Fusion protein containing mitochondrial pro-apoptotic protein and preparation method thereof

Abstract

The invention discloses a fusion protein containing mitochondrial pro-apoptotic protein and a preparation method thereof, and belongs to the technical field of biomedicine. The fusion protein is iRGD-MitoPen-Smac N55 (SEQ ID NO: 4), and a triple mechanism of'tumor targeting-cell / mitochondrial delivery-apoptosis activation 'is constructed by connecting an iRGD targeting peptide, a MitoPen bifunctional peptide and a Smac N55 active fragment in series. The preparation method comprises the steps of gene synthesis and optimization, vector construction, cell transfection expression and affinity purification, and the recombinant protein with the purity larger than or equal to 94% is obtained. The fusion protein can be specifically combined with alpha v integrin highly expressed by tumor cells and NRP-1, targeted delivery and mitochondrial apoptosis pathway activation are achieved, proliferation of tumor cells such as breast cancer and colon cancer is efficiently inhibited, in-vivo and in-vitro experiments prove that the anti-tumor effect of the fusion protein is remarkably superior to that of a Smac simulant LCL161, the fusion protein can be used for preparing anti-tumor drugs, and a new strategy is provided for tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a fusion protein containing mitochondrial pro-apoptotic proteins and a method for preparing the same. Background Technology

[0002] Malignant tumors pose a significant global health threat, with their incidence and mortality rates continuing to rise, seriously endangering human life and health. While traditional cancer treatments such as chemotherapy and radiotherapy can inhibit tumor progression to some extent, their lack of specific tumor cell recognition capabilities means they easily damage normal tissue cells while killing tumor cells, leading to severe systemic toxic side effects such as bone marrow suppression and gastrointestinal reactions. Furthermore, they easily induce tumor drug resistance, greatly limiting clinical treatment efficacy. Therefore, developing novel anti-tumor drugs that combine high targeting, strong killing power, and low toxicity has become a core research direction in the field of cancer treatment.

[0003] Mitochondria, as the cell's "energy factory" and a key organelle regulating apoptosis, are closely related to tumor development and progression when their function is abnormal. Smac (Second mitochondria-derived activator of caspase) protein, an important pro-apoptotic molecule released from mitochondria, has a mature active fragment (SmacN55) that binds with high affinity to members of the inhibitory apoptosis protein (IAP) family (such as XIAP, cIAP1, and cIAP2) via its N-terminal AVPI sequence. This releases the inhibitory effect of IAPs on caspase-3 / 7 / 9, thereby activating the intrinsic apoptosis pathway and inducing tumor cell apoptosis. This unique mechanism makes SmacN55 an important target for anti-tumor drug development, and related molecules based on the Smac protein are considered highly promising anti-tumor drug candidates.

[0004] However, natural SmacN55 faces numerous key technological bottlenecks in its clinical translation. On one hand, as a protein molecule, SmacN55 lacks tumor tissue targeting, is easily degraded by proteases in vivo, and struggles to penetrate the tumor vascular barrier and tumor cell membranes, resulting in extremely low accumulation efficiency at tumor sites and hindering its full efficacy. On the other hand, existing Smac mimics are mostly small molecule compounds. While they have solved some delivery problems, they suffer from insufficient mitochondrial targeting, limited IAP antagonistic activity, and weak penetration into the tumor microenvironment, causing their in vivo antitumor effects to fall far short of clinical expectations. Furthermore, single-functional antitumor molecules often struggle to overcome the multiple barriers created by the complex tumor microenvironment, failing to achieve the synergistic effect of "precise targeting - efficient delivery - potent killing," further limiting the clinical application prospects of Smac-related molecules.

