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

Abstract

The invention discloses a fusion protein, which comprises: telomerase activity inhibiting protein LPTS and transactivating protein TAT. The invention also discloses the nucleic acid encoding the fusion protein, the vector and host cell containing the nucleic acid, and the composition containing the fusion protein. The present invention prepares the fusion protein of TAT and LPTS for the first time, and it is proved that the fusion protein can penetrate the cell membrane and has an extremely excellent effect of inhibiting the growth of tumor cells.

Description

Fusion protein inhibiting telomerase activity, its preparation and application

technical field

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

Background technique

Telomerase (Telomerase) is a ribonucleoprotein that synthesizes and extends the telomere of cell chromosomes. It consists of two basic components: reverse transcriptase catalytic subunit hTERT and RNA component hTR. Telomerase can use its own RNA as a template to reverse-transcribe and synthesize telomere repeat sequences, which are added to the ends of chromosomes to compensate for the loss of telomere DNA during cell division and maintain the length of telomeres.

Studies have shown that telomerase activity is almost undetectable in normal human cells. Therefore, the number of normal human cell divisions is limited. Every time a cell divides, the telomere will lose 50-200bp. When the telomere is shortened to a certain extent Cell growth is inhibited, known as cellular senescence, and they die.

However, in the vast majority of malignant tumor cells (85%), telomerase activity can be detected and the activity is strong, and the resynthesis of telomeres by telomerase compensates for its continuous loss during cell reproduction, thus making the cells Can continue to divide, which is an important mechanism of cell immortalization and carcinogenesis.

Kim et al analyzed and summarized a large number of research results, detected more than 100 malignant tumor specimens, and pointed out that the sensitivity of telomerase in diagnosing tumors was 85%, the specificity was 91%, the positive predictive value was 91%, and the negative predictive value was 81%. , fully demonstrated 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 et al. believe that the activation of telomerase is one of the main factors for the occurrence of malignant tumors, and its activation and expression level are closely related to the occurrence and metastasis of tumors. Inhibiting telomerase and shortening telomeres are considered to be a key factor in inhibiting cancer cells. This mechanism makes telomerase an ideal target for tumor-targeted therapy.

At present, the research on tumor therapy with telomerase as the target mainly adopts the antisense nucleic acid technology for the RNA component of telomerase (Kondo S., Kondo Y., Li G., et al, Targeted therapy of humanmalignant 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. 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), gene therapy technology, ribozyme for cutting telomerase mRNA 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 technology for inhibiting telomerase gene transcription activity ( Meyerson, M.Counter C.M., Eaton E.N., et al.hEST2, the putative human telomerase catalytic subunit gene, isup-regulated in tumor cells and during immortalization, Cell, 1997, 90:785 795; W.C.Hahn, S.A.Brooks, M. , S.G.York, E.Eaton, A.Kurachi, R.L.Beijersbergen, J.H.Knoll, MMeyerson, R.A.Weinberg, Inhibition of telomerase limits the gr owth of human cancer cells, Nat. Med, 1999, 5: 164 1170). These technologies can shorten the telomeres of tumor cells, thereby causing the cells to enter a crisis period or die, or to significantly reduce the tumorigenicity of tumor cells.

LPTS is a protein that can inhibit the activity of tumor cell telomerase, and it is a new kind of protein preparation with important application prospects. The LPTS protein is encoded by the LPTS (Liver putative tumor suppressor) gene, which is a new liver-related candidate tumor suppressor gene (Liao C., Zhao et al. M.J.，Song H.，Uchida K.，Yokoyama K.K.，Li T.P.，Identification of the gene for a novel liver-related putativetumor suppressor at a high-frequency loss of heterozygosity region of chromosome8p23 in human hepatocellular carcinoma.Hepatology 32(2000)721 -727). The protein encoded by this gene has the activity of inhibiting cell telomerase (Zhou X.Z., Lu K.P.. The Pin2/TRF1-interacting protein PinX1 is a potent telomerase inhibitor. Cell, 2001, 107, 347-359). The LPTS gene is located on the 8p23 segment of human chromosome 8, which is frequently deleted in a variety of malignant tumor cells. Studies have shown that the expression of LPTS is very low or not expressed in liver cancer tissues and liver cancer cell lines, and the increase of telomerase activity in tumor cells may be related to the deletion or down-regulation of LPTS gene. It has been reported in the literature that the introduction of LPTS gene into liver cancer cells can inhibit the growth, proliferation and eventually 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 X.Z., Lu K.P.. The Pin2/TRF1-interacting protein PinX1 is a potent telomerase inhibitor. Cell, 2001, 107, 347-359.). Therefore, LPTS has the function of inhibiting the growth of tumor cells and leading to the death of tumor cells.

Chinese patent ZL00115395.1 discloses the gene sequence of LPTS and the amino acid sequence of its encoded protein. In previous research reports, the function of LPTS or PinX1 gene was obtained by transfecting plasmids. LPTS protein cannot enter the cell across the membrane, which limits the research and application in the form of protein.

In traditional experiments and clinical treatment, many methods have been used to deliver proteins into living cells, and these methods can be divided into two categories: viral vectors and non-viral vectors. Compared with viral vector methods, non-viral delivery strategies have great advantages in terms of biosafety and convenience of administration. Non-viral carrier methods include: microinjection, electroporation, liposome, bacterial toxin, endocytosis mediated by red blood cells and receptors, etc. However, most of these methods are inefficient or time-consuming, easily cause cell death or form intracellular vesicles, and cannot effectively transmembrane foreign proteins into cells.

