WO2000017351A1 - Nouvelle sequence codant pour le facteur de croissance derive de l'hepatome humain et polypeptide code par cette sequence d'adn, et procede de production de ceux-ci - Google Patents

Nouvelle sequence codant pour le facteur de croissance derive de l'hepatome humain et polypeptide code par cette sequence d'adn, et procede de production de ceux-ci Download PDF

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Publication number
WO2000017351A1
WO2000017351A1 PCT/CN1999/000139 CN9900139W WO0017351A1 WO 2000017351 A1 WO2000017351 A1 WO 2000017351A1 CN 9900139 W CN9900139 W CN 9900139W WO 0017351 A1 WO0017351 A1 WO 0017351A1
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Prior art keywords
sequence
hdgf2
polypeptide
seq
protein
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PCT/CN1999/000139
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English (en)
French (fr)
Inventor
Long Yu
Honglai Zhang
Qiang Fu
Yong Zhao
Qiang Tu
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Long Yu
Honglai Zhang
Qiang Fu
Yong Zhao
Qiang Tu
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Priority to US09/787,328 priority Critical patent/US6893844B1/en
Application filed by Long Yu, Honglai Zhang, Qiang Fu, Yong Zhao, Qiang Tu filed Critical Long Yu
Priority to EP99941373A priority patent/EP1123976A4/en
Priority to AU55028/99A priority patent/AU5502899A/en
Priority to JP2000574250A priority patent/JP2002526074A/ja
Priority to CA002343719A priority patent/CA2343719C/en
Publication of WO2000017351A1 publication Critical patent/WO2000017351A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/4753—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • New human liver cancer cell derived growth factor coding sequence
  • the present invention relates to the field of genetic engineering. Specifically, the present invention relates to a new human gene nucleoside ⁇ acid sequence. More specifically, the present invention relates to a cDNA sequence of a novel type II human liver cancer cell-derived growth factor (HDGF2), which is a homologue of type I HDGF. The present invention also relates to polypeptides encoded by the nucleotide sequence, applications of these polynucleotides and polypeptides, and methods of producing said polynucleotides and said polypeptides.
  • HDGF2 human liver cancer cell-derived growth factor
  • Hepatoma cell derived growth factor is a cytokine found in serum-free human liver cancer cell line HuH-7. It has heparin binding ability and can stimulate DNA synthesis in Swiss 3T3 cells (J. Biol. Chem. 269 (40): 25143-25149, 1994).
  • An object of the present invention is to provide a novel polynucleotide sequence, which encodes a HDGF homologous protein, and the HDGF homologous gene of the present invention is named human HDGF2.
  • Another object of the present invention is to provide a new protein which is named human HDGF2.
  • an isolated DNA molecule which includes: a nucleotide sequence encoding a polypeptide having human HDGF2 protein activity, the nucleotide sequence is identical to the nucleotide sequence from SEQ ID NO: 3
  • the nucleotide sequence at positions 121-732 of nucleotides has at least 70% homology; or the nucleotide sequence can be under the conditions of moderate stringency from nucleotide positions 121-732 in SEQ ID NO: 3
  • Nucleotide sequence hybridization Preferably, the sequence encodes a polypeptide having the sequence shown in SEQ ID NO: 4. More preferably, the sequence has a nucleotide sequence from nucleotides 121 to 732 in SEQ ID NO: 3.
  • an isolated HDGF2 protein polypeptide which includes: a polypeptide having the amino acid sequence of SEQ ID NO: 4, or an active fragment thereof, or an active derivative thereof.
  • the polypeptide is a polypeptide having the sequence of SEQ ID NO: 4.
  • a vector which contains the above isolated DNA.
  • a host cell transformed with the vector is provided.
  • a method for producing a polypeptide having HDGF2 protein activity includes:
  • a nucleotide sequence encoding a polypeptide having HDGF2 protein activity is operably linked to an expression control sequence to form an HDGF2 protein expression vector, and the nucleotide sequence is in accordance with SEQ ID NO: 3 from nucleotide 121 -732 nucleotide sequence has at least 70% homology;
  • step (b) transferring the expression vector in step (a) into a host cell to form a recombinant cell of HDGF2 protein;
  • step (c) culturing the recombinant cells in step (b) under conditions suitable for expressing the HDGF2 protein polypeptide; (d) isolating the polypeptide having HDGF2 protein activity.
  • the isolated polynucleotide of the present invention has a total length of 1024 nucleotides, and its detailed sequence is shown in SEQ ID NO: 3, wherein the open reading frame is located at nucleotides 121-732.
  • isolated means that the DNA or fragment has been separated from sequences flanking it in its natural state, and also means that the DNA or fragment has been Separate from components that accompany nucleic acids in their natural state, and have been separated from proteins that accompany them in cells.
  • HDGF2 protein (or polypeptide) coding sequence refers to a nucleotide sequence encoding a polypeptide having HDGF2 protein activity, such as the nucleotide sequence 121-732 in SEQ ID NO: 3 and its degenerate sequence .
  • the degenerate sequence refers to a sequence generated by replacing one or more codons with a degenerate codon encoding the same amino acid in nucleotides 121-732 of the coding frame of the sequence of SEQ ID NO: 3, Due to the degeneracy of the codons, the degenerate sequence with as little as about 70% homology to the nucleotide sequence 121-732 in SEQ ID NO: 3 can also encode the sequence described in SEQ ID NO: 4 .
  • the term also includes a nucleotide sequence from nucleotide positions 121 to 732 in SEQ ID NO: 3 that can be used under moderate stringency conditions, more preferably, under high stringency conditions. Column hybridized nucleotide sequence.
  • the term includes at least 70%, preferably at least 80%, and more preferably at least 90% homology to the nucleotide sequence from nucleotides 121 to 732 in SEQ ID NO: 3 Acid sequence.
  • the term also includes a variant form of the open reading frame sequence in SEQ ID NO: 3 capable of encoding a protein having the same function as human HDGF2.
  • variants include (but are not limited to): deletions of several (usually 1-90, preferably 1-60, more preferably 1-20, most preferably 1-10) nucleotide deletions , Insertions and / or substitutions, and adding several (usually no more than 60, preferably no more than 30, more preferably no more than 10, most preferably no more than 5) cores at the 5 and / or 3 ends Glycylic acid.
  • a "substantially pure" protein or polypeptide refers to at least 20%, preferably at least 50%, more preferably at least 80%, and most preferably at least 90% (by dry weight) of the total substance of the sample. Or wet weight meter). Purity can be measured by any suitable method, such as column purity, PAGE or HPLC. A substantially pure polypeptide is essentially free of the components that accompany it in its natural state.
  • HDGF2 protein polypeptide refers to a polypeptide having the sequence of SEQ ID NO: 4 having HDGF2 protein activity.
  • the term also includes a variant of the sequence of SEQ ID NO: 4 having the same function as human HDGF2.
  • These variants include (but are not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletions, insertions Human and / or substitutions, and the addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus.
  • substitution of amino acids with similar or similar properties usually does not change the function of the protein.
  • adding one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of HDGF2 protein.
  • Variants of the polypeptide include: homologous sequences, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with HDGF2 DNA under high or low stringency conditions, and the use of anti-HDGF2 polypeptides Antisera obtained from a peptide or protein.
  • the invention also provides other polypeptides, such as fusion proteins comprising HDGF2 polypeptides or fragments thereof. In addition to almost full-length polypeptides, the invention also includes soluble fragments of HDGF2 polypeptides.
  • the fragment has at least about 10 consecutive amino acids, typically at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids in the HDGF2 polypeptide sequence. Consecutive amino acids.
  • the invention also provides analogs of HDGF2 protein or polypeptide.
  • the difference between these analogs and the natural HDGF2 polypeptide may be a difference in the amino acid sequence, a difference in the modified form that does not affect the sequence, or both.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or Other known molecular biology techniques.
  • Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as Y-amino acids). It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modified (usually unchanged primary structure) forms include: Chemically derived forms of polypeptides in vivo or in vitro such as acetylated or carboxylated. Modifications also include glycosylation, such as those produced by glycosylation modification in the synthesis and processing of polypeptides or in further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that undergoes glycosylation, such as mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues (e.g., phosphotyrosine, phosphoserine, phosphothreonine). Also included are peptides that have been modified to increase their resistance to proteolysis or to optimize their solubility.
  • glycosylation such as those produced by glycosylation modification in the synthesis and processing of polypeptides or in further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that undergoes glycosylation, such as mammalian
  • the invention also includes antisense sequences for the HDGF2 polypeptide coding sequence. This antisense sequence can be used to inhibit HDGF2 expression in cells.
  • the invention also includes nucleic acid molecules that can be used as probes and primers.
  • the molecules typically have 8 to 100, preferably 15 to 50 consecutive nucleotides of the HDGF2 polypeptide coding sequence.
  • This probe can be used to detect the presence of a nucleic acid molecule encoding HDGF2 in a sample.
  • the present invention also includes a method for detecting a nucleotide sequence of HDGF2, which comprises hybridizing a probe with a sample as described above, and then detecting whether the probe is bound.
  • the sample is a product after PCR amplification, wherein the PCR amplification primer corresponds to the coding sequence of the HDGF2 polypeptide, and may be located on either side or in the middle of the coding sequence.
  • Primers are typically 20-50 nucleotides in length
  • vectors known in the art may be used, such as commercially available vectors.
