。混合直後に複相となる場合は、混合直後の乳化相の体積が大きいほど、また、混合後の所定時間放置後における乳化相の体積が大きいほど、乳化活性が高いと判断することができる。なお、混合直後に単相となる場合に、混合後の所定時間放置しても水性液体相及び／又は油相と分離されず安定的に乳化層が単層のままで存在する場合、及びその他の乳化層体積の大小を判断できない場合は、ミセルのサイズが小さいほど、乳化活性が高いと判断することができる。 The emulsifying activity exhibited by the immunostimulatory emulsifier of the present invention can be confirmed by obtaining an emulsion by adding an oil to an aqueous liquid containing FBA1 protein in water and mixing. Emulsions can be identified by the formation of micelles. The immunostimulatory emulsifier of the present invention tends to form, for example, oil-in-water (O / W) micelles. The magnitude of the emulsifying activity can be determined by the volume of the emulsified phase. Depending on the ratio between the amount of the aqueous liquid containing the FBA1 protein in water and the amount of the oil, the case where the emulsified phase formed immediately after mixing becomes a single phase, the case where the emulsified phase becomes a multiple phase with the aqueous liquid phase and / or the oil phase, and There is. When it becomes a single phase immediately after mixing, if it is separated from the aqueous liquid phase and / or oil phase after standing for a predetermined time after mixing, it is judged that the larger the volume of the separated emulsified phase, the higher the emulsifying activity. Can be. In the case where the emulsion becomes a double phase immediately after mixing, it can be determined that the larger the volume of the emulsified phase immediately after mixing and the larger the volume of the emulsified phase after standing for a predetermined time after mixing, the higher the emulsifying activity. In addition, when it becomes a single phase immediately after mixing, when it is left for a predetermined time after mixing, it is not separated from the aqueous liquid phase and / or the oil phase and the emulsified layer remains stably as a single layer, and When the size of the emulsified layer cannot be determined, it can be determined that the smaller the micelle size, the higher the emulsifying activity.
本発明の免疫賦活性乳化剤による乳化活性が発揮されるｐＨ及び塩濃度は特に限定されない。当該乳化活性を発揮させるｐＨとしては、例えばｐＨ２．５以上、好ましくはｐＨ３．０以上、より好ましくはｐＨ３．０〜１０．５、より好ましくはｐＨ３．０〜１０．０が挙げられる。つまり、本発明の免疫賦活性乳化剤は、酸性からアルカリ性の広い範囲に亘って良好なｐＨ耐性を有している。当該乳化活性を発揮させる塩濃度としては、塩化ナトリウム濃度として例えば０Ｍ超４Ｍ以下、好ましくは０Ｍ超３Ｍ以下、より好ましくは０Ｍ超２Ｍ以下又は０．５〜２Ｍが挙げられる。つまり、本発明の免疫賦活性乳化剤は耐塩性でもある。 The pH and salt concentration at which the emulsifying activity of the immunostimulatory emulsifier of the present invention is exhibited are not particularly limited. The pH at which the emulsifying activity is exerted is, for example, pH 2.5 or higher, preferably pH 3.0 or higher, more preferably pH 3.0 to 10.5, and more preferably pH 3.0 to 10.0. That is, the immunostimulatory emulsifier of the present invention has good pH resistance over a wide range from acidic to alkaline. The salt concentration that exerts the emulsifying activity includes, for example, a sodium chloride concentration of more than 0M to 4M or less, preferably more than 0M to 3M or less, more preferably more than 0M to 2M or 0.5 to 2M. That is, the immunostimulatory emulsifier of the present invention is also salt-resistant.
本発明の免疫賦活性乳化剤によって発揮される免疫賦活性は、免疫細胞を刺激することにより分泌されるサイトカイン量によって判断することができる。例えば、マクロファージ細胞を加えた培地に、本発明の免疫賦活性乳化剤を加えてインキュベートした後に、分泌されたＴＮＦ−αの量を測定することによって判断することができる。具体的には、ＦＢＡ１タンパク質を５０μｇ／ｍＬ濃度で加えた場合に、タンパク質を含まないバッファを同条件でインキュベートした場合におけるＴＮＦ−αの量を基準（１倍、重量基準）として、例えば２５倍以上、好ましくは５０倍以上であることを指標とすることができる。 The immunostimulatory activity exerted by the immunostimulatory emulsifier of the present invention can be determined by the amount of cytokine secreted by stimulating immune cells. For example, it can be determined by measuring the amount of secreted TNF-α after incubating the medium containing macrophage cells with the immunostimulatory emulsifier of the present invention. Specifically, when the FBA1 protein is added at a concentration of 50 μg / mL, the amount of TNF-α when a buffer containing no protein is incubated under the same conditions is used as a reference (1 time, weight basis), for example, 25 times. Above, preferably 50 times or more can be used as an index.
乳化活性及び免疫賦活性のより具体的な測定方法については、本明細書の実施例の記載に従って行うことができる。 A more specific method for measuring the emulsifying activity and the immunostimulatory activity can be performed according to the description in the Examples of the present specification.
［１−２．由来生物（真菌）］
ＦＢＡ１タンパク質の由来生物である真菌としては、酵母及び糸状真菌が挙げられ、好ましくは酵母が挙げられる。
[1-2. Origin organism (fungus)]
Fungi from which the FBA1 protein is derived include yeast and filamentous fungi, preferably yeast.
酵母としては、出芽酵母つまりサッカロマイセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）、サッカロマイセス・パストリアヌス（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｐａｓｔｒｉａｎｕｓ）、サッカロマイセス・バヤヌス（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｂａｙａｎｕｓ）、サッカロマイセス・カールスベルゲンシス（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃａｒｌｓｂｅｒｇｅｎｓｉｓ）などのサッカロマイセス属（Ｓａｃｃｈａｒｏｍｙｃｅｓ属）；ピキア・パストリス（Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ）、ピキア・アングスタ（Ｐｉｃｈｉａ ａｎｇｕｓｔａ）、ピキア・フィンランディカ（Ｐｉｃｈｉａ ｆｉｎｌａｎｄｉｃａ）、ピキア・トレハロフィラ（Ｐｉｃｈｉａ ｔｒｅｈａｌｏｐｈｉｌａ）、ピキア・コクラマエ（Ｐｉｃｈｉａ ｋｏｃｌａｍａｅ）、ピキア・メンブラナエファシエンス（Ｐｉｃｈｉａ ｍｅｍｂｒａｎａｅｆａｃｉｅｎｓ）、ピキア・マイニュータ（Ｐｉｃｈｉａ ｍｉｎｕｔａ）などのピキア属（Ｐｉｃｈｉａ属）；シゾサッカロマイセス・ポンベ（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ ｐｏｍｂｅ）などのシゾサッカロマイセス属（Ｓｃｈｉｚｏｓａｃｃｈａｒｏｍｙｃｅｓ属）；デッケラ・ブルキセレンシス（Ｄｅｋｋｅｒａ ｂｒｕｘｅｌｌｅｎｓｉｓ）、デッケラ・アノマラ（Ｄｅｋｋｅｒａ ａｎｏｍａｌａ）などのデッケラ属（Ｄｅｋｋｅｒａ属）；クルイベロマイセス・ラクティス（Ｋｌｕｙｖｅｒｏｍｙｃｅｓ ｌａｃｔｉｓ）、クルイベロマイセス・マルキシアヌス（Ｋｌｕｙｖｅｒｏｍｙｃｅｓ ｍａｒｘｉａｎｕｓ）などのクルイベロマイセス属（Ｋｌｕｙｖｅｒｏｍｙｃｅｓ属）に属するものが挙げられる。上述の酵母の中でも、サッカロマイセス属に属する酵母が好ましく、出芽酵母つまりサッカロマイセス・セレビシエがより好ましい。サッカロマイセス・セレビシエ（Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅ）細胞の菌株としては、パン酵母、ＣＢＳ ７９５９、ＣＢＳ ７９６０、ＣＢＳ ７９６１、ＣＢＳ ７９６２、ＣＢＳ ７９６３、ＣＢＳ ７９６４、ＩＺ−１９０４、ＴＡ、ＢＧ−１、ＣＲ−１、ＳＡ−１、Ｍ−２６、Ｙ−９０４、ＰＥ−２、ＰＥ−５、ＶＲ−１、ＢＲ−１、ＢＲ−２、ＭＥ−２、ＶＲ−２、ＭＡ−３、ＭＡ−４、ＣＡＴ−１、ＣＢ−１、ＮＲ−１、ＢＴ−１、及びＡＬ−１が挙げられる。 As yeast, budding yeast i.e. Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces pastorianus (Saccharomyces pastrianus), Saccharomyces bayanus (Saccharomyces bayanus), Saccharomyces (Saccharomyces genus), such as Saccharomyces carlsbergensis (Saccharomyces carlsbergensis); Pichia -Pastoris (Pichia pastoris), Pichia angusta, Pichia finlandica (Pichia finlandica), Pichia trehalophila (Pichia trehalophila), Pichia cocola Genus Pichia (genus Pichia) such as Pichia koclamae, Pichia membranaefaciens, Pichia minuta; Genus Schizosaccharomyces; genus Dekera xymuris (Keveromyces crucimer ces mira), such as Dekkera bruxellensis, Dekkera anomala, etc .; us) include those belonging to the Kluyveromyces, such as (Kluyveromyces spp.). Among the above yeasts, yeast belonging to the genus Saccharomyces is preferable, and budding yeast, ie, Saccharomyces cerevisiae, is more preferable. Saccharomyces cerevisiae cell strains include baker's yeast, CBS 7959, CBS 7960, CBS 7961, CBS 7962, CBS 7963, CBS 7964, IZ-1904, TA, BG-1, CR-1, and SA-. 1, M-26, Y-904, PE-2, PE-5, VR-1, BR-1, BR-2, ME-2, VR-2, MA-3, MA-4, CAT-1, CB-1, NR-1, BT-1, and AL-1.
糸状真菌としては、トリコデルマ・リーセイ、トリコデルマ・ロンジブラキアタム（Ｔｒｉｃｈｏｄｅｒｍａ ｌｏｎｇｉｂｒａｃｈｉａｔｕｍ）トリコデルマ・ハリジアウム（Ｔｒｉｃｈｏｄｅｒｍａ ｈａｒｚｉａｎｕｍ）、トリコデルマ・コニンギ（Ｔｒｉｃｈｏｄｅｒｍａ ｋｏｎｉｎｇｉｉ）及びトリコデルマ・ヴィリデ（Ｔｒｉｃｈｏｄｅｒｍａ ｖｉｒｉｄｅ）などのトリコデルマ属；アスペルギルス・ニガー（Ａｓｐｅｒｇｉｌｌｕｓ ｎｉｇｅｒ）、アスペルギルス・オリザエ（Ａｓｐｅｒｇｉｌｌｕｓ ｏｒｙｚａｅ）などのアルペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属；ニューロスポラ・クラッサ（Ｎｅｕｒｏｓｐｏｒａ ｃｒａｓｓａ）などのニューロスポラ（Ｎｅｕｒｏｓｐｏｒａ）属；フサリウム・グラニューム（Ｆｕｓａｒｉｕｍ ｇｒａｍｉｎｅｕｍ）、フサリウム・ベネナツム（Ｆｕｓａｒｉｕｍ ｖｅｎｅｎａｔｕｍ）などのフサリウム（Ｆｕｓａｒｉｕｍ）属；クリソスポリウム・ラクノウェンス（Ｃｈｒｙｓｏｓｐｏｒｉｕｍ ｌｕｃｋｎｏｗｅｎｓｅ）などのクリソスポリウム（Ｃｈｒｙｓｏｓｐｏｒｉｕｍ）属に属するものが挙げられる。 The filamentous fungi include Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma harzianum, Trichoderma koningi and Trichoderma kongingi, and the like. Aspergillus genus such as Aspergillus niger, Aspergillus oryzae; Neurospora genus such as Neurospora crassa; Include those belonging to chrysosporium (Chrysosporium) genus such Chrysosporium lucknowense (Chrysosporium lucknowense); sarium gramineum), Fusarium venenatum (Fusarium venenatum) Fusarium (Fusarium) genus such.
