WO2012000458A1 - 一种抑制端粒酶活性的肽及其制备方法和应用 - Google Patents

一种抑制端粒酶活性的肽及其制备方法和应用 Download PDF

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WO2012000458A1
WO2012000458A1 PCT/CN2011/076806 CN2011076806W WO2012000458A1 WO 2012000458 A1 WO2012000458 A1 WO 2012000458A1 CN 2011076806 W CN2011076806 W CN 2011076806W WO 2012000458 A1 WO2012000458 A1 WO 2012000458A1
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polypeptide
lpts
protein
telomerase
cells
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PCT/CN2011/076806
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English (en)
French (fr)
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赵慕钧
冯剑
陈国元
赵静
陈光明
答亮
李载平
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中国科学院上海生命科学研究院
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Priority to EP11800203.9A priority Critical patent/EP2589605B1/en
Priority to US13/808,059 priority patent/US8968727B2/en
Publication of WO2012000458A1 publication Critical patent/WO2012000458A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1276—RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07049—RNA-directed DNA polymerase (2.7.7.49), i.e. telomerase or reverse-transcriptase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the invention belongs to the field of biotechnology and molecular biology research.
  • the invention relates to a protein peptide segment which is highly effective in inhibiting telomerase activity, and its preparation and use. Background technique
  • Telomerase is a ribonucleoprotein that synthesizes and extends cell telomeres. It contains two basic components: the reverse transcriptase catalytic subunit hTERT and the RNA component hTR. Telomerase can synthesize telomere repeats by using self RNA as a template, and add it to the end of the chromosome to compensate for the loss of telomere DNA during cell division and maintain the length of telomeres. Studies have shown that telomerase activity is almost undetectable in normal human cells. Therefore, the number of normal somatic cell divisions is limited. When the cells are divided once, the telomere is lost by 50-200 bp, when the telomere is shortened to a certain extent.
  • telomere activity is generally detectable and active in most malignant cells (85%), and telomerase recombination of telomeres compensates for its continued loss during cell proliferation, thereby Cells can continue to divide, which is an important mechanism for cell immortalization and carcinogenesis.
  • telomerase has a sensitivity of 85%, a specificity of 91%, a positive predictive value of 91%, and a negative predictive value of 81%.
  • telomerase has a sensitivity of 85%, a specificity of 91%, a positive predictive value of 91%, and a negative predictive value of 81%.
  • Activation of telomerase is considered to be one of the major factors in the development of malignant tumors. The degree of activation and expression is closely related to the occurrence and metastasis of tumors.
  • telomerase Inhibition of telomerase and shortening of telomeres is considered to be a mechanism for inhibiting cancer cells. Therefore, telomerase is an ideal target for tumor targeted therapy. Many companies are developing telomerase inhibitors to treat tumors, of which GRN163L has begun clinical phase 2 trials, and several telomerase vaccines are about to complete clinical trials to enter the market.
  • LPTS Liver Putative Tumor Suppressor
  • LPTS Liver Putative Tumor Suppressor
  • a liver-associated candidate tumor suppressor gene obtained by the present inventors using a method of localization and cloning from a human normal liver cDNA library [C. Liao, MJ Zhao, H. Song, K. Uchida , KK Yokoyama, TP Li, Identification of the gene for a novel liver-related putative tumor suppressor at a high-frequency loss of
  • Heterozygosity region of chromosome 8p23 in human hepatocellular carcinoma is Heterozygosity region of chromosome 8p23 in human hepatocellular carcinoma.
  • the LPTS gene is located in the 8p23 segment of human chromosome 8. This segment is HF-deficient in a variety of malignant cells. Studies have shown that the expression level of LPTS in liver cancer tissues and liver cancer cell lines is extremely low or undetectable. Introducing LPTS gene into liver cancer cells can inhibit the growth, proliferation and finally death of liver cancer cells [Liao C, Zhao MJ; Mutation analysis of novel Human liver-related putative tumor suppressor gene in hepatocellular carcinoma. World J Gastroenterol, 2003, 9:89-93]].
  • the LPTS gene has been granted a Chinese invention patent in 2004 (Zhao Muyu et al.: "A liver cancer-related gene and its application", patent number: ZL 00 1 15395. 1, Patentee: Shanghai Institute of Biochemistry, Chinese Academy of Sciences; Day: October 13, 2004).
  • Dr. Lu's laboratory reported another full-length transcript of the LPTS gene, PinXl, and found that the protein encoded by PinXl binds to the telomerase catalytic subunit hTERT and inhibits telomerase activity [Zhou XZ , Lu KP ;
  • the Pin2/ TRF 1 -interacting protein PinXl is a potent telomerase inhibitor.
  • LPTS/PinXl acts as a natural telomerase inhibitor protein and inhibits tumor cell proliferation. , providing a new approach to targeted therapy of tumors.
  • the present inventors applied for an invention patent for preparing LPTS protein in 2005 (Zhao Muzhen et al.: "Preparation and purification of telomerase activity inhibitory protein", Patent Application No.: 200510030526.5, Application Date: October 14, 2005; Patentee: Shanghai Institute of Life Sciences, Chinese Academy of Sciences;).
  • LPTS LPTS protein
  • LGENE2 LPTS inhibitory telomerase active fragment LPTS 133-328
  • LPTS inhibits telomerase active fragment at its C-terminus 133-328. Amino acid residues.
  • the patent filed by the present inventors in 2008 discloses a fusion protein of TAT and LPTS 133-328 (Patent Application No.: 200810041324.4), which has been confirmed to penetrate the cell membrane and has an extremely excellent effect of inhibiting tumor cell growth.
  • LPTS protein is an important protein closely related to the growth of tumor cells, it is highly desirable to further study it to develop more effective tumor suppressing drugs for clinical applications. Summary of the invention
  • the object of the present invention is to provide a protein peptide segment capable of inhibiting telomerase activity efficiently, and preparation and application thereof
  • polypeptide in a first aspect of the invention, an isolated polypeptide (protein;) is provided, the polypeptide being:
  • amino acid sequence of SEQ ID NO: 1 is passed through one or more (eg, 1-10; preferably 1-5; a polypeptide derived from (a) which is formed by substitution, deletion or addition of an amino acid residue; and having (a) a polypeptide function; or
  • the polypeptide does not have the amino acid sequence shown in SEQ ID NO: 2 (; LPTS full-length sequence), and the amino acid sequence shown at positions 133-328 in SEQ ID NO: 2 (LPTS 13 w 28) And the amino acid sequence shown at positions 254-328 of SEQ ID NO: 2.
  • polypeptide comprises the amino acid sequence shown in 255 to 328 of SEQ ID NO: 2, or
  • amino acid sequence shown in 256th to 328th position of SEQ ID NO: 2 or the amino acid sequence shown in 257th to 328th position of SEQ ID NO: 2, or the 258th to 328th positions shown in SEQ ID NO: 2 Amino acid sequence, or
  • amino acid sequence shown in 259th to 328th position of SEQ ID NO: 2 or the amino acid sequence shown in 260th to 328th position of SEQ ID NO: 2, or the 261th to 328th positions in SEQ ID NO: 2 Amino acid sequence, or
  • amino acid sequence shown in positions 262 to 328 of SEQ ID NO: 2 or the amino acid sequence shown in positions 263 to 328 of SEQ ID NO: 2, or the positions shown in 264 to 328 of SEQ ID NO: Amino acid sequence, or
  • amino acid sequence shown in positions 265 to 328 of SEQ ID NO: 2 or the amino acid sequence shown in positions 266 to 328 of SEQ ID NO: 2, or the 267 to 328th position shown in SEQ ID NO: Amino acid sequence, or
  • amino acid sequence shown in 269 to 328 of SEQ ID NO: 2 or the amino acid sequence shown in 270 to 328 of SEQ ID NO: 2, or
  • amino acid sequence shown in positions 271 to 328 of SEQ ID NO: 2 or the amino acid sequence shown in positions 272 to 328 of SEQ ID NO: 2, or the positions shown in 273 to 328 of SEQ ID NO: Amino acid sequence, or
  • amino acid sequence shown in 274 to 328 of SEQ ID NO: 2 or the amino acid sequence shown in positions 275 to 328 of SEQ ID NO: 2, or 276 to 328 of SEQ ID NO: 2 Amino acid sequence, or
  • amino acid sequence shown in positions 277 to 328 of SEQ ID NO: 2 or comprises SEQ ID NO:
  • amino acid sequence shown in positions 278 to 328 of 2 or the amino acid sequence shown in positions 279 to 328 of SEQ ID NO: 2, or
  • amino acid sequence shown in 280th to 328th position of SEQ ID NO: 2 or the amino acid sequence shown in 281th to 328th position of SEQ ID NO: 2, or the 282th to 328th positions in SEQ ID NO: 2 Amino acid sequence, or
  • amino acid sequence shown in positions 283 to 328 of SEQ ID NO: 2 or the amino acid sequence shown in positions 284 to 328 of SEQ ID NO: 2, or the positions shown in 285 to 328 of SEQ ID NO: Amino acid sequence, or
  • amino acid sequence shown in positions 286 to 328 of SEQ ID NO: 2 or the amino acid sequence shown in positions 287 to 328 of SEQ ID NO: 2, or the positions shown in 288 to 328 of SEQ ID NO: Amino acid sequence, or
  • amino acid sequence shown in 289 to 328 of SEQ ID NO: 2 or the amino acid sequence shown in 290 to 328 of SEQ ID NO: 2 is included.
  • an isolated polynucleotide comprising a nucleotide sequence encoding said polypeptide.
  • a vector comprising the polynucleotide is provided.
  • a genetically engineered host cell comprising the vector is provided.
  • a method of preparing the polypeptide comprising:
  • the composition is for controlling a disease associated with abnormal activation of telomerase.
  • the telomerase abnormal activation-related disease is a tumor.
  • a complex is provided, the complex comprising the polypeptide, and a substance compatible with the polypeptide.
  • the complex is a fusion protein, and the polypeptide is linked to at least one functional protein (preferably via a peptide bond;), the functional protein is 5-500 One (; preferably 5 to 300; more preferably 10 to 250) amino acids.
  • the functional protein is selected from the group consisting of: a transmembrane protein (eg, a transactivator protein tagged protein (eg, GST protein), a reporter protein (eg, GFP protein), human serum albumin (extended half-life) , human IgGl: Fc fragment (extended half-life).
  • a transmembrane protein eg, a transactivator protein tagged protein (eg, GST protein)
  • a reporter protein eg, GFP protein
  • human serum albumin extended half-life
  • human IgGl Fc fragment
  • the polypeptide is directly linked to the functional protein or is linked by a linker peptide.
  • the linker peptide is 1-20 amino acids in length, more preferably 2-10 amino acids.
  • the amino acid sequence of the linked peptide is: GGS.
  • the fusion protein does not have the amino acid sequence shown in SEQ ID NO: 2 (LPTS full-length sequence), and the amino acid sequence shown at positions 133-328 in SEQ ID NO: 2 (LPTS 13 w 28) And the amino acid sequence shown at positions 254-328 of SEQ ID NO: 2.
  • the complex comprises a substance selected from the group consisting of a protein activity enhancer, a protein activity stabilizer, and a prolonged protein half-life (e.g., PEG, PEG-liposome;
  • a composition comprising a safe and effective amount of said polypeptide or said complex, and a pharmaceutically acceptable carrier.
  • the composition is for inhibiting intracellular telomerase activity.
  • the composition is for controlling telomerase activity to increase tumors.
  • a method of preparing a composition, the composition inhibiting intracellular telomerase activity comprising: administering a safe and effective amount of the polypeptide or the complex with A pharmaceutically acceptable carrier is mixed.
