WO2006082851A1 - Mucine associee a l’epiglycanine - Google Patents

Mucine associee a l’epiglycanine Download PDF

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Publication number
WO2006082851A1
WO2006082851A1 PCT/JP2006/301666 JP2006301666W WO2006082851A1 WO 2006082851 A1 WO2006082851 A1 WO 2006082851A1 JP 2006301666 W JP2006301666 W JP 2006301666W WO 2006082851 A1 WO2006082851 A1 WO 2006082851A1
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Prior art keywords
xaa
seq
independently selected
mucin
threonine
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PCT/JP2006/301666
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English (en)
Japanese (ja)
Inventor
Tatsuro Irimura
Kaori Denda
Mika Kamata
Atsuhiro Iguchi
Yuichi Itoh
Toshihisa Ogawa
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The University Of Tokyo
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Priority to JP2007501592A priority Critical patent/JPWO2006082851A1/ja
Publication of WO2006082851A1 publication Critical patent/WO2006082851A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4727—Mucins, e.g. human intestinal mucin

Definitions

  • the present invention relates to a novel mucin-like glycoprotein.
  • the present invention also provides a nucleic acid encoding the glycoprotein, a vector containing the nucleic acid, a host cell having the vector, a method for producing the glycoprotein using the host cell, an antibody against the glycoprotein, and the sugar
  • the present invention relates to a method for detecting expression of a gene encoding a protein and a kit for use in the method.
  • Mucins have traditionally been defined as highly o-glycosylated glycoproteins that are also secreted or expressed there.
  • recent results of cDNA cloning of the mucin core polypeptide have revealed the existence of a mucin family consisting of 19 members in humans.
  • These mucins are generally rich in serine, threonine and proline amino acid residues, and are also known to contain highly O-glycosylated tandem repeat regions.
  • the number of repeating units and the amino acid composition of the unit diversity among its members has been reported, and the unique biological functions of various mucins that may be attributed to such diversity are today's It offers extremely interesting research subjects and possible pharmaceutical uses in the biochemical field.
  • MUC1 a human mucin
  • the mucin is not only regarded as one of the targets of immunotherapy for tumors, but the patient serum level of the mucin is one of the most widely used clinical indicators as a diagnostic marker for breast cancer .
  • the level of mucin in the serum is the subject of examination as an indicator of ovarian tumors.
  • interesting and insights regarding other mucins include the fact that MUC2 knockout mice formed spontaneous tumors in several extinct organs. And another whip MUC6 is mainly expressed in the stomach! And is thought to confer protection against Helicobacter pylori infection.
  • the cytoplasmic regions of MUC1 and MUC4 have been found to be involved in the regulation of cell sorting and proliferation.
  • epiglycanin a mucin-like glycoprotein called epiglycanin is present in mice.
  • This protein has been observed to be present on the surface of the mouse ascites breast cancer cell line TA3-Ha cells by observing the image of fixed osmium fixed cells by transmission electron microscopy. It has been said that it can be observed as a filament protruding outward from 200 to 300 nm, and sometimes 400 nm, but there is no report that the protein was isolated. That is, as described in Non-Patent Document 1, the protein is composed of a single-chain polypeptide having a total molecular weight of about 500,000 and a force of about 1300 amino acid residues. It has been estimated that more than 500 sugar chains are attached to the chain.
  • Non-patent document 2 also shows that a glycoprotein called epiglycanin is composed of a single-chain polypeptide having a molecular weight of about 500,000 and a force of about 1300 amino acid residues, and more than 500 The glycan binding mode is determined based on the O-group between 2-acetamido 2-deoxygalatatose residue and serine or threonine residue.
  • Non-Patent Document 3 shows that a glycoprotein called TA3-MM epiglycanin from another TA3 breast cancer cell line has a molecular weight of about 500,000, and is 2.5 x 450-500 nm extended by electron microscopy. Again, the glycoprotein was obtained as a crudely purified fraction eluted near the void volume when applied to a Bio-Gel TM P-100 column or the like. And the isolation and accuracy of the shrimp glicanin It is important to disclose the physical and physical properties.
  • Non-Patent Document 4 describes the above TA3-Ha cells that can kill syngeneic mice within about 17 days with only 10 cells. Describes the protection of transplanted mice. In this document, approximately 90% long-term survival was observed by immunization with epiglycanin with prior treatment with cyclophosphamide.
  • a related document (Non-patent Document 5) is that mucins derived from deacylated sheep mandible containing a large amount of the Tn antigen, focusing on the fact that epiglycanin has a large number of Tn epitopes (GalNAc—a—SerZ Thr). Describes protection of ⁇ 3-Ha cell power.
  • Non-Patent Document 6 describes the use of Tn antigen as a tumor marker (see Non-Patent Document 7 for specific expression of Tn and T antigen in TA3-Ha cells). ).
  • epiglycanin explains the allograftability of tissues through its association with histocompatibility antigens, or provides an indication of the presence and malignancy of a tumor and protects the tumor. Has been known to provide.
  • Non-Patent Document 1 Van den Eijnden, DH, Evans, NA, Codington, JF, Reinhold, V., Silber, C., and Jeanloz, RW (1979) Biol. Chem., Vol. 254, No. 23, p. 12153— 12159
  • Non-Patent Document 2 Codington, JF, Linsley, KB, Jeanloz, RW, Irimura, T., and Osawa, T. (1975) Carbohydr. Res., Vol. 40, p.
  • Non-Patent Document 3 Codington , JF, Cooper, AG, Douglas, KM, Slayter, HS, Brown, MC, Silber, C., and Jeanloz, RW (1979) JNCI, Vol. 63, No. 1, p. 153- 161
  • Non-Patent Document 4 Fung, P.Y.S., Madej, M., Koganty, R.R., and Longen ecker, M. (1990) Cancer Res., Vol. 50, p. 4308-4314
  • Non-Patent Document 5 Singhal, A., Fohn, M., and Hakomori, S. (1991) Cancer
  • Non-Patent Document 6 Springer, GF, Taylor, CR, Howard, DR, Tegtmeyer, H., Desai, PR, Murthy, SM, Felder, B., and Scanlon, EF (1 985) Cancer, Vol. 55, No . 3, p. 561— 569
  • Non-Patent Document 7 Springer, G. F. (1984) Science, Vol. 224, p. 1198— 1206 Disclosure of the Invention
  • the present invention describes for the first time the isolation of mucin-like glycoproteins related to epiglycanin and nucleic acids encoding the proteins.
  • the present invention also reveals for the first time the existence of a human homologue of the mouse protein.
  • the mucin-like glycoprotein of the present invention obtained from the mouse ascites breast cancer cell line TA3-Ha cells contains about 2000 or more amino acid residues, and the core 'polypeptide chain alone is about 190 kDa. Gives the molecular weight.
  • amino acid sequence of the protein shows that it contains five domains, namely the N-terminal region containing the signal sequence; the serine Z threonine that provides the O-darikosili sputum site 12-15 amino acids
  • a tandem repeat region in which the amino acid sequence, which also has a residue force, is repeated about 100 times or more; a region containing a potential enzyme cleavage site; a transmembrane region that shows high homology with human homologues; and a cytoplasmic region force Therefore, it is estimated that the maximum molecular weight of the whole protein having a sugar chain added to the tandem repeat region exceeds about 100 kDa.
  • an epiglycan is defined as a molecule having a molecular weight of about 500,000, consisting of about 1300 amino acid residues, and having about 500 sugar chains bound thereto.
  • the epiglycanin-related mucin-like glycoprotein of the present invention is, in its broadest manner, an amino acid sequence represented by the following general formula (I):
  • N-terminal region selected from
  • B is a repeat unit of a tandem repeat region having an O-glycosylase site and is represented by the following SEQ ID NOs: 3 to 5:
  • Xaa is independently selected as a group force consisting of alanine, norin, glutamic acid, threonine, isoleucine, proline, serine, glycine and histidine,
  • Xaa is independently from the group consisting of serine, isoleucine, lysine, feruaranine and leucine.
  • Selected Xaa is independently selected as a group power consisting of serine, arginine and asparagine forces,
  • Xaa is independent from the group consisting of threonine, serine, isoleucine, lysine and arginine
  • Xaa is also selected independently of the group power of alanine, glycine, threonine and balinka,
  • Xaa is independently selected as a group force consisting of serine and threonine forces
  • Xaa is a group power consisting of glycine, arginine, aspartic acid, threonine, serine and alanine, an independently selected or unknown amino acid,
  • Xaa is independently selected as a group force consisting of serine, threonine and tyrosine forces
  • Xaa is threonine, methionine, serine, parin, alanine, asparagine, glutamine
  • Acid, lysine and isoleucine forces are group forces independently selected
  • Xaa is independently selected from the group power of proline, threonine, serine and leucine.
  • Xaa independently selects the group power, including threonine, serine, asparagine, and isoleucine.
  • Xaa is proline, leucine, threonine, tryptophan, arginine, glutamine and
  • Serinka is a group power independently selected
  • Is Xaa independently selected from the group consisting of threonine, proline and glutamic acid?
  • Xaa is independently selected or absent from the group power of threonine and serine
  • Xaa is independently selected from the group consisting of threonine, alanine, proline and asparagine
  • Xaa is independently selected from the group power consisting of arginine, methionine, and glycine power
  • Xaa is independently selected as a group power consisting of threonine and prolinker
  • Xaa is independently selected from the group power of isoleucine, lysine and threonka, Xaa is independently selected as a group force consisting of alanine and threonine forces,
  • Xaa is independently selected as a group force consisting of serine and feralanine forces
  • Xaa is independently selected as a group power consisting of alanine and balinka
  • Xaa is independently selected from the group consisting of serine, phenylalanine and leucine;
  • Xaa is independently selected from the group consisting of threonine and asparagine;
  • Xaa is independently selected as a group power consisting of proline and serine
  • Xaa is independently selected from the group consisting of threonine and alanine
  • Xaa is independently selected from the group consisting of serine and threonine, and
  • Xaa is independently selected from the group power of isoleucine and serine; or
  • Xaa is independently selected from the group power consisting of serine, ferrolanine and threonine,
  • Xaa is independently selected from the group consisting of serine, asparagine, histidine and arginine.
  • Xaa is independently selected from the group power of Threonine, Norin and Serinka,
  • Xaa is from threonine, valine, proline, alanine, isoleucine and asparagine
  • Xaa is independently selected as a group power consisting of serine and glutamate power
  • Xaa is independently selected as a group power consisting of serine, asparagine, threonine, and alanine.
  • Xaa is independently selected as a group force consisting of glycine and serine
  • Xaa is independently from the group consisting of alanine, isoleucine, valine, threonine and serine.
  • Xaa is independently selected as a group force consisting of serine, glycine and asparagine forces
  • Xaa is independently selected from the group consisting of threonine, isoleucine and alanine;
  • Xaa is independently selected as a group power consisting of alanine, norin, and asparagine,
  • 51 Xaa is threonine or absent
  • Xaa also has a group power consisting of asparagine, glycine, isoleucine and threonka independently.
  • Xaa is serine or absent
  • Xaa is independently selected from the group consisting of glutamic acid, glycine and aspartic acid.
