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WO2009030084A1 - NOUVELLES FONCTIONS ET UTILISATIONS DE LA PETITE PROTÉINE G HUMAINE DE TYPE ONCOGÈNE RabJ - Google Patents

NOUVELLES FONCTIONS ET UTILISATIONS DE LA PETITE PROTÉINE G HUMAINE DE TYPE ONCOGÈNE RabJ Download PDF

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Publication number
WO2009030084A1
WO2009030084A1 PCT/CN2007/070623 CN2007070623W WO2009030084A1 WO 2009030084 A1 WO2009030084 A1 WO 2009030084A1 CN 2007070623 W CN2007070623 W CN 2007070623W WO 2009030084 A1 WO2009030084 A1 WO 2009030084A1
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rabj
polypeptide
tumor
protein
cells
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French (fr)
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Taoyong Chen
Nan Li
Tao Wan
Xuetao Cao
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Second Military Medical University SMMU
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Second Military Medical University SMMU
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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
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention is in the field of biotechnology and medicine, and in particular, the present invention relates to human oncogene-like small G protein RabJ and its coding sequence.
  • the polypeptide of the present invention is a novel molecule having GTPase activity, which is involved in the regulation of cell proliferation and cell differentiation, and has an oncogene-like effect. Background technique
  • Small G proteins refer to single subunit G proteins with a molecular weight of about 20-30 kDa.
  • GTP guanosine triphosphate
  • GDP guanosine diphosphate
  • Phosphoric acid becomes GDP (ie GTPase activity;).
  • Ras Ras, Rab, Rho, Arf, Sari and Ran.
  • Ras and Rho families are extremely important small G proteins because they are involved in the regulation of signaling mechanisms, so that cells can respond correctly to external stimuli and play an important role in basic life activities such as cell survival and apoptosis.
  • Members of the Ras family include Ras, Ral, and Rap, and the most widely known of them are the Ras subfamily, including Ha-Ras, Ki-Ras, and N-Ras. Both knockout mouse models and defective cell models have demonstrated that Ras family molecules play an important role in embryogenesis and ontogeny.
  • Rho family mainly include Rho, Rac, Cdc42, etc., by regulating the functions and activities of Rho-associated kinase (ROK), myosin binding subunit (MBS) phospholipase and other target proteins. Regulate cellular activities such as cytoskeleton, cell proliferation, and stress response.
  • ROK Rho-associated kinase
  • MMS myosin binding subunit
  • Ras and tumorigenesis and their role in the regulation of cell proliferation have attracted attention.
  • mutations in the H-Ras, K-Ras or N-Ras genes and Ras mutations are the most likely in tumor-mutated genes.
  • Molecules associated with the regulation of Ras protein activity are also involved in tumorigenesis.
  • Molecules of the Rho family also regulate the formation of intracellular actin-stressing fibers, cell membrane processes, and depressions by regulating the cellular actin system.
  • NIH3T3 a mutant protein molecule that regulates the activity of the Rho family can cause cell transformation.
  • Rho family oncogene-like molecules that regulate the GTP/GDP binding activity and GTPase activity of the Rho family include Dbl, Vav, Lbc, Tiaml, Tim, Ost, Ect2, Lsc, and FGD1, which can cause abnormalities in the function of the cell actin system, causing cells. Changes in migration infiltration capacity.
  • Rab Ras-like protein in rat brain
  • the basic biochemical feature is that it can bind GTP/GDP and hydrolyze GTP. So far, the family has grown to more than 100 members, including in many species including nematodes, yeast, fruit fly, zebrafish, mice, rats, humans, etc., mainly involved in intracellular proteins.
  • the transport process regulates the fusion of membranous organelles and the process of budding, anchoring and fusion of microvesicles, regulating cell activities such as protein synthesis, endocytosis, degradation and secretion.
  • Different Rab proteins often have different subcellular localizations and thus can locally regulate the fine protein transport process.
  • Rabla, Rablb, Rab2 localize to the endoplasmic reticulum and Golgi and mediate the transport of substances between the two; Rab6, Rab8, Rabl2 are associated with the Golgi apparatus and mediate the transport of substances between different Golgi sites.
  • Another object of the invention is to provide the use of the polypeptide and the coding sequence.
  • an antagonist of a human RabJ polypeptide for use in the preparation of a composition for inhibiting tumor formation or tumor growth.
  • the antagonist is an antibody, an antisense nucleotide or an interfering RNA.
  • the RabJ polypeptide is selected from the group consisting of:
  • amino acid sequence of SEQ ID NO: 2 is formed by substitution, deletion or addition of one or more amino acid residues, and has the function of promoting the formation of clones of Hela cells or MCF-7 cells on soft agar in vitro.
  • a derived polypeptide is formed by substitution, deletion or addition of one or more amino acid residues, and has the function of promoting the formation of clones of Hela cells or MCF-7 cells on soft agar in vitro.
  • the composition is a pharmaceutical composition.
  • the antibody is a monoclonal antibody or a polyclonal antibody.
  • sequence of the interfering RNA is SEQ ID NO: 9.
  • the tumor is selected from the group consisting of lung cancer, breast cancer, liver cancer, or gastric cancer.
  • the composition is also useful for reducing tumorigenicity of tumor cells in vivo.
  • a human RabJ polypeptide or a coding sequence thereof for screening for a therapeutic agent which inhibits tumor formation or tumor growth by interaction with a human RabJ polypeptide or a coding sequence thereof .
  • a method of screening for a therapeutic agent that inhibits tumor cell growth comprising the steps of:
  • control group is a culture system of a tumor cell expressing a RabJ polypeptide or a culture system of a tumor cell to which a RabJ polypeptide is added
  • test group is added with a test substance a culture system of a tumor cell expressing a RabJ polypeptide, or a culture system of a tumor cell to which a test substance and a RabJ polypeptide are added;
  • the growth rate of the tumor cells in the test group was smaller than that of the control group, indicating that the test substance is a potential therapeutic agent for inhibiting the growth of tumor cells.
  • the test substance is an interfering RNA.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a human
  • the antagonist is an interfering RNA having the sequence SEQ ID NO: 9.
  • SEQ ID NO: 9 the sequence SEQ ID NO: 9.
  • Figure 1 is a Northern blot analysis of human RabJ protein human RabJ expression in normal human tissues of the present invention.
  • the knot prompts that the RabJ protein is selectively expressed in testicular tissue.
  • Fig. 2 is a diagram showing the expression and purification of the human RabJ protein of the present invention and the biochemical characteristics of the RabJ protein. The results showed that the RabJ protein has binding GTP/GDP activity and GTPase activity.
  • Figure 3 is a Western blot analysis of RabJ protein expressed by NIH3T3 cells transfected with eukaryotic recombinant expression vector of human RabJ of the present invention.
  • Figure 4 is an analysis of RNA interference of human RabJ protein expression in HeLa and B MCF-7 cells.
  • Figure 5 is a proliferative effect of human RabJ protein overexpression on NIH3T3 cells of the present invention. The results suggest that overexpression of RabJ protein significantly promoted the proliferation of NIH3T3 cells.
  • Fig. 6 is a graph showing the inhibitory effect on the proliferation of HeLa and MCF-7 cells after down-regulation of human RabJ protein expression of the present invention. The results suggest that down-regulation of RabJ protein expression can inhibit the proliferation of HeLa and MCF-7 cells.
  • Figure 7 is a graph showing the in vitro transformation of NIH3T3 cells induced by high expression of the human RabJ protein of the present invention. Among them, the results are expressed as the number of cells forming the focus or the number of clones/plating ⁇ 100%.
  • Figure 8 is a graph showing that the down-regulation of human RabJ protein expression of the present invention inhibits the clonality of HeLa and MCF-7 cells.
  • Figure 10 is a graph showing that down-regulation of human RabJ protein inhibits in vivo tumorigenicity (A) and tumor growth rate (B) of HeLa cells. detailed description
  • RabJ The coding sequence of RabJ is isolated from a human dendritic cell cDNA library, and its sequence is shown in SEQ ID NO: 1, which comprises a polynucleotide sequence of 1787 bases in length and an open reading frame at 25- 846 digits, the full length of the code is 273 Amino acid human RabJ protein (SEQ ID NO: 2).
  • SEQ ID NO: 1 comprises a polynucleotide sequence of 1787 bases in length and an open reading frame at 25- 846 digits, the full length of the code is 273 Amino acid human RabJ protein (SEQ ID NO: 2).
  • SEQ ID NO: 2 Amino acid human RabJ protein
  • NIH3T3 Normal fibroblasts NIH3T3 are transformed after high expression of RabJ, which is expressed as colonies on semi-solid agar plates, and RabJ-transformed NIH3T3 cells can be used in nude mice.
  • RabJ can promote cell proliferation. Blocking the expression of RabJ using RNA interference technology can inhibit the proliferation of tumor cells, resulting in decreased tumorigenicity and decreased tumor growth rate in vivo. Therefore, RabJ can be used as an effective target for tumor therapy.
  • RabJ protein refers to a protein having the testis function-related protein RabJ amino acid sequence (SEQ ID NO: 2 or 4) or Peptide. They include the testicular function-related protein RabJ with or without the initial methionine.
  • hRabJ refers to a human RabJ molecule
  • mRabJ refers to a mouse-derived homolog of hRabJ.
