A fusion protein for inhibiting angiogenesis

Abstract

The application discloses a fusion protein for inhibiting angiogenesis, and belongs to the field of genetic engineering pharmacy, and particularly relates to a fusion protein capable of serving as an angiogenesis inhibitor. The fusion protein has a long-acting effect of inhibiting angiogenesis. The technical scheme disclosed by the application improves the anti-angiogenic activity of the angiogenesis endothelial inhibitor and the inhibiting effect on melanoma, prolongs the half-life of the angiogenesis endothelial inhibitor, and further enhances the stability of the angiogenesis endothelial inhibitor.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and more specifically, relates to a fusion protein with angiogenesis-inhibiting activity, which can be applied in the field of anti-tumor drugs. Background Technology

[0002] Malignant tumors are one of the major diseases that seriously affect human health and threaten human life, constituting the top three causes of death in all countries of the world today, along with cardiovascular and cerebrovascular diseases and accidents. With the continuous development of society and economy, the incidence of tumors is rising year by year. Therefore, the World Health Organization and health departments of governments around the world have made conquering cancer a top priority.

[0003] Current treatments for tumors include physical therapy (radiotherapy, surgical removal) and chemotherapy (anti-tumor drugs). The former causes significant damage to the patient's body, while the latter often results in severe side effects, including nausea, vomiting, and hair loss, and patients are prone to developing drug resistance. None of these methods can completely cure tumors because tumor cells often exhibit invasiveness and metastasis, making treatment difficult.

[0004] The growth and metastasis of primary tumors depend on the formation of new blood vessels. Tumor blood vessels not only provide nutrition for tumor growth but also mediate tumor cell metastasis, playing a crucial role in the entire growth stage of tumors. If the formation of new blood vessels in tumors can be inhibited, it is possible to block both the nutrient supply channels for tumor cells and the channels for tumor cell metastasis, thus overcoming the disadvantage of tumors being difficult to treat due to their metastatic nature. Therefore, therapies targeting tumor angiogenesis have received much attention in recent years and have shown promising application prospects.

[0005] Studies have found that benign tumors have sparse angiogenesis and slow blood vessel growth, while most malignant tumors have dense angiogenesis and rapid blood vessel growth, indicating that the number of tumor blood vessels is closely related to the degree of tumor development. The formation of new blood vessels mainly depends on the proliferation, migration, and lumen formation of vascular endothelial cells. This is the main process of angiogenesis, a result of the coordinated interaction of pro-angiogenic and anti-angiogenic factors in the body. The specific process of tumor angiogenesis is as follows: ① Tumor cells release pro-angiogenic factors (such as VEGF, bFGF, etc.), activating pro-angiogenic factor receptors on vascular endothelial cells, activating endothelial cells, and increasing vascular permeability; ② Activated endothelial cells release proteases, degrading the basement membrane; ③ Endothelial cells proliferate and migrate, forming the surrounding matrix and budding structures; ④ The budding structures further expand into a ring shape, eventually forming a vascular lumen; ⑤ The vascular lumen matures and stabilizes, forming new vascular branches. The entire angiogenesis process is highly ordered. Based on the process of tumor angiogenesis, Judah Folkman first proposed the importance of angiogenesis for tumor growth and metastasis in 1971. To date, the "tumor angiogenesis theory" has been continuously confirmed and enriched. Research has found that tumor growth requires large amounts of oxygen and nutrients, and blood vessels are the channels that provide these nutrients to tumor cells. Furthermore, tumor blood vessels can transport harmful metabolic substances from tumor cells, stimulating tumor growth. When a tumor grows to a certain stage, it will metastasize through the connected blood vessels. Based on this process, the "anti-angiogenesis theory" was proposed, which aims to inhibit tumor growth and metastasis by inhibiting angiogenesis. Tumor invasion and metastasis are the main challenges in tumor treatment, and inhibiting tumor angiogenesis can effectively inhibit the transport of nutrients needed for tumor growth and the channels of metastasis. Therefore, targeting tumor angiogenesis as a therapeutic target to inhibit tumor growth is currently a major research direction. There are many drugs available to inhibit tumor angiogenesis, including drugs that inhibit basement membrane degradation, drugs that inhibit endothelial cells (drugs that directly inhibit endothelial cells and drugs that inhibit specific integrins on vascular endothelial cells), and drugs targeting angiogenesis factors. Drugs that inhibit endothelial cells fall into two categories: one is drugs that directly inhibit endothelial cell growth, such as endostatin; the other is drugs that inhibit specific integrins on endothelial cells. These drugs can act on different ligands of integrins, block subsequent pathways opened by integrins, inhibit endothelial cell proliferation and migration, and inhibit tumor angiogenesis.Currently, the most developed drugs target angiogenesis factor (VEGF). These drugs can inhibit VEGF receptor signaling, block the activation of subsequent signaling pathways and the secretion of VEGF, thereby achieving the goal of inhibiting tumors. Currently, drugs targeting VEGF and its receptor are developing the fastest, such as bevacizumab and endostatin. However, because tumor angiogenesis is the result of the coordination of multiple factors, its formation process is complex and involves many factors and signaling pathways. Therefore, single-target drugs cannot completely inhibit tumor growth and metastasis, and often fail to achieve the expected results in clinical practice. Therefore, multi-target and multi-pathway inhibition of angiogenesis has become one of the research directions.