［１−３．ＦＢＡ１タンパク質］
ＦＢＡ１タンパク質は、一種類のタンパク質が乳化活性と免疫賦活性とを併せ持つ。ＦＢＡ１タンパク質は、解糖系の酵素の一種であり、糖新生においてグリセルアルデヒド３−リン酸（Ｇ３Ｐ）によるジヒドロキシアセトンリン酸（ＤＨＡＰまたはグリセリンリン酸）のアルドール縮合を触媒してフルクトース１，６−ビスホスフェート（ＦＢＰ）を形成し、解糖における逆反応を触媒する。ＦＢＡ１タンパク質は、主に細胞内に局在し、その一部は細胞表面に存在することは知られているものである。しかし、ＦＢＡ１タンパク質は細胞外に分泌されることは知られていないタンパク質であり、また、免疫賦活性との関係についても知られていない。無論、ＦＢＡ１タンパク質と乳化活性との関係についても知られていない。
[1-3. FBA1 protein]
One type of FBA1 protein has both emulsifying activity and immunostimulatory activity. The FBA1 protein is a kind of glycolytic enzyme and catalyzes the aldol condensation of dihydroxyacetone phosphate (DHAP or glycerin phosphate) by glyceraldehyde 3-phosphate (G3P) in gluconeogenesis, thereby fructose 1,6 -Forms bisphosphate (FBP) and catalyzes the reverse reaction in glycolysis. It is known that the FBA1 protein is mainly localized in a cell, and a part thereof is present on the cell surface. However, the FBA1 protein is not known to be secreted extracellularly, and its relationship with immunostimulatory activity is not known. Of course, the relationship between the FBA1 protein and the emulsifying activity is not known.
ＦＢＡ１タンパク質は配列番号（ＵｎｉｐｒｏｔＫＢアクセッション番号Ｐ１４５４０）に示されるアミノ酸配列を有するタンパク質であってよい。本発明においては、ＦＢＡ１タンパク質としては配列番号１に示されるアミノ酸配列を有するタンパク質のみならず、乳化活性及び免疫賦活性を備えていれば、配列番号１に示されるアミノ酸配列において、１個又は複数個のアミノ酸残基が欠失、付加、挿入若しくは置換されていてもよい。 The FBA1 protein may be a protein having the amino acid sequence shown in SEQ ID NO: (UniprotKB Accession No. P14540). In the present invention, not only the protein having the amino acid sequence shown in SEQ ID NO: 1 but also one or more FBA1 protein in the amino acid sequence shown in SEQ ID NO: 1 as long as it has emulsifying activity and immunostimulating activity. Amino acid residues may be deleted, added, inserted or substituted.
より具体的には、ＦＢＡ１タンパク質としては、以下の（i）〜（iii）の少なくともいずれかが挙げられる。
（i）配列番号１に示されるアミノ酸配列、
（ii）配列番号１において、１個又は複数個のアミノ酸残基が欠失、付加、挿入若しくは置換されたアミノ酸配列を有し、且つ乳化活性と免疫賦活性とをするタンパク質、及び
（iii）配列番号１と７０％以上の相同性を有するアミノ酸配列を有し、且つ乳化活性と免疫賦活性とを有するタンパク質。
More specifically, the FBA1 protein includes at least one of the following (i) to (iii).
(I) an amino acid sequence represented by SEQ ID NO: 1,
(Ii) a protein having an amino acid sequence in which one or more amino acid residues have been deleted, added, inserted or substituted in SEQ ID NO: 1, and having an emulsifying activity and an immunostimulatory activity, and (iii) A protein having an amino acid sequence having 70% or more homology with SEQ ID NO: 1 and having emulsifying activity and immunostimulatory activity.
本明細書において、「１個又は複数個のアミノ酸残基が欠失、付加、挿入若しくは置換された」とは、部位特異的突然変異誘発法等の周知の方法により、または天然に生じ得る程度の複数個の数のアミノ酸の置換等により改変がなされたことを意味する。アミノ酸の改変の個数は、好ましくは１〜３０個、より好ましくは１〜１０個、さらに好ましくは１〜４個、最も好ましくは１〜２個である。改変アミノ酸配列の例は、好ましくは、そのアミノ酸が、１または複数個（好ましくは、１〜数個若しくは１、２、３、または４個）の保存的置換を有するアミノ酸配列であることができる。ここで、「保存的置換」とは、１若しくは複数個のアミノ酸残基を、別の化学的に類似したアミノ酸残基で置き換えることを意味する。例えば、ある疎水性残基を別の疎水性残基によって置換する場合、ある極性残基を同じ電荷を有する別の極性残基によって置換する場合などが挙げられる。このような置換を行うことができる機能的に類似のアミノ酸は、アミノ酸毎に当該技術分野において公知である。具体例を挙げると、非極性（疎水性）アミノ酸としては、アラニン、バリン、イソロイシン、ロイシン、プロリン、トリプトファン、フェニルアラニン、メチオニンなどが挙げられる。極性（中性）アミノ酸としては、グリシン、セリン、スレオニン、チロシン、グルタミン、アスパラギン、システインなどが挙げられる。陽電荷をもつ（塩基性）アミノ酸としては、アルギニン、ヒスチジン、リジンなどが挙げられる。また、負電荷をもつ（酸性）アミノ酸としては、アスパラギン酸、グルタミン酸などが挙げられる。 In the present specification, "one or more amino acid residues are deleted, added, inserted or substituted" means that the amino acid residues are generated by a well-known method such as site-directed mutagenesis or naturally occurring. Means that the amino acid has been modified by substitution of a plurality of amino acids. The number of amino acid modifications is preferably 1 to 30, more preferably 1 to 10, further preferably 1 to 4, and most preferably 1 to 2. An example of an altered amino acid sequence can preferably be an amino acid sequence in which the amino acid has one or more (preferably one to several or 1, 2, 3, or 4) conservative substitutions. . Here, “conservative substitution” means that one or more amino acid residues are replaced with another chemically similar amino acid residue. For example, a case where a certain hydrophobic residue is substituted with another hydrophobic residue, a case where a certain polar residue is substituted with another polar residue having the same charge, and the like can be mentioned. Functionally similar amino acids that can make such substitutions are known in the art for each amino acid. Specific examples include non-polar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, cysteine, and the like. Examples of positively charged (basic) amino acids include arginine, histidine, and lysine. Examples of negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
また、本明細書において「７０％以上の相同性を有するアミノ酸配列」は、好ましくは８０％以上、より好ましくは８５％以上、さらに好ましくは９０％以上、さらにより好ましくは９５％以上、特に好ましくは９８％以上、そして最も好ましくは９９％以上の同一性を有するアミノ酸配列であることができる。さらに、本明細書においてアミノ酸配列についての「相同性」は、比較される配列間において、各々の配列を構成するアミノ酸残基の一致の程度の意味で用いられる。本明細書において示した「相同性」の数値はいずれも、当業者に公知の相同性検索プログラムを用いて算出される数値であればよく、例えばＦＡＳＴＡ、ＢＬＡＳＴ等においてデフォルト（初期設定）のパラメータを用いることにより、容易に算出することができる。 Further, in the present specification, “amino acid sequence having 70% or more homology” is preferably 80% or more, more preferably 85% or more, further preferably 90% or more, still more preferably 95% or more, and particularly preferably. Can be an amino acid sequence having 98% or more, and most preferably 99% or more identity. Further, in the present specification, the term "homology" regarding an amino acid sequence is used to mean the degree of coincidence of amino acid residues constituting each sequence between the compared sequences. The numerical value of “homology” shown in the present specification may be any numerical value calculated using a homology search program known to those skilled in the art. For example, default (initial setting) parameters in FASTA, BLAST, etc. Can be easily calculated.
なお、ＦＢＡ１タンパク質は、グリコシル化、アミド化、カルボキシル化、リン酸化等により修飾されたものであってもよい。また、ＦＢＡ１タンパク質は、製造工程上で付与されたアフィニティタグを有するものであってもよい。 The FBA1 protein may be modified by glycosylation, amidation, carboxylation, phosphorylation, or the like. Further, the FBA1 protein may have an affinity tag added in the production process.
［１−４．用途］
本発明の免疫賦活性乳化剤は、免疫賦活性を有するバイオサーファクタントであるため、乳化剤を用いる幅広い分野において安全性の高い機能性材料として適用できる。例えば、食品、化粧品、医薬品等に用いることができる。
[1-4. Use]
Since the immunostimulatory emulsifier of the present invention is a biosurfactant having immunostimulatory activity, it can be applied as a highly safe functional material in a wide range of fields using emulsifiers. For example, it can be used for foods, cosmetics, pharmaceuticals and the like.
［２．免疫賦活性乳化組成物］
本発明の免疫賦活性乳化組成物は、上述の本発明の免疫賦活性乳化剤と油分と水とを含む。免疫賦活性乳化組成物の具体例としては、食品組成物、化粧料組成物、医薬組成物等が挙げられる。なお、免疫賦活性乳化組成物において、免疫賦活性乳化剤によって形成されるミセルのタイプは特に限定されないが、当該免疫賦活性乳化剤は、水中油型（Ｏ／Ｗ）のミセルを形成しやすい。
[2. Immunostimulatory emulsified composition]
The immunostimulatory emulsifying composition of the present invention contains the above-described immunostimulating emulsifier of the present invention, an oil component and water. Specific examples of the immunostimulatory emulsified composition include a food composition, a cosmetic composition, a pharmaceutical composition and the like. In the immunostimulatory emulsifying composition, the type of micelle formed by the immunostimulatory emulsifier is not particularly limited, but the immunostimulatory emulsifier easily forms an oil-in-water (O / W) micelle.