  • a kit comprising the polypeptide; or comprising the complex; or a composition comprising the composition.
  • the kit is for inhibiting intracellular telomerase activity.
  • the kit is for preventing telomerase activity from increasing tumors.
  • a method preferably in vitro, preferably non-therapeutic; inhibiting intracellular telomerase activity comprising administering to a subject an effective amount of said polypeptide; or said complex Or a composition as described.
  • FIG. 1 SDS-PAGE shown to induce the expression and purification of GST-LPTS 29Q _ 328 fusion protein.
  • Lane 1 is a GST-LPTS before IPTG induction of protein expression 328 29Q _ genetically engineered bacteria; lane 2
  • Lanes 3-5 are 3 tubes collected after purification
  • FIG. 2 290 -328 GST-LPTS - 133-328 Detection and comparison GST-LPTS and GST-LPTS inhibition of telomerase activity.
  • B. TRAP assay detects GST-LPTS 290-328 , GST-LPTS 133-328 and GST-LPTS proteins to inhibit telomerase activity.
  • the amount of protein used was 5, 10, 25, 50, 100, 200, 250 nM, as shown.
  • the GST protein served as a control.
  • the samples were stained with silver by 10% PAGE non-denaturing gel electrophoresis.
  • Figure 3 Effect of LPTS 29Q _ 328 on the growth of liver cancer BEL7404 cells.
  • FIG. 4 Southern Blot assay LPTS LPTS and 29 ( « Effect of telomere length 28 of hepatocellular carcinoma BEL7404 cells.
  • B. BEL7404 cells transfected with GFP or GFP-LPTS 29Q _ 328 were multiplied by FACS flow cytometry for 8 generations, and their genomic DNA was extracted and digested with Hinf l and Afa l endonuclease to repeat with single-stranded telomeres. Sequence 32 p-(TTAGGG) 6 probe hybridization. This figure shows the results after autoradiography. detailed description
  • the inventors have intensively studied for the first time to isolate the active region of the telomerase inhibiting LPTS protein (full length sequence such as SEQ ID NO: 2), which is located at positions 290-328 of the LPTS protein.
  • LPTS 29Q _ 328 This active region is more active than the full-length LPTS protein and other fragments on the LPTS and can induce tumor cell death more rapidly.
  • the present invention provides a more effective protein for inhibiting telomerase activity for targeted therapy of tumors. the term
  • 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. .
  • subject refers to any target that requires diagnosis or treatment, especially mammalian subjects, particularly humans, other subjects including cattle, dogs, cats, guinea pigs, rabbits, large Rat, mouse, horse, etc. Of particular interest are those that are abnormally activated by telomerase.
  • nucleic acid and “nucleic acid sequence” refer to nucleotides of any length in a polymeric form (ribonucleotides or deoxyribonucleotides;). It includes (but is not limited to;) single-stranded, double-stranded DNA or RNA, genomic DNA and cDNAo
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, a substance having a reasonable benefit/risk ratio .
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients or diluents.
  • an effective amount or “safe and effective amount” means that the amount administered to a subject in a single dose or in a continuous dose is effective for treatment or prevention.
  • the amount is based on the health and physiological condition of the individual being treated, The type of individual being treated (eg, non-human primates, etc.;), therapist's assessment of medical condition, and other relevant factors. This amount is expected to be in a relatively wide range and can be determined by routine experimentation.
  • containing includes “comprising”, “consisting essentially of”, “consisting essentially of”, and “consisting of”; “mainly constituted by”, “consisting essentially of” and “consisting of” belonging to “contains” , the underlying concept of "with” or “including”.
  • Polypeptide of the invention and coding gene thereof includes “comprising”, “consisting essentially of”, “consisting essentially of”, and “consisting of”; “mainly constituted by”, “consisting essentially of” and “consisting of” belonging to “contains” , the underlying concept of "with” or “including”.
  • LPTS is the first protein found to bind directly to the human telomerase catalytic subunit hTERT and inhibit telomerase catalytic activity. Based on the full-length LPTS protein, the present inventors predicted and screened a variety of LPTS sequence fragments. After repeated studies and comparisons, it was found that the active region of LPTS protein inhibiting telomerase can be reduced to the amino acid sequence of amino acids 290-328 of the protein. Within the region, this position is the most critical region for inhibiting the catalytic activity of telomerase, which is sufficient to exert a telomerase-inhibiting activity, thereby obtaining the polypeptide of the present invention.
  • the inventors expressed the GST-LPTS 29Q- 328 fusion protein by genetic engineering techniques in vitro.
  • the GST-LPTS 290-328 fusion protein is tested for inhibiting the telomerase activity of tumor cells in vitro using a TRAP (telomeric repeat amplification protocol) assay technique for determining telomerase activity.
  • the method is a PCR-based method for detecting telomerase activity, and the telomerase is derived from a liver cancer cell lysate.
  • the results showed that GST-LPTS 29() _ 328 had strong inhibition of telomerase activity.
  • the inventors further compared the activities of GST-LPTS 29Q- 328 with GST-LPTS and GST-LPTS 13 w 28 proteins. The test results indicate GST-LPTS and
  • a eukaryotic expression plasmid in which LPTS 29Q _ 328 , LPTS and green fluorescent protein GFP are fused is constructed.
  • GFP-LPTS 29 ( « 28 and GFP-LPTS.
  • GFP-LPTS 29 ( « 28 , GFP-LPTS and control GFP expression plasmids were transfected into hepatoma BEL7404 cells, respectively. After 2 weeks of G418 screening, flow cytometry was used respectively. The cells with green fluorescent protein expression were sorted by FACS and then subcultured.
  • GFP-LPTS 290 -328/7404, GFP-LPTS/7404, GFP/7404 cells were detected by western blot using rabbit anti-GFP polyclonal antibody, and the corresponding proteins were stably expressed.
  • GFP-LPTS 29 ( ⁇ 28 /7404 cells grew slower than GFP-LPTS/7404 and GFP/7404.
  • the inventors selected the above stable cell line that was multiplied by 5 generations after FACS sorting.
  • the MTT assay was performed and the growth curve was plotted.
  • the results showed that GFP-LPTS 29Q _ 328 /7404 cells grew the slowest and GFP-LPTS/7404 gradually decreased compared with the control GFP/7404 cells.
  • Description LPTS 29Q _ 328 inhibits tumors The cell's ability to grow is stronger than its full-length protein, LPTS. Overexpression of the LPTS protein in tumor cells can cause cell growth to slow down, flatten, enter a crisis, and eventually die. But this is a longer-term effect and generally requires culture. After 6 weeks, the cells died soon after transfection of LPTS 29Q _ 328.
  • LPTS 29Q _ 328 /7404 cells were further cultured. about 10 days, they have appeared symptoms of aging, and soon after all rounded off the wall of death. the results described expression LPTS 29Q _ 328 the ability to cause tumor cell death is very strong, probably more than LPTS Long protein inhibits tumor higher efficiency and more value.
  • GFP-LPTS 29 was detected by Southern Blot ( ⁇ 28 /7404,
  • GFP-LPTS/7404 telomere length of GFP/7404 cells.
  • the experimental results showed that the telomere remained stable during the passage of control GFP/7404 cells, and the length was about 4.5 kb.
  • the telomeres gradually shrank during the passage of GFP-LPTS/7404 cells, and the telomere shortened to 3.8 during the fifth passage.
  • the telomere was shortened to about 2.8 kb in the 25th generation;
  • GFP-LPTS 29Q _ 328 /7404 had a short passage time, and many cells died during the culture.
  • the telomere was shortened to about 2.5 kb. .
  • the results indicate that LPTS 29Q _ 328 has a very strong telomerase inhibitory activity, which inhibits telomere synthesis and elongation in cells, and is a tumor suppressor peptide targeting telomerase inhibition.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, a synthetic polypeptide, preferably a recombinant polypeptide.
  • polypeptides of the invention may be naturally purified products, either chemically synthesized or produced recombinantly from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells;).
  • the polypeptide of the invention may be glycosylated according to the host used in the recombinant production protocol, or may be non-glycosylated .
  • Polypeptides of the invention may also or may not include an initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of the polypeptides.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of a 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 a polypeptide encoded by the genetic code, or ( ⁇ ) a polypeptide having a substituent group in one or more amino acid residues, or () a mature polypeptide and another compound (such as a compound that extends the half-life of the polypeptide, such as polyethyl b) 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 IgG) Fragment formation of the fusion protein ;).
  • the invention also encompasses variant forms of the sequence of SEQ ID NO: 1 having the same function as the polypeptide.
  • variants include (but are not limited to, one or more (usually 1-10, preferably 1-5, more preferably 1-3, optimally 1-2;) amino acid deletions, Insertion and/or substitution, and addition of one or several (usually within 10, preferably within 5, more preferably within 3) amino acids at the C-terminus and/or N-terminus.
  • the function of the protein is usually not changed.
  • adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein.
  • active fragments and active derivatives of the polypeptides are also included.
  • Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA capable of hybridizing to the polypeptide under high or low stringency conditions And a polypeptide or protein obtained using an antiserum against the polypeptide.
  • the invention also provides other polypeptides, such as fusion proteins comprising the polypeptides or fragments thereof.
  • the invention also provides analogs of the polypeptide or polypeptide.
  • the difference between these analogs and the naturally occurring 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.
  • These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by irradiation or exposure to a mutagen, or by site-directed mutagenesis or other techniques known to molecular biology.
  • Analogs also include analogs having residues other than the native L-amino acid (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., beta, ⁇ -amino acids). It is to be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.
  • Modifications include: chemically derived forms of the polypeptide in vivo or in vitro, such as Acetylation or carboxylation. Modifications also include glycosylation, such as those produced by glycosylation modifications in the synthesis and processing of the polypeptide or in further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylation enzyme or a deglycosylation enzyme. Modified forms also include sequences having phosphorylated amino acid residues such as phosphotyrosine, phosphoserine, phosphothreonine. Also included are polypeptides modified to increase their resistance to proteolytic properties or to optimize solubility properties.
  • the polypeptide conservative variant polypeptide means having up to 10, preferably up to 5, more preferably up to 3, optimally at most as compared with the amino acid sequence of SEQ ID NO: 1. Two amino acids are replaced by amino acids of similar or similar nature to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitution according to Table 1.
  • the use of the polypeptide of the present invention includes, but is not limited to,: directly as a drug for treating diseases associated with abnormal activation of telomerase (such as tumors).
  • Fusion molecules containing or coupled to the polypeptide are also included in the present invention.
  • a fusion molecule of a polypeptide of the present invention and a targeting molecule can be constructed; such a fusion molecule can increase the concentration of a drug in a localized region without affecting other regions by including a molecule targeted to a specific tissue or organ;
  • the targeting molecules described are, for example, antibodies, ligands and the like.
  • a fusion molecule of a polypeptide of the present invention and a multimeric molecule can be constructed; the half-life of the fusion molecule is increased; and the multimeric molecule is, for example, IgG Fc.
  • Fusion genes containing the coding sequences for the polypeptides are also included in the present invention.
  • a fusion gene comprising a coding sequence for the polypeptide and a tissue or organ-specific promoter (operably linked;) can be constructed which, when applied to a body, is driven by a promoter in a particular tissue or organ. Expressed within.
  • the invention also encompasses complexes comprising a polypeptide of the invention, as well as other functional proteins or molecules linked or conjugated to the polypeptides of the invention.
  • These functional proteins (5-500; preferably 5-300; more preferably 10-250 amino acids) include, but are not limited to: transmembrane proteins, GST proteins (purification tags), GFP proteins (reporter proteins) , human serum albumin (extended half-life), human IgGl: Fc fragment (extended half-life;) and the like.
  • Other molecules may also be selected from the group consisting of protein activity promoters, protein activity stabilizers, and prolonged protein half-life preparations.