[0005] To address these issues, researchers have attempted to modify the active Smac fragment by fusing functional modules such as targeting peptides and cell-penetrating peptides. However, existing fusion molecules still have several shortcomings: some targeting peptides exhibit low binding specificity to tumor cell surface receptors, leading to off-target effects; cell-penetrating peptides have limited intracellular delivery efficiency and lack specific targeting ability to mitochondria, making it difficult for the active Smac fragment to accurately reach its target site; and some fusion proteins have unreasonable structural designs, resulting in steric hindrance between functional modules, affecting the normal functioning of each module, or causing rapid inactivation in vivo due to insufficient stability. Therefore, how to integrate highly specific tumor-targeting modules, efficient intracellular / mitochondrial delivery modules, and potent pro-apoptotic modules through rational molecular design to construct multifunctional fusion proteins with synergistic effects, overcome existing technological bottlenecks, improve anti-tumor efficacy, and reduce toxic side effects has become a key issue that urgently needs to be addressed in the current research and development of Smac-related anti-tumor drugs. Summary of the Invention

[0006] To address the technical bottlenecks of existing Smac-related antitumor molecules, such as insufficient targeting, low intracellular and mitochondrial delivery efficiency, limited apoptosis activation ability, and difficulty in overcoming the tumor microenvironment barrier, this invention first provides an iRGD-MitoPen-SmacN55 fusion protein.

[0007] In some embodiments, the fusion protein is composed of functional modules linked together by flexible linker peptides. From the N-terminus to the C-terminus, the modules are iRGD targeting peptide (SEQ ID NO:1), MitoPen bifunctional peptide (SEQ ID NO:2), and SmacN55 active fragment (SEQ ID NO:3). The modules are connected by GGGGS flexible linker peptides, and a 6×His tag (SEQ ID NO:5) is further connected to the C-terminus. The overall amino acid sequence is shown in SEQ ID NO:4. Among them, the iRGD targeting peptide retains the dual function of "RGDmotif targeting + NRP-1-mediated penetration", the MitoPen peptide has both cell penetration and mitochondrial targeting capabilities, and the SmacN55 fragment can specifically antagonize IAP family proteins. The three work together to achieve precise tumor targeting.

[0008] This invention also provides a method for preparing the fusion protein, the method comprising the following steps: 1) Based on the human preferred codon table, optimizing the synthesis of the gene encoding the fusion protein (SEQ ID NO:6), which contains an NheI restriction site at the 5' end and an XhoI restriction site at the 3' end to avoid translational stagnation caused by rare codons; 2) Digesting the synthesized encoding gene and pCDNA3.1 (+) vector with NheI and XhoI respectively, and then ligating them overnight at 16°C using T4 DNA ligase at a 3:1 molar ratio to construct a recombinant expression vector, and verifying the sequence correctness by colony PCR and Sanger bidirectional sequencing; 3) Transfecting the recombinant expression vector into HEK293T cells, replacing the culture medium with fresh medium 6 h after transfection, and adding 10 mM sodium butyrate for 24 h after 48 h; 4) Collecting cells and supernatant, extracting total protein with RIPA lysis buffer, and then lysing using HisTrap FF. The protein was purified by affinity chromatography, and the main peak fraction was collected and concentrated using a 10 kDa molecular weight cutoff ultrafiltration tube. Then, it was dialyzed three times with PBS (pH 7.4) at 4°C to remove imidazole and high salt content, yielding the target fusion protein with a purity ≥ 94%.

[0009] Finally, this invention provides the application of the fusion protein, namely, its application in the preparation of antitumor drugs; In some embodiments, the tumor is a solid tumor that highly expresses αv integrin and NRP-1, including but not limited to breast cancer and colon cancer; the drug achieves therapeutic effects by degrading XIAP and cIAP1 proteins and activating the caspase-3 / 9-mediated endogenous apoptosis pathway, thereby inhibiting tumor cell proliferation and inducing tumor cell apoptosis.

[0010] Compared with the prior art, the present invention has at least the following beneficial effects: We have innovatively constructed a triple synergistic mechanism of "tumor targeting - cell / mitochondrial delivery - apoptosis activation". Through the precise connection of functional modules, we have solved the core defects of traditional Smac molecules, such as poor targeting and low delivery efficiency, and significantly improved anti-tumor specificity and efficacy. The fusion protein was codon-optimized and subjected to efficient expression and purification processes to obtain a recombinant protein with high purity (≥94%) and high stability. In vitro and in vivo experiments confirmed that its inhibition rate of breast cancer and colon cancer cell proliferation and apoptosis induction efficiency were significantly better than the preclinical Smac mimic LCL161, with an in vivo tumor inhibition rate of 67.4%. The preparation process is simple and controllable, the carriers and cell lines used are mature and readily available, and the affinity purification and dialysis steps are mild, which can preserve protein activity to the greatest extent and is suitable for large-scale production. Its mechanism of action is clear: it reduces off-target damage to normal cells by specifically binding to tumor cell surface receptors, targeting mitochondrial delivery, and activating apoptosis pathways, thus having a higher safety profile.

