JPH06277052A - Alpha2,3-sialyltransferase - Google Patents

Abstract

PURPOSE:To obtain a novel sialyltransferase which is useful for production of physiologically active saccharides and their modified products such as sialyl Lewis a or sialyl Lewis x. CONSTITUTION:alpha2,3-Sialyltransferase containing the amino acid sequence of the formula. The cells having the recombinant vector into which cDNA encoding this novel alpha2,3-sialyltransferase is incorporated are cultured in a culture medium and the enzyme produced is collected from the culture mixture.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention contains a novel α2,3-sialyltransferase, a DNA encoding the α2,3-sialyltransferase, a recombinant vector incorporating the DNA, and the recombinant vector. The present invention relates to cells and methods for producing them. Furthermore, it relates to a method for producing a sugar chain using the α2,3-sialyltransferase and a method for producing a sugar chain by expressing the α2,3-sialyltransferase in a transformed cell. Further, the present invention relates to a method for detecting the α2,3-sialyltransferase using the DNA encoding the α2,3-sialyltransferase and a method for suppressing the production thereof. The α2,3-sialyltransferase of the present invention is useful for producing sugar chains having useful physiological activities such as sialyl Lewis a and sialyl Lewis x, and modified products thereof.

[0002]

2. Description of the Related Art Proteins produced by prokaryotes such as Escherichia coli do not have sugar chains, whereas proteins and lipids produced by eukaryotes such as yeast, mold, plant cells and animal cells In many cases, sugar chains are attached. As a sugar chain of an animal cell, an N-glycoside-linked sugar chain (also called N-glycan) that binds to an asparagine (Asn) residue in the protein, and serine (Ser) or threonine (which are added to the protein). An O-glycoside-linked sugar chain (also called O-glycan) that binds to a (Thr) residue is known. Recently, it has been clarified that certain proteins containing sugar chains are covalently bound to many proteins, and these proteins are attached to cell membranes via the lipids. This lipid containing sugar chains is a glycosyl phosphatidylinositol anch
or)).

In addition, as a sugar chain of animal cells, there is glycosaminoglycan. A compound in which a protein and a glycosaminoglycan are covalently bonded is called a proteoglycan. The glycosaminoglycan constituting the sugar chain of proteoglycan has a structure similar to that of O-glycan, which is a sugar chain of a glycoprotein, but is chemically different. Glycosaminoglycans include glucosamine or galactosamine and uronic acid (however, keratan sulfate
(keratan sulfate does not have uronic acid)] and is composed of repeating structures of disaccharide units, and has a sulfate group covalently bonded (however, hyaluronic acid does not have a sulfate group). It has features.

Furthermore, glycolipids (g
Examples include sugar chains contained in a substance called lycolipid). Glycolipids in animal cells include sphingoglycolipids in which sugars, long-chain fatty acids, and long-chain bases, sphingosine, are covalently linked, and glyceroglycolipids in which sugar chains are covalently linked to glycerol.
olipid) is known.

Recently, the functions of sugar chains have been rapidly elucidated with the progress of molecular biology and cell biology, and various functions of sugar chains have been clarified up to now.
Sugar chains play an important role in clearance of glycoproteins in blood. Erythropoietin made by transferring genes into E. coli is
It is known to be active in vitro, but rapidly cleared in vivo (Dordal et al .: Endocrino
logy), 116, 2293 (1985) and Browne et al .: Cold Spring Harbor Symposia on Quantitative Biology (Cold Spr. Harb.
Symp. Quant. Biol.) 51, 693 (1986)]. Human granulocyt / macrophage colony stimulating factor
e-macrophage colony stimulating factor; hGM-C
SF) has two N-glycoside-linked sugar chains in nature, but it is known that when the number of sugar chains is reduced, the clearance rate of rat plasma increases in proportion to that [Donahue]. Et al: Cold Spring Harbor Symposia on Quantitative
Biology (Cold Spr. Harb. Symp. Quant. Biol.) , 5
1, 685 (1986)]. The rate of clearance and the site of clearance vary depending on the structure of the sugar chain. HGM-CSF with sialic acid is cleared in the kidney, whereas hGM-CSF without sialic acid has a faster clearance rate. , Known to be cleared in the liver. Α1-acid glycoproteins with different sugar chain structures, which were biosynthesized in the presence of various asparagine-linked sugar chain biosynthesis inhibitors in the rat liver primary culture system, were cleared from the rat plasma clearance rate and rat perfusate. When the clearance rate was examined, in both cases, the clearance rate decreased in the order of high mannose type, sugar chain deficient type, hybrid type, and complex type (natural type). Tissue-type plasminogen activator (t-PA; tissue-type) already used as a thrombolytic drug
It is known that the clearance of plasminogen activator) in blood is greatly influenced by the structure of its sugar chain.

It is known that a sugar chain imparts protease resistance to a protein. For example, when the sugar chain formation of fibronectin is inhibited by tunicamycin, the resulting intracellular sugar-deficient fibronectin is degraded. The speed of will increase. It is also known that the addition of sugar chains increases the thermal stability and the antifreezing property. In erythropoietin, β-interferon and the like, it is known that sugar chains contribute to the increase in protein solubility.

[0007] Sugar chains also help proteins maintain the correct three-dimensional structure. Removal of two naturally occurring N-glycoside-linked sugar chains of the vesicular stomatitis virus membrane-bound glycoprotein inhibits transport of the protein to the cell surface, but adds a new sugar chain to the protein. It is known to recover when done. In this case, it was revealed that removal of the sugar chain induces association between protein molecules by disulfide bond, resulting in inhibition of protein transport. In addition, when a sugar chain is newly added, this association is inhibited, so that the correct three-dimensional structure of the protein is maintained, so that the protein can be transported. It has been shown that there is considerable flexibility in the position where a new sugar chain is added. On the other hand, it was also clarified that the transport of a protein having a natural sugar chain may be completely inhibited depending on the position of introduction.

An example in which a sugar chain masks an antigenic site on a polypeptide is also known. In hGM-CSF, prolactin, interferon-γ, Rauscher leukemia virus gp70 and influenza hemagglutinin,
From the experiments using a polyclonal antibody or a monoclonal antibody against a specific region on a peptide, it is considered that the sugar chains of these proteins inhibit the reaction with the antibody. It is also known that the sugar chains themselves may be directly involved in the expression of glycoprotein activity. For example, the activity of glycoprotein hormones such as luteinizing hormone, follicle-stimulating hormone, chorionic gonadotropin, etc. It is considered that sugar chains are involved in expression.

Japanese Patent Laid-Open No. 2-227075 discloses a granulocyte colony-stimulating facto (G-CSF).
r) and pro-urokinase (pro-UK; pro-urokinase) and other useful physiologically active proteins can be improved in their properties by artificially introducing sugar chains using recombinant DNA technology. It is disclosed that this can be done.

As an important function of sugar chains, sugar chains are
It is involved in the recognition phenomenon between proteins or between cells and proteins. For example, it is known that the location of clearance in vivo varies depending on the structure of sugar chains. Recently, a protein that is specifically expressed on vascular endothelial cells and promotes adhesion to neutrophils in response to inflammatory reactions
The ligand for ELAM-1 is Sialyl-Lewis
x) sugar chain [NeuAc α2-3Gal β1-4 (Fuc α1-3) G
lcNAc, NeuAc: sialic acid; Gal: galactose; Fuc
: Fucose; GlcNAc: N-acetylglucosamine], and there is a possibility that the sugar chain itself or a modified sugar chain can be used as a drug [Phillips
(Phillips) et al .: Science, 250, 1130 (199
0), Goelz et al .: Trends in Glyco Technology and Trends in Gly
coscience and Glycotechnology) , 4, 14 (1992)]. L-selectin (L-selectin), which is expressed on some T lymphocytes and neutrophils, and GMP-140 (also called P-selectin), which is expressed on the membrane surface of platelets and vascular endothelial cells by inflammatory stimulation, is ELAM. Sialyl Lewis x, which is also a ligand of ELAM-1, which is involved in inflammatory response like -1
It has been suggested that the sugar chain is similar to the (Sialyl-Lewis x) sugar chain [Rosen et al .: Torrens in.
GlycoScience and Glyco Technology (T
rends in Glycoscience and Glycotechnology) , 4, 1 (1
992), Larsen et al .: Trends in GlycoScience and Glyco Technology (Trends in
Glycoscience and Glycotechnology) , 4, 25 (1992),
Aruffo et al .: Trends in Glyco Technology
science and Glycotechnology) , 4, 146 (1992)].

Similar to the inflammatory reaction, in the metastasis of cancer,
It has been suggested that ELAM-1 and GMP-140 promote cancer metastasis by causing adhesion of cancer cells to the inner wall of blood vessels and aggregation of cancer cells with platelets [Goelz et al .: Trens. Trends in Glycoscience and Glycotechn
, 4, 14 (1992), Larsen et al .: Trends in Glycoscience and Glycotechnolog
y) , 4, 25 (1992)]. This is in agreement with the finding that the expression level of sialyl-Lewis x sugar chains is high in cancer cells with high metastatic potential [Irimura et al .: Experimental Medicine, 6, 33 (1988). ].

From these findings, sialyl Lewis x (Si
alyl-Lewis x) sugar chains or their derivatives are ELAM-
It is expected that 1, by binding to L-selectin or GMP-140, it exerts an excellent anti-inflammatory effect and suppresses cancer metastasis. Considering the above-mentioned inflammatory reaction and cancer metastasis mechanism, the inflammatory reaction is also suppressed by suppressing the expression of glycosyltransferase that synthesizes the ligand sugar chain recognized by ELAM-1, L-selectin and GMP-140. It is expected to prevent cancer metastasis. To suppress the expression of a specific gene, antisense RNA / antisense D
NA technology [Tokuhisa: Bioscience and Industry 50, 322 (1992), Murakami: Chemistry 46,
681 (1991)] or Triple Helix (Triple h
elix technology (Chubb and Hogan): Trends in Biotechnolo
gy), 10 , 132 (1992)] is useful. In order to suppress the expression of a desired glycosyltransferase using this antisense RNA / DNA technique, information on the gene or the nucleotide sequence of the gene is necessary. Therefore, cloning the gene for the desired glycosyltransferase, And it is important to analyze the nucleotide sequence information.

By examining the expression of a specific glycosyltransferase in inflammatory leukocytes or cancer cells, the malignancy of inflammatory diseases or cancer can be diagnosed. In order to investigate the expression of a desired glycosyltransferase gene, the Northern hybridization method (Sambrook, Fritsch, Maniatis (Molecular Cloning: a
Laboratory Manual (Molecular Cloning, A labo
ratory manual), 2nd Edition, Cold Spring Harbor Labo Press
ratory Press), 1989] and the polymerase chain reaction method (hereinafter abbreviated as PCR method)
[Innis et al .: PCR Protocols
ocols), Academic Press, 1990
Annual] is useful. In order to apply these methods, information on the desired glycosyltransferase gene or the nucleotide sequence of the gene is required. From this point as well, it is extremely important to clone a gene for a desired glycosyltransferase and analyze its nucleotide sequence information.

[0014] Japanese Patent Application Laid-Open No. 2-227075 discloses a granulocyte colony-stimulating facto.
r) and pro-urokinase (pro-UK; pro-urokinase) and other useful physiologically active proteins can be improved in their properties by artificially introducing sugar chains using recombinant DNA technology. It is disclosed that this can be done.

As described above, it is an industrially important task to modify the sugar chain structure of a glycoprotein or to prepare a specific sugar chain or a modified product thereof in a large amount. Means for modifying the structure of sugar chains have made remarkable progress in recent years. In particular, a highly specific enzyme (exoglycosidase) that sequentially dissociates sugar chains and a glycopeptidase or endoglycosidase that cleaves the binding point with the peptide chain without changing both the peptide chain and the sugar chain The structure of the chain could be modified, and detailed studies on the biological role of sugar chains became possible. Furthermore, an endoglycoceramidase that recently cleaves between the sugar chain of glycolipids and ceramide has been found [Ito and Yamagata:
Journal of Biological Chemistry (J.
Biol. Chem.) , 261 , 14278 (1986)], which not only facilitated the preparation of sugar chains of glycolipids, but also advanced the research to elucidate the functions of glycolipids, particularly cell surface glycolipids. .. Moreover, it has become possible to add a new sugar chain by using a glycosyltransferase. For example, sialyltransferase can add a new sialic acid to the end of a sugar chain [Sabesan and Paulson:
Journal of American Chemical Society (J. Am. Chem. Soc.), 108, 2068 (1986)]. Other various glycosyltransferase and glycosidase inhibitors [Alan et al .:
Annual Review of Biochemistry (Ann
u. Rev. Biochem.), 56 , 497 (1097)], the sugar chain to be added can be changed. However, there is no method for producing a large amount of glycosyltransferase used for sugar chain synthesis. It is desired to produce a large amount of glycosyltransferase by cloning the glycosyltransferase using recombinant DNA technology and efficiently expressing the glycosyltransferase in a host cell.

As a method of cloning glycosyltransferases, a method of purifying a protein, producing an antibody against the protein, and performing immunoscreening using the protein [Weinstein et al .: Journal of Bio Logical Chemistry (J. Biol. Chem.) , 262 , 17735
(1987)], a method of purifying a protein, determining an amino acid sequence, producing a corresponding synthetic DNA, and hybridizing the same with a probe [Narumatsu et al .: Proceeding of the National Academy of Science (Proc. Natl. Acad. Sci.), USA, 83 , 4720
(1986)] is known. A method is also known in which a gene for a glycosyltransferase having homology to the glycosyltransferase is cloned by performing hybridization using the cloned gene for the glycosyltransferase as a probe [John (B. Lowe) et al .: Journal of Biological Chemistry (J. Biol. Chem.) , 266 , 17
467 (1991)]. In addition, cloning by a direct expression cloning method using a panning method using an antibody against a sugar chain or a lectin as a screening method is also known [John (B. Lowe) et al .: Proceeding.
Of the National Academy of Sciences
(Proc.Natl.Acad.Sci.), USA , 86, 8227 (1989), Row (Jo
hn. B. Lowe) et al .: Jeans and Development (Genes Develop.), 4, 1288 (1990)].

There is no case where a glycosyltransferase could be cloned using lectin resistance as an index. From studies on various lectin-resistant mutants of CHO cells, in these lectin-resistant mutants, when a new glycosyltransferase is expressed, when the activity of a certain glycosyltransferase disappears, the synthesis of a sugar nucleotide or the Golgi body is changed. It has been shown that there may be obstacles to the migration of the population [Pamela Stanley et al .: Methods in Enzymol
ogy), 96, 157]. Therefore, it is considered possible to introduce a gene derived from a cell expressing the glycosyltransferase to be cloned into CHO cells or a lectin-resistant mutant of CHO cells, and to clone the glycosyltransferase using lectin resistance as an index. Ravindra Kumar et al .: Molecular and Cellular Biology (Mol. Cel
l. Biol.) , 9, 5713 (1989)]. Lipca (James Ripka) et al. Added A4 to a lectin-resistant mutant of CHO cells (Lec1).
Human genomic DNA derived from 31 cells was introduced, and cloning of N-acetylglucosaminyl transferase I is attempted using resistance to the lectin called concanavalin A as an index. However, they could not clone glycosyltransferases by this screening method using lectin resistance as an indicator [Lipka (James
Ripka) et al .: Biochemical and Biophysical Research Communication (Biochem. Biophys.
Res. Commun.) , 159 , 554 (1989)]. Heffernan et al. Also reported that CHO cells that produced polyoma large T antigen [Michael Heffernan et al .: Nucleic Acids Res.
s.) , 19, 85 (1991)], and the cloning of mouse sialic acid hyroxylase using the resistance to the WGA (wheat germ agglutinin) lectin as an index. (Michael Heffernan et al .: Glycoconjugate J., 8, 154
(1991)], but there is no report that the glycosyltransferase could be cloned by this screening system using lectin resistance as an index. Regarding the host, Stanley, Lipka,
Hefaran et al. All use CHO cells or lectin-resistant mutants of CHO cells as hosts.