  • RabJ may be involved in the thermal protection of testes and other protection against adverse factors; it may be used for the diagnosis and treatment of reproductive dysfunction and testicular diseases such as tumors, trauma, dysplasia, etc.; The diagnosis and treatment of abnormal diseases is also extremely meaningful.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment;).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • isolated RabJ protein or polypeptide means that the RabJ polypeptide is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. Those skilled in the art will be able to purify the RabJ protein using standard protein purification techniques. A substantially pure polypeptide produces a single major band on a non-reducing polyacrylamide gel. The purity of the RabJ polypeptide can be analyzed by amino acid sequence.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, 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 produced by recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells;
  • the polypeptide of the invention may be glycosylated, or may be non-glycosylated, depending on the host used in the recombinant production protocol.
  • Polypeptides of the invention may also or may not include an initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of RabJ polypeptides.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of a native RabJ polypeptide of the invention.
  • the polypeptide fragment, derivative or analog of the present invention may be a polypeptide in which ⁇ has one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues;), and such substituted amino acid residues may Is not also encoded by the genetic code, or (H) a polypeptide having a substituent group in one or more amino acid residues, or o a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene-2) An alcohol; a polypeptide formed by fusion of the polypeptide, or (IV) an additional amino acid sequence fused to the polypeptide sequence (such as a leader or secretion sequence or a sequence or proprotein sequence used to purify the polypeptide, or with an antigen IgG) Fragment Formation of the fusion protein ;).
  • ⁇ has one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues;), and such substituted amino acid residues may Is not also
  • RabJ polypeptide means a polypeptide having the sequence of SEQ ID NO. 2 or 4 having RabJ polypeptide activity.
  • the term also encompasses variant forms of the sequence of SEQ ID NO. 2 or 4 that have the same function as the RabJ polypeptide. These variants include (but are not limited to;): several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10;) amino acid deletions Insertion and/or substitution, and 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.
  • the function of the protein is usually not altered.
  • the addition of one or more amino acids at the C-terminus and/or N-terminus will generally not alter the function of the protein.
  • the term also encompasses active fragments and active derivatives of RabJ polypeptides.
  • Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridize to human RabJ DNA under high or low stringency conditions And a polypeptide or protein obtained using an antiserum against the RabJ polypeptide.
  • the invention also provides other polypeptides, such as fusion proteins comprising a RabJ polypeptide or a fragment thereof.
  • the present invention also encompasses soluble fragments of the RabJ polypeptide.
  • the fragment has at least about 10 contiguous amino acids of the RabJ polypeptide sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100.
  • a contiguous amino acid typically at least about 10 contiguous amino acids of the RabJ polypeptide sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100.
  • the invention also provides analogs of RabJ polypeptides or polypeptides.
  • the difference between these analogs and the native RabJ polypeptide may be a difference in amino acid sequence, a difference in the modification form which does not affect the sequence, or a combination thereof.
  • RabJ polypeptide conservative variant polypeptide means up to 10, preferably up to 8, more preferably up to 5, optimally compared to the amino acid sequence of SEQ ID NO: 2 or 4. Up to 3 amino acids are replaced by amino acids of similar or similar nature to form a polypeptide.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be either a coding strand or a non-coding strand.
  • the coding region sequence encoding the mature polypeptide may be identical to the coding region sequence shown in SEQ ID NO: l or a degenerate variant.
  • a "degenerate variant" in the present invention refers to a nucleic acid sequence which encodes a protein having SEQ ID NO: 2 but differs from the coding region sequence shown in SEQ ID NO: 1.
  • Polynucleotides encoding mature polypeptides of RabJ include: coding sequences encoding only mature polypeptides; coding sequences for mature polypeptides and various additional coding sequences; coding sequences for mature polypeptides (and optionally additional coding sequences;) and non-coding sequences .
  • polynucleotide encoding a polypeptide may be a polynucleotide comprising the polypeptide, or a polynucleotide further comprising an additional coding and/or non-coding sequence.
  • the present invention also relates to a variant of the above polynucleotide which encodes the same amino acid sequence as the present invention. Fragments, analogs and derivatives of peptides or polypeptides. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide which may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby. .
  • the invention also relates to hybridization to the sequences described above and having at least 50% between the two sequences, preferably at least
  • hybridizable polynucleotide has the same biological function and activity as the mature polypeptide of SEQ ID NO: 2.
  • the full-length human RabJ nucleotide sequence of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
  • the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then connecting them.
  • the invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered using the vectors or RabJ protein coding sequences of the invention, and methods of producing the polypeptides of the invention by recombinant techniques.
  • polynucleotide sequences of the present invention can be utilized to express or produce recombinant RabJ polypeptides by conventional recombinant DNA techniques. Generally there are the following steps:
  • the invention also relates to antagonists of RabJ polypeptides.
  • an antagonist of a RabJ polypeptide refers to a substance that is capable of combating, or reducing, the activity or expression of a RabJ polypeptide.
  • Common antagonists are antibodies, antisense nucleotides, and interfering RNA (RNAi).
  • RNAi interfering RNA
  • An antagonist or the like of the protein of the present invention when administered therapeutically (administered;), can be used for inhibiting tumor formation or for inhibiting growth of tumor cells.
  • these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8, although the pH may be The nature of the formulation and the condition to be treated vary.
  • the formulated pharmaceutical compositions can be administered by conventional routes including, but not limited to, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.
  • an antagonist of RabJ polypeptide can be directly used for the treatment of diseases, for example, for the treatment of diseases such as tumors (such as lung cancer, breast cancer).
  • diseases such as tumors (such as lung cancer, breast cancer).
  • other therapeutic agents can also be used at the same time.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a safe and effective amount of a RabJ polypeptide antagonist of the invention and a pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to;: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition 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.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably prepared under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, from about 1 microgram per kilogram of body weight to about 5 milligrams per kilogram of body weight per day.
  • a safe and effective amount of a RabJ protein antagonist is administered to the mammal, wherein the safe and effective amount is usually at least about 1 microgram per kilogram of body weight, and in most cases no more than about 8 milligrams per kilogram of body weight, Preferably, the dosage is from about 10 micrograms per kilogram of body weight to about 1 milligram per kilogram of body weight.
  • specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the corresponding antagonist can be used to inhibit tumor formation and reduce tumorigenicity for cancer treatment;
  • RabJ can be used as an effective tumor therapeutic target for screening therapeutic agents for treating tumors by interacting with RabJ.
  • the invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are only intended to illustrate the invention and not to limit the scope of the invention.
  • the experimental methods in the following examples which do not specify the specific conditions are usually carried out according to the conditions described in conventional conditions such as Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer. The suggested conditions.
  • Example 1 Cloning of human RabJ cDNA
  • RNA from human dendritic cells was extracted with Tnzol reagent (Invitrogen). Then, poly(A) mRNA is isolated from total RNA. After the reverse transcription of poly(A) mRNA to form cDNA, SuperScnptll cloning reagent was used. The cassette (Invitrogen) was inserted into the cloning site of the vector, and the DH5oc bacteria were transformed to form a cDNA plasmid library. The sequence of the 5' end of the randomly selected clone was determined by the dideoxy method. When the determined cDNA sequence was compared with an existing public DNA sequence database, it was found that the DNA sequence of one cDNA clone was a new full-length cDNA.
  • the DNA sequence contained in the new clone was bidirectionally determined by synthesizing a series of primers. Computer analysis indicated that the full-length cDNA contained in the clone was a new cDNA sequence (shown as SEQ ID NO: 1) encoding a new protein (as shown in SEQ ID NO: 2; This protein was named Rab family protein RabJ with human J domain, and its coding gene was named Rab family protein RabJ gene with human J domain.
  • SEQ ID NO: 1 is 1787 bp in length and comprises a 24 bp 5' non-coding region and a 941 bp 3' non-coding region encoding a polypeptide of 273 amino acids.
  • the unglycosylated mature molecule has a molecular weight of about 30 kD and belongs to the Rab family of molecules.
  • Example 2 Cloning of the coding sequence of human RabJ protein by RT-PCR
  • Tnzol reagent is extracted with human dendritic cells total RNA, total RNA from cells taken 5 ⁇ ⁇ 12 and 1 ⁇ ⁇ 01igo-dT - 18 were mixed and reverse transcription.
  • the reverse transcription system was 20 ⁇ l, and after the reaction was completed, 80 ⁇ l dd 3 ⁇ 40 was added for dilution.
  • the primers used for PCR amplification of RabJ were as follows: sense primer 5'-AATGGAGGCCAACATGCCG-3' (SEQ ID NO: 3), antisense primer 5'- TACTTTCTACTTGATGTT-3' (SEQ ID NO: 4), and ⁇ -actin As a positive control.
