JP2021013375A - Sugar chain-binding polypeptide, polynucleotide encoding the same and pharmaceutical composition containing the same - Google Patents
Sugar chain-binding polypeptide, polynucleotide encoding the same and pharmaceutical composition containing the same Download PDFInfo
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- JP2021013375A JP2021013375A JP2020105638A JP2020105638A JP2021013375A JP 2021013375 A JP2021013375 A JP 2021013375A JP 2020105638 A JP2020105638 A JP 2020105638A JP 2020105638 A JP2020105638 A JP 2020105638A JP 2021013375 A JP2021013375 A JP 2021013375A
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- kaa1
- sugar chain
- virus
- polypeptide
- amino acid
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Abstract
Description
本発明は、糖鎖結合性ポリペプチド、それをコードするポリヌクレオチド及びそれを含む医薬組成物に関し、特に天然のレクチンよりも糖鎖結合性が増大するように改変された糖鎖結合性ポリペプチド、それをコードするポリヌクレオチド及びそれを含む医薬組成物に関する。 The present invention relates to a sugar chain-binding polypeptide, a polynucleotide encoding the same, and a pharmaceutical composition containing the same, and in particular, a sugar chain-binding polypeptide modified so as to have higher sugar chain binding than a natural lectin. , The polynucleotide encoding it and the pharmaceutical composition containing it.
細胞表面や体液中に存在する糖タンパク質や糖脂質等の複合糖質の糖鎖は、一種の情報素子として機能し、発生、免疫、がん、感染等の重要な生命現象に深く関わっている。一方、糖鎖結合性タンパク質であるレクチンは糖鎖認識分子として機能し、糖鎖と同様に生物学的に重要な役割を担っている。 The sugar chains of complex sugars such as glycoproteins and glycolipids present on the cell surface and body fluids function as a kind of information element and are deeply involved in important biological phenomena such as development, immunity, cancer, and infection. .. On the other hand, lectin, which is a sugar chain-binding protein, functions as a sugar chain recognition molecule and plays a biologically important role like sugar chains.
これまでに、海藻類又は藻類（淡水産藍藻）から多くの種類のレクチンが単離され、その生化学的性質が明らかにされている。藻類レクチンは、その結合性に基づいて高マンノース型糖鎖特異的レクチン、複合型糖鎖特異的レクチン、両糖鎖に結合するレクチンの３グループに分類される。レクチンの一部は、例えばヒト免疫不全ウイルス（ＨＩＶ）、インフルエンザウイルス等のウイルスに特異的に結合することが知られている（非特許文献１〜１２）。藍藻Oscillatoria agardhii 由来レクチンであるＯＡＡ（Oscillatoria agardhii agglutinin）、紅藻Kappaphycus alvarezii由来レクチンであるＫＡＡ（Kappaphycus alvarezii agglutinin）、緑藻Boodlea coacta由来レクチンであるＢＣＡ（Boodlea coactaagglutinin）及び紅藻Meristotheca papulosa由来レクチンであるＭＰＬ（Meristotheca papulosa lectin）は高マンノース型糖鎖との強い結合特異性からＨＩＶやインフルエンザウイルスといった表面に高マンノース型糖鎖を有するウイルスを認識できると期待されている。特に、非特許文献１２には、ＫＡＡはＨＩＶのエンベロープ糖タンパク質であるｇｐ１２０を認識することが開示されており、抗ＨＩＶ活性を示し、医薬素材としての活用が期待されている。 So far, many types of lectins have been isolated from seaweeds or algae (freshwater cyanobacteria), and their biochemical properties have been clarified. Algal lectins are classified into three groups based on their binding properties: high mannose-type sugar chain-specific lectins, complex-type sugar chain-specific lectins, and lectins that bind to both sugar chains. It is known that a part of lectins specifically binds to viruses such as human immunodeficiency virus (HIV) and influenza virus (Non-Patent Documents 1 to 12). OAA (Oscillatoria agardhii agglutinin), a lectin derived from the blue alga Oscillatoria agardhii, KAA (Kappaphycus alvarezii agglutinin), a lectin derived from the red alga Kappaphycus alvarezii, and BCA (Boodlea coactaaglutin), a lectin derived from the green alga Boodlea coacta. MPL (Meristotheca papulosa lectin) is expected to be able to recognize viruses having high mannose type sugar chains on the surface such as HIV and influenza virus because of its strong binding specificity with high mannose type sugar chains. In particular, Non-Patent Document 12 discloses that KAA recognizes gp120, which is an envelope glycoprotein of HIV, exhibits anti-HIV activity, and is expected to be used as a pharmaceutical material.
しかしながら、上記のようなレクチンを実際に抗ウイルス薬剤等のための医薬素材として活用するためには、エンベロープ等のウイルス表面に存在する高マンノース型糖鎖とのより強い結合が求められる。このために、レクチンの糖鎖結合性を増強するようにレクチンを改変することが考えられており、そのような改変によって糖鎖結合性を増強できた報告はあるものの、未だ更に強い活性が求められている。 However, in order to actually utilize the above-mentioned lectin as a pharmaceutical material for antiviral drugs and the like, a stronger bond with a high mannose type sugar chain existing on the virus surface such as an envelope is required. For this reason, it is considered to modify the lectin so as to enhance the sugar chain binding property of the lectin, and although there are reports that such modification could enhance the sugar chain binding property, stronger activity is still required. Has been done.
本発明は、前記問題に鑑みてなされたものであり、その目的は、上記のようなウイルスが原因の疾患の治療や診断のための薬剤として利用できるように、高マンノース型糖鎖に対して顕著に高い結合性を示す改変されたレクチン変異体（ポリペプチド）を提供できるようにすることにある。 The present invention has been made in view of the above problems, and an object of the present invention is to treat a high mannose type sugar chain so that it can be used as a drug for treating or diagnosing a disease caused by a virus as described above. The purpose is to be able to provide a modified lectin variant (polypeptide) that exhibits significantly higher binding.
前記の目的を達成するために、本発明者らは、鋭意研究の結果、紅藻Kappaphycus alvarezii由来レクチンＫＡＡ１の糖鎖結合部位を高度多価化することによって、糖鎖結合性が顕著に増強することを見出して、本発明を完成した。 In order to achieve the above object, as a result of diligent research, the present inventors have significantly enhanced the sugar chain binding property by highly polyvalentizing the sugar chain binding site of the red alga Kappaphycus alvarezii-derived lectin KAA1. We found that and completed the present invention.
具体的に、本発明に係るポリペプチドは、紅藻Kappaphycus alvarezii由来レクチンＫＡＡ１（６７アミノ酸からなるドメインの４リピート配列をもつ）のタンデムリピートを有することを特徴とする。 Specifically, the polypeptide according to the present invention is characterized by having a tandem repeat of the red alga Kappaphycus alvarezii-derived lectin KAA1 (having a 4-repeat sequence of a domain consisting of 67 amino acids).
本発明に係るポリペプチドは、タンデムリピート間にリンカー配列を有してもよい。 The polypeptide according to the present invention may have a linker sequence between tandem repeats.
本発明に係るポリペプチドは、配列番号１のアミノ酸配列、または該配列番号１のアミノ酸配列において１個若しくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を少なくとも２つ含んでいてもよい。 The polypeptide according to the present invention contains at least two amino acid sequences of SEQ ID NO: 1 or amino acid sequences of SEQ ID NO: 1 in which one or several amino acids are substituted, deleted, inserted or added. You may.
本発明に係るポリペプチドは、配列番号２のアミノ酸配列、または該配列番号２つのアミノ酸配列において１個若しくは数個のアミノ酸が置換、欠失、挿入若しくは付加されたアミノ酸配列を含んでいてもよい。 The polypeptide according to the present invention may contain the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added in the amino acid sequence of SEQ ID NO: 2. ..
本発明に係るポリヌクレオチドは、上記本発明に係るポリペプチドをコードすることを特徴とする。 The polynucleotide according to the present invention is characterized by encoding the above-mentioned polypeptide according to the present invention.
本発明に係るウイルス疾患の治療用又は診断用医薬組成物は、上記本発明に係るポリペプチドを含むことを特徴とし、当該ウイルス疾患の原因ウイルスは高マンノース型糖鎖を表面に有するウイルスである。本発明に係る医薬組成物は、高マンノース型糖鎖と結合するポリペプチドの糖鎖結合性が顕著に増強された上記本発明に係るポリペプチドを含むため、高マンノース型糖鎖を表面に有するウイルスを原因とする疾患の治療や診断への利用が可能である。当該高マンノース型糖鎖を表面に有するウイルスとしては、ヒト免疫不全ウイルス（ＨＩＶ）、インフルエンザウイルス、Ｃ型肝炎ウイルス（ＨＣＶ）、ＳＡＲＳコロナウイルス（ＳＡＲＳ−ＣｏＶ）又は単純ヘルペスウイルスなどが挙げられる。 The pharmaceutical composition for treating or diagnosing a viral disease according to the present invention is characterized by containing the above-mentioned polypeptide according to the present invention, and the causative virus of the viral disease is a virus having a high mannose-type sugar chain on its surface. .. Since the pharmaceutical composition according to the present invention contains the polypeptide according to the present invention in which the sugar chain binding property of the polypeptide that binds to the high mannose type sugar chain is remarkably enhanced, the pharmaceutical composition has a high mannose type sugar chain on the surface. It can be used for the treatment and diagnosis of diseases caused by viruses. Examples of the virus having a high mannose type sugar chain on the surface include human immunodeficiency virus (HIV), influenza virus, hepatitis C virus (HCV), SARS coronavirus (SARS-CoV), and simple herpesvirus.