Therefore, it is particularly important to find a rapid and effective method to import LPTS protein into cells and maintain biological activity in cells.

Contents of the invention

The purpose of the present invention is to provide an isolated fusion protein, which includes telomerase activity inhibitory protein LPTS and transactivator protein TAT.

Another object of the present invention is to provide the use of the fusion protein for inhibiting the activity or expression of telomerase in cells, and then for inhibiting the growth of telomerase-positive tumor cells.

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

(1) telomerase activity inhibitory protein LPTS;

(2) the transactivator protein TAT; and

(3) 0-20 between (1) and (2) (preferably 0-15, more preferably 0-10, best 1-4, such as 2-3 a) connecting peptide composed of amino acids.

In another preferred example, the fusion protein basically consists of (1), (3) and (2) linked together. More preferably, the fusion protein is formed by linking (1), (3) and (2).

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

(a) the protein of the amino acid sequence shown in SEQ ID NO: 4;

(b) the protein of the amino acid sequence shown in positions 16-211 in SEQ ID NO: 2; or

(c) The amino acid sequence of the protein defined in (a) or (b) is formed by the substitution, deletion or addition of one or more amino acid residues, and has the protein function defined in (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 2-12 of SEQ ID NO:2.

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

(a1) a protein of the amino acid sequence shown in positions 16-211 of SEQ ID NO: 2; or

(b1) The amino acid sequence of the protein defined in (a1) is formed by substituting, deleting or adding 1-10 (preferably 1-6; more preferably 1-3) amino acid residues, and A protein derived from (a1) having the protein function defined by (a1).

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

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

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

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

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

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

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

In another preferred example, the cells are telomerase-positive tumor cells.

In another preferred example, the telomerase-positive cells include: liver cancer BEL-7404 cells, liver cancer HepG2 cells or cervical cancer Hela cells.

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

In the seventh aspect of the present invention, there is provided a composition for suppressing tumors, said composition comprising:

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

(ii) A pharmaceutically acceptable carrier.

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

(a) fusing the transactivator TAT with the telomerase activity inhibitory protein LPTS to obtain a fusion protein;

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

In another preferred example, the cells are telomerase positive cells.

In another preferred example, the cells are telomerase-positive tumor cells.

In another preferred example, the method for obtaining a fusion protein includes:

(i) providing a construct, which contains a gene expression cassette, the gene expression cassette contains the following operably linked elements: transactivator TAT encoding gene and telomerase activity inhibitory protein LPTS encoding gene ;

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

In another preferred example, the transactivator TAT is located at the amino terminal of the fusion protein; the telomerase activity inhibitory protein LPTS is located at the carboxy terminal of the fusion protein.

In another aspect, it also provides a method for inhibiting (such as inhibiting in vitro) the growth of tumor cells, said method comprising treating tumor cells with said fusion protein.

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

Description of drawings

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

Figure 2 shows the structural diagram of the recombinant expression vector pET24a(+)-T-LPGENE.

Figure 3 shows the induced expression of T-LPGENE in Escherichia coli and the results after separation and purification. Among them, 1: Protein Maker; 2: Before IPTG induction; 3: After IPTG induction; 4: Centrifugation supernatant after sonication; 5: T-LPGENE protein purified by SP-sephorose; 6: T-LPGENE protein purified by Superdex 75 - LPGENE protein.

Figure 4 shows that the purified T-LPGENE protein has the activity of inhibiting tumor cell telomerase in vitro. Among them, 1-5: the concentration (nmol) of the added T-LPGENE protein is respectively: 20, 40, 80, 160, 320; 6: blank control.

Figure 5 shows that T-LPGENE protein has the activity of transmembrane entry into tumor cells mediated by TAT. in,

A: Immunofluorescence experiment results, ANTI-LPTS, red fluorescence; DAPI: nucleus; PHASE: cell morphology;

B: Western-blot experiment results, Control: blank cells; 2hr, 6hr, 24hr: protein incubation for 2hr, 6hr, 24hr.

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

Fig. 6A shows the results of detecting the growth of cells in each group by MTT method at week 0 (W0) and week 6 (W6).

Fig. 6B is the cell growth rate (%) of the correspondingly treated cells in each group relative to the PBS-treated cells.

Figure 7A 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 7B shows the comparison of cell death of each cell (BEL-7404, HepG2, Saos-2 and L02) after T-LPGENE protein treatment and control treatment (PBS and TAT-GFP treatment), where the arrow points to the death at the cell.

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

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

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

Figure 8B is the volume change of the tumor after the mice transplanted with the tumor were treated with T-LPGENE protein and the control (PBS and TAT-GFP treatment);

Fig. 8C is a photo of the tumor growth in the animal body after the mice transplanted with the tumor were treated with T-LPGENE protein and the control (PBS and TAT-GFP treatment);

Fig. 8D is the weight change of the tumor after the transplanted mice were treated with T-LPGENE protein and the control (PBS and TAT-GFP treatment).

Detailed ways

After long-term research and experiments, the inventors unexpectedly found that the fusion protein formed by the fusion of the telomerase activity inhibitory protein LPTS and the transactivator protein TAT not only well retains the telomerase inhibition of the telomerase activity inhibitory protein LPTS It also significantly improves the ability of the telomerase activity inhibitory protein LPTS to enter cells, thereby greatly improving the killing effect on telomerase-positive cells, and has no significant toxic and side effects on telomerase-negative cells.

