JP4049408B2 - Method for immobilizing proteins on the surface of Gram-positive bacteria - Google Patents

Description

【００２６】
５．ＤＮＡ断片の製造
ＫＯＤ ＤＮＡポリメラーゼ（東洋紡績株式会社）を用いたＰＣＲ法により、遺伝子ＤＮＡの増幅を行った。ＳＡＫ遺伝子の増幅は、ｐＢＥ３１ＳＡＫ１を鋳型とし、プライマーＳＧＳＡＫ５とＳＡＫＳＡＬ１Ｒとの間で、９４℃３０秒、５０℃３０秒、７４℃６０秒を１サイクルとし、これを３０回繰返すことにより、行った。
【００２７】
アンカー配列の増幅は、ｐＭＱ９０８０のＤｒａＩ断片を鋳型として、プライマーＭＫ２４５，ＭＫ１６０，ＭＫ１２７，ＭＫ０９８，ＭＫ０５７，ＭＫ０３８とＡＮＣＳＰＨ１との間で、９８℃１５秒、６０℃５秒、７４℃３０秒を１サイクルとし、これを３０回繰返す条件により行った。
【００２８】
６．組換えプラスミドの製造
(1) ｐＳＡＫ−ＮＳの作製
ＰＣＲ法で増幅したＳＡＫ遺伝子を含むＤＮＡ断片を、ＮｓｐＶ，ＳａｌＩで切断し、精製した後、ｐＳＧ−ＴＥＲＭのＮｓｐＶ−ＳａｌＩサイトに挿入して、ｐＳＡＫ−ＮＳを作成した。図２はＳＡＫ−アンカー配列融合タンパク質遺伝子の構造を模式的に示す説明図である。図２のａ図に示す通り、ｐＳＡＫ−ＮＳはエリスロマイシン耐性遺伝子のプロモータの下流に、ＳＡＫ遺伝子由来のＳＤ配列（ＳＤ）、ＳＡＫシグナル配列（signal seq. ）、３’側にＳａｌＩサイトが挿入されたためにＣ末端に２アミノ酸残基が付加したＳＡＫ成熟タンパク質、終止コドン、乳酸菌プロテアーゼ由来のターミネータ配列（terminator）が繋った構造をしている。
【００２９】
(2) ｐＳＡＫ２４５，ｐＳＡＫ１６０，ｐＳＡＫ１２７，ｐＳＡＫ０９８，ｐＳＡＫ０５７，ｐＳＡＫ０３８の作製
ＰＣＲ法で増殖したアンカー配列を含むＤＮＡ断片を、ＳａｌＩ，ＳｐｈＩで切断し精製した後、ｐＳＡＫ−ＮＳのＳａｌＩ−ＳｐｈＩサイトに挿入することにより、ｐＳＡＫ２４５，ｐＳＡＫ１６０，ｐＳＡＫ１２７，ｐＳＡＫ０９８，ｐＳＡＫ０５７，ｐＳＡＫ０３８を作成した。図２のｂ図〜ｇ図に示す通り、これらのプラスミドは、ＳＡＫタンパク質のＣ末端にＳａｌＩサイトに由来する２アミノ酸残基を介して、プロテアーゼ由来のアンカー配列がＣ末端から各々、２４３，１５８，１２５，９６，５５，３６アミノ酸残基結合した融合タンパク質をコードしている。尚、得られたプラスミドについて、ｐＳＡＫ２４５は、生工研菌寄第１５９０９号として、ｐＳＡＫ０３８は生工研菌寄第１５９１０号として、各々生命工学研究所に平成０８年１０月１７日付けで寄託されている。また、他のプラスミドについては、ｐＳＡＫ２４５の所望の塩基配列を切除することにより、得られると判断したため、菌の寄託を行わなかった。
【００３０】
７．宿主への形質転換
作製したプラスミドＤＮＡは、先ず、大腸菌を宿主として、構造の確認を行った。
その後、大腸菌より精製した各々のプラスミドＤＮＡ（ｐＳＡＫ２４５，ｐＳＡＫ１６０，ｐＳＡＫ１２７，ｐＳＡＫ０９８，ｐＳＡＫ０５７，ｐＳＡＫ０３８）をエレクトロポレーション法によりラクトコッカス・ラクチス菌 YIT2018株に導入して、ＳＡＫ−ＮＳ，ＳＡＫ２４５，ＳＡＫ１６０，ＳＡＫ１２７，ＳＡＫ０９８，ＳＡＫ０５７，ＳＡＫ０３８を得た。今回の実施例で作成し、解析を行った乳酸菌クローンを前記表１にまとめた。
【００３１】
【表１】