免疫賦活性乳化組成物において、免疫賦活性乳化剤の含有量としては、乳化相を生じさせ（つまりミセルを形成させ）る程度の乳化活性と免疫賦活性とを発揮させる限り特に限定されず、例えば５μｇ／ｍＬ以上であってよい。より具体的には、例えば８μｇ／ｍＬ以上、好ましくは１０μｇ／ｍＬ以上、より好ましくは１５μｇ／ｍＬ以上、より一層好ましくは２０μｇ／ｍＬ以上、さらに好ましくは３０μｇ／ｍＬ以上、さらに一層好ましくは４０μｇ／ｍＬ以上が挙げられる。良好な耐塩性を得るために好ましい免疫賦活性乳化剤の濃度としては、たとえば２０μｇ／ｍＬ以上、好ましくは３０μｇ／ｍＬ以上、より好ましくは３５μｇ／ｍＬ以上、さらに好ましくは４０μｇ／ｍＬ以上が挙げられる。本発明の免疫賦活性乳化剤は、乳化活性及び免疫賦活性のいずれにも優れているため免疫賦活性乳化組成物中における含有量の上限としては特に限定されないが、例えば１ｇ／ｍＬ以下、９００μｇ／ｍＬ以下、５００μｇ／ｍＬ以下、３００μｇ／ｍＬ以下、又は１００μｇ／ｍＬ以下が挙げられる。 In the immunostimulatory emulsifying composition, the content of the immunostimulatory emulsifier is not particularly limited as long as the emulsifying activity and immunostimulatory activity are exerted to the extent that an emulsifying phase is formed (that is, micelles are formed). It may be 5 μg / mL or more. More specifically, for example, 8 μg / mL or more, preferably 10 μg / mL or more, more preferably 15 μg / mL or more, still more preferably 20 μg / mL or more, still more preferably 30 μg / mL or more, and still more preferably 40 μg / mL or more. mL or more. Preferred concentrations of the immunostimulatory emulsifier for obtaining good salt resistance include, for example, 20 μg / mL or more, preferably 30 μg / mL or more, more preferably 35 μg / mL or more, and even more preferably 40 μg / mL or more. Since the immunostimulatory emulsifier of the present invention is excellent in both emulsifying activity and immunostimulating activity, the upper limit of the content in the immunostimulating emulsified composition is not particularly limited, but is, for example, 1 g / mL or less, 900 μg / mL or less, 500 μg / mL or less, 300 μg / mL or less, or 100 μg / mL or less.
［２−１．食品組成物］
本発明の免疫賦活性乳化剤を含む食品組成物は、当該免疫賦活性乳化剤の作用に基づいて、食品組成物を乳化形態とするとともに、例えば腸管免疫を高める機能を備えさせる等の免疫賦活性を発揮することができる。従って、当該食品組成物は、腸管免疫増強用といった機能性食品、特定保健用食品、栄養補助食品（サプリメント）、病者用食品等において有用である。
[2-1. Food composition]
The food composition containing the immunostimulatory emulsifier of the present invention, based on the action of the immunostimulatory emulsifier, makes the food composition an emulsified form, and has an immunostimulatory activity such as having a function of enhancing intestinal immunity. Can be demonstrated. Therefore, the food composition is useful in functional foods for enhancing intestinal immunity, foods for specified health use, dietary supplements (supplements), foods for patients, and the like.
当該食品組成物の形態については、乳化組成物の形態である限り特に限定されない。例えば、液状体、ゲル状体、それらの冷凍物等が挙げられる。具体的には、飲料（炭酸飲料、清涼飲料、乳飲料、果汁飲料、茶類、栄養飲料等）、菓子類（チョコレート、グミ、ゼリー、ガム、キャンディー等）、乳製品（ヨーグルト、プリン、アイスクリーム等）、調味料（マヨネーズ、ソース、ケチャップ、ドレッシング等）等の一般食品の形態が挙げられる。また、ソフトカプセル剤、液剤、シロップ剤等の形態も挙げられる。 The form of the food composition is not particularly limited as long as it is in the form of an emulsion composition. For example, a liquid material, a gel material, a frozen product thereof, and the like can be given. Specifically, beverages (carbonated drinks, soft drinks, milk drinks, fruit drinks, teas, nutritional drinks, etc.), confectioneries (chocolate, gummy, jelly, gum, candy, etc.), dairy products (yogurt, pudding, ice cream) Creams) and seasonings (mayonnaise, sauce, ketchup, dressing, etc.). In addition, forms such as soft capsules, liquids, syrups and the like can also be mentioned.
本発明の免疫賦活性乳化剤は、耐ｐＨ性且つ耐塩性であるため、酸性を含む広いｐＨ（例えばｐＨ３．０以上、好ましくはｐＨ３．０〜１０．０）及び／又は高塩濃度を含む広い塩濃度（塩化ナトリウム濃度として例えば好ましくは０Ｍ超３Ｍ以下、より好ましくは０Ｍ超２Ｍ以下又は０．５〜２Ｍ）の食品組成物において有用である。従って、酸性（例えば３．０〜６．０、好ましくは３．０〜５．０）及び／又は高塩濃度を含む広い塩濃度（塩化ナトリウム濃度として例えば好ましくは０Ｍ超３Ｍ以下、より好ましくは０Ｍ超２Ｍ以下又は０．５〜２Ｍ）の食品組成物においても有用である。このような食品組成物としては、例えば、乳化型酸性食品（果汁飲料、ヨーグルト、マヨネーズ、ソース、ケチャップ、ドレッシング等）、乳化型高塩濃度食品（清涼飲料水、栄養飲料、マヨネーズ、ソース、ケチャップ、ドレッシング等）が挙げられ、好ましくは、乳化型酸性高塩濃度食品（マヨネーズ、ソース、ケチャップ、ドレッシング等）が挙げられる。 Since the immunostimulatory emulsifier of the present invention is pH-resistant and salt-resistant, it has a wide pH range including acidity (for example, pH 3.0 or more, preferably pH 3.0 to 10.0) and / or a broad pH range including high salt concentration. It is useful in a food composition having a salt concentration (for example, preferably as a sodium chloride concentration of preferably more than 0 M to 3 M or less, more preferably more than 0 M to 2 M or 0.5 to 2 M). Therefore, a wide salt concentration (for example, preferably more than 0M and 3M or less as sodium chloride concentration, more preferably, more than 3.0M, including acidic (for example, 3.0 to 6.0, preferably 3.0 to 5.0) and / or high salt concentration) It is also useful in food compositions of more than 0M to 2M or less or 0.5 to 2M). Examples of such food compositions include emulsified acidic foods (fruit juice, yogurt, mayonnaise, sauce, ketchup, dressing, etc.) and emulsified high salt foods (soft drinks, nutritional drinks, mayonnaise, sauces, ketchup) , Dressings and the like), preferably, emulsified acidic high salt foods (mayonnaise, sauce, ketchup, dressing, etc.).
当該食品組成物には、食品衛生学的に許容される基材や担体を含んでよく、更に必要に応じて、甘味料、酸味料、ゲル化剤、緩衝剤、保存剤、ｐＨ調節剤、溶解補助剤、懸濁化剤、等張化剤、粘稠剤、矯臭剤、香料、色素等の食品衛生学的に許容される添加剤を含有していてもよい。 The food composition may include a food hygiene-acceptable base material or carrier, and further, if necessary, a sweetener, an acidulant, a gelling agent, a buffer, a preservative, a pH adjuster, Food-hygienic acceptable additives such as dissolution aids, suspending agents, tonicity agents, thickeners, flavors, flavors, pigments and the like may be contained.
［２−２．化粧料組成物］
本発明の免疫賦活性乳化剤を含む化粧料組成物は、当該免疫賦活性乳化剤の作用に基づいて、化粧料組成物を乳化形態とするとともに、例えば外皮下にある免疫細胞を活性化する機能等の免疫賦活性を発揮することができる。従って、当該化粧料組成物は、皮膚外用医薬部外品、化粧料、皮膚洗浄料等において有用である。
[2-2. Cosmetic composition]
The cosmetic composition containing the immunostimulatory emulsifier of the present invention provides a cosmetic composition in an emulsified form based on the action of the immunostimulatory emulsifier and, for example, a function of activating immune cells located in the subcutaneous epidermis. Can exert an immunostimulatory activity. Therefore, the cosmetic composition is useful in quasi-drugs for external use on the skin, cosmetics, skin cleansers, and the like.
当該化粧料組成物の形態については、乳化組成物の形態である限り特に限定されない。例えば、液状体及びゲル状体等が挙げられる。具体的には、皮膚外用医薬部外品の場合、例えば、クリーム剤、ローション剤、ジェル剤、乳液剤、液剤、貼付剤、エアゾール剤、軟膏剤、パック剤等が挙げられ；化粧料の場合、例えば、クリーム剤、ローション剤、ジェル剤、乳液剤、液剤、軟膏剤、パック剤、入浴剤等が挙げられ；皮膚洗浄料の場合、例えば、石鹸、クレンジング、洗顔料、ボディーシャンプー、ヘアシャンプー、リンス等が挙げられる。 The form of the cosmetic composition is not particularly limited as long as it is in the form of an emulsion composition. For example, a liquid material, a gel material and the like can be mentioned. Specifically, in the case of a quasi-drug for external use on the skin, for example, creams, lotions, gels, emulsions, solutions, patches, aerosols, ointments, packs, etc .; in the case of cosmetics For example, creams, lotions, gels, emulsions, liquids, ointments, packs, baths and the like; in the case of skin cleansers, for example, soaps, cleansing, facial cleansers, body shampoos, hair shampoos And rinsing.
本発明の免疫賦活性乳化剤は、耐ｐＨ性であり、酸性からアルカリ性を含む広いｐＨ（例えばｐＨ３．０以上、好ましくはｐＨ３．０〜１０．５、より好ましくはｐＨ３．０〜１０．０）の化粧料組成物においても有用である。このような化粧料組成物としては、例えば、酸性化粧料（収斂化粧水、ピーリング用化粧水、ピーリング用皮膚洗浄料）、中性化粧料、及びアルカリ性化粧料（石鹸等のアルカリ性皮膚洗浄料）等が挙げられる。 The immunostimulatory emulsifier of the present invention is pH-resistant and has a wide pH range from acidic to alkaline (for example, pH 3.0 or higher, preferably pH 3.0 to 10.5, more preferably pH 3.0 to 10.0). It is also useful in the cosmetic composition. Such cosmetic compositions include, for example, acidic cosmetics (astringent lotion, peeling lotion, peeling skin cleanser), neutral cosmetics, and alkaline cosmetics (alkaline skin cleanser such as soap). And the like.
当該化粧料組成物には、香粧学的に許容される基材や担体を含んでよく、更に必要に応じて、緩衝剤、保存剤、ｐＨ調節剤、湿潤化剤、溶解補助剤、懸濁化剤、等張化剤、粘稠剤、矯臭剤、香料、色素等の香粧学的に許容される添加剤を含有していてもよい。 The cosmetic composition may contain a cosmetically acceptable substrate or carrier, and further, if necessary, a buffer, a preservative, a pH adjuster, a wetting agent, a solubilizer, a suspending agent, It may contain cosmetically acceptable additives such as turbidity agents, tonicity agents, thickeners, corrigents, perfumes, pigments and the like.
［２−３．医薬組成物］
本発明の免疫賦活性乳化剤を含む医薬組成物は、当該免疫賦活性乳化剤の作用に基づいて、医薬組成物を乳化形態とすると共に、例えば薬理活性成分による抗原性を増強させる機能、腸管免疫を高める機能、外皮下にある免疫細胞を活性化する機能等の免疫賦活性を発揮することができる。従って、当該医薬組成物は、ワクチン製剤、経口医薬品、皮膚外用医薬品等において有用である。当該医薬組成物においては、本発明の免疫賦活性乳化剤以外に他の薬理活性成分を含んでよい。
[2-3. Pharmaceutical composition]
The pharmaceutical composition containing the immunostimulatory emulsifier of the present invention, based on the action of the immunostimulatory emulsifier, makes the pharmaceutical composition into an emulsified form and, for example, enhances the antigenicity of the pharmacologically active ingredient, and enhances intestinal immunity It can exert immunostimulatory activities such as a function of enhancing and a function of activating immune cells located in the subepidermal region. Therefore, the pharmaceutical composition is useful in vaccine preparations, oral pharmaceuticals, skin external pharmaceuticals, and the like. The pharmaceutical composition may contain other pharmacologically active ingredients in addition to the immunostimulatory emulsifier of the present invention.
