Title of Invention

NEW HUMAN GROWTH DIFFERENTIATION FACTOR ENCODING SEQUENCE AND POLYPEPTIDE ENCODED BY SUCH DNA SEQUENCE AND PRODUCING METHOD THEREOF

Abstract The invention provides a cDNA sequence of a new human growth differentiation factor (hGDF3-2). The protein encoded by such sequence is a splice variant of hGDF3. The present invention also relates to peptides encoded by the nucleotide sequences, to uses of these polynucleotides and polypeptides, and methods for producing the said polynucleotides and polypeptides.
Full Text NEW HUMAN GROWTH DIFFERENTIATION FACTOR ENCODING SEQUENCE
AND POLYPEPTIDE ENCODED BY SUCH DNA SEQUENCE
AND PRODUCING METHOD THEREOF
Field of invention
This invention relates to the field of genetic engineering, and, in particular, relates to the nucleotide
sequence of a novel human gene. More particularly, this invention relates to the cDNA sequence of a novel
human Growth/Differentiation Factor (hGDF3-2), which is a splice variant of hGDF3. The invention also
relates to the polypeptides encoded by the nucleotide sequence, the uses of these polynucleotides and
polypeptides, and the methods for producing them.
Prior art
Transforming Growth Factor- β (TGF- β ) was discovered about 15 years ago, using biochemical
means. It is a protein with many biological regulatory activities. Shortly after the discovery, it was found
that TGF- β represented a group of growth factors with various functions. In different organisms, these
factors exert important regulatory functions on cell growth, differentiation and tissue morphogenesis.
( Handbook of Experimental Pharmacology, 1990, Vol.95, p419-475, Springer Verlag, Geidelberg). TGF- β ,
together with these related proteins, forms a superfamily named TFG-β superfamily. Up to now, the TGF- β superfamily has over 30 different members. There are four major families in the TGF- β superfamily
(Proc Soc Exp Biol Med, 1997, 214(1), 27-40), which are: (1) the Mullerian inhibitory substance (MIS)
family— MIS regulates Mullerian duct regression in male embryos; (2) the inhibin/activin family ---
Inhibins block the follicle stimulating hormone (FSH) release by the pituitary cell, and activins stimulate
FSH release; (3) Vg-related family, which includes bone morphogenic protein (BMP), dorsalin-1 (which
regulates the differentiation of neural tube), growth/differentiation factor GDF-1, DPP, Vgl in the Xenopus
and the murine homolog Vgr-1, etc.; (4) TGF- β family, which includes five isoforms of TGF-β (TGF- β 1-5).
As a representative of this superfamily, TGF- β has been extensively and intensively studied. The
investigation indicates that TGF- β is a strong endogenous mediators of tissue repair via their stimulatory
effects on chemotaxis, angiogenesis, and extracellular matrix (ECM) deposition within the wound
environment. (Clin Immunol Immunopatol, 1997,83(1), 25-30). TGF-β also regulates the growth and
differentiation of various cells (Bioessays,1997, 19(7), 581-591), either positively or negatively. Most of
the evidences suggest that TGF- β exerts its regulatory effects at the G1 phase of cell cycle. Besides, it is
reported that TGF- β can induce cell death of some sensitive cell types, including hepatoma, myeloid,
and osteoclast cells. In vitro experiments also show TGF- β regulates the differentiation of various cell
strains, though the mechanism is still unknown. The regulatory activity of TGF- β on cell growth and
differentiation naturally leads to considerations on the potential application in chemotherapy and cancer
therapy. There have been considerable amount of reports concerning these topics (Clin Immunol
Immunopathol, 1997, 83(1), 25-30; Bioessays, 1997, 19(7),581-591).
Members of TGF- β family have been found in many species, e.g., Xenopus, fowl, mice, swine,

bovine, etc. Human TGF-β (-1,-2,-3) were cloned in the late 1980's. Among them, the sequencing of TGF-
P , was finished by Derynck R et al. in 1985. (Nature, 1985, 316(6030), 701-705). By analyzing the
sequence encoding TGF- β 1, they found that functional TGF- P was produced by splicing a precursor that
was much longer than the mature protein. Later, people found this phenomenon was common in TGF- P
superfamily. In 1988, the TGF- β 2 and TGF- β 3 nucleotide sequences were obtained by Madisen L et al.
and Ten Dijke P et al., respectively. ( Proc Natl Acad Sci USA, 1988, 85(13), 4715-4719; DNA, 1988,
7(1), 1-8 ). Sequence comparison showed the homology between TGF-β 2, TGF-β 3 and TGF-β , was
70%-80%.
Along with the steady improvements of gene cloning and sequencing techniques, more and more
members of TGF-β superfamily have been cloned since 1990. Alexandra. C reported in 1993 that they
found a novel member of TGF-β superfamily — murine Growth/Differentiation Factor 3, GDF-3 (J. Biol.
Chem., 1993, 268(5), 3444-3449). The homology between GDF-3 and other members of the TGF-β
superfamily is not very high. But it still contains the unique conservative sequence of the TGF-β
superfamily. In particular, it lacks the fourth cystein of the seven conservative cysteins of the superfamily,
indicating that it might have some particular property.
The homologue of GDF-3 in human was cloned in 1998 (Oncogene, 1998, 16, 95-103). This protein is
highly homologous to the murine GDF-3, and thus named hGDF-3. Nevertheless, it is noteworthy that
hGDF-3 is much shorter than GDF-3, mainly due to the lack of nearly 50 residues in the N-terminal.
Moreover, two residues corresponding to residues 128 and 248 in the murine GDF-3 are also deleted in
hGDF-3. This change of hGDF-3 is supposed to be the result of alternative splicing variation or genetic
evolution.
Prior to this invention, no other forms of hGDF3 has been isolated or disclosed.
Summary of Invention
One purpose of the invention is to provide a new polynucleotide which encodes a splice variant of
human growth/differentiation factor hGDF3. The splice variant of hGDF3 of the invention is named
hGDF3-2.
Another purpose of the invention is to provide a novel protein, which is named hGDF3-2.
Still another purpose of the invention is to provide a new method for preparing said new hGDF3-2
protein by recombinant techniques.
The invention also relates to the uses of said hGDF3-2 protein and its coding sequence.
In one aspect, the invention provides an isolated DNA molecule, which comprises a nucleotide
sequence encoding a polypeptide having human hGDF3-2 protein activity, wherein said nucleotide
sequence shares at least 70% homology to the nucleotide sequence of nucleotides 14-1105 in SEQ ID NO:
5, or said nucleotide sequence can hybridize to the nucleotide sequence of nucleotides 14-1105 in SEQ ID
NO: 5 under moderate stringency. Preferably, said nucleotide sequence encodes a polypeptide comprising
the amino acid sequence of SEQ ID NO: 6. More preferably, the sequence comprises the nucleotide
sequence of nucleotides 14-1105 in SEQ ID NO: 5.

