Bacterial ferritin 193 and uses thereof

Abstract

The present application relates to the technical field of medicine, in particular to a kind of bacterial ferritin 193 and its application, the amino acid sequence of the bacterial ferritin 193 as shown in SEQ ID No.1, with unique structure, can specifically target human transferrin receptor 1 and cross blood-brain barrier, can be used for the targeted delivery of drug, reduce the toxicity of drug to normal cell, improve the therapeutic effect of drug;It can self-assemble to form the large nanometer particle inner cavity capacity, can realize more than 150 molecules / ferritin drug loading capacity, and can keep stable nano-particle structure under a wide range of pH conditions, suitable for application in complex physiological environment;It can be efficiently expressed in prokaryotic expression vector such as escherichia coli, obtain high-purity nanoparticles;Not only can load single drug, diagnostic reagent / tracer reagent, but also can simultaneously load multiple antitumor drugs or tracer reagent, with synergistic therapeutic effect or diagnostic effect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a bacterial ferritin 193 and its applications. Background Technology

[0002] *Acidithiobacillus ferrivorans* is an obligate acidophilic, psychrophilic chemoautotrophic bacterium belonging to the class Gammaproteobacteria. This bacterium can efficiently oxidize ferrous iron and reduced sulfides under the dual stresses of low temperature (4°C) and strong acid (pH 4.0), making it a key primary producer in acid mine drainage (AMD) ecosystems and low-temperature biometallurgical processes in cold regions. It is widely distributed in extreme habitats such as low-temperature AMD, high-latitude bioleaching dumps, and polar mining areas, playing a central role in the biogeochemical cycles of low-temperature acidic ecosystems. *A. ferrivorans* grows strictly chemoautotrophically using carbon dioxide as its sole carbon source. Through its unique physiological and genomic adaptation mechanisms to the dual stresses of low temperature and high acidity, it has become a key functional bacterium for biometallurgy and in-situ remediation of AMD in cold regions. Its mediated Fe(II) bio-oxidation process not only accelerates the evolution of acidic environment, but also drives the rapid generation of ferric hydrolysis products, thereby fixing toxic heavy metals such as arsenic, copper, and zinc through surface adsorption and co-precipitation. It has important ecological and engineering value in mining pollution control and valuable metal resource recovery.

[0003] Similar to most iron-metabolizing bacteria, *A. ferrivorans* relies heavily on its endogenous ferritin (bacterial ferritin / heme ferritin) for iron detoxification, storage, and recycling in microenvironments with high concentrations of free iron and strong oxidative stress. Classical bacterial ferritin typically consists of 24 identical or highly homologous subunits self-assembling into highly symmetrical hollow spherical nanocages. The shell surface has natural hydrophilic channels distributed along four, three, and two-fold symmetry axes, allowing small molecule drugs, metal ions, nucleic acids, or contrast agents to freely enter and exit. Therapeutic or diagnostic molecules can be precisely loaded into these cavities using strategies such as in vitro depolymerization-recombination, pH gradient methods, urea denaturation, or temperature-induced methods. Due to their excellent biocompatibility, well-defined self-assembly characteristics, easily modifiable surfaces, and potential ability to cross biological barriers, ferritin derived from extremophile microorganisms and its engineered derivatives are gradually becoming ideal platforms for nanomedicine delivery, molecular imaging tracking, and targeted diagnosis and treatment.

[0004] Ferritin carriers currently used in the medical field mainly include human, equine spleen, Helicobacter pylori, and thermophilic archaea sources. Their applications have expanded to anti-tumor therapy, gene delivery, molecular imaging, novel vaccine development, and the diagnosis and treatment or prevention of inflammatory and degenerative diseases. However, despite the unique environmental stability and structural rigidity of ferritins derived from acidophilic / psychrophilic bacteria, the following common defects still exist in the practical application of various ferritins currently disclosed: (1) Limited drug loading efficiency and versatility: Ferritin drug loading usually relies on strategies such as pH method, urea method or temperature incubation method. These methods are generally accompanied by problems such as partial protein denaturation and decreased recovery rate. Moreover, the physicochemical microenvironment of the ferritin lumen, pore size and the choice of drug loading strategy will also significantly limit the types and loading capacity of drug molecules (especially macromolecules, hydrophobic compounds or complex nucleic acids) that can be loaded; (2) Insufficient targeting ability: Except for human heavy chain ferritin, the reported bacterial ferritins all lack the natural affinity to target and bind human transferrin receptor (TfR) or other disease-specific receptors, which greatly restricts their application potential in clinical treatment (especially in complex scenarios such as solid tumor penetration, blood-brain barrier crossing or active targeted delivery).

