CN101643511A - Fusion protein for inhibiting telomerase activity, preparation and application thereof - Google Patents

Abstract

The invention discloses fusion protein. The fusion protein comprises telomerase activity telomerase inhibitory protein LPTS and trans-activation protein TAT. The invention also discloses nucleic acidfor coding the fusion protein, a vector containing the nucleic acid and a host cell, and a composition containing the fusion protein. The firstly prepared fusion protein of the TAT and the LPTS is proved to be capable of penetrating the cell membrane and to have excellent effect of inhibiting the growth of tumor cells.

Description

Fusion protein for inhibiting telomerase activity, preparation and application thereof

Technical Field

The present invention belongs to the field of biotechnology and biochemical engineering. More specifically, the invention relates to a telomerase activity inhibitory protein fused with a protein transduction structural domain, and a preparation method and application thereof.

Background

Telomerase (Telomerase) is a ribonucleoprotein that synthesizes and extends telomeres of cellular chromosomes and contains two essential components: reverse transcriptase catalyzes the subunits hTERT and RNA component hTR. The telomerase can reverse transcribe and synthesize a telomere repetitive sequence by taking the RNA of the telomerase as a template, and the telomere repetitive sequence is added to the tail end of a chromosome to make up the loss of telomere DNA during cell division and maintain the length of the telomere.

Research shows that telomerase activity can not be detected in normal human body cells, so that the number of times of human normal body cell division is limited, cells are divided once per minute, telomeres are lost by 50-200bp, and when the telomeres are shortened to a certain extent, cell growth is inhibited, namely cell senescence is called and death is achieved.

However, telomerase activity is detected in most malignant cells (85%) and is highly active, and the resynthesis of telomeres by telomerase compensates for its sustained loss during cell proliferation, allowing cells to divide continuously, which is an important mechanism for cell immortalization and canceration.

Kim et al concluded a large number of studies and examined over 100 specimens of malignant tumors, indicating that telomerase has a tumor diagnosis sensitivity of 85%, a specificity of 91%, a positive predictive value of 91%, and a negative predictive value of 81%, indicating the value of telomerase in tumor diagnosis (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.). Kim and the like think that activation of telomerase is one of main factors for malignant tumor generation, the activation and expression degree of telomerase is closely related to generation and metastasis of tumor, and inhibition of telomerase and telomere shortening are thought to be a mechanism for inhibiting cancer cells, so that telomerase becomes an ideal target for tumor targeted therapy.

At present, the research on tumor therapy targeting telomerase mainly adopts antisense nucleic acid technology (Kondo S., Kondo Y., Li G., et al, Targeted therapy of human glimment in a mouse model by 2-5A antisense polymerase RNA. oncogene, 1998, 16: 33330. Ludwig A, Saretzki, Holm PS, et al. Ribozyme clearance of telogen mRNA sensing cleavage and specificity kinase enzyme, 2001, 61: 3053-1; Feng J. Funk W.D., S.S., Wang. therapy of tumor, Science of liposome cancer. cancer Res, 2001, 61: 3053-1; RNA transcription inhibition technology of RNA of tumor cell, mRNA of tumor cell of mRNA kinase, mRNA of tumor cell of mRNA kinase, 30625, mRNA of tumor cell of mRNA kinase, mRNA of RNA transcriptional gene of mRNA of tumor cell kinase, mRNA of tumor cell of mRNA of the family ribozyme transcriptional gene of mRNA of the family mRNA of the tumor cell of the family RNA of the family ribozyme transcriptional gene of the family mRNA of the family RNA of the family ribozyme transcriptional gene of the mRNA of the family of the RNA of the family ribozyme of the family RNA of the family RNA of the, m.counter c.m., Eaton e.n., et al. hest2, the reactive human transcriptional metabolic subunit gene, isup-regulated in tumor cells and during mobilization, Cell, 1997, 90: 785795, respectively; 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 telerasemistry the growth of human cancer cells, nat.med, 1999, 5: 1641170). These techniques can shorten the telomeres of tumor cells, thereby causing the cells to enter crisis or die, or significantly reducing the tumorigenicity of tumor cells.

LPTS is a protein with the activity of inhibiting the telomerase of tumor cells, is a novel protein preparation and has important application prospect. The LPTS protein is encoded by LPTS (liver reactive tumor suppressor) gene, which is a candidate anti-cancer new gene related to liver obtained from human normal liver cDNA library by the present inventors by using the method of positional cloning (Liao C., ZHAO M.J., Song H., Uchida K., Yokoyama K.K., Li T.P., Identification of the gene for a novel liver-related promoter should be maintained at a high-frequency low of a hydrolytic tumor region 8p23 in human hepatic cellular cancer. The protein coded by the gene has the activity of inhibiting cell telomerase (Zhou X.Z., Lu K.P., the Pin2/TRF1-interacting protein PinX1 is a potential telomerase inhibitor. cell, 2001, 107, 347-359). The LPTS gene maps to the human chromosome8, segment 8p23, which is frequently deleted in a variety of malignant cells. Research shows that LPTS has very low expression level or no expression in liver cancer tissue and liver cancer cell line, and the increase of telomerase activity in tumor cell may be related to the deletion or down regulation of LPTS gene. It has been reported in the literature that introduction of LPTS gene into hepatoma cells inhibits growth, proliferation and ultimately death of hepatoma cells (Liao C, Zhao MJ, Zhao J, et al. mutation analysis of novelhuman liver cell-related purified tumor donor gene in hepatocellular cancer. world J. Gastroenterol, 2003, 9: 89-93; Zhou X. Z., Lu K. P. the Pin2/TRF1-interacting protein Pin X1 is a pore reporter inhibitor. cell, 2001, 107, 347) 359.). Thus, LPTS has the effect of inhibiting tumor cell growth and causing tumor cell death.

Chinese patent ZL00115395.1 discloses the gene sequence of LPTS and the amino acid sequence of its coded protein. In previous research reports, the functions of LPTS or PinX1 genes were obtained by means of plasmid transfection. The inability of LPTS proteins to enter cells across membranes limits research and use in protein form.