  • the term "host cell” includes prokaryotic cells and eukaryotic cells.
  • prokaryotic host cells include E. coli, Bacillus subtilis, and the like.
  • eukaryotic host cells include yeast cells, insect cells, and mammalian cells.
  • the host cell is a eukaryotic cell, such as a CHO cell, a COS cell, or the like.
  • the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, having specificity for the polypeptide encoded by HDGF2 DNA or a fragment thereof.
  • specificity means that the antibody can bind to the HDGF2 gene product or fragment.
  • it refers to those antibodies that can bind to the HDGF2 gene product or fragment but do not recognize and bind to other unrelated antigen molecules.
  • Antibodies in the present invention include those capable of binding and inhibiting HDGF2 protein, as well as those that do not affect the function of HDGF2 protein.
  • the invention also includes those antibodies that are capable of binding to a modified or unmodified form of the HDGF2 gene product.
  • the invention includes not only intact monoclonal or polyclonal antibodies, but also antibody fragments with immunological activity, such as Fab 'or (Fab) 2 fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules ( Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as those with murine antibody binding specificity but Antibodies from the human antibody portion are still retained.
  • the antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, a purified HDGF2 gene product or an antigenic fragment thereof can be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing HDGF2 or its antigenic fragments can be used to immunize animals to produce antibodies.
  • the antibody of the present invention may be a monoclonal antibody. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur. J. Immunol. 6: 5 1 1, 1976; Kohler et al., Eur. J. Immunol.
  • the antibodies of the present invention include antibodies capable of blocking HDGF2 function and antibodies that do not affect HDGF2 function.
  • Various types of antibodies of the present invention can be obtained by conventional immunological techniques using fragments or functional regions of the HDGF2 gene product. These fragments or functional regions can be prepared by recombinant methods or synthesized using a peptide synthesizer.
  • Antibodies that bind to unmodified forms of the HDGF2 gene product can be produced by immunizing animals with gene products produced in prokaryotic cells (e.g. £ Co //); antibodies that bind to post-translationally modified forms (such as glycosylation or phosphorylation) Protein or polypeptide) can be obtained by immunizing animals with gene products produced in eukaryotic cells (such as yeast or insect cells).
  • the full-length human HDGF2 nucleotide sequence or a fragment thereof of the present invention can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • PCR amplification method according to the related nucleotide sequence disclosed in the present invention, In particular, open reading frame sequences are used to design primers, and commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art are used as templates to amplify the relevant sequences. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then stitch the amplified fragments in the correct order.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning a human vector, transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • relevant methods can also be synthesized by artificial synthesis, especially when the fragment length is short. Generally, long fragments can be obtained by synthesizing multiple small fragments first and then joining them.
  • DNA sequences encoding proteins (or fragments thereof, or derivatives thereof) of the present invention can be completely synthesized by chemical synthesis. Mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • fragments of the protein of the invention can also be produced by direct synthesis of peptides using solid-phase technology (Stewart et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Merrifield J (1963) J. Am Chem. Soc 85: 2 149-2154). Protein synthesis in vitro can be performed manually or automatically. For example, an Applied Biosystems Model 43 1A Peptide Synthesizer (Foster City, CA) can be used to automatically synthesize peptides. Fragments of the protein of the invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule.
  • the coding sequence of the protein of the present invention can also be used for gene mapping. For example, by fluorescence in situ hybridization
  • FISH Fluorescence In situ hybridization
  • the mutation may be the causative factor for the disease.
  • substances that interact with HDGF2 such as receptors, inhibitors or antagonists, can be screened through various conventional screening methods.
  • the protein, the antibody, the inhibitor, the antagonist or the receptor of the present invention when administered therapeutically (administration), can provide different effects.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, and preferably about 6-8, although the pH can vary with The nature of the formulation and the condition to be treated will vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.
  • the human HDGF2 protein of the present invention can be combined with a suitable pharmaceutically acceptable carrier.
  • suitable pharmaceutically acceptable carrier include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the human HDGF2 protein of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • compositions such as injections, solutions, tablets and capsules should preferably be manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 mg / kg body weight per day.
  • the polypeptides of the invention can be used with other therapeutic agents.
  • a therapeutically effective dose of the polypeptide can be administered to a mammal, wherein the therapeutically effective dose is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 8 mg / kg body weight, preferably the dose is about 10 micrograms / kg body weight-about 1 mg / kg body weight.
  • the specific dosage should also consider factors such as the route of administration, the patient's health, and other factors, which are all within the skill of a skilled physician.
  • FIG. 1 is a homologous comparison diagram of the nucleic acid sequences of HDGF2 of the present invention and mouse HDGF2. Among them, the same nucleotides are marked with ⁇ ".
  • Figure 2 is a homology comparison diagram of the amino acid sequences of HDGF2 and mouse HDGF2 in the present invention. Among them, the same amino acid is marked with " ⁇ ", and the similar amino acid is marked with " ⁇ ".
  • the cDNA nucleotide sequence of human HDGF2 was obtained in such a way that a human testis ⁇ gtl l cDNA library ( (Purchased from Clontech) were used as templates to synthesize forward primer A1: 5'- ACCGCTCGTCCGCCCGGCTTGAG-3 'and reverse bow I A2: 5'- GATCCTAGACATGTATAAGTCTGCG C-3', and perform PCR to obtain 1024bp target fragments respectively. After sequencing, The full-length cDNA sequence of SEQ ID NO: 3 was obtained.
  • Hepatoma cell-derived growth factor is a heparin-binding protein isolated from human liver cancer cell line HuH-7. It has the activity of stimulating cell growth and can promote the growth of fibroblasts and some liver cancer cells (J. Biol. Chem. 269 (40): 25143-25149, 1994). It is expressed in various tissues of human heart, brain, lung, liver, and various cancer cell lines (J. Biol. Chem. 269 (40): 25143-25149, 1994). The expression patterns of the known members of the HDGF family are different, but they are highly enriched in the testis, and their 5 untranslated regions all have a GC ratio greater than 70% (Biochem. Biophys. Res Commun.
  • HDGF protein is mainly present in the cytoplasm (J. Biol. Chem. 269 (40): 25143-25149, 1994), the amino acid sequences of the family members contain a potential nuclear localization signal (NLS), and none of them Peptide sequences, suggesting that they may function as nuclear proteins.
  • NLS nuclear localization signal
  • HDGF acidic amino acid tail at the C-terminus of HDGF is highly homologous to HMG-1 / -2 of the HMG family, and this sequence is known in HMG-1 / -2 as a histone binding region (Biochemistry 29: 4419-4423, 1990). It is likely that HDGF exerts its cell-stimulating activity as a transcription factor after internalization (Biochem. Biophys. Res. Commun. 238: 26-32, 1997). HDGF's mitogen activity makes it extremely valuable for the treatment of acute malignant hepatitis and liver injury (Clin. Chim. Acta. 183: 273-284, 1989).
  • oligonucleotide A1 5'-
  • ACCGCTCGTCCGCCCGGCTTGAG-3 ' (SEQ ID NO: 1) is a forward primer
  • oligonucleotide A2: 5 * -GATCCTAGACATGTATAAGTCTGCGC-3' (SEQ ID NO: 2) is a reverse primer
  • PCR is performed.
  • the PCR conditions are 93 'C 4 minutes followed by 93'C for 1 minute, 68.5'C for 1 minute and 72. 35 cycles were performed at C for 1 minute, and the final 72'C was extended for 5 minutes.
  • the PCR fragment obtained by electrophoresis was a 1024 bp target fragment.
  • the PCR amplification product obtained above was ligated with pGEM-T TM vector (Promega), transformed into E. coli JM103, plasmids were extracted with QIAprep Plasmid Kit (QIAGEN), and inserted into humans with double-stranded nested deletion kit (Pharmacia) The fragments are subjected to a targeted series of deletions, and then the deletions are rapidly identified by PCR
  • the sequenced truncated deletions were sequenced using the SequiTherm EXCEL DNA sequencing kit (Epicentre Technologies), and the sequence was spliced using computer software to obtain the full-length cDNA sequence, a total of 1024 bp.
  • the detailed sequence is shown in SEQ ID NO: 3, of which the open reading frame Located at nucleotides 121-732.
  • human HDGF2 and another mouse HDGF gene (dbj
  • Hepatocellular carcinoma-derived growth factor is a heparin-binding protein isolated from the human hepatocellular carcinoma cell line HuH-7. Chem. 269 (40): 25143-25149, 1994). Although HDGF was originally found in liver cancer cells, Northern blot analysis showed that it is expressed in various tissues of human heart, brain, lung, liver, and various cancer cell lines. Whether it is expressed differently in normal cells and tumor cells needs further confirmation (J. Biol. Chem. 269 (40): 25143-25149, 1994). With the deepening of research, the role of HDGF in liver cancer cells and its impact on liver cancer treatment will continue to be revealed.