[0011] This invention is the first to synergistically integrate the originally designed MitoPen bifunctional peptide with the iRGD targeting peptide and the SmacN55 active fragment, achieving a highly efficient superposition of anti-tumor functions. This provides a novel and highly effective protein drug for the targeted therapy of solid tumors and has broad clinical application prospects. Attached Figure Description

[0012] Figure 1 SDS-PAGE electrophoresis image of the iRGD-MitoPen-SmacN55 fusion protein.

[0013] Figure 2 The inhibitory effect of the fusion protein on the proliferation of MDA-MB-231 and HCT116 cells was detected by CCK-8 assay.

[0014] Figure 3 Annexin V-FITC / PI double staining flow cytometry analysis of apoptosis rate.

[0015] Figure 4 Statistical analysis of TUNEL staining results in tumor tissues of tumor-bearing mice. Detailed Implementation

[0016] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0017] Example 1: Preparation of iRGD-MitoPen-SmacN55 fusion protein Linearized iRGD is a linear analogue of the classic cyclic iRGD (cCRGDKGPDC). It refers to the CRGDKGPDC sequence that exists in the form of a linear peptide chain after the intrachain disulfide bonds are removed. The core retains the dual function of "RGD targeting + NRP-1-mediated penetration". Its amino acid sequence is as follows: CRGDKGPDC (SEQ ID NO:1). MitoPen peptide (sequence: (R-FKFR)2-H6-RW-KK) is an original bifunctional short peptide with both high cell penetration and mitochondrial targeting capabilities. Its (R-FKFR)2 module responds to high mitochondrial membrane potential, achieving specific enrichment; H6 (hexahistidine) promotes endosome escape in the acidic tumor microenvironment; the RW motif enhances transmembrane insertion; and the C-terminal KK enhances stability and positive charge. Its amino acid sequence is as follows: RFKFRRFKFRHHHHHHRWKK (SEQ ID NO:2);

[0018] SmacN55 fusion protein is a recombinant protein constructed by fusing the mature active fragment of human Smac protein (amino acids 56–239, a total of 184 residues, often simply referred to as SmacN55) with other functional modules (such as transmembrane peptides, tumor-targeting domains, or Fc fragments). This fragment retains complete IAP binding capacity and can effectively antagonize XIAP and cIAP1 / 2, relieving their inhibition of caspase-3 / 7 / 9, thereby promoting tumor cell apoptosis. Its amino acid sequence is as follows: AVPIAQKSEPHSLSSEALMRRAVSLVTDSTSTFLSQTTYALIEAITEYTKAVYTLTSLYRQYTSLLGKMNSEEEDEVWQVIIGARAEMTSKHQEYLKLETTWMTAVGLSEMAAEAAYQTGADQASITARNHIQLVKLQVEEVHQLSRKAETKLAEAQIEELRQKTQEEGEERAESEQEAYLRED (SEQ ID NO:3);

[0019] The iRGD-MitoPen-SmacN55 fusion protein was designed based on a triple synergistic mechanism of "tumor targeting—cell / mitochondrial delivery—apoptosis activation". First, linear iRGD (CRGDKGPDC) specifically binds to αv integrins highly expressed in tumor blood vessels and on the cell surface through its RGD motif, and activates the Neuropilin-1 (NRP-1)-mediated tissue penetration pathway via its C-terminal domain, achieving selective tumor accumulation and transcellular transport. Subsequently, the MitoPen peptide (RFKFRRFKFRHHHHHHRWKK) performs a dual function: its (R-FKFR)2 module responds to the high membrane potential of tumor cell mitochondria, driving the fusion protein to target mitochondria; the H6 histidine cluster is protonated in the acidic microenvironment, promoting endosome escape; and the RW hydrophobic motif enhances transmembrane capacity, ensuring efficient intracellular delivery. Ultimately, SmacN55 (aa56–239) is released into the cytoplasm, where it binds to XIAP / cIAP1 / 2 with high affinity via the N-terminal AVPI sequence, relieving their inhibition of caspase-9 / 3 / 7 and strongly activating the mitochondrial apoptosis pathway. Its amino acid sequence is as follows:

