U.S. patent application number 10/294796 was filed with the patent office on 2003-04-24 for connective tissue growth factor-2.
This patent application is currently assigned to Human Genome Sciences, Inc.. Invention is credited to Adams, Mark D., Li, Haodong.
Application Number | 20030078391 10/294796 |
Document ID | / |
Family ID | 22242737 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030078391 |
Kind Code |
A1 |
Li, Haodong ; et
al. |
April 24, 2003 |
Connective tissue growth factor-2
Abstract
The present invention relates to a human CTGF-2 polypeptide and
DNA (RNA) encoding such polypeptide. Also provided is a procedure
for producing such polypeptide by recombinant techniques and
antibodies and antagonist/inhibitors against such polypeptide. Also
provided are methods of using the polypeptide therapeutically for
enhancing the repair of connective and support tissue, promoting
the attachment, fixation and stabilization of tissue implants and
enhancing wound healing. Diagnostic assays for identifying
mutations in nucleic acid sequence encoding a polypeptide of the
present invention and for detecting altered levels of the
polypeptide of the present invention are also disclosed.
Inventors: |
Li, Haodong; (Gaithersburg,
MD) ; Adams, Mark D.; (Rockville, MD) |
Correspondence
Address: |
HUMAN GENOME SCIENCES INC
9410 KEY WEST AVENUE
ROCKVILLE
MD
20850
|
Assignee: |
Human Genome Sciences, Inc.
Rockville
MD
|
Family ID: |
22242737 |
Appl. No.: |
10/294796 |
Filed: |
November 15, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10294796 |
Nov 15, 2002 |
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09348815 |
Jul 8, 1999 |
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09348815 |
Jul 8, 1999 |
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08459101 |
Jun 2, 1995 |
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5945300 |
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08459101 |
Jun 2, 1995 |
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PCT/US94/07736 |
Jul 12, 1994 |
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Current U.S.
Class: |
530/399 ;
435/320.1; 435/325; 435/69.1; 536/23.2; 536/23.5 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 17/00 20180101; A61K 48/00 20130101; C07K 2319/00 20130101;
A61K 38/00 20130101; C07K 14/475 20130101; A61P 43/00 20180101;
A61P 35/00 20180101 |
Class at
Publication: |
530/399 ;
536/23.2; 536/23.5; 435/69.1; 435/320.1; 435/325 |
International
Class: |
C07K 014/475; C07H
021/04; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated polynucleotide comprising a member selected from the
group consisting of: (a) a polynucleotide encoding the polypeptide
comprising amino acid 1 to amino acid 381 as set forth in SEQ ID
NO:2; (b) a polynucleotide encoding the polypeptide comprising
amino acid 25 to amino acid 381 as set forth in SEQ ID NO:2 (c) a
polynucleotide capable of hybridizing to and which is at least 70%
identical to the polynucleotide of (a) or (b); and (d) a
polynucleotide fragment of the polynucleotide of (a), (b) or
(c).
2. The polynucleotide of claim 1 wherein the polynucleotide is
DNA.
3. The polynucleotide of claim 2 which encodes the polypeptide
comprising amino acid 1 to 351 of SEQ ID NO:2.
4. An isolated polynucleotide comprising a member selected from the
group consisting of: (a) a polynucleotide which encodes a mature
polypeptide having the amino acid sequence expressed by the DNA
contained in ATCC Deposit No. 75804; (b) a polynucleotide which
encodes a polypeptide having the amino acid sequence expressed by
the DNA contained in ATCC Deposit No. 75804; (c) a polynucleotide
capable of hybridizing to and which is at least 70% identical to
the polynucleotide of (a); and (d) a polynucleotide fragment of the
polynucleotide of (a), (b) or (c).
5. The polynucleotide of claim 1 comprising the sequence as set
forth in SEQ ID No. 1 from nucleotide 1 to nucleotide 1146.
6. The polynucleotide of claim 1 comprising the sequence as set
forth in SEQ ID No. 1 from nucleotide 73 to nucleotide 1146.
7. A vector containing the DNA of claim 2.
8. A host cell genetically engineered with the vector of claim
7.
9. A process for producing a polypeptide comprising: expressing
from the host cell of claim 8 the polypeptide encoded by said
DNA.
10. A process for producing cells capable of expressing a
polypeptide comprising genetically engineering cells with the
vector of claim 7.
11. A polypeptide encoded by the polynucleotide of claim 1
comprising a member selected from the group consisting of (i) a
mature polypeptide having the deduced amino acid sequence of SEQ ID
NO:2 and fragments, analogs and derivatives thereof; and (ii) a
mature polypeptide encoded by the cDNA of ATCC Deposit No. 75804
and fragments, analogs and derivatives of said polypeptide.
12. The polypeptide of claim 11 wherein the polypeptide comprises
amino acid 25 to amino acid 381 of SEQ ID NO:2.
13. A compound which inhibits activation of the receptor for the
polypeptide of claim 11.
14. A compound which activates the receptor for the polypeptide of
claim 11.
15. A method for the treatment of a patient having need of CTGF-2
comprising: administering to the patient a therapeutically
effective amount of the polypeptide of claim 11.
16. The method of claim 15 wherein said therapeutically effective
amount of the polypeptide is administered by providing to the
patient DNA encoding said polypeptide and expressing said
polypeptide in vivo.
17. A method for the treatment of a patient having need to inhibit
a CTGF-2 polypeptide comprising: administering to the patient a
therapeutically effective amount of the compound of claim 13.
18. A process for diagnosing a disease or a susceptibility to a
disease related to an under-expression of the polypeptide of claim
11 comprising: determining a mutation in a nucleic acid sequence
encoding said polypeptide.
19. A diagnostic process comprising: analyzing for the presence of
the polypeptide of claim 11 in a sample derived from a host.
20. A method for identifying agonist or antagonist compounds to the
polypeptide of claim 11 comprising: contacting a cell expressing on
the surface thereof a receptor for the polypeptide, said receptor
being associated with a second component capable of providing a
detectable signal in response to the binding of a compound to said
receptor, with an analytically detectable compound under conditions
to permit binding to the receptor; detecting the absence or
presence of a signal generated from the interaction of the compound
with the receptor.
Description
[0001] This application is a divisional of and claims priority
under 35 U.S.C. .sctn.120 to U.S. patent application Ser. No.
09/348,815, filed Jul. 8, 1999, which is a divisional of and claims
priority under 35 U.S.C. .sctn.120 to U.S. patent application Ser.
No. 08/459,101, filed Jun. 2, 1995, now U.S. Pat. No. 5,945,300,
granted Aug. 31, 1999, which is a continuation-in-part of and
claims priority under 35 U.S.C. .sctn.120 to U.S. Patent
Application No. PCT/US94/07736, filed Jul. 12, 1994; each of which
is hereby incorporated by reference in their entireties.
[0002] This invention relates to newly identified polynucleotides,
polypeptides encoded by such polynucleotides, the use of such
polynucleotides and polypeptides, as well as the production of such
polynucleotides and polypeptides. More particularly, the
polypeptide of the present invention is connective tissue growth
factor-2 sometimes hereinafter referred to as "CTGF-2". The
invention also relates to inhibiting the action of such
polypeptides.
[0003] The CTGF polypeptides are structurally and functionally
related to a family of growth factors which include IGF
(insulin-like growth factor), PDGF (platelet-derived growth
factor), and FGF (fibroblast growth factor). This emerging family
of secreted proteins are a group of cysteine-rich proteins. This
group of growth factors are important for normal growth,
differentiation, morphogenesis of the cartilaginous skeleton of an
embryo and cell growth. Among some of the functions that have been
discovered for these growth factors are wound healing, tissue
repair, implant fixation and stimulating increased bone mass.
[0004] The extended superfamily of growth factors include TGF
(transforming growth factor), bone morphogenic factors, and
activins, among others.
[0005] The most well-known growth factor, TGF exerts a number of
different effects on a variety of cells. For example, TGF-.beta.
can inhibit the differentiation of certain cells of mesodermal
origin (Florini, J. R. et al., J. Biol. Chem., 261:1659-16513
(1986) induced the differentiation of others (Seyedine, S. M. et
al., PNAS USA, 82:2267-2271 (1987) and potently inhibit
proliferation of various types of epithelial cells, (Tucker, R. F.,
Science, 226:705-705 (1984)). This last activity has led to the
speculation that one important physiological role for TGF-.beta. is
to maintain the repressed growth state of many types of cells.
Accordingly, cells that lose the ability to respond to TGF-.beta.
are more likely to exhibit uncontrolled growth and become
tumorigenic.
[0006] Accordingly, due to amino acid sequence homology the
polypeptide of the present invention is a member of this extended
family of growth factors which has many effects on a variety of
different tissues.
[0007] In accordance with one aspect of the present invention,
there is provided a novel mature polypeptide, as well as
biologically active and diagnostically or therapeutically useful
fragments, analogs and derivatives thereof. The polypeptide of the
present invention is of human origin.
