U.S. patent application number 11/087701 was filed with the patent office on 2005-07-28 for novel serine protease bssp2.
This patent application is currently assigned to FUSO PHARMACEUTICAL INDUSTRIES, LTD.. Invention is credited to Kominami, Katsuya, Mitsui, Shinichi, Okui, Akira, Uemura, Hidetoshi, Yamaguchi, Nozomi.
Application Number | 20050165223 11/087701 |
Document ID | / |
Family ID | 18392565 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050165223 |
Kind Code |
A1 |
Uemura, Hidetoshi ; et
al. |
July 28, 2005 |
Novel serine protease BSSP2
Abstract
There are provided proteins having the amino acid sequences
represented by SEQ ID NOS: 2, 4, 6, 8 and 10; proteins having amino
acid sequences derived from these amino acid sequences by deletion,
substitution or addition of one to several amino acids; and
nucleotide sequences encoding the same; transgenic non-human
animals with altered expression level of a serine protease BSSP2;
an antibody against BSSP2; and a method for detecting BSSP2 in a
specimen by using the antibody.
Inventors: |
Uemura, Hidetoshi;
(Itami-shi, JP) ; Okui, Akira;
(Yamatokoriyama-shi, JP) ; Kominami, Katsuya;
(Osaka, JP) ; Yamaguchi, Nozomi; (Koyoto, JP)
; Mitsui, Shinichi; (Kyoto-shi, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.
624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
FUSO PHARMACEUTICAL INDUSTRIES,
LTD.
Osaka
JP
|
Family ID: |
18392565 |
Appl. No.: |
11/087701 |
Filed: |
March 24, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11087701 |
Mar 24, 2005 |
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09856371 |
May 21, 2001 |
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09856371 |
May 21, 2001 |
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PCT/JP99/06475 |
Nov 19, 1999 |
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Current U.S.
Class: |
530/388.26 |
Current CPC
Class: |
A01K 2217/05 20130101;
C12N 9/6424 20130101 |
Class at
Publication: |
530/388.26 |
International
Class: |
A61K 039/395; C07K
016/40 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 20, 1998 |
JP |
347785/1998 |
Claims
What is claimed is:
1. An antibody against a protein selected from the group consisting
of: (i) a protein having the amino acid sequence of 238 amino acids
represented by SEQ ID NO: 2, a protein having an amino acid
sequence derived from the amino acid sequence represented by SEQ ID
NO: 2 by deletion, substitution or addition of one to several amino
acids and having the same property as that of the protein having
the amino acid sequence represented by SEQ ID NO: 2, or a modified
derivative thereof; (ii) a protein having the amino acid sequence
of 273 amino acids represented by SEQ ID NO: 4, a protein having an
amino acid sequence derived from the amino acid sequence
represented by SEQ ID NO: 4 by deletion, substitution or addition
of one to several amino acids and having the same property as that
of the protein having the amino acid sequence represented by SEQ ID
NO: 4, or a modified derivative thereof; (iii) a protein having the
amino acid sequence of 311 amino acids represented by SEQ ID NO: 6,
a protein having an amino acid sequence derived from the amino acid
sequence represented by SEQ ID NO: 6 by deletion, substitution or
addition of one to several amino acids and having the same property
as that of the protein having the amino acid sequence represented
by SEQ ID NO: 6, or a modified derivative thereof; (iv) a protein
having the amino acid sequence of 455 amino acids represented by
SEQ ID NO: 8, a protein having an amino acid sequence derived from
the amino acid sequence represented by SEQ ID NO: 8 by deletion,
substitution or addition of one to several amino acids and having
the same property as that of the protein having the amino acid
sequence represented by SEQ ID NO: 8, or a modified derivative
thereof; (v) a protein having the amino acid sequence of 240 amino
acids represented by the 1st to 240th amino acids of SEQ ID NO: 10,
a protein having an amino acid sequence derived from the amino acid
sequence represented by the 1st to 240th amino acids of SEQ ID NO:
10 by deletion, substitution or addition of one to several amino
acids and having the same property as that of the protein having
the amino acid sequence represented by the 1st to 240th amino acids
of SEQ ID NO: 10, or a modified derivative thereof; (vi) a protein
having the amino acid sequence of 457 amino acids represented by
the -217th to 240th amino acids of SEQ ID NO: 10, a protein having
an amino acid sequence derived from the amino acid sequence
represented by the -217th to 240th amino acids of SEQ ID NO: 10 by
deletion, substitution or addition of one to several amino acids
and having the same property as that of the protein having the
amino acid sequence represented by the -217th to 240th amino acids
of SEQ ID NO: 10, or a modified derivative thereof; and (vii) a
protein having the amino acid sequence of 217 amino acids
represented by the -217th to -1st amino acids of SEQ ID NO: 10, a
protein having an amino acid sequence derived from the amino acid
sequence represented by the -217th to -1st amino acids of SEQ ID
NO: 10 by deletion, substitution or addition of one to several
amino acids and having the same property as that of the protein
having the amino acid sequence represented by the -217th to -1st
amino acids of SEQ ID NO: 10, or a modified derivative thereof, or
a fragment of said protein.
2. The antibody of claim 1 which is a polyclonal antibody, a
monoclonal antibody or a peptide antibody.
3. The antibody of claim 1 which is against the protein having the
amino acid sequence of 238 amino acids represented by SEQ ID NO: 2,
a protein having an amino acid sequence derived from the amino acid
sequence represented by SEQ ID NO: 2 by deletion, substitution or
addition of one to several amino acids and having the same property
as that of the protein having the amino acid sequence represented
by SEQ ID NO: 2, or a modified derivative thereof.
4. The antibody of claim 1 which is against the protein having the
amino acid sequence of 273 amino acids represented by SEQ ID NO: 4,
a protein having an amino acid sequence derived from the amino acid
sequence represented by SEQ ID NO: 4 by deletion, substitution or
addition of one to several amino acids and having the same property
as that of the protein having the amino acid sequence represented
by SEQ ID NO: 4, or a modified derivative thereof.
5. The antibody of claim 1 which is against the protein having the
amino acid sequence of 311 amino acids represented by SEQ ID NO: 6,
a protein having an amino acid sequence derived from the amino acid
sequence represented by SEQ ID NO: 6 by deletion, substitution or
addition of one to several amino acids and having the same property
as that of the protein having the amino acid sequence represented
by SEQ ID NO: 6, or a modified derivative thereof.
6. The antibody of claim 1 which is against the protein having the
amino acid sequence of 455 amino acids represented by SEQ ID NO: 8,
a protein having an amino acid sequence derived from the amino acid
sequence represented by SEQ ID NO: 8 by deletion, substitution or
addition of one to several amino acids and having the same property
as that of the protein having the amino acid sequence represented
by SEQ ID NO: 8, or a modified derivative thereof;
7. The antibody of claim 1 which is against the protein having the
amino acid sequence of 240 amino acids represented by the 1st to
240th amino acids of SEQ ID NO: 10, a protein having an amino acid
sequence derived from the amino acid sequence represented by the
1st to 240th amino acids of SEQ ID NO: 10 by deletion, substitution
or addition of one to several amino acids and having the same
property as that of the protein having the amino acid sequence
represented by the 1st to 240th amino acids of SEQ ID NO: 10, or a
modified derivative thereof.
8. The antibody of claim 1 which is against the protein having the
amino acid sequence of 457 amino acids represented by the -217th to
240th amino acids of SEQ ID NO: 10, a protein having an amino acid
sequence derived from the amino acid sequence represented by the
-217th to 240th amino acids of SEQ ID NO: 10 by deletion,
substitution or addition of one to several amino acids and having
the same property as that of the protein having the amino acid
sequence represented by the -217th to 240th amino acids of SEQ ID
NO: 10, or a modified derivative thereof.
9. The antibody of claim 1 which is against the protein having the
amino acid sequence of 217 amino acids represented by the -217th to
-1st amino acids of SEQ ID NO: 10, a protein having an amino acid
sequence derived from the amino acid sequence represented by the
-217th to -1st amino acids of SEQ ID NO: 10 by deletion,
substitution or addition of one to several amino acids and having
the same property as that of the protein having the amino acid
sequence represented by the -217th to -1st amino acids of SEQ ID
NO: 10, or a modified derivative thereof, or a fragment of said
protein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of application Ser.
No. 09/856,371, filed May 21, 2001, which is the national stage
under 35 U.S.C. .sctn.371 of international application
PCT/JP99/06475, filed Nov. 19, 1999, which designated the United
States, and which application was not published in the English
language.
FIELD OF THE INVENTION
[0002] The present invention relates to isolated polynucleotides of
human and mouse serine proteases (hereinafter referred to as
"hBSSP2" and "mBSSP2", respectively, and, in case no
differentiation thereof from each other is needed, simply referred
to as "BSSP2"), and their homologous forms, mature forms,
precursors and polymorphic variants as well as a method for
detecting thereof. Further, it relates to hBSSP2 and mBSSP2
proteins, compositions containing hBSSP2 and mBSSP2 polynucleotides
and proteins, as well as their production and use.
BACKGROUND OF THE INVENTION
[0003] In general, proteases are biosynthesized as inactive
precursors. They undergo limited hydrolysis in molecules to be
converted into activated type proteases. In so far as enzymes are
proteases, they have an activity for hydrolyzing a peptide bond,
while their actions vary according to kinds of proteases. According
to a particular kind of catalytic site, proteases are divided into
serine proteases, cysteine proteases, aspartate proteases, metal
proteases and the like. Proteases of each kind have a variety of
properties, ranging from a protease having general digestive
properties to a protease having various regulatory domains and
strict substrate specificity, thereby specifically hydrolyzing only
characteristic proteins.
[0004] Further, proteins undergo various types of processing even
after translation to produce active proteins. In many secretory
proteins, a protein is first synthesized on the ribosome in
cytoplasm as an inactive precursor (pro-form) which comprises an
active protein bearing at the N-terminus thereof a peptide of about
15 to 60 amino acids responsible for secretion (secretory signal).
This peptide region is concerned with the mechanism for passing
through the cell membrane and is removed upon cleavage by a
specific protease during the passage through the membrane, in
almost all the cases, to produce the mature form. A secretory
signal has a broad hydrophobic region comprising hydrophobic amino
acids in the middle of the sequence, and basic amino acid residues
at a site close to the N-terminus. A secretory signal is a synonym
for a signal peptide. In addition, in some proteins, a peptide
moiety which functions as a secretory signal is further attached to
the N-terminus of the inactive precursor (pro-form). Such a protein
is called a prepro-protein (prepro-form).
[0005] For example, trypsin is present in the form of a prepro-form
immediately after translation into amino acids. After being
secreted from cells, it is present in the form of a pro-form and is
then converted into active trypsin in the duodenum upon limited
hydrolysis by enteropeptidase or by trypsin itself.
[0006] The optimal pH range of serine proteases is neutral to weak
alkaline and, in general, many of them have a molecular weight of
about 30,000 or lower. All proteases relating to blood coagulation,
fibrinolysis and complement systems having a large molecular weight
belong to the family of trypsin-like serine proteases. They have
many regulator domains and form a protease cascade which is of very
importance to reactions in a living body.
[0007] Recently, cDNAs and amino acid sequences of many novel
proteases have been determined by PCR for consensus sequences of
serine proteases using oligonucleotide primers. According to this
method, novel proteases have been found by various researchers such
as Yamamura et al. (Yamanura, Y et al., Biochem. Biophys. Res.
Commun., 239, 386, 1997), Gschwend, et al. (Gschwend, T. P. et al.,
Mol. Cell. Neurosci., 9. 207, 1997), Chen et al. (Chen, Z-L, et
al., J. Neurosci., 15, 5088, 1995) and others.
[0008] SEQ ID NO: 3 of JP 9-149790 A discloses neurosin as a novel
serine protease. Neurosin has also been reported in Biochimica et
Byophysica Acta, 1350, 11-14, 1997. By this, there is provided a
method for mass production of neurosin using the serine protease
gene and a method for screening specific inhibitors using the
enzyme. In addition, the screening method has been shown to be
useful for screening medicines for treating various diseases.
[0009] Serine proteases expressed in a brain-nerve system such as
neurosin are considered to play various roles in the brain-nerve
system. Therefore, there is a possibility that isolation of a gene
encoding a novel protease expressed in a brain-nerve system and
production of a protein using the gene would be useful for
diagnosis or therapy of various diseases related to the brain-nerve
system.
[0010] Nowadays, in general, clinical diagnosis of Alzheimer's
disease is based on the diagnosis standard of DSM-IIIR and
NINCDS-ADRDA (Mckhann, G. et al., Neurology, 34. 939, 1994) or the
diagnosis standard of DSM-IV (American Psychiatric Association;
Diagnostic and statistical manuals of mental disorders, 4th ed.,
Washington D.C., American Psychiatric Association, 1994). However,
these standards are conditioned by a decline in recognition
functions which causes a severe disability in daily life or social
life. Then, it is pointed out that the diagnosis is less than
scientifically objective because the diagnosis may be influenced by
the level of an individual's social life and further the specialty
and experience of a physician who diagnoses particular conditions.
In addition, definite diagnosis of Alzheimer's disease is conducted
by pathohistological analyses and, in this respect, substantial
inconsistency between clinical diagnosis and autopsy diagnosis
exists.
[0011] At present, image diagnosis is employed as a supplemental
means in clinical diagnosis of Alzheimer's diagnosis and it is
possible to analyze brain functions, for example, decline of
metabolism and atrophy in specific sites such as hippocampus,
parietal lobe of cerebral cortex and the like which are specific
for Alzheimer's disease by PET and SPECT. However, to define
Alzheimer's disease based on lowering of a blood flow from parietal
lobe to temporal lobe is very dangerous. In addition, there is a
report showing that MRS test is useful for patients with dementia
including those of Alzheimer's disease. Further, although CT-MRI
image diagnosis is used, a lesion of white matter such as atrophy
of brain, PVL or the like is not specific for Alzheimer type
dementia. Since it has been reported that atrophy of brain proceeds
with aging, the above observation is not necessarily found in
Alzheimer type dementia. Furthermore, since an image obtained by
MRI varies according to strength of a magnetic field, performance
of the apparatus and imaging conditions, numerical data obtained in
different facilities cannot be compared with each other except for
atrophic change. In addition, there is a limit to image
measurement. Further, enlargement of the ventricle can be
recognized in vascular dementia cases and there are cases wherein
atrophy of the hippocampus is observed after ischemia of the
basilar artery.
[0012] Under these circumstances, many researchers have requested
to develop biological diagnosis markers as a means for providing
better precision and objectivity for clinical diagnosis of
Alzheimer's disease. At the same time, the following important
roles in the future will be expected.
[0013] 1) Objective judgment system of effect of medicaments for
treating Alzheimer's disease.
[0014] 2) Detection of Alzheimer's disease before a diagnosis
standard is met, or disease conditions are manifested.
[0015] Further, data obtained in different facilities can be
compared with each other by using the same diagnosis marker.
Therefore, development of biological diagnosis markers is
recognized to be a most important field among fields of Alzheimer's
disease studies and its future prospects will be expected.
Approaches to development of biological diagnosis markers up to now
are divided into those based on constitute components of
characteristic pathological changes of Alzheimer's disease such as
senile plaque and neurofibril change, and an approach based on
other measures. Examples of the former include cerebrospinal fluid
tau protein, A.beta. and its precursor, .beta.APP. Examples of the
latter include mydriasis test with cholilytic drug, Apo E and other
genes relating to Alzheimer's disease. However, no good results
have been obtained.
[0016] Serine proteases are also considered to play an important
role in cancer cells. The reason why extermination of cancer by
surgical treatment or topical irradiation of radioactive ray is
difficult is the metastatic capability of cancer. To spread solid
tumor cells in a body, they loosen their adhesion to original
adjacent cells, followed by separating from original tissue,
passing through other tissues to reach the blood vessels or lymph
nodes, entering into the circulatory system through stratum basal
and endothelial layer of the vessel, leave from the circulatory
system at somewhere in the body, and surviving and proliferating in
a new environment. While adhesion to adjacent epidermal cells is
lost when expression of cadherin which is an intercellular adhesive
molecule of epithelium is stopped, to break through tissues is
considered to depend on proteolytic enzymes which decompose an
extracellular matrix.
[0017] As enzymes which decompose the matrix, mainly, metal
proteases (Rha, S. Y. et al., Breast Cancer Research Treatment, 43,
175, 1997) and serine proteases are known. They cooperate to
decompose matrix proteins such as collagen, laminin and
fibronectin. Among the serine proteases known to be concerned in
decomposition of the matrix, in particular, there is urokinase type
plasminogen activator (U-PA). U-PA has a role as a trigger specific
for a protein decomposition chain reaction. Its direct target is
plasminogen. It is present in blood abundantly and is a precursor
of an inactive serine protease which accumulates in reconstructed
sites of tissues such as injured sites and tumors as well as
inflammatory sites. In addition, as proteases which are concerned
in metastasis and infiltration of cancers, for example, a tissue
factor, lysosomal type hydrolase and collagenase have been
known.
[0018] At present, cancer is the top cause of death in Japan and
more than 200,000 people die per year. Accordingly, specific
substances which can be used as markers for diagnosis and therapy
or prophylaxis of cancer are studied intensively. Such specific
substances are referred to as tumor markers or tumor marker
relating biomarkers. They are utilized in aid of diagnosis before
treatment of cancer, for presuming carcinogenic organ and
pathological tissue type, for monitoring effect of treatment, for
finding recurrence early, for presuming prognosis, and the like. At
present, tumor markers are essential in clinical analyses. Among
them, alpha fetoprotein (AFP) which has high specificity to
hepatocellular carcinoma and yolk sac tumor (Taketa K. et al.,
Tumour Biol., 9, 110, 1988), and carcinoembronic antigen (CEA) are
used worldwide. In the future, tumor markers will be required more
and more, and it is desired to develop, for example, organ specific
markers and tumor cell specific markers which are highly reliable
serologic diagnosis of cancer. Up to now, humunglandular kallikrein
(hK2) which is a serine protease expressed at human prostatic
epithelial cells has been reported as a marker for prostatic
cancer. And, hK2 has 78% homology with the sequence of prostatic
specific antigen (PSA) and PSA is also used widely as a biochemical
marker of prostatic cancer (Mikolajczyk, S. d. et al., Prostate,
34, 44, 1998; Pannek, J. et al., Oncology, 11, 1273, 1997; Chu, T.