当該医薬品組成物の形態については、乳化組成物の形態である限り特に限定されない。例えば、液状体及びゲル状体等が挙げられる。具体的には、ワクチン製剤の場合、抗原（水）相と油性アジュバント相とを本発明の免疫賦活性乳化剤で乳化した乳化組成物であり、例えば、注射剤、経鼻投与剤、経口剤等が挙げられ；経口医薬品の場合、液剤、シロップ剤等が挙げられ；皮膚外用医薬品の場合、例えば、クリーム剤、ローション剤、ジェル剤、乳液剤、液剤、貼付剤、エアゾール剤、軟膏剤、パック剤等が挙げられる。 The form of the pharmaceutical composition is not particularly limited as long as it is in the form of an emulsion composition. For example, a liquid material, a gel material and the like can be mentioned. Specifically, in the case of a vaccine preparation, it is an emulsified composition obtained by emulsifying an antigen (water) phase and an oily adjuvant phase with the immunostimulatory emulsifier of the present invention, for example, injection, nasal administration, oral preparation, etc. Liquid pharmaceuticals, syrups and the like in the case of oral pharmaceuticals; creams, lotions, gels, emulsions, liquids, patches, aerosols, ointments, packs in the case of skin external pharmaceuticals Agents and the like.
当該医薬品組成物には、薬学的に許容される基材や担体を含んでよく、更に必要に応じて、緩衝剤、保存剤、ｐＨ調節剤、湿潤化剤、溶解補助剤、懸濁化剤、等張化剤、無痛化剤、粘稠剤、矯臭剤、香料等の薬学的に許容される添加剤を含有していてもよい。 The pharmaceutical composition may contain a pharmaceutically acceptable base or carrier, and further, if necessary, a buffer, a preservative, a pH adjuster, a wetting agent, a solubilizing agent, a suspending agent. And pharmaceutically acceptable additives such as tonicity agents, soothing agents, thickeners, odor correctors, fragrances and the like.
［３．免疫賦活性乳化剤の製造方法］
上述の本発明の免疫賦活性乳化剤の製造方法としては特に限定されず、ＦＢＡ１タンパク質を生じさせることができるあらゆる方法が用いられる。
[3. Method for producing immunostimulating emulsifier]
The method for producing the above-mentioned immunostimulatory emulsifier of the present invention is not particularly limited, and any method capable of producing an FBA1 protein may be used.
［３−１．特定遺伝子の欠損株の培養による例］
例えば、真菌（酵母）の特定の遺伝子欠損株を培養することにより得ることができる。具体的には、ＦＢＡ１タンパク質は、GUP1遺伝子及びANP1遺伝子の二重欠損株を培養する。培養は、ソルビトール等の浸透圧保護剤を添加して浸透圧を保護した条件で行うことが好ましい。培養後に得られた菌体をリン酸緩衝生理食塩水等に懸濁させ、懸濁液を遠心し、遠心上清を精製することでＦＢＡ１タンパク質を取得することができる。
[3-1. Example of culturing a strain deficient in a specific gene]
For example, it can be obtained by culturing a specific gene-deficient strain of a fungus (yeast). Specifically, the FBA1 protein cultures a double-deficient strain of the GUP1 gene and the ANP1 gene. The cultivation is preferably performed under conditions in which an osmotic pressure protective agent such as sorbitol is added to protect the osmotic pressure. The FBA1 protein can be obtained by suspending the cells obtained after the culture in phosphate-buffered saline or the like, centrifuging the suspension, and purifying the centrifuged supernatant.
［３−２．アフィニティタグを有する融合タンパク質の発現による例］
また、ＦＢＡ１タンパク質はアミノ酸配列が公知であるため、例えば精製用のアフィニティタグが結合したＦＢＡ１タンパク質を融合タンパク質として発現可能な発現ベクターを構築し、宿主細胞に導入し、当該融合タンパク質を発現させることによって製造することができる。
[3-2. Example by Expression of Fusion Protein Having Affinity Tag]
Further, since the amino acid sequence of the FBA1 protein is known, for example, constructing an expression vector capable of expressing the FBA1 protein to which an affinity tag for purification is bound as a fusion protein, introducing the vector into a host cell, and expressing the fusion protein Can be manufactured by
具体的には、免疫賦活性乳化剤の製造方法は、ＦＢＡ１のアミノ酸配列とアフィニティタグ配列とを有するタンパク質（融合タンパク質）をコードするポリヌクレオチドを含む発現ベクターによって形質転換された細胞を作成する工程１と；前記形質転換された細胞の培養を行う工程２と；培養された細胞の抽出液を、前記アフィニティタグに特異的な担体に接触させ、発現タンパク質を捕捉する工程３と；前記捕捉された発現タンパク質を回収する工程４と、を含む。 Specifically, the method for producing an immunostimulatory emulsifier comprises the steps of: preparing a cell transformed with an expression vector containing a polynucleotide encoding a protein (fusion protein) having an amino acid sequence of FBA1 and an affinity tag sequence; Step 2 of culturing the transformed cells; Step 3 of contacting an extract of the cultured cells with a carrier specific to the affinity tag to capture an expressed protein; and Recovering the expressed protein.
［３−２−１．工程１］
工程１では、ＦＢＡ１のアミノ酸配列とアフィニティタグ配列とを有するタンパク質（融合タンパク質）をコードするポリヌクレオチドを含む発現ベクターによって形質転換された細胞を作成する。
[3-2-1. Step 1]
In step 1, cells transformed with an expression vector containing a polynucleotide encoding a protein (fusion protein) having an amino acid sequence of FBA1 and an affinity tag sequence are prepared.
［アフィニティタグ］
ＦＢＡ１タンパク質に結合させるアフィニティタグは、そのアフィニティタグに対応する（特異的な）担体と選択的に結合できる限り、どのようなアフィニティタグも用いることができる。例えば、アフィニティタグとしては、ニッケルキレートカラムに選択的に結合可能な２つ以上の連続するヒスチジン残基からなるヒスチジンタグ、不溶性セルロースに選択的に結合するセルロース結合部位、マルトース結合樹脂に選択的に結合するマルトース結合部位などが挙げられ、好ましくは、分子量の小さいヒスチジンタグが挙げられる。
[Affinity tag]
As the affinity tag to be bound to the FBA1 protein, any affinity tag can be used as long as it can selectively bind to a (specific) carrier corresponding to the affinity tag. For example, as the affinity tag, a histidine tag composed of two or more consecutive histidine residues capable of selectively binding to a nickel chelate column, a cellulose binding site selectively binding to insoluble cellulose, and a maltose binding resin selectively. The binding site includes a maltose binding site, and preferably a histidine tag having a small molecular weight.
アフィニティタグは、ＦＢＡ１タンパク質に直接的又は間接的に結合させてよい。ＦＢＡ１タンパク質に間接的に結合させる場合においては、ＦＢＡ１タンパク質の乳化活性及び免疫賦活性を大きく阻害するものでなければ、どのようなリンカー配列が介在していても構わない。 The affinity tag may be directly or indirectly linked to the FBA1 protein. In the case of indirect binding to the FBA1 protein, any linker sequence may be used as long as it does not significantly inhibit the emulsifying activity and immunostimulatory activity of the FBA1 protein.
［融合タンパク質］
ＦＢＡ１タンパク質にアフィニティタグが結合した融合タンパク質において、アフィニティタグはＦＢＡ１タンパク質のカルボキシル末端に結合していることが好ましい。融合タンパク質のアミノ酸配列としては、具体的には以下が挙げられる。
[Fusion protein]
In the fusion protein in which the affinity tag is bound to the FBA1 protein, the affinity tag is preferably bound to the carboxyl terminus of the FBA1 protein. The following are specific examples of the amino acid sequence of the fusion protein.
（i'）配列番号１に示されるアミノ酸配列と、前記アミノ酸配列のカルボキシル末端側にアフィニティタグとを有するタンパク質、
（ii'）配列番号１に示されるアミノ酸配列において、１個又は複数個のアミノ酸残基が欠失、付加、挿入若しくは置換されたアミノ酸配列と、前記アミノ酸配列のカルボキシル末端側にアフィニティタグとを有し、且つ乳化活性と免疫賦活性とを有するタンパク質、及び
（iii'）配列番号１に示されるアミノ酸配列と７０％以上の相同性を有するアミノ酸配列と、前記アミノ酸配列のカルボキシル末端側にアフィニティタグとを有し、且つ乳化活性と免疫賦活性とを有するタンパク質。
(I ′) a protein having the amino acid sequence of SEQ ID NO: 1 and an affinity tag at the carboxyl terminal side of the amino acid sequence,
(Ii ′) an amino acid sequence represented by SEQ ID NO: 1 in which one or more amino acid residues have been deleted, added, inserted or substituted, and an affinity tag at the carboxyl terminal side of the amino acid sequence. A protein having emulsifying activity and immunostimulating activity, and (iii ′) an amino acid sequence having 70% or more homology with the amino acid sequence shown in SEQ ID NO: 1, and an affinity at the carboxyl terminal side of the amino acid sequence. A protein having a tag and having emulsifying activity and immunostimulating activity.
［融合タンパク質をコードするポリヌクレオチド］
このような融合タンパク質をコードするポリヌクレオチドは、当該融合タンパク質のアミノ酸配列をコードするものであれば、どのような塩基配列からなるポリヌクレオチドであってもよい。ポリヌクレオチドは、好ましくはＤＮＡである。
[Polynucleotide encoding fusion protein]
The polynucleotide encoding such a fusion protein may be a polynucleotide having any nucleotide sequence as long as it encodes the amino acid sequence of the fusion protein. The polynucleotide is preferably DNA.
融合タンパク質をコードするポリヌクレオチドを得る方法としては特に限定されないが、例えば、人工的に化学合成することにより得ることができる。 The method for obtaining the polynucleotide encoding the fusion protein is not particularly limited. For example, it can be obtained by artificial chemical synthesis.
また、融合タンパク質をコードするポリヌクレオチドは、ＦＢＡ１タンパク質をコードする遺伝子の配列に基づいて設計されたプライマーと、ＦＢＡ１タンパク質をコードする遺伝子の配列に基づき且つアフィニティタグをコードする配列を有するプライマーと、を用い、ゲノムＤＮＡ、ｃＤＮＡ、プラスミドなど当該遺伝子が含まれるＤＮＡを鋳型としたＰＣＲにより増幅することもできる。 Further, a polynucleotide encoding the fusion protein, a primer designed based on the sequence of the gene encoding the FBA1 protein, a primer based on the sequence of the gene encoding the FBA1 protein and having a sequence encoding an affinity tag, And can be amplified by PCR using a DNA containing the gene, such as genomic DNA, cDNA, or plasmid, as a template.
さらに、融合タンパク質をコードするポリヌクレオチドは、ＦＢＡ１タンパク質をコードするポリヌクレオチドとアフィニティタグをコードするポリヌクレオチドとをそれぞれ別々に構築し、それらを連結することにより得ることもできる。この場合、ＦＢＡ１タンパク質をコードするポリヌクレオチドは、ＦＢＡ１タンパク質をコードする遺伝子の配列に基づいて設計されたプライマーを用いて、ゲノムＤＮＡ、ｃＤＮＡ、プラスミドなど当該遺伝子が含まれるポリヌクレオチドを鋳型としたＰＣＲにより増幅することができ、一方、アフィニティタグをコードするポリヌクレオチドは、アフィニティタグをコードする遺伝子が含まれるゲノムＤＮＡ、ｃＤＮＡ、プラスミドなどを鋳型としたＰＣＲにより増幅することができる。 Further, the polynucleotide encoding the fusion protein can also be obtained by separately constructing a polynucleotide encoding the FBA1 protein and a polynucleotide encoding the affinity tag, and linking them. In this case, the polynucleotide encoding the FBA1 protein is obtained by PCR using a polynucleotide containing the gene such as genomic DNA, cDNA, or plasmid as a template, using primers designed based on the sequence of the gene encoding the FBA1 protein. On the other hand, the polynucleotide encoding the affinity tag can be amplified by PCR using a genomic DNA, cDNA, plasmid, or the like containing a gene encoding the affinity tag as a template.