  • HDGF family members are known to have different expression patterns, but they are highly enriched in the testis, and the 5 untranslated regions of these genes have GC ratios greater than 70% (Biochem. Biophys. Res Commun. 238: 26-32, 1997), this feature is similar to some genes specifically expressed in the testis or embryo development, so they may have important functions in the development of male germ cells, and are related to DNA methylation, Chromatin conformation and translation regulation (J. Cell. Biol. 1 15: 887-903, 1990; Cell 62: 503-514, 1990),
  • HDGF protein mainly exists in the cytoplasm (J. Biol. Chem. 269 (40): 25 143-25149, 1994), but the amino acid sequence of the family members contains a basic region-a potential nuclear Localization signals (NLS), and no signal peptide sequence, suggest that they may function as nuclear proteins. Fibroblast growth factor (FGF) must rely on this signal region to locate it in the nucleus to exert its mitogen activity.
  • FGF Fibroblast growth factor
  • the C-terminal acidic amino acid tail of HDGF is highly homologous to HMG-1 / -2 of the HMG family, and this sequence is known to be a histone binding region in HMG-1 / -2 (Biochemistry 29: 4419-4423, 1990).
  • HDGF2 of the present invention also has a similar activity.
  • the mitogen activity of HDGF makes it extremely valuable for the treatment of acute malignant hepatitis and liver injury (Clin. Chim. Acta. 183: 273-284, 1989).
  • fibroblast growth factors have the ability to promote epithelial cell growth and can be widely used in ischemic and atherosclerotic diseases such as angiogenesis defects and nerve cell development (Blood 91 (10) : 3527- 3561, 1998; Ann. NY Acad. Sci. 545: 240-252, 1998).
  • Type I HDGF and HDGF2 of the present invention Its application in promoting fibroblast growth activity remains to be further studied.
  • the HDGF2 of the present invention can also be used to produce fusion proteins with other proteins, such as immunoglobulins to produce fusion proteins.
  • the HDGF2 of the present invention can also be fused or exchanged with other members of the family to produce new proteins, such as the N-terminus of HDGF2 of the present invention and the N-terminus of type I HDGF or mouse HDGF to generate new '' Proteins with higher sex or new properties,
  • the antibody against HDGF2 of the present invention is used for screening other members of the family, or for affinity purification of related proteins (such as other members of the family).
  • Example 3 The antibody against HDGF2 of the present invention is used for screening other members of the family, or for affinity purification of related proteins (such as other members of the family).
  • the cDNA sequence encoding HDGF2 was amplified with PCR oligonucleotide primers corresponding to the 5 'and 3' ends of the DNA sequence, and HDGF2 cDNA was obtained as an insert.
  • the 5 'oligonucleotide primer sequence used in the PCR reaction is:
  • the primer contains a restriction site for BamHI restriction enzyme, which is followed by 19 nucleotides of the HDGF2 coding sequence starting from the start code;
  • the 3 'end primer sequence is:
  • This primer contains a restriction site for a Sail restriction enzyme, a translation terminator and a partial coding sequence of HDGF2.
  • the restriction enzyme restriction site on the primer corresponds to the restriction enzyme restriction site on the bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, CA), which encodes an antibiotic resistance ( Am p r), a bacterial origin of replication (ori), an IPTG-tunable promoter / operon (P / 0), a ribosome binding site (RBS), a 6-histidine tag (6- His) and restriction enzyme cloning sites.
  • pQE-9 Qiagen Inc., Chatsworth, CA
  • the pQE-9 vector and the insert were digested with BamHI and Sail.
  • the insert was then ligated to the pQE-9 vector and the open reading frame was started at the bacterial RBS.
  • the ligation mixture was then used to transform the strain purchased from Qiagen under the trade name M15 / rep ⁇ £ .co //.
  • M15 / rep4 contained multiple copies of the plasmid pREP4, which expressed lacl repressor and carried kanamycin resistance (Kan in Transformants were screened on Amp and Kan's LB dishes, the plasmids were extracted, the size and orientation of the inserts were identified by Pstl digestion, and sequencing verified that the cDNA fragment of HDGF2 was inserted into the vector correctly.
  • HDGF2 was purified from the solution by nickel-chelation column chromatography under conditions capable of tightly binding the 6-His tag-containing protein, and eluted from the column with 6M guanidine hydrochloride (pH 5.0).
  • 6M guanidine hydrochloride pH 5.0
  • HDGF2 denaturing precipitated proteins from guanidine hydrochloride. Either the dialysis step is used to remove the guanidine hydrochloride, or the purified protein is separated from the nickel-chelated column.
  • the purified protein can be bound to a second column with a decreasing linear guanidine hydrochloride gradient.
  • the protein was denatured while bound to the column, followed by elution with guanidine hydrochloride (pH 5.0). Finally, the soluble protein was dialyzed against PBS, and the protein was stored in a stock solution with a final concentration of 10% (w / v) glycerol.
  • the molecular weight of the expressed protein was identified to be about 23 KDa.
  • conventional methods were used to sequence an amino acid of 10 amino acids in length at each of the N-terminus and C-terminus of the expressed protein, and it was found to be consistent with the sequence of SEQ ID NO: 4.
  • the cDNA sequence encoding HDGF2 was amplified with PCR oligonucleotide primers corresponding to the 5 'and 3' ends of the DNA sequence, and HDGF2 cDNA was obtained as an insertion fragment.
  • the 5 'oligonucleotide primer sequence used in the PCR reaction is:
  • the primer contains a restriction site of Hindlll restriction enzyme, and after the restriction site is 19 nucleotides of the HDGF2 coding sequence starting from the start code;
  • the 3 'end primer sequence is:
  • the primer contains a restriction site for BamHI restriction enzyme, a translation terminator and a part of the coding sequence of HDGF2.
  • the restriction enzyme restriction site on the primer corresponds to the restriction enzyme restriction site on the CHO cell expression vector pcDNA3.
  • This plasmid vector encodes antibiotic resistance (Amp f and Ne 0 r), a phage Origin of replication (fl ⁇ ), a virus replication origin (SV40 ori), a T7 promoter, a viral promoter (P-CMV), a Sp6 promoter, an SV40 promoter, an SV40 tailing signal and corresponding polyA Sequence, a BGH-tailed signal, and the corresponding polyA sequence.
  • the pcDNA3 vector and the insert were digested with Hindlll and BamHI, and the insert was subsequently ligated to the pcDNA3 vector.
  • the ligation mixture was then used to transform the £. ⁇ // 11515 0 [ strain.
  • Transformants were selected on LB dishes containing Amp, and clones containing the desired constructs were cultured overnight (0 / N) in LB liquid medium supplemented with A ⁇ (100 ⁇ g / ml). Extract the plasmid, identify the size and orientation of the insert with Pstl digestion, and sequence and verify that the cDNA fragment of HDGF2 has been inserted into the vector correctly.
  • Plasmid transfection of CHO cells was performed by lipofection using the Lipofectin kit (GiBco Life). 48 hours after transfection, cells were screened for 2-3 weeks under G418 pressure, and cells and cell supernatants were collected to determine the proteinase activity. Go to G418 and continue subculture; limit dilution of mixed cloned cells and select subclones with higher protein activity. The above-mentioned positive subclones were cultured in large numbers according to a conventional method. After 48 hours, the cells and supernatant were collected, and the cells were disrupted by ultrasonic lysis to a concentration of 0.05 ° /.
  • Triton's 50 mM Tris-HCl (pH 7.6) solution was used as the equilibration solution and eluent.
  • the pre-equilibrated Superdex G-75 column was used to collect the active peaks of the above proteins.
  • a 50 mM Tris ⁇ HCl (pH 8.0) equilibrated DEAE-Sepharose column was used, and a 50 mMTds ⁇ HCl (pH 8.0) solution containing 0-1 M NaCl was used as an eluent to perform gradient elution, and the active peaks of the proteins were collected.
  • the expressed protein solution was then dialyzed against PBS (pH 7.4) as the dialysate. Finally lyophilized and stored.
  • the molecular weight of the expressed protein was 23KDa.
  • the N-terminus and C-terminus of the expressed protein were sequenced by an amino acid having a length of 10 amino acids each at the conventional method, and were found to be identical to the sequence of SEQ ID NO: 4.
  • the recombinant proteins obtained in Examples 3 and 4 were used to immunize animals to produce antibodies, as follows.
  • the heavy components are separated by chromatography and then used. It can also be separated by SDS-PAGE gel electrophoresis.
  • the electrophoresis band is cut from the gel and emulsified with an equal volume of complete Freimd's adjuvant.
  • Mice were injected intraperitoneally with 50-100 ⁇ g / 0.2ml of emulsified protein. After 14 days, mice were injected intraperitoneally with the same antigen emulsified with incomplete Freund's adjuvant at a dose of 50-100 ⁇ g / 0.2ml to boost immunity.
  • Booster immunizations are performed every 14 days, at least three times. The specific reactivity of the obtained antiserum was evaluated by its ability to precipitate HDGF2 gene translation products in vitro.

