WO2001016175A1 - Nouveau gene rab humain et son polypeptide codant - Google Patents

Nouveau gene rab humain et son polypeptide codant Download PDF

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Publication number
WO2001016175A1
WO2001016175A1 PCT/CN2000/000251 CN0000251W WO0116175A1 WO 2001016175 A1 WO2001016175 A1 WO 2001016175A1 CN 0000251 W CN0000251 W CN 0000251W WO 0116175 A1 WO0116175 A1 WO 0116175A1
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Prior art keywords
polypeptide
rabpa
polynucleotide
human
sequence
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PCT/CN2000/000251
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English (en)
French (fr)
Inventor
Yumin Mao
Yi Xie
Xuanmao Chen
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Shanghai Bio Road Gene Development Ltd.
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Priority to AU68163/00A priority Critical patent/AU6816300A/en
Publication of WO2001016175A1 publication Critical patent/WO2001016175A1/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to newly identified polynucleotides, polypeptides encoded by the polynucleotides, and uses and preparations of these polynucleotides and polypeptides. More specifically, the present invention relates to a new human Rab protein (named RABPA) And its encoding gene Rabpa. The present invention also relates to a method for promoting and inhibiting the effect of this polypeptide.
  • GTP-binding proteins there are two types of GTP-binding proteins in the cell that play extremely important functions in signal transduction, one is a G protein in the form of a heterotrimer located under the cell membrane, and the other is a GTP-binding protein in the form of a small molecular weight monomer.
  • This GTP binding protein constitutes a superfamily, also known as the ras superfamily. It includes Ras, Rho, Alf, Sarl, Ran, and Rab.
  • the Ras family helps to transfer extracellular signals from cell surface receptors to the nucleus.
  • the MAP (mitogen-activating protein) kinase cascades to regulate gene expression. Mutations of members of the Ras family keep the cell proliferation signal constantly amplified and cannot attenuate, leading to canceration.
  • the Rlio family mainly regulates the reorganization of actin cytoskeleton; Ran regulates the nucleus Internal and external transport; Rab, Alf, Sar i and other families regulate membrane bubble transport between organelles, of which Arf ⁇ Sar 1 plays a role in the formation of donor bubble organelles, while Rab regulates the correct anchor of membrane bubble transport (Yan Feng et al. The Journal of cell biology, vol 13 1, 1995.) (Suzanne R Pfeffer, Curr opin cell biol 1994, 6: 522 -526.) (Peter novick and Patrick Brennward, cell vol 75,597-601, nov 9 1993.)
  • Eukaryotic cells are generally divided into many large and small compartments by the inner membrane of the cell. There is a material and information exchange between the compartments.
  • the main carrier of material exchange between compartments is the membrane vesicle.
  • the membrane bubble transport must also be highly selected to ensure the specificity of where the substance is coming from and where it is going.
  • Rab protein a small molecule GTP binding protein, is believed to be used to ensure Membrane vesicle carrier transport is specific.
  • Rab proteins in eukaryotic cells and different Rab proteins are also localized in different membrane compartments within the cell-each Rab protein binds to endocytosis, efflux, and cells.
  • each organelle has at least one Rab protein anchored on the cytoplasmic surface of its membrane (Philippe chavier, Lettles to nature, Vol 353, 24 oct. 1991: 769-773.) ( Peter novick and Patrick Brennward, Cell, vol 75,597-601, 1993.1 1.9) (Suzanne R Pfeffer, Curr opin cell biol 1994, 6: 522-526.).
  • Rab protein generally has the following structural characteristics: (l) Rab is quite conserved, and the Rab gene is found in yeast, Drosophila, plants and mammals, and it is quite conserved (Mustafa Benli, et al. The E BO Journal Vol 15 No 23 pp 6460-6475, 1996). (2) The molecular weight is between 20-27KD, so the CDS length of Rab gene generally does not exceed 700 nucleotides (John Armstrong et al. Journal of Cell Science 109: 1265-1274 (1996).) (Gregory Jedd, et al. The Journal of cell biology, Vol 137, No3 May 5 1997 563-580).
  • the The motif is GGTase (isoprenyl transferase) action site-GGTase, transfer fifteen-carbon farnesyl or twenty-carbon oxenyl or oxinyl to the terminal Cys residue.
  • GGTase is isoprene-modified, and modified using a highly variable C-terminal insertion On the organelle membrane. It has been shown that the highly variable C-terminus of Rab determines the position of Rab protein members on various organelle membranes, because the C-terminus can complementarily bind to birds that are specifically distributed on each organelle membrane.
  • GDF Glycoside release factor
  • Rab l7 is mainly expressed in epidermal cells
  • S 10 Also a member of Rab
  • Rab3a is mainly expressed in nerve cells
  • Rab3d is only expressed in adipocytes. Rab is found particularly in brain tissues today (John Armstrong et al.
  • Rab protein will be located in a certain compartment of the cell.
  • Rab is related to selective anchoring and fusion of membrane vesicle transport in endocytosis, efflux, and intracellular transport.
  • Different Rabs themselves are specifically anchored in different membrane systems.
  • YPT1 ie, yeast Rab l
  • Rab3a is located on the secretory vesicle
  • Rab4 is located in the early endocytosis
  • Rab7 is located in the late endocytosis
  • Rab9 is located in the "Trans Golgi Network” (Trans Golgi Network, TGN)
  • Rab5 is localized to the cytoplasmic membrane.
  • One method to determine the position of Rab protein in each membrane is immunofluorescence microscopy.
  • the principle is that Rab only uses the C-terminus to anchor on the membrane and its N-terminus is still exposed in the cytoplasm.
  • a c-myc epitope gene can be added to the N-terminus of the Rab gene.
  • the vector When the vector is transfected into cos cells, the foreign gene expression will form a c-myc-Rab fusion protein.
  • the fluorescein-labeled anti-c-myc antibody treats COS cells.
  • microscopy the position where fluorescence is anchored is the position where Rab is anchored. From this, the position of Rab on various membranes can be detected (Marino Zerial et al. Methods in enzymology.vol 219: 398-407.).
  • Rab protein functions by interacting with many protein factors, including: (1) isoprenyl transferase GGTase. After Rab synthesis, GGTase will be fifteen carbon or two Ten carbon isoprenyl group is transferred to the cysteine residue of CC or CXC motif at the end of Rab. (2) REP (Rab Escort Protein-Rab's molecular bridesmaid), will be unmodified or incompletely modified Rab is sent to the catalytic region of GGTase to complete the modification of Rab.
  • GGTase isoprenyl transferase
  • REP Rab Escort Protein-Rab's molecular bridesmaid
  • GTPase activating protein-GAP GTPase activating protein
  • GTPase activating protein GTPase activating protein
  • GDI Guanine-nucletide disassoiation inhibitor
  • GDI displacement factor Guanine-releasing factor-GDF
  • Guanine-nucleotide exchange factor (GEF), catalysis GTP replaces the GDP anchored by Rab binding to the donor membrane. Rab protein bound to GTP is only active.
  • V-Snare This factor is located on the donor membrane and is catalyzed by Rab to form membrane bubbles After moving to the target membrane, it can be combined with T-Snare on the target membrane. When T-Snare is combined with V-Snare, the membrane bubbles fuse with the target membrane to complete the material transport. (8) Rabphlin.
  • Rab affinity protein can prevent Rab from functioning (Peter novick and Patrick Brennward, cell vol 75,597-601, nov 9 1993.) (Armand Tavitian, methods in enzymology.vol 219: 387-397) (Suzanne R Pfeffer, Curr opin cell biol 1994, 6: 522-526.).
  • Rab gene has great use value in the following aspects: (1) Study of genetic diseases related to Rab gene. Chediak-Higashi syndrome is an autosomal recessive genetic disease, and its disease is a huge source in cells. In the organelle membrane of the secretory pathway. The etiology may be a homozygote of the Rab gene mutation that causes the sorting of the secreted protein of the cell to be defective. This suggests that the Rab gene can be used in the diagnosis and treatment of diseases. (Tatjana Stanrovic, Genomics 40,267-276 ( 1997).) (2) Application value of Rab in genetic engineering.
  • Rab is essential for the correct positioning and fusion of protein transport and membrane vesicle transport between eukaryotic organelles
  • some researchers have tried to play the role of Rab Genetically engineered production or use of Rab to regulate intracellular protein transport.
  • Rab protein is also related to the development of neurons, blocking the rab gene in larvae The expression in neurons can inhibit the growth of dendrites and axons. (Lukas A et al. Methods in enzymology.vol 257: 302-312) (4) Application of homologous cloning to find new Rab family member genes.
  • cDNA libraries of various tissues have been constructed, which also facilitates the search for new genes in the cDNA library. It can also be used in different species, such as mice and fruit flies. And plants to search for new Rab genes, and to explore the function and application value of genes in this species (FANGLAI, et al. Genomics 22,610-616 (1994).).
  • (5) Use yeast two-hybrid system to find interactions with Rab Gene of protein factors and try to verify its function. GGTase, REP.
  • GAP GDF
  • GDI GDI
  • GEF GGTase-isoprene transferase
  • the dienyl group is transferred to the cysteine residues of the ras protein and Rab protein, and the modified Rab and ras proteins have normal functions. Therefore, Galb MH claims that the inhibitor of GGTase will be a highly potential anticancer drug.
  • Rab affinity protein Monica Arribas, et al. European Journal of cell biology 74, 209-216 1997 ..
  • Rab affinity protein can bind to Rab protein and inhibit GTP hydrolase activity of Rab protein to completely block Rab. Function (William H. Brondyk, methods in enzymology.vol 257: 200-208). Therefore, it is of great significance to research and develop human RAB polypeptides and their agonists / inhibitors for therapeutic purposes.
  • the object of the present invention is to provide a novel human Rab polypeptide and its fragments, analogs and derivatives. Another object of the present invention is to provide a polynucleotide encoding these polypeptides.
  • Another object of the present invention is to provide a method for producing these polypeptides and the use of the polypeptides and coding sequences.
  • a novel isolated RABPA polypeptide which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant polypeptide thereof, or an active fragment thereof , Or an active derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID No. 2.
  • a polynucleotide encoding the isolated polypeptides comprises a A nucleotide sequence that is at least 80% identical to a nucleotide sequence selected from the group consisting of: (a) a polynucleotide encoding the aforementioned human RABPA polypeptide; and (b) a polynucleotide (a) a complementary polynucleotide.
  • the polynucleotide encodes a polypeptide having the amino acid sequence shown in SEQ ID No. 2. More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (A) a sequence having positions 200-838 in SEQ ID No. 1; and (b) a sequence having positions 1-920 in SEQ ID No. 1.
  • a method for preparing a polypeptide having human RABPA activity comprises: (a) culturing the above-mentioned host cell under conditions suitable for expressing human RABPA; (b) from a culture A polypeptide having human RABPA activity was isolated.
  • an antibody that specifically binds to the above-mentioned human RABPA polypeptide is provided.
  • a nucleic acid molecule that can be used for detection is also provided, which contains 10-700 nucleotides in a continuous sequence of the above-mentioned polynucleotide.