【００３２】
８．ＳＡＫタンパク質の発現の確認
(1) ＳＡＫ活性の測定
一晩培養した培養液そのままを培養液画分、これを遠心分離された菌体ペレットをM17-glucose 培地に再懸濁したものを菌体画分とし、各々１０μｌを反応溶液（chromozyme PL 0.5mM, plasminogen 0.06units/ml, polysorbate 80 0.01%, 0.1M Tris-HCl pH8.0）１００μｌと３７℃で反応させ、経時的に４０５ｎｍにおける吸光度をマイクロプレートリーダーモデル４５０（Bio Rad 社製）で測定した。
【００３３】
図３はＳＡＫ活性を測定した結果を示す線図であり、ａ図は培養液画分、ｂ図は菌体画分を示す。縦軸は吸光度、横軸は反応時間を示す。図３に示す通り、ＳＡＫ遺伝子を導入していないベクターでのみ活性が見られないほかは、全てのクローンで活性が検出された。
【００３４】
(2) ＳＤＳ−ＰＡＧＥ及びウェスタンブロッティング
ラクトコッカス・ラクチス菌クローンの一晩培養液１ｍｌを遠心分離して培養上清と菌体画分とを得た。培養上清はＴＣＡ沈殿後、２×ＳＤＳ sample buffer２０μｌに懸濁し加熱した。菌体画分も同様に２×ＳＤＳ sample buffer２０μｌに懸濁し加熱した。これらのサンプルをPhast System 20% Phast gel(Pharmacia社製) でＳＤＳ電気泳動後、ＰＶＤＦ膜に転写し、抗ＳＡＫ抗血清を１次抗体として、アルカリフォスタファーゼ標識抗ウサギＩｇＧ抗体を２次抗体として用い、ウェスタンブロッティングを行った。
【００３５】
図４はウェスタンブロッティングの結果を模式的に示す説明図であり、各クローン毎の培養上清(sup) 、菌体画分(cell)を順に示している。図に示す通り、ＳＡＫ遺伝子を導入していないベクター(Vector)では、ＳＡＫタンパク質は検出されていない。菌体画分ではＳＤＳサンプルバッファーにより菌体の表層タンパク質が抽出されていると考えられる。培養上清中にはＳＡＫ（融合）タンパク質が分解を受けたと思われる分子量の小さいバンドが多く検出された。
【００３６】
(3) アルカリフォスファターゼ標識抗体を用いた菌体表面のＳＡＫ（融合）タンパク質の検出
各プラスミド（ｐＳＡＫ２４５，ｐＳＡＫ１６０，ｐＳＡＫ１２７，ｐＳＡＫ０９８，ｐＳＡＫ０５７，ｐＳＡＫ０３８）を同様にエレクトロポレーション法により導入して、宿主菌をラクトバチルス・カゼイ(Lactobacillus casei) としたクローンを作製した。
【００３７】
ラクトコッカス・ラクチス菌クローン及びラクトバチルス・カゼイ菌クローンの一晩培養液１ｍｌを遠心分離して菌体画分を得た。得られた菌体を 50mM Tris-HCl pH7.4 １ｍｌで２回洗浄後、１％ＢＳＡ／ＴＢＳＴ 0.5mlに懸濁して室温で３０〜６０分保持し、抗ＳＡＫ抗血清１μｌを含むＴＢＳＴ 0.5mlに懸濁して室温で３０〜６０分保持した後、ＴＢＳＴ 0.5mlで３回洗浄した。その後、アルカリフォスファターゼ標識抗体ウサギＩｇＧ抗体 0.25 μl を含むＴＢＳＴ 0.5mlに懸濁して室温で３０〜６０分保持した後、ＴＢＳＴ 0.5mlで３回洗浄後、ＯＤ₆₆₀ が7.5 になるようにＴＢＳＴに懸濁し、サンプルとした。
【００３８】
得られたサンプル10μl に基質溶液（1mg p-Nitrophenyl Phosphate /10ml diethanolamine buffer ） 100μｌを加え、室温で反応させ、４０５ｎｍにおける吸光度を経時的にマイクロプレートリーダモデル４５０（Bio Rad 社製）で測定した。
【００３９】
図５はアルカリフォスファターゼ標識抗体を用いた菌体表面のＳＡＫ（融合）タンパク質の検出結果を示す線図であり、ａ図はラクトコッカス・ラクチス(Lactococcus lactis)、ｂ図はラクトバチルス・カゼイ(Lactobacillus casei) を示す。縦軸は吸光度、横軸は反応時間（ｍｉｎ）を示す。
【００４０】
図において、吸光度の活性は菌体に結合したアルカリフォスファターゼ（ＡＰ）の活性が示される。即ち、菌体表面に表出したＳＡＫにウサギ抗体ＳＡＫ抗体が結合し、更にこれにアルカリフォスファターゼによって標識されたウサギＩｇＧ抗体が結合するため、アルカリフォスファターゼの活性が高ければ、菌体表面に表出したＳＡＫの数が多いこととなる。従って、図に示したグラフの傾斜が急な方がＡＰ活性が高く、菌体に結合したＡＰ標識抗兎ＩｇＧ抗体の量が多いことを示す。このことはそのクローンにおいて菌体表層のウサギ抗ＳＡＫ抗体量が多いこと、即ち、菌体表層に提示されているＳＡＫタンパク質エピトープが多いことを意味する。
【００４１】
図に示されるように、ラクトコッカス・ラクチス菌、ラクトバチルス・カゼイ菌の何れでも、アミノ酸残基数の多いものは、ＡＰ活性、即ちＳＡＫの菌体表面への発現が高いことが確認された。３６アミノ酸残基は、Ｌ．ラクチス菌でコントロール（ベクター，ＮＳ）との差が確認できたが、Ｌ．カゼイでは差が確認できなかった。これは、融合タンパク質が菌体細胞壁内に埋没している可能性が考えられる。従って、好ましくは３６アミノ酸残基を超えたアンカー配列が必要である。
【００４２】
【発明の効果】
本発明は以上説明したとおり、ラクトコッカス・ラクチス菌由来のプロテアーゼのアンカー配列又は当該アンカー配列の部分配列を利用して、種々の任意の有用タンパク質を菌体表面に固定化し、且つ当該有用タンパク質を発現させるという効果がある。従って、本発明によれば、例えば安全性が高い乳酸菌を利用して、種々のタンパク質を固定化することが可能となるので、安全性の高い経口ワクチンなどへの利用が考えられる。
【００４３】
【配列表】