M. et al., Tumour Biology, 18, 123, 1997; Hsieh, M. et al., Cancer
Res., 57, 2651, 1997). Further, hK2 is reported to be useful as a
marker for not only prostatic cancer but also stomach cancer (Cho,
J. Y. et al. Cancer, 79, 878, 1997). Moreover, CYFRA (CYFRA 211)
for measuring cytokeratin 19 fragment in serum is reported, to be
useful for lung cancer (Sugiyama, Y. et al., Japan J. Cancer Res.,
85, 1178, 1994). Gastrin release peptide precursor (ProGRP) is
reported to be useful as a tumor marker (Yamaguchi, K. et al.,
Japan, J. Cancer Res., 86, 698, 1995).
OBJECTS OF THE INVENTION
[0019] Thus, the main object of the present invention is to provide
a novel serine protease which can be used for treating or
diagnosing various diseases such as Alzheimer's disease (AD),
epilepsy, cancer, inflammation, infertility, prostatomegaly and the
like in various tissues such as brain, lung, prostate, testicle,
skeletal muscle, liver and the like, and can be used as an
excellent marker instead of that presently used.
SUMMARY OF THE INVENTION
[0020] Under these circumstances, the present inventors have
succeeded in cloning cDNA encoding novel human and mouse serine
proteases.
[0021] In summary, the 1st feature of the present invention is the
amino acid sequences of biologically active mature serine proteases
BSSP2 and nucleotide sequences encoding the amino acid
sequences.
[0022] That is, they are the amino acid sequence composed of 238
amino acids (mature type BSSP2 (SEQ ID NO: 2)) and a nucleotide
sequence encoding the amino acid sequence (the 1st to 714th bases
of SEQ ID NO: 1). In addition, they include amino acid sequences
substantially similar to SEQ ID NO: 2 and nucleotide sequences
encoding such similar amino acid sequences. Further, they include
modified derivatives of proteins having these amino acid sequences.
An amino acid sequence substantially similar to a given amino acid
sequence used herein means an amino acid sequence derived from the
given amino acid sequence by modification such as substitution,
deletion, addition and/or insertion of one to several amino acids
with maintaining the same property as that of the protein having
the given amino acid sequence. The modified derivative of the
proteins includes, for example, phosphate adduct, sugar chain
adduct, metal adduct (e.g., calcium adduct), the protein fused to
another protein such as albumin etc., dimer of the protein, and the
like.
[0023] In the nucleotide sequences in the Sequence Listing
hereinafter, the symbol "n" represents that any of the normal bases
of a nucleic acid, i.e., adenine (a), cytosine (c), guanine (g) and
thymine (t) is present at that position.
[0024] The 2nd feature of the present invention is an amino acid
sequence composed of 273 amino acids [type 1 BSSP2 (SEQ ID NO: 4)]
wherein 35 amino acids of -35th to -1st amino acids represented by
SEQ ID NO: 4 are added to the N-terminus side of the mature BSSP2
amino acid sequence (SEQ ID NO: 2) and a nucleotide sequence
encoding the amino acid sequence (247th to 1065th bases of SEQ ID
NO: 3). In addition, this feature includes amino acid sequences
substantially similar to SEQ ID NO: 4 and nucleotide sequences
encoding these substantially similar amino acid sequences. Further,
this feature includes modified derivatives of proteins having these
amino acid sequences.
[0025] The 3rd feature of the present invention is an amino acid
sequence composed of 311 amino acids [type 2 BSSP2 (SEQ ID NO: 6)]
wherein 73 amino acids of -73rd to -1st amino acids represented by
SEQ ID NO: 6 are added to the N-terminus side of the mature BSSP2
amino acid sequence (SEQ ID NO: 2) and a nucleotide sequence
encoding the amino acid sequence (516th to 1448th bases of SEQ ID
NO: 5). In addition, this feature includes amino acid sequences
substantially similar to SEQ ID NO: 6 and nucleotide sequences
encoding these substantially similar amino acid sequences. Further,
this feature includes modified derivatives of proteins having there
amino acid sequences.
[0026] The 4th feature of the present invention is an amino acid
sequence composed of 445 amino acids [type 3 BSSP2 (SEQ ID NO: 8)]
wherein 207 amino acids of -207th to -1st amino acids represented
by SEQ ID NO: 8 are added to the N-terminus side of the mature
BSSP2 amino acid sequence (SEQ ID NO: 2) and a nucleotide sequence
encoding the amino acid sequence (116th to 1450th bases of SEQ ID
NO: 7). In addition, this feature includes amino acid sequences
substantially similar to SEQ ID NO: 8 and nucleotide sequences
encoding these substantially similar amino acid sequences. Further,
this feature includes modified derivatives of proteins having there
amino acid sequences.
[0027] The 5th feature of the present invention is an amino acid
sequence of a biologically active, mature human serine protease,
hBSSP2, and a nucleotide sequence encoding the amino acid sequence.
That is, they are an amino acid sequence [mature type hBSSP2 (SEQ
ID NO: 10) composed of 240 amino acids represented by SEQ ID NO: 10
(1st to 240th amino acids) and a nucleotide sequence encoding the
amino acid sequence (807th to 1526th bases of SEQ ID NO: 9). In
addition, this feature includes amino acid sequences substantially
similar to SEQ ID NO: 10 (1st to 240th amino acids) and nucleotide
sequences encoding these substantially similar amino acid
sequences. Further, this feature includes modified derivatives of
proteins having there amino acid sequences.
[0028] The 6th feature of the present invention is an amino acid
sequence composed of 457 amino acids (-217th to 240th amino acids
of SEQ ID NO: 10) wherein 217 amino acids of -217th to -1st amino
acids represented by SEQ ID NO: 10 are added to the N-terminus side
of the mature human serine protease hBSSP2 amino acid sequence (1st
to 240 amino acids of SEQ ID NO: 10) and a nucleotide sequence
encoding the amino acid sequence (156th to 1526th bases of SEQ ID
NO: 9). In addition, this feature includes amino acid sequences
substantially similar to SEQ ID NO: 10 and nucleotide sequences
encoding these substantially similar amino acid sequences. Further,
this feature includes modified derivatives of proteins having there
amino acid sequences.
[0029] The 7th feature of the present invention is an amino acid
sequence composed of 217 amino acids of -217th to -1st amino acids
of SEQ ID NO: 10 and a nucleotide sequence encoding the amino acid
sequence (156th to 806th bases of SEQ ID NO: 9). In addition, this
feature includes amino acid sequences substantially similar to the
amino acid composed of 217 amino acids of -217th to -1st SEQ ID NO:
10 and nucleotide sequences encoding these substantially similar
amino acid sequences. Further, this feature includes modified
derivatives of proteins having there amino acid sequences.
[0030] The present invention also relates to the nucleotide
sequences represented by SEQ ID NOS: 1, 3, 5, 7 and 9 as well as
nucleotide sequences similar to them.
[0031] The 8th feature of the present invention is a vector
comprising the nucleotide sequence according to any of the above
1st to the 7th feature, and transformant cells transformed with the
vector.
[0032] The 9th feature of the present invention is a process for
producing BSSP2 protein from the transformed cells of the 8th
feature.
[0033] The 10th feature of the present invention is a transgenic
non-human animal, wherein the expression level of BSSP2 gene has
been altered.
[0034] The 11th feature of the present invention is an antibody
against BSSP2 protein or its fragment and a process for producing
thereof.
[0035] The 12th feature of the present invention is a method for
determining BSSP2 protein or its fragment in a specimen using the
antibody of the 11th feature.
[0036] The 13th feature is a diagnostic marker of diseases
comprising BSSP2 protein.
[0037] Hereinafter, unless otherwise stated, the nucleotide
sequence represented by each SEQ ID NO: includes the
above-described various fragments thereof, and similar nucleotide
sequences and their fragments. Likewise, the amino acid sequence
represented by each SEQ ID NO: includes the above-described various
fragments thereof, similar nucleotide sequences and their
fragments, and modified derivatives thereof. In addition, unless
otherwise stated, BSSP2, hBSSP2, and mBSSP2 include proteins having
the above-described respective amino acid sequences.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 illustrates the results of northern blotting using
mRNAs prepared from mice in Example 2 hereinafter.
[0039] FIG. 2 illustrates the results of northern blotting using
mRNAs prepared from mice in Example 2 hereinafter.
[0040] FIG. 3 is a plasmid constructed by the method of Example 4
hereinafter.
[0041] FIG. 4 illustrates the construction of plasmid
pFBTrypSigTag/BSSP2 according to the method of Example 4
hereinafter.
[0042] FIG. 5 illustrates the detection of hBSSP2 mRNA by northern
hybridization.
[0043] FIG. 6 illustrates the detection of hBSSP2 mRNA by
RT-PCR.
[0044] FIG. 7 illustrates the expression of hBSSP2 by a baculovirus
system.
DETAILED DESCRIPTION OF THE INVENTION
[0045] The nucleotide sequences encoding hBSSP2 or mBSSP2 of the
present invention can be obtained by preparing mRNAs from cells
expressing the protein and converting it into double stranded DNAs
according to a conventional manner. For preparing mRNA, guanidine
isothiocyanate-calcium chloride method (Chirwin, et al.,
Biochemistry, 18, 5294, 1979) or the like can be used. For
preparing poly (A)+RNA from total RNAs, there can be used affinity
chromatography using a carrier, for example, Sepharose, latex
particles, etc., to which oligo (dT) is attached, and the like. The
above-obtained RNA can be used as a template and treated with
reverse transcriptase by using, as a primer, oligo (dT) which is
complementary to the poly (A) strand at the 3'-terminus, or a
random primer, or a synthesized oligonucleotide corresponding to a
part of the amino acid sequence of hBSSP2 or mBSSP2 to obtain a
hybrid mRNA strand comprising DNA or cDNA complementary to the
mRNA. The double stranded DNA can be obtained by treating the
above-obtained hybrid mRNA strand with E. coli RNase, E. coli DNA
polymerase and E. coli DNA ligase to convert into a DNA strand.
[0046] It is also possible to carry out cloning by RT-PCR method
using primers synthesized on the basis of the nucleotide sequence
of hBSSP2 or mBSSP2 gene and using hBSSP2 or mBSSP2 expressing cell
poly (A)+RNA as a template. Alternatively, the desired cDNA can be
obtained without using PCR by preparing or synthesizing a probe on
the basis of the nucleotide sequence of hBSSP2 or mBSSP2 gene and
screening a cDNA library directly. Among genes obtained by these
methods, the gene of the present invention can be selected by
confirming a nucleotide sequence thereof. The gene of the present
invention can also be prepared according to a conventional method
using chemical syntheses of nucleic acids, for example,
phosphoamidite method (Mattencci, M. D. et al., J. Am. Chem. Soc.,
130, 3185, 1981) and the like.
[0047] By using the thus-obtained hBSSP2 or mBSSP2 gene, their
expression in various tissues can be examined.
[0048] In case of northern blotting analysis, mBSSP2 shows the
expression in the head of a 15-20 days mouse fetus, and in the
lung, prostate and testicle of a 3 month-old mouse. hBSSP2 shows
the expression in brain, skeletal muscle and liver (see FIGS. 1, 2
and 5). In case of RT-PCR analysis, mBSSP2 shows the expression in
the brain and testicle of a 12 day-old mouse, and hBSSP2 shows the
expression in the brain and skeletal muscle. Then, the novel
proteases of the present invention are presumed to play various
roles in the brain, prostate, lung, testicle, skeletal muscle and
liver. For example, in the brain, there is a possibility that they
can be used for treatment and diagnosis of brain diseases such as
Alzheimer's disease (AD), epilepsy, brain tumor and the like.
Further, in other tissues, there is a possibility that BSSP2 of the
present invention and a gene encoding it can be used for treatment
and diagnosis of various diseases such as cancer, inflammation,
infertility, prostatomegaly and the like. Further, it is presumed
they may have a certain influence on blood coagulation,
fibrinolysis and complement systems. Furthermore, there is a
possibility that inhibitors of serine proteases can be used for
treatment and diagnosis of Alzheimer's disease, epilepsy, cancer,
inflammation, infertility, prostatomegaly and the like.
[0049] The novel mouse serine protease can be divided into types 1,
2 and 3. It has been shown that type 1 is composed of 273 amino
acids, type 2 is composed of 311 amino acids, and type 3 is
composed of 445 amino acids. These amino acid sequences contain a
common amino acid sequence of 238 amino acids whose N-terminus side
starts with Ile-Val-Gly-Gly-Gln-Ala- -Val (amino acid 1-7 of SEQ ID
NO:2) as the mature serine protease. Further, the amino acid
sequence of the mature serine protease contains a consensus
sequence having serine protease activity. Since there are two or
more amino acid sequences which are characteristic of sugar chain
binding sites, the amino acid sequence is presumed to have at least
two sugar chains.
[0050] Furthermore, in the novel human serine protease (hBSSP2),
there are a transmembrane region and a scavenger receptor cysteine
rich-like domain in the N-terminus side of hBSSP2 mature protein as
seen from SEQ ID NO: 10.
[0051] The term "pro part" used herein means a part of a pro-form,
i.e., the pro-form from which the corresponding active type protein
part is removed. The term "pre part" used herein means a part of a
prepro-form, i.e., the prepro-form from which the corresponding
pro-form is removed. The term "prepro part" used herein means a
part of a prepro-form, i.e., the prepro-form from which the
corresponding active type protein part is removed.
[0052] The amino acid sequence represented by SEQ ID NO: 2 is the
BSSP2 mature or active type protein composed of 238 amino acids,
and the nucleotide sequence encoding the amino acid sequence
represented by SEQ ID NO: 1 is composed of 714 bases. The present
inventors have shown that the serine protease activity is
maintained even when one to several amino acids of the N-terminus
of the mature type protein of the present invention is deleted or
added, while the sequence represented by SEQ ID NO: 2 is
preferred.
[0053] The amino acid sequence represented by SEQ ID NO: 4 is type
1 BSSP2 protein composed of 273 amino acids, and the nucleotide
sequence encoding the amino acid sequence represented SEQ ID NO: 3
is composed of 1685 bases. The sequence of the -35th to -1st amino
acids is the prepro or pro part and the amino acid sequence
represented by SEQ ID NO: 4 is considered to be a precursor type of
the BSSP2 protein.
[0054] The amino acid sequence represented by SEQ ID NO: 6 is type
2 BSSP 2 protein composed of 311 amino acids and the nucleotide
sequence encoding the amino acid sequence represented by SEQ ID NO:
5 is composed of 2068 bases. The sequence of the -73rd to -1st
amino acids is the prepro or pro part and the amino acid sequence
represented by SEQ ID NO: 6 is considered to be a precursor type of
BSSP2 protein.
[0055] The amino acid sequence represented by SEQ ID NO: 8 is type
3 BSSP2 protein composed of 445 amino acids and the nucleotide
sequence encoding the amino acid sequence represented by SEQ ID NO:
7 is composed of 2070 bases. The amino acid sequence of the -207th
to -1st amino acids is the prepro or pro part and the amino acid
sequence represented by SEQ ID NO: 8 is considered to be a
precursor type of BSSP2 protein.
[0056] SEQ ID NOS: 4, 6 and 8 contain the common amino acid
sequence represented by SEQ ID NO: 2 as the mature BSSP2 protein.
Further, each of amino acid sequences of -25th to 238th amino acids
in SEQ ID NOS: 4, 6 and 8 is the consensus sequence.
[0057] The amino acid sequence represented by SEQ ID NO: 10 is
hBSSP2 protein composed of 457 amino acids and the nucleotide
sequence encoding the amino acid sequence represented by SEQ ID NO:
9 is composed of 1371 bases. Since a transmembrane region and a
scavenger receptor cysteine rich-like domain are present in the
amino acid sequence of the -217th to -1st amino acids of SEQ ID NO:
10, it is considered that hBSSP2 exhibits its activity not only in
the form of the mature protein but also in the form of an adduct of
the -217th to -1st amino acids.
[0058] In general, many genes of eucaryotes exhibit polymorphism
and, sometimes, one or more amino acids are substituted by this
phenomenon. Further, even in such a case, sometimes, a protein
maintains its activity. Then, the present invention includes a gene
encoding a protein obtained by modifying a gene encoding any one of
the amino acid sequences represented by SEQ ID NOS: 2, 4, 6, 8 and
10, artificially, in so far as the protein has the characteristic
function of the gene of the present invention. Further, the present
invention includes a protein which is a modification of any one of
amino acid sequences represented by SEQ ID NOS: 2, 4, 6, 8 and 10
in so far as the protein has the characteristics of the present
invention. Modification is understood to include substitution,
deletion, addition and/or insertion. In particular, the present
inventors have shown that, even when several amino acids are added
to or deleted from the N-terminus amino acid of the BSSP2 mature
protein represented by SEQ ID NO: 2, the resultant sequence
maintains its activity.
[0059] That is, the present invention includes a protein comprising
any one of the amino acid sequences described in SEQ ID NOS: 2, 4,
6, 8 and 10; an amino acid sequence encoded 5 by any one of the
nucleotide sequences represented by SEQ ID NOS: 1, 3, 5, 7 and 9;
or one of these amino acid sequences wherein one to several amino
acids have been substituted, deleted, added and/or inserted, and
belonging to serine protease family.
[0060] Each codon for the desired amino acid itself has been known
and can be selected freely. For example, codons can be determined
according to a conventional manner by taking into consideration the
frequency of use of codons in a host to be utilized (Grantham, R.
et al., Nucleic Acids Res., 9, r43, 1989). Therefore, the present
invention also includes a nucleotide sequence appropriately
modified by taking into consideration the degeneracy of a codon.
Further, these nucleotide sequences can be modified by a site
directed mutagenesis using a primer composed of a synthetic
oligonucleotide encodinq the desired modification (Mark, D. F. et
al., Proc. Natl. Acad. Sci. USA., 81, 5662, 1984), or the like.
[0061] Furthermore, the DNA of the present invention includes DNA
which is hybridizable to any one of the nucleotide sequences
described in SEQ ID NOS: 1, 3, 5, 7 and 9 or nucleotide sequences
complementary to these nucleotide sequences in so far as the
protein encoded by the nucleotide sequence has the same properties
as those of the BSSP2 of the present invention. It is considered
that many of the sequences which are hybridizable to a given
sequence under stringent conditions have a similar activity to that
of a protein encoded by the given sequence. The stringent
conditions according to the present invention includes, for
example, incubation in a solution containing 5.times.SSC, 5%
Denhardt's solution (0.1% BSA, 0.1% Ficol 1400, 0.1% PVP), 0.5% SDS
and 20 .mu.g/ml denatured salmon sperm DNA at 37.degree. C.
overnight, followed by washing with 2.times.SSC containing 0.1% SDS
at room temperature. Instead of SSC, SSPE can be appropriately
used.
