# Patent

## Title
Polynucleotide, vector, host cell and method for producing human hepatoma-derived growth factor 5 polypeptide

## Abstract

The invention provides a cDNA sequence of human hepatoma derived growth factor 5 (HDGF5). The present invention also relates to the polypeptides encoded by the nucleotide sequences, the uses of these polynucleotides and polypeptides, and the methods for producing HDGF5 polynucleotides and polypeptides.

## Background
FIELD OF INVENTION
This invention relates to the field of genetic engineering, and, in particular, relates to the nucleotide sequence of a human gene. More particularly, this invention relates to the cDNA sequence of human Hepatoma-derived Growth Factor 5 (HDGF5). 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.
BACKGROUND OF TECHNIQUES
Growth factor, known as polypeptide growth factor customarily, is a group of proteins regulating the growth and differentiation of cells and has molecular weight from several hundred to dozens of kilodalton (KD). It is revealed that the regulation of cell growth is mediated by a series of cascade reactions triggered by the interaction between a variety of cytokines and their specific receptors on membrane surfaces. Compared to classical polypeptide and protein hormone, growth factor is released by autocrine and paracrine cells instead of endocrine gland or endocrine cells, so as to achieve the coordination of body and reaction to the environment.
Hepatoma derived growth factor (hepatoma derived growth factor, HDGF) is an important growth factor, which was first reported in paper by Klagsbrun, M et al. in 1986 (P.N.A.S. USA Vol. 33, pp 2448-2452, 1986). In this paper, Klagsbrun, M et al. reported to have isolated and purified a protein factor having 18.5-19 KD from human hepatoma cells line SK-HEP-1. And the remarkable character of this factor was its strong affinity with heparin.
In 1989, a 64 KD factor, also named as HDGF, was first partially purified from HuH-7 cells and characterized by Nakamura et. al. (Clin. Chim. Acta. 183:273-284, 1989). This research group had studied its biochemical characteristics and functions, and found that HDGF was different from PDGF, FGF, HGF. In 1997, this group found the mouse homologue of human HDGF as well as other two members of the gene family, HRP-1 and HRP-2. They all had a highly conserved N-terminal of 98 amino acids. (Biochem. Biophys. Res. Commun. 238: 26-32,1997). In 1999, this research group also cloned HRP-3 (Biochem. Biophys. Res. Commun. 266(1):81-87,1999), another member of HDGF family from human and mouse.
Prior to the publication of this invention, none has disclosed human HDGF5 of the present application.
SUMMARY OF INVENTION
One purpose of the invention is to provide a new polynucleotide which encodes a homologue of HDGF. In the invention, the gene of said homologue of HDGF is named HDGF5.
Another purpose of the invention is to provide a novel protein, which is named HDGF5 protein.
Still another purpose of the invention is to provide a new method for preparing said new HDGF5 protein by recombinant techniques.
The invention also relates to the uses of said HDGF5 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 HDGF5 protein activity, wherein said nucleotide sequence shares at least 70% homology to the nucleotide sequence of nucleotides 5-910 in SEQ ID NO: 1, or said nucleotide sequence hybridizes to the nucleotide sequence of nucleotides 5-910 in SEQ ID NO: 1 under moderate stringency. Preferably, said nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2. More preferably, the sequence comprises the nucleotide sequence of nucleotides 5-910 in SEQ ID NO: 1.
Further, the invention provides an isolated HDGF5 polypeptide, which comprises a polypeptide having the amino acid sequence of SEQ ID NO: 2, its active fragments, and its active derivatives.
Preferably, the polypeptide is selected from the group consisting of: (a) a polypeptide having the amino acid sequence of SEQ ID NO: 2. (b) the polypeptide derived from SEQ ID NO: 2 with substitution, deletion, or addition of one or more amino acids and having function of promoting cell proliferation.
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 HDGF5 protein, which comprises:
(a) forming a HDGF5 protein expression vector comprising the nucleotide sequence encoding the polypeptide having the activity of HDGF5 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 5-910 in SEQ ID NO: 1;
(b) introducing the vector of step (a) into a host cell, thereby forming a recombinant cell of HDGF5 protein;
(c) culturing the recombinant cell of step (b) under the conditions suitable for the expression of HDGF5 polypeptides;
(d) isolating the polypeptides having the activity of HDGF5 protein.
The invention provides an antibody specifically binding to HDGF5 polypeptide.
The invention further provides a probe, comprising 8-100 consecutive nucleotides shown in SEQ ID NO: 1



