Recombinant ferritin nanoparticles and methods of making the same

Abstract

The application discloses a recombinant ferritin nanoparticle and a preparation method thereof. The application belongs to the field of biological medicine and specifically relates to a recombinant ferritin nanoparticle and a preparation method thereof. The method for preparing the recombinant ferritin comprises the following steps: enabling a coding gene of the recombinant ferritin to be expressed in a prokaryotic microorganism to obtain the recombinant ferritin, wherein the recombinant ferritin is connected by a ferritin and a signal peptide, and the signal peptide can be NspA, Omp or Sip. The application matches the signal peptide suitable for expression and secretion of the ferritin sequence for the ferritin sequence, and uses a prokaryotic expression strain to perform high-efficiency protein expression, so that the defects of low expression and difficult separation and purification in the prior art are overcome, and a prokaryotic expression system and method capable of efficiently expressing the ferritin in the periplasmic space are provided.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to recombinant ferritin nanoparticles and their preparation methods. Background Technology

[0002] Escherichia coli is currently the most mature engineered bacterium used. It possesses a clear genetic background and exhibits characteristics such as short growth cycle, ease of culture, low cost, high yield, and easy purification. Therefore, it is widely used in the pharmaceutical, food, and industrial fields to produce recombinant proteins. However, despite its many advantages, E. coli lacks the post-translational processing capabilities of eukaryotic cells. The recombinant proteins it secretes often easily form inclusion bodies or are even hydrolyzed by intracellular proteases, posing a significant challenge for industrial-scale production using E. coli.

[0003] The periplasmic space of *E. coli* contains abundant disulfide bond oxidoreductases and peptidyl-prolyl isomerases, which help proteins fold correctly. The oxidative environment within the periplasmic space is conducive to the formation of disulfide bonds. Furthermore, compared to the cytoplasm, the periplasmic space contains less protein and has lower protease activity, which is beneficial for the purification and collection of recombinant proteins and avoids intracellular degradation. Therefore, the periplasmic space of *E. coli* is one of the ideal sites for recombinant protein secretion. The signal peptide is a short peptide composed of 15-30 amino acids fused to the N-terminus of the recombinant protein. Based on its structure and function, it can be divided into N-regions, H-regions, and C-regions, and plays a role in guiding the transport of recombinant proteins to the periplasmic space.

[0004] Ferritin is a class of iron-storing globular proteins widely found in organisms, assembled from 24 subunits, with an outer diameter of 12 nanometers and an internal cavity diameter of 8 nanometers. Its cage-like structure endows it with excellent thermal and chemical stability, making it easy to modify using chemical or genetic engineering methods. Human H subunit ferritin (HFn) has been shown to bind to transferrin receptor 1 (TfR1) expressed on the surface of various types of tumors, exhibiting its ability to actively target and accumulate in tumors. Furthermore, it demonstrates low immune response and toxicity after application to the body, showing great application potential.

[0005] Nanoparticles, with their small size, high specific surface area, and ease of modification, play a crucial role in targeted therapy, molecular imaging, early diagnosis, and drug delivery. As novel contrast agents and drug carriers, nanoparticles will be widely used in tumor imaging and treatment in the future. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to efficiently express ferritin nanoparticles in a prokaryotic expression system.

[0007] To address the above problems, this invention provides a ferritin, its preparation method, and its applications.

[0008] The present invention first provides a recombinant ferritin, which comprises ferritin and a signal peptide linked together.

[0009] The ferritin may include any of the following:

[0010] A1) The amino acid sequence of this protein is that of SEQ ID No. 20;

[0011] A2) A fusion protein obtained by fusion protein tagging the carboxyl terminus of the protein shown in A1);

[0012] A3) Proteins obtained by substituting and / or deleting and / or adding amino acid residues of proteins A1) or A2) have more than 80% identity with and the same function as proteins A1) or A2).

[0013] The signal peptide may be any of the following:

[0014] E1) The amino acid sequence is the polypeptide of SEQ ID No. 2;

[0015] E2) The amino acid sequence is the polypeptide of SEQ ID No. 4;

[0016] E3) The amino acid sequence is the polypeptide of SEQ ID No. 6;

[0017] E4) The amino acid sequence is the polypeptide of SEQ ID No. 8;

[0018] E5) The amino acid sequence is the polypeptide of SEQ ID No. 10;

[0019] E6) The amino acid sequence is the polypeptide of SEQ ID No. 12;

[0020] E7) is the polypeptide of SEQ ID No. 14;

[0021] The amino acid sequence of E8 is the polypeptide of SEQ ID No. 16;

[0022] E9) is the polypeptide of SEQ ID No. 18;

[0023] E10) A polypeptide with more than 80% identity and the same function as the polypeptides of E1)-E9) obtained by substituting and / or deleting and / or adding amino acid residues of the polypeptides described in E1)-E9).

[0024] The ferritin N or C segment may be tagged with tags, including FC, HIS, SpyCatcher, etc. The ferritin may also be linked to restriction enzyme sites. These linkages can be achieved via a linker or through direct connection.

[0025] According to the present invention, the preferred recombinant ferritin is composed of a signal peptide, a tag, and ferritin linked sequentially. A more preferred embodiment is composed of a signal peptide, a His tag, an enzyme cleavage site, a SpyCatcher, and ferritin linked sequentially.

[0026] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0027] In this document, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0028] In this document, the 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0029] In this article, the ferritin may be a codon-optimized protein.

[0030] In heterologous expression systems, optimization steps can enhance the host's ability to produce foreign proteins. Protein expression is controlled by a multitude of factors, including those affecting the stability and initiation of transcription, mRNA processing, and translation. Polynucleotide optimization steps can include steps to enhance the host's ability to produce foreign proteins, as well as steps to assist researchers in effectively designing expression constructs. Optimization strategies can include, for example, modifications to the translation initiation region, alterations to mRNA structural elements, and the use of different codon biases. Methods for optimizing nucleic acid sequences to enhance the expression of heterologous proteins in bacterial hosts are known in the art.