  • the present invention there are provided compounds that mimic, promote, and antagonize the activity of human RABPA polypeptides, and compounds that inhibit the expression of human RABPA polypeptides. Methods for screening and / or preparing these compounds are also provided.
  • the The compound is the antisense sequence of a human RABPA polypeptide coding sequence or a fragment thereof.
  • a method for regulating the activity of a human RABPA protein in vivo and in vitro is provided.
  • a method for detecting a disease or disease susceptibility associated with abnormal expression of a human RABPA polypeptide comprising: detecting whether a mutation exists in a nucleic acid sequence encoding the polypeptide.
  • the polypeptides and coding sequences of the present invention can be used to screen for agonists that promote the activity of human RABPA polypeptides, or to screen for antagonists that inhibit the activity of human RABPA polypeptides, or for peptide fingerprinting Map identification.
  • the coding sequence of human RABPA or a fragment thereof of the present invention can be used as a primer for a PCR amplification reaction, or as a probe for a hybridization reaction, or used to produce a gene chip or a microarray.
  • a pharmaceutical composition which contains a safe and effective amount of the human RABPA polypeptide of the present invention and a pharmaceutically acceptable carrier.
  • These pharmaceutical compositions can treat AIDS and other conditions such as acquired and inherited immunodeficiency diseases.
  • Figure 1 shows the amino acid sequence of the RABPA protein of the present invention. Standard single-letter abbreviations for amino acids are used. The full-length protein is 213 amino acids, and I, ⁇ , ⁇ , and IV are conserved GTP / GDP binding regions.
  • Figure 2 is a comparison diagram of the homology between the RABPA protein of the present invention and the known Rab2 protein (Q14964).
  • the upper sequence is the RABPA protein, and the lower sequence is the Rab2 protein (Q14964).
  • Identical amino acids are indicated by "
  • Figure 3 is an electrophoresis diagram showing RABPA expression products.
  • the first and second lanes are the empty plasmid (ie, the PBV220 expression vector containing no gene) and the cells are 42'C induced and 30'C. Uninduced protein electrophoresis map; the third lane is the standard low molecular weight protein marker (97KD, 66KD, 43 D, 31KD, 20KD, and 14KD); the fourth and fifth lanes are the genetically induced and uninduced electrophoresis images, respectively, and the arrows indicate the expressed human RABPA protein.
  • Figure 4 is the result of multiple sequence homology comparison between the RABPA protein of the present invention and the Rab family gene. Summary of the Invention
  • the polynucleotide of the present invention may be in the form of RNA or DNA.
  • DNA includes cDNA, genomic DNA and synthetic DNA.
  • DNA may be double-stranded or single-stranded, and if it is single-stranded, the single-stranded may be coding and non-coding ( Antisense) chain.
  • the coding sequence encoding the mature polypeptide may be the same as the coding sequence shown in SEQ ID NO: 1, or it may be a different coding sequence, but the different coding sequence (due to the duplication or degeneration of the genetic code) Result)
  • the encoded polypeptide is in accordance with SEQ ID NO: 2 ⁇
  • the polynucleotide encoding the SEQ ID NO: 2 polypeptide includes: the coding sequence encoding only the mature polypeptide; the coding sequence of the mature polypeptide and additional coding sequences (such as the leader sequence or secretion) Sequence or preprotein sequence); mature protein coding sequence (and optional additional coding sequence) and non-coding sequences (such as introns or non-coding sequences of the 5 'and / or 3' coding sequence of the mature polypeptide) .
  • polynucleotide encoding a polypeptide refers to a polynucleotide that is only the coding sequence of a polypeptide, and also refers to a polynucleotide that includes additional coding sequences and / or non-coding sequences.
  • the present invention also relates to a variant of the above-mentioned polynucleotide, the variant encoding a polypeptide fragment, analog, and derivative having the same amino acid sequence as the RABPA of the present invention.
  • the variant of the polynucleotide may be a naturally occurring polynucleotide Equivalent variants or non-naturally occurring variants of this polynucleotide. Therefore, the present invention includes polynucleotides encoding the same mature polypeptides as RABPA, as well as variants of these polynucleotides, and these variants encode Fragments, derivatives or analogs of the polypeptides of the invention.
  • nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
  • the polynucleotide may be SEQ ID NO :
  • an allelic variant is a replacement form of a polynucleotide, which may be a substitution of one or more nucleotides, Deletion or addition, but does not substantially change the function of the polypeptide it encodes.
  • the invention also includes a polynucleotide in which the coding sequence of a mature polypeptide can be fused to a certain polynucleotide sequence that assists the expression and secretion of the polypeptide in a host cell according to the same reading frame (for example, as a secretion sequence control polypeptide from The leader sequence transported out of the cell).
  • the polypeptide with the leader sequence is a proteinogen, and the host cell can cut off the leader sequence to form the mature form of the polypeptide.
  • Polynucleotides can also encode a proteinogen consisting of a mature protein and additional 3 'amino acid residues.
  • the mature protein of a sequencer is a proteinogen, which is an inactive form of this protein. Once the sequencer is excised, it forms a Active mature protein. Therefore, the polynucleotide of the present invention can encode a mature protein, or a sequenced protein, or a protein with both a sequence and a presequence (leader sequence).
  • the present invention also relates to a polynucleotide that is a polynucleotide that can hybridize to the above-mentioned sequence when it is at least 50%, preferably at least 70%, more preferably at least 80% identical.
  • stringent conditions means that hybrids can only hybridize when the sequences are at least 95% and preferably at least 97% identical.
  • the polypeptide encoded by the above-mentioned polynucleotide hybridized polynucleotide is substantially the same in biological function and activity as the polypeptide encoded by DNA at positions 200-838 in SEQ ID NO: 1.
  • the invention also relates to a polypeptide having the amino acid sequence of SEQ ID NO: 2 and fragments, analogs and derivatives of such polypeptides.
  • the polypeptide in the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
  • the polypeptide of the present invention may be a naturally purified product or a chemically synthesized product, or it may be obtained from a prokaryotic or eukaryotic host using recombinant technology (for example, bacteria, yeast, higher plants, insects, and mammalian cells), according to the host used in the recombinant production scheme, the polypeptide of the present invention may be glycosylated, or may be non-glycosylated.
  • the polypeptide of the present invention also The initial methionine residue may or may not be included.
  • fragment refers to a polypeptide that retains the same biological function or activity as this polypeptide. Therefore, analogs can be activated after removal of the proprotein portion To produce an active mature polypeptide protein.
  • the polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted And such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide including one substituent group in one or more amino acid residues, or (Hi) a mature polypeptide and another A polypeptide formed by fusion of a compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusing additional amino acid sequences to this polypeptide sequence (such as a leader sequence or a secreted sequence or used to purify this The sequence of a polypeptide or protease sequence). According to the teachings herein, these fragments, derivatives and analogs belong to the scope known to those skilled in the art.
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and are more preferably purified to homogeneity.
  • isolated refers to the separation of material from its original environment, for example, a naturally occurring polynucleus located in an animal
  • the nucleotides or polypeptides are not isolated, but the same polynucleotides or polypeptides isolated from a part of the natural system or all coexisting materials are isolated.
  • Such polynucleotides may be part of a vector, such polynucleotides Or the polypeptide can also be part of the composition, as long as the carrier or composition is not part of its natural environment, it is still isolated.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, a genetically engineered host cell made using the vector of the present invention, and a product of the polypeptide of the present invention produced by recombinant technology.
  • the polynucleotide sequence of the present invention may be included in Any of a number of expression vectors that express polypeptides.
  • vectors include chromosomal, non-chromosomal, and synthetic DNA sequences, such as derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, plasmids, and phages DNA combination derived vectors, viral DNA such as vaccinia virus, adenovirus, Fowlpox virus and pseudorabies virus.
  • synthetic DNA sequences such as derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, plasmids, and phages DNA combination derived vectors, viral DNA such as vaccinia virus, adenovirus, Fowlpox virus and pseudorabies virus.
  • viral DNA such as vaccinia virus, adenovirus, Fowlpox virus and pseudorabies virus.
  • any plasmid and vector can be used as long as it can replicate and survive in the host.
  • the expression vector preferably contains genes that provide phenotypic traits for screening transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance for E. coli.
  • the DNA sequence in the expression vector is linked with appropriate expression control sequences (promoters) to guide mR A synthesis.
  • promoters include LTR or SV40 promoter, E.coli.lac or ti promoter, phage lambda PL promoter and other known promoters that can control the expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • Expression vectors also include ribosome binding sites and transcription for initiation of translation Terminators. Vectors containing the appropriate DNA sequences and appropriate promoters or regulatory sequences described above can be used to transform appropriate host cells for the host to express the protein.
  • Host cells can be higher eukaryotic cells, such as mammalian cells, or lower eukaryotic cells, such as yeast cells, or prokaryotic cells, such as bacterial cells.
  • suitable hosts are: E. coli, Streptomyces; bacteria of Salmonella typhimurium; fungal cells such as yeast; insect cells such as Drosophila cells and sf9 cells; animal cells such as CHO, COS or Bowes melanoma cells; plant cells, etc.
  • the appropriate DNA sequence can be inserted into the vector by a variety of methods. Generally, DNA sequences are inserted into appropriate restriction enzyme sites by procedures well known in the art. These and other steps are well known to those skilled in the art.
  • a suitable host is transformed and grown to an appropriate cell density
  • the selected promoter can be induced by appropriate methods (such as temperature conversion or chemical induction), and the cells can be cultured for a period of time. The cells are harvested after centrifugation, and the cells are broken by physical or chemical methods. The obtained crude extract is reserved for further purification. Disintegration includes freeze-thaw method, ultrasonic method, mechanical disintegration method, or use of cell lysis reagents.
  • Precipitation with amine sulfate or ethanol, acid extraction, anion or cation exchange chromatography, phosphate fiber chromatography, hydrophobic interaction chromatography, Affinity chromatography, hydroxyapatite chromatography or phytohormone chromatography can be used to recover and purify proteins from recombinant cell cultures. If necessary, protein refolding steps can be used to form the conformation of mature proteins. Finally, high-performance liquid chromatography (HPLC) can be used to complete the final purification step.
  • HPLC high-performance liquid chromatography
  • the RABPA polypeptide or fragment or derivative thereof of the present invention can be used to treat programmed cell Death-related diseases, including but not limited to: AIDS and other acquired and hereditary immunodeficiency diseases; degenerative neurological diseases such as Alzheimer's disease, Parkinson's disease, hemi-sclerotic muscular atrophy, pigmented retinitis, Cerebellar degeneration, myelodysplastic syndromes such as aplastic anemia, local anemia injuries such as myocardial infarction, stroke and reperfusion injury, toxin-induced Diseases such as alcoholic liver poisoning, cirrhosis, and viral infections such as hepatitis C, hepatitis B and sclerosis.