本発明に係るポリペプチドは、糖鎖結合性が顕著に増強され、高マンノース型糖鎖と強く結合できるため、高マンノース型糖鎖を表面に有するウイルスを原因とする疾患の治療や診断へ利用できる可能性があり、極めて有用である。 Since the polypeptide according to the present invention has a markedly enhanced sugar chain binding property and can strongly bind to a high mannose type sugar chain, it can be used for treatment and diagnosis of a disease caused by a virus having a high mannose type sugar chain on its surface. It has the potential to be possible and is extremely useful.
以下、本発明を実施するための形態を図面に基づいて説明する。以下の好ましい実施形態の説明は、本質的に例示に過ぎず、本発明、その適用方法或いはその用途を制限することを意図するものではない。 Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. The following description of preferred embodiments is merely exemplary and is not intended to limit the invention, its application methods or its uses.
本発明の一実施形態は、紅藻Kappaphycus alvarezii由来レクチンであるＫＡＡ１における４リピート配列の直列反復（タンデムリピート）を有するポリペプチドである。ＫＡＡ１は、藍藻Oscillatoria agardhii由来レクチンＯＡＡと６０．６％の相同性を示し、同じＯＡＡファミリーに属する。ＯＡＡは、システインを含まない１３２残基のアミノ酸からなり、６７アミノ酸の２リピート配列を有し、この重複配列間には約７５％の相同性が認められている。また三次構造においては、上記重複配列間が一部入れ換わったドメインスワップ構造をとり、これにより形づくられた２つのドメインがそれぞれ１つの糖結合部位を持つ。ＫＡＡ１は約６７アミノ酸の４リピート配列を有し、ＯＡＡが２つ連なったような構造からなる。ＯＡＡ及びＫＡＡ１は、ともに極めてよく似た性状、機能を示す。すなわち、単糖結合性を示さず、認識する最少糖構造はＭａｎ（α１−３）Ｍａｎ（α１−６）Ｍａｎ（β１−４）であり、Ｎ型糖鎖のうち、Ｄ２アームの（α１−３）Ｍａｎ残基が露出した高マンノース型糖鎖に特異的に結合する。１分子あたり、ＯＡＡは２つ、ＫＡＡ１は４つの糖結合部位を持ち、細胞を架橋・凝集する。従って、それらは、エンベロープ等のウイルス表面に高マンノース型糖鎖が存在する種々のウイルスを認識できると考えられる。実際に、上記非特許文献１２には、ＫＡＡがＨＩＶのエンベロープ糖タンパク質ｇｐ１２０が有する高マンノース型糖鎖との強い結合を示し、抗ＨＩＶ活性を示すことが開示されている。ＨＩＶ以外にウイルス表面に高マンノース型糖鎖が存在するウイルスとしては、インフルエンザウイルス、ＨＣＶ、ＳＡＲＳ−ＣｏＶ、単純ヘルペスウイルス等が知られている。 One embodiment of the present invention is a polypeptide having tandem repeats of four repeat sequences in KAA1, a lectin derived from the red alga Kappaphycus alvarezii. KAA1 shows 60.6% homology with the cyanobacteria Oscillatoria agardhii-derived lectin OAA and belongs to the same OAA family. OAA consists of 132 residues of amino acids containing no cysteine, has a 2-repeat sequence of 67 amino acids, and about 75% homology is observed between the overlapping sequences. Further, in the tertiary structure, a domain swap structure in which the overlapping sequences are partially exchanged is adopted, and the two domains formed by this have one sugar binding site each. KAA1 has a 4-repeat sequence of about 67 amino acids and has a structure in which two OAAs are connected. Both OAA and KAA1 show very similar properties and functions. That is, the smallest sugar structure that does not show monosaccharide binding and is recognized is Man (α1-3) Man (α1-6) Man (β1-4), and among the N-type sugar chains, the D2 arm (α1-). 3) Mannose residues specifically bind to exposed high mannose-type sugar chains. Each molecule has two OAAs and four KAA1 sugar-binding sites, which cross-link and aggregate cells. Therefore, it is considered that they can recognize various viruses in which high mannose type sugar chains are present on the surface of the virus such as the envelope. In fact, Non-Patent Document 12 discloses that KAA exhibits strong binding to a high mannose-type sugar chain possessed by the envelope glycoprotein gp120 of HIV and exhibits anti-HIV activity. In addition to HIV, influenza virus, HCV, SARS-CoV, herpes simplex virus and the like are known as viruses having a high mannose type sugar chain on the surface of the virus.
本実施形態に係るポリペプチドは、ＫＡＡ１の４つの糖結合部位に該当する４リピート配列をタンデムリピートさせた改変がなされており、これにより、糖鎖結合部位が多価化されている。このため、本発明に係るポリペプチドの糖鎖結合性は、顕著に増大されている。上記４リピート配列を図１に示す。図１では、４リピート配列において互いに同一のアミノ酸を有するアミノ酸位置を黒色マーカーで示し、２種のアミノ酸が存在するアミノ酸位置を灰色マーカーで示している。図１に示すように、当該４リピート配列は、６５〜６８アミノ酸のリピート配列であり、互いに完全に同一の配列ではないものの、高い配列同一性を示すものである。本実施形態に係るポリペプチドは、当該４リピート配列の繰り返し配列を有するものであり、配列の反復数は特に限定されないが、２回の反復で十分に糖鎖結合性の顕著な増強が得られる。また、本実施形態に係るポリペプチドにおいて、糖鎖結合性が低減しない限り、図１に示す４リピート配列において１又は数個のアミノ酸の変異を含んでも構わない。 The polypeptide according to the present embodiment has been modified by tandem repeating the 4-repeat sequence corresponding to the four sugar-binding sites of KAA1, whereby the sugar chain-binding site is multivalued. Therefore, the sugar chain binding property of the polypeptide according to the present invention is remarkably increased. The above 4-repeat sequence is shown in FIG. In FIG. 1, amino acid positions having the same amino acids as each other in the 4-repeat sequence are indicated by black markers, and amino acid positions in which two types of amino acids are present are indicated by gray markers. As shown in FIG. 1, the 4-repeat sequence is a repeat sequence of 65 to 68 amino acids, and although they are not completely identical sequences to each other, they show high sequence identity. The polypeptide according to the present embodiment has a repeating sequence of the 4-repeat sequence, and the number of repeating sequences is not particularly limited, but two repetitions can sufficiently obtain a remarkable enhancement of sugar chain binding property. .. In addition, the polypeptide according to this embodiment may contain mutations of one or several amino acids in the four-repeat sequence shown in FIG. 1 as long as the sugar chain binding property is not reduced.
本実施形態において、反復される配列同士の間には所定のリンカー配列が挿入されていても構わない。用いられるリンカー配列は、ポリペプチドの糖鎖結合性に負の影響を及ぼさない配列であれば特に限定されず、周知の種々のリンカー配列を用いることができる。 In the present embodiment, a predetermined linker sequence may be inserted between the repeating sequences. The linker sequence used is not particularly limited as long as it does not adversely affect the sugar chain binding property of the polypeptide, and various well-known linker sequences can be used.
本実施形態に係るポリペプチドは、原核生物宿主又は真核生物宿主（例えば、細菌細胞、酵母細胞、高等植物細胞、昆虫細胞及び哺乳動物細胞を含む）から組換え技術によって産生された産物、及び化学合成手順の産物を含む。組換え産生手順において用いられる宿主に依存して、本発明に係るタンパク質は、グリコシル化され得るか又は非グリコシル化され得る。さらに、本発明に係るタンパク質は、宿主媒介プロセスの結果として、開始の改変メチオニン残基を含み得る。 The polypeptide according to this embodiment is a product produced by a recombinant technique from a prokaryotic host or a eukaryotic host (including, for example, bacterial cells, yeast cells, higher plant cells, insect cells and mammalian cells), and Includes products of chemical synthesis procedures. Depending on the host used in the recombinant production procedure, the proteins according to the invention can be glycosylated or non-glycosylated. In addition, the proteins according to the invention may contain an initiating modified methionine residue as a result of a host-mediated process.
一実施形態において、本発明に係るポリペプチドは、例えばＫＡＡ１ポリペプチドのアミノ酸配列である配列番号１のアミノ酸配列を少なくとも２つ含むことが好ましく、又は配列番号１のアミノ酸配列において１又は数個のアミノ酸の変異を含むアミノ酸配列を少なくとも２つ含んでいてもよい。さらに、本実施形態に係るポリペプチドは、配列番号２のアミノ酸配列を含むものでもよく、配列番号２のアミノ酸配列において１又は数個のアミノ酸の変異を含むアミノ酸配列を含んでいてもよい。 In one embodiment, the polypeptide according to the invention preferably comprises, for example, at least two amino acid sequences of SEQ ID NO: 1, which is the amino acid sequence of the KAA1 polypeptide, or one or several in the amino acid sequence of SEQ ID NO: 1. It may contain at least two amino acid sequences containing amino acid variants. Furthermore, the polypeptide according to the present embodiment may contain the amino acid sequence of SEQ ID NO: 2, and may contain an amino acid sequence containing mutations of one or several amino acids in the amino acid sequence of SEQ ID NO: 2.