[0008] In accordance with another aspect of the present invention,
there are provided isolated nucleic acid molecules encoding a
polypeptide of the present invention including mRNAs, DNAs, cDNAs,
genomic DNAs as well as analogs and biologically active and
diagnostically or therapeutically useful fragments thereof.
[0009] In accordance with yet a further aspect of the present
invention, there is provided a process for producing such
polypeptide by recombinant techniques comprising culturing
recombinant prokaryotic and/or eukaryotic host cells, containing a
nucleic acid sequence encoding a polypeptide of the present
invention, under conditions promoting expression of said protein
and subsequent recovery of said protein.
[0010] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing such
polypeptide, or polynucleotide encoding such polypeptide for
therapeutic purposes, for example, enhancing the repair of
connective and support tissue, promoting the attachment, fixation
and stabilization of tissue implants and enhancing wound
healing.
[0011] In accordance with yet a further aspect of the present
invention, there are provided antibodies against such
polypeptides.
[0012] In accordance with yet a further aspect of the present
invention, there is also provided nucleic acid probes comprising
nucleic acid molecules of sufficient length to specifically
hybridize to a nucleic acid sequence of the present invention.
[0013] In accordance with yet a further aspect of the present
invention, there are provided antibodies against such
polypeptides.
[0014] In accordance with another aspect of the present invention,
there are provided agonists which mimic the polypeptide of the
present invention and binds to the receptors.
[0015] In accordance with yet another aspect of the present
invention, there are provided antagonists to such polypeptides,
which may be used to inhibit the action of such polypeptides, for
example, in the treatment of CTGF dependent tumor growth.
[0016] In accordance with still another aspect of the present
invention, there are provided diagnostic assays for detecting
diseases or susceptibility to diseases related to mutations in the
nucleic acid sequences encoding a polypeptide of the present
invention.
[0017] In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing such
polypeptides, or polynucleotides encoding such polypeptides, for in
vitro purposes related to scientific research, for example,
synthesis of DNA and manufacture of DNA vectors.
[0018] These and other aspects of the present invention should be
apparent to those skilled in the art from the teachings herein.
[0019] The following drawings are illustrative of embodiments of
the invention and are not meant to limit the scope of the invention
as encompassed by the claims.
[0020] FIG. 1 depicts the cDNA sequence and corresponding deduced
amino acid sequence of CTGF-2. The standard one-letter abbreviation
for amino acids is used.
[0021] In accordance with an aspect of the present invention, there
is provided an isolated nucleic acid (polynucleotide) which encodes
for the mature polypeptide having the deduced amino acid sequence
of FIG. 1 (SEQ ID NO:2) or for the mature polypeptide encoded by
the cDNA of the clone deposited with the American Type Culture
Collection (ATCC, located at 10801 University Blvd. Manassas, Va.
20110-2209, USA) as ATCC Deposit No. 75804.
[0022] The polynucleotide of this invention was discovered in a
cDNA library derived from Human fetal lung. It is structurally
related to the IGF and PDGF family. It contains an open reading
frame encoding a protein of approximately 381 amino acid residues
of which approximately the first 24 amino acids residues are the
putative leader sequence such that the putative mature protein
comprises 357 amino acids. The protein exhibits the highest degree
of homology to Mouse CTGF with 49% identity and 67% similarity and
to Cyr61 with 89% identity and 93% similarity. Cyr61 is a growth
factor-inducible immediate early gene initially identified in
serum-stimulated mouse fibroblasts. It encodes a member of an
emerging family of cysteine-rich secreted proteins that includes a
connective tissue growth factor (O'Brien and Lau, L. F., Cell
Growth Differ., 3:645-54 (1992)).
[0023] The polynucleotide of the present invention may be in the
form of RNA or in the form of DNA, which DNA includes cDNA, genomic
DNA, and synthetic DNA. The DNA may be double-stranded or
single-stranded, and if single stranded may be the coding strand or
non-coding (anti-sense) strand. The coding sequence which encodes
the mature polypeptide may be identical to the coding sequence
shown in FIG. 1 (SEQ ID NO:1) or that of the deposited clone or may
be a different coding sequence which coding sequence, as a result
of the redundancy or degeneracy of the genetic code, encodes the
same mature polypeptide as the DNA of FIG. 1 (SEQ ID NO:1) or the
deposited cDNA.
[0024] The polynucleotide which encodes for the mature polypeptide
of FIG. 1 (SEQ ID NO:2) or for the mature polypeptide encoded by
the deposited cDNA may include: only the coding sequence for the
mature polypeptide; the coding sequence for the mature polypeptide
and additional coding sequence such as a leader or secretory
sequence or a proprotein sequence; the coding sequence for the
mature polypeptide (and optionally additional coding sequence) and
non-coding sequence, such as introns or non-coding sequence 5'
and/or 3' of the coding sequence for the mature polypeptide.
[0025] Thus, the term "polynucleotide encoding a polypeptide"
encompasses a polynucleotide which includes only coding sequence
for the polypeptide as well as a polynucleotide which includes
additional coding and/or non-coding sequence.
[0026] The present invention further relates to variants of the
hereinabove described polynucleotides which encode for fragments,
analogs and derivatives of the polypeptide having the deduced amino
acid sequence of FIG. 1 (SEQ ID NO:2) or the polypeptide encoded by
the cDNA of the deposited clone. The variant of the polynucleotide
may be a naturally occurring allelic variant of the polynucleotide
or a non-naturally occurring variant of the polynucleotide.
[0027] Thus, the present invention includes polynucleotides
encoding the same mature polypeptide as shown in FIG. 1 (SEQ ID
NO:2) or the same mature polypeptide encoded by the cDNA of the
deposited clone as well as variants of such polynucleotides which
variants encode for a fragment, derivative or analog of the
polypeptide of FIG. 1 (SEQ ID NO:2) or the polypeptide encoded by
the cDNA of the deposited clone. Such nucleotide variants include
deletion variants, substitution variants and addition or insertion
variants.
[0028] As hereinabove indicated, the polynucleotide may have a
coding sequence which is a naturally occurring allelic variant of
the coding sequence shown in FIG. 1 (SEQ ID NO:1) or of the coding
sequence of the deposited clone. As known in the art, an allelic
variant is an alternate form of a polynucleotide sequence which may
have a substitution, deletion or addition of one or more
nucleotides, which does not substantially alter the function of the
encoded polypeptide.
[0029] The polynucleotides of the present invention may also have
the coding sequence fused in frame to a marker sequence which
allows for purification of the polypeptide of the present
invention. The marker sequence may be a hexa-histidine tag supplied
by a pQE-9 vector to provide for purification of the mature
polypeptide fused to the marker in the case of a bacterial host,
or, for example, the marker sequence may be a hemagglutinin (HA)
tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag
corresponds to an epitope derived from the influenza hemagglutinin
protein (Wilson, I., et al., Cell, 37:767 (1984)).
[0030] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as
intervening sequences (introns) between individual coding segments
(exons).
[0031] Fragments of the full length gene of the present invention
may be used as a hybridization probe for a cDNA library to isolate
the full length cDNA and to isolate other cDNAs which have a high
sequence similarity to the gene or similar biological activity.
Probes of this type preferably have at least 30 bases and may
contain, for example, 50 or more bases. The probe may also be used
to identify a cDNA clone corresponding to a full length transcript
and a genomic clone or clones that contain the complete gene
including regulatory and promotor regions, exons, and introns. An
example of a screen comprises isolating the coding region of the
gene by using the known DNA sequence to synthesize an
oligonucleotide probe. Labeled oligonucleotides having a sequence
complementary to that of the gene of the present invention are used
to screen a library of human cDNA, genomic DNA or mRNA to determine
which members of the library the probe hybridizes to.
[0032] The present invention further relates to polynucleotides
which hybridize to the hereinabove-described sequences if there is
at least 90%, and more preferably at least 95% identity between the
sequences. The present invention particularly relates to
polynucleotides which hybridize under stringent conditions to the
hereinabove-described polynucleotides. As herein used, the term
"stringent conditions" means hybridization will occur only if there
is at least 95% and preferably at least 97% identity between the
sequences. The polynucleotides which hybridize to the hereinabove
described polynucleotides in a preferred embodiment encode
polypeptides which either retain substantially the same biological
function or activity as the mature polypeptide encoded by the cDNAs
of FIG. 1 (SEQ ID NO:1) or the deposited cDNA(s).
[0033] Alternatively, the polynucleotide may have at least 20
bases, preferably 30 bases, and more preferably at least 50 bases
which hybridize to a polynucleotide of the present invention and
which has an identity thereto, as hereinabove described, and which
may or may not retain activity. For example, such polynucleotides
may be employed as probes for the polynucleotide of SEQ ID NO:1,
for example, for recovery of the polynucleotide or as a diagnostic
probe or as a PCR primer.
