WO2011079431A1 - 具有端粒酶抑制活性的融合蛋白、其制备方法和用途 - Google Patents

具有端粒酶抑制活性的融合蛋白、其制备方法和用途 Download PDF

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WO2011079431A1
WO2011079431A1 PCT/CN2009/076168 CN2009076168W WO2011079431A1 WO 2011079431 A1 WO2011079431 A1 WO 2011079431A1 CN 2009076168 W CN2009076168 W CN 2009076168W WO 2011079431 A1 WO2011079431 A1 WO 2011079431A1
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protein
cells
fusion protein
cell
lpts
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French (fr)
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赵慕钧
陈光明
孙成副
许颖
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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    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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

Definitions

  • Fusion protein having telomerase inhibitory activity, preparation method and use thereof
  • the invention belongs to the fields of biotechnology and biochemical engineering. More specifically, the present invention relates to a telomerase activity inhibitory protein fused to a protein transduction domain, and a preparation method and application thereof. 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 reverse-transcribe and synthesize telomere repeats with its own RNA as a template to the end of the chromosome to compensate for the loss of telomere DNA during cell division and maintain the length of telomeres.
  • telomere activity is almost undetectable in normal human cells. Therefore, the number of normal somatic cell divisions is limited.
  • the telomere is lost by 50-200 bp, when the telomere is shortened to a certain extent.
  • Cell growth is inhibited, known as cell senescence, and goes to death.
  • telomerase activity can be detected and activity is high in most malignant cells (85%), and telomerase recombination of telomeres compensates for its continued loss during cell proliferation, resulting in cells It can be divided continuously, which is an important mechanism for cell immortalization and canceration.
  • Kim et al. analyzed a large number of studies and examined more than 100 malignant tumor specimens, indicating that telomerase has a sensitivity of 85%, a specificity of 91%, a positive predictive value of 91%, and a negative predictive value of 81%. , fully demonstrates the value of telomerase in the diagnosis of tumors (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): 201 1 -5.). Kim et al believe that activation of telomerase is one of the main factors in the development of malignant tumors.
  • telomeres The degree of activation and expression is closely related to the occurrence and metastasis of tumors. Inhibition of telomerase and shortening of telomeres is considered to be a tumor suppressor. Mechanism, telomerase is therefore an ideal target for tumor-targeted therapy.
  • telomerase-targeted tumor therapy studies mainly use antisense nucleic acid technology for telomerase RNA components (Kondo S., Kondo Y., Li G., et al, Targeted therapy of human malignant Glioma in a mouse model by 2-5A antisense directed against telomerase RNA. Oncogene, 1998, 16 :3323-3330. Ludwig A, Saretzki G, Holm PS, et al. Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase Cancer Res, 2001, 61 :3053-3061; Feng J., Funk WD, Wang SS, et al.
  • RNA component of human telomerase Science, 1995, 269: 1236-1241
  • gene therapy technology cutting telomeres Enzyme mRNA ribozyme technology (Ludwig A, Saretzki G, Holm PS, et al. Ribozyme cleavage of telomerase mRNA sensitizes Breast epithelial cells to inhibitors of topoisomerase. Cancer Res, 2001, 61 :3053-3061) and techniques for inhibiting telomerase gene transcriptional activity (Meyerson, M. Counter CM., Eaton EN, et al.
  • telomeres of tumor cells are up-regulated in tumor cells and during immortalization, Cell, 1997, 90 :785-795; WC Hahn, SA Stewart, MW Brooks, SG York, E. Eaton, A. Kurachi, RL Beijersbergen, JH Knoll, MMeyerson, RA Weinberg, Inhibition of telomerase limits the growth of human cancer cells, Nat. Med, 1999, 5: 164-1 170
  • These techniques can shorten the telomeres of tumor cells, which in turn can cause cells to enter crisis or die, or The tumorigenicity of tumor cells is significantly reduced.
  • LPTS is a protein that inhibits the telomerase activity of tumor cells. It is a new class of protein preparations with important application prospects.
  • the LPTS protein is encoded by the LPTS (Liver putative tumor suppressor) gene, which is a liver-associated candidate tumor suppressor gene (Liao C, Zhao MJ) obtained by the present inventors from a human normal liver cDNA library by localization and cloning.
  • LPTS Liver putative tumor suppressor
  • Liao C, Zhao MJ liver-associated candidate tumor suppressor gene
  • the protein encoded by this gene has the activity of inhibiting cell telomerase (Zhou XZ, Lu KP.
  • the Pin2/TRF 1 -interacting protein PinXl is a potent telomerase inhibitor.
  • Cell, 2001, 107, 347-359 The gene is located in the 8p23 segment of human chromosome 8, which is a high frequency deletion in a variety of malignant cells.
  • Studies have shown that LPTS is expressed in low or no expression in hepatocellular carcinoma and hepatocellular carcinoma cell lines, and telomeres in tumor cells The increase in enzyme activity may be related to the deletion or down-regulation of the LPTS gene.
  • liver cancer cells Due to the introduction of liver cancer cells, it can inhibit the growth, proliferation, and ultimately death of liver cancer cells (Liao C, Zhao MJ, Zhao J, et al. Mutation analysis of novel human liver-related putative tumor suppressor gene in hepatocellular carcinoma. World J Gastroenterol, 2003, 9:89-93; Zhou XZ, Lu KP.
  • the Pin2/TRF 1 -interacting protein PinXl is a potent telomerase inhibitor. Cell, 2001, 107, 347-359.) Therefore, LPTS has the effect of inhibiting tumor cell growth, And lead to the role of tumor cell death.
  • Chinese patent ZL00115395.1 discloses the gene sequence of LPTS and the amino acid sequence of the encoded protein thereof.
  • the function of the LPTS or PinX1 gene was obtained by transfecting a plasmid.
  • the LPTS protein cannot enter the cell across the membrane, which limits the research and application of protein forms.
  • Non-viral delivery strategies have significant advantages in terms of biosafety and ease of administration compared to viral vector methods.
  • Non-viral carrier package Including: microinjection, electroporation, liposome method, bacterial toxins, red blood cells and receptor-mediated endocytosis.
  • most of these methods are inefficient or time consuming, cause cell death or form intracellular vesicles, and do not allow foreign proteins to efficiently enter the cell across the membrane.
  • Another object of the present invention is to provide use of the fusion protein for inhibiting cell telomerase activity or expression, and further for inhibiting growth of telomerase-positive tumor cells.
  • an isolated fusion protein comprising:
  • telomerase activity inhibitory protein LPTS (1) telomerase activity inhibitory protein LPTS
  • the amino acid sequence of the linker peptide is: GGS.
  • the fusion protein is substantially composed of (1), (3), and (2). More preferably, the fusion protein is composed of (1), (3), and (2).
  • telomerase activity inhibitory protein LPTS is:
  • amino acid sequence of a protein as defined in (a) or (b) is formed by substitution, deletion or addition of one or more amino acid residues and has the protein function defined by (a) or (b) a protein derived from (a) or (b); or
  • the transactivator TAT is a protein having the amino acid sequence shown in positions 2 to 12 of SEQ ID NO: 2.
  • telomerase activity inhibitory protein LPTS is:
  • the transactivator TAT is located at the amino terminus of the fusion protein; the telomerase activity inhibitory protein LPTS is located at the carboxy terminus of the fusion protein.
  • a nucleic acid molecule encoding the fusion protein is provided.
  • a vector comprising the nucleic acid molecule.
  • a genetically engineered cell comprising said vector; or said nucleic acid molecule integrated in said cell genome.
  • a method of producing the fusion protein comprising: cultivating the cell, expressing and isolating the cell under conditions suitable for expressing the fusion protein Fusion protein.
  • telomerase positive cells in a sixth aspect of the invention, there is provided the use of said fusion protein for the preparation of a composition for inhibiting the growth of telomerase positive cells.
  • the cell is a telomerase-positive tumor cell, and preferably the cell is selected from the group consisting of: a liver cancer cell line, a cervical cancer cell line, a leukemia cell line, a gastric cancer cell line, or a tongue squamous cell carcinoma. Cell line.
  • the telomerase-positive cells include: BEL-7404 liver cancer cell line, HepG2 liver cancer cell line, Hela cervical cancer cell line, SMMC-7721 liver cancer cell line, HL-60 leukemia cell line, K -562 leukemia cell line, SGC-7901 gastric cancer cell line, or TCA-8113 human tongue squamous cell carcinoma cell.
  • the composition is also useful for preventing or treating a tumor.
  • composition for inhibiting tumor comprising:
  • a method of introducing a telomerase activity inhibitory protein LPTS into a cell comprising the steps of:
  • the cell is a telomerase positive cell.
  • the cell is a telomerase-positive tumor cell.
  • the method of obtaining a fusion protein comprises:
  • the transactivator TAT is located at the amino terminus of the fusion protein;
  • the telomerase activity inhibitory protein LPTS is located at the carboxy terminus of the fusion protein.
  • a method of inhibiting e.g., inhibiting in vitro
  • the method comprising treating a tumor cell with the fusion protein.
  • Figure 1 shows the structure of the T-LPGENE fusion protein.
  • Figure 2 shows the construction of the recombinant expression vector pET24a +)-T-LPGENE.
  • Figure 3 shows the induced expression of T-LPGENE in Escherichia coli and the results after isolation and purification.
  • 1 protein Maker
  • 2 before IPTG induction
  • 3 after IPTG induction
  • 4 centrifugal supernatant after sonication
  • 5 SP-sephorose purified T-LPGENE protein
  • 6 Superdex 75 purified T -LPGENEg white.
  • FIG. 4 shows that the purified T-LPGENE protein has an activity of inhibiting telomerase activity of tumor cells in vitro.
  • 1-5 the concentration (nmol) of T-LPGENE protein added was: 20, 40, 80, 160, 320; 6: blank control.
  • FIG. 5 shows that T-LPGENE protein has a transmembrane activity into tumor cells mediated by TAT.
  • A immunofluorescence experiment results, ANTI-LPTS, red fluorescence; DAPI: nucleus; PHASE: cell morphology; B: Western-blot results, Control: blank cells; 2hr, 6hr, 24hr : protein incubation for 2hr, 6hr, 24hr.
  • Figure 6 shows the effect of T-LPGENE on inhibiting the growth of telomerase-positive tumor cells and prolonging the doubling time of the cells.
  • Four kinds of telomerase-positive cells were treated with T-LPGENE, and cultured for 6-8 weeks.
  • the growth curve of each group was drawn and the multiplication algebra was calculated.
  • the logarithmic value represents the cell multiplication algebra; the abscissa: the number of days of cultivation.
  • Figure 7 shows that T-LPGENE does not affect the growth of telomerase-negative tumor cells.
  • Two kinds of telomerase-negative cells were treated with T-LPGENE, and cultured for 7 weeks.
  • the growth curve of each group was drawn and the multiplication algebra was calculated.
  • abscissa culture days.
  • Figure 8 shows the inhibition of cell proliferation by T-LPGENE after treatment of telomerase-positive tumor cells for 3 weeks.
  • A liver cancer BEL-7404 cells; B: cervical cancer HeLa cells; C: liver cancer SMMC-7901 cells; D: gastric cancer SGC-7901 cells.
  • the results in the figure are the mean and standard deviation of 5 parallel experiments.
  • Figure 9 shows the significant inhibition of cell proliferation by T-LPGENE after treatment of telomerase-positive tumor cells for 6 weeks.
  • A liver cancer BEL-7404 cells; B: liver cancer HepG2 cells; C: liver cancer SMMC-7721 Cell; D: Human tongue squamous cell carcinoma TCA-8113 cells.
  • a total of 7 days were detected and the detection wavelength was 570 nm.
  • the results in the figure are the mean and standard deviation of 5 parallel experiments.
  • FIG. 10 shows that T-LPGENE does not affect the proliferation of telomerase-negative cells.
  • the results in the figure are the mean and standard deviation of 5 parallel experiments.
  • Figure 11A shows the telomere shortening of each cell (BEL-7404, HepG2, and Saos-2) after T-LPGENE protein treatment and control treatment (PBS and TAT-GFP treatment).
  • Figure 11B shows the comparison of cell death after T-LPGENE protein treatment with control treatment (PBS and TAT-GFP treatment) for each cell (BEL-7404, HepG2, Saos-2, and L02), where the arrow points to At the death cell.
  • Figure 12 shows the survival rate of cells treated with T-LPGENE for 6 weeks as determined by flow cytometry.
  • A FCM detection map of PI staining of BEL-7404 cells, the abscissa is the fluorescence value, and the ordinate is the number of cells.
  • B The relative survival rate of BEL-7404 and HepG2 cells was counted in the PBS group, and the relative survival rate of Soas-2 and LO2 cells was counted as 100% in the PBS group. The results in the figure are the mean and standard deviation of 3 parallel experiments.
  • Figure 13 shows the death of cells induced by T-LPGENE by flow cytometry analysis.
  • the abscissa is the fluorescence value and the ordinate is the number of cells.
  • the sub-G1 region is a dead cell
  • the 2N (diploid) region is a G0/G1 phase cell
  • 4N (a tetraploid region;) is a G2/M phase cell
  • a S phase cell is between 2N and 4N.
  • B HepG2 liver cancer cells
  • C SGC-7901 gastric cancer cells
  • D L02 liver immortal cells
  • E Saos-2 myeloma cells.
  • FIG. 14 shows that the purified T-LPGENE protein has an activity of reducing the tumorigenicity of telomerase-positive cell BEL-7404 in vivo.
  • A shows that the purified T-LPGENE protein has the activity of reducing the tumorigenicity of telomerase-positive cell BEL-7404
  • B the mice after transplantation of tumor are treated with T-LPGENE protein and control treatment (PBS and TAT) Volume change of tumor after -GFP treatment
  • C Photograph of tumor growth in animals after T-LPGENE protein treatment and control treatment (; PBS and TAT-GFP treatment) of transplanted tumor
  • D Tumor weight changes after transplantation of tumor-bearing mice by T-LPGENE protein treatment and control treatment (; PBS and TAT-GFP treatment).