[0062] Probes for detecting a BSSP2 gene can be designed based on
any one of nucleotide sequences described in SEQ ID NOS: 1, 3, 5, 7
and 9. Or, primers can be designed for amplifying DNA or RNA
containing the nucleotide sequence. To design probes or primers is
carried out routinely by a person skilled in the art. An
oligonucleotide having a designed nucleotide sequence can be
synthesized chemically. And, when a suitable label is added to the
oligonucleotide, the resultant oligonucleotide can be utilized in
various hybridization assay. Or, it can be utilized in nucleic acid
synthesis reactions such as PCR. An oligonucleotide to be utilized
as a primer has, preferably, at least 10 bases, more preferably 15
to 50 bases in length. An oligonucleotide to be utilized as a probe
has, preferably, 100 bases to full length.
[0063] Moreover, it is possible to obtain a promoter region and an
enhancer region of a BSSP2 gene present in the genome based on the
cDNA nucleotide sequence of BSSP2 provided by the present
invention. Specifically, these control regions can be obtained
according to the same manner as described in JP 6-181767 A; J.
Immunol., 155, 2477, 1995; Proc. Natl. Acad. Sci., USA, 92, 3561,
1995 and the like. The promoter region used herein means a DNA
region which is present upstream from a transcription initiation
site and controls expression of a gene. The enhancer region used
herein means a DNA region which is present in an intron, a
5'-non-translated region or a 3'-non-translated region and enhances
expression of a gene.
[0064] The present invention also relates to a vector comprising
the nucleotide sequence represented by SEQ ID NO: 1 or a nucleotide
sequence encoding the amino acid sequence represented by SEQ ID NO:
2; the nucleotide sequence represented by SEQ ID NO: 3 or a
nucleotide sequence encoding the amino acid sequence represented by
SEQ ID NO: 4; the nucleotide sequence represented by SEQ ID NO: 5
or a nucleotide sequence encoding the amino acid sequence
represented by SEQ ID NO: 6; the nucleotide sequence represented by
SEQ ID NO: 7 or a nucleotide sequence encoding the amino acid
sequence represented by SEQ ID NO: 8; or the nucleotide sequence
represented by SEQ ID NO: 9 or a nucleotide sequence encoding the
amino acid sequence represented by SEQ ID NO: 10; or a nucleotide
sequence similar to them. A nucleotide sequence similar to a given
nucleotide sequence used herein means a nucleotide sequence which
is hybridizable to the given nucleotide sequence or its
complementary nucleotide sequence under the above-described
stringent conditions and which encodes a protein having the same
properties as those of the protein encoded by the nucleotide
sequence.
[0065] The vector is not specifically limited in so far as it can
express the protein of the present invention. Examples thereof
include pBAD/His, pRSETA, pcDNA2.1, pTrcHis2A, pYES2, pBlueBac4.5,
pcDNA3.1 and pSecTag2 manufacture by Invitrogen, pET and PBAC
manufactured by Novagen, pGEM manufactured by Promega,
pBluescriptII manufactured by Stratagene, PGEX and pUC18/19
manufactured by Pharmacia, PfastBAC1 manufactured by GIBCO and the
like. Preferably, a protein expression vector (described in the
specification of a patent application entitled "Protein expression
vector and its use" and filed by the same applicant on the same
day) is used. This expression vector is constructed by using
pCRII-TOPO vector described in the Examples hereinafter, or a
commercially available expression vector, for example pSecTag2A
vector or pSecTag2B vector (Invitrogen) and integrating a secretory
signal nucleotide sequence suitable for expression of the protein
of the present invention, in the 3' downstream side thereof, a Tag
nucleotide sequence, a cleavable nucleotide sequence and a cloning
site, into which a nucleotide sequence encoding a target protein
can be inserted, in this order. More specifically, it is preferred
to use trypsin signal as the secretory signal, a nucleotide
sequence encoding polyhistidine as the Tag nucleotide sequence, and
a nucleotide sequence encoding an amino acid sequence which is
susceptible to enzyme-specific cleavage, i.e., a nucleotide
sequence encoding the amino acid sequence of Asp-Asp-Asp-Asp-Lys
SEQ ID NO:42 (said amino acid sequence is recognized by
enterokinase, and the recombinant fusion protein is cleaved at the
C-terminus part thereof) as the cleavable nucleotide sequence.
[0066] Furthermore, the present invention provides transformed
cells having the nucleotide sequence of the present invention in an
expressible state by means of the above vector. Preferably, host
cells to be used for the transformed cells of the present invention
are animal cells and insect cells. However, host cells include any
cells (including those of microorganisms) which can express a
nucleotide sequence encoding the desired protein in the expression
vector of the present invention and can secrete
extracellularly.
[0067] The animal cells and insect cells used herein include cells
derived from human beings and cells derived from flies or silk
worms. For example there are CHO cells, COS cells, BHK cells, Vero
cells, myeloma cells, HEK293 cells, HeLa cells, Jurkat cells, mouse
L cells, mouse C127 cells, 10 mouse FM3A cells, mouse fibroblast,
osteablast, cartilage cells, S2, Sf9, Sf21, High Five.TM.
(registered trade mark) cells and the like.
[0068] The protein of the present invention as such can be
expressed as a recombinant fused protein so as to facilitate
isolation, purification and recognition. The recombinant fused
protein used herein means a protein expressed as an adduct wherein
a suitable peptide chain is added to the N-terminus and/or
C-terminus of the desired protein expressed by a nucleotide
sequence encoding the desired protein. The recombinant protein used
herein means that obtained by integrating a nucleotide sequence
encoding the desired protein in the expression vector of the
present invention and cut off an amino acid sequence which derived
from nucleic acids other than those encoding the desired protein
from the expressed recombinant fused protein, and is substantially
the same as the protein of the present invention.
[0069] Introduction of the above vector into host cells can be
carried out by, for example, transfection according to the
lipopolyamine method, DEAE-dextran method, Hanahan method,
lipofectin method or calcium phosphate method, microinjection,
eletroporation and the like.
[0070] As described above, the present invention also relates to a
process for producing hBSSP2 of mBSSP2 comprising culturing cells
transformed with the above nucleotide sequence of the present
invention and collecting the produced hBSSP2 of mBSSP2. The culture
of cells and separation and purification of the protein can be
carried out by a per se known method.
[0071] The present invention also relates to an inhibitor of the
novel serine protease of the present invention. Screening of the
inhibitor can be carried out according to a per se known method
such as comparing the enzyme activity upon bringing into contact
with a candidate compound with that without contact with the
candidate compound, or the like
[0072] The present invention relates to a non-human transgenic
animal whose expression level of hBSSP2 or mBSSP2 gene has been
altered. The hBSSP2 or mBSSP2 gene used herein includes cDNA,
genomic DNA or synthetic DNA encoding hBSSP2 or mBSSP2. In
addition, expression of a gene includes any steps of transcription
and translation. The non-human transgenic animal of the present
invention is useful for studies of functions or expression control
of hBSSP2 or mBSSP2, elucidation of mechanisms of diseases in which
hBSSP2 or mBSSP2 is presumed to be involved, and development of
disease model animals for screening and safety test of
medicine.
[0073] In the present invention, expression of a gene can be
modified artificially by mutagenizing at a part of several
important sites which control normal gene expression (enhancer,
promoter, intron, etc.) such as deletion, substitution, addition
and/or insertion to increase or decrease an expression level of the
gene in comparison with its inherent expression level. This
mutagenesis can be carried out according to a known method to
obtain the transgenic animal.
[0074] In a narrow sense, the transgenic animal means an animal
wherein a foreign gene is artificially introduced into reproductive
cells by gene recombinant techniques. In a broad sense, the
transgenic animal includes an antisense transgenic animal the
function of whose specific gene is inhibited by using antisense
RNA, an animal whose specific gene is knocked out by using
embryonic stem cells (ES cells), and an animal into which point
mutation DNA is introduced, and the transgenic animal means an
animal into which a foreign gene is stably introduced into a
chromosome at an initial stage of ontogeny and the genetic
character can be transmitted to the progeny.
[0075] The transgenic animal used herein should be understood in a
broad sense and includes any vertebrates other than a human being.
The transgenic animal of the present invention is useful for
studies of functions or expression control of BSSP2, elucidation of
mechanisms of diseases associated with cells expressing in a human
being, and development of disease model animals for screening and
safety test of medicine.
[0076] As a technique for creating the transgenic animal, a gene is
introduced into a nucleus in a pronucleus stage of egg cells with a
micropipette directly under a phase-contrast microscope
(microinjection, U.S. Pat. No. 4,873,191). Further, there are a
method using embryonic stem cell (ES cell), and the like. In
addition, there are newly developed methods such as a method
wherein a gene is introduced into a retroviral vector or adenoviral
vector to infect egg cells, a sperm vector method wherein a gene is
introduced into egg cells through sperms, and the like.
[0077] A sperm vector method is a gene recombinant technique
wherein a foreign gene is incorporated into sperm cells by
adhesion, electroporation, etc., followed by fertilization of egg
cells to introduce the foreign gene into the egg cells (M.
Lavitranoet et al., Cell, 57, 717, 1989). Alternatively, an in vivo
site specific gene recombinant technique such as that using
cre/loxP recombinase system of bacteriophage P1, FLP recombinase
system of Saccharomyces cerevisiae, etc. can be used. Furthermore,
introduction of a transgene of the desired protein into a non-human
animal using a retroviral vector has been reported.
[0078] For example, a method for creating a transgenic animal by
microinjection can be carried out as follows.
[0079] First, a transgene primarily composed of a promoter
responsible for expression control, a gene encoding a specific
protein and a poly A signal is required. It is necessary to confirm
expression modes and amounts between respective systems because an
expression mode and amount of a specific molecule is influenced by
a promoter activity, and transgenic animals differ from each other
according to a particular system due to the difference in a copy
number of an introduced transgene and a introduction site on a
chromosome. An intron sequence which is spliced may be previously
introduced before the poly A signal because it has been found that
an expression amount varies due to a non-translation region and
splicing. Purity of a gene to be used for introduction into
fertilized egg cells should be as high as possible. This is of
importance. Animals to be used include a mouse for collecting
fertilized eggs (5 to 6 week old), a male mouse for mating, a false
pregnancy female mouse, a seminiferous tubule-ligated mouse, and
the like.
[0080] For obtaining fertilized egg cells efficiently, ovulation
may be induced with gonadotropin or the like. Fertilized egg cells
are recovered and a gene in an injection pipette is injected into
male pronucleus of the egg cells by microinjection. For returning
the injected egg cells to a fallopian tube, an animal (false
pregnancy female mouse, etc.) is provided and about 10 to 15
eggs/mice are transplanted. Then, genomic DNA is extracted from the
end part of the tail to confirm whether the transgene is introduced
into newborn mouse or not. This confirmation can be carried out by
detection of the transgene with southern blot technique or PCR
technique, or by positive cloning wherein a marker gene, which is
activated only when homologous recombination is caused, has been
introduced. Further, transcribed products derived from the
transgene are detected by northern blot technique or RT-PCR
technique to confirm expression of the transgene. Or, western
blotting can be carried out with a specific antibody to a
protein.
[0081] The knockout mouse of the present invention is treated so
that the function of mBSSP2 gene is lost. A knockout mouse means a
transgenic mouse in which any one of its genes is destroyed by
homologous recombination technique so that its function is
deficient. A knockout mouse can be created by carrying out
homologous recombination with ES cells and selecting embryonic stem
cells wherein either of allele genes are modified or destroyed. For
example, embryonic stem cells whose genes are manipulated at the
blastocyte or morula stage of fertilized eggs are injected to
obtain a chimeric mouse wherein cells derived from the embryonic
stem cells are mixed with those derived from the embryo. The
chimeric mouse (chimeric means a single individual formed by
somatic cells based on two or more fertilized eggs) can be mated
with a normal mouse to create a heterozygote mouse wherein all of
the allele genes have been modified or destroyed. Further, a
homozygote mouse can be created by mating heterozygote mice.
[0082] Homologous recombination means recombination between two
genes whose nucleotide sequences are the same or very similar to
each other in terms of gene recombination mechanism. PCR can be
employed to select homologous recombinant cells. A PCR reaction can
be carried out by using a part of a gene to be inserted and a part
of a region where the insertion is expected as primers to find out
occurrence of homologous recombination in cells which give an
amplification product. Further, for causing homologous
recombination in a gene expressed in embryonic stem cells,
homologous recombinant cells can readily be selected by using a
known method or its modification. For example, cells can be
selected by joining a neomycin resistant gene to a gene to be
introduced to impart neomycin resistance to cells after
introduction.
[0083] The present invention also provide an antibody recognizing
hBSSP2 or mBSSP2 or a fragment thereof. The antibody of the present
invention includes an antibody against a protein having the amino
acid sequence described in any of SEQ ID NOS: 2, 4, 6, 8 and 10 or
its fragment. An antibody against hBSSP2 or mBSSP2 or a fragment
thereof (e.g., polyclonal antibody, monoclonal antibody, peptide
antibody) or an antiserum can be produced by using hBSSP2 or mBSSP2
or a fragment thereof, etc. as an antigen according to a per se
known process for producing an antibody or an antiserum.
[0084] The hBSSP2 or mBSSP2 of a fragment thereof is administered
to a site of a warm-blooded animal where an antibody can be
produced by administration thereof as such or together with a
diluent or carrier. For enhancing the antibody production, upon
administration, Freund's complete adjuvant or Freund's incomplete
adjuvant may be administrated. Normally, the administration is
carried out once every 1 to 6 weeks, 2 to 10 times in all. Examples
of the warm-blooded to be used include monkey, rabbit, dog, guinea
pig, mouse, rat, sheep, goat, chicken and the like with mouse and
rat being preferred. As rats, for example, Wistar and SD rats are
preferred. As mice, for example, BALB/c, C57BL/6 and ICR mice are
preferred.
[0085] For producing monoclonal antibody producer cells,
individuals whose antibody titer have been recognized are selected
from warm-blooded animals, e.g., a mouse immunized with an antigen.
Two to 5 days after the last immunization, the spleen or lymph node
of the immunized animal is collected and antibody producer cells
contained therein are subjected to cell fusion with myeloma cells
to prepare a monoclonal antibody producer hybridoma. The antibody
titer in an antiserum can be determined by, for example, reacting
the antiserum with a labeled hBSSP2 or mBSSP2 as described
hereinafter, followed by measurement of the activity bound to the
antibody. The cell fusion can be carried out according to a known
method, for example, that described by Koehler and Milstein
(Nature, 256, 495, 1975) or its modifications (J. Immunol. Method,
39, 285, 1980; Eur. J. biochem, 118, 437, 1981; Nature, 285, 446,
1980). As a fusion promoting agent, there are polyethylene glycol
(PEG), Sendai virus and the like. Preferably, PEG is used. Further,
for improving fusion efficiency, lectin, poly-L-lysine or DMSO can
be appropriately added.
[0086] Examples of myeloma cells include X-63Ag8, NS-1, P3U1,
SP2/0, AP-1 and the like with SP2/0 being preferred. The preferred
ratio of the number of the antibody producer cells (spleen
cells):the number of spleen cells are 1:20 to 20:1. PEG (preferably
PEG 1000 to PEG 6000) is added at a concentration of about 10 to
80% and the mixture is incubated at 20 to 40.degree. C., preferably
30 to 37.degree. C. for 1 to 10 minutes to carry out the cell
fusion efficiently. Screening of anti-hBSSP2 or mBSSP2 antibody
producer hybridomas can be carried out by various methods. For
example, a supernatant of a hybridoma culture is added to a solid
phase to which hBSSP2 or mBSSP2 antigen is adsorbed directly or
together with a carrier (e.g., microplate), followed by addition of
an anti-immunoglobulin antibody (in case that the cells used in
cell fusion are those of a mouse, anti-mouse immunoglobulin
antibody is used) or protein A to detect the anti-hBSSP2 or mBSSP2
monoclonal antibody attached to the solid phase. Or, a supernatant
of a hybridoma culture is added to a solid phase to which an
anti-immunoglobulin antibody or protein A is adsorbed, followed by
addition of hBSSP2 or mBSSP2 labeled with a radioactive substance,
an enzyme, etc., to detect the anti-hBSSP2 or mBSSP2 monoclonal
antibody attached to the solid phase.
[0087] Selection and cloning of the anti-hBSSP or mBSSP monoclonal
antibody can be carried out according to a per se known method or
its modification. Normally, a HAT (hypoxanthine, aminopterin,
thymidine)-added medium for culturing animal cells is used. Any
culture medium can be used for selection, cloning and growing up in
so far as the hybridoma can grow. For example, there can be used
RPMI culture medium containing 1 to 20%, preferably 10 to 20% fetal
bovine serum, a serum-free medium for culturing hybridomas.
Preferably, the culture is carried out at a temperature of about
37.degree. C. Normally, the culture time is 5 days to 3 weeks,
preferably 1 week to 2 weeks. Normally, the culture is carried out
under 5% CO.sub.2. The antibody titer of a supernatant of a
hybridoma culture can be measured according to the same manner as
that of the above-described measurement of anti-BSSP2 antibody
titer in an antiserum. That is, examples of the measurement to be
used include radioimmunoassay (RIA), enzyme-linked immunosorbent
assay (ELISA), FIA (fluorescence immunoassay), plaque assay,
agglutination reaction method, and the like. Among them, ELISA as
shown below is preferred.
[0088] Screening by ELISA
[0089] A protein prepared according to the same operation as that
for an immunogen is immobilized on the surface of each well of an
ELISA plate. Next, BSA, MSA, OVA, KLH, gelatin, skimmed milk, or
the like is immobilized on each well to prevent non-specific
adsorption. A supernatant of a hybridoma culture is added to each
well and is allowed to stand for a given time so that an
immunological reaction proceeds. Each well is washed with a washing
solution such as PBS or the like. Preferably, a surfactant is added
to this washing solution. An enzyme labeled secondary antibody is
added and allowed to stand for a given time. As the enzyme to be
used for the label, there can be used .beta.-galactosidase,
alkaline phosphatase, peroxidase and the like. After washing each
well with the same washing solution, a substrate solution of the
labeled enzyme used is added so that an enzymatic reaction
proceeds. When the desired antibody is present in the supernatant
of a hybridoma culture, the enzymatic reaction proceeds and the
color of the substrate solution is changed.