[0034] Thus, the present invention is directed to polynucleotides
having at least a 90% and more preferably at least a 95% identity
to a polynucleotide which encodes the polypeptide of SEQ ID NO:2 as
well as fragments thereof, which fragments have at least 30 bases
and preferably at least 50 bases and to polypeptides encoded by
such polynucleotides.
[0035] The deposit(s) referred to herein will be maintained under
the terms of the Budapest Treaty on the International Recognition
of the Deposit of Micro-organisms for purposes of Patent Procedure.
These deposits are provided merely as convenience to those of skill
in the art and are not an admission that a deposit is required
under 35 U.S.C. .sctn.112. The sequence of the polynucleotides
contained in the deposited materials, as well as the amino acid
sequence of the polypeptides encoded thereby, are incorporated
herein by reference and are controlling in the event of any
conflict with any description of sequences herein. A license may be
required to make, use or sell the deposited materials, and no such
license is hereby granted.
[0036] The present invention further relates to a polypeptide which
has the deduced amino acid sequence of FIG. 1 (SEQ ID NO:2) or
which has the amino acid sequence encoded by the deposited cDNA, as
well as fragments, analogs and derivatives of such polypeptide.
[0037] The terms "fragment," "derivative" and "analog" when
referring to the polypeptide of FIG. 1 (SEQ ID NO:2) or that
encoded by the deposited cDNA, means a polypeptide which retains
essentially the same biological function or activity as such
polypeptide. Thus, an analog includes a proprotein which can be
activated by cleavage of the proprotein portion to produce an
active mature polypeptide.
[0038] The polypeptide of the present invention may be a
recombinant polypeptide, a natural polypeptide or a synthetic
polypeptide, preferably a recombinant polypeptide.
[0039] The fragment, derivative or analog of the polypeptide of
FIG. 1 (SEQ ID NO:2) or that encoded by the deposited cDNA may be
(i) one in which one or more of the amino acid residues are
substituted with a conserved or non-conserved amino acid residue
(preferably a conserved amino acid residue) and such substituted
amino acid residue may or may not be one encoded by the genetic
code, or (ii) one in which one or more of the amino acid residues
includes a substituent group, or (iii) one in which the mature
polypeptide is fused with another compound, such as a compound to
increase the half-life of the polypeptide (for example,
polyethylene glycol), or (iv) one in which the additional amino
acids are fused to the mature polypeptide, such as a leader or
secretory sequence or a sequence which is employed for purification
of the mature polypeptide or a proprotein sequence. Such fragments,
derivatives and analogs are deemed to be within the scope of those
skilled in the art from the teachings herein.
[0040] The polypeptides and polynucleotides of the present
invention are preferably provided in an isolated form, and
preferably are purified to homogeneity.
[0041] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated
from some or all of the coexisting materials in the natural system,
is isolated. Such polynucleotides could be part of a vector and/or
such polynucleotides or polypeptides could be part of a
composition, and still be isolated in that such vector or
composition is not part of its natural environment.
[0042] The polypeptides of the present invention include the
polypeptide of SEQ ID NO:2 (in particular the mature polypeptide)
as well as polypeptides which have at least 70% similarity
(preferably at least 70% identity) to the polypeptide of SEQ ID
NO:2 and more preferably at least 90% similarity (more preferably
at least 90% identity) to the polypeptide of SEQ ID NO:2 and still
more preferably at least 95% similarity (still more preferably at
least 95% identity) to the polypeptide of SEQ ID NO:2 and also
include portions of such polypeptides with such portion of the
polypeptide generally containing at least 30 amino acids and more
preferably at least 50 amino acids.
[0043] As known in the art "similarity" between two polypeptides is
determined by comparing the amino acid sequence and its conserved
amino acid substitutes of one polypeptide to the sequence of a
second polypeptide.
[0044] Fragments or portions of the polypeptides of the present
invention may be employed for producing the corresponding
full-length polypeptide by peptide synthesis; therefore, the
fragments may be employed as intermediates for producing the
full-length polypeptides. Fragments or portions of the
polynucleotides of the present invention may be used to synthesize
full-length polynucleotides of the present invention.
[0045] The present invention also relates to vectors which include
polynucleotides of the present invention, host cells which are
genetically engineered with vectors of the invention and the
production of polypeptides of the invention by recombinant
techniques.
[0046] Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors of this invention
which may be, for example, a cloning vector or an expression
vector. The vector may be, for example, in the form of a plasmid, a
viral particle, a phage, etc. The engineered host cells can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying the
genes of the present invention. The culture conditions, such as
temperature, pH and the like, are those previously used with the
host cell selected for expression, and will be apparent to the
ordinarily skilled artisan.
[0047] The polynucleotides of the present invention may be employed
for producing polypeptides by recombinant techniques. Thus, for
example, the polynucleotide may be included in any one of a variety
of expression vectors for expressing a polypeptide. Such vectors
include chromosomal, nonchromosomal and synthetic DNA sequences,
e.g., derivatives of SV40; bacterial plasmids; phage DNA;
baculovirus; yeast plasmids; vectors derived from combinations of
plasmids and phage DNA, viral DNA such as vaccinia, adenovirus,
fowl pox virus, and pseudorabies. However, any other vector may be
used as long as it is replicable and viable in the host.
[0048] The appropriate DNA sequence may be inserted into the vector
by a variety of procedures. In general, the DNA sequence is
inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Such procedures and others are deemed
to be within the scope of those skilled in the art.
[0049] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct mRNA synthesis. As representative examples of such
promoters, there may be mentioned: LTR or SV40 promoter, the E.
coli. lac or trp, the phage lambda P promoter and other promoters
known to control expression of genes in prokaryotic or 1 eukaryotic
L
[0050] cells or their viruses. The expression vector also contains
a ribosome binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for amplifying expression.
[0051] In addition, the expression vectors preferably contain one
or more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0052] The vector containing the appropriate DNA sequence as
hereinabove described, as well as an appropriate promoter or
control sequence, may be employed to transform an appropriate host
to permit the host to express the protein.
[0053] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonella typhimurium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO,
COS or Bowes melanoma; adenoviruses; plant cells, etc. The
selection of an appropriate host is deemed to be within the scope
of those skilled in the art from the teachings herein.
[0054] More particularly, the present invention also includes
recombinant constructs comprising one or more of the sequences as
broadly described above. The constructs comprise a vector, such as
a plasmid or viral vector, into which a sequence of the invention
has been inserted, in a forward or reverse orientation. In a
preferred aspect of this embodiment, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pD10,
phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A,
pNH46A (Stratagene); ptrc99a, pKK233-3, pKK233-3, pDR540, pRIT5
(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any
other plasmid or vector may be used as long as they are replicable
and viable in the host.
[0055] Promoter regions can be selected from any desired gene using
CAT (chloramphenicol transferase) vectors or other vectors with
selectable markers. Two appropriate vectors are pKK232-8 and pCM7.
Particular named bacterial promoters include lacI, lacZ, T3, T7,
gpt, lambda P, P and trp. Eukaryotic promoters include CMV
immediate early, HSV thymidine 2 k inase R , early L
[0056] and late SV40, LTRs from retrovirus, and mouse
metallothionein-I. Selection of the appropriate vector and promoter
is well within the level of ordinary skill in the art.
[0057] In a further embodiment, the present invention relates to
host cells containing the above-described constructs. The host cell
can be a higher eukaryotic cell, such as a mammalian cell, or a
lower eukaryotic cell, such as a yeast cell, or the host cell can
be a prokaryotic cell, such as a bacterial cell. Introduction of
the construct into the host cell can be effected by calcium
phosphate transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)).
[0058] The constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Alternatively, the polypeptides of the invention can be
synthetically produced by conventional peptide synthesizers.
[0059] Mature proteins can be expressed in mammalian cells, yeast,
bacteria, or other cells under the control of appropriate
promoters. Cell-free translation systems can also be employed to
produce such proteins using RNAs derived from the DNA constructs of
the present invention. Appropriate cloning and expression vectors
for use with prokaryotic and eukaryotic hosts are described by
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which
is hereby incorporated by reference.
[0060] Transcription of the DNA encoding the polypeptides of the
present invention by higher eukaryotes is increased by inserting an
enhancer sequence into the vector. Enhancers are cis-acting
elements of DNA, usually about from 10 to 300 bp that act on a
promoter to increase its transcription. Examples include the SV40
enhancer on the late side of the replication origin bp 100 to 270,
a cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus
enhancers.
[0061] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), -factor, acid phosphatase, or heat shock proteins,
among others. The heterologous structural sequence is assembled in
appropriate phase with translation initiation and termination
sequences, and preferably, a leader sequence capable of directing
secretion of translated protein into the periplasmic space or
extracellular medium. Optionally, the heterologous sequence can
encode a fusion protein including an N-terminal identification
peptide imparting desired characteristics, e.g., stabilization or
simplified purification of expressed recombinant product.