[0006] Compared with traditional anticancer drugs, drugs that inhibit tumor angiogenesis have the following advantages: ① The growth and metastasis of solid tumors depend on tumor blood vessels, so drugs that target angiogenesis have a broad spectrum and can act on a variety of solid tumors; ② Some drugs that inhibit tumor angiogenesis act on endothelial cells, which have the advantage of a low mutation rate compared with cancer cells, resulting in a low probability of the body developing drug resistance; ③ The drugs can be transported through the bloodstream, directly reaching the target site, with a rapid onset of action and fewer toxic side effects.

[0007] In angiogenesis, endothelial cell adhesion molecules are crucial substances that bind various cellular components together, accelerating the formation and stabilization of new blood vessels. Among these, the integrin family, a type of endothelial cell adhesion molecule, are transmembrane proteins that mediate cell adhesion to the extracellular matrix and play a vital role in tumor angiogenesis and metastasis. Integrins are composed of α and β subunits; currently, 18 α subunits and 8 β subunits have been reported, resulting in 24 different integrins. Integrins regulate bidirectional signaling pathways in cells, recognizing and binding to extracellular ligands, and opening intracellular signaling pathways to mediate the adhesion and migration of endothelial cells and tumor cells. For example, integrins can participate in the MAPK signaling pathway, regulating VEGF expression and promoting VEGF binding to its receptor. Currently, αvβ3 and αvβ5 are two of the most studied integrins, and there are many reports on their involvement in tumor progression. On the one hand, integrins can regulate the expression of matrix metalloproteinases in tumor cells, accelerate the degradation of the extracellular matrix, and promote angiogenesis. On the other hand, they can promote the secretion of adhesion molecules by the tumor itself, through which tumor cells can spread and metastasize. Therefore, integrins play an important role in tumor angiogenesis and tumor cell growth and metastasis, and integrins have become one of the important targets for tumor treatment.

[0008] αvβ3 is expressed in various cell types, but it is highly expressed only on the surface of neovascular endothelial cells and tumor cells, while its expression is low on the surface of quiescent endothelial cells and normal cells. Therefore, integrin αvβ3 can serve as an important target for anti-angiogenesis. αvβ3 can recognize the Arg-Gly-Asp (RGD) sequence in its ligand, thus RGD can act as a targeting peptide for integrins in vivo. However, the RGD sequence consists of only 3 amino acids and is easily degraded by proteases in vivo if it exists alone. ZL2005100403785 introduces a fusion peptide HM-3, an integrin inhibitor, with the following amino acid sequence: Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro-Gly-Gly-Gly-Gly-Arg-Gly-Asp. HM-3 consists of two parts: the endostatin active peptide ED (Ile-Val-Arg-Arg-Ala-Asp-Arg-Ala-Ala-Val-Pro) and the integrin ligand sequence (Arg-Gly-Asp), linked by a linker (Gly-Gly-Gly-Gly), with the structural formulas ED-L-RGD and RGD-L-ED. The endostatin active peptide ED is the 60th-70th amino acid fragment of endostatin. Studies have found that this fragment has anti-angiogenic activity in vitro, but its in vivo activity is low, possibly related to the short half-life of the small peptide. HM-3 combines these two sequences, and studies have shown that this fused small peptide has a good anti-angiogenic effect, retaining the anti-angiogenic activity of ED while also exhibiting the targeting effect of RGD. Studies have shown that HM-3 targets αvβ3 and α5β1, primarily αvβ3. Both in vivo and in vitro experiments have demonstrated that this small peptide effectively inhibits the migration and adhesion of vascular endothelial cells and suppresses tumor angiogenesis, exhibiting a good antitumor effect. Currently, HM-3 has begun clinical trials in my country (registration number: CTR20150368), with the proposed clinical use being daily intravenous infusion.