In traditional experimental and clinical therapies, there have been many methods for delivering proteins into living cells, and these methods can be divided into two broad categories, viral vectors and non-viral vectors. Compared to viral vector methods, non-viral delivery strategies have great advantages in terms of biosafety and ease of administration. The non-viral vector method comprises: microinjection, electroporation, liposome, bacterial toxins, erythrocyte and receptor mediated endocytosis, and the like. However, most of these methods are inefficient or time consuming, are prone to cell death or formation of intracellular vesicles, and do not allow efficient transmembrane entry of foreign proteins into cells.

Therefore, it is important to find a method for rapidly and effectively delivering LPTS protein into cells and maintaining the biological activity in the cells.

Disclosure of Invention

The invention aims to provide an isolated fusion protein which comprises telomerase activity inhibitory protein LPTS and trans-activation protein TAT.

The invention also aims to provide the application of the fusion protein in inhibiting the activity or expression of telomerase of cells, and further inhibiting the growth of telomerase positive tumor cells.

In a first aspect of the invention, there is provided an isolated fusion protein comprising:

(1) telomerase activity inhibitory protein LPTS;

(2) transactivator TAT; and

(3) a linker peptide consisting of 0 to 20 (preferably 0 to 15, more preferably 0 to 10, most preferably 1 to 4, e.g., 2 to 3) amino acids between (1) and (2).

In another preferred embodiment, the fusion protein consists essentially of (1), (3) and (2) linked together. More preferably, the fusion protein is formed by connecting (1), (3) and (2).

In another preferred embodiment, the telomerase activity inhibitory protein LPTS is:

(a) SEQ ID NO: 4;

(b) SEQ ID NO: 2 at positions 16-211; or

(c) A protein derived from (a) or (b) and having the protein function defined in (a) or (b), which is formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of the protein defined in (a) or (b);

or

The transactivator TAT is a peptide having the sequence of SEQ ID NO: 2, 2-position 2-12.

In another preferred embodiment, the telomerase activity inhibitory protein LPTS is:

(a1) SEQ ID NO: 2 at positions 16-211; or

(b1) And (b) a protein derived from (a1) which is formed by substituting, deleting or adding 1 to 10 (preferably 1 to 6; more preferably 1 to 3) amino acid residues in the amino acid sequence of the protein defined in (a1) and has the protein function defined in (a 1).

Preferably, the transactivator TAT is located at the amino terminus of the fusion protein; the telomerase activity inhibitory protein LPTS is positioned at the carboxyl terminal of the fusion protein.

In a second aspect of the invention, there is provided a nucleic acid molecule encoding the fusion protein.

In a third aspect of the invention, there is provided a vector comprising said nucleic acid molecule.

In a fourth aspect of the invention, there is provided a genetically engineered cell comprising said vector; or

The nucleic acid molecule is integrated into the genome of the cell.

In a fifth aspect of the invention, there is provided a method of producing said fusion protein, said method comprising: culturing said cell under conditions suitable for expression of said fusion protein, expressing and isolating said fusion protein.

In the sixth aspect of the invention, the fusion protein is provided for use in preparing a composition for inhibiting the growth of telomerase positive cells.

In another preferred embodiment, the cell is a telomerase positive tumor cell.

In another preferred embodiment, the telomerase positive cell comprises: liver cancer BEL-7404 cell, liver cancer HepG2 cell or cervical cancer Hela cell.

In another preferred embodiment, the composition is also used for preventing or treating tumors.

In a seventh aspect of the present invention, there is provided a tumor-inhibiting composition comprising:

(i) an effective amount of said fusion protein; and

(ii) a pharmaceutically acceptable carrier.

In an eighth aspect of the invention, there is provided a method (non-therapeutic in vitro) of introducing telomerase activity inhibitory protein LPTS into a cell, said method comprising the steps of:

(a) fusing a trans-activation protein TAT and a telomerase activity inhibitory protein LPTS to obtain a fusion protein;

(b) co-incubating the fusion protein of (a) with a cell, whereby telomerase activity inhibitory protein LPTS is introduced into the cell.

In another preferred embodiment, the cell is a telomerase positive cell.

In another preferred embodiment, the cell is a telomerase positive tumor cell.

In another preferred embodiment, the method for obtaining a fusion protein comprises:

(i) providing a construct comprising a gene expression cassette comprising the following operably linked elements: a trans-activation protein TAT coding gene and a telomerase activity inhibitory protein LPTS coding gene;

(ii) (ii) introducing the construct of (i) into a cell expression system, thereby expressing and purifying to obtain the fusion protein.

In another preferred embodiment, the transactivator TAT is located at the amino terminus of the fusion protein; the telomerase activity inhibitory protein LPTS is positioned at the carboxyl terminal of the fusion protein.

In another aspect, a method of inhibiting (e.g., inhibiting in vitro) tumor cell growth is provided, the method comprising treating a tumor cell with the fusion protein.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 shows a schematic diagram of the structure of T-LPGENE fusion protein.

FIG. 2 shows a construction diagram of recombinant expression vector pET24a (+) -T-LPGENE.

FIG. 3 shows the induced expression of T-LPGENE in E.coli and the results after isolation and purification. Wherein, 1: protein Maker; 2: before IPTG induction; 3: after IPTG induction; 4: centrifuging the supernatant after ultrasonication; 5: SP-sephorose purified T-LPGENE protein; 6: superdex 75 purified T-LPGENE protein.

FIG. 4 shows that the purified T-LPGENE protein has the activity of inhibiting telomerase of tumor cells in vitro. Wherein, 1-5: the concentrations (nmol) of the added T-LPGENE protein were: 20, 40, 80, 160, 320; 6: blank control.

FIG. 5 shows that T-LPGENE proteins have transmembrane entry activity into tumor cells mediated by TAT. Wherein,

a: immunofluorescence experiment results, ANTI-LPTS, red fluorescence; DAPI: cell nucleus; PHASE: cell morphology;

b: western-blot experiment result, Control: a blank cell; 2hr, 6hr, 24 hr: protein incubation was 2hr, 6hr, 24 hr.

FIG. 6 shows that the purified T-LPGENE protein has the activity of inhibiting the growth of telomerase positive cells in vitro (MTT method). Wherein,

FIG. 6A shows the results of MTT assay for the growth of each group of cells at week 0 (W0) and week 6 (W6), respectively.