[0062] Useful expression vectors for bacterial use are constructed
by inserting a structural DNA sequence encoding a desired protein
together with suitable translation initiation and termination
signals in operable reading phase with a functional promoter. The
vector will comprise one or more phenotypic selectable markers and
an origin of replication to ensure maintenance of the vector and
to, if desirable, provide amplification within the host. Suitable
prokaryotic hosts for transformation include E. coli, Bacillus
subtilis, Salmonella typhimurium and various species within the
genera Pseudomonas, Streptomyces, and Staphylococcus, although
others may also be employed as a matter of choice.
[0063] As a representative but nonlimiting example, useful
expression vectors for bacterial use can comprise a selectable
marker and bacterial origin of replication derived from
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017). Such commercial
vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals,
Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis., USA).
These pBR322 "backbone" sections are combined with an appropriate
promoter and the structural sequence to be expressed.
[0064] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, the
selected promoter is induced by appropriate means (e.g.,
temperature shift or chemical induction) and cells are cultured for
an additional period.
[0065] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract
retained for further purification.
[0066] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents,
such methods are well known to those skilled in the art.
[0067] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts,
described by Gluzman, Cell, 23:175 (1981), and other cell lines
capable of expressing a compatible vector, for example, the C127,
3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors
will comprise an origin of replication, a suitable promoter and
enhancer, and also any necessary ribosome binding sites,
polyadenylation site, splice donor and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. DNA sequences derived from the SV40
splice, and polyadenylation sites may be used to provide the
required nontranscribed genetic elements.
[0068] The polypeptide can be recovered and purified from
recombinant cell cultures by methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or cation exchange
chromatography, phosphocellulose chromatography, hydrophobic
interaction chromatography, affinity chromatography,
hydroxylapatite chromatography and lectin chromatography. Protein
refolding steps can be used; as necessary, in completing
configuration of the mature protein. Finally, high performance
liquid chromatography (HPLC) can be employed for final purification
steps.
[0069] The polypeptides of the present invention may be a naturally
purified product, or a product of chemical synthetic procedures, or
produced by recombinant techniques from a prokaryotic or eukaryotic
host (for example, by bacterial, yeast, higher plant, insect and
mammalian cells in culture). Depending upon the host employed in a
recombinant production procedure, the polypeptides of the present
invention may be glycosylated or may be non-glycosylated.
Polypeptides of the invention may also include an initial
methionine amino acid residue.
[0070] The CTGF-2 polypeptides of the present invention may be
employed to enhance the repair of connective and support tissue.
For example CTGF-2 may be used to treat skin disorders such as
injuries, acne, aging, UV damage or burns. CTGF-2 may also be
employed to improve the cosmetic appearance of the skin, for
example, by treating wrinkled skin.
[0071] CTGF-2 may also be employed to promote the attachment,
fixation and stabilization of tissue implants, for example, a
prosthesis and other implants inserted during reconstructive
surgery. The CTGF-2 polypeptide of the present invention may be
employed in the healing of external wounds, by promoting growth of
epithelial and connective tissues and the synthesis of total
protein and collagen. CTGF-2 may be applied in the area of injured
or depleted bones, with regeneration occurring by promoting the
growth of connective tissue, bone and cementum and by stimulating
protein and collagen synthesis which is especially useful for
periodontal disease.
[0072] This invention provides a method for identification of the
receptor for the CTGF-2 polypeptide. The gene encoding the receptor
can be identified by expression cloning. Briefly, polyadenylated
RNA is prepared from a cell responsive to CTGF-2, and a cDNA
library created from this RNA is divided into pools and used to
transfect COS cells or other cells that are not responsive to
CTGF-2. Transfected cells which are grown on glass slides are
exposed to labeled CTGF-2. The CTGF-2 can be labeled by a variety
of means including iodidation or inclusion of a recognition site
for a site-specific protein kinase. Following fixation and
incubation, the slides are subjected to autoradiographic analysis.
Positive pools are identified and sub-pools are prepared and
retransfected using an iterative sub-pooling and rescreening
process, eventually yielding a single clone that encodes the
putative receptor. As an alternative approach for receptor
identification, labeled ligand can be photoaffinity linked with
cell membrane or extract preparations that express the receptor
molecule. Cross-linked material is resolved by PAGE and exposed to
x-ray film. The labeled complex containing the CTGF-2-receptor can
be excised, resolved into peptide fragments, and subjected to
protein microsequencing. The amino acid sequence obtained from
microsequencing would be used to design a set of generate
oligonucleotide probes to screen a cDNA library to identify the
gene encoding the putative receptor.
[0073] This invention also provides a method of screening compounds
to identify those which bind to the CTGF-2 receptor and elicit a
second messenger response (agonists) or do not elicit a second
messenger response (antagonists). As an example, a mammalian cell
or membrane preparation expressing the CTGF-2 receptor would be
incubated with a labeled compound. The response of a known second
messenger system following interaction of the compound and the
CTGF-2 receptor is then measured. Such second messenger systems
include but are not limited to, cAMP guanylate cyclase, ion
channels or phosphoinositide hydrolysis.
[0074] The present invention is also directed to antagonists
molecules of the polypeptides of the present invention, and their
use in reducing or eliminating the function of CTGF-2.
[0075] An example of an antagonist is an antibody or in some cases,
an oligonucleotide, which binds to the CTGF-2 polypeptide.
Alternatively, antagonists include closely related proteins that
have lost biological function and thereby prevent the action of
CTGF-2 since receptor sites are occupied.
[0076] Antisense technology may be employed to decrease the level
of in vivo circulation of CTGF-2. Antisense technology can be used
to control gene expression through triple-helix formation or
antisense DNA or RNA, both of which methods are based on binding of
a polynucleotide to DNA or RNA. For example, the 5' coding portion
of the polynucleotide sequence, which encodes for the mature
polypeptides of the present invention, is used to design an
antisense RNA oligonucleotide of from about 10 to 40 base pairs in
length. A DNA oligonucleotide is designed to be complementary to a
region of the gene involved in transcription (triple helix--see Lee
et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science,
241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),
thereby preventing transcription and the production of CTGF-2. The
antisense RNA oligonucleotide hybridizes to the mRNA in vivo and
blocks translation of the mRNA molecule into CTGF-2
(antisense--Okano, J. Neurochem., 56:560 (1991);
Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression,
CRC Press, Boca Raton, Fla. (1988)). The oligonucleotides described
above can also be delivered to cells such that the antisense RNA or
DNA may be expressed in vivo to inhibit production of CTGF-2.
[0077] Another example of an antagonist is a small molecule which
binds to the CTGF-2 receptors such that normal biological activity
is prevented. Examples of small molecules include but are not
limited to small peptides or peptide-like molecules.
[0078] The antagonists may be employed to prevent scar formation
due to excess proliferation of connective tissues and to prevent
CTGF-2 dependent tumor growth. The antagonists may be employed in a
composition with a pharmaceutically acceptable carrier, e.g., as
hereinabove described.
[0079] The present invention also relates to an assay for
identifying potential antagonists specific to CTGF-2. An example of
such an assay combines CTGF-2 and a potential antagonist with
membrane-bound CTGF-2 receptors or recombinant CTGF-2 under
appropriate conditions for a competitive inhibition assay. CTGF-2
can be labeled, such as by radio activity, such that the number of
CTGF-2 molecules bound to the receptor can determine the
effectiveness of the potential antagonist.
[0080] The polypeptides and antagonists and agonists of the present
invention may be employed in combination with a suitable
pharmaceutical carrier. Such compositions comprise a
therapeutically effective amount of the polypeptide, agonist or
antagonist, and a pharmaceutically acceptable carrier or excipient.
Such a carrier includes but is not limited to saline, buffered
saline, dextrose, water, glycerol, ethanol, and combinations
thereof. The formulation should suit the mode of
administration.
[0081] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use
or sale of pharmaceuticals or biological products, which notice
reflects approval by the agency of manufacture, use or sale for
human administration. In addition, the polypeptides, agonists and
antagonists of the present invention may be employed in conjunction
with other therapeutic compounds.
[0082] The pharmaceutical compositions may be administered in a
convenient manner such as by the topical, intravenous,
intraperitoneal, intramuscular, subcutaneous, intranasal or
intradermal routes. CTGF-2 is administered in an amount which is
effective for treating and/or prophylaxis of the specific
indication. In general, CTGF-2 will be administered in an amount of
at least about 10 .mu.g/kg body weight and in most cases CTGF-2
will be administered in an amount not in excess of about 8 mg/Kg
body weight per day. In most cases, the dosage is from about 10
.mu.g/kg to about 1 mg/kg body weight daily, taking into account
the routes of administration, symptoms, etc.
[0083] The polypeptides may also be employed in accordance with the
present invention by expression of such polypeptides in vivo, which
is often referred to as "gene therapy."
[0084] Thus, for example, cells from a patient may be engineered
with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo,
with the engineered cells then being provided to a patient to be
treated with the polypeptide. Such methods are well-known in the
art. For example, cells may be engineered by procedures known in
the art by use of a retroviral particle containing RNA encoding a
polypeptide of the present invention.