[0005] To address the aforementioned technical problems, this invention has discovered a bacterial ferritin 193 from *Acidithiobacillus ferrivorans*. Compared to mammalian ferritins and previously discovered bacterial ferritins, we found that bacterial ferritin 193 has a shorter primary structure, consisting of only about 147 amino acids. While its tertiary structure possesses a quadruple helix similar to that of mammalian and bacterial ferritins, it lacks the E-helix structure. However, it exhibits the ability to specifically target and bind to human transferrin receptors, enabling targeted delivery of drug molecules. With its unique structural and functional properties, bacterial ferritin 193 holds promise for providing new insights into solving the problems of ferritin drug loading efficiency and targeted delivery, potentially becoming a novel drug delivery carrier. Summary of the Invention

[0006] The primary objective of this invention is to provide a bacterial ferritin 193, the amino acid sequence of which is shown in SEQ ID No. 1.

[0007] A second object of the present invention is to provide a gene sequence encoding the bacterial ferritin 193, the gene sequence being shown in SEQ ID No. 2.

[0008] A third objective of this invention is to provide a method for preparing the bacterial ferritin 193 described above, comprising the following steps:

[0009] S1. The whole genome of His-Ftn193 was synthesized, and the nucleotide sequence of His-Ftn193 is shown in SEQ ID NO.4;

[0010] S2. Transform the recombinant expression vector obtained in step S1 into E. coli for expression, compare protein solubility, and obtain soluble expression product;

[0011] S3. The expression product obtained in step S2 was purified by affinity chromatography to obtain bacterial ferritin 193, which self-assembled into Ftn193-NPs.

[0012] A fourth object of the present invention is to provide a cage-like protein comprising a plurality of subunits that self-assemble into the cage-like protein, wherein each of the subunits is the bacterial ferritin 193.

[0013] Preferably, the cage-like protein has the ability to specifically bind to human transferrin receptor 1.

[0014] Preferably, the cage-like protein has the ability to cross the blood-brain barrier.

[0015] Preferably, the cage-like protein contains a drug, which is a chemotherapy drug, an anti-tumor traditional Chinese medicine, an anti-neurodegenerative disease drug, or an anti-inflammatory drug. The chemotherapy drug includes alkylating agents, antibiotics, alkaloids, or radiopharmaceuticals. The anti-neurodegenerative disease drug includes neurotransmitter drugs, dopamine receptor agonists, central nervous system anticholinergic drugs, cholinergic receptor agonists, serotonin system drugs, GABAergic system drugs, or glutamatergic system drugs. The anti-tumor traditional Chinese medicine includes γ-secretase inhibitors.

[0016] Preferably, the alkylating agent includes one or more of cisplatin, carboplatin, oxaliplatin, lobaplatin, and their derivatives; the antibiotic includes one or more of doxorubicin, idarubicin, and epirubicin; the alkaloid includes one or more of vinblastine, camptothecin, and podophyllotoxin; the radiopharmaceutical includes one or more of 225Ac, 177Lu, 131I, 89Sr, 32P, and 153Sm; the neurotransmitter includes carbidopa, galantamine, rivastigmine, acetylcholine, atropine, scopolamine, dopamine, and their derivatives; and the dopamine receptor agonist includes levodopa and pramipexole. The drugs include one or more of the following: ergot alkaloid derivatives or non-ergot alkaloid derivatives, such as oxaliplatin and apomorphine; the central nervous system anticholinergic drugs include haloperidol, benzalkonium chloride, or propranolol; the cholinergic receptor agonists include muscarinic acid, pilocarpine, or varenicline; the serotonin system drugs include one or more of the following: fluoxetine, sertraline, sumatriptan, buspirone, ondansetron, and mirtazapine; the GABAergic system drugs include one or more of the following: diazepam, lorazepam, phenobarbital, tiagabine, and sodium valproate; the glutamatergic system drugs include one or more of the following: memantine, ketamine, and perampanel; and the γ-secretase inhibitors are diflubenzurons.