  • the inventors unexpectedly found that the fusion protein formed by the fusion of the telomerase activity inhibitory protein LPTS and the transactivator TAT not only retains the telomerase inhibition of the telomerase activity inhibitory protein LPTS. It also significantly increased the ability of telomerase activity to inhibit the entry of protein LPTS into cells, thereby greatly enhancing the killing effect on telomerase-positive cells, and on telomerase Negative cells have no significant toxic side effects.
  • the present inventors selected TAT to carry LPTS into cells, that is, TAT and LPTS proteins were fused to prepare a TAT-LPGENE fusion protein, which was determined by experimental research. There are many ways to carry proteins into cells. It is best to choose which method, and it needs to be determined through repeated experimental studies. TAT is not suitable for all proteins and needs to be screened for the proteins it carries. Therefore, the success of the present invention is not obtained by simple imitation, but by creative labor, combined with the design of the characteristics of the LPTS protein, and repeated experimental screening.
  • LPTS protein itself does not have the ability to enter the cell interior through the membrane, and telomerase is located in the nucleus. Therefore, treatment of telomerase-positive cells with LPTS protein has no significant effect on cell growth. Therefore, when LPTS is to be administered as an anti-tumor protein drug, whether or not the protein enters the tumor cell is a key step in its efficacy.
  • telomerase activity inhibitory protein LPTS There are many methods for carrying proteins into cells known in the art, but many methods are not suitable for carrying the telomerase activity inhibitory protein LPTS into cells.
  • the inventors attempted to transport the LPTS into the cell by lipofection, the most commonly used method in the art, ie, by liposome. Embed the protein drug into the cell.
  • the inventors mixed the cationic liposome with the LPGENE protein in a certain ratio, and then incubated with the cells for 2 to 72 hours, and then used the westem-blot method to detect the presence or absence of the LPGENE protein in the cells. The results showed that no target protein appeared in the cells after 24 hours. This proves that this method cannot transport LPGENE to the inside of cells.
  • the present inventors have also attempted to connect a plurality of transmembrane proteins to LPTS and test the transmembrane effect, but have failed to successfully enter LPTS into cells. While attempting to fuse the TAT protein to LPTS, it was unexpectedly discovered that although the transactivator TAT is not suitable for all proteins (eg, for lymphocytic choriomeningitis virus nucleoprotein, LCMV-NP is not applicable), it It is the most suitable protein for linking to LPTS and increasing the ability of LPTS to enter cells.
  • TAT is a recently discovered polypeptide that can carry macromolecular substances into cells.
  • some unsuccessful cases have been reported in the field, and its functional diversity is also controversial (Green I). Et al, Trends Pharmacol Sci. 2003; Leifert JA et al, Gene Ther. 2002) 0 Therefore, although TAT can be considered as a transmembrane peptide, a large number of screenings are required for the peptides to be fused or carried to be successful. .
  • the present inventors used a genetic engineering method to link a TAT polypeptide to LPGENE to form a fusion protein, and transport the LPGENE into the cell by using the transmembrane transport function of TAT, thereby further detecting the function of the TACT-fused LPGENE protein. Whether the feature has an effect.
  • the results of the study showed that the antitumor properties of the TAT-LPGENE fusion protein and the inhibition of telomerase targeting did not change, demonstrating that the experimental design and preparation process of the present invention was successful.
  • the protein comprising about 190-200 amino acids at the C-terminus of the full length sequence of LPTS is most suitable for fusion with TAT, and the fusion protein formed is most easily transferred into telomerase-positive cells, exhibiting excellent end. Granzyme inhibition effect.
  • containing includes “comprising”, “consisting essentially of”, “consisting essentially of”, and “consisting of”; “mainly constituted by”, “consisting essentially of” and “consisting of” belonging to “contains” , “has” or
  • transmembrane proteins there are many known transmembrane proteins, including: transactivator TAT, Penetratin, signal sequence based peptides, pVEC, Transportan, Amphiphilic model peptide and Arg9 and the like.
  • TAT transactivator TAT
  • Penetratin signal sequence based peptides
  • pVEC signal sequence based peptides
  • Transportan Amphiphilic model peptide and Arg9 and the like.
  • Suitable proteins are limited by factors such as protein length, nature, and spatial structure. It has also been found that the fusion of TAT with active proteins may affect the folding of the latter, which in turn affects the biological activity of the latter. After repeated studies and comparisons, the inventors have found that TAT is particularly suitable for fusion with LPTS, and the resulting fusion protein readily enters cells and can enter the nucleus.
  • the transactivator has the amino acid sequence of positions 2-12 of SEQ ID NO: 2.
  • Telomerase activity inhibitory protein LPTS Telomerase activity inhibitory protein LPTS
  • LPTS is a protein having a telomerase activity that inhibits tumor cells and is localized to the 8p23 segment of human chromosome 8, which is a high frequency deletion in a variety of malignant cells. Studies have shown that LPTS expression in liver cancer tissues and liver cancer cell lines is extremely low or not expressed, and the increase in telomerase activity in tumor cells may be related to the deletion or down-regulation of LPTS gene.
  • the full length protein of LPTS or a biologically active fragment thereof can be used in the present invention. Any of the biologically active fragments of the LPTS protein can be used in the present invention.
  • the biologically active fragment of the LPTS protein means, as a protein fragment, which still retains all or part of the function of the intact LPTS protein (eg, at least 50% biological activity, preferably at least 70% activity, More preferably at least 90% active;).
  • the amino acid sequence of LPTS formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention.
  • the LPTS protein formed by substitution, deletion or addition of one or more amino acid residues also has a function of inhibiting cell telomerase activity or expression after fusion with TAT.
  • the modified or modified LPTS protein can also be used in the present invention, for example, an LPTS protein which is improved in order to prolong its half-life and improve its stability.
  • the inventors unexpectedly found in the experiment that it contains about 190-200 of the C-terminus in the full length sequence of LPTS.
  • the amino acid protein is most suitable for fusion with TAT, and the fusion protein formed is most easily transferred into telomerase-positive cells to exert an excellent telomerase inhibitory effect.
  • the amino acid sequence of the LPTS may be substantially the same as the sequence shown in SEQ ID NO: 4.
  • a more preferred LPTS amino acid sequence may be substantially identical to the sequence shown in positions 16-211 of SEQ ID NO: 2.
  • the TAT polypeptide and the LPTS protein or an active fragment thereof of the fusion protein of the present invention may be directly linked to each other or may be linked by a polypeptide linker (linker peptide; As a preferred mode of the present invention, the TAT and LPTS or an active fragment thereof are linked by a polypeptide linker (linker peptide) to form a fusion protein.
  • the linker comprises 0-20 amino acids; preferably 0-15 amino acids, more preferably 0-10 amino acids, most preferably 1-4 amino acids, such as 2-3.
  • the designed fusion protein comprises a protein transduction region TAT, a telomerase activity inhibiting polypeptide LPTS, and a three-amino acid linker peptide, the structure of which is as shown in Formula I:
  • the LPTS peptide is a highly potent telomerase activity inhibitory protein peptide, which is the main pharmacodynamic functional region, located at the C-terminus;
  • the TAT polypeptide is a transduction region that mediates the entry of the fusion protein into the interior of the cell, located at the N-terminus; a linker peptide consisting of three amino acids (glycine-glycine-serine, GGS) that binds to TAT and LPTS.
  • GGS glycine-glycine-serine
  • a small peptide consisting of 3 amino acids provides a certain flexibility, allowing LPGENE to rotate and fold freely to ensure its activity.
  • the commonly used TAT has a linker peptide between 12-15 amino acids in its carrier protein.
  • a small peptide consisting of 3 amino acids is used to link the two functional regions, and the interaction between the two is avoided. It can be folded normally, thus maintaining its respective structure and function, and ensuring the physiological activity of the two functional areas.
  • the linker peptide itself is very short, with only three amino acids, which minimizes its toxicity to the organism itself. Toxicity experiments have shown that T AT-LPGENE has no obvious toxic side effects on cells and animals;
  • the DNA sequence of the linker peptide contains a Bamffl cleavage site, which provides a site suitable for cleavage and assembly for the expression vector constructing TAT-LPGENE. Fusion protein
  • the present invention provides a fusion protein comprising a TAT protein, and a LPTS protein or a living organism thereof Active fragment.
  • a fusion protein comprising a TAT protein, and a LPTS protein or a living organism thereof Active fragment.
  • TAT-LPTS fusion protein refers to a protein obtained by fusing a TAT amino acid sequence and an LPTS amino acid sequence, wherein there may be either No peptide sequences were ligated.
  • the fusion protein can be used to inhibit cellular telomerase activity or expression. More preferably, the fusion protein is an isolated protein that is not associated with other proteins, polypeptides or molecules, is a purified product of recombinant host cell culture or is a purified extract.
  • the TAT polypeptide and the LPTS protein or an active fragment thereof may be directly linked to each other or may be linked by a polypeptide linker (linker peptide; As a preferred mode of the present invention, the TAT and LPTS or an active fragment thereof are linked by a polypeptide linker (linker peptide;) to form a fusion protein.
  • the linker comprises 0-20 amino acids; preferably 0-15 amino acids, more preferably 0-10 amino acids, most preferably 1-4 amino acids, such as 2-3.
  • the TAT polypeptide is located at the amino terminus (N-terminus) of the fusion protein; the LPTS protein or an active fragment thereof is located at the carboxy terminus (C-terminus) of the fusion protein.
  • the positions of the two proteins can also be interchanged.
  • the amino terminus (or carboxy terminus) of the fusion protein may also contain one or more polypeptide fragments as a protein tag.
  • Any suitable label can be used in the present invention.
  • the tags may be FLAG, HA, HA1, c-Myc, 6-His, and the like. These tags can be used to purify the fusion protein.
  • a specific example is the attachment of a 6-His structure at the C-terminus of the fusion protein. It will be understood by those skilled in the art that a cleavable structure can be provided between the amino acid sequence of the protein tag and the amino acid sequence of the TAT-LPTS fusion protein so that the tag can be separated from the fusion protein.
  • the fusion protein of the present invention is capable of both inhibiting telomerase activity and entering the interior of the cell, thus solving the limitations of LPTS application.
  • LPTS intracellular function of LPTS
  • previous studies have been carried out at the genetic level, which is a basic research, which requires the introduction of foreign genes in the genome of the cell, making it difficult to carry out practical applications.
  • the present invention proposes to introduce LPTS into cells at the protein level, LPTS can be clinically applied.
  • the invention provides an isolated nucleic acid encoding the fusion protein, or a complementary strand thereof.
  • the DNA sequence encoding the fusion protein of the present invention can be synthesized synthetically in whole sequence, or a DNA sequence encoding TAT and LPTS amino acids can be obtained by PCR amplification, and then spliced together to form a DNA sequence encoding the fusion protein of the present invention.
  • the invention also provides a vector comprising a nucleic acid molecule encoding the fusion protein.
  • the vector may further comprise an expression control sequence operably linked to the sequence of the nucleic acid molecule to facilitate expression of the fusion protein.
  • operably linked refers to a condition in which portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, if a promoter controls the transcription of a sequence, then it is operably linked to the coding sequence.
  • any suitable vector can be used, such as some vectors for cloning and expression of bacterial, fungal, yeast and mammalian cells, such as Pouwels et al., Cloning Vector: Laboratory Manual (Lasevier latest edition) describe.
  • Various carriers known in the art such as commercially available carriers can be used.
  • a commercially available vector is selected, and then the nucleotide sequence encoding the novel fusion protein of the present invention is operably linked to an expression control sequence to form a protein expression vector.
  • the vector is a prokaryotic vector, such as a pET vector.
  • recombinant cells containing a nucleic acid sequence encoding the fusion protein are also included in the present invention.
  • the term "host cell” includes prokaryotic cells and eukaryotic cells.
  • prokaryotic host cells include Escherichia coli, Bacillus subtilis, etc.; for example, E. coli cells such as Escherichia coli HMS174 (DE3), or BL21 (DE3).
  • E. coli cells such as Escherichia coli HMS174 (DE3), or BL21 (DE3).
  • Common eukaryotic host cells include yeast cells, insect cells, and mammalian cells.
  • a prokaryotic cell is used as a host cell, and a fusion protein which retains good telomerase activity and has good cell membrane permeability is obtained by expression and purification.
  • the method comprises culturing a recombinant cell comprising a fusion protein-encoding nucleic acid.
  • the fusion protein includes a TAT polypeptide and a LPTS protein or an active fragment thereof.
  • the method can include allowing the cell to express the encoded fusion protein and renaturation of the expressed fusion protein.
  • the method can further comprise isolation and/or purification of the renatured fusion protein.
  • the fusion protein obtained by the above preparation can be purified to a substantially uniform property, for example, a single band on SDS-PAGE electrophoresis.
  • a commercially available ultrafiltration membrane can be used to separate the protein, such as Millipore, Amicon, Pellicon, etc., and the supernatant is first concentrated.
  • the concentrate may be further purified by gel chromatography or by ion exchange chromatography.
  • the gel matrix may be a matrix commonly used for protein purification such as agarose, dextran, polyamide, and the like.
  • the SP group is an ideal ion exchange group.
  • the above purified product can be further purified and purified by reversed-phase high performance liquid chromatography (RP-HPLC). All of the above purification steps can utilize different combinations to ultimately achieve a substantially uniform protein purity.
  • the expressed fusion protein can be purified using an affinity chromatography column containing a specific antibody, receptor or ligand of TAT or LPTS. Depending on the characteristics of the affinity column used, conventional methods such as high can be used.
  • the fusion polypeptide bound to the affinity column is eluted by a salt buffer, a change in pH, or the like.
  • the fusion protein of the present invention can be used for the preparation of a composition for inhibiting cell telomerase activity or expression, thereby inhibiting the growth of telomerase-positive cells and reducing their tumorigenicity.
  • the fusion protein of the present invention has an excellent ability to enter cells (e.g., tumor cells;), and can stably inhibit the function of telomerase activity or expression after entering cells.
  • the fusion protein of the present invention has a superior tumoricidal effect than LPTS, and can be used to develop an effective antitumor drug.
