WO2006082851A1

WO2006082851A1 - Mucin relating to epiglycanin

Info

Publication number: WO2006082851A1
Application number: PCT/JP2006/301666
Authority: WO
Inventors: Tatsuro Irimura; Kaori Denda; Mika Kamata; Atsuhiro Iguchi; Yuichi Itoh; Toshihisa Ogawa
Original assignee: The University Of Tokyo
Priority date: 2005-02-02
Filing date: 2006-02-01
Publication date: 2006-08-10
Also published as: JPWO2006082851A1

Abstract

[PROBLEMS] To declare for the first time the isolation of a mucin-like glycoprotein relating to epiglycanin and a nucleic acid encoding this protein. To clarify for the first time the existence of a human homolog of the mouse protein as described above. [MEANS FOR SOLVING PROBLEMS] It is intended to provide a mucin-like protein having an amino acid sequence represented by the general formula: A-Bn-C-D-E (wherein A represents an N-terminal region containing a signal sequence; Bn represents a tandem repeat region with the repetition of an amino acid sequence (B) which is rich in serine/threonine providing O-glycosylation sites and consists of from about 11 to about 15 amino acid residues (n=20 to 150); C represents a potential enzyme-cleavage site-containing region; D represents a transmembrane region showing a high homology between mouse and humans; and E represents an intraplasmic region).

Description

Epiglycanin-related mucin

Technical field

[0001] 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.

Background art

[0002] Mucins have traditionally been defined as highly o-glycosylated glycoproteins that are also secreted or expressed there. In addition, 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. Regarding 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.

[0003] For example, it has been observed that changes in the expression and glycosylation of MUC1, a human mucin, correlate with the degree of malignant progression of certain tumor cells. Therefore, 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 . Also for MUC16, 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. Furthermore, the cytoplasmic regions of MUC1 and MUC4 have been found to be involved in the regulation of cell sorting and proliferation.

On the other hand, it has been suggested that 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. Furthermore, the document tried to elucidate their sugar chain structure and suggested the existence of several kinds of O-linked sugar chains in the protein. TA3—Ha cell tribsin digest was applied to a Bio-Gel ™ P-100 column and then only purified on the crude fraction collected in its void volume. . 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. Although it was identified that it contains a lycosyl bond, this study has also been carried out on the same crude fraction as in Non-Patent Document 1, especially on the core and polypeptide chains of epiglycanin! There is no mention of complete isolation of the glycoprotein, which can give certain physical properties. Furthermore, 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 ™ 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.

[0005] The above interesting findings on the biological function of epiglycanin seem to be closely related to the shielding effect against histocompatibility antigen that would be consistent with the allograftability of TA3-Ha cells expressing the protein. (See Non-Patent Document 1). Regarding TA3—MM epidariin as well, the allograftability of TA3—MM breast cancer cells that express it is said to correlate with the abundance of the glycoprotein on the cell surface. It can be explained by the hypothesis of masking histocompatibility antigens on the cell surface (see Non-Patent Document 3). Also, in relation to the malignancy of tumors, which is also related to allograftability, 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 保護 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). ).

[0006] Thus, 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.

[0007] However, despite the fact that the initial reporting ability suggesting the existence of epiglycanin has been around 30 years, any prior literature will enable accurate characterization of the protein. The clear physico-chemical properties of such epiglycanin are not definitively disclosed, and there is no description of the isolation of the protein.

[0008] Furthermore, the prior art does not even suggest the existence of a human homologue of epiglycanin.

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. 171—182 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

Res., Vol. 51, p. 1406— 1411

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

Problems to be solved by the invention

[0009] 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.

Means for solving the problem

[0010] 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. In addition, further analysis of the 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. [0011] In view of the above, the above-mentioned characteristics of the mucin-like glycoprotein of the present invention are significantly different from those of the prior art, and therefore, both of them seem to have different powers at first glance. It seems quite reasonable to conclude that the mucin-like glycoprotein of the present invention is closely related to epiglycanin due to the characteristic expression of the protein in TA3-Ha cells and the presence of massive O-glycosyl sites. It seems to be. In other words, in the above-mentioned conventional technology, 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. However, because these studies did not necessarily involve isolated epiglycanin, it included uncertainty due to the lack of effort or reliance on simple electron microscopic image analysis. It is likely that an inaccurate conclusion regarding the physico-chemical properties of the epiglycanin molecule will be drawn.

[0012] Thus, 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):

A— Bn— C D— E (I)

[Where

A is

MRRRSSLWCWLLLQILLLGSGYA (SEQ ID NO: 1) or

MKMQKGNVLLMFGLLLHLEAA (SEQ ID NO: 2)

An 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:

Aaa —Aaa — Xaa — Xaa — Xaa — Xaa — Xaa — Xaa — Xaa — Xaa — Xa

1 2 3 4 5 6 7 8 9 10 a -Xaa —Xaa —Xaa —Xaa (SEQ ID NO: 3)

11 12 13 14 15

However, in the above sequence,

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.

2

Selected Xaa is independently selected as a group power consisting of serine, arginine and asparagine forces,

Three

Xaa is independent from the group consisting of threonine, serine, isoleucine, lysine and arginine

Four

Selected

Xaa is also selected independently of the group power of alanine, glycine, threonine and balinka,

Five

Xaa is independently selected as a group force consisting of serine and threonine forces,

6

Xaa is a group power consisting of glycine, arginine, aspartic acid, threonine, serine and alanine, an independently selected or unknown amino acid,

Whether Xaa is independently selected as a group force consisting of serine, threonine and tyrosine forces

8

Noic acid,

Xaa is threonine, methionine, serine, parin, alanine, asparagine, glutamine

9

Acid, lysine and isoleucine forces are group forces independently selected,

Xaa is independently selected from the group power of proline, threonine, serine and leucine.

Ten

Xaa independently selects the group power, including threonine, serine, asparagine, and isoleucine.

11

Selected

Xaa is proline, leucine, threonine, tryptophan, arginine, glutamine and

12

Serinka is a group power independently selected

Is Xaa independently selected from the group consisting of threonine, proline and glutamic acid?

13

Or does not exist,

Xaa is independently selected or absent from the group power of threonine and serine,

14

as well as

Xaa is independently selected from the group consisting of threonine, alanine, proline and asparagine

15

Or does not exist;

Xaa —Xaa — ^> er— Xaa —Xaa —Xaa —Xaa —Xaa — ^> er— Thr— X

21 22 23 24 25 26 27

aa -Xaa Xaa — Xaa — Xaa (SEQ ID NO: 4)

28 29 30 31 32

However, in the above sequence,

Xaa is independently selected from the group power consisting of arginine, methionine, and glycine power,

twenty one

Xaa is independently selected as a group power consisting of threonine and prolinker,

twenty two

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,

twenty four

Xaa is independently selected as a group force consisting of serine and feralanine forces,

twenty five

Xaa is independently selected as a group power consisting of alanine and balinka,

26

Xaa is independently selected from the group consisting of serine, phenylalanine and leucine;

27

Xaa is independently selected from the group consisting of threonine and asparagine;

28

Xaa is independently selected as a group power consisting of proline and serine,

29

Xaa is independently selected from the group consisting of threonine and alanine,

30

Xaa is independently selected from the group consisting of serine and threonine, and

31

Xaa is independently selected from the group power of isoleucine and serine; or

32

Xaa — Xaa — Xaa — Xaa — Xaa — Xaa — Xaa — Xaa — Xaa — Xaa

41 42 43 44 45 46 47 48 49

-Xaa —Xaa —Xaa —Xaa — Xaa (SEQ ID NO: 5)

50 51 52 53 54 55

Provided that at least one serine or threonine is contained in the sequence.

Xaa is independently selected from the group power consisting of serine, ferrolanine and threonine,

41

Xaa is independently selected from the group consisting of serine, asparagine, histidine and arginine.

42

Selected

Xaa is independently selected from the group power of Threonine, Norin and Serinka,

43

Xaa is from threonine, valine, proline, alanine, isoleucine and asparagine

44

Group power consisting of independently selected,

Xaa is independently selected as a group power consisting of serine and glutamate power,

45

Xaa is independently selected as a group power consisting of serine, asparagine, threonine, and alanine.

46

And

Xaa is independently selected as a group force consisting of glycine and serine

47

Xaa is independently from the group consisting of alanine, isoleucine, valine, threonine and serine.

48

Selected

Xaa is independently selected as a group force consisting of serine, glycine and asparagine forces,

49

Xaa is independently selected from the group consisting of threonine, isoleucine and alanine;

50

Xaa is independently selected as a group power consisting of alanine, norin, and asparagine,

51 Xaa is threonine or absent,

52

Xaa also has a group power consisting of asparagine, glycine, isoleucine and threonka independently.

53

Selected or absent

Xaa is serine or absent, and

54

Xaa is independently selected from the group consisting of glutamic acid, glycine and aspartic acid.

55

Or does not exist,

n is an integer from 20 to 150,

C is

KPWE (SEQ ID NO: 6) or

ALTGMHTTSHSASTAVSEAKPGGSLVPWE (SEQ ID NO: 7)

A potential enzyme cleavage site-containing region selected from

D is

IFLITLASVIWMGLSAGLFIYV (SEQ ID NO: 8) or

IFLITLVSWAAVGLFAGLFFCV (SEQ ID NO: 9)

A transmembrane region selected from

E is

RR,

MTRI (SEQ ID NO: 10) or

RNSLSLRNTFNTAV WFWRRPVSSIAMEMSGRNSGP (SEQ ID NO: 11)

Force A region in the cytoplasm that is selected. Or a functional derivative thereof.

In another embodiment of the present invention, in the general formula (I), 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. In particular, B can be selected independently from the amino acid sequence found in mice, ie SEQ ID NO: 12 to SEQ ID NO: 138.

HISSTTTSNPSSE (SEQ ID NO: 12)

TKSIATDYETTPTTT (SEQ ID NO: 13)

ISSTASGSTPTLTTT (SEQ ID NO: 14)

ASSIASDSKPTLTTT (SEQ ID NO: 15)

ASSTASGSMSTPTTP (SEQ ID NO: 16)

ASSTASGSTPTTTTT (SEQ ID NO: 17)

ASSTASGSTPTPTTT (SEQ ID NO: 18)

ASSKASRSVPTT (SEQ ID NO: 19)

VSSTGSGSTPTPTTT (SEQ ID NO: 20)

ASSTASGSTPTLTTT (SEQ ID NO: 21)

PSSTASGSTPTPTTP (SEQ ID NO: 22)

ASSTASSSSPTPTTP (SEQ ID NO: 23)

VSSTASGSTPTPTTT (SEQ ID NO: 24)

ASSKASGSVPTT (SEQ ID NO: 25)

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)

ASSSASGSTPTLTTT (SEQ ID NO: 31)

ESSTASGSTPTWTTT (SEQ ID NO: 32)

TS STASRSTPTPTTT (SEQ ID NO: 33)

ASSTASGSTPTPTTT (SEQ ID NO: 34)

VSSTGSGSTPTLTTT (SEQ ID NO: 35)

ASRRGSGSTPTLTTT (SEQ ID NO: 36)