  • the PCR reaction volume was 50 ⁇ l, including the reverse transcription template 10 ⁇ l, 0.5 mM primer, 0.2 mM dNTP and B 1U rTaq DNA polymerase (Takara), and the amplification parameters were 95 ° C for 15 seconds, 57 ° C for 30 seconds, 72. After 30 seconds at °C, the PCR products were confirmed by 1.5% agarose gel electrophoresis after 28 cycles. The result of DNA sequence analysis indicated that the coding DNA sequence of the PCR product was identical to that of position 24-852 shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human RabJ
  • Northern blotting was performed as follows: The filter to be tested was placed in 10 ml of a pre-warmed hybridization solution at 68 ° C, pre-hybridized in a hybridization oven (Bellco) at 68 ° C for 30 minutes; the labeled cDNA probe was 95 Denature at ⁇ 100 °C for 2 to 5 minutes, add rapidly to the ice and add the hybridization solution (final concentration of cDNA probe is 2 ⁇ 10ng/ml or 1 ⁇ 2 X 10 6 cpm/ml), mix well, at 68° C hybridization for 2 hours.
  • Bellco hybridization oven
  • the filter was rinsed several times with 2xSSC, 0.05% SDS at room temperature, followed by shaking for 30 to 40 minutes, during which the lotion was changed several times. Subsequently, it was washed with 0.1 x SSC, 0.1% SDS at 50 ° C for 20 to 40 minutes. Finally, the filter was wrapped in a plastic wrap and exposed to X-ray film at -70 ° C for 24 to 48 hours.
  • the human PCR amplification product of Example 2 was used as a template, and amplification was carried out using PCR oligonucleotide primers at the 5' and 3' ends of the sequence below to obtain human RabJ DNA as an insert.
  • the 5'-end oligonucleotide primer sequence used in the PCR reaction was: 5'-GCGGATCC ATGGAGGCC AAC ATGC-3 '(SEQ ID NO: 5).
  • the 3' end primer sequence was: 5'-TCGGATCC CTACTTGATGTTTTTC -3' (SEQ ID NO: 6).
  • the obtained PCR product was purified, digested with BamH I and then recombined with the expression vector plasmid pGEX-2T (Pharmacia) according to a conventional method and transformed into competent E. coli DH5a, and the clone was picked, and the positive clone was identified by BamH I digestion.
  • the forward clones were identified by EcoR I digestion, and the products were analyzed by 0.8% agarose gel electrophoresis. After purification, it was purified and sequenced (ABI's Model 377 Sequencer, BigDye Terminator Kit, PE). It was confirmed by sequencing that the complete RabJ coding sequence has been inserted.
  • the cloning of the positive DH5a clone expressing RabJ was inoculated in 100 ml of 2xYTA medium, and cultured for 12-15 hrs at 37 °C, 300 rpm, 1 : 10 diluted in pre-warmed 2xYTA medium, and cultured for 1.5 hr, with 100 mM IPTG to O. lmM After induction at 30 ° C for 2-6 hr, 5,000 g 4 ° C centrifugation 10 mm to remove the supernatant, placed on ice with 50 ml lxPBS (0.14 M NaCl, 2.7 mM KC1, l O.
  • the RabJ protein was obtained with a molecular weight of approximately 30 kD, which was consistent with the predicted value.
  • the human RabJ recombinant protein obtained in Example 4 was used to immunize an animal to produce an antibody, and the specific method is as follows.
  • the recombinant molecules are separated by chromatography and used. Separation can also be carried out by SDS-PAGE gel electrophoresis, and the electrophoresis band is excised from the gel and emulsified with an equal volume of complete Freund's adjuvant.
  • Mice were intraperitoneally injected with 50-100 ⁇ g/0.2 ml of emulsified protein. After 14 days, mice were intraperitoneally injected with a dose of 50-100 ⁇ g/0.2 ml with the same antigen emulsified with non-complete Freund's adjuvant to boost the immunization. Enhance immunization every 14 days, at least three times.
  • the specific reactivity of the obtained antiserum was evaluated by its ability to precipitate the human RabJ gene translation product in vitro.
  • Example 6 GTP/GDP binding activity and GTPase activity analysis of human RabJ protein
  • the gel was then wrapped in a packaging film and subjected to autoradiography at -80 °C.
  • concentration of MgCl 2 was increased to 10 mM, and incubation was continued at 30 ° C.
  • Sepharose G50 column (Amersham-Phamacia) was centrifuged to remove free oc- [ 32 P]-GTP, take 1 ⁇ of eluate, spot it on polyethyleneimine cellulose filter (Sigma), perform thin layer chromatography in 1 M UC1/1 M formic acid solution, then -80 °C Autoradiography was performed.
  • the full-length plasmid DNA of Example 1 was used as a template, and amplification was carried out using PCR oligonucleotide primers at the 5' and 3' ends of the sequence below to obtain human RabJ DNA as an insert.
  • the 5' oligonucleotide primer sequence used in the PCR reaction is:
  • the 3' primer sequence is:
  • the obtained PCR product was purified, digested with BamH I-Kpn I and recombined with the eukaryotic expression vector plasmid pcDNA3.1/myc-His (-) B (Invitrogen;) by conventional methods and transformed into competent Escherichia coli DH5a. Positive clones were picked and identified, purified and sequenced (ABI's Model 377 Sequencer, B lg Dye Terminator Kit, PE). It was confirmed by sequencing that the complete RabJ coding sequence has been inserted.
  • the RabJ eukaryotic expression plasmid DNA was transfected into NIH3T3 (mouse normal fibroblast, ATCC: CRL-1658) with liposome LipofectAMINE reagent (Invitrogen) to pcDNA3.1/myc-His(-)B plasmid vector. As an irrelevant control.
  • NIH3T3 mouse normal fibroblast, ATCC: CRL-1658
  • liposome LipofectAMINE reagent Invitrogen
  • the main steps are as follows: The plasmid DNA to be transfected is mixed with the liposome LipofectAMINE in a certain ratio and allowed to react at room temperature for 45 minutes; 60-80% confluent is grown in MCF-7 cells of 6-well cell culture plate, using OPTI- After washing twice with MEM serum-free medium (Invitrogen), add the plasmid DNA-liposome mixture, incubate at 37 ° C 5% CO 2 for 6-8 hours, add an equal volume of normal medium containing 20% serum, continue Fresh medium was replaced after 6 hours of culture. Transient expression was collected 48 hours after transfection and subjected to Western blot analysis to detect transfection effects.
  • Example 8 Western blot detection
  • Cell Cellular lysate (Cell) of NIH3T3 cells transiently transfected with RabJ protein in Example 7 Signaling Inc. lyses cells. The supernatant was taken by centrifugation at 13,000 rpm x l Omin at 4 ° C, and protein quantification was performed using a BCA protein detection kit (PIERCE). The protein samples were subjected to SDS-PAGE, followed by transfer to a nitrocellulose membrane (Schleicher & Schuell) at a constant voltage of 100 V at 4 ° C, stained with Ponceau and labeled with size and orientation. Block at room temperature for 2 hours (5% skim milk powder in TBST solution), dilute the primary antibody with blocking solution, and incubate for 1 hour at room temperature.
  • PIERCE BCA protein detection kit
  • TBST (0.05% Tween 20 in TBS solution) was washed for 15 minutes, 3 times, and the secondary antibody was diluted with blocking solution and incubated for 2 hours at room temperature.
  • the TBST was washed for 15 minutes, 3 times, washed with TBS (10 mM Tns-HCl, pH 8.0, 150 mM NaCl) for 15 minutes, then added with a chemiluminescent substrate (Pierce) for 1 mm, and rapidly sealed and auto-developed.
  • the primary antibody used for Western blot detection was the anti-RabJ antibody obtained in Example 5.
  • the secondary antibody was HRP-labeled anti-rabbit IgG (Cell Signaling).
  • the expression product of RabJ has a molecular weight of 30 KD (Fig. 3).
  • Example 9 RNA interference analysis of human RabJ
  • RNAi vector pSuppressorNeo sense strand 5'- U CAU CUC CAU GGG CAA CGC -3' (SEQ ID NO: 9) and the antisense strand 5'-GCG UUG CCC AUG GAG AUG A (SEQ ID NO: 10).
  • a 21-nt long randomized control oligonucleotide was also inserted into this vector as a control: sense strand 5'-UCA GUC ACG UUA AUG GUC GUU (SEQ ID NO: 1 1) and antisense strand 5'-AAC GAC CAU UAA CGU GAC UGA (SEQ ID NO: 12).
  • ATCC: CCL-2) and MCF-7 human breast cancer cells, ATCC: HTB-22 are used to interfere with the expression of RabJ protein in cells.
  • the main steps are the same as in Example 7. 48 hours after transfection, cells were harvested for RT-PCR (main steps as in Example 2) and Western blot analysis (main steps as in Example 8) for RabJ protein expression.
  • the [ 3 H]-thymidine incorporation method was employed.
  • NIH3T3 cells overexpressing RabJ in Example He, HeLa or MCF-7 cells inhibiting RabJ expression in Example 9 were plated at 5 ⁇ 10 4 /well in 24-well culture plates (Falcon), three cells per cell. hole. After 6 h of culture, the culture was continued for 24 to 48 h in serum-free medium, and 0.5 ⁇ L was added to each well for the last 4 h.
  • ⁇ (1 Ci 37 GBq) of [ 3 H]-thymidine (Amersham-Phamacia). Then, it was washed three times with PBS, and the cells were dissolved in PBS containing 1% Triton X-100, collected with a glass fiber filter, and radioactivity was measured with a liquid scintillation meter.
