JP2001515717A - Secreted proteins and polynucleotides encoding them - Google Patents

Abstract

  (57) [Summary] Disclosed are novel polynucleotides and the proteins encoded by them.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, and therapeutic, diagnostic and research utilities for these polynucleotides and proteins.

BACKGROUND proteinaceous factors invention (e.g., lymphokines, interferons, including cytokine such as CSFs and interleukins) has methods for the purpose of finding this 10
Matured rapidly over the years. Currently, conventional hybridization and expression cloning methods rely on information directly related to the discovered protein (ie,
In the case of hybridization cloning, depending on the partial DNA / amino acid sequence of the protein; in the case of expression cloning, the activity of the protein).
The new polypeptide is cloned "directly". Signal sequence cloning (
DNA sequences are isolated based on the presence of the currently well-recognized secretory leader sequence motifs), as well as more recent "indirect" cloning methods, such as various PCR or low stringency hybridization cloning methods. A large number of DNA / amino acid sequences for proteins known to have biological activity due to their secreted nature in the case of cloning of the leader sequence, or due to the cell or tissue source in the case of the PCR method. Has enabled the situation to be advanced. The present invention is directed to these proteins and the polynucleotides that encode them.

[0003] In an embodiment of the outline 1 of the invention, the present invention is isolated to provide a composition comprising a polynucleotide selected from the group consisting of: comprising one of the nucleotide sequences: (a) SEQ ID NO: (B) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 69 to nucleotide 908; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 from nucleotide 270 to nucleotide 908; (d) A polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of clone bn971 deposited under accession number ATCC98535; (e) encoding the full-length protein encoded by the cDNA insert of clone bn971 deposited under accession number ATCC98535 (F) a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of clone bn971 deposited under accession number ATCC98535; (g) the maturation encoded by the cDNA insert of clone bn971 deposited under accession number ATCC98535 A polynucleotide encoding a protein; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 2; (i) including a fragment of the amino acid sequence of SEQ ID NO: 2 having biological activity A polynucleotide encoding a protein (the fragment is SEQ ID NO: 2 8
(J) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (g) above; (k) a species homolog of the protein of (h) or (i) above. A coding polynucleotide; and (l) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such a polynucleotide is a nucleotide sequence from nucleotide 69 to nucleotide 908 of SEQ ID NO: 1; nucleotide 2 of SEQ ID NO: 1.
Nucleotide sequence from 70 to nucleotide 908; accession number ATCC 985
Nucleotide sequence of the full length protein coding sequence of clone bn971 deposited as accession number 35; or clone b deposited as accession number ATCC 98535
n971 contains the nucleotide sequence of the mature protein coding sequence. In another preferred embodiment, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bn971 deposited under accession number ATCC 98535. In still other preferred embodiments, the present invention provides
Provided is a polynucleotide encoding a protein comprising the amino acid sequence from amino acid 1 to amino acid 83 of SEQ ID NO: 2. In a further preferred embodiment, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2 having biological activity, said fragment comprising SEQ ID NO: 2, preferably 8 More preferably, it comprises 20 and most preferably 30 contiguous amino acids) or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2 having biological activity (the fragment comprising SEQ ID NO: 2 comprising the amino acid sequence from amino acid 135 to amino acid 144). Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 1.

In another embodiment, the present invention relates to a composition comprising a protein, comprising: (a) the amino acid sequence of SEQ ID NO: 2; (b) the amino acids 1 to 83 of SEQ ID NO: 2. (C) a fragment of the amino acid sequence of SEQ ID NO: 2, comprising eight consecutive amino acids of SEQ ID NO: 2; and (d) the cDNA insert of clone bn971, deposited under accession number ATCC 98535. A composition comprising an amino acid sequence selected from the group consisting of an encoded amino acid sequence and substantially free from other mammalian proteins. Preferably, such a protein comprises the amino acid sequence of SEQ ID NO: 2 or the amino acid sequence from amino acid 1 to amino acid 83 of SEQ ID NO: 2. In a further preferred embodiment, the invention relates to a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2 having biological activity, said fragment comprising SEQ ID NO: 2, preferably 8, more preferably 20, most preferably A protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2 having biological activity (preferably comprising 30 consecutive amino acids);
2 comprising the amino acid sequence from amino acid 135 to amino acid 144).

[0005] In one embodiment, the invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3; (B) a polynucleotide comprising nucleotides 562 to 777 of SEQ ID NO: 3; (c) a polynucleotide comprising nucleotides 236 to 673 of SEQ ID NO: 3; (d) clone bn268 deposited under accession number ATCC 98535 1
A polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of 1; (e) clone bn268 1 deposited under accession number ATCC 98535
A polynucleotide encoding the full-length protein encoded by the cDNA insert of No. 1; (f) clone bn2681 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of one of the mature protein coding sequences; (g) clone bn268 1 deposited under accession number ATCC 98535
A polynucleotide encoding a mature protein encoded by the cDNA insert of 1); (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 4; (i) an amino acid of SEQ ID NO: 4 having biological activity A polynucleotide encoding a protein comprising a fragment of the sequence (the fragment comprises eight consecutive amino acid sequences of SEQ ID NO: 4); (j) an allelic variant of the polynucleotide of (a) to (g) above. (K) a polynucleotide encoding a species homolog of the protein of (h) or (i) above; and (1) any of the polynucleotides (a) to (i) under strict conditions. A polynucleotide that can hybridize to one. Preferably, such a polynucleotide is nucleotide 562 of SEQ ID NO: 3.
Nucleotide sequence from nucleotide 236 to nucleotide 673 of SEQ ID NO: 3; accession number ATCC98
The nucleotide sequence of the full length protein coding sequence of clone bn26811 deposited as 535; or the mature protein coding sequence of clone bn26811 deposited as accession number ATCC98535.
In another preferred embodiment, the polynucleotide encodes the full-length or mature protein encoded by the cDNA insert of clone bn26811 deposited under accession number ATCC 98456. In yet another preferred embodiment, the invention provides a polynucleotide encoding a protein comprising the amino acid sequence of amino acids 1 to 37 of SEQ ID NO: 4. In a further preferred embodiment, the present invention relates to a polynucleotide encoding a protein comprising a fragment of the amino acid sequence SEQ ID NO: 4 having biological activity, said fragment comprising SEQ ID NO: 4, preferably 8 More preferably, the polynucleotide encodes a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 4 that has biological activity, including 20 and most preferably 30 contiguous amino acids. 4 amino acids 31 to 40). Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 3.

In another embodiment, the present invention provides a composition comprising a protein, wherein the protein comprises: (a) the amino acid sequence of SEQ ID NO: 4; (b) amino acid 1 to amino acid 37 of SEQ ID NO: 4. (C) a fragment of the amino acid sequence of SEQ ID NO: 4, comprising eight consecutive amino acids of SEQ ID NO: 4; and (d) clone bn2681 deposited under accession number ATCC 98535.
A composition comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by one of the cDNA inserts and substantially free of other mammalian proteins. Preferably, such a protein comprises the amino acid sequence of SEQ ID NO: 4 or the amino acid sequence from amino acid 1 to amino acid 37 of SEQ ID NO: 4. In a further preferred embodiment, the invention relates to a protein comprising a fragment of the amino acid sequence SEQ ID NO: 4 having biological activity, said fragment comprising SEQ ID NO: 4, preferably 8, more preferably 20, most preferably A protein comprising a fragment of the amino acid sequence of SEQ ID NO: 4 having biological activity (preferably comprising 30 consecutive amino acids)
4 amino acids 31 to 40).

[0007] In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5; (B) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5 from nucleotide 286 to nucleotide 1686; (c) a polynucleotide comprising the nucleotide sequence of nucleotide 544 to nucleotide 1686 of SEQ ID NO: 5; (d) SEQ ID NO: A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5 from nucleotide 365 to nucleotide 1160; (e) clone cb96 10 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of (c) clone cb9610 deposited under accession number ATCC 98535.
A polynucleotide encoding the full-length protein encoded by the cDNA insert of (c) clone cb9610 deposited under accession number ATCC98535.
A polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of: (h) clone cb96 10 deposited under accession number ATCC 98535
A polynucleotide encoding a mature protein encoded by the cDNA insert of (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 6; (j) a biologically active SEQ ID NO: A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 6
(K) a polynucleotide which is an allelic variant of the polynucleotide of (a) to (h); (1) a species homolog of the protein of (i) or (j) And (m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j). Preferably, such a polynucleotide is nucleotide 286 of SEQ ID NO: 5.
Nucleotide sequence from nucleotide 544 to nucleotide 1686 of SEQ ID NO: 5; nucleotide sequence from nucleotide 365 to nucleotide 1160 of SEQ ID NO: 5; full length of clone cb9610 deposited as accession number ATCC 98535 The nucleotide sequence of the protein coding sequence; or the mature protein coding sequence of clone cb9610 deposited under accession number ATCC 98535. In another preferred embodiment, the polynucleotide has accession number ATCC.
It encodes the full-length or mature protein encoded by the cDNA insert of clone cb9610 deposited as 98456. In yet another preferred embodiment, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of amino acid 28 to amino acid 395 of SEQ ID NO: 6. In a further preferred embodiment, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 6 having biological activity, said fragment comprising SEQ ID NO: 6, preferably 8, More preferably, it comprises 20 and most preferably 30 contiguous amino acids) or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 6 having biological activity (including
The fragment comprises the amino acid sequence from amino acid 228 to amino acid 237 of SEQ ID NO: 6). Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 5.

In another embodiment, the present invention provides a composition comprising a protein, wherein the protein comprises: (a) the amino acid sequence of SEQ ID NO: 6; (b) amino acid 28 to 395 of SEQ ID NO: 6. (C) a fragment of the amino acid sequence of SEQ ID NO: 6, comprising eight consecutive amino acids of SEQ ID NO: 6; and (d) clone cb96 10 deposited under accession number ATCC 98535.
A composition comprising an amino acid sequence selected from the group consisting of: the amino acid sequence encoded by the cDNA insert of claim 1 and substantially free of other mammalian proteins. Preferably, such a protein is the amino acid sequence of SEQ ID NO: 6 or amino acids 28 to 39 of SEQ ID NO: 6.
Includes up to 5 amino acid sequences. In a further preferred embodiment, the invention relates to a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 6 having biological activity, said fragment comprising SEQ ID NO: 6, preferably 8, more preferably 20, most preferably A protein comprising a fragment of the amino acid sequence of SEQ ID NO: 6 having biological activity (preferably containing 30 consecutive amino acids); Including).

[0009] In one embodiment, the invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 7; (B) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 7 from nucleotide 99 to nucleotide 1049; (c) a polynucleotide comprising the nucleotide sequence of nucleotide 222 to nucleotide 1049 of SEQ ID NO: 7; (d) SEQ ID NO: A polynucleotide comprising a nucleotide sequence from nucleotide 632 to nucleotide 998 of 7; (e) clone cb213 1 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of 1; (f) clone cb213 1 deposited under accession number ATCC 98535
A polynucleotide encoding the full-length protein encoded by the cDNA insert of No. 1; (g) clone cb213 1 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of one of the mature protein coding sequences; (h) clone cb213 1 deposited under accession number ATCC 98535
A polynucleotide encoding a mature protein encoded by one of the cDNA inserts; (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 8; (j) a SEQ ID NO having biological activity Polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 8
(K) a polynucleotide which is an allelic variant of the polynucleotide of (a) to (h); (1) a species homolog of the protein of (i) or (j) And (m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j). Preferably, such a polynucleotide comprises the nucleotide sequence from nucleotide 99 to nucleotide 1049 of SEQ ID NO: 7; the nucleotide sequence from nucleotide 222 to nucleotide 1049 of SEQ ID NO: 7; the nucleotide sequence from nucleotide 632 to nucleotide 998 of SEQ ID NO: 7. Nucleotide sequence; accession number AT
The nucleotide sequence of the full length protein coding sequence of clone cb21311 deposited as CC98535; or the mature protein coding sequence of clone cb21311 deposited as accession number ATCC98535. In another preferred embodiment, the polynucleotide has accession number ATCC.
It encodes the full-length or mature protein encoded by the cDNA insert of clone cb21311 deposited as 98535. In yet another preferred embodiment, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 8 from amino acid 187 to amino acid 300. In yet another preferred embodiment, a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 8 having biological activity, wherein said fragment is SEQ ID NO: 8, preferably 8, more preferably Contains 20, and most preferably 30 contiguous amino acids), or a biologically active SEQ ID NO:
Provided is a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 8, wherein the fragment comprises the amino acid sequence from amino acid 153 to amino acid 162 of SEQ ID NO: 8. Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 7.

In another embodiment, the present invention relates to a composition comprising a protein, wherein the protein comprises: (a) the amino acid sequence of SEQ ID NO: 8; (b) the amino acid sequence from amino acid 187 to amino acid 300 of SEQ ID NO: 8. (C) a fragment of the amino acid sequence of SEQ ID NO: 8, comprising eight consecutive amino acids of SEQ ID NO: 8; and (d) clone cb213 1 deposited under accession number ATCC 98535.
A composition comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by one of the cDNA inserts and substantially free of other mammalian proteins. Preferably, such a protein is an amino acid sequence of SEQ ID NO: 8 or amino acids 187 to 3 of SEQ ID NO: 8.
Includes up to 00 amino acid sequences. In a further preferred embodiment, the present invention provides
A protein comprising a fragment of the amino acid sequence of SEQ ID NO: 8 having biological activity (
The fragment comprises SEQ ID NO: 8, preferably comprising 8, more preferably 20, most preferably 30 contiguous amino acids) or a fragment of the amino acid sequence SEQ ID NO: 8 having biological activity. A protein comprising the amino acid sequence from amino acid 153 to amino acid 162 of SEQ ID NO: 8.

In one embodiment, the invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9; (B) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9 from nucleotide 3003 to nucleotide 3137; (c) a polynucleotide comprising the nucleotide sequence of nucleotide 3072 to nucleotide 3137 of SEQ ID NO: 9; (d) SEQ ID NO: A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9 from nucleotide 2713 to nucleotide 3114; (e) clone cj457 4 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of (c) clone cj457 4 deposited under accession number ATCC 98535.
A polynucleotide encoding the full-length protein encoded by the cDNA insert of (c) clone cj457 4 deposited under accession number ATCC 98535.
A polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of: (h) clone cj457 4 deposited under accession number ATCC 98535
A polynucleotide encoding the mature protein encoded by the cDNA insert of (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 10; (j) a SEQ ID NO: having biological activity A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10
(K) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (h); (1) a species homolog of the protein of (i) or (j) And (m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(j). Preferably, such a polynucleotide comprises nucleotide 300 of SEQ ID NO: 9.
Nucleotide sequence from nucleotide 3072 to nucleotide 3137 of SEQ ID NO: 9; nucleotide sequence from 3 to nucleotide 3137; SEQ ID NO: 9
Nucleotide sequence of nucleotide 2713 to nucleotide 3114 of the full length protein coding sequence of clone cj4574 deposited under accession number ATCC98535; or nucleotide sequence of the mature protein coding sequence of clone cj4574 deposited under accession number ATCC98535. Including. In another preferred embodiment, the polynucleotide has accession number A
Encodes the full-length or mature protein encoded by the cDNA insert of clone cj4574, deposited as TCC98535. In yet another preferred embodiment, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence of amino acids 1 to 37 of SEQ ID NO: 10. In a further preferred embodiment, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10 having biological activity, wherein said fragment comprises preferably 8 More preferably, it comprises 20 and most preferably 30 contiguous amino acids), or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10 having biological activity (the fragment comprising SEQ ID NO: 10 amino acids 17 to 26). Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 9.

In another embodiment, the present invention provides a composition comprising a protein, wherein the protein comprises: (a) the amino acid sequence of SEQ ID NO: 10; (b) the amino acids 1 to 37 of SEQ ID NO: 10. (C) a fragment of the amino acid sequence of SEQ ID NO: 10, comprising eight consecutive amino acids of SEQ ID NO: 10; and (d) clone cj457 4 deposited under accession number ATCC 98535.
A composition comprising an amino acid sequence selected from the group consisting of: the amino acid sequence encoded by the cDNA insert of claim 1 and substantially free of other mammalian proteins. Preferably, such a protein is the amino acid sequence of SEQ ID NO: 10 or amino acids 1 to 3 of SEQ ID NO: 10.
Includes up to 7 amino acid sequences. In a further preferred embodiment, the invention relates to a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10 having biological activity (
The fragment is SEQ ID NO: 10, preferably 8, more preferably 20,
Most preferably contains 30 consecutive amino acids) or a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10 having biological activity (the fragment is an amino acid sequence from amino acid 17 to amino acid 26 of SEQ ID NO: 10) including)
I will provide a.