Further, the invention provides an isolated hGDF3-2 polypeptide, which comprises a polypeptide
having the amino acid sequence of SEQ ID NO: 6, its active fragments, and its active derivatives.
Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 6.
The invention also provides a vector comprising said isolated DNA. '"
The invention further provides a host cell transformed with said vector.
In another aspect, the invention provides a method for producing a polypeptide with the activity of
hGDF3-2 protein, which comprises:
(a) forming a hGDF3-2 protein expression vector comprising the nucleotide sequence encoding the
polypeptide having the activity of hGDF3-2 protein, wherein said nucleotide sequence is operably linked
with an expression regulatory sequences, and said nucleotide sequence shares at least 70% homology to the
nucleotide sequence of positions 14-1105 in SEQ ID NO: 5;
(b) introducing the vector of step (a) into a host cell, thereby forming a recombinant cell of hGDF3-2
protein;
(c) culturing the recombinant cell of step (b) under the conditions suitable for the expression of
hGDF3-2 polypeptides;
(d) isolating the polypeptides having the activity of hGDF3-2 protein.
In one embodiment of the present invention, the isolated polynucleotide has a full length of 1141
nucleotides, whose detailed sequence is shown in SEQ ID NO: 5. The open reading frame (ORF) is located
at nucleotides 14-1105.
In the present invention, the term "isolated" or "purified" or "substantially pure" DNA refers to a DNA
or fragment which has been isolated from the sequences which frank it in a naturally occurring state. The
term also applies to DNA or DNA fragment which has been isolated from other components naturally
accompanying the nucleic acid and from proteins naturally accompanying it in the cell.
In the present invention, the term "hGDF3-2 protein encoding sequence" or " hGDF3-2 polypeptide
encoding sequence" refers to a nucleotide sequence encoding a polypeptide having the activity of hGDF3-2
protein, such as the nucleotide sequence of positions 14-1105 in SEQ ED NO: 5 or its degenerate sequence.
The degenerate sequences means the sequences formed by replacing one or more codons in the ORF of 14-
1105 in SEQ ED NO: 5 with degenerate codes which encode the same amino acid. Because of the
degeneracy of codon, the sequence having a homology as low as about 70% to the sequence of nucleotides
14-1105 in SEQ ED NO: 5 can also encode the sequence shown in SEQ ED NO: 6. The term also refers to
the nucleotide sequences that hybridize to the nucleotide sequence of nucleotides 14-1105 in SEQ ED NO:
5 under moderate stringency or preferably under high stringency. In addition, the term also refers to the
sequences having a homology of at least 70%, preferably 80%, more preferably 90% to the nucleotide
sequence of nucleotides 14-1105 in SEQ ED NO: 5.
The term also refers to variants of the sequence in SEQ ED NO: 5, which are capable of encoding a
protein having the same function as human hGDF3-2 protein. These variants includes, but are not limited to,
deletions, insertions and/or substitutions of several nucleotides (typically 1-90, preferably 1-60, more
preferably 1-20, and most preferably 1-10) and additions of several nucleotides (typically less than 60,

preferably 30, more preferably 10, most preferably 5) at 5' end and/or 3' end.
In the present invention, "substantially pure" proteins or polypeptides refers to those which occupy at
least 20%, preferably at least 50%, more preferably at least 80%, most preferably at least 90% of the total
sample material (by wet weight or dry weight). Purity can be measured by any appropriate method, e.g., in
the case of polypeptides by column chromatography, PAGE or HPLC analysis. A substantially purified
polypeptides is essentially free of naturally associated components.
In the present invention, the term "hGDF3-2 polypeptide" or "hGDF3-2 protein" refers to a
polypeptide having the activity of hGDF3-2 protein comprising the amino acid sequence of SEQ ID NO: 6.
The term also comprises the variants of said amino acid sequence which have the same function of human
hGDF3-2. These variants include, but are not limited to, deletions, insertions and/or substitutions of several
amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and addition of
one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) at C-
terminal and/or N-terminal. For example, the protein functions are usually unchanged when an amino
residue is substituted by a similar or analogous one. Further, the addition of one or several amino acids at
C-terminal and/or N-terminal will not change the function of protein. The term also includes the active
fragments and derivatives of hGDF3-2 protein.
The variants of polypeptide include homologous sequences, allelic variants, natural mutants, induced
mutants, proteins encoded by DNA which hybridizes to hGDF3-2 DNA under high or low stringency
conditions as well as the polypeptides or proteins retrieved by antisera raised against hGDF3-2 polypeptide.
The present invention also provides other polypeptides, e.g., fusion proteins, which include the hGDF3-2
polypeptide or fragments thereof. In addition to substantially full-length polypeptide, the soluble fragments
of hGDF3-2 polypeptide are also included. Generally, these fragments comprise at least 10, typically at
least 30, preferably at least 50, more preferably at least 80, most preferably at least 100 consecutive amino
acids of hGDF3-2 polypeptide.
The present invention also provides the analogues of hGDF3-2 protein or polypeptide. Analogues can
differ from naturally occurring hGDF3-2 polypeptide by amino acid sequence differences or by
modifications which do not affect the sequence, or by both. These polypeptides include genetic variants,
both natural and induced. Induced variants can be made by various techniques, e.g., by random mutagenesis
using irradiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular
biologic techniques. Also included are analogues which include residues other than those naturally
occurring L-amino acids ( e.g., D-amino acids) or non-naturally occurring or synthetic amino acids (e.g.,
beta- or gamma-amino acids). It is understood that the polypeptides of the invention are not limited to the
representative polypeptides listed hereinabove.
Modifications ( which do not normally alter primary sequence) include in vivo, or in vitro chemical
derivation of polypeptides, e.g., acelylation, or carboxylation. Also included are modifications of
glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its
synthesis and processing or in the further processing steps, e.g., by exposing the polypeptide to enzymes
which affect glycosylation (e.g., mammalian glycosylating or deglycosylating enzymes). Also included are
sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine,