  • a clone formation assay was used. In order to examine the ability of cells to grow independently of growth, the NIH3T3 cells stably overexpressing RabJ in Example 7, and the HeLa or MCF-7 cells (5 x 10 4 ) inhibiting RabJ expression in Example 9 were suspended in 0.5% soft agar. Place in a 6-well plate containing 1% soft agar. After 3 weeks of culture, clones larger than 50 cells were recorded as positive by light microscopy.
  • the NIH3T3 cells stably expressing RabJ in Example 7, and the HeLa cells (1 x 10 6 ) inhibiting RabJ expression in Example 9 were subcutaneously injected into the right lower abdomen of Balb/C nude mice, and then the growth rate of the tumor was observed or The incidence of tumors (number of mice in which tumors appeared / number of nude mice injected with cells). Tumor size was measured with a vernier caliper (unit: mm) and the result was length X width X width (tumor volume; unit: mm 3 ). Observe for six weeks.
  • NIH3T3 cells expressing full-length RabJ could form tumor nodules (100%) in vivo, whereas wild-type and control vector-transfected NIH3T3 cells could not form tumors (0% Figure 9), while RabJ protein expression was down-regulated.
  • Post-HeLa cells showed a decrease in tumorigenicity in vivo (Fig. 10A), and the rate of tumor growth that had formed was also reduced (Fig. 10B). Discussion
  • Rab Ras-like protein in rat brain
  • Ras superfamily The basic biochemical feature is that it can bind GTP/GDP and hydrolyze GTP.
  • Rab3a is mainly distributed in neural tissue and secretory cell lines, and is mainly involved in the regulation of neurotransmitter secretion.
  • Rab3a knockout mice develop normally, but show short- and long-term synaptic plasticity defects in the mossy fiber pathway, and abnormal circadian activity cycles.
  • Rab5 is a widely distributed Rab protein that is primarily localized in early endocytosis and is involved in the regulation of endocytic pathways, including epidermal growth factor (EGF), platelet-derived growth factor (PDGF) receptor EGFR, and PDGFR endocytosis, thereby participating in signals. Regulation of conduction.
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • GnscelU syndrome is a disease caused by abnormal pigmentation and pigmentation and T lymphocyte killing dysfunction. It is found that the mutation of Rab27a is the occurrence of the disease. Genetic cause.
  • Abnormalities in the Rab7 protein can cause abnormalities in the metabolism of lipoproteins, causing hyperlipidemia and vascular diseases; some intracellular bacteria, such as Mycobacterium tuberculosis, can inhibit the function of Rab7 to infect the host and cause disease.
  • Abnormalities in Rab regulatory proteins are also the cause of certain diseases, such as choroidal disorders, X-linked neurodegenerative disorders; abnormalities in post-translational modification of Rab proteins can cause Hermansky-Pudlak syndrome.
  • Rab proteins may be involved in tumorigenesis.
  • abnormal expression of Rab protein is found in certain tumor cells or tumor types.
  • Rab25 is highly expressed in prostate cancer, and its expression level is closely related to the differentiation and clinical stage of prostate cancer.
  • Rab38 expression is also significantly increased in melanoma cell lines;
  • Rab3 is also highly expressed in pituitary adenocarcinoma.
  • Rab2 expression was significantly increased in peripheral blood mononuclear cells of hematopoietic tumors and solid tumor patients; Rab2 expression was also abnormal in mouse adrenal and mouse lung cancers.
  • Second, a deletion mutation in the Rab gene was found in the tumor.
  • a deletion of the chromosome 22ql l.2 region was found in malignant rhabdomyosarcoma, which contains the coding region of Rab36.
  • abnormalities in Rab regulatory proteins have also been found in tumors.
  • the expression of the Rab protein regulatory protein RabGDI is significantly increased.
  • the expression of Rab GTPase-activating protein, PRC17 which regulates GTP hydrolysis of Rab5 protein, can affect tumor growth characteristics.
  • the RabJ protein of the present invention is highly expressed in various tumor cells, and the normal fibroblast NIH3T3 is transformed after high expression of RabJ, which is expressed as a colony formed on a semi-solid agar plate, and RabJ-transformed NIH3T3 cells can be used in nude mice. Formation of fibroblastic sarcoma. RabJ can promote cell proliferation. Blocking the expression of RabJ using RNA interference technology can inhibit the proliferation of tumor cells, resulting in decreased tumorigenicity and decreased tumor growth rate in vivo. The above experimental results of the present invention suggest that RabJ is involved in cell proliferation and cell cycle progression, and thus RabJ can be used as an effective tumor therapeutic target.

