WO2019196790A1 - Anti-tumor fusion protein, preparation method therefor and application thereof - Google Patents

Abstract

The present invention provides an anti-tumor fusion protein, a preparation method therefor and an application thereof. Specifically, the present invention provides a fusion protein comprising a GnRH protein element, a transmembrane transport region of a PEA protein, and a P53 protein element. The fusion protein can efficiently transport a p53 protein to a cell nucleus and efficiently induce tumor cell apoptosis. The present invention also provides a nucleotide encoding the fusion protein, a method for producing the fusion protein, and a pharmaceutical composition comprising the fusion protein.

Description

Anti-tumor fusion protein and preparation method and application thereof Technical field

The invention relates to the field of biomedicine, and more particularly to an anti-tumor fusion protein and a preparation method and application thereof.

Background technique

An immunotoxin is a group of artificially constructed hybrid molecules with specific cell killing ability, consisting of a toxic part, a carrier part and a targeting part. The toxic portion may be a cytotoxin of plant, animal, or microbial origin, and the targeting moiety may be a monoclonal antibody or a cytokine. Compared with other anti-tumor drugs, immunotoxins have the advantages of strong toxicity and high specificity, and have shown great application prospects in tumor treatment.

Lyophilized recombinant human luteinizing hormone releasing hormone-exotoxin of pseudomonas aeruginosa fusion protein (LHRH-PE40) is a pair of research and production of Changchun genetic engineering drugs Tumor cells have a specific killing effect of recombinant toxins. The production method comprises the following steps: firstly, the receptor binding region (I region) encoding the Pseudomonas aeruginosa exotoxin A gene is excised by a genetic engineering method, and replaced with the LHRH gene, and then the LHRH-PE40 recombinant gene is cloned into a plasmid and passed through engineering fermentation. The LHRH-PE40 fusion protein was expressed.

The cytotoxic effect of LHRH-PE40 is exotoxin A of pseudomonas aeruginosa (PEA). Pseudomonas aeruginosa exotoxin A is the most potent toxin of Pseudomonas aeruginosa, which can block the synthesis of cellular proteins and cause apoptosis of cells. The leader in LHRH-PE40 is human luteinizing hormone releasing hormone (LHRH), which is targeted to introduce PEA into tumor cells to exert anti-tumor by binding to its type I receptor on the surface of tumor cells. effect.

There are two types of receptors for LHRH, type I and type II. LHRH has a high affinity for type I receptors. Under normal conditions, type I receptors mainly exist in the anterior pituitary, and tissues such as gonads, placenta and brain tissues also contain a certain amount of type I receptors, and other important organs do not express type I LHRH receptors. However, on the surface of some tumor cells, a large number of LHRH type I receptors are distributed due to receptor alienation, including reproductive system malignant tumors, melanoma, gastric cancer, liver cancer, pancreatic cancer, intestinal cancer, and lung cancer. Since type I LHRH receptors are generally highly expressed in tumor cells and are only localized in normal tissues, LHRH is an ideal guide for designing recombinant targeting toxins.

Type II receptors of LHRH are widely present in various tissues of the human body. LHRH has a low affinity for type II receptors. However, at very high doses, LHRH can bind to the type II receptor with low affinity, resulting in extensive cytotoxicity of LHRH-PE40. This is the theoretical basis for the toxic effects of LHRH-PE40 recombinant toxins on animals. LHRH-PE40 does not penetrate the blood-brain barrier and therefore does not cause toxic effects on the pituitary gland.

Although the immunotoxin represented by LHRH-PE40 shows a good application prospect, it still has problems such as immunogenicity and non-specific cytotoxicity, which hinders the clinical application of immunotoxin. Therefore, there is an urgent need in the art to develop new immunotoxins with good specificity and low cytotoxicity.

Summary of the invention

The object of the present invention is to provide an anti-tumor fusion protein, a preparation method and application thereof.

In a first aspect of the invention, an anti-tumor fusion protein is provided, said anti-tumor fusion protein having the structure of formula I:

D-A-B-C-E (I)

among them,

A is a GnRH protein element;

B is the transmembrane transport region of the PEA protein or is absent;

C is a P53 protein element;

E is a TAT protein element or is absent;

D is an optional signal peptide and/or leader peptide sequence;

Also, B and E are not the same at the same time;

Here, "-" means a peptide bond connecting the above elements.

In another preferred embodiment, the anti-tumor fusion protein has the structure of Formula II:

D-A-C-E (II)

among them,

A is a GnRH protein element;

C is a P53 protein element;

E is a TAT protein element;

D is an optional signal peptide and/or leader peptide sequence;

Here, "-" means a peptide bond connecting the above elements.

In another preferred embodiment, the anti-tumor fusion protein

In another preferred embodiment, the TAT is a short peptide rich in basic amino acids encoded by HIV-I.

In another preferred embodiment, the TAT protein is derived from a human or a non-human mammal.

In another preferred embodiment, the TAT protein comprises wild-type and mutant forms.

In another preferred embodiment, the TAT protein comprises a full length, mature form of P53, or an active fragment thereof.

In another preferred embodiment, the sequence of the TAT protein is shown in positions 405-416 of SEQ ID NO.: 5.

In another preferred embodiment, the anti-tumor fusion protein is selected from the group consisting of:

(A) a polypeptide having the amino acid sequence of SEQ ID NO.: 5;

(B) having ≥80% homology to the amino acid sequence shown in SEQ ID NO.: 5 (preferably, ≥90% homology; etc. preferably ≥95% homology; most preferably, ≥97 % homology, such as 98% or more, 99% or more) of the polypeptide, and the polypeptide has an activity of inhibiting tumor cell growth;

(C) A derivative polypeptide obtained by subjecting the amino acid sequence shown by SEQ ID NO: 5 to substitution, deletion or addition of 1-10 amino acid residues, and retaining the activity of inhibiting tumor cell growth.

In another preferred embodiment, the anti-tumor fusion protein has the structure of Formula II:

D-A-B-C (II)

among them,

A is a GnRH protein element;

B is a transmembrane transport region of PEA protein;

C is a P53 protein element;

D is an optional signal peptide and/or leader peptide sequence;

"-" indicates a peptide bond connecting the above elements.

In another preferred embodiment, the GnRH protein is derived from a human or a non-human mammal.

In another preferred embodiment, the GnRH protein comprises wild type and mutant form.