[0031] Therefore, optimization can address any of the various sequence characteristics of a heterologous gene. As a specific example, translational arrest caused by rare codons can lead to reduced expression of heterologous proteins. Rare codon-induced translational arrest includes the presence of codons in the target polynucleotide that are rarely used in the host organism, which can negatively impact protein translation due to their absence in the available tRNA pool. Methods to improve optimal translation in the host organism include codon optimization that can lead to the removal of rare host codons from the synthesized polynucleotide sequence.

[0032] Alternative translation initiation can also lead to reduced expression of heterologous proteins. Alternative translation initiation may involve synthetic polynucleotide sequences that incidentally contain motifs capable of functioning as ribosome-binding sites (RBS). These sites can induce translation of truncated proteins from within the gene. One approach to reducing the likelihood of truncated proteins (which may be difficult to remove during purification) involves eliminating the putative internal RBS sequence from the optimized polynucleotide sequence.

[0033] Polymerase slippage caused by repetition can lead to decreased expression of heterologous proteins. Repetition-induced polymerase slippage involves nucleotide sequence repetitions that indicate slippage in DNA polymerases capable of frameshift mutations. Such repetitive sequences can also cause slippage in RNA polymerases. In organisms with a high G+C content bias, there may be a higher degree of repetitive sequences consisting of G or C nucleotide repeats. Therefore, one approach to reducing the likelihood of RNA polymerase slippage involves altering the extended repetitive sequences of G or C nucleotides.

[0034] Interference with secondary structures can also lead to decreased expression of heterologous proteins. Secondary structures can isolate RBS sequences or start codons and are associated with decreased protein expression. Stem-loop structures can also be involved in transcriptional termination and attenuation. Optimized polynucleotide sequences can contain minimal secondary structures in the RBS and coding regions of the nucleotide sequence to allow for improved transcription and translation.

[0035] Another feature that may affect heterologous protein expression is the presence of restriction sites. The polynucleotide sequence can be optimized by removing restriction sites that might interfere with subsequent subcloning of the transcription unit into the host expression vector.

[0036] For example, the optimization process can begin by identifying the amino acid sequence required for heterologous expression by the host. Candidate polynucleotides or DNA sequences can be designed from this amino acid sequence. When designing the synthetic DNA sequence, the frequency of codon selection can be compared with the codon selection of the host expressing organism, and rare host codons can be removed from the synthetic sequence. Furthermore, the synthesized candidate DNA sequence can be modified to remove unwanted restriction enzyme sites, and to add or remove any desired signal sequences, adapters, or untranslated regions. The presence of secondary structures in the synthetic DNA sequence that might interfere with the translation process, such as G / C repeat sequences and stem-loop structures, can be analyzed. Before synthesizing the candidate DNA sequence, the optimized sequence design can be tested to confirm that the sequence correctly encodes the required amino acid sequence. Finally, the candidate DNA sequence can be synthesized using DNA synthesis techniques, such as those known in the art.

[0037] In another embodiment of the invention, universal codon selection in a host organism such as *E. coli* can be used to optimize the expression of heterologous polynucleotide sequences. The percentage and distribution of rare codons considered preferred for specific amino acids in the host expression system can be evaluated. Selection rate values ​​of 5% and 10% can be used as critical values ​​for determining rare codons.

[0038] This invention contemplates the use of any ferritin-coding sequence, including any sequence optimized for expression in the intended *E. coli* host cells. The sequence intended for use may be optimized to any degree, including, but not limited to, optimization to eliminate: codons occurring less than 5% of the *E. coli* host cells, codons occurring less than 10% of the *E. coli* host cells, translational termination caused by rare codons, presumed internal RBS sequences, extended repeat sequences of G or C nucleotides, interfering secondary structures, restriction sites, or combinations thereof.

[0039] In practice, the amino acid sequence of any useful signal peptide can be encoded by any suitable nucleic acid sequence.

[0040] The signal peptide sequences that can be used in the methods of the present invention are not limited to those disclosed in Table 1.

[0041] The present invention also provides biomaterials related to the above-mentioned recombinant ferritin.

[0042] The biomaterial related to the above-mentioned recombinant ferritin provided by this invention may be any of the following:

[0043] B1) The nucleic acid molecule that encodes the protein;

[0044] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0045] B3) A recombinant vector containing the nucleic acid molecules described in B1);

[0046] B4) A recombinant vector containing the expression cassette described in B2);

[0047] B5) Recombinant microorganisms containing the nucleic acid molecules described in B1);

[0048] B6) Recombinant microorganisms containing the expression cassette described in B2);

[0049] B7) Recombinant microorganisms containing the recombinant vector described in B3);

[0050] B8) Recombinant microorganisms containing the recombinant vector described in B4).

[0051] The vectors described herein are well-known to those skilled in the art, enabling convenient recombinant DNA procedures and the expression of nucleotide sequences. The choice of vector will generally depend on its compatibility with the host cell to which it will be introduced. Vectors can be linear or closed circular plasmids. These include, but are not limited to, plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), or viral vectors. Specifically, the pET28a vector may be used.

[0052] The vector can be a self-replicating vector, that is, a vector that exists as an extrachromosomal entity whose replication is independent of chromosome replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector can contain any means to ensure self-replication. Alternatively, the vector can be one that integrates into the genome when introduced into a host cell and replicates along with the chromosome in which the vector is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, can be used, collectively containing the complete DNA of the host cell genome to be introduced, or transposons can be used.

[0053] The vector of the present invention preferably contains one or more (e.g., several) selective markers that allow for easy selection of cells after transformation, transfection, transduction, etc. The selective markers are genes whose products provide resistance to biocides or viruses, resistance to heavy metals, protrophic resistance to auxotrophs, etc.

[0054] In the above-mentioned biological materials, the microorganisms may be bacteria, yeast, insect cells, or mammalian cells.

[0055] The microorganisms mentioned above are prokaryotic microorganisms.

[0056] In the above-mentioned biological materials, the prokaryotic microorganisms are Gram-negative bacteria.

[0057] In the above-mentioned biological materials, the Gram-negative bacteria may be Escherichia coli.

