JP4220033B2 - Novel polypeptide - Google Patents

Description

【０２５４】
【発明の効果】
本発明により、ポリ-N-アセチルラクトサミン糖鎖の合成に関与する活性を有するポリペプチド、該ポリペプチドの製造法、該ポリペプチドをコードするＤＮＡ、該ＤＮＡの製造法、該ＤＮＡを組み込んで得られる組換え体ベクター、該組換え体ベクターを保有する形質転換体、該ポリペプチドを認識する抗体、該ＤＮＡあるいは該ポリペプチドを用いるポリ-N-アセチルラクトサミン糖鎖の製造法、該ＤＮＡ、該ポリペプチドあるいは該抗体を用いた炎症または癌転移の疾病の診断および治療、該抗体を用いる本発明のポリペプチドの定量法および免疫染色、該ポリぺプチドをコードする遺伝子の発現を変動させる化合物のスクリーニング法、該ポリペプチドの有する活性を変動させる物質のスクリーニング法を提供することができる。
【０２５５】
【配列表】

【０２５６】
【配列表フリーテキスト】
配列番号３−人工配列の説明：合成ＤＮＡ
配列番号４−人工配列の説明：合成ＤＮＡ
配列番号５−人工配列の説明：合成ＤＮＡ
配列番号６−人工配列の説明：合成ＤＮＡ
配列番号７−人工配列の説明：合成ＤＮＡ
配列番号８−人工配列の説明：合成ＤＮＡ
配列番号９−人工配列の説明：合成ＤＮＡ
配列番号１０−人工配列の説明：合成ＤＮＡ
配列番号１１−人工配列の説明：合成ＤＮＡ
配列番号１２−人工配列の説明：合成ＤＮＡ
配列番号１３−人工配列の説明：合成ＤＮＡ
配列番号１４−人工配列の説明：合成ＤＮＡ
配列番号１５−人工配列の説明：合成ＤＮＡ
配列番号１６−人工配列の説明：合成ＤＮＡ
配列番号１７−人工配列の説明：市販のアミノ酸配列
【図面の簡単な説明】
【図１】発現クローニングの過程を示したものである。WM266-4およびSW1116由来のｃＤＮＡライブラリーを導入したNamalwa KJM-1細胞を、抗ｉ抗体を用いて蛍光染色した後、FACSに供し、抗ｉ抗体との反応性が高い細胞を集めた。バーで示した部分の細胞を集め、次のFACSに供した。3回のFACSの後、抗ｉ抗体との反応性が高い細胞が濃縮された。
【図２】抗ｉ抗体との反応性が高い細胞から回収されたプラスミド（１６−２−１２および16-2-31）、およびコントロールプラスミドpAMoを、それぞれNamalwa KJM-1細胞に導入し、抗ｉ抗体（太線）またはA-PBS（細線）を用いた間接蛍光抗体染色の後、FACSを用いて解析した結果である。
【図３】プラスミド16-2-12が含有するｃＤＮＡ、およびその近傍のベクター部分（太線）の制限酵素マップを示したものである。ナンバーのついたバーは、ｃＤＮＡの塩基配列を決定するためにpBluescriptIISK(+)にサブクローン化したＤＮＡ断片を示している。詳細は本文参照。
【図４】プラスミドpAMo-iの造成工程を示す図である。
【図５】ｉＧｎＴ発現プラスミドpAMo-iおよびコントロールプラスミドpAMoを、それぞれNamalwa KJM-1細胞に導入し、抗ｉ抗体（太線）またはA-PBS（細線）を用いた間接蛍光抗体染色の後、FACSを用いて解析した結果である。間接蛍光抗体染色の前に、細胞をsialidase処理したもの（+sialidase）と、しないもの（-sialidase）で反応性を比較した。
【図６】プラスミドpAMoA-FT3の造成工程を示す図である。
【図７】プラスミドpAMoA-i52Sの造成工程を示す図である。
【図８】プラスミドpAMoA-i52Sを導入したNamalwa KJM-1細胞の培養上清から、ＩｇＧセファロースを用いてプロテインＡ融合分泌型ｉＧｎＴを精製し、ＳＤＳポリアクリルアミドゲル電気泳動に供した結果である（レーン２）。コントロールとして、pAMoAを導入したNamalwa KJM-1細胞の培養上清から同様にしてサンプルを調製し、ＳＤＳポリアクリルアミドゲル電気泳動に供した（レーン１）。
【図９】プラスミドpAMoF2の造成工程を示す図である。
【図１０】プラスミドpT7B-i52S No.3の造成工程を示す図である。
【図１１】プラスミドpAMoF2-i52Sの造成工程を示す図である。
【図１２】プラスミドpAMoF2-i52Sを導入したNamalwa KJM-1細胞の培養上清から、抗Ｆｌａｇ Ｍ１アフィニティーゲルを用いてＦｌａｇペプチド融合分泌型ｉＧｎＴを精製し、ＳＤＳポリアクリルアミドゲル電気泳動に供した結果である（レーン２）。コントロールとして、pAMoF2を導入したNamalwa KJM-1細胞の培養上清から同様にしてサンプルを調製し、ＳＤＳポリアクリルアミドゲル電気泳動に供した（レーン１）。
【図１３】プラスミドpVL1393-iの造成工程を示す図である。
【図１４】プラスミドpVL1393-Ai52Sの造成工程を示す図である。
【図１５】プラスミドpVL1393-Ai52S由来の組換えウイルスが感染したSf21細胞の培養上清から、ＩｇＧセファロースを用いてプロテインＡ融合分泌型ｉＧｎＴを精製し、ＳＤＳポリアクリルアミドゲル電気泳動に供した結果である （レーン２）。コントロールとして、プラスミドpVL1393由来の組換えウイルスが感染したSf21細胞の培養上清から同様にしてサンプルを調製し、ＳＤＳポリアクリルアミドゲル電気泳動に供した（レーン１）。
【図１６】プラスミドpVL1393-F2i52Sの造成工程を示す図である。
【図１７】プラスミドpVL1393-F2i52S由来の組換えウイルスが感染したSf21細胞の培養上清から、抗Ｆｌａｇ Ｍ１アフィニティーゲルを用いてＦｌａｇペプチド融合分泌型ｉＧｎＴを精製し、ＳＤＳポリアクリルアミドゲル電気泳動に供した結果である（レーン２）。コントロールとして、プラスミドpVL1393由来の組換えウイルスが感染したSf21細胞の培養上清から同様にしてサンプルを調製し、ＳＤＳポリアクリルアミドゲル電気泳動に供した（レーン１）。
【図１８】プラスミドpBSK+ｉＧｎＴ13の造成工程を示す図である。
【図１９】プラスミドpBSK+ｉＧｎＴ13dの造成工程を示す図である。
【符号の説明】
bp：塩基対 (base pairs)
kb：キロ塩基対 (kilobase pairs)
G418 / Km ：トランスポゾン５(Tn5) 由来G418、カナマイシン耐性遺伝子
Ap：pBR322由来アンピシリン耐性遺伝子
Tc：pBR322由来テトラサイクリン耐性遺伝子
P1：pBR322由来P1プロモーター
Ptk：ヘルペス・シンプレックス・ウイルス(Herpes simplex virus; HSV) チミジンキナーゼ(tk)遺伝子プロモーター
Sp. BG：ラビットβグロビン遺伝子スプライシングシグナル
A.BG：ラビットβグロビン遺伝子ポリＡ付加シグナル
A. SE：シミアン・ウィルス (simian virus) 40 (SV40) 初期遺伝子ポリＡ付加シグナル
Atk：ヘルペス・シンプレックス・ウイルス(Herpes simplex virus ; HSV)チミジンキナーゼ(tk)遺伝子のポリＡ付加シグナル
Pse：シミアン・ウィルス (simian virus) 40 (SV40) 初期遺伝子プロモーター
Pmo：モロニー・マウス白血病ウイルスのロング・ターミナル・リピート（long terminal repeat : LTR）プロモーター
EBNA-1：エプシュタイン・バール・ウイルス（Epstein -Barr virus ）のEBNA-1遺伝子
oriP：エプシュタイン・バール・ウイルス（Epstein -Barr virus ）の複製開始点
S：ヒト顆粒球コロニー刺激因子または免疫グロブリンκのシグナルペプチドをコードする遺伝子部分
A または ProA：黄色ブドウ球菌StaphylococcusaureusのプロテインＡのＩｇＧとの結合領域をコードする遺伝子部分
F：Ｆｌａｇペプチドをコードする遺伝子部分
ｉＧｎＴ：本発明で取得したポリ-N-アセチルラクトサミン糖鎖の合成に関与するポリペプチドをコードするＤＮＡ（全長または部分長）
ｃＤＮＡ：本発明で取得したポリ-N-アセチルラクトサミン糖鎖の合成に関与するポリペプチドをコードするＤＮＡ（全長または部分長）
ｃＤＮＡ del：約0.2kbの欠失を有する、本発明で取得したポリ-N-アセチルラクトサミン糖鎖の合成に関与するポリペプチドをコードする部分長ＤＮＡ[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a polypeptide having an activity involved in the synthesis of a poly-N-acetyllactosamine sugar chain, a method for producing the polypeptide, a DNA encoding the polypeptide, a method for producing the DNA, and the DNA being incorporated. Recombinant vector, transformant containing the recombinant vector, antibody recognizing the novel polypeptide, method for producing poly-N-acetyllactosamine sugar chain using the polypeptide, the recombinant The present invention relates to a method for producing a poly-N-acetyllactosamine sugar chain using a transformant containing a vector.
[0002]
[Prior art]
In addition to being involved in life phenomena such as development / differentiation and cell recognition, sugar chains are thought to be deeply involved in the development and progression of inflammation, cancer, infectious diseases, autoimmune diseases, and many other diseases. [Kiyoyo, Hakomori Senichiro, Nagai Katsutaka, Glycobiology Series (1)-(6), Kodansha, (1993), Glycobiology,Three, 97 (1993)].
Sugar chains are added to proteins and lipids to exist as glycoproteins, proteoglycans, or glycolipids, and also exist as oligosaccharides.
[0003]
The poly-N-acetyllactosamine sugar chain targeted in the present invention is a sugar having a structure [(Galβ1-4GlcNAcβ1-3) n; n is 2 or more] in which N-acetyllactosamine is repeatedly bonded by β1,3 bonds. It is present in glycoprotein N-glycoside-linked sugar chains and O-glycoside-linked sugar chains, as well as in glycolipid sugar chains and oligosaccharides.
[0004]
Poly-N-acetyllactosamine sugar chains are synthesized by the alternating action of β1,4-galactosyltransferase and β1,3-N-acetylglucosaminetransferase. The former β1,4-galactosyltransferase gene has already been cloned, but the latter β1,3-N-acetylglucosaminyltransferase gene has not been cloned so far. Regarding β1,3-N-acetylglucosamine transferase involved in the synthesis of poly-N-acetyllactosamine sugar chain, only partial purification has been reported so far, and no amino acid sequence information has been obtained [J Biol. Chem.,268, 27118 (1993), J. Biol. Chem.,267, 2994 (1992), J. Biol. Chem.,263, 12461 (1988), Jpn. J. Med. Sci. Biol.,42, 77 (1989)].
[0005]
N-acetylglucosamine is often linked to the galactose residue in the poly-N-acetyllactosamine sugar chain via a β1,6 bond, such as Galβ1-4GlcNAcβ1-3 (Galβ1-4GlcNAcβ1-6) Galβ1-4GlcNAc A poly-N-acetyllactosamine sugar chain having a chain is synthesized. A glycosyltransferase that transfers β1,6-linked N-acetylglucosamine at this branched portion is β1,6-N-acetylglucosaminetransferase (I-branching enzyme). This enzyme gene has already been cloned. The linear poly-N-acetyllactosamine sugar chain (i antigen) is specifically produced by the anti-i antibody, and the branched poly-N-acetyllactosamine sugar chain (I antigen) is specifically produced by the anti-I antibody. Recognized [J. Biol. Chem.,254, 3221 (1979)].
[0006]
On a linear or branched poly-N-acetyllactosamine sugar chain, sugars such as fucose, sialic acid, N-acetylgalactlactosamine, galactose, or sulfate groups are added, and cell-specific or timing Various sugar chains (functional sugar chains, blood type sugar chains, cancer-related sugar chains, etc.) are formed specifically [Yo Kiso, Seiichiro Hakomori, Katsutaka Nagai, Glycobiology Series (1)-(6) ▼, Kodansha, (1993)].
It is known that a poly-N-acetyllactosamine sugar chain having a Sialyl-Lewis x sugar chain structure is present at the end of the sugar chain on granulocytes, monocytes, or activated T cells, These sugar chains function as ligands for adhesion molecules such as E-selectin and P-selectin, and are thought to be involved in the accumulation of these leukocytes in the inflammatory site [Kiyoyo, Hakomori Senichiro, Edited by Katsutaka Nagai, Glycobiology series (1)-(6), Kodansha, (1993)].
[0007]
In addition, it is known that poly-N-acetyllactosamine sugar chains having Sialyl-Lewis x sugar chains and Sialyl-Lewisa sugar chain structures are present on cancer cells such as colorectal cancer, These sugar chains also function as ligands for E-selectin and P-selectin and have been suggested to be involved in cancer metastasis [edited by Yosuke Kiso, Senichiro Hakomori, Katsutaka Nagai, Glycobiology Series ▲ 1 ▼ ~ ▲ 6 ▼, Kodansha (1993)].
[0008]
Poly-N-acetyllactosamine sugar chain structure is known to change during embryogenesis, cell differentiation, or canceration of cells [edited by Yosuke Kiso, Senichiro Hakomori, Katsutaka Nagai, glycobiology Series (1)-(6), Kodansha, (1993)]. In human fetal erythrocytes, linear poly-N-acetyllactosamine sugar chains are expressed, whereas in adult erythrocytes, branched poly-N-acetyllactosamine sugar chains are expressed [Kiyoyo・ Senichiro Hakomori and Katsutaka Nagai, Glycobiology Series (1) “Diverse world of sugar chains”, Kodansha, 1993]. An ABO blood group antigen is expressed at the end of the poly-N-acetyllactosamine sugar chain on these erythrocytes. When a blood group antigen is expressed at each end of a branched poly-N-acetyllactosamine sugar chain, it becomes a multivalent antigen, and its ability to bind an antibody to the blood group sugar chain is higher than that of a linear antigen. 10^ThreeThat will also increase.
[0009]
It is known that a series of sugar chain antigens are regularly expressed during the development process of the early mouse embryo. SSEA-1 (stage specific embryonic antigen-1) antigen is Lewis x sugar chain [Galβ1-4 (Fucα1-3) GlcNAc] existing at the end of poly-N-acetyllactosamine sugar chain. Begins in the 8-cell stage, peaks in the morula, and gradually disappears after the blastocyst stage [Kiyoyo, Henmori Senichiro, Katsutaka Nagai, Glycobiology Series (3) “Glyco in Cellular Society Biology ", Kodansha, 1993]. The morula stage is a transitional stage in which embryo cells, which have been repeatedly subjected to simple numerical increases due to cell division, move to a blastocyst stage with a differentiated “morphology” for the first time. Mulberry embryo cells gather closely together just before forming a blastocyst, causing cell compaction. Addition of oligosaccharides with SSEA-1 antigen inhibits this cell compaction and subsequent normal development [J. Exp. Med.,160, 1591 (1984)]. It is also known that the adhesion of mouse teratocarcinoma cells is inhibited by anti-SSEA-1 antibodies [edited by Yoyo Kiso, Senichiro Hakomori and Katsutaka Nagai, Glycobiology Series (3) “ Glycobiology ", Kodansha, 1993]. The above indicates that the SSEA-1 antigen acts as an adhesion molecule or sugar chain signal and plays an important role in early embryogenesis.
[0010]
Cancer cells are known to express more poly-N-acetyllactosamine sugar chains than the corresponding normal cells [J. Biol. Chem.,259, 10834 (1984), J. Biol. Chem.,261, 10772 (1986), J. Biol. Chem.,266, 1772 (1991), J. Biol. Chem.,267, 5700 (1992)]. When N-ras proto-oncogene is expressed in NIH3T3 cells, the molecular weight of the N-linked glycans on the cell surface increases and the cells acquire the invasion ability. As the amount of N-acetyllactosamine sugar chain increases, the activities of β1,4-galactose transferase and β1,3-N-acetylglucosamine transferase involved in the synthesis of poly-N-acetyllactosamine sugar chain increase [J. Biol. Chem.,266, 21674 (1991)].
[0011]
Galectins are a group of lectins that have an affinity for β-galactoside, are involved in cell adhesion and signal transduction, and have been suggested to be associated with diseases such as cancer [Trends in Glycoscience and Glycotechnology,9, 9 (1997)]. There are 10 known mammals so far. Of these, galectin-1 and -3 are known to bind with high affinity to linear poly-N-acetyllactosamine sugar chains, and specific glycoproteins containing these sugar chains are Presumed to be ligands for these galectins (Trends in Glycoscience and Glycotechnology,9, 9 (1997), Trends in Glycoscience and Glycotechnology,9, 47 (1997)].
Poly-N-acetyllactosamine sugar chain with sialic acid added to the end serves as a receptor for mycoplasma and microorganisms [Acta Paediatrica,82903 (1993)].
In this way, poly-N-acetyllactosamine sugar chains are composed of many functional sugar chains (selectin ligand sugar chains, microorganism and virus receptor sugar chains, SSEA-1 sugar chains, cancer-related sugar chains, etc.) and blood It plays an important role in forming the skeleton sugar chains of the type sugar chains and efficiently presenting those sugar chains.
Poly-N-acetyllactosamine sugar chain having Sialyl Lewis x sugar chain is expected to be a selectin antagonist and to be a drug having anti-inflammatory effect or cancer metastasis inhibiting effect.
[0012]
Oligosaccharides with polyvalent (4) Sialyl Lewis x (tetrasaccharide) bound to poly-N-acetyllactosamine sugar chain compared to non-polyvalent Sialyl Lewis x oligosaccharide (tetrasaccharide) It is known that it exhibits activity as a selectin antagonist at a low concentration of 1/100 or less [J. Exp. Med.,182, 1133 (1995), Glycobiology,6, 65 (1996), Glycobiology,7, 453 (1997), Eur. J. Immunol.,27, 1360 (1997)].
Partially purified β1,3-N-acetylglucosamine transferase was used to synthesize the poly-N-acetyllactosamine sugar chain portion of these oligosaccharides. It is difficult to synthesize N-acetyllactosamine sugar chains in large quantities [Glycobiology,7453 (1997)].
[0013]
On the other hand, poly-N-acetyllactosamine sugar chains can also be synthesized by chemical synthesis, but the synthesis requires very complicated steps [Tetrahedron Letter,twenty four, 5223 (1997)].
In view of the above, an efficient synthesis method of poly-N-acetyllactosamine sugar chain is required.
[0014]
It is known that various oligosaccharides having a poly-N-acetyllactosamine sugar chain structure exist in human milk [Acta Paediatrica,82903 (1993)]. These oligosaccharides are thought to have a function of preventing infants from being infected with viruses and microorganisms and a function of neutralizing toxins. It also has the activity of promoting the growth of bifidobacteria, which are good intestinal bacteria. On the other hand, there are few kinds of oligosaccharides present in the milk of animals such as cattle and mice, most are lactose, and oligosaccharides containing poly-N-acetyllactosamine sugar chains are hardly present [Acta Paediatrica,82, 903 (1993), J. Biol. Chem.,270, 29515 (1995)].
[0015]
If it is possible to efficiently produce various oligosaccharides containing poly-N-acetyllactosamine sugar chains contained in human milk as a mother nucleus, or milk containing them, it would be very useful industrially. But so far no such method is known.
Poly-N-acetyllactosamine sugar chains are also important for glycoprotein stabilization.
[0016]
lysosome associated membrane glycoprotein-1 (lamp-1) and lysosome associated membrane glycoprotein-2 (lamp-2) are glycoproteins that are present on lysosomes (some of which are also present on the cell surface). It ’s covered. Many sugar chains (some of which contain poly-N-acetyllactosamine sugar chains) are added to lamp-1 and lamp-2. Lamp-1 and lamp-2 are added by hydrolyzing enzymes in lysosomes. Preventing it from being disassembled. When human HL-60, a human osteoblastic cell line, is treated with dimethyl sulfoxide, it differentiates into granulocyte cells. During this differentiation, lamp-1 and lamp-2 It is known that the number of poly-N-acetyllactosamine sugar chains added to the enzyme increases, and the metabolic rate (degradation rate) of lamp-1 and lamp-2 decreases with it [J. Biol. Chem. ,265, 20476 (1990)].