  • the "tumor” of the present invention may be of various types as long as the tumor cells are telomerase-positive, and may include, for example, but not limited to: liver cancer, cervical cancer, leukemia, gastric cancer, tongue squamous cell carcinoma, and the like.
  • liver cancer cervical cancer
  • leukemia gastric cancer
  • tongue squamous cell carcinoma and the like.
  • BEL-7404 liver cancer cell line HepG2 liver cancer cell line
  • Hela cervical cancer cell line Hela cervical cancer cell line
  • SMMC-7721 liver cancer cell line HL-60 leukemia cell line
  • K-562 leukemia cell line K-562 leukemia cell line
  • SGC-7901 gastric cancer cell line or TCA- 8113 human tongue squamous cell carcinoma cell lines and so on.
  • the present invention also provides a composition for inhibiting inhibition of cellular telomerase activity or expression, the composition comprising: (i) an effective amount (e.g., 0.0001 to 50% by weight; more preferably 0.001 to 20% by weight) of the present invention
  • an effective amount e.g., 0.0001 to 50% by weight; more preferably 0.001 to 20% by weight
  • a pharmaceutically acceptable carrier e.g., a pharmaceutically acceptable carrier.
  • a "pharmaceutically acceptable” ingredient is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies;), ie, having a reasonable benefit/risk ratio substance.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • the term refers to pharmaceutical carriers which are not themselves essential active ingredients and which are not excessively toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. A full description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).
  • the pharmaceutically acceptable carrier in the composition may contain a liquid such as water, saline, glycerin and sorbitol.
  • auxiliary substances such as lubricants, glidants, wetting or emulsifying agents, pH buffering substances and stabilizers such as albumin and the like may also be present in these carriers.
  • compositions may be formulated into a variety of dosage forms suitable for mammalian administration, including, but not limited to, injections, capsules, tablets, emulsions, suppositories.
  • a safe and effective amount of a fusion protein of the invention is administered to a mammal (e.g., a human), wherein the safe and effective amount is typically at least about 0.1 microgram per kilogram of body weight, and in most cases no more than about 50
  • the mg/kg body weight preferably the dose is from about 1 microgram/kg body weight to about 10 mg/kg body weight.
  • specific doses should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.
  • the fusion protein When used to inhibit tumors in a mammal, the fusion protein can be administered systemically, or locally, depending on factors such as the type of tumor, the site of growth, and the degree of progression.
  • the compositions of the invention can be used directly to kill tumor cells. In addition, it can be used in combination with other therapeutic agents or adjuvants.
  • the main advantages of the invention are:
  • the fusion protein of TAT and LPTS was first provided and prepared, and it was confirmed that the fusion of LPTS and TAT promoted LPTS to penetrate the cell membrane without affecting the inhibitory cell telomerase activity of LPTS.
  • the fusion protein of the present invention has significantly superior antitumor activity than LPTS alone, and is available for many tumors.
  • T-LPGENE T-LPTS-C
  • T-LPGENE engineering bacteria The specific methods for constructing T-LPGENE engineering bacteria are as follows:
  • the TAT template and its primers are obtained by synthesis, and the specific sequence is as follows:
  • PCR reaction was carried out to obtain a DNA sequence encoding the TAT polypeptide (11 amino acids) with restriction enzyme sites Nde I and BamH I at both ends; the PCR reaction conditions were: PCR reaction was carried out in a 50 ⁇ system, including a ⁇ template, ⁇ dNTP mix (10 mM), 1 ⁇ 1 of upstream and downstream primers (20 ⁇ ), 5 ⁇ 1 lOxPYROBEST buffer, 0.5 ⁇ 1 PYROBEST enzyme, add deionized water to 50 ⁇ 1.
  • the reaction conditions were: 94 ° C for 3 minutes; then a total of 30 cycles, each cycle including 94 ° C for 20 seconds, 50 ° C for 20 seconds, 72 ° C for 20 seconds; finally 72 ° C for 5 minutes, 4 ° C insulation .
  • the PCR product obtained above was digested with Nde I and BamHI, and inserted into the same double-digested plasmid pET24( ) (purchased from Novagen;) to obtain a pET24-TAT plasmid.
  • the plasmid contains the full-length LPTS gene (see Hepatology, 32 (2000), 721-727), and the primers are as follows:
  • PCR reaction conditions PCR reaction in 50 ⁇ l system, including ⁇ template, ll dNTP mix (10 mM), upstream and downstream primers (20 ⁇ ) each 1 ⁇ l, 5 ⁇ l lOxPYROBEST buffer, 0.5 l PYROBEST enzyme, additional deionization Water to 50 ⁇ 1.
  • reaction conditions were: 94 ° C for 3 minutes, and then a total of 30 cycles, each cycle including 94 ° C for 30 seconds, 56 ° C for 40 seconds, 72 ° C for 60 seconds; then 72 ° C for 5 minutes, 4 ° C insulation.
  • the PCR product of the LPGENE gene obtained above was digested with BamH I and Xho I, inserted into the pET24-TAT plasmid digested with BamH I and Xho I, and finally the pET24-T-LPGENE plasmid was obtained.
  • the DNA sequence encoding T-LPGENE is shown in Figure 1.
  • the structure of the pET24-T-LPGENE expression plasmid is shown in Figure 2. After confirming the sequence, the host strain E.coli BL-21 was transformed to obtain an engineered strain expressing T-LPGENE.
  • Example 2 Induced expression and purification of T-LPGENE
  • the present inventors have used a separation method such as ion exchange to obtain a high-purity T-LPGENE protein, which can be simply divided into the following steps:
  • T-LPGENE is a potent inhibitor of telomerase activity.
  • the present inventors measured the biological activity of purified T-LPGENE by the TPAP method.
  • Telomerase is derived from SMMC-7721 liver cancer cell lysate. The specific preparation method is as follows:
  • SMMC-7721 liver cancer cells purchased from ATCC
  • culture medium was RPMI 1640, cultured at 37 ° C under 5% CO 2 , and the cells were collected after full bottle.
  • the medium was blotted dry, the cells were rinsed once with PBS, the cells were trypsinized, the cells were washed with PBS, inhaled into a centrifuge tube, and the cells were collected by centrifugation at 4000 rpm.
  • the cells were washed once with washing buffer (10 mM Hepes-KOH (pH 7.5), 1.5 mM MgCl 2 , 10 mM KCl, 1 mM DTT (dithionthreitoll)), and centrifuged. Resuspend the cells with pre-cooled lysis buffer (10 mM Tris-HCl (pH 7.5), 1 mM MgCl 2 , 1 mM EGTA, 0.1 mM PMSF, 5 mM mercaptoethanol, 0.5% CHAPS, 10% glycerol) 500 ⁇ M. The supernatant was incubated for 30 minutes at 4 ° C, 12,000 rpm for 30 minutes to determine telomerase activity. The supernatant can be stored at -70 ° C and maintains stable telomerase activity after multiple freeze-thawing.
  • washing buffer 10 mM Hepes-KOH (pH 7.5), 1.5 mM MgCl 2 , 10 mM KCl
  • TPAP assay in a 500 ⁇ l Eppendorf tube with a reaction volume of 50 ⁇ l containing 45.25 ⁇ l of a conventional reaction buffer, 0.8 ⁇ M of telomerase-containing tumor cell lysate, 0.25 l dNTP, ⁇ ⁇ ⁇ purification Protein (diluted as required).
  • ll Ts primer O.lg/ ⁇ 1, primer sequence 5, -AATCCGTCGAGCAGAGTT-3, (SEQ ID NO: 10)
  • 1 ⁇ l Taq enzyme extension at 25 °C for 30 min, 95 was added.
  • TAT-mediated activity of LPGENE protein transmembrane into tumor cells First, the inventors attempted to carry the LPTS-C protein into each tumor cell using conventional lipofection. The cationic liposome (Shanghai Biochemical Cell Research Institute;) is mixed with LPTS-C protein in a certain ratio (2:1, volume ratio;), and the protein is encapsulated and added to the normal cultured cells, and the final protein is added.
  • BEL-7404 cells and Saos-2 cells were seeded in 6 cm culture dishes in DMEM (containing 10% newborn calf serum) and Mccoy's 5A (containing 15% fetal calf serum), 37 ° C, 5 Adherent culture under %CO 2 conditions. After the cell density exceeds 30%, PBS solution (two empty;), purified LPGENE (100 mg/L) protein (3 wells, 2 hr, 6 hr and 24 hr, respectively) and purified T-LPGENE (100 mg/) were added.
  • L) protein (3 wells, incubated for 2 hr, 6 hr and 24 hr, respectively):
  • Each sample is detected by Western Blotting method, that is, after the sample is subjected to SDS-PAGE electrophoresis, The protein was transferred to a nitrocellulose membrane using a Bio-Rad electrorotator, and hybridization was carried out using a specific anti-LPGENE antibody (a rabbit antiserum immunized with LPGENE protein).
  • the secondary antibody is horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG.
  • TAT can very well mediate LPGENE protein entry into BEL-7404 cells and Saos-2 cells, and can enter the nucleus.
  • Example 5 Purified T-LPGENE protein has an activity of inhibiting telomerase-positive cell growth in vitro.
  • Test cells are provided by the Shanghai Cell Bank of the Chinese Academy of Sciences;
  • Telomerase-positive tumor cells (8 strains): BEL-7404 liver cancer cell line, HepG2 liver cancer cell line, SMMC-7721 liver cancer cell line, Hela cervical cancer cell line, HL-60 leukemia cell line, K-562 leukemia cell line , SGC-7901 gastric cancer cell line, TCA-8113 human tongue squamous cell carcinoma cell;
  • Telomerase-negative cells (3 strains): QSG-7701 liver immortalized cell line, L02 liver immortalized cell line, Saos-2 myeloma cell line.
  • test cells were seeded in a 6-well plate covered with a cover glass, and the culture medium was placed under the conditions provided by the cell bank, and cultured at 37 ° C under 5% CO 2 .
  • PBS solution, T-LPGENE (10 g/mL), T-LPGENE (40 g/mL) or T-GFP (40 g/mL, control) were added to the 6-well plate, and the number of cells was measured for each passage. The number of cells was counted according to the dilution factor, and the growth curve of each group of cells was plotted. The culture was continued for 6 to 8 weeks (green fluorescent protein GFP and green fluorescent protein T-GFP with transmembrane peptide as a test control).
  • T-LPGENE protein can inhibit the growth and proliferation of most telomerase-positive tumor cells, but has no obvious inhibitory effect on telomerase-negative cells.
  • Figure 6 shows that the growth of BEL-7404, HepG2, K-562 and HL-60 tumor cells is slowed down by T-LPGENE, and the inhibition of liver cancer BEL-7404 cells is most obvious, and this inhibition has Concentration-dependent, that is, as the concentration of the added T-LPGENE protein increases, the growth of the cells is also slower.
  • Figure 7 shows that T-LPGENE has no significant inhibitory effect on the growth of telomerase-negative Saos-2 and L02.
  • T-LPGENE protein specifically inhibits the growth of telomerase-positive tumor cells.
  • test cells were seeded in 6-well plates covered with coverslips, and the culture medium was placed under the conditions provided by the cell bank, and cultured at 37 ° C under 5% CO 2 .
  • T-GFP is at
  • GFP is linked to TAT.
  • GFP is a fluorescently labeled protein commonly used in laboratories.
  • GenBank accession number: DQ768212.
  • MTT assay After 3 or 6 weeks of continuous cell treatment, the growth activity of each group was measured by MTT assay at a wavelength of 570 nm. Specific experimental methods can be found in the literature (Situ Zhenqiang, edited by Wu Junzheng; Cell Culture [M]; Xi'an: World Book Publishing Xi'an, 2004, 250).
  • T-LPGENE protein has very good selectivity for cancer cells and can specifically inhibit the growth of telomerase-positive tumor cells.
  • the degree of inhibition is related to protein dosage and cell type, but the opposite end There was no significant effect on the growth of granzyme-negative cells.
  • Telomerase targeting Specificity for the development of therapeutic drugs and its benefits, especially 85% of tumor cells have telomerase activity, while normal cells are not or very low, meaning that T-LPGENE has no killing effect on normal cells.
  • BEL-7404 cells, HepG2 cells, Saos-2 cells and L02 cells were collected 8 weeks after administration, and the telomere length was analyzed by Southem-blot method.
  • the simple process is: Isolation of genomic DNA, digestion with the endonucleases Hinf I and Afa l, followed by separation of the digested product with a 0.8% agarose gel, after transfection, hybridization with a probe labeled ⁇ -32 ⁇ Hybridization signals were analyzed using ImageQuant.
  • the results of the experiment are shown in Figure 11A.
  • the results showed that the telomere of telomerase-positive tumor cells BEL-7404 and HepG2 were significantly shortened after 8 weeks of treatment with T-LPGENE protein, compared with the control group (PBS and TAT-GFP treatment). And the concentration group of the high concentration protein group is shorter than the concentration group.
  • the telomerase-negative tumor cell Saos-2 telomere did not significantly shorten.
  • BEL-7404 cells, HepG2 cells, Saos-2 cells and L02 cells were treated with an inverted microscope for 6 weeks, and the results were shown in Fig. 11B.
  • the results showed that the telomerase-positive tumor cells treated with T-LPGENE protein were BEL.
  • the adhesion of -7404 and HepG2 is poor, and the cells become flat and large, which is a typical crisis form.
  • telomere Every time a normal human somatic cell divides, the telomere loses 50-200 bp. When the telomere is shortened to a certain extent, cell growth is inhibited, which is called cell senescence, and it goes to death.
  • telomerase activity can be detected and activity is high in most malignant cells (85%), and telomerase recombination of telomeres compensates for its continued loss during cell proliferation, resulting in cells It can be divided continuously, which is an important mechanism for cell immortalization and canceration.
  • T-LPGENE protein can inhibit telomerase activity in cells, and the telomere of telomerase-positive tumor cells gradually shortens with cell proliferation, entering a crisis period (aging;), and it is in a crisis form.
  • the telomerase-negative tumor cells Saos-2 and immortalized cells L02 did not change significantly in morphology.