[0013] In one embodiment, the invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11; (B) a polynucleotide comprising the nucleotide sequence from nucleotide 284 to nucleotide 1357 of SEQ ID NO: 11; (c) a polynucleotide comprising the nucleotide sequence from nucleotide 603 to nucleotide 1233 of SEQ ID NO: 11; (d) accession number ATCC 98535 Clone cz6531 deposited as
A polynucleotide comprising the nucleotide sequence of one full length protein coding sequence; (e) clone cz6531 deposited under accession number ATCC 98535 1
A polynucleotide encoding the full-length protein encoded by the cDNA insert of No. 1; (f) clone cz6531 deposited under accession number ATCC 98535 1
A polynucleotide comprising the nucleotide sequence of one of the mature protein coding sequences; (g) clone cz6531 deposited under accession number ATCC 98535 1
A polynucleotide encoding a mature protein encoded by one of the cDNA inserts; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 12; (i) a SEQ ID NO having biological activity : A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 12
(J) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (g) above; (k) a species homology of the protein of (h) or (i) above. A polynucleotide encoding a body; and (l) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such a polynucleotide is nucleotide 28 of SEQ ID NO: 11.
Nucleotide sequence from nucleotide 603 to nucleotide 1233 of SEQ ID NO: 11; accession number AT
The nucleotide sequence of the full length protein coding sequence of clone cz655311 deposited as CC98535; or the mature protein coding sequence of clone cz65311 deposited as accession number ATCC98535. In another preferred embodiment, the polynucleotide has accession number ATCC.
It encodes the full-length or mature protein encoded by the cDNA insert of clone cz65311 deposited as 98535. In a further preferred embodiment, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence from amino acid 147 to amino acid 358 of SEQ ID NO: 12. In a further preferred embodiment, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 12 having biological activity, said fragment comprising SEQ ID NO: 12, preferably 8, More preferably, it comprises 20 and most preferably 30 contiguous amino acids) or a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 12 having biological activity (the fragment comprising SEQ ID NO: 12 amino acids 174 to 183
Up to and including the amino acid sequence up to). Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 11.

In another embodiment, the present invention provides a composition comprising a protein, wherein the protein comprises: (a) the amino acid sequence of SEQ ID NO: 12; (b) the amino acid 147 to amino acid 358 of SEQ ID NO: 12 (C) a fragment of the amino acid sequence of SEQ ID NO: 12, comprising eight consecutive amino acids of SEQ ID NO: 12; and (d) clone cz6531 deposited with accession number ATCC98535.
A composition comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by one of the cDNA inserts and substantially free of other mammalian proteins. Preferably, such a protein comprises the amino acid sequence of SEQ ID NO: 12 or the amino acid sequence from amino acid 147 to amino acid 358 of SEQ ID NO: 12. In a further preferred embodiment, the invention relates to a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 12 having biological activity, said fragment comprising SEQ ID NO: 12, preferably 8, more preferably 20, most preferably A protein comprising a fragment of the amino acid sequence of SEQ ID NO: 12 having biological activity (preferably containing 30 contiguous amino acids) (the fragment comprises the amino acid sequence of amino acids 174 to 183 of SEQ ID NO: 12) Including).

[0015] In one embodiment, the invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 13; (B) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 13 from nucleotide 621 to nucleotide 1763; (c) a polynucleotide comprising the nucleotide sequence of nucleotide 1461 to nucleotide 1763 of SEQ ID NO: 13; (d) accession number ATCC98535 Clone dx1384 deposited as
A polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of (e) clone dx1384 deposited under accession number ATCC 98535
A polynucleotide encoding the full-length protein encoded by the cDNA insert of (d) clone dx1384 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of: (g) clone dx1384 deposited under accession number ATCC 98535
A polynucleotide encoding the mature protein encoded by the cDNA insert of (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 14; (i) a SEQ ID NO: having biological activity A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14
(J) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (g) above; (k) a species homology of the protein of (h) or (i) above. A polynucleotide encoding a body; and (l) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such a polynucleotide is nucleotide 62 of SEQ ID NO: 13.
Nucleotide sequence from nucleotide 1461 to nucleotide 1763 of SEQ ID NO: 13; accession number A
The nucleotide sequence of the full length protein coding sequence of clone dx1384 deposited as TCC98535; or the mature protein coding sequence of clone dx1384 deposited as accession number ATCC98535. In another preferred embodiment, the polynucleotide has accession number ATCC9.
Encodes the full-length or mature protein encoded by the cDNA insert of clone dx1384 deposited as 8535. In a further preferred embodiment, the present invention provides a polynucleotide encoding a protein comprising the amino acid sequence from amino acid 83 to amino acid 229 of SEQ ID NO: 14. In a further preferred embodiment, the present invention provides a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14 having biological activity, said fragment comprising preferably 8 of SEQ ID NO: 14, A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14, which has more preferably 20 and most preferably 30 contiguous amino acids, or a fragment having the biological activity (SEQ ID NO: 14 amino acids 185 to 194). Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 13.

In another embodiment, the present invention provides a composition comprising a protein, wherein the protein comprises: (a) the amino acid sequence of SEQ ID NO: 14; (b) the amino acid sequence from amino acid 83 to amino acid 229 of SEQ ID NO: 14 (C) a fragment of the amino acid sequence of SEQ ID NO: 14, comprising eight consecutive amino acids of SEQ ID NO: 14; and (d) clone dx1384, deposited as accession number ATCC 98535.
A composition comprising an amino acid sequence selected from the group consisting of: the amino acid sequence encoded by the cDNA insert of claim 1 and substantially free of other mammalian proteins. Preferably, such a protein comprises the amino acid sequence of SEQ ID NO: 14 or the amino acid sequence from amino acid 83 to amino acid 229 of SEQ ID NO: 14. In a further preferred embodiment, the present invention relates to a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14 having biological activity, wherein said fragment comprises SEQ ID NO: 14, preferably 8, more preferably 20
, Most preferably comprising 30 contiguous amino acids) or a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14 having biological activity (the fragment comprising the amino acid sequence of amino acids 185 to 194 of SEQ ID NO: 14) Including an amino acid sequence).

In one embodiment, the present invention provides a composition comprising an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15; (B) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15 from nucleotide 119 to nucleotide 295; (c) a polynucleotide comprising the nucleotide sequence of nucleotide ID 191 to nucleotide 295 of SEQ ID NO: 15; (d) accession number ATCC98535 Clone ij167 5 deposited as
A polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of (e) clone ij1675 deposited under accession number ATCC 98535.
A polynucleotide encoding the full length protein encoded by the cDNA insert of (f) clone ij167 5 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of: (g) clone ij167 5 deposited under accession number ATCC 98535
A polynucleotide encoding a mature protein encoded by the cDNA insert of (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 16; (i) a SEQ ID NO: having biological activity A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 16
(J) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (g) above; (k) a species homology of the protein of (h) or (i) above. A polynucleotide encoding a body; and (l) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Preferably, such a polynucleotide is nucleotide 11 of SEQ ID NO: 15.
Nucleotide sequence from 9 to nucleotide 295; nucleotide sequence from nucleotide 191 to nucleotide 295 of SEQ ID NO: 15; accession number ATCC
The nucleotide sequence of the full-length protein coding sequence of clone ij1675 deposited as 98535; or the mature protein coding sequence of clone ij1675 deposited as accession number ATCC 98535.
In another preferred embodiment, the polynucleotide has accession number ATCC 9853.
No. 5 encodes the full-length or mature protein encoded by the cDNA insert of clone ij1675. In yet another preferred embodiment, the invention provides a polynucleotide encoding a protein comprising the amino acid sequence from amino acid 1 to amino acid 26 of SEQ ID NO: 16. In a further preferred embodiment, the invention relates to a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 16 which has biological activity, said fragment comprising preferably 8 of SEQ ID NO: 16, More preferably 20 and most preferably 30
SEQ ID NO: 16) having a biological activity.
(A fragment comprising the amino acid sequence from amino acid 24 to amino acid 33 of SEQ ID NO: 16). Another embodiment provides a gene corresponding to the cDNA sequence of SEQ ID NO: 15.

In another embodiment, the present invention provides a composition comprising a protein, wherein the protein comprises: (a) the amino acid sequence of SEQ ID NO: 16; (b) the amino acids 1 to 26 of SEQ ID NO: 16 (C) a fragment of the amino acid sequence of SEQ ID NO: 16, comprising eight consecutive amino acids of SEQ ID NO: 16; and (d) clone ij167 5 deposited under accession number ATCC 98535.
A composition comprising an amino acid sequence selected from the group consisting of: the amino acid sequence encoded by the cDNA insert of claim 1 and substantially free of other mammalian proteins. Preferably, such protein is the amino acid sequence of SEQ ID NO: 16 or amino acids 1 to 26 of SEQ ID NO: 16.
Up to the amino acid sequence. In a further preferred embodiment, the invention relates to a protein comprising a fragment of the amino acid sequence SEQ ID NO: 16 having biological activity, said fragment comprising SEQ ID NO: 16, preferably 8, more preferably 20, most preferably A protein comprising a fragment of the amino acid sequence of SEQ ID NO: 16 having biological activity (preferably containing 30 consecutive amino acids); Including).

[0019] In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The present invention also provides host cells, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions. Also provided by the invention are organisms having enhanced, reduced, or modified expression of a gene corresponding to a polynucleotide sequence disclosed herein.

Further, the present invention provides a method for producing a protein, comprising: (a) growing a culture of a host cell transformed with such a polynucleotide composition in a suitable medium; A method comprising purifying is also provided. The protein produced by such a method is also provided by the present invention. Preferred specific examples include those in which the protein produced by such a method is a mature form of the protein.

The protein composition of the present invention may further contain a pharmaceutically acceptable carrier. Compositions comprising antibodies specifically reactive with such proteins are also provided by the present invention.

[0022] Also provided is a method for preventing, treating or ameliorating a medical condition comprising administering to a mammalian subject a therapeutically effective amount of a composition comprising a protein of the invention and a pharmaceutically acceptable carrier. You.

[0023] Report nucleotide and amino acid sequences for each clone and protein disclosed in the detailed description Isolation proteins and polynucleotides herein below. By sequencing the deposited clones by known methods, the actual nucleotide sequence of each clone can be readily determined. The deduced amino acid sequence (both full length as well as mature) can then be determined from such nucleotide sequences. By expressing the clones in a suitable host cell, collecting the proteins, and then determining the sequence, the amino acid sequence of the protein encoded by each clone can also be determined. For each protein disclosed, Applicants have identified what has been determined to be the best identifiable reading frame using sequence information available at the time of filing. As used herein, the term "secreted" protein refers to that which is transported across a membrane when expressed in a suitable host cell, including transport as a result of a signal sequence in its amino acid sequence. “Secreted” proteins include, but are not limited to, proteins that are totally secreted (eg, soluble proteins) or partially secreted (eg, receptors) from the cell in which they are expressed. "Secreted" proteins also include, but are not limited to, those that are transported across the endoplasmic reticulum membrane.

Clone "bn971" The polynucleotide of the present invention has been identified as clone "bn971" . bn971 was isolated from an adult placenta cDNA library and identified as encoding a secreted or transmembrane protein based on computer analysis of the amino acid sequence of the encoded protein. bn971 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "bn971 protein"). The nucleotide sequence of bn971 determined this time is shown in SEQ ID NO: 1. What the applicant considers to be the correct reading frame and deduced amino acid sequence of the bn971 protein corresponding to the above nucleotide sequence is shown in SEQ ID NO: 2. Amino acid 55
To 67 are putative leader / signal sequences, the putative mature amino acid sequence starting at amino acid 68, or amino acids 55 to 67 are transmembrane domains. The EcoRI / NotI restriction fragment obtainable from the deposit containing clone bn971 should be approximately 1700 bp. The nucleotide sequence of bn971 disclosed herein was searched against the GenBank and GenSeq nucleotide sequence databases using BLASTN / BLASTX and FASTA search protocols. bn97 1 is AA046689 (zk72h06.s1 Soares pr
egnant uterus NbHPU Homo sapiens cDNA clone 488411 3 '), D30934 (Human fe
tal-lung cDNA 5'-end sequence), R78820 (yi90b03.r1 Homo sapiens cDNA clo
ne 146477 5 ') and R91687 (yq10h09.s1 Homo sapiens cDNA clone 196577 3')
Showed at least some identity to the sequence identified as bn97
The deduced amino acid sequence disclosed herein for 1 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. Estimated bn
The 971 protein showed at least some identity to the sequence identified as A10431 (Hepatitis-B virus surface antigen P31). The bn971 protein also shows some identity (30% identity, 50% conserved amino acids) to the lectin-like receptor for oxidized bovine and human LDL (low density lipoprotein). Although this homology is small, it is higher in the lectin-like domain (44% identity,
Conserved amino acids 62%). In addition, the 3 'untranslated region of the bovine receptor contains seven mRNAs.
It has a destabilizing sequence (ATTTA) and bn971 has it in its 3 'untranslated region. This oxidized LDL lectin-like receptor (named LOX-1;
Sawamura et al., 1997, Nature 386: 73-77) are integral membrane proteins that bind to oxidized low density lipoproteins, internalize them in endothelial cells, destroy them, and thus It plays an important role in the development of atherosclerosis.
Based on identity, the bn971 protein and each identical protein or peptide may share at least some activity.

Clone "bn26811" The polynucleotide of the present invention has been identified as clone "bn26811" .
bn26811 was isolated from an adult placenta cDNA library and identified as encoding a secreted or transmembrane protein based on computer analysis of the amino acid sequence of the encoded protein. bn26811 is a full-length clone,
Contains the entire coding sequence of a secreted protein (also referred to herein as "bn26811 protein"). The nucleotide sequence of bn26811 determined this time is shown in SEQ ID NO: 3.
This time, the applicant considers the correct reading frame and deduced amino acid sequence of bn26811 protein corresponding to the above nucleotide sequence to be shown in SEQ ID NO: 4. The EcoRI / NotI restriction fragment obtainable from the deposit containing clone bn26811 should be approximately 1050 bp. The nucleotide sequence of bn26811 disclosed herein was searched against the GenBank and GeneSeq databases using BLASTA / BLASTX and FASTA search protocols. bn26811 is D62832 (Human aorta cDNA 5'-end GEN-
330C09) and U20159 (Mus musculus 76kDa tyrosine pshosphoprotein SLP-76
It showed at least some identity to sequences identified as mRNA, complete cds). The deduced amino acid sequence disclosed herein for bn26811 was compared to BLAS
A search was performed against the GenPept and GeneSeq amino acid sequence databases using the TX search protocol. The putative bn26811 protein is D83171 (GDP-GTP exchange pro
It showed at least some identity to the sequence identified as tein for Rho1p [Saccharomyces cerevisiae]). Based on identity, the bn26811 protein and each protein or peptide with identity may share at least some activity. According to the TopPredII computer program, a potential transmembrane domain centering around amino acid 33 of SEQ ID NO: 4 is estimated in the bn26 811 protein sequence. This region may also function as a signal sequence.