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Description

新的人肝癌细胞衍生生长因子编码序列、
其编码的多肽及制备方法 发明领域
本发明涉及基因工程领域， 具体地， 本发明涉及一种新的人基因核苷 ·酸序 列。 更具体地说， 本发明涉及新的 II型人肝癌细胞衍生生长因子 (HDGF2)的 cDNA 序列， 该蛋白是 I型 HDGF的同系物。 本发明还涉及由该核苷酸序列编码的多肽， 这些多核苷酸和多肽的应用， 以及所述多核苷酸和所述多肽的生产方法. 背景技术
研究已表明， 细胞生长的调节是通过各种细胞因子与其特异的膜表面受体作 用后引发的一系列级联反应实现的。 在肿瘤细胞中， 某些级联反应的失控致使细 胞持续增殖。 在肝癌细胞中， 人们已经发现一些自分泌和旁分泌细胞因子 (Proc. Natl. Acad. Sci. 83:2448-2452, 1986； Proc. Natl. Acad. Sci. 86:7432-7436, 1989； Cell 61 : 1137-1146, 1990)。 肝癌细胞衍生生长因子 (HDGF)是在无血清培养的人肝 癌细胞株系 HuH-7中找到的一个细胞因子， 它具有肝素结合能力并能刺激 Swiss 3T3细胞的 DNA合成 (J. Biol. Chem. 269(40):25143-25149, 1994)。
1989年 Nakamura等人首次从 HuH-7细胞中部分纯化并鉴定了 HDGF(Clin. Chim. Acta. 183:273-284, 1989), 1994年该实验组又完整克隆了 HDGF的 cDNA序 列（J. Biol. Chem. 269(40):25143-25149, 1994), 1997年该实验组在小鼠中找到了 HDGF的同系物并发现了该基因家族的另两个成员 HRP-1、 HRP-2, 它们都具有一 个相当保守的 98个氨基酸长的氨基端序列（Biochem. Biophys. Res. Commun. 238:26-32, 1997)。 在本发明被公布之前， 尚没有任何人公开过本申请中涉及的 另一个人类 HDGF家族成员人 HDGF2。 发明概述
本发明的一个目的是提供一种新的多核苷酸序列， 该多核苷酸序列编码 HDGF同源蛋白， 本发明的 HDGF同源基因被命名为人 HDGF2。
本发明的另一个目的是提供一种新的蛋白， 该蛋白被命名为人 HDGF2。
本发明的再一个目的是提供一种利用重组技术生产所述的新的人 HDGF2蛋 白的方法。
本发明还提供了这种人 HDGF2基因序列和蛋白的应用。 在本发明的一个方面， 提供了一种分离出的 DNA分子， 它包括： 编码具有人 HDGF2蛋白活性的多肽的核苷酸序列， 所述的核苷酸序列与 SEQ ID NO: 3中从核 苷酸 121-732位的核苷酸序列有至少 70%的同源性； 或者所述的核苷酸序列能在中 度严紧条件下与 SEQ ID NO: 3中从核苷酸 121-732位的核苷酸序列杂交。 较佳地， 所述的序列编码一多肽， 该多肽具有 SEQ ID NO: 4所示的序列。 更佳地， 该序列 具有 SEQ ID NO: 3中从核苷酸 121-732位的核苷酸序列。
在本发明的另一方面，提供了一种分离的 HDGF2蛋白多肽，它包括：具有 SEQ ID NO: 4氨基酸序列的多肽、 或其活性片段， 或其活性衍生物。 较佳地， 该多肽 是具有 SEQ ID NO: 4序列的多肽。
在本发明的另一方面， 提供了一种载体， 它含有上述分离出的 DNA.
在本发明的另一方面， 提供了一种所述载体转化的宿主细胞。
在本发明的另一方面， 提供了一种产生具有 HDGF2蛋白活性的多肽的方法， 该方法包括：
(a)将编码具有 HDGF2蛋白活性的多肽的核苷酸序列可操作地连于表达调控 序列， 形成 HDGF2蛋白表达载体， 所述的核苷酸序列与 SEQ ID NO: 3中从核苷酸 121-732位的核苷酸序列有至少 70%的同源性；
(b)将步骤 (a)中的表达载体转入宿主细胞， 形成 HDGF2蛋白的重组细胞；
(c)在适合表达 HDGF2蛋白多肽的条件下， 培养步骤 (b)中的重组细胞； (d)分离出具有 HDGF2蛋白活性的多肽。
在本发明的一个具体实施方案中， 本发明的分离的多核苷酸全长为 1024个核 苷酸， 其详细序列见 SEQ ID NO: 3, 其中开放读框位于 121-732位核苷酸。
在本发明中， "分离的" 、 "纯化的" 或 "基本纯的" DNA是指， 该 DNA 或片段已从天然状态下位于其两侧的序列中分离出来， 还指该 DNA或片段已经与 天然状态下伴随核酸的组份分开， 而且已经与在细胞中伴随其的蛋白质分开。
在本发明中， 术语 "HDGF2蛋白 (或多肽)编码序列"指编码具有 HDGF2蛋白 活性的多肽的核苷酸序列， 如 SEQ ID NO: 3中 121-732位核苷酸序列及其简并序 列。 该简并序列是指， 位于 SEQ ID NO: 3序列的编码框 121-732位核苷酸中， 有一 个或多个密码子被编码相同氨基酸的简并密码子所取代后而产生的序列， 由于密 码子的简并性， 所以与 SEQ ID N〇： 3中 121-732位核苷酸序列同源性低至约 70%的 简并序列也能编码出 SEQ ID NO: 4所述的序列。 该术语还包括能在中度严紧条件 下， 更佳地， 在高度严紧条件下与 SEQ ID NO: 3中从核苷酸 121-732位的核苷酸序 列杂交的核苷酸序列。 此外， 该术语还包括与 SEQ ID NO: 3中从核苷酸 121-732 位的核苷酸序列的同源性至少 70 %， 较佳地至少 80 % , 更佳地至少 90 %的核苷酸 序列。
该术语还包括能编码具有与人 HDGF2相同功能的蛋白的、 SEQ ID NO: 3中开 放读框序列的变异形式。 这些变异形式包括 (但并不限于)： 若干个 (通常为 1-90个， 较佳地 1-60个， 更佳地 1-20个， 最佳地 1-10个)核苷酸的缺失、 插入和 /或取代， 以 及在 5和 /或 3端添加数个 (通常为 60个以内， 较佳地为 30个以内， 更佳地为 10个以 内， 最佳地为 5个以内)核苷酸。
在本发明中， "基本纯的" 蛋白质或多肽是指其至少占样品总物质的至少 20%, 较佳地至少 50%, 更佳地至少 80%, 最佳地至少 90% (按干重或湿重计)。 纯 度可以用任何合适的方法进行测量， 如用柱层析、 PAGE或 HPLC法测量多肽的纯 度。 基本纯的多肽基本上不含天然状态下的伴随其的组分。
在本发明中， 术语" HDGF2蛋白多肽"指具有 HDGF2蛋白活性的 SEQ ID NO: 4序列的多肽。 该术语还包括具有与人 HDGF2相同功能的、 SEQ ID NO: 4序列的 变异形式。 这些变异形式包括 (但并不限于)： 若干个 (通常为 1-50个， 较佳地 1-30 个， 更佳地 1-20个， 最佳地 1-10个)氨基酸的缺失、 插人和 /或取代， 以及在 C末端 和 /或 N末端添加一个或数个 (通常为 20个以内， 较佳地为 10个以内， 更佳地为 5个 以内)氨基酸。 例如， 在本领域中， 用性能相近或相似的氨基酸进行取代时， 通常 不会改变蛋白质的功能。 又比如， 在 C末端和 /或 N末端添加一个或数个氨基酸通 常也不会改变蛋白质的功能。 该术语还包括 HDGF2蛋白的活性片段和活性衍生 物。
该多肽的变异形式包括： 同源序列、 等位变异体、 天然突变体、 诱导突变体、 在高或低的严谨度条件下能与 HDGF2 DNA 杂交的 DNA所编码的蛋白、 以及利用 抗 HDGF2多肽的抗血清获得的多肽或蛋白。 本发明还提供了其他多肽， 如包含 HDGF2多肽或其片段的融合蛋白。 除了几乎全长的多肽外， 本发明还包括了 HDGF2多肽的可溶性片段。 通常， 该片段具有 HDGF2多肽序列的至少约 10个连续 氨基酸， 通常至少约 30个连续氨基酸， 较佳地至少约 50个连续氨基酸， 更佳地至 少约 80个连续氨基酸， 最佳地至少约 100个连续氨基酸。
发明还提供 HDGF2蛋白或多肽的类似物。 这些类似物与天然 HDGF2多肽的 差别可以是氨基酸序列上的差异， 也可以是不影响序列的修饰形式上的差异， 或 者兼而有之。 这些多肽包括天然或诱导的遗传变异体。 诱导变异体可以通过各种 技术得到， 如通过辐射或暴露于诱变剂而产生随机诱变， 还可通过定点诱变法或 其他已知分子生物学的技术。类似物还包括具有不同于天然 L-氨基酸的残基 (如 D- 氨基酸)的类似物， 以及具有非天然存在的或合成的氨基酸 (如 、 Y -氨基酸)的类 似物。 应理解， 本发明的多肽并不限于上述例举的代表性的多肽。
修饰 (通常不改变一级结构)形式包括： 体内或体外的多肽的化学衍生形式如 乙酰化或羧基化。 修饰还包括糖基化， 如那些在多肽的合成和加工中或进 步加 工步骤中进行糖基化修饰而产生的多肽。 这种修饰可以通过将多肽暴露于进行糖 基化的酶 (如哺乳动物的糖基化酶或去糖基化酶)而完成。 修饰形式还包括具有磷 酸化氨基酸残基 (如磷酸酪氨酸， 磷酸丝氨酸， 磷酸苏氨酸)的序列。 还包括被修 饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
本发明还包括 HDGF2多肽编码序列的反义序列。这种反义序列可用于抑制细 胞内 HDGF2的表达。
本发明还包括可用作探针和引物的核酸分子，该分子通常具有 HDGF2多肽编 码序列的 8- 100个， 较佳地 15-50个连续核苷酸。 该探针可用于检测样品中是否存 在编码 HDGF2的核酸分子。