phosphothronine, as well as sequences which have been modified to improve their resistance to proteolytic
degradation or to optimize solubility properties.
The invention also includes antisense sequence of the sequence encoding hGDF3-2 polypeptide. Said
antisense sequence can be used to inhibit expression of hGDF3-2 in cells.
The invention also includes probes, typically having 8-100, preferably 15-50 consecutive nucleotides.
These probes can be used to detect the presence of nucleic acid molecules coding for hGDF3-2 in samples.
The present invention also includes methods for detecting hGDF3-2 nucleotide sequences, which
comprises hybridizing said probes to samples, and detecting the binding of the probes. Preferably, the
samples are products of PCR amplification. The primers in PCR amplification correspond to coding
sequence of hGDF3-2 polypeptide and are located at both ends or in the middle of the coding sequence. In
general, the length of the primers is 20 to 50 nucleotides.
A variety of vectors known in the art, such as those commercially available, are useful in the invention.
In the invention, the term "host cells" includes prokaryotic and eukaryotic cells. The common
prokaryotic host cells include Escherichi coli, Bacillus subtilis, and so on. The common eukaryotic host
cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cells are eukaryotic cells,
e.g., CHO cells, COS cells, and the like.
In another aspect, the invention also includes antibodies, preferably monoclonal antibodies, which are
specific for polypeptides encoded by hGDF3-2 DNA or fragments thereof. By "specificity", it is meant an
antibody which binds to the hGDF3-2 gene products or a fragments thereof. Preferably, the antibody binds
to the hGDF3-2 gene products or a fragments thereof and does not substantially recognize nor bind to other
antigenically unrelated molecules. Antibodies which bind to hGDF3-2 and block hGDF3-2 protein and
those which do not affect the hGDF3-2 function are included in the invention. The invention also includes
antibodies which bind to the hGDF3-2 gene product in its unmodified as well as modified form.
The present invention includes not only intact monoclonal or polyclonal antibodies, but also
immunologically-active antibody fragments, e.g., a Fab' or (Fab)2 fragment, an antibody light chain, an
antibody heavy chain, a genetically engineered single chain Fv molecule (Lander, et al.,US Pat No.
4,946,778), or a chimeric antibody, e.g., an antibody which contains the binding specificity of a murine
antibody, but the remaining portion of which is of human origin.
The antibodies in the present invention can be prepared by various techniques known to those skilled
in the art. For example, purified hGDF3-2 gene products, or its antigenic fragments can be administrated to
animals to induce the production of polyclonal antibodies. Similarly, cells expressing hGDF3-2 or its
antigenic fragments can be used to immunize animals to produce antibodies. Antibodies of the invention
can be monoclonal antibodies which can be prepared by using hybridoma technique (See Kohler, et al.,
Nature, 256; 495,1975; Kohler, et al., Eur. J. Immunol. 6: 511,1976; Kohler, et al., Eur. J. Immunol. 6: 292,
1976; Hammerling, et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981).
Antibodies of the invention comprise those which block hGDF3-2 function and those which do not affect
hGDF3-2 function. Antibodies in the invention can be produced by routine immunology techniques and
using fragments or functional regions of hGDF3-2 gene product. These fragments and functional regions
can be prepared by recombinant methods or synthesized by a polypeptide synthesizer. Antibodies binding

to unmodified hGDF3-2 gene product can be produced by immunizing animals with gene products
produced by prokaryotic cells (e.g., E. coli); antibodies binding to post-translationally modified forms
thereof can be acquired by immunizing animals with gene products produced by eukaryotic cells (e.g.,
yeast or insect cells).
The full length human hGDF3-2 nucleotide sequence or its fragment of the invention can be prepared
by PCR amplification, recombinant method and synthetic method. For PCR amplification, one can obtain
said sequences by designing primers based on the nucleotide sequence disclosed in the invention,
especially the sequence of ORF, and using cDNA library commercially available or prepared by routine
techniques known in the art as a template. When the sequence is long, it is usually necessary to perform
two or more PCR amplifications and link the amplified fragments together in the correct order.
Once the sequence is obtained, a great amount of the sequences can be produced by recombinant
methods. Usually, said sequence is cloned in a vector which is then transformed into a host cell. Then the
sequence is isolated from the amplified host cells using conventional techniques.
Further, the sequence can be produced by synthesis. Typically, several small fragments are synthesized
and linked together to obtain a long sequence. At present, it is completely feasible to chemically synthesize
the DNA sequence encoding the protein of the invention, or the fragments or derivatives thereof. In
addition, the mutation can be introduced into the sequence of the protein by chemical synthesis.
In addition to recombinant techniques, the protein fragments of the invention may also be prepared by
direct chemical synthesis using solid phase synthesis techniques (Stewart et al., (1969) Solid-Phase Peptide
Synthesis, WH Freeman Co., San Francisco; Merrifield J. (1963), J. Am. Chem. Assoc. 85: 2149-2154). In
vitro protein synthesis can be performed manually or automatically, e.g., using a Model 431 Peptide
Synthesizer (Applied Biosystems, Foster City, CA). The fragments of protein of the invention can be
synthesized separately and linked together using chemical methods so as to produce full-length molecule.
The sequences encoding the protein of the present invention are also valuable for gene mapping. For
example, the accurate chromosome mapping can be performed by hybridizing cDNA clones to a
chromosome in metaphase. This technique can use cDNA as short as about 500bp, or as long as about
2000bp, or more. For details, see Verma et al., Human Chromosomes: A Manual of Basic Techniques,
Pergamon Press, New York (1988).
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 in, e.g.,
Mendelian Inheritance in Man (available on-line through Johns Hopkins University Welch Medical
Library). The relationships between genes and diseases that have been mapped to the same chromosomal
region are then identified through linkage analysis.
Then, the differences in the cDNA or genomic sequence between affected and unaffected individuals
can also be determined. If a mutation is observed in some or all of the affected individuals but not in any
normal individual, then the mutation is likely to be the causative agent of the disease.
The substances which act with the hGDF3-2, e.g., receptors, inhibitors and antagonists, can be
screened out by various conventional techniques, using the protein of the invention.
The protein, antibody, inhibitor, antagonist or receptor of the invention provide different effects when