FIG. 6B is the cell growth rate (%) of the respective treated cells versus PBS treated cells for each group.

FIG. 7A shows the telomere shortening of cells treated with T-LPGENE protein versus control (PBS and TAT-GFP) for each cell (BEL-7404, HepG2 and Saos-2).

FIG. 7B shows the cell death after treatment of each cell (BEL-7404, HepG2, Saos-2, and L02) with T-LPGENE protein compared to control treatment (PBS and TAT-GFP), where the arrow indicates the dead cell.

FIG. 7C shows the DNA content of each cell (BEL-7404, HepG2, Saos-2 and L02) after treatment with T-LPGENE protein and control treatment (PBS and TAT-GFP) using flow cytometry.

FIG. 8 shows that the purified T-LPGENE protein has the activity of reducing the tumorigenicity of telomerase positive cells BEL-7404. Wherein,

FIG. 8A shows that the purified T-LPGENE protein has the activity of reducing the tumorigenicity of telomerase positive cells BEL-7404.

FIG. 8B is the change in tumor volume following T-LPGENE protein treatment versus control treatment (PBS and TAT-GFP treatment) in mice after tumor transplantation;

FIG. 8C is a photograph showing the growth of tumors in animals treated with T-LPGENE protein and control (PBS and TAT-GFP) after tumor transplantation in mice;

FIG. 8D is the weight change of the tumor after T-LPGENE protein treatment and control treatment (PBS and TAT-GFP treatment) in mice after tumor transplantation.

Detailed Description

Through long-term research and experiments, the inventor unexpectedly finds that the fusion protein formed by fusing the telomerase activity inhibitory protein LPTS and the trans-activation protein TAT well retains the telomerase inhibitory action of the telomerase activity inhibitory protein LPTS and obviously improves the ability of the telomerase activity inhibitory protein LPTS to enter cells, thereby greatly improving the killing action on telomerase positive cells and having no obvious toxic or side effect on telomerase negative cells.

The LPTS protein basically has no capacity of entering the interior of a cell through a membrane, and the telomerase is positioned in a cell nucleus, so that the LPTS protein has no obvious effect on the growth of the cell after the telomerase positive cell is treated. In order to enable telomerase activity inhibitory protein LPTS to penetrate cell membranes and enter cells, the inventor tries to transport LPTS into cells by adopting a liposome transfection method, but the transfection effect is not good; the present inventors have also tried to link with LPTS using various transmembrane proteins (cell-penetrating proteins) and tested the effect of the membrane-penetrating, and as a result, found that the transactivator TAT is the protein most suitable for linking with LPTS and improving the ability of LPTS to enter cells. More preferably, the protein containing about 190-200 amino acids from the C-terminal of the full-length sequence of LPTS is most suitable for being fused with TAT, and the formed fusion protein is most easily transferred into cells positive to telomerase, thereby exerting excellent telomerase inhibition effect.

As used herein, the terms "comprising," "having," or "including" include "comprising," "consisting essentially of," and "consisting of,"; "consisting essentially of", "consisting essentially of" and "consisting of" belong to the subordinate concepts of "having", "having" or "including".

Transactivator TAT

There are many known transmembrane proteins, including: transactivator TAT, Pennetratin, signal sequence-based peptides, pVEC, Transportan, Amphipilic model peptide, Arg9, and the like. Although there is a precedent for using TAT to carry some proteins into cells, TAT is not suitable for mediating all kinds of proteins into the interior of cells, and suitable proteins are limited by factors such as protein length, properties, spatial structure and the like; furthermore, previous experience has also shown that TAT, when fused to an active protein, may affect the folding of the latter and, in turn, the biological activity of the latter. Through repeated research and comparison, the inventor finds that TAT is particularly suitable for being fused with LPTS, and the formed fusion protein is easy to enter cells and can enter cell nuclei.

Preferably, the transactivator has the amino acid sequence of SEQ ID NO: 2, 2-12.

Telomerase activity inhibitory protein LPTS

LPTS is a protein with telomerase inhibitory activity in tumor cells, and is localized in human chromosome8, segment 8p23, which is frequently deleted in many malignant cells. Research shows that LPTS has very low expression level or no expression in liver cancer tissue and liver cancer cell line, and the increase of telomerase activity in tumor cell may be related to the deletion or down regulation of LPTS gene.

The invention can be used with full-length proteins of LPTS or biologically active fragments thereof. Any biologically active fragment of the LPTS protein may be used in the present invention. Herein, a biologically active fragment of an LPTS protein is meant to be a protein fragment that retains all or part of the function of the intact LPTS protein (e.g., at least 50% of the biological activity, preferably at least 70% of the activity, more preferably at least 90% of the activity). 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 substituting, deleting or adding one or more amino acid residues also has the functions of penetrating cells and inhibiting the activity or expression of cell telomerase after being fused with TAT. The present invention may also employ modified or improved LPTS proteins, for example, LPTS proteins modified for their half-life and stability.

The inventor unexpectedly finds that the protein containing about 190-200 amino acids at the C terminal in the full-length sequence of LPTS is most suitable for being fused with TAT, and the formed fusion protein is most easily transferred into cells positive to telomerase and plays an excellent telomerase inhibition effect.

As a preferred embodiment of the present invention, the amino acid sequence of LPTS can be similar to SEQ ID NO: 4 are substantially identical. More preferably the amino acid sequence of LPTS may be identical to SEQ ID NO: 2, positions 16-211 are substantially identical.

Fusion proteins

The invention provides a fusion protein, which comprises TAT protein and LPTS protein or bioactive fragment thereof. The terms "fusion protein of transactivator and telomerase activity inhibitory protein", "TAT-LPTS fusion protein", "T-LPTS", or "T-LPGENE", and the like, used interchangeably, refer to a protein made by fusing a TAT amino acid sequence and an LPTS amino acid sequence, with or without a linker peptide sequence in between.

The fusion protein can be used for inhibiting the activity or expression of cell telomerase. More preferably, the fusion protein is an isolated protein, unrelated to other proteins, polypeptides or molecules, purified from recombinant host cell culture or as a purified extract.