[0020] CRGDKGPDCGGGGSGGGGSRFKFRRFKFRHHHHHHHRWKKGGGGSGGGGSAVPIAQKSEPHSLSSEALMRRAVSLVTDSTSTFLSQTTYALIEAITEYTKAVYTLTSLYRQYTSLLGKM NSEEEDEVWQVIIGARAEMTSKHQEYLKLETTWMTAVGLSEMAAEAAYQTGADQASITARNHIQLVKLQVEEVHQLSRKAETKLAEAQIEELRQKTQEEGEERAESEQEAYLRED (SEQ ID NO:4); The amino acid sequence of the target fusion protein (SEQ ID NO:4) was submitted to Nanjing Genscript Biotech Co., Ltd. The full sequence was optimized using a human-preferred codon table to avoid translational stagnation caused by rare codons (such as AGG / AGA arginine, CTA leucine, etc.). A 6×His tag (SEQ ID NO:5) was appended immediately after the SmacN55 coding region at the C-terminus to enhance tag accessibility and reduce steric hindrance, ultimately yielding a full-length 729 bp coding DNA sequence (SEQ ID NO:6). This sequence contains an NheI (GCTAGC) restriction site at the 5' end and an XhoI (CTCGAG) restriction site at the 3' end, facilitating subsequent cloning.

[0021] CRGDKGPDCGGGGSGGGGSRFKFRRFKFRHHHHHHRWKKGGGGSGGGGSAVPIAQKSEPHSLSSEALMRRAVSLVTDSTSTFLSQTTYALIEAITEYTKAVYTLTSLYRQYTSLLGKMNSE EEDEVWQVIIGARAEMTSKHQEYLKLETTWMTAVGLSEMAAEAAYQTGADQASITARNHIQLVKLQVEEVHQLSRKAETKLAEAQIEEELRQKTQEEGEERAESEQEAYLREDHHHHHH (SEQ ID NO:5); gctagctgccgcggcgataaaggcccggattgcggcggcggcggcagcggcggcggcggcagccgctttaaatttcgccgctttaaatttcgccatcatcatcatcatcatcgctggaaaaaaggcggcggcggcagcggcggcggcggcagcgcggtgccgattgcgcagaaaagcgaaccgcatagcctgagcagcgaagcgctgatgcgccgcgcggtgagcctggtgaccgatagcaccagcacctttctgagccagaccacctatgcgctgattgaagcgattaccgaatataccaaagcggtgtataccctgaccagcctgtatcgccagtataccagcctgctgggcaaaatgaacagcgaagaagaagatgaagtgtggcaggtgattattggcgcgcgcgcggaaatgaccagcaaacatcaggaatatctgaaactggaaaccacctggatgaccgcggtgggcctgagcgaaatggcggcggaagcggcgtatcagaccggcgcggatcaggcgagcattaccgcgcgcaaccatattcagctggtgaaactgcaggtggaagaagtgcatcagctgagccgcaaagcggaaaccaaactggcggaagcgcagattgaagaactgcgccagaaaacccaggaagaaggcgaagaacgcgcggaaagcgaacaggaagcgtatctgcgcgaagatcatcatcatcatcatcat ctcgag (SEQ ID NO:6); The synthesized gene fragment and the pCDNA3.1(+) vector (Thermo Fisher) were double-digested with NheI and XhoI at 37°C for 2 h. The target fragment was recovered by 1% agarose gel electrophoresis (Gene JET Gel Extraction Kit, Thermo). T4 DNA ligation was performed at a molar ratio of 3:1 (insert fragment:vector) (overnight at 16°C). The cells were transformed into DH5α competent cells and plated on LB agar plates containing 100 μg / mL ampicillin, and cultured at 37°C for 16 h. Ten single clones were randomly selected, and plasmids were extracted and subjected to colony PCR for initial screening. Positive clones were sent for Sanger sequencing (bidirectional sequencing covering the full length). Only plasmids with completely correct sequences, continuous reading frames, and no frameshifts or mutations were retained for subsequent experiments.