DESCRIPTION OF DRAWINGS
FIG. 1 shows the homologue comparison of N-terminal amino acid sequence between HDGF5 and other members of HDGF family. “*” refers to the same amino acid in this site, “:” refers to very high homologue of amino acid, and “.” refers to relatively high homologue of amino acid. The GenBankTM accession numbers of these proteins are as follows:
















Rat HRP
AAL29938



Mouse HRP1
JC5661



Mouse HDGF
JC5660



Rat HDGF
AAL47132



Human HDGF
D16431



Human HRP1
AAH18991



Human HRP3
BAA90477



Mouse HRP3
AB029493



Mouse HRP2
JC5662



Rat HDGF3
AAK50635










FIG. 1 indicates that the N-terminal of human HDGF5 (HATH region, generally including 98 amino acid residues of N-terminal) and those of other members of HDGF family share high homology.



DETAILED DESCRIPTION OF INVENTION
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 “HDGF5 protein encoding sequence” or “HDGF5 polypeptide encoding sequence” refers to a nucleotide sequence encoding a polypeptide having the activity of HDGF5 protein, such as the nucleotide sequence of positions 5-910 in SEQ ID NO: 1 or its degenerate sequence. The degenerate sequences means the sequences formed by replacing one or more codons in the ORF of 5-910 in SEQ ID NO: 1 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 5-910 in SEQ ID NO: 1 can also encode the sequence shown in SEQ ID NO: 2. The term also refers to the nucleotide sequences that hybridize to the nucleotide sequence of nucleotides 5-910 in SEQ ID NO: 1 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 5-910 in SEQ ID NO: 1.
The term also refers to variants of the sequence in SEQ ID NO: 1, which are capable of encoding a protein having the same function as human HDGF5 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 “HDGF5 polypeptide” or “HDGF5 protein” refers to a polypeptide having the activity of HDGF5 protein comprising the amino acid sequence of SEQ ID NO: 2. The term also comprises the variants of said amino acid sequence which have the same function of human HDGF5. 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 HDGF5 protein.
The variants of polypeptide include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes to HDGF5 DNA under high or low stringency conditions as well as the polypeptides or proteins retrieved by antisera raised against HDGF5 polypeptide. The present invention also provides other polypeptides, e.g., fusion proteins, which include the HDGF5 polypeptide or fragments thereof. In addition to substantially full-length polypeptide, the soluble fragments of HDGF5 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 HDGF5 polypeptide.
The present invention also provides the analogues of HDGF5 protein or polypeptide. Analogues can differ from naturally occurring HDGF5 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., acetylation, 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.
In the present invention, “the conservative variants of peptides of human HDGF5” refers to the polypeptides formed by substitutions of several amino acids (typically 10, preferably 8, more preferably 5, most preferably 3 or less). These conservative variants of polypeptides are preferably produced by substitution of amino acids shown in table 1.