LPTS protein itself basically does not have the ability to transmembrane into cells, and telomerase is located in the nucleus, so treating telomerase-positive cells with LPTS protein has no obvious effect on cell growth. In order to enable the telomerase activity inhibitory protein LPTS to penetrate the cell membrane and enter the cell, the inventors tried liposome transfection to transport LPTS into the cell, but the transfection effect was not good; the inventor also tried A variety of transmembrane proteins (cell penetrating proteins) were used to connect with LPTS and test the transmembrane effect. It was found that the transactivator TAT is the most suitable protein to connect with LPTS and improve the ability of LPTS to enter cells. More preferably, the protein containing 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 by it is most likely to be transferred into telomerase-positive cells and exerts excellent telomerase function. Granzyme inhibitory effect.

As used herein, the words "comprising", "having" or "comprising" include "comprising", "consisting mainly of", "consisting essentially of" and "consisting of"; "consisting mainly of", " Basically consist of and "consisting of " are subordinate concepts of "contain", "have" or "include".

transactivator TAT

There are many known transmembrane proteins, including: transactivator TAT, Penetratin, signal sequence-based peptides, pVEC, Transportan, Amphiphilic model peptide and Arg9, etc. Although there have been precedents of using TAT to carry certain proteins into cells, TAT is not suitable for mediating all types of proteins into cells, and suitable proteins are limited by factors such as protein length, properties, and spatial structure; and, in the past Our experience also found that after TAT is fused with an active protein, it may affect the folding of the latter, thereby affecting the biological activity of the latter. After repeated studies and comparisons, the inventors found that TAT is particularly suitable for fusion with LPTS, and the resulting fusion protein can easily enter cells and can enter the nucleus.

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

telomerase activity inhibitory protein LPTS

LPTS is a protein that inhibits telomerase activity in tumor cells. It is located on the 8p23 segment of human chromosome 8, which is frequently deleted in a variety of malignant tumor cells. Studies have shown that the expression of LPTS is very low or not expressed in liver cancer tissues and liver cancer cell lines, and the increase of telomerase activity in tumor cells may be related to the deletion or down-regulation of LPTS gene.

The present invention can use the full-length protein of LPTS or its biologically active fragments. Biologically active fragments of any LPTS protein can be used in the present invention. Here, the meaning of the biologically active fragment of the LPTS protein refers to a protein fragment that can still maintain all or part of the functions of the complete LPTS protein (such as at least 50% of the biological activity, preferably at least 70% of the activity, More preferably at least 90% 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 through the substitution, deletion or addition of one or more amino acid residues also has the function of penetrating cells and inhibiting cell telomerase activity or expression after being fused with TAT. The present invention can also use modified or improved LPTS protein, for example, can use LPTS protein modified to prolong its half-life and improve its stability.

The inventors unexpectedly found in experiments that the protein containing 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 by it is most easily transferred into telomerase-positive cells within, exhibiting an excellent telomerase inhibitory effect.

As a preferred mode of the present invention, the amino acid sequence of the LPTS can be substantially identical to the sequence shown in SEQ ID NO:4. The amino acid sequence of a more preferred LPTS may be substantially identical to the sequence shown in positions 16-211 of SEQ ID NO:2.

fusion protein

The invention provides a fusion protein, which includes TAT protein, LPTS protein or biologically active fragments thereof. The terms "transactivator fusion protein and telomerase activity inhibitory protein", "TAT-LPTS fusion protein", "T-LPTS" or "T-LPGENE" etc. are used interchangeably, and all refer to A protein fused to LPTS amino acid sequences with or without a linker peptide sequence between the two.

Said fusion protein can be used to inhibit cell telomerase activity or expression. More preferably, the fusion protein is an isolated protein, not associated with other proteins, polypeptides or molecules, and is a purified product of recombinant host cell culture or a purified extract.

The TAT polypeptide and LPTS protein or its active fragments can be directly connected, or connected through a polypeptide linker (connecting peptide). As a preferred mode of the present invention, the TAT and LPTS or their active fragments are connected through a polypeptide linker (connecting peptide) to form a fusion protein. The linker includes 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.

As a preferred manner, the TAT polypeptide is located at the amino terminal (N terminal) of the fusion protein; the LPTS protein or its active fragment is located at the carboxyl terminal (C terminal) of the fusion protein. Alternatively, the positions of the two proteins can also be swapped.

In addition, optionally, the amino terminus (or carboxyl terminus) of the fusion protein may also contain one or more polypeptide fragments as protein tags. Any suitable label can be used in the present invention. For example, the tag can be FLAG, HA, HA1, c-Myc, 6-His, etc. These tags can be used to purify fusion proteins. A specific example is a 6-His structure linked to the C-terminus of the fusion protein. Those skilled in the art should understand that an enzyme-cleavable structure can be set 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 can not only inhibit the activity of telomerase, but also can enter the inside of the cell, thus solving the limitation on the application of LPTS. For the research on the intracellular function of LPTS, the previous research was carried out at the gene level, which is a basic research, which requires the introduction of exogenous genes into the cell genome, which is difficult for practical application. However, the present invention proposes to introduce LPTS into cells to play a role at the protein level, so that LPTS can be applied clinically.