本发明还包括检测 HDGF2核苷酸序列的方法，它包括用上述的探针与样品进 行杂交， 然后检测探针是否发生了结合。 较佳地， 该样品是 PCR扩增后的产物， 其中 PCR扩增引物对应于 HDGF2多肽的编码序列， 并可位于该编码序列的两侧或 中间。 引物长度一般为 20-50个核苷酸，
在本发明中， 可选用本领域已知的各种载体， 如巿售的载体。
在本发明中， 术语 "宿主细胞" 包括原核细胞和真核细胞。 常用的原核宿主 细胞的例子包括大肠杆菌、 枯草杆菌等。 常用的真核宿主细胞包括酵母细胞， 昆 虫细胞、 和哺乳动物细胞。 较佳地， 该宿主细胞是真核细胞， 如 CHO细胞、 COS 细胞等。
另一方面， 本发明还包括对 HDGF2 DNA或是其片段编码的多肽具有特异性 的多克隆抗体和单克隆抗体， 尤其是单克隆抗体。 这里， "特异性" 是指抗体能 结合于 HDGF2基因产物或片段 较佳地， 指那些能与 HDGF2基因产物或片段结合 但不识别和结合于其它非相关抗原分子的抗体。 本发明中抗体包括那些能够结合 并抑制 HDGF2蛋白的分子， 也包括那些并不影响 HDGF2蛋白功能的抗体。 本发明 还包括那些能与修饰或未经修饰形式的 HDGF2基因产物结合的抗体。
本发明不仅包括完整的单克隆或多克隆抗体， 而且还包括具有免疫活性的抗 体片段， 如 Fab'或 (Fab)2片段； 抗体重链； 抗体轻链； 遗传工程改造的单链 Fv分子 (Ladner等人， 美国专利 No. 4,946,778); 或嵌合抗体， 如具有鼠抗体结合特异性但 仍保留来自人的抗体部分的抗体。
本发明的抗体可以通过本领域内技术人员已知的各种技术进行制备。 例如， 纯化的 HDGF2基因产物或者其具有抗原性的片段， 可被施用于动物以诱导多克隆 抗体的产生。 与之相似的， 表达 HDGF2或其具有抗原性的片段的细胞可用来免疫 动物来生产抗体。 本发明的抗体也可以是单克隆抗体。 此类单克隆抗体可 ¾利用 杂交瘤技术来制备（见 Kohler 等人， Nature 256;495, 1975 ; Kohler 等人， Eur. J.Immunol. 6:5 1 1 , 1976; Kohler等人， Eur.J.Immunol. 6:292, 1976; Hammerling等 人， In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981)。 本发明 的抗体包括能阻断 HDGF2功能的抗体以及不影响 HDGF2功能的抗体。本发明的各 类抗体可以利用 HDGF2基因产物的片段或功能区， 通过常规免疫技术获得。 这些 片段或功能区可以利用重组方法制备或利用多肽合成仪合成。 与 HDGF2基因产物 的未修饰形式结合的抗体可以用原核细胞 (例如 £. Co//)中生产的基因产物来免疫 动物而产生； 与翻译后修饰形式结合的抗体 (如糖基化或磷酸化的蛋白或多肽)， 可以用真核细胞 (例如酵母或昆虫细胞)中产生的基因产物来免疫动物而获得。
本发明的人 HDGF2核苷酸全长序列或其片段通常可以用 PCR扩增法、 重组法 或人工合成的方法获得， 对于 PCR扩增法， 可根据本发明所公开的有关核苷酸序 列， 尤其是开放阅读框序列来设计引物， 并用市售的 cDNA库或按本领域技术人 员已知的常规方法所制备的 cDNA库作为模板， 扩增而得有关序列。 当序列较长 曰寸， 常常需要进行两次或多次 PCR扩增， 然后再将各次扩增出的片段按正确次序 拼接在一起。
一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。 这通常 是将其克隆人载体， 再转入细胞， 然后通过常规方法从增殖后的宿主细胞中分离 得到有关序列。
此外， 还可用人工合成的方法来合成有关序列， 尤其是片段长度较短时。 通 常， 通过先合成多个小片段， 然后再进行连接可获得序列很长的片段。 目前， 已 经可以完全通过化学合成来编码本发明蛋白 (或其片段， 或其衍生物)的 DNA 序 列。 还可通过化学合成将突变引人本发明蛋白序列中。
除了用重组法产生之外， 本发明蛋白的片段还可用固相技术， 通过直接合成 肽而加以生产（Stewart 等人 ，（1969) Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco; Merrifield J. ( 1963) J. Am Chem. Soc 85 : 2 149-2154). 在体外合 成蛋白质可以用手工或自动进行。 例如， 可以用 Applied Biosystems的 43 1A型肽 合成仪 (Foster City, CA)来自动合成肽。 可以分别化学合成本发明蛋白的各片段， 然后用化学方法加以连接以产生全长的分子。 本发明蛋白的编码序列还可用于基因定位。 例如， 通过荧光原位杂交技术
(FISH), 将 cDNA克隆与分裂中期的染色体进行杂交， 可以准确地进行染色体定 位。 该技术可以使用短至约 500bp的 cDNA ; 也可以使用长至约 2000bp或者更 长的 cDNA。对于该技术， 可参见 Verma 等人， Human Chromosomes: A Manual of 'Basic Techniques, Pergamon Press, New York( 1988)。 一 一旦序列被定位于染色体上的某个精确位置， 将可以将序列在染色体上的物 理位置与遗传图谱数据相关联。 这些遗传图谱数据是可以获得的， 例如通过孟德 尔 (Mendelian)人遗传数据库 (可通过 Johns Hopkins University Welch Medical Library 在网上获得)。 然后， 通过连锁分析来鉴定基因与已定位于同一染色体区 域的疾病之间的相关性。
接着， 有必要确定患病个体和健康个体之间的 cDNA或基因组序列方面的差 异。 如果某一突变存在于部分或全部患病个体但不存在于正常个体， 那么该突变 可能就是该疾病的致病因素。
利用本发明蛋白， 通过各种常规筛选方法， 可筛选出与 HDGF2发生相互作 用的物质， 如受体、 抑制剂或拮抗剂等。
本发明蛋白及其抗体、 抑制剂、 拮抗剂或受体等， 当在治疗上进行施用 (给 药)时， 可提供不同的效果。 通常， 可将这些物质配制于无毒的、 惰性的和药学上 可接受的水性载体介质中， 其中 pH通常为约 5 - 8 , 较佳地 pH约为 6 - 8 , 尽管 pH值可随被配制物质的性质以及待治疗的病症而有所变化。 配制好的药物 组合物可以通过常规途径进行给药， 其中包括 (但并不限于)： 肌内、 腹膜内、 皮 下、 皮内、 或局部给药。
以本发明的人 HDGF2蛋白为例， 可以将其与合适的药学上可接受的载体联 用。 这类药物组合物含有治疗有效量的蛋白质和药学上可接受的载体或赋形剂。 这类载体包括 (但并不限于)： 盐水、 缓冲液、 葡萄糖、 水、 甘油、 乙醇、 及其组 合。 药物制剂应与给药方式相匹配。 本发明的人 HDGF2蛋白可以被制成针剂形 式， 例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。 诸如片剂和胶囊之类的药物组合物， 可通过常规方法进行制备。 药物组合物如针 剂、 溶液、 片剂和胶囊宜在无菌条件下制造。 活性成分的给药量是治疗有效量， 例如每天约 1微克 /千克体重-约 5毫克 /千克体重。 此外， 本发明的多肽还可与 其他治疗剂一起使用。
当本发明的人 HDGF2蛋白多肽被用作药物时， 可将治疗有效剂量的该多肽 施用于哺乳动物， 其中该治疗有效剂量通常至少约 10微克 /千克体重， 而且在大 多数情况下不超过约 8毫克 /千克体重， 较佳地该剂量是约 10微克 /千克体重 -约 1毫克 /千克体重。 当然， 具体剂量还应考虑给药途径、 病人健康状况等因素， 这 些都是熟练医师技能范围之内的。 附图简述
在附图中， 图 1为本发明 HDGF2与小鼠 HDGF2的核酸序列的同源比较图。 其 中， 相同的核苷酸用 Ί" 标出。
图 2为本发明 HDGF2与小鼠 HDGF2的氨基酸序列的同源比较图。 其中， 相同 的氨基酸用 Ί" 标出， 相似的氨基酸用 " ·" 标出。 在本发明的一个实例中， 人 HDGF2的 cDNA核苷酸序列是如此获得的， 以人 睾丸 λ gtl lcDNA文库（购自 Clontech公司）为模板， 合成正向引物 A1 : 5'- ACCGCTCGTCCGCCCGGCTTGAG-3' 和 反 向 弓 I 物 A2: 5'- GATCCTAGACATGTATAAGTCTGCG C-3', 进行 PCR, 分别获得 1024bp的目的 片段。 测序后得到 SEQ ID NO: 3的全长 cDNA序列。