The TAT polypeptide and the LPTS protein or active fragment thereof may be linked directly or via a polypeptide linker (linker peptide). In a preferred embodiment of the present invention, the TAT and LPTS or active fragments 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.

In a preferred embodiment, the TAT polypeptide is located at the amino terminus (N-terminus) of the fusion protein; the LPTS protein or the active fragment thereof is positioned at the carboxyl terminal (C terminal) of the fusion protein. Alternatively, the positions of the two proteins may be interchanged.

In addition, optionally, the amino-terminus (or carboxy-terminus) of the fusion protein may also contain one or more polypeptide fragments as protein tags. Any suitable label may be used in the present invention. For example, the tag may be FLAG, HA, HA1, c-Myc, 6-His, etc. These tags can be used to purify fusion proteins. A specific example is a fusion protein with a 6-His structure attached to the C-terminus. It will be appreciated by those skilled in the art that an enzyme cleavable structure may be placed between the protein tag amino acid sequence and the TAT-LPTS fusion protein amino acid sequence, such that the tag may be separated from the fusion protein.

The fusion protein can inhibit the activity of telomerase and can enter cells, so that the limitation on the application of LPTS is solved. For the study of the intracellular function of LPTS, previous studies were performed at the gene level, and are fundamental studies, which require introduction of a foreign gene into the genome of a cell and are difficult to be put into practical use. The invention provides that LPTS is introduced into cells to play a role on the protein level, so that the LPTS can be clinically applied.

In another aspect, the invention also provides an isolated nucleic acid encoding the fusion protein, and optionally the complementary strand thereof.

The DNA sequence for encoding the fusion protein can be artificially synthesized in a complete sequence, and DNA sequences for encoding amino acids TAT and LPTS can be obtained by a PCR amplification method and then spliced to form the DNA sequence for encoding the fusion protein.

After the DNA sequence encoding the fusion protein of the present invention is obtained, it is ligated into a suitable expression vector and transferred into a suitable host cell. Finally, the fusion protein of the invention is obtained by culturing the transformed host cell and separating and purifying.

Thus, 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.

As used herein, "operably linked" or "operably linked" refers to a condition in which certain portions of a linear DNA sequence are capable of affecting the activity of other portions of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.

In the present invention, any suitable vector may be used, such as some vectors for cloning and expression of bacterial, fungal, yeast and mammalian cells, e.g., Pouwels et al, cloning vectors: described in a laboratory manual (latest edition of Elsevier). Various carriers known in the art such as commercially available carriers can be used. For example, a commercially available vector can be selected and the nucleotide sequence encoding the novel fusion protein of the present invention can then be operably linked to expression control sequences to form a protein expression vector. In one embodiment of the invention, the vector is a prokaryotic vector, such as a pET vector.

In addition, recombinant cells containing nucleic acid sequences encoding the fusion proteins are also encompassed by the invention.

In the present invention, the term "host cell" includes prokaryotic cells and eukaryotic cells. Commonly used prokaryotic host cells include E.coli, Bacillus subtilis, and the like; coli cells (e.coli), such as e.coli HMS174(DE3), or BL21(DE3), may be mentioned, for example. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells.

In a preferred embodiment of the invention, prokaryotic cells are used as host cells, and the fusion protein which retains good telomerase inhibition activity and has good cell membrane penetrability is obtained after expression and purification.

Methods of producing fusion proteins are also encompassed by the present invention. The method comprises culturing a recombinant cell comprising a nucleic acid encoding a fusion protein. The fusion protein comprises TAT polypeptide and LPTS protein or active fragments thereof. The method can include allowing the cell to express the encoded fusion protein, and allowing renaturation of the expressed fusion protein. In one example, the method may further comprise isolation and/or purification of the renatured fusion protein.

The fusion protein prepared as described above can be purified to substantially uniform properties, for example, as a single band on SDS-PAGE electrophoresis. For example, when the recombinant protein is expressed for secretion, the protein can be isolated using commercially available ultrafiltration membranes, such as Millipore, Amicon, Pellicon, etc., and the expression supernatant is first concentrated. The concentrated solution can be further purified by gel chromatography or ion exchange chromatography. Such as anion exchange chromatography (DEAE etc.) or cation exchange chromatography. The gel matrix can be agarose, dextran, polyamide, etc. commonly used for protein purification. The SP group is preferably an ion exchange group. Finally, the purified product can be further purified by reversed phase high performance liquid chromatography (RP-HPLC). All of the above purification steps can be combined in different ways to achieve a substantially uniform protein purity.

The expressed fusion protein can be purified using an affinity column containing antibodies, receptors or ligands specific for TAT or LPTS. The fusion polypeptide bound to the affinity column can be eluted by conventional methods, such as high salt buffer, pH change, etc., depending on the characteristics of the affinity column used.

The fusion protein can be used for preparing a composition for inhibiting the activity or expression of cell telomerase, so that the fusion protein can be used for inhibiting the growth of telomerase positive cells and reducing the tumorigenicity of the telomerase positive cells. The fusion protein has excellent ability of entering cells (such as tumor cells), and can well inhibit the activity or expression of cell telomerase after entering the cells. Therefore, the fusion protein of the present invention has a more excellent tumor killing effect than LPTS, and thus can be used for developing effective antitumor drugs.

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 is not limited to): liver cancer BEL-7404 cell, liver cancer HepG2 cell, cervical cancer Hela cell and the like.

Composition comprising a metal oxide and a metal oxide

The invention also provides a composition for inhibiting cell telomerase activity or expression, the composition comprising: (i) an effective amount (e.g., 0.0001-50 wt%, more preferably 0.001-20 wt%) of a TAT-LPTS fusion protein of the present invention; and (ii) a pharmaceutically acceptable carrier.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Sufficient details regarding pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack pub. co., n.j.1991). Pharmaceutically acceptable carriers in the compositions may contain liquids such as water, saline, glycerin and sorbitol. In addition, 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.

The compositions may be formulated into a variety of dosage forms suitable for mammalian administration including, but not limited to: injection, capsule, tablet, emulsion, and suppository.