administrated in therapy. Usually, these substances are formulated with a non-toxic, inert and
pharmaceutically acceptable aqueous carrier. The pH typically ranges from 5 to 8, preferably from about 6
to 8, although pH may alter according to the property of the formulated substances and the diseases to be
treated. The formulated pharmaceutical composition is administrated in conventional routine including, but
not limited to, intramuscular, intraperitoneal, subcutaneous, intracutaneous, or topical administration.
As an example, the human hGDF3-2 protein of the invention may be administrated together with the
suitable and pharmaceutically acceptable carrier. The examples of carriers include, but are not limited to,
saline, buffer solution, glucose, water, glycerin, ethanol, or the combination thereof. The pharmaceutical
formulation should be suitable for the delivery method. The human hGDF3-2 protein of the invention may
be in the form of injections which are made by conventional methods, using physiological saline or other
aqueous solution containing glucose or auxiliary substances. The pharmaceutical compositions in the form
of tablet or capsule may be prepared by routine methods. The pharmaceutical compositions, e.g., injections,
solutions, tablets, and capsules, should be manufactured under sterile conditions. The active ingredient is
administrated in therapeutically effective amount, e.g., from about lug to 5mg per kg body weight per day.
Moreover, the polypeptide of the invention can be administrated together with other therapeutic agent.
When the human hGDF3-2 polypeptides of the invention are used as a pharmaceutical, the
therapeutically effective amount of the polypeptides are administrated to mammals. Typically, the
therapeutically effective amount is at least about 10 ug/kg body weight and less than about 8 mg/kg body
weight in most cases, and preferably about 10ug-1mg/kg body weight. Of course, the precise amount will
depend upon various factors, such as delivery methods, the subject health, and the like, and is within the
judgment of the skilled clinician.
Description of Drawings
Fig. 1 shows an alignment comparison of amino acid sequences of hGDF3-2 of the invention and
murine GDF3 and human GDF3 (hGDF3). The identical and similar amino acids are indicated by ":" and
"." between the sequences, respectively.
In one embodiment, the cDNA sequence of hGDF3-2 was obtained as follows: human bone marrow
λ gt 11 cDNA library (Clontech) was used as a template and the forward primer Al :5'-
GGAGCTCTCCCCGGTCTGAC-3', A2: 5'-CACTCCAGAGGCCATGCTTCG-3', and reverse primers
B1:5'-CCTAAGAACACTCCTTCTATTCC-3', B2:5'-CTAAGTGGTCATAAACCAGATTAGG-3' were
synthesized. Primers A1, B2 were first used to amplify the cDNA library of human bone marrow. Then an
additional PCR was carried out with primers A2 and B1, using the amplified product as a template. Target
fragments of 1141bp were obtained. The sequencing of the PCR product gave the full length cDNA
sequence shown in SEQ ID NO: 5. v
Homology comparison showed that the nucleotide sequence and the coded protein sequence of the
invention shared remarkable homology to murine GDF3. Noticeably, it is completely consistent with the
human GDF-3 (hGDF3) except the 5' end, and the only difference is that the encoded proteins have
different lengths of N-terminal. However, hGDF3-2 shares higher homology to murine GDF-3. According

to the expression pattern in the tissues, hGDF3-2 is supposed to be related to lymphocytopoiesis,
erythropoiesis and embryonic development of skeleton and cartilage. Moreover, the specific expression of
hGDF3 in the embryonal carcinoma (EC) stem cells indicates it may be used as a molecular marker for EC
cells and may play a role in the formation and maintenance of EC stem cells.
The invention is further illustrated by the following examples. It is appreciated that these examples are
only intended to illustrate the invention, but not to limit the scope of the invention. For the experimental
methods in the following examples, they are performed under routine conditions, e.g., those described by
Sambrook. et al., in Molecule Clone: A Laboratory Manual, New York: Cold Spring Harbor Laboratory
Press, 1989, or as instructed by the manufacturers, unless otherwise specified.
Examples
Example 1
The cloning and sequencing of hGDF3-2 cDNA sequence
1. Amplification with primers
The template was human bone marrow λ gt 11 cDNA library (commercially available from Clontech).
The PCR were carried out with two pairs of primers: forward primers Al: 5'- GGAGCTCTCC
CCGGTCTGAC-3' (SEQ ID NO:l), and A2:5'-CACTCCAGAGGCCATGCTTCG-3' (SEQ ID NO:2);
reverse primers B1: 5'- CCTAAGAACACTCCTTCTATTCC-3' (SEQ ID NO:3), and B2:5'-
CTAAGTGGTCATAAACCAGATTAGG-3' (SEQ ID NO:4). Firstly, PCR was carried out using primers
Al and B2. The PCR condition was 4 mins at 93°C; followed by 35 cycles with 1 min at 93°C, 1 min at
66°C, and 1 min at 72°C; and, finally 5 mins at 72°C. Then, using the above PCR product as a template, an
additional PCR was carried out with primers A2 and B1. The PCR condition was 4 mins at 93°C; followed
by 35 cycles with 1 min at 93°C, 1 min at 64°C, and 1 min at 72°C; and, finally, 5 mins at 72°C. The A2-B1
PCR fragments were detected by electrophoresis. The target fragment was 1141bp.
2. Sequencing PCR products
PCR products amplified by primers A2 and Bl were linked with pGEM-T™ vector (Promega) and
transformed into E. coli JM103. The plasmids were extracted using QIAprep Plasmid Kit (QIAGEN). The
oriented serial deletion of the inserted fragments was carried out with Double-Stranded Nested Deletion Kit
(Pharmacia), and the deletants were quickly identified by PCR and arranged in order. The deletants
successively cut-off were sequenced with SequiTherm EXCEL™ DNA Sequencing Kit (Epicentre
Technologies). A full length cDNA sequence of 1141bp was obtained by overlapping the sequences with
computer software. The detailed sequence is shown in SEQ ID NO: 5 with an open reading frame (ORF)
located at nucleotides 14-1105.
According to the resultant full-length cDNA sequence, the amino acid sequence of hGDF3-2 was
deduced, having 363 amino acid residues totally. See SEQ ID NO: 6 for its amino acid sequence in details.
Example 2