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Description

新的人癌基因样小 G蛋白 RabJ的功能及用途
技术领域
本发明属于生物技术和医学领域， 具体地说， 本发明涉及人癌基因样小 G 蛋白 RabJ及其编码序列。 本发明的多肽是一种新的具有 GTPase活性的分子， 它与细胞增 殖和细胞分化的调控相关， 具有癌基因样的作用。 背景技术
小 G蛋白（small G proteins)是指分子量约 20— 30 kDa的单亚基 G蛋白。 目前随 着人类基因组计划的完成， 该类蛋白质的成员已经发现有一百多种， 其共有的特征 是可以结合三磷酸鸟苷 (GTP)和二磷酸鸟苷 (GDP) ,并且能够水解 GTP γ位磷酸成为 GDP (即 GTPase 活性；)。 根据其一级结构的同源性， 可以将其分成六个家族， 包括 Ras、 Rab、 Rho、 Arf、 Sari和 Ran等六个家族。
Ras和 Rho家族的分子是极为重要的小 G蛋白， 因为他们参与信号传导机制的 调控， 从而使细胞可以对外界刺激产生正确的反应， 在细胞存活、 凋亡等基本生命 活动中具有重要作用。 Ras家族的成员包括 Ras、 Ral和 Rap等， 至今了解最多的是 Ras亚家族， 包括 Ha-Ras、 Ki-Ras和 N-Ras等。 基因敲除小鼠模型和缺陷细胞模型 的研究均证明， Ras家族分子在胚胎形成、 个体发育等方面具有重要作用。 Rho家族 的成员主要包括 Rho、 Rac、 Cdc42等，通过调控 Rho相关激酶 （Rho-associated kinase, ROK), 肌球蛋白结合亚基 (Myosin binding subunit, MBS)磷脂酶等靶蛋白的功能和活 性， 调节细胞骨架、 细胞增殖、 应激反应等细胞活动。
Ras和肿瘤发生以及其在细胞增殖调控中的作用引起人们的关注。在几乎所有类 型的肿瘤细胞或者肿瘤组织中， 都有 H-Ras、 K-Ras或者 N-Ras基因的突变， 而且在 肿瘤发生突变的基因中， Ras突变的几率最高。 与 Ras蛋白活性调控相关的分子也参 与肿瘤的发生。 Rho家族的分子也通过调控细胞肌动蛋白系统， 调节细胞内肌动蛋白 应激纤维的形成、 细胞膜突起、 凹陷的形成。 在成纤维细胞系 NIH3T3中， 突变的调 控 Rho家族活性的蛋白分子可以使细胞发生转化 (transformation)。这些调控 Rho家族 GTP/GDP结合活性和 GTPase活性的癌基因样分子包括 Dbl、 Vav、 Lbc、 Tiaml、 Tim、 Ost、 Ect2、 Lsc和 FGD1等， 可以引起细胞肌动蛋白系统功能的异常， 引起细胞迁 移浸润能力的改变。
Rab (Ras-like protein in rat brain)是一类属于 Ras超家族的小分子量 GTPase, 基 本生化特征是可以结合 GTP/GDP并水解 GTP。 到目前为止， 该家族的成员已经发展 到 100 多个， 在包括线虫、 酵母、 果蝇、 斑马鱼、 小鼠、 大鼠、 人等多种物种中均 有表达， 主要参与蛋白在细胞内的转运过程， 调控膜性细胞器的融合和微小囊泡的 出芽、 锚着和融合等过程， 调节蛋白合成、 内吞、 降解和分泌等细胞活动。 不同的 Rab蛋白往往有不同的亚细胞定位， 因此可以在局部调控细微的蛋白转运过程。例如 Rabla、 Rablb、 Rab2定位于内质网和高尔基体并且介导二者之间的物质转运； Rab6、 Rab8、 Rabl2与高尔基体相关， 介导不同高尔基部位之间的物质转运。
Rab蛋白的功能异常会引起一些病理现象和疾病。 由于 Rab家族蛋白在某些生 理过程中发挥重要调节作用， 因此本领域迫切需要开发新的人 Rab蛋白。 发明内容
本发明的目的是提供一种新的人癌基因样小 G蛋白 RabJ蛋白以及其片段、 类似 物和衍生物。
本发明的另一个目的是提供编码该多肽和编码序列的用途。 在本发明的第一方面， 提供了一种人 RabJ多肽的拮抗剂的用途， 所述的拮抗剂 用于制备抑制肿瘤形成或肿瘤生长的组合物。
在另一优选例中， 所述的拮抗剂是抗体、 反义核苷酸或干扰性 RNA。
在另一优选例中， 所述的 RabJ多肽选自下组：
(a) SEQ ID NO: 2氨基酸序列的多肽；
(b)将 SEQ ID NO: 2氨基酸序列经过一个或多个氨基酸残基的取代、 缺失或添加 而形成的，且具有体外促进 Hela细胞或 MCF— 7细胞在软琼脂上形成克隆的功能的由 (a)衍生的多肽。
在另一优选例中， 所述的组合物为药物组合物。
在另一优选例中， 所述的抗体是单克隆抗体或多克隆抗体。
在另一优选例中， 所述的干扰性 RNA的序列为 SEQ ID NO: 9。
在另一优选例中， 所述的肿瘤选自下组： 肺癌、 乳腺癌、 肝癌、 或胃癌。
在另一优选例中， 所述的组合物还用于降低肿瘤细胞在体内的致瘤性。
在本发明的第二方面， 提供了一种人 RabJ多肽或其编码序列的用途， 它们被用 于筛选通过与人 RabJ 多肽或其编码序列的相互作用， 从而抑制肿瘤形成或肿瘤生长 的治疗剂。
在本发明的第三方面， 提供了一种筛选抑制肿瘤细胞生长的治疗剂的方法， 包括 步骤：
(a) 提供一测试组和一对照组，其中所述的对照组为表达 RabJ多肽的肿瘤细胞的 培养体系或添加了 RabJ多肽的肿瘤细胞的培养体系，所述的测试组是添加了测试物质 的表达 RabJ多肽的肿瘤细胞的培养体系、 或添加了测试物质和 RabJ多肽的肿瘤细胞 的培养体系；
(b)观察测试组中所述肿瘤细胞的生长，并与对照组的所述肿瘤细胞的生长进行比 较；
其中， 测试组中所述肿瘤细胞的生长速度小于对照组， 就表示测试物质是抑制肿 瘤细胞生长的潜在治疗剂。 在另一优选例中， 所述的测试物质是干扰 RNA。
在本发明的第四方面， 提供了一种药物组合物， 它含有药学上可接受的载体和人
RabJ多肽的拮抗剂。
在另一优选例中， 所述的拮抗剂是序列为 SEQ ID NO: 9的干扰性 RNA。 本发明的其它方面由于本文的技术的公开，对本领域的技术人员而言是显而易见 的。 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是本发明的人 RabJ蛋白人 RabJ在正常人组织表达的 Northern印迹分析。 结提示人 RabJ蛋白是选择性地高表达于睾丸组织。
图 2是本发明的人 RabJ蛋白的表达纯化以及 RabJ蛋白的生化特性分析。结果显 示 RabJ蛋白具有结合 GTP/GDP活性和 GTP酶活性。
其中， （A)RabJ 及突变体 GST 融合蛋白的表达纯化。 （B)RabJ 的特异结合 GTP/GDP活性分析。 (C)Rab J GTPase活性分析。
图 3是本发明的人 RabJ的真核重组表达载体转染 NIH3T3细胞表达 RabJ蛋白 的 Western印迹分析。
图 4是 HeLa禾 B MCF-7细胞中 RNA 干扰人 RabJ蛋白表达的分析。
图 5是本发明的人 RabJ蛋白过表达对 NIH3T3细胞的促增殖作用。 结果提示人 RabJ蛋白过表达明显促进 NIH3T3细胞的增殖。
图 6是本发明的人 RabJ蛋白 表达下调后对 HeLa和 MCF-7细胞增殖的抑制作 用。 结果提示人 RabJ蛋白 表达下调可抑制促进 HeLa和 MCF-7细胞增殖。
图 7是本发明的人 RabJ蛋白高表达后诱导 NIH3T3细胞体外转化。 其中， 结果 表示为形成焦点或者克隆的个数 /铺板的细胞数 χ100%。
图 8是本发明的人 RabJ蛋白表达下调抑制 HeLa和 MCF-7细胞的克隆形成能力。 图 9是本发明的人 RabJ蛋白高表达后导致 NIH3T3细胞在裸鼠体内形成致瘤性 (n=10)。
图 10是人 RabJ蛋白表达下调抑制 HeLa细胞的体内致瘤性 (A)和肿瘤的生长速 率 (B)。 具体实施方式
本发明经过广泛而深入的研究， 克隆了一个新的 Rab家族成员 -RabJ。 RabJ的编 码序列是从人树突状细胞 cDNA文库中分离出的， 其序列如 SEQ ID NO: 1所示， 包 含的多核苷酸序列全长为 1787个碱基， 其开放读框位于 25-846位， 编码全长为 273 个氨基酸的人 RabJ蛋白 (SEQ ID NO: 2)。 实验表明， RabJ蛋白在多种肿瘤细胞内高 表达， 正常的成纤维细胞 NIH3T3在高表达 RabJ后发生转化， 表现为在半固体琼脂 平板上形成集落， 并且在裸鼠体内 RabJ转化的 NIH3T3细胞可以形成成纤维细胞肉 瘤。 RabJ可以促进细胞增殖。 使用 RNA干扰技术阻断 RabJ的表达后可以抑制肿瘤 细胞的增殖， 导致体内致瘤性降低和肿瘤生长速率下降。 因此， RabJ可作为一个有 效的肿瘤治疗靶标。
RabJ多肽
在本发明中， 术语 "RabJ蛋白" 、 "RabJ多肽"或 "睾丸功能相关蛋白 RabJ" 可互换使用， 都指具有睾丸功能相关蛋白 RabJ氨基酸序列 (SEQ ID NO:2或 4)的蛋白 或多肽。 它们包括含有或不含起始甲硫氨酸的睾丸功能相关蛋白 RabJ。 在本发明中 hRabJ指人源的 RabJ分子， 而 mRabJ指小鼠源的 hRabJ的同源物。 研究提示， RabJ 可能参与睾丸的热防护和其他对机体不利因素的防护；可能可以用于生殖功能异常和 睾丸疾病， 如肿瘤、 创伤、 发育异常等的诊断和治疗； 而且可能对于某些蛋白转运异 常疾病的诊断和治疗也极有意义。
如本文所用， "分离的"是指物质从其原始环境中分离出来 (如果是天然的物质， 原始环境即是天然环境；)。 如活体细胞内的天然状态下的多聚核苷酸和多肽是没有分 离纯化的， 但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质中分开， 则 为分离纯化的。