In another preferred embodiment, the GnRH protein comprises a full length, mature form of GnRH, or an active fragment thereof.

In another preferred embodiment, the GnRH protein comprises a type II GnRH protein and a type I GnRH protein.

In another preferred embodiment, the GnRH protein is a type II GnRH protein.

In another preferred embodiment, the sequence of the GnRH protein is as shown in 1-10 of SEQ ID NO.: 2.

In another preferred embodiment, the P53 protein is derived from a human or a non-human mammal.

In another preferred embodiment, the P53 protein comprises wild type and mutant form.

In another preferred embodiment, the P53 protein comprises a full length, mature form of P53, or an active fragment thereof.

In another preferred embodiment, the sequence of the P53 protein is shown in positions 128-520 of SEQ ID NO.: 2.

In another preferred embodiment, the transmembrane transport region of the PEA protein is derived from Pseudomonas aeruginosa.

In another preferred embodiment, the transmembrane transport region of the PEA protein is from 100 to 120 amino acids in length, preferably from 100 to 115 amino acids.

In another preferred embodiment, the sequence of the transmembrane trafficking region of the PEA protein is shown in positions 13-127 of SEQ ID NO.: 2.

In another preferred embodiment, the anti-tumor fusion protein is a recombinant protein expressed by bacteria, preferably E. coli.

In another preferred embodiment, the anti-tumor fusion protein is a protein that is not glycosylated.

In another preferred embodiment, the anti-tumor fusion protein is selected from the group consisting of:

(A) a polypeptide having the amino acid sequence of SEQ ID NO.: 2;

(B) having ≥80% homology to the amino acid sequence shown in SEQ ID NO.: 2 (preferably, ≥90% homology; etc. preferably ≥95% homology; most preferably, ≥97 % homology, such as 98% or more, 99% or more) of the polypeptide, and the polypeptide has an activity of inhibiting tumor cell growth;

(C) A derivative polypeptide obtained by subjecting the amino acid sequence of SEQ ID NO: 2 to substitution, deletion or addition of 1-10 amino acid residues, and retaining the activity of inhibiting tumor cell growth.

In another preferred embodiment, the tumor cell is a tumor cell expressing GnRHR, preferably a tumor cell expressing type I GnRHR.

In another preferred embodiment, the tumor cell is a tumor cell expressing a type II GnRHR.

In another preferred embodiment, the amino acid sequence of the anti-tumor fusion protein is set forth in SEQ ID NO: 2.

In another preferred embodiment, the anti-tumor fusion protein contains a 6xHis purification tag.

In another preferred embodiment, the anti-tumor fusion protein is capable of inhibiting tumor cell growth and/or inducing apoptosis of tumor cells.

In a second aspect of the invention, an isolated polynucleotide encoding the anti-tumor fusion protein of the first aspect of the invention is provided.

In another preferred embodiment, the sequence of the polynucleotide is as shown in SEQ ID NO.: 1.

In a third aspect of the invention, a vector comprising the polynucleotide of the second aspect of the invention is provided.

In another preferred embodiment, the vector comprises a plasmid, a viral vector.

In another preferred embodiment, the viral vector comprises a lentiviral vector, an adenoviral vector, and a yellow fever virus vector.

In another preferred embodiment, the vector comprises an expression vector.

In a fourth aspect of the invention, there is provided a host cell comprising the vector of the third aspect of the invention or the polynucleotide of the second aspect of the invention integrated in the genome.

In another preferred embodiment, the host cell comprises a prokaryotic cell and a eukaryotic cell.

In another preferred embodiment, the host cell is a bacterium, preferably E. coli.

In a fifth aspect of the invention, a method of producing an anti-tumor fusion protein comprising the steps of:

(a) cultivating the host cell of the fourth aspect of the invention under conditions suitable for expression, thereby expressing the anti-tumor fusion protein of the first aspect of the invention;

(b) isolating and purifying the anti-tumor fusion protein expressed in the step (a).

In a sixth aspect of the invention, a pharmaceutical composition comprising the anti-tumor fusion protein of the first aspect of the invention, and a pharmaceutically acceptable carrier or excipient is provided.

In a seventh aspect of the invention, there is provided a use of the anti-tumor fusion protein of the first aspect of the invention for the preparation of a medicament for treating or preventing a tumor.

In another preferred embodiment, the tumor is a tumor that expresses GnRH.

In another preferred embodiment, the tumor is selected from the group consisting of breast cancer, lung cancer, colon cancer, pancreatic cancer, ovarian cancer, prostate cancer, kidney cancer, liver cancer, brain cancer, melanoma, multiple myeloma, head and neck Tumor.

In an eighth aspect of the invention, a method for non-therapeutic inhibition of tumor cells in vitro is provided, comprising the step of culturing said tumor cells in the presence of the anti-tumor fusion protein of the first aspect of the invention.

In a ninth aspect of the invention, a method of treating a tumor comprising the step of administering the anti-tumor fusion protein of the first aspect of the invention to a subject in need thereof.

In another preferred embodiment, the anti-tumor fusion protein is administered as a monomer, a dimer and/or a tetramer, preferably the anti-tumor fusion protein is administered as a tetramer.

In another preferred embodiment, the object is a human.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 shows an SDS-PAGE electropherogram of the fusion protein of the present invention which induces expression. Wherein, the first lane is Marker, the second lane is the sample before the inducer, the third lane is the whole bacterial protein sample after the inducer, and the fourth lane is the soluble expression component after the inducer, fifth The lane is the inclusion body expression component after the inducer is added.

Figure 2 shows the killing effect of GnRH+PII+P53 on Ishikawa cells.

Figure 3 shows a schematic diagram of the molecular structure of PEA.

detailed description

The present inventors have extensively and intensively studied, and for the first time, unexpectedly discovered a fusion protein which efficiently delivers p53 protein to the nucleus and efficiently induces apoptosis. The fusion protein not only efficiently expresses, is not easily degraded, but also efficiently forms a polymer (especially a tetramer). In addition, the experimental results also show that the fusion protein (monomer or multimer) of the present invention can enter the intracellular membrane extremely efficiently and efficiently, and enter the nucleus efficiently and rapidly, thereby extremely efficiently inducing abnormal cells (such as tumors). Apoptosis of cells). The present invention has been completed on this basis.