  • n is an integer from 20 to 150
  • ALTGMHTTSHSASTAVSEAKPGGSLVPWE SEQ ID NO: 7
  • a potential enzyme cleavage site-containing region selected from
  • A is SEQ ID NO: 1, corresponding to the mucin-like glycoprotein derived from the epiglycanin-related mucin-like glycoprotein strength mouse of the present invention, and B Are selected independently, C is SEQ ID NO: 6, D is SEQ ID NO: 8, E is SEQ ID NO: 10, and n is 100 To be defined as being an integer from 1 to 50.
  • B can be selected independently from the amino acid sequence found in mice, ie SEQ ID NO: 12 to SEQ ID NO: 138.
  • ASSTASGSMSTPTTP (SEQ ID NO: 16)
  • VSSTGSGSTPTPTTT (SEQ ID NO: 20)
  • PSSTASGSTPTPTTP SEQ ID NO: 22
  • ASSTASSSSPTPTTP (SEQ ID NO: 23)
  • VSSTASGSTPTPTTT (SEQ ID NO: 24)
  • VSSTGSGSTPTLTTT (SEQ ID NO: 26)
  • VSSTVSDSTPTPTTT (SEQ ID NO: 27)
  • ASSTASGSAPTPTTT (SEQ ID NO: 28)
  • ASSTASGSMPTLTTN (SEQ ID NO: 29)
  • ASSTASGSSPTLTTT (SEQ ID NO: 30)
  • VSSTGSGSTPTLTTT (SEQ ID NO: 35)
  • ASRRGSGSTPTLTTT (SEQ ID NO: 36)
  • TSSTASRSTPTPTTT (SEQ ID NO: 124)
  • ASSTVSDSTPTPTTN (SEQ ID NO: 129)
  • AFSTASGSTPTLTTT (SEQ ID NO: 132)
  • RTSIAFASTSTPTTS (SEQ ID NO: 137)
  • GPSTASVSTLNSTSI (SEQ ID NO: 138)
  • a particular embodiment of the present invention relates to a mucin-like glycoprotein identified in the mouse ascites breast cancer cell line TA3-Ha cells, with the following amino acid sequence: MRRRSSLWCWLLL
  • NVVEMTRI (SEQ ID NO: 139) (in the above sequence, X represents an undefined amino acid).
  • the present invention further relates to a human homologue of a mouse 'epiglycanin-related mucin-like glycoprotein.
  • a human homologue of epiglycanin has even been suggested.
  • the inventors conducted a homology search by NCBI Blast based on the cDNA sequence of the mouse 'epiglycanin-related mucin-like glycoprotein revealed in the present invention. It was also clear that there was no human-derived EST or gene showing sex
  • the “theoretical” molecule has never been confirmed for its actual expression so far, but (i) the molecule is also upstream of the unique transmembrane region. The presence of a large tandem repeat region that is also rich in serine and threonine and thus provides a vast number of O-glycosyl sites, (ii) The gene is present in the MHC region of human chromosome 6, which corresponds to the region where the mouse's epiglycanin-related mucin-like glycoprotein of the present invention is located, (iii) The fact that this molecule was specifically expressed in a specific tumor tissue, etc., leads to the conclusion that the molecule is a human homologue of the mouse's epiglycanin-related mucin-like glycoprotein of the present invention. Seemed to be a strong basis.
  • a human-type epiglycanin-related mucin-like glycoprotein wherein the human-type epiglycanin-related mucin is represented by the sequence number: 2 in the above general formula (I).
  • B is SEQ ID NO: 5
  • C is SEQ ID NO: 7
  • D is SEQ ID NO: 9
  • E is SEQ ID NO: 11
  • n is an integer from 20 to 50 Can be defined.
  • B is found in human homologues and is rich in serine Z threonine.
  • amino acid sequence consisting of 15 amino acid residues ie SEQ ID NO: 140 to SEQ ID NO: 170, can be independently selected.
  • FHTTSSGISTATNSE (SEQ ID NO: 144)
  • SSTTSSGASTATNSE SEQ ID NO: 152
  • SSTTSSGASTATNSE SEQ ID NO: 153
  • the human 'epiglycanin-related mucin-like glycoprotein has the following amino acid sequence: MKMQKGNVLLMFGLLLHLEAATNSNETSTSANTGSSVI
  • GPGGNHGAPHRPRWSPNWFWRRPVSSIAMEMSGRNSGP SEQ ID NO: 171
  • its estimated maximum molecular weight can range up to 260 kDa, and contains a core 'polypeptide chain of at least about 57 kDa.
  • nucleic acid that encodes the above-described epiglycanin-related mucin-like glycoprotein Z and a nucleic acid complementary to the nucleic acid.
  • the nucleic acid can be used for recombinant production of the protein.
  • all or part of the sequence of the nucleic acid can be advantageously used for detection of the expression of the gene encoding the protein.
  • nucleic acid has an amino acid sequence represented by the following general formula (I)
  • N-terminal region selected from
  • B is a repeat unit of a tandem repeat region having an O-glycosyl cysteine site, and is represented by the following SEQ ID NOs: 3 to 5:
  • Xaa is independently selected as a group force consisting of alanine, norin, glutamic acid, threonine, isoleucine, proline, serine, glycine and histidine,
  • Xaa is independently from the group consisting of serine, isoleucine, lysine, feruaranine and leucine.
  • Xaa is independently selected as a group power consisting of serine, arginine and asparagine forces
  • Xaa is independent from the group consisting of threonine, serine, isoleucine, lysine and arginine Selected
  • Xaa is also selected independently of the group power of alanine, glycine, threonine and balinka,
  • Xaa is independently selected as a group force consisting of serine and threonine forces
  • Xaa is a group power consisting of glycine, arginine, aspartic acid, threonine, serine and alanine, an independently selected or unknown amino acid,
  • Xaa is independently selected as a group force consisting of serine, threonine and tyrosine forces
  • Xaa is threonine, methionine, serine, parin, alanine, asparagine, glutamine
  • Acid, lysine and isoleucine forces are group forces independently selected
  • Xaa is independently selected from the group power of proline, threonine, serine and leucine.
  • Xaa independently selects the group power, including threonine, serine, asparagine, and isoleucine.
  • Xaa is proline, leucine, threonine, tryptophan, arginine, glutamine and
  • Serinka is a group power independently selected
  • Is Xaa independently selected from the group consisting of threonine, proline and glutamic acid?
  • Xaa is independently selected or absent from the group power of threonine and serine
  • Xaa is independently selected from the group consisting of threonine, alanine, proline and asparagine
  • Xaa is independently selected from the group power consisting of arginine, methionine, and glycine power
  • Xaa is independently selected as a group power consisting of threonine and prolinker
  • Xaa is independently selected from the group power of isoleucine, lysine and threonka,
  • Xaa is independently selected as a group force consisting of alanine and threonine forces
  • Xaa is independently selected as a group force consisting of serine and feralanine forces
  • Xaa is independently selected as a group power consisting of alanine and balinka
  • Xaa is independently selected from the group consisting of serine, phenylalanine and leucine;
  • Xaa is independently selected as a group force consisting of threonine and asparagine forces
  • Xaa is independently selected as a group power consisting of threonine and alanine power
  • Xaa is independently selected as a group force consisting of serine and threonine forces
  • Xaa is independently selected from the group power of isoleucine and serine; or
  • Xaa is independently selected as a group power consisting of serine, ferrolanine and threonine power
  • Xaa is independently selected from the group consisting of serine, asparagine, histidine and arginine.
  • Xaa is independently selected from the group power of Threonine, Norin and Serinka,
  • Xaa is from threonine, valine, proline, alanine, isoleucine and asparagine
  • Xaa is independently selected as a group power consisting of serine and glutamate power
  • Xaa is independently selected as a group force consisting of serine, asparagine, threonine, and alanine.
  • Xaa is independently selected as a group force consisting of glycine and serine
  • Xaa is independently from the group consisting of alanine, isoleucine, valine, threonine and serine.
  • Xaa is independently selected as a group force consisting of serine, glycine and asparagine forces
  • Xaa is independently selected from the group consisting of threonine, isoleucine and alanine;
  • Xaa is independently selected as a group power consisting of alanine, norin, and asparagine,
  • Xaa is threonine or absent
  • Xaa is independently from the group consisting of asparagine, glycine, isoleucine and threonine. Selected or absent
  • Xaa is serine or absent
  • Xaa is independently selected from the group consisting of glutamic acid, glycine and aspartic acid.
  • n is an integer from 20 to 150
  • ALTGMHTTSHSASTAVSEAKPGGSLVPWE SEQ ID NO: 7
  • a potential enzyme cleavage site containing region selected by
  • A is SEQ ID NO: 1
  • B is SEQ ID NO: 3 and SEQ ID NO: 4
  • a mucin-like glycoprotein wherein C is SEQ ID NO: 6, D is SEQ ID NO: 8, E is SEQ ID NO: 10 and n is an integer from 100 to 150, or Further, there may be mentioned the group B consisting of SEQ ID NO: 12 to SEQ ID NO: 138, the ability to encode a mucin-like glycoprotein that is also independently selected, which is complementary to the nucleic acid. .
  • A is SEQ ID NO: 2
  • B is SEQ ID NO: 5
  • C is SEQ ID NO: 7 corresponding to a human-derived mucin-like glycoprotein.
  • a mucin-like glycoprotein wherein D is SEQ ID NO: 9, E is SEQ ID NO: 11 and n is an integer from 20 to 50, or further wherein B is SEQ ID NO: 140-sequence Mention may be made of those encoding mucin-like glycoproteins independently selected from the group consisting of the number: 170 or being complementary to the nucleic acid.
  • the nucleic acid corresponds to SEQ ID NO: 172 (mouse) or SEQ ID NO: 174 (human).
  • expression vectors containing the above nucleic acids preferably plasmids or viral vectors are also within the scope of the present invention, and recombinant host cells, preferably eukaryotic host cells carrying such vectors are also contemplated in the present invention.
  • the present invention includes an antibody specific for the above-mentioned epiglycanin-related mucin-like glycoprotein.
  • an antibody against human-derived epiglycanin-related mucin-like glycoprotein is interesting and preferably the antibody is specific to the extracellular domain of said epiglycanin-related mucin-like glycoprotein.
  • Such an antibody can be used as a kit for detecting the expression of the epiglycanin-related mucin-like glycoprotein of the present invention.
  • antibodies specific to the cytoplasmic region of human-derived epiglycanin-related mucin-like glycoprotein have also been shown to be suitably used for immunostaining of the protein-expressing cells and tissues.
  • the detection method comprises the whole or a part of the nucleotide sequence of the nucleic acid Z-complementary nucleic acid that codes for the epiglycanin-related glycoprotein, preferably a sequence comprising 20 consecutive nucleotides in the nucleic acid, Preferably, the sequence represented by SEQ ID NO: 176 is used as a probe or the like, but is not limited thereto. 5 '-CTTCCCATAGTG
  • nucleic acid is a kit for use in a method for detecting the expression of the above-described epiglycanin-related mucin-like glycoprotein gene which is also useful as a specific tumor marker. Can be included.
  • the epiglycanin-related mucin-like glycoprotein of the present invention is characterized by a consensus sequence represented by the general formula: A—Bn-C-D-E (I).