ESSTGSGSTPTLTTT (SEQ ID NO: 37) ASSSGSGSTPTLPTT (SEQ ID NO: 38) ESSTASGSTPTRTTT (SEQ ID NO: 39) TS STASRSTPTPTTT (SEQ ID NO: 40) ASSTASGSTPTPTTT (SEQ ID NO: 41) VSSTASGSTPTLTTT (SEQ ID NO: 42) ASRSGSGSTPTLTTT (SEQ ID NO: 43) ESSTASGSTPTPTTT (SEQ ID NO: 44) ASSTASGSAPNPTTT (SEQ ID NO: 45) VSSTGSGSTPTLTTT (SEQ ID NO: 46) ASSSGSGSTPTLTTT (SEQ ID NO: 47) ESSTASGSTPTLTTT (SEQ ID NO: 48) ASSSASGSMPTPTTT (SEQ ID NO: 49) VSSTGSGSTPTLTTT (SEQ ID NO: 50) ASSSASGSMPTPTTT (SEQ ID NO: 51) ASSG (SEQ ID NO: 52) ASSSASGSAPNPTTT (SEQ ID NO: 53) VSSTGSXXTPTPTTT (SEQ ID NO: 54) ASSKASGSVPTT (SEQ ID NO: 55) VSSTGSGSTPTLTTT (SEQ ID NO: 56) VSSTVSDSTPTPTTT (SEQ ID NO: 57) ASSTASGSAPTPTTT (SEQ ID NO: 58) ASSTASGSTPTLTTT ( SEQ ID NO: 59) ASSSGSGSTPTLPTT (SEQ ID NO: 60) ESSTASGSTPTRTTT (SEQ ID NO: 61) TS STASRSTPTPTTT (SEQ ID NO: 62) ASSTASGSTPTPTTT (SEQ ID NO: 63) VSSTGSGSTPTLTTT (SEQ ID NO: 64) ASRSGSG STPTLTTT (SEQ ID NO: 65) ESSTASGSSPTLTTT (SEQ ID NO: 66) ASSSASGSMPTPTTT (SEQ ID NO: 67) ASSTASGSSPTLTTT (SEQ ID NO: 68) ASSSASGSMPTLTTT (SEQ ID NO: 69) ASSSASGSSPTLTTT (SEQ ID NO: 70) ASSSASGSMPTPTTT (SEQ ID NO: 71) VSSTGSGSTPTLTTT (SEQ ID NO: PT) ESSG (SEQ ID NO: 73) TS STASRSTPTPTTT (SEQ ID NO: 74) ASSTASGSTPTPTTT (SEQ ID NO: 75) VSSTGSGSTPTLTTT (SEQ ID NO: 76) ASRSGSGSTPTLTTT (SEQ ID NO: 77) ESSTASGSIPTLTTA (SEQ ID NO: 78) ASSTASGSTPTPTTT (SEQ ID NO: 79) ASSTASGSMPTPTTT (SEQ ID NO: 80) ASSTGSGSTPTLTTT (SEQ ID NO: 81) ASSSGSGSTPTLTTT (SEQ ID NO: 82) ESSTASGSIPTLTSA (SEQ ID NO: 83) ASSSASGSMPTPTTT (SEQ ID NO: 84) VSSTGSGSTPTLTTT (SEQ ID NO: 85) ASSSASGSMPTPTTT (SEQ ID NO: 86) ASSTASGSSPTLTTT SEQ ID NO: 87) ASSSASGSAPNPTTT (SEQ ID NO: 88) VSSTGSGSTPTLTTT (SEQ ID NO: 89) ASSSGSGSTPTLPTT (SEQ ID NO: 90) ESSTASGSTPTRTTT (SEQ ID NO: 91) TS STASRSTPTPTTT (SEQ ID NO: 92) A SSTASGSTPTPTTT (SEQ ID NO: 93) VSSTASGSTPTLTTT (SEQ ID NO: 94) ASRSGSGSTPTLTTT (SEQ ID NO: 95) ESSTASGSTPTPTTT (SEQ ID NO: 96) ASSTASGSAPNPTTT (SEQ ID NO: 97) VSSTASGSTPTLPTT (SEQ ID NO: 98) ASSSGSGSTPTLTTT (SEQ ID NO: 99) ESSTASGSSPTLTTT (SEQ ID NO: 100) TTSASGS (SEQ ID NO: 101) ASSTASGSSPTLTTT (SEQ ID NO: 102) ASSSASGSMPTLTTT (SEQ ID NO: 103) ASSSASGSSPTLTTT (Care IJ number: 104) ASSSASGSMPTPTTT (SEQ ID NO: 105) VSSTGSGSTPTLTTT (SEQ ID NO: 106) ESSTASGSTPTWTTT (SEQ ID NO: 107) TSSTASRSTPTPTTT (SEQ ID NO: 108) ASSTASGSTPTPTTT (SEQ ID NO: 109) VSSTGSGSTPTLTTT (SEQ ID NO: 110) ASRRGSGSTPTLTTT (SEQ ID NO: 111) ESSTGSGSTPTLTTT (SEQ ID NO: 112) ASSSGSGSTPTLPTT (SEQ ID NO: 113) ESSTASGSTPTRTTT (SEQ ID NO: PT) TSSTARST (SEQ ID NO: 115) ASSTASGSTPTPTTT (SEQ ID NO: 116) VSSTASGSTPTLTTT (Alpha lj number: 117) ASRSGSGSTPILTTT (SEQ ID NO: 118) ESSTASGSTPTLTTA (SEQ ID NO: 119) ASSSASGSTPTPTTT ( IJ number: 120) VSSTGSGSTPTLTTT (SEQ ID NO: 121) ASSSGSGSTPTLTTT (SEQ ID NO: 122)

ESSTASGSTPTQTTT (SEQ ID NO: 123)

TSSTASRSTPTPTTT (SEQ ID NO: 124)

ASSTASGSIPTPTTT (SEQ ID NO: 125)

ASSIASGTTPTLTTT (SEQ ID NO: 126)

ESSTASGSSPTPTTA (SEQ ID NO: 127)

SSSSASDSKPTSTTT (SEQ ID NO: 128)

ASSTVSDSTPTPTTN (SEQ ID NO: 129)

ASSSASGSTPTQTTT (SEQ ID NO: 130)

ASRSASGSVPTLTTI (SEQ ID NO: 131)

AFSTASGSTPTLTTT (SEQ ID NO: 132)

ASNSASSSSPTPTTT (SEQ ID NO: 133)

GLSKTSASSLSLTST (SEQ ID NO: 134)

MTSITSASTFTPASI (SEQ ID NO: 135)

RTSKASASTSTSASI (SEQ ID NO: 136)

RTSIAFASTSTPTTS (SEQ ID NO: 137)

GPSTASVSTLNSTSI (SEQ ID NO: 138)

(In the above sequence, X represents an undefined amino acid.)

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

n

999T0C / 900Zdf / X3d TS8Z80 / 900Z OAV

NVVEMTRI (SEQ ID NO: 139) (in the above sequence, X represents an undefined amino acid).

[0015] The present invention further relates to a human homologue of a mouse 'epiglycanin-related mucin-like glycoprotein. To the best of the inventors' knowledge, to date, the existence of a human homologue of epiglycanin has even been suggested. Furthermore, 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

[0016] Therefore, the inventors tried a homology search focusing on various fragments from the region other than the tandem repeat region in the murine epiglycanin-related mucin-like glycoprotein of the present invention. As a result, surprisingly, only very high homology to the amino acid sequence of the transmembrane region of the mouse 'epiglycanin-related mucin (identity 47%, positive rate 55%): program default parameters (matrix = Blosum62 ; Search using the existing cost of gap = 11, gap extension cost = 1) shows the Internet site http: Z / www. Ncbi. N / m. Nih. A cDNA (approximately 2.5 kb) encoding a “theoretical” secreted protein was searched.

In other words, 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.

[0018] It should be noted that the reported amino acid sequence of the "theoretical" secreted protein was inconsistent with that actually obtained in the present invention in at least 37 amino acid residues.

[0019] Therefore, in another aspect of the present invention, a human-type epiglycanin-related mucin-like glycoprotein is provided, wherein the human-type epiglycanin-related mucin is represented by the sequence number: 2 in the above general formula (I). Yes, B is SEQ ID NO: 5, C is SEQ ID NO: 7, D is SEQ ID NO: 9, E is SEQ ID NO: 11, and n is an integer from 20 to 50 Can be defined.

[0020] In particular, B is found in human homologues and is rich in serine Z threonine.

An amino acid sequence consisting of 15 amino acid residues, ie SEQ ID NO: 140 to SEQ ID NO: 170, can be independently selected.

TNSNETSTSANTG (SEQ ID NO: 140)

SSVISSGASTATNSG (SEQ ID NO: 141)

SSVTSSGVSTATISG (SEQ ID NO: 142)

SSVTSNGVSIVTNSE (SEQ ID NO: 143)

FHTTSSGISTATNSE (SEQ ID NO: 144)

FSTASSGISIATNSE (SEQ ID NO: 145)

SSTTSSGASTATNSE (SEQ ID NO: 146)

SSTPSSGASTATNSD (SEQ ID NO: 147)

SSTTSSGASTATNSD (SEQ ID NO: 148)

SSTTSSGASTATNSE (SEQ ID NO: 149)

SSTTSSGASTATNSE (SEQ ID NO: 150)

SSTASSGASTATNSE (SEQ ID NO: 151)

SSTTSSGASTATNSE (SEQ ID NO: 152) SSTTSSGASTATNSE (SEQ ID NO: 153)

SSTTSSGASTATNSE (SEQ ID NO: 154)

SRTTSNGAGTATNSE (SEQ ID NO: 155)

SSTTSSGASTATNSE (SEQ ID NO: 156)

SSTPSSGAGTATNSE (SEQ ID NO: 157)

SSTTSSGAGTATNSE (SEQ ID NO: 158)

SSTVSSGISTVTNSE (SEQ ID NO: 159)

SSTPSSGASTATNSE (SEQ ID NO: 160)

SSTTSSGASTATNSD (SEQ ID NO: 161)

SSTTSSGASTATNSE (SEQ ID NO: 162)

SSTTSSGASTATNSE (SEQ ID NO: 163)

SSTTSSGASTATNSG (SEQ ID NO: 164)

SSTTSSGTSTATNSE (SEQ ID NO: 165)

SSTVSSGASTATTSE (SEQ ID NO: 166)

SSTTSSGASTATNSE (SEQ ID NO: 167)

SSTVSSGASTATNSE (SEQ ID NO: 168)

SSTTSSGANTATNSG (SEQ ID NO: 169)

SSVTSAGSGTA (SEQ ID NO: 170)

In a more specific embodiment, the human 'epiglycanin-related mucin-like glycoprotein has the following amino acid sequence: MKMQKGNVLLMFGLLLHLEAATNSNETSTSANTGSSVI

It is understood that it has 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.

[0021] In another aspect of the present invention, there is provided a 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. Alternatively, 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.

That is, the nucleic acid has an amino acid sequence represented by the following general formula (I)

A-Bn-C-D-E (I)

[Where

A is

MRRRSSLWCWLLLQILLLGSGYA (SEQ ID NO: 1) or

MKMQKGNVLLMFGLLLHLEAA (SEQ ID NO: 2)

An 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:

1 2 3 4 5 6 7 8 9 10 a -Xaa —Xaa —Xaa —Xaa (SEQ ID NO: 3)

11 12 13 14 15

However, in the above sequence,

2

Selected

Xaa is independently selected as a group power consisting of serine, arginine and asparagine forces,

Three

Xaa is independent from the group consisting of threonine, serine, isoleucine, lysine and arginine Selected

Five

6

8

Noic acid,

Xaa is threonine, methionine, serine, parin, alanine, asparagine, glutamine

9

Acid, lysine and isoleucine forces are group forces independently selected,

Ten

11

Selected

Xaa is proline, leucine, threonine, tryptophan, arginine, glutamine and

12

Serinka is a group power independently selected

13

Or does not exist,

14

as well as

15

Or does not exist;

Xaa —Xaa — ^> er— Xaa —Xaa —Xaa —Xaa —Xaa — ^> er— Thr— X

21 22 23 24 25 26 27

aa -Xaa Xaa — Xaa — Xaa (SEQ ID NO: 4)

28 29 30 31 32

However, in the above sequence,

twenty one

twenty two

Xaa is independently selected from the group power of isoleucine, lysine and threonka,

twenty three

Xaa is independently selected as a group force consisting of alanine and threonine forces,

twenty four

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,

26

27

Xaa is independently selected as a group force consisting of threonine and asparagine forces,

28

Is a group power independently composed of proline and serinka,

29

Xaa is independently selected as a group power consisting of threonine and alanine power,

30

Xaa is independently selected as a group force consisting of serine and threonine forces, and

31

Xaa is independently selected from the group power of isoleucine and serine; or

32

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

41 42 43 44 45 46 47 48 49

-Xaa -Xaa —Xaa —Xaa —Xaa (SEQ ID NO: 5)

50 51 52 53 54 55

Provided that at least one serine or threonine is contained in the sequence.

Xaa is independently selected as a group power consisting of serine, ferrolanine and threonine power,

41

42

Selected

43

Xaa is from threonine, valine, proline, alanine, isoleucine and asparagine

44

Independently selected from the group consisting of

45

Xaa is independently selected as a group force consisting of serine, asparagine, threonine, and alanine.

46

And

Xaa is independently selected as a group force consisting of glycine and serine

47

48

Selected

49

50

51

Xaa is threonine or absent,

52

Xaa is independently from the group consisting of asparagine, glycine, isoleucine and threonine. Selected or absent

Xaa is serine or absent, and

54

55

Or does not exist,

n is an integer from 20 to 150,

C is

KPWE (SEQ ID NO: 6) or

ALTGMHTTSHSASTAVSEAKPGGSLVPWE (SEQ ID NO: 7)

A potential enzyme cleavage site containing region selected by

D is

IFLITLASVIWMGLSAGLFIYV (SEQ ID NO: 8) or

IFLITLVSWAAVGLFAGLFFCV (SEQ ID NO: 9)

A transmembrane region selected from

E is

RR,

MTRI (SEQ ID NO: 10) or

RNSLSLRNTFNTAV WFWRRPVSSIAMEMSGRNSGP (SEQ ID NO: 11)

Force A region in the cytoplasm that is selected. Or a functional derivative thereof, or is complementary to the encoding nucleic acid.

In a preferred embodiment, corresponding to a mucin-like glycoprotein derived from mouse, in the above general formula (I), 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. . [0024] Alternatively, in the above general formula (I), A is SEQ ID NO: 2, B is SEQ ID NO: 5, and 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.