[0058] The Escherichia coli bacteria mentioned may specifically be Escherichia coli BL21(DE3) or Escherichia coli Rosetta.

[0059] This invention also provides a method for preparing recombinant ferritin.

[0060] The method for preparing recombinant ferritin provided by the present invention includes expressing the encoding gene of the recombinant ferritin in prokaryotic microorganisms to obtain the recombinant ferritin, wherein the recombinant ferritin is composed of ferritin and a signal peptide linked together.

[0061] In the above method, the ferritin may be any of the following:

[0062] A1) The amino acid sequence of this protein is that of SEQ ID No. 20;

[0063] A2) A fusion protein obtained by fusion protein tagging the carboxyl terminus of the protein shown in A1);

[0064] A3) Proteins obtained by substituting and / or deleting and / or adding amino acid residues of proteins A1) or A2) have more than 80% identity with and the same function as proteins A1) or A2).

[0065] In the above method, the signal peptide may be any of the following:

[0066] E1) The amino acid sequence is the polypeptide of SEQ ID No. 2;

[0067] E2) The amino acid sequence is the polypeptide of SEQ ID No. 4;

[0068] E3) The amino acid sequence is the polypeptide of SEQ ID No. 6;

[0069] E4) The amino acid sequence is the polypeptide of SEQ ID No. 8;

[0070] E5) The amino acid sequence is the polypeptide of SEQ ID No. 10;

[0071] E6) The amino acid sequence is the polypeptide of SEQ ID No. 12;

[0072] E7) is the polypeptide of SEQ ID No. 14;

[0073] The amino acid sequence of E8 is the polypeptide of SEQ ID No. 16;

[0074] E9) is the polypeptide of SEQ ID No. 18;

[0075] E10) A polypeptide with more than 80% identity and the same function as the polypeptides of E1)-E9) obtained by substituting and / or deleting and / or adding amino acid residues of the polypeptides described in E1)-E9).

[0076] The ferritin of this invention can be secreted into the periplasmic space of the host through a signal peptide, maintaining its native conformation, folding into nanoparticles, and facilitating purification.

[0077] The ferritin of the present invention can be secreted into the host periplasmic space through the action of one or more signal peptides with amino acid sequences of SEQ ID No. 2, 4, 6.

[0078] The ferritin host of this invention can be bacteria, yeast, insect cells, or mammalian cells. The bacteria can be from, but are not limited to, species such as *Escherichia sp.*, *Erwinia sp.*, *Agrobacterium sp.*, *Flavobacterium sp.*, *Alcaligenes sp.*, *Pseudomonas sp.*, and *Bacillus sp.*. For example, the bacteria can be *Escherichia coli*, *Bacillus subtilis*, or *Bacillus pumilus*.

[0079] In one or more embodiments of the present invention, the bacteria are preferably Escherichia coli, more preferably BL21(DE3), BL21(DE3)Star, or B834(DE3).

[0080] In the above method, expressing the coding gene of the recombinant ferritin in prokaryotic microorganisms includes introducing the coding gene into recipient Escherichia coli to obtain recombinant Escherichia coli expressing the coding gene, culturing the recombinant Escherichia coli, and expressing the recombinant ferritin.

[0081] The introduction can be achieved by transforming the host bacteria with a vector carrying the DNA molecule of the present invention through any known transformation method, such as chemical transformation or electroporation. The introduced DNA molecule can be a single copy or multiple copies. The introduction can be the integration of a foreign gene into the host chromosome or the expression of the gene extrachromosomally by a plasmid.

[0082] In one specific embodiment, the recombinant Escherichia coli is a recombinant microorganism expressing the recombinant protein obtained by introducing the pET-28a- exogenous gene into Escherichia coli BL21(DE3), and the recombinant protein is a recombinant vector obtained by replacing a small fragment between the multiple cloning recognition sites of the vector pET-28a(+) with the exogenous gene.

[0083] In the above embodiments, the polyclonal identification points are NcoI and XhoI identification sites.

[0084] In the above embodiments, the induction temperature is 18-37℃.

[0085] The inducer may be IPTG, and the concentration of the IPTG inducer may be 0.2 to 1.0 mM.

[0086] The induction time can be 3h-16h, adjusted according to the induction temperature.

[0087] The density variation of the recombinant bacteria during induction ranged from 0.2 to 0.6 absorbance units (AU). 600 .

[0088] The induced pH change range can be 6-7.5.

[0089] In the above methods, cells are cultured in a nutrient medium suitable for producing the recombinant protein using methods well known in the art. For example, cells can be cultured by shake-flask culture in a suitable medium and under conditions that allow for the expression and / or isolation of the recombinant protein, and by small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state fermentation) in laboratory or industrial fermenters.

[0090] The protein is cultured in a suitable nutrient medium containing a carbon and nitrogen source and inorganic salts, using methods known in the art. Suitable media are available from commercial suppliers or can be prepared according to publicly available compositions (e.g., in the catalogue of the U.S. Center for Type Culture Collection). If the recombinant protein is secreted into the nutrient medium, it can be recovered directly from the medium. If the recombinant protein is not secreted into the medium, it can be recovered from cell lysate.

[0091] The recombinant proteins of the present invention can be purified by a variety of methods known in the art to obtain substantially pure proteins, including but not limited to chromatography (e.g., ion exchange, affinity, hydrophobicity, chromatography focusing, and size exclusion), electrophoresis (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, for example, Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

[0092] Methods for identifying induced proteins are also known in the art. For example, proteins can be analyzed by using peptide mass fingerprinting with a MALDI-TOF mass spectrometer, N-terminal sequencing analysis, or peptide profiling.

[0093] This invention also provides any of the following applications:

[0094] Y1) Application of the above recombinant proteins in the preparation of drugs and biological products;

[0095] Y2) Application of the above-mentioned biomaterials in the preparation of drugs and biological products;

[0096] Y3) Application of the above-mentioned method for preparing recombinant ferritin in the preparation of drugs and biological products.

[0097] The preferred drug is a vaccine, but it is not limited to this.