[0017] A fifth object of the present invention is to provide the use of the bacterial ferritin 193 or the cage-like protein described herein in the preparation of pharmaceuticals, diagnostic reagents and / or tracer reagents.

[0018] Preferably, the drug is a treatment for inflammatory diseases, angiogenesis disorders, tuberculosis, HIV, Alzheimer's disease, rheumatoid arthritis, fragile plaque thin fibrous atherosclerosis, multiple sclerosis, or tumors; the tumor is selected from lymphoma, leukemia, breast cancer, esophageal cancer, gastric cancer, ovarian cancer, skin cancer, colorectal cancer, lung cancer, bladder cancer, pancreatic cancer, kidney cancer, head and neck cancer, adrenocortical carcinoma, liver cancer, brain cancer, uterine cancer, bile duct cancer, sarcoma, gallbladder cancer, cervical cancer, prostate cancer, thyroid cancer, rhabdoid tumor, bone cancer, myeloma, testicular cancer, or metastatic cancer thereof;

[0019] The diagnostic reagent is for diagnosing tumors, autoinflammatory diseases, angiogenic diseases, tuberculosis, HIV, Alzheimer's disease, rheumatoid arthritis, vulnerable plaque thin fibrous atherosclerosis, multiple sclerosis, or tumors; the tumors are selected from lymphoma, leukemia, breast cancer, esophageal cancer, gastric cancer, ovarian cancer, skin cancer, colorectal cancer, lung cancer, bladder cancer, pancreatic cancer, kidney cancer, head and neck cancer, adrenocortical carcinoma, liver cancer, brain cancer, uterine cancer, bile duct cancer, sarcoma, gallbladder cancer, cervical cancer, prostate cancer, thyroid cancer, rhabdoid tumor, bone cancer, myeloma, testicular cancer, or metastatic cancer thereof;

[0020] The tracer reagent is a tumor tracer reagent, and the tumor is selected from lymphoma, leukemia, breast cancer, esophageal cancer, gastric cancer, ovarian cancer, skin cancer, colorectal cancer, lung cancer, bladder cancer, pancreatic cancer, kidney cancer, head and neck cancer, adrenocortical carcinoma, liver cancer, brain cancer, uterine cancer, bile duct cancer, sarcoma, gallbladder cancer, cervical cancer, prostate cancer, thyroid cancer, rhabdoid tumor, bone cancer, myeloma, testicular cancer or metastatic cancer.

[0021] The beneficial effects of the present invention are: (1) The bacterial ferritin 193 provided by the present invention has a unique structure that can not only specifically target human transferrin receptor 1, but also cross the blood-brain barrier, and can be used for targeted drug delivery, reducing the toxicity of drugs to normal cells and improving the therapeutic effect of drugs.

[0022] (2) The bacterial ferritin 193 provided by the present invention can self-assemble into nanoparticles with large internal capacity, and can achieve a drug loading capacity of more than 100 molecules / ferritin.

[0023] (3) The bacterial ferritin 193 provided by the present invention can maintain a stable nanoparticle structure under a wide range of pH conditions when it is self-assembled into nanoparticles, making it suitable for application in complex physiological environments.

[0024] (4) Bacterial ferritin 193 can be efficiently expressed in prokaryotic expression vectors such as Escherichia coli, and high-purity nanoparticles can be obtained through simple purification steps, which facilitates subsequent drug loading and application.

[0025] (5) Bacterial ferritin 193 can not only load a single drug, diagnostic reagent / tracer reagent, but also load multiple anti-tumor drugs or tracer reagents at the same time, and has synergistic therapeutic or diagnostic effects. Attached Figure Description

[0026] Figure 1 This is a map of the pET24a-His-Ftn193 expression vector.