With respect to sialyltransferase,
β-galactoside α2,6-sialyltransferase (β
A gene for an enzyme having galactoside α2,6-sialyltransferase) activity has been isolated, and its nucleotide sequence has been clarified (Weinstein et al .: Journal of Biological Chemistry (J. Biol. Che
m.) , 262 , 17735 (1987)]. β-galactoside α2,3-sialyltransferase
For enzymes with transferase activity,
(Gillespie) et al. Reported cloning of a gene encoding an enzyme that adds sialic acid to galactose in an O-glycoside-linked sugar chain of sugar protein (sugar chain added to serine or threonine residue). , Its nucleotide sequence has not been revealed [Gillespie et al .: Glycoconjugate Journal
J.) , 7, 469 (1990)]. Weinstei
n) et al. from rat liver from β-galactoside α2,3-sialyltransferase (βgalactoside α2,3-sialyltrantransferase).
(Weinstein et al .: Journal of Biological Chemistry (J. Biol. Chem.) , 25 )
7 , 13835 (1982)], but this method can obtain only a very small amount of the desired enzyme. This rat liver β-galactoside α2,3-sialyltransferase gene was cloned by Wen et al. [Wen]
Et al: Journal of Biological Chemistry
(J. Biol. Chem.) , 267 , 21011 (1992)], but there is no report on the gene of human galactoside α2,3-sialyltransferase. Moreover, a method for detecting and suppressing the expression of the enzyme is not known.

[0019]

The object of the present invention is to provide a novel α2,3-sialyltransferase capable of modifying a sugar chain of a protein and efficiently producing a specific sugar chain, and the α2,3-sialyltransferase. The present invention provides a cDNA encoding the cDNA and a vector containing the cDNA. Further, it is to provide a method for detecting the active expression of the α2,3-sialyltransferase and a method for suppressing the expression of the α2,3-sialyltransferase for diagnosing and treating diseases such as cancer metastasis and inflammation. .

[0020]

[Means for Solving the Problems] The present inventors have incorporated cDNA into an expression cloning vector by incorporating a cDNA synthesized using mRNA extracted from animal cells as a template.
Cloning by constructing an A library, introducing the cDNA library into cells, culturing the obtained cells in the presence of a lectin having an activity of suppressing the growth of the cells, and isolating the growing cells When the expressed gene was introduced into a host cell and expressed, the present invention was found to express a novel α2,3-sialyltransferase, and the present invention was completed.

The present invention will be described in detail below. The present invention relates to a novel α2 containing the amino acid sequence shown by SEQ ID NO: 2,
3-sialyltransferase and cDNA encoding the α2,3-sialyltransferase and the cD
It relates to a recombinant vector containing NA. Α of the present invention
2,3-sialyltransferase is a β-galactoside α
It is a glycosyltransferase having 2,3-sialyltransferase activity, and has an activity of adding sialic acid to the end of a sugar chain that is a receptor in a binding mode of α2 → 3. Further, the α2,3-sialyltransferase of the present invention has a characteristic that it has a stronger transposition activity for β1 → 3 linked lactosamine than β1 → 4 linked lactosamine.

The DNA encoding the α2,3-sialyltransferase of the present invention includes (a) a DNA containing the nucleotide sequence shown in SEQ ID NO: 1, (b) a plurality of genetic codes for one amino acid. D, which contains a nucleotide sequence different from the nucleotide sequence shown in SEQ ID NO: 1 due to the presence of a nucleotide sequence or due to natural mutations occurring in individual animals including humans.
NA, (c) DNAs defined in (a) and (b) may have mutations such as substitution mutations, deletion mutations, insertion mutations, etc. within the range of not losing the α2,3-sialyltransferase activity of the present invention. Includes DNA having homology to the introduced DNA, for example, α2,3-sialyltransferase encoded by the DNA defined in (a) or (b), to the extent that it can be isolated by a hybridization method. . The α2,3-sialyltransferase of the present invention has the above-mentioned structure.
It includes all α2,3-sialyltransferases encoded by the DNAs defined in (a), (b) and (c). The DNA having this homology means a DNA obtained by using a colony hybridization method or a plaque hybridization method with a DNA containing the base sequence shown in SEQ ID NO: 1 as a probe. Hybridization was performed at 65 ° C in the presence of 0.7-1.0 M NaCl using a filter on which DNA derived from colonies or plaques was immobilized, and then the concentration between 0.1-fold and 2-fold SSC solution (The composition of 1-fold concentrated SSC solution is 150 mM NaCl, 15 mM sodium citrate)
, DNA that can be identified by washing the filter at 65 ° C. The hybridization method is described in Molecular Cloning: A Laboratory Manual.
anual), 2nd edition [Sambrook, Flitch (F
ritsch), Maniatis Editor, Cold Spring Harbor Laboratory Press (Cold Spring)
Harbor Laboratory Press), 1989]. The α2,3-sialyltransferase of the present invention includes all α2,3-sialyltransferases encoded by the DNA defined in (a), (b) and (c) above.

The method for producing the DNA encoding the α2,3-sialyltransferase of the present invention will be described below by taking the method for producing the DNA defined in (a) above as an example. A cDNA library is constructed by incorporating a cDNA synthesized using mRNA extracted from an animal cell as a template into an expression cloning vector. This cDNA library is introduced into animal cells or insect cells, and the cells are cultured in the presence of a lectin having an activity of suppressing the growth of the cells. Expression of a gene encoding glycosyltransferase in a cell clone into which cDNA was introduced changes the sugar chain structure recognized by the lectin, loses sensitivity to lectin, and a cell clone that proliferates in the presence of lectin appears. . This cell is isolated, and a cDNA encoding the desired α2,3-sialyltransferase is obtained from the cell.

Any animal cell can be used as the animal cell used in the above method as long as it is an animal cell producing the α2,3-sialyltransferase of the present invention. For example, human melanoma cell line WM266-4 (ATCC CR
L 1676) is used. As a vector incorporating a cDNA synthesized using mRNA extracted from these cells as a template, any vector can be used as long as the vector can incorporate and express the cDNA. For example, p
AMoERC3Sc or the like is used. Animal cells or insect cells into which the cDNA library constructed by the vector is introduced,
Any material can be used as long as it can be expressed. For example, human Namalwa cells [Hosoi et al .: Cytotechnology , 1, 151 (198
8)] etc. are used. Any lectin can be used in the present invention as long as it can suppress the growth of host cells. For example, chickpea lectin 120 or the like is used. The lectin is used at a concentration that inhibits the growth of the host cell after determining the degree of resistance of the host cell to be used to the lectin. Known methods such as the Hart method (Robert F. Margols) are used for cells grown in the presence of lectins.
kee) et al .: Molecular and Cellular Biology (Mol. Cell. Biol.), 8, 2837 (1988)], which encodes the α2,3-sialyltransferase of the present invention.
A DNA-containing plasmid or a DNA fragment containing the cDNA portion is recovered. CD encoding the enzyme of the present invention
As the plasmid containing NA, for example, pUC119-WM16
Is mentioned. Escher, an E. coli containing pUC119-WM16
ichia coli HB101 / pUC119-WM16 was deposited as FERM BP-4012 at the Institute of Microbial Science and Technology of the Agency of Industrial Science on September 22, 1992.

The DNA defined in (b) and (c) above is a hybridization method or a method for introducing a mutation into the DNA based on the DNA encoding the α2,3-sialyltransferase obtained by the above-mentioned production method. Well-known recombinant DNA technology such as [Japanese Patent Laid-Open No. 227075; Molecular Cloning: A Laboratory Manual (Molecular Cloning
Ning, A laboratory manual), 2nd edition, Cold Spring Harbor Laboratory Press (Cold Spring
Harbor Laboratory Press), 1989, etc.]. Further, the DNA encoding the α2,3-sialyltransferase of the present invention can also be produced by using a chemical synthesis method.

The α2,3- of the present invention obtained by the above method
By constructing a recombinant vector in which a DNA encoding a sialyltransferase is inserted downstream of a promoter of an appropriate vector, introducing the recombinant vector into a host cell, and culturing the obtained cell, the α2,3-sialyl of the present invention can be obtained. A transferase can be produced. As the host cell used here, any cell can be used, such as a prokaryotic cell, an animal cell, a yeast, a mold, an insect cell, etc., as long as it is a host cell used in the recombinant DNA technology so far. Examples of prokaryotic cells include Escherichia coli, and animal cells include Chinese hamster cells, CHO cells, monkey cells, COS cells, human cells, and Namalwa cells. The direct expression cloning system using Namalwa cells as the host has extremely high efficiency of introducing the cDNA library into the Namalva cells as the host,
Moreover, the introduced plasmid (cDNA library)
Is preferably used because it has the advantage that it can exist extrachromosomally and the plasmid can be easily recovered from the obtained lectin-resistant strain.

The vector for introducing the DNA encoding the α2,3-sialyltransferase of the present invention includes a DNA encoding the α2,3-sialyltransferase.
Any vector can be used as long as it can be incorporated into and can be expressed in a host cell. For example, pAGE107 [JP-A-3-22979, Miyaji et al .: Cytotechnology , 3, 133 (1990)], pA
S3-3 (JP-A-2-227075), pAMoERC3Sc,
CDM8 [Brian Seed et al .: Nature , 329 , 840 (1987)] and the like can be mentioned. In order to express the enzyme of the present invention in E. coli, trp
Foreign DNA can be inserted downstream of a promoter having strong transcription activity such as a promoter, and a Shine-Dalgarno sequence (hereinafter SD)
It is preferable to use a plasmid in which the distance between the sequence (abbreviated as "sequence") and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases). Specifically, pKYP10 (JP-A-58-110)
600), pLSA1 [Miyaji et al .: Agricultural and Biological Chemistry (Agric. Biol. Ch.
em.) , 53, 277 (1989)], pGEL1 [Sekine et al .: Proceeding of the National Academy of Science (Proc. Natl. Acad. Sci.), USA, 82 , 4306]
(1985)] and the like.

The general method of the recombinant DNA technology used in the present invention is described in JP-A-2-227075 or Sunbrook.
(Sambrook), Fritsch, Maniati
s) et al. [Molecular Cloning, A laboratory
manual), 2nd edition, Cold Spring Harbor
Laboratory Press (Cold Spring Harbor Laboratory
Press), 1989] can be used. The isolation of mRNA and the synthesis of cDNA library can be performed using many commercially available kits in addition to the above method. DN to animal cells
As a method of introducing A, any method known to date can be used. For example, the electroporation method [Miyaji et al .: Cytotechn
ology , 3, 133 (1990)], calcium phosphate method
2-227075), lipofection method [Philip L.
Philip L. Felgner et al .: Proceeding of the National Academy of Science (Proc. Natl. Acad. Sci.), USA , 84, 7413 (1987)] can be used. The transformant can be obtained and cultivated according to the method described in JP-A-2-227075 or JP-A-2-257891.

As a method for producing the cloned α2,3-sialyltransferase, there are a method of producing it inside the host cell, a method of secreting it outside the host cell, and a method of producing it on the outer membrane of the host cell. The production site depends on the type of host cell used and the form of the glycosyltransferase to be produced. When an animal cell is used as a host cell to produce a glycosyltransferase as it is, it is generally produced inside the host cell or on the outer membrane of the host cell, and a part of it is cleaved by protease and secreted extracellularly. It In the case of positive secretion outside the host cell, the method of Paulson et al. [J.
C. Paulson et al .: The Journal of Biological Chemistry (J. Biol. Chem.), 26 4, 17619 (198).
9)] and Law et al. [John. B. Lowe et al .: Proceeding of the National Academy of Science (Proc. Natl. Acad. Sci.), USA, 86 , 8227.
(1989), John. B. Lowe et al .: Jeans and Development (Genes Develop.), 4, 1288 (1990)]. It is produced in a form in which a signal peptide is added to the portion.

According to the method described in JP-A-2-227075, the production amount can be increased by utilizing a gene amplification system using a dihydrofolate reductase gene or the like. The α2,3-sialyltransferase of the present invention produced in this manner is a conventional glycosyltransferase purification method [J. Evan.
Sadler et al .: Method in Enzymology (Meth
ods of Enzymology) Vol. 83, p. 458]]. When produced in Escherichia coli, it can be efficiently purified by combining the above method with the method described in JP-A-63-267292. It is also possible to produce the enzyme of the present invention as a fusion protein with another protein and purify it using affinity chromatography using a substance having an affinity for the fused protein. For example, Lowe's method [John. B. Lowe
Et al: Proceeding of the National Academy of Science (Proc. Natl. Acad. Sci.), US
A. 86 , 8227 (1989), John. B. Lowe et al .: Jeans and Development (Genes Develop.), 4, 1288.
(1990)], the enzyme of the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using immunoglobulin G. It can also be purified by affinity chromatography using an antibody against the enzyme itself.

The activity of sialyltransferase can be measured by a known assay method [J. Evan. Sadler et al .: Methods in Enzymology, Vol. 83, 458].
Page; Naoyuki Taniguti et al .: Methods in Enzymology 179, 397]. The α2,3-sialyltransferase of the present invention can be used to synthesize sugar chains in vitro. For example, a lactosamine structure (Gal β1 → 3) possessed by glycoprotein, glycolipid or oligosaccharide
Sialic acid can be attached to the non-reducing end of the GlcNAc structure or Gal β1 → 4GlcNAc structure by α2 → 3 bond.
In addition, the α2,3-sialyltransferase of the present invention is allowed to act on a glycoprotein, glycolipid, or oligosaccharide serving as a substrate to change the sugar chain structure at the non-reducing end to a sialyl-Lewisx structure or a sialyl-Lewis a structure.
(Sialyl-Lewis a) structure. Moreover, after the α2,3-sialyltransferase of the present invention is allowed to act on an oligosaccharide having a lactosamine structure at the non-reducing end, a known α1,3 / 1,4-fucosyltransferase (fucosyltransferase) (Kuko
wska-Latallo) et al .: Jeans and Development (Genes Develop.), 4, 1288 (1990)], using Sialyl-Lewisx and Sialyl-Lewis a.
An oligosaccharide having (Sialyl-Lewis a) and its modified product at the non-reducing end can be synthesized.

The α2,3-sialyltransferase-encoding DNA of the present invention is used to produce the α2,3-sialyltransferase in an animal cell or insect cell that produces a sugar chain that is a receptor substrate for the α2,3-sialyltransferase. , 3-sialyltransferase and glycoprotein with useful physiological activity,
The α2,3-sialyltransferase produced by simultaneously producing glycolipids or oligosaccharides is allowed to act on glycoproteins, glycolipids or oligosaccharides in cells to produce glycoproteins or glycolipids with altered sugar chain structure. Alternatively, the oligosaccharide can be produced in the cell.

Further, a part of the oligosaccharide can be excised from the glycoprotein, glycolipid or oligosaccharide having a modified sugar chain structure produced by the above-mentioned method by a known enzymatic method or chemical method. The DNA encoding the α2,3-sialyltransferase of the present invention can be used not only for modification of sugar chains of proteins and glycolipids and efficient production of specific sugar chains, but also for antisense RNA /
It can also be used for treating diseases such as inflammation and cancer metastasis by using DNA technology, and can be used for diagnosing such diseases by using Northern hybridization method or PCR method.

For example, using the DNA encoding the α2,3-sialyltransferase of the present invention, antisense RNA / DNA technology [Tokuhisa: Bioscience and Industry , 50, 322-326 (1992), Murakami ( Mur
akami): Chemistry , 46, 681-684 (1991), Miller:
Biotechnology, 9, 358-362 (19
92), Cohen: Trends in Biotechnology, 10 , 87 -91 (1992)
, Agrawal: Trends in Biotechnology, 10 , 152 -158 (19
92)] or triple helix technology (Chu (Chu
bb) and Hogan: Trends in Biotechnology, 10 , 132 -136 (199)
2)] can suppress the expression of the activity of the α2,3-sialyltransferase. Specifically, DN encoding the α2,3-sialyltransferase of the present invention
A part of the base sequence of A, preferably in the translation initiation region 10
The production of the α2,3-sialyltransferase can be suppressed by designing and preparing an oligonucleotide based on a nucleotide sequence of ˜50 bases and administering it in vivo.
The base sequence of the synthetic oligonucleotide is DN that encodes the α2,3-sialyltransferase of the present invention.
A part of the base sequence of the antisense strand of A,
Alternatively, a modified product within a range that does not lose the activity of suppressing the activity expression of the α2,3-sialyltransferase can be used. When using the triple helix technique, the base sequence of the synthetic oligonucleotide is designed based on the base sequence information of both the sense and antisense strands.

The hybridization method or P
The CR method can be used to detect the expression of the α2,3-sialyltransferase of the present invention. In order to examine the production of the α2,3-sialyltransferase of the present invention using the Northern hybridization method or the PCR method, DN based on the DNA encoding the α2,3-sialyltransferase of the present invention or their nucleotide sequences is used.
Prepare A probe or synthetic oligonucleotide.
Northern hybridization method and PCR method,
Known methods (Sambrook, Fritsch, Maniatis (Molecular
Cloning: A Laboratory Manual (Molecul
ar Cloning, A laboratory manual), 2nd edition, Cold Spring Harbor Laboratory Press (Cold
Spring Harbor Laboratory Press), 1989 and Innis et al .: PCR Protocols
ocols), Academic Press, 1990
Annual publication].