  • the prolonged protein half-life preparation is, for example, PEG (which can be used to link to the amino terminus or carboxy terminus of the polypeptide of the present invention;), PEG-liposome (which can be used to embed the polypeptide of the present invention), the PEG
  • the molecular weight may be from 1,000 to 50,000; preferably from 20,000 to 40,000.
  • the polypeptide may be fused or conjugated to some molecules effective for penetrating the cell membrane to facilitate entry into the cell.
  • transmembrane proteins There are many known transmembrane proteins, including: transactivator TAT, Penetratin, signal sequence-based peptides, pVEC, Transportan, Amphiphilic model and Arg9, and the like.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the DNA form includes cDNA, genomic DNA or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be either a coding strand or a non-coding strand.
  • polynucleotide encoding a polypeptide may be a polynucleotide comprising the polypeptide, or may be a polynucleotide further comprising additional coding and/or non-coding sequences.
  • 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 polypeptides 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.
  • 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 polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • stringent conditions means: (1) hybridization and elution at a lower ionic strength and a higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C; or (2) hybridization Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) only at least 90% identity between the two sequences More preferably, hybridization occurs more than 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide of SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides or more.
  • Nucleic acid fragments can be used in nucleic acid amplification techniques (e.g., PCR) to identify and/or isolate polynucleotides encoding the polypeptides of the invention.
  • the polypeptides and polynucleotides of the invention are preferably provided in isolated form, more preferably purified to homogeneity.
  • the full-length 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.
  • DNA sequence encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNAs known in the art. Molecules (or as carriers) and cells.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • a method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • RACE method RACE-cDNA end rapid amplification method
  • primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein.
  • the amplified DNA/RNA fragment can be isolated and purified by a conventional method such as gel electrophoresis.
  • the invention also relates to vectors comprising the polynucleotides of the invention, and host cells genetically engineered using the vectors or 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 used to express or produce recombinant polypeptides of the present invention by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally there are the following steps:
  • the polynucleotide sequence of the present invention can be inserted into a recombinant expression vector.
  • recombinant expression vector refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and stabilize in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the polynucleotide sequences of the invention and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide for selection Phenotypic traits of the host cell, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes melanoma cells Animal cells, etc.
  • an enhancer sequence is inserted into the vector.
  • An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene.
  • Illustrative examples include a 100 to 270 base pair SV40 enhancer on the late side of the replication initiation site, a polyoma enhancer on the late side of the replication initiation site, and an adenovirus enhancer.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote
  • the following DNA transfection method can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc., can be cultured by conventional methods, expression A polypeptide encoded by the gene of the invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction;) and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted extracellularly.
  • the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • PCR amplification gene sequences _ 328 LPTS 29Q amplified from template plasmid pT-LPTS by designing specific primers.
  • the LPTS 29Q _ 328 gene fragment was inserted into pGEX-4T-l plasmid which can express GST fluorescent protein, to obtain pGEX-LPTS 29Q _ 328 recombinant plasmid.
  • the pGEX-LPTS 29Q _ 328 recombinant plasmid was transformed expression hosts co // BL-21 (DE3) , to obtain GST- LPTS 29Q _ 328 fusion protein engineering bacteria.
  • GST-LPTS 29Q- 328 was induced to express by IPTG in E. coli, and purified preparation of GST-LPTS 29Q- 328 fusion protein was carried out using a commercial affinity purification column GS-4B.
  • the invention also provides various compositions, particularly pharmaceutical compositions, comprising a polypeptide of the invention.
  • the composition can be used to prevent or treat diseases associated with abnormal activation of telomerase, including but not limited to tumors.
  • compositions comprising the polypeptides of the invention may comprise a buffer selected for the actual use of the polypeptide; other materials suitable for the intended use may also be included. Those skilled in the art are well versed in the selection of suitable buffers, and a variety of buffers are known in the art for the intended use.
  • the compositions may contain pharmaceutically acceptable excipients, many of which are known in the art and need not be discussed in detail herein. Various excipients that are pharmaceutically acceptable are described in various publications, including, for example, "Remington's Pharmaceutical Sciences", Remington's Pharmaceutical Sciences, 19th Edition (1995) Mack Publishing Co.).
  • composition of the present invention can be prepared into various dosage forms such as injections, granules, tablets, pills, suppositories, capsules, suspensions, sprays, suppositories, transdermal drugs (such as patches, etc.), ointments, and washes.
  • Agents, etc. Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral or topical use can be used in the formulation of various compositions containing therapeutically active compounds. Diluents known in the art include aqueous media, vegetal and animal oils and fats. Stabilizers, wetting agents and emulsifiers, salts which change the osmotic pressure or various buffers and skin penetration enhancers which maintain a suitable pH may also be used as auxiliary materials.
  • compositions of the present invention include: intramuscular, subcutaneous, intradermal, intrapulmonary, intravenous, intratumoral, nasal, oral or other parenteral routes of administration. If necessary, the route of administration can be combined, or adjusted according to the disease. It can be administered in a single dose or in multiple doses.
  • the amount of polypeptide selected is determined by the amount which produces an effect of inhibiting telomerase activity without significant side effects. Typically, about O.Ol w g-lOmg polypeptide/kg body weight is administered, preferably O. lw g-lmg polypeptide/kg body weight, more preferably 0.14 8 -10 (8 polypeptide/13 ⁇ 4 body weight). Pill box
  • the present invention also provides a kit for preventing a disease associated with abnormal activation of telomerase, which comprises the polypeptide of the present invention or a composition containing the same. Furthermore, for ease of administration, the kit may also contain a needle for injection, and/or a pharmaceutically acceptable carrier, and/or instructions for use. Advantages of the invention:
  • LPTS 29Q _ 328 has a very significant telomerase inhibitory activity in vitro and is the region with the strongest telomerase inhibition ability known in LPTS.
  • the LPTS full-length cDNA fragment was obtained by reverse transcription PCR amplification of normal primers from normal human liver tissue.
  • the PCR product was loaded into a pMD-18T vector (purchased from TaKaRa Co., Ltd.) to obtain a pT-LPTS plasmid.
  • LPTS 29Q _ 328 gene fragment was amplified by PCR using a plasmid (the plasmid containing the full-length gene cDNA fragment LPTS) from the pT-LPTS.
  • Design PCR primer P1 the plasmid containing the full-length gene cDNA fragment LPTS
  • the PCR reaction was carried out using primers PI and P2 using the pT-LPTS plasmid as a template.
  • the PCR reaction conditions were: 94 °C After 30 seconds, annealing at 55 ° C for 30 seconds, and extension at 72 ° C for 30 seconds, a total of 30 cycles were amplified.
  • the LPTS 29Q _ 328 gene fragment was obtained for the construction of the subsequent GST fusion protein and eukaryotic expression plasmid.
  • the gene sequence LPTS 29Q amino acid sequence encoded _ 328 see SEQ ID NO: 1.
  • the LPTS 133 _ 328 gene fragment used as a control was obtained by the same method as above, and the PCR primer was P3 : 5 ' - ACGCTCGAGAAGGATCTGTC ATCTCGG-3 ' (SEQ ID NO: 7) and P2.
  • the LPTS gene sequence was obtained by double digesting with EcoR I and Xho l from the plasmid pT-LPTS, and the gene encoded by the gene fragment corresponds to the full length of the LPTS protein. Construction and induction of Example 2, GST-LPTS 29 o_ 328 fusion protein engineered strain of
  • the LPTS 29Q _ 328 gene fragment and the LPTS 133 _ 328 gene fragment obtained in Example 1 were digested with EcoR I and Xho I, and then inserted into pGEX-4T-1 plasmid (purchased from Amersham Pharmacia), and the plasmid was expressed. GST protein.
  • the LPTS gene fragment obtained in Example 1 was directly inserted into the pGEX-4T-1 plasmid to obtain pGEX-LPTS 290-328 , pGEX-LPTS 133-328 and pGEX-LPTS recombinant plasmids.
  • the three recombinant plasmids were transformed into the host strain co//DH5a, and the plasmid was extracted from the co//DH5a transformed strain. After sequencing, the recombinant protein was expressed and the host strain co//BL-21 (DE3) was obtained, thereby obtaining GST.
  • -LPTS 29 « 28 , GST-LPTS 13 w 28 and GST-LPTS fusion protein expression engineered strains.
  • the fusion protein expression of pGEX-LPTS 29Q _ 328 plasmid was first expanded at 37 ° C overnight, and the next day was inoculated in 400 ml of LB medium containing Ampf antibiotics at a ratio of 1:100, and cultured at 37 ° C to OD600. When approximately equal to 0.6, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 37 ° C for 3-4 hours. The cells were collected by centrifugation at 5000 rpm for 10 minutes, the supernatant was discarded, and the remaining cells were stored at -80 ° C until use. The cells containing the target GST-LPTS 29Q _ 328 fusion protein (; see FIG. 1). Using the same method, the present inventors induced the expression of the 08 Ding-1 butyl 8 133 _ 328 and GST-LPTS fusion protein. A schematic diagram of the structure of GST and the above fusion protein is shown in Figure 2A.
  • Figure 1 shows the results of Coomassie blue staining after 10% SDS-PAGE gel electrophoresis.
  • Lane 1 is the engineered strain before IPTG induction
  • Lane 2 is the engineered strain after IPTG induction
  • a specific band is induced at 40 kD.
  • Example 3 GST-LPTS 29 () _ 328 fusion protein purification preparation
  • GST-LPTS 290 -328 preparing purified fusion proteins using commercially available affinity purification column GS-4B (; commercially available from Sigma).
  • the affinity purification column is coupled with reduced glutathione GSH and Sepharose 4B. Before loading, it is filled to a final volume of about 2 ml according to the manufacturer's instructions, and 20 to 30 ml of solution A (20 mM Tris-HCl pH 7.4). , 0.2 mM EDTA, I mM DTT, 0.5 mM PMSF, 1 M NaCl) equilibrated.
  • Example 2 The cells finally obtained in Example 2 were resuspended in 10 ml of solution A, and the bacteria were broken by ultrasonic wave (the ultrasonic instrument was manufactured by Ningbo Xinzhi;), and the ultrasonic conditions were as follows: working time 7 seconds, gap time 25 seconds, working power 400 W, 20-30 times of work).
  • the sonicated body fluid was added to Triton X-100 to a final concentration of 1%, placed on ice for 30 minutes; then centrifuged at 12000 rpm for 10 minutes at 4 ° C.
  • the supernatant after centrifugation was transferred to a new centrifuge tube and repeated centrifugation was repeated;
  • the supernatant containing the fusion protein obtained after centrifugation is connected to a well-balanced GS-4B purification column and passed through the column under a pressure difference; after the sample is completed, the column is washed with 20 to 30 ml of solution A; Wash the column with ⁇ 30 ml of solution B (20 mM Tris-HCl pH 7.4, 0.2 mM EDTA, 0.1 M NaCl); finally use 5 ml of solution C (15 mM reduced glutathione GSH, 20 mM Tris-HCl pH 7.4, Protein was eluted with 0.2 mM EDTA, 0.1 M NaCl).
  • each tube was approximately 1.5 ml, and the collected purified protein was stored at -80 °C until use.
  • the inventors purified and obtained GST-LPTS 133-328 and GST-LPTS protein for use.
  • Lanes 3, 4, and 5 in Figure 1 are the three tubes of protein collected by elution. It can be seen that the purified GST-LPTS 29Q _ 328 protein strip has a single purity of 95% or more and can be used for further applications. Collected GST-LPTS 29 ( « 28 proteins were measured by Bradford method. Example 4, GST-LPTS 29 o_ 328 fusion protein inhibited telomerase activity detection and
  • TRAP telomerase amplification protocol
  • Washing Buffer (10 mM Hepes-KOH pH 7.5, 1.5 mM MgCl 2 , 10 mM KC1, 1 mM DTT) was washed twice; Lys Buffer (10 mM Tris-HCl pH) was pre-cooled with 1 ml of ice per 10 6 cells.