  • PI propidium iodide
  • telomerase-positive cell lines namely BEL-7404 liver cancer cells and HepG2 cells
  • telomerase-negative cell lines namely liver immortalized L02 cells and myeloma Saos-2
  • the relative viable cell rate of HepG2 cells, 40 g / mL T-LPGENE dose group was 85.3%, significantly lower than the control; the relative viable cell rate of the 10 g / mL T-LPGENE dose group was 89. 1%, also significantly lower than Control group (Fig. 12B).
  • T-LPGENE can induce telomerase positive cell death, reduce their survival rate, and have a dose effect, but can not induce telomerase-negative cell death.
  • Liver cancer BEL-7404 cells, liver cancer HepG2 cells, and gastric cancer SGC-7901 cells, as well as telomerase-negative liver immortalized L02 cells and myeloma Saos-2 cells were administered for 6 weeks, and then subjected to flow cytometry analysis ( FACSCalibur, Becton Dickinson, USA).
  • the cells were stained with propidium iodide (PI), and the DNA content of the cells was detected by flow cytometry to determine the cell cycle distribution.
  • the sub-G1 region was dead cells, and the 2N (diploid) region was G0/G1.
  • Stage cells, 4N (four-fold region) are G2/M phase cells, and SN cells are between 2N and 4N. This is a quick and quantitative method for determining cell viability and cell cycle.
  • telomerase-positive tumor cells BEL-7404 and HepG2 in the sub-Gl region increased significantly after 6 weeks of continuous treatment with T-LPGENE protein, ie, the number of cell deaths or dying was significantly increased. There was no significant change in the sub-Gl region of telomerase-negative tumor cell Saos-2 and immortalized cell L02.
  • Example 6 Tumor inhibiting activity of T-LPGENE protein and reduction of tumorigenicity of telomerase-positive cells 1. Purified T-LPGENE protein can reduce the tumorigenicity of telomerase-positive cells
  • BEL-7404 cells and Saos-2 cells were seeded in a six-well plate covered with coverslips in DMEM (containing 10% newborn calf serum) and Mccoy's 5A (containing 15% fetal). Bovine serum), Adherent culture at 37 ° C, 5% CO 2 .
  • T-LPGENE can reduce the tumorigenicity of telomerase-positive cell BEL-7404, but has no inhibitory effect on reducing the tumorigenicity of telomerase-negative cell Saos-2.
  • PBS 5/5 (l.CSl, 0.S9, 0.74, 0.64, 0.53)
  • T-LPGENE 40mg/L 4/5 (0,15, 0,13, 0.1, 0.072, ⁇ 2)
  • Liver cancer cells BEL-7404 were inoculated (5 ⁇ 10 6 /only) into the right lower lumbar subcutaneous of nude mice (4 weeks old, female). After 4 weeks, the tumor was removed, cut into 2 mm 3 size, and inoculated into nude mice (4 weeks). Age, female;) The lower back of the lower part of the skin, the nude mice were randomly divided into 4 groups. After the next day, T-LPGENE protein (100 ⁇ g/400 g/K, experimental group) was injected subcutaneously, and the site was placed near the tumor. The control group was injected with PBS and TAT-GFP protein (400 ⁇ g/head;).
  • Tumor size was recorded weekly (all nude mice were photographed after 7 weeks of inoculation with a vernier caliper, nude mice were sacrificed, and tumors were weighed out.
  • T-LPGENE protein can inhibit the growth of BEL-7404 tumor cells in nude mice, which has a certain dose effect.
  • the tumor weight per injection of the 100 T-LPGENE protein group was only 57% of the PBS group (P ⁇ 0.05), while the tumor weight of the 400 ⁇ g T-LPGENE protein group was only 36% of the PBS group (P ⁇ 0.01).
  • the tumor weight of the TAT-GFP group was not significantly different from that of the PBS group, but was significantly higher than that of the two experimental groups (P ⁇ 0.05).
  • High-dose experimental group of tumors Example 7. Preparation and cell assay of fusion protein containing full length LPTS
  • the TAT-full-length LPTS (TAT-LPTS) was prepared in a manner similar to that described in Example 1, and the pET24-TAT plasmid was first constructed.
  • the pT-LPTSCDS plasmid was used as a template to obtain the full-length gene sequence of LPTS by PCR amplification (see SEQ ID NO: 3), and the restriction sites BamH I and Xho I were set at both ends, and the two enzymes were digested with the two enzymes.
  • the same double-digested pET24-TAT plasmid was inserted to obtain a pET24-T-LPTS plasmid having a DNA sequence encoding TAT-LPTS. After sequencing, the host strain co//BL-21 was transformed and the engineered bacteria were obtained.
  • TAT-LPTS has essentially the same ability to inhibit telomerase activity as T-LPGENE.
  • TAT-LPTS was able to enter BEL-7404 cells and Saos-2 cells and enter the nucleus.
  • Semi-quantitative experiments showed that compared with the case where T-LPGENE protein enters the cell, the amount of TAT-LPTS entering the nucleus is about 70% of the amount of T-LPGENE protein entering the cell. It can be seen that the transfer effect of T-LPGENE protein is more ideal. .
  • Example 8 Pharmaceutical composition
  • the fusion protein obtained by purification in Example 3 was prepared in 100 ml of normal physiological saline to obtain a fusion protein-containing composition at a concentration of 1 mg/ml.

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Description

具有端粒酶抑制活性的融合蛋白、 其制备方法和用途
技术领域
本发明属于生物技术和生物化学工程领域。 更具体地说， 本发明涉及一种 融合有蛋白转导结构域的端粒酶活性抑制蛋白， 及其制备方法和应用。 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序列， 加到染色体末端， 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***次数是有限的， 细胞每***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***， 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):201 1-5.)。 Kim等认为， 端粒 酶被激活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。
目前， 以端粒酶为靶点的肿瘤治疗研究， 主要采用了针对端粒酶 RNA组 分的反义核酸技术 (Kondo S., Kondo Y.， Li G.， et al, Targeted therapy of human malignant glioma in a mouse model by 2-5A antisense directed against telomerase RNA. Oncogene, 1998, 16 :3323-3330. Ludwig A, Saretzki G, Holm PS, et al. Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase. Cancer Res, 2001, 61 :3053-3061； Feng J., Funk W.D., Wang S.S., et al. The RNA component of human telomerase, Science, 1995, 269: 1236-1241)、 基因治疗技术、 切割端粒酶 mRNA 的核酶技术 (Ludwig A, Saretzki G, Holm PS, et al. Ribozyme cleavage of telomerase mRNA sensitizes breast epithelial cells to inhibitors of topoisomerase. Cancer Res, 2001, 61 :3053-3061)和抑制端粒酶基因转录活性的技术等 (Meyerson,M. Counter CM., Eaton E.N., et al. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization, Cell, 1997, 90 :785-795； W.C. Hahn, S.A. Stewart, M.W. Brooks, S.G. York, E. Eaton, A. Kurachi, R.L. Beijersbergen, J.H. Knoll, MMeyerson, R.A. Weinberg, Inhibition of telomerase limits the growth of human cancer cells, Nat. Med, 1999, 5: 164-1 170 这些技术可 以缩短肿瘤细胞的端粒， 进而使细胞进入危机期或者死亡， 或者使肿瘤细胞的 致瘤性显著下降。