［発現ベクター及び発現ベクターにより形質転換された宿主細胞］
融合タンパク質のアミノ酸配列をコードするＤＮＡを、宿主細胞内で複製可能で、かつ、そのＤＮＡ配列がコードするタンパク質を発現可能な状態で含む発現ベクターを構築する。発現ベクターは、自己複製ベクター、すなわち、染色体外の独立体として存在し、その複製が染色体の複製に依存しない、例えば、プラスミドを基本に構築することができる。また、発現ベクターは、宿主細胞に導入されたとき、その宿主細胞のゲノム中に組み込まれ、それが組み込まれた染色体と一緒に複製されるものであってもよい。ベクター構築の手順及び方法は、遺伝子工学の分野で慣用されているものを用いることができる。
[Expression vector and host cell transformed with expression vector]
An expression vector is constructed which is capable of replicating a DNA encoding the amino acid sequence of the fusion protein in a host cell and which can express the protein encoded by the DNA sequence. Expression vectors are self-replicating vectors, ie, exist as extrachromosomal entities, and their replication does not depend on chromosomal replication, eg, can be constructed on a plasmid basis. Further, the expression vector may be one which, when introduced into a host cell, is integrated into the genome of the host cell and replicated together with the chromosome in which it has been integrated. For the procedure and method of vector construction, those commonly used in the field of genetic engineering can be used.
発現ベクターは、これを実際に宿主細胞に導入して融合タンパク質を発現させるために、前記の融合タンパク質をコードするＤＮＡの他に、その発現を制御するＤＮＡ配列や形質転換された宿主細胞を選択するための遺伝子マーカー等を含んでいるのが望ましい。発現を制御するＤＮＡ配列としては、プロモータ、及びターミネーターをコードするＤＮＡ配列等がこれに含まれる。プロモータは宿主細胞において転写活性を示すものであれば特に限定されず、宿主細胞と同種若しくは異種のいずれかのタンパク質をコードする遺伝子の発現を制御するＤＮＡ配列として得ることができる。例えば、プロモータとしてＧＡＬ１プロモータ及びＰＧＫ１プロモータ等が挙げられ、好ましくはＰＧＫ１プロモータが挙げられる。 In order to actually express the fusion protein by introducing it into a host cell and to express the fusion protein, in addition to the DNA encoding the fusion protein, a DNA sequence controlling the expression and a transformed host cell are selected. It is desirable to include a genetic marker or the like. Examples of the DNA sequence that controls expression include a promoter and a DNA sequence that encodes a terminator. The promoter is not particularly limited as long as it exhibits transcriptional activity in the host cell, and can be obtained as a DNA sequence that controls the expression of a gene encoding a protein that is either the same or different from the host cell. For example, the GAL1 promoter and the PGK1 promoter are exemplified as the promoter, and the PGK1 promoter is preferred.
宿主細胞は特に限定されず、真菌（酵母、糸状菌）、大腸菌、放線菌等が挙げられるが、好ましくは真菌が挙げられ、より好ましくは酵母が挙げられる。また、発現ベクターによる宿主細胞の形質転換も、この分野で慣用されている方法に従い当業者が適宜実施することができる。 The host cell is not particularly limited, and includes fungi (yeasts and filamentous fungi), Escherichia coli, actinomycetes, and the like, preferably fungi, and more preferably yeast. Transformation of a host cell with an expression vector can also be appropriately performed by those skilled in the art according to a method commonly used in this field.
［３−２−２．工程２〜工程４］
工程２では、形質転換された細胞の培養を行う。融合タンパク質を発現する宿主細胞は、適当な培地で培養し、その培養物から融合タンパク質を得ることができる。融合タンパク質を発現させる宿主細胞の培養条件は、使用させる宿主細胞についての培養条件と本質的に同等であってよい。融合タンパク質は、発現する宿主細胞中に発現する。
[3-2-2. Step 2 to step 4]
In step 2, the transformed cells are cultured. The host cell expressing the fusion protein can be cultured in an appropriate medium, and the fusion protein can be obtained from the culture. Culture conditions for the host cell in which the fusion protein is expressed may be essentially the same as those for the host cell used. The fusion protein is expressed in the expressing host cell.
工程３では、培養された細胞の抽出液を、前記アフィニティタグに特異的な担体に接触させ、発現タンパク質を捕捉する。培養された細胞の抽出液は、水系液中に、培養された細胞から遊離した融合タンパク質を含むものであればよく、当業者によって適宜調製される。例えば、培養された細胞を緩衝液等の洗浄液に懸濁させて、洗浄液中に融合タンパク質を遊離させ、必要に応じてさらに遠心分離を行うことによって、上清として調製することができる。培養された細胞の抽出液は、融合タンパク質が有するアフィニティタグに対応する（特異的な）担体に接触させられ、これによって抽出液中の融合タンパク質が捕捉される。例えばアフィニティタグとしてヒスチジンタグを採用した場合は、担体としてニッケルキレートカラムを用いることとなる。 In step 3, the cultured cell extract is brought into contact with a carrier specific to the affinity tag to capture the expressed protein. The extract of the cultured cells may be any as long as it contains a fusion protein released from the cultured cells in an aqueous solution, and is appropriately prepared by those skilled in the art. For example, a supernatant can be prepared by suspending the cultured cells in a washing solution such as a buffer solution, releasing the fusion protein in the washing solution, and further performing centrifugation as needed. The extract of the cultured cells is brought into contact with a (specific) carrier corresponding to the affinity tag of the fusion protein, whereby the fusion protein in the extract is captured. For example, when a histidine tag is adopted as an affinity tag, a nickel chelate column is used as a carrier.
工程４では、捕捉された発現タンパク質を回収する。具体的には、アフィニティタグに特異的な担体で捕捉した融合タンパク質を溶出させることで選択的に回収（精製）する。融合タンパク質を溶出させるための溶出液は、融合タンパク質のアフィニティタグ及び担体に応じて、融合タンパク質を変性させない条件を当業者が適宜選択することができる。 In step 4, the captured expressed protein is recovered. Specifically, the fusion protein captured by a carrier specific to the affinity tag is eluted to be selectively recovered (purified). The eluate for eluting the fusion protein can be appropriately selected by those skilled in the art under conditions that do not denature the fusion protein, depending on the affinity tag of the fusion protein and the carrier.
以下に実施例を示して本発明をより具体的に説明するが、本発明はこれらに限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited thereto.
［実施例１］
［ＦＢＡ１タンパク質（Fba1p）の製造−１］
マンナン合成に関わるANP1遺伝子と細胞壁タンパク質を細胞壁に固定するGPIアンカーの合成に関わるGUP1遺伝子とを欠損させた株を用い、0.6 Mソルビトールを添加して浸透圧を保護した条件で培養後、遠心分離（3000xG, 5分）により菌体を得た。得られた菌体を、リン酸緩衝生理食塩水PBSに懸濁させて、細胞表層成分の一部を細胞から遊離させた。この懸濁液を遠心分離し、得られた遠心上清にケロシン油を添加すると、ミセルが形成される乳化現象が観察された。
[ Example 1 ]
[Production of FBA1 protein (Fba1p) -1]
Using a strain deficient in the ANP1 gene involved in mannan synthesis and the GUP1 gene involved in the synthesis of a GPI anchor that anchors cell wall proteins to the cell wall, culture under the condition of adding 0.6 M sorbitol to protect the osmotic pressure, followed by centrifugation (3000 × G, 5 minutes) to obtain bacterial cells. The obtained cells were suspended in phosphate-buffered saline PBS to release some of the cell surface components from the cells. When this suspension was centrifuged and kerosene oil was added to the obtained centrifugal supernatant, an emulsification phenomenon in which micelles were formed was observed.
遠心上清を陰イオン交換カラムクロマトグラフィーにより分離し、乳化活性を有する画分を中心に、SDSポリアクリルアミドゲル電気泳動で分析した。その結果、乳化活性画分中の主成分を回収し、質量分析により解析した。その結果、回収したタンパク質をFBA1タンパク質として同定した。 The centrifuged supernatant was separated by anion exchange column chromatography, and the fraction having emulsifying activity was analyzed mainly by SDS polyacrylamide gel electrophoresis. As a result, the main component in the emulsifying active fraction was recovered and analyzed by mass spectrometry. As a result, the recovered protein was identified as FBA1 protein.
［実施例２］
［ＦＢＡ１タンパク質（Fba1p）の製造−２］
（培養液中へFba1pを分泌発現するプラスミドpSP-G1(FBA1-His)の構築）
ＦＢＡ１タンパク質（Fba1p）を発現させるために、強力なプロモータ（PGK1プロモータ）の下流にFBA1遺伝子を繋いだプラスミドpSP-G1(FBA1-His)を構築した。このプラスミドpSP-G1(FBA1-His)は、具体的には以下のように、酵母染色体から取得したFBA1遺伝子を、pSP-G1プラスミド（酵母遺伝資源センターより分譲）に組み込んで構築した。
[ Example 2 ]
[Production of FBA1 protein (Fba1p) -2]
(Construction of plasmid pSP-G1 (FBA1-His) which secretes and expresses Fba1p into the culture medium)
In order to express the FBA1 protein (Fba1p), a plasmid pSP-G1 (FBA1-His) in which the FBA1 gene was linked downstream of a strong promoter (PGK1 promoter) was constructed. Specifically, this plasmid pSP-G1 (FBA1-His) was constructed by incorporating the FBA1 gene obtained from the yeast chromosome into the pSP-G1 plasmid (available from Yeast Genetic Resource Center) as follows.
Fba1pは、配列番号１からなり、以下に示すプライマーを用いてその遺伝子を酵母染色体からＰＣＲにより取得した。フォワードプライマー及びリバースプライマーは、いずれも５’末端側に制限酵素認識配列を付加し、リバースプライマーには、制限酵素認識配列に終始コドンを挟んでさらにアフィニティタグ（ヒスチジンタグ；6×His）配列を付加するようにデザインした。 Fba1p consists of SEQ ID NO: 1, and its gene was obtained from the yeast chromosome by PCR using the primers shown below. Each of the forward primer and the reverse primer has a restriction enzyme recognition sequence added to the 5 ′ end, and the reverse primer further has an affinity tag (histidine tag; 6 × His) sequence with a termination codon interposed between the restriction enzyme recognition sequence. Designed to add.