Clone "cb9610" The polynucleotide of the present invention has been identified as clone "cb9610" . cb9610 was isolated from a human fetal brain cDNA library and identified as encoding a secreted or transmembrane protein based on computer analysis of the amino acid sequence of the encoded protein. cb9610 is a full-length clone, containing the entire coding sequence of a secreted protein (also referred to herein as "cb9610 protein"). The nucleotide sequence of cb9610 determined this time is shown in SEQ ID NO: 5. This time, the applicant considers the correct reading frame and deduced amino acid sequence of cb9610 protein corresponding to the above nucleotide sequence to be shown in SEQ ID NO: 6. Amino acids 74 to 86 are a putative leader / signal sequence, and the predicted mature amino acid sequence starts at amino acid 87 or is a transmembrane domain. The EcoRI / NotI restriction fragment obtainable from the deposit containing clone cb9610 should be approximately 2100 bp. The nucleotide sequence of cb9610 disclosed herein was searched against the GenBank and GeneSeq databases using BLASTA / BLASTX and FASTA search protocols. cb96 10 is AA459012, AA459236, AA256744 (zs31h11.r1
Soares NbHTGBC Homo sapiens cDNA clone 686853 3 '), N54489 (yv40f07.s1 S
oares fetal liver spleen 1NFLS Homo sapiens cDNA clone 245221 3 ') and N
It showed at least some identity to the sequence identified as 57339 (yw81h07.r1 Homo sapiens cDNA clone 258685 5 ′). The deduced amino acid sequence disclosed herein for cb9610 was compared to GenPept and Gen using the BLASTX search protocol.
A search was performed against the eSeq amino acid sequence database. The putative cb96 10 protein is
It showed at least some identity to the sequence identified as X80036 (ascorbate peroxidase [Arabidopsis thaliana]). Based on identity, cb96
Ten proteins and each protein or peptide having identity may share at least some activity. According to the TopPredII computer program, amino acid residues 25, 80, 125 of SEQ ID NO: 6 were found in the cb96 10 protein sequence.
Seven potential transmembrane domains centered around 225, 300, 350 and 440 are estimated. Therefore, cb9610 may be an integral membrane protein with multiple helices in the membrane. cb9610 also contains a sequence motif of an actin-type actin-binding domain, which is considered to be an anchor actin for the cell membrane. Clone "cb21311" The polynucleotide of the present invention has been identified as clone "cb21311" .
cb21311 was isolated from a human fetal brain cDNA library and identified as encoding a secreted or transmembrane protein based on computer analysis of the amino acid sequence of the encoded protein. cb213 11 is a full-length clone, containing the entire coding sequence of a secreted protein (also referred to herein as "cb213 11 protein"). The nucleotide sequence of cb213_11 determined this time is shown in SEQ ID NO: 7.
This time, the applicant considers the correct reading frame and deduced amino acid sequence of cb213 11 protein corresponding to the above nucleotide sequence to be shown in SEQ ID NO: 8. Amino acid 2
9 to 41 are putative leader / signal sequences, and the predicted mature amino acid sequence starts at amino acid 42 or amino acids 29 to 41 are transmembrane domains. The EcoRI / NotI restriction fragment obtainable from the deposit containing clone cb21311 should be approximately 2400 bp. The nucleotide sequence of cb21311 disclosed herein was searched against the GenBank and GeneSeq databases using BLASTA / BLASTX and FASTA search protocols. cb213 11 is AA332165 (EST36344 Embryo, 8 week I
Homo sapiens cDNA 5 'end) and R34507 (g58a03.r1 Homo sapiens cDNA clone)
36801 showed at least some identity to the sequence identified as 5 '). The deduced amino acid sequence disclosed herein for cb21311 was searched against the GenPept and GeneSeq amino acid sequence databases using the BLASTX search protocol. The putative cb21311 protein is U39847 (AO13 ankyrin [Caenorhabditis e
legans]).
Based on the same, the cb21311 protein and each protein or peptide having identity may share at least some activity.

Clone "cj457-4" The polynucleotide of the present invention has been identified as clone "cj4574" . cj4574 was isolated from a human fetal brain cDNA library and identified as encoding a secreted or transmembrane protein based on computer analysis of the amino acid sequence of the encoded protein. cj457-4 is a full-length clone, containing the entire coding sequence of a secreted protein (also referred to herein as "cj4574 protein"). The nucleotide sequence of cj4574 determined this time is shown in SEQ ID NO: 9. SEQ ID NO: 10 shows what the applicant considers to be the correct reading frame and deduced amino acid sequence for cj4574 protein corresponding to the above nucleotide sequence. Amino acid 11
To 23 are putative leader / signal sequences, the putative mature amino acid sequence starts at amino acid 24, or amino acids 11 to 23 are transmembrane domains. The EcoRI / NotI restriction fragment obtainable from the deposit containing clone cj4574 should be approximately 3350 bp. The nucleotide sequence of cj4574 disclosed herein was searched against the GenBank and GeneSeq databases using BLASTA / BLASTX and FASTA search protocols. cj4574 was cloned from T92881 (ye22a10.s1 Homo sapiens cDNA clone).
ne 118458 3 ') and T92488 (ye21g09.r1 Homo sapiens cDNA clone 118432 5')
Showed at least some identity to the sequence identified as Based on identity, the cj4574 protein and each similar protein or peptide may share at least some activity. According to the TopPredII computer program, a potential transmembrane domain centering around amino acid 17 of SEQ ID NO: 10 is estimated in the cj4574 protein sequence. This region may also function as a signal sequence.

Clone "cz65311" The polynucleotide of the present invention has been identified as clone cz65311.
cz655311 was isolated from an adult testis cDNA library and identified as encoding a secreted or transmembrane protein based on computer analysis of the amino acid sequence of the encoded protein. cz655311 is a full-length clone,
Contains the entire coding sequence of a secreted protein (also referred to herein as "cz65311 protein"). The nucleotide sequence of cz65311 determined this time is shown in SEQ ID NO: 11. This time, the applicant considers the correct reading frame and deduced amino acid sequence of cz65311 protein corresponding to the above nucleotide sequence to be shown in SEQ ID NO: 12. The EcoRI / NotI restriction fragment obtainable from the deposit containing clone cz65311 should be approximately 1300 bp. The nucleotide sequence of cz6531 1 disclosed herein was searched against the GenBank and GeneSeq databases using the BLASTA / BLASTX and FASTA search protocols. cz6553 11 is AA024740 (ze76c90.s1 Soares fetal he
art NbHH19W Homo sapiens cDNA clone 364912 3 '), AA203204 (zx57b04.r1 Soa
res fetal liver spleen 1NFLS S1 Homo sapiens cDNA clone 446575 5 'simila
r to contains element MSR1 repetitive element), W72894 (zd59e06.s1 Soare
s fetal heart NbHH19W Homo sapiens cDNA clone 344962 3 ') and W76099 (zd
It showed at least some identity to the sequence identified as 59e06.r1 Soares fetal heart NbHH19W Homo sapiens cDNA clone 344962 5 ′). The predicted cz655311 showed similarity to various WD-40 repeat-containing proteins such as beta transducin-like protein (L28125) and coatmer, beta prime subunit (AJ006523). Homology is determined by the Trp of the beta transducin family starting at residue 262 of SEQ ID NO: 12.
-Probably due to the presence of the Asp repeat signature (WD-40). W
D-40 repeat is believed to be a protein-protein interaction domain. Based on identity, the cz65311 protein and each protein or peptide with identity may share at least some activity. According to the TopPredII computer program, the sequence of SEQ ID NO: 12 was found in the cz65311 protein sequence.
A potential transmembrane domain around amino acid 200 is predicted.

Clone "dx1384" The polynucleotide of the present invention has been identified as clone "dx1384" . dx1384 was isolated from an adult testis cDNA library and identified as encoding a secreted or transmembrane protein based on computer analysis of the amino acid sequence of the encoded protein. dx1384 is a full-length clone, including the entire coding sequence of a secreted protein (also referred to herein as "dx1384 protein"). The nucleotide sequence of dx1384 determined this time is shown in SEQ ID NO: 13.
This time, the applicant considers the correct reading frame and deduced amino acid sequence of dx1384 protein corresponding to the above nucleotide sequence to be shown in SEQ ID NO: 14. Amino acid 2
68 through 280 is a predicted leader / signal sequence, the predicted amino acid sequence starting at amino acid 281 or amino acids 268 through 280 is a transmembrane domain. The EcoRI / NotI restriction fragment obtainable from the deposit containing clone dx1384 should be approximately 2300 bp. The nucleotide sequence of dx1384 disclosed herein was searched against the GenBank and GeneSeq databases using BLASTA / BLASTX and FASTA search protocols. dx1384 is AA108970 (ml63a06.r1 Stratagene mouse te
stis # 937308) Mus musculus cDNA clone 516658 5 '), AA280976 (zs97f01.r1 S
oares NbHTGBC Homo sapiens cDNA clone 262540 3 '), H99316 (yx23a03.s1 Hom
o sapiens cDNA clone 262540 3 '), T36050 (EST96120 Homo sapiens cDNA 5'),
X85637 (H. sapiens mRNA for expressed sequence tag, clone CAM tEST517 (A)
) And Z22280 (H. sapiens DNA sequence). Based on identity, the dx1384 protein and each identical protein or peptide may share at least some activity.

Clone "ij1675" The polynucleotide of the present invention has been identified as clone ij1675. ij1675-isolated from adult blood (peripheral blood mononuclear cells treated with G-CSF in vivo) brain cDNA library and secreted protein or transmembrane based on computer analysis of the amino acid sequence of the encoded protein. It was identified as encoding a protein. ij1675 is a full-length clone, containing the entire coding sequence of a secreted protein (also referred to herein as "ij1675 protein"). The nucleotide sequence of ij1675 determined this time is shown in SEQ ID NO: 15.
The applicant has shown in SEQ ID NO: 16 the correct reading frame and deduced amino acid sequence of the ij1675 protein corresponding to the above nucleotide sequence. Amino acids 12 to 24 are a putative leader / signal sequence, which starts at amino acid 25 or is a transmembrane domain. The EcoRI / NotI restriction fragment obtainable from the deposit containing clone ij1675 should be approximately 1050 bp in length. The nucleotide sequence of ij1675 disclosed herein was searched against the GenBank and GeneSeq databases using BLASTA / BLASTX and FASTA search protocols. ij1675 is derived from N71115 (za87h10.s1 Homo sapiens cDNA clone).
ne 299587 3 '), T85491 (yd78b01.r1 Homo sapiens cDNA clone 114313 5'), W0
4374 (za43f06.r1 Soares fetal liver spleen 1NFLS Homo sapiens cDNA clone
295331 5 '), W05476 (za87h10.r1 Soares fetal lung NbHL19W Homo sapiens cD
NA clone 299587 5 ') and W40146 (zb74d09.r1 Soares fetal lung NbHL19W Ho
It showed at least some identity to the sequence identified as mo sapiens cDNA clone 309329 5 '). The deduced amino acid sequence disclosed herein as ij1675 was searched against the GenPept and GeneSeq amino acid sequence databases using the BALSTX search protocol. The putative ij1675 protein is M96653 (adenyl
cyclase, type 6 [Mus musculus]) showed at least some identity to the sequence identified. Based on identity, the ij1675 protein and each protein or peptide with identity may share at least some activity. According to the TopPredII computer program, a potential transmembrane domain centered around amino acid 40 of SEQ ID NO: 16 is expected in the ij1875 protein sequence.

Deposit of clones clones bn971, bn26811, cb9610, cb2131
1, cj457 4, cz653 11, dx138 4 and ij167 5
Will enter the American Type Culture Co. on September 4, 1997 under the Budapest Treaty.
llection (10801 University Boulevard, Manassas, Virginia 20110-2209 US
A.) and accession number ATCC 98535, from which the clone is available. All restrictions on the public use of the deposited materials are set forth in 37 CFR. F. R.
Except for the provisions of § 1.808 (b), this will be terminated by the grant of a patent, and the deposit period is 37 F. R. § 1.806. Each clone was transfected into separate bacterial cells (E. coli) as this complex deposit. EcoRI / NotI digestion (5 ′ site, EcoRI; 3)
Site, NotI) to obtain fragments of appropriate size for such clones so that each clone can be removed from the deposited vector. Each clone was deposited in either the pED6 or pNOTs vector shown in FIG. The pED6dpc2 vector (pED6) was derived from pED6dpc1 by inserting a new polylinker to facilitate cDNA cloning (Kaufman et al. (1991). NAR 19: 4485-4490). Deleting DHFR,
The pNOTs vector was transformed into pMT2 (Kaufman et al. 1989. Mol. Cell. Biol.) By inserting a new polylinker and inserting the M13 origin of replication into the ClaI site.
9: 946-958). In some cases, the deposited clones “flipped” (ie, flipped) in the deposited isolate. In such cases, the cDNA insert can still be isolated by EcoRI and NotI digestion.
However, in that case the cD in the correct orientation for expression in a suitable vector
To place the NA, NotI will create a 5 'site and EcoRI will create a 3' site. The cDNA may be expressed from a vector in which the cDNA has been deposited. Bacterial cells containing individual clones can be obtained from the complex deposit as follows: The oligonucleotide probe or probe should be designed to be against the sequence known for the individual clone. This sequence can be derived from the sequences herein or a combination of those sequences. The oligonucleotide probe sequences used to isolate each full length clone are shown below and should be most reliable in isolating the clone of interest. Clone probe sequence bn97 1 SEQ ID NO: 21 bn268 11 SEQ ID NO: 22 cb96 10 SEQ ID NO: 23 cb213 11 SEQ ID NO: 24 cj457 4 SEQ ID NO: 25 cz65311 SEQ ID NO: 26 dx1384 SEQ ID NO: 27 ij167 5 SEQ ID NO: 28

The sequences listed above contain an N at position 2, which position is a biotinylated phosphoramidite residue (eg, biotin phosphoramidite (1-dimethoxytrityloxy-2-) rather than a nucleotide in a preferred probe / primer. (Obtained by use of (N-biotinyl-4-aminobutyl) -propyl-3-O- (2-cyanoethyl)-(N, N'-diisopropyl) -phosphoramidite (Glen Research catalog number 10-1953)). Occupied. Preferably, the design of the oligonucleotide probe should follow these parameters: (a) Should be designed to be the region of the sequence where the number of undefined bases (N), if any, is minimal; (B) Tm of about 80 ° C. (2 ° C. for A or T, 4 ° C. for G or C)
(Assume ° C). Preferably, oligonucleotides should be labeled with [gamma]-< ³² > P-ATP (specific activity 6000 Ci / mmole) using T4 polynucleotide kinase, using methods widely used for oligonucleotide labeling. Other labeling methods can be used. Preferably, unincorporated label should be removed by gel filtration or other established methods. It should be quantified by measuring the amount of radioactivity incorporated into the probe with a scintillation counter. Preferably,
The specific activity of the resulting probe should be about 4e + 6 dpm / pmole. Preferably, the bacterial culture containing the pool of full-length clones should be thawed and 100 μl of the stock used to inoculate a sterile culture flask containing 25 ml of sterile L broth containing 100 μg / ml ampicillin. Preferably, the culture should be grown at 37 ° C. to reach saturation, and preferably the saturated culture should be fresh L
Should be diluted with broth. Preferably, a portion of these dilutions are plated at 37 ° C. on solid bacterial culture medium containing L-broth containing 100 μg / ml ampicillin and 1.5% agar in a 150 mm petri dish. The dilution and volume at which 5000 well-defined and well-separated colonies will grow when grown overnight should be determined. Other known methods for obtaining well-defined and well-separated colonies can also be used. The colonies should then be transferred to nitrocellulose filters using standard colony hybridization techniques and lysed, denatured, and baked. Then, preferably, 6 × SSC containing 0.5% SDS, 100 μg / ml yeast RNA, and 10 mM EDTA (205.3 × 175.3 g stock).
NaCl / liter, 88.2 g sodium citrate, pH 7.0 with NaOH
(About 10 ml per 150 mm filter) at 65 ° C. for 1 hour with gentle stirring. The probe is then preferably added to the hybridization mix so that the concentration is greater than or equal to 1e + 6 dpm / ml. The filter is then preferably washed with 500 ml of 2X SSC / 0.5% SDS without stirring at room temperature, preferably afterwards with gentle shaking at room temperature with 500 ml of 2X SSC / 0. Wash with 1% SDS. 65 ° C., 30 minutes to 1 hour, 0.1 × SSC / 0
. A third wash with 5% SDS is optimal. The filter is then preferably dried and subjected to autoradiography for a sufficient time to visualize positives on X-ray film. Other known hybridization methods can also be used. Positive colonies are picked, grown in medium, and the plasmid DNA is then isolated using standard procedures. The clone can then be verified by restriction analysis, hybridization analysis or DNA sequencing.