[0085] Similarly, cells may be engineered in vivo for expression of
a polypeptide in vivo by, for example, procedures known in the art.
As known in the art, a producer cell for producing a retroviral
particle containing RNA encoding the polypeptide of the present
invention may be administered to a patient for engineering cells in
vivo and expression of the polypeptide in vivo. These and other
methods for administering a polypeptide of the present invention by
such method should be apparent to those skilled in the art from the
teachings of the present invention. For example, the expression
vehicle for engineering cells may be other than a retrovirus, for
example, an adenovirus which may be used to engineer cells in vivo
after combination with a suitable delivery vehicle.
[0086] Retroviruses from which the retroviral plasmid vectors
hereinabove mentioned may be derived include, but are not limited
to, Moloney Murine Leukemia Virus, spleen necrosis virus,
retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus,
avian leukosis virus, gibbon ape leukemia virus, human
immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma
Virus, and mammary tumor virus. In one embodiment, the retroviral
plasmid vector is derived from Moloney Murine Leukemia Virus.
[0087] The vector includes one or more promoters. Suitable
promoters which may be employed include, but are not limited to,
the retroviral LTR; the SV40 promoter; and the human
cytomegalovirus (CMV) promoter described in Miller, et al.,
Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter
(e.g., cellular promoters such as eukaryotic cellular promoters
including, but not limited to, the histone, pol III, and
.beta.-actin promoters). Other viral promoters which may be
employed include, but are not limited to, adenovirus promoters,
thymidine kinase (TK) promoters, and B19 parvovirus promoters. The
selection of a suitable promoter will be apparent to those skilled
in the art from the teachings contained herein.
[0088] The nucleic acid sequence encoding the polypeptide of the
present invention is under the control of a suitable promoter.
Suitable promoters which may be employed include, but are not
limited to, adenoviral promoters, such as the adenoviral major late
promoter; or hetorologous promoters, such as the cytomegalovirus
(CMV) promoter; the respiratory syncytial virus (RSV) promoter;
inducible promoters, such as the MMT promoter, the metallothionein
promoter; heat shock promoters; the albumin promoter; the ApoAI
promoter; human globin promoters; viral thymidine kinase promoters,
such as the Herpes Simplex thymidine kinase promoter; retroviral
LTRs (including the modified retroviral LTRs hereinabove
described); the .beta.-actin promoter; and human growth hormone
promoters. The promoter also may be the native promoter which
controls the gene encoding the polypeptide. The retroviral plasmid
vector is employed to transduce packaging cell lines to form
producer cell lines. Examples of packaging cells which may be
transfected include, but are not limited to, the PE501, PA317,
.psi.-2, .psi.-AM, PA12, T19-14X, VT-19-17-H2, .psi.CRE, 104 CRIP,
GP+E-86, GP+envAm12, and DAN cell lines as described in Miller,
Human Gene Therapy, Vol. 1, pgs. 5-14 (1990), which is incorporated
herein by reference in its entirety. The vector may transduce the
packaging cells through any means known in the art. Such means
include, but are not limited to, electroporation, the use of
liposomes, and CaPO.sub.4 precipitation. In one alternative, the
retroviral plasmid vector may be encapsulated into a liposome, or
coupled to a lipid, and then administered to a host.
[0089] The producer cell line generates infectious retroviral
vector particles which include the nucleic acid sequence(s)
encoding the polypeptides. Such retroviral vector particles then
may be employed, to transduce eukaryotic cells, either in vitro or
in vivo. The transduced eukaryotic cells will express the nucleic
acid sequence(s) encoding the polypeptide. Eukaryotic cells which
may be transduced include, but are not limited to, embryonic stem
cells, embryonic carcinoma cells, as well as hematopoietic stem
cells, hepatocytes, fibroblasts, myoblasts, keratinocytes,
endothelial cells, and bronchial epithelial cells.
[0090] This invention is also related to the use of the gene of the
present invention as a diagnostic. Detection of a mutated form of
the gene will allow a diagnosis of a disease or a susceptibility to
a disease which results from underexpression of CTGF-2.
[0091] Individuals carrying mutations in the gene of the present
invention may be detected at the DNA level by a variety of
techniques. Nucleic acids for diagnosis may be obtained from a
patient's cells, including but not limited to blood, urine, saliva,
tissue biopsy and autopsy material. The genomic DNA may be used
directly for detection or may be amplified enzymatically by using
PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analysis.
RNA or cDNA may also be used for the same purpose. As an example,
PCR primers complementary to the nucleic acid encoding CTGF-2 can
be used to identify and analyze mutations. For example, deletions
and insertions can be detected by a change in size of the amplified
product in comparison to the normal genotype. Point mutations can
be identified by hybridizing amplified DNA to radiolabeled RNA or
alternatively, radiolabeled antisense DNA sequences. Perfectly
matched sequences can be distinguished from mismatched duplexes by
RNase A digestion or by differences in melting temperatures.
[0092] Sequence differences between the reference gene and genes
having mutations may be revealed by the direct DNA sequencing
method. In addition, cloned DNA segments may be employed as probes
to detect specific DNA segments. The sensitivity of this method is
greatly enhanced when combined with PCR. For example, a sequencing
primer is used with double-stranded PCR product or a
single-stranded template molecule generated by a modified PCR. The
sequence determination is performed by conventional procedures with
radiolabeled nucleotide or by automatic sequencing procedures with
fluorescent-tags.
[0093] Genetic testing based on DNA sequence differences may be
achieved by detection of alteration in electrophoretic mobility of
DNA fragments in gels with or without denaturing agents. Small
sequence deletions and insertions can be visualized by high
resolution gel electrophoresis. DNA fragments of different
sequences may be distinguished on denaturing formamide gradient
gels in which the mobilities of different DNA fragments are
retarded in the gel at different positions according to their
specific melting or partial melting temperatures (see, e.g., Myers
et al., Science, 230:1242 (1985)).
[0094] Sequence changes at specific locations may also be revealed
by nuclease protection assays, such as RNase and S1 protection or
the chemical cleavage method (e.g., Cotton et al., PNAS, USA,
85:4397-4401 (1985)).
[0095] Thus, the detection of a specific DNA sequence may be
achieved by methods such as hybridization, RNase protection,
chemical cleavage, direct DNA sequencing or the use of restriction
enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP))
and Southern blotting of genomic DNA.
[0096] In addition to more conventional gel-electrophoresis and DNA
sequencing, mutations can also be detected by in situ analysis.
[0097] The present invention also relates to a diagnostic assay for
detecting altered levels of the polypeptide of the present
invention in various tissues since an over-expression of the
proteins compared to normal control tissue samples can detect the
presence of disorders of the host. Assays used to detect levels of
the polypeptide of the present invention in a sample derived from a
host are well-known to those of skill in the art and include
radioimmunoassays, competitive-binding assays, Western Blot
analysis and preferably an ELISA assay. An ELISA assay initially
comprises preparing an antibody specific to the CTGF-2 antigen,
preferably a monoclonal antibody. In addition a reporter antibody
is prepared against the monoclonal antibody. To the reporter
antibody is attached a detectable reagent such as radioactivity,
fluorescence or in this example a horseradish peroxidase enzyme. A
sample is now removed from a host and incubated on a solid support,
e.g. a polystyrene dish that binds the proteins in the sample. Any
free protein binding sites on the dish are then covered by
incubating with a non-specific protein such as bovine serum
albumin. Next, the monoclonal antibody is incubated in the dish
during which time the monoclonal antibodies attached to the
polypeptide of the present invention attached to the polystyrene
dish. All unbound monoclonal antibody is washed out with buffer.
The reporter antibody linked to horseradish peroxidase is now
placed in the dish resulting in binding of the reporter antibody to
any monoclonal antibody bound to the polypeptide of the present
invention. Unattached reporter antibody is then washed out.
Peroxidase substrates are then added to the dish and the amount of
color developed in a given time period is a measurement of the
amount of the polypeptide of the present invention present in a
given volume of patient sample when compared against a standard
curve.
[0098] A competition assay may be employed wherein antibodies
specific to the polypeptide of the present invention are attached
to a solid support and labeled CTGF-2 and a sample derived from the
host are passed over the solid support and the amount of label
detected attached to the solid support can be correlated to a
quantity of the polypeptide of the present invention in the
sample.
[0099] The sequences of the present invention are also valuable for
chromosome identification. The sequence is specifically targeted to
and can hybridize with a particular location on an individual human
chromosome. Moreover, there is a current need for identifying
particular sites on the chromosome. Few chromosome marking reagents
based on actual sequence data (repeat polymorphisms) are presently
available for marking chromosomal location. The mapping of DNAs to
chromosomes according to the present invention is an important
first step in correlating those sequences with genes associated
with disease.
[0100] Briefly, sequences can be mapped to chromosomes by preparing
PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis
of the 3' untranslated region of the gene is used to rapidly select
primers that do not span more than one exon in the genomic DNA,
thus complicating the amplification process. These primers are then
used for PCR screening of somatic cell hybrids containing
individual human chromosomes. Only those hybrids containing the
human gene corresponding to the primer will yield an amplified
fragment.