[0025] In certain embodiments related to the nucleic acid, the nucleic acid corresponds to SEQ ID NO: 172 (mouse) or SEQ ID NO: 174 (human).

999T0C / 900Zdf / X3d TS8Z80 / 900Z OAV

ov o voovo ooo o oovoo voo oooovoovo

91

999T0C / 900Zdf / X3d TS8Z80 / 900Z OAV

CATCCAACAT (SEQ ID NO: 172); or

999T0C / 900Zdf / X3d TS8Z80 / 900Z OAV

TC (SEQ ID NO: 174)

Thus, 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.

[0026] Furthermore, the present invention includes an antibody specific for the above-mentioned epiglycanin-related mucin-like glycoprotein. In particular, 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.

[0027] Alternatively, 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.

[0028] In a further aspect of the present invention related thereto, there is provided a method for detecting gene expression of the epiglycanin-related mucin-like glycoprotein of the present invention. As shown here, detection of the gene expression can be used to diagnose the presence of tumor cells and their malignancy! Can be advantageously used. In a particular embodiment, 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

CCCACAGGCCCAGGTGGAGTCCTAACTGGTTC-3 ′ (SEQ ID NO: 176) All or part of such a 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.

BEST MODE FOR CARRYING OUT THE INVENTION

[0030] As described above, 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). Here, A is an N-terminal region containing a signal sequence; Bn is an amino acid sequence (B) rich in serine Z threonine that provides an O-glycosylation site and consisting of about 11 to about 15 amino acid residues (n = 20-150) Tandem repeat region; C is a region containing a potential enzyme cleavage site; D is a transmembrane region showing high homology between mouse and human; and E is an intracytoplasmic region.

[0031] Preferably, A is selected from SEQ ID NO: 1 (mouse type) or SEQ ID NO: 2 (human type), and 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), One skilled in the art will readily appreciate that any combination of the above is possible in light of the specific function of each region and the structural features that would be required for an epiglycan-like glycoprotein. Let's go.

[0032] Here, 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. One skilled in the art will also appreciate that structural features need only be met. Thus, 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. Can be defined by SEQ ID NO: 3 to SEQ ID NO: 5. More preferably, it is selected from SEQ ID NO: 12 to SEQ ID NO: 138 (mouse type) or SEQ ID NO: 140 to SEQ ID NO: 1 70 (human type).

[0033] However, in view of the functional characteristics of the repeat unit and the biochemical action of the entire tandem repeat region, 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.For example, 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).

[0034] The present invention further includes functional derivatives of the above mucin-like glycoproteins. Here, 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. The term “fragment” is intended to refer to any polypeptide subset of an epiglycanin-related mucin-like glycoprotein. The term “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. The term “analog” refers to a molecule that is functionally substantially similar to a complete epididal forcenin-related mucin-like glycoprotein molecule or fragment thereof.

[0035] 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. The amino acid sequence homology of The homology of the amino acid sequences mentioned here is the result of searching using the program default parameters (matrix = Blosum62; gap existence cost = 11, gap extension cost = 1) on the Internet site http: //www.ncbi. n / m. nih. govZegi — can be defined as the percentage of positives shown by the BLASTP algorithm, which can be implemented with ginZBLAST. 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.

[0036] The tandem repeat region of the epiglycanin-related mucin-like glycoprotein of the present invention includes Codington, JF, Linsley, Lin. Β., Jeanloz, RW, Irimura, Τ., 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 structure of the sugar chain is described in Van den Eijnden, DH, Evans, NA, Codington, JF, Reinhold, V., Silber, C., and Jeanloz, RW (1979) T. Biol. Chem., Vol. 254, No. 23, p. 12 153-12159. Therefore, the epiglycanin-related mucin-like glycoprotein of the present invention includes _α 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

[0037] [Chemical 1]

¹³ One ^Gal — ( ^{1 → 4} )-— D— Gl cAc- (l → 3)-β -D-Gal- (l → 3) One a -D-GalNAc-Ser (Thr)

^† T

(2 → 3) (2 → 6) a -NeuNAc

A sugar chain structure containing a-NeuNAc may be added.

[0038] From this information and analysis of the amino acid sequence of the tandem repeat region, 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. In addition, 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. However, since it is known that the actual amount of sugar chain binding etc. is influenced by the state of individual cells and the culture conditions, etc., the isolated gel of the epiglycanin-related mucin-like glycoprotein of the present invention is isolated. Electrophoretic prayer can give a lower molecular weight smear than the maximum molecular weight force. Such proteins with different sugar chain compositions and binding amounts are also within the scope of the present invention.

[0039] The present invention includes a nucleic acid encoding an epiglycanin-related mucin-like glycoprotein or a nucleic acid complementary to the nucleic acid. Particularly preferred nucleic acids include the sequence shown in SEQ ID NO: 172 or SEQ ID NO: 174. As used herein, the term “nucleic acid” intends cDNA, genomic DNA, and synthetic (eg, chemical synthesis or modification) DNA or RNA. In some cases, 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. Furthermore, 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.

[0040] Various codons encoding specific amino acids are known to have redundant codons. Therefore, the present invention is finally translated into the same amino acid. It relates to DNA sequences that contain alternative codons. That is, since the genetic code is degenerate, multiple codons can be used to encode a particular amino acid, so that the amino acid sequence can be encoded with any set of similar DNA 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.

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). As the antibody used in the immunoscreening method, the antibody of the present invention described later can be used. The DNA of the present invention can also be obtained by PCR using a genomic DNA library or a cDNA library as a cage. For PCR, 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. As the DNA library used in the above various methods, a DNA library having the DNA of the present invention is selected and used. As the DNA library, 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.

[0042] 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.

[0043] Vectors containing such nucleic acids of the invention are also included within the scope of the invention. For example, 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.

That is, 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. Using such a vector, bacteria, cyanobacteria are used. In addition, 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.

[0045] 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. In addition, 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 α mating promoter, etc.

It can be used depending on the properties of the host cell. Expression vectors include, but are not limited to, cloning vectors, modified cloning vectors, specially designed plasmids or viruses.

[0046] Various mammalian expression vectors can be used to express the epidermal forcenin-related mucin DNA of the present invention in mammalian host cells. In addition, 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. In addition, 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. For example, for bacterial hosts, pBR322 and pUC vectors with colicin E1-type replication initiation mechanism, λ phage derivatives such as M13, yeast For cells, there are 2 plasmids, for eukaryotic hosts, SV40, ushi papilloma virus, adenovirus and cytomegalovirus, a vector for expression control sequence, and for insects, PVL941.

[0047] 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.

[0048] 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. [0049] After the expression of 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. Several purification methods are available, such as cell lysates, by salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxyapatite adsorption chromatography, various combinations of hydrophobic interaction chromatography, or individual applications. From the cell extract, recombinant shrimp glycanin-related mucin-like glycoprotein can be purified.

[0050] Furthermore, using an immunoaffinity column produced with a polyclonal or monoclonal antibody specific for the epiglycanin-related mucin-like glycoprotein of the present invention, the recombinant ebiglycanin-related mucin-like glycoprotein is converted to other cellular protein caps. You can separate them.

[0051] Thus, 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. For example, 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. Such methods are described, for example, in CURRENT PROTOC OLS IN IMMUNOLOGY, Chapter 2.4, John Wiley & Sons, Inc., New York. Subsequently, 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.

[0052] 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.

[0053] When the antibody of the present invention is obtained as a monoclonal antibody, a laboratory animal immunized with the above-described epiglycanin-related mucin-like glycoprotein of the present invention or a partial peptide thereof using a technique well known to those skilled in the art. Preferably, 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. Next, the fused cells are cultured in vitro or in vivo, and a highly specific monoclonal antibody is collected from the culture mixture.

[0054] 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. Examples of such fragments include Fab fragments, Fab, fragments, F (ab ') fragments, F (v) fragments, H chain monomers or dimers

2

, 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.

[0055] 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.

[0056] Taking the example of Sandwich powder, which is one of the representative techniques for the detection, first, the labeled antibody is prepared. For example, antibodies can be labeled with radioactive substances, colored particles such as colloidal gold, fluorescent or chemiluminescent labels, or enzymes (for ELISA). wear. As 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. In addition, in the case of Sandwich, it is preferable that the antibody is immobilized on a solid support. For example, 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. If necessary, 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. After the inside of the well is thoroughly washed with a suitable buffer or the like, 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. In the example of the ELISA method, enzymes such as horse radish peroxidase and alkaline phosphatase, which are not particularly limited, are advantageously used. In the case of labeling with horse radish peroxidase, 3,3 ′, 5,5′-tetramethylbenzidine or the like can be used as a substrate for the enzyme. When alkaline phosphatase is used, p-trope phosphate can be cited as a substrate.

[0057] The ability of test subjects Another method suitable for detecting the expression of an epiglycanin-related mucin-like glycoprotein in a collected biological sample, particularly for detecting the expression of the protein as a tumor marker, is the mRNA in the sample. Measure the presence and amount of

[0058] That is, the detection method typically corresponds to the Northern blotting method. First, 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. As for RT-PCR, so-called competitive RT-PCR can also be used.

Furthermore, for the purpose of such amplification, 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

[0060] The use of the kit 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. In addition, 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.

[0061] Without further explanation, those skilled in the art given the above explanations fully utilize the present invention. obtain. The following examples are given for illustrative purposes only. In the present specification, unless otherwise specified, amino acid sequences are described in the direction of N-terminal force in the C-terminal direction, and nucleotide sequences in the direction of 5′-end force 3′-end.

Example 1

[0062] Example 1: Identification of murine-like epiglycanin-related mucin-like glycoprotein

1-1 · Confirmation of the presence of mucin-like glycoprotein レ by lectin

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 ™ 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 8-1½ cells are collected every 3-4 days and renewed

2

And subcultured in a fresh medium. 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. In some experiments, 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.

[0063] Next, it was confirmed whether or not a novel mucin-like glycoprotein was expressed in the cells cultured in this manner. That is, since TA3-Ha cells have been reported to be rich in T antigen and Tn antigen (for example, Non-Patent Document 7: Springer, GF (1984) Science. Vol. 224, p. 1198-1206). ), The cells were stained using biotin-VVA (recognizing T antigen) and biotin-PNA (recognizing Tn antigen), which are lectins that recognize each sugar chain. In the measurement, 0.5 to 1 X 10 ⁶ cells were washed with FCM buffer and diluted with FCM buffer. Then, biotin- PNA (1/100: VECTOR) or biotin-VVA ( lZ200: VECTOR) was 100 _i ul mosquitoes卩tut, reacted under ice-cooling for 30 minutes. Then 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. 1, high binding of both VVA and PNA was observed in Ha cells, and it was confirmed that many T antigens and Tn antigens were added. On the other hand, neither lectin binding was observed in St cells. These facts suggested that Ha cells expressed mucins with many sugar chains, whereas St cells expressed such molecules.

[0064] Using the Western blotting method, 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 Μ PMSF (iso

2

(In propyl alcohol) 500 μ1 was washed twice with 500 ml of MilliQ water). Add 5% NP-40 (SIGMA¾: ¾) / desalting buffer, stir well, solubilize at 4 ° C for 1 hour or more, and centrifuge at 15 ° C for 4 minutes at 4 ° C. Kiyoshi was used as a cell extract. The protein concentration of the extract was measured with a BCA protein quantitative reagent (Pierce).

[0065] 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. Further, 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.

[0066] In Ha cells, the cells were stained with partial forces PNA and WA (data not shown) greater than 300 kDa. On the other hand, for St cells, the high molecular weight part is not stained at all. I got it. These facts suggest that high-molecular weight proteins accompanied by T antigen or Tn antigen are expressed in Ha cells, which is considered to be mucin.

[0067] 1— 2. Confirmation of each mucin expression

Based on the above experiment, it was predicted that ΤΑ3-Ha cells would become TA3-St cells! /, And that mucin was expressed! / ヽ, so whether or not this mucin is new has been crawled so far. This was confirmed by RT-PCR of various mouse mucins.

[0068] Specifically, total RNA was extracted from TA3-Ha cells and TA3-St cells using ULTRASPEC RNA (trade name, manufactured by BIOTECX), phenol was removed with chloroform, and then precipitated with isopropanol. Thereafter, the sample was dissolved in MilliQ water and the absorbance 260 nm and Z280 nm were measured to estimate the RNA amount and purity. Next, 1 μg of the extracted total RNA was made into a saddle shape, and a single-stranded cDNA was prepared by a conventional method, and then each RT product was subjected to PCR. In this experiment, it was confirmed in advance that the density of the band observed by the PC8 of j8-actin was the same between Ha cells and St cells as an internal control, and the following primers were used for each mouse mucin. And conditions applied.