  • A is an N-terminal region containing a signal sequence
  • C is a region containing a potential enzyme cleavage site
  • D is a transmembrane region showing high homology between mouse and human
  • E is an intracytoplasmic region.
  • A is selected from SEQ ID NO: 1 (mouse type) or SEQ ID NO: 2 (human type)
  • C is selected from SEQ ID NO: 6 (mouse type) or SEQ ID NO: 7 (human type).
  • D can be selected from SEQ ID NO: 8 (mouse) or SEQ ID NO: 9 (human)
  • E can be selected from SEQ ID NO: 10 (mouse) or SEQ ID NO: 11 (human)
  • B is a force corresponding to a repeat unit in the tandem repeat region (Bn), which provides a number of O-glycosylation sites and is repeated at specific intervals.
  • the repeat unit may be defined by the minimum requirement of having serine or threonine in the sequence and including at least about 10 or more, preferably about 11 to 15 amino acid residues, and more specifically.
  • the point to be noted here is the configuration and repetition of the repeat unit in the region.
  • the pattern itself does not necessarily need to match the natural type epiglycanin-related mucin-like glycoprotein of the present invention.
  • a specific repeat unit is used preferentially, a specific repeat unit or a group of repeat units is replaced, or a specific This means that you can completely replace a repeat unit with another repeat unit, or you can delete a specific repeat unit. Therefore, the mucin-like glycoprotein of the present invention includes a sequence containing such a change, replacement, substitution, or deletion of a repeat unit.
  • the repeat number (n) of the repeat unit can be about 20 to about 150, preferably about 100 to about 150 (mouse type) or about 20 to about 50 (human type).
  • V preferably from about 120 to about 130 (mouse type), with a force of about 25 to about 35 (human type).
  • the present invention further includes functional derivatives of the above mucin-like glycoproteins.
  • the functional derivative is a compound having a biological activity (functional or structural) substantially similar to the biological activity of the epiglycanin-related mucin-like glycoprotein of the present invention. That is, the term “functional derivative” is intended to include any fragment, variant, analog and homologue, or chemical derivative having a sequence derived from the amino acid sequence of the above-mentioned epiglycanin-related mucin-like glycoprotein.
  • fragment is intended to refer to any polypeptide subset of an epiglycanin-related mucin-like glycoprotein.
  • variant is intended to refer to a molecule that is structurally, functionally and substantially similar to a complete epiglycanin-related mucin-like glycoprotein molecule or fragment thereof. If both molecules have a substantially similar structure, or if both molecules have similar biological activity, the molecules are substantially similar. So the two molecules are substantially similar If one has no structure in the other, or if the two amino acid sequences are not completely identical, the molecules are considered mutants. For example, substitution of oral ysine for valine, lysine for arginine, and glutamine for wasparagine may not change the function of the polypeptide.
  • analog refers to a molecule that is functionally substantially similar to a complete epididal forcenin-related mucin-like glycoprotein molecule or fragment thereof.
  • An example of a preferred functional derivative is 70% or more homology, more preferably 80%, particularly preferably 90% or more, with respect to the amino acid sequence of the natural-type epiglycanin-related mucin-like sugar protein of the present invention.
  • a protein having an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added in the amino acid sequence of a natural type epiglycanin-related mucin-like glycoprotein is also suitable.
  • the tandem repeat region of the epiglycanin-related mucin-like glycoprotein of the present invention includes Codington, JF, Linsley, Lin. ⁇ ., Jeanloz, RW, Irimura, ⁇ ., And Osawa. (1975) Carbohvdr. Res.. Vol. 40, p. 171— 182-O-glycosyl between 2-acetamido-2 deoxygalatatose residues and serine or threonine residues, as identified in 182 Enormous numbers of sugar chains can be added by conjugation.
  • the epiglycanin-related mucin-like glycoprotein of the present invention includes ⁇ D—GalNAc—Ser (Thr), j8—D—Gal— (1 ⁇ 3) —a—D—GalNAc—Ser (Thr), (2 Gal , 1 GlcNAc) — a— D— GalNA c-Ser (Thr), a NeuNAc— (2 ⁇ 3) j8— D— Gal— (1 ⁇ 3) a D— GalN Ac— Ser (Thr), and
  • a sugar chain structure containing a-NeuNAc may be added.
  • the maximum molecular weight of the sugar chain in the murine-type epiglycanin-related mucin-like glycoprotein is estimated to be about 830 kDa.
  • the total molecular weight of the related mucin molecule is estimated to be about 1020 kDa at maximum.
  • analysis with a human-type epiglycanin-related mucin-like glycoprotein leads to the estimation that the maximum molecular weight of the added sugar chain is about 200 kDa, and therefore the maximum value of about 260 kDa is given for the whole molecule.
  • the maximum molecular weight of the added sugar chain is about 200 kDa, and therefore the maximum value of about 260 kDa is given for the whole molecule.
  • Electrophoretic prayer can give a lower molecular weight smear than the maximum molecular weight force.
  • proteins with different sugar chain compositions and binding amounts are also within the scope of the present invention.
  • the present invention includes a nucleic acid encoding an epiglycanin-related mucin-like glycoprotein or a nucleic acid complementary to the nucleic acid.
  • nucleic acid include the sequence shown in SEQ ID NO: 172 or SEQ ID NO: 174.
  • nucleic acid intends cDNA, genomic DNA, and synthetic (eg, chemical synthesis or modification) DNA or RNA.
  • the nucleic acid may be labeled with an enzyme such as horseradish peroxidase, a radioisotope, a fluorescent substance such as Cy3 or Cy5, a chemiluminescent substance, or the like.
  • the nucleic acid molecule of the present invention can be a stable DNA derivative such as phosphorothioate or methylphosphonate, a stable RNA derivative such as 2, -O-alkyl RNA, or other epiglycanin-related mucin nucleotide analogues, Antisense oligonucleotides can be provided.
  • oligonucleotides Only one member of the set is identical to the native epiglycanin-related mucin-like glycoprotein sequence, but even mismatched DNA oligonucleotides will hybridize under appropriate stringency conditions (e.g., 3xSSC, 68 ° C, Can be hybridized to native sequence with 2xSSC, 0.1% SDS and 68 ° C) and the DNA encoding the native sequence can be identified and isolated, and such oligonucleotides are also within the scope of the present invention.
  • appropriate stringency conditions e.g., 3xSSC, 68 ° C, Can be hybridized to native sequence with 2xSSC, 0.1% SDS and 68 ° C
  • DNA encoding the native sequence can be identified and isolated, and such oligonucleotides are also within the scope of the present invention.
  • Examples of obtaining the DNA of the present invention from a DNA library include screening a suitable genomic DNA library or cDNA library by a screening method using a hybridization or an immunoscreening method using an antibody. There is a method in which clones having the DNA of the above are grown and excised using a restriction enzyme or the like. Screening by the hybridization method is performed by labeling DNA having the base sequence described in SEQ ID NO: 172 or SEQ ID NO: 174 or a part thereof with 32 P or the like as a probe, and using it as an arbitrary cDNA library. On the other hand, it can be carried out according to a known method, for example, Maniatis T., Molecular Cloning, a Laboratory Manual ⁇ Cold Spring harbor Laboratory, New York (1982).
  • the DNA of the present invention can also be obtained by PCR using a genomic DNA library or a cDNA library as a cage.
  • a sense primer and an anti-sense primer are prepared based on the nucleotide sequence set forth in SEQ ID NO: 172 or SEQ ID NO: 174, and a known method such as Michael AI is performed on an arbitrary DNA library.
  • the DNA of the present invention can also be obtained by performing PCR Protocols, a Guide to Methods and Applications, Academic Press (1990), etc.
  • a DNA library having the DNA of the present invention is selected and used.
  • any library having the DNA of the present invention can be used.
  • a commercially available DNA library can be used, or a cDNA library including the DNA of the present invention can be used.
  • Select cells suitable for production and use known methods eg Sambrook et al., Molecular Cloning, a Laboratory Man ual 2nd ed., Cold Spring Harbor Laboratory, New York (1989), a cDNA library can be prepared and used for IJ.
  • the DNA of the present invention can also be prepared by a chemical synthesis method such as the phosphoramidite method based on the sequences disclosed in the present specification.
  • clonal keviglycanin-related mucin DNA obtained by the methods described herein can be cloned by molecular cloning into an expression vector containing regulatory sequences, i.e., appropriate promoters and other appropriate transcriptional control elements. It can be expressed recombinantly and can be transferred to a prokaryotic or eukaryotic host cell to produce a recombinant shrimp glycanin-related mucin-like glycoprotein. Techniques for such operations are well described in Sambrook et al. (Supra) and are well known in the art.
  • an expression vector contains a DNA sequence necessary for transcription of a cloned copy of a gene in a suitable host and translation of the obtained mRNA.
  • bacteria, cyanobacteria are used.
  • eukaryotic genes can be expressed in various hosts such as plant cells, insect cells, fungal cells, and animal cells. Specially designed vectors can shut down DNA between hosts, eg, bacteria-yeast, bacterial animal cells, bacterial fungal cells, or bacterial invertebrate cells.
  • Appropriately constructed expression vectors are replication origins for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, possible elements for high copy number, activity Should include the appropriate promoter.
  • a promoter whether a structural promoter or a regulated promoter, is defined as a DNA sequence that binds RNA polymerase to DNA and initiates RNA synthesis. Strongly, a promoter is a promoter that initiates mRNA at a high frequency.
  • other regulatory sequences in the expression vector include transcriptional machinery and terminator, and transcriptional repressor binding, protein-mediated regulation by known mechanisms such as anti-athion. Other sequences are included.
  • SV40 or adenovirus early and late promoters lac system, trp system, TAC or TRC system, lambda phage major operator and promoter region, fd coat protein regulatory region, glycolytic enzyme (eg, 3-phosphodalase Tokina Promoter), Pho5 promoter, yeast ⁇ mating promoter, etc.
  • Expression vectors include, but are not limited to, cloning vectors, modified cloning vectors, specially designed plasmids or viruses.
  • Various mammalian expression vectors can be used to express the epidermal forcenin-related mucin DNA of the present invention in mammalian host cells.
  • epiglycanin-related mucin DNA can be expressed in bacterial host cells using various bacterial expression vectors.
  • Epiglycanin-related mucin DNA can be expressed in fungal cells using the same various fungal host cell expression vectors.
  • epiglycanin-related mucin DNA can be expressed in insect host cells using various insect cell expression vectors. Can be made.
  • Various host vectors for expression are known.
  • ⁇ phage derivatives such as M13, yeast
  • SV40 eukaryotic hosts
  • ushi papilloma virus eukaryotic hosts
  • adenovirus and cytomegalovirus a vector for expression control sequence
  • insect insect
  • Examples of host cells include Escherichia coli, Pseudomonas and Bacillus bacteria, Saccharomyces genus Pichia yeast, insect cells such as SF9, and animal cells such as CHO and COS7. Particularly preferred hosts are derived from eukaryotic cells, more preferably mammalian derived host cells. Methods for introducing a vector into a host cell include known methods such as the electopore method, the protoplast method, the alkali metal method, the calcium phosphate precipitation method, the DEAE dextran method, the microinjection method, and the method using virus particles. There is a special issue, Gene Engineering Know-how Book, published on March 20, 1991, Yodosha), but either method can be used.