上記のプライマーを用いた酵母染色体のＰＣＲにより、FBA1遺伝子にヒスチジンタグ（6×His）が付加され、且つ両端に制限酵素認識部位を有する線状ポリヌクレオチドをＰＣＲ産物として得た。当該ＰＣＲ産物及びpSP-G1プラスミドを制限酵素で処理することで粘着末端を生じさせ、両者をライゲーションにより連結し環状化させた。これによって、培養液中へFba1pを分泌発現するプラスミドpSP-G1(FBA1-His)を構築した。プラスミドpSP-G1(FBA1-His)の構造を図１に示す。プラスミドpSP-G1(FBA1-His)は、ＵＲＡ３を栄養要求性マーカーに持ち、Fba1pのＯＲＦをＰＧＫ１プロモータにより発現する。 By PCR of yeast chromosome using the above primers, a linear polynucleotide having a histidine tag (6 × His) added to the FBA1 gene and having restriction enzyme recognition sites at both ends was obtained as a PCR product. The PCR product and the pSP-G1 plasmid were treated with restriction enzymes to generate sticky ends, and both were ligated by ligation to circularize. As a result, a plasmid pSP-G1 (FBA1-His) secreting and expressing Fba1p into the culture solution was constructed. FIG. 1 shows the structure of the plasmid pSP-G1 (FBA1-His). Plasmid pSP-G1 (FBA1-His) has URA3 as an auxotrophic marker and expresses the Fba1p ORF with the PGK1 promoter.
（発現誘導及び発現Fba1pの精製）
構築したプラスミドpSP-G1(FBA1-His)を、酵母Saccharomyces cerevisiae BY4741を宿主として組み込んだ。このようにして得られた形質転換体を、グルコースを炭素源とする培地で培養し、Fba1pを発現させた。培養後、遠心分離によって上清を除去し菌体を回収した。
(Expression induction and purification of expressed Fba1p)
The constructed plasmid pSP-G1 (FBA1-His) was integrated with yeast Saccharomyces cerevisiae BY4741 as a host. The transformant thus obtained was cultured in a medium using glucose as a carbon source to express Fba1p. After the culture, the supernatant was removed by centrifugation to collect the cells.
菌体をPBSに懸濁し、懸濁液を遠心分離に供した後、遠心上清からFba1を精製した。遠心上清からのFba1pの精製には、HisTrap HP, 1 mL (Amersham Biosciences, 現GEヘルスケア バイオサイエンス)を用いた。カラムを平衡化させるため、Binding buffer（20 mMリン酸緩衝液(pH 7.4) + 30 mM Imidazole + 500mM NaCl）を5 mL通した。カラムに培養上清10 mLを通し、Fba1pをヒスチジンタグを介して捕捉した。さらに洗浄のため、カラムにBinding bufferを12 mL通した。捕捉したFba1pを溶出させるため、カラムに溶出用液Elution buffer（20 mMリン酸緩衝液(pH 7.4) + 500 mM Imidazole + 500mM NaCl）を4 mL通した。溶出したFba1p含有液画分は、500 μLずつ1.5 mLチューブに回収した。 The cells were suspended in PBS, the suspension was subjected to centrifugation, and Fba1 was purified from the centrifuged supernatant. For purification of Fba1p from the centrifugation supernatant, HisTrap HP, 1 mL (Amersham Biosciences, current GE Healthcare Bioscience) was used. To equilibrate the column, 5 mL of a binding buffer (20 mM phosphate buffer (pH 7.4) + 30 mM Imidazole + 500 mM NaCl) was passed. 10 ml of the culture supernatant was passed through the column, and Fba1p was captured via a histidine tag. For further washing, 12 mL of the binding buffer was passed through the column. To elute the captured Fba1p, 4 mL of an elution buffer Elution buffer (20 mM phosphate buffer (pH 7.4) +500 mM Imidazole + 500 mM NaCl) was passed through the column. The eluted Fba1p-containing liquid fraction was collected in 1.5 mL tubes in 500 μL portions.
［ＦＢＡ１タンパク質の発現確認試験（電気泳動及びウェスタンブロッティング）］
ＦＢＡ１タンパク質（Fba1p）の発現を確認するため、培養上清（精製前）とHis-Tag精製後の溶出液を、SDS-PAGE及びウェスタンブロッティングに供した。
[FBA1 protein expression confirmation test (electrophoresis and western blotting)]
To confirm the expression of the FBA1 protein (Fba1p), the culture supernatant (before purification) and the eluate after His-Tag purification were subjected to SDS-PAGE and Western blotting.
<使用試薬−SDS-PAGE (銀染色)>
Acetic acid (WAKO, 特級)
Methanol (WAKO, 特級)
2D-銀染色試薬・II (コスモバイオ)
(a)固定化剤 : チオ尿素
(b)前処理剤 : ジチオスレイトール、グルタルアルデヒド、チオ尿素
(c)染色液A : 硝酸銀
(d)染色液B : 水酸化アンモニウム、水酸化ナトリウム
(e)現像原液 : クエン酸、ホルムアルデヒド、チオ硫酸ナトリウム
(f)停止液 : クエン酸
2×Sample buffer（0.125 M Tris-HCl, 10% 2-Mercaptoethanol, 4% SDS, 10% Sucrose, 0.04 mg/mL Bromophenol blue）
10×Running buffer
30% Acrylamide mixture (10%ポリアクリルアミドゲルの作成に使用)
WIDE-VIEW TM Prestained Protein Size Marker III (SDS-PAGE用マーカー, WAKO)
<Reagent used-SDS-PAGE (silver staining)>
Acetic acid (WAKO, special grade)
Methanol (WAKO, special grade)
2D-Silver Staining Reagent II (Cosmo Bio)
(a) Fixing agent: thiourea
(b) Pretreatment agent: dithiothreitol, glutaraldehyde, thiourea
(c) Staining solution A: silver nitrate
(d) Staining solution B: ammonium hydroxide, sodium hydroxide
(e) Development stock solution: citric acid, formaldehyde, sodium thiosulfate
(f) Stop solution: citric acid
2 × Sample buffer (0.125 M Tris-HCl, 10% 2-Mercaptoethanol, 4% SDS, 10% Sucrose, 0.04 mg / mL Bromophenol blue)
10 × Running buffer
30% Acrylamide mixture (used for making 10% polyacrylamide gel)
WIDE-VIEWTM Prestained Protein Size Marker III (SDS-PAGE marker, WAKO)
<使用試薬−ウェスタンブロッティング>
10×TBS-T (希釈して使用)
100 mM Tris-HCl (pH 7.5)
1 M NaCl (WAKO, 特級)
0.5% Tween20 (WAKO)
1×Anode I
300 mM Tris-HCl (pH 10.4)
10% MeOH (WAKO, 特級)
1×Anode II
25 mM Tris-HCl (pH 10.4)
10% MeOH (WAKO, 特級)
1×Cathode
25 mM Tris-HCl (pH 9.6)
40 mM Glysine (WAKO, 特級)
10% MeOH (WAKO, 特級)
5%スキムミルク(WAKO, 生化学用)
一次抗体(Anti-His-tag mAb, IgG/Mouse, amg/mL, MBL)
Solution I (Signal(R) Immunoreaction Enhancer Solution, TOYOBO)
二次抗体(Anti-Mouse IgG-Alkaline Phosphatase antibody produces in goat)
Solution II (Signal(R) Immunoreaction Enhancer Solution, TOYOBO)
BCIP-NBT溶液キット(nacalai tesque)
カラーマーカー(Precision Plus Protein Standards, BIO RAD)
<Reagent used-Western blotting>
10x TBS-T (used after dilution)
100 mM Tris-HCl (pH 7.5)
1 M NaCl (WAKO, special grade)
0.5% Tween20 (WAKO)
1 × Anode I
300 mM Tris-HCl (pH 10.4)
10% MeOH (WAKO, special grade)
1 × Anode II
25 mM Tris-HCl (pH 10.4)
10% MeOH (WAKO, special grade)
1 × Cathode
25 mM Tris-HCl (pH 9.6)
40 mM Glysine (WAKO, special grade)
10% MeOH (WAKO, special grade)
5% skim milk (WAKO, for biochemistry)
Primary antibody (Anti-His-tag mAb, IgG / Mouse, amg / mL, MBL)
Solution I (Signal (R) Immunoreaction Enhancer Solution, TOYOBO)
Secondary antibody (Anti-Mouse IgG-Alkaline Phosphatase antibody produces in goat)
Solution II (Signal (R) Immunoreaction Enhancer Solution, TOYOBO)
BCIP-NBT solution kit (nacalai tesque)
Color marker (Precision Plus Protein Standards, BIO RAD)
<使用器具>
PVDFメンブレン(Immonilon-P, 0.45 μm, 20 cm × 20 cm, Millipore)
濾紙(No. 1, 240 mm, ADVANTEC)
<Equipment used>
PVDF membrane (Immonilon-P, 0.45 μm, 20 cm × 20 cm, Millipore)
Filter paper (No. 1, 240 mm, ADVANTEC)
<実験手順−SDS-PAGE及び銀染色>
10 μLの細胞抽出液（精製前）と10 μLのHis-Tag精製後のElution buffer溶出液（第１画分として0〜0.5mL画分、第２画分として0.5〜1.0mL画分、第３画分として1.0〜1.5mL画分、及び第４画分として1.5〜2.0 mL画分）をそれぞれ1.5 mLチューブに分注した。
<Experimental procedure-SDS-PAGE and silver staining>
10 μL of cell extract (before purification) and 10 μL of Elution buffer eluate after His-Tag purification (0-0.5 mL fraction as first fraction, 0.5-1.0 mL fraction as second fraction, 1.0-1.5 mL fraction as three fractions and 1.5-2.0 mL fraction as fourth fraction) were respectively dispensed into 1.5 mL tubes.
当該1.5 mLチューブに、さらに前述した2×Sample buffer10 μLを加え、ウォーターバスで変性処理(95℃, 5分)を行った。10%ポリアクリルアミドゲルにサンプルを20μLずつアプライした。WIDE-VIEW TM Prestained Protein Size Marker IIIを3 μLアプライし、電気泳動(50 mA, 約75分)を行った。 10 μL of the above-mentioned 2 × Sample buffer was further added to the 1.5 mL tube, and denaturation treatment (95 ° C., 5 minutes) was performed in a water bath. Each sample was applied to a 10% polyacrylamide gel in an amount of 20 μL. 3 μL of WIDE-VIEW ™ Prestained Protein Size Marker III was applied, and electrophoresis (50 mA, about 75 minutes) was performed.