【００４４】

【００４５】

【００４６】

【００４７】

【００４８】

【図面の簡単な説明】
【図１】プラスミドｐＳＧ−ＴＥＲＭの構造を模式的に示した説明図である。
【図２】ＳＡＫ−アンカー配列融合タンパク質遺伝子の構造を模式的に示す説明図である。
【図３】ＳＡＫ活性を測定した結果を示す線図であり、ａ図は培養液画分、ｂ図は菌体画分を示す。縦軸は吸光度、横軸は反応時間を示す。
【図４】ウェスタンブロッティングの結果を模式的に示す説明図であり、各クローン毎の培養上清(sup) 、菌体画分(cell)を順に示している。
【図５】アルカリフォスファターゼ標識抗体を用いた菌体表面のＳＡＫ（融合）タンパク質の検出結果を示す線図であり、ａ図はラクトコッカス・ラクチス(Lactococcus lactis)、ｂ図はラクトバチルス・カゼイ(Lactobacillus casei) を示す。縦軸は吸光度、横軸は反応時間（ｍｉｎ）を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an immobilization method for expressing any useful protein on the surface of a host cell by using an anchor sequence of a protease protein on the surface of the cell of Lactococcus lactis .
[0002]
[Prior art]
Many cell surface proteins of Gram-positive bacteria have a common structure called an anchor sequence, and are bound to the cells through it. Among the proteases of Lactococcus lactis, so-called 763 protease is Lactococcus lactis ssp. Lactis is a serine protease whose production information is retained on the 55 Kb plasmid (pLP 763) of the NCDO763 strain, and is expressed by an anchor sequence located at the C-terminal, taking a structure that binds to the bacterial cell surface. It is known.
[0003]
This anchor sequence is a structure possessed by many cell surface proteins of Gram-positive bacteria, consisting of (1) proline and glycine-rich repeat regions located in the peptidoglycan layer that constitutes the cell wall, and (2) amino acid residues. Consensus sequence (LPXTG motif), (3) a hydrophobic region located in the cell membrane, and (4) a positively charged region thought to be located in the cytoplasm.
[0004]
The amino acid structure of the 763 protease anchor sequence has been reported (see literature; Molecular Microbiology (1989) vol. 3 (No. 3), 359-369). In 763 protease, the above (1) The proline / glycine-rich repeat region described in 1. is not clear, and all of them are essential for the binding of proteases to cells, and if not, what part is the minimum necessary unit? Has not been fully elucidated.
[0005]
[Problems to be solved by the invention]
Expressing useful proteins on the surface of the cells can be an effective means for breeding microorganisms. Thus, as a result of diligent efforts, the present inventors have obtained the present invention by elucidating a region involved in the binding of proteases to cells in an anchor sequence derived from 763 protease.
[0006]
The present invention uses an anchor sequence of a protease derived from Lactococcus lactis or a partial sequence of the anchor sequence to immobilize various arbitrary useful proteins on the cell surface and to express the useful proteins. Objective.
[0007]
[Means for Solving the Problems]
In the method for immobilizing a protein on the surface of Gram-positive bacteria according to the invention described in claim 1, a gene sequence encoding an anchor sequence of 763 protease derived from Lactococcus lactis NCDO763 strain, Obtaining a fusion gene sequence linking a gene sequence encoding a target protein;
Introducing the fusion gene sequence into a host fungus;
In the method for immobilizing proteins to the fusion gene sequence introduced by culturing the host microorganism and a step of immobilizing a protein of the object in Gram-positive bacteria cell surface Gram-positive bacteria cell surface,
The anchor sequence has at least the following amino acid sequence.
Thr Ser Thr Asp Asp Thr Thr Asp Arg Asn Gly Gln Leu Thr Ser Gly
Lys Gly Ala Leu Pro Lys Thr Gly Glu Thr Thr Glu Arg Pro Ala Phe
Gly Phe Leu Gly Val Ile Val Val Ser Leu Met Gly Val Leu Gly Leu
Lys Arg Lys Gln Arg Glu Glu
[0009]
In the method for immobilizing a protein on the surface of a Gram-positive microbial cell according to the invention described in claim 2, the anchor sequence has excised a part of the following amino acid sequence leaving the amino acid sequence shown in claim 1 It consists of an amino acid sequence.
Lys Lys Thr Ser Leu Leu Asn Gln Leu Gln Ser Val Lys Ala Ala Leu