A fragment of the protein of the present invention that can exhibit biological activity is also included in the present invention. Protein fragments may be linear or, for example, HUSaragovi
, et al., Bio / Technology 10, 773-778 (1992) and RSMcDowell, et al., J
Amer. Chem. Soc. 114, 9245-9253 (1992) (both references are deemed to be described herein by reference) may be cyclized using known methods. . For many purposes, including increasing the number of valencies at the protein binding site,
Such a fragment may be fused to a carrier molecule along with the immunoglobulin.
For example, a protein fragment may be fused via a “linker” to the Fc portion of an immunoglobulin. For the bivalent form of the protein, such a fusion is made to the F molecule of the IgG molecule.
This can be done for part c. Such fusions may be obtained using other immunoglobulin isotypes. For example, a protein-IgM fusion will yield a ten-valent form of a protein of the invention. The invention also provides the full length and mature forms of the disclosed proteins. The full length form of such a protein has been identified in the sequence listing by translation of the nucleotide sequence of the disclosed clone. Mature forms of such proteins may be obtained by expressing the disclosed full-length polynucleotides (preferably those deposited with the ATCC) in suitable mammalian cells or other host cells. The sequence of the mature form of the protein may be determined from the amino acid sequence of the full-length form. The present invention also provides a gene corresponding to the cDNA sequence disclosed herein. "
A "corresponding gene" is a region of the genome that is transcribed to produce mRNA from which cDNA is derived and whose flanking regions (coding sequences, 5 'and 3' non-coding sequences are required for regulated expression of such gene). Translation region), or spliced exons, introns, promoters, enhancers,
As well as silencer or suppressor elements. Corresponding genes can be isolated using the sequence information disclosed herein. Corresponding genes can be isolated using the sequence information disclosed herein. Such methods include preparing probes or primers from the disclosed sequence information to identify and / or amplify genes in a suitable genomic library or other source of genomic material. An "isolated gene" is a gene that has been separated from flanking coding sequences, if present, present in the genome of the organism from which the gene was isolated.

The chromosomal location corresponding to the polynucleotide sequences disclosed herein may be determined, for example, by hybridizing an appropriately labeled polynucleotide of the invention to the chromosome in situ. By identifying significantly similar nucleotide sequences in public databases, such as expressed sequence tags (ESTs) that have already been mapped to specific chromosomal locations, the corresponding chromosomal location of the disclosed polynucleotides may be determined. For at least some of the polynucleotide sequences disclosed herein, public database sequences having at least some similarity to the polynucleotides of the present invention are listed by database accession number. A search using the GenBank accession number of these public database sequences to identify UniGene clusters of overlapping sequences was performed at the National Center for Biotechnology I
This can be done on the internet site provided by nformation, the address of which is http://www.ncbi.nlm.nih.gov/UniGene/. Many UniGene clusters so identified have already been mapped to specific chromosomal locations.

An organism is provided that has enhanced, reduced, or altered expression of a gene corresponding to a polynucleotide sequence disclosed herein. Desirable changes in gene expression are achieved by using antisense polynucleotides or by using ribozymes that bind and / or cleave mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15 (7): 250-2
54; Lavarosky et al., 1997, Biochem.Mol. Med. 62 (1): 11-22; and Hampel,
1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1-39; all of which are herein incorporated by reference). Transgenic animals having multicopy genes corresponding to the polynucleotides disclosed herein, preferably obtained by transforming cells with genetic constructs that are stably maintained in transformed cells and their progeny. A transgenic animal is provided. Also provided are transgenic animals having altered genetic control regions that increase or decrease gene expression levels or alter the temporal or spatial pattern of gene expression (see European Patent No. 0 649 464 B1; All of which are considered to be described herein by reference). Further, provided are organisms in which the gene corresponding to the polynucleotide sequence disclosed herein has been incompletely or completely inactivated by insertion of an external sequence or by deletion of all or a part of the corresponding gene. By insertion, preferably by insertion after incorrect resection of the transposable element (Plasterk, 1992, Bioessays 14 (9): 629-633; Zwaal et al., 19).
93, Proc. Natl. Acad. Sci. USA 90 (16): 7431-7435; Clark et al., 1994, Pr.
USA 91 (2): 719-722; all of which are deemed to be described herein by reference), or by homologous recombination, preferably by positive / negative genetic selection. By homologous recombination (Mansour et a
l., 1988, Nature 336: 348-352; US Patent Nos. 5,464,764; 5,487,992; 5,
5,614, 396; 5,616,491; and 5,679,523; all of which are deemed to be described herein by reference), incomplete or complete gene inactivation can be performed. These organisms with altered gene expression
Preferably they are eukaryotes, and more preferably mammals. Such organisms are useful for developing non-human models for studying diseases associated with the corresponding gene, and for developing assay systems for identifying molecules that interact with protein products from the corresponding gene. Where the protein of the invention is membrane-bound (eg, a receptor), the invention also provides soluble forms of such protein. In such a form, some or all of the intracellular and transmembrane domains of the protein are removed so that the protein is sufficiently secreted from the host in which it was expressed. The intracellular and transmembrane domains of the proteins of the present invention can be identified by known techniques for determining such domains from sequence information. The proteins and protein fragments of the present invention comprise at least 25% (more preferably at least 50%, most preferably at least 75%) of the amino acid sequence of the disclosed protein.
And has at least 60% sequence identity (more preferably at least 75% identity, most preferably at least 90%) to the disclosed proteins.
Or 95% identity). Sequence identity is determined by comparing the amino acid sequences of the proteins when the sequences are aligned to maximize overlap and identity while minimizing sequence gaps. It has at least 75% sequence identity (more preferably at least 85% identity, most preferably at least 95% identity) to any of the disclosed protein segments, preferably 8 or more ( Proteins or protein fragments containing segments comprising more preferably 20 or more, most preferably 30 or more) contiguous amino acids are also encompassed by the present invention.

In detail, WU-BLAST (Washington University BLAST) version 2. 0 software may be used to determine sequence identity, which is a publicly available unrestricted WBI based on NCBI-BLAST version 1.4.
-Built based on BLAST (Altschul and Gish, 1996, Local ali
gnment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Al
tschul et al., 1990, Basic local alignment search tool, Journal of Molec
ular Biology 215: 403-410; Gish and States, 1993, Identification of prot
ein coding regions by database similarity search, Nature Genetics 3: 266
-272; Karlin and Altschul, 1993, Applications and statistics for multipl
e high-scoring segments in molecular sequences, Proc. Natl. Acad. Sci. U
SA 90: 5873-5877 (all of these references are hereby incorporated by reference)). WU-BLAST for some UNIX platforms
Ftp://blast.wustl.edu/blast/executab version 2.0 executable
You can download from les. In addition to several support programs, a complete set of search programs (BLASTP, BLASTN, BLASTX,
TBLASTN and TBLASTX) are provided at this site. WU
-BLAST 2.0 is copyrighted and must not be sold or redistributed in any form without the permission of the author, but may be used for non-business or research purposes. Can be used for Pair (BLASTP, BLAST
N, BLASTX, TBLASTN, and TBLASTX), the gap alignment routine (gapped alignment routine)
routines) are integrated into the database search itself, resulting in highly sensitive, highly selective and easily interpretable results. Optimally, gapping can be turned off in all these programs. The default penalty (Q) for the length gap is Q = 9 for BLASTP and Q = 10 for BLASTN, but 1 to 8, 9, 10, including zero. It may be changed to any integer value such as 11, 12 to 20, 21 to 50, and 51 to 100. Residue 1 to widen the gap
The default penalty (R) per unit is R = 2 for protein and BLASTP, and R = 10 for BLASTN, but 0, 1, 2, 3
, 4, 5, 6, 7, 8, 9, 10, 11, 12 to 20, 21 to 50, 5,
It may be changed to an integer value such as 1 to 100. Any combination of Q and R may be used to compare sequences side-by-side to maximize overlap and identity and minimize sequence gaps. The default amino acid comparison matrix is BLOS
Although UM62, other amino acid comparison matrices such as PAM can be used.

[0037] Species homologs of the disclosed polynucleotides and proteins are also provided by the invention. The term "species homolog" herein is a protein or polynucleotide that relates to a species different from the source of the protein or polynucleotide, but as determined by one of skill in the art,
Those having significant sequence similarity. Preferably, the polynucleotide species homolog has at least 60% (more preferably at least 75%, most preferably at least 90%) sequence identity to the particular polynucleotide, and the protein species homolog is At least 30% (more preferably at least 45%)
%, Most preferably at least 60%). Here, sequence identity is determined by comparing the nucleotide sequence of a polynucleotide or the amino acid sequence of a protein juxtaposed such that overlap and identity are maximized and sequence gaps are minimized. Species homologs may be isolated and identified by generating appropriate probes or primers from the sequences provided herein, and then screening for an appropriate nucleic acid source from the desired species. Preferably, the species homolog is isolated from a mammalian species. Most preferably, the species homolog is, for example, Pan troglodyte
s, Gorilla gorilla, Pongo pygmaeus, Hylobates concolor, Macaca mulatta,
Papio papio, Papio hamadryas, Cercopithecus aethiops, Cebus capucinus, A
otus trivirgatus, Sanguinus oedipus, Microcebus murinus, Mus musculus, R
attus norvegicus, Crisetulus griseus, Felis catus, Mustela vison, Canis
It has been isolated from specific mammals such as familiaris, Oryctolagus cuniculus, Bos taurus, Ovis aries, Sus scrofa and Equus caballus, and has been genetically mapped to identify the genes in one species. It is possible to identify the association of gene sequence order between genomic organization and genomic organization of related genes in another species (O'Brien and Seuanez, 1988, Ann. Rev. Genet. 22: 323). -351;
O'Brien et al., 1993, Nature Genetics 3: 103-112; Johansson et al., 199
5, Genomics 25: 682-690; Lyons et al., 1997, Nature Genetics 15: 47-56;
O'Brien et al., 1997, Trends in Genetics 13 (10): 393-399; Carver and Stu
bbs, 1997, Genome Research 7: 1123-1137 (all of which are deemed to be described herein by reference)).

The present invention also relates to allelic variants of the disclosed polynucleotides, ie, the spontaneous identification of isolated polynucleotides encoding proteins identical, homologous or related to the proteins encoded by the disclosed polynucleotides. Form. Preferably, the allelic variant is at least 60% (for a particular polynucleotide)
More preferably at least 75%, most preferably at least 90%), wherein the sequence identity is such that the polynucleotides are juxtaposed such that overlap and identity are maximized and sequence gaps are minimized. By comparing the nucleotide sequences of Allelic variants may be isolated and identified by generating appropriate probes or primers from the sequences described herein and screening for an appropriate source of nucleic acid from an individual of the appropriate species. The present invention also encompasses polynucleotides having sequences complementary to the sequences of the polynucleotides disclosed herein.

The present invention also relates to a polynucleotide that is capable of hybridizing to a polynucleotide described herein under reduced stringency conditions, more preferably under stringent conditions, and most preferably under very stringent conditions. Also encompasses nucleotides. The following table shows examples of strictness conditions. The very strict conditions are, for example, at least as strict as the conditions A to F, and the strict conditions are, for example, at least as strict as the conditions GL, and the reduced conditions are, for example, For example, it is at least as strict as the conditions M to R.

[Table 1] 1): Hybrid length is the expected length for the hybridizing region (s) of the hybridizing polynucleotide. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be the length of the hybridizing polynucleotide. When polynucleotides of known sequence hybridize, the length of the hybrid can be determined by juxtaposing the polynucleotide sequences and identifying the region (s) of optimal sequence complementarity. 2) Hybridization and SSPE (1X SSPE is 0.15 M NaCl in the wash buffer, 10mM NaH ₂ PO _4, and 1.25 mM EDTA, pH 7.4 at a) the SSC (IX SSC is 0.15 M NaCl and 15mM Sodium citrate). After completion of hybridization, washing is performed for 15 minutes. * T _B -T _R : The hybridization temperature for hybrids expected to be less than 50 base pairs in length should be 5-10 ° C below the melting temperature (T _m ) of the hybrid. T _m is determined by the following equation. For hybrids less than 18 base pairs in length, T _m (° C.) = 2 (number of A + T bases) +4 (number of G + C bases). For hybrids 18 to 49 base pairs in length, T _m (° C.) = 81.5 + 16.6 (log ₁₀ [Na ⁺ ]) + 0.41 (% G + C) − (600 / N), where N is the hybrid And the sodium ion concentration in the hybridization buffer ([Na ⁺ ] for 1 × SSC = 0.165 M).

Further examples of stringency conditions for polynucleotide hybridization are described in Sambrook, J., EF Fritsch, and T. Maniatis, 1989, Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Ha
rbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology,
1995, FM Ausubel et al., Eds., John Wiley & Sons, Inc., sections 2.10
and 6.3-6.4 and are deemed to be incorporated herein by reference. Preferably, each such hybridizing polynucleotide is at least 25% of the polynucleotide of the invention to be hybridized.
(More preferably, at least 50%, most preferably at least 75%) and at least 60% sequence identity (more preferably at least 75%) to the polynucleotide of the invention to be hybridized. , Most preferably at least 90% or 95% identity). Sequence identity is determined by comparing the amino acid sequences of the proteins when the sequences are aligned to maximize overlap and identity while minimizing sequence gaps.

[0041] The isolated polynucleotide encoding the protein of the present invention was prepared according to Kaufman et al., Nucl.
The protein may be recombinantly produced by operably linking it to an expression control sequence such as the pMT2 or pED expression vector disclosed in eic Acids Res. 19, 4485-4490 (1991). Many suitable expression control sequences are known in the art. Many suitable expression control sequences are known in the art. General methods for recombinant protein expression are also known and are described in R. Kaufman, Methods in Enzymology 185, 537-566.
(1990) is a typical example. "Operably linked" as defined herein means that the isolated polynucleotide of the present invention and the expression control sequence are placed in a vector or cell and transformed (transfected) with the ligated polynucleotide / expression control sequence. It is meant to be expressed by a host cell. Many types of cells can serve as suitable host cells for expression of the proteins of the present invention.
Mammalian cells include, for example, monkey COS cells, Chinese hamster ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, and other transformed primate cells. Lines, normal diploid cells, cell lines obtained from in vitro culture of primary tissues, primary explants, HeLa cells, mouse cells, BHK, HL-60, U937HaK or Jurkat cells.

[0042] Alternatively, the protein can be produced in lower eukaryotic cells such as yeast or in prokaryotic cells such as bacteria. A potentially suitable yeast strain is Saccharomyces ce
revisiae, Schizosaccharomyces pombe, Kluyveromyces strain, Candida, or yeast strains capable of expressing heterologous proteins. A potentially suitable bacterial strain is Escherichia
E. coli, Bacillus subtilis, Salmonella typhimurium, or bacterial strains capable of expressing heterologous proteins. If the protein is produced in yeast or bacteria, it may be necessary to modify the protein produced therein, for example, by phosphorylation or glycosylation at appropriate sites to obtain a functional protein. Such covalent linkages may be made using known chemical or enzymatic methods. The isolated polynucleotides of the present invention may be operably linked to appropriate control sequences in one or more insect expression vectors and the protein obtained by using an insect expression system. Materials and methods for baculovirus / insect cell expression systems are described, for example, in In
Commercially available in kit form from Vitrogen, San Diego, California, USA (MaxBac ^R kit), such methods are well known in the art and are described by Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987).
) (Assumed to be described herein by reference). As used herein, insect cells capable of expressing a polynucleotide of the present invention have been "transformed."

The protein of the present invention may be produced by culturing the transformed host cell under culture conditions suitable for expressing the recombinant protein. The resulting expressed protein may then be purified from such culture (ie, medium or cell extract) using known purification processes, such as gel filtration and ion exchange chromatography. Purification of the protein may also be accomplished by affinity columns containing an agent that will bind to the protein; one or more column steps with an affinity column such as Concanavalin A-agarose, Heparin-Toyopearl ^R or Cibacrom blue 3GA Sepharose ^R ; One or more steps using hydrophobic interaction chromatography with a resin such as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography. Alternatively, the protein of the invention may be expressed in an easily purified form. For example, maltose binding protein (MBP), glutathione-S-tonsferase (GST)
Alternatively, it may be expressed as a fusion protein such as a fusion protein with thioredoxin (TRX). Kits for expression and purification of such fusion proteins are available from New En
Commercially available from glan BioLab (Beverly, MA), Pharmacia (Piscataway, NJ) and InVitrogen. The protein can also be tagged with an epitope and then purified by using a specific antibody directed against such epitope. One such epitope ("flag") is commercially available from Kodak (New Haeven, CT). Finally, the protein is further purified using one or more reverse phase high quality liquid chromatography (RP-PLC) steps using a hydrophobic RP-HPLC medium, such as silica gel with pendant methyl or other aliphatic groups. be able to. Some or all of the above purification steps may be used in various combinations to obtain a substantially homogeneous isolated recombinant protein. The protein thus purified is substantially free of other mammalian proteins and is defined in the present invention as "isolated protein".

The protein of the present invention can be used as a product of a transgenic animal, for example, a milk component of a transgenic cow, goat, pig, or sheep characterized by somatic cells or germ cells containing the nucleotide sequence encoding the protein. May be expressed.