In use, 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 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, preferably the dose is from about 1 microgram/kg body weight to about 10 mg/kg body weight. Of course, the particular dosage will depend upon such factors as the route of administration, the health of the patient, and the like, and is within the skill of the skilled practitioner.

When used to inhibit mammalian tumors, the fusion protein may be administered systemically or locally, depending on such factors as the type of tumor, the site of growth, the degree of progression, etc.

The composition of the invention can be directly used for killing tumor cells. In addition, it may be used in combination with other therapeutic agents or adjuvants.

The main advantages of the invention are:

(1) the fusion protein of TAT and LPTS is provided and prepared for the first time, and the fusion of LPTS and TAT is proved to promote the LPTS to penetrate cell membranes without influencing the activity of cell telomerase inhibition of LPTS.

(2) The fusion protein of the present invention has significantly more excellent antitumor activity than LPTS alone, and is useful for many tumors.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Example 1 construction of T-LPGENE (T-LPTS-C) fusion protein containing LPTS fragment and preparation of recombinant genetically engineered bacteria

The specific method for constructing the T-LPGENE engineering bacteria comprises the following steps:

(1) construction of pET24-TAT plasmid

TAT template and primer are obtained by synthesis, and the specific sequence is as follows:

template:

5-AGTTTCATATGTACGGGCGCAAGAAACGCCGCCAGCGCCGCCGCGGTGGATCCTAGAAG-3(SEQ ID NO：5)；

p1 (upstream primer):

5-AGTTTCATATGTACGGGC-3(SEQ ID NO：6)；

p2 (downstream primer):

5-CGCCACCTAGGATCTTC-3(SEQ ID NO：7)。

obtaining a DNA sequence containing coded TAT polypeptide (11 amino acids) with enzyme cutting sites Nde I and BamH I at two ends through PCR reaction; the PCR reaction conditions are as follows: the PCR reaction was carried out in a 50. mu.l system comprising 1. mu.l of template, 1. mu.l of dNTP mix (10mM), 1. mu.l each of upstream and downstream primers (20. mu.M), 5. mu.l of 10 XPYROBEST buffer, 0.5. mu.l of PYROBEST enzyme, and supplemented with deionized water to 50. mu.l. The reaction conditions are as follows: 3 minutes at 94 ℃; then 30 cycles are carried out, each cycle comprises 94 ℃ for 20 seconds, 50 ℃ for 20 seconds and 72 ℃ for 20 seconds; finally, the extension is carried out for 5 minutes at 72 ℃ and the temperature is kept at 4 ℃.

The PCR product obtained above was digested with Nde I and BamHI, and inserted into pET24(a) (available from Novagen) plasmid, which was digested with Nde I and BamHI, to obtain pET24-TAT plasmid.

(2) Construction of pET24-T-LPGENE plasmid

pT-LPTSCDS plasmid containing full-length LPTS gene (see Hepatology, 32(2000), 721-727) was used as template, and the primers were as follows:

p1 (upstream primer):

5-AAAGGATCCAAGGATCTGTCATCTCGG-3’(SEQ ID NO：8)；

p2 (downstream primer):

5-AAACTCGAGTTTGGAATCTTTCTTCTT-3(SEQ ID NO：9)。

obtaining a DNA sequence containing coded LPTS protein C-terminal 196 amino acid (LPTS-C) with enzyme cutting sites BamHI and XhoI at two ends through PCR reaction, wherein the sequence is shown in SEQ ID NO: 2 from position 16 to 211; the PCR reaction conditions are as follows: the PCR reaction was carried out in a 50. mu.l system comprising 1. mu.l of template, 1. mu.l of dNTP mix (10mM), 1. mu.l each of upstream and downstream primers (20. mu.M), 5. mu.l of 10 XPYROBEST buffer, 0.5. mu.l of PYROBEST enzyme, and supplemented with deionized water to 50. mu.l. The reaction conditions are as follows: 3 minutes at 94 ℃ after which a total of 30 cycles were carried out, each cycle comprising 30 seconds at 94 ℃, 40 seconds at 56 ℃ and 60 seconds at 72 ℃; extension was carried out at 72 ℃ for 5 minutes and incubation was carried out at 4 ℃.

After the PCR product of the LPGENE gene obtained in the above way is subjected to double enzyme digestion by BamH I and Xho I, pET24-TAT plasmid subjected to double enzyme digestion by BamH I and Xho I is inserted, and pET24-T-LPGENE plasmid is finally obtained, wherein the plasmid has a DNA sequence for coding T-LPGENE, and the sequence is shown in SEQ ID NO: 1, the structure is schematically shown in figure 1. The construction of the pET24-T-LPGENE expression plasmid is shown in FIG. 2. And (3) after the sequence is verified to be correct, transforming the host bacterium E.coli BL-21 to obtain the engineering bacterium for expressing T-LPGENE.

Example 2 inducible expression and isolation and purification of T-LPGENE

The invention adopts a separation method such as ion exchange, obtains the high-purity T-LPGENE protein, and can be simply divided into the following steps:

(1) inducing and expressing T-LPGENE protein by a conventional method;

(2) breaking the thalli by ultrasonic waves;

(3) cation exchange chromatography (SP-Sepharose);

(4) performing ultrafiltration concentration;

(5) molecular sieve chromatography (Superdex 75);

the experiment was carried out at 4 ℃. The purity and concentration of T-LPGENE were determined by SDS-PAGE, and the results are shown in FIG. 3 (lane 6), and the purity of T-LPGENE protein was more than 90%.