[0022] HEK293T cells (ATCC CRL-11268) were passaged in DMEM high-glucose medium (HyClone) + 10% FBS (Gibco, fetal bovine serum filtered through 0.22 μm) + 1% penicillin-streptomycin (10,000 U / mL) at 37°C, 5% CO2, and 95% humidity, maintaining cell viability >95%. 24 h before transfection, cells were cultured at 1.0 × 10⁶ cells / mL. 6 Cells were seeded at a density of approximately 70–80% per 10 cm dish (ensuring confluence at transfection), and transfection was performed using linear PEI (Linear Polyethylenimine, L-PEI 25 kDa).

[0023] Plasmid dosage: 10 μg / plate; PEI dosage: 30 μL (1 mg / mL, N / P molar ratio = 3); Diluent: Opti-MEM (Gibco), 250 μL each, mix after standing at room temperature for 5 min, incubate at room temperature for 20 min to form a complex; replace with fresh complete medium 6 h after transfection to avoid PEI toxicity; add 10 mM sodium butyrate after 48 h and continue culturing for 24 h to enhance protein expression.

[0024] Collect cell supernatant (pre-chilled at 4°C) and adherent cells. After washing twice with PBS, add 300 μL of pre-chilled RIPA lysis buffer (containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) to each dish, and immediately add 1× protease inhibitor mixture. Lyse on ice for 30 min, vortexing 3 times (10 s each time). Centrifuge at 12,000 × g at 4°C for 20 min, aliquot the supernatant, and store at –80°C or purify immediately.

[0025] A GE HisTrap FF 1 mL pre-packed column was connected to an AKTA pure chromatography system. All operating buffers were filtered through a 0.22 μm filter and degassed: Equilibration buffer (Buffer A): 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 20 mM Mimidazole; Wash buffer: same as Buffer A, 10 CV; Elution buffer (Buffer B): 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 500 mM Mimidazole; Elution program: 0–100% B-phase linear gradient, 10 CV, flow rate 1 mL / min.

[0026] The main peak component was collected under real-time monitoring using a UV280 microscope. Immediately after collection, it was concentrated to 1–2 mL using an Amicon Ultra-15 ultrafiltration tube (10 kDa molecular weight cutoff) and dialyzed three times (at least 4 h each time) against PBS (pH 7.4) at 4°C to completely remove imidazole and high salt content. SDS-PAGE analysis was performed. (See attached image). Figure 1 .

[0027] Figure 1 The results showed that Coomassie Brilliant Blue R-250 staining revealed a single main band with a molecular weight of approximately 26.68 kDa, as expected; Image Lab software analysis of the SDS-PAGE bands showed a purity of ≥94%.

[0028] Example 2: In vitro antitumor activity experiment of iRGD-MitoPen-SmacN55 fusion protein Cell models: Human breast cancer MDA-MB-231 cells (highly expressing αvβ3 integrin and NRP-1, with flow cytometry verification expression rates of 89.6% and 92.3%, respectively) and human colon cancer HCT116 cells (αvβ3 integrin expression rate of 78.2% and NRP-1 expression rate of 85.7%) were purchased from the Cell Bank of the Chinese Academy of Sciences. They were cultured in DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin-streptomycin at 37℃ in a 5% CO2 incubator. Cells in the logarithmic growth phase were used for experiments.