The protein may be produced by known conventional chemical synthesis. Methods for constructing the protein of the present invention by synthetic means are known to those skilled in the art. Synthetically constructed protein sequences may have common biological properties, including protein activity, by sharing primary, secondary or tertiary structure and / or conformational properties. Thus, they may be used as biological activities or immunological substitutes for naturally occurring purified proteins in immunological processes for screening therapeutic compounds and generating antibodies. The proteins described herein are characterized by an amino acid sequence similar to the amino acid sequence of the purified protein, but may be modified therein, either naturally or by derivatization. For example, modifications of the peptide or DNA sequence can be made by those skilled in the art using known methods. Modifications of interest in the protein sequence include changes, substitutions, exchanges, insertions or deletions of selected amino acid residues in the coding sequence. For example, one or more cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Methods for such changes, substitutions, exchanges, insertions or deletions are well known to those skilled in the art (see, for example, US Pat. No. 4,158,584). Preferably, such changes, substitutions, exchanges, insertions or deletions retain the desired activity of the protein. Other fragments and derivatives of the protein sequence that are expected to retain protein activity in whole or in part, and thus may be useful in screening or other immunological methods, can also be made by those skilled in the art from the disclosure herein. . Such modifications are believed to be encompassed by the present invention.

Uses and Biological Activities The polynucleotides and proteins of the present invention are believed to exhibit one or more of the following uses or biological activities, including those associated with the assays cited herein. Can be The uses or activities described with respect to the proteins of the present invention may be due to the administration or use of such proteins, or the administration or use of polynucleotides encoding such proteins (eg, such as a vector suitable for gene therapy or DNA transfer). Can be provided.

Research Uses and Utility The polynucleotides provided by the present invention can be used for various purposes by a research population. For analysis, characterization or therapeutic use, as a marker of tissue in which the corresponding protein is preferentially expressed (constitutively or at a particular stage of tissue differentiation or development or in a disease state), and To identify potential genetic diseases as compared to the patient's endogenous DNA, as chromosomal markers or tags (if labeled) for identification of chromosomes or mapping of relevant gene locations, as molecular weight markers for Southern gels The use of known sequences in the process of discovering other novel polynucleotides as a source for obtaining PCR primers for genetic fingerprinting to find new related DNA sequences to use as hybridization probes As a probe for subtraction, use a "gene chip" or other To select and generate oligomers for binding to carriers, to test expression patterns, to generate anti-protein antibodies using DNA immunization, and to generate anti-DNA antibodies, or Polynucleotides can be used as antigens to induce a response. If encoding a protein that binds or potentially binds to another protein (e.g., as in a receptor-ligand interaction), identify the other protein that binds, and / or an inhibitor of the binding interaction Polynucleotides can be used in an interaction trap assay (e.g., as described in Gyuris et al., Cell 75: 791-803 (1993)) to identify The proteins provided by the present invention may be used in assays to determine biological activity, including a group of multiple proteins for high-throughput screening; to generate antibodies or induce other immune responses; As a reagent (including a labeling reagent) in an assay designed to quantitatively determine the level of a protein (or its receptor) in a biological fluid; the corresponding protein is preferentially expressed (constitutively Or expressed at a particular stage of tissue differentiation or development, or in a disease state) as a marker for tissue; and, of course, may be used to isolate relevant receptors or ligands. Where a protein binds or potentially binds to another protein, the protein can be used to identify other proteins that bind, or to identify inhibitors of the binding interaction. Using proteins involved in these binding interactions, screening for peptides or small molecular inhibitors or agonists for the binding interaction can also be performed. Any or all of these research applications can be evolved into reagent grade or kit formats for commercialization as research products. Methods for performing the above uses are well known to those skilled in the art. A literature disclosing such a method can be found in "Molecular Cloning: A Laboratory Manual", 2nd ed., Cold Sp.
ring Harbor Laboratory Press, Sambrook, J., EF Fritsch and T. Maniatis
eds., 1989, and "Methods in Enzymology: Guide to Molecular Cloning Tec
hniques ", Academic Press, Berger, SL and AR Kimmel eds., 1987.

Use as Nutrition The polynucleotides and proteins of the present invention can be used as nutrient sources or additives. Such uses include, but are not limited to, use as a protein or amino acid additive, use as a carbon source, use as a nitrogen source, and use as a carbohydrate source. In such cases, the proteins or polynucleotides of the present invention may be added as food for a particular organism, or may be administered as a separate solid liquid formulation such as in the form of a powder, pill, solution, suspension or capsule. . In the case of a microorganism, the protein or polynucleotide of the present invention can be added to a medium in which the microorganism is cultured.

Cytokines and Cell Proliferation / Differentiation Activity The proteins of the present invention may exhibit cytokine activity, cell proliferation activity (induction or inhibition) or cell differentiation activity (induction or inhibition), or produce other cytokines in certain cell populations. Can be induced. Many proteinaceous factors (including all known cytokines) that have been found to date have demonstrated activity in one or more factor-dependent cell proliferation assays, thus making the assay a convenient method of confirming cytokine activity. Serve as. The activity of the protein of the present invention can be measured in cell lines (32D, DA2, DA1G, T10, B9, B9 / 11, BaF3, MC9 / G, M + (preB M +), 2E8, RB5, DA1, 123, T1165, HT2, CTLL2 , TF-1, Mo7e and CMK, including, but not limited to, any of the conventional factor-dependent cell proliferation assays. The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for T cell or thymocyte proliferation are described in Current Protocols in I.
mmunology, Ed by JE Coligan, AM Kruisbeek, DH Margulies, EM She
vach, W Strober, Pub.Green Publishing Associates and Wiley-Interscienc
e (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Ch
apter 7, Immunologic studies in Humans); Takai et al., J. Immunol. 137: 3
494-3500, 1986; Bertagnolli et al., J. Immunol. 145: 1706-1712, 1990; Ber
tagnolli et al., Cellular Immunology 133: 327-341, 1991; Bertagnolli, et
al., J. Immunol. 149: 3778-3783, 1992; Bowman et al., J. Immunol. 152: 17
56-1761, 1994, including, but not limited to. Assays for cytokine production and / or proliferation of spleen cells, lymph node cells or thymocytes are described in Polyclonal T cell stimulation, Kruisbeek, AM
d Shevach, EM In Current Protocols in Immunology. JEea Coligan eds
Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; and Meas
urement of mouse and human Interferon g, Schreiber, RD In Current Pro
tocols in Immunology. JEea Coligan eds. Vol 1 pp. 6.8.1-6.8.8, John
Includes, but is not limited to, those described in Wiley and Sons, Toronto. 1994. Assays for proliferation and differentiation of hematopoietic and lymphogenic cells are described in the Measurement of Human and Murine Interleukin 2 and Interleukin 4, Bottoml
y, K., Davis, LS and Lipsky, PE In Current Protocols in Immunology.
JEea Coligan eds.Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toron
to. 1991; deVries et al., J. Exp.Med. 173: 1205-1211, 1991; Moreau et al.
., Nature 336: 690-692, 1988; Greenberger et al., Proc. Natl. Acad. Sci.
USA 80: 2931-2938, 1983; Measurement of mouse and human interleukin 6
-Nordan, R. In Current Protocols in Immunology. JEea Coligan eds. V
ol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991; Smith et al.,
Proc. Natl. Acad. Sci. USA 83: 1857-1861, 1986; Measurement of human I
nterleukin 11-Bennett, F., Giannotti, J., Clark, SC and Turner, K. J
. In Current Protocols in Immunology. JEea Coligan eds. Vol 1 pp.
6.15.1 John Wiley and Sons, Toronto. 1991; Measurement of mouse and hum
an Interleukin 9-Ciarletta, A., Giannotti, J., Clark, SC and Turner,
KJ In Current Protocols in Immunology. JEea Coligan eds. Vol 1 p
p. 6.13.1, including but not limited to those described in John Wiley and Sons, Toronto. 1991. Assays for the response of T cell clones to antigens, specifically identifying APC-T cell interactions as well as direct T cell effects by measuring proliferation and cytokine production, are described in Current Protocols in Immunology, Ed by JE.
Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W Strober, Pub.
Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro
assays for Mouse Lymphocyte Function; Chapter 6, Cytokines and their ce
llular receptors; Chapter 7, Immunologic studies in Humans); Weinberger
Natl. Acad. Sci. USA 77: 6091-6095, 1980; Weinberger et al.
, Eur. J. Immun. 11: 405-411, 1981; Takai et al., J. Immunol. 137: 3494-35.
00, 1986; Takai et al., J. Immunol. 140: 508-512, 1988, but are not limited thereto.

Immunostimulatory or Suppressive Activity The protein of the present invention may also exhibit immunostimulatory or immunosuppressive activity, including but not limited to the activity in the assays described herein. Protein is
It may be useful in a variety of deficiencies and disorders, including severe immunodeficiency complications (SCID), eg, in up-regulating or down-regulating the proliferation of T and / or B lymphocytes, and in N
It may be useful in activating the cytolytic activity of K cells and other cell populations. These immunodeficiencies can be genetic and can include viral (
For example, it may be caused by HIV) and bacterial or fungal infections, or may be caused by an autoimmune disease. More particularly, infectious diseases caused by viruses, bacteria, fungi or other infectious agents, such as various fungal infections such as HIV, hepatitis virus, herpes virus, mycobacteria, Leishmania, malaria and Candida species, It can be treated with the protein of the present invention. Of course, in this regard, the proteins of the present invention may be used where a boost to the immune system is needed, ie, in the treatment of cancer. Autoimmune diseases that may be treated using the protein of the present invention include, for example, multiple sclerosis,
Systemic deep elitomades, rheumatoid arthritis, autoimmune pneumonia, Guillain-Barre syndrome, autoimmune thyroiditis, insulin-dependent diabetes mellitus, myasthenia gravis, graft-versus-host disease and autoimmune inflammatory Eye diseases. Such proteins of the invention may be used in the treatment of allergic reactions and conditions, such as asthma or other respiratory diseases. Other conditions for which immunosuppression is desired, including, for example, asthma (particularly allergic asthma) and related respiratory problems may be treated with the proteins of the invention. Other conditions for which immunosuppression is desired (including, for example, organ transplantation)
May be treated using the protein of the present invention.

The proteins of the present invention can also be used to enable an immune response in a number of ways. Down-regulation may be in the form of inhibiting or blocking an immune response already in progress, or may involve interfering with the induction of an immune response. The function of activated T cells may be inhibited by suppressing T cell responses, or by inducing specific resistance in T cells, or both. In general, immunosuppression of a T cell response is an active, non-antigen-specific process that requires continuous exposure of T cells to an inhibitor. Tolerance means non-responsiveness or anergy in T cells, and differs from immunosuppression in that it is generally antigen-specific and persists after exposure to the tolerizing agent is stopped.
Operationally, resistance may be indicated by a lack of a T cell response when exposed to a specific antigen in the absence of a tolerizing agent. Down-regulating or preventing one or more antigen functions, including but not limited to B-lymphocyte antigen functions (such as, for example, B7), eg, high levels of lymphokines by activated T cells Interference with synthesis may be useful in tissue, skin and organ transplantation and graft versus host disease (GVHD). For example, blocking T cell function should reduce tissue destruction in tissue transplantation. Typically, in tissue transplantation, graft rejection is initiated by the recognition of the tissue piece as foreign by T cells, followed by an immune response that destroys the graft. A monomeric form having the activity of a molecule that inhibits or blocks the interaction of a B7 lymphocyte antigen with its native ligand on immune cells (another B lymphocyte antigen (eg, B7-1, B7-3)) Mixed with peptides,
Alternatively, pre-transplant administration of a soluble, monomeric form of a peptide with B7-2 activity alone, induces binding of the molecule to its native ligand on immune cells without transmitting a responsive costimulatory signal. there is a possibility. Blocking B lymphocyte antigen function in this regard prevents cytokine synthesis by immune cells, such as T cells, and thus acts as an immunosuppressant. Moreover, the lack of costimulation may be sufficient to cause a loss of T cell response. The induction of long-term tolerance by B lymphocyte antigen blocking reagents may obviate the need for repeated administration of these blocking reagents. To achieve sufficient immunosuppression or resistance in a subject, it may also be necessary to block the function of the B lymphocyte antigen combination.

[0052] The effectiveness of individual blocking reagents in preventing organ transplant rejection or GVHD can be evaluated using animal models that can predict efficacy in humans. Examples of suitable systems that can be used include allogeneic heart grafts in rats and xenogeneic pancreatic islet cell grafts in mice, both of which are used to test the immunosuppressive effects of CTLA4Ig fusion proteins in vivo. (Lensch
ow et al., Science 257: 789-792 (1992) and Turka et al., Proc Natl. Acad.
Sci. USA, 89: 11102-11105 (1992)). In addition, a rat model of GVHD (Paul ed., Fundamental Immunology, Ravan Press, New York, 1
989, pp. 846-847) can be used to determine the blocking effect of B lymphocyte antigen function on the progression of the disease in vivo. Also, blocking antigen function may be therapeutically useful for treating autoimmune diseases. Many autoimmune diseases are the result of inappropriate activation of T cells that are reactive to self tissue and promote the production of cytokines and autoantibodies that are involved in the pathology of the disease. Interfering with the activation of autoreactive T cells may reduce or eliminate the symptoms of the disease. Receptors for B lymphocyte antigens: autoantibodies or T cell-derived that inhibit T cell activation using administration of reagents that block T cell costimulation by disrupting ligand interactions and may be involved in the disease process Can interfere with the production of cytokines. In addition, blocking reagents can induce antigen-specific resistance of autoreactive T cells that can lead to long-term remission of the disease. The effectiveness of a blocking reagent in preventing or ameliorating an autoimmune disease can be determined using a number of well-characterized animal models for human autoimmune diseases. Examples include murine experimental autoimmune encephalitis, systemic deep erythematosus in MRL lpr / lpr or NZB hybrid mice, murine autoimmune collagen arthritis, diabetes in NOD mice and BB rats, and experimental murine myasthenia gravis. (Paul ed., Fundamental Immunology, Raven Pres
s, New York, 1989, pp. 840-856). Up-regulation of antigen function (preferably B-lymphocyte antigen function) as a means to up-regulate the immune response is also therapeutically useful.
Up-regulation of the immune response may be in the form of promoting an existing immune response or eliminating the initial immune response. For example, enhancing an immune response by stimulating B lymphocyte antigen function may be useful in the case of a viral infection.
In addition, systemic viral diseases such as influenza, common cold, and encephalitis may be ameliorated by systemic administration of a stimulatory form of B lymphocyte antigen.

Alternatively, T cells are taken from a patient and co-stimulated in vitro with T cells with viral antigens to which ACPs expressing the peptide of the invention have been added, or combined with a stimulatory form of a soluble peptide of the invention, The T-cells activated in vitro may then be reintroduced into the patient to enhance the anti-viral immune response in the infected patient. Another method of promoting an antiviral immune response is
The infected cells are removed from the patient and transfected with them a nucleic acid encoding a protein of the invention as described herein so that the cells express all or part of the protein on their surface. Will be re-introduced. The infected cells would then be able to transmit the costimulatory signal to the T cells in vivo and thereby activate the T cells. In another application, up-regulation or promotion of antigen function (preferably B lymphocyte antigen function) may be useful in inducing tumor immunity. Administering a tumor cell (eg, sarcoma, melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleic acid encoding at least one peptide of the invention to a tumor-specific tumor in the subject Can overcome resistance. If desired, tumor cells can be transfected to express the peptide combination. For example, an expression vector that directs the expression of tumor cells obtained from a patient in combination with only a peptide having B7-2-like activity or a peptide having B7-1-like activity and / or B7-3-like activity It can be transfected ex vivo. The transfected tumor cells are returned to the patient and the peptide is expressed on the surface of the transfected cells. Alternatively, gene therapy can be used to target tumor cells for transfection in vivo. The presence of a peptide of the invention having the activity of a B lymphocyte antigen on the surface of tumor cells provides the necessary costimulatory signals for T cells to induce an immune response to transfected tumor cells mediated by T cells I do. In addition, MHC
Tumor cells lacking class I or MHC class II molecules, or sufficient MHC
Class I or tumor cells can not be re-expressed MHC class II molecules, MHC class Iα chain protein and beta ₂ microglobulin protein or an MHC class IIα chain Contact and MHC class IIβ chain all or part of the protein (e.g., truncations protein The nucleic acid encoding the cytoplasmic domain can be transfected, thereby allowing the expression of class I or class II MHC proteins on the cell surface. Expression of appropriate Class I or Class II HMC in combination with a peptide having the activity of a B lymphocyte antigen (eg, B7-1, B7-2, B7-3) is transfected by T cells. Induces an immune response against tumor cells. If desired, a gene encoding an antisense construct that blocks the expression of an MHC class II binding protein, such as an invariant chain, can be co-transfected with a DNA encoding a peptide having B lymphocyte antigen activity. It can also enhance the presentation of tumor-associated antigens and induce tumor-specific immunity. Thus, induction of an immune response mediated by T cells in a human subject may be sufficient to overcome tumor-specific resistance in the subject.