Fusion protein

The term "active ingredient" as used herein refers to an anti-tumor fusion protein of the invention.

As used herein, the terms "fusion protein of the invention", "fusion protein", "anti-tumor fusion protein" are used interchangeably and refer to the fusion protein of the first aspect of the invention.

In a preferred embodiment, the fusion protein has a GnRH+PII+P53 structure, and the sequence is shown in SEQ ID NO.: 2.

In another preferred embodiment, the fusion protein having the structure of GnRH + P53 + TAT, the sequence shown in (QHWSYGLRPGHMEEPQSDPSVEPPLSQETFSDLWKLLPENNVLSPLPSQAMDDLMLSPDDIEQWFTEDPGPDEAPRMPEAAPRVAPAPAAPTPAAPAPAPSWPLSSSVPSQKTYQGSYGFRLGFLHSGTAKSVTCTYSPALNKMFCQLAKTCPVQLWVDSTPPPGTRVRAMAIYKQSQHMTEVVRRCPHHERCSDSDGLAPPQHLIRVEGNLRVEYLDDRNTFRHSVVVPYEPPEVGSDCTTIHYNYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRNSFEVHVCACPGRDRRTEEENLRKKGEPHHELPPGSTKRALSNNTSSSPQPKKKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSRAHSSHLKSKKGQSTSRHKKLMFKTEGPDSDGRKKRRQRRRPQ) SEQ ID NO.:5, which is of GnRH 1-10, 12-404 of bit P53, the first Bits 405-416 are TAT.

The two fusion proteins of the present invention can be used in combination after mixing.

Pharmaceutical composition

Since the fusion protein of the present invention has excellent inhibitory activity against tumor cell growth, the fusion protein of the present invention and a pharmaceutical composition containing the fusion protein of the present invention as a main active ingredient can be used for (a) prevention Or inhibit tumor growth, metastasis or tumor cell growth, migration, or (b) induce apoptosis in human tumor cells.

The pharmaceutical compositions of the present invention comprise a fusion protein of the invention and a pharmaceutically acceptable excipient or carrier in a safe and effective amount. By "safe and effective amount" it is meant that the amount of fusion protein is sufficient to significantly improve the condition without causing serious side effects. In general, the pharmaceutical compositions contain from 1 to 2000 mg of the fusion protein per agent of the invention, more preferably from 10 to 200 mg of the compound of the invention per agent. Preferably, the "one dose" is a capsule or tablet.

"Pharmaceutically acceptable carrier" means: one or more compatible solid or liquid fillers or gel materials which are suitable for human use and which must be of sufficient purity and of sufficiently low toxicity. By "compatibility" it is meant herein that the components of the composition, as well as the components and the active ingredients of the present invention, can be incorporated into each other without significantly reducing the efficacy of the active ingredient. Examples of pharmaceutically acceptable carriers are cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate). , calcium sulfate, vegetable oil (such as soybean oil, sesame oil, olive oil, etc.), polyol (such as propylene glycol, glycerin, sorbitol, etc.), emulsifier (such as Tween

), a wetting agent (such as sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, a pyrogen-free water, and the like.

Application method

The administration mode of the fusion protein or the pharmaceutical composition thereof according to the present invention is not particularly limited, and representative administration methods include, but are not limited to, oral, intratumoral, rectal, and parenteral (intravenous, intramuscular or subcutaneous) ).

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or mixed with: (a) a filler or compatibilizer, for example, Starch, lactose, sucrose, and silicic acid; (b) binders, for example, hydroxymethylcellulose, gelatin, sucrose, and acacia; (c) humectants, for example, glycerin; (d) disintegrants, for example , agar, calcium carbonate, potato starch or tapioca starch, and sodium carbonate; (e) a slow solvent such as paraffin; (f) an absorption accelerator, for example, a quaternary amine compound; (g) a wetting agent such as cetyl alcohol and a single Glyceryl stearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, sodium lauryl sulfate, or a mixture thereof. In capsules, tablets and pills, the dosage form may also contain a buffer.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other materials known in the art. They may contain opacifying agents and the release of the active ingredient in such compositions may be released in a portion of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric and waxy materials. If necessary, the active ingredient may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances.

In addition to these inert diluents, the compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.

In addition to the active ingredient, the suspension may contain suspending agents, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these and the like.

Compositions for parenteral injection may comprise a physiologically acceptable sterile aqueous or nonaqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion. Suitable aqueous and nonaqueous vehicles, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.

The fusion proteins of the invention may be administered alone or in combination with other pharmaceutically acceptable compounds (or tumor suppressors).

The other pharmaceutically acceptable compound comprises an antitumor drug selected from the group consisting of an alkylating agent, an antimetabolite, a folic acid analog, a pyrimidine analog, a purine analog, a vinblastine, and epipodophyllotoxin. ), antibiotics, L-aspartate, topoisomerase inhibitor, interferon, platinum coordination complex, emodin-substituted urea, methyl hydrazine derivative, adrenocortical inhibitor, adrenal corticosteroid, pregnancy Hormones, estrogens, antiestrogens, androgens, antiandrogens, and or gonadotropin-releasing hormone analogs.

Preferably, the anti-tumor drug is selected from the group consisting of 5-fluorouracil (5-FU), formyltetrahydrofolate, irinotecan, oxaliplatin, capecitabine, paclitaxel, docetaxel, Or a combination thereof.

When a pharmaceutical composition is used, a safe and effective amount of the fusion protein of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective effective dose for a person weighing 60 kg. In general, the daily dose is usually from 1 to 2000 mg, preferably from 20 to 500 mg. Of course, specific doses should also consider factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled physician.

PEA

PEA is a single-stranded toxin protein consisting of 613 amino acids with a molecular weight of 66 kD. It consists of three structural functional zones: Zone I, Zone II and Zone III. The I region is in the antiparallel beta structure at the N-terminus of the PEA molecule. The I region is further divided into an Ia region and an Ib region, which are separated in the DNA sequence but close together in the three-dimensional structure. The Ia region contains amino acid 1-252, which is responsible for binding to the target cell surface receptor-cell binding function; the Ib region contains amino acids 365-399, and the deletion of most of the region (amino acids 365-380) does not affect PEA. Biological activity. The region II is the central region, including amino acids 253-364, and has six consecutive alpha helices, which are responsible for transmembrane translocation function. When the region II is deleted, although its cell-binding function and ADP ribosylation activity remain, However, loss of cytotoxicity indicates that zone II is required for toxin translocation. Region III includes amino acids 400-613, which have two functions: one is to catalyze ADP ribosylation of EF-2; the other is that its C-terminal specific amino acid sequence mediates toxin fragments into the endoplasmic reticulum. The series consists of five amino acid residue fragments (Arg609Glu610Asp611Leu612Lys613 or REDLK), and its deletion causes the cytotoxicity of PE to be lost, and the sequence adjustment can significantly improve the ADP ribosylation efficiency of the toxin. Table 1 below shows the structure and function of PE molecules.