変異体としては、欠失、挿入、逆転、反復、及びタイプ置換（例えば、親水性の残基の別の残基への置換、しかし通常は強く親水性の残基を強く疎水性の残基には置換しない）を含む変異体が挙げられる。特に、ポリペプチドにおける「中性」アミノ酸置換は、一般的にそのポリペプチドの活性にほとんど影響しない。 Variants include deletions, insertions, reversals, repetitions, and type substitutions (eg, substitution of a hydrophilic residue with another, but usually a strongly hydrophilic residue and a strongly hydrophobic residue. Does not replace). In particular, "neutral" amino acid substitutions in a polypeptide generally have little effect on the activity of that polypeptide.
ポリペプチドのアミノ酸配列中のいくつかのアミノ酸が、このポリペプチドの構造又は機能に有意に影響することなく容易に改変され得ることは、当該分野において周知である。さらに、人為的に改変させるだけではく、天然のタンパク質において、当該タンパク質の構造又は機能を有意に変化させない変異体が存在することもまた周知である。 It is well known in the art that some amino acids in the amino acid sequence of a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide. Furthermore, it is also well known that there are variants of intrinsically disordered proteins that are not only artificially modified but do not significantly alter the structure or function of the protein.
当業者は、周知技術を使用してポリペプチドのアミノ酸配列において１又は数個のアミノ酸を容易に変異させることができる。例えば、公知の点変異導入法に従えば、ポリペプチドをコードするポリヌクレオチドの任意の塩基を変異させることができる。また、ポリペプチドをコードするポリヌクレオチドの任意の部位に対応するプライマーを設計して欠失変異体又は付加変異体を作製することができる。さらに、本明細書中に記載される方法を用いれば、作製した変異体が、糖鎖結合性を有する所望の変異体であるか否かを容易に決定し得る。 One of ordinary skill in the art can easily mutate one or several amino acids in the amino acid sequence of a polypeptide using well-known techniques. For example, according to a known point mutation introduction method, any base of the polynucleotide encoding the polypeptide can be mutated. In addition, a primer corresponding to an arbitrary site of the polynucleotide encoding the polypeptide can be designed to prepare a deletion mutant or an addition mutant. Furthermore, by using the methods described herein, it can be easily determined whether or not the produced mutant is a desired mutant having sugar chain binding property.
上記「１又は数個のアミノ酸」の変異とは、部位特異的突然変異誘発法等の公知の変異ポリペプチド作製法により置換、欠失、挿入、若しくは付加できる程度の数（好ましくは１から１０個、より好ましくは１から７個、さらに好ましくは１個から５個、特に好ましくは１個から３個）のアミノ酸が置換、欠失、挿入若しくは付加されていることを意味する。 The above-mentioned mutation of "1 or several amino acids" is a number (preferably 1 to 10) that can be replaced, deleted, inserted, or added by a known mutagenesis method such as a site-specific mutagenesis method. This means that 1, 7, more preferably 1 to 7, more preferably 1 to 5, and particularly preferably 1 to 3) amino acids are substituted, deleted, inserted or added.
他の実施形態において、本発明に係るポリペプチドは、融合タンパク質のような改変された形態で組換え発現され得る。例えば、本発明に係るポリペプチドの付加的なアミノ酸、特に荷電性アミノ酸の領域が、宿主細胞内での、精製の間又は引き続く操作及び保存の間の安定性及び持続性を改善するために、ポリペプチドのＮ末端に付加され得る。 In other embodiments, the polypeptides of the invention can be recombinantly expressed in modified forms such as fusion proteins. For example, regions of additional amino acids, especially charged amino acids, of the polypeptides according to the invention may improve stability and persistence in the host cell during purification or subsequent manipulation and storage. It can be added to the N-terminus of the polypeptide.
組換え生成は、当該分野において周知の方法を使用して行なうことができ、例えば、以下に詳述されるようなベクター及び細胞等を用いて行なうことができる。 Recombinant production can be carried out using methods well known in the art, for example using vectors and cells as detailed below.
本発明は、上述したように、本発明に係るポリペプチドをコードするポリヌクレオチドを提供する。本明細書中で使用される場合、用語「ポリヌクレオチド」は「核酸」又は「核酸分子」と交換可能に使用され、ヌクレオチドの重合体が意図される。本明細書中で使用される場合、用語「塩基配列」は、「核酸配列」又は「ヌクレオチド配列」と交換可能に使用され、デオキシリボヌクレオチド（Ａ、Ｇ、Ｃ及びＴと省略される）の配列として示される。 The present invention provides a polynucleotide encoding a polypeptide according to the present invention, as described above. As used herein, the term "polynucleotide" is used interchangeably with "nucleic acid" or "nucleic acid molecule" and is intended to be a polymer of nucleotides. As used herein, the term "base sequence" is used interchangeably with "nucleic acid sequence" or "nucleotide sequence" and is a sequence of deoxyribonucleotides (abbreviated as A, G, C and T). Shown as.
本発明に係るポリヌクレオチドは、ＲＮＡ（例えば、ｍＲＮＡ）の形態、又はＤＮＡの形態（例えば、ｃＤＮＡ又はゲノムＤＮＡ）で存在し得る。ＤＮＡは、二本鎖又は一本鎖であり得る。一本鎖ＤＮＡ又はＲＮＡは、コード鎖（センス鎖としても知られる）であり得、又は、非コード鎖（アンチセンス鎖としても知られる）であり得る。 The polynucleotide according to the invention can be present in the form of RNA (eg, mRNA) or DNA (eg, cDNA or genomic DNA). The DNA can be double-stranded or single-stranded. The single-stranded DNA or RNA can be a coding strand (also known as the sense strand) or a non-coding strand (also known as the antisense strand).
本明細書中で使用される場合、用語「オリゴヌクレオチド」は、ヌクレオチドが数個ないし数十個結合したものが意図され、「ポリヌクレオチド」と交換可能に使用される。オリゴヌクレオチドは、短いものはジヌクレオチド（二量体）、トリヌクレオチド（三量体）といわれ、長いものは３０マー又は１００マーというように重合しているヌクレオチドの数で表される。オリゴヌクレオチドは、より長いポリヌクレオチドのフラグメントとして生成されても、化学合成されてもよい。 As used herein, the term "oligonucleotide" is intended to be a combination of several to dozens of nucleotides and is used interchangeably with "polynucleotide". The short ones are called dinucleotides (dimers) and trinucleotides (trimers), and the long ones are represented by the number of polymerized nucleotides such as 30 mer or 100 mer. Oligonucleotides may be produced as fragments of longer polynucleotides or chemically synthesized.
また、本発明に係るポリヌクレオチドは、その５’側又は３’側で上述のタグ標識（タグ配列又はマーカー配列）をコードするポリヌクレオチドに融合され得る。 In addition, the polynucleotide according to the present invention can be fused to a polynucleotide encoding the above-mentioned tag label (tag sequence or marker sequence) on the 5'side or 3'side thereof.
本発明はさらに、本発明に係るポリペプチドをコードするポリヌクレオチドの変異体に関する。変異体は、天然の対立遺伝子変異体のように、天然に生じ得る。「対立遺伝子変異体」によって、生物の染色体上の所定の遺伝子座を占める遺伝子のいくつかの交換可能な形態の１つが意図される。天然に存在しない変異体は、例えば当該分野で周知の変異誘発技術を用いて生成され得る。 The present invention further relates to variants of the polynucleotide encoding the polypeptide according to the invention. Mutants can occur naturally, like natural allelic variants. By "allelic variants", one of several exchangeable forms of a gene that occupies a given locus on an organism's chromosome is intended. Non-naturally occurring variants can be produced, for example, using mutagenesis techniques well known in the art.
このような変異体としては、本発明に係るポリペプチドをコードするポリヌクレオチドの塩基配列において１又は数個の塩基が欠失、置換、又は付加した変異体が挙げられる。変異体は、コード若しくは非コード領域、又はその両方において変異され得る。コード領域における変異は、保存的若しくは非保存的なアミノ酸欠失、置換、又は付加を生成し得る。 Examples of such a mutant include a mutant in which one or several bases are deleted, substituted, or added in the base sequence of the polynucleotide encoding the polypeptide according to the present invention. Mutants can be mutated in the coding and / or non-coding regions. Mutations in the coding region can result in conservative or non-conservative amino acid deletions, substitutions, or additions.
本発明はさらに、ストリンジェントなハイブリダイゼーション条件下で、本発明に係るポリペプチドをコードするポリヌクレオチド又は当該ポリヌクレオチドにハイブリダイズするポリヌクレオチドを含む、単離したポリヌクレオチドを提供する。 The present invention further provides an isolated polynucleotide comprising a polynucleotide encoding a polynucleotide according to the invention or a polynucleotide hybridizing to the polynucleotide under stringent hybridization conditions.