[0017]
Since poly-N-acetyllactosamine sugar chains contribute to protein stabilization, it is possible to stabilize proteins by artificially adding poly-N-acetyllactosamine sugar chains to any protein. Conceivable. In addition, since the clearance rate of blood protein from the kidney decreases as the effective molecular weight of the protein increases, the effective molecular weight is increased by artificially adding poly-N-acetyllactosamine sugar chains to any protein. Thus, it is considered that the clearance rate from the kidney can be reduced and the blood stability can be increased. Furthermore, by adding a poly-N-acetyllactosamine sugar chain, there is a possibility that any protein can be targeted to a specific cell.
[0018]
When F9 cells were treated with retinoic acid or Swiss 3T3 cells were treated with TGF-β, it was shown that poly-N-acetyllactosamine sugar chains were added to the sugar chains of cell membrane-bound glycoproteins. [J. Biol. Chem.,268, 1242 (1993), Biochim.
Biophys. Acta.,1221, 330 (1994)].
When N-ras proto-oncogene is expressed in NIH3T3 cells, the activities of β1,4-galactosyltransferase and β1,3-N-acetylglucosaminetransferase involved in the synthesis of poly-N-acetyllactosamine sugar chains are increased. It is known that the amount of poly-N-acetyllactosamine sugar chain in the N-linked sugar chain of the membrane protein increases [J. Biol. Chem.,266, 21674 (1991)].
[0019]
When the core 2β1,6-N-acetylglucosaminyltransferase gene is expressed in the T cell line EL-4, the molecular weight of CD43, CD45, or CD44, which is a membrane protein on the cell surface, increases [J. Biol. Chem. ,271, 18732 (1996)]. This is because the sugar chain synthesized by core 2β1,6-N-acetylglucosamine transferase is a good substrate for β1,3-N-acetylglucosamine transferase, which is involved in the synthesis of poly-N-acetyllactosamine sugar chain. It is thought to be.
[0020]
It is also known that when HL-60 cells are cultured at 27 ° C., the amount of poly-N-acetyllactosamine sugar chain added to lamp-1 or lamp-2 increases (J. Biol. Chem. ,266, 23185 (1991)].
However, it is not clear whether the above method is effective in a host cell suitable for production of recombinant glycoprotein. Therefore, the development of methods for increasing the ability to synthesize poly-N-acetyllactosamine sugar chains in host cells (eg Namalwa cells, Namalwa KJM-1 cells, CHO cells, etc.) suitable for the production of recombinant glycoproteins This is an important issue.
[0021]
Considering the mechanism of inflammatory reaction and cancer metastasis described above, it is expected that the inflammatory reaction can be suppressed or cancer metastasis can be prevented by suppressing the expression of poly-N-acetyllactosamine sugar chain. However, a method for efficiently suppressing the expression of poly-N-acetyllactosamine sugar chain has not been known so far.
In addition, genes involved in the synthesis of poly-N-acetyllactosamine sugar chains in inflammatory leukocytes, cancer cells, or serum (for example, β1,3-, which is a key enzyme for poly-N-acetyllactosamine sugar chain synthesis) N-acetylglucosamine transferase genes and the like), or the expression of polypeptides encoded by them, is expected to diagnose the malignancy of inflammatory diseases and cancer. However, no such method has been known so far.
[0022]
[Problems to be solved by the invention]
The present invention provides pharmaceuticals such as anti-inflammation, anti-infection, and cancer metastasis suppression, foods such as dairy products, protein improvement methods, and diagnostic methods for malignancy of inflammatory diseases and cancer.
[0023]
[Means for Solving the Problems]
The present invention relates to a polypeptide having an activity involved in the synthesis of a poly-N-acetyllactosamine sugar chain, a method for producing the polypeptide, a DNA encoding the polypeptide, a method for producing the DNA, and incorporating the DNA Recombinant vector obtained, transformant harboring the recombinant vector, antibody recognizing the polypeptide, method for producing the DNA or poly-N-acetyllactosamine sugar chain using the polypeptide, the DNA Diagnosis and treatment of diseases such as inflammation or cancer using the polypeptide or the antibody, quantification method and immunostaining of the polypeptide of the present invention using the antibody, and changing the expression of the gene encoding the polypeptide The present invention relates to a method for screening a compound and a method for screening a substance that varies the activity of the polypeptide.
[0024]
Hereinafter, the present invention will be described in detail.
The DNA of the present invention is a DNA encoding a polypeptide having an activity involved in the synthesis of poly-N-acetyllactosamine sugar chain, for example, encoding a polypeptide having the amino acid sequence represented by SEQ ID NO: 1. Synthesis of a poly-N-acetyllactosamine sugar chain having an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence of DNA or a polypeptide having the amino acid sequence represented by SEQ ID NO: 1. DNA that encodes a polypeptide that participates in the DNA, or DNA that hybridizes with the DNA under stringent conditions and encodes a polypeptide that is involved in the synthesis of poly-N-acetyllactosamine sugar chains .
[0025]
Examples of the polypeptide having an activity involved in the synthesis of a poly-N-acetyllactosamine sugar chain include a polypeptide having β1,3-N-acetylglucosaminyltransferase activity.
DNA that can hybridize under stringent conditions means DNA obtained by using colony hybridization method, plaque hybridization method, Southern blot hybridization method, or the like using the DNA as a probe. Specifically, hybridization was performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then the concentration was 0.1 to 2 times. Examples of the DNA that can be identified by using an SSC solution (the composition of a 1-fold concentration SSC solution is 150 mM sodium chloride and 15 mM sodium citrate) and washing the filter at 65 ° C. can be mentioned. Hybridization can be performed according to the method described in Molecular Cloning 2nd edition. Specifically, the DNA capable of hybridizing is DNA having at least 60% homology with the base sequence of DNA encoding the polypeptide having the amino acid sequence represented by SEQ ID NO: 1, preferably 80% or more homology. And DNA having homology, more preferably DNA having homology of 95% or more.
[0026]
Examples of the polypeptide of the present invention include the polypeptide encoded by the above-described DNA. Specifically, the polypeptide having the amino acid sequence represented by SEQ ID NO: 1, or the amino acid represented by SEQ ID NO: 1 A polypeptide having an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the polypeptide having a sequence and having an activity involved in the synthesis of a poly-N-acetyllactosamine sugar chain, etc. I can give you.
[0027]
A polypeptide having an amino acid sequence in which one or more amino acids of the polypeptide amino acid sequence are substituted, deleted or added, and involved in the synthesis of a poly-N-acetyllactosamine sugar chain is Nucleic Acids Research,Ten, 6487 (1982), Proc. Natl. Acad. Sci., USA,79, 6409 (1982), Proc. Natl. Acad. Sci., USA,81, 5662 (1984), Science,224, 1431 (1984), PCT WO85 / 00817 (1985), Nature,316, 601 (1985), Gene,34, 315 (1985), Nucleic Acids Research,13, 4431 (1985), Current Protocols in Molecular Biology, Chapter 8, Mutagenesis of Cloned DNA, John Wiley & Sons, Inc. (1989) can do.
Examples of the antibody of the present invention include antibodies that recognize the aforementioned polypeptides.
[0028]
DETAILED DESCRIPTION OF THE INVENTION
(1) Method for producing DNA encoding a polypeptide involved in the synthesis of poly-N-acetyllactosamine sugar chain
A cDNA library is prepared from cells expressing poly-N-acetyllactosamine sugar chains by a conventional method.
[0029]
cDNA library preparation methods include Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology, Supplements 1-34 (Supplement 1-34), and Green Publishing Associates.・ Methods described in Greene Publishing Associates and Wiley-Interscience, 1987-1996 (hereinafter abbreviated as Current Protocols in Molecular Biology Supplements 1-34), etc., or Commercially available kits such as Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Gibco BRL) or Zapp-cDNA Seshisu Kit [ZAP-cDNA Synthesis Kit, Stratagene] a method using thereof.
[0030]
As the cell expressing poly-N-acetyllactosamine sugar chain, any cell expressing poly-N-acetyllactosamine sugar chain can be used, for example, human melanoma cell Strain WM266-4 (ATCC CRL 1676), human colon cancer cell line SW1116 (ATCC CRL 233), human monocytic cell line THP-1 (ATCC TIB 202), human monocytic cell line U-937 (ATCC CRL 1593) ), Human granulocyte cell line HL-60 (ATCC CCL 240) and the like can be used.
[0031]
As a cloning vector for preparing a cDNA library, any vector such as a phage vector or a plasmid vector can be used as long as it can autonomously replicate in E. coli K12. Specifically, ZAP Express (Stratagies, made by Stratagene,Five, 58 (1992)], pBluescript II SK (+) [Nucleic Acids Research,17, 9494 (1989)], λzap II (Stratagene), λgt10, λgt11 (DNA cloning, A Practical Approach),1, 49 (1985)], Lambda BlueMid (Clontech), λExCell (Pharmacia), pT7T3 18U (Pharmacia), pcD2 (Mol. Cell. Biol.),Three, 280 (1983)], pUC18 [Gene,33, 103 (1985)], pAMo [J. Biol. Chem.,268, 22782-22787 (1993), also known as pAMoPRC3Sc (Japanese Patent Laid-Open No. 05-336963)].
[0032]
As the host microorganism, any microorganism belonging to E. coli can be used. In particular,Escherichia coli XL1-Blue MRF '(Stratagies, made by Stratagene,Five, 81 (1992)),Escherichia coli C600 (Genetics,39, 440 (1954)),Escherichia coli Y1088 (Science,222, 778 (1983)),Escherichia co li Y1090 (Science,222, 778 (1983)),Escherichia coli NM522 [J. Mol. Biol.,166, 1 (1983)],Escherichia coli K802 [J. Mol. Biol.,16, 118 (1966)) andEscherichia coli JM105 (Gene,38, 275 (1985)].
[0033]
As a cDNA library, cDNA was synthesized from mRNA derived from WM266-4 or SW1116 using a cDNA Synthesis System kit manufactured by GIBCO BRL, and both ends of the DNA were synthesized.SfiAfter adding the I linker, the cloning vector pAMoSfiUsing a plasmid created by inserting into the I site,E.coli A cDNA library obtained by transforming LE392 can be mentioned.
[0034]
The inserted DNA is excised from the cDNA library and incorporated into a vector that can be expressed in animal cells or insect cells. When a vector that can be expressed in animal cells or insect cells is used at the time of preparation of the cDNA library, it can be used as an expression vector described below without performing this operation.
As the vector, any vector can be used as long as it can be expressed by incorporating the cDNA. For example, pcDNAI / Amp, pcDNAI, pCDM8 (all are commercially available from Funakoshi), pAGE107 [Japanese Patent Laid-Open No. 3-22979, Cytotechnology,Three, 133 (1990)], pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem.,101, 1307 (1987)], pAMo, pAMoA [J. Biol. Chem.,268, 22782-22787 (1993), also known as pAMoPRSA (Japanese Patent Laid-Open No. 05-336963)], pAS3-3 (Japanese Patent Laid-Open No. 2-227075), pVL1392 (manufactured by Invitrogen), pVL1393 (manufactured by Invitrogen), pBlueBacIII (manufactured by Invitrogen) Etc. can be used.
[0035]
An expression vector into which DNA excised from the cDNA library is incorporated is introduced into animal cells or insect cells capable of expressing the target DNA using the expression vector to obtain transformed cells.
As the method for introducing the expression vector, any method can be used as long as it is a method for introducing DNA into animal cells or insect cells. For example, electroporation [Cytotechnology,Three, 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), lipofection method [Proc. Natl. Acad. Sci. USA,84, 7413 (1987)), Baculovirus Expression Vectors, W.H.Freeman and Company, New York (1992), Molecular Biology, A Laboratory Manual, Current Protocols in Molecular Biology, Bio / Technology,6, 47 (1988).
[0036]
Animal cells or insect cells include human cells, Namalwa cells, monkey cells, COS cells, Chinese hamster cells, CHO cells, HBT5637 (Japanese Patent Laid-Open No. 63-299),SpodopterafrugiperdaOvary cells Sf9, Sf21 (Baculovirus Expression Vectors (1992)),TrichoplusianiHigh 5 (manufactured by Invitrogen) and the like, which are ovarian cells of the above, can be used. Preferred host cells include Namalwa cells.
[0037]
The obtained transformed cells are fluorescently stained with an anti-poly-N-acetyllactosamine sugar chain antibody or a lectin that recognizes poly-N-acetyllactosamine sugar chains, and then a fluorescence activated cell sorter. (Fluorescence Activated Cell Sorter; hereinafter abbreviated as FACS) is used to concentrate and separate cells that have increased binding amount of antibodies or lectins that recognize linear poly-N-acetyllactosamine sugar chains.
[0038]
As the antibody against poly-N-acetyllactosamine sugar chain, any antibody that reacts with poly-N-acetyllactosamine sugar chain can be used, for example, linear poly-N-acetyl Anti-i antibody [J. Biol. Chem., Which is an antibody that recognizes lactosamine sugar chain,254, 3221 (1979)]. Alternatively, a lectin that recognizes a poly-N-acetyllactosamine sugar chain can be used instead of an antibody, for example,DaturastramoniumLectin (DSA),LycopersiconesculentumLectin (LEA),PhytolaccaamericanaLectin (PWA) can be mentioned.
[0039]
A method known from the cells thus concentrated and separated, for example, the heart method [Mol. Cell. Biol.,8, 2837 (1988)], the plasmid containing the DNA of the present invention can be recovered and a DNA fragment containing the DNA can be obtained.
An example of a plasmid containing the DNA of the present invention is pVL1393-i. E. coli containing pVL1393-iEscherichia coli MM294 / pVL1393-i was deposited as FERM BP-6145 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology on October 16, 1997.
[0040]
The base sequence of the DNA obtained by the above-mentioned method is obtained by cleaving the DNA fragment as it is or with a suitable restriction enzyme and then incorporating the DNA fragment into a vector by a conventional method, for example, a base sequence analysis method commonly used, such as Sanger et al. Act [Proc. Natl. Acad. Sci. USA,74, 5463 (1977)] or a 373A DNA sequencer (manufactured by Perkin Elmer) or the like.
[0041]
Examples of the DNA obtained by the method include a DNA encoding the peptide represented by SEQ ID NO: 1, and specifically a DNA having the base sequence represented by SEQ ID NO: 2 be able to.
The target DNA can also be prepared by chemical synthesis with a DNA synthesizer based on the determined DNA base sequence. Examples of the DNA synthesizer include a DNA synthesizer manufactured by Shimadzu Corporation using the thiophosphite method, a DNA synthesizer model 392 manufactured by Perkin Elmer using the phosphoramidite method, and the like.
[0042]
In addition, using the oligonucleotides described below as sense and antisense primers, and using cDNA prepared from mRNA of cells expressing mRNA complementary to these DNAs as a template, polymerase chain reaction (hereinafter referred to as Polymerase Chain Reaction) , Abbreviated as PCR) [Molecular Cloning Second Edition and PCR Protocols Academic Press (1990)] and the like can also be used to prepare the target DNA.
[0043]
Oligonucleotides such as antisense oligonucleotides and sense oligonucleotides having a partial sequence of the DNA of the present invention using the DNA and DNA fragments of the present invention obtained by the above-described method, using a conventional method or a DNA synthesizer Can be prepared. Examples of the oligonucleotide include DNA having the same sequence as 10 to 50 bases in the base sequence of the DNA or DNA having a sequence complementary to the DNA. Specifically, SEQ ID NO: Examples thereof include DNA having the same sequence as 10 to 50 consecutive bases in the DNA having the base sequence represented by 2, or DNA having a sequence complementary to the DNA. When used as a sense primer and an antisense primer, the above-mentioned oligonucleotides in which the melting temperature (Tm) and the number of bases of both do not change extremely are preferable.
[0044]
Such oligonucleotides can also be mentioned as the DNA of the present invention.
Furthermore, derivatives of these oligonucleotides can also be mentioned as the DNA of the present invention. The derivative DNA includes a derivative DNA in which a phosphodiester bond in DNA is converted to a phosphorothioate bond, and a derivative DNA in which a phosphodiester bond in DNA is converted to an N3′-P5 ′ phosphoramidate bond. Derivative DNA in which ribose and phosphodiester bonds in DNA are converted to peptide nucleic acid bonds, Derivative DNA in which uracil in DNA is replaced with C-5 propynyluracil, and uracil in DNA is replaced with C-5 thiazole uracil Derivative DNA, derivative DNA in which cytosine in DNA is substituted with C-5 propynylcytosine, derivative DNA in which cytosine in DNA is substituted with phenoxazine-modified cytosine, ribose in DNA is 2 Derivative DNA substituted with '-O-propylribose, or D Can be mentioned derivative DNA ribose in A is substituted with 2'-methoxyethoxy ribose or the like [Cell Engineering,16, 1463 (1997)].
[0045]
(2) Method for producing a polypeptide having an activity involved in the synthesis of poly-N-acetyllactosamine sugar chain
In order to produce the polypeptide of the present invention by expressing the DNA of the present invention obtained by the above method in a host cell, Molecular Cloning 2nd Edition, Current Protocols in Molecular Biology Supplement 1 ~ 34 etc. can be used.
[0046]
That is, a recombinant vector in which the DNA of the present invention is inserted downstream of a promoter of an appropriate expression vector is constructed, and the vector is introduced into a host cell to obtain a transformant that expresses the polypeptide of the present invention. The polypeptide of the present invention can be produced by culturing the transformant.
As the host cell, any prokaryotic cell, yeast, animal cell, insect cell, plant cell or the like can be used so long as it can express the target gene. Moreover, an animal individual or a plant individual can be used.
[0047]
The expression vector contains a promoter at a position suitable for transcription of a gene involved in the synthesis of poly-N-acetyllactosamine sugar chain, which can be autonomously replicated in the above host cell or can be integrated into the chromosome. Is used.
When a prokaryote such as a bacterium is used as a host cell, an expression vector for a gene involved in the synthesis of a poly-N-acetyllactosamine sugar chain is capable of autonomous replication in a prokaryote, and at the same time a promoter, a ribosome binding sequence, It is preferably composed of a gene involved in the synthesis of a poly-N-acetyllactosamine sugar chain and a transcription termination sequence.
A gene that controls the promoter may also be included.
[0048]
Examples of expression vectors include pBTrp2, pBTac1, pBTac2 (all commercially available from Boehringer Mannheim), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN), pKYP10 (special 58-110600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci , USA, 82, 4306 (1985)], pBluescript II SK (-) (STRATAGENE), pBluescript II SK (+) (STRATAGENE), pTrs30 (FERM BP-5407), pTrs32 (FERM BP-5408), pGHA2 (FERM BP-400), pGKA2 (FERM BP-6798), pTerm2 (Japanese Patent Laid-Open No. 3-22979, US Pat. No. 4,686,191, US Pat. No. 4,939,09) 4, US 5,160,735), pKK233-2 (Pharmacia), pGEX (Pharmacia), pET system (Novagen), pSupex, pUB110, pTP5, pC194, pTrxFus (Invitrogen), pMAL-c2. (New England Biolabs), pEG400 [J. Bacteriol.,172, 2392 (1990)].
[0049]
Any promoter may be used as long as it functions in host cells such as E. coli. For example,trpPromoter (Ptrp),lacPromoter (Plac), P_LPromoter, P_RPromoter, PletI promoter, P_SEExamples of the promoter include promoters derived from E. coli and phages, SPO1 promoter, SPO2 promoter, penP promoter, and the like. In addition, a promoter (Ptrpx2),tacAn artificially designed and modified promoter such as a promoter can also be used.
It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence and the start codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
Although the transcription termination sequence is not necessarily required for the expression of the DNA of the present invention, it is preferable to arrange the transcription termination sequence immediately below the structural gene.