Table 1: Relationship between structure and function of PE molecules

The structure of the PEA molecule is shown in Figure 3.

GnRH

GnRH was isolated and purified from animals by Schally in 1971, and its structure was synthesized and then synthesized, and the Nobel Prize of 1976 was obtained. GnRH is a decapeptide containing no free amino acid and carboxyl group, and its molecular structure is: P-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂ , of which amino acids 4-6 It forms a β-turn, which is hairpin-shaped and is suitable for binding to receptors. Positions 2 and 3 are important for biological activity, and position 6 plays an important role in maintaining hairpin conformation. The first and fourth amino acids are involved. Receptor binding, if substituted for the above amino acid residues, can result in loss of vigor or geometrical enhancement.

Natural GnRH is easily degraded by proteolytic enzymes in the body, so its half-life is only 4-8 min. The main sites of action of the hydrolase peptidase are Gly6-Leu7 and Pro9-Gly10-NH2. To find efficient and long lasting GnRH analogs, more than 3,000 GnRH analogs have been synthesized by picking up or replacing amino acids in their peptide chain structures. Since the synthetic GnRH has a longer half-life and is more potent, it is more suitable for the treatment of patients than natural GnRH. The requirement for the synthesis of long-acting GnRH agonists is to stabilize the molecular structure, making it less susceptible to enzymatic hydrolysis, increasing binding to circulating proteins and membranes, and increasing affinity for the GnRH receptor. For example, the 6 position is an analog of D-amino acid and the substituted Gly10 amide group. This GnRH agonist is not only resistant to protease hydrolysis but also has a high affinity for receptors. The introduction of a bulky hydrophobic group at position 6 further increases the affinity for the receptor. Such substitutions stabilize the "active" configuration of the release hormone analog, increasing binding to circulating proteins, thereby extending half-life.

The normal human gonadotropin-releasing hormone (GnRH) receptor is mainly present in the anterior pituitary, and there is a small amount of GnRH receptor distribution in the pituitary tissues such as the gonad placenta, although it can also be found in important organs such as liver, kidney, heart and skeletal muscle. A certain level of mRNA for the GnRH receptor was detected. However, using the method of quantitative analysis of receptors - radioligand analysis (RLA) to detect these organ tissues, only negative results can be obtained.

Current research shows that GnRH can basically be divided into two types, namely

GnRH I, the primary structure is as follows:

pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂

GnRH II, the primary structure is as follows:

pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH ₂

The corresponding receptors are also divided into two types, the type I GnRH receptor and the type II GnRH receptor. The main differences between the two types of GnRH receptors are:

1 The genes exist in different positions: the type I GnRH receptor gene is present on chromosome 8, and the type II GnRH receptor gene is present on chromosome 20.

2 The transcription direction of the gene is different: the transcription direction of the type I GnRH receptor gene is in the sense direction, and the transcription direction of the type II GnRH receptor gene is in the antisense direction, so the difference between the RNA cleavage and the termination site is also large. the difference.

3 Amino acid composition is different: The amino acid homology of the amino acid expressed by exons 2 and 3 of the type II GnRH receptor gene and the same site of the type I receptor are 45% and 41%, respectively.

4 Molecular structure is different: Type II GnRH receptor structure has a C-terminal cytoplasmic tail. The type I GnRH receptor is absent.

5 The selectivity to ligand binding is different: ligand binding specificity tests performed on functional cells indicate that both receptors have clear ligand selectivity, and type II receptors have very high reactivity with type II GnRH, whereas The response capacity to type I GnRH is very low, and the difference between the two is 420 times.

6 Distribution in tissues: Using RT-PCR, GnRHRI mRNA is mainly distributed in pituitary and a few reproductive system tissues, while GnRHRII mRNA is widely distributed in almost all organ tissues. The significance of the widespread and abundant presence of type II GnRH receptors in human tissues is still unclear.

7 different ligands are different from the signals produced by binding to different receptors.

P53

Wtp53 (wild-type p53) is the most potent tumor suppressor found to date. Studying how to use wtp53 to treat tumors has become a hot spot in the field of cancer research. Because p53 protein has a short half-life and is a non-secretory protein, there is no p53 receptor or ligand on the cell membrane, making it difficult for p53 protein to break through the cell membrane and enter the cell. Play a role.

At present, most studies at home and abroad focus on introducing p53 gene into cells through a vector (such as viral vector AAV), but the introduced vector has unsafe factors, so how to make p53 protein enter the cell, and it does not affect p53. The biological activity of the protein, and avoiding the unsafeness of the introduced vector, is a problem that must be solved in the treatment of tumors with p53.

The p53 gene is located on human chromosome 17p13 and is about 20 kb in length. It consists of 11 exons and 10 introns and is transcribed into 2.5 kb mRNA, encoding a protein of 393 amino acids with a relative molecular mass of 53×10 ³ . The protein is a nuclear-binding protein with three major functional regions: 1) an N-terminal transcriptional activation region that activates transcription and mediates protein-protein interactions, which can also bind to p53 negative regulators; 2) Central DNA core binding region, which has specific binding to DNA function and is a hot spot for tumor cell mutation; 3) C-terminal non-specific DNA binding region, including nuclear localization signal region and nuclear export signal region

P53 protein is a phosphorylated protein that is easily hydrolyzed in cells and has a half-life of only about 30 minutes, which is almost undetectable in the nucleus of normal cells. However, when cells are stimulated by external factors, such as hypoxia, ultraviolet radiation or certain drugs, DNA damage, p53 protein acts as a transcription factor, inhibiting cell proliferation by transactivation; on the other hand, p53 protein It can directly interact with components in the DNA replication mechanism to inhibit DNA replication and ensure genetic stability. Specifically, P53 has the following functions:

P53 regulates cell cycle

P53 can effectively prevent malignant transformation of cells, monitor the integrity of the genome, and identify various abnormalities that may cause tumors. It is called "Geneguards." Various damages in DNA can regulate p53 function through specific post-translational modifications, including phosphorylation and acetylation. Regulators such as viral-encoded proteins, intracellular proteins, and transcriptional repressors can also affect their function. By interacting with different synergistic molecules, P53 induces activation of different target genes, regulates the cell cycle, and stops the cell cycle at specific checkpoints.