Homologous comparison
First, the conservative sequence of TGF- β superfamily is found in the sequence of hGDF3-2 protein:

wherein (L/I/V/M) represents any amino acid of L, I, V, and M; X2 represents for any two amino acids.
The sequence corresponding to the conservative sequence in hGDF3-2 is: 281IIAPKGFMANYCHGEC296.
The full length cDNA sequence of hGDF3-2 and the coded protein were used for homologous
searching Non-redundant GenBank + EMBL + DDBJ + PDB and on-redundant GenBank CDS translations
+ PDB + SwissProt + Spupdate + PIR databases by BLAST algorithm. The result showed that they shared
high homology to murine GDF3 and human hGDF3. On the protein level, the amino acid sequence of
hGDF3-2 shares 69.1% identity and 76.5% similarity with GDF3, when analyzed by PCGENE software.
Furthermore, hGDF3-2 was identical to hGDF3 except that hGDF3-2 had nearly 50 additional amino acid
residues at N-terminal. The relationship of the three proteins is shown in Figure 1. Based on the homology
between GDF-3, hGDF3 and hGDF3-2 and the length of the proteins, it is strongly suggested that hGDF3-2
and hGDF3 are encoded by the same gene, and the difference is resulted from the different splicing of said
gene. Moreover, it is reasonable to state that hGDF3-2, instead of hGDF3, is the real human homologous
gene for mice GDF-3. Therefore, hGDF3-2 performs many functions identical to or similar to those of
GDF-3 or hGDF3.
The transcripts of GDF-3 gene were found only in a few tissues, e.g., thymus, spleen, bone marrow,
etc., in adult mice, which suggested GDF-3 may function in lymphocytopoiesis and erythropoiesis (J. Biol.
Chem., 1993, 268(5), 3444-3449). Jones found that GDF-3 expressed in skeletal and cartilaginous tissues in
embryos during the gestation, which indicated that GDF-3 might be involved in the skeletal development as
well (Mol. Endo., 1992, 6, 1961-1968). Similarly, the expression of hGDF3 was also tissue specific
(Oncogene, 1998, 16, 95-103).
Further, GDF-3 and hGDF3 were also expressed specifically in EC stem cells. Studies have shown that,
regardless of their differentiation ability or nutritional dependence of the cell strains, it was easy to detect
the transcript of hGDF3 in all of the tested EC cell strains. Retinoic acid could induce cellular
differentiation. Interestingly, the expression amount of hGDF3 in pluripotent EC cells drop to a very low
level after a long time treatment of retinoic acid. However, the treatment of retinoic acid did not lead to a
decreased hGDF3 expression in nullipotent EC cell strains. Therefore, the expression of hGDF3 in human
EC cell strains was directly related to the phenotype of EC stem cells. Other reports demonstrated that the
expression of hGDF3 was EC-cell specific, suggesting that hGDF3 might be a molecular marker of EC
cells. This property can be used to identify and screen particular cell types. Noticeably, hGDF3 is located
in human chromosome 12p, which is a focused area because of the overexpression in CIS (carcinoma in
situ), TGCT and TGCT derivative cell strains. Summing up, although the biological meaning of the
association of GDF-3 and EC cells is still unclear, the above phenomena reasonably suggest that hGDF3
functions in the formation and maintenance of EC stem cells (Oncogene, 1998, 16, 95-103).
The hGDF3-2 of the invention can be used not only as a member of the superfamily in the study of
function, but also to produce fusion proteins with other proteins, such as immunoglobulins. Besides,
hGDF3-2 can be fused with or exchange fragments with other members of the superfamily to form new

proteins. For example, the N terminal of hGDF3-2 can exchange with the N terminal of hGDF3 or mice
GDF-3 to produce proteins which are more active or have new properties.
The antibodies against hGDF3-2 can be used to screen other members of the superfamily or to purify
the related proteins such as other members of the superfamily through affinity purification.
Example 3
Expression of hGDF3-2 in E. coli
In this example, the cDNA sequence encoding hGDF3-2 (GenBank Accession No. AF064257. The
gene sequence was not available to the public prior to the filing of the application because of the secrecy
protection.) was amplified with oligonucleotide PCR primers corresponding to 5'- and 3'-end of said DNA
sequence. The resultant hGDF3-2 cDNA was used as an insertion fragment.
The sequence of 5'-end oligonucleotide primer was:
5'-ATACGGATCCATGCTTCGTTTCTTGCCAG-3' (SEQ ID NO: 7).
This primer contained a cleavage site of restriction endonuclease BamH I, followed by 19 nucleotides
of hGDF3-2 coding sequence starting from the start codon.
The sequence of 3'-end primer was:
5'-GTTAGTCGACCTACCCACACCCACATTCAT-3'(SEQ ID NO: 8).
This primer contained a cleavage site of restriction endonuclease SalI, a translation terminator and
partial hGDF3-2 coding sequence.
These cleavage sites of restriction endonuclease in primers corresponded to the cleavage sites in
bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, CA). Vector pQE-9 encodes an antibiotic
resistance (Amp'), a bacterial replication origin (ori), an IPTG-adjustable promotor/operon (P/O), a
ribosome-binding site (RBS), a six-hisitine tag (6-His) and cloning sites of restriction endonuclease.
Vector pQE-9 and insertion fragments were digested by BamHI and SalI, and then linked together,
ensuring that the open reading frame started from the bacterial RBS. Then, the linkage mixture was used to
transform E.coli M15/rep4 (Qiagen) containing multi-copy of plasmid pREP4 which expressed repressor of
lacI and was resistant to kanamycin (Kan'). Transformants were screened out in LB medium containing
Amp and Kan. The plasmids were extracted. The size and direction of the inserted fragments were verified
by BamHI digestion. The sequencing confirmed that hGDF3-2 cDNA fragment was correctly inserted into
the vector.
The positive clones of transformant were cultured overnight in LB liquid medium supplemented with
Amp (100µg/ml) and Kan (25µg/ml). The overnight culture was 1:100-1:250 diluted, inoculated into large
volume medium, and cultured until the 600nm optical density (OD600) reached 0.4-0.6. IPTG
(isopropylthio-beta-D-galactoside) was added to final concentration of ImM. By deactivating repressor of
LacI, IPTG induced and promoted P/O, thereby increasing the expression of gene. The cells were cultured
for another 3-4 hours, and then centrifuged (6000 X g, 20 mins). The cultures were sonicated, and cell
lysate was collected and diluted with 6M guanidine hydrochloride. After clarification, the dissolved
hGDF3-2 in solution were purified by nickel-chelated column chromatography under the conditions
suitable for the tight binding of 6-His tagged protein and column. hGDF3-2 was eluted with 6M guanidine