肝癌细胞衍生生长因子 (HDGF)是从人的肝癌细胞株 HuH-7中分离到的肝素 结合蛋白,它具有刺激细胞生长的活性，能够促进成纤维细胞和一些肝癌细胞的生 长 (J. Biol. Chem. 269(40):25143-25149, 1994)。 它在人的心、 脑、 肺、 肝等各个 组织及各种癌细胞株中均有表达 (J. Biol. Chem. 269(40):25143-25149, 1994)。 HDGF家族的已知各成员的表达模式是不同的， 但是它们在精巢中都有很高程度 的富集， 而且它们的 5非翻译区均有高于 70%的 GC比 (Biochem. Biophys. Res. Commun. 238:26-32, 1997), 因而可能在雄性生殖细胞发育过程中有重要功能， 并和 DNA甲基化， 染色质构象以及翻译调控相关 (J. Cell. Biol. 115:887-903, 1990； Cell 62:503-514 , 1990)。 尽管 HDGF蛋白主要存在于细胞质（J. Biol. Chem. 269(40):25143-25149, 1994), 但是该家族成员的氨基酸序列中都含有一个潜在的 核定位信号 (NLS), 并且都无信号肽顺序， 提示它们可能作为核蛋白起作用。 另 外， HDGF的 C端的酸性氨基酸尾和 HMG家族的 HMG- 1/-2高度同源， 而这段序列 在 HMG- 1/-2中已知是组蛋白结合区域 (Biochemistry 29:4419-4423, 1990)。 很有可 能 HDGF在内化后作为转录因子发挥其刺激细胞生长的活性 (Biochem. Biophys. Res. Commun. 238:26-32, 1997)。 HDGF的有丝分裂原活性使其在急性恶性肝炎和 肝损伤的治疗上存在着极大的应用价值 (Clin. Chim. Acta. 183:273-284, 1989). 研 究表明， 许多成纤维细胞生长因子能够广泛应用于局部缺血症及动脉粥样硬化症 等血管形成方面有缺陷的疾病以及神经细胞的发育 (Blood 91(10):3527-3561 , 1998； Ann. N. Y. Acad. Sci. 545:240-252, 1998). 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明 本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规条件如 Sambrook等人， 分子克隆： 实验室手册 (New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建 i义的条 件。
实施例
实施例 1
HDGF2的 cDNA序列的克隆和测定
1. 引物扩增
以人睾丸 λ gtl lcDNA文库（购自 Clontech公司）为模板， 用寡核苷酸 A1 : 5'-
ACCGCTCGTCCGCCCGGCTTGAG-3' (SEQ ID NO: 1)为正向引物， 寡核苷酸 A2: 5*-GATCCTAGACATGTATAAGTCTGCGC-3' (SEQ ID NO: 2)为反向引物， 进行 PCR, PCR条件为 93 'C 4分钟， 随之以 93 'C 1分钟、 68.5 'C 1分钟和 72。C 1分钟进行 35个循环，最后 72'C延伸 5分钟。 电泳检测得到的 PCR片段， 为 1024bp的目的片段。
2. PCR产物的测序
将如上获得的 PCR扩增产物与 pGEM-T™载体 (Promega)连接， 转化大肠杆菌 JM103, 用 QIAprep Plasmid试剂盒 (QIAGEN)提取质粒， 用双链嵌套式缺失试剂盒 (Pharmacia)对插人片段进行定向系列缺失， 然后用 PCR对缺失子进行快速鉴定及
TM
排序。 用 SequiTherm EXCEL DNA测序试剂盒 (Epicentre Technologies)对依次截 短的缺失子进行测序， 最后用电脑软件拼接顺序， 获得全长 cDNA序列， 共 1024bp, 详细序列见 SEQ ID NO: 3, 其中开放读框位于 121-732位核苷酸。
根据得到的全长 cDNA序列推导出 HDGF2的氨基酸序列， 共 203个氨基酸残 基， 其氨基酸序列详见 SEQ ID NO: 4。 实施例 2
同源比较
用人 HDGF2的全长 cDNA序列及其编码蛋白在 Non-redundant GenBank+ EMBL +DDBJ+PDB数据库及 Non-redundant GenBank CDS translations+PDB ÷SwissProt+Spupdate+PIR数据库中用 BLAST进行核酸和蛋白同源检索。 结果发现 它们与小鼠 HDGF(dbjlD63707|MUSHDGF)基因及其编码蛋白具有极高同源性， 用 PCGENE软件比较发现， 它们在核酸水平上的同一性达到了 68.7%, 在蛋白水平 上的同一性达到了 53.7%, 并还有 9.4%的氨基酸相似 (图 1和图 2)。 特别是在保守的 由 98个氨基酸所构成的氨基末端， 与小鼠 HDGF的同源性高达 90 %。 此外， 人 HDGF2和另一个小鼠 HDGF基因（dbj|D63850|D63850)以及另一个人的 HDGF基因 (dbj|D 1643 1 |HUMHDGF)也有一定的同源性。 上述这些基因被认为构成一—个家 族， 所以可以从已知的这些基因或蛋白的功能来推测人 HDGF2的功能。
肝癌细胞衍生生长因子 (HDGF)是从人的肝癌细胞株 HuH-7中分离到的肝素 结合蛋白,它具有刺激细胞生长的活性,能够促进成纤维细胞和一些肝癌细胞的生 长 (J. Biol. Chem. 269(40):25143-25149, 1994)。 尽管 HDGF最初在肝癌细胞中被发 现， 但 Northern杂交分析显示它在人的心、 脑、 肺、 肝等各个组织及各种癌细胞 株中均有表达。 它在正常细胞和肿瘤细胞中是否存在着表达差异， 还需进一步实 验证明 (J. Biol. Chem. 269(40):25143-25149, 1994)。 随着研究的深入， HDGF在肝 癌细胞中的作用及其对肝癌治疗的影响也会不断被揭示。 HDGF家族已知各成员 的表达模式是不同的， 但是它们在精巢中都有很高程度的富集， 而且这些基因的 5非翻译区均有高于 70%的 GC比 (Biochem. Biophys. Res. Commun. 238:26-32, 1997), 这个特点和一些在精巢或胚胎发育中特异表达的基因相类似， 因而它们可 能在雄性生殖细胞发育过程中有重要功能， 并和 DNA甲基化， 染色质构象以及翻 译调控相关 (J. Cell. Biol. 1 15 :887-903, 1990； Cell 62:503-514, 1990),
荧光免疫实验显示 HDGF蛋白主要存在于细胞质 (J. Biol. Chem. 269(40):25 143-25149, 1994), 但是该家族成员的氨基酸序列中都含有一段碱性区 域——一个潜在的核定位信号 (NLS), 并且都无信号肽顺序， 提示它们可能作为 核蛋白起作用。 成纤维细胞生长因子 (FGF)必须依靠这段信号区域， 使之定位于 核内而发挥其有丝分裂原的活性。 另外， HDGF的 C端的酸性氨基酸尾和 HMG家 族的 HMG- 1/-2高度同源， 而这段序列在 HMG- 1/-2中已知是组蛋白结合区域 (Biochemistry 29:4419-4423 , 1990)。 综合上述现象， 很有可能 HDGF在内化后作 为转录因子发挥其刺激细胞生长的活性 (Biochem. Biophys. Res. Commun. 238:26- 32, 1997)。 本发明的 HDGF2也同样具有类似活性。
HDGF的有丝分裂原活性使其在急性恶性肝炎和肝损伤的治疗上存在着极大 的应用价值 (Clin. Chim. Acta. 183 :273-284, 1989)。 研究表明， 许多成纤维细胞生 长因子具有促进上皮细胞生长的能力， 能够广泛应用于局部缺血症及动脉粥样硬 化症等血管形成方面有缺陷的疾病以及神经细胞的发育 (Blood 91 ( 10):3527- 3561 , 1998; Ann. N. Y. Acad. Sci. 545:240-252, 1998)。 I型 HDGF及本发明 HDGF2 的在促成纤维细胞生长活性方面的应用尚有待于进一步研究。
本发明的 HDGF2除了可作为该家族一员用于进一步的功能研究，还可用于与 其他蛋白一起产生融合蛋白， 比如与免疫球蛋白一起产生融合蛋白。 此外， 本发 明 HDGF2还可以与该家族的其他成员进行融合或交换片段， 以产生新的蛋白， 如 将本发明 HDGF2的 N端与 I型 HDGF或鼠 HDGF的 N端进行交换，以产生新的' 性更 高或具有新特性的蛋白，
针对本发明 HDGF2的抗体， 用于筛选该家族的其他成员， 或者用于亲和纯化 相关蛋白 (如该家族的其他成员)。 实施例 3
HDGF2在大肠杆菌中的表达
在该实施例中，将编码 HDGF2的 cDNA序列用对应于该 DNA序列的 5'和 3'端的 PCR寡核苷酸引物进行扩增， 获得 HDGF2 cDNA作为插入片段。
PCR反应中使用的 5'寡核苷酸引物序列为：
5 -CCACGGATCCATGGCGCGTCCGCGGCCCC-3'(SEQ ID NO: 5)
该引物含有 BamHI限制性内切酶的酶切位点， 在该酶切位点之后是由起始密 码子开始的 HDGF2编码序列的 19个核苷酸；
3'端引物序列为：
5 -ATCCGTCGACTTAGGTCCCTTCACTGGTT-3 (SEQ ID NO: 6) 该引物含有 Sail限制性内切酶的酶切位点、 翻译终止子和 HDGF2的部分编码 序列。
引物上的限制性内切酶的酶切位点对应于细菌表达载体 pQE-9(Qiagen Inc., Chatsworth, CA)上的限制性内切酶酶切位点， 该质粒载体编码抗生素抗性 (Ampr)、 一个细菌复制起点 (ori)、 一个 IPTG-可调启动子 /操纵子 (P/0)、 一个核糖 体结合位点 (RBS)、 一个 6-组氨酸标记物 (6-His)以及限制性内切酶克隆位点。
用 BamHI和 Sail消化 pQE-9载体及插入片段， 随后将插入片段连接到 pQE-9载 体并保持开放读框在细菌 RBS起始。 随后用连接混合物转化购自 Qiagen, 商品名 为 M15/rep^ £.co//菌株， M15/rep4含有多拷贝的质粒 pREP4, 其表达 lacl阻遏物 并携带卡那霉素抗性 (Kan 在含有 Amp和 Kan的 LB培养皿上筛选转化子， 抽提 质粒， 用 Pstl酶切鉴定插入片段大小及方向， 并测序验证 HDGF2的 cDNA片段已正 确插入了载体。