メタノール25 mL、酢酸5 mL、純水20 mLを混合した(固定液I)。ゲルを固定液Iに浸し、振盪(10分)した。メタノール15 mL、酢酸5 mL、(a)固定化剤2.5 mL、純水27.5 mLを混合した(固定液II)。固定液Iを捨て、ゲルを固定液IIに浸して振盪(15分)した。メタノール25 mL、(b)前処理剤2.5 mL、純水22.5 mLを混合した(前処理液)。固定液IIを捨て、ゲルを前処理液に浸して振盪(10分)した。前処理液を捨て、ゲルを純水50 mLに浸して振盪(5分)した。(c)染色液A 2.5 mL、(d)染色液B 2.5 mL、純水45 mLを混合した(銀染色液)。純水を捨て、ゲルを銀染色液に浸して振盪(15分)した。銀染色液を回収し、純水50 mLで2分間、計3回洗浄した(銀染色液は塩酸を加えて塩化銀にしてから、廃液として回収した)。(e)現像原液2.5 mL、純水47.5 mLを混合した(現像液)。純水を捨て、ゲルを現像液に浸して振盪した。適度なバンドが確認できたら、(f)停止液を2.5 mL加えて振盪した。反応が停止したら、ゲルを水で5分程度2、3回洗浄した。その後、ゲルを観察した。 25 mL of methanol, 5 mL of acetic acid, and 20 mL of pure water were mixed (fixing solution I). The gel was immersed in fixative I and shaken (10 minutes). 15 mL of methanol, 5 mL of acetic acid, (a) 2.5 mL of the fixing agent, and 27.5 mL of pure water were mixed (fixing solution II). The fixative I was discarded, and the gel was immersed in the fixative II and shaken (15 minutes). 25 mL of methanol, 2.5 mL of the pretreatment agent (b) and 22.5 mL of pure water were mixed (pretreatment liquid). The fixative II was discarded, and the gel was immersed in the pretreatment solution and shaken (10 minutes). The pretreatment liquid was discarded, and the gel was immersed in 50 mL of pure water and shaken (5 minutes). (c) 2.5 mL of the staining solution A, (d) 2.5 mL of the staining solution B, and 45 mL of pure water were mixed (silver staining solution). The pure water was discarded, and the gel was immersed in a silver staining solution and shaken (15 minutes). The silver staining solution was collected and washed with 50 mL of pure water for 2 minutes for a total of three times (the silver staining solution was converted into silver chloride by adding hydrochloric acid, and then collected as a waste solution). (e) 2.5 mL of a stock solution for development and 47.5 mL of pure water were mixed (developer). The pure water was discarded, and the gel was immersed in a developer and shaken. When an appropriate band was confirmed, (f) 2.5 mL of a stop solution was added and the mixture was shaken. After the reaction was stopped, the gel was washed with water for 2 to 3 times for about 5 minutes. Thereafter, the gel was observed.
<実験手順−ウェスタンブロッティング>
上述と同様にSDS-PAGEによる電気泳動を行い、以下のようにして、His抗体を用いて、Fba1pをHisタグを介して検出することによりその存在を確認した。ゲルのサイズより一回り大きいPVDFメンブレン(80 mm × 90 mm)を用意し、100% MeOHに20秒浸して親水化し、その後すぐに純水に浸した。3つのプラスチック容器に、1×Anode I、1×Anode II、1×Cathodeを加え、それぞれ4・2・6枚の濾紙(80 mm × 90 mm)を浸した。SDS-PAGE後、新しいプラスチック容器に1×Cathodeを加え、ゲルを浸した。セミドライ式転写装置の陽極にAnode Iの濾紙、Anode IIの濾紙、メンブレン、ゲル、Cathodeの濾紙を順に重ねた。泡が入らないように濾紙の上に次の溶液をマイクロピペットで少量撒いた。陰極を重ね、泡を抜くように陰極を手の平で押した。陰極の上に1 kg程度の重りを置き、転写を行った(60 mA, 110分)。メンブレンのマーカーと余分な部分を切り取り、表裏が分かるよう左上に切れ込みを入れた。メンブレンのサイズに合わせた容器をパラフィルムで作り、5%のスキムミルク溶液20 mLを用いてブロッキングを行った(一晩振盪, 4℃)。20 mLの1×TBSTでメンブレンを洗浄した(5分振盪, 3回, 液はアスピレーターでよく取り除く)。一次抗体をSolution Iで1000倍希釈したもの5 mLをメンブレンに撒き、振盪(1 h)した。その後20 mLの1×TBSTでメンブレンを洗浄した(5分振盪, 3回)。二次抗体をSolution IIで5000倍希釈したもの5 mLをメンブレンに撒き、振盪(30分)した。その後、20 mLの1×TBSTでメンブレンを洗浄した(5分振盪, 3回)。BCIP-NBT溶液キットの緩衝液5 mLと発色原液50 μLとを直前に混合し、メンブレンに添加し、目的の位置に青紫色のバンドが現れるまで振盪した。適度に染色されたら、すぐに純水でメンブレンを洗浄し、反応を停止させた。その後、メンブレンを観察した。
<Experimental procedure-Western blotting>
Electrophoresis by SDS-PAGE was performed in the same manner as described above, and the presence thereof was confirmed by detecting Fba1p via a His tag using a His antibody as described below. A PVDF membrane (80 mm × 90 mm) slightly larger than the size of the gel was prepared, immersed in 100% MeOH for 20 seconds to make it hydrophilic, and then immediately immersed in pure water. 1 × Anode I, 1 × Anode II, and 1 × Cathode were added to three plastic containers, and 4.2.6 sheets of filter paper (80 mm × 90 mm) were soaked in each. After SDS-PAGE, 1 × Cathode was added to a new plastic container, and the gel was soaked. Anode I filter paper, Anode II filter paper, membrane, gel, and Cathode filter paper were sequentially stacked on the anode of the semi-dry transfer device. A small amount of the following solution was sprinkled on a filter paper with a micropipette to prevent bubbles from entering. The cathode was overlaid, and the cathode was pressed with the palm of the hand to remove bubbles. Transfer was performed by placing a weight of about 1 kg on the cathode (60 mA, 110 minutes). We cut off the marker and extra parts of the membrane, and made a cut in the upper left so that the front and back could be seen. A container corresponding to the size of the membrane was made of parafilm, and blocking was performed using 20 mL of a 5% skim milk solution (shaking overnight, 4 ° C.). The membrane was washed with 20 mL of 1 × TBST (shake for 5 minutes, 3 times, and the solution was removed well with an aspirator). 5 mL of the primary antibody diluted 1000-fold with Solution I was spread on a membrane and shaken (1 h). Thereafter, the membrane was washed with 20 mL of 1 × TBST (shake for 5 minutes, 3 times). 5 mL of the secondary antibody diluted 5000 times with Solution II was spread on a membrane, and shaken (30 minutes). Thereafter, the membrane was washed with 20 mL of 1 × TBST (5 minutes shaking, 3 times). 5 mL of the buffer solution of the BCIP-NBT solution kit and 50 μL of the chromogenic stock solution were mixed immediately before, added to the membrane, and shaken until a blue-violet band appeared at the desired position. As soon as the staining was adequate, the membrane was washed with pure water to stop the reaction. Thereafter, the membrane was observed.
銀染色の結果とウェスタンブロッティングの結果とを図２に示す。図２において、左のゲル写真は銀染色の結果であり、右のゲル写真はウェスタンブロッティングの結果である。図２においては、プラスミドpSP-G1(FBA1-His)で形質転換した酵母Saccharomyces cerevisiae (BY4741)の培養上清の各分画（溶出分画１〜３）に加え、精製前分画、スルー分画及び洗浄分画も併せて示している。最左レーンはタンパク質サイズマーカーである。図２に示すように、精製前分画と、プラスミドpSP-G1(FBA1-His)で形質転換した酵母の培養上清の各画分（溶出分画１〜３）のうちの溶出分画２とにFba1pの存在が確認された。 FIG. 2 shows the results of silver staining and the results of western blotting. In FIG. 2, the left gel photograph is the result of silver staining, and the right gel photograph is the result of western blotting. In FIG. 2, in addition to each fraction (elution fractions 1 to 3) of the culture supernatant of yeast Saccharomyces cerevisiae (BY4741) transformed with plasmid pSP-G1 (FBA1-His), The fraction and the wash fraction are also shown. The leftmost lane is a protein size marker. As shown in FIG. 2, the fraction before purification and the eluted fraction 2 among the fractions (eluted fractions 1 to 3) of the culture supernatant of the yeast transformed with the plasmid pSP-G1 (FBA1-His) The presence of Fba1p was confirmed.
［乳化活性試験］
精製前分画（Fba1p精製前）及び溶出分画２（Fba1p精製後）それぞれから、Fba1p濃度が５μｇ／ｍＬ、１０μｇ／ｍＬ、２０μｇ／ｍＬ、３０μｇ／ｍＬ、４０μｇ／ｍＬ、５０μｇ／ｍＬ、７５μｇ／ｍＬ及び１００μｇ／ｍＬを含む画分を調製した。画分それぞれのFba1p濃度は、BCA法によって測定した。これらのFba1p含有画分（Fba1p水溶液）に、その半分の体積のケロシン油を加え、３０秒ボルテックスした。その結果を図３に示す。図３に示すように、精製前分画（Fba1p精製前）及び溶出分画２（Fba1p精製後）のすべてに対して乳化相が生じた。乳化相が占める体積に着目すると、溶出分画２（Fba1p精製後）の方が乳化活性に優れていることが確認され、これらの中でも、１０μｇ／ｍＬ以上である場合に好ましい乳化活性が確認され、２０μｇ／ｍＬ以上である場合により好ましい乳化活性が確認され、３０μｇ／ｍＬ以上である場合にさらに好ましい乳化活性が確認された。４０μｇ／ｍＬ以上の場合においても、濃度に応じて乳化活性が好ましく得られる傾向が確認された。
[Emulsification activity test]
From the pre-purification fraction (before Fba1p purification) and the eluted fraction 2 (after Fba1p purification), the Fba1p concentration was 5 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 75 μg / ML and fractions containing 100 μg / mL. The Fba1p concentration of each fraction was measured by the BCA method. To these Fba1p-containing fractions (Fba1p aqueous solution), half the volume of kerosene oil was added and vortexed for 30 seconds. The result is shown in FIG. As shown in FIG. 3, an emulsified phase was generated in all of the pre-purification fraction (before Fba1p purification) and the eluted fraction 2 (after Fba1p purification). Focusing on the volume occupied by the emulsified phase, it is confirmed that the elution fraction 2 (after Fba1p purification) has better emulsifying activity, and among them, a preferable emulsifying activity is confirmed when the emulsifying fraction is 10 μg / mL or more. , 20 μg / mL or more, more preferable emulsifying activity was confirmed, and when it was 30 μg / mL or more, more preferable emulsifying activity was confirmed. Even in the case of 40 μg / mL or more, it was confirmed that the emulsifying activity was preferably obtained according to the concentration.
なお、Fba1pによる乳化相（１時間静置後）を、1 mL程度の水に滴下すると、乳化相は広がりうすめられた。一方、Fba1pによる乳化相（１時間静置後）を1 mL程度のケロシン油に滴下すると、乳化相はケロシン油中で液滴を形成した。これらの結果から、Fba1pによる乳化相のエマルジョンの型は水中油滴（O/W）型であると判明した。 In addition, when the emulsified phase by Fba1p (after standing for 1 hour) was dropped into about 1 mL of water, the emulsified phase spread and was thinned. On the other hand, when the emulsified phase by Fba1p (after standing for 1 hour) was dropped into about 1 mL of kerosene oil, the emulsified phase formed droplets in the kerosene oil. From these results, it was found that the emulsion type of the emulsified phase by Fba1p was an oil-in-water (O / W) type.
［ｐＨ耐性試験］
Fba1pを含む培養上清をヒスタグラムカラム精製に供し、精製画分とｐＨが異なるクエン酸緩衝液、リン酸緩衝液、Tris-HCl緩衝液、及び炭酸−重炭酸緩衝液とを用い、Fba1pを２０μｇ／ｍＬ又は４０μｇ／ｍＬの濃度で含み且つｐＨが異なる水溶液を調製した。得られた水溶液のｐＨは、３．０、４．０、５．０（クエン酸緩衝液を用いた場合）；６．０、７．０（リン酸緩衝液を用いた場合）；８．０、９．０（Tris-HCl緩衝液を用いた場合）；及び１０（炭酸−重炭酸緩衝液を用いた場合）であった。これらのFab1水溶液に、その半分の体積のケロシン油を加え、３０秒ボルテックスした。
[PH tolerance test]
The culture supernatant containing Fba1p was subjected to histogram column purification, and Fba1p was purified using a citrate buffer, a phosphate buffer, a Tris-HCl buffer, and a carbonate-bicarbonate buffer having different pH from the purified fraction. Aqueous solutions were prepared containing 20 μg / mL or 40 μg / mL at different concentrations and different pH. The pH of the resulting aqueous solution is 3.0, 4.0, 5.0 (when a citrate buffer is used); 6.0, 7.0 (when a phosphate buffer is used); 0, 9.0 (when using a Tris-HCl buffer); and 10 (when using a carbonate-bicarbonate buffer). To these Fab1 aqueous solutions, half the volume of kerosene oil was added and vortexed for 30 seconds.