[0098] This study successfully expressed ferritin with a fused specific signal peptide and optimized codons using a prokaryotic expression system. After purification using a nickel column, ferritin with a natural nanoparticle structure was obtained. This ferritin exhibits uniform particle size and stable structure, and can be expressed by fusion with protein drugs or antigens, overcoming the shortcomings of low expression levels and difficult isolation and purification in existing technologies. This invention provides a prokaryotic expression system and method for efficiently expressing ferritin suitable for biological experiments or production in periplasmic space, offering a reference for the research and application of ferritin nanoparticles in the biomedical field. Attached Figure Description

[0099] Figure 1 An overview of the strategy for constructing vector clones for prokaryotic expression of ferritin.

[0100] Figure 2This study describes the low-level induced expression of ferritin carrying a signal peptide. Lane 1 is a molecular weight marker (Solarbio, catalog number PR1910, hereinafter the same); Lane 2 is the uninduced recombinant strain BL21-pET28a transfected with the empty pET28a vector; Lane 3 is the induced recombinant strain BL21-pET28a transfected with the empty pET28a vector; Lane 4 is the uninduced recombinant strain BL21-pET28a-Ferritin transfected with the nanoparticle plasmid pET28a-Ferritin without the signal peptide; and Lane 5 is the induced recombinant strain BL21-pET28a-Ferritin transfected with the nanoparticle plasmid pET28a-Ferritin without the signal peptide. Lane 6 is the recombinant strain BL21-pET28a-Ferritin-FlgI, which was transfected with a nanoparticle plasmid containing the fusion FlagI signal peptide without induction; lane 7 is the recombinant strain BL21-pET28a-Ferritin-FlgI, which was transfected with a nanoparticle plasmid containing the fusion FlagI signal peptide without induction; lane 8 is the recombinant strain BL21-pET28a-Ferritin-NspA, which was transfected with a nanoparticle plasmid containing the fusion NspA signal peptide without induction; and lane 9 is the recombinant strain BL21-pET28a-Ferritin-NspA, which was transfected with a nanoparticle plasmid containing the fusion NspA signal peptide without induction.

[0101] Figure 3 Small-scale expression of ferritin fused with different signal peptides was induced. Figure 3Lane A: Lane 1 is a molecular weight marker; Lane 2 is the uninduced recombinant strain BL21-pET28a transfected with the empty pET28a vector; Lane 3 is the induced recombinant strain BL21-pET28a transfected with the empty pET28a vector; Lane 4 is the uninduced recombinant strain BL21-pET28a-Ferritin-NspA transfected with a nanoparticle plasmid fused with the NspA signal peptide; Lane 5 is the induced recombinant strain BL21-pET28a-Ferritin-NspA transfected with a nanoparticle plasmid fused with the NspA signal peptide; Lane 6 is the uninduced recombinant strain BL21-pET28a-Ferritin-DsbA transfected with the nanoparticle plasmid pET28a-Ferritin; Lane 7 is the induced recombinant strain transfected with the DsbA signal peptide transfected with the nanoparticle plasmid pET28a-Ferritin. The recombinant strain BL21-pET28a-Ferritin-DsbA contains the peptide nanoparticle plasmid pET28a-Ferritin. Lane 8 is the uninduced recombinant strain BL21-pET28a-Ferritin-Omp containing the nanoparticle plasmid fusion with the Omp (OmpA) signal peptide. Lane 9 is the induced recombinant strain BL21-pET28a-Ferritin-Omp containing the nanoparticle plasmid fusion with the Omp signal peptide. Lane 10 is the uninduced recombinant strain BL21-pET28a-Ferritin-Yral containing the nanoparticle plasmid fusion with the Yral signal peptide. Lane 11 is the induced recombinant strain BL21-pET28a-Ferritin-Yral containing the nanoparticle plasmid fusion with the Yral signal peptide. Figure 3Lane B: Lane 1 is a molecular weight marker. Lane 1 is the recombinant strain BL21-pET28a-Ferritin-NspA, induced to carry a nanoparticle plasmid containing the fusion NspA signal peptide. Lane 2 is the uninduced recombinant strain BL21-pET28a-Ferritin-CcmH, induced to carry a nanoparticle plasmid containing the fusion CcmH signal peptide. Lane 3 is the induced bacterial cell BL21-pET28a-Ferritin-CcmH, induced to carry a nanoparticle plasmid containing the fusion CcmH signal peptide. Lane 4 is the uninduced recombinant strain BL21-pET28a-Ferritin-Tort, induced to carry a nanoparticle plasmid containing the fusion Tort signal peptide. Lane 5 is the induced strain containing a nanoparticle plasmid containing the fusion Tort signal peptide. The recombinant strain BL21-pET28a-Ferritin-Tort contains nanoparticle plasmids. Lane 6 is the uninduced recombinant strain BL21-pET28a-Ferritin-Sip containing nanoparticle plasmids fused with the Sip signal peptide. Lane 7 is the induced recombinant strain BL21-pET28a-Ferritin-Sip containing nanoparticle plasmids fused with the Sip signal peptide. Lane 8 is the uninduced recombinant strain BL21-pET28a-Ferritin-NikA containing nanoparticle plasmids fused with the NikA signal peptide. Lane 9 is the induced recombinant strain BL21-pET28a-Ferritin-NikA containing nanoparticle plasmids fused with the NikA signal peptide.

[0102] Figure 4Small-scale ferritin expression was induced at different temperatures. In the gel electrophoresis diagrams under induction conditions of 18℃ and 25℃, lane 1 is the molecular weight marker; lanes 2 and 4 are the uninduced recombinant strain BL21-pET28a transfected with the empty pET28a vector; lanes 3 and 5 are the induced recombinant strain BL21-pET28a transfected with the empty pET28a vector; lanes 6 and 8 are the uninduced recombinant strain BL21-pET28a-Ferritin-NspA transfected with a nanoparticle plasmid containing the fused NspA signal peptide; and lanes 7 and 9 are the induced recombinant strain BL21-pET28a-Ferritin-NspA transfected with a nanoparticle plasmid containing the fused NspA signal peptide. n-NspA; In the gel electrophoresis diagram under 37℃ induction conditions, lane 1 is the molecular weight marker, lane 2 is the uninduced recombinant strain BL21-pET28a transfected with the empty pET28a vector, lane 3 is the induced recombinant strain BL21-pET28a transfected with the empty pET28a vector, lane 4 is the uninduced recombinant strain BL21-pET28a-Ferritin-NspA transfected with a nanoparticle plasmid containing the fused NspA signal peptide, and lane 5 is the induced recombinant strain BL21-pET28a-Ferritin-NspA transfected with a nanoparticle plasmid containing the fused NspA signal peptide.