[0029] Experimental reagents: iRGD-MitoPen-SmacN55 fusion protein (prepared from Example 1, purity verified by HPLC ≥95%, endotoxin content <0.1 EU / μg); Smac mimic LCL161 (Selleck Chemicals, Cat. No. S7009); GFP-His unrelated protein (purchased from GenScript, catalog number 10404-H08H); CCK-8 assay kit (purchased from DOJINDO, catalog number CK04-3000T); Annexin V-FITC / PI apoptosis detection kit (purchased from Wuhan Pronosei Biotechnology Co., Ltd., catalog number P-CA-201); anti-cleaved caspase-3 (purchased from Cell Signaling Technology, catalog number 9661), cleaved caspase-9 (purchased from Cell Signaling Technology, catalog number 9505), PARP (purchased from Cell Signaling Technology, catalog number 9542), XIAP (purchased from Cell... Signaling Technology (catalog number 2042), cIAP1 (purchased from Cell Signaling Technology, catalog number 4952) antibody and HRP-labeled secondary antibody (goat anti-rabbit IgG-HRP) were purchased from Cell Signaling Technology, catalog number 7074.

[0030] Two types of tumor cells were seeded at 5 × 10³ cells / well in 96-well plates (for CCK-8 assay), and at 2 × 10³ cells / well. 5 Cells were seeded per well in 6-well plates (for flow cytometry apoptosis detection and Western blot). After culturing for 24 h to allow cell adhesion, the cells were randomly divided into four groups (n=6 / group, 3 independent replicates): Blank control group: Only an equal volume of serum-free DMEM medium (containing a final concentration of 0.1% BSA to maintain protein stability) was added. Negative control group: Add an equimolar concentration (1 μM) of GFP-His unrelated protein (dissolved in serum-free DMEM containing 0.1% BSA). Positive control group: Smac mimic LCL161 (dissolved in DMSO, final DMSO concentration ≤0.1%, no cytotoxicity) was added to a final concentration of 10 μM. Experimental group: Add 1 μM of iRGD-MitoPen-SmacN55 fusion protein (dissolved in serum-free DMEM containing 0.1% BSA).

[0031] All groups were incubated in a 37℃, 5% CO2 incubator, and the corresponding indicators were measured at 24 h, 48 h, and 72 h.

[0032] Cell viability assay (CCK-8 assay): After culturing to the set time point, add 10 μL of CCK-8 reagent to each well and continue incubation for 2 h. Detect the absorbance (OD) at 450 nm using a Thermo Multiskan FC microplate reader. 450 ), calculate cell viability: viability (%) = (experimental group OD) 450 - Blank Hole OD 450 ) / (Blank control group OD 450 - Blank Hole OD 450 ×100%. Statistical analysis was performed using GraphPad Prism 9.0. One-way ANOVA was used for comparisons between groups. P < 0.05 was considered statistically significant. See [link to relevant documentation]. Figure 2 .

[0033] CCK-8 assay results showed that in both types of tumor cells, the cell survival rate in the experimental group decreased significantly with prolonged culture time, reaching its lowest value at 72 h. The survival rates in both the blank control group and the negative control group were >90%, with no significant difference between the groups (P>0.05). Statistical analysis indicated that the cell survival rate in the experimental group was significantly lower than that in the positive control group (P<0.01), demonstrating that the iRGD-MitoPen-SmacN55 fusion protein had a superior inhibitory effect on tumor cell proliferation compared to the preclinical Smac mimic LCL161.

[0034] Apoptosis rate detection (Annexin V-FITC / PI double staining flow cytometry): After 48 h of culture, cells from each group (including suspended cells in the supernatant) were collected from 6-well plates. Cells were washed twice with PBS, resuspended in 100 μL binding buffer, and then 5 μL Annexin V-FITC and 5 μL PI staining solution were added sequentially. Cells were incubated at room temperature in the dark for 15 min, and then 400 μL binding buffer was added. Detection was performed using a flow cytometer (BD FACSCanto II) within 1 h. Annexin V-FITC positive and PI negative cells were considered early apoptotic cells, and Annexin V-FITC positive and PI positive cells were considered late apoptotic cells. Total apoptosis rate = early apoptosis rate + late apoptosis rate. (See [link to relevant documentation]). Figure 3 .