  • programmed cell Death-related diseases including but not limited to: AIDS and other acquired and hereditary immunodeficiency diseases; degenerative neurological diseases such as Alzheimer's disease, Parkinson's disease, hemi-sclerotic muscular atrophy, pigmented retinitis, Cerebellar degeneration, myel
  • RABPA polypeptides or fragments or derivatives thereof can be added to cell lines to stimulate cell proliferation, and can also be directly introduced into living cells by means of liposomes, electroporation and other means.
  • the polypeptide of the present invention can be used in combination with a suitable pharmaceutically acceptable carrier.
  • This composition comprises a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient.
  • suitable pharmaceutically acceptable carrier include, but are not limited to, saline, buffered saline, Glucose, water, glycol, ethanol and mixtures thereof. These preparations should be suitable for the mode of administration.
  • the present invention also provides a kit or kit containing one or more containers in which one or more of the present invention are contained Pharmaceutical composition ingredients. Together with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts reflect government agency approvals for production, use, or sale It is administered on the human body.
  • the polypeptide of the present invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route.
  • RABPA should be effective to treat and / or prevent specific indications
  • the amount and range of RABPA administered to a patient will depend on many factors, such as the mode of administration, the physical conditions of the person to be treated, and the judgment of the diagnostician.
  • polypeptides, fragments or derivatives thereof, or analogs thereof, or cells expressing them of the present invention can be used as immunogens, used to produce antibodies thereof, used for diagnosis or used as antagonists. These antibodies can be, for example, multiple Cloning of Hang body or monoclonal antibody.
  • the present invention also includes chimeric chain antibodies, single chain antibodies, and humanized antibodies, as well as Fab fragments, or products of Fab expression libraries, which can be produced by a variety of methods known in the art.
  • Antibodies generated against the polypeptide corresponding to the sequence of the present invention can be obtained by directly injecting the polypeptide into a human animal (preferably a non-human animal). The antibody thus obtained will then bind to its polypeptide. In this manner, even the polypeptide encoding the polypeptide fragment Sequences can also be used to generate antibodies that bind entire natural polypeptides. These antibodies can then be used to isolate the polypeptide from tissues expressing the polypeptide.
  • any technique for producing antibodies by continuous cell line culture can be used. Examples of these techniques include hybridoma technology (Kohler and Milstein, 1975, Nature, 256: 495-497), trioma technology, human B-cell hybridoma technology (Kozbor et al., 1983, Immuolgy Today 4:72), and EBV hybridoma technology that produces human monoclonal antibodies (Cole et al., 1985, Monoclonal Antibodies and Cancer ⁇ erapy, Alan R. Liss, Inc., pp77-96).
  • the technology for producing single-chain antibodies (USP 4,946,778) can be improved to produce single-chain antibodies of the immune polypeptide products of the present invention.
  • the invention also relates to diagnostic test methods for quantitative and localized detection of RABPA levels. These tests are present It is well known in the art, and includes FISH measurement and radioimmunoassay.
  • the level of RABPA detected in the test can be used to explain the importance of RABPA in various diseases and to diagnose diseases related to RABPA.
  • the present invention also provides screening drugs In order to identify agents that increase (agonist) or suppress (antagonist) RABPA. Agonists improve the biological functions of RABPA (such as stimulating cell proliferation, etc.), while antagonists prevent and treat disorders related to excessive cell proliferation, such as various Cancer.
  • mammalian cells or ABPA-expressing membranes can be cultured with labeled RABPA in the presence of a drug. The ability of the drug to increase or block this interaction can then be determined.
  • Agonists / inhibitors can also be used (e.g., as described above) in compositions containing a pharmaceutically acceptable carrier.
  • polypeptides of the present invention can also be used by expressing these polypeptides in vivo, which is commonly referred to as "gene therapy".
  • a patient's cells can be genetically engineered with a polynucleotide (DNA or RNA) encoding a RABPA polypeptide in vitro, The engineered cells are then provided to a patient to be treated with this polypeptide.
  • DNA or RNA polynucleotide
  • cells can be processed in vitro by genetic engineering techniques, and RABPA polypeptides can be expressed in vivo by, for example, methods known in the art.
  • production cells producing retroviral particles containing RNA encoding a polypeptide of the invention, They can be administered to patients, produce engineered cells in vivo, and express polypeptides in vivo.
  • the expression vector of engineered cells in addition to retroviruses, may be an adenovirus used to produce engineered cells in vivo after being combined with a suitable delivery vector.
  • the polynucleotides of the present invention can be used to design antisense DNA or RNA, as inhibitors to treat and prevent various disorders related to cell proliferation (such as tumors, etc.) and to treat and prevent various inflammations.
  • Formation or antisense DNA by triploid Or RA both methods are based on the binding of a polynucleotide to DNA or RA
  • antisense technology can be used to control gene expression.
  • the 5 'coding portion of a polynucleotide sequence encoding a mature polypeptide of the present invention can be used to design an antisense RNA oligonucleotide of about 10-40 base pairs in length.
  • Antisense RNA oligonucleotides hybridize with mRNA in vivo and inhibit translation of mR A molecules into RABPA. Antisense RNA and DNA can be transported to cells and inhibit RABPA production in vivo. In addition, antisense RNA and DNA can contain phosphate sulfide bonds or peptide lipid bonds to extend their half-life in vivo.
  • sequences of the invention are also valuable for chromosome identification. This sequence will be specific to someone Chromosome specific location and can be crossed with it. At present, the specific location of each gene on the chromosome needs to be identified. Currently, only few chromosome markers based on actual sequence data (repeating polymorphisms) can be used to mark the chromosome location. According to The present invention, to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on the chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be mapped on chromosomes. These primers are then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those containing corresponding Hybrid cells based on the human gene of the primer will produce amplified fragments.
  • the somatic hybrid cell PCR mapping method is a fast method for mapping DNA to a specific chromosome.
  • oligonucleotide primers of the present invention by a similar method, a group of fragments from a specific chromosome or a large number of genomic clones can be used. Realize sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inheritance in Man The Medical Library is available online. Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions. Next, the cDNA or genomic sequence differences between the affected and unaffected individuals need to be determined. Or a mutation is observed in all diseased individuals, and the mutation is not observed in any normal individuals, then the mutation may be the cause of the disease.
  • Comparing diseased and non-diseased individuals usually involves first looking for structures in the chromosome Changes, such as deletions or translocations that are visible at the chromosomal level or detectable by cDNA sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping techniques, cDNAs are precisely mapped to disease-related chromosomal regions, Can be one of 50 to 500 potentially pathogenic genes (assuming 1 trillion bases And mapping each 20kb resolving power corresponding to a gene).
  • polypeptides and polynucleotides of the present invention such as identification of peptide fingerprints of peptides of the present invention.
  • the coding sequence of human RABPA or fragments thereof of the present invention can be used as primers for PCR amplification reactions, or as probes For hybridization reactions, or for the production of gene chips or microarrays. According to the teachings of the present invention, these applications will be apparent to those skilled in the art.
  • an isolated nucleotide (polynucleotide) is provided, which encodes a mature polypeptide having the amino acid sequence shown in SEQ ID NO: 2.
  • the polynucleotide of the present invention is from human fetal brain at 18 weeks. cDNA library development Now. It contains an open reading frame encoding a protein of approximately 213 amino acids in length, which is structurally 73.2 ° to the Rab gene (99962) at the nucleic acid level. Identical; 75.6% identity and 82..6% similarity to the protein (Q14964) encoded by the Rab gene (X99962).
  • the RABPA polypeptide of the present invention contains a characteristic motif, as shown in FIG.
  • domains I, II, III, and IV are GTP / GDP binding regions
  • the effector domain is a GTPase activating protein binding region
  • the underlined protein is a hypervariable C-terminus. Region and CXC domain.
  • the molecular weight of the RABPA protein by computer analysis is 24566.89 Daltons, and the isoelectric point is 7.43.
  • the human RABPA of the present invention has a natural amino acid sequence derived from humans, it is expected to have higher activity and / or lower side effects when administered to humans compared to homologous proteins derived from other species ( (E.g. less or no immunogenicity in the human body).
  • the determined cDNA sequence was compared with the existing public DNA sequence database, and it was found that the DNA sequence of clone 067D10 was a new DNA.
  • a series of primers were used to determine the DNA sequence of the 067D10 clone in both directions.
  • Computer analysis showed that the full-length cDNA contained in the 067D10 clone was a new DNA sequence (as shown in SEQ ID NO: l), with one from 200bp to 841bp. 641bp ORF, encoding a new protein of 213 amino acids (as shown in SEQ ID NO: 2).
  • SEQ ID NO: 2 RT-PCR Methods to clone human Rabpa gene
  • CDNA was synthesized using fetal brain cell total RNA as a template and oligo-dT as a primer for reverse transcription reaction.
  • PCR amplification was performed with the following primers: forward primer F1 5-GGAATTCATGGAGGCCAT CTGGCTGTAC -3 (SEQ ID NO: 3); reverse primer R1: 5- GGAAGCTTCATGCCGTATGCAGCAGCCA -3 (SEQ ID NO: 4).
  • the reaction volume of 50 ⁇ 1 contains 50mmol / L KCl, 10mmol / L Tris-Cl (pH8.5), 1.5mmol / L MgCl2, 20 ( ⁇ mol / L dNTP, each 25pmoI primer, 2.5U Taq DNA polymerase.
  • the pBV220 expression vector containing the Rabpa gene was transferred into a human DH5CC host strain, and transformants were identified by the ability to grow on LB plates, and ampicillin-resistant colonies were screened out. Plasmid DNA was isolated and determined by restriction analysis. Clones of the constructs were grown overnight in liquid medium in LB medium supplemented with Amp (100 ug / ml). Overnight cultures were used to inoculate a large number of cultures at a ratio of 1: 100-1: 250. The cells were grown to an optical density of 600 (O..D. 60O) between 0.4 and 0.6, and expression was induced at 42 ° C 3 hours, 12% SDS-PAGE gel to identify the expression products.
  • O..D. 60O optical density
  • the protein expression electrophoresis diagram is shown in Figure 3.
  • the first and second lanes are the empty plasmid (that is, the PBV220 expression vector containing no gene). Induced protein electrophoresis map; lane 3 is standard low molecular weight protein markers (97KD, 66KD, 43KD, 31KD, 20KD, and 14KD molecular weight from top to bottom respectively); lanes 4 and 5 are gene-induced and uninduced electrophoresis
  • the arrow refers to the expressed human RABPA protein with a molecular weight of about 25kDa.
  • Northern blot analysis was performed to determine the expression level of RABPA in human cells.
  • Total cell RA samples were isolated using the BNAzol TM B system (Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX 77033). Isolate approximately 10ug of total RNA from each specific human tissue, isolate on a 1% agarose gel, and Blot onto a nylon filter (Sambrook, fritsch and Maniatis, Molecular Cloning, Cold Spring Harbor Press, (1989).
  • a Stratagene Prime-It kit was used with a 50ng DNA fragment for the labeling reaction.
  • the labeled DNA was purified using a Select-G-50 column ( 5 Prime-3 prime, Inc. 5603 Arapahoe Road, Boulder, Co 80303).