なお、上記「ストリンジェントな条件」とは、少なくとも９０％以上の同一性、好ましくは少なくとも９５％以上の同一性、最も好ましくは９７％以上の同一性が配列間に存在する時にのみハイブリダイゼーションが起こることを意味する。 The above-mentioned "stringent condition" means that hybridization occurs only when at least 90% or more identity, preferably at least 95% or more identity, and most preferably 97% or more identity exists between sequences. Means to happen.
上記ハイブリダイゼーションは、Sambrookら、Molecular Cloning,A Laboratory Manual,2d Ed.,Cold Spring Harbor Laboratory（1989）に記載されている方法のような周知の方法で行なうことができる。通常、温度が高いほど、塩濃度が低いほどストリンジェンシーは高くなり（ハイブリダイズし難くなる）、より相同なポリヌクレオチドを取得することができる。 The hybridization can be performed by a well-known method such as that described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory (1989). Generally, the higher the temperature and the lower the salt concentration, the higher the stringency (difficulty in hybridizing), and more homologous polynucleotides can be obtained.
ハイブリダイゼーションの条件としては、従来公知の条件を好適に用いることができ、特に限定しないが、例えば、４２℃、６×ＳＳＰＥ、５０％ホルムアミド、１％ＳＤＳ、１００μｇ／ｍｌ サケ精子ＤＮＡ、５×デンハルト液（ただし、１×ＳＳＰＥ；０．１８Ｍ 塩化ナトリウム、１０ｍＭリン酸ナトリウム、ｐＨ７．７、１ｍＭ ＥＤＴＡ。５×デンハルト液；０．１％牛血清アルブミン、０．１％フィコール、０．１％ポリビニルピロリドン）が挙げられる。 Conventionally known conditions can be preferably used as the hybridization conditions, and the conditions are not particularly limited. For example, 42 ° C., 6 × SSPE, 50% formamide, 1% SDS, 100 μg / ml salmon sperm DNA, 5 × Denhardt's solution (however, 1 x SSPE; 0.18 M sodium chloride, 10 mM sodium phosphate, pH 7.7, 1 mM EDTA. 5 x Denhardt's solution; 0.1% bovine serum albumin, 0.1% ficol, 0.1% Polyvinylpyrrolidone) can be mentioned.
本発明に係るポリヌクレオチド又はオリゴヌクレオチドは、２本鎖ＤＮＡのみならず、それを構成するセンス鎖及びアンチセンス鎖といった各１本鎖ＤＮＡやＲＮＡを包含する。またＤＮＡには例えばクローニングや化学合成技術又はそれらの組み合わせで得られるようなｃＤＮＡやゲノムＤＮＡなどが含まれる。さらに、本発明に係るポリヌクレオチド又はオリゴヌクレオチドは、非翻訳領域（ＵＴＲ）の配列やベクター配列（発現ベクター配列を含む）などの配列を含むものであってもよい。 The polynucleotide or oligonucleotide according to the present invention includes not only double-stranded DNA but also single-stranded DNA and RNA such as the sense strand and antisense strand constituting the double-stranded DNA. In addition, DNA includes, for example, cDNA and genomic DNA obtained by cloning, chemical synthesis technology, or a combination thereof. Furthermore, the polynucleotide or oligonucleotide according to the present invention may include a sequence such as a sequence of an untranslated region (UTR) or a vector sequence (including an expression vector sequence).
本発明に係るポリヌクレオチド又はオリゴヌクレオチドを取得する方法として、公知の技術により、本発明に係るポリヌクレオチド又はオリゴヌクレオチドを含むＤＮＡ断片を単離し、クローニングする方法が挙げられる。例えば、本発明におけるポリヌクレオチドの塩基配列の一部と特異的にハイブリダイズするプローブを調製し、ゲノムＤＮＡライブラリーやｃＤＮＡライブラリーをスクリーニングすればよい。このようなプローブとしては、本発明に係るポリヌクレオチドの塩基配列又はその相補配列の少なくとも一部に特異的にハイブリダイズするプローブであれば、いずれの配列及び／又は長さのものを用いてもよい。 Examples of the method for obtaining the polynucleotide or oligonucleotide according to the present invention include a method for isolating and cloning a DNA fragment containing the polynucleotide or oligonucleotide according to the present invention by a known technique. For example, a probe that specifically hybridizes with a part of the nucleotide sequence of the polynucleotide in the present invention may be prepared, and a genomic DNA library or a cDNA library may be screened. As such a probe, any sequence and / or length can be used as long as it is a probe that specifically hybridizes to at least a part of the nucleotide sequence of the polynucleotide according to the present invention or its complementary sequence. Good.
あるいは、本発明に係るポリヌクレオチドを取得する方法として、ＰＣＲ等の増幅手段を用いる方法を挙げることができる。例えば、本発明におけるポリヌクレオチドのｃＤＮＡのうち、５’側及び３’側の配列（又はその相補配列）の中からそれぞれプライマーを調製し、これらプライマーを用いてゲノムＤＮＡ（又はｃＤＮＡ）等を鋳型にしてＰＣＲ等を行い、両プライマー間に挟まれるＤＮＡ領域を増幅することで、本発明に係るポリヌクレオチドを含むＤＮＡ断片を大量に取得できる。 Alternatively, as a method for obtaining the polynucleotide according to the present invention, a method using an amplification means such as PCR can be mentioned. For example, among the cDNAs of the polynucleotide in the present invention, primers are prepared from the 5'side and 3'side sequences (or complementary sequences thereof), and genomic DNA (or cDNA) or the like is used as a template using these primers. By performing PCR or the like to amplify the DNA region sandwiched between the two primers, a large amount of DNA fragment containing the polynucleotide according to the present invention can be obtained.
また、本発明は上記ポリペプチドを含むことを特徴とする高マンノース型糖鎖を表面に有するウイルスを原因とする疾患の治療用又は診断用医薬組成物を提供する。 The present invention also provides a pharmaceutical composition for treating or diagnosing a disease caused by a virus having a high mannose-type sugar chain on the surface, which is characterized by containing the above-mentioned polypeptide.
本発明に係る医薬組成物は経口製剤、非経口製剤のいずれであってもよい。また、剤型は特に限定されるものではなく、常法に従い、錠剤、顆粒剤、散剤、カプセル剤、エリキシル剤、シロップ剤、マイクロカプセル剤あるいは懸濁液剤等に製剤化して用いることができる。 The pharmaceutical composition according to the present invention may be either an oral preparation or a parenteral preparation. The dosage form is not particularly limited, and can be formulated into tablets, granules, powders, capsules, elixirs, syrups, microcapsules, suspensions and the like according to a conventional method.
非経口的に投与する場合には、例えば、本発明に係るポリペプチドを含有する溶液を点鼻噴霧することや、注射剤として投与することができる。経口的に投与する場合には、食前、食後、食間のいずれに投与してもよい。 When administered parenterally, for example, a solution containing the polypeptide according to the present invention can be nasally sprayed or administered as an injection. When administered orally, it may be administered before, after, or between meals.
本発明に係る医薬組成物は、必要に応じて、担体、賦形剤、結合剤、膨化剤、潤滑剤、甘味剤、香味剤、防腐剤、安定剤、被覆剤等の材料を含有することができる。 The pharmaceutical composition according to the present invention contains materials such as a carrier, an excipient, a binder, a swelling agent, a lubricant, a sweetener, a flavoring agent, a preservative, a stabilizer, and a coating agent, if necessary. Can be done.
本発明に係る医薬組成物において、例えば錠剤、カプセル剤等に含有することができる具体的な成分としては、トラガント、アラビアゴム、コーンスターチ及びゼラチンのような結合剤；微晶性セルロース、結晶セルロースのような賦形剤； コーンスターチ、前ゼラチン化デンプン、アルギン酸、デキストリンのような膨化剤； ステアリン酸マグネシウムのような潤滑剤；微粒二酸化ケイ素のような流動性改善剤； グリセリン脂肪酸エステルのような滑沢剤； ショ糖、乳糖及びアスパルテームのような甘味剤； ペパーミント、ワニラ香料及びチェリーのような香味剤等を挙げることができる。 In the pharmaceutical composition according to the present invention, specific components that can be contained in, for example, tablets, capsules and the like include binders such as tragant, arabic rubber, corn starch and gelatin; microcrystalline cellulose and crystalline cellulose. Excipients such as; swelling agents such as corn starch, pregelatinized starch, alginic acid, dextrin; lubricants such as magnesium stearate; fluidity improvers such as fine silicon dioxide; glides such as glycerin fatty acid esters Agents; sweeteners such as sucrose, lactose and aspartame; flavors such as peppermint, crocodile and cherry.
調剤単位形態がカプセル剤である場合には上記のタイプの材料にさらに油脂のような液
状担体を含有することができる。
When the dispensing unit form is a capsule, the above-mentioned type of material can further contain a liquid carrier such as fats and oils.
また、種々の他の材料を、被覆剤として又は調剤単位の物理的形態を変化させるために含有させることができる。錠剤の被覆剤としては、例えば、シェラック、砂糖又はその両方が挙げられる。シロップ剤又はエリキシル剤は、例えば、甘味剤としてショ糖、防腐剤としてメチルパラベン及びプロピルパラベン、色素及びチェリー又はオレンジ香味等を含有することができる。その他、各種ビタミン類、各種アミノ酸類を含有しても良い。 Also, various other materials can be included as a coating or to change the physical form of the dispensing unit. Tablet coatings include, for example, shellac, sugar, or both. The syrup or elixir can contain, for example, sucrose as a sweetener, methylparaben and propylparaben as preservatives, pigments and cherry or orange flavors and the like. In addition, various vitamins and various amino acids may be contained.