On the other hand, the present invention also provides an isolated nucleic acid encoding the fusion protein, or its complementary chain.

The DNA sequence encoding the fusion protein of the present invention can be artificially synthesized in its entirety, or the DNA sequences encoding TAT and LPTS amino acids can be respectively obtained by PCR amplification, and then spliced together to form the DNA sequence encoding the fusion protein of the present invention.

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

Therefore, the present invention also provides a vector comprising a nucleic acid molecule encoding said fusion protein. The vector may also contain an expression control sequence operably linked to the sequence of the nucleic acid molecule, so as to facilitate the expression of the fusion protein.

As used herein, "operably linked" or "operably linked to" refers to the condition that certain parts of a linear DNA sequence are capable of affecting the activity of other parts 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 can be used, such as some vectors for cloning and expression of bacteria, fungi, yeast and mammalian cells, such as Pouwels et al., Cloning Vectors: Laboratory Manual (Elsevier latest edition) describe. Various vectors known in the art such as commercially available vectors can be used. For example, select a commercially available vector, and then operably link the nucleotide sequence encoding the new fusion protein of the present invention to the expression control sequence to form a protein expression vector. In one embodiment of the present invention, the vector is a prokaryotic vector, such as pET vector.

In addition, recombinant cells containing the nucleic acid sequence encoding the fusion protein are also included in the present 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, etc.; for example, E. coli cells (E. coli), such as E. coli HMS174 (DE3), or BL21 (DE3). Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells.

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

Methods of producing fusion proteins are also included in the present invention. The method includes culturing a recombinant cell containing a fusion protein-encoding nucleic acid. The fusion protein includes TAT polypeptide and LPTS protein or active fragments thereof. The method can include allowing the cell to express the encoded fusion protein, and renaturation of the expressed fusion protein. In one example, the method may further include isolation and/or purification of the refolded fusion protein.

The fusion protein prepared above can be purified to a substantially uniform nature, for example, a single band on SDS-PAGE electrophoresis. For example, when the recombinant protein is secreted and expressed, commercial ultrafiltration membranes can be used to separate the protein, such as products from companies such as Millipore, Amicon, and Pellicon, and the expression supernatant is first concentrated. The concentrate can be further purified by gel chromatography or by ion exchange chromatography. For example anion exchange chromatography (DEAE etc.) or cation exchange chromatography. The gel matrix can be agarose, dextran, polyamide and other commonly used matrix for protein purification. The SP group is an ideal ion exchange group. Finally, methods such as reverse-phase high-performance liquid chromatography (RP-HPLC) can be used to further refine and purify the above-mentioned purified product. All the above-mentioned purification steps can be combined in different ways, so that the protein purity can be basically uniform.

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

The fusion protein of the invention can be used to prepare 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 (such as tumor cells), and can well inhibit cell telomerase activity or expression after entering cells. Therefore, the fusion protein of the present invention has a better anti-tumor effect than LPTS, so it can be used to develop effective anti-tumor drugs.

The "tumor" of the present invention can be of various types, as long as the tumor cells are telomerase positive, for example may include (but not limited to): liver cancer BEL-7404 cells, liver cancer HepG2 cells, cervical cancer Hela cells and the like.

combination

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

As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation and allergic reactions), ie, has a reasonable benefit/risk ratio. The term "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, by themselves, are not essential active ingredients and which are not unduly toxic upon administration. Suitable vectors 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). Pharmaceutically acceptable carriers in compositions can contain liquids such as water, saline, glycerol and sorbitol. In addition, there may also be auxiliary substances in these carriers, such as lubricants, glidants, wetting agents or emulsifiers, pH buffer substances and stabilizers, such as albumin.

The composition can be made into various dosage forms suitable for administration to mammals, and the dosage forms include but not limited to: injections, capsules, tablets, emulsions, suppositories.

When used, a safe and effective amount of the fusion protein of the present invention is administered to mammals (such as humans), wherein the safe and effective amount is usually at least about 0.1 μg/kg body weight, and in most cases no more than about 50 μg/kg body weight. mg/kg body weight, preferably the dosage is about 1 microgram/kg body weight to about 10 mg/kg body weight. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

When used to inhibit tumors in mammals, the fusion protein can be administered systemically or locally, depending on factors such as the type of tumor, growth site, and degree of progression.

The composition of the present invention can be directly used to kill tumor cells. In addition, it can also be used in combination with other therapeutic agents or adjuvants at the same time.

The main advantages of the present invention are:

(1) The fusion protein of TAT and LPTS was provided and prepared for the first time, and it was confirmed that the fusion of LPTS and TAT promoted LPTS to penetrate the cell membrane without affecting the activity of LPTS to inhibit cell telomerase.

(2) The fusion protein of the present invention has significantly better anti-tumor activity than LPTS alone, and can be used for many tumors.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as Sambrook et al., molecular cloning: the conditions described in the laboratory guide (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Example 1. Construction of T-LPGENE (T-LPTS-C) fusion protein containing LPTS fragments and preparation of recombinant genetically engineered bacteria

The specific method of constructing T-LPGENE engineering bacteria is as follows:

(1) Construction of pET24-TAT plasmid

Both the TAT template and its primers were synthesized, and the specific sequences are as follows:

template:

5-AGTTT CATATG TACGGGCGCAAGAAACGCCGCCAGCGCCGCCGCGGTGGATCCTAGAAG-3 (SEQ ID NO: 5);

P1 (upstream primer):

5-AGTTT CATATG TACGGGC-3 (SEQ ID NO: 6);

P2 (downstream primer):

5-CGCCA CCTAGG ATCTTC-3 (SEQ ID NO: 7).