[0069] Mouse β

Sense: 5, — GCTCCTCGTTGCCGGTCCACA— 3, (SEQ ID NO: 179) Antisense: 5,-TAGG AGCC AG AGC AGTAATCTCCT-3, (SEQ ID NO: 18

0)

Mouse Mucl

Sense: 5,-AGGCTCCGTGGTGGTAG AAT-3 '(SEQ ID NO: 181) Antisense: 5, — GGCATGAACAGAATACCAGA— 3, (SEQ ID NO: 182) Mouse Muc2

Sense: 5,-CAATGACAAGGTGTCCTGCC-3 '(SEQ ID NO: 183) Antisense: 5, — GTGCTCTCCAAACTCTCTGG— 3, (SEQ ID NO: 184) Mouse Muc3

Sense: 5,-CCGATGTCACCACTTCTGCTG-3 '(SEQ ID NO: 185) Antisense: 5,-GCAGAGCAAGGCGTG ATACAG-3' (SEQ ID NO: 186) Mouse Muc4 Sense: 5, -TGATGGAACAACCACCTCAC-3 '(SEQ ID NO: 187) Antisense: 5,-GGATGCAGGTGAGGTATTC-3' (SEQ ID NO: 188) Mouse Muc5AC

Sense: 5,-CCAGCACCACCTATACAACC-3 '(SEQ ID NO: 189) Antisense: 5, — GGTGACAGAGTCTCTCTCCCC-3, (SEQ ID NO: 190)

Mouse Muc9

Sense: 5, — CATCCCAAGCATCATCCATAC-3, (SEQ ID NO: 191) Antisense: 5, — GATTCTGTCTGCACCAAGAGC-3, (SEQ ID NO: 192) Mouse MuclO

Sense: 5, — GC AAC AACTG ACTGTTCC AAA-3, (SEQ ID NO: 193) Antisense: 5, — GCG ACTG ATC AGC ATTAAGGT-3, (SEQ ID NO: 194) In other words, in the above experiment, After preparing each reaction solution in the same manner, using 1 μ1 of 5 times diluted template, heat denaturation at 95 ° C for 9 minutes, and then 95 mMuc9. C for 30 seconds, 50. C for 30 seconds, 72. C for 1 minute 45-55 cycles, 72. The reaction was carried out with C for 10 minutes. For mucins other than mMuc9, perform annealing at 95 ° C for 30 seconds, annealing for 1 minute, 72 ° C for 1 minute and 30 seconds in 35-55 cycles, and 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 μl of the reaction product was run on 1% Seakem ME agarose containing 0.5 μg Zml ethylene bromide, and the band was confirmed.

[0070] The results are shown in FIG. It was shown that mMucl, mMuc2 and mMuc5AC are expressed in large amounts in St cells. In addition, mMuc3 and mMuclO were not expressed in both. On the other hand, it was found that mMuc4 and mMuc9 were also expressed at high frequency in Ha cells. Nevertheless, other mucin-like glycoproteins are specifically expressed in Ha cells because the molecular weights of these mucins are different from those predicted in the literature. I couldn't deny that

[0071] 1— 3. Acquisition and decoding of cDNA fragment by subtraction method

mMuc4 and mMuc9! /, expression level between TA3-1-1½ cells and Ding-8-3-St cells However, if the expression level of the mucin-like glycoprotein of the present invention is still significantly different between the two, the TA3-H It was anticipated that it would be possible to obtain a highly expressed cDNA fragment.

[0072] Thus, mRNA was purified from each of Ha cells and St cells, and a double-stranded cDNA library was prepared by reverse transcription reaction.

[0073] Specifically, 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 μg of the obtained mRNA as a saddle and performing RT reaction from Oligo d T primer using Timesaver ™ cDNA synthesis kit (Amersham Bioscience). cDNA was prepared. NICK (trademark) column (manufactured by Amersham Bioscience) 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). Phenolic (Wako Pure Chemical Industries): Chloroform = 1: 1: The protein was removed three times, and after precipitation with EtOH, it was dissolved in MilliQ water to obtain a double-stranded cDNA library.

[0074] 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. As a result, only the cDNA present in excess in Ha cells was amplified, and a slightly dark band-like portion was observed in the smear. 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).

[0075] Specifically, the above two synthesized from mRNA of TA3-Ha cells and TA3-St cells Each strand cDNA library was treated with Sau3AI, and the resulting fragment was subjected to R Bgl 24: 5, — AGCACTCTCCAGCCTCTCACCGCA—3, using the TAKARA DN A Ligation Kit ver. 2 (trade name, manufactured by TAKARA). 195), and R Bgl 12: 5′-G ATCTGCGGTG A-3, (SEQ ID NO: 196) was ligated.

[0076] The above ligation product was divided into 30 equal parts as templates, and 10 1 10 X PCR buffer (15 mM MgCl 2, 10 ^ 1 dNTP mix (each 2 mM))

2, 60 pmolZ 1 R Bgl 24, 0

. 5 ^ 1 AmpliTaq (trademark) DNA Polymerase (Roche) containing) MilliQ water is added to make 100 1 into PCR tube, using thermal cycler at 72 ° C for 5 minutes, then 95 ° The reaction was performed at C for 1 minute, 72 ° C for 3 minutes for 20 cycles, and 72 ° C for 10 minutes. Of the reaction products, 51 was electrophoresed in 3% Nusieve agarose (manufactured by TAKARA) containing 0.5 g / ml ethylene bromide to confirm whether the PCR reaction was successful.

[0077] The above PCR product was treated with phenol: chloroform, and after EtOH precipitation, dissolved in TE (lm M Tris-HCl pH 8.0, 0. ImM EDTA). Sau3AI is removed from the PCR product by removing the R Bgl 24 adapter, dissolved in TE after EtOH precipitation, electrophoresed on 3% Nusieve agarose, excised from 100 to 1500 bp, and DNA extracted with QIAEX II gel ext raction kit Extracted. The extracted DNA was treated with phenol: chloroform, dissolved in MilliQ water after EtOH precipitation, the absorbance was measured, and the amount of DNA was estimated. TA 3—St cells were used as driver amplicons for future RDA.

[0078] For TA3—Ha cells, using TAKARA DNA Ligation Kit ver. 2, J Bgl 24: 5 '-ACCG ACGTCG ACTATCC ATG AAC A-3 (SEQ ID NO: 197) Bgl 12: 5' -GATCTGTTCATG 3, (SEQ ID NO: 198) was ligated into a tester amplicon. Add 100 times the amount of the driver amplicon of the tester amplicon, treat it with phenol: chloroform, treat with chloroform, and then precipitate with EtOH, and then precipitate the precipitate with the hybridization buffer (1M EPPS p H8. 25 (SIGMA 3 ml) and 0.5M EDTA pH 8 0.6 ml in 100 ml of MilliQ water. 30 μ1 mineral oil (manufactured by SIGMA) was overlaid and treated at 100 ° C. for 3 minutes, and then 1 μ 1 of 5M NaCl preheated to 67 ° C. was added and stirred with the aqueous layer. After that, 1 晚 hybridization was performed at 67 ° C. The next day, 4 5 μl of ice-cold TE was added and treated with chloroform to remove mineral oil. A 10 x PCR buffer (15 mM MgCl

2, 10 μ \ dNTP mix (each 2 mM), 60 pmolZl of J Bgl 24, 0.5 ^ 1 AmpliTaq (trademark) containing DNA Polymerase, add MilliQ water to 100 / zl, and add to PCR tube Using a thermal cycler, the reaction was carried out at 72 ° C for 5 minutes, then at 93 ° C for 1 minute, at 70 ° C for 3 minutes for 20 cycles, and at 70 ° C for 10 minutes. Ten reaction products were collectively collected by EtOH precipitation, and then treated with 30 U mung-bean nuclease (TAKARA) at 37 ° C for 30 minutes. Add 200 1 of 0.05 M Tris (pH 8.9), 95. After incubating at C for 5 minutes, a 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 μl of the PCR product was run on 3% Nusieve agarose containing 0.5 μg 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. As with 1st round RDA, N Bgl 24: 5 '-AGGCAACTGTGCTATCCGAGGGAA 3' (SEQ ID NO: 199) and N Bgl 12: 5 '-using TAKARA DNA Ligation Kit ver. 2 for the 1st round RDA product GATCTTCCCTCG 3 ′ (SEQ ID NO: 200) was ligated to form a tester amplicon, and 2nd round RDA was performed. In addition, a 3rd round RDA was performed using J Bgl 24] Bgl 12 again. After 3rd round dDA, the band was cut out and DNA was extracted using QIAEX II gel extraction kit.

[0079] In the subsequent subtractive process, the above band was cut out and subcloned into a vector, and about 200 clones were picked up, and the entire base sequence was resolved.

[0080] In other words, the band DNA purified after the 3rd round RDA reaction was converted into pGEM—T easy ve ctor (trade name, manufactured by Promega) and ligation product 51 obtained was mixed with E. coli JM109 (TAKARA) 60 μ 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 μm filter). ) And 50 μ 1 X-gal (TAKARA, 100 mgZ5 ml—dimethylformamide) coated with 5 μgZ ml Ampicilliirs LB pre-to-to (bacto trypton 5 g, bacto yeast extract 2.5 g, NaCl 5 g and bacto agar (Nacalai Testa) 7g in MilliQ water (500ml, high-pressure steam sterilization) was sprinkled on 2 plates and cultured at 37 ° C for 1cm. After culturing, the white single mouth proliferated was picked up with a toothpick and cultured in 3 ml of LB medium containing 5 / zg / ml Ampicillin for more than 8 hours at 37 ° C. After culturing, plasmid DNA was extracted with Nucleo Spin ™ 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.

[0081] 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). Using plasmid DNA as a template, add MilliQ water to 2 1 of 10 X PCR buffer (15 mM MgCl, 4 μ \ Premix, 3.2 pmol primer included)

2

Put 20 1 in the PCR tube and use a thermal cycler at 96 ° C for 10 seconds and then 96. C for 10 seconds, 50. C for 5 seconds, 60. The reaction was carried out for 25 minutes for 4 minutes at C. Gel filtration purification was performed using Sepha dex (trademark) G50 Fine DNA Grade (Pharmacia), followed by heat denaturation at 95 ° C for 2 minutes, and then the nucleotide sequence was analyzed.

[0082] The sequence reaction in the analysis by Aloka was performed using 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 μ 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. After completion of the reaction, S top / Loading Buffer (Basic Fuchsin (Wako Pure Chemical Industries, Ltd.) 1.5 ^ 1, Bromo Phe nol Blue (Wako Pure Chemical Industries, Ltd.) in small amount (0.01%), 0.5M EDTA 10 1 and deionized formamide (manufactured by Wako Pure Chemical Industries, Ltd.) 475 μ1 containing 500 μ1 MilliQ water) were added to each tube 4 by 1. After heat denaturation at 95 ° C for 2 minutes, each 1.2 1 was subjected to electrophoresis gel (50% Long Ranger ™ 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. 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.

[0083] Among the clones picked up above, even though NCBI Blast was searched at about 100 bp, there was no homologous molecule, and it was thought that this was derived from a new molecule (data Is not shown). As a result of predicting the amino acid sequence of this nucleotide sequence, it is possible that almost all amino acids are serine, threonine, and proline in the open reading frame without a stop codon, and that this cDNA fragment might be a part of the tandem repeat of mucin. It was done.

[0084] 1-4. Analysis by Northern blotting

Furthermore, whether or not the expression of the cDNA fragment obtained by the subtraction method in TA3-Ha cells is actually higher than that in TA3-St cells was examined by Northern blotting.

[0085] The mRNA of TA3-Ha cells and TA3-St cells was electrophoresed in 1% formaldehyde agarose gels at 1 μg 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.

[0086] Next, the 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.

[0087] More specifically, a probe incorporated in the above-described pGEM-T easy vector was used.

In other words, 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 α- Ρ — 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. 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 as a secondary wash, add 0.1 X SSC-C0.01% SDS solution. Washing was performed twice at 68 ° C for 15 minutes. The plate was exposed to an imaging plate (Fuji Film) and developed with BAS2000 or BAS1800 (Fuji Film).

[0088] As a result of analysis using G3PDH as an internal control probe to compare the amount of mRNA, a highly expressed sequence was found in Ha cells. Henceforth, based on this sequence, the full length was obtained. Determine this cDNA fragment epil-1: CCCGCTCTGACCACCAC

It was named TGCATCTAGCACTGCCTCAG (SEQ ID NO: 201).

[0089] Next, in order to accurately estimate the size of the molecule from which the cDNA fragment was derived, mRNA was run on a large gel and Northern blotting was performed. Total RNA as a marker Based on the position of ribosomal RNA at the time of electrophoresis and the position of the band when G3PDH was used as a probe, the size of the mRNA that epil-1 was amplified was calculated. As a result of swimming in the large gel and increasing the degree of separation, 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.