  • telomerase-containing liver cancer BEL7404 cell lysate 7.5, 1 mM MgCl 2 , 1 mM EGTA, 0.1 mM PMSF, 5 mM mercaptoethanol, 0.5% CHAPS, 10% glycerol
  • the cell lysate can be stored in a -80 ° C refrigerator.
  • TRAP reaction first take l L cell lysate, add GST-LPTS 29Q _ 328 protein or other to be tested The protein was fixed on ice for 10 minutes; then l L Ts primer (0.1 g/L, sequence 5 '-AATCCGTCGAGCAGAGTT-3 '), 0.25 10 mM dNTP, 42 reaction buffer (20 mM Tris-HCl pH8) was added.
  • Fig. 2B shows the results of the TRAP experiment. It can be seen from the figure that GST-LPTS and GST-LPTS 13 ⁇ 328 protein showed inhibition of telomerase activity at 50 nM, and inhibition activity was stronger at the time of ⁇ , but the reaction could not be completely inhibited. Telomerase in the system; while GST-LPTS 29Q _ 328 has a strong inhibitory activity at 50 nM, and completely inhibits telomerase activity in the system at the time of ⁇ .
  • LPTS 29Q _ 328 has stronger telomerase inhibitory activity than full-length LPTS and LPTS 133 _ 328 , and is a functional domain in which LPTS protein inhibits telomerase activity.
  • Example 5 LPTS 29() _ 328 inhibits the growth of liver cancer cell BEL7404 and causes its death.
  • the inventors constructed
  • LPTS 29Q _ 328 An eukaryotic expression plasmid in which LPTS 29Q _ 328 , LPTS and green fluorescent protein GFP are fused.
  • LPTS obtained in Example 1 of the embodiment 29Q _ 328 gene through the EcoR I and Xho l digested plasmid was inserted into pEGFP-C2 (; commercially available from Clontech;), to give pEGFP-LPTS 29 ( «28 Expression
  • the cDNA fragment of the LPTS gene was digested with EcoR I and Xho I, and inserted into the pEGFP-C2 plasmid to obtain the pEGFP-LPTS expression plasmid.
  • the above plasmid was transfected with BEL7404. The corresponding protein can be expressed after the cell.
  • GFP-LPTS 29Q _ 328 , GFP-LPTS and control GFP expression plasmids were transfected into BEL7404 cells, and after 2 weeks of G418 screening, cells with green fluorescent protein expression were selected by flow cytometry FACS. Subculture is then carried out.
  • the GFP-LPTS 29Q _ 328 /7404, GFP-LPTS/7404, GFP/7404 cells were sorted by Western Blot using rabbit anti-GFP polyclonal antibody, and the corresponding proteins were stably expressed, as shown in Figure 3A. Shown in .
  • GFP-LPTS/7404 and GFP/7404 are slower.
  • the inventors selected FACS sorting and multiplied by 5 generations. The stable cell strain was described, the MTT assay was performed, and a growth curve was drawn. The results demonstrated that GFP-LPTS 29 ( ⁇ 28 /7404 cells grew the slowest, GFP-LPTS/7404 gradually, compared to control GFP/7404 cells, see Figure 3B).
  • LPTS 29Q _ 328 inhibits the growth of tumor cells more strongly than its full-length protein LPTS.
  • LPTS 29Q _ 328 Overexpression of the LPTS protein in tumor cells can cause cell growth to slow down, flatten, enter a crisis, and eventually die. This is because the LPTS protein inhibits the telomerase activity of tumor cells, making the telomeres of the cells unable to extend and become shorter, leading to cell aging and death. But this is a longer-term effect that usually takes about 6 weeks to develop. However, the cells died soon after transfection of LPTS 29Q _ 328. After 2 weeks of G418 screening, only a small number of cells were obtained for FACS sorting. The LPTS 29Q _ 328 /7404 cells obtained by sorting were further cultured for about 10 days. Symptoms of aging appear one after another, and soon all of them become rounded and then die off, see Figure 3C.
  • LPTS 29Q _ 328 has a strong ability to cause tumor cell death, and is more efficient and more useful than LPTS full-length protein in inhibiting tumors.
  • Example 6 LPTS 29() _ 328 shortens telomere length of liver cancer BEL7404 cells
  • the inventors detected the GFP-LPTS 29 by the Southern Blot method.
  • -328 /7404, GFP-LPTS/7404, GFP/7404 cell telomere length After FACS sorting, the cells were collected according to the culture algebra shown in Fig. 4A, and the genomic DNA of the cells was extracted, digested with Hinf l and Afa l endonuclease, and isotopically labeled 32 P-(TTAGGG) 6
  • the probe is a telomere single-stranded DNA repeat (TTAGGG) and the telomere length of the cells is analyzed.
  • telomere long-sighted autoradiography The gradation of the telomere long-sighted autoradiography and the position of the strip are comprehensively judged. The closer the strip is to the upper end of the sample well, the longer the telomere of the corresponding cell. As can be seen from Fig. 4A, the telomere remained stable during the passage of control GFP/7404 cells, and the length was about 4.5 kb; the telomeres gradually shortened during the passage of GFP-LPTS/7404 cells, and the telomeres in the fifth passage of culture.
  • telomere was shortened to about 2.8 kb in the 25th generation; GFP-LPTS 29Q _ 328 /7404 had a short passage time, and many cells died during the culture. In the 8th generation, the telomere was shortened to 2.5. Kb is around (Fig. 4B;).

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Abstract

本发明提供一种了具有端粒酶抑制活性的多肽，所述多肽在肿瘤细胞内表达可显著抑制肿瘤细胞端粒酶活性，抑制肿瘤细胞生长，并导致细胞死亡。本发明进一步提供了上述多肽的制备方法和在肿瘤靶向治疗中的应用。

Description

一种抑制端粒酶活性的肽及其制备方法和应用
技术领域
本发明属于生物技术和分子生物学研究领域。 发明涉及一种高效抑制端粒酶 活性的蛋白肽段及其制备和应用。 背景技术
端粒酶 (Telomerase)是一种合成和延伸细胞染色体端粒的核糖核蛋白， 它包 含两种基本成分：逆转录酶催化亚基 hTERT和 RNA组分 hTR。端粒酶能以自身 RNA为模板， 反转录合成端粒重复列， 加到染色体的末端， 以弥补细胞分裂时 端粒 DNA的丢失， 维持端粒的长度。 研究表明在正常人体细胞内端粒酶活性几 乎检测不到， 因此， 人正常体细胞分裂次数是有限的， 细胞每分裂一次， 端粒便 丢失 50-200bp， 当端粒缩短到一定程度时细胞生长受到抑制， 即称为细胞衰老， 并走向死亡。 然而， 在绝大多数恶性肿瘤细胞 (85%)中普遍可以检测到端粒酶活 性且活性较强， 端粒酶对端粒的重新合成补偿了它在细胞繁殖过程中的持续丢 失， 从而使得细胞可以不断的分裂， 这是细胞永生化和癌变的一个重要机制。
Kim等分析总结了大量研究结果， 检测了 100多种恶性肿瘤标本， 指出端粒 酶诊断肿瘤的敏感性为 85%， 特异性为 91%， 阳性预测值为 91%， 阴性预测值 为 81%， 充分表明端粒酶在肿瘤诊断中的价值 (Kim NW， Piatyszek MA, Prowse KR, et al. Specific association of human telomerase activity with immortal cells and cancer. Science. 1994 Dec 23; 266(5193):2011-5.)。 端粒酶的激活被认为是发生恶 性肿瘤的主要因素之一， 其激活及表达程度与肿瘤的发生和转移密切相关， 抑制 端粒酶并使端粒缩短被认为是抑制癌细胞的一种机制，因此端粒酶成为肿瘤靶向 治疗的理想靶点。 有许多公司正在开发端粒酶抑制剂治疗肿瘤， 其中 GRN163L 已经开始了临床 2期实验， 几个端粒酶疫苗也即将完成临床试验进入市场。
LPTS (Liver Putative Tumor Suppressor) 是本发明人利用定位克隆的方法 从人的正常肝 cDNA文库中得到的一个肝相关的候选抑癌新基因 [ C. Liao， M.J. Zhao, H. Song, K. Uchida, K.K. Yokoyama, T.P. Li, Identification of the gene for a novel liver-related putative tumor suppressor at a high-frequency loss of
heterozygosity region of chromosome 8p23 in human hepatocellular carcinoma.