  • the protein of the present invention can also be expressed as a fusion protein with another protein or tag, such as dartathione S transferase, protein A, hexahistidine tag, or FLAG tag.
  • the expressed fusion form can be excised using an appropriate protease such as thrombin, and sometimes the protein can be prepared more advantageously.
  • an epiglycanin-related mucin-like glycoprotein or its fusion protein in a host cell the protein can be recovered and the protein can be obtained in a purified form.
  • the recombinant ebiglycanin-related mucin-like glycoprotein is converted to other cellular protein caps. You can separate them.
  • the present invention also contemplates antibodies specific for epiglycanin-related mucin-like glycoproteins.
  • Such antibodies can be produced according to any method known to those skilled in the art.
  • an immunogen containing an adjuvant that is, the epiglycanin-related mucin-like glycoprotein of the present invention or a partial peptide thereof is subcutaneously administered to an immunized animal. Then, it is repeated at an appropriate interval (for example, 1 week) a predetermined number of times (for example, 5 times), whole blood is collected after the final immunization, and antiserum is obtained by separating it.
  • the purification of the polyclonal antibody from the antiserum is carried out by using the epiglycanin-related mucin-like glycoprotein used for animal immunization or a partial peptide thereof for chromatography, such as CNBr-activated Sepharose or HiTrap N HS— activated (both manufactured by Amersham Pharmacia) and covalently immobilized, the antiserum is subjected to the immobilized serum to specifically adsorb the antibody in the antiserum on the resin, This can also be achieved by eluting and recovering the antibody adsorbed on the resin using an appropriate buffer or chaotropic ion, but is not limited thereto.
  • a suitable immunogen may be composed of a partial peptide containing an epitope related to the transmembrane region or extracellular domain of the epiglycanin-related mucin-like glycoprotein of the present invention. It is specific for the extracellular domain of the epiglycanin-related mucin-like glycoprotein.
  • a non-limiting example of the partial peptide is SSSTPTPTTTASST (SEQ ID NO: 177) in the tandem repeat region of the mouse protein.
  • the force that can cite a fragment containing an epitope present in NHTGTPVMEVKPSGS (SEQ ID NO: 178) located just outside the transmembrane region is not limited thereto.
  • the antibody of the present invention is obtained as a monoclonal antibody
  • the mouse spleen cells of rodents such as hamsters are fused with parental cells for cell fusion such as myeloma cell lines, and the resulting neutralized hybridoma is selected and cloned.
  • the fused cells are cultured in vitro or in vivo, and a highly specific monoclonal antibody is collected from the culture mixture.
  • the antibody of the present invention also includes an antigen-binding fragment of the antibody as obtained by enzymatic digestion of the antibody described above.
  • antigen-binding fragments include Fab fragments, Fab, fragments, F (ab ') fragments, F (v) fragments, H chain monomers or dimers
  • L chain monomers or dimers 1 H chain and 1 L chain dimer.
  • the fragment can be obtained by, for example, the ability to digest a complete antibody with a protease such as pepsin or papain, and after treatment with a reducing agent, if necessary.
  • the H chain and L chain monomers can also be obtained by treating a complete antibody with a reducing agent such as dithiothreitol and then separating the purified chain.
  • the antibody can be advantageously used for detection of epiglycanin-related mucin-like glycoprotein in a biological sample collected by the subject of the test, for example, a body fluid such as a cell or tissue or blood. That is, as shown in the present specification, it has been confirmed that the epiglycanin-related mucin-like glycoprotein of the present invention is specifically expressed in thyroid tumor cells and testicular tumor cells. Useful as a tumor marker. Therefore, the presence of the epiglycanin-related mucin-like glycoprotein of the present invention can be a diagnostic indicator of tumors in those organs.
  • the labeled antibody is prepared.
  • antibodies can be labeled with radioactive substances, colored particles such as colloidal gold, fluorescent or chemiluminescent labels, or enzymes (for ELISA). wear.
  • a method for labeling antibodies can force s to employ any method known to those skilled in the art, e.g.,].
  • Biochem. Vol. 11, pp 395 ⁇ 399 (1979), J. Biochem. Vol. 14, 41 ⁇ 5 (1982), Immunofluorescence and Related Techniques ⁇ Elsevier / North Holland Biomedical Press, pages 215 to 225 (1978) can be used.
  • the antibody is immobilized on a solid support.
  • the antibody is dissolved in a carbonate buffer (about pH 8.6). This can be achieved by adding the solution to the wells of the microplate and incubating for a predetermined time.
  • a carbonate buffer about pH 8.6
  • one of the above-mentioned antibodies is used for coating a solid support such as the bottom of the well to provide a capillary side antibody, and the other antibody is labeled with a radioactive substance, a colored particle or an enzyme and is detected. I will provide a. After the biological sample having the ability of the subject to be tested is added to the well having the antibody on the side of the capsule and incubated for a predetermined time, the sample is also removed.
  • the antibody on the detection side is added to the well. After the predetermined incubation, the inside of the tool is washed to detect the formation of the capture antibody detection object-detection antibody complex. Detection depends on the nature of the labeling substance labeled on the detection-side antibody. If radioactive labeling is used, the radiation dose is high, if it is a colored particle label, the color development amount or absorbance, and if it is enzyme labeling (ELISA), Furthermore, an appropriate substrate is added to the well, and the absorbance after a predetermined incubation is detected.
  • ELISA enzyme labeling
  • enzymes such as horse radish peroxidase and alkaline phosphatase, which are not particularly limited, are advantageously used.
  • horse radish peroxidase 3,3 ′, 5,5′-tetramethylbenzidine or the like can be used as a substrate for the enzyme.
  • alkaline phosphatase When alkaline phosphatase is used, p-trope phosphate can be cited as a substrate.
  • the detection method typically corresponds to the Northern blotting method.
  • the nucleic acid of the present invention preferably the entire nucleic acid represented by SEQ ID NO: 172 or SEQ ID NO: 174, or Preparing a probe based on a part, hybridizing the probe and RNA from a sample under stringent conditions, and detecting the probe-RNA complex formed.
  • Other typical examples include PCR amplification (RT-PCR) after reverse transcription of the RNA of interest prior to hybridization, or using transcription-based amplification typified by NASBA, in which case The amplification product may be detected according to the Southern blotting method.
  • RT-PCR so-called competitive RT-PCR can also be used.
  • amplification is performed based on the nucleic acid of the present invention, preferably the whole or part of the nucleic acid represented by SEQ ID NO: 172 or SEQ ID NO: 174 or the sequence of their complementary strands.
  • Primers can be designed and such design would be easy for those skilled in the art. Accordingly, preferred probes and primers contain 10 or more consecutive nucleotides of the nucleic acid sequence shown in SEQ ID NO: 172 or SEQ ID NO: 174, more preferably 15 or more, and even more preferably 20 or more consecutive nucleotides. More preferably, the primer can amplify the unique transmembrane region of the present invention and the probe can recognize the region.
  • Non-limiting examples of such probes are: CTTCCCATAGTGCATCTACT
  • GCCCAGGTGGAGTCCTAACTGGTTC (SEQ ID NO: 176) can be mentioned
  • kits for detecting the expression of an epiglycanin-related mucin-like glycoprotein of the present invention is convenient when a measurer performs a test.
  • the kit may include a probe designed on the basis of the sequence of the whole or part of the nucleic acid represented by SEQ ID NO: 172 or SEQ ID NO: 174 or a complementary strand thereof.
  • the kit includes a RNase H inhibitor, reverse transcriptase, TaqDNA polymerase, and the like, and the construction and production method of such kits are well known to those skilled in the art.
  • Example 1 Identification of murine-like epiglycanin-related mucin-like glycoprotein
  • TA3-Ha cells and TA3-St cells were purchased from AZJ mice (6 weeks old, female; SLC, and bred under the SPF conditions at the University of Tokyo Faculty of Pharmacy. For experiments, mice pre-bred for 1 week after purchase were used. )) In the abdominal cavity or petri dish.
  • TA3-Ha cells and TA3-St cells were provided by Dr. Codington, Bergen University, and were used after confirming that there was no mycoplasma infection using Mycoplasma Plus TM PCR Primer Set. Specifically, TA3-Ha cells and TA3-St cells were cultured in DZF-IOG / O FCS medium in the presence of 37 ° C. and 5% CO. Ding 8-11 ⁇ 2 cells are collected every 3-4 days and renewed
  • TA3 St cells are washed with PBS every 3-4 days, and then Trp — EDTA solution (0.05% Trypsin / 0.02% EDTA—PBS) is added and removed. C, incubated for 5 minutes, and then subcultured from the culture dish.
  • Trp EDTA solution
  • C incubated for 5 minutes, and then subcultured from the culture dish.
  • about 2 ⁇ 10 6 TA3-Ha cells were suspended in HANKS 'solution and injected intraperitoneally into AZJ mice. One week later, the cells were collected for peritoneal force. After suspension in 2 ml of 0.15 M PBS, 6 ml of MilliQ water was added, suspended for about 20 seconds, and immediately 2 ml of 3.5 M NaCl. With this procedure, red blood cells were removed and used for the experiment.
  • biotin- PNA (1/100: VECTOR) or biotin-VVA ( lZ200: VECTOR) was 100 i ul mosquitoes ⁇ tut, reacted under ice-cooling for 30 minutes.
  • F After washing the cells with CM buffer, FITC—strept avidin (manufactured by ZYMED) diluted 100-fold with FCM buffer was added at 100 / zl and allowed to react for 30 minutes under ice cooling. Further, the cells were washed with FCM buffer, suspended again in FCM buffer, passed through a nylon mesh, and the fluorescence intensity was measured with a flow cytometer. As shown in FIG.
  • the molecular weight of the protein produced by T-antigen or Tn-antigen was estimated. Specifically, Ha cells and St cells were collected, washed once with PBS, and further desalting buffer (Sucrose (Wako Pure Chemicals) 42. 79g, 1M Tris — HC1 pH7.25 ml, 50 mM CaCl (Wako Pure Chemicals) 500 / zl, 0.1 ⁇ PMSF (iso
  • the obtained cell extract was subjected to SDS-denaturation 4% polyacrylamide electrophoresis in the presence of 2-ME and semi-drited onto a PVDF membrane (Immunobilon (trademark); manufactured by Millipore). After that, the membrane after blotting was blocked with 2% BSA (in PBS) and diluted 2,000 times with 0.1% Tween20 and 2% BSA (in PBS). Reacted with VVA for 2 hours. After the reaction, the PVDF membrane was washed 4 times with 0.1% Tween20 (in PBS), and diluted 5,000 times with 0.1% Tween20 and 2% BSA (in PBS). HRP — streptavidin (manufactured by ZYMED) Reacted for 1 hour.
  • the membrane was washed 4 times with 0.1% Tween20 (in PBS), then developed with ECL Plus (trademark) Western Blotting Detection kit (Amersham Bioscience), and hyperfilm ECL (Amersham Bioscience) was developed. I was exposed.