[0050]
Examples of host cells include microorganisms belonging to the genus Escherichia, Serratia, Corynebacterium, Brevibacterium, Pseudomonas, Bacillus, Microbacterium, etc., specifically,Escherichia coli XL1-Blue,Escherichia coli XL2-Blue,Escherichia coli DH1,Escherichia coli MC1000,Escherichia coli KY3276,Escherichia coli W1485,Escherichia coli JM109,Escherichia coli HB101,Escherichia coli No. 49,Escherichia coli W3110,Escherichia coli NY49,Escherichia coli GI698,Escherichia coli GI724,Escherichia coli TB1,Bacillussubtilis,Bacillusamyloliquefacines,Brevibacteriumammmoniagenes,BrevibacteriumimmariophilumATCC14068,BrevibacteriumsaccharolyticumATCC14066,Brevibacteriumflavum ATCC14067,Brevibacteriumlactofermentum ATCC13869,Corynebacterium glutamicum ATCC13032,Corynebacterium glutamicum ATCC14297,Corynebacterium glutamicum ATCC13869,Corynebacterium acetoacidophilum ATCC13870,MicrobacteriumammoniaphilumATCC15354,Pseudomonas putida,Pseudomonassp. D-0110,Serratiamarcescens,Serratiaficaria,Serratiafonticola,SerratialiuefaciensEtc.
[0051]
As a method for introducing the recombinant vector, any method can be used as long as it is a method for introducing DNA into the host cell, for example, a method using calcium ions [Proc. Natl. Acad. Sci. USA,69, 2110 (1972)], protoplast method (Japanese Patent Laid-Open No. 63-248394), Gene,17, 107 (1982) and Molecular & General Genetics,168, 111 (1979).
When yeast strains are used as host cells, examples of expression vectors include YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like.
[0052]
Any promoter can be used as long as it can be expressed in a yeast strain. For example, promoters such as PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, CUP1 promoter and the like can be mentioned.
Examples of host cells include yeast strains belonging to the genus Kluyveromyces, Saccharomyces, Schizosaccharomyces, Trichosporon, Swaniomyces, and the like.Saccharomyces cerevisiae,Schizosaccharomyces pombe,Kluyveromyces lactis,Trichosporonpullulans,SchwanniomycesalluviusEtc.
As a method for introducing the recombinant vector, any method for introducing DNA into yeast can be used. For example, the electroporation method [Methods Enzymol.,194, 182 (1990)], spheroplast method [Proc. Natl. Acad. Sci. USA,75, 1929 (1978)], lithium acetate method [J. Bacteriol.,153, 163 (1983)], Proc. Natl. Acad. Sci. USA,75, 1929 (1978).
[0053]
When animal cells are used as host cells, examples of expression vectors include pcDNAI, pcDM8 (commercially available from Funakoshi), pAGE107 [Japanese Patent Laid-Open No. 3-22979; Cytotechnology,Three, 133, (1990)], pAS3-3 (JP-A-2-27075), pCDM8 [Nature,329, 840, (1987)], pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 [J. Biochem.,1011307 (1987)], pAGE210 and the like.
[0054]
Any promoter can be used so long as it functions in animal cells. For example, cytomegalovirus (human CMV) IE (immediate early) gene promoter, SV40 early promoter, Moloney Mullin Leukemia Examples thereof include a virus (Moloney Murine Leukemia Virus) long terminal repeat promoter, a retrovirus promoter, a heat shock promoter, an SRα promoter, and a metallothionein promoter. In addition, an enhancer of human CMV IE gene may be used together with a promoter. Host cells include mouse myeloma cells, rat myeloma cells, mouse hybridoma cells, Chinese hamster CHO cells, BHK cells, African green monkey kidney cells, human cells Namalwa cells, human fetal kidney cells And human leukemia cells, HBT5637 (Japanese Patent Laid-Open No. 63-299), and the like.
[0055]
As mouse myeloma cells, SP2 / 0, NSO, etc., as rat myeloma cells, YB2 / 0, etc., as human embryonic kidney cells, HEK293 (ATCC: CRL-1573), 293, etc., as human leukemia cells, BALL -1, etc. Examples of African green monkey kidney cells include COS-1, COS-7, and the like.
As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into animal cells. For example, an electroporation method [Cytotechnology,Three, 133 (1990)], calcium phosphate method (JP-A-2-27075), lipofection method [Proc. Natl. Acad. Sci. USA,84, 7413 (1987)], Virology,52456 (1973).
[0056]
When insect cells are used as hosts, for example, Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), Molecular Biology Laboratory -Manual (Molecular Biology, A Laboratory Manual), Current Protocols in Molecular Biology Supplement 1-38 (Current Protocols in Molecular Biology), Bio / Technology,6, 47 (1988), etc., can be used to express the protein.
[0057]
That is, the recombinant gene transfer vector and baculovirus are co-introduced into insect cells to obtain the recombinant virus in the insect cell culture supernatant, and then the recombinant virus is further infected into insect cells to express the protein. it can.
Examples of gene transfer vectors used in the method include pVL1392, pVL1393, pBlueBacIII (all manufactured by Invitrogen) and the like.
[0058]
As the baculovirus, for example, Autographa californica nuclear polyhedrosis virus, which is a virus that infects the night stealing insects, can be used.
As an insect cell,SpodopterafrugiperdaOvary cells,TrichoplusianiOvary cells, cultured cells derived from silkworm ovary, and the like can be used.
[0059]
SpodopterafrugiperdaAs ovary cells, Sf9, Sf21 (Baculovirus Expression Vectors Laboratory Manual), etc.TrichoplusianiAs the ovarian cells of High 5, BTI-TN-5B1-4 (manufactured by Invitrogen), etc.Bombyxmori N4 etc. can be raised. Examples of the method for co-introducing the recombinant gene introduction vector and the baculovirus into insect cells for preparing a recombinant virus include, for example, the calcium phosphate method (JP-A-2-27075), the lipofection method [Proc. Natl. Acad Sci. USA,84, 7413 (1987)].
[0060]
In addition, DNA can be introduced into insect cells using a method similar to the method for introducing DNA into animal cells. For example, electroporation [Cytotechnology,Three, 133 (1990)], calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), lipofection method [Proc. Natl. Acad. Sci. USA,84, 7413 (1987)].
When a plant cell or plant individual is used as a host, a known method [tissue culture, 20 (1994), tissue culture, 21 (1995), Trends in Biotechnology,15, 45 (1997)] can be produced.
[0061]
Any promoter can be used as long as it can be expressed in plant cells, for example, cauliflower mosaic virus (CaMV) 35S promoter, rice actin 1 promoter and the like. In addition, gene expression efficiency can be increased by inserting intron 1 of a maize alcohol dehydrogenase gene between the promoter and the gene to be expressed.
[0062]
Examples of host cells include plant cells such as potato, tobacco, corn, rice, rape, soybean, tomato, wheat, barley, rye, alfalfa and flax.
As a method for introducing a recombinant vector, any method for introducing DNA into plant cells can be used. For example, Agrobacterium (Agrobacterium), Electroporation [Cytotechnology,Three, 133 (1990)], a method using a particle gun (gene gun), and the like.
[0063]
Plant cells and organs into which a gene has been introduced can be cultured in large quantities using a jar fermenter. In addition, a plant individual into which a gene has been introduced (transgenic plant) can be created by redifferentiating the plant cell into which the gene has been introduced.
The protein of the present invention can also be produced using an animal individual. For example, a known method [American Journal of Clinical Nutrition,63, 639S (1996), American Journal of Clinical Nutrition,63, 627S (1996), Bio / Technology,9, 830 (1991)], the protein of the present invention can be produced in an animal into which a gene has been introduced.
[0064]
Any promoter can be used as long as it can be expressed in animals. For example, α-casein promoter, β-casein promoter, β-lactoglobulin promoter, whey acidic protein promoter, etc., which are specific for mammary cells are suitable. Used for.
A transformant having a recombinant vector incorporating the DNA encoding the polypeptide of the present invention is cultured according to a normal culture method, the polypeptide is produced and accumulated, and the polypeptide is collected from the culture. Thus, the polypeptide can be produced.
[0065]
When the transformant is an animal individual or plant individual, the polypeptide is reared or cultivated according to a normal method, the polypeptide is produced and accumulated, and the polypeptide is collected from the animal individual or plant individual. Can be manufactured. That is, in the case of an individual animal, for example, a non-human transgenic animal carrying the DNA of the present invention is bred and has an activity involved in the synthesis of a poly-N-acetyllactosamine sugar chain encoded by the recombinant DNA. A polypeptide having activity involved in the synthesis of poly-N-acetyllactosamine sugar chain can be produced by generating and accumulating the polypeptide in the animal and collecting the polypeptide from the animal. . Examples of the production / accumulation place in the animal include milk and eggs of the animal.
[0066]
In the case of a plant individual, for example, a transgenic plant carrying the DNA of the present invention is cultivated, and a polypeptide having an activity involved in the synthesis of a poly-N-acetyllactosamine sugar chain encoded by the recombinant DNA is A polypeptide having an activity involved in the synthesis of a poly-N-acetyllactosamine sugar chain can be produced by producing and accumulating in a plant and collecting the polypeptide from the plant.
When the transformant for producing the polypeptide of the present invention is a prokaryote such as Escherichia coli or a eukaryote such as yeast, the medium for culturing these organisms includes a carbon source, a nitrogen source, inorganic salts that can be assimilated by the organism Any of a natural medium and a synthetic medium may be used as long as the medium can efficiently culture the transformant.
[0067]
Any carbon source may be used as long as it can be assimilated by each microorganism. Glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, organic acids such as acetic acid and propionic acid, ethanol Alcohols such as propanol are used.
Examples of nitrogen sources include ammonium salts of various inorganic and organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, and casein. A hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells, digested products thereof, and the like are used.
[0068]
As the inorganic salt, monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used. The culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40 ° C., and the culture time is usually 16 hours to 7 days. During the culture, the pH is maintained at 3.0 to 9.0. The pH of the medium is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.
[0069]
Moreover, you may add antibiotics, such as an ampicillin and a tetracycline, to a culture medium as needed during culture | cultivation.
When culturing a microorganism transformed with an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium as necessary.
For example,lacWhen culturing a microorganism transformed with an expression vector using a promoter, isopropyl-β-D-thiogalactopyranoside (IPTG), etc.,trpWhen culturing a microorganism transformed with an expression vector using a promoter, indoleacrylic acid (IAA) or the like may be added to the medium.
[0070]
When the transformant for producing a polypeptide of the present invention is an animal cell, a medium for culturing the cell is a commonly used RPMI 1640 medium [The Journal of the American Medical Association,199519 (1967)], Eagle's MEM medium [Science,122, 501 (1952)], DMEM medium [Virology,8, 396 (1959)], 199 medium [Proceeding of the Society for the Biological Medicine,73, 1 (1950)] or media obtained by adding fetal calf serum or the like to these media.
[0071]
Culture is usually pH 6-8, 30-40 ° C., 5% CO₂Perform for 1-7 days under conditions such as presence.
Moreover, you may add antibiotics, such as kanamycin and penicillin, to a culture medium as needed during culture | cultivation.
As a medium for culturing a transformant obtained by using insect cells as host cells, generally used TNM-FH medium (Pharmingen), Sf-900 II SFM medium (Life Technologies) ), ExCell400, ExCell405 (both manufactured by JRH Biosciences), Grace's Insect Medium [Grace, TCC, Nature,195, 788 (1962)] and the like.
[0072]
The pH is 6 to 7, the culture temperature is 25 to 30 ° C., and the culture time is usually 1 to 5 days.
Moreover, you may add antibiotics, such as a gentamicin, to a culture medium as needed during culture | cultivation.
As a gene expression method, in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning 2nd edition.
[0073]
As a method for producing the polypeptide of the present invention, there are a method for producing in the host cell, a method for secreting it outside the host cell, or a method for producing it on the outer membrane of the host cell. The method can be selected by changing the structure.
When the polypeptide of the present invention is produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem.,264, 17619 (1989)], Law et al. [Proc. Natl. Acad. Sci., USA,86, 8227 (1989), Genes Develop.,Four, 1288 (1990)], or by applying the method described in JP-A Nos. 05-336963 and 06-823021, the polypeptide can be actively secreted outside the host cell.
[0074]
That is, by using a gene recombination technique, the polypeptide of the present invention can be actively expressed outside the host cell by expressing in a form in which a signal peptide is added in front of the polypeptide containing the active site of the polypeptide of the present invention. Can be secreted.
Further, in accordance with the method described in JP-A-2-27075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.
In order to isolate and purify the polypeptide of the present invention from the culture of the transformant for producing the polypeptide of the present invention, the usual enzyme isolation and purification methods can be used.
[0075]
For example, when the polypeptide of the present invention accumulates in the dissolved state in the cells of the transformant for producing the polypeptide of the present invention, the culture is centrifuged to collect the cells in the culture, and the cells After washing, the cells are crushed with an ultrasonic crusher, French press, Manton Gaurin homogenizer, dyno mill or the like to obtain a cell-free extract.
[0076]
From the supernatant obtained by centrifuging the cell-free extract, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, diethylaminoethyl (DEAE) -sepharose, DIAION HPA-75 (Mitsubishi Kasei Co., Ltd.) Anion exchange chromatography method using resin, cation exchange chromatography method using resin such as S-Sepharose FF (Pharmacia), resin such as butyl sepharose, phenyl sepharose The purified sample can be obtained by using a method such as hydrophobic chromatography, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing.
[0077]
In addition, when the polypeptide is expressed by forming an insoluble substance in the cell, the polypeptide is similarly collected from the precipitate fraction obtained by crushing and centrifuging, and the polypeptide is obtained by a usual method. After the recovery, the insoluble substance of the polypeptide is solubilized with a polypeptide denaturant. The solubilized solution was diluted or dialyzed into a dilute solution not containing a polypeptide denaturant or having a polypeptide denaturant concentration that does not denature the polypeptide, thereby forming the polypeptide into a normal three-dimensional structure. Thereafter, a purified preparation can be obtained by the same isolation and purification method as described above.
When the polypeptide is secreted extracellularly, the culture is treated by a technique such as centrifugation to obtain a soluble fraction. From the soluble fraction, a purified preparation of the polypeptide can be obtained by a method similar to the method of isolation and purification from the cell-free extract supernatant.
[0078]
A glycosyltransferase purification method can also be used. Examples of the purification method include, for example, a method [Methods Enzymol., Used for partial purification of β1,3-N-acetylglucosaminyltransferase.83, 458 (1982), J. Biol. Chem.,268, 27118 (1993), J. Biol. Chem.,267, 2994 (1992), J. Biol. Chem.,263, 12461 (1988), Jpn.J.Med.Sci.Biol,42, 77 (1989)].
[0079]
In addition, the polypeptide of the present invention can be produced as a fusion protein with another protein and purified using affinity chromatography using a substance having affinity for the fused protein. For example, the method of Law et al. [Proc. Natl. Acad. Sci., USA,86, 8227 (1989), Genes Develop.,Four, 1288 (1990)], affinity chromatography using immunoglobulin G, which produces the polypeptide of the present invention as a fusion protein with protein A according to the methods described in JP-A Nos. 05-336963 and 06-823021 Can be purified. Moreover, the polypeptide of the present invention can be produced as a fusion protein with a Flag peptide and purified by affinity chromatography using an anti-Flag antibody [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989). Genes Develop., 4, 1288 (1990)]. Furthermore, it can also be purified by affinity chromatography using an antibody against the polypeptide itself.
[0080]
When the polypeptide of the present invention has β1,3-N-acetylglucosaminyltransferase activity, a known measurement method [J. Biol. Chem.,268, 27118 (1993), J. Biol. Chem.,267, 2994 (1992), J. Biol. Chem.,263, 12461 (1988), Jpn. J. Med. Sci. Biol,42, 77 (1989)], the activity of β1,3-N-acetylglucosaminyltransferase can be measured.
[0081]
(3) Production method of glycoprotein, glycolipid, or oligosaccharide to which poly-N-acetyllactosamine sugar chain or its modified sugar chain is added
A transformant capable of producing the polypeptide of the present invention described in (2) above, wherein the transformant capable of synthesizing sugar chains is cultured in a culture solution, and a sugar containing poly-N-acetyllactosamine in the culture Glycoproteins with added chains, glycolipids with added sugar chains containing poly-N-acetyllactosamine or oligosaccharides containing poly-N-acetyllactosamine are generated and accumulated, and poly- Collecting a glycoprotein with a sugar chain containing N-acetyllactosamine, a glycolipid with a sugar chain containing poly-N-acetyllactosamine or an oligosaccharide containing poly-N-acetyllactosamine To contain a glycoprotein with a sugar chain containing poly-N-acetyllactosamine, a glycolipid with a sugar chain containing poly-N-acetyllactosamine or poly-N-acetyllactosamine. It can be produced oligosaccharides.
The culture can be performed according to the above (2).
[0082]
When a transformant simultaneously expresses a glycosyltransferase gene (for example, β1,4-galactosetransferase gene), a glycoprotein and a sugar to which a sugar chain containing poly-N-acetyllactosamine is added more efficiently. Lipids or oligosaccharides can be produced.
The recombinant glycoprotein contains poly-N-acetyllactosamine by simultaneously producing the polypeptide of the present invention and any recombinant glycoprotein (for example, a recombinant protein for pharmaceutical use) in a transformant. Can be added.
[0083]
Furthermore, when the above transformant also expresses a glycosyltransferase gene (for example, β1,4-galactosyltransferase gene), a group to which a sugar chain containing poly-N-acetyllactosamine is added more efficiently. Can produce a modified glycoprotein.
A part of the oligosaccharide can be excised from the glycoprotein, glycolipid or oligosaccharide having a sugar chain containing poly-N-acetyllactosamine produced by the above method by a known enzymatic method or chemical method. [Japan Biochemical Society, Second Biochemistry Experiments, Volume 4, Complex Carbohydrate Research Methods I and II, Tokyo Chemical Doujin (1986), Supervised by Naoyuki Taniguchi, Akemi Suzuki, Kiyoshi Furukawa and Kazuyuki Sugawara, Glycobio Rosie Experiment Protocol, Shujunsha, (1996)].
[0084]
When the polypeptide of the present invention is a polypeptide having β1,3-N-acetylglucosamine transferase activity, the polypeptide of the present invention, UDP-GlcNAc, and the lactosamine structure (Galβ1-4GlcNAc structure) are sugar chains. An aqueous acceptor substrate selected from glycoproteins having non-reducing ends of glycoproteins, glycolipids having lactosamine structures at the non-reducing ends of sugar chains, oligosaccharides having lactosamine structures at the non-reducing ends of sugar chains, and lactose (Galβ1-4Glc) In the aqueous medium, galactose at the end of the lactosamine structure of the acceptor substrate generates and accumulates a reaction product to which N-acetylglucosamine is added with β1,3 bonds. By collecting the reaction product, N-acetylglucosamine is imparted to the galactose at the end of the lactosamine structure of the acceptor substrate through a β1,3 bond. It may be prepared the reaction product.
[0085]
Further, the product obtained by the production method, UDP-Gal and β1,4-galactosyltransferase are present in an aqueous medium, and N-acetylglucosamine in the product is added to β1,4 in the aqueous medium. A reaction product to which galactose has been added is generated and accumulated by binding, and the reaction product is collected from the aqueous medium. Can be manufactured.
[0086]
In addition, the polypeptide of the present invention, UDP-GlcNAc, UDP-Gal, β1,4-galactosyltransferase, a glycoprotein having a lactosamine structure at the non-reducing end of the sugar chain, and a lactosamine structure at the non-reducing end of the sugar chain An acceptor substrate selected from oligosaccharides and lactose having a glycolipid, a lactosamine structure at the non-reducing end of a sugar chain, and poly-N at the end of the lactosamine structure of the acceptor substrate in the aqueous medium. By generating and accumulating a reaction product to which -acetyllactosamine sugar chain [(Galβ1-4GlcNAcβ1-3) n; n is 2 or more] is collected and collecting the reaction product from the aqueous medium, A reaction product having a poly-N-acetyllactosamine sugar chain attached to the end of the lactosamine structure can be produced.
[0087]
As β1,4-galactosyltransferase, an enzyme purified from bovine or human milk (commercially available from Sigma) or a recombinant enzyme (commercially available from Calbiochem) can be used.
In addition, using an individual animal or plant individual, a glycoprotein containing a sugar chain containing poly-N-acetyllactosamine or a sugar chain containing poly-N-acetyllactosamine according to the method of (2) above An oligosaccharide containing a glycolipid or poly-N-acetyllactosamine can be produced.