TABLE 1









Preferred



Initial residue
Representative substitution
substitution









Ala (A)
Val; Leu; Ile
Val



Arg (R)
Lys; Gln; Asn
Lys



Asn (N)
Gln; His; Lys; Arg
Gln



Asp (D)
Glu
Glu



Cys (C)
Ser
Ser



Gln (Q)
Asn
Asn



Glu (E)
Asp
Asp



Gly (G)
Pro; Ala
Ala



His (H)
Asn; Gln; Lys; Arg
Arg



Ile (I)
Leu; Val; Met; Ala; Phe
Leu



Leu (L)
Ile; Val; Met; Ala; Phe
Ile



Lys (K)
Arg; Gln; Asn
Arg



Met (M)
Leu; Phe; Ile
Leu



Phe (F)
Leu; Val; Ile; Ala; Tyr
Leu



Pro (P)
Ala
Ala



Ser (S)
Thr
Thr



Thr (T)
Ser
Ser



Trp (W)
Tyr; Phe
Tyr



Tyr (Y)
Trp; Phe; Thr; Ser
Phe



Val (V)
Ile; Leu; Met; Phe; Ala
Leu










The invention also includes antisense sequences of the HDGF5 encoding sequence or its fragments. Said antisense sequence can be used to inhibit expression of HDGF5 in cells.
The invention also includes probes, typically having 8-100, preferably 15-50 consecutive nucleotides of HDGF5 coding sequence. These probes can be used to detect the presence of nucleic acid molecules coding for HDGF5 in samples.
The present invention also includes methods for detecting HDGF5 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 HDGF5 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.
In the present invention, various vectors known in the art such as commercially available vectors could be used. For example, a commercially available vector can be operably linked to the nucleic acid sequence encoding the new fusion protein in the present invention under an expression regulatory sequence, and thus forms the expression vector.
As used herein, the term “operably linked” refers to such a condition where a certain part of a linear DNA sequence could affect the activity of another certain part in the same linear DNA sequence. For example, if a signal peptide plays a role in the secretion of polypeptides, then the DNA sequence encoding the signal peptide (precursor sequence of secretion) is operably linked to the DNA of the polypeptide; if a promoter controls the transcription of a sequence, then it is operably linked to the encoding sequence. If a ribosomal binding site is located at a position which can initiate translation, then it is operably linked to the encoding sequence. Generally, “operably linked” means adjacency, while in the precursor sequence of secretion it means adjacency in reading frame.
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 HDGF5 DNA or fragments thereof. By “specificity”, it is meant an antibody which binds to the HDGF5 gene products or a fragments thereof. Preferably, the antibody binds to the HDGF5 gene products or a fragments thereof and does not substantially recognize nor bind to other antigenically unrelated molecules. Antibodies which bind to HDGF5 and block HDGF5 protein and those which do not affect the HDGF5 function are included in the invention. The invention also includes antibodies which bind to the HDGF5 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, or a chimerical 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 HDGF5 gene products, or its antigenic fragments can be administrated to animals to induce the production of polyclonal antibodies. Similarly, cells expressing HDGF5 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 HDGF5 function and those which do not affect HDGF5 function. Antibodies in the invention can be produced by routine immunology techniques and using fragments or functional regions of HDGF5 gene product. These fragments and functional regions can be prepared by recombinant methods or synthesized by a polypeptide synthesizer. Antibodies binding to unmodified HDGF5 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 HDGF5 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 431A Peptide Synthesizer (Applied Biosystems, Foster City, Calif.). 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 FISH technique can use cDNA as short as about 500 bp, or as long as about 2000 bp, 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 HDGF5, 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 HDGF5 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 HDGF5 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 1 ug to 10 mg per kg body weight per day. Moreover, the polypeptide of the invention can be administrated together with other therapeutic agent.
When the human HDGF5 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 1 ug/kg body weight and less than about 10 mg/kg body weight in most cases, and preferably about 10 ug-1 mg/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.
In one embodiment, the cDNA sequence of HDGF5 was obtained as follows: human testis λgt11 cDNA library (ClontechTM) was used as a template and PCR was carried out with the synthetic forward primer A1: 5′-CGCTATGTCTTGCTTCAGCCG-3′(SEQ ID NO: 3) and reverse primer A2:5′-GGCCCTAGCGGGTTTCCCAAG-3′(SEQ ID NO:4). The PCR condition are 4 min at 93° C., followed by 35 cycles with 1 min at 93° C., 1 min at 68° C., 1 min at 72° C., and finally 5 min at 72° C. The target fragments of about 1000 bp were obtained. The detailed sequence of 990 bp nucleotides was shown in SEQ ID NO: 1 and the ORF comprises the nucleotides 5-910.
Hepatoma-derived Growth Factor (HDGF) is a hepatin-binding protein isolated from human hepatoma-derived cell line HuH-7. HDGF has the activity of stimulating cell growth and promoting the growth of fibroblast and some heptoma cells. The mitogenic activity of HDGF implied the great application value of HDGF in treating pernicious oxyhepatitis and liver injury.
It is indicated in the researches that although HDGF is derived from the hepatoma cell lines, it is widely expressed both in normal cells and tumor cell lines. Therefore, HDGF not only affects the tumor cells, and also plays important roles in the physiological and developmental process of normal tissues and cells. For example, HDGF stimulates cell division of SMC, COS-7, Swiss-3T3, HuH-7, 7.1.1, promotes and regulates the growth of blood vessel endothelium cells and leiomyocytes, and plays physiological roles as secretory growth factor or nucleus factor. The expression patterns of the HDGF gene family members are different. However, they are all enriched in testis and the 5′-untranslated region contains GC-rich nucleotide sequences (GC content>70%), suggesting their potential important roles in male germ-cell development. They may also relate to DNA methylation, chromatin conformation, and translational regulation.
HDGF5 in the present invention can promote the proliferation of liver cell after partial liver tissue had been cut. The small molecule antagonists or inhibitors against HDGF5 can be found and produced as medicines to suppress the growth of hepatoma cells and induce apoptosis, and therefore, slow or control the symptom of hepatocellular carcinoma. Because HDGF5 has different expression pattern in various cells, the HDGF5 protein, its coding nucleotide sequence or its active part can be developed into special kits to diagnose the early stage of carcinoid and malignant tumor and evaluate the treatment effect and prognosis after HCC surgery.
HDGF5 stimulates mitosis of various cells in varying degrees. It can also be used in regulating the growth of blood vessel endothelium cells and leiomyocytes so that HDGF5 has certain application value in promoting wound healing, treating injury of acute inflammation, malignant tumor other than liver cancer.
In addition, human HDGF5 of the invention has human derived natural amino acid sequence. So compared with HDGF proteins derived from other species, it will have higher activity or less by-effect (e.g. lesser or no immunogenicity in human body) in treating human disease.
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.
EXAMPLE 1
The Cloning and Sequencing of HDGF5 cDNA Sequence
1. Amplification With Primers
The template was human testis λgt 11 cDNA library (commercially available from CLONTECHTM). PCR was carried out with the forward primer A1: 5′-CGCTATGTCTTGCTTCAGCCG-3′ (SEQ ID NO: 3) and reverse primer A2: 5′-GGCCCTAGCGGGTTTCCCAAG-3′ (SEQ ID NO: 4). The PCR condition was 4 mins at 93° C.; followed by 35 cycles with 1 min at 93° C., 1 min at 68° C., and 1 min at 72° C.; and, finally 5 mins at 72° C. The PCR fragments were detected by electrophoresis. The target fragment was about 1000 bp.
2. Sequencing PCR Products
The above obtained PCR products were linked with pGEM-TTM vector (Promega) and transformed into E. coli JM103. The plasmids were extracted using QIAprep Plasmid Kit (QIAGENTM). 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 EXCELTM DNA Sequencing Kit (Epicentre Technologies). A full length cDNA sequence of 990 bp was obtained by overlapping the sequences with computer software. The detailed sequence is shown in SEQ ID NO: 1.