Examples of the present invention will be shown below.

[0037]

【Example】

Example 1 1. Direct Expression Cloning Vector (Expression Cloni
ng Vector) pAMoERC3Sc and pAMoPR
Creation of C3Sc: pAMoPRC3Sc is shown below.
It was constructed according to the steps (1) to (15). (1) Construction of pAGEL106 (See Fig. 1) Simian virus (simian virus)
virus 40 (SV40) early gene promoter and human T-cell leukemia virus type-
1: HTLV-1) long terminal repeat (long te
The plasmid pAGEL106 having a promoter in which the R region of rminal repeat (LTR) and a part of the U5 region were fused was constructed. A DNA fragment containing the R region and part of the U5 region [BanII-Sa
u3A fragment (0.27 kb)] was excised from pATK03 and inserted between BglI and BamHI of pAGE106 via a synthetic linker.

1 μ of pAGE106 (Japanese Patent Laid-Open No. 2-227075)
g was dissolved in 30 μl of a buffer solution (hereinafter abbreviated as Y-100 buffer solution) consisting of 10 mM Tris-hydrochloric acid (pH 7.5), 6 mM magnesium chloride, 100 mM sodium chloride, 6 mM 2-mercaptoethanol, and 10 units of Bgl were dissolved. I (manufactured by Takara Shuzo Co., Ltd., unless otherwise specified, restriction enzymes manufactured by Takara Shuzo Co., Ltd. were used) and 10 units of BamHI were added, and a digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis,
A DNA fragment of about 4.9 kb was recovered.

PATK03 [Shimizu et al .: Proc. Natl. Acad. Sci.], USA, 80 , 3618
(1983)] 1 μg was dissolved in 30 μl of Y-100 buffer, 10 units of Ban II was added, digestion reaction was carried out at 37 ° C. for 2 hours, and after electrophoresis on agarose gel, a DNA fragment of about 0.4 kb was recovered. . The recovered DNA fragment was dissolved in 30 μl of Y-100 buffer, 10 units of Sau3AI was added, and the digestion reaction was performed at 37 ° C. for 2 hours. After agarose gel electrophoresis, about 0.27 kb of DNA was added.
The fragments were collected.

Separately, the following DNA linker was synthesized as a linker for connecting the BglI cleavage site and the BanII cleavage site.

[0041]

[Chemical 1]

The 5 mer and 6 mer single-stranded DNAs of this DNA linker are respectively applied to Applied Biosystems (Appl.
It was synthesized using a 380A DNA synthesizer manufactured by ied Biosystems). 0.2 μg of each synthesized DNA, 50 mM
Tris-hydrochloric acid (pH 7.5), 10 mM magnesium chloride, 5 mM dithiothreitol (hereinafter abbreviated as DTT), 0.1 nMED
30 units of T4 polynucleotide kinase (Takara Shuzo Co., Ltd.) Was added to carry out phosphorylation reaction at 37 ° C. for 2 hours.

Bgl from pAGE106 obtained above
0.2 μg of I-BamHI fragment (4.9 kb) and Ba from pATK03
n II-Sau3A fragment (0.27kb) 0.01 μg of 66 mM Tris-HCl
(pH 7.5), 6.6 mM magnesium chloride, 10 mM DTT and 0.
Dissolve in 30 μl of a solution consisting of 1 mM adenosine triphosphate (hereinafter abbreviated as ATP) (hereinafter abbreviated as T4 ligase buffer), and 0.01 μg of the above DNA linker and 175 units of T4 DNA ligase (Takara Shuzo, the same hereafter) In addition, 16 at 12 ℃
A time binding reaction was performed.

Using the reaction solution, Escherichia coli HB101 strain [Bolivar et al .: Gene 2,75 (1988)] was subjected to the method of Cohen et al. [SN Cohen et al .: Proceeding of・ The National Academy ・
Of Science (Proc. Natl. Acad. Sci.), USA , 6
9, 2110 (1972)] (hereinafter, this method is used for transformation of Escherichia coli) to obtain an ampicillin-resistant strain. A known method from this transformant [HC Birnboim et al .: Nucleic Acids Res., 7, 1513 (197
9)] (hereinafter, this method was used for plasmid isolation) to isolate the plasmid. This plasmid is called pAGE
It was named L106 and its structure was confirmed by restriction enzyme digestion.

(2) Construction of pASLB3-3-1 (see FIG. 2) According to the method described below, SV40 early gene promoter and HTLV-1 long terminal repeat (LTR) R region and part of U5 region Expression plasmid pA of human granulocyte colony stimulating factor (hG-CSF) having a promoter fused with
SLB3-3-1 was created.

0.5 of pAGEL106 obtained in (1)
A buffer solution consisting of 10 mM Tris-hydrochloric acid (pH 7.5), 6 mM magnesium chloride, 20 mM potassium chloride, 6 mM 2-mercaptoethanol (hereinafter abbreviated as K-20 buffer solution) 30
It was dissolved in μl, 10 units of SmaI was added, and digestion reaction was carried out at 37 ° C. for 2 hours. After ethanol precipitation, the precipitate was dissolved in 30 μl of T4 ligase buffer, and SalI linker (5'-pGGTCGACC-
3 ': Takara Shuzo Co., Ltd.) (0.01 μg) and 175 units of T4 DNA ligase were added, and a binding reaction was performed at 12 ° C for 16 hours. After ethanol precipitation, it was dissolved in 30 μl of a buffer solution (hereinafter referred to as Y-175 buffer solution) consisting of 10 mM Tris-hydrochloric acid (pH 7.5), 6 mM magnesium chloride, 175 mM sodium chloride, 6 mM 2-mercaptoethanol, and 10 units were dissolved. SalI and 10 units of MluI were added, and a digestion reaction was carried out at 37 ° C for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 1.7 kb was recovered.

On the other hand, 1 μg of pAS3-3 (JP-A-2-227075) was dissolved in 30 μl of Y-175 buffer to give 10 units of S.
alI and 10 units of MluI were added, and the digestion reaction was performed at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, about 6.7 kb
The DNA fragment was recovered. PAGEL10 obtained above
6-derived MluI-SalI fragment (1.7 kb) 0.1 μg and pAS3-
0.2 μg of MluI-SalI fragment (6.7 kb) derived from 3 was dissolved in 30 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and a ligation reaction was carried out at 12 ° C. for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution by the method of Cohen et al. To obtain a kanamycin resistant strain. A plasmid was isolated from this transformant according to a known method. This plasmid was named pASLB3-3-1 and its structure was confirmed by digestion with restriction enzymes.

(3) Construction of pASLB3-3 (see FIG. 3) In order to construct the plasmid pASLB3-3 in which the ampicillin resistance gene was introduced into pASLB3-3-1, the ampicillin resistance of pAS3-3 was constructed. A DNA fragment containing the gene [XhoI-MluI fragment (7.26 kb)] was added to pASLB3-3-1 XhoI-MluI.
Introduced in between. Of pASLB3-3-1 obtained in (2)
1 μg was dissolved in 30 μl of a buffer solution (hereinafter referred to as Y-150 buffer solution) consisting of 10 mM Tris-hydrochloric acid (pH 7.5), 6 mM magnesium chloride, 150 mM sodium chloride, 6 mM 2-mercaptoethanol, and 10 units of XhoI And 10 units of MluI were added, and the digestion reaction was performed at 37 ° C for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 7.26 kb was recovered.

On the other hand, 1 μg of pAS3-3 was added to Y-150.
Dissolve in 30 μl of buffer and add 10 units of XhoI and 10 units of MluI.
Was added and the digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 2.58 kb was recovered. XhoI-M derived from pASLB3-3-1 obtained above
0.2 μg of luI fragment (7.26 kb) and XhoI-Ml derived from pAS3-3
0.1 μg of uI fragment (2.58 kb) in 30 μl of T4 ligase buffer
And 175 units of T4 DNA ligase, and add 16
A time binding reaction was performed. E. coli HB101 using the reaction solution
The 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 is called pASLB
It was named 3-3 and its structure was confirmed by digestion with restriction enzymes.

(4) Construction of pASLBE3-3 (Fig. 4
Refer to the following method to remove the dihydrofolate reductase (dhfr) expression unit in pASLB3-3 and to simultaneously perform Epstein-Barr virus (Epstein-Barrvirus)
Construction of the plasmid pASLBE3-3 into which the EBNA-1 gene, which is a factor that acts in trans on oriP and its origin of replication, was introduced. with oriP
The EBNA-1 gene is p201 (Bill Sugden).
Et al., Nature, 313 , 812 (198
5)] at the NarI site of pUC12 [Messi (Messi
ng) et al .: Methods in Enzymology (Methods
in Enzymology) , 101 , 20 (1983)] and the SmaI-HaeIII fragment containing the multi-cloning site was excised and used from p220.2.

1 μg of p220.2 was added to Y-100 buffer solution.
It was dissolved in 30 μl, 20 units of EcoRI was added, and a digestion reaction was carried out at 37 ° C. for 2 hours. After ethanol precipitation, 30 μl of DNA polymerase I buffer [50 mM Tris-HCl (pH 7.5), 10 mM
Magnesium chloride, 0.1 mM dATP (deoxyadenosine 3
Phosphoric acid), 0.1 mM dCTP (deoxycytidine triphosphate),
0.1 mM dGTP (deoxyguanosine triphosphate), 0.1 mM TT
P (thymidine triphosphate)] and dissolved in 6 units of E. coli DN
A polymerase I Klenow fragment was added and reacted at 37 ° C for 60 minutes to change the 5'protruding end generated by EcoRI digestion into a blunt end. The reaction was stopped by phenol extraction, followed by chloroform extraction and ethanol precipitation, followed by dissolution in 20 μl of T4 ligase buffer, and XhoI linker (5'-pCCT
CGAGG-3 ': manufactured by Takara Shuzo Co., Ltd.) was added to 0.05 μg and 175 units of T4 DNA ligase, and a ligation reaction was performed at 12 ° C. for 16 hours. After ethanol precipitation, it was dissolved in 30 μl of Y-100 buffer, 10 units of BamHI was added, and digestion reaction was carried out at 37 ° C. for 2 hours. After ethanol precipitation, 30 μl DNA polymerase I
It was dissolved in a buffer solution, 6 units of Escherichia coli DNA polymerase I Klenow fragment was added, and the reaction was carried out at 37 ° C for 60 minutes to change the 5'protruding end generated by BamHI digestion into a blunt end. The reaction was stopped by phenol extraction, followed by chloroform extraction and ethanol precipitation, followed by dissolution in 30 μl of Y-100 buffer,
10 units of XhoI was added, and the digestion reaction was carried out at 37 ° C for 2 hours.
After the reaction solution was subjected to agarose gel electrophoresis, about 4.9 kb of DNA was
The fragments were collected.

1 μ of pASLB3-3 obtained in (3)
g was dissolved in 30 μl of Y-100 buffer, 20 units of XhoI was added, and digestion reaction was carried out at 37 ° C. for 2 hours. After ethanol precipitation, dissolve in 30 μl of DNA polymerase I buffer,
Unit E. coli DNA polymerase I Klenow fragment,
The reaction was carried out at 37 ° C for 60 minutes, and the 5'protruding end generated by the XhoI digestion was changed to a blunt end. The reaction was stopped by phenol extraction, and after chloroform extraction and ethanol precipitation, 10
mM Tris-HCl (pH 7.5), 6 mM magnesium chloride, 6 m
A buffer solution consisting of M 2-mercaptoethanol (hereinafter Y-
It was dissolved in 30 μl of 0 buffer, 20 units of KpnI was added, and a digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 1.3 kb was recovered.

In addition, pAGE107 [Japanese Patent Application No. 1-156]
302, Miyaji et al .: Site Technology (Cytotechnolog
y), 3 , 133 (1990)] 1 μg of Y-0 buffer 3
It was dissolved in 0 μl, 20 units of KpnI was added, and a digestion reaction was carried out at 37 ° C. for 2 hours. After that, the sodium chloride concentration is 100
Sodium chloride was added to make mM and 20 units of Xh
oI was added, and the digestion reaction was further performed at 37 ° C for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 6.0 kb was recovered.

XhoI-B derived from p220.2 obtained above
AmHI (blunt end) fragment (4.9 kb) 0.2 μg and pASLB3
-3 derived XhoI (blunt end) -KpnI fragment (1.3 kb) 0.1 μ
KpnI-XhoI fragment (6.0k from g and pAGE107)
b) 0.2 μg was dissolved in 30 μl of T4 ligase buffer,
After adding 175 units of T4 DNA ligase, the binding reaction was performed at 12 ° C for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution 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 pASLBE3-3, and its structure was confirmed by digestion with restriction enzymes.

(5) Construction of pASLBC (see FIG. 5) According to the method described below, the hG-CSF gene in pASLB3-3 was removed, and instead, a plasmid pASLBC into which a multicloning site was introduced was constructed. The multi-cloning site was created using synthetic DNA.

1 μ of pASLB3-3 obtained in (3)
g was dissolved in 30 μl of Y-175 buffer and 20 units of SalI was added.
20 units of MluI was added and a digestion reaction was carried out at 37 ° C for 2 hours.
After the reaction solution was subjected to agarose gel electrophoresis, about 3.1 kb of DNA was
The fragments were collected. Also, 1 μg of the same plasmid was dissolved in 30 μl of Y-0 buffer, 20 units of KpnI was added, and the mixture was incubated at 37 ° C. for 2 hours.
A time digestion reaction was performed. Then, sodium chloride was added so that the sodium chloride concentration was 150 mM, 20 units of MulI was added, and the digestion reaction was further carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, about 6.0 kb of
The DNA fragment was recovered.

Separately, the following DNA linker was synthesized as a linker for connecting the SalI cleavage site and the KpnI cleavage site. In this linker, HindIII, EcoR
V, SfiI, StuI, and NotI restriction enzyme cleavage sites are incorporated.

[0058]

[Chemical 2]

This DNA linker 52mer (SEQ ID NO: 3)
And single-stranded DNAs of 44 mer (SEQ ID NO: 4) were respectively synthesized using Applied Biosystems 380A DNA synthesizer. 0.2 μg of each synthesized DNA
Each of them was dissolved in 20 μl of T4 kinase buffer, 30 units of T4 polynucleotide kinase (Takara Shuzo, the same applies hereinafter) was added, and phosphorylation reaction was carried out at 37 ° C. for 2 hours.

Sa derived from pASLB3-3 obtained above
lI-MluI fragment (3.1 kb) 0.1 μg
0.2 μg of KpnI-MluI fragment (6.0 kb) was dissolved in 30 μl of T4 ligase buffer, and 0.01 μg of the above DNA linker and T4 were added.
175 units of DNA ligase was added and the binding reaction was carried out at 12 ° C for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution 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. We named this plasmid pASLBC,
The structure was confirmed by restriction enzyme digestion.

(6) Construction of pASLBEC (see FIG. 6) According to the method described below, the dihydrofolate reductase (dhfr) expression unit in pASLBC was removed to construct a plasmid pASLBEC into which oriP and EBNA-1 gene were introduced. ..

1 μg of pASLBE3-3 was added to Y-150.
It was dissolved in 30 μl of buffer, 20 units of MluI and 20 units of XhoI were added, and digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 1.3 kb was recovered. In addition, 1 μg of the same plasmid was added to 30 μl of Y-0 buffer.
20 units of KpnI was added, and a digestion reaction was carried out at 37 ° C. for 2 hours. Then, sodium chloride was added so that the sodium chloride concentration was 150 mM, 5 units of MluI was added, and a partial digestion reaction was further carried out at 37 ° C. for 20 minutes. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 9.6 kb was recovered.

Next, 1 of pASLBC obtained in (5)
μg was dissolved in 30 μl of Y-0 buffer, 20 units of KpnI was added, and digestion reaction was carried out at 37 ° C. for 2 hours. Then, sodium chloride was added so that the concentration of sodium chloride was 100 mM, 20 units of XhoI was added, and the digestion reaction was further carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 0.6 kb was recovered.

Derived from pASLBE3-3 obtained above
0.2 μg of MluI-XhoI fragment (1.3 kb) and 0.2 μg of KpnI-MluI fragment (9.6 kb) derived from the same plasmid, and pASL
0.05 μg of BC-derived KpnI-XhoI fragment (0.6 kb) was dissolved in 30 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and a ligation reaction was carried out at 12 ° C. for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution 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 pASLBEC, and its structure was confirmed by restriction enzyme digestion.