[0027] Figure 2 The results are obtained from SDS-PAGE electrophoresis detection of protein expression and purification.

[0028] Figure 3 It is a Native-PAGE electrophoresis assay used to detect the particle size of assembled proteins.

[0029] Figure 4 It is a result of yeast hybridization. Detailed Implementation

[0030] The above solution will be further described below with reference to specific embodiments. The invention will be further described in detail through the following embodiments, but it should be understood that the invention is not limited to the following content.

[0031] It should be noted that, unless otherwise specified, the methods and steps not described in detail in the following embodiments are performed in accordance with conventional experimental methods and procedures in the art.

[0032] It should be noted that, unless otherwise specified, the reagents and consumables used in the following embodiments are commercially available.

[0033] It should be noted that in this invention, bacterial ferritin 193 is abbreviated as Ftn193, and the two have the same meaning.

[0034] Example 1: Construction of the pET24a-His-Ftn193 fusion protein vector

[0035] The amino acid sequence of Ftn193 is shown in SEQ ID NO.1, and the nucleotide sequence of Ftn193 is shown in SEQ ID NO.2. The His-Ftn193 gene fragment was synthesized, and the nucleotide sequence encoding His-Ftn193 is shown in SEQ ID NO.4. The amino acid sequence of the His-Ftn193 protein is shown in SEQ ID NO.3. The molecular weight of Ftn193 is approximately 16.78 kDa. Sequence information is as follows:

[0036] The amino acid sequence of Ftn193 (SEQ ID NO.1):

[0037] MANDPRMTGYLMRALSAEMAAVQQYLTQSSLSAMWQLKEYSGRFRRDAEEELGHAQRLIEHMMLMLGIPFNGTQLPPIRPGRSLEEMLLIDRDLEIDVIRLYEEAAHYCARVRAEGLQALFAGLLQEELGHLRDLDHLLTEMHQGAIA

[0038] The nucleotide sequence of Ftn193 (SEQ ID NO.2):

[0039] ATGGCGAACGATCCGCGTATGACCGGCTACCTGATGCGTGCGCTGAGCGCGGAAATGGCGGCGGTTCAGCAGTACCTGACCCAGAGCAGCCTGAGCGCGATGTGGCAGCTGAAAGAATACAGCGGCCGCTTCCGTCGTGATGCGGAAGAAGAACTGGGTCACGCGCAGCGTCTGATCGAACACATGCTGATGCTGGGCATCCCGTTCAACGGCACCCAGCTGCCGCCGATCCGTCCGGGCCGTAGCCTGGAAGAAATGCTGCTGATCGATCGTGATCTGGAAATCGATGTTATCCGTCTGTACGAAGAAGCGGCGCACTACTGCGCGCGTGTTCGTGCGGAAGGCCTGCAGGCGCTGTTCGCGGGCCTGCTGCAGGAAGAACTGGGCCACCTGCGTGATCTGGATCACCTGCTGACCGAAATGCACCAGGGCGCGATCGCG

[0040] Amino acid sequence of His-Ftn193 protein (SEQ ID NO.3):

[0041] HHHHHHMANDPRMTGYLMRALSAEMAAVQQYLTQSSLSAMWQLKEYSGRFRRDAEEELGHAQRLIEHMLMLGIPFNGTQLPPIRPGRSLEEMLLIDRDLEIDVIRLYEEAAHYCARVRAEGLQALFAGLLQEELGHLRDLDHLLTEMHQGAIA

[0042] Nucleotide sequence of His-Ftn193 (SEQ ID NO.4):

[0043] CACCATCACCACCACCACATGGCGAACGATCCGCGTATGACCGGCTACCTGATGCGTGCGCTGAGCGCGGAAATGGCGGCGGTTCAGCAGTACCTGACCCAGAGCAGCCTGAGCGCGATGTGGCAGCTGAAAGAATACAGCGGCCGCTTCCGTCGTGATGCGGAAGAAGAACTGGGTCACGCGCAGCGTCTGATCGAACACATGCTGATGCTGGGCATCCCGTTCAACG GCACCCAGCTGCCGCCGATCCGTCCGGGCCGTAGCCTGGAAGAAATGCTGCTGATCGATCGTGATCTGGAAATCGATGTTTATCCGTCTGTACGAAGAAGCGGCGCACTACTGCGCGCGTGTTCGTGCGGAAGGCCTGCAGGCGCTGTTCGCGGGCCTGCTGCAGGAAGAACTGGGCCACCTGCGTGATCTGGATCACCTGCTGACCGAAATGCACCAGGGCGCGATCGCG