[0103] Figure 5 To induce ferritin expression in different expression strains. Figure 5 Lane A: Lane 1 is a molecular weight marker; Lane 2 is uninduced recombinant E. coli BL21-pET28a transfected with the empty pET28a vector; Lane 3 is induced recombinant E. coli BL21-pET28a transfected with the empty pET28a vector; Lane 4 is uninduced recombinant E. coli BL21-pET28a-Ferritin-Omp transfected with a nanoparticle plasmid containing the fusion Omp signal peptide; Lane 5 is induced recombinant E. coli BL21-pET28a-Ferritin-Omp transfected with a nanoparticle plasmid containing the fusion Omp signal peptide; Lane 6 is uninduced recombinant E. coli transfected with the fusion Omp signal peptide nanoparticle plasmid containing the empty pET28a vector. Recombinant E. coli BL21-pET28a-Ferritin-Sip transfected with nanoparticle plasmid containing the Sip signal peptide; Lane 7 is recombinant E. coli BL21-pET28a-Ferritin-Sip transfected with nanoparticle plasmid containing the fused Sip signal peptide; Lane 8 is recombinant E. coli BL21-pET28a-Ferritin-NspA transfected with nanoparticle plasmid containing the fused NspA signal peptide; Lane 9 is recombinant E. coli BL21-pET28a-Ferritin-NspA transfected with nanoparticle plasmid containing the fused NspA signal peptide. Figure 5Lane B: Lane 1 is a molecular weight marker; Lane 2 is uninduced recombinant E. coli Rosetta-pET28a transfected with the empty pET28a vector; Lane 3 is induced recombinant E. coli Rosetta-pET28a transfected with the empty pET28a vector; Lane 4 is uninduced recombinant E. coli Rosetta-pET28a-Ferritin-Omp transfected with a nanoparticle plasmid containing the fusion Omp signal peptide; Lane 5 is induced recombinant E. coli Rosetta-pET28a-Ferritin-Omp transfected with a nanoparticle plasmid containing the fusion Omp signal peptide; Lane 6 is uninduced recombinant E. coli transfected with the fusion Omp signal peptide. Recombinant E. coli Rosetta-pET28a-Ferritin-Sip transparticles containing the Sip signal peptide nanoparticle plasmid are shown in lanes 7, 8, and 9.

[0104] Figure 6Western blotting was used to detect ferritin-induced expression of different signal peptide fusions. Lane 1 is a molecular weight marker; lane 2 is the uninduced recombinant strain BL21-pET28a transfected with the empty pET28a vector; lane 3 is the induced recombinant strain BL21-pET28a transfected with the empty pET28a vector; lane 4 is the uninduced recombinant strain BL21-pET28a-Ferritin-FlgI transfected with a nanoparticle plasmid containing the fused FlgI signal peptide; lane 5 is the induced recombinant strain BL21-pET28a-Ferritin-FlgI transfected with a nanoparticle plasmid containing the fused FlgI signal peptide; lane 6 is the uninduced recombinant strain BL21-pET28a-Ferritin-NspA transfected with a nanoparticle plasmid containing the NspA signal peptide; and lane 7 is the induced recombinant strain BL21-pET28a-Ferritin-NspA transfected with a nanoparticle plasmid containing the NspA signal peptide. Lane 8 is the recombinant strain BL21-pET28a-Ferritin-NspA, which is a nanoparticle plasmid containing the fusion Omp (OmpA) signal peptide. Lane 9 is the recombinant strain BL21-pET28a-Ferritin-Omp, which is a nanoparticle plasmid containing the fusion Omp signal peptide after induction. Lane 10 is the recombinant strain BL21-pET28a-Ferritin-Sip, which is a nanoparticle plasmid containing the fusion Sip signal peptide after induction. Lane 11 is the recombinant strain BL21-pET28a-Ferritin-Sip, which is a nanoparticle plasmid containing the fusion Sip signal peptide after induction.

[0105] Figure 7 SDS-PAGE analysis was performed on the supernatant of the recombinant strain Rosetta-pET28a-Ferritin-NspA to observe protein expression after staining.

[0106] Figure 8 SDS-PAGE analysis was performed on the cell lysate supernatant of recombinant strain Rosetta-pET28a-Ferritin-NspA after purification by nickel column, and the protein expression was observed after staining.

[0107] Figure 9 It is in the nanoparticle state of ferritin. Detailed Implementation

[0108] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0109] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0110] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0111] Example 1: Construction of plasmids for prokaryotic expression system

[0112] Construction strategy of prokaryotic ferritin expression vector clones, such as Figure 1 As shown.

[0113] The design of this application's sequence: To facilitate purification and subsequent removal of the His tag, this invention adds a His tag and an enzyme cleavage site after the signal peptide, and incorporates a SpyCatcher tag to bind to the target gene, thereby obtaining ferritin nanoparticles tagged with SpyCatcher. These nanoparticles can play an important role in targeted therapy, molecular imaging, early diagnosis, and drug delivery. They are linked using commonly used linkers in the field, such as GSS and GGSGGS.

[0114] In summary, the sequence structures of nanoparticle plasmids without signal peptides are as follows: His tag (6H) restriction site - linker SpyCatcher Linker - ferritin; the sequence structures of nanoparticle plasmids with signal peptides are as follows: signal peptide - His tag (6H) restriction site - linker SpyCatcher Linker - ferritin, where the signal peptides are shown in Table 1.