[0090] Normally, cloning is carried out by a per se known method
such as semi-solid agar method, limiting dilution method and the
like. Specifically, after confirming a well in which the desired
antibody is produced by the above-described method, cloning is
carried out to obtain a single clone. For cloning, it is preferred
to employ limiting dilution method wherein hybridoma cells are
diluted so that one colony is formed per one well of a culture
plate. For cloning by limiting dilution method, feeder cells can be
used, or a cell growth factor such as interleukin 6, etc. can be
added to improve colony forming capability. In addition, cloning
can be carried out by using FACS and single cell manipulation
method. The cloned hybridoma is preferably cultured in a serum-free
culture medium and an optimal amount of an antibody is added to its
supernatant. The single hybridoma thus obtained can be cultured in
a large amount by using a flask or a cell culture device, or
cultured in the abdominal cavity of an animal (J. Immunol. Meth.,
53, 313, 1982) to obtain a monoclonal antibody. When culturing in a
flask, there can be used a cell culture medium (e.g., IMDM, DMEM,
RPMI1640, etc.) containing 0 to 20% of FCS. When culturing in the
abdominal cavity of an animal, the animal to be used is preferably
the same species or the same line as that from which the myeloma
cells used in the cell fusion are derived, a thymus deficient nude
mouse or the like, and the hybridoma is transplanted after
administration of a mineral oil such as pristane, etc. After 1 to 2
weeks, myeloma cells are proliferated in the abdominal cavity to
obtain ascites containing a monoclonal antibody.
[0091] The monoclonal antibody of the present invention which does
not cross-react with other proteins can be obtained by selecting a
monoclonal antibody which recognizes an epitope specific to hBSSP2
or mBSSP2. In general, an epitope presented by an amino acid
sequence composed of at least 3, preferably 7 to 20 successive
amino acid residues in an amino acid sequence which constitutes a
particular protein is said to be an inherent epitope of the
protein. Then, a monoclonal antibody recognizing an epitope
constituted by a peptide having an amino acid sequence composed of
at least 3 successive amino acid residue selected from the amino
acid residues disclosed in any of SEQ ID NOS: 2, 4, 6 and 8 can be
said to be the monoclonal antibody specific for BSSP2 of the
present invention. An epitope common to BSSP2 family can be
selected by selecting an amino acid sequence conservative among the
amino acid sequences described in SEQ ID NOS: 2, 4, 6, 8 and 10.
Or, in case of a region containing an amino acid sequence specific
for each sequence, a monoclonal antibody which can differentiate
respective proteins can be selected.
[0092] Separation and purification of the anti-hBSSP2 or mBSSP2
monoclonal antibody, like a conventional polyclonal antibody, can
be carried out according to the same manner as those of
immunoglobulins. As a known purification method, there can be used
a technique, for example, salting out, alcohol precipitation,
isoelectric precipitation, electrophoresis, ammonium sulfate
precipitation, absorption and desorption with an ion exchange
material (e.g., DEAE), ultrafiltration, gel filtration, or specific
purification by collecting only an antibody with an
antibody-binding solid phase or an active adsorber such as protein
A or protein G, etc., and dissociating the binding to obtain the
antibody. For preventing formation of aggregates during
purification or decrease in the antibody titer, for example, human
serum albumin is added at a concentration of 0.05 to 2%.
Alternatively, amino acids such as glycine, .alpha.-alanine, etc.,
in particular, basic amino acids such as lysine, arginine,
histidine, etc., saccharides such as glucose, mannitol, etc., or
salts such as sodium chloride, etc. can be added. In case of IgM
antibody, since it is very liable to be aggregated, it may be
treated with .beta.-propionilactone and acetic anhydride.
[0093] The polyclonal antibody of the present invention can be
produced according to a per se known method or its modification.
For example, an immunogen (protein antigen) per se or a complex
thereof with a carrier protein is prepared and, according to the
same manner as that in the above monoclonal antibody production, a
warm-blooded animal is immunized. A material containing an antibody
against the protein of the present invention or its fragment is
collected from the immunized animal and the antibody is separated
and purified to obtain the desired antibody. As for a complex of an
immunogen and a carrier protein for immunizing a warm-blooded
animal, the kind of a carrier protein and the mixing ratio of a
carrier and a hapten are not specifically limited in so far as an
antibody against hapten immunized by cross-linking with the carrier
is efficiently produced. For example, there can be used about 0.1
to 20, preferably about 1 to 5 parts by weight of bovine serum
albumin, bovine cycloglobulin, hemocyanin, etc. coupled with one
part by weight of a hapten. For coupling a carrier and a hapten,
various condensing agents can be used. Examples thereof include
glutaraldehyde, carbodiimide or maleimide active ester, active
ester agents having thiol group or dithiopyridyl group, and the
like. The condensed product is administered as such or together
with a carrier or diluent to a site of a warm-blooded animal where
an antibody can be produced. For enhancing the antibody production,
upon administration, Freund's complete adjuvant or Freund's
incomplete adjuvant may be administered. Normally, the
administration is carried out once every 2 to 6 weeks, 3 to 10
times in all. The polyclonal antibody can be collected from blood,
ascites, or the like, preferably blood of the immunized animal. The
polyclonal antibody titer in an antiserum can be measured according
to the same manner as measurement of the above monoclonal antibody
titer in the antiserum. Separation and purification of the
polyclonal antibody, like the above monoclonal antibody, can be
carried out according to the same manner as those of
immunoglobulins.
[0094] The monoclonal antibody and polyclonal antibody against
hBSSP2 or mBSSP2 or a fragment thereof can be utilized for
diagnosis and treatment of diseases associated with cells
expressing hBSSP2 or mBSSP2. By using these antibodies, hBSSP2 or
mBSSP2 or a fragment thereof can be determined based on their
immunological binding to hBSSP2 or mBSSP2 or a fragment thereof of
the present invention. Specifically, examples of a method for
determining hBSSP2 or mBSSP2 or a fragment thereof in a specimen by
using these antibodies include a sandwich method wherein the
antibody attached to an insoluble carrier and the labeled antibody
are reacted with hBSSP2 or mBSSP2 or a fragment thereof to form a
sandwich complex and the sandwich complex is detected, as well as a
competitive method wherein labeled hBSSP2 or mBSSP2, and hBSSP2 or
mBSSP2 or a fragment thereof in the specimen are competitively
reacted with the antibody and hBSSP2 or mBSSP2 or a fragment
thereof in the specimen is determined based on the amount of the
labeled antigen reacted with the antibody.
[0095] As a sandwich method for determining hBSSP2 or mBSSP2 or a
fragment thereof, there can be used a two step method, a one step
method and the like. In the two step method, first, the immobilized
antibody is reacted with hBSSP2 or mBSSP2 or a fragment thereof and
then unreacted materials are completely removed by washing,
followed by addition of the labeled antibody to form immobilized
antibody-hBSSP2 or mBSSP2-labeled antibody. In the one step method,
the immobilized antibody, labeled antibody and hBSSP2 or mBSSP2 or
a fragment thereof are added at the same time.
[0096] Examples of an insoluble carrier used for the determination
include synthetic resins such as polystyrene, polyethylene,
polypropylene, polyvinyl chloride, polyester, polyacrylate, nylon,
polyacetal, fluorine plastic, etc.; polysaccharides such as
cellulose, agarose, etc.; glass; metal; and the like. An insoluble
carrier may be shaped in various forms, for example, tray, sphere,
fiber, rod plate, container, cell, test tube, and the like. The
antibody adsorbed by a carrier is stored at a cold place in the
presence of an appropriate preservative such as sodium azide or the
like.
[0097] For immobilization of the antibody, a known chemical bonding
method or a physical adsorption can be used. Examples of a chemical
bonding method include a method using glutaraldehyde; maleimide
method using N-succinimidyl-4-(N-maleimidomethyl)
cyclohexane-1-carboxylate, N-succinimdyl-2-maleimide acetate or the
like; carbodiimide method using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; or the
like. In addition, there are maleimidobenzoyl-N-hydroxysuccinimide
ester method, N-succinimidyl-3-(2-pyridylthio)propionic acid
method, bisdiazobenzidine method, and dipalmityllysine method. Or,
it is possible to capture a complex formed beforehand by reacting a
material to be tested with two antibodies, whose epitopes are
different, with an immobilized a 3rd antibody against the
antibody.
[0098] For labeling, it is preferred to use an enzyme, fluorescent
substance, luminous substance, radioactive substance, metal
chelate, or the like. Examples of the enzyme include peroxidase,
alkaline phosphatase, .beta.-D-galactosidase, malate dehydrogenase,
Staphylococcus nuclease, .delta.-5-steroidisomerase,
.alpha.-glycerol phosphate dehydrogenase, triose phosphate
isomerase, horseradish peroxidase, asparaginase, glucose oxidase,
ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,
glucoamylase, acetylcholinesterase and the like. Examples of the
fluorescent substance include fluorescein isothiocyanate,
phycobiliprotein, rhodamine, phycoerythrin, phycocyanin,
allophycocyanin, o-phthalaldehyde, and the like. Examples of the
luminous substance include isoluminol, lucigenin, luminol, aromatic
acridinium ester, imidazole, acrdinium salt and its modified ester,
luciferin, luciferase, aequorin and the like. Examples of the
radioactive substance include .sup.125I, .sup.127I, .sup.131I,
.sup.14C, .sup.3H, .sup.32P, .sup.35S and the like. The labeling
material is not limited to them and any material which can be used
for immunological determination can be used. Further, a low
molecular weight hapten such as biotin, dinitrophenyl, pyridoxal or
fluorescamine may be attached to the antibody. Preferably,
horseradish peroxidase is used as a labeling enzyme. This enzyme
can be reacted with various substrates and can readily be attached
to the antibody by periodate method.
[0099] When an enzyme is used as a labeling material, a substrate
and, if necessary, a coloring enzyme is used for measuring its
activity. In case of using peroxidase as the enzyme, H.sub.2O.sub.2
is used as a substrate and, as a coloring agent, there can be used
2,2'-azino-di-[3-ethylbenzthi- azoline sulfonic acid] ammonium salt
(ABTS), 5'-aminosalicylic acid, o-phenylenediamine,
4-aminoantipyrine, 3,3',5,5'-tetramethylbenzidine and the like. In
case of using alkaline phosphatase as the enzyme,
o-nitrophenylphosphate, p-nitrophenylphosphoric acid, or the like
can be used as a substrate. In case of using .beta.-D-galactosidase
as the enzyme, fluorescein-d-(.beta.-D-galactopyranoside),
4-methylumbelliphenyl-.beta.-D-galactopyranoside, or the like can
be used as a substrate. The present invention also includes a kit
comprising the above monoclonal antibody, polyclonal antibody and
reagents.
[0100] As a cross-linking agent, a known cross-linking agent such
as N,N'-o-phenylenedimaleimide, 4-(N-maleimidomethyl)
cyclohexanoate-N-succinimide ester,
6-maleimidohexanoate-N-succineimide ester, 4,4'-dithiopyridine or
the like can be utilized. The reaction of these cross-linking
agents with enzymes and antibodies can be carried out by a known
method according to properties of a particular cross-linking agent.
Further, as the antibody, a fragment thereof, for example, Fab',
Fab, F(b'2) can be used as the case may be. A labeled enzyme can be
obtained by the same treatment regardless of whether the antibody
is polyclonal or monoclonal. When the above labeled enzyme obtained
by using a cross-linking agent is purified by a known method such
as affinity chromatography or the like, an immunoassay system
having more higher sensitivity can be obtained. The enzyme labeled
and purified antibody is stored in a dark cold place with addition
of a stabilizer such as thimerosal, glycerin or after
lyophilization.
[0101] An objective to be determined is not specifically limited in
so far as it is a sample containing BSSP2 or a fragment thereof, or
a sample containing a precursor of BSSP2 or a fragment thereof and
includes body fluids such as plasma, serum, blood, serum, urine,
tissue fluid, cerebrospinal fluid and the like.
[0102] The following Examples further illustrate the present
invention in detail but are not construed to limit the scope
thereof.
EXAMPLE 1
Cloning of Novel Serine Protease mBSSP2 Gene
[0103] The cloning was carried out by PCR using a mouse brain cDNA
library (Clontech) as a template and nucleotide sequences
corresponding to an amino acid sequence common to serine proteases
represented by
1 Primer 1: GTG CTC ACN GCN GCB CAY TG (SEQ ID NO: 20) Primer 2:
CCV CTR WSD CCN CCN GGC GA (SEQ ID NO: 21)
[0104] as primers. Namely, 5 .mu.l of the template, 5 .mu.l of
10.times.ExTaq buffer, 5 .mu.l of dNTP, 10 pmol of each of the
above primers and 0.5 .mu.l of ExTaq (TAKARA) were added and the
total volume was adjusted to 50 .mu.l with sterilized water. PCR
was carried out by repeating a cycle of heating at 94.degree. C.
for 0.5 minute, at 55.degree. C. for 0.5 minute and then at
72.degree. C. for 1 minute, 30 times. The PCR product was mixed
with pCR II-TOPO vector attached to TOPO TA cloning kit
(Invitrogen) and the mixture was allowed to stand at room
temperature for 5 minutes. Then, according to a conventional
manner, E. coli Top 10 attached to the kit was transformed and
applied to a LB (Amp+) plate (containing 100 .mu.g/ml of
ampicillin). According to a conventional manner, a plasmid was
extracted from each colony obtained and its nucleotide sequence was
determined by cycle sequencing method with a fluorescence sequencer
(ABI). Homology of the sequence of each clone was examined by means
of GenBank. Regarding an unknown sequence, i.e., BSSP2 gene, the
full length cDNA was obtained by 5' RACE and 3' RACE and, according
to the same manner as described above, the nucleotide sequence was
determined. Namely, BSSP2 clone specific primers, GSP1 primers
[mBSSP2.2 (SEQ ID NO: 27) or mBSSP2.0 (SEQ ID NO: 22)] and GSP2
primers [mBSSP2R2 (SEQ ID NO: 28) or mBSSP2.1 (SEQ ID NO: 23)] were
prepared. PCR was carried out by using mouse brain Marathon-Ready
cDNA (Clontech), AP1 primer attached to this reagent and either of
the above GSP1 primers and heating at 94.degree. C. for 2 minutes
once and repeating a cycle of heating at 94.degree. C. for 30
seconds, at 60.degree. C. for 30 seconds and then at 72.degree. C.
for 30 seconds 35 times. Then, 5 .mu.l of the PCR product diluted
to {fraction (1/100)}, 5 .mu.l of 10.times.buffer, 5 .mu.l of dNTP,
10 pmol of either of 10 .mu.M of the above GSP2 primer, 10 pmol of
AP2 primer attached to the above reagent and 0.5 unit of ExTaq were
admixed and adjusted to 50 .mu.l with sterilized water. Then,
according to the same manner as the above, PCR was carried out. The
PCR product was cloned by the above TOPO TA cloning kit and
sequenced to obtain the upstream and downstream regions of the
above clone. At this time, as for a clone which seemed not to cover
the full length of a protein, the specific primers shown
hereinafter were prepared based on the newly found nucleotide
sequence. Further, based on this sequence, the primers capable of
amplifying ORF as shown hereinafter [mBSSPF7 (SEQ ID NO: 26),
mBSSP2R/E (SEQ ID NO: 29)] were prepared and PCR carried out using
mouse brain Marathon-ready cDNA as a template to confirm that these
clones were identical. This was cloned into pCR II-TOPO vector
attached to TOPO TA cloning kit to obtain the plasmid pCR II/mBSSP2
containing the full length cDNA clone. The nucleotide sequence of
DNA contained in this plasmid is shown in SEQ ID NO: 7 and the
amino acid sequence of mSSP2 protein deduced from the nucleotide
sequence is shown in SEQ ID NO: 8. Further, two different types of
clones were obtained. The nucleotide sequences of these DNA are
shown in SEQ ID NOS: 3 and 5, respectively. The amino acid
sequences of mBSSP2 proteins deduced from these nucleotide
sequences are shown in SEQ ID NOS: 4 and 6. These novel proteases
are divided into types 1, 2 and 3. Type 1 is composed of 273 amino
acids, type 2 is composed of 311 amino acids and type 3 is composed
of 445 amino acids. These amino acid sequences contained the common
amino acid sequence composed of 238 amino acids whose N-terminus
side started with Ile-Val-Gly-Gly-Gln-Ala-Val (amino acid 1-7 of
SEQ ID NO:2) as the mature serine protease. Further, in the amino
acid sequence of the mature serine protease, a consensus sequence
having a serine protease activity was contained. Furthermore, since
there were two or more amino acid sequences specific for a sugar
chain bonding site, it was presumed that the amino acid sequence
had at least two sugar chains.
2TABLE 1 SEQ ID Name of NO: primer Direction Sequence Use 22
mBSSP2.0 Forward ATGGTGGAGAAGATCATTCCT RACE 23 mBSSP2.1 Forward
TACAGTGCCCAGAACCATG RACE 24 mBSSPF4 Forward CTCAACTCTCTGCTAGACCG
RACE 25 mBSSP2F5 Forward ATAGTTGGCGGCCAAGCTGT mature 26 mBSSPF7
Forward CCCAGCAGAACTTACTGCCT FL* 27 mBSSP2.2 Reverse
TGTTGCAGAGGTGGGTGCTG RACE 28 mBSSP2R2 Reverse TACCATTGTGTCCTGCAGTGT
RACE 29 mBSSP2R5/E Reverse TGAATTCTGCTGCTTCTTCGGCTAGCG FL* *for
full length
EXAMPLE 2
Expression mBSSP2 Gene in Mice Internal Organs
[0105] According to the protocol of QuickPrep Micro mRNA
purification Kit (Amersham-Pharmacia), mRNAs were isolated from
various internal organs of Balb/c mice or their fetuses. They were
subjected to electrophoresis according to a conventional manner and
transcribed to a nylon membrane. A probe was prepared separately by
isolating a part of a nucleotide sequence encoding the mature
protein of mBSSP2 from pCR II/mBSSP2, purifying it and labeling it
with .alpha.-32P dCTP. The probe was diluted with 5.times.SSC and
reacted with the above membrane filter at 65.degree. C. for a whole
day and night. Then, the filter was washed twice each with
2.times./0.1% SDS at room temperature for 30 minutes, 1.times./0.1%
SDS at room temperature for 30 minutes and 0.1.times./0.1% SDS at
65.degree. C. for 30 minutes. The filter was exposed to an imaging
plate for FLA2000 (Fuji Film) for one day to analyze the
expression. The results shown in the drawings are those obtained by
using mRNAs prepared from head of fetuses of mice and mRNAs
prepared from brain of 5-day-, 10-day-, 14-day-, 18-day-, 30-day-,
3-month-, 7-month and 1-year-old mice (FIG. 1) and mRNAs prepared
from various internal organs of 3-month-old mice (FIG. 2). In
addition, the mRNAs of mice prepared above were subjected to RT-PCR
by using Ready To Go RT-PCR Beads (Amersham-Pharmacia) and mBSSP2
gene specific primers (SEQ ID NOS: 25 and 29) according to the
protocol attached to the kit.