  • annealing temperatures are mMucl: 56 ° C, mMuc2: 58 ° C, mMuc3 : 49 ° C, mMuc4: 58 ° C, mMuc5AC: 54 ° C, mMuc9: 50 ° C, mMuclO: 48 ° C. 10 ⁇ l of the reaction product was run on 1% Seakem ME agarose containing 0.5 ⁇ g Zml ethylene bromide, and the band was confirmed.
  • mRNA was purified from each of Ha cells and St cells, and a double-stranded cDNA library was prepared by reverse transcription reaction.
  • mRNA was extracted from TA3-Ha cells and TA3-St cells using mMACS mRNA Isolation kit (Daiichi Kagaku Co., Ltd.), precipitated with EtOH, and then dissolved in Milli Q water. The absorbance was measured at 260 nm and Z280 nm to estimate the amount and purity of mRNA.
  • the double-stranded cDN A library is a double-stranded cDNA, using 5 ⁇ g of the obtained mRNA as a saddle and performing RT reaction from Oligo d T primer using Timesaver TM cDNA synthesis kit (Amersham Bioscience). cDNA was prepared.
  • NICK (trademark) column removes short DNA fragments, etc., precipitates EtOH, dissolves in MilliQ water and contains 0.5 g / ml ethylene bromide to 0.7% Sea kern ME agarose.
  • the DNA was extracted from the gel using QIAEX (trademark) II gel extraction kit (QIAGEN).
  • the next step of the subtraction method is to digest the above library with Sau3AI to produce a double-stranded cDNA fragment of approximately lOObp, and then to adapter Z primer at both ends of the cDNA fragment derived from Ha cells.
  • the tester amplicons are combined with the tester amplicons, while the cDNA fragments derived from St cells are converted into driver amplicons without binding adapter Z primers, denatured, and hybridized with each other. It was.
  • PCR of this amplicon was performed 3 times, changing each adapter and Z primer. Concentration increased with each successive round, and a single band was detected after 3 reactions (data not shown).
  • PCR reaction solution was prepared in the same manner as described above, and PCR reaction was carried out using a template obtained by dividing this into 5 equal parts. The cycle was performed 30 times. 5 ⁇ l of the PCR product was run on 3% Nusieve agarose containing 0.5 ⁇ g Zml ethyl bromide to confirm whether DNA concentration occurred (concentration after 1st round RDA: data shown) ) The PCR products were combined, treated with phenol: chloroform, EtOH precipitated and dissolved in TE.
  • the JBgl24 adapter was removed by treatment with Sau3AI, dissolved in TE after EtOH precipitation, electrophoresed in 3% Nusieve agarose, excised from 100 to 1500 bp, and DNA was extracted with QIAEX II gel extraction kit.
  • Phenolic Treated with chloroform, dissolved in MilliQ water after EtOH precipitation, measured for absorbance, and estimated the amount of DNA.
  • N Bgl 24 5 '-AGGCAACTGTGCTATCCGAGGGAA 3' (SEQ ID NO: 199) and N Bgl 12: 5 '-using TAKARA DNA Ligation Kit ver.
  • the band DNA purified after the 3rd round RDA reaction was converted into pGEM—T easy vector (trade name, manufactured by Promega) and ligation product 51 obtained was mixed with E. coli JM109 (TAKARA) 60 ⁇ 1 and incubated on ice for 20 minutes. I put it in the bath for 50 seconds and then put it back on ice for more than 2 minutes. This was cultured at 37 ° C for 1 hour in 100 i ul of SOC medium (TAKARA), then sterilized by filtration with 51 IPTG (TAK ARA, 2gZl0ml—MilliQ water, 0.22 ⁇ m filter).
  • TAKARA E. coli JM109
  • Plasmid DNA was extracted with Nucleo Spin TM Plasmid (manufactured by MACHEREY-NAGE L). EcoRI (TOYOBO) treatment and 0.5 / zg / m 1 containing ethylene bromide 1.5% Seakem ME agarose was used to confirm the presence of inserts.
  • the confirmed base sequence of the insert was based on analysis by ABI (ABI PRISMR 3100 Genetic Analyzer (trade name)) and analysis by Aloka (LI- COR dNA Sequencer 4200 (trade name)).
  • the sequence reaction in ABI was performed using BigDye (trademark) Terminator ycie sequencing v2.0 Ready Reaction (manufactured by PE Biosystem).
  • plasmid DNA as a template, add MilliQ water to 2 1 of 10 X PCR buffer (15 mM MgCl, 4 ⁇ ⁇ Premix, 3.2 pmol primer included)
  • Thermo Sequenase Cycle Sequencing Kit manufactured by Amersham Pharmacia Biotech. Plasmid DNA 300-600 fmol as template, 2 1 10 X PCR buffer (15 mM MgC 1, 2 1 IRD800-labeled Primer (1. Opmol / l: Aloka) or IRD700 -Labeled Primer (1.
  • OpmolZ 1 Aloka
  • L 1 2.5 mM dNTP 2 ⁇ 1
  • Thermo Sequenase (trademark) DNA polymerase containing MilliQ water is added to 17 1 and placed in PCR tube Using a thermal cycler, the reaction was carried out for 30 cycles of 95 ° C for 5 minutes, 95 ° C for 30 seconds, 50 ° C for 30 seconds, and 70 ° C for 1 minute.
  • each 1.2 1 was subjected to electrophoresis gel (50% Long Ranger TM Gel solution 4.5 ml (manufactured by Bio Whittaker Molecular Applications), 10 XTBE 6 ml, Urea (manufactured by Wako Pure Chemical Industries, Ltd.) 25. 2 g and ammonium sulfate persulfate (manufactured by Wako Pure Chemical Industries, Ltd.) 40 mg in MilliQ water 60 ml were stirred for about 1 hour, degassed, and then TEMED (Nacalai (Tester Co., Ltd.) 40 1 was poured into a cake gel plate and left to stand for 4 hours or more.
  • electrophoresis gel 50% Long Ranger TM Gel solution 4.5 ml (manufactured by Bio Whittaker Molecular Applications), 10 XTBE 6 ml, Urea (manufactured by Wako Pure Chemical Industries, Ltd.) 25. 2 g and ammonium sulfate per
  • the above 10 XTBE used was 108 g of Tris, 55 g of boric acid (manufactured by Wako Pure Chemical Industries, Ltd.) and 8.3 g of EDTA '2Na dissolved in 1 L of MilliQ water and autoclaved under high pressure steam.
  • RNA of TA3-Ha cells and TA3-St cells was electrophoresed in 1% formaldehyde agarose gels at 1 ⁇ g Zlane of mRNA, and the gel after electrophoresis was treated with 0.05N NaOH for 20 minutes. After rinsing with water, soak twice in 20 X SSC (NaCl 175.3 g and Trisodiu m Citrate '2H O (Wako Pure Chemicals) 88. 2 g in 1 L of MilliQ water) for 20 minutes. It was. Nylon membrane (manufactured by PALL) was blotted with 1 kg of mildew, and the membrane was treated at 80 ° C for 2 hours to immobilize RNA.
  • RNA immobilized on the nylon membrane as described above was labeled with 32 P using the novel sequence cDNA fragment obtained by the subtraction method as a probe, and hybridization was performed. It was.
  • E. coli containing the vector is cultured in 5 ml of LB medium containing 5 gZml Ampicillin for at least 8 hours at 37 ° C, and the plasmid DNA is obtained using the culture force QIAGEN plasmid mini kit (trade name, manufactured by QIAGEN). After extraction, the plasmid DNA was first treated with E ⁇ RI, then electrophoresed with 2% Seakem ME agarose containing 0.5 gZml ethylene bromide, and the insert was excised from the gel. QIAquick gel extraction kit (QIAGEN) The DNA of the insert was extracted as a probe.
  • Hybridization was performed by prehybridizing for about 2 hours at 68 ° C with 2 ml of Perfect Hybri (trademark, manufactured by TOYOBO) after the above membrane was squeezed with 3 X SSC. After labeling with ⁇ - ⁇ — dCTP by the product Rediprime (trade name, manufactured by Amersham pharmacia biotech), separate with NICK column, measure the count and calculate 10 6 cpm / ml, 95 ° C, denatured for 10 minutes, and then allowed to incubate at 68 ° C for 1 mm.
  • RNA was run on a large gel and Northern blotting was performed.
  • Total RNA was calculated.
  • a part of the noisy hybridization pattern that was smeared in the minigel was separated until it became a band (Fig. 3). The larger one was estimated as 8.8 kb and the smaller one was estimated as 6 kb.
  • the 5 'side was strong without any expansion even when the conditions were variously improved.
  • a slightly strong band-like part was obtained in the smear, so that part was cut out and subcloned into pGEM—T easy vector according to the method described above, and the nucleotide sequence was changed. Deciphered.
  • This race product contains a poly A tail signal and poly A tail, and when converted to an amino acid sequence, several tandem repeats, a part thought to be a transmembrane site rich in hydrophobic amino acids, and that part Including the subsequent intracellular site, the stop codon was also confirmed (data not shown). From this, the 3 'race product is considered to be the third side of a novel mouse mucin-like glycoprotein highly expressed in TA3-Ha cells, and this sequence was named 3, race3.
  • mice other than the A strain also have the gene for the mucin-like glycoprotein. It was confirmed by PCR. Thus, to date, mouse epiglycanin has been expressed in TA3-Ha cells derived from A strain of mice. It is reported that! However, confirming that the mouse mucin-like glycoprotein gene of the present invention also exists in mice other than the A strain was considered to be effective for the undiscovered 5 'side analysis. .
  • the gene of C57BLZ6N mouse or 129SVJ mouse which is more frequently used in animal experiments than A strain mice and has a gene library and BAC clone, etc., is used as a template, and epintmF: 5, — TTCTCATCACCCTAGCCTCTG—3, (SEQ ID NO: 202) and epintmR: 5 '— TATCCTCCTTCGCGTGTCTGG— 3' (SEQ ID NO: 203), or epil— 5: 5,-TCTG ACC ACC ACT ACTGC ATCC AGC ACT- 3. PCR was performed using (SEQ ID NO: 204) and epintmR as primers (Fig. 4).
  • the cells were further cultured at 37 ° C for 1 cm. The next day, the culture is centrifuged at 3, OOOrpm for 5 minutes, and the pelleted Escherichia coli is suspended in 5 ml of 10 mM MgSO.
  • phage solution 1 or more were mixed in a tube and incubated at 37 ° C for 20 minutes. So After that, add the mixture to Top agarose (2g bacto tryptone 3.2g, bacto yeast extract 2g, NaCl lg and bacto agar 1.4g in 1L MilliQ water, autoclaved) and add to 2X YT plate A plaque was prepared by incubating at 37 ° C for 1 hour. Elution of the formed plaque-powered phage was performed using SM buffer (NaCl 5.8 g, MgSO ⁇ 7 ⁇ O 2) on a plate with plaques on the entire surface (containing approximately 300,000 plaques).
  • SM buffer NaCl 5.8 g, MgSO ⁇ 7 ⁇ O 2
  • the phage eluted from the plate that was found to contain the phage having the mucin-like glycoprotein gene of the present invention by PCR was spread on several new plates, and plaques were again made on the entire plate surface. Similarly, PCR was performed. By performing this operation several times, positive phages were concentrated.