在补加 ΑΓηρ(100 μ g/ml)和 Kan(25 μ g/ml)的 LB液体培养基中过夜培养（Ο Ν) 含所需构建物的阳性转化子克隆。 过夜 (0/N)培养物以 1 : 100-1： 250的稀释率稀 释， 然后接种到大体积培养基中， 培养细胞生长至 600光密度 (OD6QQ)为 0.4-0.6时， 加入 IPTG( "异丙基硫代 - β -D-半乳糖苷" ）至终浓度为 lmM。 通过使 lacl阻遏物失 活， IPTG诱导启动 P/0导致基因表达水平提高。 继续培养细胞 3-4小时， 随后离心 (6000 - g, 20分钟)。 超声裂解培养物， 收集细胞裂解液并将其稀释于 6M的盐酸 胍中。 澄清后， 通过在能使含 6-His标记物蛋白紧密结合的条件下， 用镍-螯合柱 层析从溶液中纯化溶解的 HDGF2 , 用 6M盐酸胍 (pH5.0)从柱中洗脱 HDGF2. 可用 几种方法从盐酸胍中变性沉淀蛋白。 或者使用透析步骤除去盐酸胍， 或者从镍- 螯合柱中分离出纯化蛋白， 纯化后的蛋白可以结合到第二个柱中， 该柱中具有递 减的线性盐酸胍梯度。 在结合到该柱时蛋白质变性， 随后用盐酸胍 (pH5.0)洗脱。 最后， 将可溶的蛋白质对含 PBS进行透析,然后将蛋白质保存在终浓度为 10%(w/v) 甘油的贮存液中。
用 12%的 SDS-PAGE胶进行电泳， 鉴定表达蛋白的分子量大小为约 23KDa。 此外， 用常规方法对表达蛋白的 N端和 C端各 10个氨基酸长度的氨基酸进行 测序， 发现与 SEQ ID NO: 4的序列一致. 实施例 4
HDGF2在真核细胞 (CHO细胞株）中的表达
在该实施例中，将编码 HDGF2的 cDNA序列用对应于该 DNA序列的 5'和 3'端的 PCR寡核苷酸引物进行扩增， 获得 HDGF2 cDNA作为插人片段。
PCR反应中使用的 5'寡核苷酸引物序列为：
5 -CCCTAAGCTTATGGCGCGTCCGCGGCCCC-3'(SEQ ID NO: 7)
该引物含有 Hindlll限制性内切酶的酶切位点， 在该酶切位点之后是由起始密 码子开始的 HDGF2编码序列的 19个核苷酸；
3'端引物序列为：
5 - TTTCGGATCCTTAGGTCCCTTCACTGGTT-3 (SEQ ID NO: 8)
该引物含有 BamHI限制性内切酶的酶切位点、一个翻译终止子和 HDGF2的部 分编码序列。
引物上的限制性内切酶的酶切位点对应于 CHO细胞表达载体 pcDNA3上的限 制性内切酶酶切位点， 该质粒载体编码抗生素抗性 (Ampf和 Ne0r)、 一个噬菌体复 制起点 (fl ο )、 一个病毒复制起点 (SV40 ori)、 一个 T7启动子、 一个病毒启动子 (P-CMV), 一个 Sp6启动子、 一个 SV40启动子、 一个 SV40加尾信号和相应的 polyA 顺序、 一个 BGH加尾信号和相应的 polyA顺序。
用 Hindlll和 BamHI消化 pcDNA3载体及插入片段， 随后将插入片段连接到 pcDNA3载体。 随后用连接混合物转化£.^// 0115 0[菌株。 在含有 Amp的 LB培养皿 上筛选转化子， 在补加 Απιρ(100 μ g/ml)的 LB液体培养基中过夜培养 (0/N)含所需 构建物的克隆。 抽提质粒， 用 Pstl酶切鉴定插入片段大小及方向， 并测序 -验证 HDGF2的 cDNA片段已正确插入了载体，
质粒转染 CHO细胞是用脂转染法， 用 Lipofectin试剂盒 (GiBco Life)进行。 转 染 48小时后， 经 2-3周的持续 G418加压筛选， 收集细胞及细胞上清测定表达蛋白 酶活力。 去 G418, 连续传代培养； 对混合克隆细胞极限稀释， 选择具有较高蛋白 活性的细胞亚克隆。 按常规方法大量培养上述阳性亚克隆。 48小时后， 开始收集 细胞及上清，用超声裂解方法破碎细胞，以含 0.05°/。Triton的 50mMTris -HCl(pH7.6) 溶液为平衡液及洗脱液， 用经预平衡的 Superdex G-75柱收集上述蛋白的活性峰。 再用 50mMTris · HCl(pH8.0)平衡的 DEAE-Sepharose柱， 以含 0-1M NaCl的 50mMTds · HCl(pH8.0)溶液为洗脱液进行梯度洗脱， 收集上述蛋白的活性峰。 然 后以 PBS(pH7.4)为透析液对表达蛋白溶液进行透析。 最后冻干保存。
用 12%的 SDS-PAGE胶进行电泳， 鉴定表达蛋白的分子量大小为 23KDa。 此外， 用常规方法对表达蛋白的 N端和 C端各 10个氨基酸长度的氨基酸进行 测序， 发现与 SEQ ID NO: 4的序列一致。 实施例 5
制备抗体
将实施例 3和 4获得的重组蛋白用来免疫动物以产生抗体， 具体如下。 重组分 子用层析法进行分离后备用。 也可用 SDS-PAGE凝胶电泳法进行分离， 将电泳条 带从凝胶中切下， 并用等体积的完全 Freimd s佐剂乳化。 用 50- 100 μ g/0.2ml乳化 过的蛋白， 对小鼠进行腹膜内注射。 14天后， 用非完全 Freund s佐剂乳化的同样 抗原， 对小鼠以 50-100 μ g/0.2ml的剂量进行腹膜内注射以加强免疫。 每隔 14天进 行一次加强免疫， 至少进行三次。 获得的抗血清的特异反应活性用它在体外沉淀 HDGF2基因翻译产物的能力加以评估。
在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被 单独引用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本 领域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所 附权利要求书所限定的范围。 序 列 表
(1)一般信息:
(ii)发明名称： 新的人肝癌细胞衍生生长因子编码序列、 其编码的多肽及制备方法
(iii)序列数目： 8
(2)SEQ ID NO: 1的信息
(i)序列特征
(A)长度： 23碱基
(B)类型： 核酸
(C)链性： 单链
(D)拓扑结构： 线性
(ii)分子类型： 寡核苷酸
(xi)序列描述： SEQ ID NO: 1
ACCGCTCGTC CGCCCGGCTT GAG
(2)SEQ ID NO: 2的信息
(i)序列特征
(A)长度： 26碱基
(B)类型： 核酸
(C)链性： 单链
(D)拓扑结构： 线性
(ii)分子类型： 寡核苷酸
(xi)序列描述： SEQ ID NO: 2
GATCCTAGAC ATGTATAAGT CTGCGC
(2)SEQ ID NO: 3的信息：
(i)序列特征
(A)长度： 1024bp (B)类型： 核酸
(C)链性： 双链
(D)拓扑结构： 线性
(ii)分子类型： cDNA
(xi)序列描述： SEQ ID NO: 3
1 ACCGCTCGTC CGCCCGGCTT GAGGCCCGCG GGGAGCGCGC GCAATTCGTC GGCCCGCGGG
61 GGGGCGGCCT CCCGGCATCT TCGCGGCGAC CAAGGACTAC CAGGAAGGGG AGCGGCTGGG
121 ATGGCGCGTC CGCGGCCCCG CGAGTACAAA GCGGGCGACC TGGTCTTCGC CAAGATGAAG
181 GGCTACCCGC ACTGGCCGGC CCGGATTGAT GAACTCCCAG AGGGCGCTGT GAAGCCTCCA
241 GCAAACAAGT ATCCTATCTT CTTrTTTGGC ACCCATGAAA CTGCA丌 TCT AGGTCCCAAA
301 GACCTTTTTC CATATAAGGA GTACAAAGAC AAGTTTGGAA AGTCAAACAA ACGGAAAGGA
361 丌 TAACGAAG GATTGTGGGA AATAGAAAAT AACCCAGGAG TAAAG丌 TAC TGGCTACCAG
421 GCAA丌 CAGC AACAGAGCTC TTCAGAAACT GAGGGAGAAG GTGGAAATAC TGCAGATGCA
481 AGCAGTGAGG AAGAAGGTGA TAGAGTAGAA GAAGATGGAA AAGGCAAAAG AAAGAATGAA
541 AAAGCAGGCT CAAAACGGAA AAAGTCATAT AC丌 CAAAGA AATCCTCTAA ACAGTCCCGG
601 AAATCTCCAG GAGATGAAGA TGACAAAGAC TGCAAAGAAG AGGAAAACAA AAGCAGCTCT
661 GAGGGTGGAG ATGCGGGCAA CGACACAAGA AACACAACTT CAGACTTGCA GAAAACCAGT
721 GAAGGGACCT AACTACCATA ATGAATGCTG CATATTAAGA GAAACCACAA GAAGG丌 ATA
781 TGTTTGGTTG TCTAATATTC 丌 GGAT丌 GA TATGAACCAA CACATAGTCC TTGTTGTCAT
841 TGACAGAACC CCAG丌 TGTA TGTACATTAT TCATATTCCT CTCTGTTGTG 丌 TCGGGGGG
901 AAAAGACA1T TTAGCCTTTT TTAAAAGTTA CTGATTTAAT 丌 CATG丌 AT TTGGTTGCAT
961 GAAGTTGCCC TTAACCACTA AGGATTATCA AGA丌丌 TGC GCAGACTTAT ACATGTCTAG