  • the filter was radiolabeled with a full-length RABPA gene at 65 'C at 1,000,000 cpm / ml in 0.5 M NaPO 4 , ⁇ ⁇ 7 ⁇ Hybridization in 4 and 7% SDS overnight. After washing twice with 0.5 x SSC, 0.1% SDS at room temperature and twice at 60'C, the filter was exposed to -70'C with the enhanced fluorescent screen.
  • RABPA's Messenger RNA is rich in activated and inactivated T cells, monocytes, and T cell lines.
  • the sequence of the Rabpa gene of the present invention and the encoded protein sequence are used for homology search in databases such as Genbank, Swissport, etc.
  • the program used for searching is called Blast (Basic local Alignment search tool) (1993 Proc Nat Acad Sci 90: 5873-5877 ), Blast can find many genes that are homologous to Rabpa, among which the gene most homologous to the Rabpa gene of the present invention is a known human RAB protein (Genbank accession number X99962), the protein encoded by X99962 is in Genbank's accession number is Q14964. These retrieved gene or protein sequences can be retrieved from the Genbank database.
  • the retrieved sequences can be paired using the Pileup (multi-sequence) and Gap (two-sequence) programs in the GCG software package. Comparison. The results show that X99962 and 067D10 have the highest homology, and the similarity at the nucleotide level is 73%. Comparing the proteins encoded by the two ( Figure 2), the results show that the two are highly similar Source, its similarity is 82.6%; the sameness is 75.5%.
  • the Pileup program was used to perform multiple sequence homology comparison between the protein of the present invention and the Rab family gene.
  • the results are shown in Figure 4.
  • the protein encoded by the Rabpa of the present invention was subjected to motif analysis using the GCG software package, and the results showed that Rabpa
  • the encoded protein belongs to the Rab GTPase family and has structural features common to GTPase.
  • the specific results and characteristics of Rabpa-encoded proteins are shown in Figure 1. See Figure 1, where I, II, III, and IV are GTP / GDP binding regions, and the effector domain is a GTPase activating protein, that is, an effector binding region, underlined The C-terminal hypervariable region and CXC domain of the protein.
  • the molecular weight of the novel human Rabpa protein of the present invention is 24566.89 Daltons and the isoelectric point is 7.43.
  • Example 6 Production of anti-human RABPA antibodies
  • a peptide synthesizer (PE-ABI) was used to synthesize the following human RABPA-specific peptides: NH2- MetGluAlalleTrpLeuTyrGlnPheArgLeuIleVallleGly-COOH.
  • the peptide was coupled with hemocyanin and bovine serum albumin to form a complex, respectively.
  • Avrameas. 6:43 Immunize rabbits with 4mg of the above hemocyanin peptide complex plus complete Freund's adjuvant, and then use blood blue after 15 days Protein-peptide complex plus incomplete Freund's adjuvant to boost immunity once.

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Description

新的人 Rab某因及其编码的名胩
术领域
本发明涉及新鉴别的多核苷酸、 由此多核苷酸编码的多肽以及这些多核苷酸和 多肽的用途和制备. 更具体地说， 本发明涉及一种新的人 Rab蛋白 (命名为 RABPA) 及其编码基因 Rabpa. 本发明还涉及促进和抑制此多肽作用的方法.
技术背景
细胞内有两种 GTP结合蛋白在信号传导中起极其重要的功能，其一是位于细 胞膜下的异源三聚体形式的 G蛋白， 另一种是小分子量单体形式的 GTP结合蛋 白. 这种 GTP结合蛋白构成一个超级家族， 也称 ras超级家族. 它包括 Ras、 Rho、 Alf、 Sarl、 Ran和 Rab等家族， 其中 Ras家族帮助把细胞外信号从细胞表面受体 传到细胞核，它通过 MAP (促分裂原激活蛋白)激酶级联放大来调节基因表达， Ras 家族成员的突变使细胞增殖信号一直放大不能衰减从而导致癌变； Rlio家族主要 调节肌动蛋白细胞骨架的再组织； Ran调节细胞核内外的运输； Rab、 Alf、 Sar i 等家族调节细胞器之间膜泡的运输， 其中 Arf^Sar 1起作用是在供体细胞器膜泡 的形成阶段， 而 Rab则调节膜泡的运输的正确锚定. (Yan Feng et al. The Journal of cell biology ,vol 13 1 , 1995. )(Suzanne R Pfeffer , Curr opin cell biol 1994 ,6:522- 526.)(Peter novick and Patrick Brennward , cell vol 75,597-601 ,nov 9 1993.)
真核细胞一般被细胞内膜分隔成许多大大小小的不同的区室， 各区室之间 存在着物质和信息的交流. 区室间的物质交流的主要载体是膜泡， 由于区室是 多种多样的， 所以膜泡运输也必须是高度选择的， 以保证物质从何处来运到何 处去这一特异性. Rab蛋白——一种小分子 GTP结合蛋白， 据认为是用来确保膜 泡载体运输的特异性的. 真核细胞内有多种 Rab蛋白， 不同的 Rab本身也定位于 细胞内不同的膜区室——每种 Rab蛋白都结合到与内吞、 外排及细胞内运输过程 相关的细胞器膜上， 一般每种细胞器在其膜的胞质面上至少锚定有一种 Rab蛋白 (Philippe chavier， Lettles to nature ,Vol 353 ,24 oct. 1991 :769-773.)(Peter novick and Patrick Brennward， Cell, vol 75,597-601, 1993.1 1.9)(Suzanne R Pfeffer , Curr opin cell biol 1994 ,6:522-526.).
Rab蛋白一般具有以下的结构特征： （l)Rab相当保守， 在酵母、 果蝇、 植 物和哺乳类中都发现有 Rab基因 ，而且相当保守（Mustafa Benli, et al. The E BO Journal Vol 15 No 23 pp 6460-6475, 1996). (2) 分子量大小在 20-27KD之 间， 所以 Rab基因的 CDS的长度一般不超过 700个核苷酸 (John Armstrong et al. Journal of Cell Science 109: 1265-1274(1996). )(Gregory Jedd,et al. The Journal of cell biology ,Vol 137,No3 May 5 1997 563-580). (3)具有四个分别为 I 、 Π 、 ΙΠ、 IV的保守的 GTP/GDP结合区域， 这也是 ras 超家族的特点. （4) 1 、 Π之间还有 保守的效应子（GAP)结合区域（Janouei-Lerssey I "two -hybrid system screen with the small GTP-binding protein Rab 6 identification of a novel mouse GDP dissociation inhibitor isoform and two other potential partners of Rab6 .). (5)C末 端为高变区域， 但尾部都有 CC、 CXC或 CXXX基序， 该基序为 GGTase (异戊二烯 基转移酶)作用位点一 GGTase, 把十五碳法尼基或二十碳找牛儿找牛儿基转移 至末端 Cys 残基上, Rab合成后首先由 GGTase进行异戊二烯化修饰， 修饰后利用 高度变化的 C末端插入细胞器的膜上. 已证明： Rab的高度变化的 C末端决定了 Rab蛋白成员在各个不同的细胞器膜上的位置， 原因是其 C末端可以互补地结合 到在各细胞器膜上特异性分布的鸟苷释放因子 (GDF)上. 如果 Rab 的 C末端被去 除， 则其膜结合特异性消失，各种 Rab功能是可以互换 (Philippe chavier , Lettles to nature ,Vol 353 ,24 oct. 1991 :769-773.) . (6)Rab基因的表达大部分没有组织特 异性， 但也有不少基因经 Northern分析表明只表达于脑、 表皮和肝脏等组织. 如 Rab l7主要表达于表皮细胞中， S 10 (也是 Rab成员）只表达于淋巴细胞系， Rab3a主 要表达于神经细胞, Rab3d只表达于脂肪细胞中. 现今在脑组织发现的 Rab特别多 (John Armstrong et al. Journal of Cell Science 109: 1265-1274(1996).)(Mustafa Benli, et al. The EMBO Journal Vol 15 No 23 pp 6460-6475, 1996)( Gregory Jedd, ,et al. The Journal of cell biology ,Vol 137,No. 3 May 5 1997 563- 580)(Janouei-Lerssey I "two -hybrid system screen with the small GTP-binding protein Rab 6 dentification of a novel mouse GDP dissociation inhibitor isoform and two other potential partners of Rab6 .).
Rab蛋白会定位于在细胞一定区室。 Rab与内吞、 外排及细胞内运输的膜泡 转运的选择性锚定和融合有关， 不同的 Rab自身也特异性地锚定于不同的膜系统 中. 例如， YPT1 (即酵母的 Rab l)定位于高尔基复合体 (Golgi complex); Rab3a 定位于分泌泡膜上； Rab4定位于早期内吞体； Rab7定位于晚期内吞体； Rab9定 位于 "高尔基体外侧网络" （Trans Golgi Network, TGN); Rab5定位于细胞质膜. 一种确定 Rab蛋白在各膜的位置的方法是免疫荧光显微镜检技术. 其原理是 Rab 只利用 C末端锚定在膜上而它的 N-末端仍然露在细胞质中， 所以构建 Rab基因的 真核细胞表达载体时可以在 Rab基因的 N未端附加一个 c-myc的抗原表位基因， 当 载体转染 cos细胞后外源基因表达则形成 c-myc-Rab融合蛋白，而后用带有荧光素 标记的抗 c-myc抗体处理 COS细胞， 显微镜检时， 有萤光的位置便是 Rab锚定的 位置. 由此可检测出 Rab在各种膜的位置（Marino Zerial et al. methods in enzymology.vol 219:398-407.) .
在膜泡运输过程中， Rab蛋白通过与许多蛋白因子相互作用而实现其功能， 这些蛋白因子包括： （1) 异戊二烯基转移酶 GGTase. 当 Rab合成后， GGTase将 十五碳或二十碳的异戊二烯基转至 Rab 末尾的 CC或 CXC基序的半胱氨酸残基 上. （2) REP(Rab Escort Protein—— Rab的分子伴娘)， 将未修饰或未完全修饰的 Rab送至 GGTase的催化区域使 Rab完成修饰. （3) GTP酶激活蛋白 -GAP(GTPase activating protein), 当膜泡与目标膜融合后激活 Rab的 GTPase活性， 使 Rab催化的 GTP水解成 GDP。 （4)鸟苷解离抑制因子 -GDI(Guanine-nucletide disassoiation inhibitor), 将结合 GDP形式的 Rab从目标膜解离下， 并与 Rab共溶于细胞质中. （5) 鸟苷释放因子 -GDF(GDI displacement Factor), 促使 Rab从 GDI上解脱出而与自身 结合， 并使 Rab特异性的锚定在供体膜上. （6) 鸟苷交换因子 -GEF(guanine- nucleotide exchange factor), 催化 GTP将锚定于供体膜的 Rab结合的 GDP替换下. 结合 GTP形式的 Rab蛋白才具有活性. （7) V-Snare. 该因子位于供体膜上， 受 Rab 的催化，在形成膜泡后移至目标膜，可与目标膜上的 T-Snare相互结合； 当 T-Snare 与 V-Snare结合后， 膜泡与目标膜发生融合， 完成物质运输。 （8) Rab亲和蛋白 (Rabphlin) . Rab亲和蛋白可以阻碍 Rab的功能发挥（Peter novick and Patrick Brennward , cell vol 75,597-601,nov 9 1993.)(Armand Tavitian, methods in enzymology.vol 219:387-397)(Suzanne R Pfeffer , Curr opin cell biol 1994 ,6:522- 526.).