Through PCR reaction, obtain the DNA sequence that contains coding TAT polypeptide (11 amino acids) with restriction sites Nde I and BamH I at both ends; PCR reaction conditions are: PCR reaction is carried out in 50 μ l system, includes 1 μ l template, 1 μ l dNTP mix (10mM), 1 μl each of upstream and downstream primers (20 μM), 5 μl 10×PYROBEST buffer, 0.5 μl PYROBEST enzyme, and add deionized water to 50 μl. The reaction conditions were: 94°C for 3 minutes; then a total of 30 cycles were performed, each cycle including 94°C for 20 seconds, 50°C for 20 seconds, and 72°C for 20 seconds; finally, 72°C for 5 minutes and 4°C for heat preservation.

After the PCR product obtained above was digested with Nde I and BamHI, it was inserted into the pET24(a) (purchased from Novagen) plasmid that had undergone the same double digestion to obtain the pET24-TAT plasmid.

(2) Construction of pET24-T-LPGENE plasmid

Using the pT-LPTSCDS plasmid as a template, which contains the full-length LPTS gene (see Hepatology, 32 (2000), 721-727), the primers are as follows:

P1 (upstream primer):

5-AAA GGATCC AAGGATCTGTCATCTCGG-3' (SEQ ID NO: 8);

P2 (downstream primer):

5-AAA CTCGAG TTTGGAATCTTTCTTCTT-3 (SEQ ID NO: 9).

Through the PCR reaction, the DNA sequence containing the 196 amino acids (LPTS-C) encoding the C-terminal of the LPTS protein with restriction sites BamHI and XhoI at both ends is obtained. The sequence is shown in amino acids 16-211 in SEQ ID NO: 2 Sequence; PCR reaction conditions are as follows: PCR reaction is carried out in 50 μl system, including 1 μl template, 1 μl dNTP mix (10 mM), 1 μl each of upstream and downstream primers (20 μM), 5 μl 10×PYROBEST buffer, 0.5 μl PYROBEST enzyme, supplemented with Ionized water to 50 μl. The reaction conditions were: 94°C for 3 minutes, followed by a total of 30 cycles, each cycle including 94°C for 30 seconds, 56°C for 40 seconds, 72°C for 60 seconds; extension at 72°C for 5 minutes, and heat preservation at 4°C.

The PCR product of the LPGENE gene obtained above was inserted into the pET24-TAT plasmid that was digested by BamH I and Xho I after double digestion with BamH I and Xho I, and finally the pET24-T-LPGENE plasmid was obtained. For the DNA sequence encoding T-LPGENE, see SEQ ID NO: 1 for the sequence, and see Figure 1 for the schematic structure. The structure of pET24-T-LPGENE expression plasmid is shown in Figure 2. After the sequence was verified to be correct, the host strain E.coli BL-21 was transformed to obtain an engineering strain expressing T-LPGENE.

Induced expression and separation and purification of embodiment 2.T-LPGENE

The present inventor has adopted separation methods such as ion exchange to obtain high-purity T-LPGENE protein, which can be simply divided into the following steps:

(1) Conventional methods induce the expression of T-LPGENE protein;

(2) Ultrasonic disruption of bacteria;

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

(4) ultrafiltration concentration;

(5) Molecular sieve chromatography (Superdex 75);

The experiments were carried out at 4°C. The purity and concentration of T-LPGENE were detected by SDS-PAGE, the results are shown in Figure 3 (swimming lane 6), and the purity of T-LPGENE protein can reach 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 used the TPAP method to measure the biological activity of the purified T-LPGENE. Telomerase comes from the lysate of SMMC-7721 liver cancer cells, and the specific preparation method is as follows:

SMMC-7721 liver cancer cells (purchased from ATCC) were inoculated in 10ml culture flasks, the culture medium was RPMI 1640, cultured on the wall at 37°C and 5% CO ₂ , and the cells were collected after the flask was full. The culture medium was aspirated dry, the cells were rinsed once with PBS, the cells were digested with trypsin, the cells were rinsed with PBS, sucked into a centrifuge tube, and centrifuged at 4000rpm to collect the cells. The cells were washed again 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 in 500μl of pre-cooled lysis buffer (10mM Tris-HCl (pH 7.5), 1mM MgCl ₂ , 1mM EGTA, 0.1mMPMSF, 5mM mercaptoethanol, 0.5% CHAPS, 10% glycerol), put on ice for 30 minutes, 4 Centrifuge at 12,000 rpm for 30 minutes, and the supernatant obtained can be used to measure telomerase activity. The supernatant can be stored at -70°C, and the stable telomerase activity can still be maintained after repeated freezing and thawing.