[0035] Flow cytometry results showed that at 48 h, the total apoptosis rate in the experimental group was significantly higher than that in other groups, while the total apoptosis rate in the blank control group and the negative control group was <10%. Figure 3Intergroup comparisons showed that the apoptosis rate in the experimental group was 7-8 times that of the blank control group and 1.9-2.1 times that of the positive control group, with statistically significant differences (P<0.01), confirming that the iRGD-MitoPen-SmacN55 fusion protein can efficiently induce tumor cell apoptosis.

[0036] Caspase activation and IAP protein expression detection (Western blot): After culturing for 48 h, cells were collected and RIPA lysis buffer (containing 1% PMSF and 1% protease inhibitor cocktail) was added. Lysis was performed on ice for 30 min, followed by centrifugation at 12000 rpm for 15 min at 4 °C to extract total protein. Protein concentration was determined by BCA method. 50 μg of total protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 h, and then incubated overnight at 4 °C with primary antibody (1:1000 dilution). The membrane was washed three times with TBST (10 min each time), and then incubated with HRP-labeled secondary antibody (1:5000 dilution) at room temperature for 1 h. After three TBST washes, the membrane was developed using an ECL chemiluminescence kit, and the band grayscale value was quantified using ImageJ software. The relative expression level of the target protein was calculated using GAPDH as an internal control.

[0037] The Western blot results showed that the relative expression levels of cleaved caspase-3, cleaved caspase-9, and PARP cleavage products were significantly increased in the experimental group. In MDA-MB-231 cells, the relative expression level of cleaved caspase-3 was 4.8 ± 0.6 times that of the blank control group, cleaved caspase-9 was 5.2 ± 0.7 times, and the PARP cleavage product was 4.5 ± 0.5 times. In HCT116 cells, the above indexes were 4.5 ± 0.5 times, 4.9 ± 0.6 times, and 4.2 ± 0.4 times that of the blank control group, respectively. At the same time, the protein expression levels of XIAP and cIAP1 in the experimental group were significantly downregulated, being 0.28 ± 0.05 times and 0.32 ± 0.06 times that of the blank control group (MDA-MB-231) and 0.31 ± 0.04 times and 0.35 ± 0.07 times (HCT116), respectively. Although the positive control group could upregulate the expression of cleaved caspase-3 / 9 and PARP cleavage products (about 2.0 - 2.5 times that of the blank control group) and downregulate the expression of XIAP / cIAP1 (about 0.55 - 0.65 times that of the blank control group), the effect was significantly weaker than that of the experimental group (P < 0.01). There was no significant difference in the above protein expression between the blank control group and the negative control group (P > 0.05). These results confirmed that the iRGD-MitoPen-SmacN55 fusion protein induced tumor cell apoptosis by degrading XIAP and cIAP1 to relieve the inhibition of caspase and activate the endogenous apoptotic pathway.

[0038] Example 3. Pharmacodynamic study of iRGD-MitoPen-SmacN55 fusion protein in tumor-bearing mice Experimental animals: Female BALB / c nude mice, 6 - 8 weeks old, weighing 18 - 22 g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animal license number was: SCXK (Beijing) 2021-0006. They were housed in a SPF-level animal room at a temperature of 22 - 26°C, a humidity of 50% - 60%, with a 12-hour light-dark cycle, and free access to food and water. After 1 week of adaptive feeding, they were used for the experiment.

[0039] Model construction: MDA-MB-231 cells in the logarithmic growth phase were taken, and the cell concentration was adjusted to 5×10 6 cells / 100 μL with PBS and injected subcutaneously into the right axilla of nude mice. After inoculation, the status of the mice was observed daily, and the tumor long diameter (L) and short diameter (W) were measured twice a week to calculate the tumor volume (V = 0.5 × L × W²). When the tumor volume reached 100 ± 20 mm³, the nude mice were randomly divided into 4 groups (n = 8 / group) to ensure that there was no significant difference in the tumor volume among the groups (P > 0.05).