Hepatology 2000, 32 721-727] 。 LPTS基因定位于人第 8号染色体 8p23区段， 该区段在多种恶性肿瘤细胞中高频缺失。研究表明 LPTS在肝癌组织及肝癌细胞 系中表达量极低或检测不到， 将 LPTS基因导入肝癌细胞， 能抑制肝癌细胞的生 长、 增殖、 最后引起死亡 [Liao C， Zhao MJ; Mutation analysis of novel human liver-related putative tumor suppressor gene in hepatocellular carcinoma. World J Gastroenterol, 2003， 9:89-93]]。 LPTS基因于 2004年已获得中国发明专利授权 (赵 慕钧等： "一种肝癌相关基因及其应用"， 专利号： ZL 00 1 15395. 1， 专利权人： 中国科学院上海生物化学研究所； 授权公告日： 2004年 10月 13 日）。 2001年 Lu博士的实验室报导了 LPTS基因的另一个全长转录本 PinXl ,发现 PinXl编码 的蛋白可以结合端粒酶催化亚基 hTERT并抑制端粒酶活性 [Zhou X.Z. , Lu K.P ； The Pin2/TRF 1 -interacting protein PinXl is a potent telomerase inhibitor. Cell, 2001， 107， 347-359] ， 从机制上第一次证明了 LPTS/PinXl作为一种天然的端 粒酶抑制蛋白可抑制肿瘤细胞的增殖， 为肿瘤的靶向治疗提供了新的途径。本发 明人在 2005年申请了一项有关制备 LPTS蛋白的发明专利 (赵慕钧等： "端粒酶 活性抑制蛋白的制备和纯化" ， 专利申请号： 200510030526.5， 申请日： 2005 年 10月 14日； 专利权人： 中国科学院上海生命科学研究院；)。 在该专利中提供 了 LPTS蛋白（LPGENEl)禾卩 LPTS抑制端粒酶的活性片段 LPTS 133-328(LPGENE2) 的制备方法， 并提供了 LPTS抑制端粒酶的活性片段位于其 C端的 133-328氨基 酸残基。 本发明人在 2008年申请的专利揭示了 TAT 与 LPTS 133-328 的融合蛋 白 （专利申请号： 200810041324.4)， 经证实该融合蛋白可穿透细胞膜的且具有极 其优异的抑制肿瘤细胞生长的效果。
鉴于 LPTS蛋白是一种与肿瘤细胞生长密切相关的重要的蛋白， 因此非常需 要对其进行进一步地深入研究， 以开发出更为有效的肿瘤抑制药物， 满足临床应 用所需。 发明内容
本发明的目的在于提供一种高效抑制端粒酶活性的蛋白肽段及其制备和应用
在本发明的第一方面， 提供一种分离的多肽 (蛋白；)， 所述多肽是：
(a) 包含 SEQ ID NO: 1所示氨基酸序列的多肽；
(b) SEQ ID NO: 1氨基酸序列经过一个或多个 (如 1-10个； 较佳地 1-5个； 更 佳地 1-3个；)氨基酸残基的取代、 缺失或添加而形成的， 且具有 (a)多肽功能的由 (a)衍生的多肽； 或
(c) 与 (a)限定的多肽序列有 90% (；较佳地 95%， 更佳地 98%， 最佳地 99%)以 上相同性且具有 (a) 多肽功能的由 (a)衍生的多肽。
在另一优选例中， 所述的多肽不具有 SEQ ID NO: 2所示氨基酸序列 (； LPTS全 长序列）， SEQ ID NO: 2中第 133-328位所示氨基酸序列 (LPTS13w28)和 SEQ ID NO: 2中第 254-328位所示氨基酸序列。
在另一优选例中， 所述的多肽包含 SEQ ID NO: 2 中第 255〜328 位所示氨 基酸序列， 或
包含 SEQ ID NO: 2 中第 256〜328 位所示氨基酸序列，或包含 SEQ ID NO: 2 中第 257〜328 位所示氨基酸序列， 或包含 SEQ ID NO: 2 中第 258〜328 位 所示氨基酸序列， 或
包含 SEQ ID NO: 2 中第 259〜328 位所示氨基酸序列，或包含 SEQ ID NO: 2 中第 260〜328 位所示氨基酸序列， 或包含 SEQ ID NO: 2 中第 261〜328 位 所示氨基酸序列， 或
包含 SEQ ID NO: 2 中第 262〜328 位所示氨基酸序列，或包含 SEQ ID NO: 2 中第 263〜328 位所示氨基酸序列， 或包含 SEQ ID NO: 2 中第 264〜328 位 所示氨基酸序列， 或
包含 SEQ ID NO: 2 中第 265〜328 位所示氨基酸序列，或包含 SEQ ID NO: 2 中第 266〜328 位所示氨基酸序列， 或包含 SEQ ID NO: 2 中第 267〜328 位 所示氨基酸序列， 或
包含 SEQ ID NO: 2 中第 268〜328 位所示氨基酸序列，或包含 SEQ ID NO:
2 中第 269〜328 位所示氨基酸序列， 或包含 SEQ ID NO: 2 中第 270〜328 位 所示氨基酸序列， 或
包含 SEQ ID NO: 2 中第 271〜328 位所示氨基酸序列，或包含 SEQ ID NO: 2 中第 272〜328 位所示氨基酸序列， 或包含 SEQ ID NO: 2 中第 273〜328 位 所示氨基酸序列， 或
包含 SEQ ID NO: 2 中第 274〜328 位所示氨基酸序列，或包含 SEQ ID NO: 2 中第 275〜328 位所示氨基酸序列， 或包含 SEQ ID NO: 2 中第 276〜328 位 所示氨基酸序列， 或
包含 SEQ ID NO: 2 中第 277〜328 位所示氨基酸序列，或包含 SEQ ID NO: 2 中第 278〜328 位所示氨基酸序列， 或包含 SEQ ID NO: 2 中第 279〜328 位 所示氨基酸序列， 或
包含 SEQ ID NO: 2 中第 280〜328 位所示氨基酸序列，或包含 SEQ ID NO: 2 中第 281〜328 位所示氨基酸序列， 或包含 SEQ ID NO: 2 中第 282〜328 位 所示氨基酸序列， 或
包含 SEQ ID NO: 2 中第 283〜328 位所示氨基酸序列，或包含 SEQ ID NO: 2 中第 284〜328 位所示氨基酸序列， 或包含 SEQ ID NO: 2 中第 285〜328 位 所示氨基酸序列， 或
包含 SEQ ID NO: 2 中第 286〜328 位所示氨基酸序列，或包含 SEQ ID NO: 2 中第 287〜328 位所示氨基酸序列， 或包含 SEQ ID NO: 2 中第 288〜328 位 所示氨基酸序列， 或
包含 SEQ ID NO: 2 中第 289〜328 位所示氨基酸序列，或包含 SEQ ID NO: 2 中第 290〜328 位所示氨基酸序列。 在本发明的另一方面， 提供一种分离的多核苷酸， 它含有一核苷酸序列， 该 核苷酸序列编码所述的多肽。 在本发明的另一方面， 提供一种载体， 它含有所述的多核苷酸。 在本发明的另一方面， 提供一种遗传工程化的宿主细胞， 它含有所述的载体
在本发明的另一方面， 提供一种所述的多肽的制备方法， 该方法包括：
(a) 在适合表达的条件下， 培养所述的宿主细胞；
(b) 从培养物中分离出所述的多肽。 在本发明的另一方面， 提供所述的多肽的用途， 用于制备抑制细胞内端粒酶 活性的组合物。
在另一优选例中， 所述的组合物用于防治端粒酶异常激活相关疾病。
在另一优选例中， 所述的端粒酶异常激活相关疾病是肿瘤。 在本发明的另一方面， 提供一种复合物， 所述的复合物包含所述的多肽， 以 及与该多肽相容的物质。
在另一优选例中， 所述的复合物是融合蛋白， 所述的多肽与至少一条功能性 蛋白相连接 (；较佳地通过肽键连接；)， 所述的功能性蛋白有 5-500个 (；较佳地 5-300 个； 更佳地 10-250个)氨基酸。
在另一优选例中， 所述的功能性蛋白选自： 穿膜蛋白 (如反式激活蛋白 ΤΑΊ 标签蛋白 (如 GST蛋白）， 报告蛋白 (如 GFP蛋白）， 人血清白蛋白 (延长半衰期)， 人 IgGl : Fc片段 (延长半衰期)。
在另一优选例中， 所述的多肽与所述的功能性蛋白直接相连， 或通过连接肽 连接。 所述连接肽的长度为 1-20个氨基酸， 更佳为 2-10个氨基酸。 如所述连接 肽的氨基酸序列为： GGS。
在另一优选例中， 所述的融合蛋白不具有 SEQ ID NO: 2所示氨基酸序列 (LPTS全长序列）， SEQ ID NO: 2中第 133-328位所示氨基酸序列 (LPTS13w28)和 SEQ ID NO: 2中第 254-328位所示氨基酸序列。
在另一优选例中， 所述的复合物包含选自以下的物质： 蛋白活性促进剂、 蛋 白活性稳定剂、 延长蛋白半衰期的制剂 (如 PEG, PEG-脂质体；)。 在本发明的另一方面， 提供一种组合物， 它含有安全有效量的所述的多肽或 所述的复合物， 以及药学上可接受的载体。
在另一优选例中， 所述的组合物用于抑制细胞内端粒酶活性。
在另一优选例中， 所述的组合物用于防治端粒酶活性增高肿瘤。 在本发明的另一方面， 提供一种制备组合物的方法， 所述组合物抑制细胞内 端粒酶活性， 所述方法包括： 将安全有效量的所述的多肽或所述的复合物与药学 上可接受的载体混合。 在本发明的另一方面， 提供一种药盒， 所述药盒中含有所述的多肽； 或含有 所述的复合物； 或含有所述的组合物。
在另一优选例中， 所述的药盒用于抑制细胞内端粒酶活性。
在另一优选例中， 所述的药盒用于防治端粒酶活性增高肿瘤。 在本发明的另一方面， 提供一种 (优选体外， 优选非治疗性地;)抑制细胞内端 粒酶活性的方法， 包括给予受试者有效量的所述的多肽； 或所述的复合物； 或所 述的组合物。 本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而 易见的。 附图说明
图 1、 SDS-PAGE显示 GST-LPTS29Q_328融合蛋白的诱导表达及纯化。
泳道 1为 IPTG诱导前的 GST-LPTS29Q_328基因工程菌表达的蛋白； 泳道 2为
IPTG诱导后该工程菌表达的蛋白； 泳道 3-5为纯化后收集到的 3管
GST-LPTS290-328蛋白。 图 2、 GST-LPTS290-328 - GST-LPTS133-328以及 GST-LPTS抑制端粒酶活性的检 测与比较。
A、 GST-LPTS290-328 - GST-LPTS133_328以及 GST-LPTS融合蛋白结构示意图;
B、 TRAP法检测 GST-LPTS290-328， GST-LPTS133-328以及 GST-LPTS蛋白抑制 端粒酶的活性。 蛋白的用量分别为 5、 10、 25、 50、 100、 200、 250nM， 如图所 示。 GST蛋白作为对照。 检测样品经 10%PAGE非变性胶电泳后银染结果。 图 3、 LPTS29Q_328对肝癌 BEL7404细胞生长的影响。
A、 Western Blot检测 GFP、 GFP-LPTS、 GFP-LPTS290-328在其稳定转染细胞 株 GFP/7404 , GFP-LPTS/7404和 GFP-LPTS29Q-328/7404中的表达。 兔源 anti-GFP 抗体作为杂交探针；
B、 MTT法绘制各稳定细胞株的生长曲线图；
C、 GFP/7404, GFP-LPTS/7404和 GFP-LPTS29。-328/7404等细胞培养过程中细 胞状态图。 图中箭头所指为处于危机期、 衰老症状的细胞，
D、 为脱壁死亡后的 GFP-LPTS29(«28/7404细胞。 图 4、 Southern Blot实验检测 LPTS和 LPTS29(«28对肝癌 BEL7404细胞端粒 长度的影响。 A、 转染 GFP或 GFP-LPTS的 BEL7404细胞经 FACS分选后持续培养， 选取 不同的培养代数 (； PD)细胞如图所示， 抽取其基因组 DNA， 经 Hinf l和 Afa l内切 酶消化后与单链端粒重复序列 32p-(TTAGGG)6探针杂交；
B、转染 GFP或 GFP-LPTS29Q_328的 BEL7404细胞经 FACS流式细胞仪分选后 倍增 8代， 抽取其基因组 DNA， 经 Hinf l和 Afa l内切酶消化后与单链端粒重复 序列 32p-(TTAGGG)6探针杂交。 本图为放射自显影后的结果。 具体实施方式
本发明人经过深入的研究，首次分离到 LPTS蛋白 （全长序列如 SEQ ID NO: 2) 的抑制端粒酶的活性区域， 该活性区域位于 LPTS蛋白的第 290-328位
(LPTS29Q_328)。 该活性区域的活性超过全长 LPTS蛋白及 LPTS上的其它片段， 并 能更快地诱导肿瘤细胞死亡。本发明为肿瘤的靶向治疗提供了更为有效的抑制端 粒酶活性的蛋白。 术语