The activity of the protein of the invention may be measured, inter alia, by the following method: Suitable assays for thymocyte or spleen cell cytotoxicity are described in Current Protocols
in Immunology, Ed by JE Coligan, AM Kruisbeek, DH Margulies, EM
Shevach, W Strober, Pub. Greene Publishing Associates and Wiley-Intersc
ience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19
; Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl
Acad. Sci. USA 78: 2488-2492, 1981; Herrmann et al., J. Immunol. 128:
1968-1974, 1982; Handa et al., J. Immunol. 135: 1564-1572, 1985; Takai e
t al., J. Immunol. 137: 3494-3500, 1986; Takai et al., J. Immunol. 140:
508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA 78: 2488-2492.
Herrmann et al., J. Immunol. 128: 1968-1974, 1982; Handa et al., 1981;
J. Immunol. 135: 1564-1572, 1985; Takai et al., J. Immunol. 137: 3494-3
500, 1986; Bowmanet al., J. Virology 61: 1992-1998; Takai et al., J. Imm
unol. 140: 508-512, 1988; Bertagnolli et al., Cellular Immunology 133: 3
27-341, 1991; including but not limited to the assays described in Brown et al., J. Immunol. 153: 3079-3092, 1994.

Assays for T cell-dependent immunoglobulin responses and isotype switching, particularly those that modulate T cell-dependent antibody responses and identify proteins that affect Th1 / Th2 profiles, are described by Maliazewski, J. Immunol. .144: 3028-3033, 1990, including but not limited to assays for B cell function, as described in In vitro antibody production, Mond, JJand Brunswick, M. In Current.
Protocols in Immunology JEColigan eds.Val 1 pp.3.8.1-3.8.16, John Wile
y and Sons, Tronto 1994. Mixed lymphocyte reaction (MLR) assays, which identify proteins that primarily produce Th1 and CTL responses, are described in Current Protocols in Immunology, Ed by
JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W Strober,
Pub.Green Publishing Associates and Wiley-Interscience (Chapter 3, In
Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunol
ogic studies in Humans); Takai et al., J. Immunol. 137: 3494-3500, 1986;
Takai et al., J. Immunol. 140: 508-512, 1988; Bertagnolli et al., J. Im.
munol. 149: 3778-3783, 1992, but is not limited thereto.

The dendritic cell-dependent assay (particularly identifying proteins expressed by dendritic cells that activate intact T cells) is described by Guery et al., J. Immunol. 134: 536-544.
, 1995; Inaba et al., Journal of Experimental Medicine 173: 549-559, 199
1; Macatonia et al., Journal of Immunology 154: 5071-5079, 1995; Porgado
r et al., Journal of Experimental Medicine 182: 255-260, 1995; Nair et a
l., Journal of Virology 67: 4062-4069, 1993; Huang et al., Science 264:
961-965, 1994; Macatonia et al., Journal of Experimental Medicine 169:
1255-1264, 1989; Bhardwaj et al., Journal of Clinical Investigation 94:
797-807, 1994; and Inaba et al., Journal of Experimental Medicine 172:
631-640, 1990, including, but not limited to. Assays for lymphocyte survival / apoptosis, specifically identifying proteins that prevent apoptosis following superantibody induction, as well as proteins that regulate lymphocyte homeostasis, are described by Darzynkiewicz et al., Cytometry 13: 795-808, 1992; Gorczyca et al.
, Leukemia 7: 659-670, 1993; Gorczyca et al., Cancer Research 53: 1945-1
951, 1993; Itoh et al., Cell 66: 233-243, 1991; Zacharchuk, Journal of I
mmunology 145: 4037-4045, 1990; Zamai et al., Cytometry 14: 891-897, 199
3; including but not limited to the assays described in Gorczyca et al., International Journal of Oncology 1: 639-648, 1992. Early stage T cell commitment and development assays
Antica et al., Blood 84: 111-117, 1994; Fine et al., Cellular Immunology 15
5: 111-122, 1994; Galy et al., Blood 85: 2770-2778, 1995; Toki et al., Pr
Natl. Acad. Sci. USA 88: 7548-7551, 1991, including, but not limited to.

Hematopoietic modulatory activity The proteins of the present invention are useful in the regulation of hematopoiesis and therefore in the treatment of bone marrow and lymphocyte deficiencies. Even minimal biological activity that supports colony forming cells or factor-dependent cell lines has been implicated in regulating hematopoiesis.
For example, supporting the growth of erythroid progenitor cells alone, or in combination with other cytokines, for example, showing utility in treating various anemias, or in combination with radiation therapy / chemotherapy, erythroid progenitor cells And / or stimulating the production of erythroblasts; supporting the proliferation of bone marrow cells such as granulocytes and monocytes / macrophages (ie, traditional CSF activity), eg, in combination with chemotherapy, Useful in preventing myelosuppression that occurs; supporting proliferation of megakaryocytes and then platelets, and thereby preventing or treating various platelet disorders such as thrombocytopenia; It is also commonly used instead of or in complement to platelet infusion; and / or hematopoietic stem cells (mature Becomes all of hematopoietic cells, therefore, a variety of stem cell disease (
It is usually a disease that is treated by transplantation, and is useful in supporting the growth of, for example, aplastic anemia and paroxysmal nocturnal hemoglobinuria), and is also useful in vivo or ex vivo. It is also useful in repopulating the stem cell compartment as normal cells after radiation / chemotherapy with (ie, combined with bone marrow transplantation), or after being genetically engineered for gene therapy. In particular, the activity of the protein of the present invention may be measured by the following method. Assays suitable for the proliferation and differentiation of various hematopoietic cell lines have already been cited. Assays for embryonic stem cell differentiation, particularly those that identify proteins that affect embryonic hematopoiesis, are described in Johansson et al. Cellular Biology 15: 141-151, 1995; Keller et al.
, Molecular and Cellular Biology 13: 473-486, 1993; McClanahan et al., B
lood 81: 2903-2915, 1993, including, but not limited to. Assays for stem cell survival and differentiation (particularly identifying proteins that regulate lympho-hematopoiesis) are described in Methylcellulose colony forming assays, Freshney, MG In Cultu
re of Hematopoietic Cells.RI Freshney, et al. eds.Vol pp. 265-268,
Wiley-Liss, Inc., New York, NY. 1994; Hirayama et al., Proc. Natl. Acad.
Sci. USA 89: 5907-5911, 1992; Primitive hematopoietic colony forming ce
lls with high proliferative potential, McNiece, IK and Briddell, RA
In Culture of Hematopoietic Cells.RI Freshney, et al. Eds.Vol pp. 2
3-39, Wiley-Liss, Inc., New York, NY. 1994; Neben et al., Experimental H
ematology 22: 353-359, 1994; Cobblestone area forming cell assay, Ploema
cher, RE In Culture of Hematopoietic Cells.RI Freshney, et al. eds.
Vol pp. 1-21, Wiley-Liss, Inc., New York, NY. 1994; Long term bone ma
rrow cultures in the presence of stromal cells, Spooncer, E., Dexter, M.
and Allen, T. In Culture of Hematopoietic Cells.RI Freshney, et al.
eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, NY. 1994; Long term c
ulture initiating cell assay, Sutherland, HJ In Culture of Hematopoiet
ic Cells.RI Freshney, et al. eds.Vol pp. 139-162, Wiley-Liss, Inc.,
Includes, but is not limited to, the assays described in New York, NY. 1994.

Tissue Proliferation Activity The protein of the present invention is useful in compositions used for the growth or regeneration of bone, cartilage, keys, ligaments and / or nervous tissue, as well as for wound healing and tissue repair, and in the treatment of burns, lacerations and ulcers. Has utility. The protein of the present invention, which induces cartilage and / or bone growth in an environment where bone is not normally formed, has use in healing fractures and cartilage damage or defects in humans and other animals. Such formulations using the protein of the present invention are useful for reducing closed and open fractures and for improving fixation of artificial joints. De novo bone formation induced by osteogenic agents contributes to the repair of congenital, traumatic or oncological resection-induced craniofacial defects, and is also useful in cosmetic surgery. The protein of the present invention may be used in the treatment of periodontal disease and other tooth restoration processes. Such agents provide an environment for attracting osteogenic cells, stimulate the growth of osteogenic cells,
Alternatively, it can induce differentiation of osteogenic cell precursors. The protein of the invention may be osteoporotic or osteoarthritis, for example, by stimulating bone and / or cartilage repair or by blocking the process of tissue destruction mediated by inflammation or inflammatory processes (collagenase activity, osteoclast activity, etc.). May also be useful in the treatment of Another category of tissue development activity that may be due to the proteins of the present invention is key / ligament formation. Proteins of the invention that induce the formation of key / ligament-like or other tissues in an environment in which such tissues are not normally formed can be used to prevent key or ligament laceration, deformation and other key or ligament defects in humans and other animals. Applied to healing, Such formulations using the key / ligament-like tissue inducing protein may be used to prevent damage to the key or ligament tissue as well as to improve the fixation of the key or ligament to bone or other tissue. The de novo key / ligament-like tissue formation induced by the compositions of the present invention contributes to the repair of congenital, traumatic or oncological resection-induced craniofacial defects, and furthermore the attachment of keys or ligaments Or it is also useful in cosmetic surgery for repair. The compositions of the present invention provide an environment for attracting key- or ligament-forming cells, stimulate the growth of key- or ligament-forming cells, and induce the differentiation of key- or ligament-forming cell precursors. Or induce the growth of key / ligament cells or progenitors ex vivo so as to effect tissue repair when returned to vivo.
The compositions of the present invention are also useful for keratitis, carpal tunnel syndrome and other key or ligament defects. The compositions of the present invention may include a suitable matrix and / or sequestering agent, such as a carrier well known in the art.

The protein of the present invention is useful for the growth of nerve cells and the regeneration of nerve and brain tissues, ie, central and peripheral nervous system diseases and neuropathies and mechanical diseases and traumatic diseases (degeneration, death or death to nerve cells or nerve tissues). (Including trauma). More particularly, diseases of the peripheral nervous system, such as peripheral nerve injury, peripheral and localized neuropathy, and Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral spinal sclerosis and Shy-Drager
The protein may be used in the treatment of diseases of the central nervous system, such as the syndrome. Additional conditions that may be treated according to the present invention include mechanical and traumatic diseases such as spinal cord diseases, cerebrovascular diseases such as head trauma and stroke. Peripheral neuropathy caused by chemotherapy or other medical therapies can be treated with the proteins of the present invention. The protein of the present invention is also useful for promoting more preferable and prompt closure of non-healing wounds including (but not limited to) pressure ulcers, ulcers associated with vascular dysfunction, surgical and trauma wounds, and the like. It is possible. The protein of the present invention may also be used in other tissues such as organs (including pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth muscle, skeletal muscle or cardiac muscle), and vascular (including vascular endothelium) tissue. Or an activity for promoting the growth of cells constituting such a tissue. Part of the desired effect may be the regeneration of normal tissue by suppressing fibrotic scarring. The proteins of the present invention may also exhibit angiogenic activity. The proteins of the present invention may also be useful in protecting or regenerating the intestine and treating conditions caused by lung or liver fibrosis, reperfusion injury of various tissues, and systemic cytokine damage. The protein of the present invention promotes or suppresses differentiation of the above-mentioned tissue from precursor tissue or cells;
Alternatively, it may be useful for suppressing the growth of the tissue. The activity of the protein of the invention may be measured, inter alia, by the following method: Tissue regeneration activity assay is described in WO 95/16035 (bone, cartilage, key).
Including, but not limited to, the assays described in WO 95/05846 (nerves, neurons); WO 91/07471 (skin, endothelium). Wound healing activity assays are described in Winter, Epidermal Wound Healing, pps. 71-112.
(Maibach, HI and Rovee, DT, eds.), Year Book Medical Publishers, Inc.,
Including, but not limited to, those described in Chicago (modified by Eaglstein and Mertz, J. Invest. Dermatol 71: 382-384 (1978)).

Activin / Inhibin Activity The protein of the present invention may also exhibit activin- or inhibin-related activity. Inhibins are characterized by their ability to inhibit follicle stimulating hormone (FSH) release,
Activins are characterized by their ability to stimulate the release of follicle stimulating hormone (FSH). Thus, the protein of the present invention, alone or in the form of a heterodimer with a member of the inhibin α family, reduces the fertility of female mammals and reduces the spermatogenesis of male mammals. Can be useful as Administration of a sufficient amount of another inhibin can induce infertility in these mammals. Alternatively, the protein of the present invention, as a homodimer or as a heterodimer with subunits of other proteins of the inhibin-β group, can be used to stimulate fertility based on the ability of activin molecules to stimulate FSH release from anterior pituitary cells. May be useful as therapeutic agents that induce See, for example, U.S. Pat. No. 4,798,885. The proteins of the invention may also be useful for improving reproduction in sexually immature mammals, resulting in increased fertility during the life of livestock such as cattle, sheep and pigs. The activity of the protein of the invention may be measured, inter alia, by the following method: Activin / inhibin activity assays are described in Vale et al., Endocrinology 91:
562-572, 1972; Ling et al., Nature 321: 779-782, 1986; Vale et al., Nat
ure 321: 776-779, 1986; Mason et al., Nature 318: 659-663,1985; Forage e
Natl. Acad. Sci. USA 83: 3091-3095, 1986, including but not limited to the assays described in US Pat.

Chemotaxis / chemokinesis activity The protein of the present invention can be used for the chemotaxis or chemokinesis activity of mammalian cells such as monocytes, neutrophils, T cells, mast cells, eosinophils and / or endothelial cells (for example, (Acting as a chemokine). Chemotactic and chemokinetic proteins can be used to recruit or attract a desired cell population to a desired site of action. Chemotaxis or chemokinesis proteins are particularly advantageous for treating injuries and other trauma to tissues and for treating local infections. For example, attracting lymphocytes, monocytes or neutrophils to a tumor or site of infection can improve the immune response to the tumor or infectious agent. Certain proteins or peptides have chemotactic activity on a particular cell population and, when stimulable, direct or indirectly direct the movement of such cell population. Preferably, the protein or peptide has the ability to directly stimulate the directed movement of the cell. Whether a particular protein has chemotactic activity on a cell population can be readily determined by using such a protein or peptide in a known assay for cell chemotaxis. The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for chemotactic activity (assays to identify proteins that induce or interfere with chemotaxis) induce migration across cell membranes. Assays that measure the ability of a protein to induce adhesion of one cell population to another cell population as well as the ability of the protein to adhere to another cell population. Suitable assays for migration and adhesion are described in Current Protocols in Immunology, Ed by JE
Coligan, AM Kruisbeek, DH Margulies, EM Shevach, W. Strober, Pub.
reene Publishing Associates and Wiley-Interscience (Chapter 6.12, Measur
ement of alpha and beta Chemokines 6.12.1-6.12.28; Taub et al. J. Clin.
Invest. 95: 1370-1376, 1995; Lind et al. APMIS 103: 140-146, 1995; Mulle
r et al Eur. J. Immunol. 25: 1744-1748; Gruber et al. J. of Immunol. 152
: 5860-5867, 1994; Johnston et al. J. of Immunol. 153: 1762-1768, 1994, but not limited thereto.

Hemostatic and thrombolytic activity The proteins of the present invention may also exhibit hemostatic or thrombolytic activity. As a result, such proteins are useful in the treatment of various coagulation disorders, including genetic disorders such as hemophilia, or in the treatment of injuries caused by trauma, surgery or other causes and other hemostasis. Can be promoted. The protein of the present invention may be useful for the treatment and prevention of thrombolysis or inhibition of thrombus formation and conditions resulting therefrom (eg, myocardial infarction and central nervous system infarction (eg, stroke)). The activity of the protein of the invention may be measured, inter alia, by the following method: Assays for haemostatic and thrombolytic activities are described in Linet et al., J. Clin. Pharmacol.
26: 131-140,1986; Burdick et al., Thrombosis Res. 45: 413-419, 1987; Hum
phrey et al., Fibrinolysis 5: 71-79 (1991); Schaub, Prostaglandins 35: 4
67-474, 1988, including, but not limited to.