Glu Thr Asp Leu Gly Asn Gln Thr Asp Ser Ser Thr Gly Lys Thr Phe
Thr Ala Ala Leu Asp Asp Leu Val Ala Gln Ala Gln Ala Gly Thr Gln
Thr Asp Asp Gln Leu Gln Ala Thr Leu Ala Lys Val Leu Asp Ala Val
Leu Ala Lys Leu Ala Glu Gly Ile Lys Ala Ala Thr Pro Ala Glu Val
Gly Asn Ala Lys Asp Ala Ala Thr Gly Lys Thr Trp Tyr Ala Asp Ile
Ala Asp Thr Leu Thr Ser Gly Gln Ala Ser Ala Asp Ala Ser Asp Lys
Leu Ala His Leu Gln Ala Leu Gln Ser Leu Lys Thr Lys Val Ala Ala
Ala Val Glu Ala Ala Lys Thr Val Gly Lys Gly Asp Gly Thr Thr Gly
Thr Ser Asp Lys Gly Gly Gly Gln Gly Thr Pro Ala Pro Thr Pro Gly
Asp Ile Gly Lys Asp Lys Gly Asp Glu Gly Ser Gln Pro Ser Ser Gly
Gly Asn Ile Pro Thr Asn Pro Ala Thr Thr Thr Ser Thr Ser Thr Asp
Asp Thr Thr Asp Arg Asn Gly Gln Leu Thr Ser Gly Lys Gly Ala Leu
Pro Lys Thr Gly Glu Thr Thr Glu Arg Pro Ala Phe Gly Phe Leu Gly
Val Ile Val Val Ser Leu Met Gly Val Leu Gly Leu Lys Arg Lys Gln
Arg Glu Glu
[0010]
The target protein in the present invention refers to a useful protein that is expressed on the surface of a lactic acid bacterium according to the culture of the lactic acid bacterium serving as a host. For example, enzymes such as staphylokinase (SAK) shown in Examples described later, partial proteins of pathogenic bacteria, physiologically active proteins, and the like are also considered. That is, there is no particular limitation as long as a gene for an arbitrary protein has been isolated and can be combined with the anchor sequence using a general gene recombination technique to create a gene for a fusion protein.
[0011]
In addition, the host bacterium in the present invention is a substance that takes a structure that binds to the microbial cells on the surface of the bacterium by the anchor sequence of 763 protease derived from Lactococcus l actis NCDO763 strain and is expressed on the microbial surface. If it is. Specifically, Lactococcus lactis , Lactobacillus casei , Streptococcus thermophilus , Enterococcus faecalis, Enterococcus faecalis , Staphylococcus phy Streptococcus mutans (Streptococcus mutans), lactic acid bacteria such as Streptococcus pyogenes (Streptococcus pyogenes), and the like.
[0012]
Preparation of a fusion gene sequence linking a gene sequence encoding an anchor sequence and a gene sequence encoding a target protein, introduction of the fusion gene sequence into a host bacterium, host bacterium into which the fusion gene sequence has been introduced The culture can be performed by a conventional method.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
According to the present invention, a fusion gene sequence obtained by linking a gene sequence encoding a 763 protease anchor sequence derived from Lactococcus lactis NCDO763 strain and a gene sequence encoding a target protein is obtained. A gene sequence is introduced into a host fungus and cultured to immobilize the target protein on the surface of the fungus body.
[0014]
As a result, for example, the enzyme is expressed on the surface of the host cell and immobilized, or a partial protein of a pathogenic bacterium is used to have a vaccine effect, or a physiologically active protein is used for pharmaceutical purposes. It is possible to use it. In Examples described later, a staphylokinase (SAK) protein having an excellent thrombolytic action was used as a model, but various modifications can be made depending on the target protein.
[0015]
Introduction of a gene sequence encoding a fusion protein of an anchor sequence and a target protein into a host bacterium can be performed using a general gene recombination technique. For example, the gene sequence can be retained in a plasmid that can be introduced into a lactic acid bacterium serving as a host bacterium, and this plasmid can be introduced into the host bacterium.
[0016]
Regarding the anchor sequence of 763 protease derived from Lactococcus lactis NCDO763 strain, it is confirmed for the first time by the present invention that the 243 amino acid residue sequence to 36 amino acid residue sequence shown in SEQ ID NOs: 1 to 6 can be used. It was done. In particular, in the present invention, it was confirmed that the 55 amino acid residue sequence shown in SEQ ID NO: 5 can be used in Lactococcus lactis and Lactobacillus casei.