[0090] 1-5. cDNA elongation and decoding by race reaction

Using the Marathon method, we tried epil- 1 force and 5 'race and 3' race reactions to try to elongate.

[0091] Specifically, using the Marathon (trademark) cDNA Amplification Kit (manufactured by TOYOBO), the 3'—marathon race and the 5—marathon race? T A reverse transcription reaction was carried out from the modified Oligo dT primer from mRNA purified from TA3-Ha Itoda using SUPERSCRIPT ™ 11 Reverse Transcriptase (Invitrogen). Subsequently, double-stranded cDNA was synthesized, Marathon cDNA adaptor was bound, and PCR was performed using a primer that binds to this adaptor part and a gene-specific primer.

[0092] The 5 'side was strong without any expansion even when the conditions were variously improved. On the other hand, on the 3 'side, 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.

[0093] 1— 6. Mouse gene PCR

By the way, prior to the elucidation of the 5 ′ side of the mouse mucin-like glycoprotein, in order to obtain not only the cDNA of this glycoprotein but also the gene, 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. .

[0094] Specifically, 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). Compared with the band amplified by RT-PCR of TA3—Ha cells, a band was obtained at a larger position, so it was considered that an intron was included, and when the nucleotide sequence was read, an intron that met the AGZGT rule was found. One power station was included.

[0095] 1— 7. Screening analysis of 129SVT gene library

In order to obtain the above genes, screening was performed using a phage library of 129SVJ gene (1FIXII-129 / SVJ Mouse genomic library (STRATAGENE)).

[0096] First, spread the above-mentioned phage library on several plates, create phage plaques on the entire plate surface, elute the phages, denature them, and perform PCR. went. Specifically, XL1-Blue MRA was dissolved in 2 XYT plates (bacto tryptone 16g, bacto yeast extract 10g, NaCl 5g and bacto agar 14g in 1L of MilliQ water and then high-pressure steam sterilized. And then used in a petri dish with a diameter of 10 cm) and incubated at 37 ° C for 1 mm. Pick colonies the next day and add 10 mM MgSO (Wako Pure Chemical) in 10 ml of 2 XYT medium (bacto tryptone 16 g, bacto ye ast extract lOg and NaCl 5 g dissolved in 1 L of MilliQ water and then autoclaved). In medium containing 2% Maltose (Wako Pure Chemical Industries)

Four

Then, 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.

Four

1 and 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 · 7Η O 2) on a plate with plaques on the entire surface (containing approximately 300,000 plaques).

4 2

0g, 1M Tris (pH7.5) 50ml and bact gelatin (Nacalai Testa) 0.lg was dissolved in 1L of MilliQ water and used under high pressure steam sterilization) 5ml was added and shaken at room temperature for 2 hours or more . The buffer was recovered in a tube, centrifuged at 3,000 rpm for 5 minutes at 4 ° C, and the supernatant was used as a phage solution. 2 ml of chloroform was added and vigorously stirred and stored. The PCR of the phage DNA is 2 1 10 X PCR buffer (15 mM MgCl, 2 μ Ι dNTP mix (2 mM each) ゝ

2

1 μ 1 (7) 5 μ Μ primer—sense, 1 1 5 Μ primer—anti—sense, 0.5 1 A mpliTaq ™ Gold (Roche) and 1 μ 1 template (100 ° after dilution with lZlO) (Including 5 minutes treatment with C), add MilliQ water to 20 μ1, place in PCR tube, and perform PCR reaction using thermal cycler. The primers used were epintmF, epintmR or epil-5. When reacting with epintmF and epintmR, heat denaturation at 94 ° C for 10 minutes, then 94 ° C for 30 seconds, 51 ° C for 30 seconds, 72 ° C for 1 minute 45 cycles, 72 ° The reaction was carried out at C for 10 minutes. In addition, when reacting with epil-5 and epintmR, it is 30 seconds at 94 ° C, 57. C for 30 seconds, 72. C for 1 minute to 45 cycles. Of the reaction products, 101 was electrophoresed on 1% Seakem ME agarose containing 0.5 μg, ml ethylene bromide, and the band was confirmed. Subsequently, 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.

In this way, 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 ™ at 68 ° C for about 2 hours. After labeling the probe with α- 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)

It was exposed to AR (35 × 43 cm: from Amersham Pharmacia) and developed with an automatic processor. 3. Align the developed film with the phage-produced plate, pick up the top agarose part containing positive plaques with a toothpick, and place it in SM buffer 100 1. After centrifuging at 3,000 rpm for 5 minutes at C, the supernatant was used as a phage solution, and chloroform 101 was added, mixed and stored.

According to the above procedure, 6 positive phage clones (a ~; 0 were obtained, and a large amount of each clone was sprinkled to prepare plaques, and DNA was recovered. After preparing a large amount, add 5 ml of SM buffer, shake at room temperature for 2 hours or more, collect in a tube, centrifuge at 3,000 rpm for 5 minutes at 4 ° C, and remove the supernatant into a new tube. Further, 2 ml of chloroform was added thereto and stirred vigorously, followed by centrifugation at 3,000 rpm for 5 minutes at 4 ° C, and the upper layer was used as a phage solution. DNA was purified using a QIAGEN Lamda kit (trade name, manufactured by QIAGEN).

[0099] 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

After acid treatment in HC1 and rinsing with MilliQ water, it was soaked twice in 1.5M NaCl, 0.5M NaOH for 15 minutes. Continued! /, Soaked in 3M NaCl, 0.5M Tris pH7.4 for 30 minutes, and transferred to nylon membrane for 1 kg. The next day, the membrane was air-dried and treated at 80 ° C for 2 hours to immobilize DNA on the membrane. The membrane was subjected to Southern blotting according to the procedure described above. The results are shown in FIG. Since the probe did not bind to clones c and f, it was judged to be falsely positive that plaques appeared to be bound by plaque hybridization. b, d and e were used.

[0100] Among the bands obtained by restriction enzyme treatment of the DNA of these clones, the band to which the probe was bound by Southern blotting was cut out, and DNA was extracted with the QIAEX II gel extraction kit. The pBluescrpit II SK + vector (Invitrogen) was digested with restriction enzymes according to the restriction enzyme sites at both ends of the insert, and the above DNA was ligated there using TAKARA ligation kit ver. 2 (TAKARA). The ligation product and E. coli JM109 were mixed and incubated on ice for 20 minutes, then placed in a 42 ° C water bath for 50 seconds, and then returned to ice again for 2 minutes or longer. This was cultured in SOC medium at 37 ° C for 1 hour, then plated on a 5 gZml Ampicillin-containing LB plate coated with IPTG 51 and X-gal 50 1, and cultured at 37 ° C for 1 mm. White single colonies were picked with a toothpick and cultured in 3 ml of LB medium containing 5 g / ml Ampicillin at 37 ° C for 8 hours or more. Plasmid DNA was extracted with Nucleo Spin ™ plasmid. After treatment with each restriction enzyme, electrophoresis was carried out with 0.6% Seakem ME agarose containing 0.5 g / ml ethylene bromide, and the presence of the target insert was confirmed. Since the DNA of about 10kb is subcloned, the base sequence is decoded little by little from both ends, and the mucin-like glycoprotein of the present invention Whether the gene was included was examined. As a result, several clones containing the mucin-like glycoprotein gene of the present invention were obtained for the mHI-treated DNA in which the restriction enzyme sites exist in the cDNA sequence obtained so far.

[0101] 1— 8. Gene mapping

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. However, the undeciphered area was included in the middle part of the tandem repeat. By comparing the length of the obtained gene clone with the registered sequence, 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. In practice, tandem repeats could be repeated over 100 times. There is a stop codon and methionine as the start codon in the approximately 300 bp upstream of the tandem repeat, as well as a signal sequence and an expected sequence. 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.

[0102] 1-9. Search for 5 'end 1

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.

[0103] Before designing the primer for the Race reaction, it was confirmed by RT-PCR of TA3-Ha cells whether this approximately 300 bp portion was actually cDNA. Specifically, single-stranded cDNA synthesized from total RNA of TA3-Ha cells was used as a template, and the following were used as primers. sense:

epicDNAl: 5, — CACCCATGCAATGAATTTAACAAAGG-3, (SEQ ID NO: 205)

Antisense:

epitmR: TGGATGCAGTGGTGGTCAGGGTGG 3 '(SEQ ID NO: 206) epiracerl: GTGGCTATGCTCTTCGTTTCTGATGAAGG-3, (SEQ ID NO: 207)

epiracer2: GTGGTGGAGGAAATATGGGCATAACC-3, (SEQ ID NO: 208

)

2 ^ 1 (10 Χ ΡΟΚ buffer (15 mM MgCl, 2 μ Ι dNTP mix (2 mM each), 1 1

2

(7) 5 μM primer—sense ^ 1 μ1 (7) 5 μM primer—anti-sense, 0.5 1 Ampli Taq ™ Gold, including 1 μ1 template (moderately diluted) MilliQ water was added to make 210, put into a PCR tube, and PCR was performed using a thermal cycler. 9 After heat denaturation at 4 ° C for 10 minutes, reaction was performed at 94 ° C for 30 seconds, 59 ° C for 30 seconds, 72 ° C for 1 minute for 50 cycles, and 72 ° C for 10 minutes. . Among the reaction products, 101 was electrophoresed on 1% Seakem ME agarose containing 0.5 / z gZml ethylene bromide to confirm the band. The results are shown in FIG. A band was observed in the size expected to be amplified by the primers used.

[0104] Subsequently, a primer for a race reaction was designed in the cDNA portion confirmed by RT-PCR, and a gene-specific reverse transcription reaction and a race reaction were performed. By using the GeneRacer (trademark) kit, 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.

[0105] 1 10. 5 'End Search 2

As described above, it has been found that the 5 'end of the mouse's epiglycanin-related mucin-like glycoprotein of the present invention cannot be determined only by the 5' race reaction, which is one of the most feasible methods in the related technology. did.

[0106] Therefore, 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)

Subsequently, 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.

[0108] The putative splicing donor site was checked against the genomic sequence of lOkb upstream of the tandem repeat region. For the exon region where the force can be estimated, 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. As a result, 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 ™ 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.

[0109] Reading the sequence of the obtained PCR product confirmed that splicing had occurred in the predicted sequence, and this caused the exon 'intron present in the upstream part of the tandem repeat region of this gene. First elucidated. In other words, this exon has a TATA box upstream and methionine in the reading frame. The upstream sequence of the glycanin-related mucin-like glycoprotein was reliable and could be presumed to be the first exon. However, to determine if this region is really the first exon and whether this methionine is really the translation start point,

It was necessary to identify the transcription start position by RACE method.

Therefore, 5 ′ rece reaction was performed using 4.4 μg of total RNA generated from TA3-Ha cells as a raw material using GeneRacer ™ Kit, and the transcription start position was identified. In this experiment, the 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! For the tandem repeat region, an antisense primer for reverse transcription was designed as follows, and the RNA was reversely transcribed gene-specifically using SUPERSCRIPT ™ III (Invitrogen) in a saddle shape.

RTGeneTandeml: GCTGTCAACGATACGCTACGTAACGGCATGACA

GCTGGATACAGTGGTGGTCGGG 3 '(SEQ ID NO: 212)

Next, nested PCR was performed on the generated single-stranded cDNA using the following primers. Sense primers are provided in the kit for GeneRacer ™ 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 ™ 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 ™ 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 the same.

[0111] In addition, in order to investigate whether there is a splicing norant that has not spliced at the previous splicing donor site, further nested PCR was performed using the cDNA prepared by this 5'race reaction as a template. went. The following antisense primers were designed upstream from the tandem upstream splicing donor site.

[0112] EpiAS 15130: 5 '-AG ACC ATG ATAAGTACCC AGGGC-3, (SEQ ID NO: 215)

As a sense primer, GeneRacer (trademark) 5, Primer was used for the first time and Gene Racer (trademark) 5, NestedPrimer was used for the second time, and Epi AS15130 was used for the antisense primer in both reactions. According to the standard protocol of KOD—plus ™, at 94 ° C for 2 minutes! 94 ° C for 15 seconds: 54 ° C for 30 seconds: 68 ° C for 5 minutes, repeated 30 times Later, a system of 10 minutes at 68 ° C was used for both PCR reactions. When the nucleotide sequence of the PCR product was analyzed, it was confirmed that it was the DNA of the present invention, and a splicing nootropic was confirmed. The first exon of this splicing noriant was the same as the previous variant. The transcription start position was 61b upstream from the previous noreant, and the splicing donor site was the same as the previous noreant.

[0113] In this way, 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

[0114] 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

2- 1. Production of polyclonal pile

For the purpose of obtaining an antibody that specifically recognizes the novel mucin-like glycoprotein of the present invention, it is a part of a sequence considered to be a tandem repeat based on the sequence of the above cDNA. Epitm peptide: SSSTPTPTTTASST (SEQ ID NO: 177) and epintm peptide: NHTGTPVMEVKPSGS (SEQ ID NO: 178), which is a part of the portion considered not to be a tandem repeat just outside the transmembrane region, were prepared. A local antibody was prepared.