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， "对象" 、 "个体" 、 或 "患者" 指需要进行诊断或治疗的 任何目标， 尤其是哺乳动物对象， 特别是人， 其它对象包括牛、 狗、 猫、 豚鼠、 兔、 大鼠、 小鼠、 马等。 特别受关注的是那些端粒酶异常激活的对象。
如本文所用， "核酸"和 "核酸序列"指聚合形式的任意长度的核苷酸 (核糖 核苷酸或脱氧核糖核苷酸；)。 它包括 (；但不限于;)单链、 双链的 DNA或 RNA， 基因 组 DNA和 cDNAo
如本文所用， "药学上可接受的"的成分是适用于人和 /或哺乳动物而无过度 不良副反应 （如毒性、 剌激和变态反应） 的， 即具有合理的效益 /风险比的物质。 术语 "药学上可接受的载体"指用于治疗剂给药的载体， 包括各种赋形剂或稀释 剂。
如本文所用， "有效量" 或 "安全有效量" 指以单剂或连续剂一部分给予个 体的量对治疗或预防是有效的。 该用量根据所治疗个体的健康状况和生理状况、 所治疗个体的类别 （如非人灵长类等；)、 治疗医师对医疗状况的评估、 及其它的 相关因素而定。 预计该用量将在相对较宽的范围内， 可通过常规实验来确定。
如本文所用， 所述的 "含有" ， "具有" 或 "包括"包括了 "包含" 、 "主 要由 ......构成" 、 "基本上由 ......构成" 、 和 "由 ......构成" ； "主要由 ......构 成" 、 "基本上由 ......构成"和 "由 ......构成"属于 "含有" 、 "具有 "或 "包 括" 的下位概念。 本发明的多肽及其编码基因
LPTS是第一个被发现可以直接和人端粒酶催化亚基 hTERT结合并抑制端 粒酶催化活性的蛋白。 本发明人以全长的 LPTS蛋白为基础， 预测并筛选了多种 LPTS序列片段， 经过反复研究比较， 发现 LPTS蛋白抑制端粒酶的活性区域可 以缩小到该蛋白的第 290-328位氨基酸序列区域内， 该位置才是抑制端粒酶催化 活性的最关键区域， 其足以发挥抑制端粒酶催化活性的作用， 从而获得了本发明 的多肽。
为了验证所述的多肽的功能， 本发明人在体外通过基因工程技术表达了 GST-LPTS29Q_328融合蛋白。 在本发明的一个实施例中， 采用测定端粒酶活性的 TRAP(telomeric repeat amplification protocol)实验技术， 检测了 GST-LPTS290-328融 合蛋白在体外抑制肿瘤细胞端粒酶的活性。 该方法是一种基于 PCR技术的端粒酶 活性检测方法，端粒酶来源于一种肝癌细胞裂解液。检测结果发现 GST-LPTS29()_328 有很强的抑制端粒酶的活性。 为此， 本发明人进一步比较了 GST-LPTS29Q_328与 GST-LPTS和 GST-LPTS13w28蛋白的活性。 检测结果表明 GST-LPTS和
GST-LPTS133_328蛋白在 50nM的时候显示有抑制端粒酶的活性， 100nM的时候抑 制活性较强，但不能完全抑制反应体系中的端粒酶； 而 LPTS29Q_328在 50nM的时候 抑制活性已很强， 在 ΙΟΟηΜ的时候可完全抑制体系中的端粒酶活性。 以上结果说 明 LPTS29Q_328具有比全长 LPTS和 LPTS133_328更强的端粒酶抑制活性， 是 LPTS蛋 白抑制端粒酶活性的功能域。
为了检测 1^1829()_328在体内抑制肿瘤细胞的活性， 在本发明的一个实施例中， 构建了 LPTS29Q_328、 LPTS与绿色荧光蛋白 GFP融合的真核表达质粒，
GFP-LPTS29(«28和 GFP-LPTS。 在肝癌 BEL7404细胞中分别转染 GFP-LPTS29(«28， GFP-LPTS以及对照 GFP表达质粒， 经 2周的 G418筛选之后， 分别采用流式细胞 仪 FACS分选出有绿色荧光蛋白表达的细胞， 然后进行传代培养。 分选得到的 GFP-LPTS290-328/7404, GFP-LPTS/7404, GFP/7404细胞， 利用兔源 anti-GFP多克 隆抗体进行 western blot检测， 发现都可以稳定表达相应的蛋白。 在细胞传代培养 的过程中， GFP-LPTS29(«28/7404细胞的生长较 GFP-LPTS/7404和 GFP/7404的要 慢。 本发明人选取 FACS分选后倍增 5代的上述稳定细胞株， 进行了 MTT试验， 并绘制生长曲线。 结果证明， 与对照 GFP/7404细胞相比较， GFP-LPTS29Q_328/7404 细胞生长最慢， GFP-LPTS/7404渐次。 说明 LPTS29Q_328抑制肿瘤细胞的生长能力 较其全长蛋白 LPTS要强。 在肿瘤细胞中过表达 LPTS蛋白， 可以导致细胞生长变 慢、 变扁平、 进入危机期， 最终死亡。 但这是一个较长期的效应， 一般需要培养 6 周后出现。 细胞在转染 LPTS29Q_328后， 很快发生死亡， 经 2周的 G418筛选之后只 能获得少量细胞进行 FACS分选， 分选获得的 LPTS29Q_328/7404细胞继续培养 10 天左右， 就相继出现衰老症状， 并且很快就全部变圆后脱壁死亡。 结果说明过表 达 LPTS29Q_328导致肿瘤细胞死亡的能力很强，可能比 LPTS全长蛋白抑制肿瘤的效 率要高， 更有应用价值。
为了证明 LPTS29Q_32^ 制肿瘤细胞是靶向抑制细胞端粒的合成， 在本发明的 一个实施例中， 采用 Southern Blot方法检测了 GFP-LPTS29(«28/7404，
GFP-LPTS/7404, GFP/7404细胞端粒的长度。 实验结果表明， 对照 GFP/ 7404细 胞传代过程中端粒保持稳定， 长度在 4.5kb左右； GFP-LPTS/7404细胞在传代过程 中端粒逐渐缩段， 在培养第 5代时端粒缩短至 3.8kb左右， 第 25代的时候端粒缩 短到 2.8kb左右； GFP-LPTS29Q_328/7404细胞传代时间短， 培养过程中细胞死亡多， 在第 8代时端粒已经缩短至 2.5kb左右了。 结果说明， LPTS29Q_328有着非常强的端 粒酶抑制活性， 其在细胞体内能抑制端粒的合成和延伸， 是一种靶向抑制端粒酶 的肿瘤抑制肽。
由于本发明揭示了抑制端粒酶催化活性的最关键区域， 因此可以理解， 一些 蛋白（比如一些包含有本发明的多肽的融合蛋白；)， 只要它们包含有该最关键区域、 且不包含影响该关键区域结构或活性的因素 (可通过有限次实验方便地验证；)，也将 具有抑制端粒酶催化活性的作用， 这些蛋白也被包含在本发明中。 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。
本发明还包括所述多肽的片段、 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所述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 (ϋ)在一个或多个氨基酸残 基中具有取代基团的多肽，或 ( )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 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。
本发明还包括具有与所述多肽相同功能的、 SEQ ID NO: 1序列的变异形式。 这些变异形式包括 (但并不限于 一个或多个 (通常为 1-10个， 较佳地 1-5个， 更佳地 1-3个， 最佳地 1-2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 10个以内， 较佳地为 5个以内， 更佳地为 3 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。该术语还包括所述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所述多肽杂交的 DNA所编码的 蛋白、 以及利用抗所述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， 如包含所述多肽或其片段的融合蛋白。
发明还提供所述多肽或多肽的类似物。 这些类似物与天然所述多肽的差别可 以是氨基酸序列上的差异， 也可以是不影响序列的修饰形式上的差异， 或者兼而 有之。这些多肽包括天然或诱导的遗传变异体。诱导变异体可以通过各种技术得 到， 如通过辐射或暴露于诱变剂而产生随机诱变， 还可通过定点诱变法或其他已 知分子生物学的技术。类似物还包括具有不同于天然 L-氨基酸的残基 (如 D-氨基 酸)的类似物， 以及具有非天然存在的或合成的氨基酸 (如 β、 Υ -氨基酸)的类似 物。 应理解， 本发明的多肽并不限于上述例举的代表性的多肽。
修饰 (通常不改变一级结构)形式包括： 体内或体外的多肽的化学衍生形式如 乙酰化或羧基化。修饰还包括糖基化， 如那些在多肽的合成和加工中或进一步加 工步骤中进行糖基化修饰而产生的多肽。这种修饰可以通过将多肽暴露于进行糖 基化的酶 (如哺乳动物的糖基化酶或去糖基化酶)而完成。 修饰形式还包括具有磷 酸化氨基酸残基 (如磷酸酪氨酸， 磷酸丝氨酸， 磷酸苏氨酸)的序列。 还包括被修 饰从而提高了其抗蛋白水解性能或优化了溶解性能的多肽。
在本发明中， "所述多肽保守性变异多肽"指与 SEQ ID NO: 1的氨基酸序列 相比， 有至多 10个， 较佳地至多 5个， 更佳地至多 3个， 最佳地至多 2个氨基 酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据 表 1进行氨基酸替换而产生。
Figure imgf000012_0001
Val (V) lie; Leu; Met; Phe; Ala Leu 本发明所述多肽的用途包括 (但不限于；)： 直接做为药物治疗端粒酶异常激活 相关的疾病 (如肿瘤)。
含有或偶联有所述多肽的融合分子也包括在本发明中。 例如， 可构建本发明 的多肽与靶向分子的融合分子；该种融合分子由于包含了靶向于特定组织或器官 的分子而可实现在局部区域增加药物的浓度， 而不影响其它区域； 所述的靶向分 子例如是： 抗体、 配体等。 又例如， 可构建本发明的多肽与多聚分子的融合分子 ； 该融合分子的半衰期增长； 所述的多聚分子例如是 IgG Fc。
含有所述多肽的编码序列的融合基因也包括在本发明中。 例如， 可构建含有 所述多肽的编码序列以及组织或器官特异性启动子 (两者操作性连接；)的融合基 因， 所述基因当施加于体内后， 被启动子驱动在特定的组织或器官内表达。
本发明还包含一些复合物， 该复合物含有本发明的多肽， 以及与本发明的多 肽相连接或偶联的其它功能性蛋白或分子。 这些功能性蛋白 (有 5-500个； 较佳 地 5-300个； 更佳地 10-250个氨基酸)包括但不限于： 穿膜蛋白， GST蛋白 (纯化 标签)， GFP蛋白 (报告蛋白）， 人血清白蛋白 (延长半衰期)， 人 IgGl : Fc片段 (延 长半衰期;)等。 其它分子还可以是选自以下的物质： 蛋白活性促进剂、 蛋白活性 稳定剂、 延长蛋白半衰期的制剂。 所述的延长蛋白半衰期的制剂例如是 PEG (可 用于与本发明的多肽的氨基端或羧基端联接；)， PEG-脂质体 (可用于包埋本发明的 多肽）， 所述的 PEG的分子量可以是 1000-50000; 较佳地 20000-40000。
作为本发明的一种实施方式， 所述的多肽可与一些对于穿透细胞膜有效的分 子融合或偶联，从而更方便地进入到细胞内发挥作用。已知的穿膜蛋白有许多种， 包括： 反式激活蛋白 TAT、 Penetratin、 基于信号序列的肽、 pVEC、 Transportan, Amphiphilic model peptide禾口 Arg9等等。 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。
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： （1)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。
本发明还涉及与上述的序列杂交的核酸片段。 如本文所用， "核酸片段" 的 长度至少含 15个核苷酸， 较好是至少 30个核苷酸， 更好是至少 50个核苷酸， 最好是至少 100个核苷酸以上。核酸片段可用于核酸的扩增技术 (如 PCR)以确定 和 /或分离编码本发明所述多肽的多聚核苷酸。