[0017]
In particular,
(1) 243 amino acid residue sequence shown in SEQ ID NO: 1
(2) 158 amino acid residue sequence shown in SEQ ID NO: 2
(3) 125 amino acid residue sequence shown in SEQ ID NO: 3
(4) 96 amino acid residue sequence shown in SEQ ID NO: 4
(5) 55 amino acid residue sequence shown in SEQ ID NO: 5
(6) It has been confirmed that the 36 amino acid residue sequence shown in SEQ ID NO: 6 can be used, and other partial sequences can be used within these ranges.
[0018]
More specifically, the sequence shown in SEQ ID NO: 2 has a structure in which 85 amino acid residues on the N-terminal side of SEQ ID NO: 1 have been excised. Similarly, the sequence shown in SEQ ID NO: 3 has a structure in which 118 amino acid residues on the N-terminal side of SEQ ID NO: 1 have been excised. Similarly, the sequence shown in SEQ ID NO: 4 has a structure in which 147 amino acid residues on the N-terminal side of SEQ ID NO: 1 have been excised. The sequence shown in SEQ ID NO: 5 has a structure in which 198 amino acid residues on the N-terminal side of SEQ ID NO: 1 have been excised. Similarly, the sequence shown in SEQ ID NO: 6 has a structure in which 27 amino acid residues on the N-terminal side of SEQ ID NO: 1 have been excised.
[0019]
In addition, the fusion protein according to the present invention includes a gene encoding a protein for the purpose of active expression (in the examples, a SAK protein) and a gene encoding an amino acid sequence used as an anchor sequence in a conventional manner. It is a combination.
[0020]
By integrating this fusion protein into an arbitrarily selected vector (for example, pSG-TERM) and transforming it into a suitable host as a plasmid, a microorganism having the target protein immobilized on the surface of the cell can be obtained. The gene binding, amplification, transformation method to the host, etc. can be performed by combining well-known methods in the field of gene recombination research.
[0021]
【Example】
With the aim of creating an expression system on the cell surface via anchor sequences using food-grade lactic acid bacteria, secretory protein staphylokinase (SAK) and protease produced by Lactococcus lactis NCDO763 ( 763 protease) and a fusion protein with the anchor sequence part were constructed, and the function as an anchor was examined.
[0022]
1. Material Escherichia coli (E. Coli) JM109 strain and (Toyobo Co., Ltd.), and Lactococcus lactis (Lactococcus lactis) YIT2081 strain, and Lactobacillus casei (Lactobacillus casei) YIT9029 strain was used as the host. Escherichia coli was cultured in L-broth, and lactic acid bacteria were cultured in M17-glucose medium. As a vector, plasmid pSG-TERM was used. PBE31SAK1 and pMQ9080 were used as template DNA for PCR.
[0023]
2. Shuttle vector pSG-TERM
FIG. 1 is an explanatory view schematically showing the structure of plasmid pSG-TERM. This plasmid has an Acc-SmaI fragment of about 0.21 kb deleted from shuttle vector pBE31 (see JP-A-6-253861) that functions in Escherichia coli and lactic acid bacteria, and is derived from an erythromycin-resistant gene at the BamHI and SphI sites. About 0 including the promoter (Em promoter), SD sequence and signal sequence derived from 763 protease gene (763 protease signal seq.), Multiple restriction enzyme recognition sequence (MCS), and terminator sequence derived from 763 protease gene (763 protease terminator) A .46 kb DNA fragment was inserted into pSG-TERM.
[0024]
3. pMQ9080
A 3.1 kb BglII fragment derived from pLP763 containing the C-terminus of 763 protease was cloned into the BamHI site of E. coli plasmid pUC119 to obtain pMQ9080.
[0025]
4). Each primer sequence and synthesis DNA primers having the following sequences were synthesized using a DNA synthesizer (Model 392, Abride Biosystems).