Specifically, 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 μl 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. Subsequently, 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) is linearly replaced with buffer B {0.05% TFA in propanol: acetonitorile (from PE Biosystems) = 7: 3} up to 50% over 30 minutes, then Elution with buffer B was performed for 5 minutes. Subsequently, equilibration with buffer A was performed for 25 minutes. 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. The purified peptide bound to KLH. That is, KLH 10 mg (from CALBIOCHEM) and 2 ml of 10 mM KPi buffer pH 7.0 (mixed with 0.2 ml KH PO 39 ml and 0.2 ml K HPO 61 ml), 63 1 MBS solution (MBS (SIGM

2 4 2 4

Company A) lmgZ67 μ 1 DMF (Wako Pure Chemical Industries, Ltd.)} was added and shaken at room temperature for 30 minutes to bind KLH and MBS. The reaction solution was separated by Sephadex (trademark) G25 (Pharmacia fine Chemicals) with 0.1 M KPi buffer pH 6.0 (0.2 M KH PO 87.7 ml and 0.2 M

twenty four

Gel filtration using 12.3 ml of K HPO). Measure the absorbance of each fraction

twenty four

Then, a fraction containing KLH—MBS was obtained. Thereafter, 6 mg of the lyophilized peptide obtained above was dissolved in 1 ml of 0.1 M Sodium Borate buffer (pH 9.0). Add the collected KLH-MBS to the mixture with slow stirring, and adjust the pH with HC1. 7. Adjusted to 0-7.5. The mixture was allowed to react at room temperature for 3 hours and dialyzed against PBS 1L at 4 ° C for 1 hour. This was aliquoted into 1 mg of peptide and stored at 20 ° C.

[0116] Japanese white rabbits (2.5 kg, female) used as immunized animals were purchased from the Japan Institute of Animal Science, and bred at the University of Tokyo School of Pharmacy. The experiment used rabbits bred one week after purchase. To the immunized animal, the peptide lmg solution covalently bonded to KLH and Freund complete adjuvant (NAKARAI CHEMICALS, LTD) were mixed to prepare an emulsion, which was administered subcutaneously. Immunization was carried out every 2 weeks, and from the second time onwards, it was mixed with Freund incomplete adjuvant (NAKARAI CHEMICALS, LTD.) To prepare an emulsion and immunized. After each immunization, blood is collected from the rabbit ear vein, and the blood is incubated at 37 ° C for 30 minutes, then allowed to stand at 4 ° C for 1 minute to clot, and centrifuged at 15, OOOrpm for 15 minutes. Kiyoshi was used as an antiserum. The antibody titer was measured by ELISA using the peptide as an antigen. Specifically, in a 96-well ELISA plate, 20 g / ml synthetic peptide dissolved in 0.05 M NaHCO (pH 9.6) as an antigen was placed at 4 ° C.

Three

Left for more than 24 hours. After washing twice with 0.05% Tween 20 (in PBS), blocking was performed with 3% BSA (in PBS) at room temperature for 2 hours. After washing three times with 0.05% Tween 20 (in PBS), a rabbit antiserum diluted with 1% BSA (in PBS) was added and allowed to react at room temperature for 2 hours. After washing 3 times with 0.05% Tween 20 (in PBS) and diluted 1,000-fold with 1% BSA (in PBS), HRP—Goat Anti Rabbit IgG antibody (ZYMED) was used as a secondary antibody at room temperature. Reacted for 1 hour. 0. After washing 5 times with 05% Tween 20 (in PBS), develop color with ImM ABTS 1 μ 1 / ml H 2 O and measure absorbance at 405 nm.

twenty two

It was. The results are shown in FIG.

[0117] Although the antibody titer against the Epitm peptide and the antibody titer against the epintm peptide increased as the number of immunizations increased, the antibody titer did not increase so much after three immunizations. Ended. Anti-epintm peptide antibodies tended to have higher antibody titers than anti-epitm antibodies.

[0118] 2-2. Detection of mouse mucin-like glycoprotein by polyclonal antibody

It was confirmed that the rabbit antibody prepared above bound to the peptide.

. Subsequently, this polyclonal antibody is subjected to flow cytometry or Western blotting. Check whether it can be used for

[0119] First, 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 μl 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.

[0120] As shown in Fig. 9, 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.

[0121] Next, Ha cell and St cell lysates were electrophoresed, blotted onto a PVDF membrane, and stained with a polyclonal antibody. When 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. Specifically, 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. Centrifuge at 10,000 rpm for 10 minutes at 4 ° C, suspend the pellet in a minimal amount of PBS, dialyze against PBS 1L at 4 ° C for 4 hours, and then add 1 ° C against fresh PBS 1L at 4 ° C. He was dialyzed. After ammonium sulfate precipitation, 100 μl of Sepharose ™ 4B (Amersham Bioscience) was added per ml, and the mixture was stirred at 4 ° C. for 2 hours or more. After centrifuging at 13,000 rpm and 4 ° C for 1 minute, the supernatant was used as a crude purified polyclonal antibody, and the activity was examined by ELISA and used in subsequent experiments. Next, Western blotting was performed according to the Western blotting method described in the section “1-1. Confirmation of presence of novel mucin-like glycoprotein by lectin”. Briefly, each cell extract was subjected to SDS-denatured 4% or 15% polyacrylamide electrophoresis in the presence of 2-ME and semi-drited onto a PVDF membrane. Block with 1% NGS-2% BSA (in PBS) and 0.1% Tween20-1% NGS and 2% BSA (PBS The above-mentioned crudely purified rabbit antibody diluted 500 times in the middle) was reacted with the PVDF membrane for 90 minutes. After the reaction, 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.

[0122] As shown in FIG. 10, particularly with the anti-epintm peptide antibody, the large part of the Ha cell molecular weight was stained smear. From these results, it was shown that the produced anti-peptide antibody can detect the epiglycanin-related mucin-like glycoprotein of the present invention.

[0123] 2- 3. Epiglycanin 本 in the mouse normal tissue;

Expression of the epiglycanin-related mucin-like glycoprotein of the present invention in the whole body of the mouse was analyzed by Northern blotting.

[0124] Specifically, whole body organs of C57BLZ6N mice were isolated, and total RNA was isolated from each of them by a conventional method. Briefly, the organs of the whole body of mice fasted the day before the dissection were isolated and frozen in liquid nitrogen. ULTRASPEC RNA was added and homogenized to extract total RNA. After removing the protein with PCI, the phenol was removed with chloroform, then precipitated with isopropanol, dissolved in water, and the absorbance was measured at 260 nm, Z, and 280 nm to estimate the RNA amount and purity.

[0125] Next, Northern blotting was performed according to the above-described experimental method using the epil-1 as a probe (Fig. 11). The force with which the binding of the probe was confirmed in many tissues and the expression of this mucin was confirmed. Particularly, strong binding was observed at a relatively large position in the spleen, cecum, large intestine, and lymph node, and the thymus, heart, liver, cerebrum, and muscle. A relatively strong bond was also seen in the skin. On the other hand, no binding was seen in the kidney, indicating that this molecule may not be expressed. From these results, this mucin was found to be very distinctive in that it was strongly expressed not only in the epithelial tissues such as the digestive tract and trachea but also in the immune system.

[0126] 2-4. Mouse cancer cell line and the epiglycanin-related whip of the present invention in breast cancer tissue -Like glycoprotein expression

Next, we analyzed whether this new mouse mucin was expressed in cancer cells other than TA3-Ha cells. Since TA3—Ha cells are breast cancer cells, we think that 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. In addition, 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

Three

1, 0. Prepared by adding 5 ml of ImM NEAA. Further, the immobilized FCS was added to a final concentration of 10% and used (MEM—NEAA—10% FCS medium). ES medium was purchased from Nissui Pharmaceutical, and FCS was added to a final concentration of 10% (ES—10% FCS medium). Others RPMI1640-10% FCS medium and DZF-10% FCS medium were similarly prepared and used. The mouse colon cancer cell line Colon38, 26 cells used for the experiment was provided by Dr. Takashi Tsuruo of the Institute for Molecular Biology at the University of Tokyo. Mouse lymphoma cell line RMA cells were kindly provided by Dr. Olivera J. Finn of Pittsburgh University. Mouse Renal Cancer Cell Line Renca cells were kindly provided by Dr. Ikuo Seki of Toyama Medical and Pharmaceutical University. 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. Mel-18 + Z—A mouse cell line MMK3-7 cell pellet that was also established in breast cancer was provided by Dr. Masamoto Kanno of Hiroshima University. Regarding the passage methods of these mouse cancer cell lines, 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

2

After washing, a Trp—EDTA solution (0.05% Trypsin / 0.02% EDTA—in PBS) was added thereto, incubated at 37 ° C. for 5 minutes, and then subcultured from the culture dish. RM A, MM2, MM46, MM48 are RPMI1640—10% FCS medium, 37 ° C, 5% CO

2 Cultured in the presence and passaged every 3-4 days. 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,

2

The 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

2

The cells were cultured in CS medium at 37 ° C in the presence of 5% CO and subcultured every 3 to 4 days. Ehrlich

2

Approximately 2 × 10 ⁶ cells were suspended in HANKS 'solution and administered intraperitoneally to ICR mice. One week later, peritoneal cells were collected. The collected cells were suspended in 2 ml of 0.15 M PBS, and then 6 ml of MilliQ water was added thereto, suspended for about 20 seconds, and 2 ml of 3.5 M NaCl was immediately added. By this operation, erythrocytes were removed and used in subsequent experiments. MRNA 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.In particular, 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. In addition, KLN205 cells, which are lung squamous cell carcinoma cells, showed a very strong binding. However, none of the cells showed stronger expression than TA3-Ha cells.

Example 3

[0129] Example 3: Identification of human 'epiglycanin-related mucin-like glycoprotein

3- 1- RT-PCR analysis of mRNA expression of human epiglycan 閗; carcin mucin-like glycoprotein mRNA

First, after extracting total RNA from a plurality of human cell lines, cDNA corresponding to each total RNA was prepared by RT reaction. Next, 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. As a result, 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.

[0130] (1) Cell 'passage

Nine cell lines, 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). Each cell was confirmed to be free of mycoplasma infection by Mycoplasma Plus ™ PCR Primer Set (Stratagene). Of these, EBC-1, 8505C, 8305C, and ME-180 were donated by Tohoku University Institute of Force and Age Medicine.

[0131] 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. MRK— nu— 1, KATO

2

III, U-937 was collected every 3-4 days and suspended in fresh medium. SK-BR-3, HepG2, and EBC-1 are washed with PBS every 3 to 4 days, and then Trp-EDTA solution (0.0 5% Trypsin / 0.02% EDTA-in PBS) is prepared. After incubating at ° C for 5 minutes, the cells were removed from the culture dish. 8505C, 8305C, and ME-180 were washed with PBS every 3-4 days, added with 0.02% EDTA-PBS solution, incubated at 37 ° C for 5 minutes, and then removed from the culture dish.

[0132] (2) Total RNA extraction and RT

Total RNA was extracted from the above nine types of cells using SV Total RNA Isolation Kit (trade name, Promega). 1 g of the extracted total RNA is used as a bowl, and SUPERSCRIPT ™ II Reverse Transcriptase (Invitrogen) is used in the presence of RNase in hibitor (from Ambion) ヽ, Oligo α 丄, primer (Amersham Pharmacia Bio A reverse transcription reaction was performed from tech), and RNA was removed from it using RNaseH (TOYOBO) to produce single-stranded cDNA.

[0133] (3) PCR

For control human 'G3PDH, 2 1 10 X PCR buffer (15 mM MgCl, 2 μm

2

MilliQ in 1 dNTP mix (2 mM each), 5 5 5 Μ primer—sense, 1 1 5 5 Μ primer-anti-sense, 0.1 ^ 1 AmpliTaq ™ Gold, 1 μ 1 cDNA template) Add water to 20 μ1 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 μg / ml ethylene bromide to confirm the band.

[0134] Regarding the human 'epiglycanin-related mucin-like glycoprotein of the present invention, the above G3PDH

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.