[0115] The SpyCatcherde sequence (sequence 21 in the sequence list) mentioned above is as follows:

[0116] GAMVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFY LYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT

[0117] The sequence of the ferritin mentioned above (sequence 20 in the sequence listing) is as follows:

[0118] DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKSGS

[0119] Table 1. Signal peptide sequences of ferritin nanoparticles

[0120]

[0121]

[0122] Nanoparticle plasmid sequences with and without the signal peptides shown in Table 1 were designed, and 10 designed sequences were inserted into the NcoI and XhoI sites of the pET28a expression vector via gene synthesis. To ensure complete reading frame accuracy, the NcoI site on the vector was disrupted during gene synthesis (only CC was retained in the 5' flanking sequence, and ATGG was deleted). The synthesized plasmids were named BL21-pET28a-Ferritin (no signal), BL21-pET28a-Ferritin-NspA, BL21-pET28a-Ferritin-Omp, BL21-pET28a-Ferritin-Sip, BL21-pET28a-Ferritin-FIgI, BL21-pET28a-Ferritin-DsbA, BL21-pET28a-Ferritin-TerT, BL21-pET28a-Ferritin-CcmH, BL21-pET28a-Ferritin-Yral, and BL21-pET28a-Ferritin-NikA, a total of 10 plasmids (all synthesized, prepared, and sequenced by Nanjing Genscript Biotech Co., Ltd.). Reference Figure 1 .

[0123] Example 2: Effects of different signal peptides on ferritin expression

[0124] Ten prokaryotic expression plasmids (without and with different signal peptides) and one empty vector pET28a obtained in Example 1 were transformed into E. coli BL21(DE3) competent cells (purchased from Beijing Polymer Biotechnology Co., Ltd., catalog number MF040). Positive clones were screened, and the recombinant strains of positive clones that were verified by sequencing were named as follows: BL21-pET28a-Ferritin (no signal), BL21-pET28a-Ferritin-NspA, BL21-pET28a-Ferritin-Omp, and BL21-pE Eleven strains were identified: T28a-Ferritin-Sip, BL21-pET28a-Ferritin-FIgI, BL21-pET28a-Ferritin-DsbA, BL21-pET28a-Ferritin-TerT, BL21-pET28a-Ferritin-CcmH, BL21-pET28a-Ferritin-Yral, BL21-pET28a-Ferritin-NikA, and BL21-pET28a (recombinant E. coli transformed into the empty pET28a vector).

[0125] Take 3 mL of LB medium and culture the recombinant strain and the empty vector recombinant strain BL21-pET28a (empty vector control strain) obtained above at 37℃ and 220 rpm until OD200. 600nm Approximately 0.6-0.8 g of IPTG (purchased from INALCO, catalog number 1758-1400) was added to a concentration of 1 mM. The induction temperature was 18°C, and bacterial cells were collected after 5 hours of induction. The cells were resuspended in PBS buffer (purchased from Solarbio, catalog number P1020), and protein loading buffer was added. The mixture was incubated at 95°C for 10 min, then centrifuged at 12000 rpm for 20 min, and the supernatant was collected. The supernatant was analyzed by SDS-PAGE, and staining was performed to observe protein expression.

[0126] SDS-PAGE analysis of protein expression results are as follows: Figure 2 and Figure 3As shown, the recombinant strains BL21-pET28a-Ferritin-NspA, BL21-pET28a-Ferritin-Omp, and BL21-pET28a-Ferritin-Sip, containing the signal peptides NspA, Omp, and Sip, clearly expressed the target band (at 35 kDa), indicating that the recombinant strains BL21-pET28a-Ferritin-NspA, BL21-pET28a-Ferritin-Omp, and BL21-pET28a-Ferritin-Sip can better express the nanoparticles of this invention. The recombinant strains BL21-pET28a-Ferritin (no signal), BL21-pET28a-Ferritin-FIgI, BL21-pET28a-Ferritin-DsbA, BL21-pET28a-Ferritin-TerT, BL21-pET28a-Ferritin-CcmH, BL21-pET28a-Ferritin-Yral, BL21-pET28a-Ferritin-NikA, and BL21-pET28a, which do not contain the signal peptide, did not show expression of the target gene.

[0127] This shows that signal peptides are very important for the expression of the nanoparticles by the strain, and different signal peptides have different effects on the expression of the target gene. Among them, nanoparticle sequences with signal peptides NspA, Omp, and Sip can significantly promote the expression of the target gene under induced expression.

[0128] Example 3: Effect of different induction temperatures on protein expression in nanoparticles

[0129] Take 3 ml of LB medium and culture the recombinant strain BL21-pET28a-Ferritin-NspA obtained in Example 2 and the empty vector recombinant strain BL21-pET28a (empty vector control strain) at 37°C until OD200. 600 Approximately 0.6-0.8. Induction was performed using 1 mM IPTG (INALCO, catalog number 1758-1400) at temperatures of 18℃, 25℃, and 37℃. Bacterial cells were collected after 5 hours. The cells were resuspended in 100 μL of PBS buffer (0.01 M, pH 7.2-7.4, Solarbio, catalog number P1020). 20 μL of 5× protein loading solution was added, and the mixture was centrifuged at 95℃ for 10 min. The supernatant was analyzed by SDS-PAGE, and staining was performed to observe protein expression.

[0130] SDS-PAGE analysis of protein expression results are as follows: Figure 4As shown, BL21-pET28a-Ferritin-NspA can induce recombinant ferritin nanoparticles at induction temperatures of 18℃, 25℃, and 37℃.

[0131] Example 4: Effects of different expression strains on protein expression in nanoparticles

[0132] 1. Construction of different expression strains

[0133] The plasmids pET28a-Ferritin-NspA, pET28a-Ferritin-Omp, and pET28a-Ferritin-Sip, as well as the empty vector pET28a, constructed in Example 1, were transformed into Escherichia coli BL21 competent cells (purchased from Beijing Polymer Biotechnology Co., Ltd., catalog numbers MF040 and MF040-R). Positive clones were screened and verified by PCR electrophoresis or sequencing results. The successfully verified positive clone recombinant strains were named recombinant strains BL21-pET28a-Ferritin-NspA, BL21-pET28a-Ferritin-Omp, BL21-pET28a-Ferritin-Sip, and BL21-pET28a, respectively.