[0009] Daily injections not only cause significant suffering for patients but also increase treatment costs because HM-3 is a short peptide with a short half-life. To address this issue, patent 201110370529.9 discloses a method for polyethylene glycol modification of HM-3, resulting in HM-3 with a longer half-life. However, PEGylated small peptides are chemically synthesized products, inevitably suffering from product inhomogeneity and difficulty in strictly controlling product quality, posing challenges to clinical practice. In 2018, Beijing Saisheng Pharmaceutical disclosed a long-acting HM-3 technology that modifies HM-3 with fatty acids and maleimide groups, retaining its anti-tumor activity while extending its half-life. Studies have shown that polyethylene glycol modification, fatty acid modification, and maleimide group modification are all chemical modifications, complex in operation, prone to product inhomogeneity, and cannot be used for biological expression to reduce costs.

[0010] Given the problems arising from chemical modifications, researchers have sought superior drug modification methods to extend drug half-life. In recent years, fusion protein technology has matured, with Fc fusion protein drugs and HSA-related fusion protein technologies developing rapidly. HSA, in particular, has attracted significant attention due to its slight targeting specificity compared to Fc and its ability to achieve monomeric fusion. Studies have found that HSA-related fusion proteins, due to HSA's large molecular weight (being a long peptide), significantly increase the molecular weight of the original drug peptide when linked to a drug. This leads to low renal clearance efficiency for large-molecule drugs, thus reducing the body's clearance efficiency of the fusion protein drug and prolonging its half-life. Furthermore, HSA, being an endogenous molecule, can modify the structure of small-molecule peptide drugs when bound to them, weakening the body's immune rejection response to exogenous small-molecule peptides. This delays drug clearance and reduces cytotoxicity caused by exogenous small-molecule peptides.

[0011] Despite extensive research, only two HSA fusion protein drugs have been marketed to date. Undeniably, fusion proteins can extend the half-life of small molecule peptide drugs to some extent. However, unsatisfactorily, the efficacy of fusion proteins against specific tumors is not as good as using short peptides alone. Therefore, when using fusion proteins with target short peptides, increasing the expression efficiency and enhancing the efficacy of the fusion protein is key to improving its drug-likeness. Summary of the Invention

[0012] This invention constructs a fusion protein of angiogenic endostatin HM-3, which not only solves the problems of reduced efficacy and unsatisfactory expression levels caused by the HSA fusion strategy in existing technologies, but also improves the low activity of existing angiogenic endostatin. The inventors also unexpectedly discovered that although the small molecule peptide HM-3 has no inhibitory effect on the proliferation of melanoma cells B16F10 in vitro, the HM-3 fusion protein can significantly inhibit the proliferation of melanoma cells B16F10 in vitro. To solve the above problems, the technical solution adopted in this invention is as follows:

[0013] The present invention provides a fusion protein, the amino acid sequence of which is shown in SEQ ID NO:1, and the DNA sequence encoding the amino acid sequence is shown in SEQ ID NO:2.

[0014] The fusion protein was prepared by expression in yeast cells.

[0015] The yeast mentioned is *Pichia pastoris*.

[0016] The method for preparing the fusion protein includes the following steps:

[0017] ① DNA for synthesizing fusion proteins from the entire genome;

[0018] ② By connecting the target fragment to the vector using genetic engineering techniques, a recombinant yeast expression vector containing a DNA sequence encoding the fusion protein is obtained;

[0019] ③ The recombinant yeast expression vector described in step ② is transformed into competent Escherichia coli cells. After successful sequencing, the plasmid is transformed into a host expression system for expression to obtain the fusion protein. The host expression system is Pichia pastoris.

[0020] Another object of the present invention is to provide a recombinant expression vector containing a DNA sequence encoding the fusion protein.

[0021] Another object of the present invention is to provide a host expression system containing the recombinant expression vector.

[0022] Another object of the present invention is to provide the use of the fusion protein in the preparation of antitumor drugs.

[0023] Preferably, the fusion protein of the present invention is used to prepare anti-tumor drugs, especially in the preparation of drugs that inhibit solid tumor cells such as melanoma cells, liver cancer, lung cancer, pancreatic cancer cells, and cervical cancer cells.