(7) Construction of pASLBEC2 (see FIG. 7) According to the method shown below, a plasmid pASLBEC2 was constructed by introducing a BamHI linker into the StuI site of the multicloning site of pASLBEC. In pASLBEC2, the StuI site in the multicloning site has disappeared.

1 μg of pASLBEC obtained in (6)
Was dissolved in 30 μl of Y-100 buffer, 5 units of StuI was added, and a partial digestion reaction was carried out at 37 ° C. for 20 minutes. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 11.5 kb was recovered. The recovered DNA fragment was dissolved in 30 μl of T4 ligase buffer, and BamHI linker (5′-pCCGGATCCG
G-3 ': 0.01 μg of Takara Shuzo Co., Ltd. and T4DNA Ligase 175
The unit was added and the binding reaction was performed at 12 ° C. for 16 hours. After ethanol precipitation, it was dissolved in 30 μl of Y-100 buffer, 20 units of BamHI was added, and digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 11.5 kb was recovered. The recovered DNA fragment was dissolved in 20 μl of T4 ligase buffer, and 175 units of T4 DNA ligase was added.
The binding reaction was performed at 16 ° C for 16 hours. E. coli using the reaction solution
The HB101 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. PA of this plasmid
It was named SLBEC2 and its structure was confirmed by restriction enzyme digestion.

(8) Construction of pAMoEC2 (see FIG. 8) According to the method described below, the promoter in pASLBEC2 [SV40 early gene promoter and R region of long terminal repeat (LTR) of HTLV-1] The plasmid pAMoEC2 was constructed by replacing the promoter [a part of the U5 region] with the promoter of the long terminal repeat (LTR) of Moloney murine leukemia virus. The promoter of Moloney murine leukemia virus LTR is the plasmid Molp-1 (Akinori Ishimoto).
Et al., Virology , 141 , 30 (1985)].

1 μ of pASLBEC2 obtained in (7)
g is a buffer solution consisting of 10 mM Tris-hydrochloric acid (pH 7.5), 6 mM magnesium chloride, 50 mM potassium chloride, 6 mM 2-mercaptoethanol (hereinafter referred to as K-50 buffer solution) 30
It was dissolved in μl, 20 units of HindIII and 20 units of AatII (manufactured by Toyobo Co., Ltd.) were added, and the digestion reaction was carried out at 37 ° C. for 2 hours.
After the reaction mixture was subjected to agarose gel electrophoresis, about 4.8 kb of DNA was
The fragments were collected.

Further, 1 μg of the plasmid was dissolved in 30 μl of K-50 buffer, 20 units of AatII was added, and the mixture was incubated at 37 ° C. for 2 hours.
A time digestion reaction was performed. Then add 5 units of XhoI,
Further, a partial digestion reaction was carried out at 37 ° C. for 20 minutes. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 6.1 kb was recovered. Next, the following DNA linker was synthesized as a linker for connecting the XhoI cleavage site and the ClaI cleavage site.

[0070]

[Chemical 3]

Single-stranded D of the above DNA linkers 9mer and 7mer
NA is Applied Biosystems 380 each
It was synthesized using an A.DNA synthesizer. 0.2 μg of each synthesized DNA was dissolved in 40 μl of T4 kinase buffer, and 30 units of T4 polynucleotide kinase was added.
Phosphorylation was performed at 37 ° C for 2 hours. Separately, Mol
p-1 [Akinori Ishimoto, et al.
Virology , 141 , 30 (1985)] 1 μg of Y-
Dissolve in 30 μl of 50 buffer, add 20 units of ClaI, and incubate at 37 ℃
The digestion reaction was carried out for 2 hours. 30 μl after ethanol precipitation
Dissolved in T4 ligase buffer solution of
μg and 175 units of T4 DNA ligase were added, and the binding reaction was performed at 12 ° C for 16 hours. After ethanol precipitation, K-20 buffer
It was dissolved in 30 μl, 20 units of SmaI was added, and digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 0.6 kb was recovered. The recovered DNA fragment was dissolved in 30 μl of T4 ligase buffer and 0.03 of HindIII linker (5'-pCAAGCTTG-3 ': manufactured by Takara Shuzo) was used.
μg and 175 units of T4 DNA ligase were added, and the binding reaction was performed at 12 ° C for 16 hours. After ethanol precipitation, it was dissolved in 30 μl of a buffer solution (hereinafter abbreviated as Y-50 buffer solution) consisting of 10 mM Tris-hydrochloric acid (pH 7.5), 6 mM magnesium chloride, 50 mM sodium chloride, 6 mM 2-mercaptoethanol, and 10 units were dissolved. HindIII was added and digestion reaction was carried out at 37 ° C for 2 hours.
Then add sodium chloride so that the sodium chloride concentration is 100 mM, add 10 units of XhoI, and
The digestion reaction was carried out at 37 ° C for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 0.6 kb was recovered.

Hi derived from pASLBEC2 obtained above
0.2 μg of ndIII-AatII fragment (4.8 kb) and 0.2 μg of AatII-XhoI fragment (6.1 kb) derived from the same plasmid, and Mol
0.05 μg of HindIII-XhoI fragment (0.6 kb) derived from p-1 was dissolved in 30 μl of T4 ligase buffer, and T4 DNA ligase 17
5 units were added and the binding reaction was performed at 12 ° C for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution 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 pAMoEC2, and its structure was confirmed by digestion with restriction enzymes.

(9) Construction of pAMoEC3 (see FIG. 9) According to the method described below, the packed DNA (Stuffer
As a DNA), a DNA fragment containing the tetracycline resistance gene of pBR322 [DraI-PvuII fragment (2.5 kb)] was inserted to construct a plasmid pAMoEC3.

1 μg of pAMoEC2 obtained in (8)
Was dissolved in 30 μl of Y-100 buffer, 20 units of BamHI was added, and a digestion reaction was carried out at 37 ° C. for 2 hours. After ethanol precipitation, dissolve in 30 μl of DNA polymerase I buffer and add 6 units of E. coli DNA polymerase I. Klenow fragment,
5'generated by BamHI digestion after reacting at 37 ° C for 60 minutes
The protruding ends were changed to blunt ends. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 11.5 kb was recovered.

In addition, pBR322 [Bolivar]
Et al., Gene, 2, 95 (1977)] 1 μg dissolved in 30 μl Y-50 buffer, 20 units DraI and 20 units PvuII.
Was added and the digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 2.5 kb was recovered. 0.1 μg of BamHI (blunt end) fragment (11.5 kb) derived from pAMoEC2 obtained above and DraI derived from pBR322
-0.2 μg of PvuII fragment (2.5 kb) in T4 ligase buffer 30
Dissolve in μl, add 175 units of T4 DNA ligase, and stir at 12 ℃.
The binding reaction was carried out for 16 hours. E. coli HB using the reaction solution
The 101 strain was transformed by the method of Cohen et al. To obtain a strain resistant to ampicillin and tetracycline. A plasmid was isolated from this transformant according to a known method.
This plasmid was named pAMoEC3, and its structure was confirmed by restriction enzyme digestion.

(10) Construction of pAMoERC3 (Fig. 1
According to the method shown below, a plasmid pAMoERC3 in which the orientation of the units of oriP and EBNA-1 gene in pAMoEC3 was reversed was constructed.

1 μm of pAMoEC3 obtained in (9)
g was dissolved in 30 μl of Y-100 buffer, 20 units of XhoI was added, and digestion reaction was carried out at 37 ° C. for 2 hours. Thereafter, 30 μl of 1M Tris-hydrochloric acid (pH 8.0) and 1 unit of Escherichia coli alkaline phosphatase (Takara Shuzo) were added, and dephosphorylation reaction was carried out at 37 ° C. for 2 hours. After ethanol precipitation, it was dissolved in 30 μl of a buffer solution (hereinafter, abbreviated as TE buffer solution) consisting of 10 mM Tris-hydrochloric acid (pH 8.0) and 1 mM EDTA (sodium ethylenediamine tetraacetate) and subjected to agarose gel electrophoresis to about 9.1. A kb DNA fragment was recovered.

Also, 1 μg of the same plasmid was dissolved in 30 μl of Y-100 buffer, 20 units of XhoI was added, and the mixture was incubated at 37 ° C. for 2 hours.
A time digestion reaction was performed. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 4.9 kb was recovered. 0.1 μg of XhoI fragment (9.1 kb) derived from pAMoEC3 obtained above and 0.2 μg of XhoI fragment (4.9 kb) derived from the same plasmid were dissolved in 30 μl of T4 ligase buffer, and 175 units of T4 DNA ligase was added, and the mixture was added at 12 ° C. The binding reaction was performed for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution 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 pAMoERC3 and its structure was confirmed by restriction enzyme digestion.

(11) Construction of pAGE207 (Fig. 11)
Reference) According to the method described below, a plasmid pAGE207 in which the G418 resistance gene in pAGE107 was replaced with the hygromycin (hyg) resistance gene was constructed. hyg resistance gene is p201
(Bill Sugden et al., Nature (Natu
re), 313, 812 (1985)).

1 μg of pAGE107 (JP-A-3-22979)
Was dissolved in 30 μl of Y-50 buffer, 20 units of ClaI was added, and a digestion reaction was carried out at 37 ° C. for 2 hours. Then, sodium chloride was added so that the sodium chloride concentration would be 150 mM, 20 units of MluI was added, and the digestion reaction was further carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis,
A DNA fragment of about 4.6 kb was recovered.

P201 [Bill Sugden]
Et al: Nature, 313, 812 (1985)] 0.5 μg
Was dissolved in 30 μl of Y-50 buffer and 20 units of NarI [New England Biolab]
[Manufactured by the company] was added and a digestion reaction was carried out at 37 ° C. for 2 hours. After ethanol precipitation, it was dissolved in 30 μl of DNA polymerase I buffer solution, 6 units of Escherichia coli DNA polymerase I Klenow fragment was added, and the mixture was reacted at 37 ° C. for 60 minutes to change the 5 ′ protruding end generated by NarI digestion to a blunt end. . The reaction was stopped by phenol extraction, followed by chloroform extraction and ethanol precipitation, followed by dissolution in 20 μl of T4 ligase buffer solution, and 0.05 μg of ClaI linker (5'p CATCGAGTG3 ': manufactured by Takara Shuzo).
And 175 units of T4 DNA ligase were added and the binding reaction was performed at 12 ° C for 16 hours. After ethanol precipitation, Y-50 buffer solution 30μ
It was dissolved in 10 ml, added with 10 units of ClaI, and digested at 37 ° C. for 2 hours. After that, the sodium chloride concentration is 150 mM
Sodium chloride was added thereto so that 10 units of MluI was added, and the digestion reaction was further carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 1.6 kb was recovered.

ClaI derived from pAGE107 obtained above
-MluI fragment (4.6 kb) 0.2 μg and p201-derived ClaI
-0.1 μg of MluI fragment (1.6 kb) in 30 μl of T4 ligase buffer
Dissolve in l, add 175 units of T4 DNA ligase, and add at 12 ° C.
The binding reaction was performed for 16 hours. E. coli HB10 using the reaction solution
One 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 is called pAGE
It was named 207 and its structure was confirmed by restriction enzyme digestion.

(12) Construction of pAGE207ScN
(See FIG. 12) In order to remove the sequence similar to the SfiI site existing in the rabbit β globin gene, a plasmid pAGE207ScN in which a ScaI linker was inserted into the BalI site of pAGE207 was constructed. pA
The number of ScaI linkers inserted in GE207ScN is not clear.

0.5 of pAGE207 obtained in (11)
μg was dissolved in 30 μl of Y-0 buffer, 10 units of BalI was added, and digestion reaction was carried out at 37 ° C. for 2 hours. After ethanol precipitation, it was dissolved in 20 μl of T4 ligase buffer and 0.01 μg of ScaI linker (5'p AAGACTT3 ': manufactured by Takara Shuzo) was added.
After adding 175 units of T4 DNA ligase, the binding reaction was performed at 12 ° C for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution 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 designated as pAGE207ScN.
And its structure was confirmed by restriction enzyme digestion.

(13) Construction of pAMoC3Sc (Fig. 1
3) In order to remove the similar sequence of the SfiI site existing in the rabbit β globin gene in pAMoERC3 according to the method shown below, the rabbit β globin gene in pAMoERC3 was
Replaced by the rabbit β globin gene in pAGE207ScN, which already had its similar sequences removed, the plasmid pAMoERC3
Created Sc. For convenience of creation, first create pAMoC3Sc,
Next, pAMoERC3Sc was constructed. In pAGE207ScN described above, the number of ScaI linkers inserted to remove the similar sequence at the SfiI site is not clear, but pAMoERC3
In the case of Sc, pAGE207ScN was once cleaved with ScaI at the time of construction, so it is estimated that the number of inserted ScaI sites is one.

PAGE207ScN obtained in (12)
1 μg of KpnI was dissolved in 30 μl of Y-0 buffer.
Was added and the digestion reaction was carried out at 37 ° C. for 2 hours. Then, sodium chloride was added so that the sodium chloride concentration was 100 mM, 20 units of ScaI was added, and a digestion reaction was further carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 0.7 kb was recovered.

Further, 1 μg of the same plasmid was dissolved in 30 μl of Y-100 buffer, 20 units of ScaI and 20 units of ClaI were added, and a digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 0.9 kb was recovered. Separately, 1 μg of pAMoERC3 obtained in (10) was dissolved in 30 μl of Y-0 buffer, 20 units of KpnI was added, and digestion reaction was carried out at 37 ° C. for 2 hours. Then, sodium chloride was added so that the concentration of sodium chloride was 100 mM, 20 units of XhoI was added, and the digestion reaction was further carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 3.2 kb was recovered.

Next, pAGE107 (Japanese Patent Laid-Open No. 2-227075)
Was dissolved in 30 μl of Y-100 buffer, 20 units of XhoI and 20 units of ClaI were added, and a digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, about 4.3k
The DNA fragment of b was recovered. PAGE20 obtained above
0.1 μg of KpnI-ScaI fragment (0.7 kb) derived from 7ScN and 0.1 μg of ScaI-ClaI fragment (0.9 kb) derived from the same plasmid, p
KpnI-XhoI fragment (3.2 kb) 0.3 μm from AMoERC3
g, and the XhoI-ClaI fragment from pAGE107 (4.3
kb) 0.3 μg is dissolved in 30 μl of T4 ligase buffer, and T4D
175 units of NA ligase was added and the binding reaction was performed at 12 ° C for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution 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 pAMoC3Sc, and its structure was confirmed by digestion with restriction enzymes.

(14) Construction of pAMoERC3Sc
(See FIG. 14) 1 μg of pAMoERC3 obtained in (10) was added to Y-0.
Dissolve in 30 μl of buffer, add 20 units of KpnI, and add 2 at 37 ℃.
A time digestion reaction was performed. Then, sodium chloride was added so that the sodium chloride concentration would be 150 mM, 20 units of MluI was added, and the digestion reaction was further carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, about 6.8 kb
The DNA fragment was recovered.

Further, 1 μg of the same plasmid was dissolved in 30 μl of Y-150 buffer, 20 units of XhoI and 20 units of MluI were added, and digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 1.3 kb was recovered. Separately, 1 of pAMoC3Sc obtained in (3)
μg was dissolved in 30 μl of Y-0 buffer, 20 units of KpnI was added, and digestion reaction was carried out at 37 ° C. for 2 hours. Then, sodium chloride was added so that the concentration of sodium chloride was 100 mM, 20 units of XhoI was added, and the digestion reaction was further carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis,
A DNA fragment of about 5.9 kb was recovered.

Kp derived from pAMoERC3 obtained above
0.2 μg of nI-MluI fragment (6.8 kb) and Xh derived from the same plasmid
oI-MluI fragment (1.3 kb) 0.05 μg, and pAMoC3
0.2 μg of KpnI-XhoI fragment (5.9 kb) derived from Sc was dissolved in 30 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and a ligation reaction was carried out at 12 ° C. for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution 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 pAMoERC3Sc, and its structure was confirmed by digestion with restriction enzymes.

PAMoERC3Sc is a long terminal repeat of Moloney murine leukemia virus as a promoter for expressing a heterologous gene.
repeat). In addition, for efficient expression of the heterologous gene, the rabbit β globin gene splicing signal, the rabbit β globin gene poly A addition signal and the SV40 early gene poly A addition signal are designed to be added after the inserted heterologous gene. ing. Also, G41 is used as a drug resistance marker for animal cells.
It has a kanamycin resistance gene (the same as the G418 resistance gene) and an ampicillin resistance gene as drug resistance markers for E. coli. further,
Epstein-Barr vi
rus) has an origin of replication (oriP) and an EBNA-1 gene, which is a factor that causes trans to act on oriP and causes replication, so that it is integrated into the chromosome in many cells excluding rodents including Namalwa cells. Can exist in the plasmid state without.