Human 'G3PDH:

Sense hG3PDH— F: 5, — GACCCCTTCATTGACCTCAACTAC— 3, (sequence number: 216)

Antisense hG3PDH-R: 5 '-CAGTGATGGCATGGACTGTGGT-3' (SEQ ID NO: 217)

Human Epiglycanin related mucin

Sense hepintm— F: 5, — CTTCCCATAGTGCATCTACTGC-3, (SEQ ID NO: 218)

Antisense hepintm -R: 5,-GAACCAGTTAGGACTCCACCTGGGC C-3 '(SEQ ID NO: 219)

(3) Sequence confirmation of PCR products

The band confirmed above was excised and the PCR product was recovered with QIAquick gel extraction kit (trade name, QI AGEN). Recovered PCR product using standard method pGEM-T Subcloning into easy (trade name, Promega), and the sequence was analyzed by ABI (ABI PRISM (trademark) 3 100 Genetic Analyzer). This sequence reaction was performed using BigDye (trademark) Terminator Cycle Sequencing vl. 1 Ready Reaction (Applied Biosystems). Using plasmid DNA as a template, 2 μ 1 of 10 X PCR buffer (15 mM MgCl, 4 μ \ Premix, 3.2 pmol primer (Τ7

2,

(Including SP6)) Add MilliQ water to 20 μl, add to PCR tube, use thermal cycler for 1 minute at 96 ° C, then 10 seconds at 96 ° C, 5 seconds at 50 ° C The reaction was carried out at 60 ° C for 4 minutes for 25 cycles. The PCR reaction product was purified by gel filtration using Sephadex (trademark) G50 Fine DNA Grade (Pharmacia), heat-denatured at 95 ° C for 2 minutes, and then analyzed for nucleotide sequence to confirm the sequence of the PCR product. did.

As shown in FIG. 13, 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.

[0136] (4) Confirmation of expression in normal human tissues

Expression of the epiglycanin-related mucin-like glycoprotein of the present invention in normal human tissues by the above PCR using cDNA panels derived from various human organs (trade name: human Multiple Tissue cDNA panels I and II, BD Biosciences Clontech) We also confirmed about. 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.

[0137] 3-2. Isolation of cDNA encoding human 'epiglycanin-related mucin-like glycoprotein by Nested PCR method

From the mRNA of the cervical cancer cell line ME-180, which was found to be highly expressing the target gene in the above, cDNA was prepared by RT reaction using a specific primer corresponding to the 3 'region of the gene, Next, 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.

[0138] (l) Extraction of mRNA and RT MRNA was extracted from ME-180 cell line using z MACS mRNA Isolation kit (Miltenyi Biotec). Using 0.35 μg of the extracted mRNA in a saddle type, SUPERSCRIPT ™ III Reverse Transcriptase (Invitrogen) was used in the presence of RNase inhibitor (Ambion), and the human 'epiglycanin-related mucin-like glycoprotein of the present invention was 3. A primer corresponding to the region: Reverse transcription reaction was performed from CCTATAACTGAGTTCATCTCAGAGC (SEQ ID NO: 220)! RNA was removed by RNaseH (TOYOBO) to prepare single-stranded cDNA.

(2) Amplification of the region encoding human 'epiglycanin-related mucin-like glycoprotein by Nested PCR method

2 1 10 X PCR buffer, 2 μ 1 dNTP mix (2 mM each), 1.2 μ \ σ) 5 μ prim prim er— sense— 1, 1. 2 1 5 μ M primer— anti— sense— 1, 0.5 1 KOD—plu s ™, 0.48 1 25 mM MgSO, 1 μ 1 cDNA template with MilliQ water

Four

20 1 and put into PCR tube, heat denaturation at 95 ° C for 5 minutes using thermal cycler, 25 cycles of 95 ° C for 30 seconds, 68 ° C for 3 minutes, 68 ° C And reacted for 10 minutes. Precipitate 10 1 of the resulting PCR products with ethanol, add 11.3 μ \ MilliQ water to dissolve, and use the total amount as a bowl to make 2 1 10 X PCR buffer, 2 μ 1 dNTP mix (each 2 mM ;), 1.2 μ \ (Ό5 μ Μ primer— sense— 2, 1.2 μ \ (Ό5 μ Μ primer— ant i— sense— 2, 0.5 ^ 1 KOD— plus (trademark), 0. 8 Add 1 of 25 mM MgSO 20

Four

1) Place in a PCR tube and heat denature at 95 ° C for 5 minutes using a thermal cycler, then at 95 ° C for 30 seconds, 68 ° C for 3 minutes 25 cycles at 68 ° C The reaction was carried out for 10 minutes (nested-PCR). Run the whole reaction product in 0.5% Seam em ME agarose containing 0.5 g / ml ethylene bromide, cut out the band confirmed around 2kbp and place it in QIAquick gel extraction kit (trade name, QIAGEN) And recovered. The collected PCR product was subcloned into pCR4Blunt—TOPO vector (Invitrogen), and the sequence was analyzed by ABI (AB I PRISM (trademark) 3100 Genetic Analyzer). This sequence reaction was performed using BigDye (trademark) Terminator Cycle Sequencing vl. 1 Ready Reaction (Applied Biosystems). Using plasmid DNA as a template, prepare 2 × 10 X PCR buffer (15 mM MgCl, 4 μΙ 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. Gel filtration purification was performed using Sephade x (trademark) G50 Fine DNA Grade (Pharmacia), followed by heat denaturation at 95 ° C. for 2 minutes, and then the nucleotide sequence was analyzed. Each of the above primers was as follows.

Human Epiglycanin related mucin

Sense-1 clonest5- l F: 5 '-AAGCCTAAGGAACCCAGGCATCCAG CTGCCCACGCC— 3' (SEQ ID NO: 221)

Antisense-1 clonest3— 1 R: 5, — GTTCATCTCAGAGCTAGATACC C ACCCCGGGGC-3 '(SEQ ID NO: 222)

Sense 1 2 clonest5 1 2 F: 5, CCCAGGAACACAAACGTAGGAGACC CACGCTCCTG— 3 '(SEQ ID NO: 223)

Antisense— 2 clonest3- 2 R: 5 '-C AGGATCTCAAACAAAGCTGG GAGGGGTCTCCTGGG 3' (SEQ ID NO: 224)

Sequence analysis primer

hepiseq5— F: 5, — GGTCTACTATTGCATTTAGAAGCTGC-3 '(SEQ ID NO: 225)

hepiseq3-R: 5′—TTGTGTGCATTCCAGTCAGAGC—3 (SEQ ID NO: 226) As a result of the analysis, the human 'epiglycanin-related mucin-like glycoprotein cDNA of the present invention having the sequence shown in SEQ ID NO: 174 could be isolated.

Example 4

Example 4: Specific expression of human 'epiglycanin-related mucin-like glycoprotein gene in tumor tissue

Using a probe designed based on the transmembrane region segment (approx. 270 bp) from the human 'epiglycanin-related mucin-like glycoprotein cDNA of the present invention using Cancer Profiling Arrayll (trade name) of BD Biosciences Clontech The gene expression profile between human normal tissue and tumor tissue was analyzed. This membrane 'base' array In this figure, several types of tumor tissues from each individual and the total mRNA expressed in the corresponding normal tissues are spotted in parallel, and by observing the hybridization of specific probes with them. The specific expression profile of the specific gene can be analyzed. Details were as follows.

[0141] (1) Preparation of probe

An amplified probe incorporated into pGEM-T easy vector (supra) 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).

[0142] The probe sequence (269 bp) is

TC-3 '(SEQ ID NO: 176)

(The underlined portion is the sequence corresponding to hepintm—F and R).

[0143] (2) Analysis using Cancer Profiling Arrayll

Using a Cancer Profiling Arrayll (BD Biosciences Clontech), noise reduction was performed. Specifically, 30 ml at 68 ° C for 10 ml of Express Hyb solution (trade name, BD Biosciences Clontech) supplemented with lmg sheared salmon testis DNA (SIGMA) denatured at 95 ° C for 5 minutes. The array membrane was prehybridized for more than a minute. On the other hand, the above probe was labeled with α- ³² P-dCTP by Rediprime (trade name, Amersham pharmacia biotech), Separated by NICK column (trade name, Amer sham pharmacia biotech). Measure the count of the isolate and adjust it to 2 x 10 ⁶ cpmZml. Mix with 5 ml of fresh Express Hyb soluti on with 0.15 mg s heared salmon testis DNA (SIGMA), 95 ° C After 10 minutes of denaturation, the membrane was hybridized with the above membrane at 68 ° C for 1 hour. The next day, wash with 2 X SSC—0.5% SDS solution at 68 ° C for 30 minutes three times, and as a secondary wash, use 0.2 X SSC-0.5% SDS solution at 68 ° C. Washed once for 30 minutes. After removing SDS with 2 X SSC solution, the plate was exposed to an imaging plate (Fuji Film) and developed with BAS 1800 (Fuji Film).

[0144] As shown in FIG. 14, in the comparison between each tumor tissue and normal tissue, the force that was most pronounced in normal thread and tissue of the eclampsia and cervix was constant between individuals. I'm not doing it. What is noteworthy about the tissue is that when the tumor and normal tissue pair for each individual is compared, the expression in the normal tissue decreases when the expression is significant in the tumor tissue, and vice versa. It was.

[0145] 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.

[0146] From this, it was confirmed that 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

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. Flow cytometry to detect the expression of epiglycanin-related mucin-like glycoprotein on the cells in question combines anti-FLAG—M2 antibody (Sigma) and FITC-labeled goat anti-mouse IgGl antibody (Zymed Lab oratories) Epics Coulter XL (trade name) flow cytometer (Beckmann Coulter) was used. On the other hand, for the detection of mucin-like glycoprotein specific sugar chain epitope on the cell surface, Piotin KVicia villosa (Velved Fujixa) Agurchun B4 (: WA-B4) and Biochemical Industries, Ltd. from Vector Laboratories Pyotinylated Arachis hvpogaea (peanut) aglucun (: PNA) was used with phycoerythrin labeled streptavidin (eBioscience). Dead cells were excluded.

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. The cell lysate was 1% (ν / ν) 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 ™, Millipore). After blocking the membrane with 3% BSA in PBS containing 0.1% Tween 20, Tris buffered saline containing 3% BSA and 0.1% Tween 20 (50 mM Tris-HCU pH 7.5, 150 mM) It was incubated for 2 hours at room temperature with piotinylated anti-FLAG-M2 antibody (Sigma, 1Z1000 dilution) diluted with NaCl), 2.5 gZml of pyotinylated WA-B4, or 2.5 g Zml of Pyotin チン PNA. An anti-mouse IgGl antibody (Zumed) was used as a negative control for antibody staining under the same conditions. Western sage peroxidase-streptavidin 'conjugate (Zymed) diluted 1000-fold with Tris buffered saline containing 3% BSA and 0.1% Tween 20 on a Western' plot, alkaliphosphatase 1 streptavidin ' Conjugate (Vector) Each was used in a lot to examine binding. The bound antibody and lectin were visualized with an ECL detection kit (Amersham) and an alkaline phosphatase substrate kit (Vector), respectively.

[0149] As shown in Fig. 16, 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. In addition, 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). In SDS-PAGE of the lysate of the transformed cells of the present invention, anti-FLAG antibody bound significantly to a molecule that migrated to about 200-230 kDa, but this binding was not observed in control transformed cells. . The same results were obtained with the WA-B4 and PNA lectin 'plots, but with the ConA plot, no binding was observed (Fig. 16B). This demonstrates that O-linked glycans bind to the gene product of the present invention. In addition, at least in this expression system, the apparent molecular weight of the gene product was estimated to be about 230 kDa, which was found to be much larger than the molecular weight of the peptide portion of about 57 kDa.

Example 6

[0150] Example 6: Cell and tissue staining using anti-serum specific to human epiglycanin-related mucin-like glycoprotein

6- 1. Human 'epiglycanin-related mucin-like glycoprotein with synthetic peptide as immunogen ^^ rrfnj 胄の胄

(1) Preparation of synthetic peptide

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. As the F-moc amino acid used in the synthesis, 0.4 mmol each of OH form (Applied Biosyst ems) was used. After the synthesis, the peptide was removed by reacting the resin with a peptide elimination reagent (0.7 g pheno 1, 0. ami thioanisoU 0.2 ml ethaneditniol, 9.5 ml tnfiuoro acetic acid) at room temperature for 2 hours to perform deprotection. The resin was removed by filtration with 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.

[0151] (2) Purification

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. After applying 4mg of pre-purified synthetic peptide to the column, solvent A (0. 05% TFAZMilliQ (trademark) water) was added over 30 minutes using Jasco PU — 1580 pump (trade name, JASCO Corporation). Solvent B (0. 05% TFAZ propanol): Acetonitrile (PE Biosystems) = 50%: 35%: Replace with 15%, then over 35 minutes, gradually replace solvent B: Solvent C = 70:30 The elution was carried out. Thereafter, elution was performed with the same composition for 5 minutes, followed by 15 minutes of equilibration with solvent A. The collected fraction was subjected to mass spectrometry using MALDI-TOF MS spectrum (VoyagerElite (trade name), PE Biosystems) to collect the desired fraction. The purified peptide was lyophilized and stored at 20 ° C.