Cell cycle G1 pause

P53 regulates the p21 gene and regulates it with p21, which plays an important regulatory role in the G1/S phase arrest caused by DNA damage.

Another blockade of P53 in the G1 phase of cell cycle arrest is through a non-transcriptional mechanism. CDK-activating kinase (CAK), a cyclin-dependent protein kinase, binds to and activates cyclins, phosphorylates target proteins, and regulates cell cycle progression.

Cell cycle S phase pause

P21waf1 binds to proliferating cell nuclear antigen (PCNA), a protein involved in eukaryotic cell replication, and inhibits PCNA activity. PCNA only exists in proliferating cells and tumor cells, and p21waf1 binds to PCNA, and the complex formed prevents the extension of DNA replication and affects the progression of the cell cycle. During DNA replication, PCNA and replication factor C (RFC) recognize primer-template primers to promote polymerase δ (polδ) loading, and PCNA can also bind to polymerase ε (polε). The PCNA-RFC-polδ complex slides the DNA in the loop it forms, allowing the DNA leader strand to be continuously synthesized, accelerating the progress of the DNA replication elongation phase. Direct binding of P21waf1 to PCNA results in rapid dissociation of the PCNA-RFC-polδ complex from the DNA replication fork, attenuating the initiation of PCNA cell proliferation, thereby preventing DNA replication synthesis. In addition, p53 protein can directly interact with PCNA, inhibit DNA replication and prevent cell division.

Cell cycle G2 pause

In the S phase of the cell cycle, p21waf1 disappeared in the nucleus, and in the late phase of the G2 phase, p21waf1 re-entered the nucleus and stayed for a short time. Similar to p53 blocking the G1 arrest by a non-transcriptional mechanism, p21waf1 also binds to the cyclin A and B complexes. P21waf1 prevents the protein substrate cdk2 from being activated by CAK, or directly inhibits cdk2 activity, preventing cells from passing through the checkpoint and interfering with cell cycle progression. P21waf1 also forms a complex with cdk2/cyclinA, and the p21waf1-cdk2/cyclinA complex blocks the interaction of the substrate with cdk2/cyclinA. In addition, when cdk2 forms a complex with p21waf1, the activation of cdk2 positively regulates the cdk1/cyclin B complex is weakened, making it impossible for cells to enter the mitosis phase.

P53 and apoptosis

P53 can induce tumor cell growth inhibition and promote apoptosis through two mechanisms. On the one hand, as a transcription factor, p53 induces transcription-induced apoptosis such as Bax, Bcl-2, and p53-induced apoptosis-inducing proteins in the nucleus. Bax is an essential signal for initiating apoptosis. The p53 binding site is present on the Bax promoter. The p53 recognition binding site acts directly on the Bax gene. p53 induces apoptosis by inducing Bax transcription.

Another mechanism by which P53 promotes apoptosis is anchoring to the mitochondria directly in the cytoplasm, inducing mitochondria-dependent apoptosis. Studies have shown that p53 mitochondrial anchoring mainly acts through the E3 ligase murine double minute 2 (mdm2). As an important tumor suppressor gene, P53 can regulate the occurrence and development of tumor through various pathways. In the interaction with p53, mdm2 gene mediates the degradation of p53, inhibits the transcriptional activation of p53, and down-regulates its tumor growth inhibitory activity. On the other hand, mdm2 induced by p53 can stabilize the function of p53 protein.

Promote cell self-phagocytosis

The cell-regulated autophagy modulator (DRAM) is mainly involved in the autophagy of cells. The DRAM contains a p53-binding sequence and is a newly discovered p53 downstream target gene. In the absence of cell nutrition, DRAM can activate the cell's self-phagocytic function and decompose long-acting proteins to stabilize the cell's morphology and maintain its basic life. DRAM induces apoptosis when cells are stimulated. The study found that when p53 is deleted and only DRAM is expressed, the killing effect of DRAM on cells is only 2% to 4% of the original killing effect, and when DRAM and p53 are co-transfected, the killing ability is greatly improved. Therefore, it is concluded that the role of DRAM in inducing programmed cell death is dependent on p53, which can induce autophagic death by p53.

Angiogenesis inhibition

After the tumor develops to a certain extent, pro-angiogenic factors can be formed through the autocrine pathway, which promotes neovascularization and is conducive to rapid tumor growth. This phenomenon is caused by the expression level of thrombospondin-1 (TSP-1) gene. The result of the decline. TSP-1 is a potent inhibitor of angiogenesis. p53 up-regulates TSP-1 gene expression, activates the endogenous TSP-1 gene, and positively regulates the promoter sequence of TSP-1. The study also found that p53 can activate a (II) collagen prolyl-4-hydroxylase (a2PH) transcription, resulting in the synthesis and secretion of full-length collagen, and the production of anti-angiomotin, induced protein hydrolysis at the substrate level, increased Synthesis and decomposition of collagen-derived anti-angiogenic collagen. P53 inhibits tumor angiogenesis by inhibiting the expression of angiogenic genes by stimulation. At the same time, studies have shown that there is a significant correlation between p53 and vascular endothelial growth factor (VEGF). After p53 mutation, it can up-regulate VEGF and increase the number of microvessels to promote tumor angiogenesis.

DNA repair

P53 protein plays an important role in DNA repair process, mainly in the case of DNA damage, p53 protein prevents DNA replication, and gains time for DNA repair. If repair fails, p53 protein activates apoptosis-inducing mechanism and promotes programmed cell death. To maintain the stability of the body. P53 can also interact with DNA repair factors such as RPA, PCNA and other genes directly involved in DNA damage repair process. At the same time, p53 can interact with the components of Nucleotide Excision Repair (NER) to make NER also aggregate here for nucleotide repair.