[0088]
That is, in the case of an animal individual, for example, a non-human transgenic animal carrying the DNA of the present invention is bred, and a glycoprotein to which a sugar chain containing poly-N-acetyllactosamine is added, poly- Glycolipids containing sugar chains containing N-acetyllactosamine or oligosaccharides containing poly-N-acetyllactosamine are generated and accumulated, and sugar chains containing poly-N-acetyllactosamine from the animal Poly-N-acetyllactosamine is collected by collecting glycoproteins with added sugar, glycolipids with added sugar chains containing poly-N-acetyllactosamine or oligosaccharides containing poly-N-acetyllactosamine Glycoprotein with added sugar chain, glycolipid with added sugar chain containing poly-N-acetyllactosamine or oligosaccharide containing poly-N-acetyllactosamine It can be produced. Examples of the production / accumulation place in the animal include milk and eggs of the animal.
[0089]
In the case of a plant individual, for example, a transgenic plant having the DNA of the present invention is cultivated, and a glycoprotein, poly-N-acetyl lactate, to which a sugar chain containing poly-N-acetyllactosamine is added, is added to the plant. Glycolipids containing sugar chains containing samine or oligosaccharides containing poly-N-acetyllactosamine are produced and accumulated, and sugars containing poly-N-acetyllactosamine are added from the plant. Glycolipids containing poly-N-acetyllactosamine by collecting glycolipids with added sugar chains containing proteins, poly-N-acetyllactosamine or oligosaccharides containing poly-N-acetyllactosamine Glycoproteins with added sugar, glycolipids with added sugar chains containing poly-N-acetyllactosamine or oligosaccharides containing poly-N-acetyllactosamine Can.
[0090]
(4) Use of DAN or oligonucleotide of the present invention for treatment or diagnosis of diseases
The DNA of the present invention comprises antisense RNA / DNA technology [Bioscience and Industry,50, 322 (1992), chemistry,46, 681 (1991), Biotechnology,9, 358 (1992), Trends in Biotechnology,Ten, 87 (1992), Trends in Biotechnology,Ten, 152 (1992), Cell engineering,16, 1463 (1997)] or triple helix technology (Trends in Biotechnology,Ten, 132 (1992)] for the treatment of diseases such as inflammation and cancer, and the diagnosis of those diseases using the Northern hybridization method or the PCR method.
[0091]
For example, the production of the polypeptide of the present invention can be suppressed by administering the oligonucleotide of the present invention or the derivative thereof described in (1) above.
In addition, the expression level of the DNA encoding the polypeptide of the present invention can be quantified by the Northern hybridization method or the PCR method using the DNA of the present invention or the oligonucleotide prepared from the DNA.
[0092]
Furthermore, using the DNA of the present invention as a probe, the promoter region of the gene can be determined using a known method (Edition of Cancer Research, University of Tokyo, New Cell Engineering Experiment Protocol, Shujunsha (1993)). It is possible to obtain.
Examples of the promoter region include all promoter regions involved in transcription of a gene encoding the polypeptide of the present invention in mammalian cells. For example, in human melanoma cells, human colon cancer cells, or human white blood cells, a promoter region involved in transcription of a gene encoding the polypeptide of the present invention can be mentioned. The promoter can be used in the screening method described below.
[0093]
(5) Preparation of an antibody that recognizes the polypeptide of the present invention
(I) Preparation of polyclonal antibody
Using the full-length or partial fragment purified preparation of the polypeptide of the present invention obtained by the method described in (2) above as an antigen, a suitable adjuvant [for example, Freund's complete adjuvant (Complete Freund's Adjuvant) or aluminum hydroxide gel And pertussis vaccine, etc.], and polyclonal antibodies can be produced by administration to animals. A peptide having a partial amino acid sequence of the polypeptide of the present invention synthesized by a peptide synthesizer can also be used as an antigen.
[0094]
As animals to be administered, rabbits, goats, 3 to 20-week-old rats, mice, hamsters and the like can be used.
The dose of the antigen is preferably 50 to 100 μg per animal.
When a peptide is used, it is desirable that the peptide is covalently bound to a carrier protein such as keyhole limpet haemocyanin or bovine thyroglobulin as an antigen.
[0095]
The antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. Three to seven days after each administration, blood was collected from the fundus venous plexus, and the reaction of the serum with the antigen used for immunization was determined by enzyme immunoassay [enzyme immunoassay (ELISA): 1976, published by Medical Shoin. Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988)].
Polyclonal antibodies can be obtained by obtaining serum from a non-human mammal whose serum showed a sufficient antibody titer against the antigen used for immunization, and separating and purifying the serum.
[0096]
Methods for separating and purifying antibodies include centrifugation, salting out with 40-50% saturated ammonium sulfate, precipitation of caprylic acid (Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, (1988)), or DEAE-Sepharose column, Examples thereof include a method of treating chromatography or the like using an ion exchange column, protein A or G-column or gel filtration column alone or in combination.
[0097]
(Ii) Production of monoclonal antibodies
(a) Preparation of antibody-producing cells
Rats whose serum showed a sufficient antibody titer against the partial fragment polypeptide of the polypeptide of the present invention used for immunization are used as a source of antibody-producing cells.
The spleen is removed 3 to 7 days after the final administration of the antigenic substance to the rat showing the antibody titer.
[0098]
The spleen is shredded in MEM medium (Nissui Pharmaceutical Co., Ltd.), loosened with tweezers, centrifuged at 1,200 rpm for 5 minutes, and the supernatant is discarded.
The spleen cells of the obtained precipitate fraction were treated with Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove erythrocytes and then washed 3 times with MEM medium. Used as a cell.
[0099]
(b) Preparation of myeloma cells
As myeloma cells, cell lines obtained from mice or rats are used. For example, 8-azaguanine resistant mice (derived from BALB / c) myeloma cell line P3-X63Ag8-U1 (hereinafter abbreviated as P3-U1) [Curr. Topics. Microbiol. Immunol.,81, 1 (1978), Europ. J. Immunol.,6, 511 (1976)], SP2 / 0-Ag14 (SP-2) (Nature,276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunol.,one two Three, 1548 (1979)], P3-X63-Ag8 (X63) [Nature,256, 495 (1975)]. These cell lines consisted of 8-azaguanine medium [RPMI-1640 medium with glutamine (1.5 mM), 2-mercaptoethanol (5 × 10 5^-FiveM), gentamicin (10 μg / ml) and fetal calf serum (FCS) (manufactured by CSL, 10%) were further added with 8-azaguanine (15 μg / ml). Medium], but cultured in normal medium 3 to 4 days before cell fusion, and the cells were fused at 2 × 10⁷Use more than one.
[0100]
(c) Production of hybridoma
The antibody-producing cells obtained in (a) and the myeloma cells obtained in (b) are mixed with MEM medium or PBS (1.83 g of disodium phosphate, 0.21 g of monopotassium phosphate, 7.65 g of sodium chloride, 1 liter of distilled water). Wash well with pH 7.2), mix so that the number of cells is antibody-producing cells: myeloma cells = 5 to 10: 1, centrifuge at 1,200 rpm for 5 minutes, and discard the supernatant.
[0101]
Thoroughly loosen the cell group of the obtained precipitate fraction and stir the cell group at 37 ° C. with stirring.⁸0.2 to 1 ml of a solution prepared by mixing 2 g of polyethylene glycol-1000 (PEG-1000), 2 ml of MEM and 0.7 ml of dimethyl sulfoxide (DMSO) is added to each antibody-producing cell, and MEM medium 1 is added every 1 to 2 minutes. Add ~ 2 ml several times.
[0102]
After the addition, MEM medium is added to prepare a total volume of 50 ml.
The preparation is centrifuged at 900 rpm for 5 minutes, and the supernatant is discarded.
Loosely loosen the cells of the resulting precipitate fraction, and then gently suck in and blow out with a pipette to gently add HAT medium [hypoxanthine (10%^-FourM), thymidine (1.5 × 10^-FiveM) and aminopterin (4 × 10^-7Medium supplemented with M)] Suspend in 100 ml.
[0103]
The suspension was dispensed at 100 μl / well into a 96-well culture plate, and 5% CO 2 was added.₂Incubate in an incubator at 37 ° C for 7-14 days.
After culturing, a part of the culture supernatant is taken and the enzyme immunoassay described in Antibodies, A Laboratory manual, Cold Spring Harbor Laboratory, Chapter 14 (1988) etc. A hybridoma that specifically reacts with the partial fragment polypeptide is selected.
[0104]
The following methods can be given as specific examples of the enzyme immunoassay.
During immunization, a partial fragment polypeptide of the polypeptide of the present invention used as an antigen is coated on an appropriate plate, and the hybridoma culture supernatant or the purified antibody obtained in (d) described below is reacted as a first antibody, After reacting with anti-rat or anti-mouse immunoglobulin antibody labeled with biotin, enzyme, chemiluminescent substance or radiation compound as the second antibody, it reacts according to the labeling substance and reacts specifically with the polypeptide of the present invention. Is selected as a hybridoma producing the polypeptide monoclonal antibody of the present invention.
[0105]
Using this hybridoma, cloning was repeated twice by the limiting dilution method (the first time was HT medium (a medium obtained by removing aminopterin from HAT medium), the second time was using a normal medium), a stable and strong antibody Those having a recognized valence are selected as anti-polypeptide antibody-producing hybridoma strains of the polypeptide of the present invention.
[0106]
(d) Preparation of monoclonal antibody
Obtained in (c) from 8-10 week old mice or nude mice treated with pristane [0.5 ml of 2,6,10,14-tetramethylpentadecane (Pristane) intraperitoneally and bred for 2 weeks] The polypeptide monoclonal antibody-producing hybridoma cells of the present invention 5-20 × 10⁶Cells / animals are injected intraperitoneally. The hybridoma becomes ascites tumor in 10 to 21 days.
[0107]
Ascites is collected from the mouse with ascites tumor, and centrifuged at 3,000 rpm for 5 minutes to remove solids.
From the obtained supernatant, the monoclonal antibody can be purified and obtained by the same method as that used for the polyclonal antibody. Monoclonal antibodies can also be purified and obtained from the culture supernatant of hybridoma strains producing monoclonal antibodies by the same method.
[0108]
Determination of antibody class and subclass is performed using a mouse monoclonal antibody typing kit or a rat monoclonal antibody typing kit. Protein mass is calculated from the Raleigh method or absorbance at 280 nm.
The antibody class is an antibody isotype. In humans, IgG, IgA, IgM, IgD, and IgE are listed. The subclass refers to an isotype within the class, and includes IgG1, IgG2a, IgG2b, IgG3 in mice, and IgG1, IgG2, IgG3, IgG4 in humans.
[0109]
(6) Use of the antibody of the present invention
(A) The polypeptide of the present invention can be detected using the antibody of the present invention. Specifically, the antibody of the present invention can be used in a detection method using an ELISA method, a fluorescent antibody method, a Western blot method, or the like using a microtiter plate.
(B) The antibody of the present invention can be used for immunohistochemical staining of cells expressing the polypeptide of the present invention.
(C) The antibody of the present invention can be used for diagnosis and treatment of diseases such as inflammation or cancer.
[0110]
(7) Application to screening methods.
Since the polypeptide of the present invention has an activity involved in the synthesis of poly-N-acetyllactosamine sugar chain, a compound that enhances or inhibits the activity of the polypeptide can be used to produce poly-N-acetyllactide in cells. It is possible to increase or decrease the synthesis amount of samine sugar chains.
[0111]
In addition, a compound that promotes or suppresses the transcription process of a gene encoding the polypeptide or the translation process from a transcription product to a protein controls the expression of the polypeptide, and the poly-N-acetyllactosamine sugar chain in the cell It is possible to control the amount of synthesis.
The above compounds are useful for the treatment of diseases such as inflammation and cancer and the synthesis of poly-N-acetyllactosamine sugar chains.
The compound can be obtained by the methods shown in the following (a) to (e).
[0112]
(A) The polypeptide of the present invention described in (2) above, a cell extract capable of synthesizing a poly-N-acetyllactosamine sugar chain, a test sample, UDP-GlcNAc, UDP-Gal, and a receptor substrate In the presence of a sugar chain (a sugar chain having a lactosamine structure at the end), a known method [J. Biol. Chem.,268, 27118 (1993), J. Biol. Chem.,267, 2994 (1992), J. Biol. Chem.,263, 12461 (1988), Jpn. J. Med. Sci. Biol,42, 77 (1989)], and a compound that increases or decreases the amount of the product produced by the reaction is selected and obtained.
[0113]
(B) When the polypeptide of the present invention is a polypeptide having β1,3-N-acetylglucosaminyltransferase activity, the polypeptide prepared by using the method described in (2) above (the polypeptide The cell transformant or purified product of the transformant to be expressed) is used as an enzyme, and β1,3-N-acetylglucosaminyltransferase activity is measured using the method described above in the presence of the test sample, and β1,3-N -Select and obtain compounds that have activity to increase or decrease acetylglucosamine transferase activity.
[0114]
(C) The cell expressing the polypeptide of the present invention or the transformant described in (2) above is cultured in the presence of the test sample for 2 hours to 1 week by the culture method described in (2) above. The amount of poly-N-acetyllactosamine sugar chain on the cell surface is measured using an anti-i antibody, and a compound having an activity to increase or decrease the sugar chain amount is selected and obtained.
Examples of the measurement method using an anti-i antibody include a detection method using an ELISA method / fluorescent antibody method using a microtiter plate, a Western blot method, immunohistochemical staining, and the like.
[0115]
(D) After culturing cells expressing the polypeptide of the present invention in the presence of the test sample by the culture method described in (2) above for 2 hours to 1 week, the amount of the polypeptide in the cells is Measurement is performed using the antibody of the present invention described in (5), and a compound having an activity of increasing or decreasing the amount of the polypeptide is selected and obtained.
Examples of the measurement method using the antibody of the present invention include a detection method using an ELISA method / fluorescent antibody method using a microtiter plate, a Western blot method, immunohistochemical staining, and the like.
[0116]
(E) a gene encoding the polypeptide in the cell after culturing the cell expressing the polypeptide of the present invention in the presence of the test sample in the culture method described in (2) above for 2 hours to 1 week. The amount of the transcription product is measured using the method such as the Northern hybridization method and the PCR method described in the above (4), and a compound having the activity to increase or decrease the transcription product amount is selected and obtained.
[0117]
(F) A plasmid incorporating a reporter gene-linked DNA downstream of the promoter obtained in (4) above is prepared by a known method, and the animal cell described in (2) above is applied to the method described in (2) above. Introduce accordingly, and obtain a transformant. The transformant is cultured in the presence of the test sample by the culture method described in (2) above for 2 hours to 1 week, and the expression level of the reporter gene in the cell is determined by a known method [Medical Research Institute, University of Tokyo]. Cancer Research Department, New Cell Engineering Experimental Protocol, Shujunsha (1993), Biotechniques,20, 914 (1996), J. Antibiotics,49, 453 (1996), Trends in Biochemical Sciences, 20, 448 (1995), Cell engineering,16, 581 (1997)], and a compound having an activity to increase or decrease the expression level is selected and obtained.
Examples of the reporter gene include chloramphenicol acetyltransferase gene, β-galactosidase gene, luciferase gene, green fluorescent protein (GFP) gene and the like.
[0118]
【Example】
Examples are shown below. As a genetic manipulation technique, a known method described in Molecular Cloning 2nd edition was used unless otherwise specified.
[0119]
Example 1 Cloning of a gene (cDNA) that increases the reactivity with anti-i antibody (Den)
(1) Acquisition of mRNA from human melanoma cell line WM266-4 and human colon cancer cell line SW1116
1 × 10⁸Fast Track (product number K1593), an mRNA extraction kit manufactured by Invitrogen, from human melanoma cell line WM266-4 (ATCC CRL 1676) and human colon cancer cell line SW1116 (ATCC CRL 233) -02), about 30 μg of mRNA was obtained for each. Specific reagents and methods followed the instructions given in the kit.
[0120]
(2) Preparation of cDNA library of WM266-4 and SW1116
Double-stranded cDNA was synthesized using oligo dT as a primer using 8 μg each of the mRNA derived from WM266-4 and SW1116 obtained in (1) above and a cDNA synthesis system kit manufactured by GIBCO BRL. However, the reverse transcriptase was not the Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase included in the kit, but its Super ScriptTM RNAse H-Reverse Transcriptase.
[0121]
At the both ends of these double-stranded cDNAs,SfiI linker was applied. [SfiGiving I linker]
The single-stranded DNA represented by SEQ ID NO: 3 and the single-stranded DNA represented by SEQ ID NO: 4 were synthesized using an Applied Biosystems 380A DNA synthesizer. 50 μg each of the synthetic single-stranded DNA, 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂Dissolve in 50 μl of a buffer solution (hereinafter abbreviated as T4 kinase buffer) containing 5 mM dithiothreitol (hereinafter abbreviated as DTT), 0.1 mM EDTA and 1 mM ATP, and 30 units of T4 polynucleotide kinase (Takara Shuzo) And a phosphorylation reaction was carried out at 37 ° C. for 16 hours to obtain 11-base and 8-base linkers.
[0122]
After dissolving 11 base linker 4 μg, 8-base linker 2.9 μg and the double-stranded cDNA synthesized above in T4 ligase buffer 45 μl, add 1050 units of T4 DNA ligase, and react at 16 ° C. for 16 hours. Each strand DNASfiI linker was applied.
The obtained reaction solution was subjected to agarose gel electrophoresis, and DNA fragments of about 1.6 kb or more were collected.
[0123]
In addition, 8 μg of the WM266-4-derived mRNA obtained above was used to synthesize a double-stranded cDNA using a cDNA synthesis system kit manufactured by GIBCO BRL with a random primer as a primer. In this synthesis reaction, Super ScriptTM RNAse H-Reverse Transcriptase was also used as the reverse transcriptase.
At both ends of the cDNA, the same method as aboveSfiAfter adding the I linker, agarose gel electrophoresis was performed to recover a DNA fragment of about 1.2 kb or more.
[0124]
PAMo [J. Biol. Chem., A direct expression cloning vector]268, 22782 (1993), also known as pAMoPRC3Sc (Japanese Patent Laid-Open No. 05-336963)] 24 μg, 10 mM Tris-HCl (pH 7.5), 6 mM MgCl₂, 50 mM NaCl, 6 mM 2-mercaptoethanol buffer solution (hereinafter abbreviated as Y-50 buffer solution) 590 μl,SfiI (manufactured by Takara Shuzo Co., Ltd., unless otherwise specified, the restriction enzyme was manufactured by Takara Shuzo Co., Ltd.) and digestion reaction was carried out at 37 ° C. for 16 hours.
[0125]
40 units of the reaction solutionBamHI was added and digestion was performed at 37 ° C. for 2 hours.
The reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment of about 8.8 kb was recovered.
Prepared aboveSfiThree types of DNA (I mRNA derived from 8 μg) to which I linker was added were separately dissolved in 250 μl of T4 ligase buffer, and 2 μg of the approximately 8.8 kb DNA fragment and 2000 units of T4 DNA ligase were added to each lysate, The binding reaction was performed at 16 ° C. for 16 hours.
[0126]
After the reaction, 5 μg of transfer RNA (tRNA) was added to each reaction solution, and after ethanol precipitation, the resulting precipitate was buffered with 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (ethylenediaminetetraacetic acid sodium acetate) , Abbreviated as TE buffer).
Using the reaction solution, an electroporation method [Nucleic Acids Res.,16, 6127 (1988)] by transforming Escherichia coli LE392 strain (Molecular Cloning Second Edition) and using DNA derived from WM266-4 using oligo dT primer, about 260,000 random primers were used. Approximately 300,000 transformants with ampicillin resistance obtained when using DNA derived from WM266-4 used, and about 300,000 when using DNA derived from SW1116 using oligo dT primer Then, cDNA libraries derived from the respective DNAs were constructed.
[0127]
(3) Cloning of a gene (cDNA) that increases the reactivity with anti-i antibody (Den)
For each cDNA library obtained in (2) above, a plasmid was prepared using a plasmid preparation kit / plasmid / maxi kit (product number 41031) manufactured by Qiagen.
The plasmid was precipitated in ethanol and dissolved in TE buffer so as to be 1 μg / μl.
[0128]
Using the plasmid lysate, serum-free medium-conditioned Namalwa cells (Namalwa KJM-1 cells) [Cytotechnology,1, 151 (1988)]. For the introduction of the plasmid, Lipofectin (manufactured by GIBCO BRL) was used, and a method according to the lipofection method described in the product instructions was used. In this method, as the serum-free medium, RPMI1640 • ITPSGF medium [7.5% NaHCO 3 containing no penicillin / streptomycin is used._Three1/40, 200 mM L-glutamine solution (GIBCO) 3%, penicillin / streptomycin solution (GIBCO 5000 units / ml penicillin, 5000 μg / ml streptomycin) 0.5%, N-2-hydroxyethyl Piperazine-N'-2-ethanesulfonic acid (N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid; HEPES) (10 mM), insulin (3 μg / ml), transferrin (5 μg / ml), pyruvin RPMI 1640 medium (manufactured by Nissui Pharmaceutical Co., Ltd.) supplemented with sodium acid (5 mM), sodium selenite (125 nM) and galactose (1 mg / ml) was used.