hydrochloride (pH 5.0). The denaturalized proteins in guanidine hydrochloride were precipitated by several
methods. First, guanidine hydrochloride was separated by dialysis. Alternatively, the purified protein,
which was isolated from nickel-chelated column, bound to the second column with decreased linear
gradient of guanidine hydrochloride. The proteins were denatured when binding to the column. Then, the
proteins were eluted with guanidine hydrochloride (pH 5.0). Finally, the soluble proteins were dialyzed
with PBS, then preserved in glycerol stock solution with the final glycerol concentration of 10% (w/v).
The molecular weight of the expressed protein was about 41 kDa, as identified by 12% SDS-PAGE.
Moreover, the sequencing results of the 10 amino acids at the N- and C-terminal of the expressed
protein indicated that they were identical to those in SEQ ID NO: 6.
Example 4
Expression of hGDF3-2 in eukaryotic cells (CHO cell line)
In this example, the cDNA sequence encoding hGDF3-2 (GenBank Accession No. AF064257) was
amplified with oligonucleotide PCR primers corresponding to 5'- and 3'-end of said DNA sequence. The
resultant product was used as an insertion fragment.
The sequence of 5'-end oligonucleotide primer was:
5'- ATACGGATCCATGCTTCGTTTCTTGCCAG -3'(SEQ ID NO: 7),
This primer contained a cleavage site of restriction endonuclease BamHI, followed by 19 nucleotides
of hGDF3-2 coding sequence starting from the start codon.
The sequence of 3'-end primer was:
5'-GTTAGAATTCCTACCCACACCCACATTCAT-3' (SEQ ID NO: 9)
This primer contained a cleavage site of restriction endonuclease EcoRI, a translation stop codon, and
partial hGDF3-2 coding sequence.
These cleavage sites of restriction endonuclease in primers corresponded to the cleavage sites in
expression vector pcDNA3 for CHO cell. This vector encoded two kinds of antibiotic resistance (Ampr and
Neo'), a phage replication origin (fl ori), a virus replication origin (SV40 ori), a T7 promoter, a virus
promoter (P-CMV), a Sp6 promoter, a polyadenylation signal of SV40 and the corresponding polyA
sequence thereof, a polyadenylation signal of BGH and the corresponding poly A sequence thereof.
The vector pcDNA3 and insertion fragment were digested with BamHI and EcoRI, and linked together.
Subsequently, E.coli strand DH5 α was transformed with linkage mixture. Transformants were screened
out in LB medium containing Amp. The clones containing the needed constructs were cultured overnight in
LB liquid medium supplemented with Amp (100 ug/ml). Plasmids were extracted. The size and direction of
the inserted fragments were verified by PstI digestion. The sequencing indicated that hGDF3-2 cDNA
fragment was correctly inserted into the vector.
Plasmids were transfected into CHO cells by lipofection with Lipofectin Kit (GEBco Life). After
transfecting the cells for 48 hours and screening the cells with G418 for 2-3 weeks, the cells and cell
supernatant were collected and the activity of the expressed protein was measured. G418 was removed and
the transformants were subcultured continuously. The mixed clonal cells were limiting diluted and the
subclones with higher protein activity were selected. The positive subclones were mass cultured by routine

methods. 48 hours later, the cells ana supernatant were collected. The cells were ultrasonicated. Using
50mM Tris-HCl (pH7.6) solution containing 0.05% Triton as an equilibrium solution and eluent, the active
peek of the protein was collected with a pre-balanced Superdex G-75 column. Then, using 50mM Tris-HCl
(pH8.0) solution containing 0-1 M NaCl as an eluent, the protein was gradiently washed on a DEAE-
Sepharose column balanced with 50mM Tris-HCl (pH8.0) solution. The active peek of the protein was
collected. The solution of the expressed protein was dialyzed with PBS (pH7.4), and finally lyophilized and
preserved.
The molecular weight of the expressed protein was about 41 kDa as identified by 12% SDS-PAGE.
Moreover, the sequencing results of the 10 amino acids at the N- and C-terminal of the expressed
protein indicated that they were identical to those in SEQ ID NO: 6.
Example 5
Antibody preparation
Antibodies were produced by immunizing animals with the recombinant proteins obtained in Examples
3 or 4. The method was as follows: the recombinant proteins were isolated by chromatography, and stored
for use. Alternatively, the protein was isolated by SDS-PAGE electrophoresis, and obtained by cutting
eletrophoretic bands from gel. The protein was emulsified with Freund's complete adjuvant of the same
volume. The emulsified protein was injected intraperitoneally into mice at a dosage of 50-100ug/0.2ml. 14
days later, the same antigen was emulsified with Freund's incomplete adjuvant and injected
intraperitoneally into mice at a dosage of 50-100ug/0.2ml for booster immunization. Booster immunization
was carried out every 14 days, for at least three times. The specific activity of the obtained antiserum was
evaluated by its ability of precipitating the translation product of hGDF3-2 gene in vitro.
All the documents cited herein are incorporated into the invention as reference, as if each of them is
individually incorporated. Further, it is appreciated that, in the above teaching of the invention, the skilled
in the art can make certain changes or modifications to the invention, and these equivalents are still within
the scope of the invention defined by the appended claims of the present application.