[0026]
5. Production of DNA Fragment Amplification of gene DNA was performed by the PCR method using KOD DNA polymerase (Toyobo Co., Ltd.). Amplification of the SAK gene was performed by using pBE31SAK1 as a template and repeating the cycle 30 times at 94 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 74 ° C. for 60 seconds between the primers SGSAK5 and SAKSAL1R.
[0027]
For amplification of the anchor sequence, one cycle of primers MK245, MK160, MK127, MK098, MK057, MK038 and ANCSPH1 at 98 ° C for 15 seconds, 60 ° C for 5 seconds, and 74 ° C for 30 seconds using the DraI fragment of pMQ9080 as a template And this was performed under the condition of repeating 30 times.
[0028]
6). Production of recombinant plasmids
(1) Preparation of pSAK-NS A DNA fragment containing the SAK gene amplified by PCR is cleaved with NspV and SalI, purified, and then inserted into the NspV-SalI site of pSG-TERM to produce pSAK-NS. did. FIG. 2 is an explanatory view schematically showing the structure of a SAK-anchor sequence fusion protein gene. As shown in FIG. 2a, pSAK-NS has a SAK gene-derived SD sequence (SD), a SAK signal sequence (signal seq.), And a SalI site 3 ′ inserted downstream of the promoter of the erythromycin resistance gene. Therefore, it has a structure in which a SAK mature protein with 2 amino acid residues added to the C-terminal, a stop codon, and a terminator sequence (terminator) derived from lactic acid bacteria protease are connected.
[0029]
(2) Preparation of pSAK245, pSAK160, pSAK127, pSAK098, pSAK057, and pSAK038 The DNA fragment containing the anchor sequence grown by PCR was cleaved with SalI and SphI, and then inserted into the SalI-SphI site of pSAK-NS. As a result, pSAK245, pSAK160, pSAK127, pSAK098, pSAK057, and pSAK038 were prepared. As shown in FIGS. 2b to 2g, these plasmids have a protease-derived anchor sequence from the C terminus to 243,158 via two amino acid residues derived from the SalI site at the C terminus of the SAK protein, respectively. , 125, 96, 55, and 36 amino acid residues are encoded. As for the obtained plasmids, pSAK245 was deposited as Seikenken No. 15909, and pSAK038 was deposited as Seikenken 15910 as of October 17, 2008, respectively. ing. In addition, the other plasmids were not deposited because they were judged to be obtained by excising the desired base sequence of pSAK245.
[0030]
7). Transformation of the host plasmid DNA was first confirmed for its structure using Escherichia coli as a host.
Thereafter, each plasmid DNA purified from E. coli (pSAK245, pSAK160, pSAK127, pSAK098, pSAK057, pSAK038) was introduced into the Lactococcus lactis YIT2018 strain by electroporation, and SAK-NS, SAK245, SAK160, SAK127. , SAK098, SAK057, SAK038 were obtained. The lactic acid bacteria clones prepared and analyzed in this example are summarized in Table 1 above.
[0031]
[Table 1]