[0024] The antitumor drug is available in dosage forms such as injections, capsules, tablets, pills, nasal sprays, or aerosols. Compared to existing technologies, the advantages of this invention are:

[0025] (1) This invention has discovered an angiogenic endostatin fusion protein with more significant tumor proliferation inhibitory activity;

[0026] (2) The present invention uses a yeast expression system, which can process proteins, most of which can form the correct higher-order structure. Moreover, the yeast expression protein has a high yield, high purity, and is easy to purify, thus increasing the host expression efficiency.

[0027] (3) In vitro experiments of the fusion protein of the present invention have demonstrated that the fusion protein has a selective inhibitory effect on the proliferation of tumor cells.

[0028] (4) In vitro experiments of the fusion protein of the present invention have shown that the fusion protein has little effect on normal cells.

[0029] (5) In vitro experiments of the fusion protein of the present invention have demonstrated that the fusion protein has an inhibitory effect on tumor cell migration. Attached Figure Description

[0030] Figure 1 pPinkα-HC vector spectrum

[0031] Figure 2 pPinkα-HC / Fusion protein gene map

[0032] Figure 3 SDS-PAGE electrophoresis was used to detect protein expression.

[0033] Figure 4 SDS-PAGE of the purified fusion protein

[0034] Figure 5 The fusion protein inhibits tumor cell proliferation; CK represents the blank control.

[0035] Figure 6 The fusion protein inhibits the proliferation of normal cells; CK represents the blank control.

[0036] Figure 7 Tanswell results for the fusion protein, CK represents the blank control. Detailed Implementation

[0037] Example 1

[0038] I. Construction of pPINKα-HC / fusion protein vector

[0039] ① A fusion protein gene fragment was synthesized from the whole genome, wherein the amino acid sequence of the fusion protein is shown in SEQ ID NO:1, and the DNA sequence encoding the amino acid sequence is shown in SEQ ID NO:2;

[0040] The pPINKα-HC plasmid (Invitrogen product) and fusion protein fragment were digested with KpnI and StUI. The pPINKα-HC (KpnI / StuI) vector and protein fragment were recovered via gel extraction. The recovered pPINKα-HC (KpnI / StuI) vector fragment and fusion protein gene fragment were then subjected to a recombination reaction to obtain a recombinant yeast expression vector containing the DNA sequence encoding the fusion protein. This vector was transformed into *E. coli* competent cells TOP10, plated on ampicillin-resistant LB plates, and incubated overnight at 37°C. Positive clones were screened. The obtained clones were sent to a company for sequencing.

[0041] ② The correctly sequenced pPINKα-HC / fusion protein plasmid DNA was recovered by Afl II restriction enzyme digestion to obtain a linearized recombinant plasmid fragment, which was transformed into Pichia pastoris. The transformed bacterial culture was then inoculated onto PAD plates and cultured at 30°C for 5-7 days. Positive clones were picked. The obtained positive clones were inoculated into BMGY liquid medium and cultured at 30°C for 48 hours. Then, they were transferred to BMMY medium to induce expression. After 96 hours, the culture was centrifuged at 8000 rpm for 30 minutes, and the supernatant was collected for SDS-PAGE electrophoresis to detect protein expression.

[0042] The results are as follows Figure 3 As shown, the molecular weight of the fusion protein is approximately 68 kDa, and it exhibits high expression levels and good purity.

[0043] Example 2: Purification of the fusion protein

[0044] Based on the properties of the fusion protein, DEAE Sepharose Fast Flow was first selected for purification. The experimental procedure is as follows:

[0045] ① Sample pretreatment: Dialyze the expression supernatant using 20 mM PB buffer; ② Column equilibration: Load the DEAE Sepharose fast flow purification packing into the purification column and equilibrate with 20 mM PB buffer; ③ Sample loading: Load an appropriate amount of sample, collect the flow-through sample, and name it LC; ④ Rinse the packing with an appropriate amount of 20 mM PB buffer, collect the flow-through, and name it W; ⑤ Elution: Elute with 0.1 M NaCl, 0.3 M NaCl, 0.5 M NaCl, and 1 M NaCl respectively, collect the eluents, and name them E1, E2, E3, and E4; ⑥ Wash the packing with 0.1 M NaOH, collect the flow-through, and name it N; ⑦ Rinse the packing with plenty of distilled water, and store the packing with 20% ethanol. Analyze the protein samples using SDS-PAGE.