LPTS 是一种具有抑制肿瘤细胞端粒酶活性的蛋白， 是一类新的蛋白质制 剂， 有重要的应用前景。 LPTS蛋白是由 LPTS(Liver putative tumor suppressor) 基因编码， 该基因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 (Liao C, Zhao M.J., Song H.， Uchida K.， Yokoyama K.K., Li T.P., 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 32 (2000) 721-727 该基因 编码的蛋 白具有抑制细胞端粒酶的活性（Zhou X.Z., Lu K.P.. The Pin2/TRF 1 -interacting protein PinXl is a potent telomerase inhibitor. Cell, 2001, 107, 347-359)。 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 极低或不表达， 肿瘤细胞中端粒酶活性的升高， 可能与 LPTS基因的缺失或表 达下调有关。 有文献报道， 将 LPTS基因导入肝癌细胞， 能抑制肝癌细胞的生 长、增殖、最终引起死亡 (Liao C, Zhao MJ, Zhao J, et al. Mutation analysis of novel human liver-related putative tumor suppressor gene in hepatocellular carcinoma. World J Gastroenterol, 2003, 9:89-93 ； Zhou X.Z., Lu K.P.. The Pin2/TRF 1 -interacting protein PinXl is a potent telomerase inhibitor. Cell, 2001, 107, 347-359.)。 因此， LPTS 具有抑制肿瘤细胞生长， 并导致肿瘤细胞死亡的 作用。
中国专利 ZL00115395.1公开了 LPTS的基因序列及其编码蛋白的氨基酸序 列。 在以往的研究报告中， LPTS或 PinXl基因的功能是通过转染质粒的方式 获得的。 LPTS蛋白不能跨膜进入细胞内， 这就限制了蛋白形式的研究和应用。
在传统的实验和临床治疗上， 已经有许多方法用于将蛋白质递送进入活细 胞中， 这些方法可以分成病毒载体和非病毒载体两大类。 较之病毒载体法， 非 病毒递送策略在生物安全和给药方便性方面具有很大的优点。 非病毒载体法包 括： 显微注射法， 电穿孔法， 脂质体法， 细菌毒素， 红细胞及受体介导的胞吞 作用等。 但是这些方法大多效率不高或费时、 容易引起细胞死亡或形成胞内小 泡而不能使外源蛋白有效地跨膜进入细胞。
因此， 寻找一种迅速、 有效的将 LPTS蛋白输入细胞并在细胞内保持生物 活性的方法显得尤为重要。 发明内容
本发明的目的在于提供一种分离的融合蛋白， 所述的融合蛋白包括端粒酶 活性抑制蛋白 LPTS和反式激活蛋白 TAT。
本发明的另一目的在于提供所述融合蛋白的用途， 用于抑制细胞端粒酶活 性或表达， 进而可用于抑制端粒酶阳性肿瘤细胞的生长。
在本发明的第一方面， 提供一种分离的融合蛋白， 所述的融合蛋白包括：
(1) 端粒酶活性抑制蛋白 LPTS ;
(2) 反式激活蛋白 TAT; 和
(3) 位于 (1)和 (2)之间的由 0-20个 (较佳的为 0-15个， 更佳的为 0-10个， 最佳的 1-4个， 如 2-3个；)氨基酸构成的连接肽。
在一个优选例中， 所述连接肽的氨基酸序列为： GGS。
在另一优选例中， 所述的融合蛋白基本上由（1)、 (3), (2)相连接而构成。 更佳地， 所述的融合蛋白由（1)、 （3)、 （2)相连接而构成。
在另一优选例中， 所述的端粒酶活性抑制蛋白 LPTS是：
(a) SEQ ID NO: 4所示的氨基酸序列的蛋白；
(b) SEQ ID NO: 2中第 16-211位所示氨基酸序列的蛋白； 或
(c) 将 （a) 或 （b) 所限定的蛋白的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) 或 （b) 所限定的蛋白功能的由 （a) 或 （b) 衍生的蛋白； 或者
所述的反式激活蛋白 TAT是具有 SEQ ID NO: 2中第 2-12位所示氨基酸序 列的蛋白。
在另一优选例中， 所述的端粒酶活性抑制蛋白 LPTS是：
(al) SEQ ID NO: 2中第 16-211位所示氨基酸序列的蛋白； 或
(bl) 将 （al) 所限定的蛋白的氨基酸序列经过 1-10个 （较佳的 1-6个； 更 佳的 1-3个） 氨基酸残基的取代、缺失或添加而形成的， 且具有 （al) 所限定的 蛋白功能的由 （al) 衍生的蛋白。
较佳的， 所述的反式激活蛋白 TAT 位于融合蛋白的氨基端； 所述的端粒 酶活性抑制蛋白 LPTS位于融合蛋白的羧基端。 在本发明的第二方面， 提供一种编码所述的融合蛋白的核酸分子。
在本发明的第三方面， 提供一种载体， 它含有所述的核酸分子。
在本发明的第四方面， 提供一种基因工程化的细胞， 所述的细胞含有所述 的载体； 或所述的细胞基因组中整合有所述的核酸分子。
在本发明的第五方面， 提供一种产生所述的融合蛋白的方法， 所述的方法 包括： 在适合表达所述融合蛋白的条件下， 培养所述的细胞， 表达和分离出所 述的融合蛋白。
在本发明的第六方面， 提供所述的融合蛋白的用途， 用于制备抑制端粒酶 阳性细胞生长的组合物。
在另一优选例中， 所述的细胞是端粒酶阳性的肿瘤细胞， 优选所述细胞选 自： 肝癌细胞株、 ***细胞株、 白血病细胞株、 胃癌细胞株、 或舌鳞状细胞 癌细胞株。
在另一优选例中， 所述的端粒酶阳性细胞包括： BEL-7404 肝癌细胞株、 HepG2肝癌细胞株、 Hela***细胞株、 SMMC-7721肝癌细胞株、 HL-60 白 血病细胞株、 K-562白血病细胞株、 SGC-7901胃癌细胞株、 或 TCA-8113人舌 鳞状细胞癌细胞。
在另一优选例中， 所述的组合物还用于预防或***。
在本发明的第七方面， 提供一种抑制肿瘤的组合物， 所述的组合物含有：
(i) 有效量的所述的融合蛋白； 和
(ϋ) 药学上可接受的载体。
在本发明的第八方面， 提供一种将端粒酶活性抑制蛋白 LPTS导入细胞的 方法 (体外非治疗性地)， 所述方法包括以下步骤：
(a)将反式激活蛋白 TAT与端粒酶活性抑制蛋白 LPTS融合，获得融合蛋白；
(b)将 (a)的融合蛋白与细胞共孵育， 从而端粒酶活性抑制蛋白 LPTS被导入 细胞内。
在另一优选例中， 所述的细胞是端粒酶阳性的细胞。
在另一优选例中， 所述的细胞是端粒酶阳性的肿瘤细胞。
在另一优选例中， 所述获得融合蛋白的方法包括：
(i) 提供一构建物， 所述的构建物中含有一基因表达盒， 所述基因表达盒 含有以下操作性相连的元件：反式激活蛋白 TAT编码基因与端粒酶活性抑制蛋 白 LPTS编码基因；
(ii) 将 （i) 的构建物导入细胞表达***， 从而表达和纯化获得所述融合蛋 白。
在另一优选例中， 所述的反式激活蛋白 TAT 位于融合蛋白的氨基端； 所 述的端粒酶活性抑制蛋白 LPTS位于融合蛋白的羧基端。
另一方面， 还提供一种抑制（如体外抑制）肿瘤细胞生长的方法， 所述的方 法包括利用所述的融合蛋白处理肿瘤细胞。
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显示 T-LPGENE融合蛋白的结构示意图。
图 2显示重组表达载体 pET24a +)-T-LPGENE的构造图。
图 3显示 T-LPGENE在大肠肝菌中的诱导表达以及分离纯化后的结果。 其 中， 1 : 蛋白 Maker; 2： IPTG诱导前； 3 : IPTG诱导后； 4: 超声破碎后的离心 上清； 5 : SP-sephorose纯化后的 T-LPGENE蛋白； 6 : Superdex 75 纯化后的 T-LPGENEg白。
图 4显示纯化后的 T-LPGENE蛋白具有体外抑制肿瘤细胞端粒酶的活性。其 中， 1-5 : 加入 T-LPGENE蛋白的浓度 (nmol)分别为： 20， 40， 80， 160， 320； 6： 空白对照。
图 5显示在 TAT的介导下 T-LPGENE蛋白具有跨膜进入肿瘤细胞内的活 性。其中， A:免疫荧光实验结果， ANTI-LPTS,红色荧光； DAPI:细胞核； PHASE: 细胞形态； B: Western-blot 实验结果， Control: 空白细胞； 2hr， 6hr， 24hr: 蛋白孵育 2hr， 6hr， 24hr。
图 6显示 T-LPGENE抑制端粒酶阳性肿瘤细胞的生长并延长细胞的倍增时 间的作用。 用 T-LPGENE处理 4种端粒酶阳性细胞， 连续培养 6〜8周， 绘制 各组细胞的生长曲线， 计算倍增代数； 纵坐标： 细胞数 (log2): 细胞数目以 2 为底， 计算其对数值， 代表细胞倍增代数； 横坐标： 培养天数。
图 7显示 T-LPGENE不影响端粒酶阴性肿瘤细胞的生长。 用 T-LPGENE 处理 2种端粒酶阴性细胞， 连续培养 7周， 绘制各组细胞的生长曲线， 计算倍 增代数； 纵坐标： 细胞数 (log2): 细胞数目以 2为底， 计算其对数值， 代表细胞 倍增代数； 横坐标： 培养天数。
图 8显示 T-LPGENE处理端粒酶阳性肿瘤细胞 3周后对细胞增殖的抑制作 用。 A: 肝癌 BEL-7404细胞； B : *** HeLa细胞； C: 肝癌 SMMC-7901细 胞； D: 胃癌 SGC-7901细胞。 共检测 5〜7天， 检测波长为 570 nm。 图中结果 为 5次平行实验的平均值及其标准差。
图 9显示 T-LPGENE处理端粒酶阳性肿瘤细胞 6周后对细胞增殖的显著抑 制作用。 A: 肝癌 BEL-7404细胞； B: 肝癌 HepG2细胞； C: 肝癌 SMMC-7721 细胞； D: 人舌鳞状细胞癌 TCA-8113细胞。 共检测 7天， 检测波长为 570 nm。 图中结果为 5次平行实验的平均值及其标准差。
图 10显示 T-LPGENE不影响端粒酶阴性细胞的增殖。 A: 肝永生化 L02 细胞 (6周后；)； B : 肝永生化 QSG-7701细胞 (3周后；)； C: 骨髓瘤 Saos-2细胞 (6 周后；)。 共检测 7天， 检测波长为 570 nm。 图中结果为 5次平行实验的平均值 及其标准差。
图 11A显示了各细胞 (BEL-7404、 HepG2和 Saos-2) 经 T-LPGENE蛋白处 理与对照处理 （PBS和 TAT-GFP处理） 后， 细胞端粒缩短情况。
图 11B显示了各细胞 （BEL-7404、 HepG2、 Saos-2禾卩 L02) 经 T-LPGENE 蛋白处理与对照处理 （PBS和 TAT-GFP处理） 后细胞的死亡情况比较，其中箭 头所指处是死亡细胞处。
图 12显示用流式细胞法测定的 T-LPGENE处理细胞 6周后的存活率。 A: BEL-7404细胞单染 PI的 FCM检测图， 横坐标为荧光值， 纵坐标为细胞数目。 B: 以 PBS组细胞存活数为 100%， 统计 BEL-7404和 HepG2细胞的相对存活 率； C: 以 PBS组细胞存活数为 100%， 统计 Soas-2和 LO2细胞的相对存活率。 图中结果为 3次平行实验的平均值及其标准差。
图 13显示流式细胞仪分析的由 T-LPGENE诱导细胞的死亡。 横坐标为荧 光值， 纵坐标为细胞数目。 sub-Gl区为死亡细胞， 2N(2倍体)区为 G0/G1期细 胞， 4N(4倍体区;)为 G2/M期细胞， 2N〜4N之间为 S期细胞。 A: BEL-7404 肝癌细胞； B: HepG2肝癌细胞； C: SGC-7901 胃癌细胞； D: L02肝永生化 细胞； E: Saos-2骨髓瘤细胞。
图 14 显示纯化后的 T-LPGENE 蛋白具有体内降低端粒酶阳性细胞 BEL-7404致瘤性的活性。 其中， A: 显示纯化后的 T-LPGENE蛋白具有体降低 端粒酶阳性细胞 BEL-7404致瘤性的活性； B : 移植肿瘤后的小鼠经 T-LPGENE 蛋白处理与对照处理 （PBS和 TAT-GFP处理)后， 肿瘤的体积变化； C: 移植肿 瘤后的小鼠经 T-LPGENE蛋白处理与对照处理 （； PBS和 TAT-GFP处理)后， 肿 瘤在动物体内生长情况的照片； D: 移植肿瘤后的小鼠经 T-LPGENE蛋白处理 与对照处理 （； PBS和 TAT-GFP处理)后， 肿瘤的重量变化。 具体实施方式
本发明人经过长期的研究和试验， 意外地发现端粒酶活性抑制蛋白 LPTS 和反式激活蛋白 TAT 融合在一起形成的融合蛋白不仅良好地保留了端粒酶活 性抑制蛋白 LPTS的端粒酶抑制作用，又显著提高了端粒酶活性抑制蛋白 LPTS 进入细胞的能力， 从而大大提高了对端粒酶阳性细胞的杀伤作用， 且对端粒酶 阴性的细胞没有显著的毒副作用。
本发明人选择 TAT携带 LPTS进入细胞， 即 TAT与 LPTS蛋白融合， 制 备成 TAT-LPGENE 融合蛋白， 是经过实验研究确定。 将蛋白携带进细胞的方 法也有多种， 选择那一种方法最好， 需经过反复实验研究确定。 而 TAT也并非 对所有蛋白都适用， 需要对其携带的蛋白进行筛选。 因此， 本发明的成功， 并 不是通过简单的模仿获得的， 而是经创造性的劳动， 结合 LPTS蛋白的特性设 计、 经反复实验筛选而获得的。
LPTS 蛋白本身基本上不具备跨膜进入细胞内部的能力， 而端粒酶位于细 胞核内，因而用 LPTS蛋白处理端粒酶阳性细胞后对细胞的生长没有明显作用。 因此， LPTS 要作为抗肿瘤的蛋白药物施用时， 该蛋白能否进入肿瘤细胞是其 行使药效的关键步骤。
本领域中已知的将蛋白携带入细胞的方法有很多种， 但很多方法对携带端 粒酶活性抑制蛋白 LPTS进入细胞并不适用。 例如， 为了使端粒酶活性抑制蛋 白 LPTS能够穿透细胞膜进入到细胞内， 本发明人尝试了采用本领域最常用的 方法 脂质体转染法将 LPTS转运入细胞内， 即通过脂质体包埋携带蛋白质 药物进入细胞内。 发明人将阳离子脂质体按一定比例与 LPGENE蛋白混合， 再 与细胞一起孵育 2〜72小时， 然后用 westem-blot方法检测细胞内有无 LPGENE 蛋白， 结果显示 24 小时后细胞内没有目的蛋白出现， 从而证明了这种方法不 能将 LPGENE运输到细胞内部。