Construction of a cDNA library using pAMoERC3Sc was carried out by adding SfiI linkers to both ends of the cDNA and then adding SfiI in pAMoERC3Sc.
This can be done by incorporating it into the site.

(15) Construction of pAMoPRC3Sc
(See FIG. 15) When a cell originally expressing the EBNA-1 gene such as Namalwa cell is used as a host, the plasmid introduced into the host is chromosomally integrated even if the EBNA-1 gene in the plasmid pAMoERC3Sc is not present. It is believed possible to exist in the plasmid state without being integrated. Therefore, EB from pAMoERC3Sc
Plasmid pAMoPRC3 with NA-1 gene removed
Sc was produced as follows. pAMoPRC
3Sc can be directly used as an expression cloning vector in the same manner as pAMoERC3Sc.

PAMoERC3Sc obtained in (14)
Dissolved in 30 μl of Y-50 buffer to give 20 units of N
siI [New England Biolabs (New Engla
ndBiolabs) was added and the digestion reaction was carried out at 37 ° C. for 2 hours. After ethanol precipitation, 30 μl DNA polymerase I
It was dissolved in a buffer solution, 6 units of E. coli DNA polymerase I Klenow fragment was added, and the mixture was reacted at 37 ° C for 60 minutes to change the 3'protruding end generated by NsiI digestion into a blunt end. The reaction was stopped by phenol extraction, and after chloroform extraction and ethanol precipitation, it was dissolved in 30 μl of Y-100 buffer,
20 units of NotI was added and a digestion reaction was carried out at 37 ° C for 2 hours.
After the reaction solution was subjected to agarose gel electrophoresis, about 8.1 kb of DNA was
The fragments were collected.

2 μg of the plasmid was dissolved in 30 μl of Y-100 buffer, 20 units of XhoI was added, and the mixture was incubated at 37 ° C. for 2 hours.
A time digestion reaction was performed. After ethanol precipitation, 30 μl DN
6 units of E. coli DNA dissolved in A polymerase I buffer
Polymerase I-Klenow fragment was added and reacted at 37 ° C for 60 minutes to change the 5'protruding end generated by XhoI digestion into a blunt end. The reaction was stopped by phenol extraction, chloroform extraction and ethanol precipitation, followed by Y-100 buffer solution.
It was dissolved in 30 μl, 20 units of NotI was added, and a digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 3.2 kb was recovered.

NsiI (blunt end) -NotI fragment (8.1 kb) derived from pAMoERC3Sc obtained above and 0.1 μg of XhoI (blunt end) -NotI fragment (3.2 kb) derived from the same plasmid.
1 μg was dissolved in 30 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and the binding reaction was carried out at 12 ° C. for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution 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 pAMoPRC3Sc, and its structure was confirmed by digestion with restriction enzymes.

2. Namaluba cell bean lectin 12
Examination of degree of resistance to 0 Namalwa cells conditioned by serum-free medium (KJM-1 strain) [Hosoi et al., Cytotechnology, 1, 151 (198)
8)] was cultured in the presence of various concentrations of Lima bean lectin 120, and the degree of resistance of KJM-1 strain to Lima bean lectin 120 was examined. KJM-1 strain as RPMI164
0-ITPSGF medium [7.5% sodium bicarbonate 1/40
Volume, 200 mM L-glutamine solution (GIBCO) 3%, penicillin-streptomycin solution (GIBCO, 5000
units / ml penicillin, 5000 μg / ml streptomycin) 0.5%, N-2-hydroxyethylpiperazine-
N'-2-ethanesulfonic acid (N-2-hydroxyethylpipera
zine-N'-2-hydroxypropane-3-sulfonic acid; HEPES)
(10 mM), insulin (3 μg / ml), transferrin (5 μg / ml), sodium pyruvate (5 mM), sodium aselenate (125 nM), galactose (1 mg / m)
l), Pluronic F68 (0.1% w / v) in RPMI1640 medium (Nissui Pharmaceutical Co., Ltd.)]
The cells were suspended to a concentration of 10 ⁴ cells / ml, and 200 μl of each was dispensed into a 96-well microtiter plate. Various concentrations of Himame lectin 120 (manufactured by Seikagaku Corporation)
Each / 100 amount was added and the cells were cultured in a carbon dioxide gas incubator at 37 ° C for 3 weeks. As a result, the minimum concentration of Himame lectin 120, which completely inhibits the growth of KJM-1 strain, was 50 ng / m 2.
It was l. When 4 million KJM-1 strains were examined, no spontaneous appearance of Himame lectin 120 resistant strain was observed at this concentration.

3. WM266-4, a human melanoma cell
Lima bean lectin 120 resistance gene (WM16) from cells
(1) Obtaining mRNA from human melanoma cell line WM266-4 cells 1 × 10 ⁸ WM266-4 cells (ATCC CRL 1676) with Invitrogen mRNA extraction kit A Fast Track; Item No. K1593-0
Using 2), about 30 μg of mRNA was obtained. The specific reagents and methods were in accordance with the instructions attached to the kit.

(2) Preparation of cDNA library From 8 μg of the mRNA obtained above, a cDNA synthesis system (cDNA Synthesis System) which is a kit manufactured by GIBCO BRL is used.
m) and double-stranded cD using oligo dT as a primer
NA was synthesized. In that case, reverse transcriptase in the kit
Moloney MurineLeukemia Virus (M-MLV) reverse trans
Instead of criptase, its Super Script ^TM RNase H
-Reverse Transcriptase was used. Then, SfiI linkers were added to both ends of the cDNA as described below, and the cDNA was fractionated by size by agarose gel electrophoresis to recover a cDNA fragment of about 1.2 kb or more.

[0101]

[Chemical 4]

11mer of the above SfiI linker (SEQ ID NO: 5)
Single-stranded DNAs of 8 mer and 8 mer were respectively synthesized using Applied Biosystems 380A DNA synthesizer.
50 μg of each of the synthesized DNAs was dissolved separately in 50 μl of T4 kinase buffer, 30 units of T4 polynucleotide kinase (Takara Shuzo) was added, and phosphorylation reaction was carried out at 37 ° C. for 16 hours before use. Double-stranded cD obtained above
NA and the above phosphorylated linker (4 μg for 11mer and 2.9 μg for 8mer) in T4 ligase buffer
Dissolve in 45 μl, add 1050 units of T4 DNA ligase and
The binding reaction was performed at 16 ° C for 16 hours. After the reaction solution was subjected to agarose gel electrophoresis, a cDNA fragment of about 1.2 kb or more was recovered.

In addition, a direct expression cloning vector (Ex
pAMoPRC3S which is the compression Cloning Vector)
24 μg of c was dissolved in 590 μl of Y-50 buffer, 80 units of SfiI was added, and digestion reaction was carried out at 37 ° C. for 16 hours. After taking 5 μl from this reaction mixture and confirming that the digestion was completed by agarose gel electrophoresis, add 40 units of BamHI to reduce the amount of clones without the cDNA insert when the cDNA library was constructed. , 37 ℃
The digestion reaction was carried out for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 8.8 kb was recovered.

2 μg of the SfiI fragment (8.8 kb) derived from pAMoPRC3Sc obtained above and the cDNA purified above were transformed into T4.
Dissolve in 250 μl of ligase buffer and use T4 DNA Ligase 2000
The unit was added and the binding reaction was carried out at 16 ° C. for 16 hours. Thereafter, 5 μg of transfer RNA (tRNA) was added, precipitated with ethanol, and then dissolved in 20 μl of TE buffer. Escherichia coli LE392 strain [Maniat (Maniat
edited by is) et al .: Molecular Cloning (Molecular
Cloning), 2.58, Cold Spring Harbor, 1989]
Electroporation method [William J.
William J. Dower et al .: Nucleic Acids Res., 16 , 6127
(1988)] to obtain about 260,000 ampicillin-resistant strains.

(3) α2,3-sialyltransferase
After mixing about 260,000 ampicillin-resistant strains (cDNA library) obtained by cloning (2) of cDNA (WM16), a plasmid preparation kit (plasmid <maxi) manufactured by Qiagen was used. kit (
A plasmid was prepared using the product number 41031). The obtained plasmid was precipitated with ethanol and then dissolved in TE buffer at 1 μg / μl.

The above plasmid was prepared by the electroporation method [Miyaji et al .: Cytotechnolog
y), 3 , 133 (1990)]. After introducing 4 μg of plasmid per 1.6 × 10 ⁶ cells, the cells were suspended in 8 ml of RPMI1640.ITPSGF medium and cultured in a carbon dioxide gas incubator at 37 ° C. for 24 hours. Then, G418 (manufactured by Gibco) was added to 0.5 mg / ml, and the cells were further cultured for 7 days to obtain a transformant. The obtained transformant strain is
RPMI1640 / ITPSGF containing 50 ng / ml)
The cells were suspended in the medium at 5 × 10 ⁴ cells / ml, and 200 μl each was dispensed to a 96-well microtiter plate.

After culturing at 37 ° C. for 4 weeks in a carbon dioxide gas incubator, a Spodoptera frugiperda 120 resistant strain was obtained. After culturing the resistant strain, about 5 × 10 ⁶ cells were subjected to the Hart method (Robert FM Margolsky).
(argolskee) et al .: Molecular and Cellular Biology (Mol. Cell. Biol.), 8, 2837 (1988)]. The recovered plasmid was introduced into E. coli LE392 by electroporation [William J. Dower et al .: Nucleic Acids Res., 16 , 6127 (1988)], An ampicillin resistant strain was obtained. Qiagen from the transformant
When a plasmid was prepared using a plasmid preparation kit manufactured by the company, and its structure was cleaved with various restriction enzymes and examined,
It was revealed to contain a cDNA of about 2.2 kb. The plasmid containing this cDNA was named pAMoPRWM16, and was introduced into the KJM-1 strain again in the same manner as described above. Since it became resistant to Himammelectin 120 again, this cDNA produced α2,3-sialyltransferase. Presumed to be the encoding cDNA.

4. α2,3-sialyltransferase cD
Determination of the nucleotide sequence of NA (WM16) (1) α2,3-sialyltransferase cDNA (WM16)
Incorporation of pAMoPRWM16 into pUC119 (see FIG. 16) 2 μg of pAMoPRWM16 obtained in item (3)
Dissolve in 50 μl of -100 buffer and add 30 units of EcoRV and 3
0 unit of Asp718 (Boehringer Mannheim (Boehringer
Mannheim) was added and a digestion reaction was carried out at 37 ° C. for 2 hours. After ethanol precipitation, 30 μl DNA polymerase I
It was dissolved in a buffer solution, 6 units of Escherichia coli DNA polymerase I Klenow fragment was added, and the reaction was carried out at 37 ° C for 60 minutes to change the 5'protruding end generated by Asp718 digestion into a blunt end. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 2.3 kb was recovered.

In addition, pUC119 [Messing (Messin
g) et al: Methods in Enzymology (Methods i
n Enzymology), 153, 3 (1987)] in Y-10.
Dissolve in 30 μl of 0 buffer solution, add 20 units of HincII, 37 ℃
The digestion reaction was carried out for 2 hours. After that, 1M Tris-hydrochloric acid
30 μl of (pH8.0) and 1 unit of Escherichia coli alkaline phosphatase (Takara Shuzo) were added, and dephosphorylation reaction was carried out at 37 ° C. for 2 hours. After ethanol precipitation, it was dissolved in 30 μl of TE buffer and subjected to agarose gel electrophoresis.
The DNA fragment was recovered.

The pAMoPRWM16-derived EcoRV-Asp718 (blunt end) fragment (2.3 kb) 0.05 μg and the pUC119-derived HincII fragment (3.16 kb) 0.05 μg obtained above were
4 Dissolve in 30 μl of ligase buffer and use T4 DNA ligase175
The unit was added and the binding reaction was performed at 12 ° C. for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution by the method of Cohen et al. To obtain an ampicillin resistant strain. Plasmids were isolated from these transformants according to known methods.
As a result, EcoRV- Asp718 derived from pAMoPRWM16
Different orientation of the (blunt end) fragment in pUC119 2
Seed plasmids were isolated and p.
UC119-WM16 and pUC119-WM16R
And its structure was confirmed by restriction enzyme digestion.

(2) Construction of Deletion Mutation Plasmid for Determining Nucleotide Sequence (Deletion Plasmid)
Dissolve in 30 μl of 0 buffer, add 50 units of KpnI, and incubate at 37 ℃
The digestion reaction was performed for 16 hours. After that, add sodium chloride so that the concentration of sodium chloride is 100 mM, and
A unit of NotI was added, and the digestion reaction was further performed at 37 ° C. for 2 hours. After ethanol precipitation, it was dissolved in 100 μl of Exo III buffer solution (provided with Takara Shuzo Co., Ltd.'s deletion kit for kilosequencing). Exo III after ethanol precipitation
It was dissolved in 100 μl of buffer.

The KpnI-BamHI fragment and pUC119-WM16 derived from pUC119-WM16 obtained above.
Dozens of deletion mutant plasmids were prepared from the KpnI-BamHI fragment derived from R using a deletion kit for kilosequencing manufactured by Takara Shuzo. The specific reagents and methods were in accordance with the instructions attached to the kit. The nucleotide sequence of the deletion plasmid obtained above is the nucleotide sequence determination kit (Taq DyeDeoxy ^™ Terminator Cycle Sequencing Kit of Applied Biosystems);
It was determined using the product number 401113). The determined nucleotide sequence is shown in SEQ ID NO: 1. As a result, it was revealed that the α2,3-sialyltransferase cDNA (WM16) encodes a protein consisting of 375 amino acids.
The amino acid sequence also revealed that this protein has a structure common to glycosyltransferase (GT). That is, the N-terminal 8 amino acids are exposed to the cytoplasmic side, followed by membrane-bound in a hydrophobic region consisting of 20 amino acids, and most of the remaining C-terminal portion (including the catalytic site) is exposed in the Golgi body lumen. It is thought that it has such a structure.

5. α2,3-sialyltransferase
Α2,3-strain of KJM-1 strain introduced with gene expression plasmid
Measurement of allyltransferase activity Plasmid pAMoPRC3Sc obtained above and
pAMoPRWM16 manufactured by Qiagen
> Plasmid <maxi kit (product
No. 41031). The obtained plasmid is
After ethanol precipitation, add TE buffer to 1 μg / μl
Dissolved in. After that, both plasmids were electroporated.
Ration method [Miyaji et al .: Site Technology (Cytotec
hnology), 3,133 (1990)] respectively, Namalba K
It was introduced into JM-1 strain. 1.6 x 10 ⁶4 μg per cell
After introducing the plasmid, 8 ml of RPMI1640.ITP
Suspend in SGF medium and in a carbon dioxide incubator at 37 ℃
The cells were cultured for 24 hours. After that, G418 (manufactured by Gibco) is 0.5m
The cells were added to g / ml and cultured for 7 days. Then 22
ml RPMI1640-ITPSGF medium (0.5 mg / ml
(Including G418) and cultured for 5 days
Got The obtained transformants each contained 0.5 mg of G418 /
5 x in 30 ml of RPMI1640 / ITPSGF medium containing 5 ml
Ten^FourSuspend cells / ml and incubate with carbon dioxide gas.
The cells were cultivated at 37 ° C for 8 days. Then, centrifuge (160 ×
 cells for 10 minutes, and then add PBS [8g / l sodium chloride
Thorium, 0.2g / l potassium chloride, 1.15g / l phosphoric anhydride
Sodium monohydrogen, 0.2 g / l potassium dihydrogen phosphate] 10 ml
After washing with, the cells were collected by centrifugation again. The collected cells are used
Stored at -80 ° C until use.

The two types of cells obtained above were about 1.0 × 10 ⁷
50 μl of 1% Triton X-100 was added to each and suspended, and then ultrasonically disrupted (Bioruptor; COSMO BI
The cells were disrupted using O.) and the supernatant was obtained by centrifugation (550 x g, 10 minutes).