The TPAP assay was carried out in a 500 μl Eppendorf tube with a reaction volume of 50 μl, containing 45.25 μl of conventional reaction buffer, 0.8 μl of tumor cell lysate containing telomerase, 0.25 μl of dNTP, and 1 μl of purified protein (diluted as required). After reacting on ice for 10 minutes, add 1 μl Ts primer (0.1 μg/μl, primer sequence is 5-AATCCGTCGAGCAGAGTT-3 (SEQ ID NO: 10)), 1 μl Taq enzyme, extend at 25°C for 30 minutes, inactivate at 95°C for 5 minutes, and then add Add Taq enzyme (0.5 μl), Acx primer (1 μl, 0.1 μg/μl, the primer sequence is 5-(CCCTTA) ₃ CCCTAA-3 (SEQ ID NO: 11)), and perform PCR reaction (94°C for 30s, 50°C for 40s , 72°C 40s, 33 cycles; 72°C 2min). The PCR reaction product was identified by 10% polyacrylamide gel electrophoresis, and the electrophoresis system was TBE buffer solution. After electrophoresis (120 volts, 2 hr), silver staining was used to develop the color.

The results of the TRAP experiment show that the T-LPGENE protein purified by the present invention has a high telomerase inhibitory activity in vitro, and at about 320nmol/L, T-LPGENE can completely inhibit the activity of telomerase, see Figure 4.

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

First, the inventors used a conventional lipofection method to deliver LPTS-C protein into each tumor cell. The test results showed that the entry of LPTS-C protein into cells was basically not detected. Therefore, the liposome method is not suitable for the transfer of LPTS-C protein.

In order to verify whether TAT can mediate LPGENE protein into cells, the inventors chose two experimental methods: immunofluorescence experiment and Western-blot experiment. The specific operation method of 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 covered with coverslips, and the medium was DMEM ( Contain 10% newborn calf serum) and McCoy's 5A (contain 15% fetal calf serum), and culture at 37°C under 5% CO ₂ conditions. After the cells adhere to the wall, add PBS solution (two empty) and purified T-LPGENE (100mg/L) protein (three empty, incubate for 2hr, 6hr and 24hr respectively):

(2) After incubation, wash once with PBS, add 1mL-20°C pre-cooled methanol to each well, fix for 3min; discard methanol, wash twice with PBS, then add 1mL PBS (containing 1% newborn goat Serum), blocked for 10min;

(3) Take out the fixed slide and put it into a closed container, add dropwise the primary antibody (rabbit antiserum immunized with LPGENE protein) appropriately diluted with PBS (containing 1% neonatal goat serum), react at room temperature for 2 hours, One of the slides treated with PBS culture was only dripped with PBS (containing 1% newborn goat serum), as a blank control;

(4) wash with PBS (containing 1% neonatal goat serum) 3 times, each time for 5min;

(5) Add secondary antibody (FITC-labeled goat anti-rabbit serum) appropriately diluted with PBS (containing 1% newborn goat serum) dropwise, react at room temperature in the dark for 1 hr; wash with PBS (containing 1% newborn goat serum) for 3 times, 5 minutes each time;

(6) Put the slide back into the 6-well plate, add 1mL PBS (containing 1% neonatal goat serum) and 10μL 33258 reagent to each well, and stain the nucleus for 10min;

(7) Wash 3 times with PBS, 5 min each time;

(8) Take a clean glass slide, add 50 μL cat oil dropwise, place the treated cover glass with the cell side down on the glass slide, there is no air bubble between the cover glass and cat oil, keep it away from light for 4 hours at room temperature or overnight.

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

(1) Inoculate BEL-7404 cells and Saos-2 cells in a 6cm culture dish with 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%, add PBS solution (two empty), purified LPGENE (100mg/L) protein (3 wells, incubate 2hr, 6hr and 24hr respectively) and purified T-LPGENE (100mg/L) Protein (3 wells, incubated for 2hr, 6hr and 24hr respectively):

(2) After washing twice with PBS, digest with trypsin for 10 minutes;

(3) The cells were collected by centrifugation, and 100 μL of SDS-PAGE electrophoresis loading buffer was added for every 10 ⁶ cells;

(4) Detect each sample with the Western Blotting method, that is, after the sample is subjected to SDS-PAGE electrophoresis, the protein is transferred to the nitrocellulose membrane with the electroporation instrument of Bio-Rad Company, and the specific anti-LPGENE antibody (the home of LPGENE protein immunization) rabbit antiserum) for hybridization. The secondary antibody was goat anti-rabbit IgG labeled with horseradish peroxidase (HRP).

The results of immunofluorescence experiments and western-blot experiments are shown in Figure 5. The results show that TAT can mediate LPGENE protein into BEL-7404 cells and Saos-2 cells extremely well, and can enter the nucleus.

Example 5. The purified T-LPGENE protein has the activity of inhibiting the growth of telomerase positive cells in vitro

MTT analysis

(1) BEL-7404 cells and Saos-2 cells were inoculated in 6-well plates covered with coverslips, and the medium was DMEM (containing 10% newborn calf serum) and McCoy's 5A (containing 15% fetal calf serum) ), 37°C, 5% CO ₂ for adherent culture; HepG2 cells (purchased from Shanghai Biochemical Cell Bank) and L02 cells (purchased from Shanghai Biochemical Cell Bank) were also inoculated on 6 In the well plates, the culture media used were DMEM+10% FBS (fetal bovine serum) and RPMI1640+10% NBS (newborn calf serum). Add the PBS solution and the aforementioned purified T-LPGENE (40mg/L) and TAT-GFP (40mg/L) (T-GFP) to the 6-well plate respectively (using the full-length sequence of GFP, see the GenBank accession number for the GFP protein sequence: DQ768212; the C-terminal of TAT is connected with the N-terminal of GFP) protein;

(2) After continuous culture for 6 weeks, the growth activity of cells in each group was detected by MTT method. For specific experimental methods, see the literature (Situ Zhenqiang, edited by Wu Junzheng; Cell Culture [M]; Xi'an: World Book Publishing Xi'an Company, 2004, 250). During the detection, the cells in each group continued to add the corresponding protein, and the detection wavelength was 570nm.