本发明人还尝试了采用多种穿膜蛋白与 LPTS相连接并测试穿膜效果， 却 无法成功使得 LPTS进入细胞。 而在尝试将 TAT蛋白与 LPTS融合后， 却意外 发现， 虽然反式激活蛋白 TAT并非对所有蛋白质都适用 (例如对淋巴细胞性脉 络丛脑膜炎病毒核蛋白， LCMV-NP不适用）， 但它却是最适合与 LPTS相连接 并提高 LPTS进入细胞能力的蛋白。
具体而言， TAT 是最近研究发现的一种能携带大分子物质进入细胞的多 肽， 但是， 本领域中也报道了一些不成功的事例， 对它的功能多样性也存在一 定的争议 (Green I等， Trends Pharmacol Sci. 2003； Leifert JA等， Gene Ther. 2002) 0 因此， 尽管 TAT可看作是一种跨膜肽， 但针对所融合或携带的肽段， 需进行大量的筛选才能获得成功。
本发明人利用基因工程的方法， 把 TAT多肽与 LPGENE连接在一起， 组 成一个融合蛋白， 利用 TAT的跨膜转运功能， 把 LPGENE运送到细胞内部， 进一步检测了融合了 TAT对 LPGENE蛋白的功能和特性是否发生影响。 研究 结果表明， TAT-LPGENE融合蛋白的抗肿瘤特性和抑制端粒酶的靶向性没有发 生改变， 证明本发明的实验设计和制备工艺是成功的。 更佳地， 含有 LPTS全长序列中 C末端的约 190-200个氨基酸的蛋白最适 合于与 TAT融合，其形成的融合蛋白最易于转入到端粒酶阳性的细胞内， 发挥 优异的端粒酶抑制效果。
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。 反式激活蛋白 TAT
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和 Arg9等等。 尽 管以往有利用 TAT来携带某些蛋白进入细胞的先例， 然而 TAT并非适合于介 导所有种类的蛋白进入到细胞内部， 适合的蛋白受到蛋白长度、 性质、 空间结 构等因素的限制； 并且， 以往的经验也发现， TAT与活性蛋白融合后， 可能会 影响后者的折叠， 进而影响后者的生物活性。 经过反复研究和比较， 本发明人 发现， TAT特别适合于与 LPTS融合， 形成的融合蛋白易于进入到细胞， 并且 可以进入到细胞核内。
较佳的，所述的反式激活蛋白具有 SEQ ID NO: 2中第 2-12位的氨基酸序列。 端粒酶活性抑制蛋白 LPTS
LPTS是一种具有抑制肿瘤细胞端粒酶活性的蛋白， 其定位于人第 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不表达， 肿瘤细胞中端粒酶活性的升高， 可 能与 LPTS基因的缺失或表达下调有关。
本发明可用 LPTS的全长蛋白或其生物活性片段。 任何一种 LPTS蛋白的生 物活性片段都可以应用到本发明中。在这里， LPTS蛋白的生物活性片段的含义 是指作为一种蛋白片段， 其仍然能保持完整的 LPTS蛋白的全部或部分功能 （如 至少 50%的生物活性， 较佳的至少 70%的活性， 更佳的至少 90%的活性；)。 经过 一个或多个氨基酸残基的取代、缺失或添加而形成的 LPTS的氨基酸序列也包括 在本发明中。 所述的经过一个或多个氨基酸残基的取代、 缺失或添加而形成的 LPTS蛋白在与 TAT融合后也具有穿透细胞且具有抑制细胞端粒酶活性或表达 的功能。 本发明也可采用经修饰或改良的 LPTS蛋白， 比如， 可采用为了延长其 半衰期、 改善其稳定性而改良的 LPTS蛋白。
本发明人在试验中意外地发现， 含有 LPTS全长序列中 C末端的约 190-200 个氨基酸的蛋白最适合于与 TAT融合， 其形成的融合蛋白最易于转入到端粒酶 阳性的细胞内， 发挥优异的端粒酶抑制效果。
作为本发明的一种优选方式，所述的 LPTS的氨基酸序列可以与 SEQ ID NO: 4所示的序列基本上相同。 更优选的 LPTS的氨基酸序列可以与 SEQ ID NO: 2中 第 16-211位所示序列基本上相同。 连接肽
本发明的融合蛋白中 TAT多肽和 LPTS蛋白或其活性片段之间可以直接相 连接， 或者通过多肽连接子 (连接肽;)连接。 作为本发明的一种优选的方式， 所 述的 TAT和 LPTS或其活性片段通过多肽连接子 (连接肽)连接， 从而形成融合蛋 白。 所述的连接子包括 0-20个氨基酸； 较佳地为 0-15个氨基酸， 更佳地为 0-10 个氨基酸， 最佳的是 1-4个氨基酸， 如 2-3个。
例如， 在本发明的优选实施方式中， 所设计的融合蛋白包括蛋白转导区 TAT、 端粒酶活性抑制多肽 LPTS以及三个氨基酸组成的连接肽三个部分， 其结 构如式 I所示：
TAT-连接肽 -LPTS (式 I)
其中， LPTS肽段是一个高效的端粒酶活性抑制蛋白肽， 是主要的药效功能 区， 位于 C端；
TAT多肽是一个能介导融合蛋白进入细胞内部的转导区， 位于 N端； 在 TAT和 LPTS起到连接作用的是由三个氨基酸 (甘氨酸 -甘氨酸-丝氨酸， GGS)组成的连接肽。 采用该连接肽具有如下优点：
1. 采用 3个氨基酸组成的小肽， 提供一定的柔性， 使 LPGENE能自由转动 和折叠， 保证其活性。 而常用的 TAT于其携带蛋白之间的连接肽都在 12-15氨基 酸， 采用 3个氨基酸组成的小肽， 把两个功能区连接起来， 又可避免二者之间 的相互影响， 使它们能够正常折叠， 从而能维持各自的结构和功能， 保证两个 功能区的生理活性。 体内、 体外试验证明了融合蛋白中 TAT的穿膜功能和 LPGENE抑制肿瘤的功能都得到了保证；
2.连接肽自身非常短小， 只有三个氨基酸， 可尽量减少它对生物体本身的 毒性。 毒性实验证明 T AT-LPGENE对细胞和动物没有明显的毒副作用；
3.连接肽的 DNA序列含有一个 Bamffl酶切位点， 这为构建 TAT-LPGENE的 表达载体提供一个适合于切割和组装的位点。 融合蛋白
本发明提供一种融合蛋白， 该蛋白包括 TAT蛋白， 和 LPTS蛋白或其生物 活性片段。 术语 "反式激活蛋白的融合蛋白和端粒酶活性抑制蛋白 " 、
" TAT-LPTS 融合蛋白" 、 " T-LPTS " 或 " T-LPGENE" 等可互换使用， 都指 由 TAT氨基酸序列和 LPTS氨基酸序列融合而成的蛋白，其中在两者之间可以 有或者没有连接肽序列。
所述的融合蛋白能用于抑制细胞端粒酶活性或表达。 更优选的， 所述的融 合蛋白是一种分离的蛋白， 与其它蛋白、 多肽或分子无联系， 是重组宿主细胞 培养的纯化产物或作为一种纯化的提取物。
所述的 TAT多肽和 LPTS蛋白或其活性片段之间可以直接相连接，或者通 过多肽连接子 (连接肽;)连接。 作为本发明的一种优选的方式， 所述的 TAT 和 LPTS或其活性片段通过多肽连接子 (连接肽;)连接， 从而形成融合蛋白。 所述的 连接子包括 0-20个氨基酸；较佳地为 0-15个氨基酸，更佳地为 0-10个氨基酸， 最佳的是 1-4个氨基酸， 如 2-3个。
作为一种优选的方式， 所述的 TAT多肽位于融合蛋白的氨基端 (N端)； 所 述的 LPTS蛋白或其活性片段位于融合蛋白的羧基端 (C端)。可选择地， 也可互 换两种蛋白所处的位置。
此外， 可选择地， 所述的融合蛋白的氨基端 (或羧基端)还可含有一个或多 个多肽片段， 作为蛋白标签。 任何合适的标签都可以用于本发明。 例如， 所述 的标签可以是 FLAG, HA， HA1， c-Myc， 6-His等。 这些标签可用于对融合蛋 白进行纯化。 一个具体的例子是在融合蛋白的 C末端连接有 6-His结构。 本领域 人员应理解， 可以在蛋白标签氨基酸序列与 TAT-LPTS融合蛋白氨基酸序列之 间设置可酶切结构， 从而可将标签从融合蛋白上分离。
本发明的融合蛋白既能够抑制端粒酶活性， 又能够进入细胞内部， 这样就 解决了 LPTS应用上的限制。 对于 LPTS细胞内功能的研究， 以前的研究是在基 因水平上进行的， 属于基础性的研究， 其需要在细胞基因组中引入外源基因， 难以进行实际应用。 而本发明提出了蛋白水平上将 LPTS引入到细胞内发挥作 用， 从而使得 LPTS可在临床上被应用。
另一方面， 本发明还提供了编码所述的融合蛋白的分离的核酸， 也可以是 其互补链。
编码本发明融合蛋白的 DNA序列， 可以全序列人工合成， 也可用 PCR扩 增的方法分别获得编码 TAT和 LPTS氨基酸的 DNA序列，然后将其拼接起来， 形成编码本发明融合蛋白的 DNA序列。
在获得了编码本发明融合蛋白的 DNA序列之后， 将其连入合适的表达载 体， 再转入合适的宿主细胞。 最后通过培养转化后的宿主细胞， 通过分离纯化 得到本发明的融合蛋白。 因此， 本发明还提供了包含编码所述融合蛋白的核酸分子的载体。 所述的 载体还可包含与所述核酸分子的序列操作性相连的表达调控序列， 以便于所述 融合蛋白的表达。
如本文所用， "操作性相连 "或 "可操作地连于 "指这样一种状况， 即线 性 DNA序列的某些部分能够影响同一线性 DNA序列其它部分的活性。 例如， 如果启动子控制序列的转录， 那么它就是可操作地连于编码序列。
在本发明中， 任何合适的载体都可以使用， 比如一些用于细菌、 真菌、 酵 母和哺乳动物细胞的克隆和表达的载体， 如 Pouwels等， 克隆载体： 实验室手 册 (Elsevier最新版)中所描述的。 可选用本领域已知的各种载体如市售的载体。 比如， 选用市售的载体， 然后将编码本发明新融合蛋白的核苷酸序列可操作地 连于表达调控序列， 形成蛋白表达载体。 在本发明的一种实施方式中， 所述的 载体为原核载体， 如 pET载体。
此外， 含有编码所述融合蛋白的核酸序列的重组细胞也包括在本发明中。 在本发明中， 术语 "宿主细胞"包括原核细胞和真核细胞。 常用的原核宿 主细胞包括大肠杆菌、 枯草杆菌等； 例如可为大肠杆菌细胞 (E. coli)， 如大肠杆 菌 HMS174(DE3)、 或 BL21(DE3)。 常用的真核宿主细胞包括酵母细胞、 昆虫细 胞和哺乳动物细胞。
在本发明的优选实施方式中， 采用原核细胞作为宿主细胞， 经表达、 纯化 后获得保留了良好的抑制端粒酶活性且细胞膜穿透性良好的融合蛋白。
生产融合蛋白的方法也已包括在本发明中。 所述方法包括培养含有融合蛋 白编码核酸的重组细胞。所述的融合蛋白包括 TAT多肽以及 LPTS蛋白或其活性 片段。 所述方法可包括让细胞表达编码的融合蛋白， 以及使表达的融合蛋白的 复性。 在一个实例中， 所述方法还可包括复性的融合蛋白的分离和 /或纯化。
可将上述制备获得的融合蛋白纯化为基本均一的性质， 例如在 SDS-PAGE 电泳上呈单一条带。 例如， 当重组蛋白为分泌表达时， 可以采用商品化的超滤 膜来分离所述蛋白， 例如 Millipore、 Amicon、 Pellicon等公司产品， 首先将表达 上清浓缩。 浓缩液可采用凝胶层析的方法进一步加以纯化， 或采用离子交换层 析的方法纯化。例如阴离子交换层析 (DEAE等；)或阳离子交换层析。凝胶基质可 为琼脂糖、 葡聚糖、 聚酰胺等常用于蛋白纯化的基质。 SP基团是较为理想的离 子交换基团。 最后， 还可用反相高效液相色谱 (RP-HPLC)等方法对上述纯化产 物进一步精制纯化。 上述所有纯化步骤可利用不同的组合， 最终使蛋白纯度达 到基本均一。
可利用含有 TAT或 LPTS的特异性抗体、受体或配体的亲和层析柱对表达的 融合性蛋白进行纯化。 根据所使用的亲和柱的特性， 可利用常规的方法， 如高 盐缓冲液、 改变 pH等方法洗脱结合在亲和柱上的融合性多肽。
本发明的融合蛋白可用于制备抑制细胞端粒酶活性或表达的组合物， 从而 用于抑制端粒酶阳性细胞的生长， 降低其致瘤性。 本发明的融合蛋白具有优异 的进入细胞 (如肿瘤细胞；)的能力， 且进入细胞后可良好地抑制细胞端粒酶活性 或表达的功能。 因而， 本发明的融合蛋白具有比 LPTS更优异的杀肿瘤效果， 从 而可用于开发有效的抗肿瘤药物。
本发明的 "肿瘤"可以是多种类型的， 只要肿瘤细胞是端粒酶阳性的， 例 如可包括 (；但不限于；)： 肝癌、 ***、 白血病、 胃癌、 舌鳞状细胞癌等， 例如 BEL-7404肝癌细胞株、 HepG2肝癌细胞株、 Hela***细胞株、 SMMC-7721 肝癌细胞株、 HL-60白血病细胞株、 K-562白血病细胞株、 SGC-7901胃癌细胞株、 或 TCA-8113人舌鳞状细胞癌细胞株等等。 组合物
本发明还提供一种抑制抑制细胞端粒酶活性或表达的组合物， 所述的组合 物含有： （i) 有效量 (如 0.0001-50wt%； 更佳的是 0.001-20^%)的本发明所述的 TAT与 LPTS的融合蛋白； 和 (ii) 药学上可接受的载体。
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***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， 包括各种赋形剂和稀 释剂。 该术语指这样一些药剂载体： 它们本身并不是必要的活性成分， 且施用 后没有过分的毒性。 合适的载体是本领域普通技术人员所熟知的。 在 Remington's Pharmaceutical Sciences(Mack Pub. Co. , N.J. 1991) 中可找到关于 药学上可接受的载体的充分说明。 在组合物中药学上可接受的载体可含有液 体， 如水、 盐水、 甘油和山梨醇。 另外， 这些载体中还可能存在辅助性的物质， 如润滑剂、 助流剂、 润湿剂或乳化剂、 pH缓冲物质和稳定剂， 如白蛋白等。
可将所述的组合物制成各种适合于哺乳动物给药的剂型， 所述剂型包括但 不限于： 注射剂、 胶囊剂、 片剂、 乳剂、 栓剂。
在使用时， 是将安全有效量的本发明所述的融合蛋白施用于哺乳动物 (如 人)， 其中该安全有效量通常至少约 0.1微克 /千克体重， 而且在大多数情况下不 超过约 50毫克 /千克体重， 较佳地该剂量是约 1微克 /千克体重-约 10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。
在用于抑制哺乳动物肿瘤时， 所述的融合蛋白可全身性施用， 或者局部施 用， 具体可视肿瘤的种类、 生长部位、 进展程度等因素决定。 本发明的组合物可直接用于杀伤肿瘤细胞。 此外， 还可同时与其它治疗剂 或辅剂联合使用。 本发明的主要优点在于：
(1) 首次提供且制备出 TAT与 LPTS的融合蛋白， 经证实 LPTS与 TAT的融合 在促进 LPTS穿透细胞膜的同时不影响 LPTS的抑制细胞端粒酶活性。
(2) 本发明的融合蛋白比单用 LPTS 具有显著更优异的抗肿瘤活性， 并且 对于许多肿瘤均可用。 实施例
下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说 明本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方 法， 通常按照常规条件如 Sambrook等人， 分子克隆： 实验室指南 (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。 除非另外说明， 否则百分比和份数按重量计算。 实施例 1. 含有 LPTS片段的 T-LPGENE (T-LPTS-C) 融合蛋白的构建以 及重组基因工程菌的制备