[0129]
Specifically, 1.0 × 10 5 in RPMI1640 • ITPSGF medium without penicillin / streptomycin.⁶To 0.8 ml of cell suspension prepared to cells / ml, add 3.25 μl of lipofectin and 2 μg of plasmid,₂The cells were cultured overnight in an incubator (37 ° C).
After the culture, 6.5 ml of RPMI1640 · ITPSGF medium was added, and further cultured for 2 days. Then, 10 times the amount of RPMI1640 · ITPSGF medium and G418 (GIBCOBRL; final concentration 0.5 mg / ml) were added, Further, the cells were cultured for 10 days to obtain stably transformed cells.
[0130]
By this method, about 33 μg of plasmid was used for the WM266-4 derived cDNA library using oligo dT primer, about 39 μg of plasmid was used for the WM266-4 derived cDNA library using random primer, and oligo dT primer was used. With respect to the SW1116-derived cDNA library, about 60 μg of plasmid was introduced into Namalwa KJM-1 cells to obtain stable transformed cells.
The obtained transformed cells are mixed for each library, and then anti-i antibody (Den) [J. Biol. Chem., Which is an antibody that recognizes a linear poly-N-acetyllactosamine sugar chain.254, 3221 (1979)]. A specific method is shown below.
[0131]
About 4 × 10⁷The stably transformed cells were taken in a 50 ml centrifuge tube (2059 tube: manufactured by Falcon), and the cells were collected by centrifugation (130 × g, 10 minutes).
The cells were washed with phosphate buffer PBS containing 0.1% sodium azide (A-PBS: 8 g / l NaCl, 0.2 g / l KCl, 1.15 g / l Na₂HPO_Four(Anhydrous), 0.2 g / lKH₂PO_Four, 0.1% sodium azide).
[0132]
To the washed cells, 0.5 ml of anti-i antibody (Den) diluted 100-fold with A-PBS was added and suspended, and reacted at 4 ° C. for 1 hour.
After the reaction, the cells were washed once with 20 ml of A-PBS, then suspended by adding 300 μl of a 30-fold diluted solution of anti-human IgM antibody (manufactured by DAKO) fluorescently labeled with fluorescein isothiocyanate (FITC) in A-PBS. The reaction was performed at 4 ° C. for 30 minutes.
[0133]
After the reaction, the cells were washed once with A-PBS, suspended in 1 ml of A-PBS, and fluoresce activated cell sorter [EPICS Elite Flow Cytometer; Coulter ( COULTER)] was used to aseptically separate and collect cells with high fluorescence intensity (upper 2.8%) (FIG. 1).
[0134]
The recovered cells were cultured and grown in RPMI1640 · ITPSGF medium containing 0.5 mg / ml G418, and then cells with high fluorescence intensity were aseptically separated and recovered by the same operation as described above. This operation was repeated again to separate and concentrate cells with high fluorescence intensity.
In the second operation, cells with high fluorescence intensity (upper 1.3%) were separated and in the third operation, cells with higher fluorescence intensity (upper 1.1%) were separated and recovered (FIG. 1).
[0135]
By the separation operation, cells with increased fluorescence intensity, that is, cells with increased expression level of linear poly-N-acetyllactosamine sugar chains were obtained (see FIG. 1).
After culturing the cells in RPMI1640 · ITPSGF medium containing 0.5 mg / ml G418, about 2 × 10⁶From the individual cells, the Heart method [Mol. Cell. Biol.,8, 2837 (1988)].
[0136]
The plasmid was electroporated [Nucleic Acids Res.,16, 6127 (1988)] and introduced into Escherichia coli MC1061A strain to obtain a transformant having ampicillin resistance.
Plasmids were prepared from 75 of the transformants using an automated plasmid separator PI-100 manufactured by KURABO, and each plasmid was treated with a restriction enzyme (HindIII andAspThe structure of cDNA inserted by cutting in 718) was examined.
[0137]
As a result, the obtained plasmids were classified into 18 types.
The 18 kinds of plasmids were extracted with phenol / chloroform (1: 1) and precipitated with ethanol, dissolved in 10 μl of TE buffer, and electroporation [Cytotechnology,Three, 133 (1990)], each plasmid was introduced into Namalwa KJM-1 cells.
That is, 1.0 to 1.6 × 10⁶Each cell (200 μl) was introduced with 3 μl of the plasmid prepared above or 4 μg of pAMo (control plasmid), suspended in 8 ml of RPMI1640 · ITPSGF medium,₂The cells were cultured at 37 ° C. for 24 hours in an incubator.
[0138]
After the culture, G418 (manufactured by GIBCOBRL) was added to a concentration of 0.5 mg / ml, and the cells were further cultured for 10 to 14 days to obtain transformed cells.
The obtained transformed cells were subjected to indirect fluorescent antibody staining using an anti-i antibody (Den). As a result, a cell introduced with a plasmid designated 16-2-12 was compared with a cell introduced with pAMo. In addition, the reactivity to anti-i antibody (Den) (the peak value of the histogram) increased about 7 times (FIG. 2). On the other hand, the reactivity of the transformed cells introduced with other plasmids to the anti-i antibody (Den) hardly changed compared to the cells introduced with the control plasmid (pAMo) (FIG. 2).
[0139]
From the above results, it was revealed that the reactivity of the transformed cells to the anti-i antibody (Den) is increased by the cDNA inserted in the plasmid 16-2-12.
Indirect fluorescent antibody staining using anti-i antibody (Den) was performed as follows.
About 1 × 10⁶Individual transformed cells were collected in a microtube (1.5 ml: Eppendorf), and the cells were collected by centrifugation (550 × g, 7 minutes).
[0140]
The cells were washed with 0.9 ml of A-PBS, 20 μl of anti-i antibody (Den) diluted 100-fold with A-PBS was added to the washed cells, and the cells were reacted at 4 ° C. for 1 hour.
[0141]
After the reaction, the cells were washed once with 0.9 ml of A-PBS, then suspended by adding 20 μl of a 30-fold diluted solution of anti-human IgM antibody (manufactured by DAKO) fluorescently labeled with FITC in A-PBS, 4 ° C. For 30 minutes.
After the reaction, the cells were washed once with 0.9 ml of A-PBS, suspended in 0.6 ml of A-PBS, and then used with a fluorescence activated cell sorter (FACSCaliber; Becton Dickinson Immunocytometry Systems USA). Analysis. As a control experiment, the same analysis was performed using A-PBS instead of the anti-i antibody (Den) (thin line in FIG. 2).
[0142]
(4) Determination of base sequence of cDNA inserted into plasmid 16-2-12 After preparing a restriction enzyme map of cDNA contained in plasmid 16-2-12 obtained in (3), DNA derived from the cDNA The fragment was subcloned into pBluescript II SK (+), and the entire nucleotide sequence of the cDNA was determined.
That is, by preparing plasmid 16-2-12 using a plasmid preparation kit / plasmid / maxi kit (product number 41031) manufactured by Qiagen, and cleaving the plasmid with various restriction enzymes, A restriction enzyme map of cDNA contained in the plasmid was prepared. The results are shown in FIG.
[0143]
Of the plasmid 16-2-12HindIII-StuI fragment (approximately 0.8 kb: No. 2 fragment in FIG. 3) andHindIII-SmaAn I fragment (about 0.6 kb: No. 12 fragment in FIG. 3) was isolated from pBluescript II SK (+)HindIII-HincSubcloned between II. In addition, plasmid 16-2-12HindIII-SmaThe I fragment (about 1.0 kb: No. 13 fragment in FIG. 3) was isolated and the pBluescript II SK (+)HindIII-HincSubcloned between II. In addition, plasmid 16-2-12HindIII-Asp718 fragment (about 0.6 kb: No. 7 fragment in FIG. 3) andHindIII-Asp718 fragment (about 0.4 kb: No. 11 fragment of FIG. 3) was isolated and pBluescript II SK (+)HindIII-AspSubcloned between 718. In addition, plasmid 16-2-12StuI-SacI fragment (about 0.7 kb: No. 19 fragment of FIG. 3) was isolated and pBluescript II SK (+)SacI-HincSubcloned between II.
[0144]
Using the six types of subcloned plasmids prepared as described above as templates, the nucleotide sequences of the cDNA fragments contained in them were determined using a DNA sequencer 377 from PerkinElmer. The base sequence was determined using a Perkin Elmer kit according to the instructions of the kit. By joining the results, the entire nucleotide sequence (2011 bp) of the cDNA contained in plasmid 16-2-12 was determined.
[0145]
The base sequence of the cDNA is shown in SEQ ID NO: 2.
The cDNA encoded a polypeptide consisting of 415 amino acids having a structure characteristic of glycosyltransferase. The polypeptide did not show homology with the amino acid sequences of known proteins whose structures have been elucidated so far. The amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.
[0146]
The polypeptide has 8 amino acids at the N-terminus to the cytoplasm side, followed by a hydrophobic region consisting of 28 amino acids and bound to the membrane, and the remaining C-terminal part (including the catalytic site) is attached to the Golgi body lumen. It is thought that the structure is exposed to the sun.
Hereinafter, the cDNA is referred to as iGnT cDNA, and the protein encoded by the cDNA is referred to as iGnT.
[0147]
Example 2 Synthesis of poly-N-acetyllactosamine sugar chain in Namalwa KJM-1 cells into which the expression plasmid of iGnT cDNA obtained in Example 1 was introduced
(1) Construction of plasmid pAMo-i for expressing iGnT cDNA in animal cells (FIG. 4)
1 μg of pAMo is dissolved in 20 μl of Y-50 buffer and 20 units ofHindIII was added and digestion was performed at 37 ° C. for 2 hours.
[0148]
After the reaction, NaCl was added to 150 mM, and 20 units ofNotI was added, and a digestion reaction was further performed at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, about 8.7 kbHindIII-NotI-treated DNA fragments were recovered.
Also, 1 μg of the plasmid 16-2-12 obtained in Example 1 was dissolved in 20 μl of Y-50 buffer to obtain 20 units.HindIII was added and digestion reaction was performed at 37 ° C. for 2 hours.
[0149]
After the reaction, NaCl was added to 150 mM, and 20 units ofBstXI was added, and a digestion reaction was further performed at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, about 0.5 kbHindIII-BstThe XI-treated DNA fragment was recovered.
In addition, plasmid 16-2-12 (1 μg) was mixed with 10 mM Tris-HCl (pH 7.5), 6 mM MgCl.₂, 150 mM NaCl, 6 mM 2-mercaptoethanol buffer solution (hereinafter abbreviated as Y-150 buffer solution) 30 μl,BstXI and 20 unitsNotAdd I and digest at 37 ° C for 2 hourswent. After the reaction solution was subjected to agarose gel electrophoresis, about 1.5 kb of BstXI-NotI-treated DNA fragments were recovered.
[0150]
About 8.7 kb obtained aboveHindIII-NotI-treated DNA fragment 0.02 μg, about 0.5 kbHindIII-BstXI-treated DNA fragment 0.02 μg, and about 1.5 kbBstXI-Not0.05 μg of I-treated DNA fragment was dissolved in 20 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and a binding reaction was performed at 12 ° C. for 16 hours.
[0151]
Escherichia coli MM294 strain was transformed with the reaction solution by the method of Cohen et al. To obtain a transformant having ampicillin resistance.
A plasmid was isolated from the transformant according to a known method, and its structure was confirmed by restriction enzyme digestion. Hereinafter, this plasmid is called pAMo-i (FIG. 4).
[0152]
(2) Synthesis of poly-N-acetyllactosamine sugar chain in Namalwa KJM-1 cells introduced with pAMo-i (FIG. 5)
Plasmids pAMo and pAMo-i were each dissolved in TE buffer so as to be 1 μg / μl, and then introduced into Namalwa KJM-1 cells by the lipofection method described in Example 1, (3), and transformed cells. Got.
Anti-i antibody (Den), an antibody that recognizes linear poly-N-acetyllactosamine sugar chains, is difficult to recognize poly-N-acetyllactosamine sugar chains with sialic acid added to the non-reducing end [Roelcke et al .: Vox Sanguinis,48, 181-183 (1985)]. Therefore, the transformed cells obtained above are treated with sialidase to remove terminal sialic acid, and then subjected to indirect fluorescent antibody staining with anti-i antibody (Den), and the cells directly The expression of chain poly-N-acetyllactosamine sugar chain was examined.
[0153]
Namalwa KJM-1 cells transfected with pAMo-i or control plasmid pAMo (5 × 10 5⁶) 20mUClostridium perfringensOf the neuraminidase (neuraminidase, N2133, manufactured by Sigma) was suspended in 100 μl of PBS and reacted at 37 ° C. for 1 hour, whereby the transformed cells were treated with sialidase (+ sialidase). Control cells were prepared by reacting for 1 hour at 37 ° C. in PBS without neuraminidase (-sialidase).
About 1 × 10 of these⁶Cells were stained with indirect fluorescent antibody using anti-i antibody (Den) or A-PBS, and the expression of linear poly-N-acetyllactosamine sugar chain was examined.
[0154]
Indirect fluorescent antibody staining was performed according to the method described in Example 1 (3). The results are shown in FIG.
As the sialidase treatment increases the reactivity of Namalwa KJM-1 cells introduced with pAMo-i or control plasmid pAMo with anti-i antibody (Den), these transformed cells have sialic acid at the non-reducing end. It can be seen that the added linear poly-N-acetyllactosamine sugar chain was expressed.
[0155]
In Namalwa KJM-1 cells into which control plasmid pAMo has been introduced, the fluorescence intensity of cells stained with anti-i antibody (Den) (FIG. 5 bold line) is the fluorescence intensity of control cells stained with A-PBS (FIG. 5 thin line). The strength is relatively high, indicating that Namalwa KJM-1 cells originally expressed linear poly-N-acetyllactosamine sugar chains.
On the other hand, the reactivity of Namalwa KJM-1 cells transfected with pAMo-i with anti-i antibody (Den) is similar to that of Namalwa KJM-1 cells transfected with control plasmid pAMo, in both sialidase-treated and untreated conditions. It is stronger than that.
[0156]
This is because a linear poly-N-acetyllactosamine sugar chain is newly synthesized on a glycoprotein or glycolipid sugar chain on the cell surface by expressing iGnT cDNA in Namalwa KJM-1 cells. Furthermore, it can be understood that a linear poly-N-acetyllactosamine sugar chain can be newly synthesized also in a sugar chain of a glycoprotein secreted from a cell in which iGnT cDNA is expressed.
As described above, by secreting and producing a useful glycoprotein using the cell expressing the cDNA as a host, a sugar chain containing poly-N-acetyllactosamine is added to the secreted glycoprotein. Is possible.
[0157]
Example 3 Secretory production of protein A fusion type iGnT using Namalwa KJM-1 cells as a host
(1) Construction of iGnT secretion expression plasmid pAMoA-i52S (FIGS. 6 and 7) From the primary sequence of cloned iGnT, an N-terminal cytoplasmic region (8 amino acids) followed by a membrane-binding region (28 amino acids) and Since it is presumed to consist of a C-terminal catalytic activity region (379 amino acids), the cytoplasmic region and membrane-bound region on the N-terminal side of iGnT are removed, and the signal sequence of human granulocyte colony stimulating factor and yellow Staphylococcus (StaphylococcusaureusThe secretory expression of iGnT was attempted by adding the IgG binding region of protein A.
[0158]
A DNA region encoding a region considered to have catalytic activity [from the 52nd serine to the 415th cysteine of SEQ ID NO: 1] was prepared using PCR, and α1,3-fucose transferase (Fuc) described below was prepared. The plasmid pAMoA-i52S for secreting and expressing iGnT is obtained by replacing the Fuc-TIII gene part in pAMoA-FT3, which is a plasmid for secreting and expressing -TIII), with DNA encoding the putative catalytic region of iGnT. Created.
[0159]
pAMoA-FT3 was constructed by the following method (FIG. 6).
1 μg of pAMoA in 10 mM Tris-HCl (pH 7.5), 6 mM MgCl₂, 100 mM NaCl, 6 mM 2-mercaptoethanol buffer solution
Dissolved in 20 μl (hereinafter abbreviated as Y-100 buffer)StuI was added and digestion reaction was performed at 37 ° C. for 2 hours.
[0160]
After the reaction, NaCl was added to 150 mM, and 20 units ofNotI was added, and a digestion reaction was further performed at 37 ° C. for 2 hours.
After subjecting the reaction solution to agarose gel electrophoresis, about 9.1 kbStuI-NotI-treated DNA fragments were recovered.
In addition, pAMo-FT3 [Sasaki et al .: Journal of Biological Chemistry (J. Biol. Chem.),269, 14730-14737 (1994)] Dissolve 2 μg in 20 μl of Y-100 buffer,BamHI was added and digestion was performed at 37 ° C. for 2 hours.
[0161]
After the reaction, ethanol precipitation is performed, the obtained precipitate is dissolved in 30 μl of DNA polymerase I buffer, 6 units of E. coli DNA polymerase I / Klenow fragment is added, and the mixture is reacted at 37 ° C. for 60 minutes.BamThe 5 ′ overhang generated by HI digestion was changed to a blunt end.
The reaction was stopped by phenol extraction, chloroform extraction and ethanol precipitation were performed, then dissolved in 30 μl of Y-150 buffer, and 20 units ofNotI was added and digestion reaction was performed at 37 ° C. for 2 hours.
[0162]
After the reaction solution was subjected to agarose gel electrophoresis, about 1.8 kbBamHI (blunt end)-NotI-treated DNA fragments were recovered.
About 9.1 kb obtained aboveStuI-NotI fragment 0.05μg and about 1.8kbBamHI (blunt end)-Not0.05 μg of I fragment was dissolved in 20 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and a binding reaction was performed at 12 ° C. for 16 hours.
[0163]
Escherichia coli MM294 strain was transformed with the reaction solution by the method of Cohen et al. To obtain a transformant having ampicillin resistance.
A plasmid was isolated from the transformant according to a known method, and the structure of the plasmid was confirmed by restriction enzyme digestion. This plasmid is called pAMoA-FT3.
[0164]
Plasmid pAMoA-i52S for secreting and expressing iGnT was constructed by the following method (FIG. 7).
As primers for PCR, DNA represented by SEQ ID NO: 5 (hereinafter referred to as C12-7) and DNA represented by SEQ ID NO: 6 (hereinafter referred to as C12-9) were synthesized (purchased from Sawaddy Technology). Is also possible).
In C12-7BamHI site is in C12-9NotSince the I site is designed to be introduced, the DNA fragment amplified by PCR isBamHI andNotAfter cutting with I, pAMoA-FT3BamWith HI siteNotIt can be incorporated between I sites.
[0165]
PCR was performed using a kit (GeneAmp ™ DNA Amplification Reagent Kit with AmpliTaq ™ Recombinant Taq DNA Polymerase) manufactured by Takara Shuzo. The reaction solution was prepared according to the method of the kit. Using a DNA thermal cycler (PERKIN ELMER CETUS DNA Thermal Cycler; sold by Takara Shuzo Co., Ltd.), the reaction was performed at 94 ° C. for 30 seconds, 65 ° C. for 1 minute, 72 ° C. for 2 minutes for 10 minutes. After cycling, the reaction was further continued at 72 ° C. for 7 minutes. As a template, 10 ng of the plasmid pAMo-i constructed in Example 2 was used.
[0166]
After completion of the reaction, chloroform extraction and ethanol precipitation were performed, and then dissolved in 30 μl of Y-150 buffer to obtain 20 units.BamHI and 20 unitsNotI was added and digestion was performed at 37 ° C. for 3 hours.
After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 1.1 kb was recovered.
In addition, 1 μg of the pAMoA-FT3 obtained above was dissolved in 30 μl of Y-150 buffer, and 20 units ofBamHI andNotI was added and digestion reaction was performed at 37 ° C. for 2 hours.
[0167]
After subjecting the reaction solution to agarose gel electrophoresis, about 9.1 kbBamHI-NotI-treated DNA fragments were recovered.