10 of each of the two types of supernatants obtained above
Using a final volume of 30 μl assay solution (0.1 M cacodylic acid-hydrochloric acid (pH 7.5), 0.01 M manganese chloride, 0.45%
Triton X-100, 0.1 mM substrate, 5 mM CMP-sialic acid (added or not added)], and allowed to react at 37 ° C for 2 hours, then identify the product using high performance liquid chromatography (HPLC). Α2,3- in each supernatant by
Sialyltransferase activity was measured. The BCA Protein Assay is used to quantify the amount of protein in the supernatant used for activity measurement.
Reagent (manufactured by PIERCE) was used. As a substrate, sugar chains fluorescently labeled with aminopyridine [Gal β1-4GlcNA
c β1-3 Gal β1-4Glc (LNT) -aminopyridine and Ga
l β1-3GlcNAc β1-3 Gal β1-4Glc (LNnT) -aminopyridine] was used. Fluorescent labeling of the substrate was carried out using lacto-N-tetraose and lacto-N-neotetraose (both manufactured by Oxford Glycosystems) in a conventional manner [Akihiro Kondo et al .: Agricultural.
And Biological Chemistry (Agric. Biol.
Chem.), 54 , 2169 (1990)]. After reacting each supernatant with an assay solution containing the sugar donor CMP-sialic acid and an assay solution not containing it, the reaction solutions were separated by HPLC,
The product was a peak that appeared only in the assay solution containing CMP-sialic acid. After the reaction is completed,
After treatment at 5 ° C. for 5 minutes, centrifugation was performed at 10,000 × g for 10 minutes, and 10 μl of the supernatant was subjected to HPLC. HPLC is T
SKgel ODS-80TM column (4.6mm x 30cm;
Tosoh Corporation) and 0.02M ammonium acetate buffer
Elution was performed using (pH 4.0) at an elution temperature of 50 ° C. and a flow rate of 1 ml / min. Fluorescence detection (Fl
uorescence HPLC MonitorRF-535T) (excitation wavelength 320 nm, emission wavelength 400 nm). The product was identified by sialyllact-N-neotetraose c (NeuAc
α2-6Gal β1-4GlcNAc β1-3 Gal β1-4Glc; Seikagaku Corporation) or sialyllact-N-neotetraose a
(NeuAc α2-3Gal β1-4GlcNAc β1-3 Gal β1-4Glc) or sialyllact-N-tetraose a (NeuAc α
2-3Gal β1-3GlcNAc β1-3 Gal β1-4 Glc; Seikagaku Kogyo Co., Ltd.) was used in the same manner as the standard, but the elution time was compared with these, and when the product was treated with sialidase. This was done by regenerating the substrate. The product was quantified in the same manner using lactose aminopyridylated as a standard,
This was done by comparing the fluorescence intensities. As a result,
As shown in 7, when lacto-N-neotetraose was used as a substrate, peak 1 and peak 2 were detected as products. Peak 1 is NeuAcα2-6Galβ1-4GlcNAcβ1-3 because the elution time matches that of the standard and the substrate is regenerated when the product is treated with sialidase.
Gal β1-4Glc- aminopyridine, peak 2 is NeuAcα
It was revealed that it is 2-3Gal β1-4GlcNAc β1-3 Gal β1-4Glc-aminopyridine. Further, as shown in FIG. 18, when lacto-N-tetraose was used as a substrate, peak 3 was detected as a product. Peak 3 shows that the elution time matches that of the standard, and that the substrate is regenerated when the product is treated with sialidase, so NeuA
c α2-3Gal β1-3GlcNAc β1-3Gal β1-4Glc-Aminopyridine was revealed. The sialidase treatment of the product was performed as follows. Supernatant after enzymatic reaction
30 μl of each fraction was subjected to HPLC to separate peak 1, peak 2 or peak 3, freeze-dried and then 20 mM Tris-
It was dissolved in 50 μl of a buffer solution containing maleic acid (pH 6.0) and 1 mM calcium citrate. Then add 20 μl of the lysate
400mU / ml sialidase (neuraminidase, manufactured by Sigma),
2 μl of N-2133) was added and the reaction was carried out at 37 ° C. for 16 hours. Also, as a control, use water instead of sialidase.
2 μl was added and the reaction was performed in the same manner. The solution after the reaction was treated at 100 ° C. for 5 minutes and then centrifuged at 10,000 × g for 10 minutes, and 10 μl of the supernatant was subjected to the above-mentioned HPLC.

Table 1 shows the amount of the product and the relative activity of the α2,3-sialyltransferase of the present invention to LNnT when the activity to LNT was 100. Also,
Known α2, which has been reported to be purified from rats,
The activity of 3-sialyltransferase on LNT was 10
Relative activity with respect to LNnT at 0 (Weinstein
(Weinstein) et al: Journal of Biological
Chemistry (J. Biol. Chem.) , 257 , 13845 (1982)] is also shown in Table 1.

[0117]

[Table 1]

KJM-introduced with pAMoPRWM16
In one strain, the α2,3-sialyltransferase activity was significantly increased when lacto-N-tetraose was used as a substrate, as compared with the KJM-1 strain into which the vector pAMoPRC3Sc was introduced. In addition, regarding the α2,3-sialyltransferase activity when lacto-N-neotetraose was used as a substrate, in the KJM-1 strain into which pAMoPRWM16 was introduced, the vector pAMoPRC3S was used.
It was increased compared to the KJM-1 strain into which c was introduced. From the above results, WM16 encodes α2,3-sialyltransferase, and α2,3-sialyltransferase encoded by WM16 has a substrate specificity for lacto-N-tetraose rather than lacto-N-neotetraose. It was shown to be high and that the α2,3-sialyltransferase encoded by the cDNA can be used to produce an oligosaccharide having sialic acid.

Example 2 Sialyl Lewis x (Sial in KJM-1 strain into which α2,3-sialyltransferase expression plasmid was introduced
yl Lewis x) Synthesis of sugar chain pAMoPRWM16 (α2,3-sialyltransferase expression plasmid) obtained in Example 1, or p
RPMI16 containing 0.5 mg / ml of G418 was added to the KJM-1 strain introduced with AMoPRC3Sc (control plasmid).
After culturing in 40-ITPSGF medium, each about 1 x
10 ⁶ cells were placed in a microtube (1.5 ml: manufactured by Eppendorf) and centrifuged at 550 × g for 7 minutes to collect the cells. Next, the cells were washed with 1 ml of a phosphate buffer PBS (hereinafter abbreviated as A-PBS) containing 0.1% sodium azide, and then CSLEX1 [Fukushima et al .: Cancer. research
(Cancer Res.), 44 , 5279 (1984)] and KM93 [F
uruya et al .: Anticancer Research
Res.), 12 , 27 (1992)], and the expression of sialyl Lewis x sugar chain in these cells was examined by indirect fluorescent antibody staining.

CSLEX1 or KM93 was added to each cell in an amount of 50 μl (10 μg / ml), and the cells were suspended and reacted at 4 ° C. for 1 hour. Then, the cells are A-PB
After washing 3 times with S, fluorescein isothiocyanate (F
ITC) fluorescently labeled anti-mouse IgG antibody and IgM antibody (Kappel, 16-fold diluted with A-PBS) 20
μl was added to suspend, and the mixture was reacted at 4 ° C. for 30 minutes. After the reaction, the cells were washed 3 times with A-PBS and then again with A-PB.
Suspended in S, Epics Elite Flow Cytme
Type [EPICS Elite Flow Cytometer; Coulter (COUL
TER) product]. As a control, CSLEX
1 or KM93 instead of normal mouse serum (A-PB
The same analysis as above was carried out by using 500-fold diluted with S). The results are shown in Fig. 19. It can be seen that in the KJM-1 strain into which the direct expression cloning vector pAMoPRC3Sc has been introduced, the fluorescence intensity of cells stained with CSLEX1 or KM93 is stronger than that of the control. This means that the KJM-1 strain was originally
It shows that the Lewis x sugar chain is expressed. Also,
KJM-1 into which the plasmid pAMoPRWM16 expressing the α2,3-sialyltransferase of the present invention has been introduced
The fluorescence intensity of cells stained with CSLEX1 or KM93 is KJM-1 into which pAMoPRC3Sc was introduced.
It was even stronger than the stock. This indicates that the α2,3-sialyltransferase of the present invention synthesizes sialyl Lewis x sugar chains in cells.

Example 3 Production of α2,3-sialyltransferase by animal cells 1. Construction of plasmid pAMoPRSAW16 for α2,3-sialyltransferase secretion expression (1) Construction of pAGE147 (see FIG. 20) According to the method shown below, the SV40 early gene promoter of pAGE107 was expressed as a long terminal repeat (long) of Moloney murine leukemia virus. terminal repea
t; LTR) plasmid replaced with pA
Creation of GE147 was performed.

2 μg of the plasmid pPMOL1 obtained by the method described in JP-A-1-63394 was dissolved in 30 μl of Y-0 buffer, 20 units of SmaI was added, and digestion reaction was carried out at 30 ° C. for 3 hours. Then, sodium chloride was added to 50 mM, 20 units of ClaI was added, and a digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, Moloney
About 0.6k containing mouse leukemia virus LTR promoter
The DNA fragment of b was recovered.

Next, 2 synthesized in item 1 (8) of Example 1 was used.
Seed synthetic DNA

[0124]

[Chemical 5]

25 pmoles (pmoles) of each was dissolved in 10 μl of T4 kinase buffer, 5 units of T4 DNA kinase was added, and the reaction was carried out at 37 ° C. for 30 minutes to phosphorylate the 5 ′ end. ClaI-S derived from pPMOL1 obtained above
0.05 μg of maI fragment (0.6 kb), two kinds of 5'-phosphorylated synthetic DNA (1 picomole each) and HindIII linker (5'-pCAAGCTTG-3 '; Takara Shuzo) (1 picomole) were added to T4 ligase buffer. It was dissolved in 30 μl, 200 units of T4 DNA ligase was added, and the binding reaction was performed at 12 ° C for 16 hours. After recovering the DNA fragment by ethanol precipitation, it was dissolved in Y-100 buffer and added with 10 units of HindIII and 10 units of XhoI.
The digestion reaction was carried out at 37 ° C for 2 hours. The reaction was stopped by phenol-chloroform extraction, and the DNA fragment was recovered by ethanol precipitation.

On the other hand, pAGE107 [JP-A-3-229]
79, Miyaji et al .: Site Technology (Cytotechnolog
y) , 3,133 (1990)] 1 μg was dissolved in 30 μl of Y-100 buffer, and 10 units of HindIII and 10 units of XhoI were added.
The digestion reaction was carried out at ℃ for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 6.0 kb containing the G418 resistance gene and the ampicillin resistance gene was recovered.

Hind from pAGE107 obtained above
III-XhoI fragment (6.0 kb) 0.3 μg and pPMOL1-derived Hind
Add 0.01 μg of III-XhoI fragment (0.6 kb) to 20 μL of T4 ligase buffer.
Dissolve in l, add 200 units of T4 DNA ligase, and add 16 units at 12 ℃.
A time binding reaction was performed. E. coli HB101 using the reaction solution
The 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 is called pAGE1
It was named 47 and its structure was confirmed by restriction enzyme digestion.

(2) Construction of pAGE247 (see FIG. 21) According to the method described below, the SV40 early gene promoter of pAGE207 was replaced with the long terminal repeat of Moloney murine leukemia virus.
t: LTR) plasmid pA replaced with promoter
Creation of GE247 was performed.

2 μm of pAGE147 obtained in the above (1)
g was dissolved in 30 μl of Y-100 buffer and 10 units of Hind
III and 10 units of XhoI were added and a digestion reaction was carried out at 37 ° C for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, the reaction solution containing the LTR promoter of Moloney murine leukemia virus was about 0.
A 63 kb DNA fragment was recovered. On the other hand, item 1 (1
2 μg of pAGE207 obtained in 1) was added to 30 μl of Y-
Dissolve in 100 buffer, 10 units HindIII and 10 units Xh
oI was added and the digestion reaction was performed at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 5.84 kb containing a hyg resistance gene and an ampicillin resistance gene was recovered.

Hind from pAGE147 obtained above
III-XhoI fragment (0.63 kb) 0.05 μg and derived from pAGE207
0.1 μg of HindIII-XhoI fragment (5.84 kb) in T4 ligase buffer
Dissolve in 30 µl, add 100 units of T4 DNA ligase, and
The binding reaction was carried out for 16 hours. E. coli HB using the reaction solution
The 101 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 is pAG
It was named E247 and its structure was confirmed by restriction enzyme digestion.

(3) Construction of pAMN6hyg (Fig. 22)
Reference) According to the following method, a human granulocyte colony-stimulating factor derivative expression plasmid pAMN6hyg having LTR of Moloney murine leukemia virus as a promoter and a hyg resistance gene as a marker was constructed.

2 μm of pAGE247 obtained in the above (2)
g was dissolved in 30 μl of Y-50 buffer, 20 units of ClaI was added, and digestion reaction was carried out at 37 ° C. for 2 hours. Then, sodium chloride was added to 175 mM, 20 units of SalI was added, and a digestion reaction was carried out at 37 ° C for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, the LTR promoter of Moloney murine leukemia virus, ampicillin resistance gene and hy
A DNA fragment of about 4.8 kb containing the g resistance gene was recovered.

On the other hand, 2 μg of the plasmid pASN6 obtained by the method described in JP-A-2-227075 was added to a Y-50 buffer solution.
It was dissolved in 30 μl, 20 units of ClaI was added, and a digestion reaction was carried out at 37 ° C. for 2 hours. Then add sodium chloride to 175 mM and add 20 units SalI and 20 units MluI.
The digestion reaction was carried out at 37 ° C for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 5.0 kb containing a human granulocyte colony stimulating factor derivative gene was recovered.

ClaI from pAGE247 obtained above
-SalI fragment (4.8 kb) 0.1 μg and pASN6-derived ClaI-S
0.1 μg of alI fragment (5.0 kb) was dissolved in 20 μl of T4 ligase buffer, 200 units of T4 DNA ligase was added, and a ligation reaction was performed at 12 ° C. for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution 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 is called pAMN6hy
The structure was confirmed by restriction enzyme digestion.

(4) Construction of pAMoERSA (FIG. 23)
Refer to the following method for Staphylococcus
aureus) protein A immunoglobulin G (Ig
G) Secretory expression vector pAMo for secretory expression of any protein as a fusion protein with the binding region to
Created ERSA.

2 of pAMN6hyg obtained in (3) above
μg was dissolved in 30 μl of Y-50 buffer and 20 units of SnaBI
Was added and the digestion reaction was carried out at 37 ° C. for 2 hours. Then add sodium chloride to 100 mM and add 20 units of XbaI.
Was added to carry out a digestion reaction at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 0.33 kb containing a signal sequence of human granulocyte colony stimulating factor was recovered.

Also, pPrAS1 [Saito et al .: Protein Engineering , 2, 481
(1989)] in 30 μl of Y-50 buffer,
20 units of ClaI was added and a digestion reaction was carried out at 37 ° C. for 2 hours.
After ethanol precipitation, it was dissolved in 30 μl of DNA polymerase I buffer solution, 6 units of Escherichia coli DNA polymerase I Klenow fragment was added, and the mixture was reacted at 37 ° C. for 60 minutes to change the 5 ′ protruding end generated by ClaI digestion into a blunt end. . The reaction was stopped by phenol extraction, followed by chloroform extraction and ethanol precipitation, followed by dissolution in 30 μl of Y-100 buffer, 20 units of BamHI was added, and a digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, Ig of protein A was
A DNA fragment of about 0.21 kb containing the G-binding region was recovered.

Further, pA obtained in item (14) of Example 1
2 μg of MoERC3Sc was added to 30 μl of Y-100 buffer.
Dissolve in, add 20 units of XbaI and 20 units of BamHI and incubate at 37 ℃
The digestion reaction was carried out for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 12.1 kb was recovered. PAMN6hyg-derived SnaBI-XbaI obtained in (3) above
Fragment (0.33 kb) 0.05 μg and pPrAS1-derived ClaI (blun
t)-BamHI fragment (0.21 kb) 0.05 μg, and pAMo
XbaI-BamHI fragment (12.1 kb) from ERC3Sc 0.1 μg
Is dissolved in 30 μl of T4 ligase buffer and T4 DNA ligase
175 units were added and the binding reaction was carried out at 12 ° C for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution 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 pAMoERSA, and its structure was confirmed by restriction enzyme digestion.

(5) Construction of pAMoPRSA (Fig. 24)
EBNA- in pAMoERSA according to the following method
A plasmid pAMoPRSA having one gene removed was constructed. Similar to pAMoERSA, pAMoPRSA can be used as a secretory expression vector.

2 μg of pAMoERSA was added to 10 mM Tris-
Dissolve in 30 μl of a buffer solution (hereinafter abbreviated as Y-80 buffer solution) consisting of hydrochloric acid (pH 7.5), 6 mM magnesium chloride, 80 mM sodium chloride, 6 mM 2-mercaptoethanol,
20 units of XbaI and 20 units of Asp718 (manufactured by Boehringer Mannheim) were added, and a digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 1.3 kb was recovered.