[0134] The plasmids pET28a-Ferritin-NspA, pET28a-Ferritin-Omp, and pET28a-Ferritin-Sip, as well as the empty pET28a vector, constructed in Example 1 were transformed into E. coli Rosetta competent cells (purchased from Beijing Polymer Biotechnology Co., Ltd., catalog numbers MF040 and MF040-R). Positive clones were screened and verified by PCR electrophoresis or sequencing results. The successfully verified positive clone recombinant strains were named Rosetta-pET28a-Ferritin-NspA, Rosetta-pET28a-Ferritin-Omp, Rosetta-pET28a-Ferritin-Sip, and Rosetta-pET28a, respectively.

[0135] 2. Effects of different expression strains on protein expression in nanoparticles

[0136] Take 3 ml of LB medium and culture the recombinant strains BL21-pET28a-Ferritin-NspA, BL21-pET28a-Ferritin-Omp, BL21-pET28a-Ferritin-Sip, BL21-pET28a, Rosetta-pET28a-Ferritin-NspA, Rosetta-pET28a-Ferritin-Omp, Rosetta-pET28a-Ferritin-Sip, and Rosetta-pET28a at 37℃ and 220 rpm until OD280. 600 Approximately 0.6-0.8. Add 1 mM IPTG (purchased from INALCO, catalog number 1758-1400), incubate at 37℃, and collect bacterial cells after 5 hours of induction. Resuspend the bacterial cells in PBS buffer (0.01 M, pH 7.2-7.4, purchased from Solarbio, catalog number P1020), add 20 μL of protein loading solution, incubate at 95℃ for 10 min, centrifuge, and collect the supernatant. Perform SDS-PAGE analysis on the supernatant to observe protein expression.

[0137] SDS-PAGE analysis of protein expression results are as follows: Figure 5 As shown, around 35 kDa, the recombinant strains BL21-pET28a-Ferritin-NspA, BL21-pET28a-Ferritin-Omp, BL21-pET28a-Ferritin-Sip, Rosetta-pET28a-Ferritin-NspA, Rosetta-pET28a-Ferritin-Omp, and Rosetta-pET28a-Ferritin-Sip all exhibited the target band, indicating that both the recombinant expression strains Escherichia coli BL21 and Rosetta can induce the expression of recombinant ferritin nanoparticles.

[0138] 3. Western Blot Analysis of Nanoparticle Proteins

[0139] The above-mentioned BL21-pET28a-Ferritin, BL21-pET28a-Ferritin-FIgI, Rosetta-pET28a-Ferritin-NspA, Rosetta-pET28a-Ferritin-Omp, and Rosetta-pET28a-Ferritin-Sip were analyzed by Western blotting, and the steps are as follows:

[0140] 1) SDS-PAGE electrophoresis: Prepare a 10% SDS-PAGE gel with a thickness of 1.0 mm. Perform gel electrophoresis in 1×SDS electrophoresis buffer. Load 20 μL of protein sample. Use 80V voltage until the sample enters the separating gel, then switch to 130V.

[0141] 2) Semi-dry transfer: Prepare one PVDF membrane of the same size as the separating gel and six sheets of filter paper. Wet the membrane with 1× membrane transfer buffer (39mM glycine, 48mM Tris, 0.037% SDS, 20% methanol). After electrophoresis, cut off the excess stacking and separating gels. Construct a graphite electrode-transfer membrane gel complex in the following direction: anode electrode - three layers of wetted filter paper - PVDF membrane - protein gel - three layers of wetted filter paper - cathode electrode. Connect the power supply and apply 1.0 mA / cm² according to the gel area. 2 Constant current transfer membrane for 60 min.

[0142] 3) Blocking: Soak the membrane in PBST blocking buffer containing 5% skim milk powder and block at 37°C for 1 hour.

[0143] 4) Primary antibody incubation: Immerse the sealed membrane in 1×PBST buffer containing the primary antibody (Proteintech, His-Tag Monoclonal Antibody, catalog number: 66005-1-Ig) and incubate at 37°C for 60 min. After incubation, wash the membrane three times with 1×PBST on a shaker at 70 rpm for 10 min each time.

[0144] 5) Secondary antibody incubation: Add secondary antibody (Cell Signaling Technology, catalog number: 7076S) diluted with 1×PBST and incubate at 37°C for 45 min. After incubation, wash the membrane three times for 10 min each time with 1×PBST on a shaker at 70 rpm.

[0145] 6) Color development: Color development was performed using the DAB color development kit (Solarbio, catalog number: DA1016).

[0146] The Western Blot results are as follows: Around 35 kDa, the target bands appeared in the recombinant strains Rosetta-pET28a-Ferritin-NspA, Rosetta-pET28a-Ferritin-Omp, and Rosetta-pET28a-Ferritin-Sip, indicating that the bands expressed by the recombinant expression strain Escherichia coli Rosetta were confirmed to be the designed recombinant ferritin nanoparticles.

[0147] Example 5: Isolation of Periplasmic Space Proteins

[0148] Methods: Periplasmic space proteins were separated by disruption and centrifugation.

[0149] Take 1 L of LB medium and culture the recombinant strain Rosetta-pET28a-Ferritin-NspA at 37℃ and 220 rpm until OD. 600 Approximately 0.6 μL of the bacterial suspension was retained, and 3 mL was centrifuged to collect the supernatant, yielding the uninduced sample. 1 mM IPTG (purchased from INALCO, catalog number 1758-1400) was added, and the induction temperature was adjusted to 18°C. After 15 hours of induction, the bacterial cells were collected. The cells were centrifuged at 8000 rpm for 10 min at room temperature, yielding approximately 1.2 g of bacteria. The cells were resuspended in 60 mL of TB Buffer (TB Buffer: 500 mM NaCl, 20 mM Tris-HCl, 1 mM MPMSF, pH 8). The cells were then disrupted using a cell disruptor (50% power, disruption time 20 min), followed by centrifugation at 8000 rpm, 4°C, for 45 min. The disrupted precipitate and supernatant were obtained. Approximately 50 mL of the supernatant was collected for further purification.