About 1.1 kb obtained aboveBamHI-Not0.1 μg of I-treated DNA fragment and about 9.1 kbBamHI-Not0.1 μg of I-treated DNA fragment was dissolved in 30 ml of T4 ligase buffer, 175 units of T4 DNA ligase was added, and a binding reaction was performed at 12 ° C. for 16 hours.
[0168]
Escherichia coli MM294 was transformed with the reaction solution by the method of Cohen et al. To obtain a transformant having ampicillin resistance.
A plasmid was isolated from the transformant according to a known method, and its structure was confirmed by restriction enzyme digestion. This plasmid is called pAMoA-i52S.
[0169]
(2) Expression of secreted iGnT in Namalwa KJM-1 cells and acquisition of secreted iGnT
The control plasmid pAMoA and the plasmid pAMoA-i52S for secretory expression of iGnT constructed as described above were prepared using a plasmid preparation kit (/ plasmid / maxi kit; trademark number 41031) manufactured by Qiagen.
The prepared and obtained plasmid was dissolved in TE buffer so as to be 1 μg / μl after ethanol precipitation.
After lysis, both plasmids were introduced into Namalwa KJM-1 cells by the lipofection method described in Example 1 to obtain transformants.
[0170]
The obtained transformant is 5 × 10 5 in 30 ml of RPMI1640 · ITPSGF medium containing 0.5 mg / ml of G418.^FourSuspend to cells / ml, CO₂The cells were cultured at 37 ° C. for 9 days in an incubator.
After culturing, the cells were removed by centrifugation under conditions of 160 × g, 10 minutes and 1500 × g, 10 minutes, and the supernatant was collected.
The culture supernatant can be stored at −80 ° C. and can be thawed before use.
[0171]
IGnT encoded by the plasmid pAMoA-i52S is S. aureus (StaphylococcusaureusThe protein A is secreted and expressed as a fusion protein with the IgG binding region of protein A, and thus can be easily purified using IgG Sepharose.
After adding sodium azide to the culture supernatant obtained above to a final concentration of 0.1%, 30 μl of IgG sepharose (Pharmacia) pretreated according to the product instructions was added, and overnight at 4 ° C. Stir slowly.
[0172]
After stirring, IgG Sepharose was recovered by centrifugation at 160 × g for 10 minutes, and the Sepharose was mixed with 1 ml of a buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM sodium chloride, 0.05% Tween20. Washed twice.
After washing, the protein adsorbed to IgG sepharose was eluted with 30 μl of 0.5 M acetate buffer (pH adjusted to 3.4 with ammonium acetate), and then IgG sepharose was centrifuged at 160 × g for 10 minutes. Excluded.
9 μl of 2M Tris was added to the eluate to adjust the pH to 7.0.
[0173]
SDS-PAGE was performed using 15 μl of the eluate thus prepared, and then silver staining was performed using a silver staining kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.) (FIG. 8).
When an eluate derived from the culture supernatant of Namalwa KJM-1 cells introduced with pAMoA-i52S was used, a band of about 57 kD was confirmed. On the other hand, when an eluate derived from the culture supernatant of Namalwa KJM-1 cells introduced with the vector pAMoA was used, a band of about 57 kD was not detected.
From the above results, secretory iGnT can be secreted and produced in the culture supernatant as a fusion protein with the IgG binding region of S. aureus protein A, and the fusion protein can be easily purified using IgG sepharose. It was shown that there is.
[0174]
Example 4 Secretion production of Flag-fused iGnT using Namalwa KJM-1 cells as a host
(1) Construction of Flag fusion type secretion vector pAMoF2 (FIG. 9)
A secretory vector pAMoF2 for secreting and expressing a Flag peptide (SEQ ID NO: 17) added to the N-terminus of an arbitrary protein was constructed. The signal sequence of immunoglobulin κ and the DNA encoding the Flag peptide were prepared using 6 types of synthetic DNA.
[0175]
1 μg of pAMo was added to 10 mM Tris-HCl (pH 7.5), 6 mM MgCl.₂, 80 mM NaCl, 6 mM 2-mercaptoethanol buffer solution (hereinafter abbreviated as Y-80 buffer solution) 20 μl,HindIII and 20 unitsAsp718 was added and digestion reaction was performed at 37 ° C. for 2 hours.
After the reaction solution was subjected to agarose gel electrophoresis, about 8.7 kbHindIII-AspThe 718-treated DNA fragment was recovered.
[0176]
Separately,HindIII cleavage site andAspThe following 6 types of DNA [IgK-1 (SEQ ID NO: 7), IgK-2 (SEQ ID NO: 8), IgK-3 (SEQ ID NO: 9), IgK-4 (SEQ ID NO: 7)] are used as linkers for linking 718 cleavage sites. 10), IgK-5 (SEQ ID NO: 11), IgK-6 (SEQ ID NO: 12)]. In the linker constructed by these DNAs,PmaCI,StuI,SnaEach restriction enzyme cleavage site of BI is incorporated. Each of the six DNAs was synthesized using an Applied Biosystems 380A DNA synthesizer. 0.2 μg of each synthesized DNA was dissolved in 20 μl of T4 kinase buffer, 30 units of T4 polynucleotide kinase (Takara Shuzo Co., Ltd., hereinafter the same) was added, and phosphorylation was performed at 37 ° C. for 2 hours.
[0177]
Six types of phosphorylated synthetic DNA (5 ng each) obtained above and about 8.7 kbHindIII-Asp0.05 μg of 718-treated DNA fragment was dissolved in 20 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and a binding reaction was performed at 12 ° C. for 16 hours.
Escherichia coli MM294 strain was transformed with the reaction solution by the method of Cohen et al. To obtain a transformant having ampicillin resistance.
A plasmid was isolated from the transformant according to a known method, and its structure was confirmed by restriction enzyme digestion and nucleotide sequencing. Hereinafter, this plasmid is called pAMoF2 (FIG. 9).
[0178]
(2) Construction of Flag-fused iGnT secretion expression plasmid pAMoF2-i52S (FIGS. 10 and 11)
Since the cloned iGnT is presumed from its primary sequence, it consists of an N-terminal cytoplasmic region (8 amino acids) followed by a membrane-bound region (28 amino acids) and a C-terminal catalytic activity region (379 amino acids). The cytoplasmic region and the membrane-bound region on the N-terminal side of iGnT were removed, and an immunoglobulin signal sequence and a Flag peptide were added to the removed region to attempt secretory expression of iGnT.
[0179]
First, a DNA fragment (prepared in Example 1) encoding a region considered to have iGnT catalytic activity (from the 52nd serine of SEQ ID NO: 1 to the 415th cysteine) was prepared from the T-vector pT7Blue (R ) (Manufactured by Novagen), the plasmid pT7B-i52S No. 3 was created.
pT7B-i52S No. 3 was formed by the following method (FIG. 10).
[0180]
0.1 μg of the approximately 1.1 kb PCR amplified DNA fragment prepared in (1) of Example 3 and 0.02 μg of T-vector pT7Blue (R) (manufactured by Novagen) were dissolved in 30 μl of T4 ligase buffer, and T4 DNA was dissolved. 175 units of ligase was added and a binding reaction was performed at 12 ° C. for 16 hours.
Escherichia coli MM294 was transformed with the reaction solution by the method of Cohen et al. To obtain a transformant having ampicillin resistance.
A plasmid was isolated from the transformant according to a known method, and its structure was confirmed by restriction enzyme digestion. This plasmid was designated as pT7B-i52S No. 3 (FIG. 10).
[0181]
Subsequently, the construction of a flag fusion type iGnT secretion expression plasmid pAMoF2-i52S was performed (FIG. 11).
1 μg of pAMoF2 is dissolved in 20 μl of Y-100 buffer and 20 units ofStuI and 20 unitsBanIII was added and digestion was performed at 37 ° C. for 2 hours.
After subjecting the reaction solution to agarose gel electrophoresis, about 7.2 kbStuI-BanIII-treated DNA fragments were recovered.
Also, 1 μg of pAMo is dissolved in 20 μl of Y-100 buffer, and 20 units ofBanIII was added and digestion was performed at 37 ° C. for 2 hours.
[0182]
After the reaction, NaCl was added to 150 mM, and 20 units ofNotI was added, and a digestion reaction was further performed at 37 ° C. for 2 hours.
After the reaction solution was subjected to agarose gel electrophoresis, about 1.7 kbBanIII-NotI-treated DNA fragments were recovered.
In addition, 1 μg of pT7B-i52S No. 3 is dissolved in 20 μl of Y-100 buffer and 20 units ofBamHI was added and digestion was performed at 37 ° C. for 2 hours.
[0183]
After the reaction, ethanol precipitation is performed, the obtained precipitate is dissolved in 30 μl of DNA polymerase I buffer, 6 units of E. coli DNA polymerase I / Klenow fragment is added, and the mixture is reacted at 37 ° C. for 60 minutes.BamThe 5 ′ overhang generated by HI digestion was changed to a blunt end.
The reaction was stopped by phenol extraction, chloroform extraction and ethanol precipitation were performed, then dissolved in 30 μl of Y-150 buffer, and 20 units ofNotI was added and digestion reaction was performed at 37 ° C. for 2 hours.
[0184]
After the reaction solution was subjected to agarose gel electrophoresis, about 1.1 kbBamHI (blunt end)-NotI-treated DNA fragments were recovered.
The 7.2 kb obtained above.StuI-BanIII-treated DNA fragment of 0.05 μg and about 1.7 kbBanIII-Not0.01 μg of I-treated DNA fragment, and about 1.1 kbBamHI (blunt end)-Not0.05 μg of I fragment was dissolved in 20 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and a binding reaction was performed at 12 ° C. for 16 hours.
[0185]
Escherichia coli MM294 strain was transformed with the reaction solution by the method of Cohen et al. To obtain a transformant having ampicillin resistance.
A plasmid was isolated from the transformant according to a known method, and the structure of the plasmid was confirmed by restriction enzyme digestion. This plasmid is called pAMoF2-i52S (FIG. 11).
[0186]
(2) Expression of Flag fusion secretory iGnT in Namalwa KJM-1 cells
The control plasmid pAMoF2 and the iGnT Flag fusion secretion expression plasmid pAMoF2-i52S constructed as described above were prepared using a plasmid preparation kit (/ plasmid / maxi kit; trademark number 41031) manufactured by Qiagen.
The prepared and obtained plasmid was dissolved in TE buffer so as to be 1 μg / μl after ethanol precipitation.
[0187]
After lysis, both plasmids were introduced into Namalwa KJM-1 cells by the lipofection method described in Example 1 to obtain transformants.
The obtained transformant was 5 × 10 5 in 30 ml of RPMI1640 medium containing 0.5 mg / ml of G418 and 2% of fetal calf serum.^FourSuspend to cells / ml, CO₂The cells were cultured at 37 ° C. for 9 days in an incubator.
[0188]
After culturing, the cells were removed by centrifugation under conditions of 160 × g, 10 minutes and 1500 × g, 10 minutes, and the supernatant was collected.
The culture supernatant can be stored at −80 ° C. and can be thawed before use.
Since iGnT encoded by the plasmid pAMoF2-i52S is secreted and expressed as a fusion protein with the Flag peptide, it can be easily obtained using an anti-Flag M1 Affinity Gel (Cosmo Bio). Purification is possible.
[0189]
After adding sodium azide, sodium chloride, and calcium chloride to the culture supernatant obtained above to a final concentration of 0.1%, 150 mM, and 2 mM, respectively, anti-Flag M1 Affinity Gel (Anti-Flag M1 Affinity Gel) Cosmo Bio) was added, and the mixture was stirred slowly at 4 ° C. overnight.
After stirring, the anti-Flag M1 affinity gel was recovered by centrifuging at 160 × g for 10 minutes, and the gel was collected with 1 ml of a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 1 mM calcium chloride. Washed twice.
[0190]
After washing, 30 μl of a buffer solution containing 50 mM Tris-hydrochloric acid (pH 7.4), 150 mM sodium chloride, and 2 mM EDTA was added to the gel, followed by treatment at 4 ° C. for 30 minutes to elute the protein adsorbed on the gel. Thereafter, the supernatant was obtained by centrifugation at 160 × g for 10 minutes. To the gel, 30 μl of a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, and 2 mM EDTA was added again, treated at 4 ° C. for 10 minutes, and then centrifuged at 160 × g for 10 minutes. Acquired. Then, said operation was performed again and elution operation was performed 3 times in total.
To the eluate, 1M calcium chloride was added to a final concentration of 4 mM.
[0191]
SDS-PAGE was performed using 15 μl of the first eluate prepared as described above, and then silver staining was performed using a silver staining kit Wako (manufactured by Wako Pure Chemical Industries, Ltd.) (FIG. 12).
When an eluate derived from the culture supernatant of Namalwa KJM-1 cells introduced with pAMoF2-i52S was used, a band of about 49 kD was confirmed. On the other hand, when an eluate derived from the culture supernatant of Namalwa KJM-1 cells into which the vector pAMoF2 was introduced, a band of about 49 kD was not detected.
From the above results, it was shown that Flag-fused iGnT is secreted and produced in the culture supernatant and can be purified using an anti-FlagM1 affinity gel.
[0192]
Example 5 Preparation of recombinant virus solution for iGnT production using insect cells as hosts
(1) Production of recombinant virus for expressing iGnT in insect cells
A step of incorporating a DNA encoding a target protein into a special plasmid called a transfer vector (step 1), a transfer vector incorporating the target DNA prepared in step 1 and a wild type virus are cotransfected into insect cells, and homologous Recombinant virus was produced in two steps of obtaining recombinant virus by recombination (step 2).
This step was performed according to the following procedure according to the manual of the kit using a Pharmingen Baculo Gold Starter Kit (Product No. PM-21001K).
[0193]
(Step 1) Integration of iGnT cDNA into transfer vector (FIG. 13)
Of the multiple cloning site present in the transfer vector pVL1393SmaWith I siteBglA plasmid pVL1393-i incorporating iGnT cDNA was constructed between the II sites.
1 μg of pVL1393 was added to 25 μl of 10 mM Tris-HCl (pH 7.5), 6 mM MgCl.₂, 20 mM KCl, 6 mM 2-mercaptoethanol in a buffer solution (hereinafter abbreviated as K-20 buffer solution)SmaI was added and digestion was performed at 37 ° C. for 2 hours.
[0194]
After the reaction, NaCl was added to 80 mM, and 20 units ofBstPI was added, and a digestion reaction was further performed at 60 ° C. for 2 hours.
After the reaction solution was subjected to agarose gel electrophoresis, about 3.2 kbSmaI-BstThe PI-treated DNA fragment was recovered.
Also, 1 μg of pVL1393 was dissolved in 25 μl of Y-80 buffer, and 20 units ofBglII was added and digestion was performed at 37 ° C. for 2 hours.
[0195]
After the reaction, 20 unitsBstPI was added, and a digestion reaction was further performed at 60 ° C. for 2 hours.
After the reaction solution was subjected to agarose gel electrophoresis, about 6.4 kb was obtained.BglII-BstThe PI-treated DNA fragment was recovered.
1 μg of pAMo-i is dissolved in 25 μl of Y-150 buffer and 20 units ofBglII and 20 unitsEcoRV was added and digestion was performed at 37 ° C. for 2 hours.
[0196]
After the reaction solution was subjected to agarose gel electrophoresis, about 2.0 kbEcoRV-BglII-treated DNA fragments were recovered.
About 3.2 kb derived from pVL1393 obtained aboveSmaI-BstPI-treated DNA fragment 0.1 μg and about 6.4 kb derived from pVL1393BglII-Bst0.2 μg of PI-treated DNA fragment, and about 2.0 kb derived from pAMo-iEcoRV-BglII-treated DNA fragment (0.2 μg) was dissolved in 30 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and a binding reaction was performed at 12 ° C. for 16 hours.
Using the reaction solution, E. coli MM294 strain was transformed by the method of Cohen et al. To obtain an ampicillin resistant strain. A plasmid was isolated from this transformant according to a known method. This plasmid was named pVL1393-i, and its structure was confirmed by restriction enzyme digestion (FIG. 13).
[0197]
(Step 2) Production of recombinant virus
Insect cells Sf9 (manufactured by Pharmingen) cultured using TNM-FH insect medium (manufactured by Pharmingen) have linear baculovirus DNA (BaculoGold baculovirus DNA), Pharmingen And the plasmid pVL1393-i was prepared by the lipofectin method [protein nucleic acid enzyme,37, 2701 (1992)], a recombinant baculovirus was prepared as follows.
[0198]
1-5 μg of pVL1393-i and 15 ng of linear baculovirus DNA were dissolved in 12 μl of distilled water, and then 6 μl (6 μg) of Lipofectin (GIBCO BRL) and 6 μl of distilled water were mixed and added at room temperature. Left for 15 minutes.
About 2 × 10⁶Sf9 cells were suspended in 2 ml of Sf900-II medium (GIBCO BRL), placed in a plastic dish for cell culture with a diameter of 35 mm, and then the total amount of a mixed solution of pVL1393-i, linear baculovirus DNA, and lipofectin Was added and cultured at 27 ° C. for 3 days.
[0199]
1 ml of the culture supernatant containing the recombinant virus was collected from the culture solution.
1 ml of TNM-FH insect medium was added to the petri dish from which the culture supernatant was obtained, and further cultured at 27 ° C. for 4 days. After the culture, 1.5 ml of a culture supernatant containing the recombinant virus was obtained in the same manner.
[0200]
(2) Acquisition of recombinant virus solution
About 8 × 10⁶Individual Sf9 cells were suspended in 5 ml of Sf900-II medium,²Place in a flask (manufactured by Greiner) and let stand at room temperature for 30 minutes to allow cells to adhere to the flask, remove the supernatant, and add 1 ml of Sf900-II medium and the recombinant virus obtained in (1) above to the flask. 1 ml of culture supernatant containing was added.
After the addition, the cells and the virus were sufficiently brought into contact by gently shaking at room temperature for 1 hour, and then 4 ml of TNM-FH insect medium was added and cultured at 27 ° C. for 4 days.
[0201]
The culture solution was centrifuged at 1500 × g for 10 minutes to obtain Sf9 cells infected with the recombinant virus and 5.5 ml of the recombinant virus solution.
About 2 × 10⁷Individual Sf9 cells are suspended in 15 ml of Sf900-II medium,²Place in a flask (manufactured by Greiner) and leave at room temperature for 30 minutes to allow cells to adhere to the flask. Remove the supernatant and add 5 ml of Sf900-II medium and 1 ml of the recombinant virus solution obtained above to the flask. did.
[0202]
After the addition, the cells and the virus were brought into sufficient contact by gently shaking at room temperature for 1 hour, and then 10 ml of TNM-FH insect medium was added and cultured at 27 ° C. for 4 days.
The culture solution was centrifuged at 1500 × g for 10 minutes to obtain Sf9 cells infected with the replacement virus and 15 ml of the recombinant virus solution.
[0203]
The virus titer of the recombinant virus solution was calculated by the following method (Pharmingen Baculo Gold Starter Kit Manual).
About 6 × 10⁶Each Sf9 cell was suspended in 4 ml of Sf900-II medium, placed in a plastic petri dish for cell culture having a diameter of 60 mm, and allowed to stand at room temperature for 30 minutes to allow the cells to adhere to the petri dish. Sf900-II medium 400 μl and Sf900-II medium 10^-FourOr 10^-Five100 μl of the above-mentioned recombinant virus solution diluted in 1 ml was added.
[0204]
After the addition, the petri dish was gently shaken at room temperature for 1 hour to sufficiently bring the cells and the virus into contact.
After contact, the medium was removed from the petri dish, and 2 ml of Sf900-II medium (incubated at 42 ° C.) containing 2% low melting point agarose (Agarplaque Agarose; manufactured by Pharmingen) in the petri dish. A mixture of 2 ml of TNM-FH insect medium (kept at 42 ° C.) was poured and left at room temperature for 15 minutes.
[0205]
After leaving, the petri dish was covered with vinyl tape to prevent drying, and the petri dish was placed in a sealable plastic container and cultured at 27 ° C. for 5 days.
After culturing, 1 ml of PBS buffer containing 0.01% neutral red was added to the petri dish, and after further culturing for 1 day, the number of plaques that appeared was counted.
From the above operation, the recombinant virus solution is about 1.2 × 10⁸It was found to contain plaque forming unit (PFU) / ml virus.
Further, using the transfer vector pVL1393, a recombinant virus for control and Sf9 cells infected with the virus were prepared. Recombinant virus solution for control is about 1.4 × 10⁸It was found to contain plaque forming unit (PFU) / ml virus.