本発明に係る医薬組成物の投与量は、適用対象が必要とする量を確保できるように設定すればよく、製剤化して用いたり、飲食品に配合して上記医薬組成物を用いることができる。 The dose of the pharmaceutical composition according to the present invention may be set so as to secure the amount required by the application target, and the above-mentioned pharmaceutical composition can be used by formulating or blending with foods and drinks. ..
以下に、本発明に係るポリペプチド及びそれをコードするポリヌクレオチドについて詳細に説明するための実施例を示す。 Examples of the polypeptide according to the present invention and the polynucleotide encoding the same will be described below in detail.
［実施例１：ＫＡＡ１のタンデムリピートによる糖鎖結合部位の高度多価化ポリペプチド（ＫＡＡ１／ＫＡＡ１）の作製］
（ＫＡＡ１／ＫＡＡ１発現大腸菌株の構築）
ＫＡＡ１／ＫＡＡ１発現コンストラクトを構築するための発現ベクターとしてｐＥＴ−２８ａ（＋）（Ｎｏｖａｇｅｎ）及びｐＣｏｌｄＩ（タカラバイオ）、発現用大腸菌株としてＳＨｕｆｆｌｅ Ｔ７ Ｅｘｐｒｅｓｓ（Ｎｏｖａｇｅｎ）を用いた。
[Example 1: Preparation of highly multivalent polypeptide (KAA1 / KAA1) at the sugar chain binding site by tandem repeat of KAA1]
(Construction of KAA1 / KAA1-expressing Escherichia coli strain)
PET-28a (+) (Novagen) and pColdI (Takara Bio) were used as expression vectors for constructing the KAA1 / KAA1 expression construct, and SHuffle T7 Express (Novagen) was used as the expression Escherichia coli strain.
まず、ＫＡＡ１発現コンストラクトを鋳型に、高正確性ＤＮＡポリメラーゼＫＯＤ Ｐｌｕｓ Ｎｅｏ（ＴＯＹＯＢＯ）及び下記表１に記載の各プライマーを用い、ＫＡＡ１／ＫＡＡ１コード領域を増幅した。なお、ＫＡＡ１発現コンストラクトとしては、上記非特許文献１２に記載のｐＥＴ２８ａ−ｒＫＡＡ１を用い、それは、ｐＥＴ−２８ａ（＋）ベクターに６−Ｈｉｓ及びトロンビン切断サイトをＮ末端側に含むＫＡＡ１をコードする配列が挿入されたものである。これにより発現される６−Ｈｉｓ及びトロンビン切断サイトをＮ末端側に含むＫＡＡ１のアミノ酸配列は配列番号３に示す。ＫＡＡ１／ＫＡＡ１コード領域の増幅において、ＫＡＡ１／ＫＡＡ１（ｐＥＴ２８ａ（＋））発現コードＤＮＡはＫＡＡ−ｐＥＴ−１Ｆ及びＫＡＡ−ｐＥＴ−１Ｒ、ＫＡＡ−ｐＥＴ−２Ｆ及びＫＡＡ−ｐＥＴ−２Ｒを、ＫＡＡ１／ＫＡＡ１（ｐＣｏｌｄＩ）はＫＡＡ−ｐＣｏｌｄ−１Ｆ及びＫＡＡ−ｐＥＴ−１Ｒ、ＫＡＡ−ｐＥＴ−２Ｆ及びＫＡＡ−ｐＣｏｌｄ−２Ｒを用い増幅を行った。ＰＣＲの反応系はＫＯＤ ｐｌｕｓ ＮｅＯ用１０×ＰＣＲ緩衝液を５μｌ、２ｍＭのｄＮＴＰｍｉｘを５μｌ、１０μＭフォワードプライマー及び１０μＭリバースプライマーをそれぞれ１．５μｌ、２５ｍＭのＭｇＳＯ４を３μｌ、ＫＡＡ１発現コンストラクトを１μｌ、ＫＯＤ Ｐｌｕｓ Ｎｅｏを１μｌ及び超純水を加えて反応液を５０μｌとし、十分に混合した後、ＰＣＲに供した。ＰＣＲはＴ Ｇｒａｄｉｅｎｔｅｒ ９６ Ｔｈｅｒｍｏｃｙｃｌｅｒ（Ｂｉｏｍｅｔｒａ）を用いて９４℃で２分間、９８℃で１０秒間、６０℃で３０秒間及び増幅反応を６８℃で１５秒のサイクルを３５回繰り返し行った。最後に６８℃で５分間保持し、反応を終了した。 First, the KAA1 / KAA1 coding region was amplified using the highly accurate DNA polymerase KOD Plus Neo (TOYOBO) and each primer shown in Table 1 below, using the KAA1 expression construct as a template. As the KAA1 expression construct, pET28a-rKAA1 described in Non-Patent Document 12 is used, and it is a sequence encoding KAA1 containing 6-His and a thrombin cleavage site on the N-terminal side in the pET-28a (+) vector. Is inserted. The amino acid sequence of KAA1 containing the 6-His and thrombin cleavage sites expressed thereby on the N-terminal side is shown in SEQ ID NO: 3. In the amplification of the KAA1 / KAA1 coding region, the KAA1 / KAA1 (pET28a (+)) expression coding DNA was KAA-pET-1F and KAA-pET-1R, KAA-pET-2F and KAA-pET-2R, and KAA1 / KAA1. (PColdI) was amplified using KAA-pCold-1F and KAA-pET-1R, KAA-pET-2F and KAA-pCold-2R. The reaction system for PCR 5μl the KOD plus NeO for 10 × PCR buffer, 2mM of 5μl of dNTPmix, 10 [mu] M forward primer and 10 [mu] M reverse primer respectively 1.5 [mu] l, 3 [mu] l of MgSO 4 in 25 mM, 1 [mu] l of KAA1 expression construct, KOD 1 μl of Plus Neo and ultrapure water were added to make 50 μl of the reaction solution, and the mixture was thoroughly mixed and then subjected to PCR. PCR was performed using a T Gradienter 96 Thermal cycler (Biometera) at 94 ° C. for 2 minutes, 98 ° C. for 10 seconds, 60 ° C. for 30 seconds, and an amplification reaction at 68 ° C. for 15 seconds, repeated 35 times. Finally, the reaction was completed by holding at 68 ° C. for 5 minutes.
太字：FactorXa認識配列 波下線：終止コドン
斜体：レクチン遺伝子相同配列 点下線：フレームシフト防止挿入配列
Ｉｎ−Ｆｕｓｉｏｎ ＨＤ Ｃｌｏｎｉｎｇ Ｋｉｔ（タカラバイオ）を用いて、制限酵素ＮｄｅＩ及びＸｈｏＩにより切断した発現用ベクターｐＥＴ−２８ａ（＋）（Ｎｏｖａｇｅｎ）及びｐＣｏｌｄＩ（タカラバイオ）へ増幅産物を挿入し、得られたコンストラクトを用いて大腸菌株ＳＨｕｆｆｌｅ Ｔ７ Ｅｘｐｒｅｓｓ及び大腸菌株ＳＨｕｆｆｌｅ Ｅｘｐｒｅｓｓを形質転換した。各形質転換体をＬＢ／Ｋａｎ＋（ｐＥＴ−２８ａ（＋））又はＬＢ／Ａｍｐ＋寒天培地（ｐＣｏｌｄＩ）に適量塗布し、３７℃で一晩培養した。単一コロニーを対象にインサートチェックを行った。 Obtained by inserting amplification products into expression vectors pET-28a (+) (Novagen) and pColdI (Takara Bio) cleaved with restriction enzymes NdeI and XhoI using In-Fusion HD Cloning Kit (Takara Bio). The construct was used to transform Escherichia coli Strain T7 Express and Escherichia coli Strain SHuffle Express. Each transformant was applied in an appropriate amount to LB / Kan + (pET-28a (+)) or LB / Amp + agar medium (pColdI) and cultured at 37 ° C. overnight. An insert check was performed on a single colony.