SEQUENCE LISTING
(l)General information:
(ii)Title of invention: NEW HUMAN GROWTH DIFFERENTIATION FACTOR ENCODING SEQUENCE AND
POLYPEPTIDE ENCODED BY SUCH DNA SEQUENCE AND PRODUCING METHOD THEREOF
(iii)Number of Sequences: 9
(2) INFORMATION FOR SEQ ID NO: 1:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH: 20bp
(B)TYPE: nucleic acid
(C)STRANDEDNESS: single
(D)TOPOLOGY: linear
(ii)MOLECULAR TYPE: oligonucleotide
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GGAGCTCTCC CCGGTCTGAC 20
(2) INFORMATION FOR SEQ ID NO: 2:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH: 21bp
(B)TYPE: nucleic acid
(C)STRANDEDNESS: single
(D)TOPOLOGY: linear
(ii)MOLECULAR TYPE: oligonucleotide
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 2:
CACTCCAGAG GCCATGCTTC G 21
(2) INFORMATION FOR SEQ ID NO: 3:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH: 23bp
(B)TYPE: nucleic acid
(OSTRANDEDNESS: single
(D)TOPOLOGY: linear
(ii)MOLECULAR TYPE: oligonucleotide
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 3:
CCTAAGAACA CTCCTTCTAT TCC 23
(2) INFORMATION FOR SEQ ID NO: 4:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH: 24bp
(B)TYPE: nucleic acid
(OSTRANDEDNESS: single
(D)TOPOLOGY: linear
(ii)MOLECULAR TYPE: oligonucleotide
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CTAAGTGGTC ATAAACCAGA TTAGG 24
(2) INFORMATION FOR SEQ ID NO: 5:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH: 1141bp

(B)TYPE: nucleic acid
(C)STRANDEDNESS: double
(D)TOPOLOGY: linear
(ii)MOLECULAR TYPE: cDNA
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 5:
1 CACTCCAGAC GCCATGCTTC GTTTCTTGCC AGATTTGGCT TTCAGCTTCC TGTTAATTCT
61 GGCTTTGGGC CAGGCAGTCC AATTTCAAGA ATATGTCTTT CTCCAATTTC TGGGTTAGA
121 TAAGGCGCCT TCACCCCACA AGTTCCAACC TGTGCCTTAT ATCTTGAAGA AAATTTTCCA
181 GGATCGCGAG GCAGCGGCGA CCACTGGGGT CTCCCGAGAC TTATGCTACG TAAAGGAGCT
241 GGGCGTCCGC GGGAATGTAC TTCGCTTTCT CCCAGACCAA GGTTTCTTTC TTTACCCAAA
301 GAAAATTTCC CAAGaTCCT CCTGCaGCA GAAGCTCCTC TACTTTAACC TGTCTGCCAT
361 CAAAGAAAGG GAACAGCTGA CATTGGCCCA GCTGGTGGAC TTGGGGCCCA ATTCTTACTA
421 TAACCTGGGA CCAGAGCTGG AACTGGCTCT GTTCCTGGTT CAGGAGCCTC ATGTGTGGCG
481 CCAGACCACC CCTAAGCCAG GTAAAATGTT TGTGTTGCGG TCAGTCCCAT GGCCACAAGG
541 TGCTGTTCAC TTCAGCCTGC TGGATGTAGC TAAGGATTGG AATGACAACC CCCGGAAAAA
601 TTTCGGGTTA TTCCTGGAGA TACTGGTCAA AGAAAATAGA GACTCAGGGG TGAATTTTCA
661 GCCTGAAGAC ACCTGTGCCA GACTAAGATG CTCCCTTCAT GCTTCCCTGC TGGTGGTGAC
721 TCTCAACCCT GATCAGTGCC ACCCTTCTCG GAAAAGGAGA GCAGCCATCC CTGTCCCCAA
781 GCTTTCTTGT AAGAACCTCT GCCACCGTCA CCAGCTATTC ATTAACTTCC GGGACCTGGG
841 TTGGCACAAG TGGATCATTG CCCCCAAGGG TTTCATGGCA AATTACTGCC ATGGAGAGTG
901 TCCCTTCTCA CTGACCATCT CTCTCAACAG CTCCAATTAT GCTTTCATGC AAGCCCTGAT
961 GCATGCCGTT GACCCAGAGA TCCCCCAGGC TGTGTGTATC CCCACCAAGC TGTCTCCCAT
1021 TTCCATGCTC TACCAGGACA ATAATGACAA TGTCATTCTA CGACATTATG AAGACATGGT
1081 AGTCGATGAA TGTGGGTGTG GGTAGGATGT CAGAAATGGG AATAGAAGGA GTGTTCTTAG
1141 G
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 363 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: lineal
(ii) MOLECULE TYPE: polypeptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
1 Met Leu Arg Phe Leu Pro Asp Leu Ala Phe Ser Phe Leu Leu Ile
16 Leu Ala Leu Gly Gln Ala Val Gln Phe Gln Glu Tyr Val Phe Leu
31 Gln Phe Leu Gly Leu Asp Lys Ala Pro Ser Pro His Lys Phe Gln
46 Pro Val Pro Tyr Ile Leu Lys Lys Ile Phe Gln Asp Arg Glu Ala
61 Ala Ala Thr Thr Gly Val Ser Arg Asp Leu Cys Tyr Val Lys Glu
76 Leu Gly Val Arg Gly Asn Val Leu Arg Phe Leu Pro Asp Gln Gly
91 Phe Phe Leu Tyr Pro Lys Lys Ile Ser Gln Ala Ser Ser Cys Leu
106 Gln Lys Leu Leu Tyr Phe Asn Leu Ser Ala Ile Lys Glu Arg Glu
121 Gln Leu Thr Leu Ala Gln Leu Val Asp Leu Gly Pro Asn Ser Tyr
136 Tyr Asn Leu Gly Pro Glu Leu Glu Leu Ala Leu Phe Leu Val Gln
151 Glu Pro His Val Trp Arg Gln Thr Thr Pro Lys Pro Gly Lys Met
166 Phe Val Leu Arg Ser Val Pro Trp Pro Gln Gly Ala Val His Phe
181 Ser Leu Leu Asp Val Ala Lys Asp Trp Asn Asp Asn Pro Arg Lys
196 Asn Phe Gly Leu Phe Leu Glu Ile Leu Val Lys Glu Asn Arg Asp
211 Ser Gly Val Asn Phe Gln Pro Glu Asp Thr Cys Ala Arg Leu Arg
226 Cys Ser Leu His Ala Ser Leu Leu Val Val Thr Leu Asn Pro Asp
241 Gln Cys His Pro Ser Arg Lys Arg Arg Ala Ala Ile Pro Val Pro
256 Lys Leu Ser Cys Lys Asn Leu Cys His Arg His Gln Leu Phe Ile