[0032]
8). Confirmation of SAK protein expression
(1) Measurement of SAK activity The culture broth cultured overnight was used as the culture broth fraction, and the bacterial pellet obtained by centrifuging this was resuspended in M17-glucose medium. The reaction solution (chromozyme PL 0.5 mM, plasminogen 0.06 units / ml, polysorbate 80 0.01%, 0.1 M Tris-HCl pH 8.0) was reacted with 100 μl at 37 ° C., and the absorbance at 405 nm over time was measured with a microplate reader model 450 (Bio Rad).
[0033]
FIG. 3 is a diagram showing the results of measuring SAK activity, where a shows the culture broth fraction, and b shows the fungus body fraction. The vertical axis represents absorbance, and the horizontal axis represents reaction time. As shown in FIG. 3, the activity was detected in all clones except that the activity was not observed only with the vector into which the SAK gene was not introduced.
[0034]
(2) SDS-PAGE and Western blotting Lactococcus lactis clones 1 ml of an overnight culture were centrifuged to obtain a culture supernatant and cell fraction. The culture supernatant was suspended in 20 μl of 2 × SDS sample buffer after TCA precipitation and heated. The bacterial cell fraction was similarly suspended in 20 μl of 2 × SDS sample buffer and heated. These samples were subjected to SDS electrophoresis with Phast System 20% Phast gel (Pharmacia), transferred to a PVDF membrane, anti-SAK antiserum as the primary antibody, and alkaline phosphatase-labeled anti-rabbit IgG antibody as the secondary antibody. Used for Western blotting.
[0035]
FIG. 4 is an explanatory view schematically showing the results of Western blotting, showing the culture supernatant (sup) and the cell fraction (cell) for each clone in order. As shown in the figure, no SAK protein was detected in the vector into which no SAK gene was introduced. In the cell fraction, it is considered that the surface protein of the cell is extracted by the SDS sample buffer. In the culture supernatant, a large number of low molecular weight bands that were considered to have been degraded by the SAK (fusion) protein were detected.
[0036]
(3) Detection of SAK (fusion) protein on the cell surface using an alkaline phosphatase labeled antibody Each plasmid (pSAK245, pSAK160, pSAK127, pSAK098, pSAK057, pSAK038) was similarly introduced by electroporation, A clone was prepared using Lactobacillus casei .
[0037]
1 ml of an overnight culture of Lactococcus lactis clone and Lactobacillus casei clone was centrifuged to obtain a cell fraction. The obtained cells were washed twice with 1 ml of 50 mM Tris-HCl pH7.4, suspended in 0.5 ml of 1% BSA / TBST, kept at room temperature for 30 to 60 minutes, and 0.5 ml of TBST containing 1 μl of anti-SAK antiserum. And suspended at room temperature for 30 to 60 minutes, and then washed 3 times with 0.5 ml of TBST. Then, suspended in 0.5 ml of TBST containing 0.25 μl of alkaline phosphatase-labeled antibody rabbit IgG antibody and kept at room temperature for 30-60 minutes, washed 3 times with 0.5 ml of TBST and suspended in TBST so that OD ₆₆₀ becomes 7.5. It became cloudy and used as a sample.
[0038]
To 10 μl of the obtained sample, 100 μl of a substrate solution (1 mg p-Nitrophenyl Phosphate / 10 ml diethanolamine buffer) was added, reacted at room temperature, and absorbance at 405 nm was measured with a microplate reader model 450 (manufactured by Bio Rad) over time.
[0039]
FIG. 5 is a diagram showing the results of detection of SAK (fusion) protein on the cell surface using an alkaline phosphatase-labeled antibody. FIG. 5 shows a Lactococcus lactis and FIG. 5B shows a Lactobacillus casei ( Lactobacillus casei). casei ). The vertical axis represents absorbance, and the horizontal axis represents reaction time (min).
[0040]
In the figure, the activity of absorbance indicates the activity of alkaline phosphatase (AP) bound to the cells. That is, the rabbit antibody SAK antibody binds to the SAK expressed on the surface of the bacterial cell, and further, the rabbit IgG antibody labeled with alkaline phosphatase binds to this, so that if the alkaline phosphatase activity is high, the antibody is expressed on the cell surface. There will be a large number of SAKs. Therefore, the steeper slope of the graph shown in the figure indicates that the AP activity is higher, and the amount of AP-labeled anti-peptidic IgG antibody bound to the cells is larger. This means that in the clone, the amount of rabbit anti-SAK antibody on the cell surface is large, that is, there are many SAK protein epitopes displayed on the cell surface.
[0041]
As shown in the figure, it was confirmed that any of Lactococcus lactis bacteria and Lactobacillus casei bacteria having a large number of amino acid residues has high AP activity, that is, expression of SAK on the cell surface. . 36 amino acid residues are Although the difference from the control (vector, NS) was confirmed in Lactis, In Casei, the difference could not be confirmed. This may be because the fusion protein is buried in the cell wall. Therefore, an anchor sequence preferably exceeding 36 amino acid residues is required.
[0042]
【The invention's effect】
As described above, the present invention uses the anchor sequence of a protease derived from Lactococcus lactis or a partial sequence of the anchor sequence to immobilize various arbitrary useful proteins on the cell surface, There is an effect of expressing. Therefore, according to the present invention, it is possible to immobilize various proteins using, for example, lactic acid bacteria with high safety, so that it can be used for oral vaccines with high safety.
[0043]
[Sequence Listing]