如本文所用， "分离的 RabJ蛋白或多肽"是指 RabJ多肽基本上不含天然与其相 关的其它蛋白、 脂类、 糖类或其它物质。 本领域的技术人员能用标准的蛋白质纯化技 术纯化 RabJ蛋白。 基本上纯的多肽在非还原聚丙烯酰胺凝胶上能产生单一的主带。 RabJ多肽的纯度能用氨基酸序列分析。
本发明的多肽可以是重组多肽、 天然多肽、 合成多肽， 优选重组多肽。 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。
本发明还包括 RabJ多肽的片段、 衍生物和类似物。 如本文所用， 术语 "片段"、 "衍生物"和 "类似物"是指基本上保持本发明的天然 RabJ多肽相同的生物学功能 或活性的多肽。 本发明的多肽片段、 衍生物或类似物可以是 ω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 (H)在一个或多个氨基酸残基中具有取代 基团的多肽， 或 o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， 或与抗原 IgG片段 的形成的融合蛋白；)。 根据本文的教导， 这些片段、 衍生物和类似物属于本领域熟练 技术人员公知的范围。
在本发明中， 术语 "RabJ多肽"指具有 RabJ多肽活性的 SEQ ID NO. 2或 4序 列的多肽。 该术语还包括具有与 RabJ多肽相同功能的、 SEQ ID NO. 2或 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、 插入和 /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 RabJ多肽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、 保守性变异体、 等位变异体、 天然突变体、 诱导突变体、在高或低的严紧度条件下能与人 RabJ DNA杂交的 DNA所编码的蛋白、 以及利用抗 RabJ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 RabJ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 RabJ多 肽的可溶性片段。 通常， 该片段具有 RabJ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 RabJ多肽或多肽的类似物。这些类似物与天然 RabJ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。
在本发明中， "RabJ多肽保守性变异多肽"指与 SEQ ID NO: 2或 4的氨基酸序 列相比， 有至多 10个， 较佳地至多 8个， 更佳地至多 5个， 最佳地至多 3个氨基酸 被性质相似或相近的氨基酸所替换而形成多肽。
RabJ的编码序列
本发明的多核苷酸可以是 DNA形式或 RNA形式。 DNA形式包括 cDNA、 基因 组 DNA或人工合成的 DNA。 DNA可以是单链的或是双链的。 DNA可以是编码链或 非编码链。 对于人 RabJ而言， 编码成熟多肽的编码区序列可以与 SEQ ID NO: l所示 的编码区序列相同或者是简并的变异体。 如本文所用， "简并的变异体"在本发明中 是指编码具有 SEQ ID NO:2的蛋白质， 但与 SEQ ID NO: l所示的编码区序列有差别 的核酸序列。
编码 RabJ的成熟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 的编码序列和各种附加编码序列； 成熟多肽的编码序列 (和任选的附加编码序列;)以及 非编码序列。
术语 "编码多肽的多核苷酸"可以是包括编码此多肽的多核苷酸， 也可以是还包 括附加编码和 /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，其编码与本发明有相同的氨基酸序列的多 肽或多肽的片段、 类似物和衍生物。 此多核苷酸的变异体可以是天然发生的等位变异 体或非天然发生的变异体。 这些核苷酸变异体包括取代变异体、 缺失变异体和插入变 异体。 如本领域所知的， 等位变异体是一个多核苷酸的替换形式， 它可能是一个或多 个核苷酸的取代、 缺失或插入， 但不会从实质上改变其编码的多肽的功能。
本发明还涉及与上述的序列杂交且两个序列之间具有至少 50%， 较佳地至少
70%, 更佳地至少 80%相同性的多核苷酸。 本发明特别涉及在严格条件下与本发明所 述多核苷酸可杂交的多核苷酸。 在本发明中， "严格条件"是指： 在较低离子强度 和较高温度下的杂交和洗脱， 如 0.2 X SSC, 0.1%SDS, 60°C ; 或 (2)杂交时加有变性剂, 如 50%(v/v)甲酰胺， 0.1%小牛血清 /0.1% Ficoll， 42°C等； 或 (3)仅在两条序列之间的 相同性至少在 90%以上,更好是 95%以上时才发生杂交。并且， 可杂交的多核苷酸编码 的多肽与 SEQ ID NO:2所示的成熟多肽有相同的生物学功能和活性。
本发明的人 RabJ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。 当序列较长时， 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 其克隆入载体， 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。
本发明也涉及包含本发明的多核苷酸的载体， 以及用本发明的载体或 RabJ蛋白 编码序列经基因工程产生的宿主细胞， 以及经重组技术产生本发明所述多肽的方法。
通过常规的重组 DNA技术， 可利用本发明的多聚核苷酸序列可用来表达或生产 重组的 RabJ多肽。 一般来说有以下步骤：
(1) .用本发明的编码 RabJ多肽的多核苷酸 (或变异体；)， 或用含有该多核苷酸的重 组表达载体转化或转导合适的宿主细胞；
(2) .在合适的培养基中培养的宿主细胞；
(3) .从培养基或细胞中分离、 纯化蛋白质。
RabJ多肽的拮抗剂
本发明还涉及 RabJ多肽的拮抗剂。如本文所用， RabJ多肽的拮抗剂指能够对抗、 或降低 RabJ多肽的活性或表达的物质。 常见的拮抗剂是抗体、 反义核苷酸以及干扰 性 RNA(RNAi)。 药物组合物和施用方式
本发明蛋白的拮抗剂等， 当在治疗上进行施用 (给药；)时， 可用于抑制肿瘤形成或 用于抑制肿瘤细胞的生长。 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通常约为 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、 静脉内 、 皮下、 皮内、 或局 部给药。
在本发明中， RabJ多肽的拮抗剂可直接用于疾病治疗， 例如， 用于肿瘤 (如肺癌、 乳腺癌;)等疾病的治疗。 在使用本发明 RabJ蛋白的拮抗剂时， 还可同时使用其他治疗 剂。
本发明还提供了一种药物组合物， 它含有安全有效量的本发明 RabJ多肽拮抗剂 以及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。 本发明的药物组 合物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。
使用药物组合物时， 是将安全有效量的 RabJ蛋白拮抗剂施用于哺乳动物， 其中 该安全有效量通常至少约 1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。 本发明的主要优点在于：
(a)基于 RabJ多肽及其编码多核苷酸与肿瘤形成和致瘤性的相关性， 可用相应的 拮抗剂来抑制肿瘤形成、 降低致瘤性从而用于癌症治疗；
(b)基于 RabJ与细胞增殖的相关性， RabJ可作为一个有效的肿瘤治疗靶标，用于 筛选通过与 RabJ相互作用而治疗肿瘤的治疗剂。 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 RabJ cDNA的克隆
用 Tnzol试剂 （Invitrogen)提取人树突状细胞总 RNA。 然后， 从总 RNA中分离 poly(A) mRNA。 将 poly(A) mRNA经逆转录形成 cDNA后， 用 SuperScnptll克隆试剂 盒 (Invitrogen)将 cDNA 片段定向插入到载体的多克隆位点上， 转化 DH5oc细菌形成 cDNA质粒文库。 用双脱氧法测定随机挑选克隆的 5'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 cDNA克隆的 DNA序列 为新的全长 cDNA。通过合成一系列引物对新克隆所含的 DNA序列进行双向测定。计 算机分析表明，克隆所含的全长 cDNA是一个新的 cDNA序列 (如 SEQ ID NO: 1 所示)， 编码一个新的蛋白质 (如 SEQ ID NO: 2所示;)。此蛋白质被命名为人具有 J功能域的 Rab 家族蛋白 RabJ， 其编码基因命名为人具有 J功能域的 Rab家族蛋白 RabJ基因。
SEQ ID NO: 1全长为 1787bp， 包括 24bp的 5'端非编码区和 941bp 的 3'端非编 码区， 编码含 273个氨基酸的多肽。 理论上计算未糖基化的成熟分子的分子量约为 30kD , 属于 Rab家族分子。 实施例 2: 用 RT-PCR方法克隆人 RabJ蛋白的编码序列
用 Tnzol 试剂提取处于人树突状细胞总 RNA， 取 5μ§ 细胞总 RNA 与 1 μ§ 01igo-dT12-18混合， 进行反转录。 反转录体系为 20μ1， 反应结束后加 80μ1 dd¾0进行 稀 释 。 PCR 扩 增 RabJ 所 用 的 引 物 如 下 ： 有 义 引 物 5'-AATGGAGGCCAACATGCCG-3'(SEQ ID NO ： 3) , 反 义 引 物 5'- TACTTTCTACTTGATGTT-3'(SEQ ID NO : 4)， 同时以 β-actin作为阳性对照。 PCR反 应体积为 50μ1， 其中含反转录模板 10μ1、 0.5mM引物、 0.2mM dNTP禾 B 1U rTaq DNA 聚合酶 (Takara公司）， 扩增参数为 95 °C 15秒、 57 °C 30秒、 72°C 30秒， 28个循环后 PCR产物行 1.5%琼脂糖凝胶电泳初步确认。 DNA序列分析结果表明该 PCR产物的编 码 DNA序列与 SEQ ID ΝΟ: 1所示的 24-852位完全相同。 实施例 3: 人 RabJ的 Northern 印迹分析