The experimental results are shown in Figure 6. The results show that T-LPGENE protein can inhibit the growth of telomerase-positive cells BEL-7404 and HepG2, but has no obvious inhibitory effect on reducing the growth of telomerase-negative cells Saos-2 and immortalized cell L02 .

Southern-blot analysis

The cells after continuous culture for 8 weeks were collected, and the telomere length was analyzed by Southern-blot method. For specific experimental methods, please refer to the literature (Damm K. et al., A highly selective telomerase inhibitor limiting human cancer cell proliferation; EMBO J 2001; 20: 6958- 6968). The simple process is as follows: isolate genomic DNA, double-digest with endonucleases Hinf I and Afa I, then use 0.8% agarose gel to separate the digested product, transfer to the membrane, hybridize with the probe labeled γ-32P, and finally Hybridization signals were analyzed with ImageQuant.

The experimental results are shown in Figure 7A. The results showed that after 8 weeks of T-LPGENE protein treatment, compared with the control group (PBS and TAT-GFP treatment), the telomeres of telomerase-positive tumor cells BEL-7404 and HepG2 were significantly shortened, And the telomere of the high concentration protein group is shorter than that of the low concentration group. However, the telomeres of Saos-2 tumor cells with negative telomerase were not significantly shortened.

Cell Morphology Detection

After the cells were continuously cultured for 6 weeks, photographs were taken with an inverted microscope, and the results are shown in Figure 7B. The results showed that the adhesion ability of the telomerase-positive tumor cells BEL-7404 and HepG2 treated with T-LPGENE protein became poor, and the cells became flat and flat. Big, this is a typical crisis (crisis) form.

Every time a normal human body cell divides, the telomere will lose 50-200bp. When the telomere is shortened to a certain extent, the cell growth will be inhibited, which is called cell senescence, and it will die. However, in the vast majority of malignant tumor cells (85%), telomerase activity can be detected and the activity is strong, and the resynthesis of telomeres by telomerase compensates for its continuous loss during cell reproduction, thus making the cells Can continue to divide, which is an important mechanism of cell immortalization and carcinogenesis. T-LPGENE protein can inhibit the activity of intracellular telomerase, so that the telomere of telomerase-positive tumor cells gradually shortens with cell proliferation, enters the crisis period (senescence), and presents a crisis form in morphology. However, the morphology of the telomerase-negative tumor cell Saos-2 and the immortalized cell L02 did not change significantly.

DNA content detected by flow cytometry

After the cells were continuously cultured for 6 weeks, 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 cells in G2/M phase is tetraploid, and the DNA of dead or dying cells is broken, and the flow signal is in a region less than diploid (sub-G1 region).

The experimental results are shown in Figure 7C. The results showed that after 6 weeks of continuous treatment with T-LPGENE protein, the content of the sub-G1 region of the telomerase-positive tumor cells BEL-7404 and HepG2 increased significantly, that is, the number of dead or dying cells increased significantly. ; while the sub-G1 region of the telomerase-negative tumor cell Saos-2 and the immortalized cell L02 did not change significantly.

Example 6.Tumor inhibitory 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

(1) Inoculate BEL-7404 cells and Saos-2 cells in a six-well plate covered with coverslips, and the medium is DMEM (containing 10% newborn calf serum) and McCoy's 5A (containing 15% fetal calf serum) ), adherent culture at 37°C, 5% CO ₂ . Add PBS solution and the purified T-LPGENE (final concentration in the culture medium is 40mg/L) and TAT-GFP (40mg/L) protein respectively:

(2) After continuous culture for 6 weeks, the cells were collected and inoculated subcutaneously in nude mice at 8×10 ⁶ ;

(3) After inoculation, observe the tumor growth and measure the tumor size every week;

(4) After 6 weeks of inoculation, photographs were taken, and the tumors were taken out and weighed.

The experimental results are shown in Fig. 8A and Table 1. The results show that T-LPGENE protein can reduce the tumorigenicity of the telomerase-positive cell BEL-7404, but has no inhibitory effect on reducing the tumorigenicity of the telomerase-negative cell Saos-2. However, the fusion protein TAT-GFP protein of TAT and green fluorescent protein has basically no effect on the tumorigenicity of telomerase-positive cells BEL-7404.

Table 1

2. Antitumor activity of purified T-LPGENE protein

Hepatocellular carcinoma cell BEL-7404 was inoculated (5×10 ⁶ /head) subcutaneously into the right waist of nude mice (4-week-old, female). After 4 weeks, the tumor was removed, cut into 2 mm ³ size, and then inoculated into nude mice (4-week-old age, female) under the skin of the right waist, and the nude mice were randomly divided into 4 groups. The next day, subcutaneous injections of T-LPGENE protein (100 μg, 400 μg/mouse, experimental group) were started, near the tumor, and PBS and TAT-GFP protein (400 μg/mouse) were injected into the control group. Injections were given every 2 days for the first 2 weeks, and every 3 days for the next 3 weeks, for a total of 14 injections. The tumor size was recorded every week (measured with a vernier caliper), and all nude mice were photographed 7 weeks after inoculation, and the nude mice were sacrificed, and the tumors were taken out and weighed.