In addition, 2 μg of pAMoPRC3Sc obtained in item (15) of Example 1 was added to Y-100 buffer solution.
Dissolve in μl, add 20 units of XbaI and 20 units of Asp718,
The digestion reaction was carried out at 37 ° C for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 8.5 kb was recovered.

0.05 μg of XbaI-Asp718 fragment (1.3 kb) derived from pAMoERSA obtained in (4) above and pAMo
XbaI-Asp718 fragment (8.5 kb) from PRC3Sc 0.1 μ
g was dissolved in 30 μl of T4 ligase buffer, 175 units of T4 DNA ligase was added, and the binding reaction was carried out at 12 ° C. for 16 hours.
Escherichia coli 101 strain was transformed with the reaction solution 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 pAMoPRSA, and its structure was confirmed by digestion with restriction enzymes.

(6) Construction of pAMoPRSAW16
(Refer to FIG. 25) The cloned α2,3-sialyltransferase outputs the N-terminal 8 amino acids to the cytoplasmic side from its primary sequence,
It is presumed that the structure is such that the subsequent 20-amino acid-rich region, which is rich in hydrophobicity, binds to the membrane and exposes most of the remaining C-terminal portion (including the catalytic site) to the Golgi body lumen. Therefore, the α2,3-sialyltransferase was removed by removing a portion presumed to be a membrane-bound region as described below and adding a signal sequence of human granulocyte colony-stimulating factor and a binding region of IgG of protein A instead. Was produced.

A DNA encoding a portion presumed to be a membrane-bound region [from the 32nd Leu to the 375th Ile of SEQ ID NO: 1] was prepared by the PCR method, and the above (4) was prepared.
It was incorporated into the secretory expression vector pAMoPRSA constructed in 1. As primers for PCR, the following two kinds of synthetic DNA [W16-A (32L) (38mer; SEQ ID NO: 6) and
W16-C (37mer; SEQ ID NO: 7)] was synthesized using an Applied Biosystems 380A DNA synthesizer.

[0145]

[Chemical 6]

Since the StuI site was introduced into W16-A (32L) and the Asp718 site was introduced into W16-C, the DNA fragment amplified by PCR contained StuI and Astu.
After cutting with p718, the StuI site of pAMoPRSA
Can be incorporated between Asp718 sites. The PCR reaction is based on the Takara Shuzo kit (GeneAmpTM DNA Amplification
Reagent Kit with AmpliTaq ^TM Recombinant Taq DNA Po
lymerase). The reaction solution is prepared according to the instructions attached to the kit, and the thermal cycler (PERKIN ELMER CETUS) manufactured by Perkin Elmer Cetus is used.
DNA Thermal Cycler) at 94 ℃ for 1 minute, 5
After 30 cycles of reaction at 5 ° C. for 1 minute and 72 ° C. for 3 minutes, further reaction was performed at 72 ° C. for 7 minutes. As template, 1 ng of plasmid pUC119-WM16 was used. After completion of the reaction, the solution was extracted with chloroform and precipitated with ethanol, and then dissolved in 30 μl of Y-100 buffer, and
A unit of StuI and 20 units of Asp718 were added, and a digestion reaction was carried out at 37 ° C. for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 1.0 kb was recovered.

In addition, 2 μg of pAMoPRSA was added to Y-1.
Dissolve in 30 μl of 00 buffer and add 20 units of StuI and 20 units of
Asp718 was added, and a digestion reaction was carried out at 37 ° C for 2 hours. After the reaction solution was subjected to agarose gel electrophoresis, a DNA fragment of about 9.06 kb was recovered. PCR-amplified DNA-derived StuI-Asp718 fragment (1.0 kb) 0.1 μg and pAMoPR
0.1 μg of StuI-Asp718 fragment (9.06 kb) derived from SA to T4
It was dissolved in 30 μl of ligase buffer, 175 units of T4 DNA ligase was added, and the binding reaction was performed at 12 ° C. for 16 hours. Escherichia coli HB101 strain was transformed with the reaction solution 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 pAMoPRSAW16, and its structure was confirmed by digestion with restriction enzymes.

2. Secretory production of α2,3-sialyltransferase using Namalwa KJM-1 cells as hosts pAMoPRSA (secretion expression vector; control) and pAMoPRSAW1 obtained above
> Plasmid <maxi kit (product number 41031), a plasmid preparation kit manufactured by Qiagen for 6 (α2,3-sialyltransferase secretory expression plasmid)
Was prepared using. The obtained plasmid was precipitated with ethanol and then dissolved in TE buffer at 1 μg / μl.
Then, both plasmids were electroporated [Miyaji et al .: Cytotechnology , 3,
133 (1990)], and introduced into the Namalba KJM-1 strain. After introducing 4 μg of plasmid per 1.6 × 10 ⁶ cells, the cells were suspended in 8 ml of RPMI1640.ITPSGF medium and cultured at 37 ° C. for 24 hours in a carbon dioxide gas incubator. Then, G418 (manufactured by Gibco) was added to 0.5 mg / ml and the cells were cultured for 7 days. After that, 22 ml of RPMI
1640 ITPSGF medium (containing 0.5 mg / ml G418)
Was added and the cells were further cultured for 5 days to obtain a transformant. The obtained transformants were RPM containing 0.5 mg / ml of G418, respectively.
5 × 10 ⁴ cells / ml in 30 ml of I1640 / ITPSGF medium
Suspend to 37 ° C in a carbon dioxide incubator
The cells were cultured for 8 days. After that, the cells are removed by centrifugation (160 × g, 10 minutes), the supernatant is recovered, and centrifugation (15
(00 × g, 10 minutes), the supernatant was collected. The culture supernatant thus obtained was stored at -80 ° C until use.

Since the protein encoded by the plasmid pAMoPRSAW16 is secreted and expressed as a fusion protein with the binding region of protein A with IgG, IgG
It can be easily purified using Sepharose. Therefore, 100 μl of IgG sepharose [Pharmacia] pretreated according to the attached instructions was added to the culture supernatant obtained as above so that sodium azide was added to a final concentration of 0.1%.
Add and stir gently at 4 ° C. overnight. After that, IgG sepharose was recovered by centrifugation (160 × g, 10 minutes), and washed 3 times with 1 ml of a buffer solution containing 50 mM Tris-hydrochloric acid (pH 7.6), 150 mM sodium chloride, and 0.05% Tween20.
Then 0.5 M acetate buffer (pH 3 with ammonium acetate.
The protein adsorbed on IgG sepharose was eluted with 100 μl (adjusted to 4), and the IgG sepharose was removed by centrifugation (160 × g, 10 minutes). The pH was adjusted to 7. by adding 2M Tris-HCl (pH 8.0) to this eluate.
After adjusting to 0, water was added to bring the final volume to 1 ml. Sialyltransferase activity was measured using 5 μl of the eluate thus prepared. 30 μl of assay solution [0.1 M cacodylic acid-hydrochloric acid (pH 6.5), 0.01
M manganese chloride, 0.45% Triton X-100, 0.1 mM substrate,
IgG above Sepharose (5 μl), 5 mM CMP-sialic acid
(With or without addition)] at 37 ° C. for 30 minutes, and then the product was identified by high performance liquid chromatography (HPLC). Lacto-N as a substrate
-Neo-lactose (Lacto-N-neotetraose; LNnT), lacto-N-lactose (Lacto-N-tetraose; LNT) (both are manufactured by Oxford Glycosystems and their structures are as shown in Table 2). Was fluorescently labeled with aminopyridine. The fluorescent labeling of the substrate is carried out by a conventional method [Akihiro Kondo et al .: Agricultural and Biological Chemistry (Agric.
hem.), 54 , 2169 (1990)]. For each IgG sepharose, after reacting with an assay solution containing CMP-sialic acid (sugar donor) and an assay solution not containing CMP-sialic acid, analyzed by HPLC to generate a peak that appears only in the assay solution containing CMP-sialic acid. It was a thing. The assay solution after the reaction was treated at 100 ° C. for 5 minutes and then centrifuged (10,000 × g, 10 minutes), and 10 μl of the supernatant was subjected to HPLC. HPLC is TSKgel ODS
Elution was carried out using 0.02M ammonium acetate buffer (pH 4.0) under the conditions of an elution temperature of 50 ° C. and a flow rate of 1 ml / min using a 80TM column (4.6 mm × 30 cm; manufactured by Tosoh Corporation). Fluorescence detector (Fluorescence detector manufactured by Shimadzu Corporation)
HPLC Monitor RF-535T) (excitation wavelength 320
nm, emission wavelength 400 nm). The product was identified by matching the elution time with the standard and by regenerating the substrate by sialidase treatment of the product. The product was quantified by comparing the fluorescence intensities using lactose similarly aminopyridylated as a standard. Table 2 shows the amount of the product and the relative activity of the α2,3-sialyltransferase of the present invention to LNnT when the activity to LNT is 100. In addition, α2,3-, which has been reported to be purified from rats so far,
100 sialyltransferase activity against LTN
Relative activity to LNnT (Weinstein (W
Einstein) et al .: Journal of Biological Chemistry (J. Biol. Chem.) , 257 , 13845 (1982)] is also shown in Table 2.

[0150]

[Table 2]

When IgG sepharose derived from the culture supernatant of Namalwa cells into which pAMoPRSAW16 had been introduced was used, α2,3-sialyltransferase activity was detected regardless of which sugar chain was used as the substrate. On the other hand, when IgG sepharose derived from the culture supernatant of Namalwa cells into which the vector pAMoPRSA was introduced was used, no activity was detected using any sugar chain as a substrate.

From the above results, the α2,3-sialyltransferase of the present invention is secreted and produced in the culture supernatant as a fusion protein with the binding region of Staphylococcus aureus protein A to IgG, and its secretory product. Was easily recovered and purified using IgG sepharose.

Since the α2,3-sialyltransferase of the present invention can utilize not only lacto-N-lactose but also lacto-N-neolactose as a substrate, not only Sialyl Lewis a sugar chain in vitro but also
It was shown that Sialyl Lewis-x sugar chains can also be synthesized. Furthermore, using the α2,3-sialyltransferase obtained this time, the sugar chain terminal structure was changed to NeuAc α2-3Galβ1-3GlcNAc.
Alternatively, after converting to NeuAc α2-3Galβ1-4GlcNAc, α
1,4-fucosyltransferas
e) or α1,3-fucosyltransferase (fucos
Sylyl Lewis a (Sialyl Le
wis a) Sugar chain or Sialyl Lewis-
x) Can synthesize sugar chains.

[0154]

INDUSTRIAL APPLICABILITY The present invention provides a novel α2,3-sialyltransferase useful for producing sugar chains having useful physiological activities such as sialyl Lewis a and sialyl Lewis x and modified products thereof.

[0155]

[Sequence list]

 SEQ ID NO: 1 Sequence Length: 2232 Sequence Type: Nucleic Acid Number of Strands: Double Strand Sequence Type: cDNA to mRNA Origin: Organism Name: Human Strain Name: WM266-4 Cell Cell Type: Melanoma Sequence: CGCGTTGTGG GCTCCCGCCG GGGTCCCCCG CGGCTGTCGC CGCCGCCTAC GCCGCTGCCT 60 CCGCCTTCCT GCCCCGCGTC GGGCCGGGCG CCACCTCCC CCTGCCTCCC TCTCCGCTGT 120 GGTCATTTAG GAAATCGTAA ATCATGTGAC CTC GTC CTC GTC CTC GTC CTC TTG GTA TTT GTG 172 Leu Met Gly 220 Arg Asn Leu Leu Leu Ala Leu Cys Leu Phe Leu Val Leu Gly Phe Leu 10 15 20 TAT TAT TCT GCG TGG AAG CTA CAC TTA CTC CAG TGG GAG GAG GAC TCC 268 Tyr Tyr Ser Ala Trp Lys Leu His Leu Leu Gln Trp Glu Glu Asp Ser 25 30 35 AAT TCA GTG GTT CTT TCC TTT GAC TCC GCT GGA CAA ACA CTA GGC TCA 316 Asn Ser Val Val Leu Ser Phe Asp Ser Ala Gly Gln Thr Leu Gly Ser 40 45 50 55 GAG TAT GAT CGG TTG GGC TTC CTC CTG AAT CTG GAC TCT AAA CTG CCT 364 Glu Tyr Asp Arg Leu Gly Phe Leu Leu Asn Leu Asp Ser Lys Leu Pro 60 65 70 GCT GAA TTA GCC ACC AAG TAC GCA AAC TTT TCA GAG GGA GCT TGC AAG 412 Ala Glu Leu Ala Thr Lys Tyr Ala Asn Phe Ser Glu Gly Ala Cys Lys 75 80 85 CCT GGC TAT GCT TCA GCC TTG ATG ACG GCC ATC TTC CCC CGG TTC TCC 460 Pro Gly Tyr Ala Ser Ala Leu Met Thr Ala Ile Phe Pro Arg Phe Ser 90 95 100 AAG CCA GCA CCC ATG TTC CTG GAT GAC TCC TTT CGC AAG TGG GCT AGA 508 Lys Pro Ala Pro Met Phe Leu Asp Asp Ser Phe Arg Lys Trp Ala Arg 105 110 115 ATC CGG GAG TTC GTG CCG CCT TTT GGG ATC AAA GGT CAA GAC AAT CTG 556 Ile Arg Glu Phe Val Pro Pro Phe Gly Ile Lys Gly Gln Asp Asn Leu 120 125 130 135 ATC AAA GCC ATC TTG TCA GTC ACC AAA GAG TAC CGC CTG ACC CCT GCC 604 Ile Lys Ala Ile Leu Ser Val Thr Lys Glu Tyr Arg Leu Thr Pro Ala 140 145 150 TTG GAC AGC CTC CGC TGC CGC CGC TGC ATC ATC GTG GGC AAT GGA GGC 652 Leu Asp Ser Leu Arg Cys Arg Arg Cys Ile Ile Val Gly Asn Gly Gly 155 160 165 GTT CTT GCC AAC AAG TCT CTG GGG TCA CGA ATT GAC GAC TAT GAC ATT 700 Val Leu Ala Asn Lys Ser Leu Gly Ser Arg Ile Asp Asp Tyr Asp Ile 170 175 180 GTG GTG AGA CTG AAT TCA GCA CCA GTG AAA GGC TTT GAG AAG GAC GTG 748 Val Val Arg Leu Asn Ser Ala Pro Val Lys Gly Phe Glu Lys Asp Val 185 190 195 GGC AGC AAA ACG ACA CTG CGC ATC ACC TAC CCC GAG GGC GCC ATG CAG 796 Gly Ser Lys Thr Thr Leu Arg Ile Thr Tyr Pro Glu Gly Ala Met Gln 200 205 210 215 CGG CCT GAG CAG TAC GAG CGC GAT TCT CTC TTT GTC CTC GCC GGC TTC 844 Arg Pro Glu Gln Tyr Glu Arg Asp Ser Leu Phe Val Leu Ala Gly Phe 220 225 230 AAG TGG CAG GAC TTT AAG TGG TTG AAA TAC ATC GTC TAC AAG GAG AGA 892 Lys Trp Gln Asp Phe Lys Trp Leu Lys Tyr Ile Val Tyr Lys Glu Arg 235 240 245 GTG AGT GCA TCG GAT GGC TTC TGG AAA TCT GTG GCC ACT CGA GTG CCC 940 Val Ser Ala Ser Asp Gly Phe Trp Lys Ser Val Ala Thr Arg Val Pro 250 255 260 AAG GAG CCC CCT GAG ATT CGA ATC CTC AAC CCA TAT TTC ATC CAG GAG 988 Lys Glu Pro Pro Glu Ile Arg Ile Leu Asn Pro Tyr Phe Ile Gln Glu 265 270 275 GCC GCC TTC ACC CTC ATT GGC CTG CCC TTC AAC AAT GGC CTC ATG GGC 1036 Ala Ala Phe Thr Leu Ile Gly Leu Pro Phe Asn Asn Gly Leu Met Gly 280 285 290 295 CGG GGG AAC ATC CCT ACC CTT GGC AGT GTG GCA GTG ACC ATG GCA CTA 1084 Arg Gly Asn Ile Pro Thr Leu Gly Ser Val Ala Val Thr Met Ala Leu 300 305 310 CAC GGC TGT GAC GAG GTG GCA GTC GCA GGA TTT GGC TAT GAC ATG AGC 1132 His Gly Cys Asp Glu Val Ala Val Ala Gly Phe Gly Tyr Asp Met Ser 315 320 325 ACA CCC AAC GCA CCC CTG CAC TAC TAT GAG ACC GTT CGC ATG GCA GCC 1180 Thr Pro Asn Ala Pro Leu His Tyr Tyr Glu Thr Val Arg Met Ala Ala 330 335 340 ATC AAA GAG TCC TGG ACG CAC AAT ATC CAG CGA GAG AAA GAG TTT CTG 1228 Ile Lys Glu Ser Trp Thr His Asn Ile Gln Arg Glu Lys Glu Phe Leu 345 350 355 CGG AAG CTG GTG AAA GCT CGC GTC ATC ACT GAT CTA AGC AGT GGC ATC 1276 Arg Lys Leu Val Lys Ala Arg Val Ile Thr Asp Leu Ser Ser Gly Ile 360 365 370 375 TER CCAGCACCAC CTACACAGGA GTCTTCAGACCCAGAGAA 1389 GGCAGCAAGG CCTTGGTGGA GCAGCCAGAG CTGTGCCTGC TCAGCAGCCA GTCTCAGAGA 1449 CCAGCACTCA GCCTCATTCA GCATGGGTCC TTGATGCCAG AGGGCCAGCA GGCTCCTGGC 1509 TGTG CCCAGC AGGCCCAGCA TGCAGGTGGT GGGACACTGG GCAGCAAGGC TGCTGCCGGA 1569 ATCACTTCTC CAATCAGTGT TTGGTGTATT ATCATTTTGT GAATTTGGGT AGGGGGGAGG 1629 GTAGGGATAA TTTATTTTTA AATAAGGTTG GAGATGTCAA GTTGGGTTCA CTTGCCATGC 1689 AGGAAGAGGC CCACTAGAGG GCCCATCAGG CAGTGTTACC TGTTAGCTCC CTGTGGGGCA 1749 GGAGTGCCAG GACCAGCCTG TACCTTGCTG TGGGGCTACA GGATGGTGGG CAGGATCTCA 1809 AGCCAGCCCC CTCCAGCTCA TGACACTGTT TGGCCTTTCT TGGGGAGAAG GCGGGGTATT 1869 CCCACTCACC AGCCCTAGCT GTCCCATGGG GAAACCCTGG AGCCATCCCT TCGGAGCCAA 1929 CAAGACCGCC CCAGGGCTAT AGCAGAAAGA ACTTTAAAGC TCAGGAGGGT GACGCCCAGC 1989 TCCGCCTGCT GGGAAGAGCT CCCCTCCACA GCTGCAGCTG ATCCATAGGA CTACCGCAGG 2049 CCCGGACTCA CCAACTTGCC ACATGTTCTA GGTTTCAGCA ACAAGACTGC CAGGTGGTTG 2109 GGTTCTGCCT TTAGCCTGGA CCAAAGGGAA GTGAGGCCCA AGGAGCTTAC CCAAGCTGTG 2169 GCAGCCGTCC CAGGCCACCC CCATGGAAGC AATAAAGCTC TTCCCTGTAA AAAAAAAAAA 2229 AAA 2232