インサートチェックはｐＥＴ−２８ａ（＋）又はｐＣｏｌｄＩベクターに特異的なプライマー対を用いたＰＣＲにより行った。具体的に、ｐＥＴ−２８ａ（＋）ベクター特異的プライマー対として、Ｔ７プライマー（５’−ＴＡＡＴＡＣＧＡＣＴＣＡＣＴＡＴＡＧＧＧ−３’：配列番号１０）及びＴ７ターミネータプライマー（５’−ＧＣＴＡＧＴＴＡＴＴＧＣＴＣＡＧＣＧＧ−３’ ：配列番号１１）を用い、ただし、ｐＣｏｌｄＩベクター特異的プライマー対としてｐＣｏｌｄ−Ｆプライマー（５’−ＡＣＧＣＣＡＴＡＴＣＧＣＣＧＡＡＡＧＧ−３’：配列番号１２）及びｐＣｏｌｄ−Ｒプライマー（５’−ＧＧＣＡＧＧＧＡＴＣＴＴＡＧＡＴＴＣＴＧ−３’ ：配列番号１３）を用いた。ＰＣＲの反応系はＢｌｅｎｄ Ｔａｑ用１０×ＰＣＲ緩衝液を５μｌ、２ｍＭのｄＮＴＰｍｉｘ溶液を５μｌ、１０μＭのプライマーをそれぞれ１μｌ、ＤＮＡ合成酵素Ｂｌｅｎｄ Ｔａｑ（ＴＯＹＯＢＯ）を０．５μＬ及び超純水を加えて反応液を５０μｌとし、十分に混合した後、１０μｌずつ分注し、コロニーから一部採取した菌体を懸濁し、ＰＣＲに供した。ＰＣＲはＴ Ｇｒａｄｉｅｎｔｒ ９６ Ｔｈｅｒｍｏｃｙｃｌｅｒ（Ｂｉｏｍｅｔｒａ）を用いて９４℃で５分間の熱変性の後、熱変性を９４℃で３０秒間、アニーリング６０℃で３０秒間及び伸長反応を７２℃で１分間のサイクルを３５回繰り返し行った。最後に７２℃で５分間保持し、反応を終了した。 The insert check was performed by PCR using a primer pair specific for pET-28a (+) or pColdI vector. Specifically, as the pET-28a (+) vector-specific primer pair, T7 primer (5'-TAATACGACTCATCATAGGG-3': SEQ ID NO: 10) and T7 terminator primer (5'-GCTAGATTATTGCTCAGGCG-3': SEQ ID NO: 11) were used. However, pCold-F primers (5'-ACGCCATATCGCCGAAAGG-3': SEQ ID NO: 12) and pCold-R primers (5'-GGCAGGGATCTTAGATTTCTG-3': SEQ ID NO: 13) were used as pColdI vector-specific primer pairs. For the PCR reaction system, add 5 μl of 10 × PCR buffer for Blend Taq, 5 μl of 2 mM dNTPmix solution, 1 μl of 10 μM primers, 0.5 μL of DNA synthase Blend Taq (TOYOBO), and ultrapure water. The solution was made into 50 μl, mixed well, and then dispensed in 10 μl increments, and the cells partially collected from the colony were suspended and subjected to PCR. PCR is performed using a T Gradentr 96 Thermocycler (Biometera) at 94 ° C. for 5 minutes, followed by a cycle of heat denaturation at 94 ° C. for 30 seconds, annealing at 60 ° C. for 30 seconds, and extension reaction at 72 ° C. for 1 minute. It was repeated 35 times. Finally, the reaction was completed by holding at 72 ° C. for 5 minutes.
得られたＰＣＲ産物５μｌを１０×ローディング緩衝液と混合し、アガロース電気泳動用の試料とした。泳動用緩衝液としてＴＡＥ（４０ｍＭ Ｔｒｉｓ−ＨＣｌ、４０ｍＭ 酢酸、１ｍＭ ＥＤＴＡ）を、泳動用ゲルとしてエチジウムブロマイドを含む１％アガロースゲルを用いて、一定電圧１００Ｖで電気泳動に供し、ＵＶトランスイルミネーターを用いてバンドの検出を行った。目的サイズにバンドが検出された陽性クローンにつき、Ｈｉ Ｙｉｅｌｄ Ｐｌａｓｍｉｄ Ｍｉｎｉ Ｋｉｔを用い、添付のマニュアルに従ってプラスミドを精製した。精製プラスミドを鋳型に、ベクター特異的プライマーを用いてＢｉｇ Ｄｙｅ Ｔｅｒｍｉｎａｔｏｒ ｖ３．１ Ｃｙｃｌｅ Ｓｅｑｕｅｎｃｉｎｇ Ｋｉｔによりラベリング後、ジェネティックアナライザ３１３０ｘｌ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ）を用いて塩基配列分析に供した。なお、塩基配列解析は、Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ Ｓｅｑｕｅｎｃｅ Ｓｃａｎｎｅｒ Ｓｏｆｔｗａｒｅ ｖｅｒ．１．０により行った。塩基配列を確認したクローンにつき、ＬＢ／Ｋａｎ＋（ｐＥＴ−２８ａ（＋））又はＬＢ／Ａｍｐ＋（ｐＣｏｌｄＩ）液体培地に植菌し３７℃で一晩培養後、等量の４０％グリセロール（終濃度２０％）を加えてグリセロールストックを調製し、使用するまで−８０℃で保存した。 5 μl of the obtained PCR product was mixed with 10 × loading buffer to prepare a sample for agarose gel electrophoresis. Using TAE (40 mM Tris-HCl, 40 mM acetic acid, 1 mM EDTA) as a running buffer and 1% agarose gel containing ethidium bromide as a running gel, electrophoresis was performed at a constant voltage of 100 V, and a UV transilluminator was used. Bands were detected using. For positive clones in which a band was detected in the desired size, a plasmid was purified using a Hi Yield plasmid Mini Kit according to the attached manual. Using the purified plasmid as a template, it was labeled with Big Dye Terminator v3.1 Cycle Sequencing Kit using vector-specific primers, and then subjected to nucleotide sequence analysis using a genetic analyzer 3130 xl (Applied Biosystems). In addition, the base sequence analysis is performed by Applied Biosystems Sequence Scanner Software ver. It was done according to 1.0. The clones whose nucleotide sequences were confirmed were inoculated into LB / Kan + (pET-28a (+)) or LB / Amp + (pColdI) liquid medium and cultured overnight at 37 ° C., and then an equal amount of 40% glycerol (final). A glycerol stock was prepared by adding (concentration 20%) and stored at −80 ° C. until use.
（ＫＡＡ１／ＫＡＡ１の発現及び精製）
上記のようにして得られたＫＡＡ１／ＫＡＡ１発現株のグリセロールストックにつき、ＬＢ／Ｋａｎ＋（ｐＥＴ−２８ａ（＋））又はＬＢ／Ａｍｐ＋液体培地（ｐＣｏｌｄＩ）３ｍｌに植菌し３７℃で一晩培養した。同終夜培養液をＬＢ／Ｋａｎ＋又はＬＢ／Ａｍｐ＋液体培地２５０ｍＬにそれぞれ加え、さらに３７℃で対数増殖期中期になるまで振とう培養した。ＯＤ６００が０．５に達したところで、終濃度が０．５ｍＭとなるようにＩｓｏｐｒｏｐｙｌ β−Ｄ−１−ｔｈｉｏｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅ（ＩＰＴＧ）を添加することで発現誘導を開始し、ＫＡＡ１／ＫＡＡ１発現株（ｐＥＴ−２８ａ（＋））は２０℃で１６時間、ＫＡＡ１／ＫＡＡ１発現株（ｐＣｏｌｄＩ）は１５℃で２４時間振とう培養した。これを遠心分離（１０，０００×ｇ、４℃、１５分）により集菌し、培養液に対し１／２０容の超音波破砕用緩衝液（２０ｍＭリン酸緩衝液（ｐＨ ７．４）、５００ｍＭ ＮａＣｌ、２０ｍＭ イミダゾール）に懸濁した後、超音波破砕を行った。破砕後、再度遠心分離（１０，０００×ｇ、４℃、１５分）し、上清を可溶性画分、残渣を不溶性画分として回収した。
(Expression and purification of KAA1 / KAA1)
The glycerol stock of the KAA1 / KAA1 expression strain obtained as described above was inoculated into 3 ml of LB / Kan + (pET-28a (+)) or LB / Amp + liquid medium (pColdI) and overnight at 37 ° C. It was cultured. The same overnight culture solution was added to 250 mL of LB / Kan + or LB / Amp + liquid medium, respectively, and further cultured at 37 ° C. with shaking until the middle logarithmic growth phase was reached. When the OD 600 reached 0.5, expression induction was started by adding Isopropanol β-D-1-thiogalactopylanoside (IPTG) so that the final concentration became 0.5 mM, and the KAA1 / KAA1 expression strain (pET) was started. −28a (+)) was shake-cultured at 20 ° C. for 16 hours, and the KAA1 / KAA1 expression strain (pColdI) was shake-cultured at 15 ° C. for 24 hours. This was collected by centrifugation (10,000 × g, 4 ° C., 15 minutes), and 1/20 volume of the culture solution was used as an ultrasonic crushing buffer solution (20 mM phosphate buffer solution (pH 7.4)). After suspending in 500 mM NaCl, 20 mM imidazole), ultrasonic disruption was performed. After crushing, the mixture was centrifuged again (10,000 × g, 4 ° C., 15 minutes), and the supernatant was recovered as a soluble fraction and the residue as an insoluble fraction.