Rab基因在以下方面有很大的利用价值， （1)研究与 Rab基因相关的遗传性疾 病. Chediak-Higashi综合症是一种的常染色体隐性遗传病， 其病症是细胞内有巨 大的来源于分泌路径的细胞器膜. 该病病因可能为 Rab基因突变的纯合体导致细 胞的分泌蛋白分选发生缺陷. 这提示 Rab基因可应用于疾病的诊断和治疗. (Tatjana Stanrovic , Genomics 40,267-276(1997).) (2)Rab在基因工程中的应用价 值. 既然 Rab对于真核细胞细胞器间的蛋白转运和膜泡运输的正确定位和融合至 关重要，因此已有学者尝试着发挥 Rab的作用进行基因工程生产或利用 Rab调节细 胞内蛋白的转运. 例如， Chishi A.等人报道， Rab3D的超表达加强转基因小鼠腺 泡受控淀粉酶的分泌。 （3)Rab蛋白也与神经元的发育有关， 阻断 rab基因在幼体 神经元内的表达会使树突和轴突的生长受到抑制. （Lukas A et al. methods in enzymology.vol 257:302-312) (4)应用同源克隆法寻找新的 Rab家族成员基 因. 由于一些相关 Rab基因的基序都已弄清楚， 各种组织的 cDNA文库都已构建 完成，这也方便了在 cDNA文库中寻找新基因. 同时也可在不同的物种， 如小鼠， 果蝇及植物中搜寻新的 Rab基因， 并在该物种中探寻基因的功能和应用价值 (FANGLAI,et al . Genomics 22,610-616(1994).). (5)利用酵母双杂交系统寻找与 Rab相互作用的蛋白因子的基因，并努力验证其功能. GGTase, REP. GAP、 GDF、 GDI、 GEF等许多因子对于 Rab功能发挥起着重要作用， 尤其是 GGTase-异戊二烯 转移酶， 催化将异戊二烯基转移至 ras蛋白和 Rab蛋白的半胱氨酸残基上， 修饰后 的 Rab、 ras蛋白才具有正常的功能， 所以 Galb M.H.称 GGTase的抑制物将是极具 潜在价值的抗癌药物. 最近又发现一种新的作用因子叫 Rab亲和蛋白 (Monica Arribas , et al. European Journal of cell biology 74,209-216 1997 ..), 该蛋白可以与 Rab蛋白结合， 抑制 Rab蛋白的 GTP水解酶活性从而完全阻断 Rab的功能 (William H. Brondyk， methods in enzymology.vol 257:200-208). 因此， 为治疗目的研究和 开发人 RAB多肽及其激动剂 /抑制剂有重要意义.
发明目的
本发明的目的是提供一种新的人 Rab多肽以及其片段、 类似物和衍生物. 本发明的另一目的是提供编码这些多肽的多核苷酸.
本发明的另一目的是提供生产这些多肽的方法以及该多肽和编码序列的用 途.
发明概要
在本发明的第一方面， 提供新颍的分离出的 RABPA 多肽， 该多肽是人源的， 它包含： 具有 SEQ ID No. 2氨基酸序列的多肽、 或其保守性变异多肽、 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. 2氨基酸序列的多肽. 在本发明的第二方面， 提供编码分离的这些多肽的多核苷酸， 该多核苷酸包 含一核苷酸序列， 该核苷酸序列与选自下组的一种核苷酸序列有至少 80%相同 性： （a)编码上述人 RABPA多肽的多核苷酸； 和 (b)与多核苷酸 (a)互补的多核苷酸. 较佳地， 该多核苷酸编码具有 SEQ ID No. 2所示氨基酸序列的多肽. 更佳地， 该多核苷酸的序列是选自下组的一种： （a)具有 SEQ ID No. 1中 200-838位的序列； 和 (b)具有 SEQ ID No. 1中 1- 920位的序列。
在本发明的第三方面， 提供了含有上述的多核苷酸的载体， 以及被该载体转 化或转导的宿主细胞或者上述的多核苷酸直接转化或转导的宿主细胞. 在本发明的第四方面， 提供了制备具有人 RABPA活性的多肽的制备方法， 该 方法包含： （a)在适合表达人 RABPA的条件下， 培养上述的宿主细胞； （b)从培养 物中分离出具有人 RABPA活性的多肽.
在本发明的第五方面， 提供了与上述的人 RABPA多肽特异性结合的抗体. 还 提供了可用于检测的核酸分子， 它含有上述的多核苷酸中连续的 10-700个核苷 酸.
在本发明的第六方面， 提供了模拟、 促进、 拮抗人 RABPA多肽活性的化合物， 以及抑制人 RABPA多肽的表达的化合物. 还提供了筛选和 /或制备这些化合物的方 法. 较佳地， 该化合物是人 RABPA多肽的编码序列或其片段的反义序列.
在本发明的第七方面， 提供了上述化合物来调节人 RABPA蛋白在体内、 体外活 性的方法.
在本发明的第八方面， 提供了一种检测与人 RABPA多肽异常表达相关的疾病或 疾病易感性的方法， 该方法包括： 检测编码所述多肽的核酸序列中是否存在突变. 在本发明的第九方面， 提供了本发明多肽和编码序列的用途. 例如本发明多肽 可被用于筛选促进人 RABPA多肽活性的激动剂，或者筛选抑制人 RABPA多肽活性的 拮抗剂、 或者被用于肽指纹图谱鉴定. 本发明的人 RABPA的编码序列或其片段， 可 被作为引物用于 PCR扩增反应， 或者作为探针用于杂交反应， 或者用于制造基因芯片 或微阵列.
在本发明的第十方面， 提供了一种药物组合物， 它含有安全有效量的本发明的 人 RABPA多肽以及药学上可接受的载体。 这些药物组合物可治疗 AIDS和其它的获 得性和遗传性的免疫缺陷病等病症.
附图说明
下列附图用于说明本发明的具体实施方案， 而不用于限定由权利要求书所界定 的本发明范围.
图 1显示了本发明 RABPA 蛋白的氨基酸序列. 采用标准的氨基酸单字母缩写。 全长蛋白是 213个氨基酸， I 、 Π、 ΙΠ、 IV为保守的 GTP/GDP结合区域.
图 2是本发明 RABPA蛋白和已知的 Rab2蛋白 (Q14964)的同源性比较图。 上方序 列是 RABPA蛋白， 下方序列是 Rab2蛋白 (Q14964). 相同氨基酸用 " | " 表示， 相似氨 基酸用 "：" 或 " . " 表示.
图 3是显示 RABPA表达产物的电泳图. 图中， 从左到右是第一、 二泳道分别是 空质粒 (即含没装基因的 PBV220表达载体)的菌体 42'C诱导和 30'C未诱导蛋白电泳图； 第三泳道为标准低分子量蛋白标记物 (从上到下分子量分别为 97KD、 66KD、 43 D、 31KD、 20KD和 14KD);第四、 五泳道分别为基因诱导和未诱导电泳图， 箭头所指为 表达的人 RABPA蛋白.
图 4是本发明的 RABPA蛋白与 Rab家族基因的多序列同源比较结果。 发明内容
本发明的多核苷酸可以是 RNA形式或 DNA形式， DNA包括 cDNA、基因组 DNA 和合成 DNA. DNA可以是双链或单链的， 且如果是单链， 单链可以是编码链和非编 码 (反义)链. 编码成熟多肽的编码序列可以与 SEQ ID NO: 1中所示的编码序列相同， 也可以是不同的编码序列， 但该不同的编码序列 (由于基因密码的重复或简并的结果) 编码的多肽与 SEQ ID NO: 2的相 · 编码 SEQ ID NO: 2多肽的多核苷酸包括： 只编码 成熟多肽的编码序列； 成熟多肽的编码序列和附加编码序列 (如前导序列或分泌序列 或前蛋白序列)； 成熟蛋白的编码序列 (和可有可无的附加编码序列)和非编码序列 (如 成熟多肽的 5'和 /或 3'编码序列的内含子或非编码序列).
术语 "编码多肽的多核苷酸" 是指仅为多肽的编码序列的多核苷酸， 也指包括 附加编码序列和 /或非编码序列的多核苷酸.
本发明还涉及上述多核苷酸的变异体， 该变异体编码具有本发明 RABPA相同的 氨基酸序列的多肽片断、 类似物和衍生物. 此多核苷酸的变异体可以是此多核苷酸 天然存在的等效变异体或此多核苷酸的非天然存在的变异体. 因此， 本发明包括编 码与 RABPA同样的成熟多肽的多核苷酸， 还包括这些多核苷酸的变异体， 而这些变 异体编码本发明所述的多肽的片段、 衍生物或类似物. 这些核苷酸变异体包括缺失 变异体、取代变异体和添加或插人变异体.如上述所示,此多核苷酸可以是 SEQ ID NO: 1所示编码序列的天然存在的等位变异体的编码序列， 正如本领域所公知的， 等位变 异体是多核苷酸的替换形式， 它可以是一个或多个核苷酸的取代、 缺失或添加， 但 不会从实质上改变其编码的多肽的功能.
本发明还包括这样的多核苷酸， 其中成熟多肽的编码序列可以按相同的阅读框 架而融合于某个协助多肽在宿主细胞中表达和分泌的多核苷酸序列 (比如， 作为分泌 序列控制多肽从细胞中运输出来的前导序列). 有前导序列的多肽是一个蛋白原， 宿 主细胞可以切掉其前导序列， 从而形成此多肽的成熟形式。 多核苷酸还可以编码一 个由成熟蛋白和附加的 3'氨基酸残基组成的蛋白原. 有序列原的成熟蛋白是蛋白 原， 是此蛋白的非活性形式. 一旦序列原被切除就形成了一个有活性的成熟蛋白. 因此， 本发明的多核苷酸可以编码成熟蛋白， 或有序列原的蛋白， 或同时具有序列 原和前序列 (前导序列)的蛋白.
本发明还涉及这样的多核苷酸， 即当与上述的序列有至少 50%, 较佳地至少 70%, 更佳地至少 80 %相同时可与之杂交的多核苷酸。 特别涉及在严格条件下与上述 多核苷酸杂交的多核苷酸. 如本文所用， "严格条件" 这个术语是指只有当序列 之间有至少 95%和最好至少 97%相同时才能杂交. 在一优选的实施方案中， 与上述多 核苷酸杂交的多核苷酸所编码的多肽， 与 SEQ ID NO: 1中 200-838位的 DNA编码的 多肽在生物功能和活性方面实质上相同.
本发明还涉及具有 SEQ ID NO: 2的氨基酸序列的多肽和这种多肽的片段、 类似 物和衍生物.