[0044] The vector selected is the universal prokaryotic expression vector pET-24a(+). Figure 1 The sequencing results of the synthesized sequence were consistent with the template, and the expression strain P1 was obtained.

[0045] 10 μL of P1 glycerol bacteria was added to 10 ml of kanamycin-resistant LB broth and incubated overnight at 37°C with shaking at 220 rpm until OD600 > 1.0. 1 ml of this culture was then inoculated into 100 ml of kanamycin-resistant LB broth and incubated at 37°C with shaking at 220 rpm until OD600 ≈ 0.6. Then, 240 μL of 50 mg / ml IPTG was added to a final concentration of 0.5 mM, and expression was induced at 16°C with shaking at 220 rpm for 16 h. Afterward, the cells were centrifuged at 4°C, 10000 rpm for 5 min, the culture medium was discarded, and the cells were resuspended in 30 ml of disruption buffer (20 mM PB, 5 mM imidazole). The cells were then sonicated until clear and transparent, and the expression of the target protein in the supernatant was detected by SDS-PAGE electrophoresis.

[0046] The results are as follows Figure 2 As shown, the protein showed a distinct band at around 15KD after induction, indicating high expression after induction. The target band was more prominent in the supernatant after lysis than in the precipitate, indicating that the supernatant showed dominant expression, which meets the conditions for the next purification step.

[0047] Example 2: Purification of His-Ftn193 protein

[0048] The cleavage buffer containing His-Ftn193 protein obtained in Example 1 was purified by affinity chromatography. The experimental procedure is as follows:

[0049] 1. Affinity chromatography purification

[0050] (1) Equilibrate with 20 mmol pH 7.4 and 5 mM imidazole phosphate buffer, then elute with 20 mmol pH 7.4 and different concentrations of imidazole phosphate buffer, collect the eluent, the 20 mmol pH 7.4 and 250 mM imidazole eluent is the component with the highest protein content in the first step of purification.

[0051] (2) SDS-PAGE was used to analyze protein samples. Figure 2 The results showed that pET24a-Ftn193 had a high purity (≥95%).

[0052] 2. Assembly of nanoparticles

[0053] (1) The purified target collection solution was concentrated through a 10 kDa ultrafiltration tube and centrifuged at 4000 rpm at 4℃ to concentrate the protein. The concentrated protein sample was placed in a 3.5 kDa dialysis bag and then placed in 2L of assembly buffer (20 mM PB, pH 7.4). Dialysis was performed at 4℃ for 14-16 h to remove imidazole from the protein concentrate and allow it to fully assemble. The size of the assembled particles was then observed by Native-PAGE.

[0054] (2) Native-PAGE analysis of the assembled samples Figure 3 The results showed that His-Ftn193 could be assembled into nanoparticles of about 400 kDa.

[0055] Example 3: Yeast Two-Hybrid Experiment

[0056] To demonstrate the specific binding of bacterial ferritin 193 to the TFR1 receptor, a membrane-based yeast two-hybrid assay was performed. Bacterial ferritin 193 and human ferritin heavy chain were homologously ligated into the pPR3-N vector via BamHI and EcoRI restriction sites. The amino acid sequence of bacterial ferritin 193 is shown in SEQ ID NO. 1. The receptor was constructed into the pBT3-N vector via sfil restriction site digestion and ligation. After successful sequencing, plasmids were extracted using a plasmid miniprep kit for subsequent yeast two-hybrid experiments.