cgctatgtct tgcttcagcc gcccaaaata caagaccggg gacctggtgt ttgccaaatt
60
(SEQ ID NO: 1)



 


aaagggctat gcccattggc cagcgaggat tgaacatgtc actgaaccca accgctacca
120


 


ggtgttcttc ttcgggaccc atgagaccgc cctgctgggc cccaagcacc tttttcctta
180


 


tgaggagtcc aaggagaggt tcggcaagcc taacaagagg cgcggcttca gtgaggggct
240


 


gtgggagatc gagcacgacc ctatggctga ggcctcccct tgcctgtgcc cagatgagga
300


 


gcagctttgt gccgaggagc cagggccagg agaggagcca gagccggggc aggagctgga
360


 


gccggaatcc aggcctgagc tggaatccat gcctgagctg gaggcagaac cgaggcctga
420


 


gaaagagtgt gagcaggagc cggagcagga gccggagcag gagctggagc aggagccgga
480


 


gctggagccg gagccggagc cggagccgga gccggagccg gagcccgagc ccgagccgga
540


 


gccggagccc cagcctgcct atgacctact ggatgccaag gaggagcctg gcctcattga
600


 


ggccgagcca ggagatcagc aagccgagca agtgcgagag cagcacgctg aagctgaggt
660


 


catggctgta gtggaggagc cggagagtct gaagaggagc gcggaggatg aacagcctca
720


 


cagtcctccc aaacggccca gggaggcggc gcctggcgcg ctggagatgg agccggctgg
780


 


agagegegag gcagaggcct gccccttcgt ggaggagcct gaccaagccc aggaacagca
840


 


gactccgttg gaagaagagg ccacagagga ggcagtccag ggcctgatgg ttggagaaat
900


 


cgaaggcctg tagtcacggt gtctgtaaaa gagccctctc tacccgttcc tggtgccacc
960


 


tggctgtggc ttgggaaacc cgctagggcc. 990






According to the resultant full-length cDNA sequence, the amino acid sequence of HDGF5 was deduced, having 302 amino acid residues totally. See SEQ ID NO:2 for its amino acid sequence in details.