1021 GATC
(2)SEQ ID NO: 4的信息：
(i)序列特征
(A)长度： 203个氨基酸
(B)类型： 氨基酸
(D)拓扑结构： 线性
(H)分子类型： 多肽
(xi)序列描述： SEQ ID NO: 4 1 Met Ala Arg Pro Arg Pro Arg Glu Tyr Lys Ala Gly Asp Leu Val
16 Phe Ala Lys Met Lys Gly Tyr Pro His Trp Pro Ala Arg lie Asp
31 Glu Leu Pro Glu Gly Ala Val Lys Pro Pro Ala Asn Lys Tyr Pro
46 He Phe Phe Phe Gly Thr His Glu Thr Ala Phe Leu Gly Pro Lys
61 Asp Leu Phe Pro Tyr Lys Glu Tyr Lys Asp Lys Phe Gly Lys Ser
76 Asn Lys Arg Lys Gly Phe Asn Glu Gly Leu Trp Glu lie Glu Asn
91 Asn Pro Gly Val Lys Phe Thr Gly Tyr Gin Ala He Gin Gin Gin
106 Ser Ser Ser Glu Thr Glu Gly Glu Gly Gly Asn Thr Ala Asp Ala
121 Ser Ser Glu Glu Glu Gly Asp Arg Val Glu Glu Asp Gly Lys Gly
136 Lys Arg Lys Asn Glu Lys Ala Gly Ser Lys Arg Lys Lys Ser T r
151 Th「 Ser Lys Lys Ser Ser Lys Gin Ser Arg Lys Ser Pro Gly Asp
166 Glu Asp Asp Lys Asp Cys Lys Glu Glu Glu Asn Lys Ser Ser Ser
181 Glu Gly Gly Asp Ala Gly Asn Asp Th「 Arg Asn Thr Thr Ser Asp
196 Leu Gin Lys Th「 Ser Glu Gly Thr
(2)SEQ ID NO: 5的信息
(i)序列特征
(A)长度： 29碱基
(B)类型: 核酸
(C)链性： 单链
(D)拓扑结构： 线性
(ii)分子类型： 寡核苷酸
(xi)序列描述： SEQ ID NO: 5
CCACGGATCC ATGGCGCGTC CGCGGCCCC
(2)SEQ IDNO: 6的信息
(i)序列特征
(A)长度： 29碱基
(B)类型： 核酸
(C)链性: 单链
(D)拓扑结构： 线性
(Π)分子类型： 寡核苷酸
- !5 - (xi)序列描述： SEQ ID NO: 6
ATCCGTCGAC TTAGGTCCCT TCACTGGTT
(2)SEQ ID NO: 7的信息
(i)序列特征
(A)长度： 29碱基
(B)类型： 核酸
(C)链性： 单链
(D)拓扑结构： 线性
(Π)分子类型： 寡核苷酸
(xi)序列描述： SEQ ID NO: 7
CCCTAAGCTT ATGGCGCGTC CGCGGCCCC
(2)SEQ ID NO: 8的信息
(i)序列特征
(A)长度： 29碱基
(B)类型： 核酸
(C)链性： 单链
(D)拓扑结构： 线性
(Π)分子类型： 寡核苷酸
(xi)序列描述： SEQ ID NO: 8
TTTCGGATCC TTAGGTCCCT TCACTGGTT

Claims

权 利 要 求 书
1. 一种分离出的 DNA分子， 其特征在于， 它包括： 编码具有人 HDGF2蛋白 活性的多肽的核苷酸序列，
所述的核苷酸序列与 SEQ ID NO: 3中从核苷酸 121-732位的核苷酸序歹 Jl有至 少 70%的同源性； 或者
所述的核苷酸序列能在中度严紧条件下与 SEQ ID NO: 3中从核苷酸 121-732 位的核苷酸序列杂交。
2.如权利要求 1所述的 DNA分子， 其特征在于， 所述的序列编码一多肽， 该 多肽具有 SEQ ID NO: 4所示的序列。
3.如权利要求 1所述的 DNA分子， 其特征在于， 该序列具有 SEQ ID NO: 3中核 苷酸 121-732位的序列。
4.一种分离的 HDGF2蛋白多肽， 其特征在于， 它包括： 具有 SEQ ID NO: 4氨 基酸序列的多肽、 或其活性片段， 或其活性衍生物。
5.如权利要求 4所述的多肽， 其特征在于， 该多肽是具有 SEQ ID NO: 4序列的 多肽。
6.—种载体， 其特征在于， 它含有权利要求 1所述的 DNA。
7.—种用权利要求 6所述载体转化的宿主细胞。
8.如权利要求 7所述的宿主细胞， 其特征在于， 该细胞是大肠杆菌。
9.如权利要求 7所述的宿主细胞， 其特征在于， 该细胞是真核细胞。
10.—种产生具有 HDGF2蛋白活性的多肽的方法， 其特征在于， 该方法包括：
(a)将编码具有 HDGF2蛋白活性的多肽的核苷酸序列可操作地连于表达调控 序列， 形成 HDGF2蛋白表达载体， 所述的核苷酸序列与 SEQ ID NO: 3中从核苷酸 121-732位的核苷酸序列有至少 70%的同源性；
(b)将步骤 (a)中的表达载体转入宿主细胞， 形成 HDGF2蛋白的重组细胞；
(c)在适合表达 HDGF2蛋白多肽的条件下， 培养步骤 (b)中的重组细胞；
(d)分离出具有 HDGF2蛋白活性的多肽。
1 1.如权利要求 10所述的方法， 其特征在于， 该序列为 SEQ ID NO: 3中从核苷 酸 121-732位.
12.—种能与权利要求 4所述的 HDGF2蛋白多肽特异性结合的抗体。
13.—种核苷酸分子， 其特征在于， 它是权利要求 1所述 DNA分子的反义序 列。
14.一种探针分子，其特征在于，它含有权利要求 1所述的 DNA分子中约 8- 100 个连续核苷酸。
PCT/CN1999/000139 1998-09-22 1999-09-06 Nouvelle sequence codant pour le facteur de croissance derive de l'hepatome humain et polypeptide code par cette sequence d'adn, et procede de production de ceux-ci WO2000017351A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
US09/787,328 US6893844B1 (en) 1998-09-22 1999-06-09 DNA encoding a new human hepatoma derived growth factor and producing method thereof
EP99941373A EP1123976A4 (en) 1998-09-22 1999-09-06 NOVEL SEQUENCE ENCODING THE GROWTH FACTOR DERIVED FROM HUMAN HEPATOMA AND POLYPEPTIDE ENCODED BY THIS DNA SEQUENCE, AND METHOD FOR THE PRODUCTION THEREOF
AU55028/99A AU5502899A (en) 1998-09-22 1999-09-06 New human hepatoma-derived growth factor encoding sequence and polypeptide encoded by such dna sequence and producing method thereof
JP2000574250A JP2002526074A (ja) 1998-09-22 1999-09-06 新規のヒト肝臓癌誘導生長因子のコード配列、そのコードされるポリペプチド、およびこれらの製造方法。
CA002343719A CA2343719C (en) 1998-09-22 1999-09-06 New human hepatoma-derived growth factor encoding sequence and polypeptide encoded by such dna sequence and producing method thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN98119758.2 1998-09-22
CN98119758 1998-09-22

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WO2000017351A1 true WO2000017351A1 (fr) 2000-03-30

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JP5559159B2 (ja) * 2008-06-11 2014-07-23 バイオニュークレオン ソチエタ・レスポンサビリタ・リミタータ 修飾オリゴヌクレオチドを使用するｈｒｐ−３の阻害

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AU5502899A (en) 2000-04-10
CA2343719A1 (en) 2000-03-30
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