[0150] All precipitated samples were resuspended in 100 μL of PBS buffer, and 20 μL of 5× protein loading solution was added. 100 μL of all supernatant samples were aspirated and 20 μL of 5× protein loading solution was added. All samples were treated in a metal bath at 95°C for 10 min, then centrifuged at 12000 rpm for 10 min. The supernatant was collected for SDS-PAGE analysis, and staining was performed to observe protein expression.

[0151] Depend on Figure 7 As can be seen, there are clear target-sized bands in the supernatant (lane 6 from left to right) after the recombinant strain Rosetta-pET28a-Ferritin-NspA was broken up.

[0152] Example 5: Purification of Ferritin

[0153] The cell lysate supernatant from the fermentation broth was purified using Ni packing material.

[0154] Bacterial lysis buffer NW Rose Ni FF, 100ml (Suzhou Nanomicro, batch number: L07D2903-1), XK16 chromatography column (Cytiva), equilibration buffer: 20mM PB + 0.5M NaCl + 5mM imidazole, pH 7.4, elution buffer: 20mM PB + 0.5M NaCl + 0.5M imidazole, pH 7.4.

[0155] Experimental Procedure: The cell lysis supernatant of Rosetta-pET28a-Ferritin-NspA from Example 4 was centrifuged at 8000 rpm for 1 h to obtain approximately 50 ml of solution. Equilibration buffer was added to a final volume of 200 ml. The chromatography column was equilibrated with equilibration buffer, and the sample was loaded using an A1 pump at a flow rate of 2 ml / min. After loading, the column was washed with equilibration buffer until the absorbance returned to its pre-loading value and stabilized. Gradient elution was performed at a flow rate of 2 ml / min, 0-100% B, for 50 min. The elution peak was collected. The supernatant (concentrated 10-fold), elution buffer, and 5× protein loading solution (20 μL) were added, and the column was treated at 95°C for 10 min followed by centrifugation. The supernatant was analyzed by SDS-PAGE, and staining was performed to observe protein expression. Figure 8 From left to right, lane 1 contains the molecular weight marker, lane 2 contains the supernatant before purification (concentrated 10 times), and lane 3 contains the elution buffer. Figure 8 As can be seen, the target protein can be purified from the supernatant released in the periplasmic space.

[0156] Mass spectrometry confirmation procedure: The bands at position 35Kd in lane 2 (before purification) and lane 3 (elution) of the Coomassie Brilliant Blue stained SDS-PAGE gel of the purified Rosetta-pET28a-Ferritin-NspA product were cut and sent to the detection provider (Sangon Biotech (Shanghai) Co., Ltd.). The detection method was selected as Maldi-TOF-TOF. After mass spectrometry analysis, it was confirmed to be the designed target protein sequence.

[0157] Example 6: Nanoparticle state of ferritin

[0158] The purified ferritin obtained in Example 5 was imaged using transmission electron microscopy to determine its particle morphology.

[0159] To obtain electron micrographs, the purified ferritin particles were stained before microscopy. The specific negative staining procedure was as follows: The ultrathin carbon membrane was pre-vacuumed for 3 minutes using a Harrick Basic Plasma Cleaner PDC-32G-2 instrument, followed by glow discharge at medium setting for 30 seconds, and then removed. A 4 μm sample was pipetted onto the carbon membrane, placed horizontally for 1 minute, blotted dry with filter paper, and then 7 μm of 2% uranium acetate was added. After standing for 1 minute, the membrane was blotted dry with filter paper, and then electron microscopy was performed.

[0160] Transmission electron microscopy observation results as follows Figure 8 As shown, when magnified to 150,000 times, spherical particles that meet the target size (particle size of about 10-15 nm) can be observed, and most particles show a relatively uniform distribution.

[0161] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Recombinant ferritin, characterized in that: The recombinant ferritin is composed of ferritin and a signal peptide that guides ferritin expression. The ferritin is any one of the following: A1) The amino acid sequence of this protein is that of SEQ ID No. 20; A2) A fusion protein obtained by fusion protein tagging the carboxyl terminus of the protein shown in A1); The signal peptide is any one of the following: E1) The amino acid sequence of this polypeptide is that of SEQ ID No. 2; E2) The amino acid sequence of this polypeptide is that of SEQ ID No. 4; E3) is a polypeptide with the amino acid sequence of SEQ ID No.

6.

2. A biomaterial related to the recombinant ferritin of claim 1, wherein the biomaterial is any one of the following: B1) A nucleic acid molecule encoding the protein of claim 1; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecules described in B1); B4) A recombinant vector containing the expression cassette described in B2); B5) Recombinant microorganisms containing the nucleic acid molecules described in B1); B6) Recombinant microorganisms containing the expression cassette described in B2); B7) Recombinant microorganisms containing the recombinant vector described in B3); B8) Recombinant microorganisms containing the recombinant vector described in B4); The recombinant microorganism is Escherichia coli.

3. A method for preparing recombinant ferritin, comprising expressing the gene encoding the recombinant ferritin in a prokaryotic microorganism to obtain the recombinant ferritin, wherein the recombinant ferritin is the recombinant ferritin according to claim 1; and the prokaryotic microorganism is Escherichia coli.

4. The method according to claim 3, characterized in that: Expressing the recombinant ferritin encoding gene of claim 1 in prokaryotic microorganisms includes introducing the recombinant ferritin encoding gene linked to the signal peptide into recipient Escherichia coli to obtain recombinant Escherichia coli expressing the encoding gene, culturing the recombinant Escherichia coli, and expressing the recombinant ferritin.

5. The method according to claim 3 or 4, characterized in that: The expression was induced by an inducer, namely IPTG.

6. The method according to claim 5, characterized in that: The induction time can be 3 hours. The induction process lasted 16 hours, with the induction temperature being 18-37℃.

7. Any of the following applications: Y1) The use of the recombinant ferritin described in claim 1 in the preparation of vaccines; Y2) The application of the biomaterial described in claim 2 in the preparation of vaccines; Y3) The application of the method described in claim 3 or 4 in the preparation of a vaccine.

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