[0044]

[0045]

[0046]

[0047]

[0048]

[Brief description of the drawings]
FIG. 1 is an explanatory view schematically showing the structure of a plasmid pSG-TERM.
FIG. 2 is an explanatory view schematically showing the structure of a SAK-anchor sequence fusion protein gene.
FIG. 3 is a diagram showing the results of measuring SAK activity, where a shows the culture broth fraction and b shows the bacterial cell fraction. The vertical axis represents absorbance, and the horizontal axis represents reaction time.
FIG. 4 is an explanatory diagram schematically showing the results of Western blotting, showing a culture supernatant (sup) and a cell fraction (cell) for each clone in order.
FIG. 5 is a diagram showing the detection results of SAK (fusion) protein on the bacterial cell surface using an alkaline phosphatase-labeled antibody. FIG. 5a shows Lactococcus lactis , and FIG. B shows Lactococcus lactis ( Lactococcus lactis ). Lactobacillus casei ). The vertical axis represents absorbance, and the horizontal axis represents reaction time (min).

Claims (2)

Obtaining a fusion gene sequence in which a gene sequence encoding the anchor sequence of 763 protease derived from Lactococcus lactis NCDO763 strain and a gene sequence encoding the target protein are obtained; Gram-positive bacterial cell surface comprising a step of introducing into a host cell, and a step of culturing the host cell into which the fusion gene sequence has been introduced and immobilizing the target protein on the surface of the Gram-positive cell In the method of immobilizing proteins to
A method for immobilizing a protein on the surface of a Gram-positive bacterial cell, wherein the anchor sequence has at least the following amino acid sequence.
Thr Ser Thr Asp Asp Thr Thr Asp Arg Asn Gly Gln Leu Thr Ser Gly
Lys Gly Ala Leu Pro Lys Thr Gly Glu Thr Thr Glu Arg Pro Ala Phe
Gly Phe Leu Gly Val Ile Val Val Ser Leu Met Gly Val Leu Gly Leu
Lys Arg Lys Gln Arg Glu Glu Said anchor sequence is fixed of the protein to the Gram-positive bacterial cell surface according to claim 1, leaving the amino acid sequence shown in claim 1, characterized in that the amino acid sequence was excised part following amino acid sequence Method.
Lys Lys Thr Ser Leu Leu Asn Gln Leu Gln Ser Val Lys Ala Ala Leu
Glu Thr Asp Leu Gly Asn Gln Thr Asp Ser Ser Thr Gly Lys Thr Phe
Thr Ala Ala Leu Asp Asp Leu Val Ala Gln Ala Gln Ala Gly Thr Gln
Thr Asp Asp Gln Leu Gln Ala Thr Leu Ala Lys Val Leu Asp Ala Val
Leu Ala Lys Leu Ala Glu Gly Ile Lys Ala Ala Thr Pro Ala Glu Val
Gly Asn Ala Lys Asp Ala Ala Thr Gly Lys Thr Trp Tyr Ala Asp Ile
Ala Asp Thr Leu Thr Ser Gly Gln Ala Ser Ala Asp Ala Ser Asp Lys
Leu Ala His Leu Gln Ala Leu Gln Ser Leu Lys Thr Lys Val Ala Ala
Ala Val Glu Ala Ala Lys Thr Val Gly Lys Gly Asp Gly Thr Thr Gly
Thr Ser Asp Lys Gly Gly Gly Gln Gly Thr Pro Ala Pro Thr Pro Gly
Asp Ile Gly Lys Asp Lys Gly Asp Glu Gly Ser Gln Pro Ser Ser Gly
Gly Asn Ile Pro Thr Asn Pro Ala Thr Thr Thr Ser Thr Ser Thr Asp
Asp Thr Thr Asp Arg Asn Gly Gln Leu Thr Ser Gly Lys Gly Ala Leu
Pro Lys Thr Gly Glu Thr Thr Glu Arg Pro Ala Phe Gly Phe Leu Gly
Val Ile Val Val Ser Leu Met Gly Val Leu Gly Leu Lys Arg Lys Gln
Arg Glu Glu

Images