[0057] (1) Preparation of competent yeast cells

[0058] a. Take the NMY51 yeast culture out of the -80℃ freezer, streak it on YPDA medium, and incubate at 30℃ for 3-5 days;

[0059] b. Pick a single colony in good condition from the culture medium and inoculate it into YPDA liquid medium, shake overnight until OD600>1.0;

[0060] c. Take 1 ml of bacterial culture, add YPDA medium to make up to 50 ml, pour into an Erlenmeyer flask, and incubate with shaking until OD600 = 0.5;

[0061] d. In a clean bench, pour the shaken bacterial culture into a 50 ml centrifuge tube, centrifuge, discard the supernatant, add sterile water to resuspend, centrifuge again and discard the supernatant;

[0062] e. Resuspend the bacterial cells in 1 ml of 1.1×TE / LiAc, centrifuge, and discard the supernatant;

[0063] f. Resuspend the bacterial cells in 1.1×TE / LiAc. The preparation of competent cells is now complete.

[0064] (2) Plasmids are transferred into competent states

[0065] a. Add 5 μl each of the receptor and ligand plasmid to a 1.5 ml centrifuge tube, add 5 μl of the prepared carrier DNA, and then add 100 μl of yeast competent cells and mix gently.

[0066] b. Let stand for 10 minutes;

[0067] c. Add 40% PEG / LiAc solution, mix gently, and incubate for 30-60 min;

[0068] d. Add DMSO, mix thoroughly by blowing, and heat shock for 5 min;

[0069] e. Centrifuge and discard the supernatant.

[0070] f. Add 1 ml of sterile water, mix well, centrifuge and discard the supernatant;

[0071] g. Add 200 μl of sterile water, mix well by pipetting, spread evenly on SD-Leu / -Trp medium, and incubate for about 5 days.

[0072] (3) Observe the interaction between SD-Ade / -His / -Leu / -Trp and SD-Ade / -His / -Trp.

[0073] a. Pick a certain number of single colonies and add them to 20 μL of sterile physiological saline and mix well;

[0074] b. Dilute each component separately to create three gradients (10⁻¹⁰). 0 10 1 10 2 );

[0075] c. Take 10 μl of bacterial suspension and spot it onto the labeled SD-Ade / -His / -Trp and SD-Ade / -His / -Leu / -Trp media, respectively.

[0076] d. After drying the culture medium, invert it at 30℃ for 4-5 days and observe the growth of yeast.

[0077] Figure 4 The results show that yeast cells containing bacterial ferritin 193, WT, G, and HFn grew on triple-deficient medium (containing 10 mM 3-AT), indicating successful transformation of the two hybrid plasmids and the ability to synthesize Trp and Leu. WT and Hfn served as positive controls; WT's growth was guaranteed to support yeast growth, while Hfn, containing the human ferritin sequence in the Prey plasmid, also grew. G served as a negative control, containing the empty Prey vector, confirming the correctness of the experimental system and its inability to grow on triple-deficient and quadruple-deficient plates (with added 3-AT inhibitors). Simultaneously, bacterial ferritin 193, HFn, and TfR1 interacted. The growth of yeast cells containing bacterial ferritin 193, WT, and HFn on quadruple-deficient medium indicated reporter gene activation, demonstrating a strong interaction between these three proteins and TfR1. The wild-type showed strong interaction, while the wild-type mutant showed no interaction, indicating that the experimental system of this invention meets the requirements. Bacterial ferritin 193 exhibits a strong interaction with TfR1.

[0078] Example 4: Drug Loading Experiment with Ftn-193-NPs

[0079] 1. Doxorubicin loading experiment

[0080] (1) Take 1 mL (0.5 mg / mL) of Ftn-193-NPs and add it to a centrifuge tube. Add DOX solution (1 mg / mL) to the Ftn-193-NPs solution at a ratio of 2:1. Incubate the Ftn-193-NPs and DOX mixture in a metal bath at 25 °C for 8 h, 16 h, and 24 h to obtain DOX-loaded Ftn-193 nanoparticles, named Ftn-193@DOX.

[0081] (2) Remove the Ftn-193@DOX mixture and cool it to room temperature. Centrifuge at 12,000 rpm for 20 min at 4°C to remove the precipitate. Dialyze in PBS for 12 h to remove free DOX. Remove the dialysis bag solution and centrifuge at 5,000 rpm for 5 min at 4°C to remove the precipitate.