Example 3 detection of T-LPGENE in vitro inhibition of telomerase Activity

T-LPGENE is a strong inhibitor of telomerase activity. The present inventors determined the biological activity of purified T-LPGENE using the TPAP method. The telomerase is from SMMC-7721 liver cancer cell lysate, and the preparation method comprises the following steps:

SMMC-7721 hepatoma cells (purchased from ATCC) were inoculated in 10ml culture flasks in RPMI1640, 37 ℃ and 5% CO₂Culturing the cells on the wall under the condition, and collecting the cells after the bottle is full. The culture medium is sucked dry, cells are rinsed once by PBS, the cells are digested by pancreatin, washed by PBS, sucked into a centrifugal tube, and centrifuged at 4000rpm to collect the cells. Then washing buffer (10mM Hepes-KOH (pH7.5), 1.5mM MgCl₂Cells were washed once with 10mM KCl, 1mM DTT (dithionthreitol)) and centrifuged. With precooled lysis buffer (10mM Tris-HCl (pH7.5), 1mM MgCl₂1mM EGTA, 0.1mM PMSF, 5mM mercaptoethanol, 0.5% CHAPS, 10% glycerol) 500. mu.l of the resuspended cells, placed on ice for 30 minutes, centrifuged at 12,000rpm for 30 minutes at 4 ℃ and the supernatant obtained can be used for measuring telomerase activity. The supernatant can be stored at-70 deg.C, and can maintain stable telomerase activity after multiple times of freeze-thawing.

TPAP assay was performed in 500. mu.l Eppendorf tubes in a reaction volume of 50. mu.l, containing 45.25. mu.l of conventional reaction buffer, 0.8. mu.l of telomerase-containing tumor cell lysate, 0.25. mu.l of dNTP, 1. mu.l of purified protein (diluted as required). After 10min reaction on ice, 1. mu.l of Ts primer (0.1. mu.g/. mu.l, primer sequence 5-AATCCGTCGAGCAGAGTT-3(SEQ ID NO: 10)) was added, 1. mu.l of Taq enzyme was extended at 25 ℃ for 30min, inactivated at 95 ℃ for 5min, and then supplemented with Taq enzyme (0.5. mu.l), Acx primer (1. mu.l, 0.1. mu.g/. mu.l, primer sequence 5- (CCCTTA)₃CCCTAA-3(SEQ ID NO: 11)), and performing PCR (94 ℃ for 30s, 50 ℃ for 40s, 72 ℃ for 40s, 33 cycles; 72 ℃ for 2 min). And (3) identifying the PCR reaction product by adopting 10% polyacrylamide gel electrophoresis, wherein an electrophoresis system is TBE buffer solution, and after electrophoresis (120 volts and 2 hours), silver staining and color development are carried out.

TRAP experiment results show that the purified T-LPGENE protein has high telomerase inhibitory activity in vitro, and the T-LPGENE can completely inhibit the telomerase activity when the concentration is about 320nmol/L, which is shown in figure 4.

Example 4 Activity detection of TAT-mediated transmembrane entry of LPGENE proteins into tumor cells

First, the present inventors used conventional lipofection to deliver LPTS-C protein into each tumor cell. The results of the assay showed that essentially no LPTS-C protein was detected entering the cells. Therefore, the liposome method is not suitable for transporting LPTS-C protein.

To verify whether TAT mediates entry of LPGENE proteins into cells, the inventors chose two experimental approaches: immunofluorescence experiments and Western-blot experiments. The specific operation method of the immunofluorescence experiment is as follows:

(1) BEL-7404 cells (purchased from Shanghai Biochemical cell Bank) and Saos-2 cells (Shanghai Biochemical cell Bank) were seeded in six-well plates plated with coverslips, in DMEM (containing 10% newborn calf serum) and Mccoy's 5A (containing 15% fetal calf serum), 37 ℃ and 5% CO, respectively₂Culturing the cells on the wall under the condition. After cell attachment, PBS solution (two voids) and purified T-LPGENE (100mg/L) protein (3 voids, incubated for 2hr, 6hr and 24hr, respectively) were added:

(2) after incubation, washing with PBS once, adding 1mL of methanol precooled at-20 ℃ into each well, and fixing for 3 min; discarding methanol, washing twice with PBS, adding 1mL PBS (containing 1% newborn goat serum) per well, and sealing for 10 min;

(3) taking out fixed slide, placing into a sealed container, adding primary antibody (LPGENE protein-immune rabbit antiserum) diluted with PBS (containing 1% newborn goat serum), reacting at room temperature for 2hr, and adding PBS (containing 1% newborn goat serum) as blank control;

(4) washing with PBS (containing 1% newborn goat serum) for 3 times, each for 5 min;

(5) adding secondary antibody (FITC labeled goat anti-rabbit serum) diluted with PBS (containing 1% newborn goat serum) dropwise, and reacting at room temperature in dark for 1 hr; washing with PBS (containing 1% newborn goat serum) for 3 times, each for 5 min;

(6) the slide is put back into a 6-well plate, 1mL PBS (containing 1% newborn goat serum) and 10 μ L33258 reagent are added into each well, and the core is stained for 10 min;

(7) washing with PBS for 5min for 3 times;

(8) taking a clean glass slide, dripping 50 mu L of cat oil, placing the processed cover glass on the glass slide with the cell side facing downwards, and keeping the glass slide and the cat oil from light at room temperature for 4hr or staying overnight.

The specific operation method of the Western-blot experiment is as follows:

(1) BEL-7404 cells and Saos-2 cells were seeded in 6cm dishes in DMEM (containing 10% newborn calf serum) and Mccoy's 5A (containing 15% fetal calf serum), 37 ℃ and 5% CO, respectively₂Culturing the cells on the wall under the condition. After the cell density exceeded 30%, PBS solution (two empty), purified LPGENE (100mg/L) protein (3 wells, incubated for 2hr, 6hr and 24hr, respectively) and purified T-LPGENE (100mg/L) protein (3 wells, incubated for 2hr, 6hr and 24hr, respectively) were added:

(2) washing with PBS twice, and digesting with pancreatin for 10 min;

(3) cells were collected by centrifugation at 10 intervals⁶Adding 100 mu L SDS-PAGE electrophoresis loading buffer solution into the cells;

(4) each sample was examined by Western Blotting method, i.e., after SDS-PAGE, proteins were transferred to nitrocellulose membrane using an electrotransfer from Bio-Rad, and hybridization was carried out using a specific anti-LPGENE antibody (rabbit antiserum immunized with LPGENE protein). The secondary antibody is goat anti-rabbit IgG labeled by horseradish peroxidase (HRP).

The results of immunofluorescence experiments and western-blot experiments are shown in FIG. 5, and the results show that TAT can excellently mediate LPGENE protein to enter BEL-7404 cells and Saos-2 cells and enter cell nuclei.