Receptor / Ligand Activity The protein of the present invention may also exhibit activity as a receptor, receptor ligand or inhibitor or agonist of receptor / ligand interaction. Examples of such receptors and ligands include cytokine receptors and their ligands, receptor kinases and their ligands, receptor phosphatases and their ligands, cells-
Receptors involved in cell interactions and their ligands (such as cell adhesion molecules (selectins, integrins and their ligands)) and receptors / ligands involved in antigen presentation, antigen recognition, development of cellular and humoral immune responses But not limited thereto. Receptors and ligands are also useful for screening potential peptide or small molecule inhibitors for related receptor / ligand interactions. The proteins of the present invention, including but not limited to receptor and ligand fragments, may themselves be useful as inhibitors of receptor / ligand interaction. The activity of the protein of the invention may be measured, inter alia, by the following method: Suitable assays for receptor-ligand activity are described in Current Protocols in Immunolog.
y, Ed by JE Coligan, AM Kruisbeek, DH Margulies, EM Shevach,
W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (C
hapter 7.28, Measurement of Cellular Adhesion under static conditions 7.
28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84: 6864-6868, 1
987; Bierer et al., J. Exp.Med. 168: 1145-1156, 1988; Rosenstein et al.
, J. Exp. Med. 169: 149-160 1989; Stoltenborg et al., J. Immunol. Metho.
ds 175: 59-68, 1994; Stitt et al., Cell 80: 661-670, 1995, including, but not limited to.

Anti-inflammatory activity The protein of the present invention can exhibit anti-inflammatory activity. Anti-inflammatory activity involves stimulating cells that are involved in the stimulus response, resulting in cell-cell interactions (eg, attachment).
Inhibits or promotes the chemotaxis of cells involved in the inflammatory process, thereby inhibiting or promoting cell extravasation,
Alternatively, it is exerted by stimulating or suppressing the production of other factors that more directly inhibit or promote the inflammatory response. Symptoms of inflammation (
Includes chronic or acute symptoms, infection-related inflammation (such as septic shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, lethal endotoxin injury, arthritis, complement mediated Super-acute rejection,
Nephritis, lung injury induced by cytokines or chemokines, inflammatory bowel disease, Crohn's disease, or diseases resulting from overproduction of cytokines such as TNF or IL-1). . The proteins of the invention may also be useful for treating anaphylaxis and hypersensitivity to antigenic substances or materials.

Cadherin / Tumor Inhibition Inhibitory Activity Cadherin is a calcium-dependent adhesion molecule and appears to play a major role during development, particularly in determining specific cell types. Loss or alteration of normal cadherin expression can induce changes to cell adhesion properties associated with tumor growth and metastasis. Cadherin dysfunction has also been implicated in other human diseases, such as pemphigus vulgaris and pemphigus foliaceus (autoimmune cutaneous bullous disease), Crohn's disease, and some developmental abnormalities. The cadherin superfamily contains over 40 members, each with a different expression pattern. All members of the superfamily share common conserved extracellular repeats (cadherin domains), but show structural differences in other parts of the molecule. Calcium is required for their attachment, since the cadherin domains bind to calcium to form their tertiary structure. Since the small number of amino acids in the first cadherin domain provides the basis for homophilic attachment, modification of this recognition site can alter the specificity of cadherin, thereby recognizing only itself Instead, the mutant molecule will be able to bind to different cadherins. In addition, some cadherins are involved in heterophilic attachment with other cadherins. E-cadherin, a member of the cadherin superfamily, is expressed in epithelial cell types. Pathologically, if E-cadherin expression is lost in the tumor, the malignant cells become invasive and the cancer metastasizes. Transfecting a polynucleotide expressing E-cadhedrin into a cancer cell line restores the altered cell morphology, restores cell-to-cell and adherence to other substrates, reduces cell growth rates, By dramatically reducing the growth of dependent cells,
The changes associated with cancer were reversed. Thus, reintroducing E-cadhedrin expression returns the carcinoma to a less advanced stage. Other cadherins may have the same inhibitory role in carcinomas from other tissue types. Therefore, cancer can be treated using the protein of the present invention having cadherin activity and the polynucleotide of the present invention encoding such a protein. Introducing such proteins or polynucleotides into cancer cells may reduce or eliminate the cancerous changes observed in these cells by providing normal cadherin expression. Cancer cells are also known to express cadherins of different tissue types than originally, so these cancer cells can invade different tissues and metastasize. The protein of the present invention having cadherin activity, and the polynucleotide of the present invention encoding such a protein replaces cadherin inappropriately expressed in these cells, restores normal cell adhesion properties, and reduces the tendency of cells to metastasize. Or can be removed.

Furthermore, an antibody that recognizes and binds to cadherin can be obtained using the protein of the present invention having cadherin activity and the polynucleotide of the present invention encoding such a protein. Such antibodies can also be used to block the attachment of inappropriately expressed tumor cells cadherin, so that the cells do not form tumors elsewhere. Such anti-cadhedrin antibodies can be used as markers for cancer grade, pathological type, and prognosis. That is, as the cancer progresses, the expression of cadherin decreases, and by using a cadherin-binding antibody, the decrease of cadherin expression can be detected. Using a fragment of a protein of the present invention having cadherin activity, preferably a decapeptide at the cadherin recognition site, and a polynucleotide of the present invention encoding such a protein fragment, to bind to cadherin and prevent cadherin binding that produces undesirable effects Can also block cadherin function. Further, using a fragment of the protein of the present invention having cadherin activity, preferably a truncated soluble cadherin fragment known to be stable in the circulatory system of cancer patients, and a polynucleotide encoding such a protein fragment, Correct cell-cell attachment can also be disrupted. Assays for cadherin adhesion and entry inhibitory activity are described in Hortsch et al. J Bio
l Chem 270 (32): 18809-18817, 1995; Miyaki et al. Oncogene 11: 2547-2552
, 1995; Ozawa et al. Cell 63: 1033-1038, 1990.

Tumor Inhibitory Activity In addition to the activities described above for the immunological treatment or prevention of tumors, the proteins of the present invention may exhibit other antitumor activities. Certain proteins directly or indirectly affect tumor growth (eg,
(Via ADCC). Certain proteins act on tumor tissue or tumor precursor tissue to inhibit the formation of tissue necessary to support tumor growth (eg, by inhibiting angiogenesis) and to inhibit tumor growth. Tumor inhibitory activity may be exhibited by causing the production of a factor, agent or cell type, or by removing or inhibiting a factor, agent or cell type that promotes tumor growth.

Other Activities The protein of the present invention may exhibit one or more of the following additional activities or effects: Infectious agents, including but not limited to bacteria, viruses, fungi, and other parasites. Killing; height, weight, hair color, eye color, skin or other tissue pigmentation, or the size or form of an organ or body part (eg, breast augmentation or vice versa)
Effects on body characteristics (suppression or promotion); effects on digestion of consumed fat, protein or carbohydrates; appetite, libido, stress, cognition (including cognitive disorders), depression (including depressive disorders) ) And effects on behavioral characteristics including, but not limited to, violent behavior; providing analgesic or other pain-relieving effects; promoting differentiation and proliferation of embryonic stem cells in non-hematopoietic lineages; hormones or endocrine Activity; for enzymes,
Correcting enzyme deficiency and treating related disorders; treating hyperproliferative disorders (eg, psoriasis); immunoglobulin-like activity (eg, the ability to bind antibodies or complement);
And the ability to act as an antigen in a vaccine composition to generate an immune response to such proteins or other substances or entities that cross-react with such proteins.

Administration and Dose The protein of the present invention (which may be from any source, including but not limited to recombinant and non-recombinant methods) is mixed with a pharmaceutically acceptable carrier. May be used in pharmaceutical compositions. Such compositions may contain (in addition to proteins and carriers) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable"
A non-toxic substance that does not interfere with the effectiveness of the biological activity of the active ingredient. The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the present invention comprises M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL -9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2
, G-CSF, Meg-CSF, thrombopoietin, stem cell factor, and cytokines such as erythropoietin, lymphokines, or other hematopoietic factors. The pharmaceutical composition may further contain other agents that enhance protein activity or supplement its activity or utility in therapy. Such additional factors and / or agents may be included in the pharmaceutical composition to exert a synergistic effect with the protein of the invention or to minimize side effects. Conversely, specific cytokines, lymphokines, other hematopoietic factors, thrombolytic factors or antithrombotic factors, or by incorporating the protein of the present invention into the formulation of anti-inflammatory agents, cytokines, lymphokines, other hematopoietic factors, thrombolytic factors or Side effects of antithrombotic factors or anti-inflammatory agents may be minimized. The protein of the invention may be active in multimers (eg, heterodimers or homodimers) or in complexes with itself or other proteins. Consequently, the pharmaceutical composition of the present invention may contain the multimeric or complexed form of the protein of the present invention. The pharmaceutical composition of the present invention may be in the form of a complex of the protein of the present invention and a protein or peptide antigen. Protein and / or peptide antigens can be
It will deliver a stimulus signal to the lymphocytes. B lymphocytes will respond to antigen through their surface immunoglobulin receptor. T lymphocytes will respond to antigen through the T cell receptor (TCR) after presentation of the antigen by MHC proteins.
MHC and structurally related proteins, including those encoded by the host cell class I and class II MHC genes, will be useful in presenting peptide antigens to T lymphocytes. The antigen component can be supplied as a purified MHC-peptide complex alone or with a costimulatory molecule that can directly signal T cells. Alternatively, surface immunoglobulins on B cells and T cells on T cells
Antibodies that can bind CR and other molecules can also be mixed with the pharmaceutical compositions of the present invention. The pharmaceutical composition of the present invention may be in the form of a liposome, in which the protein of the present invention further contains another pharmaceutically acceptable carrier, an amphipathic agent such as a lipid (in the form of an aggregate such as micelles in an aqueous solution). , Present as an insoluble monolayer, liquid crystal or lamellar layer). Suitable lipids for liposome formulations include, but are not limited to, monoglycerides, diglycerides, sulfatide, lysolecithin, phospholipids, saponins, bile acids, and the like. The preparation of such liposome formulations is within the level of ordinary skill in the art and is described, for example, in U.S. Patent Nos. 4,235,871, 4501728, 4837028,
And 4737323, all of which are herein incorporated by reference.

As used herein, the term “therapeutically effective amount” refers to each of the pharmaceutical compositions or methods sufficient to show significant patient benefit, eg, amelioration of symptoms of such symptoms, healing, increased rate of healing. Means the total amount of active ingredients. When used for an individual active ingredient administered alone, the term refers only to that ingredient. When used in a combination of active ingredients, it refers to the total amount of active ingredients that produces a therapeutic effect regardless of sequential or simultaneous administration. In practicing the treatment method of the present invention, a therapeutically effective amount of the protein of the present invention is administered to a mammal having the condition to be treated. According to the method of the invention, the protein of the invention may be administered alone or together with other therapeutic agents such as cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, the proteins of the invention may be administered simultaneously or sequentially with cytokines, lymphokines, other hematopoietic factors, thrombolytic or antithrombotic factors. If administered sequentially, the attending physician will determine the appropriate order of administration for combinations of the proteins of the present invention with cytokines, lymphokines, other hematopoietic, thrombolytic or antithrombotic factors. The administration of the protein of the present invention in the pharmaceutical composition of the present invention or used in the practice of the present invention can be performed by various conventional methods, for example, oral feeding, inhalation, or intradermal, subcutaneous, or intravenous injection. Intravenous injection into a patient is preferred. When a therapeutically effective amount of a protein of the invention is administered orally, the protein of the invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the present invention may further contain a solid carrier such as gelatin or an adjuvant. Tablets, capsules, and powders contain about 5 to 95% of a protein of the present invention,
It preferably contains about 25-90% of the protein of the invention. When administered in liquid form, water, petroleum, oils of animal or vegetable origin, such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. Liquid form pharmaceutical compositions may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains about 0.5 to 90% by weight of a protein of the invention, preferably about 1 to 50%. When a therapeutically effective amount of the protein of the present invention is administered by intravenous, intradermal or subcutaneous injection,
The protein of the present invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions having appropriate pH, isotonicity, and stability is within the skill of the art. Preferred pharmaceutical compositions for intravenous, intradermal, or subcutaneous injection are, in addition to the protein of the present invention, sodium chloride for injection, Ringer's solution, dextrose for injection, dextrose and sodium chloride for injection, and Ringer's solution containing lactic acid for injection. Or it should contain other carriers known in the art. The pharmaceutical composition of the present invention may contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those skilled in the art.

The amount of the protein of the invention in the pharmaceutical composition of the invention depends on the nature and severity of the condition to be treated,
As well as the nature of the treatment the patient has already received. Ultimately, your doctor
The amount of the protein of the invention to be treated for each patient will be determined. First, the attending physician administers a low dose of the protein of the invention and observes the patient's response. Higher doses of the protein of the invention may be administered until the optimal therapeutic effect is obtained, and once the optimal therapeutic effect has been obtained, the dose is not further increased. Various pharmaceutical compositions for carrying out the method of the present invention contain about 0.01 μg to about 100 mg of the protein of the present invention per kg of body weight (preferably,
It should contain from about 0.1 μg to about 10 mg per kg body weight, more preferably from 0.1 μg to about 1 mg per kg body weight. The duration of treatment by intravenous administration using the compositions of the present invention will vary with the severity of the disease to be treated and the condition and response of each patient. It is believed that the duration of each application of the protein of the invention ranges from 12 to 24 hours for continuous intravenous administration. Ultimately, the attending physician will decide the duration of intravenous treatment using the pharmaceutical composition of the present invention.

An animal may be immunized using the protein of the present invention to obtain polyclonal and monoclonal antibodies that specifically react with the protein of the present invention. Such antibodies may be obtained using whole proteins or fragments thereof as immunogens. The peptide immunogen may further include a cysteine residue at the carboxy terminus and binds to haptens such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, RP Merrifield, J. Amer. Che.
m. Soc. 85, 2149-2154 (1963); JL Krstenansky, et. al., FEBS Lett. 211
, 10 (1987). A monoclonal antibody that binds to the protein of the present invention may be a useful diagnostic agent for immunodetection of the protein of the present invention. Neutralizing antibodies that bind to the protein of the present invention can be useful as therapeutics for conditions related to the protein of the present invention,
In addition, it may be useful in the treatment of certain tumors in the treatment of some forms of cancer in which aberrant expression of the protein of the invention is involved. For cancer cells or white blood cells,
Neutralizing monoclonal antibodies against the protein of the present invention may be useful for detecting and preventing metastatic spread of cancer cells mediated by the protein of the present invention.

For compositions of the invention useful for the regeneration of bone, cartilage, tendon or ligament, the method of treatment comprises:
It involves administering the composition locally, systemically, or locally, such as an implant or device. When administered, the therapeutic compositions used in the present invention are, of course, in a pyrogen-free, physiologically acceptable form. In addition, if desired, the composition may be encapsulated or injected in a viscous form and delivered to the site of bone, cartilage or tissue damage. Topical administration is suitable for wound healing and tissue repair. Therapeutically useful agents other than the protein of the present invention, which may be contained in the above composition, may be alternatively or additionally administered simultaneously or sequentially with the composition in the method of the present invention. Preferably, for bone and / or cartilage formation, the composition delivers the protein-containing composition to the site of bone and / or cartilage damage, provides a structure for bone and cartilage development, and more optimally. Will contain a matrix that can be absorbed into the body. Such matrices may be made of materials currently used for other implanted medical devices. The choice of matrix material is based on biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. The appropriate formulation will be determined by the particular application of the composition. Possible matrices for the composition may be biodegradable and chemically known calcium sulfate, tricalcium phosphate, hydroxyapatite, polylactic acid, polyglycolic acid and polyanhydride. Other usable materials are biodegradable and biologically well known, such as bone or skin collagen. Further, the matrix may contain pure proteins or extracellular matrix components.
Other possible matrices are not biodegradable, such as sintered hydroxyapatite, bioglass, aluminate, or other ceramics, but are chemically known. The matrix may comprise a combination of materials of the above type, for example polylactic acid and hydroxyapatite or collagen and tricalcium phosphate. The bioceramics may be modified in the composition, for example in calcium-aluminate-phosphate, and processed to pore size, particle size,
The particle shape and biodegradability may be varied. A 50:50 (molar weight) copolymer of lactic acid and glycolic acid in the form of porous particles having a diameter in the range of 150 to 800 microns is presently preferred. In some applications, it may be useful to use a sequestering agent, such as carboxymethylcellulose or an autologous clot, to prevent dissociation of the protein composition from the matrix.