  • plaque hybridization was performed using phages concentrated to some extent. That is, Southern blotting was performed by rolling several new plates at a concentration that allows identification of each plaque, transferring the plaque DNA to a nylon membrane. Thereafter, phage clones were collected from positive plaques, and Southern blotting was similarly performed on several new plates. How many times do this work? Thus, a clone of a positive phage was obtained. Specifically, XL1-Blue MRA solution 51 and phage solution were mixed in a tube and incubated at 37 ° C for 20 minutes. After that, about 4 ml of Top agarose was mixed, seeded on 2 XYT plates, and cultured at 37 ° C for 1 cm to prepare cinder plaques. The next day, nylon membrane filter BIODYNE (trademark) A
  • the plaques were copied by bringing TRANSFER MEMBRANE (PALL) into close contact, and the needles were placed three times to mark the plate and membrane so that they could be handled. Remove the peeled membrane in alkaline solution (0.5M NaOH, 1.5M NaCl), neutralization solution (0.5M Tris pH7.5, 1.5M NaCl), and washing solution (2 X SSC) for about 10 minutes each. After treatment and air drying, phage DNA was immobilized on the filter by treatment at 80 ° C for 2 hours. The probe used for Southern blotting was prepared according to the section “1-4. Analysis by Northern blotting”.
  • the membrane was immersed in 3 X SSC 0.5% SDSa (Wako Pure Chemicals) solution at 65 ° C for 30 minutes, and then prehybridized with 2 ml of Perfect Hybri TM at 68 ° C for about 2 hours. After labeling the probe with ⁇ - 32 P-dCTP using Rediprime, separating it with NICK column, measuring the count and calculating to 3 X 10 6 cpm / ml, at 68 ° C I'm happy. The next day, 2 x SSC 0.1% SDS solution was washed twice at 68 ° C for 5 minutes, and if the count was high, then secondary washing was performed with 0.1 X SSC 0.001% SDS solution. Washing was performed twice at 68 ° C for 15 minutes. Kodak Sceintific Imaging Film X—OMAT (trademark)
  • the purified DNA was treated with 2il, BamHI, Hindlll, and E ⁇ RI, respectively, electrophoresed, blotted on a nylon membrane, and then Southern blotted to see if the mucin gene was actually contained. It was confirmed. Specifically, DNA was digested with the above restriction enzymes and electrophoresed with 0.6% Seakem ME agarose. Gel, 0.2 N
  • the Ensembl Genome Browser was searched using partial sequences that could be decoded so far, including the sequence of the acquired gene clone, and it was revealed that this mucin gene was present in the MHC region of mouse chromosome 17. It was. In addition, a longer gene sequence including the full length of this mucin gene clone has been registered, and by analyzing this sequence in detail, the gene sequence and cDNA sequence corresponding to the 5 'side of the 3' race3 sequence are revealed. (Fig. 6). On the 5 'side, over 4 kb or more, no stop codon was observed, and it was a continuous exon. The tandem repeat consisting of 15 amino acids was repeated more than 60 times.
  • the undeciphered area was included in the middle part of the tandem repeat.
  • the undecoded region is expected to be about 1.5 kb to 2 kb, and all of this part is thought to be tandemly repeated.
  • tandem repeats could be repeated over 100 times.
  • This partial force is also a force if this new mucin is translated. It was thought to be smaller than the mRNA size estimated by Northern blotting. Therefore, it was predicted that a splice would occur in this 30 Obp portion.
  • a splice occurs between about 300 bp upstream of the tandem repeat part, and another exon is expected upstream, and a primer is designed in this about 300 bp part. , Tried to extend to the end.
  • epicDNAl 5 — CACCCATGCAATGAATTTAACAAAGG-3, (SEQ ID NO: 205)
  • epitmR TGGATGCAGTGGTGGTCAGGGTGG 3 '(SEQ ID NO: 206) epiracerl: GTGGCTATGCTCTTCGTTTCTGATGAAGG-3, (SEQ ID NO: 207)
  • epiracer2 GTGGTGGAGGAAATATGGGCATAACC-3, (SEQ ID NO: 208
  • RNA that does not have a 5 'end is in principle not extended, and the obtained cDNA is expected to start with a 5' end force. 5 'It was impossible to estimate the end.
  • the mouse genomic DNA sequence around the upstream end of the tandem repeat region is expressed as NCBI. And then, by examining this, a presumed splicing acceptor sequence was found upstream of the tandem repeat region. In addition, when a base sequence upstream of this sequence was examined, a termination codon appeared in any reading frame. Therefore, it was certain that splicing actually occurred in this estimated splicing receptor sequence. Therefore, two new antisense primers shown below were designed downstream of the estimated splicing acceptor sequence and upstream of the tandem repeat region. SEQ ID NO: 209) and 3 '(SEQ ID NO: 210)
  • the upstream exon of the mouse's epiglycanin-related mucin-like glycoprotein of the present invention was searched by reverse transcription PCR. Specifically, the above primer was used for antisense, and the sense primer was determined as follows.
  • the putative splicing donor site was checked against the genomic sequence of lOkb upstream of the tandem repeat region.
  • the encoded amino acid sequence is examined in all reading frames, and a sequence having a certain length that the stop codon is removed is used as an exon candidate region, and a sense primer is designed therein. did.
  • Thirteen regions were selected and examined for the presence of cDNA by RT-PCR.
  • the target PCR product could be obtained only when PCR was performed using EpiRT12 000: 5′-GGAGAAGCAGCCTCTGGTGCTGGCTGC 3 ′ (SEQ ID NO: 211) as a sense primer and the above-mentioned EpiAMP as an antisense primer.
  • the PCR reaction conditions were as follows: AmpliTaq TM Gold standard protocol, 94 ° C for 10 minutes, 94 ° C for 30 seconds: 60 ° C for 30 seconds: 72 ° C The 5 minute cycle was repeated 30 times, finally at 72 ° C for 5 minutes.
  • RNA generated from TA3-Ha cells was a raw material using GeneRacer TM Kit, and the transcription start position was identified.
  • kit manufacturer's instructions were followed except for the following two points: 1) phenolic after CIP treatment, 1 phenol insertion before chloroform extraction, 2) by Vortex There are several instructions in the manual to stir, but no vortexing was performed and the tube was stirred up and down. In this way, ol igo RNA was bound only to RNA having a CAP structure!
  • an antisense primer for reverse transcription was designed as follows, and the RNA was reversely transcribed gene-specifically using SUPERSCRIPT TM III (Invitrogen) in a saddle shape.
  • RTGeneTandeml GCTGTCAACGATACGCTACGTAACGGCATGACA
  • Sense primers are provided in the kit for GeneRacer TM 5 'Primer: 5, CGACTGGAGCACGAGGACACTGA-3 (SEQ ID NO: 213) and 061 ⁇ ! ⁇ acer (trademark) 5 ′
  • NestedPrimer 5′—GGACACTGACATGGACTGAAGGAGT A—3 ′ (SEQ ID NO: 214) was used, and the above EpiREV and EpiAMP were used for antisense.
  • the reaction conditions were as follows.
  • the first PCR uses GeneRacer TM 5, Primer and EpiREV as sense and antisense, respectively, at 94 ° C for 10 minutes, then at 94 ° C for 30 seconds: at 60 ° C for 30 seconds: A cycle of 3 minutes at 72 ° C was repeated 30 times and 72 ° C for 5 minutes.
  • the second round of PCR uses GeneRacer TM 5 'NestedPrimer and EpiAMP as sense and antisense, respectively, at 94 ° C for 10 minutes, then at 94 ° C for 30 seconds: 68 ° C for 30 seconds : A cycle of 3 minutes at 72 ° C was repeated 30 times, and at 72 ° C for 5 minutes. Analyzing the base sequence of the obtained PCR product, 4 types of transcription start positions Identified. Of the 9 samples, 6 samples started from the same position, and this position was determined as the transfer start position. This exon was identical to the first exon predicted by RT-PCR earlier, and the splicing donor site and the splicing acceptor sequence were
  • EpiAS 15130 5 '-AG ACC ATG ATAAGTACCC AGGGC-3, (SEQ ID NO: 215)
  • the murine epiglycanin-related mucin-like glycoprotein cDNA of the present invention having the sequence shown in SEQ ID NO: 172 including the variant could be isolated.
  • Example 2 Preparation of polyclonal antibody against mouse 'epiglycanin-related mucin-like glycoprotein, detection of the mucin with the antibody, and identification of expression distribution of mouse-epigencanin-related mucin-like sugar protein in mice
  • the peptide was synthesized by F-moc solid-phase synthesis method using Peptide Synthesis System (Pioneer). All F-moc amino acids used in the synthesis were OH (Applied Biosystems). After synthesis, to remove the peptide from the resin and deprotect it, 0.7 g phenol, 500 ⁇ l Milli Q water, TFA (from PE Biosystems) 9.5 ml And shake for 1 hour at room temperature. The resin was removed by filtration through a glass filter and washed several times with jetyl ether to precipitate the peptide. The precipitated peptide was dissolved in a minimum amount of MilliQ water.
  • the peptide was purified using reverse phase HP LC (manufactured by JASCO Corporation).
  • the column was a 5C18 column (Cosmosil (trademark), manufactured by Nacalai Testa Co., Ltd.), and separation was performed at a column temperature of 40 ° C and a flow rate of 2 mlZmin.
  • Buffer A 0.05% TFA in MilliQ water
  • buffer B ⁇ 0.05% TFA in propanol
  • acetonitorile from PE Biosystems
  • the collected fraction was subjected to mass spectrometry using MALDI-TOF MS spectrum (Voyager Elite: manufactured by PE Biosystems).
  • the purified peptide was lyophilized and stored at -20 ° C.
  • this polyclonal antibody is subjected to flow cytometry or Western blotting. Check whether it can be used for
  • TA3-Ha cells and TA3-St cells were stained with a polyclonal antibody, and fluorescence was measured with a flow cytometer. Specifically, 0.5 to 1 X 10 6 TA3-Ha cells and TA3-St cells were washed with FCM buffer, and then 100 1 Usagi antiserum diluted with FCM buffer was added to it and kept on ice. Reacted for 30 minutes. After washing the cells reacted with FCM buffer, 100 ⁇ l of FITC-goat anti rabbit IgG (ZYMED) diluted 100 times with FCM buffer was added and allowed to react for 30 minutes under ice cooling. The cells were washed with FCM buffer, suspended in FCM buffer, passed through a nylon mesh, and the fluorescence intensity was measured with a flow cytometer.
  • ZYMED FITC-goat anti rabbit IgG
  • both antibodies showed higher binding to Ha cells compared to binding to St cells, and it was confirmed that they recognized proteins expressed on the Ha cell surface. It was. In addition, the binding force of anti-epitm antibody tended to be lower than that of anti-epintm peptide antibody.
  • Ha cell and St cell lysates were electrophoresed, blotted onto a PVDF membrane, and stained with a polyclonal antibody.
  • staining with a polyclonal antibody non-specific binding was found to be very high in both cells, so the polyclonal antibody was crudely purified and stained for strength.
  • the amount of the above-mentioned rabbit antiserum was weighed, ammonium sulfate was added so as to become 50% saturation, and the mixture was vigorously stirred at 4 ° C for 60 minutes or more.