[0046] Phenyl Sepharose Fast Flow was selected for subsequent purification, and the experimental procedure is as follows:

[0047] ① Sample pretreatment: The sample was the E1 eluent collected after DEAE Sepharose Fast Flow purification. Ammonium sulfate was added to make the sample contain 1.5M (NH4)2SO4.

[0048] ② Column equilibration: Phenyl Sepharose Fast Flow purification packing material was loaded into the purification column and equilibrated with 1.5M (NH4)2SO4 + 0.1M NaCl buffer;

[0049] ③ Sample loading: Load an appropriate amount of sample, collect the flow-through sample, and name it LC;

[0050] ④ Rinse the packing material with an appropriate amount of 1.5M (NH4)2SO4 + 0.1M NaCl buffer, collect the flow-through liquid, and name it W;

[0051] ⑤ Elution: Elute with 1.0M (NH4)2SO4, 0.8M (NH4)2SO4, 0.6M (NH4)2SO4, 0.5M (NH4)2SO4, 0.3M (NH4)2SO4, 0.1M (NH4)2SO4, and H2O respectively, and collect the eluents, naming them E1, E2, E3, E4, E5, E6, and E7.

[0052] ⑥ Clean the packing material with 0.1M NaOH, collect the flow-through liquid, and name it N;

[0053] ⑦ Rinse the packing material with plenty of distilled water and store it in 20% ethanol. Analyze the protein samples using SDS-PAGE. Concentrate the protein samples in 10kDa concentration tubes and determine the protein concentration using the Lowry method.

[0054] The results are as follows Figure 4 As shown, a fusion protein with a purity >90% was obtained.

[0055] Example 3: Inhibitory effect of fusion protein on tumor cells

[0056] HM-3 was synthesized by Shanghai Jier Peptide Co., Ltd. (HM-3 amino acid sequence: IVRRADRAAVPGGGGRGD), with a purity of over 95%.

[0057] The MTT assay was used to detect the inhibitory effect of the fusion protein on the proliferation of B16F10 (mouse melanoma cells), SMMC-7721 (human hepatocellular carcinoma cells), A549 (human lung cancer cells), HeLa (human cervical cancer cells), and PANC-1 (human pancreatic cancer cells) tumor cells. Tumor cells were cultured at 37℃ and 5% CO2 until the cell density reached over 90%, then digested with trypsin and collected. The cells were resuspended in culture medium and counted under a microscope. The cell concentration was adjusted to 3.0 × 10⁻⁶ cells / year. 4 Cell suspension was seeded at 100 μL / mL into 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. The fusion protein was diluted with PBS to predetermined concentrations. After complete cell adhesion, 100 μL of each dilution was added to each well of the 96-well plate. The plate with the added fusion protein served as the treatment group, HM-3 as the control group, and an equal volume of PBS as the blank control group. The plates were incubated at 37°C in a 5% CO2 incubator for 48 hours. 15 μL of 5 mg / mL MTT was added to each well of the 96-well plate, and the plates were incubated for another 4 hours. The culture medium was removed, and 100 μL of DMSO was added to each well to dissolve the cells. The cells were then analyzed using a microplate reader at 490 nm, and the growth inhibition rate was calculated. The experiment was repeated three times. The experimental results are shown below. Figure 5 As shown.

[0058] Tumor cells were used in this experiment. Different concentrations of drugs were administered, and the absorbance was measured after 48 hours. The MTT assay showed that the small peptide HM-3 and protein HSA had no significant inhibitory effect on the proliferation of the five types of tumor cells, while the fusion protein had a selective inhibitory effect on the proliferation of tumor cells. Among them, the inhibitory effect on the proliferation of B16F10 cells was the most obvious, and it was concentration- and time-dependent.