Example 5 purified T-LPGENE protein has the Activity to inhibit the growth of telomerase positive cells in vitro

MTT assay

(1) BEL-7404 cells and Saos-2 cells were seeded in coverslipped 6-well plates in DMEM (10% newborn calf serum) and Mccoy's 5A (15% fetal calf serum), 37 ℃ and 5% CO, respectively₂Culturing the seeds on a wall under the condition; HepG2 cells (purchased from shanghai biochemical cell bank) and L02 cells (purchased from shanghai biochemical cell bank) were also seeded in 6-well coverslipped plates in DMEM + 10% FBS (fetal bovine serum) and RPMI1640+ 10% NBS (neonatal calf serum), respectively. Adding PBS solution and the purified T-LPGENE (40mg/L) and TAT-GFP (40mg/L) (T-GFP) (using GFP full-length sequence, GFP protein sequence see GenBank accession No. DQ 768212; C terminal of TAT is connected with N terminal of GFP) proteins into 6-well plate;

(2) after 6 weeks of continuous culture, the growth activity of each group of cells was examined by MTT method, and the specific experimental methods were described in the literature (Staunton, Wu-Yu-Zheng-Shu; cell culture [ M ]; Xian: Xian-Shu-Xian Co., Ltd., 2004, 250). During detection, corresponding protein is continuously added into each group of cells, and the detection wavelength is 570 nm.

The experimental result is shown in figure 6, and the result shows that the T-LPGENE protein can inhibit the growth of telomerase positive cells BEL-7404 and HepG2, but has no obvious inhibition effect on reducing the growth of telomerase negative cells Saos-2 and immortalized cells L02.

Southern-blot analysis

Cells were harvested after 8 weeks of continuous culture and analyzed for telomere length using the Southern-blot method, as described in the literature (Damm K. et al., A high selective telomerase inhibition or limitinghuman cell promotion; EMBO J2001; 20: 6958-6968-cost 6968). The simple process is as follows: genomic DNA was isolated, double digested with endonucleases Hinf I and Afa I, then the digested products were separated on a 0.8% agarose gel, after membrane transfer, hybridized with a probe labeled γ -32P and finally the hybridization signal was analyzed with ImageQuant.

The results are shown in FIG. 7A, and show that after 8 weeks of treatment with T-LPGENE protein, telomeres of telomerase positive tumor cells BEL-7404 and HepG2 were significantly shortened compared to the control group (PBS and TAT-GFP treated), and the telomeres were shorter in the high concentration protein group than in the concentration group. While the telomerase negative tumor cell, Saos-2 telomere, did not shorten significantly.

Cell morphology detection

After 6 weeks of continuous culture, the cells were photographed by an inverted microscope, and the results are shown in FIG. 7B, which shows that T-LPGENE protein-treated telomerase positive tumor cells BEL-7404 and HepG2 were poor in anchorage ability, and the cells became flat and large, which is a typical crisis (crisis) morphology.

Human normal somatic cells divide once per minute, telomeres are lost by 50-200bp, and when the telomeres are shortened to a certain degree, cell growth is inhibited, namely cell senescence is called and death is promoted. However, telomerase activity is detected in most malignant cells (85%) and is highly active, and the resynthesis of telomeres by telomerase compensates for its sustained loss during cell proliferation, allowing cells to divide continuously, which is an important mechanism for cell immortalization and canceration. The T-LPGENE protein can inhibit telomerase activity in cells, so that telomeres of telomerase positive tumor cells are gradually shortened along with cell proliferation, enter a crisis stage (senescence) and are in crisis forms in form. The morphology of the telomerase negative tumor cell Saos-2 and the immortalized cell L02 is not obviously changed.

Flow cytometry for detecting DNA content

After 6 weeks of continuous cell culture, samples were taken for flow cytometry analysis (FACSCalibur, Becton Dickinson, USA). Cells were stained with Propidium Iodide (PI) and different signals represent DNA size. The DNA of normal cells is diploid, the DNA of G2/M phase cells is tetraploid, the DNA of dead or dying cells is broken, and the flow signal is in a region smaller than that of the diploid (sub-G1 region).

The experimental result is shown in figure 7C, and the result shows that after the T-LPGENE protein is continuously treated for 6 weeks, the content of the sub-G1 region of telomerase positive tumor cells BEL-7404 and HepG2 is obviously increased, namely the death or imminent death number of the cells is obviously increased; while the sub-G1 region of the telomerase negative tumor cell Saos-2 and the immortalized cell L02 did not change significantly.

Example 6 Studies on tumor suppressor Activity of T-LPGENE protein and reduction of tumorigenicity of telomerase Positive cells

1. The purified T-LPGENE protein can reduce the tumorigenicity of telomerase positive cells

(1) BEL-7404 cells and Saos-2 cells were seeded in coverslipped six-well plates in DMEM (10% serum from newborn calf) and Mccoy's 5A (15% serum from fetal calf), 37 ℃ and 5% CO₂Culturing the cells on the wall under the condition. PBS solution and the purified T-LPGENE (40mg/L final concentration in the culture medium) and TAT-GFP (40mg/L) proteins were added:

(2) after 6 weeks of continuous culture, cells were collected at 8X 10⁶Inoculating to the skin of a nude mouse;

(3) after inoculation, tumor growth was observed weekly and tumor size was measured;

(4) after 6 weeks of inoculation, pictures were taken and tumors were removed and weighed.

The experimental result is shown in figure 8A and table 1, and the result shows that the T-LPGENE protein can reduce the tumorigenicity of telomerase positive cells BEL-7404, and has no inhibition effect on reducing the tumorigenicity of telomerase negative cells Saos-2. And the TAT-GFP protein which is the fusion protein of TAT and green fluorescent protein has no influence on the tumorigenicity of telomerase positive cells BEL-7404.