MSCFSRPKYK TGDLVFAKLK GYAHWPARIE HVTEPNRYQV FFFGTHETAL LGPKHLFPYE
60
(SEQ ID NO: 2)



 


ESKERFGKPN KRRGFSEGLW ELEHDPMAEA SPCLCPDEEQ LCAEEPGPGE EPEPGQELEP
120


 


ESRPELESMP ELEAEPRPEK ECEQEPEQEP EQELEQEPEL EPEPEPEPEP EPEPEPEPEP
180


 


EPQPAYDLLD AKEEPGLIEA EPGDQQAEQV REQHAEAEVM AVVEEPESLK RSAEDEQPHS
240


 


PPKRPREAAP GALEMEPAGE REAEACPFVE EPDQAQEQQT PLEEEATEEA VQGLMVGEIE
300


 


GL 302






EXAMPLE 2
Homologous Comparison
The full length cDNA sequence of human HDGF5 and the encoded 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 N-terminal of the HDGF family has high conservative region (HATH region, for homologous to the amino terminus of the HDGF). This region is regarded as the signature sequence. The N-terminal of human HDGF5 shared high homologous with those of other HDGF family members (See the homologous comparison of N-terminal sequence between HDGF5 and other members of HDGF family in FIG. 1). Therefore, it was indicated that HDGF5 was a member of HDGF family and had functions similar to those of other members.
Hepatoma-derived Growth Factor (HDGF) is a hepatin-binding protein isolated from human hepatoma-derived cell line HuH-7. HDGF has the activity of stimulating cell growth and promoting the growth of fibroblast and some heptoma cells (J. Biol. Chem. 269(40): 25143-25149, 1994). The mitogenic activity of HDGF implied the great application value of HDGF in treating pernicious oxyhepatitis and liver injury.
It is indicated in the researches that although HDGF is derived from the hepatoma cell lines, it is widely expressed both in normal cells and tumor cell lines. Therefore, HDGF not only affects the tumor cells, and also plays important roles in the physiological and developmental process of normal tissues and cells. For example, HDGF stimulates cell division of SMC, COS-7, Swiss-3T3, HuH-7, 7.1.1, promotes and regulates the growth of blood vessel endothelium cells and leiomyocytes, and plays physiological roles as secretory growth factor or nucleus factor.
The HDGF5 of the invention can be used to produce fusion proteins with other proteins, such as immunoglobulins. Besides, HDGF5 can be fused with or exchange fragments with other members of the family to form new proteins. For example, the N terminal of HDGF5 can exchange with the N terminal of other HDGFs or mice HDGF to produce proteins, which are more active or have new properties.
The antibodies against HDGF5 can be used to screen other members of the family or to purify the related proteins such as other members of the family through affinity purification.
Moreover, HDGF5 nucleic acid (coding sequence and antisense sequence) could be induced into cell to enhance the expression level of HDGF5 or inhibit the over-expression of HDGF5. Human HDGF5 protein or its active polypeptide fragment could be used to treat patients whose HDGF5 was lacking, no function or abnormal. In addition, the nucleic acid sequence or antibody of the invention could be used in diagnosis or prognosis judgment.
EXAMPLE 3
Expression of HDGF5 in E. coli 
In this example, the cDNA sequence encoding HDGF5 was amplified with oligonucleotide PCR primers corresponding to 5′- and 3′-end of said DNA sequence.
The sequence of 5′-end oligonucleotide primer was:








(SEQ ID NO: 5)











E1: 5′-GCAGGATCCATGTCTTGCTTCAGCCGC-3′.







This primer contained a cleavage site of restriction endonuclease BamHI, followed by partial nucleotides of HDGF5 coding sequence starting from the start codon.
The sequence of 3′-end primer was:








(SEQ ID NO: 6)











E2: 5′-CCGAAGCTTCAGGCCTTCGATTTCTCC-3′.







This primer contained a cleavage site of restriction endonuclease HindIII, a translation terminator and partial HDGF5 coding sequence.
These cleavage sites of restriction endonuclease in primers corresponded to the cleavage sites in bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, Calif.). Vector pQE-9 encodes an antibiotic resistance (Ampr), 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 HindIII, and then linked together. 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 (Kanr). 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 EcoNI digestion. The sequencing confirmed that HDGF5 cDNA fragment was correctly inserted into the vector.
The positive clones of transformant were cultured overnight in LB liquid medium supplemented with Amp (100 ug/ml) and Kan (25 ug/ml). The overnight culture was 1:100-1:250 diluted, inoculated into large volume medium, and cultured until the 600 nm optical density (OD600) reached 0.4-0.6. IPTG (isopropylthio-beta-D-galactoside) was added to final concentration of 1 mM. 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×g, 20 mins). The cultures were sonicated, and cell lysate was collected and diluted with 6M guanidine hydrochloride. After clarification, the dissolved HDGF5 in solution were purified by nickel-chelated column chromatography under the conditions suitable for the tight binding of 6-His tagged protein and column. HDGF5 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 34148 Da, 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: 2.
EXAMPLE 4
Expression of HDGF5 in Eukaryotic Cells (CHO Cell Line)
In this example, the cDNA sequence encoding HDGF5 was amplified with oligonucleotide PCR primers corresponding to 5′- and 3′-end of DNA sequence. The resultant product was used as an insertion fragment.
The sequence of 5′-end oligonucleotide primer was:








(SEQ ID NO: 7)











C1: 5′-GCAAAGCTTATGTCTTGCTTCAGCCGC-3′







This primer contained a cleavage site of restriction endonuclease HindIII, followed by 18 nucleotides of HDGF5 coding sequence starting from the start codon.
The sequence of 3′-end primer was:








(SEQ ID NO: 8)











C2: 5′-CCGGGATCCCAGGCCTTCGATTTCTCC-3′







This primer contained a cleavage site of restriction endonuclease BamHI, a translation stop codon, and partial HDGF5 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 Neor), 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 polyA sequence thereof.
The vector pcDNA3 and insertion fragment were digested with HindIII and BamHI, and linked together. Subsequently, E. coli strand DH5α 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 EcoNI digestion. The sequencing indicated that HDGF5 cDNA fragment was correctly inserted into the vector.
Plasmids were transfected into CHO cells by lipofection with LipofectinTM Kit (GIBco 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 and supernatant were collected. The cells were ultrasonicated. Using 50 mM 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 50 mM 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 50 mM 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 34 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: 2.
EXAMPLE 5
Antibody Preparation
Antibodies were produced by immunizing animals with the recombinant proteins obtained in Examples 3 and 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 electrophoretic 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 50-100 ug/0.2 ml. 14 days later, the same antigen was emulsified with Freund's incomplete adjuvant and injected intraperitoneally into mice at a dosage of 50-100 ug/0.2 ml 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 HDGF5 gene in vitro. The result showed that the antibody specifically bound to HDGF5 protein of the invention.
EXAMPLE 6
Promotion Effect of HDGF5 on Cell Proliferation
The promotion effect of HDGF5 on cell proliferation effect was tested by adding various concentrations of HDGF5. The detailed protocols were as follows: The mouse endothelium cell Berd-3 was planted into 96-hole panel with 10% 1640 medium to final concentration of 1-1.2×104 cells/ml. The cells were incubated in the incubator (5% CO2) at 37° C. overnight. The primary medium was substituted with serum-free medium and the cells were incubated in the incubator (5% CO2) at 37° C. overnight again. The purified HDGF5 protein prepared in Example 4 was added into the holes containing the mouse endothelium cells. The concentrations of HDGF5 were gradiently diluted in each holes, i.e., 0 (blank control), 0.1 ng/ml, 0.5 ng/ml, 2.5 ng/ml, 25 ng/ml, 100 ng/ml. After adding various concentrations of HDGF5, these cells were cultured again at 37° C. and 5% CO2, and stopped at 24, 48, 72 hours respectively to test MTS.
The test results indicated that as the content of HDGF5 increased, the cell number was increased corresponding.
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.

## Claims

1. An isolated DNA molecule comprising a nucleotide sequence encoding a human hepatoma-derived growth factor 5 (HDGF5) polypeptide having an activity of proliferation of mouse endothelium cells, wherein said nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2.

2. The DNA molecule of claim 1 wherein said nucleotide sequence encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

3. The DNA molecule of claim 1 wherein said nucleotide sequence comprises nucleotides 5-910 in SEQ ID NO: 1.

4. A vector containing the DNA molecule of claim 1.

5. A host cell transformed by the vector of claim 4.

6. A method for producing a human hepatoma-derived growth factor 5 (HDGF5) polypeptide having the activity of proliferation of mouse endothelium cells, which comprises the steps of:
(a) forming an expression vector comprising a nucleotide sequence encoding the HDGF5 polypeptide having the activity of proliferation of mouse endothelium cells, wherein said nucleotide sequence is operably linked with an expression regulatory sequences, and said nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2;
(b) introducing the vector of step (a) into a host cell, thereby forming a recombinant cell;
(c) culturing the recombinant cell of step (b) under the conditions suitable for expression of HDGF5 polypeptide; and
(d) isolating the HDGF5 polypeptides so produced.

7. The method of claim 6 wherein said nucleotide sequence comprises nucleotides 5-910 in SEQ ID NO: 1.