本发明中的多肽可以是重组多肽、 天然多肽或合成多肽， 优选是重组多肽. 本 发明的多肽可以是天然纯化的产物， 或是化学合成的产物， 或使用重组技术从原核 或真核宿主 (例如， 细菌、 酵母、 高等植物、 昆虫和哺乳动物细胞)中产生， 根据重组 生产方案所用的宿主， 本发明的多肽可以是糖基化的， 或可以是非糖基化的. 本发 明的多肽还可包括或不包括起始的甲硫氨酸残基.
当指本发明的多肽时， 术语 "片段" 、 "衍生物" 和 "类似物" 指保留与此多 肽相同的生物学功能或活性的多肽. 因此， 类似物包括切除蛋白原部分后能被激活 以产生活性成熟多肽的蛋白原. 本发明的多肽片断、 衍生物或类似物可以是 (i)有一 个或多个保守或非保守性氨基酸残基 (优选保守性氨基酸残基)被取代的多肽，而这样 的取代的氨基酸残基可以是也可以不是由遗传密码编码的， 或 (ii)在一个或多个氨基 酸残基中包括一个取代基团的多肽，或 (Hi)成熟多肽与另一个化合物 (比如延长多肽半 衰期的化合物， 例如聚乙二醇)融合所形成的多肽， 或 (iv )附加的氨基酸序列融合到 此多肽序列而形成的多肽 (如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原 序列)。 根据本文的教导， 这些片断、 衍生物和类似物属于本领域熟练技术人员公知 的范围.
本发明中的多肽和多核苷酸优选以分离的形式提供， 更佳地被纯化至均质. 术语 "分离的" 是指材料从其原始环境分离出来， 例如， 位于动物体内的天然 存在的多核苷酸或多肽不是分离的， 而从天然系统中的一部分或所有共存材料中分 离出来的同样的多核苷酸或多肽就是分离的. 这样的多核苷酸可以是载体的一部 分， 这样的多核苷酸或多肽也可以是组合物的一部分， 只要这些载体或组合物不是 其天然环境的一部分， 则其仍是分离的.
本发明还涉及包含本发明的多核苷酸的载体， 用本发明的载体制造的遗传工程 宿主细胞和用重组技术生产的本发明的多肽的产品. 本发明的多核苷酸序列可以包 含在用于表达多肽的众多表达载体中的任一种中. 这些载体包括染色体的、 非染色 体的和合成的 DNA序列， 比如 SV40的衍生物、 细菌质粒、 噬菌体 DNA、 杆状病毒、 酵母质粒、质粒和噬菌体 DNA组合衍生而来的载体、病毒 DNA如牛痘病毒、腺病毒、 禽痘病毒和假狂犬病病毒. 总之， 只要能在宿主体内复制存活， 任何质粒和载体都 可以用.
表达载体最好含有提供筛选转化的宿主细胞的表型性状的基因， 如真核细胞培 养用的二氢叶酸还原酶或新霉素抗性， 或用于大肠杆菌的四环素或氨苄青霉素抗 性.
表达载体中的 DNA序列与合适的表达调控序列 (启动子)连接在一起以指导 mR A合成. 这类启动子的一些有代表性的例子有： LTR或 SV40启动子， E.coli.lac 或 ti 启动子， 噬菌体 λ PL启动子和其他一些己知的可控制基因在原核或真核细胞或 其病毒中的表达启动子. 表达载体还包括有翻译起始用的核糖体结合位点和转录终 止子. 含有上述适当 DNA序列和适当启动子或调控序列的载体可以用来转化适当宿 主细胞以使宿主表达此蛋白.
宿主细胞可以是高等真核细胞， 如哺乳动物细胞， 或是低等真核细胞， 如酵母 细胞， 或是原核细胞， 如细菌细胞. 适当宿主的一些有代表性的例子有： 如大肠杆 菌， 链霉菌； 鼠伤寒沙门氏菌的细菌； 诸如酵母之类的真菌细胞； 昆虫细胞， 例如 果蝇细胞和 sf9细胞； 动物细胞如 CHO, COS或 Bowes黑素瘤细胞； 植物细胞等. 通过本文的讲授， 适当宿主细胞的选择是本领域熟练技术人员所公知的.
适当的 DNA序列可以通过各种不同方法插入载体。 一般来说， DNA序列通过 本领域公知的程序被插入合适的限制性内切酶位点. 这些和其他步骤对本领域技术 人员是公知的. 当合适的宿主被转化并生长到适当的细胞密度后， 选用的启动子即 可用适当的方法 (如温度转换或化学诱导)诱导， 细胞再培养一段时间. 细胞经离心后 收获， 用物理或化学的方法破碎细胞， 得到的粗提物留作进一步纯化用. 破碎包括 冻融法、 超声波法、 机械破碎法、 或使用细胞裂解试剂. 用硫酸胺或乙醇沉淀， 酸 抽提， 阴离子或阳离子交换层析， 磷酸纤维层析， 疏水相互作用层析， 亲和层析， 羟基磷灰石层析或植物凝激素层析等方法， 可将蛋白从重组细胞培养物中回收并纯 化出来. 如果需要， 可以使用蛋白质再折叠步骤以形成成熟蛋白的构象. 最后， 可 以使用高效液相层析 (HPLC)来完成最后的纯化步骤.
因为 Rab基因表达的下降会导致致命的细胞程序性死亡（美国专利 5892012). 因此， 作为一种新的 RAB蛋白， 本发明的 RABPA多肽或其片段或其 衍生物可以用来治疗与细胞程序性死亡相关的疾病， 其中包括但不限于： AIDS 和其它的获得性和遗传性的免疫缺损病； 退行性神经性疾病如老年性痴呆， 帕 金森病， 半侧硬化肌萎缩， 色素性视网膜炎， 小脑退化， 脊髓发育不良综合症 如再生障碍性贫血， 局部贫血伤害如心肌梗塞、 中风和再灌流受伤， 毒素引发 的疾病如酒精性肝中毒、 肝硬变， 病毒性感染如丙肝， 乙肝和骨硬化病.
RABPA多肽或其片段或其衍生物加到细胞系中可以刺激细胞增殖， 还可以 借助脂质体、 电穿孔等手段直接引人到活体细胞内.
本发明的多肽可以与合适的药用载体组合使用. 这种组合物包含治疗有效 量的多肽， 和药学上可接受的载体或赋型剂. 这样的载体包括但不限于： 盐水、 缓冲盐水、 葡萄糖、 水、 甘醇、 乙醇及其混合物. 这些制剂应适合于给药方式. 本发明还提供含有一个或多个容器的药盒或试剂盒， 在这些容器中装有一 种或多种本发明的药用组合物成分. 与这些容器一起， 可以有由制造、 使用或 销售药品或生物制品的政府管理机构所给出的指示性提示， 该提示反映出生产、 使用或销售的政府管理机构许可其在人体上施用. 此外， 本发明的多肽可以与 其它的治疗化合物结合使用.
药物组合物可以以方便的方式给药， 如通过局部、 静脉内、 腹膜内、 肌内、 皮下、 鼻内或皮内的途径给药. RABPA宜以有效地治疗和 /或预防具体的适应症 的量来给药. 给药于患者的 RABPA的量和剂量范围将取决于许多因素， 如给药 方式、 欲治疗者的身体条件和诊断医生的判断.
本发明的多肽、 其片段或其衍生物、 或其类似物、 或表达它们的细胞， 可 以用作免疫原， 用来生产其抗体， 用于诊断或用作拮抗剂. 这些抗体可以是例 如多克隆杭体或单克隆抗体。 本发明还包括嵌合链抗体、 单链抗体， 和人源化 抗体， 以及 Fab片段， 或 Fab表达库的产物， 可以用本领域各种已知的方法来生 产这些抗体和片段.
针对相应于本发明序列的多肽产生的抗体， 可以通过直接将多肽注射人动 物 (优选非人动物)来获得. 这样获得的抗体然后会与其多肽结合. 在这种方式 中， 甚至编码多肽片段的序列也可以用来产生结合整个天然多肽的抗体。 然后， 这些抗体可以用来将多肽从表达此多肽的组织中分离出来.
为制备单克隆杭体， 可以使用任何通过连续细胞系培养物而生产抗体的技 术，这些技术的例子包括杂交瘤技术 (Kohler和 Milstein, 1975,Nature,256:495-497), trioma技术、 人 B细胞杂交瘤技术 (Kozbor等， 1983,Immuolgy Today 4:72), 和生 产人单克隆抗体的 EBV杂交瘤技术 （Cole等， 1985, Monoclonal Antibodies and Cancer†erapy,Alan R.Liss,Inc.,pp77-96).
可以对生产单链抗体的技术 (USP4,946,778)加以改进， 用来生产本发明的免 疫多肽产物的单链抗体.
本发明还涉及定量和定位检测 RABPA水平的诊断试验方法. 这些试验是本 领域所熟知的， 且包括 FISH测定和放射免疫测定. 试验中检测的 RABPA水平可 以用作解释 RABPA在各种疾病中的重要性和用于诊断与 RABPA相关的疾病. 本发明也提供了筛选药物以鉴定提高 (激动剂)或阻遏 (拮抗剂) RABPA的药 剂的方法. 激动剂提高 RABPA的生物功能 (例如刺激细胞增殖等)， 而拮抗剂阻止 和治疗与细胞过度增殖有关的紊乱如各种癌症. 例如， 能在药物的存在下， 将 哺乳动物细胞或表达 ABPA的膜与标记的 RABPA—起培养. 然后测定药物提高 或阻遏此相互作用的能力.
激动剂 /抑制剂也可以用在 (例如上面所述的)含有药学上可接受载体的组合 物中.
本发明的多肽也可以通过在活体表达这些多肽来使用， 这通常称作 "基因 治疗" .例如，患者的细胞可以在体外用编码 RABPA多肽的多核苷酸 (DNA或 RNA) 进行基因工程操作， 然后将工程化的细胞提供给欲用此多肽治疗的患者. 这些 方法是本领域熟知的。 例如， 细胞可以通过使用含有编码本发明多肽的 RNA的 逆转录病毒， 用本领域已知的基因工程方法操作.
同样， 细胞可以在体外经基因工程技术处理， 在体内通过例如本领域已知 的方法表达 RABPA多肽. 正如本领域所知的， 生产含有编码本发明多肽的 RNA 的逆转录病毒颗粒的生产细胞， 可以被施用于患者体内， 在体内产生工程细胞， 并且在体内表达多肽. 施用本发明多肽的这些和其它的方法， 在本发明的教导 基础上， 对本领域的技术人员来说是显而易见的。 例如， 工程细胞的表达载体 除了逆转录病毒之外， 可以是在与合适的运送载体组合后用来在体内产生工程 细胞的腺病毒.