按如下常规方法进行 Northern印迹：待检滤膜置于 10ml 经 68°C 预热的杂交液， 在杂交炉 (Bellco)中于 68°C预杂交 30分钟； 将标记好的 cDNA探针于 95〜100°C变 性 2〜5分钟，置冰上迅速冷却后加入杂交液 (cDNA探针终浓度为 2〜10ng/ml或 1〜2 X 106cpm/ml) ,充分混匀，于 68°C 杂交 2小时。杂交结束后，滤膜用 2xSSC、 0.05%SDS 室温淋洗数次，继振荡冲洗 30〜40分钟，其间更换洗液数次。随后用 0.1xSSC、0.1%SDS 于 50°C振荡冲洗 20〜40分钟。 最后滤膜用塑料保鲜膜包裹， 于 -70°C曝光 X线胶片 24〜48小时。
Northern显示人 RabJ优势高表达于在正常人睾丸组织， 在卵巢和大脑组织中可 见微弱表达， 在所检测的其他组织中未见表达 (图 1)。 实施例 4: 人 RabJ蛋白的原核重组表达
在该实施例中， 以实施例 2中的人 PCR扩增产物为模板， 用序列如下的 5'和 3' 端的 PCR寡核苷酸引物进行扩增， 获得人 RabJDNA作为插入片段。 PCR 反应 中使用 的 5'端寡核苷酸引 物序列为 ： 5'- GCGGATCC ATGGAGGCC AAC ATGC-3 '(SEQ ID NO : 5)。 3'端引物序列为： 5'-TCGGATCC CTACTTGATGTTTTTC -3'(SEQ ID NO : 6)。
将获得的 PCR产物纯化后经 BamH I酶切再与表达载体质粒 pGEX-2T(Pharmacia 公司)按常规方法重组并转化至感受态大肠杆菌 DH5a，挑取克隆， 阳性克隆用 BamH I 酶切鉴定， 正向克隆用 EcoR I酶切鉴定， 产物行 0.8%琼脂糖凝胶电泳分析。 鉴定后 纯化并测序 (ABI公司的 377型测序仪， BigDye Terminator试剂盒， PE公司）。 经测序 证实， 已插入了完整的 RabJ编码序列。
挑表达 RabJ的阳性 DH5a克隆接种于 100ml 2xYTA培养基中， 37 °C 300rpm振 荡培养 12-15hr， 1 : 10稀释于预热的 2xYTA培养基继续振荡培养 1.5hr，加 l OOmM IPTG 至 O. lmM后 30°C诱导 2-6hr，5,000g 4°C 离心 10mm去上清，置冰上用 50ml lxPBS (0.14M NaCl, 2.7 mM KC1, l O. l mM Na2HP04, 1.8mM KH2P04, pH7.3) 重悬， 超声 (B. Braun Labsonic U)破碎后再加入 20% Triton X-100至 1%轻摇 30min，然后 12,000g 4 °C离心 10min，上清用 0.8μηι滤膜过滤后，过 1ml 50%谷胱甘肽 Sepharose 4B层析柱， lxPBS 充分洗涤后， 加入 500ul 谷胱甘肽洗脱缓冲液 （10 mM 谷胱甘肽， 50 mM Tris-HCl, pH 8.0)室温静置 30分钟后收集洗脱液，重复洗脱 2-3 次，得到人 RabJ-GST 融合蛋白。 （图 2A)
经凝血酶 (thrombinXSigma公司）酶切去除 GST后得到 RabJ 蛋白， 分子量约为 30kD , 与预测值相符。
用 Edams水解法进行 N-氨基酸序列分析， 证实其 N端序列与 SEQ ID NO:2所示 的 N端序列相符。 实施例 5: 抗人 RabJ抗体的产生
将实施例 4中获得的人 RabJ重组蛋白用来免疫动物以产生抗体， 具体方法如下。 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 Freund'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人 RabJ基因 翻译产物的能力加以评估。
结果发现， 制备的抗体可特异性地与本发明蛋白发生结合。 实施例 6: 人 RabJ蛋白的 GTP/GDP结合活性及 GTPase活性分析
用 10 μ§ 人 RabJ-GST融合蛋白， 以 GST蛋白或 BSA作为对照， 在含 25 nM a-[32P]-GTP(Amersham-Phamacia公司）的 50 mM Tris-HCl , 2 mM EDTA, 1 mM DTT， 5 mM MgCl2， pH8.0 的 50 μΐ反应体系中于 30°C作用 30 min。或者在缓冲液中分别加 入 100 μΜ的 GTP、 GDP、 GTP Y S或 ATP于上述缓冲液中 30°C作用 30 min。 用 1 M EDTA终止反应后，取 10 μΐ溶于 10 μΐ非变性蛋白上样缓冲液中，用 15%的 SDS-PAGE 分离。然后将凝胶包在包装膜中，在 -80°C进行放射自显影。结合反应结束后，将 MgCl2 浓度提高到 10 mM, 继续在 30°C孵育， 在不同的时间点用 1 M EDTA终止反应后， Sepharose G50 层析柱 (Amersham-Phamacia公司）离心去除游离 oc-[32P]-GTP， 取出 1 μΐ 洗脱液， 点样于聚乙烯亚胺纤维素滤膜 (Sigma) , 在 1 M UC1/1 M 甲酸溶液中进行薄 层层析， 然后 -80°C进行放射自显影。
结果显示， RabJ可以结合 p32标记的 GTP , 该结合反应可以被超量的 GDP、 未 标记的 GTP和 GTP类似物 GTPyS阻断， 却不能被 ATP阻断， 提示 RabJ是一个可 以特异结合 GTP/GDP 的小 G 蛋白。 去除氨基端 209 个氨基酸的蛋白 RabJ(209-273)-GST不能结合 GTP/GDP , 提示这种 GTP/GDP结合活性是由 Rab功能 域介导的。此外 RabJ表现出明显的 GTP水解活性，而且该活性也由 Rab功能域所介 导。 因此， RabJ是一个具有 GTP酶活性的小 G蛋白。 （图 2B和 2C) 实施例 7: 人 RabJ真核表达载体的构建和真核细胞基因转染
在该实施例中， 以实施例 1中的全长质粒 DNA为模板， 用序列如下的 5'和 3'端 的 PCR寡核苷酸引物进行扩增， 获得人 RabJ DNA作为插入片段。
PCR反应中使用的 5'端寡核苷酸引物序列为：
5'- CGGGATCC AATGGAGGCCAACATGCCGA -3'(SEQ ID NO: 7)
3'端引物序列为：
5'- GCGGTACC GACTACTTGATGTTTTTCAG -3'(SEQ ID NO: 8)
将获得的 PCR产物纯化后经 BamH I- Kpn I 酶切再与真核表达载体质粒 pcDNA3.1/myc-His (-) B(Invitrogen 公司;)按常规方法重组并转化至感受态大肠杆菌 DH5a , 挑取阳性克隆酶切鉴定后纯化并测序 (ABI 公司的 377 型测序仪， BlgDye Terminator 试剂盒， PE公司）。 经测序证实， 已插入了完整的 RabJ编码序列。
将该 RabJ真核表达质粒 DNA以脂质体 LipofectAMINE试剂 （Invitrogen公司） 转染 NIH3T3 (小鼠正常成纤维细胞， ATCC : CRL-1658) , 以 pcDNA3. l/myc-His(-)B 质粒载体作为无关对照。 按照说明书操作。 主要步骤为： 待转染的质粒 DNA与脂质 体 LipofectAMINE按一定比例混合， 室温作用 45分钟； 处 60-80%汇合 (confluent)生 长于 6孔细胞培养板的 MCF-7细胞， 用 OPTI-MEM无血清培养基 (Invitrogen公司） 洗两遍后， 加入质粒 DNA-脂质体混合物， 置 37°C 5% C02培养 6-8小时， 加等体积 含 20%血清的正常培养基， 继续培养 6小时后更换新鲜培养基。 瞬时表达于转染后 48小时收集细胞进行 Western印迹分析， 检测转染效果。 实施例 8: Western印迹检测
将实施例 7 中经瞬时转染过表达 RabJ蛋白的 NIH3T3 细胞用细胞裂解液 (Cell Signaling公司)裂解细胞。 4°C离心 13,000 rpm x l Omin取上清， 利用 BCA蛋白检测 试剂盒 (PIERCE公司)进行蛋白定量。将蛋白样品行 SDS-PAGE, 随后以 100V恒电压 于 4°C 转至硝酸纤维素膜上 (Schleicher & Schuell公司）， 丽春红染色并标记大小和方 向。 室温阻断 2小时 (5%脱脂奶粉的 TBST溶液)， 以阻断液稀释一抗， 室温孵育 1小 时。 TBST(0.05% Tween 20的 TBS溶液)洗 15分钟、 3次， 以阻断液稀释二抗， 室温 孵育 2小时。 TBST洗 15分钟、 3次， TBS(10mM Tns-HCl , pH8.0 , 150mM NaCl) 洗 15分钟， 然后加入化学发光底物 (Pierce公司)作用 l mm， 并迅速封膜和自显影。 用于 Western印迹检测的一抗为实施例 5中获得的抗 RabJ抗体。 二抗为 HRP标记抗 兔 IgG (Cell Signaling公司）。
结果显示， 转染后 48小时在转染了人 RabJ真核表达载体的细胞中可检测到人
RabJ的表达产物， 分子量为 30KD (图 3)。 实施例 9: 人 RabJ的 RNA干扰 (RNA interference)分析
体外合成 19-nt 长的 RabJ寡核苷酸片段 (上海生工公司；)， 插入真核 RNAi载体 pSuppressorNeo(IMGENEX公司）： 有义链 5'- U CAU CUC CAU GGG CAA CGC -3' (SEQ ID NO : 9)和反义链 5'-GCG UUG CCC AUG GAG AUG A(SEQ ID NO : 10)。 21 -nt长的随机对照寡核苷酸也插入该载体作为对照： 有义链 5'-UCA GUC ACG UUA AUG GUC GUU (SEQ ID NO : 1 1)和反义链 5'-AAC GAC CAU UAA CGU GAC UGA(SEQ ID NO : 12)。
利用以脂质体 Lipofect AMINE试剂 （Invitrogen公司)转染 HeLa (人宫颈癌细胞，