[0106] As seen from FIGS. 1 and 2, in the case of northern blotting
analysis, the expression of mBSSP2 was recognized in the head of
15th to 20th day fetuses of mice and, as to the 3-month-old mice,
the expression was recognized in the prostate and testicle.
Further, according to the results of RT-PCR, the expression was
recognized in the head of 12-day-old mice and the testicle of
3-month-old mice.
EXAMPLE 3
Expression of Novel Serine Protease Mature Protein Encoded by
mBSSP2 Gene
[0107] (1) Construction of Expression Plasmid
[0108] A cDNA region encoding the mature protein of BSSP2 protein
was amplified by PCR using the plasmid pCR II/mBSSP2 as a template
(the sequence of the 1st to 717th bases of SEQ ID NO: 1 was
amplified by using the primers having the sequences represented by
SEQ ID NOS: 25 and 29). The PCR product was ligated to pTrc-HisB
(Invitrogen) which had been digested with BamHI and blunted with
mung bean nuclease. E. coli JM109 was transformed by the resultant
and colonies formed were analyzed by PCR to obtain E. coli
containing the desired serine protease expressing plasmid
pTrcHis/mBBSP2.
[0109] The resultant E. coli was designated E. coli pTrcHis/mBSSP2
and deposited at National Institute of Bioscience and
Human-Technology (NIBH), Agency of Industrial Science &
Technology of 1-1-3 Higashi, Tsukuba-shi, Ibaraki-ken, Japan on
Oct.29, 1998 under the accession numbers of FERM P-17033.
[0110] (2) Expression of Protein by E. coli Containing Expression
Plasmid
[0111] A single colony of E. coli having the expression plasmid was
inoculated in 10 ml of LB (Amp+) culture medium and incubated at
37.degree. C. overnight. This was inoculated in 250 ml of LB (Amp+)
culture medium and incubated at 37.degree. C. When the absorbance
at 600 nm became 0.5, 250 .mu.l of 0.1 M IPTG
(isopropyl-.beta.-D-(-)-thiogalact- opyranoside) was added and the
incubation was continued for additional 5 hours. The E. coli was
centrifuged and suspended in a cell disruption buffer (10 mM
phosphate buffer pH 7.5, 1 mM EDTA) and sonicated on ice to disrupt
E. coli. This was centrifuged at 14,000 r.p.m. for 20 minutes to
obtain a precipitate. The precipitate was washed twice with a cell
disruption buffer containing 0.5% Triton X-100TM and washed with
water to remove Triton X-100TM. Then, the resultant mixture was
dissolved by soaking in a denaturation buffer containing 8 M urea
(8 M urea, 50 mM Tris pH8.5, 20 mM 2 ME) at 37.degree. C. for 1
hour. The solution was passed through TALON metal affinity resin
(Clontech), washed with the denaturation buffer containing 10 mM
imidazole, and then eluted with the denaturation buffer containing
100 mM imidazole to purify the solution. The purified product was
dialyzed against PBS for 3 days with exchanging the buffer every
other night to obtain the protein mBSSP2-His.
EXAMPLE 4
Expression of Novel Serine Protease Mature Protein Encoded by
mBSSP2 Gene by Using pFBTrypSigTag/BSSP2
[0112] (1) Construction of pFBTrypSigTag/BSSP2
[0113] The sequences represented by SEQ ID NOS: 11 and 12 were
subjected to annealing and digested with NheI and BamHI. The
resultant fragment was inserted into pSecTag2A (Invitrogen) to
obtain pSecTrypHis. Twenty units of BAmHI was added to 5 .mu.g of
pSecTrypHis vector and the vector was cleaved at 37.degree. C. over
4 hours. Then, 6 units of mung bean nuclease (TAKARA) was added
thereto and reacted at room temperature (25.degree. C.) for 30
minutes to blunt the terminal ends. Further, the 3'-terminus side
of the cloning site was digested cleaved with 20 units of XhoI, 1
unit of bacterial alkaline phosphatase (TAKARA) was added thereto
and the reaction was carried out at 65.degree. C. for 30
minutes.
[0114] According to the same manner as that described in JP
9-149790 A or Biochim. Biophys. Acta, 1350, 11, 1997, mRNA was
prepared from COLO201 cells and cDNA was synthesized to obtain the
plasmid pSPORT/neurosin. cDNA of an active region of neurosin was
obtained from pSPORT/neurosin by PCR using primers having the
sequences represented by SEQ ID NOS: 13 and 14. Ten units of XhoI
was reacted with the PCR product at 37.degree. C. for 3 hours to
cleave XhoI site at the 3'-side thereof. This was inserted into
pSecTrypHis by TAKARA ligation kit to obtain pSecTrypHis/neursoin
(FIG. 3).
[0115] Amplification was carried out by using the primers having
the sequences represented by SEQ ID NOS: 15 and 16 so that the
peptide of Leu-Val-His-Gly (SEQ ID NO:43) was present at the
C-terminus of the part from trypsin signal to the enterokinase
recognition site of pSecTrypHis/neurosin. This was inserted between
NheI and HindIII sites of pSecTag2A to construct the plasmid
pTrypSig.
[0116] One .mu.g (0.1 .mu.l) of the plasmid pSecTab2A was treated
with the restriction enzymes NheI and BamHI to completely remove a
region encoding the leader sequence of IgGk. One hundred pmol
portions of DANs represented by SEQ ID NOS: 40 and 41 were added to
the resultant solution and the mixture was heated at 70.degree. C.
for 10 minutes and subjected to annealing by allowing to stand at
room temperature for 30 minutes. Two .mu.l of I solution of DNA
ligation kit Ver. 2 (TAKARA) was added to 1 .mu.l portions of His
secretory signal sequence treated by NheI and BamHI and pSecTag2A
and the reaction was carried out at 16.degree. C. for 30
minutes.
[0117] To the reaction mixture was add 0.1 ml of E. coli competent
cell XL1-Blue (STRATAGENE) and reacted on ice for 30 minutes. Then,
the reaction mixture was subjected to heat shock at 42.degree. C.
for 60 seconds. After standing on ice for 2 minutes, 0.9 ml of SOC
culture medium (Toyo Boseki K.K.) was added thereto and the mixture
was shaken with a shaker at 37.degree. C. for 1 hour. The mixture
was centrifuged at 5,000 r.p.m. for 1 minutes and the supernatant
was discarded. The precipitated competent cells were suspended in
the liquid remained in the centrifuge tube and the suspension was
applied to 2 ampicillin LB plates containing 100 .mu.g/ml of
ampicillin in the ratio of 1:10. The plates were incubated at
37.degree. C. for one night. Among the colonies formed, a colony
into which DNA of His secretory signal was inserted was selected by
PCR to obtain pTrypHis.
[0118] A sequence of about 200 bp containing His Tag region of
pTrypHis was amplified by using primers having the sequence
represented by SEQ ID NOS: 16 and 17 and a fragment of about 40 bp
containing His Tag and enterokinase recognizing site formed by
digestion of HindIII and BamHI was inserted into pTrypSig to
construct pTrypSigTag (FIG. 4A).
[0119] cDNA was prepared by PCR of the sequence from trypsin signal
to enterokinase recognizing site of pTrypSigTag using primers
having the sequences represented by SEQ ID NOS 14 and 18 and cut
out by digestion with BglII and BamHI. It was inserted into BamHI
site of pFastBAC1. The insertion direction was confirmed by PCR
using primers having the sequences represented by SEQ ID NOS: 14
and 19. A clone into which the cDNA was inserted in the direction
toward transcription and translation was selected to obtain
pFBTrypSigTag.
[0120] Twenty units of BamHI was added to 5 .mu.g of pFBTrypSigTag
vector and the vector was cleaved at 37.degree. C. over 4 hours,
followed by addition of 6 units of mung bean nuclease (TAKARA) and
reaction at room temperature (25.degree. C.) for 30 minutes to
blunt the terminal ends. Further, the 3'-side of the cloning site
was cleaved by 20 units of EcoRI, followed by addition of 1 unit of
bacterial alkaline phosphatase (TAKARA). The reaction was carried
out at 65.degree. C. for 30 minutes.
[0121] cDNA of the active region of mBSSP2 was obtained by PCR
according to a conventional manner using pTrcHis/mBSSP2 or
pCRII/mBSSP2 prepared from E. coli pTrcHis/mBSSP2 (accession No.
FERM P-17033). The resultant cDNA was inserted into pFBTrypSigTag
to obtain pFBTrypSigTag/mBSSP2 (FIG. 4B). At this time, correct
insertion of mBSSP2 was confirmed by determining the sequence.
[0122] Bacmid DNA was transformed with PFBTrypSigTag/mBSSP2
according to a protocol of Gibco BRL BAC-TO-BAC baculovirus
expression system to prepare a recombinant bacmid having chimera
BSSP2 fused with trypsinogen signal peptide, HisTag and
enterokinase recognizing site. When this was expressed in Sf-9 cell
according to a manual of BAC-TO-BAC baculovirus expression system,
it was secreted in the culture supernatant from 2 days after
infection of the virus.
[0123] (2) Determination of Enzyme Activity
[0124] The recombinant fused protein mSSP2 obtained in the culture
supernatant was passed through a chelate column to purify it and,
after dialysis, its enzyme activity was determined. First, the
culture supernatant was applied to a chelate column
(Ni-NTA-Agarose, Qiagen) with PBS buffer and eluted stepwise with a
solution of imidazole (Wako Pure Chemical Industries, Ltd.)
dissolved in PBS. The resultant imidazole-eluted fraction was
applied to a PD-10 column (Pharmacia) to exchange to PBS buffer.
Fifty .mu.l of this sample was mixed with 10 .mu.l of enterokinase
(1 U/1 .mu.l, Invitrogen) and the reaction was carried out at room
temperature for 60 minutes. Each of various synthetic substrates
(Peptide Laboratory, Boc-Gln-Ala-Arg-MCA, Boc-Phe-Ser-Arg-MCA,
Bz-Arg-MCA, Boc-Val-Leu-Lys-MCA, Pyr-Gly-Arg-MCA, Pro-Phe-Arg-MCA,
Boc-Val-Pro-Arg-MCA, Z-Arg-Arg-MCA, Arg-MCA, Z-Phe-Arg-MCA) was
dissolved in DMSO and diluted with 1 M Tris-HCl (pH 8.0) to obtain
a substrate solution. Fifty .mu.l of 0.2 M substrate solution was
added thereto and further the reaction was carried out at
37.degree. C. After one hour, the fluorescence of AMC
(7-amino-4-methylcoumalin) formed by the enzymatic reaction was
measured at 380 nm of excitation wavelength and 460 nm of
fluorescence wavelength to determine the activity.
[0125] As a result, the recombinant fused protein mBSSP2 has been
shown to have serine protease activity.
EXAMPLE 5
Cloning of hBSSP2 Gene
[0126] Reverse transcription of 1 .mu.g of mRNA of human fetus
brain (Clontech) was carried out by using Superscript II (Gibco
BRL) and oligo dT-Not I primer (5' GGCCACGCGTCGACTAGTA C(T).sub.17
3' SEQ ID NO:44) to obtain cDNA. By using this as a template, PCR
was carried out with primes prepared from mBSSP2 nucleotide
sequence and represented by SEQ ID NOS: 30 and 31 to obtain a cDNA
fragment of hBSSP2. Namely, 5 .mu.l of the template, 5 .mu.l of
10.times.ExTaq buffer (TAKARA), 5 .mu.l of dNTPs, 10 pmol portions
of the above primers and 0.5 .mu.l of ExTaq (TAKARA) were adjusted
to 50 .mu.l with sterilized water and PCR was carried out by
repeating a cycle of heating at 94.degree. C. for 0.5 minute, at
55.degree. C. for 0.5 minute and then at 72.degree. C. for 1
minute, 35 times. The PCR reactions described hereinafter were
carried out according to the same manner as the above composition
and conditions except the template and primers. The PCR product was
mixed with pGEM-T Easy vector (Promega) and Takara Ligation
Solution I (TAKARA) and the reaction was carried out at 16.degree.
C. for 2 hours. Then, according to the same manner, E. coli JM109
was transformed and applied to a LB (Amp.sup.+) plate. A plasmid
was extracted from each colony formed according to a conventional
manner and its nucleotide sequence was determined by dideoxy
method. As for a clone having homology to mBSSP2, full length cDNA
was obtained by 5' RACE and 3' RACE and its sequence was determined
as described above. PCR was carried out by using the above cDNA as
a template and primers having the sequences represented by SEQ ID
NOS: 30 and 37. 3' RACE was carried out by PCR using a {fraction
(1/100)} dilution of the above PCR product as a template and
primers having the sequences represented by SEQ ID NOS: 32 and 37.
As for 5' RACE, cDNA for RACE was prepared from human fetal brain
mRNA (Clontech) by using Superscript II and SMART RACE cDNA
amplification kit (Clontech). PCR of this cDNA was carried out by
using a primer of 10.times.Universal Primer Mix (attached to the
kit) and a primer having the sequence represented by SEQ ID NO: 33.
Further, PCR was carried out by using the {fraction (1/100)}
dilution of the latter PCR product, a template, Nested PCR Primer
(attached to the kit) and a primer having the sequence represented
by SEQ ID NO: 34. The finally obtained PCR product was subjected to
TA cloning as described above and the nucleotide sequence was
determined to obtain the upstream and downstream regions of the
above clone. In addition, primers for amplifying the full length
cDNA as represented by SEQ ID NOS: 35 and 36 were prepared based on
the resultant nucleotide sequence and PCR was carried out by using
the above synthetic cDNA as a template. This PCR product was cloned
into pGEM-T Easy vector to obtain the plasmid pGEM-TE/hBSSP2
containing the full length cDNA clone. The DNA sequence contained
in this plasmid is shown in SEQ ID NO: 9 and hBSSP2 protein deduced
from the nucleotide sequence is shown in SEQ ID NO:10.
[0127] E. coli containing this plasmid was designated E. coli
pGEM-TE/hBSSP2 and deposited at National Institute of Bioscience
and Human-Technology (NIBH), Agency of Industrial Science &
Technology of 1-1-3 Higashi, Tsukuba-shi, Ibaraki-ken, Japan on
Jul. 27, 1999 under the accession numbers of FERM P-17487.
3TABLE 2 SEQ ID Name of NO: primer Direction Sequence Use 30
BSSP2SPF Forward ACTGCTGCCCACTGCATG for part 31 BSSP2SPR Reverse
CAGGGGTCCCCCGCTGTCTCC for part 32 hBSSP2F11 Forward
GCTCTCAACTTCTCAGACAC RACE 33 hBSSP2R12 Reverse ACTCAGCTACCTTGGCGTAG
RACE 34 hBSSP2R11 Reverse CCTGGAGCATATCCGAGCTG RACE 35 hBSSR2F12
Forward GCTTTACAACAGTGCTAC WB* 36 hBSSP2R13/E Reverse
TGGAATTCGAGGAAACAGCAGGACTCAG WB* 37 TACTAGTCGACGCGTGGCC *whole
body
EXAMPLE 6
Detection of hBSSP2 mRNA by Northern Blotting
[0128] Poly A+RNA extracted from respective tissues of human adults
and fetuses were blotted on a membrane (Clontech) and the membrane
was subjected to northern hybridization with a hBSSP2 probe. The
probe was labeled by Takara BcaBEST random labeling kit (TAKARA)
according to random priming method using a cDNA fragment which was
amplified by using the full length of hBSSP2 as a template and the
sequences represented by SEQ ID NOS: 34 and 35 as primers. The
hybridization was carried out at 60.degree. C. overnight and the
filter was finally washed with 0.1.times.SSC and 0.1% SDS. The
radioactivity was detected by FLA-2000 (Fuji Film). The signal
corresponding to the adult brain was recognized at about 2.4 kb,
the signal corresponding to the adult skeletal muscle was
recognized at 7 kb and 1.3 kb and further the signal of the fetus
liver was recognized at 7 kb (FIG. 5). The signal of the adult
brain is considered to correspond to the exact nucleotide sequence
and the others are considered to correspond to polymorphic forms
resulted from the difference in splicing.
EXAMPLE 7
Detection of hBSSP2 mRNA by RT-PCR
[0129] mRNAs of human tissues purchased from Clontech were
subjected to RT-PCR against hBSSP2 by using Ready To Go RT-PCR
Beads (Amersham-Pharmacia) according to the protocol attached to
the kit. Expression of hBSSP2 was recognized in brain and skeletal
muscle (FIG. 6). No clear band was obtained in pancreas due to the
combination of primers. This is considered to be non-specific
amplification by a large amount of a serine protease present in
pancreas.
EXAMPLE 8
Expression of hBSSP2 by Baculovirus System
[0130] The signal sequence of human trypsinogen 2 and (His)6 Tag
and a sequence encoding the cleavage site of enterokinase were
inserted into pFastBac1 (Gibco BRL) to obtain the plasmid
pFBTrypSigTag. The mature form of hBSSP2 was inserted into the
plasmid pFBTrypSigTag so that it was located in the flame (FIG.
4B). The mature form of hBSSP2 amplified by the sequences
represented by SEQ ID NOS: 38 and 36 was cleaved by EcoRI and,
according to the same manner as described with respect to mBSSP2,
it was inserted into pFBTrySigTag to construct
pFastBacTrypSigTag/hBSSP2. At this time, correct insertion of BSSP2
was confirmed by determining the nucleotide sequence by using the
fluorescent labeled sequence represented by SEQ ID NO: 39. Bacmid
DNA was transformed with PFBTrypSigTag/hBSSP2 according to a
protocol of Gibco BRL BAC-TO-BAC baculovirus expression system to
prepare a recombinant bacmid having chimera BSSP2 fused with
trypsinogen signal peptide, HisTag and enterokinase recognizing
site. When this was expressed in Sf-9 cell according to a manual of
BAC-TO-BAC baculovirus expression system and the culture
supernatant from 3 days after infection of the virus subjected to
western blot technique with anti-DDDDK antibody, a specific band
was detected to confirm expression of hBSSP2 (FIG. 7).