[0082] (3) Detection of DOX content by fluorescence method: The DOX content in the final Ftn-193-NPs-DOX complex was determined. First, the DOX stock solution (1 mg / mL) was diluted to prepare DOX standard solutions of different concentrations. The DOX fluorescence intensity was measured using an ELISA reader, and a DOX fluorescence intensity-concentration curve was plotted. Doxorubicin absorbs light at 485 nm and emits light at 580 nm. Then, the pH of the complex was adjusted to about 2.0 with 1 M hydrochloric acid, mixed well, and incubated. After passing through a 0.22 μm filter membrane, 200 μL was added to a 96-well plate with a black background. The obtained fluorescence value was substituted into the standard curve to obtain the DOX content.

[0083] (4) Ferritin content determination: The concentration of ferritin was determined using the Bradford kit.

[0084] The results are shown in Table 1. On average, one molecule of Ftn-193-NPs was loaded with 111 DOX molecules at 8 h and 153 DOX molecules at 16 h.

[0085] Table 1. DOX loading experiments of Ftn-193-NPs

[0086] In summary, this invention provides a bacterial ferritin 193, whose amino acid sequence is shown in SEQ ID NO.1. Its unique structure not only specifically targets human transferrin receptor 1 but also allows it to cross the blood-brain barrier, enabling targeted drug delivery, reducing drug toxicity to normal cells, and improving therapeutic efficacy. The bacterial ferritin 193 provided by this invention can self-assemble into nanoparticles with a large lumen capacity, achieving a drug loading capacity exceeding 150 molecules / ferritin. The self-assembled nanoparticles of the bacterial ferritin 193 provided by this invention maintain a stable nanoparticle structure over a wide pH range, making them suitable for application in complex physiological environments. Bacterial ferritin 193 can be efficiently expressed in prokaryotic expression vectors such as *E. coli*, and high-purity nanoparticles can be obtained through simple purification steps, facilitating subsequent drug loading and application. Yeast double hybridization results demonstrate that Ftn193 can be taken up by TfR1-expressing tumor cells via TfR1-mediated endocytosis, delivering loaded DOX antitumor drugs to tumor cells, achieving targeted killing of tumor cells without affecting normal cells. Bacterial ferritin 193 can not only load single drugs, diagnostic reagents / tracer reagents, but also load multiple anti-tumor drugs or tracer reagents simultaneously, exhibiting synergistic therapeutic or diagnostic effects.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A bacterial ferritin 193, characterized in that, The amino acid sequence of bacterial ferritin 193 is shown in SEQ ID No.

1.

2. The gene sequence encoding bacterial ferritin 193 as described in claim 1, characterized in that, The gene sequence is shown in SEQ ID No.

2.

3. A method for preparing bacterial ferritin 193 as described in claim 1, characterized in that, Includes the following steps: S1. The whole genome of His-Ftn193 was synthesized, and the nucleotide sequence of His-Ftn193 is shown in SEQ ID NO.4; S2. Transform the recombinant expression vector obtained in step S1 into E. coli for expression, compare protein solubility, and obtain soluble expression product; S3. The expression product obtained in step S2 was purified by affinity chromatography to obtain bacterial ferritin 193, which self-assembled into Ftn193-NPs.

4. A cage-like protein, characterized in that, The cage-like protein comprises multiple subunits, which self-assemble into the cage-like protein, wherein each subunit is bacterial ferritin 193 as described in claim 1.

5. The cage-like protein as described in claim 4, characterized in that, The cage-like protein has the ability to specifically bind to human transferrin receptor 1.

6. The cage-like protein as described in claim 4, characterized in that, The cage-like protein has the ability to cross the blood-brain barrier.