  • the membrane was washed 4 times or more with 0.1% Tween20 (in PBS) and then diluted 2,000 times with 0.1% Tween20—1% NGS and 2% BSA (in PBS). The mixture was reacted with rabbit IgG (ZYMED) for 45 minutes. Furthermore, the membrane was washed 4 times or more with 0.1% Tween20 (in PBS), and then diluted 3,000 times with 0.1% Tween20-1% NGS and 2% BSA (in PBS) for 30 minutes with HRP-streptavidin. Reacted. Color was developed with ECL Plus Western Detection kit and exposed to Hyper film ECL.
  • TA3—Ha cells are breast cancer cells
  • this new mucin mucin is mainly expressed in breast cancer cells, and we have established a variety of breast cancer cell lines and breast cancer tissue power generated by spontaneous carcinogenesis.
  • the obtained cell lines were examined.
  • epithelial cancer cells derived from commonly used mice such as C57BLZ6N mice and BalbZc mice were also examined.
  • ICR mice five weeks old, female, experimental animals used for culturing mouse cancer cell lines, were purchased from Oriental Yeast and bred under SPF conditions at the University of Tokyo Faculty of Pharmacy. Mice pre-bred for one week after purchase were used for the experiment.
  • the medium used for the culture was Minimun Essential Medium (MEM: GIBCO BRL) 9.4 g dissolved in 1 L of MilliQ water, autoclaved and sterilized 3 ml L-glutamine 5 ml, 10 % NaHCO 10m
  • Mouse breast cancer cell lines MM2, 46, 48, 102 cells, Ehrich cells, FM3A cells, and FM3AR cells, as well as lung squamous cell line KLN205 cells were provided by Tohoku University Institute for Aging Medicine.
  • Colon38, Colon26 and Renca were cultured in DZF-10% FCS medium in the presence of 37 ° C and 5% CO. These are PBS every 3-4 days
  • Trp—EDTA solution 0.05% Trypsin / 0.02% EDTA—in PBS
  • Trp—EDTA solution 0.05% Trypsin / 0.02% EDTA—in PBS
  • MM102 was cultured in RPMI1640-10% FCS medium at 37 ° C in the presence of 5% CO. After washing with PBS every 3-4 days,
  • Trp-EDTA solution was added thereto, incubated at 37 ° C for 5 minutes, and then removed from the culture dish and subcultured.
  • KLN205 was cultured in MEM-NEAA-10% FCS medium at 37 ° C in the presence of 5% CO and passaged every 3-4 days.
  • FM3A, FM3AZR is ES— 10% F
  • the cells were cultured in CS medium at 37 ° C in the presence of 5% CO and subcultured every 3 to 4 days.
  • Ehrlich
  • RNA from the above mouse cancer cell line was extracted using z MACS mRNA Isolation Kit (trade name), precipitated with EtOH, dissolved in water, and measured for absorbance 260nmZ28 Onm. It was prepared by determining the RNA amount and purity. Finally, according to the section “1-4. Analysis by Northern blotting”, 100 g of total RNA or 1 g of mRNA was electrophoresed to perform Northern blotting.
  • the results are shown in FIG. The ability of the probe to bind to several breast cancer cells and the expression of this mucin to be confirmed.
  • the breast cancer cell line FM3A cells also FM3AR cells, and the breast cancer tissue force generated by spontaneous carcinogenesis.
  • MMK7 cells showed relatively strong binding.
  • KLN205 cells which are lung squamous cell carcinoma cells, showed a very strong binding.
  • none of the cells showed stronger expression than TA3-Ha cells.
  • Example 3 Identification of human 'epiglycanin-related mucin-like glycoprotein
  • cDNA corresponding to each total RNA was prepared by RT reaction.
  • mouse 'epiglycanin-related mucin-like sugar tongue 3 'sequencing ability including the portion related to the transmembrane domain of the protein Using the designed primers, the cDNA obtained above was amplified by PCR, and the PCR product was subcloned for sequence analysis and confirmation. went.
  • expression analysis of mRNA encoding the human biglycanin-related mucin-like glycoprotein of the present invention in each human cell line was performed. The details will be described below.
  • MRK-nu-1, SK-BR-3 breast cancer cell line
  • He pG2 liver cancer cell line
  • EBC-1 lung cancer cell line
  • KATOIII gastric cancer cell line
  • 8505C, 8305C above thyroid cancer cell line
  • ME-180 cervical cancer cell line
  • U-937 monocyte lymphoma cell line.
  • EBC-1, 8505C, 8305C, and ME-180 were donated by Tohoku University Institute of Force and Age Medicine.
  • MRK—nu—1, SK—BR—3, EBC-1, KATOIII, 8305C, ME—180, U—937 are RPMI 1640-10% FCS medium, HepG2 is DMEM—HG—10% FCS — 50 OU / ml penycilin, lmg / ml streptomycin medium, 8505C was cultured in Eagle, s ME M—10% FCS medium at 37 ° C in the presence of 5% CO.
  • RNase in hibitor from Ambion
  • Oligo ⁇ ⁇ Oligo ⁇ ⁇
  • primer A reverse transcription reaction was performed from tech
  • MilliQ in 1 dNTP mix (2 mM each), 5 5 5 ⁇ primer—sense, 1 1 5 5 ⁇ primer-anti-sense, 0.1 ⁇ 1 AmpliTaq TM Gold, 1 ⁇ 1 cDNA template) Add water to 20 ⁇ 1 and place in a PCR tube. Use a thermal cycler to heat denature at 95 ° C for 1 minute, then 95 ° C for 30 seconds, 58 ° C for 30 seconds, 72 ° The reaction was carried out at C for 45 seconds for 30 cycles and at 72 ° C for 10 minutes. Of the reaction products, 101 was electrophoresed with 1.5% Agarose S containing 0.5 ⁇ g / ml ethylene bromide to confirm the band.
  • a reaction solution was prepared in the same manner as PCR. After heat denaturation at 95 ° C for 10 minutes, a reaction was performed at 95 ° C for 1 minute, 52 ° C for 30 seconds, 72 ° C for 1 minute for 45 cycles, and 72 ° C for 10 minutes. Ten of the reaction products were run on 1.5% Seakem ME agarose containing 0.5 g / ml ethylene bromide. In addition, each primer used was as follows.
  • Antisense hG3PDH-R 5 '-CAGTGATGGCATGGACTGTGGT-3' (SEQ ID NO: 217)
  • Sense hepintm— F 5, — CTTCCCATAGTGCATCTACTGC-3, (SEQ ID NO: 218)
  • Antisense hepintm -R 5,-GAACCAGTTAGGACTCCACCTGGGC C-3 '(SEQ ID NO: 219)
  • a clear band was observed around 270 bp in breast cancer cell lines, lung cancer cell lines, monocytic lymphoma cell lines, and particularly cervical cancer cell lines.
  • the cDNA panel includes standardized first strand cDNA prepared from heart, brain, placenta, lung, liver, skeletal muscle, kidney, spleen, spleen, thyroid, prostate, testis, ovary, small intestine, large intestine and leukocyte tissue. It was. As shown in FIG. 15, it was suggested that the gene is also expressed in normal tissues of the lung, thyroid and large intestine.
  • cDNA was prepared by RT reaction using a specific primer corresponding to the 3 'region of the gene.
  • cDNA encoding the human's epiglycanin-related mucin-like glycoprotein of the present invention was isolated by nested PCR using the obtained cDNA as a saddle type. The procedure was as follows.
  • This sequence reaction was performed using BigDye (trademark) Terminator Cycle Sequencing vl. 1 Ready Reaction (Applied Biosystems).
  • plasmid DNA as a template, prepare 2 ⁇ 10 X PCR buffer (15 mM MgCl, 4 ⁇ Premix, 3.2 Add pmol primer (including T7, T3, hepiseq5-F, hepiseq3-R) to MilliQ water, put it into 201, put it in a PCR tube, use a thermal cycler for 1 minute at 96 ° C, The reaction was performed for 25 cycles of 96 ° C for 10 seconds, 50 ° C for 5 seconds, and 60 ° C for 4 minutes.
  • pmol primer including T7, T3, hepiseq5-F, hepiseq3-R
  • Sense-1 clonest5- l F 5 '-AAGCCTAAGGAACCCAGGCATCCAG CTGCCCACGCC— 3' (SEQ ID NO: 221)
  • Example 4 Specific expression of human 'epiglycanin-related mucin-like glycoprotein gene in tumor tissue
  • An amplified probe incorporated into pGEM-T easy vector was used. That is, E. coli containing the vector was cultured in 3 ml of LB medium containing 5 g / ml Ampicillin at 37 ° C for 8 hours or more. Next, the plasmid DNA was extracted from the culture with NucleoSpin Plasmid (trade name, Machere y —Nagel), treated with EcoRI for 3 hours, and containing 0.5 / zg / ml ethylene bromide with 1.5% Seakem ME agarose. Electrophoresis was performed, and the insert portion was cut out. The target probe DNA was extracted with a QIAquick gel extraction kit (trade name, QIAGEN).
  • tumor tissue In particular, in the thyroid gland and testis, the expression of tumor tissue always exceeded that of normal tissue. Regarding lung, stomach, vulva, rectum and skin, expression was observed in tumor tissues or normal tissues such as several solid forces. In breast tumor tissue, the expression of the human epidariin force-related mucin-like glycoprotein of the present invention was not observed.
  • the epiglycanin-related mucin-like glycoprotein of the present invention is useful as a tumor marker for at least these thyroid tumor cells and testicular tumor cells.
  • Example 5 Expression of human 'epiglycanin-related mucin-like glycoprotein in transformed host cells
  • the coding sequence of the human-epiglycanin-related mucin-like glycoprotein with a FLAG epitope tag added to the N-terminal side was inserted into a mammalian expression vector pcDNA3.1 (-) (Invitrogen). Further, a plasmid not containing the coding sequence was used as a control. These plasmids were introduced into the human myeloid leukemia cell line K562 obtained from ATCC by electoporation. Selected by dieneticin (G418 sulfate; CalbioChem) Thereafter, the transformed cells were cloned by the limiting dilution method.
  • Cells were harvested and washed twice with PBS for Western 'blotting. After washing, the cells were treated with 10 mM Tris—HCl (pH 7.4; 0.5% Nonidet P—40, 0.25M Sucrose, 0.05 mM salt cannoleum, 2 mM EDTA and proteinase inhiibitor cocktail (1 Z1000 dilution, Sigma))), and centrifuged at 14,000 rpm at 4 ° C for 20 minutes. The supernatant was collected and quantified by BCA protein assays (Pierce) to obtain a cell lysate corresponding to 25 g protein.
  • Tris—HCl pH 7.4; 0.5% Nonidet P—40, 0.25M Sucrose, 0.05 mM salt cannoleum, 2 mM EDTA and proteinase inhiibitor cocktail (1 Z1000 dilution, Sigma)
  • the cell lysate was 1% ( ⁇ / ⁇ ) 2-mercapto in buffer (0.25 M Tris-HCl (pH 6.8), 40% glycerol, 8% SDS, 0.001% bromphenol blue). It was boiled with ethanol, and after 8% polyacrylamide 'gel electrophoresis, it was transferred to a polyvinylidene fluoride membrane (Immobilon-P TM, Millipore).