271 Asn Phe Arg Asp Leu Gly Trp His Lys Trp Ile Ile Ala Pro Lys
286 Gly Phe Met Ala Asn Tyr Cys His Gly Glu Cys Pro Phe Ser Leu
301 Thr Ile Ser Leu Asn Ser Ser Asn Tyr Ala Phe Met Gln Ala Leu
316 Met His Ala Val Asp Pro Glu Ile Pro Gln Ala Val Cys Ile Pro
331 Thr Lys-Leu Ser Pro Ile Ser Met Leu Tyr Gln Asp Asn Asn Asp
346 Asn Val Ile Leu Arg His Tyr Glu Asp Met Val Val Asp Glu Cys
361 Gly Cys Gly
(2) INFORMATION FOR SEQ ID NO: 7:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH: 29bp
(B)TYPE: nucleic acid
(C)STRANDEDNESS: single
(D)TOPOLOGY: linear
(ii)MOLECULAR TYPE: oligonucleotide
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 7:
ATACGGATCC ATGCTTCGTT TCTTGCCAG 29
(2) INFORMATION FOR SEQ ID NO: 8:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH: 30bp
(B)TYPE: nucleic acid
(C)STRANDEDNESS: single
(D)TOPOLOGY: linear
(ii)MOLECULAR TYPE: oligonucleotide
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 8:
GTTAGTCGAC CTACCCACAC CCACATTCAT 30
(2) INFORMATION FOR SEQ ID NO: 9:
(i)SEQUENCE CHARACTERISTICS:
(A)LENGTH: 29bp
(B)TYPE: nucleic acid
(C)STRANDEDNESS: single
(D)TOPOLOGY: linear
(ii)MOLECULAR TYPE: oligonucleotide
(xi)SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GTTAGAATTC CTACCCACAC CCACATTCAT 29

WE CLAIM:
1. A vector containing the DNA sequence encoding a polypeptide comprising the amino acid
sequence of SEQ ID NO: 6.
2. A method for producing a human growth differentiation factor (hGDF3-2) protein, which
comprises the steps of;
(a) forming an expression vector comprising the nucleotide sequence encoding the hGDF3-2
protein having the amino acid sequence of SEQ ID NO: 6, wherein said nucleotide sequence is
operably linked with an expression regulatory sequence;
(b) introducing the vector of step (a) into a host cell, thereby forming a recombinant cell of
hGDF3-2 protein;
(c) culturing the recombinant cell of step (b) under conditions suitable for expression of
hGDF3-2 protein; and
(d) isolating the hGDF3-2 protein.
3. The method as claimed in Claim 2, wherein said nucleotide sequence comprises nucleotides
14-1105 of SEQ ID NO: 5.

The invention provides a cDNA sequence of a new human growth differentiation factor (hGDF3-2).
The protein encoded by such sequence is a splice variant of hGDF3. The present invention also relates to
peptides encoded by the nucleotide sequences, to uses of these polynucleotides and polypeptides, and
methods for producing the said polynucleotides and polypeptides.

Documents:

in-pct-2001-342-kol-assignment.pdf

IN-PCT-2001-342-KOL-CORRESPONDENCE 1.1.pdf

in-pct-2001-342-kol-correspondence.pdf

in-pct-2001-342-kol-examination report.pdf

in-pct-2001-342-kol-form 18.pdf

in-pct-2001-342-kol-form 3.pdf

in-pct-2001-342-kol-form 5.pdf

IN-PCT-2001-342-KOL-FORM-27.pdf

in-pct-2001-342-kol-gpa.pdf

in-pct-2001-342-kol-granted-abstract.pdf

in-pct-2001-342-kol-granted-claims.pdf

in-pct-2001-342-kol-granted-description (complete).pdf

in-pct-2001-342-kol-granted-drawings.pdf

in-pct-2001-342-kol-granted-form 1.pdf

in-pct-2001-342-kol-granted-specification.pdf

in-pct-2001-342-kol-others.pdf

in-pct-2001-342-kol-priority document.pdf

in-pct-2001-342-kol-reply to examination report.pdf

in-pct-2001-342-kol-translated copy of priority document.pdf


Patent Number 246697
Indian Patent Application Number IN/PCT/2001/342/KOL
PG Journal Number 10/2011
Publication Date 11-Mar-2011
Grant Date 10-Mar-2011
Date of Filing 26-Mar-2001
Name of Patentee YU LONG
Applicant Address HANDAN ROAD 220, INSTITUTE OF GENETICS, FUDAN UNIVERSITY, SHANGHAI
Inventors:
# Inventor's Name Inventor's Address
1 YU LONG HANDAN ROAD 220, INSTITUTE OF GENETICS, FUDAN UNIVERSITY, SHANGHAI 200433
2 ZHANG HONGLAI HANDAN ROAD 220, INSTITUTE OF GENETICS, FUDAN UNIVERSITY, SHANGHAI 200433
3 FU QIANG HANDAN ROAD 220, INSTITUTE OF GENETICS, FUDAN UNIVERSITY, SHANGHAI 200433
4 DAI FANGYAN HANDAN ROAD 220, INSTITUTE OF GENETICS, FUDAN UNIVERSITY, SHANGHAI 200433
5 ZHAO SHOUYUAN HANDAN ROAD 220, INSTITUTE OF GENETICS, FUDAN UNIVERSITY, SHANGHAI 200433
PCT International Classification Number C07K 14/495
PCT International Application Number PCT/CN1999/00138
PCT International Filing date 1999-09-06
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 98119759.0 1998-09-22 China