构建 T-LPGENE工程菌具体方法如下：
(1) 构建 pET24-TAT质粒
TAT模板以及其引物均由合成获得， 具体序列如下：
模板：
GTGGATCCTAGAAG-3 ' (SEQ ID NO: 5)；
Pl(上游引物)：
5'-AGTTTCATATGTACGGGC-3' (SEQ ID NO: 6)；
P2(下游引物）：
5'-CGCCACCTAGGATCTTC-3' (SEQ ID NO: 7)。
通过 PCR反应， 获得两端带有酶切位点 Nde I和 BamH I的含有编码 TAT 多肽 (11个氨基酸)的 DNA序列； PCR反应条件为： PCR反应在 50 μΐ体系中进 行， 包括 Ιμΐ 模板， Ιμΐ dNTP mix(lOmM) , 上下游引物 (20μΜ)各 1μ1， 5μ1 lOxPYROBEST 缓冲液， 0.5μ1 PYROBEST酶， 补加去离子水至 50μ1。反应条件 为： 94°C 3分钟； 之后共进行 30个循环， 每循环包括 94 °C 20秒， 50°C 20秒， 72°C20秒； 最后再 72°C延伸 5分钟， 4°C保温。 将如上获得的 PCR产物， 经过 Nde I和 BamHI双酶切后， ***经过同样 双酶切的 pET24( ) (；购自 Novagen公司;)质粒， 得到 pET24-TAT质粒。
(2)构建 pET24-T-LPGENE质粒
以 pT-LPTSCDS质粒为模板，该质粒含有全长 LPTS基因 (；参见 Hepatology, 32 (2000)， 721-727), 引物如下：
Pl(上游引物)：
5'-AAAGGATCCAAGGATCTGTCATCTCGG-3 ' (SEQ ID NO: 8)；
P2(下游引物）：
5'-AAACTCGAGTTTGGAATCTTTCTTCTT-3 ' (SEQ ID NO: 9)。
通过 PCR反应， 获得两端带有酶切位点 BamHI和 Xhol的含有编码 LPTS 蛋白 C端 196氨基酸 （LPTS-C) 的 DNA序列，序列见 SEQ ID NO: 2中第 16-211 位所示氨基酸序列； PCR反应条件为： PCR反应在 50 μ1体系中进行， 包括 Ιμΐ 模板， l l dNTP mix(10mM)， 上下游引物 (20μΜ)各 1μ1， 5μ1 lOxPYROBEST 缓 冲液， 0.5 l PYROBEST酶， 补加去离子水至 50μ1。 反应条件为： 94°C 3分钟， 之后共进行 30个循环，每循环包括 94 °C 30秒， 56 °C 40秒， 72°C60秒；再 72°C 延伸 5分钟， 4°C保温。
将如上获得的 LPGENE基因的 PCR产物，经过 BamH I和 Xho I双酶切后, ***经过 BamH I 和 Xho I 双酶切的 pET24-TAT 质粒， 最后得到 pET24-T-LPGENE质粒，该质粒上有编码 T-LPGENE的 DNA序列，序列见 SEQ ID NO: 1， 结构示意图见图 1。 pET24-T-LPGENE表达质粒的构造见图 2。 经测 序验证序列无误后转化宿主菌 E.coli BL-21， 获得表达 T-LPGENE的工程菌。 实施例 2. T-LPGENE的诱导表达和分离纯化
本发明人采用了离子交换等分离方法， 获得了高纯度的 T-LPGENE蛋白， 可简单分为以下几个步骤：
(1)常规方法诱导表达 T-LPGENE蛋白；
(2)超声波破碎菌体；
(3)阳离子交换层析 (SP-Sepharose);
(4)超滤浓缩；
(5)分子筛层析 (Superdex 75)；
实验在 4°C条件下进行。 SDS-PAGE检测 T-LPGENE纯度和浓度， 结果见 图 3(泳道 6)， T-LPGENE蛋白的纯度可达 90%以上。 实施例 3. T-LPGENE体外抑制端粒酶活性的检测
T-LPGENE是一种很强的端粒酶活性抑制剂。 本发明人采用 TPAP法测定 纯化的 T-LPGENE的生物学活性。 端粒酶来自 SMMC-7721肝癌细胞裂解液， 具体制备方法如下：
将 SMMC-7721 肝癌细胞 (购自 ATCC)接种在 10ml 培养瓶中， 培养基为 RPMI 1640, 37°C， 5%CO2条件下贴壁培养， 满瓶后收集细胞。 即将培养基吸 干， 用 PBS 漂洗一遍细胞， 用胰酶消化细胞， PBS 冲洗细胞， 吸入离心管， 4000rpm离心收集细胞。 再用洗涤缓冲液 （10 mM Hepes-KOH (pH7.5)， 1.5mM MgCl2， 10 mM KCl， 1 mM DTT (dithionthreitoll )) 洗一遍细胞， 离心。 用预冷 裂解缓冲液（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% 甘油 ）500 μ ΐ重悬细胞， 置冰上 30分钟， 4°C， 12,000rpm离心 30分钟， 获得上清可用来测定端粒酶活性。 上 清液可保存在 -70°C， 经多次冻溶仍可保持稳定的端粒酶活性。
TPAP法测定在 500 μ 1 Eppendorf管中进行， 反应体积为 50 μ 1， 含有常规 的反应缓冲液 45.25 μ 1， 0.8 μ ΐ含有端粒酶的肿瘤细胞裂解液， 0.25 l dNTP， ΐ μ ΐ纯化蛋白（根据要求稀释）。 冰上反应 lOmin后， 加入 l l Ts 引物 (O. l g/ μ 1， 引物序列为 5，-AATCCGTCGAGCAGAGTT-3， (SEQ ID NO: 10))， 1 μ 1 Taq 酶， 25 °C延伸 30min， 95°C灭活 5min， 再补力卩 Taq酶 (0.5 μ 1)、 Acx 引物（1 μ 1， 0.1μδ/ μ 1, 引物序列为 5，-(CCCTTA)3CCCTAA-3，（SEQ ID NO: 11))， 进行 PCR 反应 （94°C 30s， 50 °C 40s, 72 °C 40s, 33个循环； 72°C 2min) 。 采用 10%聚丙 烯酰胺凝胶电泳对 PCR反应产物进行鉴定，电泳体系为 TBE缓冲液，电泳 （120 伏， 2hr) 后， 银染显色。
TRAP实验结果表明， 本发明纯化所得 T-LPGENE蛋白在体外有很高的端 粒酶抑制活性，在约为 320nmol/L时， T-LPGENE就能完全抑制端粒酶的活性， 参见图 4。 实施例 4. TAT介导 LPGENE蛋白跨膜进入肿瘤细胞内的活性检测 首先， 本发明人尝试了采用常规的脂质体转染法将 LPTS-C蛋白运载到各 肿瘤细胞内。即将阳离子脂质体 (上海生化细胞研究所;)按一定比例 (2: 1，体积比；) 与 LPTS-C蛋白混合， 检测蛋白被包封后， 添加到正常培养的细胞中， 最终的 蛋白浓度为 100 mg/L， 37°C， 5%CO2条件下一起孵育 24小时。 用 western-blot 方法 (；同本实施例下文中的表述;)检测细胞内有无 LPGENE蛋白。 结果显示细胞 内没有目的蛋白出现。 由此证明了， 脂质体法不适合转运 LPTS-C和 LPTS蛋 白。 为了验证 TAT是否能够介导 LPGENE蛋白进入细胞内， 本发明人选用了 两种实验方法： 免疫荧光实验和 Westem-blot实验。 免疫荧光实验具体操作方 法如下：
(1) 将 BEL-7404细胞 (；购自上海生化细胞所细胞库)和 Saos-2细胞 (；上海生 化细胞所细胞库;)接种在铺有盖玻片的六孔板中， 培养基分别为 DMEM (含 10% 新生小牛血清)和 Mccoy，s 5A (含 15% 胎牛血清）， 37°C， 5%CO2条件下贴壁培 养。 细胞贴壁后， 分别加入 PBS 溶液 (两个空)和纯化的 T-LPGENE(100mg/L) 蛋白（3个空， 分别孵育 2hr， 6hr和 24hr):
(2) 孵育后，用 PBS洗涤一次，每孔加入 lmL -20°C预冷的甲醇，固定 3min; 弃去甲醇，再用 PBS洗涤两遍，接着每孔加入 lmL PBS (；含 1%的新生山羊血清；)， 封闭 lOmin;
(3) 将固定好的玻片取出放入一封闭容器中， 滴加用 PBS (；含 1%的新生山 羊血清;)适当稀释的一抗 (； LPGENE蛋白免疫的家兔抗血清 室温反应 2hr， 其 中一个用 PBS培养处理过的玻片只滴加 PBS (；含 1%的新生山羊血清；)， 作为空 白对照；
(4) 用 PBS (含 1%的新生山羊血清)洗涤 3次， 每次 5min;
(5) 滴加用 PBS (；含 1%的新生山羊血清；)适当稀释的二抗 (； FITC标记的山羊 抗兔血清；)， 室温避光反应 lhr; 用 PBS (；含 1%的新生山羊血清；)洗涤 3次， 每次 5min;
(6) 玻片放回 6孔板， 每孔加入 lmL PBS (；含 1%的新生山羊血清)和 ΙΟμ 的 33258试剂， 染核 lOmin;
(7) 用 PBS洗涤 3次， 每次 5min;
(8) 取一洁净载玻片， 滴加 50 L猫油， 将处理好的盖玻片有细胞的一面 朝下置于载玻片上， 盖玻片与猫油间无气泡， 室温避光 4hr或过夜。
Western-blot实验具体操作方法如下：
(1) 将 BEL-7404 细胞和 Saos-2 细胞接种在 6cm培养皿中培养基分别为 DMEM (含 10%新生小牛血清)和 Mccoy's 5A (含 15%胎牛血清）， 37°C， 5%CO2 条件下贴壁培养。 细胞密度超过 30%后， 分别加入 PBS溶液 (；两个空；)、 纯化的 LPGENE(100mg/L)蛋白（3 个孔， 分别孵育 2hr， 6hr 和 24hr)和纯化的 T-LPGENE(100mg/L)蛋白（3个孔， 分别孵育 2hr， 6hr和 24hr):
(2) PBS洗涤两次后， 用胰酶消化 lOmin;
(3) 离心收集细胞，按每 106细胞加入 100 μ L SDS-PAGE电泳上样缓冲液；
(4) 用 Western Blotting方法检测各个样品， 即样品经 SDS-PAGE电泳后， 用 Bio-Rad公司的电转仪将蛋白转至硝酸纤维素膜上， 用特异的抗 LPGENE抗 体 (； LPGENE 蛋白免疫的家兔抗血清;)进行杂交。 二抗为辣根过氧化物酶 (； HRP) 标记的羊抗兔 IgG。
免疫荧光实验和 westem-blot实验结果见图 5， 结果表明 TAT能够极其良 好地介导 LPGENE蛋白进入 BEL-7404细胞和 Saos-2细胞内， 并能进入细胞核 内。 实施例 5. 纯化后的 T-LPGENE蛋白具有体外抑制端粒酶阳性细胞生长的 活性
(1) 受试细胞 (细胞株均由中科院上海细胞库提供；)：
端粒酶阳性的肿瘤细胞 (8株)： BEL-7404肝癌细胞株、 HepG2肝癌细胞株、 SMMC-7721 肝癌细胞株、 Hela ***细胞株、 HL-60 白血病细胞株、 K-562 白血病细胞株、 SGC-7901胃癌细胞株、 TCA-8113人舌鳞状细胞癌细胞；
端粒酶阴性细胞 (3株)： QSG-7701肝永生化细胞株、 L02肝永生化细胞株、 Saos-2骨髓瘤细胞株。
(2) 生长曲线实验分析 T-LPGENE对细胞的生长抑制作用
将检测细胞接种在铺有盖玻片的 6孔板中， 培养基按细胞库提供的条件配 置， 37 °C， 5% CO2条件下贴壁培养。 分别向 6 孔板中加入 PBS 溶液、 T-LPGENE(10 g/mL)、 T-LPGENE(40 g/mL)或 T-GFP(40 g/mL， 对照）， 每次 传代测量细胞数目， 并按稀释倍数统计细胞数， 绘制各组细胞的生长曲线。 连 续培养 6〜8周 (绿色荧光蛋白 GFP和带有跨膜肽的绿色荧光蛋白 T-GFP作为试 验对照)。
实验结果： T-LPGENE蛋白能够抑制大多数端粒酶阳性肿瘤细胞的生长和 增殖， 而对端粒酶阴性细胞没有明显抑制作用。 图 6显示： BEL-7404、 HepG2、 K-562 和 HL-60 肿瘤细胞的生长受 T-LPGENE 的抑制而变缓， 其中对肝癌 BEL-7404细胞的抑制作用最为明显， 并且这种抑制作用具有浓度依赖性， 即随 着加入的 T-LPGENE 蛋白的浓度提高， 细胞的生长也更加缓慢。 图 7 显示： T-LPGENE对端粒酶阴性的 Saos-2和 L02的生长没有明显抑制作用。
该结果表明： T- LPGENE蛋白能专一性抑制端粒酶阳性肿瘤细胞的生长。
(3) MTT法分析 T-LPGENE对细胞的生长抑制作用
将检测细胞接种在铺有盖玻片的 6孔板中， 培养基按细胞库提供的条件配 置， 37°C，5% CO2条件下贴壁培养。分别向 6孔板中加入 15 μ1ΡΒ8溶液 (ρΗ 7.4)、 T-LPGENE(10ug/mL)、 T-LPGENE (40ug/mL)或 T-GFP (40ug/mL)。 T-GFP是在
GFP的 N端与 TAT相连接而成， GFP是实验室常用的荧光标记蛋白， 其全长 序列参见 GenBank, 登录号： DQ768212。 细胞连续处理 3或 6周后， 用 MTT 法检测各组细胞的生长活性，检测波长为 570 nm。具体的实验方法参见文献 (司 徒镇强，吴均正主编；细胞培养 [M] ;西安： 世界图书出版西安公司， 2004， 250)。
实验结果： MTT试验结果同样表明 T-LPGENE蛋白对肿瘤细胞有明显的 抑制用。 图 8和图 9显示 LP-GENE蛋白对 8种肿瘤细胞均明显的抑制作用， 抑制程度与 LP-GENE蛋白用量及细胞种类有关。 但是对端粒酶阴性细胞的生 长没有明显的作用（图 10)。 实验 (2)-(；3；)小结： 通过生长曲线法和 MTT法检测的 T-LPGENE蛋白对 11 株细胞的抑制活性结果， 概括如下 (见表 1):
Figure imgf000019_0001
+ : 抑制率为 10〜30%； ++： 抑制率为 30〜50%； +++： 抑制率为 50〜70%； ++++： 抑制率为 70〜90%； -: 抑制率小于 10%。 通过对 11株细胞的检测， 证明了 T-LPGENE蛋白对癌症细胞的选择性非 常好， 能专一性抑制端粒酶阳性肿瘤细胞的生长， 抑制程度与蛋白用量和细胞 种类有关， 但是对端粒酶阴性细胞的生长没有明显的作用。 这种靶向端粒酶的 专一性作为， 为开发为治疗药物及其有利， 特别是 85%的肿瘤细胞具有端粒酶 活性，而正常细胞没有或非常低，意味着 T-LPGENE对正常细胞没有杀伤作用。
(4) Southern-blot分析细胞端粒的长度
收集给药处理后 8周后的 BEL-7404细胞、 HepG2细胞、 Saos-2细胞和 L02 细胞， 用 Southem-blot方法分析端粒长度， 具体实验方法参见文献 (Damm K. et al.， A highly selective telomerase inhibitor limiting human cancer cell proliferation; EMBO J 2001 ; 20:6958-6968)。简单的过程为： 分离基因组 DNA， 用内切酶 Hinf I和 Afa l双酶切， 然后用 0.8%的琼脂糖凝胶分离消化产物， 转 膜后， 用标有 γ-32Ρ的探针杂交最后用 ImageQuant分析杂交信号。