4TABLE 3 SEQ ID Name of NO: primer Direction Sequence Use 38
hBSSP2F13 Forward ACTGCTGCCCACTGCATG for part 39 FBTrypSigTagF5
GCGCTAGCAGATCTCCATGAATCTACTCCTGATCC NS* *nucleotide sequence
INDUSTRIAL UTILITY
[0131] According to the present invention, there are provided
isolated human and mouse serine protease (hBSSP2 and mBSSP2)
polynucleotides, their homologous forms, mature forms, precursors
and polymorphic variants. Further, according to the present
invention, there are provided hBSSP2 and mBSSP2 proteins as well as
compositions containing hBSSP2 and mBssP2 polynucleotides and
proteins, their production and use.
[0132] Sequence Listing Free Text
[0133] SEQ ID NO: 11: Designed oligonucleotide to construct plasmid
pSecTrypHis.
[0134] SEQ ID NO: 12: Designed oligonucleotide to construct plasmid
pSecTrypHis.
[0135] SEQ ID NO: 13: Designed oligonucleotide primer to amplify
neurosin-encoding sequence.
[0136] SEQ ID NO: 14: Designed oligonucleotide primer to amplify
neurosin-encoding sequence.
[0137] SEQ ID NO: 15: Designed oligonucleotide primer to amplify a
portion of plasmid pSecTrypHis/Neurosin.
[0138] SEQ ID NO: 16: Designed oligonucleotide primer to amplify a
portion of plasmid pSecTrypHis/Neurosin.
[0139] SEQ ID NO: 17: Designed oligonucleotide primer to amplify a
portion of plasmid pTrypHis.
[0140] SEQ ID NO: 18: Designed oligonucleotide primer to amplify a
portion of plasmid pTrypSigTag.
[0141] SEQ ID NO: 19: Designed oligonucleotide primer to amplify a
portion of plasmid pFBTrypSigTag.
[0142] SEQ ID NO: 20: Designed oligonucleotide primer to amplify
conserved region of serine proteases-encoding sequence; n is a, c,
g or t.
[0143] SEQ ID NO: 21: Designed oligonucleotide primer to amplify
conserved region of serine proteases-encoding sequence; n is a, c,
g or t.
[0144] SEQ ID NO: 22: Designed oligonucleotide primer designated as
mBSSP2.0 for RACE for mBSSP2 (forward).
[0145] SEQ ID NO: 23: Designed oligonucleotide primer designated as
mBSSP2.1 for RACE for mBSSP2 (forward).
[0146] SEQ ID NO: 24: Designed oligonucleotide primer designated as
mBSSPF4 for RACE for mBSSP2 (forward).
[0147] SEQ ID NO: 25: Designed oligonucleotide primer designated as
mBSSP2F5 to amplify mature mBSSP2-encoding region (forward).
[0148] SEQ ID NO: 26: Designed oligonucleotide primer designated as
mBSSPF7 to amplify full-length mBSSP2-encoding mRNA (forward).
[0149] SEQ ID NO: 27: Designed oligonucleotide primer designated as
mBSSP2.2 for RACE for mBSSP2 (reverse).
[0150] SEQ ID NO: 28: Designed oligonucleotide primer designated as
mBSSP2R2 for RACE for mBSSP2 (reverse).
[0151] SEQ ID NO: 29: Designed oligonucleotide primer designated as
mBSSP2R5/E to amplify full-length mBSSP2-encoding mRNA
(reverse).
[0152] SEQ ID NO: 30: Designed oligonucleotide primer designated as
BSSP2SPF to amplify a portion of hBSSP2 (forward).
[0153] SEQ ID NO: 31: Designed oligonucleotide primer designated as
BSSP2SPR to amplify a portion of hBSSP2 (reverse).
[0154] SEQ ID NO: 32: Designed oligonucleotide primer designated as
hBSSP2F11 for RACE for hBSSP2 (forward).
[0155] SEQ ID NO: 33: Designed oligonucleotide primer designated as
hBSSP2R12 for RACE for hBSSP2 (reverse).
[0156] SEQ ID NO: 34: Designed oligonucleotide primer designated as
hBSSP2R11 for RACE for hBSSP2 (reverse).
[0157] SEQ ID NO: 35: Designed oligonucleotide primer designated as
hBSSP2F12 to amplify full length hBSSP2 (forward).
[0158] SEQ ID NO: 36: Designed oligonucleotide primer designated as
hBSSP2R13/E to amplify full length hBSSP2 (reverse).
[0159] SEQ ID NO: 37: Designed oligonucleotide primer for RACE for
hBSSP2.
[0160] SEQ ID NO: 38: Designed oligonucleotide primer designated as
hBSSP2F13 to amplify a portion of hBSSP2 (forward).
[0161] SEQ ID NO: 39: Designed oligonucleotide primer designated as
FBTrpsigtagF5 to detect hBSSP2.
[0162] SEQ ID NO: 40: Designed oligonucleotide to construct plasmid
pTrypHis.
[0163] SEQ ID NO: 41: Designed oligonucleotide to construct plasmid
pTrypHis.
Sequence CWU 1
1
44 1 717 DNA Mus sp. CDS (1)..(717) 1 ata gtt ggc ggc caa gct gtg
gct tct ggg cgc tgg cca tgg caa gct 48 Ile Val Gly Gly Gln Ala Val
Ala Ser Gly Arg Trp Pro Trp Gln Ala 1 5 10 15 agc gtg atg ctt ggc
tcc cgg cac acg tgt ggg gcc tct gtg ttg gca 96 Ser Val Met Leu Gly
Ser Arg His Thr Cys Gly Ala Ser Val Leu Ala 20 25 30 cca cac tgg
gta gtg act gct gcc cac tgc atg tac agt ttc agg ctg 144 Pro His Trp
Val Val Thr Ala Ala His Cys Met Tyr Ser Phe Arg Leu 35 40 45 tcc
cgc cta tcc agc tgg cgg gtt cat gca ggg ctg gtc agc cat ggt 192 Ser
Arg Leu Ser Ser Trp Arg Val His Ala Gly Leu Val Ser His Gly 50 55
60 gct gtc cga caa cac cag gga act atg gtg gag aag atc att cct cat
240 Ala Val Arg Gln His Gln Gly Thr Met Val Glu Lys Ile Ile Pro His
65 70 75 80 cct ttg tac agt gcc cag aac cat gac tat gat gtg gct ctg
ctg cag 288 Pro Leu Tyr Ser Ala Gln Asn His Asp Tyr Asp Val Ala Leu
Leu Gln 85 90 95 ctc cgg aca cca atc aac ttc tca gac acc gtg gac
gct gtg tgc ttg 336 Leu Arg Thr Pro Ile Asn Phe Ser Asp Thr Val Asp
Ala Val Cys Leu 100 105 110 ccg gcc aag gag cag tac ttt cca tgg ggg
tcg cag tgc tgg gtg tct 384 Pro Ala Lys Glu Gln Tyr Phe Pro Trp Gly
Ser Gln Cys Trp Val Ser 115 120 125 ggc tgg ggc cac acc gac ccc agc
cat act cat agc tca gat aca ctg 432 Gly Trp Gly His Thr Asp Pro Ser
His Thr His Ser Ser Asp Thr Leu 130 135 140 cag gac aca atg gta ccc
ctg ctc agc acc cac ctc tgc aac agc tca 480 Gln Asp Thr Met Val Pro
Leu Leu Ser Thr His Leu Cys Asn Ser Ser 145 150 155 160 tgc atg tac
agt ggg gca ctt aca cac cgc atg ttg tgt gct ggc tac 528 Cys Met Tyr
Ser Gly Ala Leu Thr His Arg Met Leu Cys Ala Gly Tyr 165 170 175 ctg
gat gga agg gca gac gca tgc cag gga gac agc ggg gga ccc ctg 576 Leu
Asp Gly Arg Ala Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu 180 185
190 gta tgt ccc agt ggt gac acg tgg cac ctt gta ggg gtg gtc agc tgg
624 Val Cys Pro Ser Gly Asp Thr Trp His Leu Val Gly Val Val Ser Trp
195 200 205 ggt cgt ggc tgt gca gag ccc aat cgc cca ggt gtc tat gcc
aag gta 672 Gly Arg Gly Cys Ala Glu Pro Asn Arg Pro Gly Val Tyr Ala
Lys Val 210 215 220 gca gag ttc ctg gac tgg atc cat gac act gtg cag
gtc cgc tag 717 Ala Glu Phe Leu Asp Trp Ile His Asp Thr Val Gln Val
Arg 225 230 235 2 238 PRT Mus sp. 2 Ile Val Gly Gly Gln Ala Val Ala
Ser Gly Arg Trp Pro Trp Gln Ala 1 5 10 15 Ser Val Met Leu Gly Ser
Arg His Thr Cys Gly Ala Ser Val Leu Ala 20 25 30 Pro His Trp Val
Val Thr Ala Ala His Cys Met Tyr Ser Phe Arg Leu 35 40 45 Ser Arg
Leu Ser Ser Trp Arg Val His Ala Gly Leu Val Ser His Gly 50 55 60
Ala Val Arg Gln His Gln Gly Thr Met Val Glu Lys Ile Ile Pro His 65
70 75 80 Pro Leu Tyr Ser Ala Gln Asn His Asp Tyr Asp Val Ala Leu
Leu Gln 85 90 95 Leu Arg Thr Pro Ile Asn Phe Ser Asp Thr Val Asp
Ala Val Cys Leu 100 105 110 Pro Ala Lys Glu Gln Tyr Phe Pro Trp Gly
Ser Gln Cys Trp Val Ser 115 120 125 Gly Trp Gly His Thr Asp Pro Ser
His Thr His Ser Ser Asp Thr Leu 130 135 140 Gln Asp Thr Met Val Pro
Leu Leu Ser Thr His Leu Cys Asn Ser Ser 145 150 155 160 Cys Met Tyr
Ser Gly Ala Leu Thr His Arg Met Leu Cys Ala Gly Tyr 165 170 175 Leu
Asp Gly Arg Ala Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu 180 185
190 Val Cys Pro Ser Gly Asp Thr Trp His Leu Val Gly Val Val Ser Trp
195 200 205 Gly Arg Gly Cys Ala Glu Pro Asn Arg Pro Gly Val Tyr Ala
Lys Val 210 215 220 Ala Glu Phe Leu Asp Trp Ile His Asp Thr Val Gln
Val Arg 225 230 235 3 1685 DNA Mus sp. CDS (247)..(1065) 3
ctcacatgta tctttcagaa taaatggaga ggatcttctg cttcaagtac aagtaagagc
60 tcggccagac tggctcctgg tatgccatga gggccggagc ccagccctgg
gcatgcacat 120 ctgcaagagt cttgggcata tcaggcttac tcaacacaag
gccgtgaatc tgtctgacat 180 caagctcaac agatcccagg agtttgctca
actctctgct agaccgggag gccttgtaga 240 ggaggc atg gaa gcc cag gta ggg
ctt ctg tgg gtt agc gct aac tgt 288 Met Glu Ala Gln Val Gly Leu Leu
Trp Val Ser Ala Asn Cys -35 -30 -25 cct tct ggc cga att gtt tct ctc
aaa tgt tct gag tgt ggg gca agg 336 Pro Ser Gly Arg Ile Val Ser Leu
Lys Cys Ser Glu Cys Gly Ala Arg -20 -15 -10 cct ctg gct tct cga ata
gtt ggc ggc caa gct gtg gct tct ggg cgc 384 Pro Leu Ala Ser Arg Ile
Val Gly Gly Gln Ala Val Ala Ser Gly Arg -5 -1 1 5 10 tgg cca tgg
caa gct agc gtg atg ctt ggc tcc cgg cac acg tgt ggg 432 Trp Pro Trp
Gln Ala Ser Val Met Leu Gly Ser Arg His Thr Cys Gly 15 20 25 gcc
tct gtg ttg gca cca cac tgg gta gtg act gct gcc cac tgc atg 480 Ala
Ser Val Leu Ala Pro His Trp Val Val Thr Ala Ala His Cys Met 30 35
40 tac agt ttc agg ctg tcc cgc cta tcc agc tgg cgg gtt cat gca ggg
528 Tyr Ser Phe Arg Leu Ser Arg Leu Ser Ser Trp Arg Val His Ala Gly
45 50 55 ctg gtc agc cat ggt gct gtc cga caa cac cag gga act atg
gtg gag 576 Leu Val Ser His Gly Ala Val Arg Gln His Gln Gly Thr Met
Val Glu 60 65 70 75 aag atc att cct cat cct ttg tac agt gcc cag aac
cat gac tat gat 624 Lys Ile Ile Pro His Pro Leu Tyr Ser Ala Gln Asn
His Asp Tyr Asp 80 85 90 gtg gct ctg ctg cag ctc cgg aca cca atc
aac ttc tca gac acc gtg 672 Val Ala Leu Leu Gln Leu Arg Thr Pro Ile
Asn Phe Ser Asp Thr Val 95 100 105 gac gct gtg tgc ttg ccg gcc aag
gag cag tac ttt cca tgg ggg tcg 720 Asp Ala Val Cys Leu Pro Ala Lys
Glu Gln Tyr Phe Pro Trp Gly Ser 110 115 120 cag tgc tgg gtg tct ggc
tgg ggc cac acc gac ccc agc cat act cat 768 Gln Cys Trp Val Ser Gly
Trp Gly His Thr Asp Pro Ser His Thr His 125 130 135 agc tca gat aca
ctg cag gac aca atg gta ccc ctg ctc agc acc cac 816 Ser Ser Asp Thr
Leu Gln Asp Thr Met Val Pro Leu Leu Ser Thr His 140 145 150 155 ctc
tgc aac agc tca tgc atg tac agt ggg gca ctt aca cac cgc atg 864 Leu
Cys Asn Ser Ser Cys Met Tyr Ser Gly Ala Leu Thr His Arg Met 160 165
170 ttg tgt gct ggc tac ctg gat gga agg gca gac gca tgc cag gga gac
912 Leu Cys Ala Gly Tyr Leu Asp Gly Arg Ala Asp Ala Cys Gln Gly Asp
175 180 185 agc ggg gga ccc ctg gta tgt ccc agt ggt gac acg tgg cac
ctt gta 960 Ser Gly Gly Pro Leu Val Cys Pro Ser Gly Asp Thr Trp His
Leu Val 190 195 200 ggg gtg gtc agc tgg ggt cgt ggc tgt gca gag ccc
aat cgc cca ggt 1008 Gly Val Val Ser Trp Gly Arg Gly Cys Ala Glu
Pro Asn Arg Pro Gly 205 210 215 gtc tat gcc aag gta gca gag ttc ctg
gac tgg atc cat gac act gtg 1056 Val Tyr Ala Lys Val Ala Glu Phe
Leu Asp Trp Ile His Asp Thr Val 220 225 230 235 cag gtc cgc
tagccgaaga agcagcagca gccacctgtg acgccgagct 1105 Gln Val Arg
gtggatcgcc catggatcac cccagtctgg gggccagcat ctgggtcact gggcctctcc
1165 ccaaaggctc tgacttcgag ttcatctttc tcatctgaga acctccacaa
caggaaaagg 1225 agtctgcggc tagattggga atgatggtga gaggaaggga
taggaggaca gaagagacag 1285 cagaggcttc tggaagcatc tgggagactg
ctcctctgct ccccccacac cccacgtgca 1345 tccactgggg gatgctggag
atgcccaatc cttgtttctt gtggggccac tggaaggcta 1405 agtccaactt
tagaggatgc cctgtctcga gagttactag gcagataagg ttaaggttgg 1465
acaagctcag gtaaaggcac ggaagtcaag atcccctctc ccccgtgcgg tcctgttctg
1525 aggtaagcta atagccccgc accaggcaga ggtctacagg gtaagaagga
tgcagttggg 1585 ctacacgacg ctatttttca aatgatgttt ctgtaaattg
gttgagagag ttttgttatt 1645 aaacagaaat tatgtataaa aaaaaaaaaa
aaaaaaaaaa 1685 4 273 PRT Mus sp. 4 Met Glu Ala Gln Val Gly Leu Leu
Trp Val Ser Ala Asn Cys Pro Ser -35 -30 -25 -20 Gly Arg Ile Val Ser
Leu Lys Cys Ser Glu Cys Gly Ala Arg Pro Leu -15 -10 -5 Ala Ser Arg
Ile Val Gly Gly Gln Ala Val Ala Ser Gly Arg Trp Pro -1 1 5 10 Trp
Gln Ala Ser Val Met Leu Gly Ser Arg His Thr Cys Gly Ala Ser 15 20
25 Val Leu Ala Pro His Trp Val Val Thr Ala Ala His Cys Met Tyr Ser
30 35 40 45 Phe Arg Leu Ser Arg Leu Ser Ser Trp Arg Val His Ala Gly
Leu Val 50 55 60 Ser His Gly Ala Val Arg Gln His Gln Gly Thr Met
Val Glu Lys Ile 65 70 75 Ile Pro His Pro Leu Tyr Ser Ala Gln Asn
His Asp Tyr Asp Val Ala 80 85 90 Leu Leu Gln Leu Arg Thr Pro Ile
Asn Phe Ser Asp Thr Val Asp Ala 95 100 105 Val Cys Leu Pro Ala Lys
Glu Gln Tyr Phe Pro Trp Gly Ser Gln Cys 110 115 120 125 Trp Val Ser
Gly Trp Gly His Thr Asp Pro Ser His Thr His Ser Ser 130 135 140 Asp
Thr Leu Gln Asp Thr Met Val Pro Leu Leu Ser Thr His Leu Cys 145 150