P53 has multiple abilities to interact with DNA, allowing p53 to be directly involved in DNA repair, such as binding to DNA repair factors, or directly involved in DNA repair through p53 protein-protein interactions, which bind p53 to damaged DNA. It is of great significance. Under the action of DNA damage factors, the damaged DNA is detected at the C-terminus of p53, and binds to form a p53-DNA complex. P53 binds to damaged DNA and acts as a transcription factor, binds to sequence-specific DNA, and participates in and enhances DNA repair by trans-activating target genes.

Inhibition of tumor cell movement

Cell migration depends on the formation of filopodia and the integrity of the extracellular matrix, and the cytoskeleton and its binding proteins are the material basis of this process. P53 protein negatively regulates cell spreading and fibronectin formation. The Ras (P21) protein is located inside the cell membrane and plays an important role in transmitting cell growth and differentiation signals. Ras regulates the role of Rasomolog gene fami ly member A (RhoA). On the one hand, Ras can anchor the membrane of RhoA; on the other hand, Ras promotes tyrosine phosphorylation of p190Rho GTPase activating protein (Rho GAP), which promotes the hydrolysis of RhoA-GTP to inactive RhoA-GDP, resulting in decreased RhoA activity. P53 is an essential factor for Ras to promote Rho GAP phosphorylation, independent of membrane anchoring of RhoA. After P53 deletion, the phosphorylation of p190Rho GAP is reduced, which reduces the loading of RhoA GTP, which greatly promotes the cell's ability to move.

TAT

Human immunodeficiency virus type I (HIV-I) is the causative agent of acquired immunodeficiency syndrome (AIDS). Tat is a short peptide rich in basic amino acids encoded by HIV-I, and its sequence is YGRKKRRQRRR (SEQ ID NO.: 5, positions 405-416), which is one of the six regulatory proteins it encodes. The regulatory protein, and the core region of the Tat transduction domain is composed of these 11 amino acid residues. As a newly discovered protein transduction domain, Tat protein can efficiently mediate DNA, peptide, protein and other molecules covalently linked to it into almost all tissues and cells, and even pass the blood-brain barrier, and the transduction efficiency is high. It has almost no damage to cells and maintains the biological activity of the protein. The Tat fusion protein system is considered to be a promising and efficient delivery vehicle with broad application prospects in basic medical research and clinical treatment.

Transduction of Tat protein

Tat protein can guide a variety of peptides and proteins into almost all target cells, which is the transduction of Tat protein, also known as internalization. The transduction of Tat protein depends mainly on the concentration of the polypeptide or protein, and is different from the general channel, receptor, and endocytosis mode of entry. Because of this function, Tat can be used as a carrier for mediating foreign proteins through cell membranes, and is therefore receiving increasing attention.

Tat protein mediates foreign matter into cells by cavern pathway

Caveolae is a small depression on the cell membrane, about 50-70 nm. Eguchi performed a bacteriomycin test on Tat-mediated exogenous gene transduction, which inhibited the entry of foreign substances into cells via a cavelet pathway, and found that Tat-mediated foreign protein transduction was inhibited. Therefore, it is concluded that Tat enters the cell in a small nest by destroying the plasma membrane. This method is simple to operate, free from external factors such as temperature, and is not affected by other internal environment, does not produce toxicity, and can be directly applied to cells in the body. The induction effect of this method is significantly higher than other transfection methods, the speed is fast, the transduction efficiency is high, and the biological activity of the protein is retained during the transduction process. Using another drug, fillipin, which inhibits the caveola pathway, redistributes a small fraction of caveolin-1 on the cell surface, affecting the cavelet pathway, and found that Tat-mediated foreign protein delivery is inhibited, proving Tat Protein is transmembrane through the cavern pathway.

Preparation of fusion protein

The development of biotechnology provides an effective way to produce recombinant human lysozyme, a typical method for producing anti-tumor fusion proteins, including steps:

(a) cultivating the host cell of the fourth aspect of the invention under conditions suitable for expression, thereby expressing the anti-tumor fusion protein of the first aspect of the invention;

(b) isolating and purifying the anti-tumor fusion protein expressed in the step (a).

In another preferred embodiment, the host cell is a bacterium, preferably E. coli.

In another preferred embodiment, the method for preparing GnRH+PII+P53 provided by the present invention includes the following aspects:

1) Based on the amino acid sequence of GnRH+PII+P53, the gene encoding GnRH+PII+P53 was synthesized by sequence optimization and codon degeneracy, and the high expression of this gene in E. coli was obtained.

2) Add the SUMO of the recognition site 98AA of ULP1 to the GnRH+PII+P53 coding sequence on the basis of 1) to ensure that the fusion protein can obtain the same theoretical design as GnRH+PII+P53 after ULP1 enzymatic hydrolysis. Terminal residue sequence;

3) On the basis of 2), a sequence which is favorable for expression and purification was added, and finally cloned into expression vector pET21 by NdeI and Hind III to obtain expression construct pET21-6xhis-SUMO-GnRH+PII+P53, named pET220 -GnRH+PII+P53;

4) The expression vector pET220-GnRH+PII+P53 was transferred into E. coli strain BL21(DE3)RP plus to obtain an engineering strain;

5) The engineered strain expresses the fusion protein 6xhis-SUMO-GnRH+PII+P53 under the induction of IPTG, and the fusion protein is expressed in the form of inclusion bodies;

6) The fusion expression of 6xhis-SUMO-GnRH+PII+P53 is obtained by the steps of lysis, inclusion body dissolution, metal chelation purification and the like;

7) the fusion protein is renatured by dropwise addition to the renaturation fluid;

8) The fused protein fusion product is subjected to dialysis, enzyme digestion, affinity chromatography, etc. to obtain crude GnRH+PII+P53;

9) GnRH+PII+P53 crude product was purified by molecular sieve, and finally GnRH+PII+P53 with purity greater than 95% was obtained, which showed a single band on SDS-PAGE electrophoresis and tetramer protein on molecular sieve;

10) The pure GnRH+PII+P53 obtained in step 9) was tested for biological activity by ISHIKAWA cells, and >95% of the cells disintegrated through the apoptotic pathway at a final protein concentration of 0.5 μg/ml or more.

The main advantages of the invention include:

(a) The fusion protein of the present invention is not easily degraded, and a tetramer can be formed naturally and efficiently.

(b) The fusion protein (tetramer) of the present invention can efficiently enter target cells and rapidly enter the nuclear region to induce apoptosis of the mutant cells (such as tumor cells).