实验结果见图 11A， 结果表明， 经 T-LPGENE蛋白处理 8周后， 与对照组 (PBS和 TAT-GFP处理） 相比， 端粒酶阳性肿瘤细胞 BEL-7404和 HepG2的端 粒明显缩短， 并且高浓度蛋白组的端粒比的浓度组的短。 而端粒酶阴性肿瘤细 胞 Saos-2端粒没有明显的缩短。
(5) 细胞形态检测
BEL-7404细胞、 HepG2细胞、 Saos-2细胞和 L02细胞经给药处理 6周后， 用倒置显微镜拍照， 结果见图 11B， 结果表明， 经 T-LPGENE蛋白处理的端粒 酶阳性肿瘤细胞 BEL-7404和 HepG2的贴壁能力变差， 细胞变得扁平而大， 这 是典型的危机 (crisis)形态。
人正常体细胞每***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***， 这是 细胞永生化和癌变的一个重要机制。 T-LPGENE蛋白能够抑制细胞内端粒酶活 性， 使端粒酶阳性肿瘤细胞的端粒随着细胞增殖而逐渐缩短， 进入危机期 (衰 老；)， 在形态上呈危机形态。 而端粒酶阴性肿瘤细胞 Saos-2和永生化细胞 L02 在形态在并没有明显变化。
(6) PI染色后流式细胞仪检测 T-LPGENE诱发肿瘤细胞死亡
细胞经碘化丙啶 (； PI)染色后， 流式细胞仪检测 T-LPGENE蛋白诱导细胞死 亡的情况。 PI能与细胞中的 DNA和 RNA结合， 使之着色。 但是 PI不能穿过 活细胞的细胞膜， 故细胞拒染。 PI 能穿过死细胞的细胞膜， 使细胞着色。 PI 鉴别细胞存活与否的灵敏度很高。 用 T-LPGENE蛋白处理细胞 6周后， 收集细胞， PI染色后用流式细胞仪 (FACSCalibur, Becton Dickinson, USA)进行检测。 本实验检测了 2株端粒酶阳 性细胞株， 即 BEL-7404肝癌细胞和 HepG2细胞， 2株端粒酶阴性细胞株， 即 肝永生化 L02细胞和骨髓瘤 Saos-2。
结果： 与对照组相比， 40 g/mL T-LPGENE处理的 BEL-7404细胞， 死亡 数显著增加，细胞的存活率明显下降 (图 12A)。以空白对照的活细胞数为 100%， 分析细胞的存活率。 BEL-7404细胞， 40 g/mL T-LPGENE剂量组的活细胞率 (相 对存活率;)约为 65.6%,显著低于对照组； 10 g/mL T-LPGENE组的相对存活率 也有明显下降， 其值约为 78.8%。 HepG2细胞， 40 g/mL T-LPGENE剂量组的 相对活细胞率为 85.3%， 显著低于对照； 10 g/mL T-LPGENE剂量组的相对活 细胞率为 89. 1%， 也显著低于对照组 (图 12B)。 端粒酶阴性的肝永生化 L02细 胞和骨髓瘤 Saos-2细胞, 40 g/mL的 T-LPGENE对它们的相对存活率没有影响 (图 12C)。
总结以上数据， 我们可以得出这样的结论， T-LPGENE能够诱导端粒酶阳 性细胞死亡， 降低它们的存活率， 并且具有一定的剂量效应， 但是不能诱导端 粒酶阴性细胞死亡。
(7) 流式细胞仪检测 DNA含量
肝癌 BEL-7404细胞、 肝癌 HepG2细胞、 和胃癌 SGC-7901细胞， 以及端 粒酶阴性的肝永生化 L02细胞和骨髓瘤 Saos-2细胞经给药 6周后，取样进行流 式细胞仪分析 (FACSCalibur, Becton Dickinson, USA)。 细胞用碘化丙啶 (PI)染 色， 通过流式细胞仪对细胞的 DNA含量进行检测， 确定细胞的周期分布， 其 中 sub-Gl区为死亡细胞， 2N(2倍体)区为 G0/G1期细胞， 4N(4倍体区）为 G2/M 期细胞， 2N〜4N之间为 S期细胞。 这是一种快速、 定量确定细胞活力和细胞 周期的方法。
实验结果见图 13， 结果表明， 经 T-LPGENE蛋白连续处理 6周后， 端粒 酶阳性肿瘤细胞 BEL-7404和 HepG2的 sub-Gl区含量显著增加， 即细胞的死 亡或者濒临死亡数量显著增加； 而端粒酶阴性肿瘤细胞 Saos-2 和永生化细胞 L02的 sub-Gl区没有明显变化。 实施例 6. T-LPGENE蛋白的抑瘤活性和降低端粒酶阳性细胞致瘤性研究 1. 纯化后的 T-LPGENE蛋白可降低端粒酶阳性细胞致瘤性
(1) 将 BEL-7404细胞和 Saos-2细胞接种在铺有盖玻片的六孔板中， 培养 基分别为 DMEM (含 10%新生小牛血清)和 Mccoy' s 5A (含 15% 胎牛血清）， 37°C， 5%CO2条件下贴壁培养。分别加入 PBS溶液和所述纯化的 T-LPGENE (；在 培养基中的终浓度是 40mg/L)和 TAT-GFP(40mg/L)蛋白：
(2) 连续培养 6周后， 收集细胞， 按 8 X 106接种到裸鼠皮下；
(3) 接种后， 每周观察肿瘤生长情况， 并测量肿瘤大小；
(4) 接种 6周后， 拍照， 取出肿瘤称重。
实验结果见图 14A、 表 2， 结果表明 T-LPGENE蛋白能够降低端粒酶阳性 细胞 BEL-7404的致瘤性， 而对降低端粒酶阴性细胞 Saos-2的致瘤性没有抑制 作用。 而 TAT 与绿色荧光蛋白的融合蛋白 TAT-GFP 蛋白对端粒酶阳性细胞 BEL-7404的致瘤性基本没有影响。
表 2
BEL-7404 肿瘤发生频率和瘤重（g)
PBS 5/5 (l.CSl, 0.S9, 0.74， 0.64, 0.53)
TAT-GFR 40mg/L 4/5 (0.99, 0,54, 0.24} 0.19)
l^LPGENE, 40iiig/L 4/5 (0.Q94, 0.074, 0.026, 0.019)
S&OS-2 肿瘤发生频率和瘤重（g)
PBS 5/5 (0.2, 0.1, D.098. 0.09, 0.067)
T-LPGENE, 40mg/L 4/5 (0,15, 0,13, 0.1, 0.072, Θ 2)
2. 纯化后的 T-LPGENE蛋白的抑瘤活性
肝癌细胞 BEL-7404接种 (5 X 106/只）到裸鼠 (4周龄， 雌性)右侧腰部皮下， 4 周后， 取出肿瘤， 切成 2mm3大小， 再接种到裸鼠 (4周龄， 雌性;)右侧腰部皮下， 把裸鼠随机分成 4组。 隔天后， 开始皮下注射 T-LPGENE蛋白（100 μ g/只、 400 μ g/K , 实验组）， 部位在肿瘤附近， 对照组注射 PBS和 TAT-GFP蛋白 (400 μ g/ 只；)。 开始的 2周每 2天注射一次， 随后的 3周每 3天注射一次， 共注射 14次。 每周 记录肿瘤大小 (用游标卡尺测量 接种 7周后给所有裸鼠拍照， 处死裸鼠， 取出 肿瘤称重。
实验结果见图 14B-D , 结果表明， T-LPGENE蛋白可以抑制裸鼠体内 BEL-7404肿瘤细胞的生长， 这种具有一定的剂量效应。 每只注射 100 T-LPGENE蛋白组的肿瘤重量只有 PBS组的 57%(P<0.05)， 而 400 μ g T-LPGENE 蛋白组的肿瘤重量只有 PBS组的 36% (； P<0.01)。 而 TAT-GFP组的肿瘤重量与 PBS 组没有明显差别， 但是明显重于两个实验组的 (P<0.05)。 高剂量实验组的肿瘤 实施例 7. 含全长 LPTS的融合蛋白的制备和细胞试验
按照实施例 1中记载的类似的方法来制备表达 TAT-全长 LPTS (TAT-LPTS), 首先构建 pET24-TAT质粒。 以 pT-LPTSCDS质粒为模板， PCR扩 增获得 LPTS的全长基因序列 (；见 SEQ ID NO: 3), 在两端设置酶切位点 BamH I和 Xho I， 用该两种酶双酶切后***经同样双酶切的 pET24-TAT质粒， 得到 pET24-T-LPTS质粒， 该质粒上有编码 TAT-LPTS的 DNA序列。 经测序验证序列 无误后转化宿主菌 co// BL-21， 获得表达的工程菌。
采用如前述实施例 2所述的方法诱导表达和分离纯化， 获得纯度 80%以上 TAT-LPTS蛋白。 体外试验 (；如实施例 3)发现， TAT-LPTS抑制端粒酶活性的能 力与 T-LPGENE基本相同。
采用如前述实施例 4所述的方法进行免疫荧光实验和 westem-blot实验， 检测 TAT-LPTS进入细胞的情况。 结果显示， TAT-LPTS 能够进入 BEL-7404 细胞和 Saos-2 细胞内， 并能进入细胞核内。 半定量试验发现， 与 T-LPGENE 蛋白进入细胞的情况对比， TAT-LPTS进入细胞核的量是 T-LPGENE蛋白进入 细胞的量的约 70%左右， 可见 T-LPGENE蛋白的转入效果更为理想。 实施例 8. 药物组合物
将实施例 3 中纯化获得的融合蛋白 lOOmg配制于 100ml的常规生理盐水 中， 获得浓度为 lmg/ml的含有融合蛋白的组合物。
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. 一种分离的融合蛋白， 其特征在于， 所述的融合蛋白包括：
(1) 端粒酶活性抑制蛋白 LPTS ;
(2) 反式激活蛋白 TAT; 和
(3) 位于 (1)和 (2)之间的由 0-20个氨基酸构成的连接肽。
2. 如权利要求 1所述的融合蛋白， 其特征在于， 所述的端粒酶活性抑制蛋 白 LPTS是：
(a) SEQ ID NO: 4所示的氨基酸序列的蛋白；
(b) SEQ ID NO: 2中第 16-211位所示氨基酸序列的蛋白； 或
(c) 将 （a) 或 （b) 所限定的蛋白的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) 或 （b) 所限定的蛋白功能的由 （a) 或 （b) 衍生的蛋白；
或者
所述的反式激活蛋白 TAT是具有 SEQ ID NO: 2中第 2-12位所示氨基酸序 列的蛋白。
3. 如权利要求 1所述的融合蛋白， 其特征在于， 所述的端粒酶活性抑制蛋 白 LPTS是：
(al) SEQ ID NO: 2中第 16-211位所示氨基酸序列的蛋白； 或
(bl) 将 （al) 所限定的蛋白的氨基酸序列经过 1-10个氨基酸残基的取代、 缺失或添加而形成的， 且具有 （al) 所限定的蛋白功能的由 （al) 衍生的蛋白。
较佳的， 所述连接肽的长度为 0-10个氨基酸， 更佳为 1-4个氨基酸。 在一 个优选例中， 所述连接肽的氨基酸序列为： GGS。
较佳的， 所述反式激活蛋白 TAT位于融合蛋白的氨基端 (N端)； 所述端粒 酶活性抑制蛋白 LPTS位于融合蛋白的羧基端 (C端)。
4. 一种核酸分子， 其特征在于， 所述的核酸分子编码权利要求 1-3任一所 述的融合蛋白。
5. —种载体， 其特征在于， 它含有权利要求 4所述的核酸分子。
6. 一种基因工程化的细胞， 其特征在于， 所述的细胞含有权利要求 5所述的载体； 或
所述的细胞基因组中整合有权利要求 4所述的核酸分子。
7. —种产生权利要求 1所述的融合蛋白的方法， 其特征在于， 所述的方法 包括： 在适合表达所述融合蛋白的条件下， 培养权利要求 6所述的细胞， 表达 和分离出所述的融合蛋白。
8. 权利要求 1-3任一所述的融合蛋白的用途， 其特征在于， 用于制备抑制 端粒酶阳性细胞生长的组合物。
较佳的， 所述的细胞是端粒酶阳性的肿瘤细胞， 优选所述细胞选自： 肝癌 细胞株、 ***细胞株、 白血病细胞株、 胃癌细胞株、 或舌鳞状细胞癌细胞株。
较佳的， 所述的端粒酶阳性细胞包括： BEL-7404肝癌细胞株、 HepG2肝 癌细胞株、 Hela***细胞株、 SMMC-7721肝癌细胞株、 HL-60 白血病细胞 株、 K-562白血病细胞株、 SGC-7901胃癌细胞株、 或 TCA-8113人舌鳞状细胞 癌细胞株。
9. 一种抑制肿瘤的组合物， 其特征在于， 所述的组合物含有：
(i) 有效量的权利要求 1-3任一所述的融合蛋白； 和
(ϋ) 药学上可接受的载体。
10. 一种将端粒酶活性抑制蛋白 LPTS 导入细胞的方法， 其特征在于， 所 述方法包括以下步骤：
(a) 将反式激活蛋白 TAT与端粒酶活性抑制蛋白 LPTS融合， 获得融合蛋 白；
(b) 将 (a)的融合蛋白与细胞共孵育， 从而端粒酶活性抑制蛋白 LPTS被导 入细胞内。
PCT/CN2009/076168 2009-12-29 2009-12-29 具有端粒酶抑制活性的融合蛋白、其制备方法和用途 WO2011079431A1 (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019134574A1 (zh) * 2018-01-05 2019-07-11 杭州端丽生物技术有限公司 防治端粒功能异常相关疾病的多肽及医药用途

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1318645A (zh) * 2000-04-14 2001-10-24 中国科学院上海生物化学研究所 一种肝癌相关基因及其应用
CN1948339A (zh) * 2005-10-14 2007-04-18 中国科学院上海生命科学研究院 端粒酶活性抑制蛋白的制备和纯化
CN101643511A (zh) * 2008-08-04 2010-02-10 中国科学院上海生命科学研究院 抑制端粒酶活性的融合蛋白、其制备及应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1318645A (zh) * 2000-04-14 2001-10-24 中国科学院上海生物化学研究所 一种肝癌相关基因及其应用
CN1948339A (zh) * 2005-10-14 2007-04-18 中国科学院上海生命科学研究院 端粒酶活性抑制蛋白的制备和纯化
CN101643511A (zh) * 2008-08-04 2010-02-10 中国科学院上海生命科学研究院 抑制端粒酶活性的融合蛋白、其制备及应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIAO, C. ET AL.: "Over-expression of LPTS-L in hepatocellular carcinoma cell line SMMC-7721 induces crisis.", vol. 8, no. 6, 15 December 2002 (2002-12-15), pages 1050 - 1052 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019134574A1 (zh) * 2018-01-05 2019-07-11 杭州端丽生物技术有限公司 防治端粒功能异常相关疾病的多肽及医药用途
US11365217B2 (en) 2018-01-05 2022-06-21 Hangzhou Duanli Biotechnology Company Limited Peptides for treating telomere dysfunction-associated diseases and uses thereof

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