[0206]
Example 6 Secretory production of protein A-fused iGnT using insect cells as hosts
Secretory expression of the protein A fusion type iGnT shown in Example 3 in insect cells was performed.
(1) Preparation of recombinant virus for secretory expression of protein A fusion type iGnT in insect cells (Step 1) Incorporation of DNA encoding protein A fusion type iGnT into a transfer vector (FIG. 14)
Of the multiple cloning site present in the transfer vector pVL1393SmaWith I siteNotA plasmid pVL1393-Ai52S incorporating the DNA encoding protein A fusion secretory iGnT shown in Example 3 was constructed between the I sites.
[0207]
1 μg of pVL1393 is dissolved in K-20 buffer and 20 units ofSmaI was added and digestion was performed at 37 ° C. for 2 hours.
After the reaction, NaCl was added to 80 mM, and 20 units ofBstPI was added, and a digestion reaction was further performed at 60 ° C. for 2 hours.
After the reaction solution was subjected to agarose gel electrophoresis, about 3.2 kbSmaI-BstPI-treated DNA fragments were recovered.
Also, 1 μg of pVL1393 was dissolved in 25 μl of Y-80 buffer, and 20 units ofBstPI was added and digestion was performed at 60 ° C. for 2 hours.
[0208]
After the reaction, NaCl was added to 150 mM, and 20 units ofNotI was added, and a digestion reaction was further performed at 37 ° C. for 2 hours.
[0209]
After the reaction solution was subjected to agarose gel electrophoresis, about 6.4 kb was obtained.NotI-BstPI-treated DNA fragments were recovered.
[0210]
1 μg of pAMoA-i52S (made in Example 3) was dissolved in 25 μl of Y-100 buffer and 20 units ofSalI was added and digestion was performed at 37 ° C. for 2 hours.
After the reaction, ethanol precipitation is performed, the obtained precipitate is dissolved in 30 μl of DNA polymerase I buffer, 6 units of E. coli DNA polymerase I / Klenow fragment is added, and the mixture is reacted at 37 ° C. for 60 minutes.SalThe 5 ′ overhang generated by I digestion was changed to a blunt end.
[0211]
The reaction was stopped by phenol extraction, chloroform extraction and ethanol precipitation were performed, then dissolved in 30 μl of Y-150 buffer, and 20 units ofNotI was added and digestion reaction was performed at 37 ° C. for 2 hours.
After the reaction solution was subjected to agarose gel electrophoresis, about 1.4 kbSalI (blunt end)-NotI-treated DNA fragments were recovered.
[0212]
About 3.2 kb derived from pVL1393 obtained aboveSmaI-BstPI-treated DNA fragment 0.1 μg and about 6.4 kb derived from pVL1393NotI-Bst0.2 μg of PI-treated DNA fragment, and about 1.4 kb derived from pAMoA-i52SSalI (blunt end)-Not0.2 μg of I-treated DNA fragment was dissolved in 30 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and a binding reaction was performed at 12 ° C. for 16 hours.
[0213]
Escherichia coli MM294 strain was transformed with the reaction solution by the method of Cohen et al. To obtain a transformant having ampicillin resistance. A plasmid was isolated from the transformant according to a known method. This plasmid was named pVL1393-Ai52S, and its structure was confirmed by restriction enzyme digestion (FIG. 14).
[0214]
(Step 2) Production of recombinant virus
In the same manner as in Example 5 (Step 2), 1.5 ml of a recombinant baculovirus solution derived from pVL1393-Ai52S was obtained.
(2) Acquisition of recombinant virus solution and Sf9 cells infected with recombinant virus
In the same manner as the method shown in (2) of Example 5, about 8 × 10⁶Sf9 cells were infected with the recombinant virus obtained in the above (1) to obtain Sf9 cells infected with the recombinant virus and 5.5 ml of the recombinant virus solution.
[0215]
Further, in the same manner as the method shown in (2) of Example 5, approximately 2 × 10⁷Individual Sf9 cells were infected with the recombinant virus obtained above to obtain Sf9 cells infected with the recombinant virus and 15 ml of the recombinant virus solution.
Further, using the transfer vector pVL1393, a recombinant virus for control and Sf9 cells infected with the virus were prepared.
[0216]
(3) Secretion production and purification of protein A fusion type iGnT polypeptide
IGnT encoded by the recombinant virus derived from plasmid pVL1393-Ai52S is S. aureus (StaphylococcusaureusThe protein A is secreted and expressed as a fusion protein with the IgG binding region of protein A, and thus can be easily purified using IgG Sepharose.
About 2 × 10⁷Individual Sf21 cells were suspended in 15 ml of Sf900-II medium,²Place in a flask (manufactured by Greiner) and let stand at room temperature for 30 minutes to allow cells to adhere to the flask. Then remove the supernatant and add 5 ml of Sf900-II medium and the recombinant virus solution obtained in (2) above to the flask. 1 ml was added.
[0217]
After the addition, the cells and the virus were brought into sufficient contact by gently shaking at room temperature for 1 hour, and then 10 ml of TNM-FH insect medium was added and cultured at 27 ° C. for 4 days.
The culture solution was centrifuged at 1500 × g for 10 minutes to obtain 15 ml of a culture supernatant that was thought to contain secreted iGnT.
After adding sodium azide to a final concentration of 0.1% to 10 ml of the culture supernatant obtained above, 100 μl of IgG sepharose (manufactured by Pharmacia) pretreated according to the product instructions was added, and 4 ° C. Stir slowly overnight.
[0218]
After stirring, IgG Sepharose was recovered by centrifugation at 160 × g for 10 minutes, and the Sepharose was mixed with 1 ml of a buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM sodium chloride, 0.05% Tween20. Washed twice.
After washing, 100 μl of 0.5 M acetate buffer (pH adjusted to 3.4 with ammonium acetate) was used to elute the protein adsorbed to IgG sepharose, and then centrifuged at 160 × g for 10 minutes to obtain IgG sepharose. Excluded.
30 μl of 2M Tris was added to the eluate to adjust the pH to 7.0.
[0219]
SDS-PAGE was performed using 15 μl of the eluate thus prepared, and then staining was performed using Coomassie Brilliant Blue (FIG. 15).
When an eluate prepared from the culture supernatant of Sf21 infected with the recombinant virus derived from pVL1393-Ai52S was used, a band of about 50 kD was confirmed. On the other hand, when an eluate prepared from the culture supernatant of Sf21 infected with a recombinant virus derived from the vector pVL1393 was used, a band of about 50 kD was not detected.
From the above results, secreted iGnT can be secreted and produced in the culture supernatant as a fusion protein with the IgG binding region of S. aureus protein A, and the secreted protein can be purified using IgG sepharose. It was shown that.
[0220]
Example 7 Secretory production of Flag peptide-fused iGnT using insect cells as hosts
Secreted expression in insect cells of the Flag peptide-fused iGnT shown in Example 4 was performed.
(1) Preparation of recombinant virus for secretory expression of Flag peptide-fused iGnT in insect cells (Step 1) Incorporation of DNA encoding Flag peptide-fused secretory iGnT into a transfer vector (FIG. 16)
Of the multiple cloning site present in the transfer vector pVL1393SmaWith I siteNotA plasmid pVL1393-F2i52S incorporating the DNA encoding the Flag peptide fusion secretory iGnT shown in Example 4 was constructed between the I sites.
[0221]
PAMoF2-i52S prepared in Example 4 was dissolved in 1 μg, 25 μl of Y-80 buffer, and 20 units ofHindIII was added and digestion was performed at 37 ° C. for 2 hours.
After the reaction, ethanol precipitation is performed, the obtained precipitate is dissolved in 30 μl of DNA polymerase I buffer, 6 units of E. coli DNA polymerase I / Klenow fragment is added, and the mixture is reacted at 37 ° C. for 60 minutes.HinThe 5 ′ overhang generated by dIII digestion was changed to a blunt end.
[0222]
The reaction was stopped by phenol extraction, chloroform extraction and ethanol precipitation were performed, then dissolved in 30 μl of Y-150 buffer, and 20 units ofNotI was added and digestion reaction was performed at 37 ° C. for 2 hours.
After the reaction solution was subjected to agarose gel electrophoresis, about 1.2 kbHindIII (blunt end)-NotI-treated DNA fragments were recovered.
[0223]
About 1.2 kb obtained aboveHindIII (blunt end)-Not0.2 μg of I-treated DNA fragment and about 3.2 kb derived from pVL1393 obtained in (1) of Example 5SmaI-BstPI-treated DNA fragment 0.1 μg and about 6.4 kb derived from pVL1393 obtained in Example 5 (1)NotI-Bst0.2 μg of the PI-treated DNA fragment was dissolved in 30 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and a binding reaction was performed at 12 ° C. for 16 hours.
Using the reaction solution, E. coli MM294 strain was transformed by the method of Cohen et al. To obtain an ampicillin resistant strain. A plasmid was isolated from this transformant according to a known method. This plasmid was named pVL1393-F2i52S (FIG. 16), and its structure was confirmed by restriction enzyme digestion.
[0224]
(Step 2) Production of recombinant virus
In the same manner as in Example 5 (Step 2), 1.5 ml of a recombinant baculovirus solution derived from pVL1393-F2i52S was obtained.
(2) Acquisition of recombinant virus solution and Sf9 cells infected with recombinant virus
In the same manner as the method shown in (2) of Example 5, about 8 × 10⁶Sf9 cells were infected with the recombinant virus obtained in the above (1) to obtain Sf9 cells infected with the recombinant virus and 5.5 ml of the recombinant virus solution.
In the same manner as the method shown in (2) of Example 5, approximately 2 × 10⁷Individual Sf9 cells were infected with the recombinant virus obtained above to obtain Sf9 cells infected with the recombinant virus and 15 ml of the recombinant virus solution.
Further, using the transfer vector pVL1393, a recombinant virus for control and Sf9 cells infected with the virus were prepared.
[0225]
(3) Secretion production and purification of Flag peptide-fused iGnT polypeptide
Since iGnT encoded by a recombinant virus derived from the plasmid pVL1393-F2i52S is secreted and expressed as a fusion protein with the Flag peptide, an anti-Flag M1 affinity gel (Cosmo Bio) was used. And can be easily purified.
About 2 × 10⁷Individual Sf21 cells were suspended in 15 ml of Sf900-II medium,²Place in a flask (manufactured by Greiner) and let stand at room temperature for 30 minutes to allow cells to adhere to the flask. Then remove the supernatant and add 5 ml of Sf900-II medium and the recombinant virus solution obtained in (2) above to the flask. 1 ml was added.
[0226]
After the addition, the cells and the virus were brought into sufficient contact by gently shaking at room temperature for 1 hour, and then 10 ml of TNM-FH insect medium was added and cultured at 27 ° C. for 4 days. The culture solution was centrifuged at 1500 × g for 10 minutes to obtain 15 ml of a culture supernatant that was thought to contain secreted iGnT.
After adding sodium azide, sodium chloride, and calcium chloride to 10 ml of the culture supernatant obtained above to a final concentration of 0.1%, 150 mM, and 2 mM, respectively, anti-FlagM1 affinity gel (Anti-Flag M1 Affinity Gel) ; Cosmo Bio) was added and slowly stirred at 4 ° C. overnight.
[0227]
After stirring, the anti-Flag M1 affinity gel was recovered by centrifuging at 160 × g for 10 minutes, and the gel was collected with 1 ml of a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, 1 mM calcium chloride. Washed twice.
After washing, 80 μl of a buffer solution containing 50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, and 2 mM EDTA was added to the gel, followed by treatment at 4 ° C. for 30 minutes to elute the protein adsorbed on the gel. Thereafter, the supernatant was obtained by centrifugation at 160 × g for 10 minutes. To the gel, 80 μl of a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM sodium chloride, and 2 mM EDTA was added again, treated at 4 ° C. for 10 minutes, and then centrifuged at 160 × g for 10 minutes. Acquired. Then, said operation was performed again and elution operation was performed 3 times in total.
[0228]
To the eluate, 1M calcium chloride was added to a final concentration of 4 mM.
After performing SDS-PAGE using 15 μl of the eluate thus prepared, staining was performed using Coomassie Brilliant Blue (FIG. 17).
When an eluate prepared from the culture supernatant of Sf21 infected with the recombinant virus derived from pVL1393-F2i52S was used, a band of about 43 kD was confirmed. On the other hand, when an eluate prepared from the culture supernatant of Sf21 infected with a recombinant virus derived from the vector pVL1393 was used, a band of about 43 kD was not detected.
From the above results, it was shown that Flag-fused iGnT is secreted and produced in the culture supernatant and can be easily purified using an anti-Flag M1 affinity gel.
[0229]
Example 8 Establishment of Quantification Method of iGnT Transcript by PCR Method and Examination of Expression Level in Various Cells Quantification of iGnT transcript was performed by quantitative PCR method [Proc. Natl. Acad. Sci. USA.,87, 2725 (1990)]. The amount of iGnT transcript in various cells and cell lines was expressed as a relative value when the amount of β-actin transcript thought to be expressed to the same extent in all cells was taken as 100.
[0230]
(1) Construction of standard plasmid pBSK + iGnT13 (FIG. 18)
One of the plasmids for determining the iGnT cDNA sequence (named pBSK + iGnT13) constructed in (4) of Example 1 was used as a standard plasmid. The plasmid is plasmid 16-2-12.HindIII-SmaThe I fragment (about 1.0 kb: No. 13 fragment in FIG. 3) of pBluescript II SK (+)HindIII-HincPrepared by subcloning between II. The method for constructing the plasmid is shown below.
[0231]
1 μg of plasmid 16-2-12, 25 μl of 10 mM Tris-HCl (pH 7.5), 6 mM MgCl₂, 60 mM KCl, 6 mM 2-mercaptoethanol.HindIII and 20 unitsSmaI was added and digestion reaction was performed at 37 ° C. for 2 hours.
After the reaction solution was subjected to agarose gel electrophoresis, about 1.0 kbHinThe dIII-SmaI treated DNA fragment was recovered.
[0232]
Dissolve pBluescript II SK (+) in 1 μg, 25 μl of Y-50 buffer,HindIII and 20 unitsHincII was added and digestion was performed at 37 ° C. for 2 hours.
After the reaction solution was subjected to agarose gel electrophoresis, about 2.95 kbHindIII-HincII-treated DNA fragments were recovered.
[0233]
About 1.0 kb obtained aboveHindIII-Sma0.1 μg of I-treated DNA fragment and about 2.95 kbHindIII-HincII-treated DNA fragment 0.05 μg was dissolved in 20 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and a binding reaction was performed at 12 ° C. for 16 hours.
Escherichia coli JM105 strain was transformed with the reaction solution by the method of Cohen et al. To obtain a transformant having ampicillin resistance.
A plasmid was isolated from the transformant according to a known method.
The plasmid was named pBSK + iGnT13, and its structure was confirmed by restriction enzyme digestion and nucleotide sequence determination. The structure of the plasmid is shown in FIG.
[0234]
(2) Construction of plasmid pBSK + iGnT13d for internal control (FIG. 19)
Plasmid pBSK + iGnT13d having a deletion mutation in iGnT cDNA contained in pBSK + iGnT13 constructed in (1) above was constructed.
[0235]
pBSK + iGnT13 was dissolved in 1 μg, 25 μl of Y-100 buffer, and 20 units ofXhoI and 20 unitsStuI was added and digestion reaction was performed at 37 ° C. for 2 hours.
[0236]
After the reaction solution was subjected to agarose gel electrophoresis, about 3.1 kbXhoI-StuI-treated DNA fragments were recovered.
pBSK + iGnT13 was dissolved in 1 μg, 25 μl of Y-100 buffer, and 20 units ofXhoI and 20 unitsHincII was added and digestion was performed at 37 ° C. for 2 hours.
After the reaction solution was subjected to agarose gel electrophoresis, about 0.6 kbXhoI-HincII-treated DNA fragments were recovered.
[0237]
About 3.1 kb obtained aboveXhoI-Stu0.05 μg of I-treated DNA fragment and about 0.6 kbXhoI-HincII-treated DNA fragment (0.1 μg) was dissolved in 20 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and a binding reaction was performed at 12 ° C. for 16 hours.
Escherichia coli MM294 strain was transformed with the reaction solution by the method of Cohen et al. To obtain a transformant having ampicillin resistance.
A plasmid was isolated from the transformant according to a known method.
The plasmid was named pBSK + iGnT13d, and its structure was confirmed by restriction enzyme digestion. The structure of the plasmid is shown in FIG.
[0238]
(3) Quantification of iGnT transcripts in various cells and cell lines using quantitative PCR
(A) Synthesis of single-stranded cDNA (used as a template for quantitative PCR) derived from various cells and cell lines
As cell lines, Namalwa KJM-1 cells, WM266-4 cells, THP-1 cells, HL-60 cells, U-937 cells, Colo205 cells, LS180 cells, SW1116 cells and Jurkat cells were used.
[0239]
WM266-4 cells, THP-1 cells, HL-60 cells, U-937 cells, Colo205 cells and LS180 cells were obtained from the American Type Culture Collection (ATCC). SW1116 cells (available from ATCC) and Jurkat cells (RIKEN Gene Bank; available from RIKEN GENE BANK) were obtained from Dr. Takahashi of Aichi Cancer Center.
[0240]
In addition, polymorphonuclear leukocytes and mononuclear cells were obtained from peripheral blood of healthy adults using Polymorphprep ™, a kit manufactured by Nycomed Pharma.
The obtained mononuclear cells can be obtained by conventional methods (J. Immunol.,130, 706 (1983)], were further separated into monocytes and lymphocytes.
The total RNA of each cell is measured using a conventional method [Biochemistry,18, 5294 (1977)].
[0241]
Synthesis of single-stranded cDNA from total RNA was performed using a kit (SUPERSCRIPT ™ Preamplification System; manufactured by BRL).
Single-stranded cDNA was synthesized from 5 μg of total RNA for cell lines and 1 μg of total RNA for blood cells, diluted 50-fold and 10-fold with water, respectively, and used as a PCR template.
[0242]
(B) Preparation of standards and internal controls for quantitative PCR
The pBSK + iGnT13 and pBSK + iGnT13d obtained in (1) and (2) above were cleaved with a restriction enzyme that cleaves the cDNA portion and converted into linear DNA, and then used as a standard for quantifying iGnT transcripts and an internal control, respectively.
1 μg each of BSK + iGnT13 and pBSK + iGnT13d was dissolved in 36 μl of Y-100 buffer, and 20 units ofXhoI and 20 unitsBamHI was added and digestion was performed at 37 ° C. for 2 hours.
[0243]
A 10 μl portion of the reaction solution was subjected to agarose gel electrophoresis, and after confirming that it was completely cleaved, it was used by diluting stepwise with water containing 1 μg / ml of yeast transfer RNA.
As a standard for quantifying β-actin transcripts, a restriction enzyme (pUC119-ACT) that cleaves the cDNA portion (HindIII andAsp718) and converted to linear DNA [J. Biol. Chem.,26914730 (1994), JP 06-181759].
[0244]
As an internal control for quantifying β-actin transcripts, a restriction enzyme that cleaves pUC119-ACTd from the cDNA portion (HindIII andAsp718) and converted to linear DNA [J. Biol. Chem.,26914730 (1994), JP 06-181759].
[0245]
(C) Quantification of iGnT transcript using quantitative PCR
Internal control prepared in (b) (XhoI andBamIn the presence of 5fg of pBSK + iGnT13d) cleaved with HI, PCR was performed using single-stranded cDNAs derived from the various cells and cell lines prepared in (a) as templates.
As primers for PCR, the DNA shown in SEQ ID NO: 13 (hereinafter abbreviated as C12-3) and the DNA shown in SEQ ID NO: 14 (hereinafter abbreviated as C12-4) were synthesized (purchased from Sawasday Technology). Can also be).
[0246]
The PCR reaction was performed using a kit (GeneAmp ™ DNA Amplification Reagent Kit with AmpliTaq ™ Recombinant Taq DNA Polymerase) manufactured by Takara Shuzo.
The reaction solution was prepared according to the method of the kit. At that time, dimethyl sulfoxide was added to a final concentration of 5%.
[0247]
After treatment with 29 μl of a reaction solution other than Taq DNA polymerase at 97 ° C. for 5 minutes using PERKIN ELMER CETUS DNA Thermal Cycler (sold by Takara Shuzo) in ice, Quenched quickly.