(c) The fusion protein of the present invention has no toxicity in animal experiments due to the use of P53.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

Example 1

Optimization design and whole gene synthesis of the GnRH+PII+P53 gene sequence encoding the present invention

According to the designed amino acid sequence of GnRH+PII+P53, through gene software analysis, the rare codon and the secondary structure of the gene, as well as the use of synonymous codon, were fully considered, and finally the preferred GnRH+PII+P53 encoding nucleic acid sequence was obtained. As shown in SEQ ID NO.: 1, the specific nucleic acid sequence is:

The GnRH+PII+P53 protein sequence is shown in SEQ ID NO.: 2, and the specific protein sequence is:

The restriction endonuclease NdeI site and the sequence encoding the purified and purified sequence and the ulp1 recognition sequence sumo were added to the N-terminus of the optimized sequence, and the restriction endonuclease Hind III site was added at the C-terminus. As shown in SEQ ID NO.: 3, the specific nucleic acid sequence is:

The sequence was synthesized by commercial gene synthesis company Shanghai Shenggong Bioengineering Co., Ltd. and cloned into PUC19 vector. After sequencing, it was named as PUC19-GnRH+PII+P53.

Example 2

Construction of vector expressing GnRH+PII+P53

The preferred expression vector for expression of GnRH+PII+P53 in E. coli was PET21, and Pet21 and PUC19-GnRH+PII+P53 were digested with restriction endonucleases Nde I and Hind III, and the digested products were agarose-condensed. After gel electrophoresis separation, the gel was recovered, and then ligated with T4 ligase, and the ligated product was transformed into Escherichia coli DH5α. A positive clone was picked and the plasmid was extracted, and after sequencing, it was named pET220-GnRH+PII+P53.

The pET220-GnRH+PII+P53 protein sequence is shown in SEQ ID NO.: 4, and the specific protein sequence is:

Example 3

Construction of engineered bacteria expressing GnRH+PII+P53 protein and its induced expression analysis

The plasmid pET220-GnRH+PII+P53 was transformed into E. coli host BL21 Codon Plus (DE3) RP. After centrifugation, the bacteria were coated on LB plates containing 100 μg/ml ampicillin and cultured overnight at 37 ° C on LB plates. Monoclonal culture was carried out and identified by colony PCR to obtain monoclonal transformant pET220-GnRH+PII+P53/BL21Codon Plus(DE3)RP inoculated with monoclonal transformant pET220-GnRH+PII+P53/BL21Codon Plus(DE3)RP. Incubate in 3 ml of LB medium containing 100 μg/ml ampicillin at 37 ° C overnight; inoculate E. coli in 2% inoculation amount into fresh LB medium and incubate at 37 ° C, when the bacteria density is OD600 When the ratio was between 0.6 and 1.0, the expression of the fusion protein was induced by adding IPTG at a final concentration of 0.5 mM, and after continuing to culture for 3.5 hours, the cells were collected. Protein expression was analyzed by SDS-PAGE.

The results are shown in Figure 1. The fusion protein is expressed in the form of inclusion bodies, and the expression of the fusion protein accounts for about 25% of the total bacterial protein.

Example 4

Fermentation, purification and purification of GnRH+PII+P53 protein

1. Fermentation production

pET220-GnRH+PII+P53/BL21Codon Plus(DE3)RP was inoculated in LB medium and cultured overnight at 37 °C. The next day, 2% of the inoculum was used to access fresh TB (glycerol 5 g/L, peptone 12 g). / liter, yeast extract 24 g / liter, K2HPO4 12.54 g / liter, KH2PO42.31 g / liter) in the fermentation liquid culture medium, cultured at 37 ° C until the bacterial OD600 value reached 1, adding a final concentration of 0.5 mM IPTG induced fusion protein Expression, the cells were collected after 4 hours of incubation.

2, inclusion body acquisition

The fusion protein was expressed in the form of inclusion bodies. In order to obtain inclusion bodies, the cells were resuspended in a ratio of 1:10 with PBS, and the homogenate pressure was set to 750 Pa, homogenized twice, and the precipitated fraction was collected by centrifugation at 15,000 g; Part of the inclusion body protein is in the precipitated fraction of the collection.

3. Dissolution and purification of fusion protein

The inclusion bodies were fully dissolved in a ratio of 1:20 with solution 1 (20 Mm Tris-HCL, 500 mM NaCl, 20 mM Imidazole, 8 M Urea 20 mM 2-Mercaptoethanol pH 8.0), centrifuged at 15000 g for 20 minutes under centrifugation, and the supernatant fraction was taken. Further purification

The supernatant fraction was purified by denaturing conditions by NI ²⁺ metal chelating chromatography. The fusion protein was placed in a denaturation solution containing 8 M Urea, and the fusion protein was treated with DTT at a final concentration of 5 mM, and stirred at room temperature overnight.

4. Refolding of denatured proteins (protein renaturation)

The treated denatured protein was added dropwise to the reconstitution solution (reconstitution solution was 100 mM Tris-HCL, 500 mM NaCl, 0.5 M Arginine, 1% triton X-100, 10% glycerol, 1 mM EDTA, 1 mM GSH, 0.5 mM). GSSG pH 8.0) After the addition is completed, place in the refrigerator at 4 ° C for 48-72 hours, fully renatured.

5. Recovery of protein after renaturation

The fully refolded protein solution was placed in a dialysis bag and dialyzed against a buffer (20 Mm Tris. CL, Ph 8.5) for 4 hours each time for a total of 3 times of dialysis; after dialysis, the target protein was recovered by an anion column Q.

6, fusion protein intestinal ULP1 digestion

The ULP1 enzyme of 20IU/mg fusion protein was added to the fusion protein recovered by anion column Q, and stirred slowly by adding a magnetic stirrer to ensure sufficient digestion. The enzyme digestion conditions were: 4 ° C, 16-24 hours.

7. Purification after digestion

The digested fusion protein solution is mainly composed of two components: fusion expression tag protein and GnRH+PII+P53, and a small amount of undigested intact fusion protein, wherein the fusion expression tag protein and the intact fusion protein both contain a 6xHis purification tag. The two parts of the protein can be combined by NI ²⁺ metal chelating chromatography, and GnRH+PII+P53 cannot be bound to the NI ²⁺ metal chelating column and exists in the flow-through portion.