[0101] PCR mapping of somatic cell hybrids is a rapid procedure for
assigning a particular DNA to a particular chromosome. Using the
present invention with the same oligonucleotide primers,
sublocalization can be achieved with panels of fragments from
specific chromosomes or pools of large genomic clones in an
analogous manner. Other mapping strategies that can similarly be
used to map to its chromosome include in situ hybridization,
prescreening with labeled flow-sorted chromosomes and preselection
by hybridization to construct chromosome specific-cDNA
libraries.
[0102] Fluorescence in situ hybridization (FISH) of a cDNA clone to
a metaphase chromosomal spread can be used to provide a precise
chromosomal location in one step. This technique can be used with
cDNA having at least 50 or 60 bases. For a review of this
technique, see Verma et al., Human Chromosomes: a Manual of Basic
Techniques, Pergamon Press, New York (1988).
[0103] Once a sequence has been mapped to a precise chromosomal
location, the physical position of the sequence on the chromosome
can be correlated with genetic map data. Such data are found, for
example, in V. McKusick, Mendelian Inheritance in Man (available on
line through Johns Hopkins University Welch Medical Library). The
relationship between genes and diseases that have been mapped to
the same chromosomal region are then identified through linkage
analysis (coinheritance of physically adjacent genes).
[0104] Next, it is necessary to determine the differences in the
cDNA or genomic sequence between affected and unaffected
individuals. If a mutation is observed in some or all of the
affected individuals but not in any normal individuals, then the
mutation is likely to be the causative agent of the disease.
[0105] With current resolution of physical mapping and genetic
mapping techniques, a cDNA precisely localized to a chromosomal
region associated with the disease could be one of between 50 and
500 potential causative genes. (This assumes 1 megabase mapping
resolution and one gene per 20 kb).
[0106] The polypeptides, their fragments or other derivatives, or
analogs thereof, or cells expressing them can be used as an
immunogen to produce antibodies thereto. These antibodies can be,
for example, polyclonal or monoclonal antibodies. The present
invention also includes chimeric, single chain, and humanized
antibodies, as well as Fab fragments, or the product of an Fab
expression library. Various procedures known in the art may be used
for the production of such antibodies and fragments.
[0107] Antibodies generated against the polypeptides corresponding
to a sequence of the present invention can be obtained by direct
injection of the polypeptides into an animal or by administering
the polypeptides to an animal, preferably a nonhuman. The antibody
so obtained will then bind the polypeptides itself. In this manner,
even a sequence encoding only a fragment of the polypeptides can be
used to generate antibodies binding the whole native polypeptides.
Such antibodies can then be used to isolate the polypeptide from
tissue expressing that polypeptide.
[0108] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, 1975, Nature, 256:495-497), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., 1983, Immunology
Today 4:72), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0109] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce
single chain antibodies to immunogenic polypeptide products of this
invention. Also, transgenic mice may be used to express humanized
antibodies to immunogenic polypeptide products of this
invention.
[0110] The present invention will be further described with
reference to the following examples; however, it is to be
understood that the present invention is not limited to such
examples. All parts or amounts, unless otherwise specified, are by
weight.
[0111] In order to facilitate understanding of the following
examples certain frequently occurring methods and/or terms will be
described.
[0112] "Plasmids" are designated by a lower case p preceded and/or
followed by capital letters and/or numbers. The starting plasmids
herein are either commercially available, publicly available on an
unrestricted basis, or can be constructed from available plasmids
in accord with published procedures. In addition, equivalent
plasmids to those described are known in the art and will be
apparent to the ordinarily skilled artisan.
[0113] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 .mu.g of
plasmid or DNA fragment is used with about 2 units of enzyme in
about 20 .mu.l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 .mu.g of DNA
are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubation
times of about 1 hour at 37.degree. C. are ordinarily used, but may
vary in accordance with the supplier's instructions. After
digestion the reaction is electrophoresed directly on a
polyacrylamide gel to isolate the desired fragment.
[0114] Size separation of the cleaved fragments is performed using
8 percent polyacrylamide gel described by Goeddel, D. et al.,
Nucleic Acids Res., 8:4057 (1980).
[0115] "Oligonucleotides" refers to either a single stranded
polydeoxynucleotide or two complementary polydeoxynucleotide
strands which may be chemically synthesized. Such synthetic
oligonucleotides have no 5' phosphate and thus will not ligate to
another oligonucleotide without adding a phosphate with an ATP in
the presence of a kinase. A synthetic oligonucleotide will ligate
to a fragment that has not been dephosphorylated.
[0116] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Maniatis,
T., et al., Id., p. 146). Unless otherwise provided, ligation may
be accomplished using known buffers and conditions with 10 units to
T4 DNA ligase ("ligase") per 0.5 .mu.g of approximately equimolar
amounts of the DNA fragments to be ligated.
[0117] Unless otherwise stated, transformation was performed as
described in the method of Graham, F. and Van der Eb, A., Virology,
52:456-457 (1973).
EXAMPLE 1
[0118] Cloning and Expression of CTGF-2 in a Baculovirus Expression
System
[0119] The DNA sequence encoding the full length CTGF-2 protein,
ATCC #75804, was amplified using PCR oligonucleotide primers
corresponding to the 5' and 3' sequences of the gene:
[0120] The 5' primer has the sequence CGCGGGATCCTGCGCGACACAATGAGCT
(SEQ ID NO:3) and contains a BamHI restriction enzyme site (in
bold) followed by 18 nucleotides resembling an efficient signal for
the initiation of translation in eukaryotic cells (J. Mol. Biol.
1987, 196, 947-950, Kozak, M.). The initiation codon for
translation "ATG" is underlined.
[0121] The 3' primer has the sequence
CGCGGGTACCAGGTAGCATTTAGTCCCTAA (SEQ ID NO:4) and contains the
cleavage site for the restriction endonuclease Asp781 and 20
nucleotides complementary to the 3' non-translated sequence of the
CTGF-2 gene. The amplified sequences were isolated from a 1%
agarose gel using a commercially available kit ("Geneclean", BIO
101 Inc., La Jolla, Calif.). The fragment was then digested with
the endonucleases BamHI and Asp781 and then purified by isolation
on a 1% agarose gel. This fragment is designated F2.
[0122] The vector pRG1 (modification of pVL941 vector, discussed
below) is used for the expression of the CTGF-2 protein using the
baculovirus expression system (for review see: Summers, M. D. and
Smith, G. E. 1987, A manual of methods for baculovirus vectors and
insect cell culture procedures, Texas Agricultural Experimental
Station Bulletin No. 1555). This expression vector contains the
strong polyhedrin promoter of the Autographa californica nuclear
polyhedrosis virus (AcMNPV) followed by the recognition sites for
the restriction endonucleases BamHI and Asp781. The polyadenylation
site of the simian virus (SV)40 is used for efficient
polyadenylation. For an easy selection of recombinant viruses the
beta-galactosidase gene from E.coli is inserted in the same
orientation as the polyhedrin promoter followed by the
polyadenylation signal of the polyhedrin gene. The polyhedrin
sequences are flanked at both sides by viral sequences for the
cell-mediated homologous recombination of cotransfected wild-type
viral DNA. Many other baculovirus vectors could be used in place of
pRG1 such as pAc373, pVL941 and pAcIM1 (Luckow, V. A. and Summers,
M. D., Virology, 170:31-39).
[0123] The plasmid was digested with the restriction enzymes BamHI
and Asp781 and then dephosphorylated using calf intestinal
phosphatase by procedures known in the art. The DNA was then
isolated from a 1% agarose gel. This vector DNA is designated
V2.
[0124] Fragment F2 and the dephosphorylated plasmid V2 were ligated
with T4 DNA ligase. E.coli HB101 cells were then transformed and
bacteria identified that contained the plasmid (pBacCTGF-2) with
the CTGF-2 gene using the enzymes BamHI and Asp781. The sequence of
the cloned fragment was confirmed by DNA sequencing.
[0125] 5 .mu.g of the plasmid pBacCTGF-2 were cotransfected with
1.0 .mu.g of a commercially available linearized baculovirus
("BaculoGold baculovirus DNA", Pharmingen, San Diego, Calif.) using
the lipofection method (Felgner et al. Proc. Natl. Acad. Sci. USA,
84:7413-7417 (1987)).
[0126] 1 .mu.g of BaculoGold virus DNA and 5 .mu.g of the plasmid
pBacCTGF-2 were mixed in a sterile well of a microtiter plate
containing 50 .mu.l of serum free Grace's medium (Life Technologies
Inc., Gaithersburg, Md.). Afterwards 10 .mu.l Lipofectin plus 90
.mu.l Grace's medium were added, mixed and incubated for 15 minutes
at room temperature. Then the transfection mixture was added
dropwise to the Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm
tissue culture plate with 1 ml Grace' medium without serum. The
plate was rocked back and forth to mix the newly added solution.