  • the anti-FLAG antibody binds only to cells transformed with the human 'epiglycanin-related mucin-like glycoprotein of the present invention (K562ZMUC21) and to the transformed cells (K562ZMock) of the control plasmid. Did not combine. This indicates that the gene product is ubiquitously expressed on the cell surface.
  • VVA-B4 and PNA were found to bind significantly more to cells transformed with the human 'epiglycanin-related mucin-like glycoprotein of the present invention than to the plasmid-transformed cells, which is characterized by This shows that many O-linked sugar chains were presented on the surface of the transformed cells (FIG. 16A).
  • Example 6 Cell and tissue staining using anti-serum specific to human epiglycanin-related mucin-like glycoprotein
  • N-terminal of 17 amino acid residues of partial sequence located in the cytoplasmic region of the human 'epiglycanin-related mucin-like glycoprotein of the present invention (17 amino acid residues that also have positions 580-596 in the amino acid sequence of SEQ ID NO: 171)
  • a synthetic peptide having cysteine added thereto was prepared.
  • Peptide Synthesis System (Pioneer) is used for peptide synthesis. Done by law.
  • F-moc amino acid used in the synthesis 0.4 mmol each of OH form (Applied Biosyst ems) was used.
  • the peptide was removed by reacting the resin with a peptide elimination reagent (0.7 g pheno 1, 0.
  • the synthetic peptide was purified using reverse layer HPLC (manufactured by JASCO Corporation).
  • the column was a 5C18 column (Cosmosil (trademark), Nacalai Tester), and the separation was performed at a column temperature of 40 ° C. and a flow rate of 2 mlZ min.
  • solvent A (0. 05% TFAZMilliQ (trademark) water
  • Jasco PU — 1580 pump trade name, JASCO Corporation.
  • Solvent B (0.
  • KLH (CalbioChem) was dissolved in 1 ml of 0.01 M sodium phosphate buffer ( ⁇ 7.2) and dialyzed overnight against the same buffer.
  • MBS solution (5 mg MBS (SIGMA) Z 17ml DMF (Wako Pure Chemical Industries)) was added to the dialyzed solution and shaken at room temperature for 30 minutes to bind KLH and MBS.
  • the reaction solution was subjected to gel filtration with an econo-pac GD10 column (trade name, Bio-Rad) using 0.05M phosphate buffer (pH 6.0). The absorbance of each fraction was measured to obtain a fraction containing KLH-MBS.
  • Japanese white rabbits (2 to 2.5 kg, female) were purchased from the Japan Institute for Animal Science, and were bred at the University of Tokyo School of Pharmacy. The experiment used a rabbit that had been bred for one week after purchase.
  • a 0.2 mg portion of peptide covalently bound to KLH and Freund complete adj uvant (NAKARAI CHEMICALS) were mixed to prepare an emulsion and administered subcutaneously to a rabbit. Immunization was carried out every 2 weeks, and the second and subsequent boosters were immunized by mixing Freund incompl ete adjuvant with 0.1 lmg of peptide covalently bound to KLH to prepare an emulsion.
  • blood was collected from the rabbit ear vein. After incubating at 37 ° C for 1 hour, the mixture was allowed to stand at 4 ° C for 1 hour for blood coagulation, centrifuged at 15, 0 OOrpm for 15 minutes, and the supernatant was used as serum.
  • the cell was surrounded with pap pen (trade name, Daido Sangyo) and then immersed in PBS for 5 minutes to swell.
  • 2% NGS and 3% BSA in PBS 100 / zl were added and blocking was performed for 30 minutes at room temperature.
  • the human Epigeglycanin-related mucin-like glycoprotein-specific rabbit serum 100 1 diluted 5000-fold with a PBS solution of 2% NGS and 3% BSA was added, and 1 ° C at 4 ° C. Reacted.
  • a 5 ⁇ m section was prepared with a microtome and adhered onto a MAS-coated glass slide (Matsunami Glass). Tissue sections were dried at 37 ° C for 1 cm and force was also used for staining.
  • the epiglycanin-related mucin-like glycoprotein of the present invention explains the transplantability of tissues in relation to histocompatibility antigens, or serves as an indicator of the presence of tumor and its malignancy. As well as providing protection against the tumor, it is useful in such applications in the diagnostic and pharmaceutical industries.
  • Fig. 1 shows confirmation of surface sugar chains of TA3-Ha cells and TA3-St cells.
  • TA3-Ha cells and TA3-St cells were stained with PNA and WA, and fluorescence was detected with a flow cytometer.
  • FIG. 2 shows the expression of various mucins in TA3-Ha cells and TA3-St cells.
  • Total RNA was extracted from TA3 Ha cells and TA3-St cells, and the presence or absence of expression of various mucins in the cells was confirmed by RT-PCR.
  • FIG. 3 shows the expression of the mucin-like glycoprotein of the present invention in TA3-Ha cells and TA3-St cells.
  • MRNA was extracted from TA3-Ha cells and TA3-St cells, and the presence or absence of expression of the mucin-like glycoprotein of the present invention was confirmed by Northern blotting.
  • a Electrophoresis with mini gel.
  • b migration on a large gel.
  • FIG. 4 shows detection of mucin-like glycoprotein gene of the present invention by PCR of mouse DNA.
  • DNA was extracted from the tails of C57BL / 6N mice and 129SVJ mice, respectively, and the mucin-like glycoprotein gene of the present invention was amplified by PCR.
  • FIG. 5 shows detection of a mucin-like glycoprotein gene of the present invention by Southern blotting of phage clone DNA.
  • DNA was extracted plaques hybrida I See Farr acquired by Chillon method Jikuron (a ⁇ f), after treatment with various restriction enzymes to detect the mucin-like glycoprotein gene of the present invention by Southern blotting.
  • FIG. 6 shows the structure of the mouse mucin-like glycoprotein of the present invention clawed.
  • FIG. 7 shows the expression of the mucin-like glycoprotein of the present invention in TA3-Ha cells.
  • RNA was extracted from Ha cells, and cDNA of the mucin-like glycoprotein of the present invention was confirmed by RT-PCR.
  • FIG. 8 shows detection of anti-epitm antibody and anti-epintm antibody in immunized rabbit rabbit serum. Specific antibodies in rabbit serum immunized several times with epitm peptide or epintm peptide were detected by ELISA using the peptide as an antigen. Solid diamonds and circles indicate results from post-immunization, blank diamonds and circles indicate results from pre-immune serum.
  • FIG. 9 shows the results of flow cytometry analysis of TA3-Ha cells and TA3-St cells using anti-epitm and anti-epintm polyclonal antibodies.
  • TA3-Ha cells and TA3-St cells were stained with each polyclonal antibody, and fluorescence was detected by flow cytometry.
  • FIG. 10 shows Western blotting of TA3-Ha cells and TA3-St cells with anti-epitm and anti-epintm polyclonal antibodies.
  • the cell extracts from TA3-11 ⁇ 2 cells and Dingpachi 3-St cells were electrophoresed and stained with a polyclonal antibody by Western blotting.
  • FIG. 11 shows the expression of the mucin-like glycoprotein of the present invention in C57BLZ6N mouse tissues. Extract total RNA from each tissue of C57BLZ6N mice and Northern blotting The presence or absence of expression of the mucin-like glycoprotein of the present invention was confirmed by the method.
  • FIG. 12 shows the expression of the mucin-like glycoprotein of the present invention in a mouse cancer cell line.
  • the mRNA was extracted from each mouse cancer cell line and the presence or absence of expression of the mucin-like glycoprotein of the present invention was confirmed by Northern blotting.
  • FIG. 13 shows the expression of the human mucin-like glycoprotein of the present invention in nine types of human cell lines.
  • Total RNA was extracted from each human cell line, and the presence or absence of expression of the human mucin-like glycoprotein of the present invention was confirmed by RT-PCR.
  • FIG. 14 shows the expression analysis results of the human 'mucin-like glycoprotein of the present invention in human normal and tumor tissues.
  • N indicates normal tissue
  • T indicates tumor tissue.
  • FIG. 15 shows the expression of the epiglycanin-related mucin-like glycoprotein of the present invention in normal human tissues.
  • the upper row is a probe for the gene of the present invention, and the lower row is a G3pdh probe.
  • FIG. 16 shows the expression of human 'epiglycanin-related mucin-like glycoprotein in transformed host cells.
  • A Flow cytometry analysis results (vertical axis is cell number, horizontal axis is fluorescence intensity).
  • B Western and lectin. Blot analysis results (values are in kDa).
  • Anti—FLAG mAb Anti-FLAG—M2 antibody.
  • VVA-B4 Vicia villosa agglutinin— B4 PNA: Arachis hvOogaea agrcun
  • K562 / MUC21 (MUC21) A cell transformed with the human epiglycan-related mucin-like glycoprotein of the present invention.
  • K562ZMock (Mock): transformed cells with control plasmid.
  • ⁇ 17 Shows specific antibody staining in a human 'epiglycanin-related mucin-like glycoprotein-expressing cell line.
  • A Staining result of negative control cells (K562ZMock) with rabbit serum before immunization.
  • B Staining result of a cell line (K562 / MUC21) that strongly expresses human epiglycanin-related mucin-like glycoprotein with pre-immune rabbit serum.
  • C Staining results of negative control cells with human 'epiglycanin-related mucin-like glycoprotein-specific rabbit antiserum.
  • D Staining results of strongly expressing cell lines with human ebbig licanin-related mucin-like glycoprotein-specific rabbit antiserum. Among the sites where expression was detected, representative ones are indicated by arrows.
  • FIG. 18 shows specific antibody staining in papillary thyroid cancer.
  • A Staining result of the tissue with pre-immune rabbit serum.
  • B Staining result of the tissue with human epiglycanin-related mucin-like glycoprotein specific rabbit antiserum. Of the sites where expression was detected, Is indicated by an arrow.

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Abstract

Le problème à résoudre dans le cadre de cette invention consiste à déclarer pour la première fois l’isolement d’une glycoprotéine de type mucine associée à l’épiglycanine et d’un acide nucléique encodant cette protéine. Il consiste également à clarifier pour la première fois l’existence d’un homologue humain de la protéine murine telle que décrite précédemment. La solution proposée consiste à fournir une protéine de type mucine ayant une séquence d’acides aminés représentée par la formule générale : A-Bn-C-D-E (où A représente une région N-terminale contenant une séquence signal ; Bn représente une région de séquences répétées en tandem avec la répétition d’une séquence d’acides aminés (B) qui est riche en sérine/thréonine fournissant des sites d’O-glycosylation et comprend entre environ 11 et environ 15 résidus d’acides aminés (n=20 à 150) ; C représente une région contenant un site de clivage enzymatique potentiel ; D représente une région transmembranaire montrant une forte homologie entre la souris et les humains ; et E représente une région intraplasmique).
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WO2021142245A1 (fr) * 2020-01-10 2021-07-15 Translate Bio, Inc. Composés, compositions pharmaceutiques et méthodes pour moduler l'expression de la muc5b dans des cellules et des tissus pulmonaires
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CN113063761B (zh) * 2021-03-17 2023-02-10 北京化工大学 一种用于检测muc1粘蛋白的荧光适配体传感器及其应用方法

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