[0248]
After quenching, 1 μl of Taq DNA polymerase diluted to 1 / 6.7 is added to the reaction solution, and then used at 94 ° C. for 30 seconds, 60 ° C. for 1 minute, 72 hours using a thermal cycler manufactured by Perkin Elmer Citas. The reaction at -2 minutes was carried out for 27 cycles.
[0249]
A 7 μl portion of the reaction solution was subjected to agarose gel electrophoresis, and then the gel was stained with SYBR Green I nucleic acid stain (Molecular Probes). The amount of the amplified DNA fragment was measured by analyzing the pattern of the amplified DNA fragment with a fluoroimager (FluorImager SI; manufactured by Molecular Dynamics).
In addition, instead of the single-stranded cDNA derived from various cells and cell lines, the standard (b) prepared (XhoI andBamPCR was performed in the same manner using pBSK + iGnT13) cleaved with HI as a template to prepare a calibration curve.
[0250]
The size of the iGnT transcript and the standard-derived DNA fragment was 615 bp, the size of the DNA fragment derived from the internal control was 416 bp, and the amount (in moles) of the iGnT transcript was calculated from the quantitative ratio of the two DNA fragments. In order to more accurately quantify transcripts, similar PCR was performed again on each sample using an internal control in an amount close to the amount of transcripts obtained as described above. The number of PCR cycles was varied according to the amount of internal control.
[0251]
Similarly, the β-actin transcript was quantified by two-step PCR. As described in (b) for internal controlHindIII andAspPUC119-ACTd cleaved at 718 was described in (b) as a standard.HindIII andAspPUC119-ACT cut at 718 was used.
As a primer for PCR, the DNA shown in SEQ ID NO: 15 (hereinafter abbreviated as Ac-1) and the DNA shown in SEQ ID NO: 16 (hereinafter abbreviated as Ac-3) were applied Biosystems 380A DNA synthesizer. What was synthesize | combined using was used.
[0252]
The first PCR was performed with 17 cycles using 10 pg of internal control.
In the case of β-actin, dimethyl sulfoxide was not added to the PCR reaction solution. The final expression level of the iGnT transcript was determined as a relative value (%) when the amount of β-actin transcript was 100.
The results are shown in Table 1.
It was shown that the amount of iGnT transferred can be measured by the above method.
[0253]
[Table 1]

[0254]
【The invention's effect】
According to the present invention, a polypeptide having an activity involved in the synthesis of a poly-N-acetyllactosamine sugar chain, a method for producing the polypeptide, a DNA encoding the polypeptide, a method for producing the DNA, and incorporating the DNA Recombinant vector obtained, transformant harboring the recombinant vector, antibody recognizing the polypeptide, method for producing the DNA or poly-N-acetyllactosamine sugar chain using the polypeptide, the DNA Diagnosis and treatment of diseases of inflammation or cancer metastasis using the polypeptide or the antibody, quantification method and immunostaining of the polypeptide of the present invention using the antibody, and changing the expression of the gene encoding the polypeptide A screening method for a compound and a screening method for a substance that changes the activity of the polypeptide can be provided.
[0255]
[Sequence Listing]

[0256]
[Sequence Listing Free Text]
SEQ ID NO: 3 Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 4-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 5-description of artificial sequence: synthetic DNA
SEQ ID NO: 6 Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 7--Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 8-Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 9—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 10—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 11—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 12—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 13—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 14—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 15—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 16—Description of Artificial Sequence: Synthetic DNA
SEQ ID NO: 17—Description of artificial sequence: commercially available amino acid sequence
[Brief description of the drawings]
FIG. 1 shows the process of expression cloning. Namalwa KJM-1 cells into which cDNA libraries derived from WM266-4 and SW1116 were introduced were fluorescently stained with an anti-i antibody and then subjected to FACS to collect cells highly reactive with the anti-i antibody. The portion of cells indicated by the bar was collected and subjected to the next FACS. After three FACS, cells highly reactive with anti-i antibody were concentrated.
[Fig. 2] Plasmids (16-2-12 and 16-2-31) recovered from cells highly reactive with anti-i antibody and control plasmid pAMo were introduced into Namalwa KJM-1 cells, respectively. It is the result of analyzing using FACS after indirect fluorescent antibody staining using i antibody (thick line) or A-PBS (thin line).
FIG. 3 shows a restriction enzyme map of cDNA contained in plasmid 16-2-12 and a vector portion (bold line) in the vicinity thereof. Numbered bars indicate DNA fragments subcloned into pBluescriptIISK (+) to determine the base sequence of the cDNA. See text for details.
FIG. 4 is a diagram showing a construction process of plasmid pAMo-i.
FIG. 5: iGnT expression plasmid pAMo-i and control plasmid pAMo were introduced into Namalwa KJM-1 cells, respectively. After indirect fluorescent antibody staining with anti-i antibody (thick line) or A-PBS (thin line), FACS It is the result of having analyzed using. Prior to indirect fluorescent antibody staining, the reactivity of cells treated with sialidase (+ sialidase) and not (-sialidase) was compared.
FIG. 6 is a diagram showing a construction process of plasmid pAMoA-FT3.
FIG. 7 is a diagram showing a construction process of plasmid pAMoA-i52S.
FIG. 8 shows the result of purifying protein A fusion secreted iGnT from the culture supernatant of Namalwa KJM-1 cells introduced with plasmid pAMoA-i52S using IgG sepharose and subjecting it to SDS polyacrylamide gel electrophoresis ( Lane 2). As a control, a sample was prepared in the same manner from the culture supernatant of Namalwa KJM-1 cells into which pAMoA had been introduced, and subjected to SDS polyacrylamide gel electrophoresis (lane 1).
FIG. 9 is a diagram showing a construction process of plasmid pAMoF2.
FIG. 10 is a diagram showing a construction process of plasmid pT7B-i52S No. 3.
FIG. 11 is a diagram showing a construction process of plasmid pAMoF2-i52S.
FIG. 12 shows the result of purifying Flag peptide fusion secreted iGnT from the culture supernatant of Namalwa KJM-1 cells introduced with plasmid pAMoF2-i52S using an anti-Flag M1 affinity gel and subjecting it to SDS polyacrylamide gel electrophoresis. (Lane 2). As a control, a sample was prepared in the same manner from the culture supernatant of Namalwa KJM-1 cells into which pAMoF2 had been introduced, and subjected to SDS polyacrylamide gel electrophoresis (lane 1).
FIG. 13 is a diagram showing a construction process of plasmid pVL1393-i.
FIG. 14 is a diagram showing a construction process of plasmid pVL1393-Ai52S.
FIG. 15 shows the result of purifying protein A fusion secreted iGnT using IgG sepharose from the culture supernatant of Sf21 cells infected with a recombinant virus derived from plasmid pVL1393-Ai52S and subjecting it to SDS polyacrylamide gel electrophoresis. Yes (lane 2). As a control, a sample was prepared in the same manner from the culture supernatant of Sf21 cells infected with a recombinant virus derived from plasmid pVL1393, and subjected to SDS polyacrylamide gel electrophoresis (lane 1).
FIG. 16 is a diagram showing a construction process of plasmid pVL1393-F2i52S.
[Fig. 17] Flag peptide fusion secreted iGnT was purified from the culture supernatant of Sf21 cells infected with a recombinant virus derived from plasmid pVL1393-F2i52S using anti-Flag M1 affinity gel, and subjected to SDS polyacrylamide gel electrophoresis. (Lane 2). As a control, a sample was prepared in the same manner from the culture supernatant of Sf21 cells infected with a recombinant virus derived from plasmid pVL1393, and subjected to SDS polyacrylamide gel electrophoresis (lane 1).
FIG. 18 is a diagram showing a construction process of plasmid pBSK + iGnT13.
FIG. 19 is a diagram showing a construction process of plasmid pBSK + iGnT13d.
[Explanation of symbols]
bp: base pairs
kb: kilobase pairs
G418 / Km: G418 derived from transposon 5 (Tn5), kanamycin resistance gene
Ap: pBR322-derived ampicillin resistance gene
Tc: pBR322-derived tetracycline resistance gene
P1: P1 promoter derived from pBR322
Ptk: Herpes simplex virus (HSV) thymidine kinase (tk) gene promoter
Sp. BG: Rabbit β-globin gene splicing signal
A.BG: Rabbit β-globin gene poly A addition signal
A. SE: Simian virus 40 (SV40) early gene poly A addition signal
Atk: Herpes simplex virus (HSV) thymidine kinase (tk) gene poly A addition signal
Pse: Simian virus 40 (SV40) early gene promoter
Pmo: Moloney murine leukemia virus long terminal repeat (LTR) promoter
EBNA-1: EBNA-1 gene of Epstein-Barr virus
oriP: Epstein-Barr virus replication origin
S: Gene part encoding human granulocyte colony-stimulating factor or immunoglobulin κ signal peptide
A or ProA: Staphylococcus aureusStaphylococcusaureusPart of the gene encoding the binding region of IgG of protein A
F: part of the gene encoding the Flag peptide
iGnT: DNA encoding a polypeptide involved in the synthesis of the poly-N-acetyllactosamine sugar chain obtained in the present invention (full length or partial length)
cDNA: DNA (full length or partial length) encoding a polypeptide involved in the synthesis of the poly-N-acetyllactosamine sugar chain obtained in the present invention
cDNA del: partial length DNA encoding a polypeptide involved in the synthesis of the poly-N-acetyllactosamine sugar chain obtained in the present invention having a deletion of about 0.2 kb

Claims (42)

A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1, a polypeptide comprising an amino acid sequence from which the amino acid from the N-terminal to the 51st amino acid has been deleted in the amino acid sequence represented by SEQ ID NO: 1, or a sequence amino acid sequence and one or several amino acid polypeptide consisting of the amino acid sequence represented by numbers 1, deletion, or addition to the amino acid sequence, and of the cell surface when expressed in a cell poly - A polypeptide having an activity of increasing the amount of N -acetyllactosamine sugar chain . The polypeptide according to claim 1, wherein the activity to increase the amount of poly -N -acetyllactosamine sugar chain on the cell surface when expressed in a cell is β1,3-N-acetylglucosamine transferase activity. . A DNA encoding the polypeptide of claim 1 or 2, or a poly -N -acetyllactosamine sugar chain on the cell surface when hybridized with the DNA under stringent conditions and expressed in a cell A DNA encoding a polypeptide having an activity of increasing the amount of . The DNA according to claim 3, wherein the DNA is a DNA consisting of the 80th to 1324th base sequences of the base sequence represented by SEQ ID NO: 2.   Recombinant DNA obtained by incorporating the DNA according to claim 3 or 4 into a vector.   The recombinant DNA according to claim 5, wherein the recombinant DNA is plasmid pVL1393-i.   A transformant having the recombinant DNA according to claim 5 or 6.   The transformant according to claim 7, wherein the transformant is a non-human transgenic animal or a transgenic plant. The transformant according to claim 7, wherein the transformant is a microorganism belonging to the genus Escherichia . The transformant according to claim 9, wherein the microorganism is Escherichia coli MM294 / pVL1393-i (FERMBP-6145).   The transformant according to claim 7, wherein the transformant is an animal cell or an insect cell.   The animal cell is an animal cell selected from mouse myeloma cell, rat myeloma cell, mouse hybridoma cell, CHO cell, BHK cell, African green monkey kidney cell, Namalwa cell, human fetal kidney cell and human leukemia cell. Item 12. A transformant according to Item 11. The transformant according to claim 11, wherein the insect cell is an insect cell selected from Spodoptera frugiperda ovary cells, Trichoplusiani ovary cells and silkworm ovary cells. When the transformant harboring the recombinant DNA according to claim 5 or 6 is cultured in a culture solution and expressed in cells encoded by the recombinant DNA , poly -N- acetyl on the cell surface a polypeptide having the activity of increasing the amount of lactosamine oligosaccharide to produce and accumulate in the culture, and collecting the polypeptide from in the culture, to come and was expressed in cells A method for producing a polypeptide having an activity of increasing the amount of poly -N -acetyllactosamine sugar chain on the cell surface . Poly -N- of the cell surface when the claim 5 carrying the recombinant DNA of breeding a non-human transgenic animal of claim 8, were expressed in cells encoded by the recombinant DNA A polypeptide having an activity of increasing the amount of acetyllactosamine sugar chain is produced and accumulated in the animal, and the polypeptide is collected from the animal . When expressed in a cell, the polypeptide is collected. A method for producing a polypeptide having an activity of increasing the amount of poly -N -acetyllactosamine sugar chain on the cell surface .   16. The process according to claim 15, wherein the production / accumulation is in animal milk. A transgenic plant according to claim 8 having the recombinant DNA according to claim 5 cultivated and expressed in cells encoded by the recombinant DNA , poly -N- acetyl lactate on the cell surface a polypeptide having the activity of increasing the amount of the summing sugar chains is produced and accumulated in the plant, and collecting the polypeptide from the plant in, of the cell surface when expressed in cells A method for producing a polypeptide having an activity of increasing the amount of a poly -N -acetyllactosamine sugar chain . A cDNA library is constructed by incorporating cDNA synthesized using mRNA extracted from animal cells as a template into an expression cloning vector, the cDNA library is introduced into the cells, and poly-N-acetyllactosamine sugar chains are obtained from the resulting cells. A cell having an activity of increasing the amount of the poly -N -acetyllactosamine sugar chain on the cell surface when cells that strongly react with an antibody against (anti-i antibody) are selected and expressed in the cell. The method for producing DNA according to claim 3 or 4, wherein DNA encoding a peptide is collected.   A transformant according to claim 7, wherein said transformant is cultured in a culture solution, and a glycoprotein to which a sugar chain containing poly-N-acetyllactosamine is added, a sugar containing poly-N-acetyllactosamine in the culture Generating and accumulating oligosaccharides containing a chain-attached glycolipid or poly-N-acetyllactosamine, and collecting the glycoprotein, the glycolipid or the oligosaccharide from the culture, Production of glycoproteins with added sugar chains containing poly-N-acetyllactosamine, glycolipids with added sugar chains containing poly-N-acetyllactosamine or oligosaccharides containing poly-N-acetyllactosamine Law.   The polypeptide of claim 1 or 2, UDP-GlcNAc, a glycoprotein having a lactosamine structure at the non-reducing end of the sugar chain, a glycolipid having a lactosamine structure at the non-reducing end of the sugar chain, and a lactosamine structure of the sugar chain An acceptor substrate selected from oligosaccharide and lactose having a non-reducing end is present in an aqueous medium, and in this aqueous medium, N-acetylglucosamine is bonded to galactose at the end of the lactosamine structure of the acceptor substrate by β1,3 linkage. The reaction product is generated and accumulated, and the reaction product is collected from the aqueous medium. The galactose at the end of the lactosamine structure of the acceptor substrate is bound to N-acetylglucosamine with β1,3 linkage. A method for producing a given reaction product.   The product obtained by the production method according to claim 20, UDP-Gal and β1,4-galactosyltransferase are present in an aqueous medium, and in the aqueous medium, N1, -acetylglucosamine is added to β1, Producing and accumulating a reaction product to which galactose has been added by 4 bonds, collecting the reaction product from the aqueous medium, and adding galactose by β1,4 bond to N-acetylglucosamine of the product Of the reaction product produced.   A polypeptide according to claim 1 or 2, UDP-GlcNAc, UDP-Gal, β1,4-galactosyltransferase, a glycoprotein having a lactosamine structure at the non-reducing end of a sugar chain, and a lactosamine structure being non-reducing sugar chain An acceptor substrate selected from glycolipids at the ends, oligosaccharides having a lactosamine structure at the non-reducing end of the sugar chain, and lactose is present in an aqueous medium, and a polysacyl at the end of the lactosamine structure of the acceptor substrate is present in the aqueous medium. A reaction product to which -N-acetyllactosamine sugar chain is added is generated and accumulated, and the reaction product is collected from the aqueous medium. Poly-N- at the end of the lactosamine structure of the acceptor substrate A method for producing a reaction product provided with an acetyllactosamine sugar chain.   A non-human transgenic animal according to claim 8, which holds the recombinant DNA according to claim 5, and a glycoprotein to which a sugar chain containing poly-N-acetyllactosamine is added, poly Glycolipids containing sugar chains containing N-acetyllactosamine or oligosaccharides containing poly-N-acetyllactosamine are generated and accumulated, and the glycoprotein, glycolipid or oligosaccharide is produced from the animal. A glycoprotein with a sugar chain containing poly-N-acetyllactosamine, a glycolipid with a sugar chain containing poly-N-acetyllactosamine, or poly-N-acetyl A method for producing an oligosaccharide containing lactosamine.   The production method according to claim 23, wherein the production / accumulation is in animal milk.   A transgenic plant according to claim 8, which holds the recombinant DNA according to claim 5, and a glycoprotein to which a sugar chain containing poly-N-acetyllactosamine is added, poly-N -Glycolipids containing sugar chains containing acetyllactosamine or oligosaccharides containing poly-N-acetyllactosamine are generated and accumulated, and the glycoprotein, glycolipid or oligosaccharide is collected from the plant A glycoprotein with an added sugar chain containing poly-N-acetyllactosamine, a glycolipid with an added sugar chain containing poly-N-acetyllactosamine, or poly-N-acetyllactosamine Of oligosaccharides containing   A method for quantifying the expression level of a gene encoding the polypeptide according to claim 1 or 2, wherein the DNA according to claim 3 or 4 is used. DNA consisting claim 3, wherein the DNA and SEQ ID NO: 2 the same sequence as consecutive 10-50 bases in the base sequence possessed by a DNA selected from the DNA consisting of the nucleotide sequence represented by, from the DNA sequence complementary to comprising DNA or derivatives DNA of these DNA,.   Derivative DNA in which a phosphodiester bond in DNA is converted to a phosphorothioate bond, a derivative DNA in which a phosphodiester bond in DNA is converted to an N3′-P5 ′ phosphoramidate bond, DNA Derivative DNA in which the ribose and phosphodiester bonds are converted to peptide nucleic acid bonds, the uracil in the DNA is replaced with C-5 propynyluracil, and the uracil in the DNA is replaced with C-5 thiazole uracil Derivative DNA, derivative DNA in which cytosine in DNA is substituted with C-5 propynylcytosine, derivative DNA in which cytosine in DNA is substituted with phenoxazine-modified cytosine, and ribose in DNA is 2′- Derivative DNA substituted with O-propyl ribose and ribo in DNA Scan is a derivative DNA selected from derivatives DNA substituted with 2'-methoxyethoxy ribose, claim 27 DNA according.   A method for quantifying the expression level of a gene encoding the polypeptide according to claim 1 or 2, wherein the DNA according to claim 27 or 28 is used.   A method for detecting inflammation or cancer using the method according to claim 29.   A method for suppressing transcription of a DNA encoding a polypeptide according to claim 1 or 2 or translation of mRNA, using a DNA selected from the DNAs according to claim 3, 4, 27 and 28. A method for detecting inflammation or cancer using a DNA selected from the DNAs according to claim 3, 4, 27 and 28 .   An antibody that recognizes the polypeptide according to claim 1 or 2.   The immunological detection method of the polypeptide of Claim 1 or 2 using the antibody of Claim 33.   An immunohistochemical staining method, wherein the antibody according to claim 33 is detected using the antibody according to claim 33.   An immunohistochemical staining agent containing the antibody according to claim 33, wherein the polypeptide according to claim 1 or 2 is detected.   A screening method for a substance that varies the activity of the polypeptide, which comprises contacting the polypeptide according to claim 1 or 2 with a test sample.   A cell expressing the polypeptide according to claim 1 or 2 is contacted with a test sample, and the poly-N-acetyllactosamine sugar chain content is measured using an anti-i antibody. A screening method for compounds that alter the expression of a gene encoding a peptide.   A cell expressing the polypeptide according to claim 1 or 2 is contacted with a test sample, and the polypeptide content according to claim 1 or 2 is measured using the antibody according to claim 33. A method for screening a compound that changes the expression of a gene encoding the polypeptide.   An animal cell is transformed with a plasmid containing a promoter existing upstream of the gene encoding the polypeptide of claim 1 and a reporter gene linked downstream of the promoter, and the transformant A method for screening a compound that varies the expression of a gene encoding the polypeptide, which comprises contacting a test sample and measuring the translation product content of the reporter gene. 41. The screening method according to claim 40 , wherein the promoter is a promoter that functions in human melanoma cells, human colon cancer cells, or human white blood cells. 41. The screening method according to claim 40 , wherein the reporter gene is a gene selected from a chloramphenicol acetyltransferase gene, a β-galactosidase gene, a luciferase gene, and a green fluorescent protein gene.

Images