The plate was then incubated for 5 hours at 27.degree. C. After 5
hours the transfection solution was removed from the plate and 1 ml
of Grace's insect medium supplemented with 10% fetal calf serum was
added. The plate was put back into an incubator and cultivation
continued at 27.degree. C. for four days.
[0127] After four days the supernatant was collected and a plaque
assay performed similar as described by Summers and Smith (supra).
As a modification an agarose gel with "Blue Gal" (Life Technologies
Inc., Gaithersburg) was used which allows an easy isolation of blue
stained plaques. (A detailed description of a "plaque assay" can
also be found in the user's guide for insect cell culture and
baculovirology distributed by Life Technologies Inc., Gaithersburg,
page 9-10).
[0128] Four days after the serial dilution of the viruses was added
to the cells, blue stained plaques were picked with the tip of an
Eppendorf pipette. The agar containing the recombinant viruses was
then resuspended in an Eppendorf tube containing 200 .mu.l of
Grace's medium. The agar was removed by a brief centrifugation and
the supernatant containing the recombinant baculoviruses was used
to insect Sf9 cells seeded in 35 mm dishes. Four days later the
supernatants of these culture dishes were harvested and then stored
at 4.degree. C.
[0129] Sf9 cells were grown in Grace's medium supplemented with 10%
heat-inactivated FBS. The cells were infected with the recombinant
baculovirus V-CTGF-2 at a multiplicity of infection (MOI) of 2. Six
hours later the medium was removed and replaced with SF900 II
medium minus methionine and cysteine (Life Technologies Inc.,
Gaithersburg). 42 hours later 5 .mu.Ci of .sup.35S-methionine and 5
.mu.Ci .sup.35S cysteine (Amersham) were added. The cells were
further incubated for 16 hours before they were harvested by
centrifugation and the labelled proteins visualized by SDS-PAGE and
autoradiography.
EXAMPLE 2
[0130] Expression of Recombinant CTGF-2 in COS Cells
[0131] The expression of plasmid, CTGF-2 HA is derived from a
vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of
replication, 2) ampicillin resistance gene, 3) E.coli replication
origin, 4) CMV promoter followed by a polylinker region, a SV40
intron and polyadenylation site. A DNA fragment encoding the entire
CTGF-2 precursor and a HA tag fused in frame to its 3' end was
cloned into the polylinker region of the vector, therefore, the
recombinant protein expression is directed under the CMV promoter.
The HA tag correspond to an epitope derived from the influenza
hemagglutinin protein as previously described (I. Wilson, H. Niman,
R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell
37, 767). The infusion of HA tag to our target protein allows easy
detection of the recombinant protein with an antibody that
recognizes the HA epitope.
[0132] The plasmid construction strategy is described as
follows:
[0133] The DNA sequence encoding for CTGF-2, ATCC #75804, was
constructed by PCR on the full-length clone using two primers: the
5' primer 5' AAAGGATCCACAATGAGCTCCCGAATC (SEQ ID NO:5) 3' contains
a Bam HI site followed by 18 nucleotides of CTGF-2 coding sequence
starting from the -3 position relative to the initiation codon; the
3' sequence 5'
CGCTCTAGATTAAGCGTAGTCTGGGACGTCGTATGGGTATTGGAACAGCCTGT AGAAG 5' (SEQ
ID NO:6) contains complementary sequences to an Xba I site,
translation stop codon, HA tag and the last 19 nucleotides of the
CTGF-2 coding sequence (not including the stop codon). Therefore,
the PCR product contains a Bam HI site, CTGF-2 coding sequence
followed by an HA tag fused in frame, a translation termination
stop codon next to the HA tag, and an Xba I site. The PCR amplified
DNA fragment and the vector, pcDNAI/Amp, were digested with Bam HI
and Xba I restriction enzymes and ligated. The ligation mixture was
transformed into E. coli strain SURE (available from Stratagene
Cloning Systems, 11099 North Torrey Pines Road, La Jolla, Calif.
92037) the transformed culture was plated on ampicillin media
plates and resistant colonies were selected. Plasmid DNA was
isolated from transformants and examined by restriction analysis
for the presence of the correct fragment. For expression of the
recombinant CTGF-2, COS cells were transfected with the expression
vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T.
Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring
Laboratory Press, (1989)). The expression of the CTGF-2 HA protein
was detected by radiolabelling and immunoprecipitation method. (E.
Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours
with .sup.35S-cysteine two days post transfection. Culture media
were then collected and cells were lysed with detergent (RIPA
buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM
Tris, pH 7.5). (Wilson, I. et al., Id. 37:767 (1984)). Both cell
lysate and culture media were precipitated with a HA specific
monoclonal antibody. Proteins precipitated were analyzed on 15%
SDS-PAGE gels.
EXAMPLE 3
[0134] Expression Via Gene Therapy
[0135] Fibroblasts are obtained from a subject by skin biopsy. The
resulting tissue is placed in tissue-culture medium and separated
into small pieces. Small chunks of the tissue are placed on a wet
surface of a tissue culture flask, approximately ten pieces are
placed in each flask. The flask is turned upside down, closed tight
and left at room temperature over night. After 24 hours at room
temperature, the flask is inverted and the chunks of tissue remain
fixed to the bottom of the flask and fresh media (e.g., Ham's F12
media, with 10% FBS, penicillin and streptomycin, is added. This is
then incubated at 37.degree. C. for approximately one week. At this
time, fresh media is added and subsequently changed every several
days. After an additional two weeks in culture, a monolayer of
fibroblasts emerge. The monolayer is trypsinized and scaled into
larger flasks.
[0136] pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988)
flanked by the long terminal repeats of the Moloney murine sarcoma
virus, is digested with EcoRI and HindIII and subsequently treated
with calf intestinal phosphatase. The linear vector is fractionated
on agarose gel and purified, using glass beads.
[0137] The cDNA encoding a polypeptide of the present invention is
amplified using PCR primers which correspond to the 5' and 3' end
sequences respectively. The 5' primer containing an EcoRI site and
the 3' primer further includes a HindIII site. Equal quantities of
the Moloney murine sarcoma virus linear backbone and the amplified
EcoRI and HindIII fragment are added together, in the presence of
T4 DNA ligase. The resulting mixture is maintained under conditions
appropriate for ligation of the two fragments. The ligation mixture
is used to transform bacteria HB101, which are then plated onto
agar-containing kanamycin for the purpose of confirming that the
vector had the gene of interest properly inserted.
[0138] The amphotropic pA317 or GP+am12 packaging cells are grown
in tissue culture to confluent density in Dulbecco's Modified
Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and
streptomycin. The MSV vector containing the gene is then added to
the media and the packaging cells are transduced with the vector.
The packaging cells now produce infectious viral particles
containing the gene (the packaging cells are now referred to as
producer cells).
[0139] Fresh media is added to the transduced producer cells, and
subsequently, the media is harvested from a 10 cm plate of
confluent producer cells. The spent media, containing the
infectious viral particles, is filtered through a millipore filter
to remove detached producer cells and this media is then used to
infect fibroblast cells. Media is removed from a sub-confluent
plate of fibroblasts and quickly replaced with the media from the
producer cells. This media is removed and replaced with fresh
media. If the titer of virus is high, then virtually all
fibroblasts will be infected and no selection is required. If the
titer is very low, then it is necessary to use a retroviral vector
that has a selectable marker, such as neo or his.
[0140] The engineered fibroblasts are then injected into the host,
either alone or after having been grown to confluence on cytodex 3
microcarrier beads. The fibroblasts now produce the protein
product.
[0141] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, within the scope of the appended claims, the invention
may be practiced otherwise than as particularly described.