A preferred family of sequestering agents is cellulosic materials such as alkylcellulose (including hydroxyalkylcellulose), including methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and carboxymethylcellulose; Most preferred are cationic salts of (CMC).
Other preferred sequestering agents include hyaluronic acid, sodium alginate, poly (ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymers and poly (vinyl alcohol). The amount of sequestrant useful in the present invention is 0.5 to 20%, preferably 1 to 10% by weight, based on the total formulation weight,
An amount that prevents detachment of the protein from the polymer matrix and allows for proper handling of the composition, but provides an opportunity for the progenitor cells to infiltrate the matrix, thereby promoting the osteogenic activity of the progenitor cells. In order not to give too much. In a further composition, the protein of the invention may be mixed with other agents that are beneficial for treating bone and / or cartilage defects, wounds, or tissues. These agents include epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factors (TGF-α and TGF-β), and insulin-like growth factor (IGF). At present, the therapeutic compositions are also valuable for veterinary use. For details,
In addition to humans, livestock and thoroughbred horses are desirable patients for such treatments using the proteins of the present invention. The administration rules of the protein-containing pharmaceutical composition used for the regeneration of the tissue are controlled by various factors that alter the action of the protein, such as the tissue weight desired to be formed, the site of damage, the state of the damaged tissue, the size of the wound, The physician will decide on the type of tissue required (eg, bone), patient age, gender, and diet, severity of infection, duration of administration, and other clinical factors. Dosages may vary depending on the type of matrix used for reconstitution and the inclusion of other proteins in the pharmaceutical composition. For example, IGF
The addition of other known growth factors, such as I (insulin-like growth factor I), to the final composition can also affect the dose. Progress can be monitored by periodically assessing tissue / bone growth and / or repair, for example, by X-ray, histomorphological examination and tetracycline labeling.

The polynucleotide of the present invention can also be used for gene therapy. Such polynucleotides can be introduced into cells in vivo or ex vivo and expressed in a mammalian subject. The polynucleotides of the present invention may be administered by other known methods for introducing nucleic acids into cells or organisms, including but not limited to viral vectors or in the form of naked DNA. ). The cells may be cultured and grown ex vivo in the presence of the protein of the present invention, or have a desired effect on such cells, or have a desired activity in such cells. The treated cells can then be introduced in vivo for therapeutic purposes. The patents and publications cited herein are considered to be entirely incorporated herein by reference.

[0076]

[Sequence list]

[Brief description of the drawings]

FIGS. 1A and 1B schematically show the pED6 and pNOTs vectors, respectively, used for depositing the clones disclosed herein.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 A61K 37/18 C12N 5/00 B (81) Designated country EP (AT, BE, CH, CY) , DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW) , ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG , MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor John M. McCoy, United States 01867 Reading, Massachusetts, 56, Howard Street (72) Inventor Edward R. Lavary, United States 01451 Harvard, Mass. 113, Ann Lee Road Inventor Lisa A. Lacy United States 01720 Acton, Mass., School Street 124 (72) Inventor Cheryl Evans United States 0 1801 Locust Street Number 41, 111, Woburn, Mass. (72) Inventor David Marberg, United States 01720 Acton, Mass., Orchard Drive No. 2 (72) Inventor, Maurice Tracy United States 02167 Chesnut Hill, Mass., USA Walcott Road 93, 72 (72) Inventor Michael Jay Agostino Walcott Avenue 26, Andover, Mass. 18810, United States of America Inventor Vicki Spalding United States 01821 Billorica, Mass., Meadowbank Road 11F Terms (Reference) 4B024 AA01 AA11 BA80 CA02 CA04 DA01 DA02 DA05 DA11 FA02 4B065 AA01X AA57X AA58X AA72X AA88X AA90X AA93Y AB01 AC14 BA01 CA24 CA43 CA44 CA46 4C084 AA06 AA07 BA01 BA20 CA53 DA31 ZC78A FA74

Claims (32)

[Claims] 1. A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1; (b) a polynucleotide comprising the nucleotide sequence from nucleotide 69 to nucleotide 908 of SEQ ID NO: 1; A polynucleotide comprising the nucleotide sequence from nucleotide 270 to nucleotide 908; (d) a polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of clone bn971 deposited under accession number ATCC98535; (e) a polynucleotide comprising accession number ATCC98535 A polynucleotide encoding the full-length protein encoded by the cDNA insert of clone bn971; (f) the nucleotide sequence of the mature protein coding sequence of clone bn971 deposited under accession number ATCC 98535 (G) a polynucleotide encoding the mature protein encoded by the cDNA insert of clone bn971 deposited under accession number ATCC 98535; (h) a protein comprising the amino acid sequence of SEQ ID NO: 2 (I) a polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 2 having biological activity (the fragment comprises 8 of SEQ ID NO: 2)
(J) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (g) above; and (k) a species homolog of the protein of (h) or (i) above. An encoding polynucleotide; and (l) an isolated polynucleotide selected from the group consisting of: a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides (a) to (i). . 2. The polynucleotide of claim 1, operably linked to at least one expression control sequence. 3. A host cell transformed with the polynucleotide of claim 2. 4. The host cell according to claim 3, wherein said cell is a mammalian cell. 5. The polynucleotide encoded by the polynucleotide of claim 2, comprising: (a) growing the culture of the host cell of claim 3 in a suitable medium; and (b) purifying the protein from the culture. A method for producing a protein. 6. A protein produced by the method of claim 5. 7. (a) the amino acid sequence of SEQ ID NO: 2; (b) the amino acid sequence from amino acid 1 to amino acid 83 of SEQ ID NO: 2; A fragment of the amino acid sequence of SEQ ID NO: 2; and (d) a protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by the cDNA insert of clone bn971 deposited under accession number ATCC 98535, A protein substantially free of other mammalian proteins. 8. The protein of claim 7, comprising the amino acid sequence of SEQ ID NO: 2. 9. The protein according to claim 7, which comprises the amino acid sequence from amino acid 1 to amino acid 83 of SEQ ID NO: 2. 10. A composition comprising the protein of claim 7 and a pharmaceutically acceptable carrier. 11. An isolated gene corresponding to the cDNA sequence of SEQ ID NO: 1. 12. (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3; (b) a polynucleotide comprising from nucleotide 562 to nucleotide 777 of SEQ ID NO: 3; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 3 A polynucleotide comprising up to nucleotide 673; (d) clone bn2681 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of the full-length protein coding sequence of 1; (e) clone bn268 1 deposited under accession number ATCC 98535
A polynucleotide encoding the full-length protein encoded by the cDNA insert of No. 1; (f) clone bn2681 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of one of the mature protein coding sequences; (g) clone bn268 1 deposited under accession number ATCC 98535
A polynucleotide encoding a mature protein encoded by the cDNA insert of 1); (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 4; (i) an amino acid of SEQ ID NO: 4 having biological activity A polynucleotide encoding a protein comprising a fragment of the sequence (the fragment comprises eight consecutive amino acid sequences of SEQ ID NO: 4); (j) an allelic variant of the polynucleotide of (a) to (g) above. (K) a polynucleotide encoding a species homolog of the protein of (h) or (i) above; and (1) any of the polynucleotides shown in (a) to (i) under strict conditions. An isolated polynucleotide selected from the group consisting of polynucleotides capable of hybridizing together. 13. (a) an amino acid sequence of SEQ ID NO: 4; (b) an amino acid sequence from amino acid 1 to amino acid 37 of SEQ ID NO: 4; (c) comprising eight consecutive amino acids of SEQ ID NO: 4 A fragment of the amino acid sequence of SEQ ID NO: 4; and (d) clone bn2681 deposited under accession number ATCC98535.
A protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by one of the cDNA inserts, wherein the protein is substantially free of other mammalian proteins. 14. An isolated gene corresponding to the cDNA sequence of SEQ ID NO: 3. 15. A polynucleotide comprising the nucleotide sequence of SEQ ID NO: 5; (b) a polynucleotide comprising the nucleotide sequence of nucleotide 286 to nucleotide 1686 of SEQ ID NO: 5; A polynucleotide comprising the nucleotide sequence from nucleotide 544 to nucleotide 1686; (d) a polynucleotide comprising the nucleotide sequence from nucleotide 365 to nucleotide 1160 of SEQ ID NO: 5; (e) clone cb96 10 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of (c) clone cb9610 deposited under accession number ATCC 98535.
A polynucleotide encoding the full-length protein encoded by the cDNA insert of (c) clone cb9610 deposited under accession number ATCC98535.
A polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of: (h) clone cb96 10 deposited under accession number ATCC 98535
A polynucleotide encoding a mature protein encoded by the cDNA insert of (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 6; (j) a biologically active SEQ ID NO: A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 6
(K) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (h); (1) a species homolog of the protein of (i) or (j) And (m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) to (j). . 16. (a) the amino acid sequence of SEQ ID NO: 6; (b) the amino acid sequence from amino acid 28 to amino acid 395 of SEQ ID NO: 6; (c) comprising eight consecutive amino acids of SEQ ID NO: 6 A fragment of the amino acid sequence of SEQ ID NO: 6; and (d) clone cb96 10 deposited under accession number ATCC 98535.
A protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by the cDNA insert of claim 1, wherein the protein is substantially free of other mammalian proteins. 17. An isolated gene corresponding to the cDNA sequence of SEQ ID NO: 5. 18. A polynucleotide comprising: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 7; (b) a polynucleotide comprising the nucleotide sequence from nucleotide 99 to nucleotide 1049 of SEQ ID NO: 7; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 7 A polynucleotide comprising the nucleotide sequence from nucleotide 222 to nucleotide 1049; (d) a polynucleotide comprising the nucleotide sequence from nucleotide 632 to nucleotide 998 of SEQ ID NO: 7; (e) clone cb213 1 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of 1; (f) clone cb213 1 deposited under accession number ATCC 98535
A polynucleotide encoding the full-length protein encoded by the cDNA insert of No. 1; (g) clone cb213 1 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of one of the mature protein coding sequences; (h) clone cb213 1 deposited under accession number ATCC 98535
A polynucleotide encoding a mature protein encoded by one of the cDNA inserts; (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 8; (j) a SEQ ID NO having biological activity Polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 8
(K) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (h); (1) a species homolog of the protein of (i) or (j) And (m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) to (j). . 19. (a) an amino acid sequence of SEQ ID NO: 8; (b) an amino acid sequence from amino acid 187 to amino acid 300 of SEQ ID NO: 8; (c) comprising eight consecutive amino acids of SEQ ID NO: 8 A fragment of the amino acid sequence of SEQ ID NO: 8; and (d) clone cb213_1 deposited under accession number ATCC 98535.
A protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by one of the cDNA inserts, wherein the protein is substantially free of other mammalian proteins. 20. An isolated gene corresponding to the cDNA sequence of SEQ ID NO: 7. 21. (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9; (b) a polynucleotide comprising the nucleotide sequence from nucleotide 3003 to nucleotide 3137 of SEQ ID NO: 9; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 9 A polynucleotide comprising the nucleotide sequence from nucleotide 3072 to nucleotide 3137; (d) a polynucleotide comprising the nucleotide sequence from nucleotide 2713 to nucleotide 3114 of SEQ ID NO: 9; (e) clone cj457 4 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of (c) clone cj457 4 deposited under accession number ATCC 98535.
A polynucleotide encoding the full-length protein encoded by the cDNA insert of (c) clone cj457 4 deposited under accession number ATCC 98535.
A polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of: (h) clone cj457 4 deposited under accession number ATCC 98535
A polynucleotide encoding the mature protein encoded by the cDNA insert of (i) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 10; (j) a SEQ ID NO: having biological activity A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 10
(K) a polynucleotide which is an allelic variant of the polynucleotide of (a) to (h); (1) a species homolog of the protein of (i) or (j) And (m) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) to (j). . 22. (a) an amino acid sequence of SEQ ID NO: 10; (b) an amino acid sequence from amino acid 1 to amino acid 37 of SEQ ID NO: 10; (c) comprising eight consecutive amino acids of SEQ ID NO: 10 A fragment of the amino acid sequence of SEQ ID NO: 10; and (d) clone cj457 4 deposited under accession number ATCC 98535.
A protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by the cDNA insert of claim 1, which is substantially free of other mammalian proteins. 23. An isolated gene corresponding to the cDNA sequence of SEQ ID NO: 9. 24. (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 11; (b) a polynucleotide comprising the nucleotide sequence from nucleotide 284 to nucleotide 1357 of SEQ ID NO: 11; A polynucleotide comprising a nucleotide sequence from nucleotide 603 to nucleotide 1233; (d) clone cz6531 deposited under accession number ATCC 98535 1
A polynucleotide comprising the nucleotide sequence of one full length protein coding sequence; (e) clone cz6531 deposited under accession number ATCC 98535 1
A polynucleotide encoding the full-length protein encoded by the cDNA insert of No. 1; (f) clone cz6531 deposited under accession number ATCC 98535 1
A polynucleotide comprising the nucleotide sequence of one of the mature protein coding sequences; (g) clone cz6531 deposited under accession number ATCC 98535 1
A polynucleotide encoding a mature protein encoded by one of the cDNA inserts; (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 12; (i) a SEQ ID NO having biological activity : A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 12
(J) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (g) above; (k) species homology of the protein of (h) or (i) above. And (l) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Polynucleotide. 25. (a) an amino acid sequence of SEQ ID NO: 12; (b) an amino acid sequence from amino acid 147 to amino acid 358 of SEQ ID NO: 12; (c) comprising eight consecutive amino acids of SEQ ID NO: 12 A fragment of the amino acid sequence of SEQ ID NO: 12; and (d) clone cz6531_1 deposited under accession number ATCC 98535.
A protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by one of the cDNA inserts, wherein the protein is substantially free of other mammalian proteins. 26. An isolated gene corresponding to the cDNA sequence of SEQ ID NO: 11. (27) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 13 from nucleotide 621 to nucleotide 1763; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 13 from nucleotide 621 to nucleotide 1763; A polynucleotide comprising the nucleotide sequence from nucleotide 1461 to nucleotide 1763; (d) clone dx1384, deposited as accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of (e) clone dx1384 deposited under accession number ATCC 98535
A polynucleotide encoding the full-length protein encoded by the cDNA insert of (d) clone dx1384 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of: (g) clone dx1384 deposited under accession number ATCC 98535
A polynucleotide encoding the mature protein encoded by the cDNA insert of (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 14; (i) a SEQ ID NO: having biological activity A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 14
(J) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (g) above; (k) species homology of the protein of (h) or (i) above. And (l) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Polynucleotide. 28. (a) the amino acid sequence of SEQ ID NO: 14; (b) the amino acid sequence from amino acid 83 to amino acid 229 of SEQ ID NO: 14; (c) comprising eight consecutive amino acids of SEQ ID NO: 14 A fragment of the amino acid sequence of SEQ ID NO: 14; and (d) clone dx1384 deposited under accession number ATCC 98535.
A protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by the cDNA insert of claim 1, which is substantially free of other mammalian proteins. 29. An isolated gene corresponding to the cDNA sequence of SEQ ID NO: 13. (30) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15 from nucleotide 119 to nucleotide 295; (c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 15 from nucleotide 119 to nucleotide 295; A polynucleotide comprising a nucleotide sequence from nucleotide 191 to nucleotide 295; (d) clone ij167 5 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of the full length protein coding sequence of (e) clone ij1675 deposited under accession number ATCC 98535.
A polynucleotide encoding the full length protein encoded by the cDNA insert of (f) clone ij167 5 deposited under accession number ATCC 98535
A polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of: (g) clone ij167 5 deposited under accession number ATCC 98535
A polynucleotide encoding a mature protein encoded by the cDNA insert of (h) a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 16; (i) a SEQ ID NO: having biological activity A polynucleotide encoding a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 16
(J) a polynucleotide that is an allelic variant of the polynucleotide of (a) to (g) above; (k) species homology of the protein of (h) or (i) above. And (l) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). Polynucleotide. 31. (a) the amino acid sequence of SEQ ID NO: 16; (b) the amino acid sequence from amino acid 1 to amino acid 26 of SEQ ID NO: 16; (c) comprising eight consecutive amino acids of SEQ ID NO: 16 A fragment of the amino acid sequence of SEQ ID NO: 16; and (d) clone ij167 5 deposited under accession number ATCC 98535.
A protein comprising an amino acid sequence selected from the group consisting of the amino acid sequence encoded by the cDNA insert of claim 1, which is substantially free of other mammalian proteins. 32. An isolated gene corresponding to the cDNA sequence of SEQ ID NO: 15.