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。 本发明的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。
目前， 已经可以完全通过化学合成来得到编码本发明蛋白 (或其片段， 或其衍 生物;)的 DNA序列。然后可将该 DNA序列引入本领域中已知的各种现有的 DNA 分子 (或如载体)和细胞中。 此外， 还可通过化学合成将突变引入本发明蛋白序列 中。
应用 PCR技术扩增 DNA/RNA的方法 (Saiki, et al. Science 1985;230： 1350-1354) 被优选用于获得本发明的基因。特别是很难从文库中得到全长的 cDNA时， 可优 选使用 RACE法 (RACE-cDNA末端快速扩增法；)，用于 PCR的引物可根据本文所 公开的本发明的序列信息适当地选择， 并可用常规方法合成。可用常规方法如通 过凝胶电泳分离和纯化扩增的 DNA/RNA片段。 载体和细胞
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 (Science, 1984； 224： 1431)， 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本发明的多肽。 一般来说有以下步骤：
(1) .用本发明的编码所述多肽的多核苷酸 (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。
本发明中， 本发明的多核苷酸序列可插入到重组表达载体中。 术语 "重组表 达载体"指本领域熟知的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动 物细胞病毒如腺病毒、 逆转录病毒或其他载体。 只要能在宿主体内复制和稳定， 任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动 子、 标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含本发明的多核苷酸序列和合适的 转录 /翻译控制信号的表达载体。 这些方法包括体外重组 DNA技术、 DNA合成 技术、 体内重组技术等。 所述的 DNA序列可有效连接到表达载体中的适当启动 子上， 以指导 mRNA合成。 这些启动子的代表性例子有： 大肠杆菌的 lac或 trp 启动子； λ噬菌体 PL启动子； 真核启动子包括 CMV立即早期启动子、 HSV胸 苷激酶启动子、 早期和晚期 SV40启动子、 反转录病毒的 LTRs和其他一些已知 的可控制基因在原核或真核细胞或其病毒中表达的启动子。表达载体还包括翻译 起始用的核糖体结合位点和转录终止子。
此外， 表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择转 化的宿主细胞的表型性状， 如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以 及绿色荧光蛋白 (GFP)， 或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当 DNA序列以及适当启动子或者控制序列的载体， 可以用于 转化适当的宿主细胞， 以使其能够表达蛋白质。
宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高等真核细胞， 如哺乳动物细胞。 代表性例子有： 大肠杆菌， 链霉菌属； 鼠 伤寒沙门氏菌的细菌细胞； 真菌细胞如酵母； 植物细胞； 果蝇 S2或 Sf9的昆虫 细胞； CHO、 COS, 293细胞、 或 Bowes黑素瘤细胞的动物细胞等。
本发明的多核苷酸在高等真核细胞中表达时， 如果在载体中插入增强子序列 时将会使转录得到增强。 增强子是 DNA的顺式作用因子， 通常大约有 10到 300 个碱基对， 作用于启动子以增强基因的转录。可举的例子包括在复制起始点晚期 一侧的 100到 270个碱基对的 SV40增强子、在复制起始点晚期一侧的多瘤增强 子以及腺病毒增强子等。
本领域一般技术人员都清楚如何选择适当的载体、 启动子、 增强子和宿主细 胞。
用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。 当宿 主为原核生物如大肠杆菌时，能吸收 DNA的感受态细胞可在指数生长期后收获， 用 CaCl2法处理， 所用的步骤在本领域众所周知。 另一种方法是使用 MgCl2。 如 果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的 DNA 转染方法： 磷酸钙共沉淀法， 常规机械方法如显微注射、 电穿孔、 脂质体包装等 获得的转化子可以用常规方法培养， 表达本发明的基因所编码的多肽。 根据 所用的宿主细胞， 培养中所用的培养基可选自各种常规培养基。在适于宿主细胞 生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法 (如 温度转换或化学诱导;)诱导选择的启动子， 将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、 或在细胞膜上表达、 或分泌到细胞 外。 如果需要， 可利用其物理的、 化学的和其它特性通过各种分离方法分离和纯 化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并 不限于： 常规的复性处理、 用蛋白沉淀剂处理 (盐析方法)、 离心、 渗透破菌、 超 处理、 超离心、 分子筛层析 (凝胶过滤)、 吸附层析、 离子交换层析、 高效液相层 析 (HPLC)和其它各种液相层析技术及这些方法的结合。 作为本发明的一个具体实施方式， 主要通过 PCR扩增获得 LPTS29Q_328基因 序列， 通过特异设计引物从模板 pT-LPTS质粒扩增。 将 LPTS29Q_328基因片段， 插入 pGEX-4T-l质粒， 该质粒可表达 GST荧光蛋白， 得到 pGEX-LPTS29Q_328重 组质粒。 将 pGEX-LPTS29Q_328重组质粒转化表达宿主菌 co// BL-21(DE3)， 从而 得到 GST- LPTS29Q_328融合蛋白表达工程菌。 GST-LPTS29Q_328在大肠肝菌中通过 IPTG进行诱导表达，使用商品化的亲和纯化柱 GS-4B进行 GST-LPTS29Q_328融合 蛋白的纯化制备。 组合物
本发明还提供了包含本发明的多肽的各种组合物， 特别是药物组合物。 该组 合物可用于预防或治疗端粒酶异常激活相关疾病， 所述疾病包括 (但不限于 肿 瘤。
包含本发明的多肽的各种组合物可以包含按多肽的实际用途所选用的缓冲剂 ； 还可包含适用于预定用途的其它物质。本领域技术人员都善于选择合适的缓冲 剂， 本领域已知有多种缓冲剂适用于预定用途。 在有些实例中， 该组合物可含有 药学上可接受的赋形剂， 本领域已知有多种而无需在此详细讨论。药学上可接受 的各种赋形剂在多种出版物已有详述， 包括如 "Remington's Pharmaceutical Sciences" ( 《雷明顿药物科学》 ， 第 19版（1995)Mack Publishing Co.)。
可将本发明的组合物制备成各种剂型， 如注射剂、 粒剂、 片剂、 丸剂、 栓剂、 胶囊、 悬浮液、 喷雾、 栓剂、 透皮药物 (如贴片等；)、 油膏、 洗剂等。 适用于口服 或局部使用的药用级别的有机或无机载体和 /或稀释剂， 可用于配制包含治疗活 性化合物的各种组合物。本领域已知的稀释剂包括水性介质、植物性和动物性油 和脂肪。 还可用稳定剂、 润湿剂和乳化剂、 改变渗透压的盐类或维持合适 pH值 的各种缓冲剂和皮肤渗透增强剂等作为辅助性材料。
给予本发明组合物的常规和药学上可接受的途径包括： 肌内、 皮下、 皮内、 肺内、 静脉内、 瘤内、 经鼻、 经口服或其它肠胃外给药途径。 如果需要可以组合 给药途径， 或按疾病情况进行调节。 可以单剂量或多剂量给予。
所选用的多肽的量， 是按可产生抑制端粒酶活性的效果而无明显的副作用的 量而定。 通常， 给予约 O.Ol w g-lOmg多肽 /kg体重， 优选 O. l w g-lmg多肽 /kg体 重， 更优选0.1 4 8-10(^ 8多肽/1¾体重。 药盒
本发明还提供了一种防治端粒酶异常激活相关疾病的药盒， 其中含有本发明 的多肽或含有该多肽的组合物。 此外， 为了方便给药， 所述的药盒中还可含有注 射用的针， 和 /或药学上可接受的载体， 和 /或使用说明书。 本发明的优点：
1、首次发现 LPTS29Q_328在体外具有非常显著的端粒酶抑制活性，而且是 LPTS 中已知的具有端粒酶抑制能力最强的区域。
2、首次发现异源表达 LPTS29Q_328可以抑制肿瘤细胞生长， 导致细胞端粒缩短 并最终死亡。 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： 实验室指南 （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。
除非另行定义， 文中所使用的所有专业与科学用语与本领域熟练人员所熟悉 的意义相同。此外， 任何与所记载内容相似或均等的方法及材料皆可应用于本发 明中。 文中所述的较佳实施方法与材料仅作示范之用。 实施例 1、 LPTS29o_328基因片段的制备
pT-LPTS质粒的构建： 设计引物 L1 :
5 ' - AGGAATTC ATGTCT ATGCTGGCTGAACG-3 ' (SEQ ID NO: 3)； 禾卩 L2:
5'-ACGCTCGAGCTTTGGAATCTTTCTTCTTCT-3'(SEQ ID NO: 4)。 利用该对引 物从正常人肝组织中反转录 PCR扩增获得 LPTS全长 cDNA片段。 PCR产物装 入 pMD-18T载体 (购自 TaKaRa公司）， 从而获得 pT-LPTS质粒。
LPTS29Q_328基因片段通过 PCR方法从 pT-LPTS质粒扩增 (该质粒含有 LPTS 基因全长 cDNA片段)。 设计 PCR引物 P1 :
5 ' - AGGAATTC ACCCTGAAGCCCAAAAAGAGG -3，（SEQ ID NO: 5)和 P2:
5'-ACGCTCGAGCTTTGGAATCTTTCTTCTTCTTCT -3'(SEQ ID NO: 6)。 以 pT-LPTS质粒为模板， 用引物 PI和 P2进行 PCR反应。 PCR反应条件为： 94 °C 30秒， 55°C退火 30秒， 72°C延伸 30秒， 共扩增 30个循环。 PCR扩增后得到的 DNA产物经 DNA测序确定无误后， 即获得 LPTS29Q_328基因片段，用于后续 GST 融合蛋白和真核表达质粒的构建。该 LPTS29Q_328基因序列所编码的氨基酸序列见 SEQ ID NO: 1。
作为对照用的 LPTS133_328基因片段通过上述相同方法获得， PCR引物为 P3 : 5 ' - ACGCTCGAGAAGGATCTGTC ATCTCGG-3 ' (SEQ ID NO: 7)和 P2。LPTS基因 序列从质粒 pT-LPTS中通过 EcoR I和 Xho l双酶切后得到， 该基因片段编码的 蛋白对应于 LPTS蛋白的全长。 实施例 2、 GST-LPTS29o_328融合蛋白表达工程菌的构建和诱导
实施例 1中得到的 LPTS29Q_328基因片段和 LPTS133_328基因片段均经 EcoR I 和 Xho I双酶切， 然后插入 pGEX-4T-l质粒 (购自 Amersham Pharmacia公司）， 该质粒能表达 GST蛋白。 实施例 1中得到的 LPTS基因片段可直接插入 pGEX-4T-l质粒，获得 pGEX-LPTS290-328、 pGEX-LPTS133-328禾卩 pGEX-LPTS重组 质粒。将这三种重组质粒分别转化宿主菌 co// DH5a， 再从 co// DH5a转化菌 株中抽取质粒， 测序验证无误后再转化蛋白表达宿主菌 co// BL-21(DE3)， 从 而得到 GST-LPTS29(«28、 GST-LPTS13w28和 GST-LPTS融合蛋白表达工程菌株。
GST-LPTS29Q-328在大肠肝菌 BL-21 (DE3) 中的诱导表达。 含
pGEX-LPTS29Q_328质粒的融合蛋白表达工程菌先 37°C过夜扩增培养，第二天按照 1: 100比例接种于 400毫升含 Ampf抗生素的 LB培养基中， 37°C继续培养至 OD600约等于 0.6时加入 IPTG至终浓度 0.5 mM， 37°C诱导表达 3-4小时。 5000 rpm， 10分钟离心收集菌体， 弃上清， 剩下的菌体保存于 -80°C备用。 该菌体含 有目标 GST-LPTS29Q_328融合蛋白 (；见图 1)。 采用同样的方法， 本发明人诱导表达 了 08丁-1^丁8133_328和 GST-LPTS融合蛋白。 GST以及上述融合蛋白的结构示意 图见图 2A。