The experimental results are shown in Fig. 8B-D, and the results show that T-LPGENE protein can inhibit the growth of BEL-7404 tumor cells in nude mice, which has a certain dosage effect. The tumor weight of each injected 100 μg T-LPGENE protein group was only 57% (P<0.05) of the PBS group, while the tumor weight of the 400 μg 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 heavier than that of the two experimental groups (P<0.05). The tumor weight of the high-dose experimental group was also significantly smaller than that of the low-dose experimental group (P<0.05).

Example 7. Preparation and cell test of fusion protein containing full-length LPTS

The expression TAT-full-length LPTS (TAT-LPTS) was prepared according to the similar method described in Example 1, and the pET24-TAT plasmid was constructed first. Using the pT-LPTSCDS plasmid as a template, the full-length gene sequence of LPTS (see SEQ ID NO: 3) was amplified by PCR, and restriction sites BamH I and Xho I were set at both ends, and after double digestion with these two enzymes Insert the pET24-TAT plasmid that has been cut with the same double restriction enzymes to obtain the pET24-T-LPTS plasmid, which has a DNA sequence encoding TAT-LPTS. After the sequence was verified to be correct, the host strain E.coli BL-21 was transformed to obtain the expressed engineering strain.

The TAT-LPTS protein with a purity of more than 80% was obtained by inducing expression and separating and purifying the method as described in the foregoing Example 2.

In vitro tests (such as Example 3) found that the ability of TAT-LPTS to inhibit telomerase activity is basically the same as that of T-LPGENE.

Immunofluorescence experiments and western-blot experiments were performed using the method described in Example 4 above to detect the entry of TAT-LPTS into cells. The results showed that TAT-LPTS could enter BEL-7404 cells and Saos-2 cells, and could enter the nucleus. The semi-quantitative test found that compared with the situation of T-LPGENE protein entering the cell, the amount of TAT-LPTS entering the nucleus is about 70% of the amount of T-LPGENE protein entering the cell, which shows that the transfer effect of T-LPGENE protein is more ideal .

Embodiment 8. Preparation and cell test of T-LPGENE protein without connecting peptide

Compared with the T-LPGENE protein in Example 1 (the protein of the sequence shown in SEQ ID NO: 2), the T-LPGENE protein without connecting peptide lacks the 13th-15th in SEQ ID NO: 2 sequence of bits. After artificially synthesizing the gene sequence encoding the T-LPGENE protein without the connecting peptide, insert the sequence into the pET24 plasmid, transform into engineering bacteria, express and purify to obtain the T-LPGENE protein without the connecting peptide.

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

Example 9. Pharmaceutical composition

100 mg of the fusion protein purified in Example 3 was prepared in 100 ml of regular physiological saline to obtain a composition containing the fusion protein at a concentration of 1 mg/ml.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

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Claims (10)

1. a fusion protein of separation, is characterized in that, described fusion protein comprises: (1) telomerase activity inhibitory protein LPTS; (2) the transactivator protein TAT; and (3) A connecting peptide consisting of 0-20 amino acids located between (1) and (2). 2. fusion protein as claimed in claim 1, is characterized in that, described telomerase activity inhibitory protein LPTS is: (a) the protein of the amino acid sequence shown in SEQ ID NO: 4; (b) the protein of the amino acid sequence shown in positions 16-211 in SEQ ID NO: 2; or (c) The amino acid sequence of the protein defined in (a) or (b) is formed by the substitution, deletion or addition of one or more amino acid residues, and has the protein function defined in (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 2-12 of SEQ ID NO:2. 3. fusion protein as claimed in claim 1, is characterized in that, described telomerase activity inhibitory protein LPTS is: (a1) a protein of the amino acid sequence shown in positions 16-211 of SEQ ID NO: 2; or (b1) A protein derived from (a1) formed by substituting, deleting or adding 1-10 amino acid residues to the amino acid sequence of the protein defined in (a1) and having the protein function defined in (a1) . 4. A nucleic acid molecule, characterized in that the nucleic acid molecule encodes the fusion protein according to 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 cells contain the carrier of claim 5; or The nucleic acid molecule of claim 4 is integrated in the genome of the cell. 7. A method for producing the fusion protein according to claim 1, characterized in that, the method comprises: under conditions suitable for expressing the fusion protein, culturing the cell according to claim 6, expressing and isolating The fusion protein. 8. The use of the fusion protein according to any one of claims 1-3, characterized in that it is used to prepare a composition for inhibiting the growth of telomerase positive cells. Preferably, the cells are telomerase positive tumor cells. Preferably, the telomerase-positive cells include: liver cancer BEL-7404 cells, liver cancer HepG2 cells or cervical cancer Hela cells. 9. A composition for suppressing tumors, characterized in that, said composition contains: (i) an effective amount of the fusion protein according to any one of claims 1-3; and (ii) A pharmaceutically acceptable carrier. 10. A method for introducing telomerase activity inhibitory protein LPTS into cells, characterized in that the method comprises the following steps: (a) fusing the transactivator TAT with the telomerase activity inhibitory protein LPTS to obtain a fusion protein; (b) co-incubating the fusion protein of (a) with the cells, so that the telomerase activity inhibitory protein LPTS is introduced into the cells.

Images