本发明的多核苷酸可用来设计反义 DNA或 RNA, 作为抑制剂治疗和预防与细胞 增殖有关的各种紊乱 (如肿瘤等)及治疗和预防各种炎症. 通过三螺旋体形成或反义 DNA或 R A (两方法均是以多核苷酸与 DNA或 R A结合为基础)， 反义技术可以用来 控制基因表达。 例如， 编码本发明成熟多肽的多核苷酸序列的 5'编码部分， 可以用来 设计约 10-40个碱基对长度的反义 RNA寡核苷酸。 设计 DNA寡核苷酸， 互补于涉及转 录的基因区域 (三螺旋体——参见 Lee等， Nucl.Acids Res.,6:3073(1979); Cooney等，
Figure imgf000012_0001
Science,251:1360(1991))，由此防止转录和 RABPA 的产生. 反义 RNA寡核苷酸与 mRNA在体内杂交， 并且阻遏 mR A分子翻译成 RABPA。 反义 RNA和 DNA可以转运到细胞， 在体内抑制 RABPA的产生. 另外， 反 义 RNA和 DNA可以含磷酸硫脂键或肽脂键以延长其在体内的半衰期.
本发明的序列对染色体鉴定也是有价值的。 该序列会特异性地针对某条人 染色体具体位置且并可以与其杂交. 目前， 需要鉴定染色体上的各基因的具体 位点. 现在， 只有很少的基于实际序列数据 (重复多态性)的染色体标记物可用于 标记染色体位置. 根据本发明， 将这些序列与疾病相关基因相关联， 其重要的 第一步就是将这些 DNA序列定位于染色体上.
简而言之， 根据 cDNA制备 PCR引物 (优选 15-35bp), 可以将序列定位于染色 体上. 然后， 将这些引物用于 PCR筛选含各条人染色体的体细胞杂合细胞. 只有 那些含有相应于引物的人基因的杂合细胞会产生扩增的片段.
体细胞杂合细胞的 PCR定位法， 是将 DNA定位到具体染色体的快捷方法. 使用本发明的的寡核苷酸引物， 通过类似方法， 可利用一组来自特定染色体的 片段或大量基因组克隆而实现亚定位. 可用于染色体定位的其它类似策略包括 原位杂交、 用标记的流式分选的染色体预筛选和杂交预选， 从而构建染色体特 -异的 cDNA库.
将 cDNA克隆与中期染色体进行荧光原位杂交 (FISH), 可以在一个步骤中精 确地进行染色体定位. 此技术的综述， 参见 Verma等， Human Chromosomes:a Manual of Basic Techniques,Pergamon Press, New York(1988).
—旦序列被定位到准确的染色体位置， 此序列在染色体上的物理位置就可 以与基因图数据相关联.这些数据可见于例如， V.Mckusick,Mendelian Inheritance in Man (可通过与 Johns Hopkins University Welch Medical Library联机获得）. 然后 可通过连锁分析， 确定基因与业已定位到染色体区域上的疾病之间的关系. 接着， 需要测定患病和未患病个体间的 cDNA或基因组序列差异. 如果在一 些或所有的患病个体中观察到某突变， 而该突变在任何正常个体中未观察到， 则该突变可能是疾病的病因. 比较患病和未患病个体， 通常涉及首先寻找染色 体中结构的变化， 如从染色体水平可见的或用基于 cDNA序列的 PCR可检测的缺 失或易位. 根据目前的物理作图和基因定位技术的分辨能力， 被精确定位至与 疾病有关的染色体区域的 cDNA,可以是 50至 500个潜在致病基因间之一种 (假定 1 兆碱基作图分辨能力和每 20kb对应于一个基因).
本发明多肽和多核苷酸的其他用途， 例如将本发明多肽用于肽指纹图谱鉴 定. 本发明的人 RABPA的编码序列或其片段， 可被作为引物用于 PCR扩增反应， 或者作为探针用于杂交反应， 或者用于制造基因芯片或微阵列. 根据本发明的 教导， 这些应用对于本领域技术人员而已是显而易见的.
在本发明的一个实例中， 提供一种分离的核苷酸 (多核苷酸） 它编码具有 SEQ ID NO: 2所示氨基酸序列的成熟多肽. 本发明的多核苷酸是从 18周人胎脑 cDNA文库发 现的。 它含有编码大致 213个氨基酸长度的蛋白的开放读框， 它结构上与 Rab基因 ( 99962)在核酸水平上73.2°/。相同； 与 Rab基因 (X99962)编码的蛋白 (Q14964)有 75.6 %相同性和 82..6 %相似性。 本发明的 RABPA多肽含有特征基序， 参见图 2, 其中结构 域 I、 II、 III、 IV区是 GTP/GDP结合区域， 效应子结构域为 GTPase激活蛋白结合区域， 下划线为蛋白 C端高变区域及 CXC结构域. 另外， 经计算机分析 RABPA蛋白的分子 量为 24566.89道尔顿，等电点为 7.43.
此外， 由于本发明的人 RABPA具有源自人的天然氨基酸序列， 因此， 与来 源于其他物种的同族蛋白相比，预计在施用于人时将具有更高的活性和 /或更低的 副作用 (例如在人体内的免疫原性更低或没有).
实施例
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议的条件. 实施例 1 : 人 Rabpa基因 cDNA的克隆
用异硫氰酸胍 /酚 /氯仿一步法提取人胎脑总 RNA. 用 Quick mR A Isolation Kit (Qiegene)从总 RNA中分离 poly(A) mRNA. 2ug poly(A) mRNA经逆转录形成 cDNA. 用 Smart cDNA克隆试剂盒 (购自 Clontech)将 cDNA片段定向插人到 pBS- SK载体的多克隆位点上， 转化 DH5c细菌形成 cDNA文库. 共获得约 2800个克隆。 用双脱氧法测定所有克隆的 5'和 3'末端的序列. 将测定的 cDNA 序列与已有的公 共 DNA序列数据库进行比较， 结果发现有一个克隆 067D10的 DNA序列为新的 DNA. 通过合成一系列引物对 067D10克隆所含的 DNA序列进行双向测定. 计算 机分析表明， 067D10克隆所含的全长 cDNA是一个新的 DNA序列 (如 SEQ ID NO: l 所示)， 从第 200bp至 841bp有一个 641bp的 ORF, 编码一个 213个氨基酸的新的蛋白 质 (如 SEQ ID NO: 2所示). 我们将此基因命名为人 Rabpa基因， 所编码的蛋白命 名为 RABPA蛋白. 实施例 2: 用 RT-PCR方法克隆人 Rabpa基因
用胎脑细胞总 RNA为模板， 以 oligo-dT为引物进行逆转录反应合成 cDNA. 用
Qiagen的试剂盒纯化后，用下列引物进行 PCR扩增： 正向引物 F1 5 - GGAATTCATGGAGGCCAT CTGGCTGTAC -3 (SEQ ID NO: 3); 反向引物 Rl: 5 - GGAAGCTTCATGCCGTATGCAGCAGCCA -3 (SEQ ID NO: 4) . 扩增反应的条件： 在 50μ1的反应体积中含有 50mmol/L KCl,10mmol/L Tris-Cl(pH8.5),1.5mmol/L MgCl2,20(^mol/L dNTP, 各 25pmoI引物， 2.5U的 Taq DNA聚合酶. 在 PE9600型 DNA 热循环仪上按下列条件反应 25个周期： 94 "C 30sec; 55'C , 30sec; 72'C 2min.在 RT-PCR 时同时设 β-肌动蛋白为阳性对照和模板空白为阴性对照. 扩增产物 QIAGEN试剂盒纯 化后， 用 ΤΑ克隆试剂盒连接到 pBV220表达载体， 并测定 DNA序列. 结果： PCR产物的 DNA序列与 SEQ ID NO: 1的 200-841bp完全相同，并且在两端分别含有 EcoRI和 Hindlll 酶切位点. 实施例 3 人 Rabpa基因的重组表达
把装有 Rabpa基因的 pBV220表达载体转人 DH5CC宿主菌中， 通过在 LB板上的 生长能力识别转化体， 筛选出氨苄青霉素抗性菌落. 质粒 DNA通过限制性分析分 离和确定.将含有所需构建物的克隆在补充有 Amp(100ug/ml)的 LB介质中的液体 培养基中生长过夜。 用过夜培养物以 1: 100—1： 250的比率来接种大量的培养物. 将细胞生长到光学密度 600(O..D.60O)为 0. 4和 0.6之间， 42°C诱导表达 3小时， 12% SDS-PAGE胶鉴定表达产物.
因 Rabpa基因表达的蛋白在 42 'C生长环境中不能稳定存在， 因此本发明人尝 试在表达载体的 SD序列后且在新基因起始密码子 ATG前插入 51bp的人工合成的 干扰素寡核昔酸片段 (ATGtgctact gccaggaccc gtacgttaaa gaagctgaaa acctggaatt c(SEQ ID NO: 5) (在 SEQ ID NO: 5后面为包括 ATG在内的 920bp的 RABPA编码序 列))， 使干扰素片段与 Rabpa基因一起融合表达， 实验证明： 融合表达的蛋白能稳 定存在于细菌体内。 蛋白表达电泳图如图 3所示. 图中， 从左到右是第一、 二泳 道分别是空质粒 (即含没装基因的 PBV220表达载体)的菌体 42 'C诱导和 30 'C未诱 导蛋白电泳图；第三泳道为标准低分子量蛋白标记物（从上到下分子量分别为 97KD、 66KD、 43KD、 31KD、 20KD和 14KD);第四、 五泳道分别为基因诱导和未 诱导电泳图， 箭头所指的就是表达的人 RABPA蛋白， 分子量约 25kDa. 实施例 4
人细胞中 RABPA的表达方式
进行 Northern印迹分析， 测定人细胞中 RABPA的表达水平。 用 BNAzolTM B 体系（ Biotecx Laboratories, Inc.6023 South Loop East,Houston,TX 77033)分离总细胞 R A样品。 从各具体的人组织分离约 lOug的总 RNA, 在 1%琼脂糖凝胶上分离， 并且 印迹到尼龙滤器上（ Sambrook,fritsch和 Maniatis, Molecular Cloning,Cold Spring HarborPress,(1989). 用 Stratagene Prime-It试剂盒， 用 50ngDNA片段做标记反应. 标记 的 DNA用 Select-G-50柱纯化（5 Prime-3 prime,Inc.5603 Arapahoe Road,Boulder,Co 80303). 然后， 滤器用放射标记的全长 RABPA基因， 在 65 'C下， 以 l，000,000cpm/ml 在 0.5 M NaPO4、 ρΗ7· 4和 7% SDS中杂交过夜. 用 0.5 χ SSC、 0.1%SDS在室温下冲 洗两次和在 60'C冲洗两次后， 将滤器与强化荧光屏一起暴露在 -70 'C下. RABPA的信 使 RNA富含于活化和失活的 T细胞、 单核细胞和 T细胞系中. 实施例 5 cDNA克隆的同源检索
用本发明的 Rabpa基因的序列及其编码的蛋白序列在 Genbank,Swissport等数据 库进行同源检索.用于检索的程序叫 Blast(Basic local Alignment search tool)(1993 Proc Nat Acad Sci 90:5873-5877), Blast 可以找出与 Rabpa同源的许多基因， 其中与本发明 的 Rabpa基因同源性最大的基因为一种已知的人 RAB蛋白 （Genbank准入号为 X99962), X99962编码的蛋白在 Genbank 的准入号为 Q14964. 这些检索到的基因或 蛋白序列可以从 Genbank数据库中调出. 调出的序列可以用 GCG软件包中的 Pileup (多 序列)和 Gap (两序列)程序做连配比较. 结果显示， X99962与 067D10具有最高的同源 性， 在核苷酸水平上之间的相似性为 73%. 将两者编码的蛋白质进行比较 (图 2), 结 果表明， 两者高度同源， 其相似性为 82.6% ； 相同性为 75.5%.