155 Asn Ser Ser Cys Met Tyr Ser Gly Ala Leu Thr His Arg Met Leu Cys
160 165 170 Ala Gly Tyr Leu Asp Gly Arg Ala Asp Ala Cys Gln Gly Asp
Ser Gly 175 180 185 Gly Pro Leu Val Cys Pro Ser Gly Asp Thr Trp His
Leu Val Gly Val 190 195 200 205 Val Ser Trp Gly Arg Gly Cys Ala Glu
Pro Asn Arg Pro Gly Val Tyr 210 215 220 Ala Lys Val Ala Glu Phe Leu
Asp Trp Ile His Asp Thr Val Gln Val 225 230 235 Arg 5 2068 DNA Mus
sp. CDS (516)..(1448) 5 ctggctgggc tgttgaatca atcccgacat gaggacagga
gcctcaccct gcccagcaga 60 acttactgcc ttatatcagt gcagctgact
catatgagtc caacactgga tgaccaaagc 120 ccaatggaga ttcggtgcac
ggaagagggt gctgggcctg ggatcttcag aatggagttg 180 ggagaccaga
ggcaatccat ttctcagtcc caacgctggt gctgcctgca acgtggctgt 240
gtaatactgg gcgtcctggg gctgctggct ggagcaggca ttgcttcatg gctcttagtg
300 ttgtatctat ggccggctgc ctctccatcc atctctggga cgttgcagga
ggaggagatg 360 actttgaact gtccaggagt gagctgtgag gaagagctcc
ttccatctct tcccaaaaca 420 gaataaatgg aggggatctt ctgcttcaag
tacaagtaag agctcggcca gactggctcc 480 tggtctgcca tgagggctgg
agccccgccc tgggc atg cac atc tgc aag agt 533 Met His Ile Cys Lys
Ser -70 ctt ggg cat atc agg ctt act caa cac aag gcc gtg aat ctg tct
gac 581 Leu Gly His Ile Arg Leu Thr Gln His Lys Ala Val Asn Leu Ser
Asp -65 -60 -55 atc aag ctc aac aga tcc cag gag ttt gct caa ctc tct
gct aga ccg 629 Ile Lys Leu Asn Arg Ser Gln Glu Phe Ala Gln Leu Ser
Ala Arg Pro -50 -45 -40 gga ggc ctt gta gag gag gca tgg aag ccc agc
gct aac tgt cct tct 677 Gly Gly Leu Val Glu Glu Ala Trp Lys Pro Ser
Ala Asn Cys Pro Ser -35 -30 -25 -20 ggc cga att gtt tct ctc aaa tgt
tct gag tgt ggg gca agg cct ctg 725 Gly Arg Ile Val Ser Leu Lys Cys
Ser Glu Cys Gly Ala Arg Pro Leu -15 -10 -5 gct tct cga ata gtt ggc
ggc caa gct gtg gct tct ggg cgc tgg cca 773 Ala Ser Arg Ile Val Gly
Gly Gln Ala Val Ala Ser Gly Arg Trp Pro -1 1 5 10 tgg caa gct agc
gtg atg ctt ggc tcc cgg cac acg tgt ggg gcc tct 821 Trp Gln Ala Ser
Val Met Leu Gly Ser Arg His Thr Cys Gly Ala Ser 15 20 25 gtg ttg
gca cca cac tgg gta gtg act gct gcc cac tgc atg tac agt 869 Val Leu
Ala Pro His Trp Val Val Thr Ala Ala His Cys Met Tyr Ser 30 35 40 45
ttc agg ctg tcc cgc cta tcc agc tgg cgg gtt cat gca ggg ctg gtc 917
Phe Arg Leu Ser Arg Leu Ser Ser Trp Arg Val His Ala Gly Leu Val 50
55 60 agc cat ggt gct gtc cga caa cac cag gga act atg gtg gag aag
atc 965 Ser His Gly Ala Val Arg Gln His Gln Gly Thr Met Val Glu Lys
Ile 65 70 75 att cct cat cct ttg tac agt gcc cag aac cat gac tat
gat gtg gct 1013 Ile Pro His Pro Leu Tyr Ser Ala Gln Asn His Asp
Tyr Asp Val Ala 80 85 90 ctg ctg cag ctc cgg aca cca atc aac ttc
tca gac acc gtg gac gct 1061 Leu Leu Gln Leu Arg Thr Pro Ile Asn
Phe Ser Asp Thr Val Asp Ala 95 100 105 gtg tgc ttg ccg gcc aag gag
cag tac ttt cca tgg ggg tcg cag tgc 1109 Val Cys Leu Pro Ala Lys
Glu Gln Tyr Phe Pro Trp Gly Ser Gln Cys 110 115 120 125 tgg gtg tct
ggc tgg ggc cac acc gac ccc agc cat act cat agc tca 1157 Trp Val
Ser Gly Trp Gly His Thr Asp Pro Ser His Thr His Ser Ser 130 135 140
gat aca ctg cag gac aca atg gta ccc ctg ctc agc acc cac ctc tgc
1205 Asp Thr Leu Gln Asp Thr Met Val Pro Leu Leu Ser Thr His Leu
Cys 145 150 155 aac agc tca tgc atg tac agt ggg gca ctt aca cac cgc
atg ttg tgt 1253 Asn Ser Ser Cys Met Tyr Ser Gly Ala Leu Thr His
Arg Met Leu Cys 160 165 170 gct ggc tac ctg gat gga agg gca gac gca
tgc cag gga gac agc ggg 1301 Ala Gly Tyr Leu Asp Gly Arg Ala Asp
Ala Cys Gln Gly Asp Ser Gly 175 180 185 gga ccc ctg gta tgt ccc agt
ggt gac acg tgg cac ctt gta ggg gtg 1349 Gly Pro Leu Val Cys Pro
Ser Gly Asp Thr Trp His Leu Val Gly Val 190 195 200 205 gtc agc tgg
ggt cgt ggc tgt gca gag ccc aat cgc cca ggt gtc tat 1397 Val Ser
Trp Gly Arg Gly Cys Ala Glu Pro Asn Arg Pro Gly Val Tyr 210 215 220
gcc aag gta gca gag ttc ctg gac tgg atc cat gac act gtg cag gtc
1445 Ala Lys Val Ala Glu Phe Leu Asp Trp Ile His Asp Thr Val Gln
Val 225 230 235 cgc tagccgaaga agcagcagca gccacctgtg acgccgagct
gtggatcgcc 1498 Arg catggatcac cccagtctgg gggccagcat ctgggtcact
gggcctctcc ccaaaggctc 1558 tgacttcgag ttcatctttc tcatctgaga
acctccacaa caggaaaagg agtctgcggc 1618 tagattggga atgatggtga
gaggaaggga taggaggaca gaagagacag cagaggcttc 1678 tggaagcatc
tgggagactg ctcctctgct ccccccacac cccacgtgca tccactgggg 1738
gatgctggag atgcccaatc cttgtttctt gtggggccac tggaaggcta agtccaactt
1798 tagaggatgc cctgtctcga gagttactag gcagataagg ttaaggttgg
acaagctcag 1858 gtaaaggcac ggaagtcaag atcccctctc ccccgtgcgg
tcctgttctg aggtaagcta 1918 atagccccgc accaggcaga ggtctacagg
gtaagaagga tgcagttggg ctacacgacg 1978 ctatttttca aatgatgttt
ctgtaaattg gttgagagag ttttgttatt aaacagaaat 2038 tatgtataaa
aaaaaaaaaa aaaaaaaaaa 2068 6 311 PRT Mus sp. 6 Met His Ile Cys Lys
Ser Leu Gly His Ile Arg Leu Thr Gln His Lys -70 -65 -60 Ala Val Asn
Leu Ser Asp Ile Lys Leu Asn Arg Ser Gln Glu Phe Ala -55 -50 -45 Gln
Leu Ser Ala Arg Pro Gly Gly Leu Val Glu Glu Ala Trp Lys Pro -40 -35
-30 Ser Ala Asn Cys Pro Ser Gly Arg Ile Val Ser Leu Lys Cys Ser Glu
-25 -20 -15 -10 Cys Gly Ala Arg Pro Leu Ala Ser Arg Ile Val Gly Gly
Gln Ala Val -5 -1 1 5 Ala Ser Gly Arg Trp Pro Trp Gln Ala Ser Val
Met Leu Gly Ser Arg 10 15 20 His Thr Cys Gly Ala Ser Val Leu Ala
Pro His Trp Val Val Thr Ala 25 30 35 Ala His Cys Met Tyr Ser Phe
Arg Leu Ser Arg Leu Ser Ser Trp Arg 40 45 50 55 Val His Ala Gly Leu
Val Ser His Gly Ala Val Arg Gln His Gln Gly 60 65 70 Thr Met Val
Glu Lys Ile Ile Pro His Pro Leu Tyr Ser Ala Gln Asn 75 80 85 His
Asp Tyr Asp Val Ala Leu Leu Gln Leu Arg Thr Pro Ile Asn Phe 90 95
100 Ser Asp Thr Val Asp Ala Val Cys Leu Pro Ala Lys Glu Gln Tyr Phe
105 110 115 Pro Trp Gly Ser Gln Cys Trp Val Ser Gly Trp Gly His Thr
Asp Pro 120 125 130 135 Ser His Thr His Ser Ser Asp Thr Leu Gln Asp
Thr Met Val Pro Leu 140 145 150 Leu Ser Thr His Leu Cys Asn Ser Ser
Cys Met Tyr Ser Gly Ala Leu 155 160 165 Thr His Arg Met Leu Cys Ala
Gly Tyr Leu Asp Gly Arg Ala Asp Ala 170
175 180 Cys Gln Gly Asp Ser Gly Gly Pro Leu Val Cys Pro Ser Gly Asp
Thr 185 190 195 Trp His Leu Val Gly Val Val Ser Trp Gly Arg Gly Cys
Ala Glu Pro 200 205 210 215 Asn Arg Pro Gly Val Tyr Ala Lys Val Ala
Glu Phe Leu Asp Trp Ile 220 225 230 His Asp Thr Val Gln Val Arg 235
7 2070 DNA Mus sp. CDS (116)..(1450) 7 cccagcagaa cttactgcct
tatatcagtg cagctgactc atatgccctg gtgtggggct 60 gctggatctt
caaccactat ttctccagag tccaacactg gatgaccaaa gccca atg 118 Met gag
att cgg tgc acg gaa gag ggt gct ggg cct ggg atc ttc aga 163 Glu Ile
Arg Cys Thr Glu Glu Gly Ala Gly Pro Gly Ile Phe Arg -205 -200 -195
atg gag ttg gga gac cag agg caa tcc att tct cag tcc caa cgc 208 Met
Glu Leu Gly Asp Gln Arg Gln Ser Ile Ser Gln Ser Gln Arg -190 -185
-180 tgg tgc tgc ctg caa cgt ggc tgt gta ata ctg ggc gtc ctg ggg
253 Trp Cys Cys Leu Gln Arg Gly Cys Val Ile Leu Gly Val Leu Gly
-175 -170 -165 ctg ctg gct gga gca ggc att gct tca tgg ctc tta gtg
ttg tat 298 Leu Leu Ala Gly Ala Gly Ile Ala Ser Trp Leu Leu Val Leu
Tyr -160 -155 -150 cta tgg cca gct gcc tct cca tcc atc tct ggg acg
ttg cag gag 343 Leu Trp Pro Ala Ala Ser Pro Ser Ile Ser Gly Thr Leu
Gln Glu -145 -140 -135 gag gag atg act ttg aac tgt cca gga gtg agc
tgt gag gaa gag 388 Glu Glu Met Thr Leu Asn Cys Pro Gly Val Ser Cys
Glu Glu Glu -130 -125 -120 ctc ctt cca tct ctt ccc aaa aca gta tct
ttc aga ata aat gga 433 Leu Leu Pro Ser Leu Pro Lys Thr Val Ser Phe
Arg Ile Asn Gly -115 -110 -105 gag gat ctt ctg ctt caa gta caa gta
aga gct cgg cca gac tgg ctc 481 Glu Asp Leu Leu Leu Gln Val Gln Val
Arg Ala Arg Pro Asp Trp Leu -100 -95 -90 ctg gtc tgc cat gag ggc
tgg agc ccc gcc ctg ggc atg cac atc tgc 529 Leu Val Cys His Glu Gly
Trp Ser Pro Ala Leu Gly Met His Ile Cys -85 -80 -75 -70 aag agt ctt
ggg cat atc agg ctt act caa cac aag gcc gtg aat ctg 577 Lys Ser Leu
Gly His Ile Arg Leu Thr Gln His Lys Ala Val Asn Leu -65 -60 -55 tct
gac atc aag ctc aac aga tcc cag gag ttt gct caa ctc tct gct 625 Ser
Asp Ile Lys Leu Asn Arg Ser Gln Glu Phe Ala Gln Leu Ser Ala -50 -45
-40 aga ccg gga ggc ctt gta gag gag gca tgg aag ccc agc gct aac tgt
673 Arg Pro Gly Gly Leu Val Glu Glu Ala Trp Lys Pro Ser Ala Asn Cys
-35 -30 -25 cct tct ggc cga att gtt tct ctc aaa tgt tct gag tgt ggg
gca agg 721 Pro Ser Gly Arg Ile Val Ser Leu Lys Cys Ser Glu Cys Gly
Ala Arg -20 -15 -10 cct ctg gct tct cga ata gtt ggc ggc caa gct gtg
gct tct ggg cgc 769 Pro Leu Ala Ser Arg Ile Val Gly Gly Gln Ala Val
Ala Ser Gly Arg -5 -1 1 5 10 tgg cca tgg caa gct agc gtg atg ctt
ggc tcc cgg cac acg tgt ggg 817 Trp Pro Trp Gln Ala Ser Val Met Leu
Gly Ser Arg His Thr Cys Gly 15 20 25 gcc tct gtg ttg gca cca cac
tgg gta gtg act gct gcc cac tgc atg 865 Ala Ser Val Leu Ala Pro His
Trp Val Val Thr Ala Ala His Cys Met 30 35 40 tac agt ttc agg ctg
tcc cgc cta tcc agc tgg cgg gtt cat gca ggg 913 Tyr Ser Phe Arg Leu
Ser Arg Leu Ser Ser Trp Arg Val His Ala Gly 45 50 55 ctg gtc agc
cat ggt gct gtc cga caa cac cag gga act atg gtg gag 961 Leu Val Ser
His Gly Ala Val Arg Gln His Gln Gly Thr Met Val Glu 60 65 70 75 aag
atc att cct cat cct ttg tac agt gcc cag aac cat gac tat gat 1009
Lys Ile Ile Pro His Pro Leu Tyr Ser Ala Gln Asn His Asp Tyr Asp 80
85 90 gtg gct ctg ctg cag ctc cgg aca cca atc aac ttc tca gac acc
gtg 1057 Val Ala Leu Leu Gln Leu Arg Thr Pro Ile Asn Phe Ser Asp
Thr Val 95 100 105 gac gct gtg tgc ttg ccg gcc aag gag cag tac ttt
cca tgg ggg tcg 1105 Asp Ala Val Cys Leu Pro Ala Lys Glu Gln Tyr
Phe Pro Trp Gly Ser 110 115 120 cag tgc tgg gtg tct ggc tgg ggc cac
acc gac ccc agc cat act cat 1153 Gln Cys Trp Val Ser Gly Trp Gly
His Thr Asp Pro Ser His Thr His 125 130 135 agc tca gat aca ctg cag
gac aca atg gta ccc ctg ctc agc acc cac 1201 Ser Ser Asp Thr Leu
Gln Asp Thr Met Val Pro Leu Leu Ser Thr His 140 145 150 155 ctc tgc
aac agc tca tgc atg tac agt ggg gca ctt aca cac cgc atg 1249 Leu
Cys Asn Ser Ser Cys Met Tyr Ser Gly Ala Leu Thr His Arg Met 160 165
170 ttg tgt gct ggc tac ctg gat gga agg gca gac gca tgc cag gga gac
1297 Leu Cys Ala Gly Tyr Leu Asp Gly Arg Ala Asp Ala Cys Gln Gly
Asp 175 180 185 agc ggg gga ccc ctg gta tgt ccc agt ggt gac acg tgg
cac ctt gta 1345 Ser Gly Gly Pro Leu Val Cys Pro Ser Gly Asp Thr
Trp His Leu Val 190 195 200 ggg gtg gtc agc tgg ggt cgt ggc tgt gca
gag ccc aat cgc cca ggt 1393 Gly Val Val Ser Trp Gly Arg Gly Cys
Ala Glu Pro Asn Arg Pro Gly 205 210 215 gtc tat gcc aag gta gca gag
ttc ctg gac tgg atc cat gac act gtg 1441 Val Tyr Ala Lys Val Ala
Glu Phe Leu Asp Trp Ile His Asp Thr Val 220 225 230 235 cag gtc cgc
tagccgaaga agcagcagca gccacctgtg acgccgagct 1490 Gln Val Arg
gtggatcgcc catggatcac cccagtctgg gggccagcat ctgggtcact gggcctctcc
1550 ccaaaggctc tgacttcgag ttcatctttc tcatctgaga acctccacaa
caggaaaagg 1610 agtctgcggc tagattggga atgatggtga gaggaaggga
taggaggaca gaagagacag 1670 cagaggcttc tggaagcatc tgggagactg
ctcctctgct ccccccacac cccacgtgca 1730 tccactgggg gatgctggag
atgcccaatc cttgtttctt gtggggccac tggaaggcta 1790 agtccaactt
tagaggatgc cctgtctcga gagttactag gcagataagg ttaaggttgg 1850
acaagctcag gtaaaggcac ggaagtcaag atcccctctc ccccgtgcgg tcctgttctg
1910 aggtaagcta atagccccgc accaggcaga ggtctacagg gtaagaagga
tgcagttggg 1970 ctacacgacg ctatttttca aatgatgttt ctgtaaattg
gttgagagag ttttgttatt 2030 aaacagaaat tatgtataaa aaaaaaaaaa
aaaaaaaaaa 2070 8 445 PRT Mus sp. 8 Met Glu Ile Arg Cys Thr Glu Glu
Gly Ala Gly Pro Gly Ile Phe -205 -200 -195 Arg Met Glu Leu Gly Asp
Gln Arg Gln Ser Ile Ser Gln Ser Gln -190 -185 -180 Arg Trp Cys Cys
Leu Gln Arg Gly Cys Val Ile Leu Gly Val Leu -175 -170 -165 Gly Leu
Leu Ala Gly Ala Gly Ile Ala Ser Trp Leu Leu Val Leu -160 -155 -150
Tyr Leu Trp Pro Ala Ala Ser Pro Ser Ile Ser Gly Thr Leu Gln -145
-140 -135 Glu Glu Glu Met Thr Leu Asn Cys Pro Gly Val Ser Cys Glu
Glu -130 -125 -120 Glu Leu Leu Pro Ser Leu Pro Lys Thr Val Ser Phe
Arg Ile Asn -115 -110 -105 Gly Glu Asp Leu Leu Leu Gln Val Gln Val
Arg Ala Arg Pro Asp Trp -100 -95 -90 Leu Leu Val Cys His Glu Gly
Trp Ser Pro Ala Leu Gly Met His Ile -85 -80 -75 Cys Lys Ser Leu Gly