8. Heparin affinity chromatography of GnRH+PII+P53, molecular sieve refining

The column was filled with GnRH+PII+P53 in the flow-through portion (step 7) by affinity chromatography packed with heparin affinity packing purchased from GE Healthcare, and the concentrated GnRH+PII+P53 was refined by SUPERDEX 200. The protein solution is sterile filtered and used for subsequent experiments.

Example 5

Killing effect of GnRH+PII+P53 on different tumor cells

Each of the cells in Table 2 below was treated with the GnRH+PII+P53 fusion protein prepared in Example 4, and the IC50 value was determined.

The results are shown in Table 2. GnRH+PII+P53 has no killing effect on normal cells with IC50>100μg/ml, and GnRH+PII+P53 has good killing effect on various tumor cells. The IC50 is less than 5μg/ml.

Table 2 Killing effect of GnRH+PII+P53 on different tumor cells

Cell Chinese name	Cell line	IC50 (μg/ml)
Vascular endothelial cells (normal cells)	HAoSMC	>100
Vagina (normal cells)	VK2/E6E7	>100
Human lung (normal cell)	WI-38	>100
Human embryonic kidney (normal cell)		>100
African colobus monkey kidney (normal cell)	Vero	>100
Esophageal cancer	KYSE-150	2.6068
Breast cancer	MDA-MB-231	4.6218
Breast cancer	CAL120	1.01376
Breast cancer	HCCC1954	4.9032
Ovarian cancer	SK-OV-3	23.212
Ovarian cancer	SW626	3.164
Ovarian cancer	A2780	1.8671
Cervical cancer	HeLa	4.9189
Cervical cancer	HeLa229	3.5206
Colorectal cancer	SW480	2.373
Colorectal cancer	NCI-H716	0.5262
Colorectal cancer	COLO205	0.7427
Liver cancer	HuCCT1	3.8609
Liver cancer	HepG2	2.7328
Endometrial cancer	Ishikawa	0.3544
Endometrial cancer	HEC-1B	0.152
Uterine leiomyoma	SK-UT-1	10.562
Uterine sarcoma	MES-SA	2.464
Choriocarcinoma	JEG-3	0.5463
Kidney cancer	A498	0.938
Adrenal cortical carcinoma	SW13	0.2038

Squamous cell carcinoma	NCI-H520	0.9105
Lung cancer	A549	5.482
Bladder Cancer	5637	3.863
Prostate cancer	DU145	2.4673
Prostate cancer	RWPE-1	0.722
Tongue cancer	CAL-27	0.1508
Tongue cancer	SCC-4	1.3657
Tongue cancer	OSC-19	0.7453
Tongue cancer	SCC-25	0.3807
Tongue cancer	SCC-15	0.8436

Example 6

Determination of the killing effect of GnRH+PII+P53 on Ishikawa cells

The GnRH+PII+P53 fusion protein Slg001 prepared in Example 4 was used as an example to demonstrate its killing effect on Ishikawa cells. The detection results are shown in Figure 2. The high concentration of cells is lethal, and there are no normal cells. Particle fragments. details as follows:

After 72 hours of treatment with Igkawa cells Slg001, the medium in the 20μg/ml Slg001 treated plate was red, and a large number of granular cell debris were observed by microscopic observation. The medium in the 10μg/ml Slg001 treated plate was light red, microscopic observation Some cells and cell debris were observed, and the cells treated with low concentration of Slg001 were not significantly different from the control group;

As the treatment time was extended to 8 days, cells treated with 0.4-20 μg/ml Slg001 had different degrees of cell debris in different control cells. The blank control medium was yellow under 5% CO ₂ and the pH was 6.0, while the cells treated with 20 μg/ml Slg001 were purple-red after 8 days and the pH was around 8.0.

Example 7

Effectiveness of GnRH+PII+P53 in intravenous administration of tumor-bearing mice inoculated with tongue squamous cell carcinoma CAL-27

The GnRH+PII+P53 fusion protein prepared in Example 4 was intravenously administered to three groups of CAL-27 tumor-bearing mice, and administered every other day for five times. The doses were 250 μg/kg, 500 μg/kg, and 1000 μg, respectively. /kg, 2000μg/kg, 4000μg/kg, observe whether the tumor-bearing mice have toxic reaction and whether the drug is effective. As a result, no toxic reaction was observed, and the tumor was not enlarged in the first 4 needles, and the tumor inhibition rate was 72% after the fifth needle.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (10)

1. An anti-tumor fusion protein, characterized in that the fusion protein has the structure of formula I:

   D-A-B-C-E (I)

   among them,

   A is a GnRH protein element;

   B is the transmembrane transport region of the PEA protein or is absent;

   C is a P53 protein element;

   E is a TAT protein element or is absent;

   D is an optional signal peptide and/or leader peptide sequence;

   Also, B and E are not the same at the same time;

   Here, "-" means a peptide bond connecting the above elements.
2. The fusion protein according to claim 1, wherein said fusion protein has the structure of formula II:

   D-A-B-C (II)

   among them,

   A is a GnRH protein element;

   B is a transmembrane transport region of PEA protein;

   C is a P53 protein element;

   D is an optional signal peptide and/or leader peptide sequence;

   "-" indicates a peptide bond connecting the above elements.
3. The fusion protein according to claim 1, wherein said GnRH protein comprises a type II GnRH protein and a type I GnRH protein.
4. The fusion protein according to claim 1, wherein the fusion protein is a recombinant protein expressed by bacteria, preferably E. coli.
5. An isolated polynucleotide, characterized in that the polynucleotide encodes the fusion protein of claim 1.
6. A vector comprising the polynucleotide of claim 5.
7. A host cell comprising the vector of claim 6 or a polynucleotide in which the polynucleotide of claim 5 is integrated.
8. A method for producing an anti-tumor fusion protein, comprising the steps of:

   (a) cultivating the host cell of claim 7 under conditions suitable for expression, thereby expressing the fusion protein of claim 1;

   (b) isolating and purifying the anti-tumor fusion protein expressed in the step (a).
9. A pharmaceutical composition comprising the fusion protein of claim 1 and a pharmaceutically acceptable carrier or excipient.
10. Use of an anti-tumor fusion protein according to claim 1, which is for use in the preparation of a medicament for treating or preventing a tumor.

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