本実施例において得られた組換え体はいずれもＨｉｓタグ融合体として発現される（得られた組換え体のアミノ酸配列は、配列番号２で示される。）。そこで、可溶性画分に含まれるＨｉｓタグ融合組換え体を、ニッケルキレートカラム（Ｖｔ＝１ｍｌ、Ｈｉｓ Ｇｒａｖｉ Ｔｒａｐ、ＧＥ ヘルスケア）により精製した。すなわち、平衡化緩衝液（２０ｍＭ リン酸緩衝液（ｐＨ ７．４）、５００ｍＭ ＮａＣｌ、２０ｍＭイミダゾール）にて十分に平衡化したカラムへ可溶性画分を添加し、組換え体を結合させた。同平衡化緩衝液で十分に洗浄後、溶出用緩衝液（２０ｍＭリン酸緩衝液（ｐＨ ７．４）、５００ｍＭ ＮａＣｌ、５００ｍＭイミダゾール）を５ｍｌ添加し組換え体を溶出させた。イミダゾール除去のため、超純水で十分に透析したものを精製標品とし、以下の試験に供した。なお、ＫＡＡ１／ＫＡＡ１（ｐＥＴ−２８ａ（＋））及びＫＡＡ１／ＫＡＡ１（ｐＣｏｌｄＩ）精製標品の収量は、培養液１Ｌあたりそれぞれ５２ｍｇ及び４２ｍｇであったので収量がより多かったＫＡＡ１／ＫＡＡ１（ｐＥＴ−２８ａ（＋））を以降の試験に供した。 All of the recombinants obtained in this example are expressed as His tag fusions (the amino acid sequence of the obtained recombinants is shown by SEQ ID NO: 2). Therefore, the His tag fusion recombinant contained in the soluble fraction was purified by a nickel chelate column (Vt = 1 ml, His Gravi Trap, GE Healthcare). That is, the soluble fraction was added to a column sufficiently equilibrated with an equilibration buffer (20 mM phosphate buffer (pH 7.4), 500 mM NaCl, 20 mM imidazole), and the recombinant was bound. After thorough washing with the same equilibration buffer, 5 ml of an elution buffer (20 mM phosphate buffer (pH 7.4), 500 mM NaCl, 500 mM imidazole) was added to elute the recombinant. In order to remove imidazole, a refined product that had been sufficiently dialyzed with ultrapure water was used for the following tests. The yields of the KAA1 / KAA1 (pET-28a (+)) and KAA1 / KAA1 (pColdI) purified preparations were 52 mg and 42 mg, respectively, per 1 L of the culture solution, so that the yields were higher than that of KAA1 / KAA1 (pET-). 28a (+)) was subjected to subsequent tests.
［実施例２：ＫＡＡ１／ＫＡＡ１に対するＳＤＳ−ＰＡＧＥを用いた分析］
（方法）
ＳＤＳ−ＰＡＧＥはＳｃｈａｇｇｅｒ ａｎｄ Ｊａｇｏｗ（１９８７）の方法に準じ、電気泳動装置（ＡＥ−６５３０、ＡＴＴＯ）に電気泳動用アクリルアミドゲル（１２．５％）をセットし、１００Ｖの定電圧で行った。分子量マーカーにはタンパク質分子量マーカーＩＩ（テフコ）を用いた。試料液としての上記精製製品に等量の２×ＳＤＳローディング緩衝液（４％（ｗ／ｖ）ＳＤＳ、１２％（ｗ／ｖ）グリセロール、及び０．０１％（ｗ／ｖ）ブロモフェノールブルーを含む５０ｍＭ トリス−塩酸緩衝液（ｐＨ ６．８））を添加した後、１００℃で５分間加熱処理し、非還元下での泳動用試料を調製した。また、還元条件下での泳動用試料としては、上記等量の２×ＳＤＳローディング緩衝液、及び２％（ｗ／ｖ）メルカプトエタノールを加えたものを用いた。それぞれの試料を泳動後、ゲルをクマシーブリリアントブルー（ＣＢＢ）Ｒ−２５０で染色し、タンパク質バンドを確認した。なお、泳動には、精製標品は３μｇ相当量を用いた。その結果を図１に示す。図１のＭは分子量マーカー、１は非還元下のＫＡＡ１、２は非還元下のＫＡＡ１／ＫＡＡ１、３は還元下のＫＡＡ１、４は還元下のＫＡＡ１／ＫＡＡ１をそれぞれ示す。
[Example 2: Analysis of KAA1 / KAA1 using SDS-PAGE]
(Method)
SDS-PAGE was carried out at a constant voltage of 100 V by setting an electrophoresis acrylamide gel (12.5%) on an electrophoresis apparatus (AE-6530, ATTO) according to the method of Schager and Jago (1987). A protein molecular weight marker II (Tefco) was used as the molecular weight marker. Equal amounts of 2 × SDS loading buffer (4% (w / v) SDS, 12% (w / v) glycerol, and 0.01% (w / v) bromophenol blue) were added to the purified product as a sample solution. After adding 50 mM Tris-hydrochloric acid buffer (pH 6.8)), the sample was heat-treated at 100 ° C. for 5 minutes to prepare a sample for electrophoresis under non-reduction. As a sample for electrophoresis under reducing conditions, the same amount of 2 × SDS loading buffer and 2% (w / v) mercaptoethanol were added. After electrophoresis of each sample, the gel was stained with Coomassie Brilliant Blue (CBB) R-250 to confirm the protein band. For the electrophoresis, an amount equivalent to 3 μg of the purified sample was used. The result is shown in FIG. In FIG. 1, M indicates a molecular weight marker, 1 indicates a non-reduced KAA1, 2 indicates a non-reduced KAA1 / KAA1, 3 indicates a reduced KAA1, and 4 indicates a reduced KAA1 / KAA1.
（結果）
図１に示すように、ＫＡＡ１／ＫＡＡ１は、還元下及び非還元下のいずれのＳＤＳ−ＰＡＧＥにおいても約６０ｋＤａの付近にバンドが確認された。野生型ＫＡＡ１（Ｈｉｓタグ融合体）の分子量が３０．２ｋＤａであり、その直列反復の構造を持つＫＡＡ１／ＫＡＡ１の理論分子量は５８．９ｋＤａであることから、得られた上記精製標品は確かにＫＡＡ１／ＫＡＡ１であると確認された。
(result)
As shown in FIG. 1, a band of KAA1 / KAA1 was confirmed in the vicinity of about 60 kDa in both reduced and non-reduced SDS-PAGE. Since the molecular weight of wild-type KAA1 (His tag fusion) is 30.2 kDa and the theoretical molecular weight of KAA1 / KAA1 having a structure of its series repeat is 58.9 kDa, the obtained purified preparation is certainly. It was confirmed to be KAA1 / KAA1.
［実施例３：ＫＡＡ１／ＫＡＡ１の赤血球凝集活性の測定］
（方法）
赤血球凝集活性（ｈｅｍａｇｇｌｕｔｉｎａｔｉｏｎ ａｃｔｉｖｉｔｙ（ＨＡ））はマイクロタイター法を用いて測定した。すなわち、各試料の１６μＭ溶液から生理食塩水中連続２倍希釈液各２５μｌをマイクロタイタープレート上に作製し、各希釈液に２％トリプシン処理ウサギ赤血球浮遊液（ｔｒｙｐｓｉｎ−ｔｒｅａｔｅｄ ｒａｂｂｉｔ ｂｌｏｏｄ ｃｅｌｌ（ＴＲＢＣ））を２５μｌ加えて軽く攪拌し、室温にて３時間以上静置後、凝集能を観察した。凝集能は肉眼で判定し、赤血球の５０％以上が凝集している場合を陽性とした。ＨＡの強さは凝集素価（力価）、すなわち陽性を示した検液の希釈倍率で表した。なお、ＴＲＢＣは以下のように調製した。市販されているウサギ無菌保存血（広島動物実験所）につき赤血球２ｍｌ相当量を約５０ｍｌの生理食塩水で３回洗浄後、生理食塩水を加えて、４５ｍｌにメスアップし、２％ウサギ赤血球浮遊液を調製した。これに１／１０容の０．５％トリプシンを含む生理食塩水を加え、３７℃で６０分静置（３０分ごとに攪拌）した。トリプシン処理赤血球を５０ｍｌの生理食塩水で４回洗浄後、生理食塩水を加えて４５ｍｌとし、２％ＴＲＢＣとした。赤血球凝集活性試験の結果を表２に示す。
[Example 3: Measurement of hemagglutination activity of KAA1 / KAA1]
(Method)
Hemagglutination activity (HA) was measured using the microtiter method. That is, 25 μl of each of 25 μl of a continuous 2-fold diluted solution in physiological saline was prepared from a 16 μM solution of each sample on a microtiter plate, and 2% trypsin-treated rabbit erythrocyte suspension (TRBC) was added to each diluted solution. Was added 25 μl and stirred lightly, and after allowing to stand at room temperature for 3 hours or more, the agglutinating ability was observed. The agglutinating ability was judged with the naked eye, and the case where 50% or more of erythrocytes were agglutinating was regarded as positive. The strength of HA was expressed by the aggregation titer (titer), that is, the dilution ratio of the test solution showing a positive result. The TRBC was prepared as follows. 2 ml of red blood cells equivalent to commercially available rabbit sterile preserved blood (Hiroshima Animal Research Institute) is washed 3 times with about 50 ml of physiological saline, then physiological saline is added, and the volume is increased to 45 ml, and 2% rabbit red blood cells float. The solution was prepared. A physiological saline solution containing 1/10 volume of 0.5% trypsin was added thereto, and the mixture was allowed to stand at 37 ° C. for 60 minutes (stirred every 30 minutes). The trypsin-treated erythrocytes were washed 4 times with 50 ml of physiological saline, and then the physiological saline was added to make 45 ml to obtain 2% TRBC. The results of the hemagglutination activity test are shown in Table 2.