用 Pileup程序， 将本发明的蛋白与 Rab家族基因进行多序列同源比较. 结果如图 4 所示. 将本发明的 Rabpa编码的蛋白用 GCG软件包进行基序 (motif)分析， 结果表明 Rabpa编码的蛋白属于 Rab GTPase家族，具有 GTPase共有的结构特征。 Rabpa编码的蛋 白的具体结果特征如图 1所示. 参见图 1 , 图中 I、 II、 III、 IV分别为 GTP/GDP结合区 域， 效应子结构域为 GTPase激活蛋白即效应子结合区域， 下划线为蛋白 C端高变区域 及 CXC结构域.
另外经计算机分析，本发明的新的人 Rabpa蛋白的分子量为 24566.89道尔顿，等电 点为 7.43, 实施例 6抗人 RABPA抗体的产生
用多肽合成仪（PE-ABI)合成下述人 RABPA特异性的多肽： NH2- MetGluAlalleTrpLeuTyrGlnPheArgLeuIleVallleGly-COOH. 将该多肽分别与血蓝蛋白 和牛血清白蛋白偶合形成复合物， 方法参见： Avrameas. Immunochemistry,1969; 6:43. 用 4mg上述血蓝蛋白多肽复合物加上完全弗氏佐剂免疫家兔， 15天后再用血蓝 蛋白多肽复合物加不完全弗氏佐剂加强免疫一次. 采用经 15μ§/ιηΙ牛血清白蛋白多肽 复合物包被的滴定板做 ELISA测定兔血清中抗体的滴度. 用蛋白 A-Sepharose从抗体 阳性的家兔血清中分离总 IgG。 将多肽结合于溴化氰活化的 Sepharose 4B柱上， 用亲 和层析法从总 IgG中分离抗多肽抗体. 免疫沉淀法证明纯化的抗体可特异性地与人 RABPA结合. 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 Rab基 及其编码的多肽
(iii)序列数目： 5
(2) SEQ ID NO: 1的信息：
(i)序列特征：
(A)长度： 920bp
(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 1:
1 6CAI 1 I CATC ACC I 1 I GCGA GCGCAGCATC CATCCCTCCG CTCTCCCGGC
51 GCCTGGGCCT ACCCAGCTTC GGGCTCCCAG GCCAGCGATG CGCTCGCGGC
101 TGAGCTAGAT CCTGCCGAGC CGCGCTCTCT GAGGCGTCGG CGGGGCGCCC
151 CCTCCCGCCG TCCCCGGTCC GGGCCAAGGA GACCTGCAGA GCCGCGGCCA
201 TGGAGGCCAT CTGGCTGTAC CAGTTCCGGC TCATTGTCAT CGGGGATTCC
251 ACAGTGGGCA AGTCCTGCCT GATCCGCCGC TTCACCGAGG GTCGC I 1 I GC
301 CCAGGTTTCT GACCCCACCG TGGGGGTGGA U N I I CTCC CGCHGGTGG
351 AGATCGAGCC AGGAAAAAGC ATCAAGCTCC AGATCTGGGA TACCGCGGGT
401 CAAGAGAGGT TCAGATCCAT CACTCGCGCC TACTACAGGA ACTCAGTAGG
451 TGGTCHCTC TTATTTGACA nACTAACCG CAGGTCCTTC CAGAATGTCC
501 ATGAGTGGH AGAAGAGACC AAAGTACACG 丌 CAGCCCTA CCAAATTGTA
551 nTGTTCTGG TGGGTCACAA GTGTGACCTG GATACACAGA GGCAAGTGAC
601 TCGCCACGAG GCCGAGAAAC TGGCTGCTGC ATACGGCATG AAGTACAHG
651 AAACGTCAGC CCGAGATGCC ATTAATGTGG AGAAAGCC丌 CACAGACCTG
701 ACAAGAGACA TATATGAGCT GGTTAAAAGG GGGGAGATTA CAATCCAGGA
751 GGGCTGGGAA GGGGTGAAGA GTGGAI 1 I GT ACCAAATGTG GHCACTCn
801 CAGAAGAGGT TGTCAAATCA GAGAGGAGAT GTTTGTGCTA GTCAGTTCTT
851 AACATGCTCT CCTACHGAA CTGAAAAGTA AGAGAAATAA ATAGAATCH TGTGTAACTG
(2)SEQ ID NO: 2的信息：
(i)序列特征：
(A)长度： 213个氨基酸
(B)类型 '· 氨基酸
(D)拓扑结构： 线性
(ii)分子类型： 多肽
(xi)序列描述： SEQIDNO:2:
Met Glu Ala Ile Trp Leu Tyr Gin Phe Arg Leu He Val Ile Gly 15
Asp Ser Thr Val Gly Lys Ser Cys Leu Ile Arg Arg Phe Thr Glu 30
Gly Arg Phe Ala Gin Val Se「 Asp Pro Thr Val Gly Val Asp Phe 45
Phe Ser Arg Leu Val Glu Ile Glu Pro Gly Lys Thr Ile Lys Leu 60
Gin He Trp Asp Thr Ala Gly Gin Glu Arg Phe Arg Ser Ile Thr 75
Arg Ala Ty「 Tyr Arg Asn Ser Val Gly Gly Leu Leu Leu Phe Asp 90 ile Th「 Asn Arg Arg Ser Phe Gin Asn Val His Glu Trp Leu Glu 105
Glu Thr Lys Val His Val Gin Pro Tyr Gin Ile Val Phe Val Leu 120
Val Gly His Lys Cys Asp Leu Asp Thr Gin Arg Gin Val Thr Arg 135
His Glu Ala Glu Lys Leu Ala Ala Ala Tyr Gly Met Lys Tyr Ile 150
Glu Thr Ser Ala Arg Asp Ala Ile Asn Val Glu Lys Ala Phe Thr 165
Asp Leu Thr Arg Asp He Tyr Glu Leu Val Lys Arg Gly Glu Ile 180
Thr Ile Gin Glu Gly Trp Glu Gly Val Lys Ser Gly Phe Val Pro 195
Asn Val Val His Ser Ser Glu Glu Val Val Lys Ser Glu Arg Arg 210
Cys Leu Cys 213
(2)SEQ ID NO: 3的信息
W序列特征
(A)长度： 28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 ： 3
GGAATTCATG GAGGCCATCT GGCTGTAC
(2)SEQ ID NO: 4的信息
(i)序列特征
(A)长度： 28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: 4:
GGAAGCTTCA TGCCGTATGC AGCAGCCA
(2)SEQ ID NO: 5的信息
(i)序列特征
(A)长度： 51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 :
ATGTGCTACT GCCAGGACCC GTACGTTAAA GAAGCTGAAA ACCTGGAATT C 51

Claims

权 利 要 求
I .—种分离的人 RABPA多肽， 它包含： 具有 SEQ ID No. 2氨基酸序列的多肽、 或其保守性变异多肽、 或其活性片段、 或其活性衍生物.
2.如权利要求 1所述的多肽， 其特征在于， 该多肽是具有 SEQ ID No. 2氨基酸 序列的多肽.
3.—种分离的多核苷酸， 其特征在于， 它包含一核苷酸序列， 该核苷酸序列 与选自下组的一种核苷酸序列有至少 80%相同性：
(a)编码如权利要求 1和 2所述多肽的多核苷酸；
(b)与多核苷酸 (a)互补的多核苷酸.
4.如权利要求 3所述的多核苷酸，其特征在于，该多核苷酸编码具有 SEQ ID No. 2所示氨基酸序列的多肽.
5.如权利要求 3所述的多核苷酸， 其特征在于， 该多核苷酸的序列选自下组的 一种：
(a)具有 SEQ ID No. 1中 200-838位的序列；
(b)具有 SEQ ID No. 1中 1- 920位的序列.
6.—种载体， 其特征在于， 它含有权利要求 3所述的多核苷酸.
7.—种遗传工程化的宿主细胞， 其特征在于， 它是选自下组的一种宿主细胞： (a)用权利要求 6所述的载体转化或转导的宿主细胞；
(b)用权利要求 3所述的多核苷酸转化或转导的宿主细胞。
8- 一种具有人 RABPA活性的多肽的制备方法， 其特征在于， 该方法包含：
(a)在适合表达人 RABPA的条件下， 培养权利要求 7所述的宿主细胞；
(b)从培养物中分离出具有人 RABPA活性的多肽.
9.一种能与权利要求 1所述的人 RABPA多肽特异性结合的抗体.
10.—种核酸分子， 它含有权利要求 3所述的多核苷酸中连续的 10-700个核苷 酸.
I I.—种化合物， 其特征在于， 它选自下组：
(a)模拟权利要求 1所述多肽的活性的化合物，
(b)促进权利要求 1所述多肽的活性的化合物，
(c)拮抗权利要求 1所述多肽的活性的化合物，
(d)抑制权利要求 1所述多肽的表达的化合物.
12.如权利要求 11所述的化合物， 其特征在于， 它是 SEQ ID NO: 1所示的多核苷 酸序列或其片段的反义序列。
13.—种应用权利要求 1 1所述化合物来调节人 RABPA蛋白在体内、体外活性的方 法.
14.一种检测与权利要求 1所述的多肽异常表达相关的疾病或疾病的易感性的方 法， 其特征在于， 包括检测编码所述多肽的核酸序列中的突变.
15.如权利要求 1所述的多肽的用途， 其特征在于， 它被用于筛选促进人 RABPA 多肽活性的激动剂， 或者筛选抑制人 RABPA多肽活性的拮抗剂、 或者被用于肽指紋 图谱鉴定.
16.如权利要求 10所述的核酸分子的用途， 其特征在于， 它被作为引物用于 PCR 扩增反应， 或者作为探针用于杂交反应， 或者用于制造基因芯片或微阵列.
17.—种药物组合物， 其特征在于， 它含有安全有效量的权利要求 1所述的人 RABPA多肽以及药学上可接受的载体.
PCT/CN2000/000251 1999-08-31 2000-08-28 Nouveau gene rab humain et son polypeptide codant WO2001016175A1 (fr)

Priority Applications (1)

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AU68163/00A AU6816300A (en) 1999-08-31 2000-08-28 A novel human rab gene and its coding polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN99118398.3 1999-08-31
CN99118398A CN1286263A (zh) 1999-08-31 1999-08-31 新的人Rab基因及其编码的多肽

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GENOMICS, vol. 40, no. 2, 1997, pages 267 - 276 *

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CN1286263A (zh) 2001-03-07

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