图 1是 10%SDS-PAGE胶电泳后考马斯亮蓝染色后的结果， 泳道 1为 IPTG 诱导前的工程菌， 泳道 2为 IPTG诱导后的工程菌， 在 40kD处有特异条带被诱 导出， 为 GST-LPTS29(«28蛋白。 实施例 3、 GST-LPTS29()_328融合蛋白的纯化制备
GST-LPTS290-328融合蛋白的纯化制备使用商品化的亲和纯化柱 GS-4B (；购自 Sigma公司）进行。 该亲和纯化柱为还原性谷胱苷肽 GSH和 Sepharose 4B耦合而 成，上样前先按照厂商说明装填至终体积 2毫升左右，并用 20〜30 ml溶液 A (20 mM Tris-HCl pH 7.4, 0.2 mM EDTA, I mM DTT, 0.5mM PMSF, 1 M NaCl)平 衡。 实施例 2中最终获得的菌体用 10 ml溶液 A重悬， 超声波破碎细菌 (；超声仪 为宁波新芝制造;)，超声条件如下：工作时间 7秒，间隙时间 25秒，工作功率 400W, 工作次数 20-30次）。 超声后的菌体液加入 TritonX-100至终浓度 1%， 冰上放置 30分钟； 然后 12000 rpm， 4°C下离心 10分钟， 离心后的上清转移到新的离心管 继续重复离心一次； 再次离心后得到的含融合蛋白的上清液， 连到平衡好的 GS-4B纯化柱， 在压力差下过柱； 样品过柱完后， 用 20〜30 ml溶液 A洗柱子； 然后继续用 20〜30 ml溶液 B (20 mM Tris-HCl pH 7.4， 0.2 mM EDTA， 0.1 M NaCl) 洗柱子； 最终用 5 ml溶液 C (15 mM还原型谷胱苷肽 GSH， 20 mM Tris-HCl pH 7.4， 0.2 mM EDTA, 0.1 M NaCl )洗脱蛋白， 共收集 3管， 每管约 1.5毫升， 收 集的纯化后蛋白可于 -80°C保存备用。 采用同样的方法， 本发明人纯化获得了 GST-LPTS133-328禾卩 GST-LPTS蛋白备用。
图 1中泳道 3， 4， 5为洗脱收集到的 3管蛋白。 可以看到， 纯化后得到的 GST-LPTS29Q_328蛋白条带单一， 纯度可以达到 95%以上， 可以用来做进一步的应 用。 收集到的 GST-LPTS29(«28蛋白经 Bradford法测定浓度。 实施例 4、 GST-LPTS29o_328融合蛋白抑制端粒酶活性的检测以及与
GST-LPTS, GST-LPTS133-328蛋白抑制活性的比较
为了检测 08^1^1829()_328融合蛋白抑制端粒酶的活性， 本发明人采用了体 外 TRAP实验。 TRAP(telomeric repeat amplification protocol)是一种基于 PCR技 术的端粒酶活性检测方法。 首先制备含有端粒酶的肝癌 BEL7404细胞裂解液， 将处于对数生长旺盛期的 BEL7404细胞 (购自中国科学院上海细胞库;)用
Washing Buffer(10 mM Hepes-KOH pH7.5， 1.5mM MgCl2， 10 mM KC1， 1 mM DTT) 洗 2遍； 每 106个细胞用 1毫升冰预冷的 Lysis Buffer ( 10 mM Tris-HCl pH 7.5， 1 mM MgCl2， 1 mM EGTA， 0.1 mM PMSF, 5 mM巯基乙醇， 0.5% CHAPS, 10% glycerol )重悬， 置冰上裂解 30分钟； 然后 15000rpm， 4°C高速离心 30分钟， 得 到的上清即为含有端粒酶的肝癌 BEL7404细胞裂解液。 该细胞裂解液可保存在 -80°C冰箱。
TRAP反应先取 l L 细胞裂解液， 加入 GST-LPTS29Q_328蛋白或者其他待测 定蛋白， 冰上混合 10分钟； 然后再加入 l L Ts 引物 （0.1 g/ L， 序列为 5 ' -AATCCGTCGAGCAGAGTT-3 ' )， 0.25 10 mM dNTP， 42 反应缓冲 液 （20 mM Tris-HCl pH8.3， 1.5 mM MgCl2， 63 mM KC1， 0.005% Tween-20, 1 mM EGTA， 0.1 mg/ml BSA)， 以上总体积一共为 50 μ ■, 然后在 25 °C下延伸反应 30 分钟， 90°C灭活 3分钟； 再加入 l L Cx 引物 (序列为
5， -GCGCGG(CCCTTA)3CCCTAA-3， )， 0.5 μ (2 U) Taq酶，进行 PCR反应 (94 。C 40秒， 50°C退火 40 秒， 72°C延伸 1分钟， 扩增 30个循环）； PCR产物进行
10% PAGE非变性胶分离后银染显色。 显色结果中条带越多说明体系中的端粒 酶活性越高， 反之说明体系中的端粒酶活性被抑制的越多。
图 2B为 TRAP实验结果， 从图中可以看出 GST-LPTS和 GST-LPTS13^328蛋 白在 50nM的时候显示有抑制端粒酶的活性， ΙΟΟηΜ的时候抑制活性较强， 但不 能完全抑制反应体系中的端粒酶；而 GST-LPTS29Q_328在 50nM的时候抑制活性已 很强， 在 ΙΟΟηΜ的时候可完全抑制体系中的端粒酶活性。
以上结果说明， LPTS29Q_328具有比全长 LPTS和 LPTS133_328更强的端粒酶抑 制活性， 是 LPTS蛋白抑制端粒酶活性的功能域。 实施例 5、 LPTS29()_328抑制肝癌细胞 BEL7404的生长并导致其死亡 为了检测 1^1829()_328在体内抑制肿瘤细胞的活性， 本发明人构建了
LPTS29Q_328、 LPTS与绿色荧光蛋白 GFP融合的真核表达质粒。 具体操作如下： 将实施例 1中得到的 LPTS29Q_328基因片段， 经 EcoR I和 Xho l双酶切后插入 pEGFP-C2质粒 (；购自 Clontech；)， 得到 pEGFP-LPTS29(«28表达质粒。 利用 EcoR I 和 Xho I双酶切 pT-LPTS质粒得到 LPTS基因 cDNA片段， 同样插入 pEGFP-C2 质粒， 得到 pEGFP-LPTS表达质粒。 pEGFP-C2可表达 GFP蛋白作为对照。 上 述质粒转染 BEL7404细胞后可以表达相应的蛋白。
在肝癌 BEL7404细胞中分别转染 GFP-LPTS29Q_328，GFP-LPTS以及对照 GFP 表达质粒， 经 2周的 G418筛选之后， 分别采用流式细胞仪 FACS分选出有绿色 荧光蛋白表达的细胞， 然后进行传代培养。 分选得到的 GFP-LPTS29Q_328/7404， GFP-LPTS/7404, GFP/7404细胞，利用兔源 anti-GFP多克隆抗体进行 Western Blot 检测， 发现都可以稳定表达相应的蛋白， 如图 3A中所显示。
在细胞传代培养的过程中， GFP-LPTS29Q_328/7404细胞的生长较
GFP-LPTS/7404和 GFP/7404的要慢。 本发明人选取 FACS分选后倍增 5代的上 述稳定细胞株，进行了 MTT试验，并绘制生长曲线。结果证明，与对照 GFP/7404 细胞相比较， GFP-LPTS29(«28/7404细胞生长最慢， GFP-LPTS/7404渐次， 见图 3B。
说明 LPTS29Q_328抑制肿瘤细胞的生长能力较其全长蛋白 LPTS要强。
在肿瘤细胞中过表达 LPTS蛋白， 可以导致细胞生长变慢、 变扁平、 进入危 机期， 最终死亡。 这是由于 LPTS蛋白抑制了肿瘤细胞的端粒酶活性， 使细胞的 端粒不能延伸而变短， 导致细胞衰老而死亡。 但这是一个较长期的效应， 一般需 要培养 6周后出现。但细胞在转染 LPTS29Q_328后，很快发生死亡，经 2周的 G418 筛选之后只能获得少量细胞进行 FACS分选， 分选获得的 LPTS29Q_328/7404细胞 继续培养 10天左右， 就相继出现衰老症状， 并且很快就全部变圆后脱壁死亡， 见图 3C。
以上结果说明， 过表达 LPTS29Q_328导致肿瘤细胞死亡的能力很强， 比 LPTS 全长蛋白抑制肿瘤的效率要高， 更有应用价值。 实施例 6、 LPTS29()_328缩短肝癌 BEL7404细胞端粒长度
为了证明 LPTS29Q_32^ 制肿瘤细胞是靶向抑制细胞端粒的合成，本发明人采 用 Southern Blot方法检测了 GFP-LPTS29。-328/7404， GFP-LPTS/7404, GFP/7404 细胞端粒的长度。 采用 FACS分选后后的细胞， 按图 4A所示的培养代数收集细 胞， 抽取细胞的基因组 DNA， 用 Hinf l和 Afa l内切酶消化后， 与同位素标记的 32P-(TTAGGG)6 探针杂交， 该探针为端粒单链 DNA重复序列 (TTAGGG), 分析 细胞的端粒长度。 端粒长短视放射自显影后的条带灰度以及处于的位置综合判 断， 条带离上端加样孔的位置越近， 对应细胞的端粒越长。从图 4A中可以看到， 对照 GFP/7404细胞传代过程中端粒保持稳定，长度在 4.5kb左右； GFP-LPTS/7404 细胞在传代过程中端粒逐渐缩短， 在培养第 5代时端粒缩短至 3.8kb左右， 第 25 代的时候端粒缩短到 2.8kb左右； GFP-LPTS29Q_328/7404细胞传代时间短， 培养过 程中细胞死亡多， 在第 8代时端粒已经缩短至 2.5kb左右了 (；图 4B；)。
结果说明， LPTS29Q_328有着非常强的端粒酶抑制活性， 其在细胞体内能靶向 抑制端粒的合成和延伸。 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
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<120> 一种抑制端粒酶活性的肽及其制备方法和应用
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Claims

权 利 要 求
1. 一种分离的多肽， 其特征在于， 所述多肽是：
(a) 包含 SEQ ID NO: 1所示氨基酸序列的多肽；
(b) SEQ ID NO: 1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 (a)多肽功能的由 (a)衍生的多肽； 或
(c) 与 (a)限定的多肽序列有 90%以上相同性且具有 (a)多肽功能的由 (a)衍生的 多肽。
2. 一种分离的多核苷酸， 其特征在于， 它含有一核苷酸序列， 该核苷酸序列 编码权利要求 1所述的多肽。
3. 一种载体， 其特征在于， 它含有权利要求 2所述的多核苷酸。
4.一种遗传工程化的宿主细胞， 其特征在于， 它含有权利要求 3所述的载体
5. —种权利要求 1的多肽的制备方法， 其特征在于， 该方法包括：
(a) 在适合表达的条件下， 培养权利要求 4所述的宿主细胞；
(b) 从培养物中分离出权利要求 1的多肽。
6. 权利要求 1所述的多肽的用途， 用于制备抑制细胞内端粒酶活性的组合物
7. 如权利要求 6所述的多肽的用途， 所述的组合物用于防治端粒酶异常激活 相关疾病。
8. 如权利要求 7所述的用途， 其特征在于， 所述的端粒酶异常激活相关疾病 是肿瘤。
9. 一种复合物， 所述的复合物包含权利要求 1所述的多肽， 以及与该多肽相 容的物质。
10. 如权利要求 9所述的复合物， 其特征在于， 所述的复合物是融合蛋白， 权利要求 1所述的多肽与至少一条功能性蛋白相连接， 所述的功能性蛋白有 5-500个氨基酸。
11. 如权利要求 9所述的复合物， 其特征在于， 所述的复合物包含选自以下 的物质： 蛋白活性促进剂、 蛋白活性稳定剂、 延长蛋白半衰期的制剂。
12. 一种组合物， 其特征在于， 它含有安全有效量的权利要求 1所述的多肽 或权利要求 9所述的复合物， 以及药学上可接受的载体。
13. 一种制备组合物的方法， 所述组合物抑制细胞内端粒酶活性， 所述方法 包括：将安全有效量的权利要求 1所述的多肽或权利要求 9所述的复合物与药学 上可接受的载体混合。
14. 一种药盒， 其特征在于， 所述药盒中含有权利要求 1所述的多肽； 或含 有权利要求 9所述的复合物； 或含有权利要求 12所述的组合物。
PCT/CN2011/076806 2010-07-02 2011-07-04 一种抑制端粒酶活性的肽及其制备方法和应用 WO2012000458A1 (zh)

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