（結果）
表２に示すように、ＫＡＡ１／ＫＡＡ１は濃度が高くなるにつれて、劇的に活性が上昇することが確認された。０．２５μＭ溶液では、ＫＡＡ１の力価が２７であるのに対し、ＫＡＡ１／ＫＡＡ１は２８であり、大きな差は見られなかったが、８μＭ溶液では、ＫＡＡ１の力価が２１９であるのに対し、ＫＡＡ１／ＫＡＡ１の力価は２５８であった。さらに、１６μＭ溶液では、ＫＡＡ１の力価が２２０であるのに対し、ＫＡＡ１／ＫＡＡ１の力価は＞２１４４とさらなる活性強度の劇的な上昇が認められた。
(result)
As shown in Table 2, it was confirmed that the activity of KAA1 / KAA1 increased dramatically as the concentration increased. In 0.25μM solution, whereas the titer of KAA1 is 2 7, KAA1 / KAA1 is 2 8, a large difference was not observed, the 8μM solution, titer of KAA1 is 2 19 whereas, the titer of KAA1 / KAA1 was 2 58. Furthermore, in the 16μM solution, whereas the titer of KAA1 is 2 20, the titer of KAA1 / KAA1 is> 2 144 and a dramatic further increase in activity intensity was observed.
［実施例４：ＫＡＡ１／ＫＡＡ１のＳＡＲＳコロナウイルス不活化効果の検討］
次に、実際にＫＡＡ１／ＫＡＡ１がウイルスの不活化作用を示すか否かについて検討するために、ＳＡＲＳコロナウイルス（ＳＡＲＳ−ＣｏＶ）に対してＫＡＡ１／ＫＡＡ１を処理してＳＡＲＳ−ＣｏＶの細胞感染能を評価した。その方法及び結果を以下に説明する。
[Example 4: Examination of SARS coronavirus inactivating effect of KAA1 / KAA1]
Next, in order to examine whether or not KAA1 / KAA1 actually exhibits a virus inactivating effect, SARS coronavirus (SARS-CoV) is treated with KAA1 / KAA1 to infect SARS-CoV cells. Was evaluated. The method and results will be described below.
まず、１０μＬのＳＡＲＳ−ＣｏＶ−２（２０１９−ｎＣｏＶ／Ｊａｐａｎ／ＡＩ／Ｉ−００４／２０２０株（国立感染症研究所）：２．０×１０８ＴＣＩＤ５０／ｍＬ）と、９０μＬのＫＡＡ１（１０μＭ）又はＫＡＡ１／ＫＡＡ１（１０μＭ）とを混合した。なお、これらとは別にコントロールとしてレクチンを含まないＤＭＥＭとＳＡＲＳ−ＣｏＶ−２とを混合した。その後、３０分間室温で反応させた後に、細胞維持液ＤＭＥＭで１０倍に稀釈して反応を停止した。さらに１０段階系列希釈して、種々の濃度のウイルス含有液を準備した。そして、予め９６ウェルプレートに播種されたＶｅｒｏ／ＴＭＰＲＳＳ２細胞（ＪＣＲＢ１８１９（国立研究開発法人 医薬基盤・健康・栄養研究所ＪＣＲＢ細胞バンク））に当該各濃度のウイルス含有液を接種して（５０μｌ／ｗｅｌｌ、各濃度４ｗｅｌｌずつ）、１時間吸着させた後に、接種液を吸引除去して、１００μｌ／ｗｅｌｌのＤＭＥＭを加えた。なお、その３日後に細胞変性効果が広がったところで、各ウェルを顕微鏡により観察して感染の有無を評価し、感染価を測定した。感染価の単位は、５０％細胞感染濃度（ＴＣＩＤ５０）／ｍｌである。その結果を図３に示す。 First, 10 μL of SARS-CoV-2 (2019-nCoV / Japan / AI / I-004 / 2020 strain (National Institute of Infectious Diseases): 2.0 × 10 8 TCID50 / mL) and 90 μL of KAA1 (10 μM). Alternatively, KAA1 / KAA1 (10 μM) was mixed. Separately from these, DMEM containing no lectin and SARS-CoV-2 were mixed as a control. Then, after reacting at room temperature for 30 minutes, the reaction was stopped by diluting 10-fold with cell maintenance solution DMEM. Further 10-step serial dilution was performed to prepare virus-containing solutions having various concentrations. Then, Vero / TMPRSS2 cells (JCRB1819 (National Institute of Biomedical Innovation, Health and Nutrition JCRB Cell Bank)) seeded in advance on a 96-well plate are inoculated with the virus-containing solution at each concentration (50 μl / well). After adsorbing for 1 hour (4 well at each concentration), the inoculum was removed by suction, and DMEM of 100 μl / well was added. When the cytopathic effect spread 3 days later, each well was observed with a microscope to evaluate the presence or absence of infection, and the infection titer was measured. The unit of infectious titer is 50% cell infection concentration (TCID50) / ml. The result is shown in FIG.
図３に示すように、ＫＡＡ１又はＫＡＡ１／ＫＡＡ１により処理されたＳＡＲＳ−ＣｏＶ−２はコントロール（ＤＭＥＭ）と比較して、不活化して感染価が低下した。また、ＫＡＡ１を処理した場合と比較して、ＫＡＡ１／ＫＡＡ１を処理した場合では、その感染価がより低いことが明らかとなった。ＫＡＡ１又はＫＡＡ１／ＫＡＡ１によりＳＡＲＳ−ＣｏＶ−２が不活化して感染価が低下しているのは、ＫＡＡ１又はＫＡＡ１／ＫＡＡ１がＳＡＲＳ−ＣｏＶ−２粒子の表面の糖鎖に結合して、立体障害又はウイルス同士の凝集が生じたためであると考えられる。ＫＡＡ１と比べてＫＡＡ１／ＫＡＡ１の不活化能が強いのはＫＡＡ１と比較して糖鎖結合部位が多い（２倍）ためであり、ＳＡＲＳ−ＣｏＶ−２の凝集を強く起こしているためであると考えられる。この結果から、ＫＡＡ１／ＫＡＡ１は、ＳＡＲＳ−ＣｏＶに起因する疾患の治療又は診断に有用であると考えられ、また、ＳＡＲＳ−ＣｏＶと同様に高マンノース型糖鎖を表面に有するウイルス疾患の治療又は診断に有用であると考えられる。 As shown in FIG. 3, SARS-CoV-2 treated with KAA1 or KAA1 / KAA1 was inactivated and reduced infectious titer as compared with control (DMEM). In addition, it was revealed that the infectious titer was lower when KAA1 / KAA1 was treated than when KAA1 was treated. The reason why SARS-CoV-2 is inactivated by KAA1 or KAA1 / KAA1 and the infectious titer is lowered is that KAA1 or KAA1 / KAA1 binds to the sugar chain on the surface of SARS-CoV-2 particles, resulting in steric hindrance. Alternatively, it is considered that this is because the viruses have aggregated with each other. The reason why the inactivating ability of KAA1 / KAA1 is stronger than that of KAA1 is that there are more sugar chain binding sites (twice) than that of KAA1 and that SARS-CoV-2 is strongly aggregated. Conceivable. From this result, KAA1 / KAA1 is considered to be useful for the treatment or diagnosis of diseases caused by SARS-CoV, and also for the treatment of viral diseases having a high mannose type sugar chain on the surface like SARS-CoV. It is considered to be useful for diagnosis.
以上の結果から、ＫＡＡ１の糖鎖結合部位のタンデムリピートにより高度多価化されたＫＡＡ１／ＫＡＡ１は、野生型ＫＡＡ１と比較して糖鎖結合性が劇的に増強されることが示唆された。ＫＡＡ１は、上述のようにＨＩＶのエンベロープ糖タンパク質ｇｐ１２０が有する高マンノース型糖鎖と結合するため、糖鎖結合性が顕著に増強されたＫＡＡ１／ＫＡＡ１は、上記高マンノース型糖鎖と強く結合できてＨＩＶ治療やＨＩＶ診断への利用可能性があるので極めて有用である。さらに、ＨＩＶ以外のＳＡＲＳ−ＣｏＶ等の高マンノース型糖鎖を表面に有するウイルス疾患にも有用であると考えられる。 From the above results, it was suggested that KAA1 / KAA1, which was highly multivalent by tandem repeat of the sugar chain binding site of KAA1, had dramatically enhanced sugar chain binding as compared with wild-type KAA1. Since KAA1 binds to the high mannose-type sugar chain of the HIV enveloped glycoprotein gp120 as described above, KAA1 / KAA1 whose sugar chain binding property is remarkably enhanced can strongly bind to the high mannose-type sugar chain. It is extremely useful because it can be used for HIV treatment and HIV diagnosis. Furthermore, it is considered to be useful for viral diseases having high mannose-type sugar chains such as SARS-CoV on the surface other than HIV.
Claims (7)
The pharmaceutical composition according to claim 6, wherein the virus is a human immunodeficiency virus (HIV), an influenza virus, a hepatitis C virus (HCV), a SARS corona virus (SARS-CoV), or a simple herpes virus. Stuff.
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