[0059] Example 4: Inhibitory effect of fusion protein on normal cells

[0060] The MTT assay was used to detect the inhibitory effect of the fusion protein on the proliferation of normal cells, including BEAS-2B (normal human lung epithelial cells), H9c2 (rat cardiomyocytes), HIEC (normal human intestinal epithelial cells), and HL-7702 (normal human hepatocytes). Cells were cultured at 37°C and 5% CO2 until the cell density reached over 90%, then collected by trypsin digestion. The cells were resuspended in culture medium and counted under a microscope. The cell concentration was adjusted to 3.0 × 10⁻⁶ cells / year. 4Cell suspension was seeded at a rate of 100 μL / mL into 96-well plates and incubated overnight at 37°C in a 5% CO2 incubator. The fusion protein was diluted with PBS to the predetermined concentrations. After complete cell adhesion, 100 μL of each dilution was added to each well of the 96-well plate. The plate with the added fusion protein served as the treatment group, HM-3 as the control group, and an equal volume of PBS as the blank control group. The plates were incubated at 37°C in a 5% CO2 incubator for 48 hours. 15 μL of 5 mg / mL MTT was added to each well of the 96-well plate, and the plates were incubated for another 4 hours. The culture medium was removed, and 100 μL of LDMSO was added to each well to dissolve the cells. The cells were then analyzed using a microplate reader at 490 nm, and the growth inhibition rate was calculated. The experiment was repeated three times. The experimental results are as follows: Figure 6 As shown.

[0061] This experiment used human normal tissue cells and rat normal tissue cells. Different concentrations of drugs were administered, and the absorbance was measured after 48 hours. The MTT assay showed that the three drugs had significantly lower inhibitory effects on the proliferation of the four normal cell types than the tumor cells B16F10, indicating that the fusion protein has low toxicity to normal cells.

[0062] Example 5: Detection of the activity of fusion protein in inhibiting tumor cell migration using Transwell assay

[0063] B16F10 cells were cultured in 1640 medium containing 10% fetal bovine serum. When the confluence reached over 80% in an incubator at 37°C and 5% CO2, the Transwell assay was used to detect the inhibitory activity of the fusion protein on tumor cell migration. The specific procedure is as follows:

[0064] ① Cells cultured to the logarithmic growth phase were digested with trypsin containing 0.2% EDTA, collected, washed with PBS, diluted with serum-free 1640 medium, counted under a microscope, and the cell concentration was adjusted to 5 × 10⁻⁶ cells / year. 5 cells / mL;

[0065] ② The cells were seeded into the upper chamber of the Transwell at 100 μL / well, and the experimental solutions for each group were added to the upper chamber. The fusion protein was added as the drug administration group, and the small peptide HM-3 (4.5 μM) was added as the positive control group. An equal volume of physiological saline was added as the blank control group. 0.6 mL of 1640 cell culture medium containing 10% fetal bovine serum was added to the lower chamber to stimulate cell migration. The cells were incubated at 37°C for 48 h in 5% CO2.

[0066] ③ Discard the culture medium in the wells, fix with 4% paraformaldehyde at room temperature for 30 min, stain with 0.1% crystal violet at room temperature for 10 min, rinse with PBS 3 times, gently wipe away the unmigrated cells on the upper layer with a cotton swab, observe under a microscope and select four fields of view to take pictures and count, calculate the migration inhibition rate, and repeat the experiment 3 times.

[0067] According to literature reports, 4.5 μM HM-3 achieved the highest migration inhibition rate; therefore, we chose 4.5 μM HM-3 as a control. Transwell assay ( Figure 7 This study demonstrated that the inhibition rate of B16F10 cell migration increased with increasing dosage of the fusion protein drug. The inhibition rate reached 32.2% at 250 nM, while the migration inhibition rate of 4.5 μM HM-3 was 33.6%, comparable to the efficacy of the 250 nM HM-3-HSA fusion protein, proving that the fusion protein was more effective than the small peptide HM-3.

Claims

1. A fusion protein, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ ID NO:1, and the DNA sequence encoding the amino acid sequence is shown in SEQ ID NO:

2.

2. A method for preparing the fusion protein as described in claim 1, characterized in that, The method includes the following steps: ① DNA for synthesizing fusion proteins from the entire genome; ② By connecting the target fragment to the vector using genetic engineering techniques, a recombinant yeast expression vector containing a DNA sequence encoding the fusion protein is obtained; ③ The recombinant yeast expression vector described in step ② is transformed into competent Escherichia coli cells, the plasmid is extracted, and the plasmid is transformed into a Pichia pastoris expression system for expression to obtain the fusion protein.

3. A recombinant expression vector containing a DNA sequence encoding the fusion protein as described in claim 1.

4. A host expression system comprising the recombinant expression vector of claim 3.

5. The use of the fusion protein as described in claim 1 in the preparation of an anti-melanoma drug.

6. The use of the fusion protein as described in claim 5 in the preparation of an anti-melanoma drug, characterized in that... Anti-melanoma drugs are available in the form of injections, capsules, tablets, pills, nasal sprays, or aerosols.

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