TABLE 1

2. Anti-tumor activity of purified T-LPGENE protein

Inoculation of hepatoma cells BEL-7404 (5X 10)⁶One) to the right side of nude mice (female, 4 weeks old) subcutaneously, after 4 weeks, tumors were removed and cut into 2mm pieces³Size, and then inoculated subcutaneously into the right side of the lumbar region of nude mice (4 weeks old, female), and the nude mice were randomly divided into 4 groups. On alternate days, subcutaneous injections of T-LPGENE protein (100. mu.g/mouse, 400. mu.g/mouse, experimental group) were initiated with sites near the tumor, and PBS and TAT-GFP protein (400. mu.g/mouse) were injected into the control group. The initial 2 weeks were injected every 2 days, followed by 3 weeks every 3 days for a total of 14 injections. Tumor size was recorded weekly (measured with a vernier caliper), all nude mice were photographed 7 weeks after inoculation, sacrificed, tumors removed and weighed.

The experimental results are shown in FIGS. 8B-D, and the results show that the T-LPGENE protein can inhibit the growth of BEL-7404 tumor cells in nude mice, and the T-LPGENE protein has certain dose effect. The tumor weight of 100. mu.g of the T-LPGENE protein group per injection was only 57% (P < 0.05) of the PBS group, whereas the tumor weight of the 400. mu. g T-LPGENE protein group was only 36% (P < 0.01) of the PBS group. The TAT-GFP group showed no significant difference in tumor weight from the PBS group, but significantly heavier than the two experimental groups (P < 0.05). The tumor weight in the high dose group was also significantly less than that in the low dose group (P < 0.05).

Example 7 preparation of fusion proteins containing full-Length LPTS and cell assays

TAT-full-length LPTS (TAT-LPTS) was prepared by a similar method as described in example 1, and pET24-TAT plasmid was first constructed. pT-LPTSCDS plasmid is taken as a template, PCR amplification is carried out to obtain the full-length gene sequence of LPTS (shown in SEQ ID NO: 3), enzyme cutting sites BamH I and Xho I are arranged at two ends, the two enzymes are used for double enzyme cutting, and pET24-TAT plasmid which is subjected to the same double enzyme cutting is inserted into the plasmid to obtain pET24-T-LPTS plasmid, and the plasmid is provided with a DNA sequence for coding TAT-LPTS. And (3) after the sequence is verified to be correct by sequencing, transforming the host bacterium E.coli BL-21 to obtain the expressed engineering bacterium.

The TAT-LPTS protein with the purity of more than 80 percent is obtained by adopting the method of inducing expression, separating and purifying as described in the previous example 2.

In vitro assays (e.g., example 3) found TAT-LPTS was substantially identical to T-LPGENE in its ability to inhibit telomerase activity.

Immunofluorescence and western-blot assays were performed as described in example 4 above to detect the entry of TAT-LPTS into cells. The results show that TAT-LPTS can enter BEL-7404 cells and Saos-2 cells and can enter cell nuclei. Semi-quantitative tests show that compared with the condition that the T-LPGENE protein enters cells, the TAT-LPTS enters the cell nucleus by about 70 percent of the amount of the T-LPGENE protein entering the cells, and the transfer effect of the T-LPGENE protein is more ideal.

Example 8 preparation and cellular assays of T-LPGENE proteins without linker peptide

The T-LPGENE protein without the linker peptide lacks the sequence shown in SEQ ID NO: 2, positions 13-15. After the gene sequence of the T-LPGENE protein without the connecting peptide is artificially synthesized, the sequence is inserted into pET24 plasmid, and is transferred into engineering bacteria to be expressed and purified to obtain the T-LPGENE protein without the connecting peptide.

Immunofluorescence experiments and western-blot experiments were performed as described in example 4 above, and it was found that the T-LPGENE protein without the linker peptide had a transfer effect close to that of the T-LPGENE protein of example 1.

Example 9 pharmaceutical compositions

100mg of the fusion protein purified in example 3 was formulated in 100ml of a normal physiological saline to obtain a fusion protein-containing composition at a concentration of 1 mg/ml.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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Claims (10)

1. An isolated fusion protein, said fusion protein comprising:

(1) telomerase activity inhibitory protein LPTS;

(2) transactivator TAT; and

(3) a connecting peptide consisting of 0-20 amino acids and positioned between (1) and (2).

2. The fusion protein of claim 1, wherein the telomerase activity inhibitory protein LPTS is:

(a) SEQ ID NO: 4;

(b) SEQ ID NO: 2 at positions 16-211; or

(c) A protein derived from (a) or (b) and having the protein function defined in (a) or (b), which is formed by substituting, deleting or adding one or more amino acid residues in the amino acid sequence of the protein defined in (a) or (b);

or

The transactivator TAT is a peptide having the sequence of SEQ ID NO: 2, 2-position 2-12.

3. The fusion protein of claim 1, wherein the telomerase activity inhibitory protein LPTS is:

(a1) SEQ ID NO: 2 at positions 16-211; or

(b1) And (b) a protein derived from (a1) and having the protein function defined in (a1), which is formed by substituting, deleting or adding 1 to 10 amino acid residues in the amino acid sequence of the protein defined in (a 1).

4. A nucleic acid molecule encoding the fusion protein of any one of claims 1-3.

5. A vector comprising the nucleic acid molecule of claim 4.

6. A genetically engineered cell characterized in that,

the cell comprising the vector of claim 5; or

The cell genome having integrated therein the nucleic acid molecule of claim 4.

7. A method of producing the fusion protein of claim 1, comprising: culturing the cell of claim 6 under conditions suitable for expression of the fusion protein, and expressing and isolating the fusion protein.

8. Use of a fusion protein according to any one of claims 1 to 3 for the preparation of a composition for inhibiting the growth of telomerase positive cells.

Preferably, the cell is a telomerase positive tumor cell.

Preferably, the telomerase positive cell comprises: liver cancer BEL-7404 cell, liver cancer HepG2 cell or cervical cancer Hela cell.

9. A composition for inhibiting a tumor, said composition comprising:

(i) an effective amount of the fusion protein of any one of claims 1-3; and

(ii) a pharmaceutically acceptable carrier.

10. A method for introducing telomerase activity inhibitory protein LPTS into a cell, comprising the steps of:

(a) fusing a trans-activation protein TAT and a telomerase activity inhibitory protein LPTS to obtain a fusion protein;

(b) co-incubating the fusion protein of (a) with a cell, whereby telomerase activity inhibitory protein LPTS is introduced into the cell.

Images