His Ile Arg Leu Thr Gln His Lys Ala Val Asn -70 -65 -60 -55 Leu Ser
Asp Ile Lys Leu Asn Arg Ser Gln Glu Phe Ala Gln Leu Ser -50 -45 -40
Ala Arg Pro Gly Gly Leu Val Glu Glu Ala Trp Lys Pro Ser Ala Asn -35
-30 -25 Cys Pro Ser Gly Arg Ile Val Ser Leu Lys Cys Ser Glu Cys Gly
Ala -20 -15 -10 Arg Pro Leu Ala Ser Arg Ile Val Gly Gly Gln Ala Val
Ala Ser Gly -5 -1 1 5 10 Arg Trp Pro Trp Gln Ala Ser Val Met Leu
Gly Ser Arg His Thr Cys 15 20 25 Gly Ala Ser Val Leu Ala Pro His
Trp Val Val Thr Ala Ala His Cys 30 35 40 Met Tyr Ser Phe Arg Leu
Ser Arg Leu Ser Ser Trp Arg Val His Ala 45 50 55 Gly Leu Val Ser
His Gly Ala Val Arg Gln His Gln Gly Thr Met Val 60 65 70 Glu Lys
Ile Ile Pro His Pro Leu Tyr Ser Ala Gln Asn His Asp Tyr 75 80 85 90
Asp Val Ala Leu Leu Gln Leu Arg Thr Pro Ile Asn Phe Ser Asp Thr 95
100 105 Val Asp Ala Val Cys Leu Pro Ala Lys Glu Gln Tyr Phe Pro Trp
Gly 110 115 120 Ser Gln Cys Trp Val Ser Gly Trp Gly His Thr Asp Pro
Ser His Thr 125 130 135 His Ser Ser Asp Thr Leu Gln Asp Thr Met Val
Pro Leu Leu Ser Thr 140 145 150 His Leu Cys Asn Ser Ser Cys Met Tyr
Ser Gly Ala Leu Thr His Arg 155 160 165 170 Met Leu Cys Ala Gly Tyr
Leu Asp Gly Arg Ala Asp Ala Cys Gln Gly 175 180 185 Asp Ser Gly Gly
Pro Leu Val Cys Pro Ser Gly Asp Thr Trp His Leu 190 195 200 Val Gly
Val Val Ser Trp Gly Arg Gly Cys Ala Glu Pro Asn Arg Pro 205 210 215
Gly Val Tyr Ala Lys Val Ala Glu Phe Leu Asp Trp Ile His Asp Thr 220
225 230 Val Gln Val Arg 235 9 2265 DNA Homo sapiens CDS
(156)..(1526) 9 acgcgggata cagggagggg ccatgtgcga accagggaga
cctcatcttc caaccaagct 60 tgctgggctt gcatttaatc aatgcatggc
cagagaacag gagcggaaca ttgcctagta 120 gaccctgagg ctttacaaca
gtgctactga cccct atg agc ctg atg ctg gat 173 Met Ser Leu Met Leu
Asp -215 gac caa ccc cct atg gag gcc cag tat gca gag gag ggc cca
gga 218 Asp Gln Pro Pro Met Glu Ala Gln Tyr Ala Glu Glu Gly Pro Gly
-210 -205 -200 cct ggg atc ttc aga gca gag cct gga gac cag cag cat
ccc att 263 Pro Gly Ile Phe Arg Ala Glu Pro Gly Asp Gln Gln His Pro
Ile -195 -190 -185 tct cag gcg gtg tgc tgg cgt tcc atg cga cgt ggc
tgt gca gtg 308 Ser Gln Ala Val Cys Trp Arg Ser Met Arg Arg Gly Cys
Ala Val -180 -175 -170 ctg gga gcc ctg ggg ctg ctg gcc ggt gca ggt
gtt ggc tca tgg 353 Leu Gly Ala Leu Gly Leu Leu Ala Gly Ala Gly Val
Gly Ser Trp -165 -160 -155 ctc cta gtg ctg tat ctg tgt cct gct gcc
tct cag ccc att tcc 398 Leu Leu Val Leu Tyr Leu Cys Pro Ala Ala Ser
Gln Pro Ile Ser -150 -145 -140 ggg acc ttg cag gat gag gag ata act
ttg agc tgc tca gag gcc 443 Gly Thr Leu Gln Asp Glu Glu Ile Thr Leu
Ser Cys Ser Glu Ala -135 -130 -125 agc gct gag gaa gct ctg ctc cct
gca ctc ccc aaa aca gta tct 488 Ser Ala Glu Glu Ala Leu Leu Pro Ala
Leu Pro Lys Thr Val Ser -120 -115 -110 ttc aga ata aac agc gaa gac
ttc ttg ctg gaa gcg caa gtg agg gat 536 Phe Arg Ile Asn Ser Glu Asp
Phe Leu Leu Glu Ala Gln Val Arg Asp -105 -100 -95 cag cca cgc tgg
ctc ctg gtc tgc cat gag ggc tgg agc ccc gcc ctg 584 Gln Pro Arg Trp
Leu Leu Val Cys His Glu Gly Trp Ser Pro Ala Leu -90 -85 -80 -75 ggg
ctg cag atc tgc tgg agc ctt ggg cat ctc aga ctc act cac cac 632 Gly
Leu Gln Ile Cys Trp Ser Leu Gly His Leu Arg Leu Thr His His -70 -65
-60 aag gga gta aac ctc act gac atc aaa ctc aac agt tcc cag gag ttt
680 Lys Gly Val Asn Leu Thr Asp Ile Lys Leu Asn Ser Ser Gln Glu Phe
-55 -50 -45 gct cag ctc tct cct aga ctg gga ggc ttc ctg gag gag gcg
tgg cag 728 Ala Gln Leu Ser Pro Arg Leu Gly Gly Phe Leu Glu Glu Ala
Trp Gln -40 -35 -30 ccc agg aac aac tgc act tct ggt caa gtt gtt tcc
ctc aga tgc tct 776 Pro Arg Asn Asn Cys Thr Ser Gly Gln Val Val Ser
Leu Arg Cys Ser -25 -20 -15 gag tgt gga gcg agg ccc ctg gct tcc cgg
ata gtt ggt ggg cag tct 824 Glu Cys Gly Ala Arg Pro Leu Ala Ser Arg
Ile Val Gly Gly Gln Ser -10 -5 -1 1 5 gtg gct cct ggg cgc tgg ccg
tgg cag gcc agc gtg gcc ctg ggc ttc 872 Val Ala Pro Gly Arg Trp Pro
Trp Gln Ala Ser Val Ala Leu Gly Phe 10 15 20 cgg cac acg tgt ggg
ggc tct gtg cta gcg cca cgc tgg gtg gtg act 920 Arg His Thr Cys Gly
Gly Ser Val Leu Ala Pro Arg Trp Val Val Thr 25 30 35 gct gca cat
tgt atg cac agt ttc agg ctg gcc cgc ctg tcc agc tgg 968 Ala Ala His
Cys Met His Ser Phe Arg Leu Ala Arg Leu Ser Ser Trp 40 45 50 cgg
gtt cat gcg ggg ctg gtc agc cac agt gcc gtc agg ccc cac caa 1016
Arg Val His Ala Gly Leu Val Ser His Ser Ala Val Arg Pro His Gln 55
60 65 70 ggg gct ctg gtg gag agg att atc cca cac ccc ctc tac agt
gcc cag 1064 Gly Ala Leu Val Glu Arg Ile Ile Pro His Pro Leu Tyr
Ser Ala Gln 75 80 85 aat cat gac tac gac gtc gcc ctc ctg agg ctc
cag acc gct ctc aac 1112 Asn His Asp Tyr Asp Val Ala Leu Leu Arg
Leu Gln Thr Ala Leu Asn 90 95 100 ttc tca gac act gtg ggc gct gtg
tgc ctg ccg gcc aag gaa cag cat 1160 Phe Ser Asp Thr Val Gly Ala
Val Cys Leu Pro Ala Lys Glu Gln His 105 110 115 ttt ccg aag ggc tcg
cgg tgc tgg gtg tct ggc tgg ggc cac acc cac 1208 Phe Pro Lys Gly
Ser Arg Cys Trp Val Ser Gly Trp Gly His Thr His 120 125 130 cct agc
cat act tac agc tcg gat atg ctc cag gac acg gtg gtg ccc 1256 Pro
Ser His Thr Tyr Ser Ser Asp Met Leu Gln Asp Thr Val Val Pro 135 140
145 150 ttg ttc agc act cag ctc tgc aac agc tct tgc gtg tac agc gga
gcc 1304 Leu Phe Ser Thr Gln Leu Cys Asn Ser Ser Cys Val Tyr Ser
Gly Ala 155 160 165 ctc acc ccc cgc atg ctt tgc gct ggc tac ctg gac
gga agg gct gat 1352 Leu Thr Pro Arg Met Leu Cys Ala Gly Tyr Leu
Asp Gly Arg Ala Asp 170 175 180 gca tgc cag gga gat agc ggg ggc ccc
cta gtg tgc cca gat ggg gac 1400 Ala Cys Gln Gly Asp Ser Gly Gly
Pro Leu Val Cys Pro Asp Gly Asp 185 190 195 aca tgg cgc cta gtg ggg
gtg gtc agc tgg ggg cgt gcg tgc gca gag 1448 Thr Trp Arg Leu Val
Gly Val Val Ser Trp Gly Arg Ala Cys Ala Glu 200 205 210 ccc aat cac
cca ggt gtc tac gcc aag gta gct gag ttt ctg gac tgg 1496 Pro Asn
His Pro Gly Val Tyr Ala Lys Val Ala Glu Phe Leu Asp Trp 215 220 225
230 atc cat gac act gct cag gac tcc ctc ctc tgagtcctgc tgtttcctcc
1546 Ile His Asp Thr Ala Gln Asp Ser Leu Leu 235 240 agtctcactg
cacaccactg cctcatgctt cctggggcct ccagcagctc cactaatgga 1606
ggagaggcag tagcctccga cacagaacgc atggacctcc tactactgtg tgtgaggaac
1666 agtcactacc cactggccag ccacccagcc aacaggtctc tcctcttggg
ccctgatttc 1726 agagtcctct ttctcactag agactcaatg acagaagaga
ggctgggact tggttgggca 1786 tgctgtggtt gctgagggat gagggggagg
agagaggtag gagctggaga tgaagagact 1846 gctagaagca gcaggaagcc
tgcccttctg ccctctcccc tccctgcccc tgtgtgagtc 1906 ttttagggag
ggtgactggg aggtgccccc cgtcccacct ttttcctgtg ctctaggtgg 1966
gctaagtgcc tccctagagg actccatggc tgagaggctc ctgggcagat ggggtcaagg
2026 ctgggccagt cccagatgaa gcctatggga gtcaggaccc tctccactct
ccctctccac 2086 tccccttcct gttctcacct ggctgtggct ggccctgtgt
ggggtgggta cactggaaaa 2146 caagaaggtt ggagttggtc taggacattg
gttttaaatg acagttctgt gaactggtcc 2206 aaggaggttc tgttattaaa
gtgatatatg gtcttgaaaa aaaaaaaaaa aaaaaaaaa 2265 10 457 PRT Homo
sapiens 10 Met Ser Leu Met Leu Asp Asp Gln Pro Pro Met Glu Ala Gln
Tyr -215 -210 -205 Ala Glu Glu Gly Pro Gly Pro Gly Ile Phe Arg Ala
Glu Pro Gly -200 -195 -190 Asp Gln Gln His Pro Ile Ser Gln Ala Val
Cys Trp Arg Ser Met -185 -180 -175 Arg Arg Gly Cys Ala Val Leu Gly
Ala Leu Gly Leu Leu Ala Gly -170 -165 -160 Ala Gly Val Gly Ser Trp
Leu Leu Val Leu Tyr Leu Cys Pro Ala -155 -150 -145 Ala Ser Gln Pro
Ile Ser Gly Thr Leu Gln Asp Glu Glu Ile Thr -140 -135 -130 Leu Ser
Cys Ser Glu Ala Ser Ala Glu Glu Ala Leu Leu Pro Ala -125 -120 -115
Leu Pro Lys Thr Val Ser Phe Arg Ile Asn Ser Glu Asp Phe Leu -110
-105 -100 Leu Glu Ala Gln Val Arg Asp Gln Pro Arg Trp Leu Leu Val
Cys His -95 -90 -85 Glu Gly Trp Ser Pro Ala Leu Gly Leu Gln Ile Cys
Trp Ser Leu Gly -80 -75 -70 His Leu Arg Leu Thr His His Lys Gly Val
Asn Leu Thr Asp Ile Lys -65 -60
-55 -50 Leu Asn Ser Ser Gln Glu Phe Ala Gln Leu Ser Pro Arg Leu Gly
Gly -45 -40 -35 Phe Leu Glu Glu Ala Trp Gln Pro Arg Asn Asn Cys Thr
Ser Gly Gln -30 -25 -20 Val Val Ser Leu Arg Cys Ser Glu Cys Gly Ala
Arg Pro Leu Ala Ser -15 -10 -5 Arg Ile Val Gly Gly Gln Ser Val Ala
Pro Gly Arg Trp Pro Trp Gln -1 1 5 10 15 Ala Ser Val Ala Leu Gly
Phe Arg His Thr Cys Gly Gly Ser Val Leu 20 25 30 Ala Pro Arg Trp
Val Val Thr Ala Ala His Cys Met His Ser Phe Arg 35 40 45 Leu Ala
Arg Leu Ser Ser Trp Arg Val His Ala Gly Leu Val Ser His 50 55 60
Ser Ala Val Arg Pro His Gln Gly Ala Leu Val Glu Arg Ile Ile Pro 65
70 75 His Pro Leu Tyr Ser Ala Gln Asn His Asp Tyr Asp Val Ala Leu
Leu 80 85 90 95 Arg Leu Gln Thr Ala Leu Asn Phe Ser Asp Thr Val Gly
Ala Val Cys 100 105 110 Leu Pro Ala Lys Glu Gln His Phe Pro Lys Gly
Ser Arg Cys Trp Val 115 120 125 Ser Gly Trp Gly His Thr His Pro Ser
His Thr Tyr Ser Ser Asp Met 130 135 140 Leu Gln Asp Thr Val Val Pro
Leu Phe Ser Thr Gln Leu Cys Asn Ser 145 150 155 Ser Cys Val Tyr Ser
Gly Ala Leu Thr Pro Arg Met Leu Cys Ala Gly 160 165 170 175 Tyr Leu
Asp Gly Arg Ala Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro 180 185 190
Leu Val Cys Pro Asp Gly Asp Thr Trp Arg Leu Val Gly Val Val Ser 195
200 205 Trp Gly Arg Ala Cys Ala Glu Pro Asn His Pro Gly Val Tyr Ala
Lys 210 215 220 Val Ala Glu Phe Leu Asp Trp Ile His Asp Thr Ala Gln
Asp Ser Leu 225 230 235 Leu 240 11 99 DNA Artificial Sequence
Synthetic 11 aagcttggct agcaacacca tgaatctact cctgatcctt acctttgttg
ctgctgctgt 60 tgctgccccc tttgacgacg atgacaagga tccgaattc 99 12 99
DNA Artificial Sequence Synthetic 12 gaattcggat ccttgtcatc
gtcgtcaaag ggggcagcaa cagcagcagc aacaaaggta 60 aggatcagga
gtagattcat ggtgttgcta gccaagctt 99 13 15 DNA Artificial Sequence
Synthetic 13 ttggtgcatg gcgga 15 14 27 DNA Artificial Sequence
Synthetic 14 tcctcgagac ttggcctgaa tggtttt 27 15 35 DNA Artificial
Sequence Synthetic 15 gcgctagcag atctccatga atctactcct gatcc 35 16
29 DNA Artificial Sequence Synthetic 16 tgaagcttgc catggaccaa
cttgtcatc 29 17 26 DNA Artificial Sequence Synthetic 17 ccaagcttca
ccatcaccat caccat 26 18 17 DNA Artificial Sequence Synthetic 18
gcacagtcga ggctgat 17 19 17 DNA Artificial Sequence Synthetic 19
caaatgtggt atggctg 17 20 20 DNA Artificial Sequence Synthetic 20
gtgctcacng cngcbcaytg 20 21 20 DNA Artificial Sequence Synthetic 21
ccvctrwsdc cnccnggcga 20 22 21 DNA Artificial Sequence Synthetic 22
atggtggaga agatcattcc t 21 23 19 DNA Artificial Sequence Synthetic
23 tacagtgccc agaaccatg 19 24 20 DNA Artificial Sequence Synthetic
24 ctcaactctc tgctagaccg 20 25 20 DNA Artificial Sequence Synthetic
25 atagttggcg gccaagctgt 20 26 20 DNA Artificial Sequence Synthetic
26 cccagcagaa cttactgcct 20 27 20 DNA Artificial Sequence Synthetic
27 tgttgcagag gtgggtgctg 20 28 21 DNA Artificial Sequence Synthetic
28 taccattgtg tcctgcagtg t 21 29 27 DNA Artificial Sequence
Synthetic 29 tgaattctgc tgcttcttcg gctagcg 27 30 18 DNA Artificial
Sequence Synthetic 30 actgctgccc actgcatg 18 31 21 DNA Artificial
Sequence Synthetic 31 caggggtccc ccgctgtctc c 21 32 20 DNA
Artificial Sequence Synthetic 32 gctctcaact tctcagacac 20 33 20 DNA
Artificial Sequence Synthetic 33 actcagctac cttggcgtag 20 34 20 DNA
Artificial Sequence Synthetic 34 cctggagcat atccgagctg 20 35 18 DNA
Artificial Sequence Synthetic 35 gctttacaac agtgctac 18 36 28 DNA
Artificial Sequence Synthetic 36 tggaattcga ggaaacagca ggactcag 28
37 19 DNA Artificial Sequence Synthetic 37 tactagtcga cgcgtggcc 19
38 18 DNA Artificial Sequence Synthetic 38 actgctgccc actgcatg 18
39 35 DNA Artificial Sequence Synthetic 39 gcgctagcag atctccatga
atctactcct gatcc 35 40 117 DNA Artificial Sequence Synthetic 40
aagcttggct agcaacacca tgaatctact cctgatcctt acctttgttg ctgctgctgt
60 tgctgccccc tttcaccatc accatcacca tgacgacgat gacaaggatc cgaattc
117 41 117 DNA Artificial Sequence Synthetic 41 gaattcggat
ccttgtcatc gtcgtcatgg tgatggtgat ggtgaaaggg ggcagcaaca 60
gcagcagcaa caaaggtaag gatcaggagt agattcatgg tgttgctagc caagctt 117
42 5 PRT Artificial Sequence Synthetic 42 Asp Asp Asp Asp Lys 1 5
43 4 PRT Artificial Sequence Synthetic 43 Leu Val His Gly 1 44 37
DNA Artificial Sequence Synthetic 44 ggccacgcgt cgactagtac
tttttttttt ttttttt 37
* * * * *