[0040] Experimental reagents: iRGD-MitoPen-SmacN55-His fusion protein (same as in vitro experiment in Example 2); SmacN55-His protein (containing only the SmacN55 domain, without iRGD and MitoPen modules, purity ≥95%); LCL161 (same as in vitro experiment in Example 2); TUNEL apoptosis detection kit (purchased from Wuhan Sunncell Biotechnology Co., Ltd. (Sunncell®), catalog number SNK-019); Blank control group: 100 μL of PBS was injected via the tail vein once every other day for 10 consecutive times; Negative control group: SmacN55-His protein (5 mg / kg, dissolved in 100 μL PBS) was injected via tail vein once every other day for 10 consecutive times; Positive control group: LCL161 (30 mg / kg, dissolved in physiological saline containing 5% DMSO, 100 μL / animal) was injected intraperitoneally once every other day for 10 consecutive times; Experimental group: iRGD-MitoPen-SmacN55-His fusion protein (5 mg / kg, dissolved in 100 μL PBS) was injected via tail vein once every other day for 10 consecutive times.

[0041] Tumor growth indicators: The tumor inhibition rate was calculated at the experimental endpoint (day 21): Tumor inhibition rate (%) = (average tumor weight of control group - average tumor weight of experimental group) / average tumor weight of control group × 100%; see Table 1.

[0042] Table 1. Comparison of tumor growth-related indicators in nude mice of different groups on day 21 of the experiment (x±s, n=8) Note: Compared with the blank control group, P a >0.05, P b <0.01; compared with the positive control group, P c <0.01 Table 1 shows that the tumor inhibition rate in the experimental group was significantly higher than that in the positive control group (P<0.01), while the tumor inhibition effect in the negative control group was weak and there was no significant difference compared with the blank control group (P>0.05). These results confirm that the introduction of the iRGD and MitoPen modules can significantly improve the tumor-targeted delivery efficiency of SmacN55 and enhance its in vivo anti-tumor effect.

[0043] Tumor tissue apoptosis detection (TUNEL staining): At the experimental endpoint (day 21), mice were sacrificed, tumor tissue was dissected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (5 μm). Following the TUNEL kit instructions, nuclei were stained with DAPI, and observed under a fluorescence microscope. Five high-power fields (×400) were randomly selected, and the number of TUNEL-positive cells (apoptotic cells) and the total number of cells were counted. The apoptosis index (AI) was calculated as (number of TUNEL-positive cells / total number of cells) × 100%. (See [reference needed]). Figure 4 ;

[0044] Figure 4 The results showed that the number of apoptotic cells in the tumor tissue of the experimental group was significantly increased, and the AI ​​of the experimental group was significantly higher than that of the positive control group (P<0.01), confirming that the iRGD-MitoPen-SmacN55 fusion protein can efficiently induce tumor cell apoptosis in vivo.

[0045] In vitro experiments confirmed that the iRGD-MitoPen-SmacN55 fusion protein can specifically inhibit the proliferation of MDA-MB-231 and HCT116 tumor cells and efficiently induce apoptosis. Its effect is significantly better than that of the preclinical Smac mimic LCL161, and the mechanism of action is clear (degradation of XIAP / cIAP1 and activation of the caspase apoptosis pathway). In vivo experiments showed that the fusion protein can significantly inhibit the growth of MDA-MB-231 xenografts in nude mice, with a tumor inhibition rate of 67.4%. At the same time, it can efficiently induce tumor tissue apoptosis. The tumor targeting function of iRGD and the mitochondrial delivery function of MitoPen are the key to enhancing the bioactivity of SmacN55.

[0046] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An iRGD-MitoPen-SmacN55 fusion protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

4.

2. A gene comprising encoding the fusion protein of claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

6.

3. A recombinant expression vector containing the gene of claim 2, characterized in that, The gene was inserted using pCDNA3.1 (+) as the vector backbone via NheI and XhoI restriction sites.

4. The method for preparing the fusion protein according to claim 1, characterized in that, Includes the following steps: (1) Synthesize the optimized coding gene; (2) Construct and validate the recombinant expression vector; (3) Transfect HEK293T cells and induce expression with sodium butyrate; (4) Purify the target protein by His tag affinity chromatography and dialysis.

5. The use of the fusion protein of claim 1 in the preparation of a drug for inhibiting the proliferation of breast cancer or colon cancer cells.

6. The use of the fusion protein of claim 1 in the preparation of an antitumor drug that induces tumor cell apoptosis.

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