[0152] (3) Covalent bond with carrier protein

20 mg of KLH (CalbioChem) was dissolved in 1 ml of 0.01 M sodium phosphate buffer (ρΗ7.2) 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. 1 mg of the above freeze-dried synthetic peptide was dissolved in 0.6 ml of 0.01 M phosphate buffer (pH 7.2), and the KLH-MBS fractionated above was slowly added to it with stirring. The pH was adjusted to 7.0-7.5. The reaction was allowed to proceed at room temperature for 3 hours, and the reaction solution was dialyzed against PBS (IL) at 4 ° C for 1 hour. This was dispensed and stored at 20 ° C. Similarly for BSA A bonded body was created.

[0153] (4) Immunization and serum collection

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. One week after the booster immunization, 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.

[0154] (5) Measurement of antibody titer by ELISA

96-well ELISA plate dissolved in 0.05M NaHCO (pH 9.6) as antigen 10

Three

mgZml of BSA-binding synthetic peptide, 20 mgZml of KLH and 20 mgZml of BSA were added, and the mixture was allowed to stand at 4 ° C. After washing three times with 0.05% Tween 20 (in PBS), blocking was performed with 3% BSA (in PBS) at room temperature for 2 hours. After washing three times with 0.05% Tween 20 (in PBS), the above rabbit sera diluted with 1% BSA (in PBS) was collected and reacted at room temperature for 2 hours. After washing 3 times with 0.05% Tween 20 (in PBS), HRP—Goat Anti Rabbit IgG antibody (Zymed) diluted 1000 times with 1% BSA (in PBS) at room temperature as a secondary antibody 1 Reacted for hours. After washing 5 times with 0.05% Tween 20 (in PBS), color was developed with 1 / z lZml H 2 O solution of ImM ABTS and absorbed.

twenty two

A degree of 405Z492nm was measured.

[0155] 6—2. Using the human Xepiglycanin-related mucin-like glycoprotein-specific antiserum, the l X 10 ⁴ cell of the human-epigeglycanin-related mucin-like glycoprotein overexpression cell line (K562ZMUC21) With FCS and cyto funnel do uble (trade name, Thermo) immobilized on a glass slide, Cy-spin3 (trade name, Thermo) and poly-L-lvsine hydrobromide lmg / ml (Wako Pure Chemicals) On the applied slide glass Pasted. After air-drying at room temperature for 1 hour or more, it was fixed and permeabilized with acetone for 10 minutes and air-dried again. 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. Thereafter, as a primary antibody, 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. After washing 3 times with PBS for 5 minutes, AP-goat anti rabbit IgG (H + L) (Zymed) was diluted 100-fold with PBS solution of 2% NGS and 3% BSA as a secondary antibody. The reaction was performed at / zl for 30 minutes at room temperature. After washing with PBS for 5 minutes, once; TBS for 5 minutes twice, the sections were reacted with HistMark® ED (Kirkegaad & Perry Laboratories Inc.) 100 1 for about 10 minutes. After soaking in PBS and stopping the enzyme reaction, it was sealed with Aqua Poly Mount (PolyScience). As a control cell, K562ZMock was used.

The results are shown in FIG. Cells expressing the glycoprotein were specifically stained with the human epiglycanin-related mucin-like glycoprotein-specific rabbit serum of the present invention.

[0157] 6- 3. Human 'biglycanin wheeler mucin-like glycoprotein's special stake

¾ color

(1) Specimen sample

A paraffin-embedded block of papillary thyroid cancer borrowed from the Department of Pathology, University of Tokyo Hospital was used as a sample.

(2) Section preparation

A 5 _μm 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.

(3)

For deparaffin, xylene 10 min, 3 times, 2 min 100% ethanol, 2 times, 100%, 90%, 80 ^_0/0 and 70 ^_0/0 of Etanonore [this respectively 2 minutes, immersion once And then washed with water for more than 10 minutes. In order to activate the antigen, it was immersed in 10 mM sodium citrate buffer (PH 6.0) boiled in a microwave oven for 15 minutes and then allowed to cool at room temperature for 20 minutes. After washing 3 times with PBS for 5 minutes, 1% HO (mi Soaked in Hi—Q (water) for 30 minutes. After washing 3 times with PBS for 5 minutes, the cells were surrounded by pap pen (Daido Sangyo), and avidinZbiotin blocking kit (Vector) avi din D solution 4drops / ml was prepared 2% NGS and 3% 50 Oml of BSA in PBS was added and blocked at room temperature for 30 minutes. After that, add 300 μl of Usagi serum diluted 2000 times with PBS solution of biotin solution 4drops / ml caro 7% 2% NGS and 3% BSA of avidin Zbiotin blocking kit (Vector), 4 ° Incubate with C for 1 hour. After washing 3 times with PBS for 5 minutes, biotin-goat anti rabbit IgG (H + L) (Zymed) was diluted 200-fold with 2% NGS and 3% BSA in PBS as the secondary antibody. The reaction was performed at 300 / zl for 30 minutes at room temperature. After washing 3 times with PBS for 5 minutes, as a tertiary antibody, react with ZyMax ™ streptavidin—HRP conjugate (Zymed) 100-fold diluted with PBS solution of 2% NGS and 3% BSA for 30 minutes at room temperature. It was. After washing 4 times with PBS for 5 minutes, the reaction was carried out for 5 minutes with 200 μ1 of AEC—Substrate Chromogen Kit (Zymed). After washing once with PBS for 5 minutes, nuclear staining was performed with Mayer's Hematoxylin (Muto Jun Chemical Co., Ltd.) for 2 minutes at room temperature, followed by washing with water for 10 minutes or more. Sealed with Aqua Poly Mount (PolyScience). The results are shown in Fig. 18.

Industrial applicability

[0158] 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.

Brief Description of Drawings

[0159] 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. TA3

— Total 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 TA3-1½ 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. In each organ, N indicates normal tissue and 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.

圆 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.

Claims

An isolated mucin-like glycoprotein, the protein comprising:

(1) An amino acid sequence represented by the following general formula (I) from the N-terminus to the C-terminus:

A-Bn-C-D-E (I)

[Where

A is

MRRRSSLWCWLLLQILLLGSGYA (SEQ ID NO: 1) or

MKMQKGNVLLMFGLLLHLEAA (SEQ ID NO: 2)

An N-terminal region selected from

Xaa — Xaa — Xaa — Xaa — Xaa — Xaa — Xaa — Xaa — Xaa — Xaa — Xa

1 2 3 4 5 6 7 8 9 10 a -Xaa —Xaa —Xaa —Xaa (SEQ ID NO: 3)

11 12 13 14 15

However, in the above sequence,

2

Selected

Three

Four

Selected

Five

6

8

Noic acid,

Xaa is threonine, methionine, serine, parin, alanine, asparagine, glutamine Acid, lysine and isoleucine forces are group forces independently selected,

Ten

11

Selected

Xaa is proline, leucine, threonine, tryptophan, arginine, glutamine and

12

Serinka is a group power independently selected

13

Or does not exist,

14

as well as

15

Or does not exist;

Xaa —Xaa — ^> er— Xaa —Xaa —Xaa —Xaa —Xaa — ^> er— Thr— X

21 22 23 24 25 26 27

aa -Xaa Xaa — Xaa — Xaa (SEQ ID NO: 4)

28 29 30 31 32

However, in the above sequence,

twenty one

twenty two

twenty three

twenty four

twenty five

26

27

28

29

30

31

Xaa is independently selected from the group power of isoleucine and serine; or

32

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

41 42 43 44 45 46 47 48 49 -Xaa -Xaa — Xaa — Xaa — Xaa (SEQ ID NO: 5)

50 51 52 53 54 55

Provided that at least one serine or threonine is included in the above sequence.

41

42

Selected

43

Xaa is from threonine, valine, proline, alanine, isoleucine and asparagine

44

Independently selected from the group consisting of

45

46

And

Xaa is independently selected as a group force consisting of glycine and serine

47

48

Selected

49

50

51

Xaa is threonine or absent,

52

Xaa is independently from the group consisting of asparagine, glycine, isoleucine and threonine.

53

Selected or absent

Xaa is serine or absent, and

54

55

Or does not exist,

n is an integer from 20 to 150,

C is

KPWE (SEQ ID NO: 6) or A potential enzyme cleavage site-containing region selected from ALTGMHTTSHSASTAVSEAKPGGSLVPWE (SEQ ID NO: 7),

D is

IFLITLASVIWMGLSAGLFIYV (SEQ ID NO: 8) or

IFLITLVSWAAVGLFAGLFFCV (SEQ ID NO: 9)

A transmembrane region selected from

E is

RR,

MTRI (SEQ ID NO: 10) or

RNSLSLRNTFNTAV WFWRRPVSSIAMEMSGRNSGP (SEQ ID NO: 11)

An intracytoplasmic region selected from ]

Have or

(2) The mucin-like glycoprotein characterized by having a sequence in which one or several amino acids are deleted, inserted, substituted or added to the amino acid sequence represented by the general formula (I). .

[2] In the general formula (I) according to claim 1, A is SEQ ID NO: 1, B is a group force consisting of SEQ ID NO: 3 and SEQ ID NO: 4 and is independently selected, and C is SEQ ID NO: The mucin-like glycoprotein according to claim 1, wherein D is SEQ ID NO: 8, E is SEQ ID NO: 10, and n is an integer of 100 to 150.

[3] The mucin-like glycoprotein according to claim 2, wherein the group power consisting of B: SEQ ID NO: 12 to SEQ ID NO: 138 is also independently selected.

[4] The mucin-like glycoprotein according to claim 3, which is derived from a mouse.

[5] Characterized by having a sequence represented by SEQ ID NO: 139, or a sequence in which one or several amino acids are deleted, inserted, substituted or added to the amino acid sequence represented by SEQ ID NO: 139 The mucin-like glycoprotein according to claim 3.

[6] In the general formula (I) according to claim 1, A is SEQ ID NO: 2, B is SEQ ID NO: 5, C is SEQ ID NO: 7, and D is SEQ ID NO: 9. E is SEQ ID NO: 11, and The mucin-like glycoprotein according to claim 1, wherein n is an integer of 20 to 50.

[7] The mucin-like glycoprotein according to claim 6, wherein the group power consisting of SEQ ID NO: 140 to SEQ ID NO: 170 is also independently selected.

[8] The mucin-like glycoprotein according to claim 7, which is derived from human.

[9] It is characterized by having a sequence represented by SEQ ID NO: 171 or a sequence in which one or several amino acids are deleted, inserted, substituted or added to the amino acid sequence represented by SEQ ID NO: 171 The mucin-like glycoprotein according to claim 7.

[10] A nucleic acid encoding the mucin-like glycoprotein according to claim 1, or a nucleic acid complementary to the nucleic acid.

[11] The nucleic acid according to claim 10, which is represented by SEQ ID NO: 172.

[12] The nucleic acid according to claim 10, which is represented by SEQ ID NO: 174.

[13] An expression vector comprising the nucleic acid according to claim 10 operably linked to a regulatory sequence capable of directing gene expression in a host cell.

[14] A genetically modified host cell containing the expression vector according to claim 13.

15. The genetically modified host cell according to claim 14, which is derived from a eukaryote.

[16] A method for producing a mucin-like glycoprotein according to claim 1, wherein the method comprises

The host cell according to 5 is cultured under conditions allowing the expression of the gene introduced into the host cell, and the cell is! A positive ij 'self method, characterized by isolating the recombinant gene product expressed in advance.

[17] An antibody against the mucin-like glycoprotein according to claim 1.

[18] The antibody according to claim 17, which is specific for a mucin-like glycoprotein derived from human.

[19] The antibody according to claim 17 or 18, which is specific for an extracellular domain of the mucin-like glycoprotein.

[20] A mucin-like glycoprotein expression detection and Z or quantification method according to claim 1 in a sample, the method comprising:

(1) preparing a probe comprising all or part of the nucleic acid according to claim 10;

(2) a step of allowing the probe in the step (1) and RNA from the sample or a reverse transcription product of the RNA to be subjected to a stringent condition and to be hybridized; and (3) The method as described above, comprising a step of detecting the probe RNA complex or the probe reverse transcript complex formed in the step (2).

[21] The method of claim 20, wherein the probe comprises at least 20 consecutive nucleotides in the nucleic acid of claim 10.

[22] The probe includes a nucleotide sequence represented by SEQ ID NO: 176

21. The method of claim 20.

[23] A kit for use in detection and Z or quantification of mucin-like glycoprotein expression according to claim 1, wherein the kit also comprises all or part of the nucleic acid according to claim 10. Alternatively, the kit comprising a primer.

[24] A kit for use in detection and Z or quantification of mucin-like glycoprotein expression according to claim 1, wherein the kit comprises the antibody according to any one of claims 17 to 19. The kit described above.

[25] An ex vivo detection method for mucin-like glycoprotein expression according to claim 1 in a cell or tissue, the method using an antibody specific for a cytoplasmic region of the protein. The method comprising immunostaining.

26. The method according to claim 25, wherein the tissue is thyroid cancer tissue.