結果を図４に示す。図４に示すように、いずれのFba1p濃度及びいずれのｐＨでも乳化相が確認されており、乳化活性が確認された。乳化相が占める体積はｐＨに影響されていないため、Fab1が極めてｐＨ耐性に優れていることが確認できた。 FIG. 4 shows the results. As shown in FIG. 4, the emulsified phase was confirmed at any Fba1p concentration and any pH, and the emulsifying activity was confirmed. Since the volume occupied by the emulsified phase was not affected by the pH, it was confirmed that Fab1 was extremely excellent in pH resistance.
［塩耐性試験］
Fba1pを含む培養上清をヒスタグラムカラム精製に供し、精製画分と塩化ナトリウムとを用い、Fba1pを２０μｇ／ｍＬ又は４０μｇ／ｍＬを含み且つ塩化ナトリウム濃度が異なる水溶液を調製した。得られた水溶液における、精製画分に添加した塩化ナトリウムの終濃度は、０Ｍ、１Ｍ、２Ｍ及び３Ｍであった。これらのFab1水溶液に、その半分の体積のケロシン油を加え、３０秒ボルテックスした。
[Salt tolerance test]
The culture supernatant containing Fba1p was subjected to histogram column purification, and an aqueous solution containing Fba1p containing 20 μg / mL or 40 μg / mL and having a different sodium chloride concentration was prepared using the purified fraction and sodium chloride. The final concentrations of sodium chloride added to the purified fraction in the obtained aqueous solution were 0M, 1M, 2M and 3M. To these Fab1 aqueous solutions, half the volume of kerosene oil was added and vortexed for 30 seconds.
結果を図５に示す。図５に示すように、いずれのFba1p濃度及びいずれの塩化ナトリウム濃度でも乳化相が確認されており、乳化活性が確認された。本試験に供されたFba1p水溶液は、その調製に用いられたFba1p精製画分に予め含まれていた塩の量を考慮すると、図５中に示された濃度よりも高い塩濃度を有しているが、少なくとも、３Ｍ以下で乳化活性が得られ、２Ｍ以下でより好ましい乳化活性が得られたことが認められる。 FIG. 5 shows the results. As shown in FIG. 5, the emulsified phase was confirmed at any Fba1p concentration and any sodium chloride concentration, and the emulsifying activity was confirmed. The aqueous Fba1p solution used in this test has a salt concentration higher than the concentration shown in FIG. 5 in consideration of the amount of salt previously contained in the Fba1p purified fraction used for its preparation. However, it is recognized that emulsifying activity was obtained at least at 3M or less, and more preferable emulsifying activity was obtained at 2M or less.
［免疫賦活性試験］
溶出画分２を、溶出用液を用いてＦＢＡ１タンパク質（Fba1p）が所定濃度（１０μｇ／ｍＬ、２５μｇ／ｍＬ及び５０μｇ／ｍＬ）となるように調製したFba1p含有液と；比較用に、免疫賦活性と乳化活性との両方を有するタンパク質として本発明者が別途見出したＧＡＳ１タンパク質（Gas1p、ＵｎｉｐｒｏｔＫＢアクセッション番号Ｐ２２１４６）を、同様の方法で溶出用液を用いて所定濃度（１０μｇ／ｍＬ、２５μｇ／ｍＬ及び５０μｇ／ｍＬ）となるように調製したGas1p含有液、β−グルカンが溶出用液中に所定濃度（５０μｇ／ｍＬ、２５０μｇ／ｍＬ、及び５００μｇ／ｍＬ）となるように均一分散させたβ−グルカン含有液、リン酸緩衝液、及び実施例２で用いた培地と、を試験サンプルとして用い、これら試験サンプルに対し、以下のようにしてマクロファージ活性化能（TNF-α分泌量）の測定を行った。なお、試験サンプル中のFba1p等のタンパク質濃度は、BCA法によって測定した。
[Immunostimulatory test]
The eluted fraction 2 was used as an Fba1p-containing solution prepared by using the eluate so that the FBA1 protein (Fba1p) had a predetermined concentration (10 μg / mL, 25 μg / mL, and 50 μg / mL); GAS1 protein (Gas1p, UniprotKB Accession No. P22146), which was separately found by the present inventors as a protein having both properties and emulsifying activity, was subjected to a predetermined concentration (10 µg / mL, 25 µg / and the β-glucan uniformly dispersed in the eluate to a predetermined concentration (50 μg / mL, 250 μg / mL, and 500 μg / mL). -The glucan-containing solution, the phosphate buffer and the medium used in Example 2 were used as test samples. A manner was measured macrophage activating activity (TNF-alpha secretion). The protein concentration of Fba1p and the like in the test sample was measured by the BCA method.
継代培養したマクロファージ細胞を、遠心分離により回収し、新しいRPMI-1640培地に懸濁し、細胞濃度を1.0 x 106 cells/mLに調整した。細胞懸濁溶液を500μLずつ24穴の細胞培養プレートにまき、24時間、37℃、5% CO2下で培養した。その後、培地を除き細胞に新しいRPMI-1640培地437.5μLと試験サンプル62.5μLとを加えて混合した。6時間インキュベートした後、遠心分離(4℃, 3000 × g, 5分)により上清を回収した。ELISAキット(Quantikine Mouse TNF-α ELISA kit, R＆D Systems)を用いて、マクロファージによって分泌され上清に含まれるTNF-α量を測定した。TNF-αのstandardを用いて検量線を作成し、サンプル中のTNF-α量を算出した。なお、ELISAの操作はキットに記載されている方法に従った。結果を図６に示す。図６において、縦軸は、TNF-αの量（ｐｇ／ｍＬ）を示す。 The subcultured macrophage cells were collected by centrifugation, suspended in fresh RPMI-1640 medium, and the cell concentration was adjusted to 1.0 × 10 6 cells / mL. Each 500 μL of the cell suspension was spread on a 24-well cell culture plate, and cultured at 37 ° C. under 5% CO 2 for 24 hours. Thereafter, the medium was removed, and 437.5 μL of a new RPMI-1640 medium and 62.5 μL of a test sample were added to the cells and mixed. After incubation for 6 hours, the supernatant was collected by centrifugation (4 ° C., 3000 × g, 5 minutes). The amount of TNF-α secreted by macrophages and contained in the supernatant was measured using an ELISA kit (Quantikine Mouse TNF-α ELISA kit, R & D Systems). A calibration curve was prepared using the TNF-α standard, and the amount of TNF-α in the sample was calculated. In addition, the operation of ELISA followed the method described in the kit. FIG. 6 shows the results. In FIG. 6, the vertical axis indicates the amount (pg / mL) of TNF-α.
図６に示すように、比較用のリン酸緩衝液（buffer）に比べ、Fba1p含有液で刺激した場合において、TNF-αの分泌量の明らかな増加が認められた。また、免疫賦活性を有することが知られているβ−グルカン濃度が５０μｇ／ｍＬである場合と、Fba1p濃度が５０μｇ／ｍＬである場合とで比較した場合においても、Fba1pによるTNF-αの分泌量がはるかに高い（免疫賦活性がはるかに高い）ことが確認された。さらに、免疫賦活性と乳化活性との両方を有するGas1pが５０μｇ／ｍＬである場合と、Fba1p濃度が５０μｇ／ｍＬである場合とで比較した場合においても、Fba1pによるTNF-αの分泌量が顕著に高い（免疫賦活性が顕著に高い）ことが確認された。 As shown in FIG. 6, when stimulated with the Fba1p-containing solution, a clear increase in the secretion amount of TNF-α was observed as compared to the phosphate buffer for comparison (buffer). In addition, when the β-glucan concentration known to have immunostimulatory activity is 50 μg / mL and the Fba1p concentration is 50 μg / mL, the secretion of TNF-α by Fba1p is also observed. The amount was found to be much higher (the immunostimulation was much higher). Furthermore, even when Gas1p having both immunostimulatory activity and emulsifying activity is 50 μg / mL and Fba1p concentration is 50 μg / mL, the secretion amount of TNF-α by Fba1p is remarkable. (The immunostimulatory activity was remarkably high).
配列番号２及び３は、プライマーである。 SEQ ID NOs: 2 and 3 are primers.
Claims (7)
（i）配列番号１に示されるアミノ酸配列、
（ii）配列番号１において、１個又は複数個のアミノ酸残基が欠失、付加、挿入若しくは置換されたアミノ酸配列を有し、且つ乳化活性と免疫賦活性とを有するタンパク質、及び
（iii）配列番号１と７０％以上の相同性を有するアミノ酸配列を有し、且つ乳化活性と免疫賦活性とを有するタンパク質
の少なくともいずれかである、請求項１に記載の免疫賦活性乳化剤。 The FBA1 protein is
(I) an amino acid sequence represented by SEQ ID NO: 1,
(Ii) a protein having an amino acid sequence in which one or more amino acid residues are deleted, added, inserted or substituted in SEQ ID NO: 1, and having an emulsifying activity and an immunostimulatory activity, and (iii) The immunostimulatory emulsifier according to claim 1, which has an amino acid sequence having 70% or more homology with SEQ ID NO: 1 and is at least one of a protein having emulsifying activity and immunostimulating activity.
（ii'）配列番号１に示されるアミノ酸配列において、１個又は複数個のアミノ酸残基が欠失、付加、挿入若しくは置換されたアミノ酸配列とアフィニティタグとを有し、且つ乳化活性と免疫賦活性とを有するタンパク質、及び
（iii'）配列番号１に示されるアミノ酸配列と７０％以上の相同性を有するアミノ酸配列とアフィニティタグとを有し、且つ乳化活性と免疫賦活性とを有するタンパク質
の少なくともいずれかのタンパク質をコードするポリヌクレオチドを含む発現ベクターによって形質転換された細胞を作成する工程、
前記形質転換された細胞の培養を行う工程、
培養された細胞の抽出液を、前記アフィニティタグに特異的な担体に接触させ、発現タンパク質を捕捉する工程、及び
前記捕捉された発現タンパク質を回収する工程、
を含む、免疫賦活性乳化剤の製造方法。 (I ′) a protein having the amino acid sequence represented by SEQ ID NO: 1 and an affinity tag,
(Ii ′) the amino acid sequence represented by SEQ ID NO: 1, which has an amino acid sequence in which one or more amino acid residues have been deleted, added, inserted, or substituted, and an affinity tag, and has emulsifying activity and immunostimulation. And (iii ′) a protein having an amino acid sequence having at least 70% homology with the amino acid sequence shown in SEQ ID NO: 1 and an affinity tag, and having an emulsifying activity and an immunostimulatory activity. Producing a cell transformed by an expression vector comprising a polynucleotide encoding at least one of the proteins,
Culturing the transformed cells,
Contacting the extract of the cultured cells with a carrier specific to the affinity tag, and capturing the expressed protein; and recovering the captured expressed protein,
A method for producing an immunostimulatory emulsifier, comprising:
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