ATCC : CCL-2)和 MCF-7 (人乳腺癌细胞， ATCC : HTB-22) , 用于干扰细胞中 RabJ 蛋白的表达。 主要步骤同实施例 7。 转染后 48小时， 收集细胞用作 RT-PCR (主要步 骤同实施例 2)和 Western印迹分析 (主要步骤同实施例 8)RabJ蛋白表达情况。
通过 RT-PCR和 Western印迹检测，结果显示 RabJ RNA干扰成功阻抑了人 RabJ 的 mRNA 及蛋白在 HeLa和 MCF-7细胞中的表达， 而 21nt的随机对照对 RabJ的 mRNA 及蛋白的表达没有影响（图 4)。 实施例 10: 细胞增殖分析
采用 [3H]-胸腺嘧啶掺入法。 将实施例 Ί中过表达 RabJ的 NIH3T3细胞、 实施例 9中 RabJ 表达抑制的 HeLa或 MCF-7细胞以 5xl 04/孔铺于 24孔培养板 (Falcon公司） 中，每种细胞铺三个孔。培养 6h 后，在无血清培养基中继续培养 24-48h， 在最后 4h， 每孔加入 0.5 μ。ι (1 Ci = 37 GBq) 的 [3H]-胸腺嘧啶 (Amersham-Phamacia公司）。 然后 用 PBS洗三遍， 细胞溶于含 1% Triton X-100的 PBS中， 用玻璃纤维滤膜收集， 放射 性用液闪仪进行测定。
结果显示， 在无血清条件下， 未转染的野生型 NIH3T3细胞增长缓慢， 但是转染 了 RabJ 的细胞能够迅速增长， 是对照细胞 (未转染的野生型和空白载体转染对照)的 25-35倍， 提示 NIH3T3细胞在 RabJ过表达后获得了自主生长能力（图 5)。 而在 RabJ蛋白表达下调后， HeLa和 MCF-7细胞的生长速率明显下降 (图 6)。 实施例 11: 体外转化活性分析
采用克隆形成试验。为了检测细胞接触非依赖生长的能力，将实施例 7中稳定过 表达 RabJ的 NIH3T3细胞、实施例 9中 RabJ表达抑制的 HeLa或 MCF-7细胞 (5 x 104) 混悬于 0.5%软琼脂的，铺于底层含 1%软琼脂的 6孔板中。培养 3周后，光镜下观察， 大于 50个细胞的克隆记为阳性。
结果显示，稳定表达 RabJ的 ΝΙΗ3Τ3细胞在在软琼脂上可以形成细胞克隆 (图 7)， 而未转染的正常野生型细胞和空载体对照细胞却不能。 而在 RabJ蛋白表达下调后， HeLa和 MCF-7细胞体外软琼脂上形成克隆的能力受到抑制 (图 8)。 实施例 12: 体内致瘤性试验
将实施例 7中稳定过表达 RabJ的 NIH3T3细胞、 实施例 9中 RabJ 表达抑制的 HeLa细胞 (1 x 106)皮下注射到 Balb/C裸鼠的右下侧腹部， 然后观察肿瘤的生长速度 或者肿瘤的发生率 (出现肿瘤的小鼠个数 /注射细胞的裸鼠个数)。肿瘤大小用游标卡尺 测量 (单位： mm)， 结果用长 X宽 X宽 (肿瘤体积； 单位： mm3)。 观察六周。
结果显示， 表达全长 RabJ的 NIH3T3细胞在体内可以形成肿瘤结节 (100%), 而 野生型和对照载体转染的 NIH3T3细胞却不能形成肿瘤 (0% 图 9)，而在 RabJ蛋白表 达下调后 HeLa细胞表现为体内致瘤性下降 (图 10A), 而且已形成的肿瘤生长速率也 降低（图 10B)。 讨论
Rab (Ras-like protein in rat brain)是一类属于 Ras超家族的小分子量 GTPase, 基 本生化特征是可以结合 GTP/GDP并水解 GTP。
不同的 Rab蛋白有不同的功能。 在细胞中 Rab3a主要分布在神经组织和分泌功 能旺盛的细胞系，主要参与调控神经递质的分泌。 Rab3a基因敲除小鼠可以正常发育， 但是却显示苔藓纤维通路 (mossy fiber pathway)中短期和长期突触可塑性缺陷、 昼夜 运动活动周期异常。 Rab5是广泛分布的 Rab蛋白,主要定位于早期内吞体， 参与调节 内吞通路， 包括表皮生长因子 (EGF)、 血小板来源的生长因子 (PDGF)受体 EGFR 和 PDGFR的内吞， 从而参与信号传导的调控。 Rab 蛋白及其调控蛋白和效应蛋白的异 常也可以引起疾病的发生， 如某些出血和色素沉着异常的疾病 (GnscelU综合征；)、 抑 郁症、 Charcot-Mane-Tooth神经症、 肾脏疾病（肾小管硬化；)、 失明 (无脉络膜症;)等疾 病均与 Rab相关蛋白的功能异常相关； 在血管、 肺、 甲状腺疾病的某些病理过程中 往往伴随 Rab蛋白的高表达。 GnscelU综合征是一种由色素转运异常引起的伴随色素 沉着和 T淋巴细胞杀伤功能异常的疾病， 研究发现， Rab27a的突变是该疾病发生的 遗传学病因。 Rab7 蛋白的异常可以造成脂蛋白的代谢异常， 引起高脂血症和血管性 疾病； 一些胞内细菌， 如结核杆菌， 可以通过抑制 Rab7的功能达到感染宿主和致病 的目的。 Rab调控蛋白的异常也是某些疾病的病因， 例如无脉络膜症、 X染色体连锁 的神经抑郁症； Rab蛋白的翻译后修饰异常可以引起 Hermansky-Pudlak综合征。
一些研究还提示， 某些 Rab蛋白可能参与肿瘤的发生。 首先， 在某些肿瘤细胞 或肿瘤类型中发现有 Rab蛋白的表达异常。 Rab25 在前列腺癌中表达比较高， 而且 其表达水平与前列腺癌的分化、 临床分期等密切相关； Rab38 在黑色素瘤细胞系内 的表达也明显升高； Rab3 在垂体腺癌中的表达也很高； 在造血系肿瘤和实体瘤病人 外周血单个核细胞中， Rab2的表达明显增高； 在小鼠肾上腺癌和小鼠肺癌中， Rab2 的表达也表现为异常。 其次， 在肿瘤中发现有 Rab基因的缺失突变。 在恶性横紋肌 肉瘤中发现有染色体 22ql l .2区域的缺失， 该区域包含 Rab36 的编码区。 另外， 在 肿瘤中也发现有 Rab调控蛋白的异常。 在神经母细胞瘤和造血系肿瘤中， Rab蛋白 的调控蛋白 RabGDI的表达升高明显。在前列腺癌中， 调控 Rab5蛋白的 GTP水解的 Rab GTPase活化蛋白一 PRC17表达增高， 可以影响肿瘤的生长特性。
本发明的 RabJ蛋白在多种肿瘤细胞内高表达， 正常的成纤维细胞 NIH3T3在高表 达 RabJ后发生转化， 表现为在半固体琼脂平板上形成集落， 并且在裸鼠体内 RabJ转 化的 NIH3T3细胞可以形成成纤维细胞肉瘤。 RabJ可以促进细胞增殖。 使用 RNA干 扰技术阻断 RabJ的表达后可以抑制肿瘤细胞的增殖， 导致体内致瘤性降低和肿瘤生 长速率下降。 本发明的上述实验结果提示， RabJ与细胞增殖以及细胞周期进程有关， 因此 RabJ可作为一个有效的肿瘤治疗靶标。
此外，人 RabJ蛋白的拮抗剂 (如抗体或干扰性 RNA)可用于抑制肿瘤细胞的生长， 因而具有巨大的应用前景。 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。

Claims

权 利 要 求
1 . 一种人 RabJ多肽的拮抗剂的用途， 其特征在于， 所述的拮抗剂用于制备抑制 肿瘤形成或肿瘤生长的组合物。
2.如权利要求 1所述的用途， 其特征在于， 所述的拮抗剂是抗体、 反义核苷酸或 干扰性 RNA。
3.如权利要求 1所述的用途， 其特征在于， 所述的 RabJ多肽选自下组：
(a) SEQ ID NO: 2氨基酸序列的多肽；
(b)将 SEQ ID NO: 2氨基酸序列经过一个或多个氨基酸残基的取代、 缺失或添加 而形成的，且具有体外促进 Hela细胞或 MCF— 7细胞在软琼脂上形成克隆的功能的由
(a)衍生的多肽。
4.如权利要求 1所述的用途， 其特征在于， 所述的组合物为药物组合物。
5.如权利要求 2所述的用途， 其特征在于， 所述的干扰性 RNA的序列为 SEQ ID NO: 9。
6.如权利要求 1所述的用途， 其特征在于， 所述的肿瘤选自下组： 肺癌、 乳腺癌、 肝癌、 或胃癌。
7.如权利要求 1所述的用途， 其特征在于， 所述的组合物还用于降低肿瘤细胞在 体内的致瘤性。
8.—种人 RabJ多肽或其编码序列的用途， 其特征在于， 用于筛选通过与人 RabJ 多肽或其编码序列的相互作用， 从而抑制肿瘤形成或肿瘤生长的治疗剂。
9.一种筛选抑制肿瘤细胞生长的治疗剂的方法， 其特征在于， 包括步骤：
(a) 提供一测试组和一对照组，其中所述的对照组为表达 RabJ多肽的肿瘤细胞的 培养体系或添加了 RabJ多肽的肿瘤细胞的培养体系，所述的测试组是添加了测试物质 的表达 RabJ多肽的肿瘤细胞的培养体系、 或添加了测试物质和 RabJ多肽的肿瘤细胞 的培养体系；
(b)观察测试组中所述肿瘤细胞的生长，并与对照组的所述肿瘤细胞的生长进行比 较；
其中， 测试组中所述肿瘤细胞的生长速度小于对照组， 就表示测试物质是抑制肿 瘤细胞生长的潜在治疗剂。
10. 一种药物组合物， 其特征在于， 它含有药学上可接受的载体和人 RabJ多肽 的拮抗剂。
PCT/CN2007/070623 2007-09-04 2007-09-04 NOUVELLES FONCTIONS ET UTILISATIONS DE LA PETITE PROTÉINE G HUMAINE DE TYPE ONCOGÈNE RabJ Ceased WO2009030084A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1408724A (zh) * 2001-09-21 2003-04-09 第二军医大学免疫学研究所 新的睾丸功能相关蛋白及其用途
CN1948483A (zh) * 2005-10-12 2007-04-18 中国人民解放军第二军医大学 抑制人RabJ基因表达的siRNA及其应用
CN1948482A (zh) * 2005-10-12 2007-04-18 中国人民解放军第二军医大学 抑制人RabJ基因表达的反义寡核苷酸序列及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1408724A (zh) * 2001-09-21 2003-04-09 第二军医大学免疫学研究所 新的睾丸功能相关蛋白及其用途
CN1948483A (zh) * 2005-10-12 2007-04-18 中国人民解放军第二军医大学 抑制人RabJ基因表达的siRNA及其应用
CN1948482A (zh) * 2005-10-12 2007-04-18 中国人民解放军第二军医大学 抑制人RabJ基因表达的反义寡核苷酸序列及其应用

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