7. The cage-like protein as described in claim 4, characterized in that, The cage-like protein contains a drug, which is a chemotherapy drug, an anti-tumor traditional Chinese medicine, a drug for treating neurodegenerative diseases, or an anti-inflammatory drug. The chemotherapy drug includes alkylating agents, antibiotics, alkaloids, or radiopharmaceuticals. The drug for treating neurodegenerative diseases includes neurotransmitter drugs, dopamine receptor agonists, central nervous system anticholinergic drugs, cholinergic receptor agonists, serotonin system drugs, GABAergic system drugs, or glutamatergic system drugs. The anti-tumor traditional Chinese medicine includes γ-secretase inhibitors.

8. The cage-like protein as described in claim 7, characterized in that, The alkylating agents include one or more of cisplatin, carboplatin, oxaliplatin, lobaplatin, and their derivatives; the antibiotics include one or more of doxorubicin, idarubicin, and epirubicin; the alkaloids include one or more of vinblastine, camptothecin, and podophyllotoxin; the radiopharmaceuticals include one or more of 225Ac, 177Lu, 131I, 89Sr, 32P, and 153Sm; the neurotransmitters include carbidopa, galantamine, rivastigmine, acetylcholine, atropine, scopolamine, dopamine, and their derivatives; and the dopamine receptor agonists include levodopa, pramipexole, and... Apomorphine and other ergot alkaloid derivatives or non-ergot alkaloid derivatives are one or more of the following: the central nervous system anticholinergic drugs include haloperidol, benztropine, or propiconazole; the cholinergic receptor agonists include muscarinic acid, pilocarpine, or varenicline; the serotonin system drugs include fluoxetine, sertraline, sumatriptan, buspirone, ondansetron, and mirtazapine are one or more of the following: the GABAergic system drugs include diazepam, lorazepam, phenobarbital, tiagabine, and sodium valproate are one or more of the following: the glutamatergic system drugs include memantine, ketamine, and perampanel are one or more of the following: the γ-secretase inhibitor is a diflubenzuron.

9. Use of the bacterial ferritin 193 as claimed in claim 1 or the cage-like protein as claimed in claim 4 in the preparation of pharmaceuticals, diagnostic reagents and / or tracer reagents.

10. The application as described in claim 9, characterized in that, The drug is for the treatment of inflammatory diseases, angiogenesis disorders, tuberculosis, HIV, Alzheimer's disease, rheumatoid arthritis, fragile plaque thin fibrous atherosclerosis, multiple sclerosis, or tumors; the tumor is selected from lymphoma, leukemia, breast cancer, esophageal cancer, gastric cancer, ovarian cancer, skin cancer, colorectal cancer, lung cancer, bladder cancer, pancreatic cancer, kidney cancer, head and neck cancer, adrenocortical carcinoma, liver cancer, brain cancer, uterine cancer, bile duct cancer, sarcoma, gallbladder cancer, cervical cancer, prostate cancer, thyroid cancer, rhabdoid tumor, bone cancer, myeloma, testicular cancer, or metastatic cancer thereof; The diagnostic reagent is for diagnosing tumors, autoinflammatory diseases, angiogenic diseases, tuberculosis, HIV, Alzheimer's disease, rheumatoid arthritis, vulnerable plaque thin fibrous atherosclerosis, multiple sclerosis, or tumors; the tumors are selected from lymphoma, leukemia, breast cancer, esophageal cancer, gastric cancer, ovarian cancer, skin cancer, colorectal cancer, lung cancer, bladder cancer, pancreatic cancer, kidney cancer, head and neck cancer, adrenocortical carcinoma, liver cancer, brain cancer, uterine cancer, bile duct cancer, sarcoma, gallbladder cancer, cervical cancer, prostate cancer, thyroid cancer, rhabdoid tumor, bone cancer, myeloma, testicular cancer, or metastatic cancer thereof; The tracer reagent is a tumor tracer reagent, and the tumor is selected from lymphoma, leukemia, breast cancer, esophageal cancer, gastric cancer, ovarian cancer, skin cancer, colorectal cancer, lung cancer, bladder cancer, pancreatic cancer, kidney cancer, head and neck cancer, adrenocortical carcinoma, liver cancer, brain cancer, uterine cancer, bile duct cancer, sarcoma, gallbladder cancer, cervical cancer, prostate cancer, thyroid cancer, rhabdoid tumor, bone cancer, myeloma, testicular cancer or metastatic cancer.

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