Sequence CWU 1
1
6 1 1146 DNA Homo sapiens CDS (1)..(1146) 1 atg agc tcc cgc atc gcc
agg gcg ctc gcc tta gtc gtc acc ctt ctc 48 Met Ser Ser Arg Ile Ala
Arg Ala Leu Ala Leu Val Val Thr Leu Leu 1 5 10 15 cac ttg acc agg
ctg gcg ctc tcc acc tgc ccc gct gcc tgc cac tgc 96 His Leu Thr Arg
Leu Ala Leu Ser Thr Cys Pro Ala Ala Cys His Cys 20 25 30 ccc ctg
gag gcg ccc aag tgc gcg ccg gga gtc ggg ctg gtc cgg gac 144 Pro Leu
Glu Ala Pro Lys Cys Ala Pro Gly Val Gly Leu Val Arg Asp 35 40 45
ggc tgc ggc tgc tgt aag gtc tgc gcc aag cag ctc aac gag gac tgc 192
Gly Cys Gly Cys Cys Lys Val Cys Ala Lys Gln Leu Asn Glu Asp Cys 50
55 60 agc aaa acg cag ccc tgc gac cac acc aag ggg ctg gaa tgc aac
ttc 240 Ser Lys Thr Gln Pro Cys Asp His Thr Lys Gly Leu Glu Cys Asn
Phe 65 70 75 80 ggc gcc agc tcc acc gct ctg aag ggg atc tgc aga gct
cag tca gag 288 Gly Ala Ser Ser Thr Ala Leu Lys Gly Ile Cys Arg Ala
Gln Ser Glu 85 90 95 ggc aga ccc tgt gaa tat aac tcc aga atc tac
caa aac ggg gaa agt 336 Gly Arg Pro Cys Glu Tyr Asn Ser Arg Ile Tyr
Gln Asn Gly Glu Ser 100 105 110 ttc cag ccc aac tgt aaa cat cag tgc
aca tgt att gat ggc gcc gtg 384 Phe Gln Pro Asn Cys Lys His Gln Cys
Thr Cys Ile Asp Gly Ala Val 115 120 125 ggc tgc att cct ctg tgt ccc
caa gaa cta tct ctc ccc aac ttg ggc 432 Gly Cys Ile Pro Leu Cys Pro
Gln Glu Leu Ser Leu Pro Asn Leu Gly 130 135 140 tgt ccc aac cct cgg
ctg gtc aaa gtt acc ggg cag tgc tgc gag gag 480 Cys Pro Asn Pro Arg
Leu Val Lys Val Thr Gly Gln Cys Cys Glu Glu 145 150 155 160 tgg gtc
tgt gac gag gat agt atc aag gac ccc atg gag gac cag gac 528 Trp Val
Cys Asp Glu Asp Ser Ile Lys Asp Pro Met Glu Asp Gln Asp 165 170 175
ggc ctc ctt ggc aag gag ctg gga ttc gat gcc tcc gag gtg gag ttg 576
Gly Leu Leu Gly Lys Glu Leu Gly Phe Asp Ala Ser Glu Val Glu Leu 180
185 190 acg aga aac aat gaa ttg att gca gtt gga aaa ggc agc tca ctg
aag 624 Thr Arg Asn Asn Glu Leu Ile Ala Val Gly Lys Gly Ser Ser Leu
Lys 195 200 205 cgg ctc cct gtt ttt gga atg gag cct cgc atc cta tac
aac cct tta 672 Arg Leu Pro Val Phe Gly Met Glu Pro Arg Ile Leu Tyr
Asn Pro Leu 210 215 220 caa ggc cag aaa tgt att gtt caa aca act tca
tgg tcc cag tgc tca 720 Gln Gly Gln Lys Cys Ile Val Gln Thr Thr Ser
Trp Ser Gln Cys Ser 225 230 235 240 aag acc tgt gga act ggt atc tcc
aca cga gtt acc aat gac aac cct 768 Lys Thr Cys Gly Thr Gly Ile Ser
Thr Arg Val Thr Asn Asp Asn Pro 245 250 255 gag tgc cgc ctt gtg aaa
gaa acc cgg att tgt gag gtg cgg cct tgt 816 Glu Cys Arg Leu Val Lys
Glu Thr Arg Ile Cys Glu Val Arg Pro Cys 260 265 270 gga cag cca gtg
tac agc agc ctg aaa aag ggc aag aaa tgc agc aag 864 Gly Gln Pro Val
Tyr Ser Ser Leu Lys Lys Gly Lys Lys Cys Ser Lys 275 280 285 acc aag
aaa tcc ccc gaa cca gtc agg ttt act tac gct gga tgt ttg 912 Thr Lys
Lys Ser Pro Glu Pro Val Arg Phe Thr Tyr Ala Gly Cys Leu 290 295 300
agt gtg aag aaa tac cgg ccc aag tac tgc ggt tcc tgc gtg gac ggc 960
Ser Val Lys Lys Tyr Arg Pro Lys Tyr Cys Gly Ser Cys Val Asp Gly 305
310 315 320 cga tgc tgc acg ccc cag ctg acc agg act gtg aag atg cgg
ttc cgc 1008 Arg Cys Cys Thr Pro Gln Leu Thr Arg Thr Val Lys Met
Arg Phe Arg 325 330 335 tgc gaa gat ggg gag aca ttt tcc aag aac gtc
atg atg atc cag tcc 1056 Cys Glu Asp Gly Glu Thr Phe Ser Lys Asn
Val Met Met Ile Gln Ser 340 345 350 tgc aaa tgc aac tac aac tgc ccg
cat gcc aat gaa gca gcg ttt ccc 1104 Cys Lys Cys Asn Tyr Asn Cys
Pro His Ala Asn Glu Ala Ala Phe Pro 355 360 365 ttc tac agg ctg ttc
aat gac att cac aaa ttt agg gac taa 1146 Phe Tyr Arg Leu Phe Asn
Asp Ile His Lys Phe Arg Asp 370 375 380 2 381 PRT Homo sapiens 2
Met Ser Ser Arg Ile Ala Arg Ala Leu Ala Leu Val Val Thr Leu Leu 1 5
10 15 His Leu Thr Arg Leu Ala Leu Ser Thr Cys Pro Ala Ala Cys His
Cys 20 25 30 Pro Leu Glu Ala Pro Lys Cys Ala Pro Gly Val Gly Leu
Val Arg Asp 35 40 45 Gly Cys Gly Cys Cys Lys Val Cys Ala Lys Gln
Leu Asn Glu Asp Cys 50 55 60 Ser Lys Thr Gln Pro Cys Asp His Thr
Lys Gly Leu Glu Cys Asn Phe 65 70 75 80 Gly Ala Ser Ser Thr Ala Leu
Lys Gly Ile Cys Arg Ala Gln Ser Glu 85 90 95 Gly Arg Pro Cys Glu
Tyr Asn Ser Arg Ile Tyr Gln Asn Gly Glu Ser 100 105 110 Phe Gln Pro
Asn Cys Lys His Gln Cys Thr Cys Ile Asp Gly Ala Val 115 120 125 Gly
Cys Ile Pro Leu Cys Pro Gln Glu Leu Ser Leu Pro Asn Leu Gly 130 135
140 Cys Pro Asn Pro Arg Leu Val Lys Val Thr Gly Gln Cys Cys Glu Glu
145 150 155 160 Trp Val Cys Asp Glu Asp Ser Ile Lys Asp Pro Met Glu
Asp Gln Asp 165 170 175 Gly Leu Leu Gly Lys Glu Leu Gly Phe Asp Ala
Ser Glu Val Glu Leu 180 185 190 Thr Arg Asn Asn Glu Leu Ile Ala Val
Gly Lys Gly Ser Ser Leu Lys 195 200 205 Arg Leu Pro Val Phe Gly Met
Glu Pro Arg Ile Leu Tyr Asn Pro Leu 210 215 220 Gln Gly Gln Lys Cys
Ile Val Gln Thr Thr Ser Trp Ser Gln Cys Ser 225 230 235 240 Lys Thr
Cys Gly Thr Gly Ile Ser Thr Arg Val Thr Asn Asp Asn Pro 245 250 255
Glu Cys Arg Leu Val Lys Glu Thr Arg Ile Cys Glu Val Arg Pro Cys 260
265 270 Gly Gln Pro Val Tyr Ser Ser Leu Lys Lys Gly Lys Lys Cys Ser
Lys 275 280 285 Thr Lys Lys Ser Pro Glu Pro Val Arg Phe Thr Tyr Ala
Gly Cys Leu 290 295 300 Ser Val Lys Lys Tyr Arg Pro Lys Tyr Cys Gly
Ser Cys Val Asp Gly 305 310 315 320 Arg Cys Cys Thr Pro Gln Leu Thr
Arg Thr Val Lys Met Arg Phe Arg 325 330 335 Cys Glu Asp Gly Glu Thr
Phe Ser Lys Asn Val Met Met Ile Gln Ser 340 345 350 Cys Lys Cys Asn
Tyr Asn Cys Pro His Ala Asn Glu Ala Ala Phe Pro 355 360 365 Phe Tyr
Arg Leu Phe Asn Asp Ile His Lys Phe Arg Asp 370 375 380 3 28 DNA
Artificial sequence 5' primer sequence containing a BamHI
restriction enzyme site followed by 18 nucleotides resembling an
efficient signal for the initiation of translation in eukaryotic
cells 3 cgcgggatcc tgcgcgacac aatgagct 28 4 30 DNA Artificial
sequence 3' primer sequence containing the cleavage site for the
restriction endonuclease Asp781 and 20 nucleotides complementary
tothe 3' non-translated sequence of the CTGF-2 gene 4 cgcgggtacc
aggtagcatt tagtccctaa 30 5 27 DNA Artificial sequence 5' primer
sequence containing a BamHI site followed by 18 nucleotides of
CTGF-2 coding sequence 5 aaaggatcca caatgagctc ccgaatc 27 6 58 DNA
Artificial sequence 3' primer sequence containing complementary
sequences to an XbaI site, translation stop codin, HA tag and the
last 19 nucleotides of the CTGF-2 coding sequence 6 cgctctagat
taagcgtagt ctgggacgtc gtatgggtat tggaacagcc tgtagaag 58
* * * * *