SEQ ID NO: 2 Sequence Length: 375 Sequence Type: Amino Acid Number of Chains: Linear Sequence Type: Protein Origin: Organism Name: Human Strain Name: WM266-4 Cell Cell Type: Melanoma Sequence: Met Gly Leu Leu Val Phe Val Arg Asn Leu Leu Leu Ala Leu Cys 1 5 10 15 Leu Phe Leu Val Leu Gly Phe Leu Tyr Tyr Ser Ala Trp Lys Leu 20 25 30 His Leu Leu Gln Trp Glu Glu Asp Ser Asn Ser Val Val Leu Ser 35 40 45 Phe Asp Ser Ala Gly Gln Thr Leu Gly Ser Glu Tyr Asp Arg Leu 50 55 60 Gly Phe Leu Leu Asn Leu Asp Ser Lys Leu Pro Ala Glu Leu Ala 65 70 75 Thr Lys Tyr Ala Asn Phe Ser Glu Gly Ala Cys Lys Pro Gly Tyr 80 85 90 Ala Ser Ala Leu Met Thr Ala Ile Phe Pro Arg Phe Ser Lys Pro 95 100 105 Ala Pro Met Phe Leu Asp Asp Ser Phe Arg Lys Trp Ala Arg Ile 110 115 120 Arg Glu Phe Val Pro Pro Phe Gly Ile Lys Gly Gln Asp Asn Leu 125 130 135 Ile Lys Ala Ile Leu Ser Val Thr Lys Glu Tyr Arg Leu Thr Pro 140 145 150 Ala Leu Asp Ser Leu Arg Cys Arg Arg Cys Ile Ile Va l Gly Asn 155 160 165 Gly Gly Val Leu Ala Asn Lys Ser Leu Gly Ser Arg Ile Asp Asp 170 175 180 Tyr Asp Ile Val Val Arg Leu Asn Ser Ala Pro Val Lys Gly Phe 185 190 195 Glu Lys Asp Val Gly Ser Lys Thr Thr Leu Arg Ile Thr Tyr Pro 200 205 210 Glu Gly Ala Met Gln Arg Pro Glu Gln Tyr Glu Arg Asp Ser Leu 215 220 225 Phe Val Leu Ala Gly Phe Lys Trp Gln Asp Phe Lys Trp Leu Lys 230 235 240 Tyr Ile Val Tyr Lys Glu Arg Val Ser Ala Ser Asp Gly Phe Trp 245 250 255 Lys Ser Val Ala Thr Arg Val Pro Lys Glu Pro Pro Glu Ile Arg 260 265 270 Ile Leu Asn Pro Tyr Phe Ile Gln Glu Ala Ala Phe Thr Leu Ile 275 280 285 Gly Leu Pro Phe Asn Asn Gly Leu Met Gly Arg Gly Asn Ile Pro 290 295 300 Thr Leu Gly Ser Val Ala Val Thr Met Ala Leu His Gly Cys Asp 305 310 315 Glu Val Ala Val Ala Gly Phe Gly Tyr Asp Met Ser Thr Pro Asn 320 325 330 Ala Pro Leu His Tyr Tyr Glu Thr Val Arg Met Ala Ala Ile Lys 335 340 345 Glu Ser Trp Thr His Asn Ile Gln Arg Glu Lys Glu Phe Leu Arg 350 355 360 Lys Leu Val Lys Ala Arg Val Ile Thr Asp Le u Ser Ser Gly Ile 365 370 375

SEQ ID NO: 3 Sequence Length: 52 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TCGACAAGCT TGATATCGGC CTGTGAGGCC TCACTGGCCG CGGCCGCGGT AC 52

SEQ ID NO: 4 Sequence Length: 44 Sequence Type: Nucleic Acid Number of Strands: Duplex Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CGCGGCCGCG GCCAGTGAGG CCTCACAGGC CGATATCAAG CTTG 44

SEQ ID NO: 5 Sequence Length: 11 Sequence Type: Nucleic Acid Number of Strands: Duplex Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CTTTAGACA A 11

SEQ ID NO: 6 Sequence Length: 38 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CTCTGTAGGC CTTACTCCAG TGGGAGGAGG ACTCCAAT 38

SEQ ID NO: 7 Sequence Length: 37 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GACTCAGGTA CCACTCAGAT GCCACTGCTT AGATCAG 37

[Brief description of drawings]

FIG. 1 is a diagram showing a construction process of a plasmid pAGEL106.

FIG. 2 is a diagram showing a construction step of plasmid pASLB3-3-1.

FIG. 3 is a diagram showing a construction process of a plasmid pASLB3-3.

FIG. 4 is a diagram showing a construction process of plasmid pASLBE3-3.

FIG. 5 is a diagram showing a construction process of a plasmid pASLBC.

FIG. 6 is a diagram showing a construction process of a plasmid pASLBEC.

FIG. 7 is a diagram showing a construction process of a plasmid pASLBEC2.

FIG. 8 is a diagram showing a construction process of a plasmid pAMoEC2.

FIG. 9 is a diagram showing a construction process of a plasmid pAMoEC3.

FIG. 10 is a diagram showing a construction process of plasmid pAMoERC3.

FIG. 11 is a diagram showing a construction step of plasmid pAGE207.

FIG. 12 is a diagram showing a construction process of a plasmid pAGE207ScN.

FIG. 13 is a diagram showing a construction process of a plasmid pAMoC3Sc.

FIG. 14 is a diagram showing a construction process of a plasmid pAMoERC3Sc.

FIG. 15 is a diagram showing a construction process of a plasmid pAMoPRC3Sc.

FIG. 16 is a diagram showing the construction steps of plasmid pUC119-WM16.

FIG. 17 is a diagram showing the results of sialyltransferase activity measurement when LNnT was used as a substrate. a,
b is KJM-1 into which pAMoPRC3Sc (control) was introduced
C and d of the strain cells are the sialyltransferase activity of the KJM-1 strain cells into which pAMoPRWM16 was introduced.
It is an HPLC pattern. a and c are CM when using an assay solution that does not contain CMP-sialic acid as a sugar donor, and b and d are CM
It is an HPLC pattern when an assay solution containing P-sialic acid is used.

FIG. 18 is a diagram showing the results of sialyltransferase activity measurement using LNT as a substrate. a,
b is KJM-1 into which pAMoPRC3Sc (control) was introduced
C and d of the cell lines are the sialyltransferase activity of the KJM-1 cell line into which pAMoPRWM16 was introduced.
It is an HPLC pattern. a and c are CM when using an assay solution that does not contain CMP-sialic acid as a sugar donor, and b and d are CM
It is an HPLC pattern when an assay solution containing P-sialic acid is used.

FIG. 19 shows an EPICS Elite Flow Cytom after staining with indirect fluorescent antibody.
eter; manufactured by COULTER)] is a diagram showing the results of analysis. “A” shows the result of indirect fluorescent antibody staining using CSLEX1 for the KJM-1 strain into which pAMoPRC3Sc (control plasmid) or pAMoPRWM16 was introduced. b shows the result of indirect fluorescent antibody staining using KM93 for the KJM-1 strain into which pAMoPRC3Sc or pAMoPRWM16 was introduced. In both cases, pAMoPRC3
Regarding the KJM-1 strain into which Sc has been introduced, the results of indirect fluorescent antibody staining using normal mouse serum are shown as a control.

FIG. 20 is a diagram showing a construction step of plasmid pAGE147.

FIG. 21 is a diagram showing a construction step of plasmid pAGE247.

FIG. 22 is a diagram showing a construction process of a plasmid pAMN6hyg.

FIG. 23 is a diagram showing a construction process of a plasmid pAMoERSA.

FIG. 24 is a diagram showing a construction step of plasmid pAMoPRSA.

FIG. 25 is a diagram showing a construction step of plasmid pAMoPRSAW16.

[Explanation of symbols]

dhfr: dihydrofolate reductase gene hG-CSF: human granulocyte colony stimulating factor gene bp: base pairs kb: kilobase pairs G418 / Km: transposon 5 (Tn5) -derived G418, kanamycin resistance gene hyg: Hygromycin resistance gene Ap: pBR322-derived ampicillin resistance gene Tc: pBR322-derived tetracycline resistance gene P1: pBR322-derived P1 promoter Ptk: Herpes simplex virus (Herpes)
simplex virus; HSV) Thymidine kinase (tk) gene promoter Sp. βG: rabbit β globin gene splicing signal A. βG: rabbit β globin gene poly A addition signal A. SE: simian virus 40 (SV)
40) Early gene poly A addition signal Atk: Herpes simplex virus (Herpes
simplex virus; HSV) Poly-A addition signal Pse of thymidine kinase (tk) gene: simian virus 40 (SV4
0) Early gene promoter Pmo: Moloney murine leukemia virus long terminal repeat (LTR) promoter HTLV-1: human T-cell leukemia virus
ukemia virus type-1: HTLV-1) gene EBNA-1: Epstein Barr virus (Epstei)
n-Barr virus) EBNA-1 gene oriP: Epstein-Barr virus
-Barr virus) origin of replication ori: Origin of replication of pUC119 lac'Z: Part of β-galactosidase gene of Escherichia coli IG: M13 Intergenic region of phage DNA (int
ergenic region) G-CSF der .: Human granulocyte colony stimulating factor derivative gene S: Gene part A or ProA encoding the signal peptide of human granulocyte colony stimulating factor: Staphylococcus au
Gene part encoding reus protein A IgG binding region WM16: α2,3-sialyltransferase gene (full length or active region gene) obtained from WM266-4 cells

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12N 15/54 C12P 19/26 7432-4B 21/00 A 8214-4B C12Q 1/68 Z 7823- 4B // (C12N 9/10 C12R 1:91) (C12N 1/21 C12R 1:19) (C12N 5/10 C12R 1:91) (C12N 15/54 C12R 1:91) (72) Inventor Tatsuya Nishi 3-9-13 Nakamachi, Machida-shi, Tokyo

Claims (23)

[Claims] 1. An α containing the amino acid sequence of SEQ ID NO: 2.
2,3-sialyltransferase. 2. A cDNA encoding the α2,3-sialyltransferase according to claim 1, or a DNA having homology with the cDNA. 3. A DN containing the nucleotide sequence set forth in SEQ ID NO: 1.
A. 4. A recombinant vector incorporating the cDNA encoding the α2,3-sialyltransferase according to claim 1. 5. A DNA containing the nucleotide sequence of SEQ ID NO: 1.
A recombinant vector in which is incorporated. 6. A cDNA library is constructed by incorporating a cDNA synthesized using an mRNA extracted from an animal cell as a template into an expression cloning vector, and the cDNA library is constructed.
The NA library is introduced into cells, the obtained cells are cultured in the presence of a lectin having an activity of suppressing the growth of the cells, the proliferating cells are isolated, and the α2,3-sialyltransferase is encoded from the cells. 4. The DNA according to claim 2 or 3, characterized in that
Manufacturing method. 7. A cDNA library is constructed by incorporating a cDNA synthesized using an mRNA extracted from an animal cell as a template into an expression cloning vector, and
The DNA library is introduced into cells, and the resulting cells are
The cells are cultured in the presence of a lectin having an activity of suppressing the growth of the cells, cells that grow are selected, and α2,3-
The method for producing a recombinant vector according to claim 4 or 5, wherein cDNA encoding sialyltransferase is isolated and the cDNA is introduced downstream of the promoter in the vector. 8. A cell carrying the recombinant vector according to claim 4 or 5 is cultured in a medium to produce and accumulate α2,3-sialyltransferase in the culture, and α2,3-sialyl is produced from the culture. The method for producing α2,3-sialyltransferase according to claim 1, wherein the transferase is collected. 9. The method for producing DNA according to claim 6, wherein the animal cell is human melanoma WM266-4 cell. 10. The animal cell is a human melanoma WM266-4.
The method for producing a recombinant vector according to claim 7, which is a cell. 11. The method for producing DNA according to claim 6, wherein the lectin is Himaimae lectin 120. 12. The method for producing a recombinant vector according to claim 7, wherein the lectin is Lima bean lectin 120. 13. The plasmid pUC119-WM16. 14. A cell containing the recombinant vector according to claim 4 or 5. 15. A method of imparting sialic acid to the non-reducing end of the lactosamine structure of a glycoprotein sugar chain or glycolipid by α2 → 3 bond using the cell according to claim 14. 16. The sialyl Lewis a (Sial) on the sugar chain of a glycoprotein or glycolipid using the cell according to claim 14.
yl-Lewis a) structure or Sialyl-Lew
is x) How to introduce the structure. 17. A method for imparting sialic acid to the non-reducing end of the lactosamine structure of a glycoprotein sugar chain or glycolipid by an α2 → 3 bond using the α2,3-sialyltransferase according to claim 1. 18. A sialyl Lewis a (Sialyl-Lewis a) structure or a sialyl Lewis x (Sialyl-Lewis x) structure is introduced onto the sugar chain of a glycoprotein or glycolipid by using the α2,3-sialyltransferase according to claim 1. how to. 19. The α according to claim 1, which is obtained by the hybridization method using the DNA according to claim 2 or 3.
A method for detecting 2,3-sialyltransferase. 20. The α according to claim 1, which is obtained by the polymerase chain reaction method using an oligonucleotide based on the nucleotide sequence of the DNA according to claim 2 or 3.
A method for detecting 2,3-sialyltransferase. 21. A method for suppressing the production of α2,3-sialyltransferase according to claim 1, which uses an oligonucleotide containing a part or all of the nucleotide sequence of the DNA according to claim 2 or 3. 22. Escherichia coli containing the recombinant vector according to claim 4 or 5. 23. Escherichia coli HB101 / pUC119-WM16
(FERM BP-4012).