Artificially evolved protein cage nanodrugs, preparation method and application thereof

Abstract

This application belongs to the field of nanomedicine technology, and particularly relates to an artificially evolved protein cage nanomedicine, its preparation method, and its application. The artificially evolved protein cage nanomedicine provided by this application includes an artificially evolved protein cage and a nucleic acid-based biomolecular drug. The artificially evolved protein cage is artificially obtained by an artificial evolution system from a 240-polymer nanoprotein cage C27-A with PDB number 6NJ8. The amino acid sequence of the protein subunit of the artificially evolved protein cage is shown in SEQ ID NO.1. The artificially evolved protein cage can efficiently encapsulate nucleic acid-based biomolecular drugs and has advantages such as high protein yield, good stability, good tumor efficacy, and excellent safety. It can solve the technical problem of low drug encapsulation efficiency in existing protein cage nanomedicines.

Description

Technical Field

[0001] This application belongs to the field of nanomedicine technology, and in particular relates to an artificially evolved protein cage nanomedicine, its preparation method and application. Background Technology

[0002] Macromolecular drugs are classified into vaccines, blood and blood products. Based on their source, peptides, proteins, antibodies, polysaccharides and nucleic acids are clinically classified as biological macromolecular drugs.

[0003] Nucleic acid drugs among biological macromolecules have broad prospects in the field of gene therapy. For example, mRNA drugs release mRNA after reaching cells, which is translated into target proteins to exert drug effects. siRNA drugs use RNA interference to silence abnormally expressed genes, opening up new pathways for disease treatment. However, nucleases such as DNase and RNase present in the blood and cells can rapidly decompose nucleic acid drugs.

[0004] Nanomedicine refers to drug delivery systems designed and fabricated using nanotechnology. These systems precisely deliver drug molecules to lesions via nanoscale carriers, enabling highly effective treatment or diagnosis in vivo. Nanomedicine can improve the stability of drug molecules and protect nucleic acid drugs from degradation. Protein cages are also an important delivery tool for nanomedicine. Protein cages are hollow cage-like structures assembled from multiple protein subunits. The hollow nanoscale cavities inside the cage structure encapsulate drug molecules, overcoming the vulnerability of nucleic acid drugs to degradation by nucleases. Furthermore, protein cages exhibit good biocompatibility and immunogenicity. Protein cages offer advantages such as low toxicity, ensuring safe drug delivery. Furthermore, their high modifiability allows for chemical modification to achieve targeted and responsive functions. Their uniform size also ensures stable and consistent drug delivery, and suitable protein cage structures can be designed to meet various needs. However, both traditional and artificially designed protein cages for drug encapsulation generally suffer from low encapsulation efficiency. Encapsulation efficiency plays a crucial role in achieving effective drug concentrations in vivo and ultimately, therapeutic efficacy. Therefore, further improving the encapsulation efficiency of protein cage delivery tools is a pressing challenge. Summary of the Invention

[0005] In view of this, this application provides an artificially evolved protein cage nanomedicine, its preparation method and application, to solve the technical problem of low drug encapsulation efficiency in the prior art.

[0006] The first aspect of this application provides an artificially evolved protein cage nanomedicine, comprising an artificially evolved protein cage and a biological macromolecule drug;

[0007] The amino acid sequence of the protein subunit of the artificially evolved protein cage is shown in SEQ ID NO.1;

[0008] The biological macromolecular drugs are selected from nucleic acid drugs.

[0009] Preferably, the molecular weight of the artificially evolved protein cage is 9117.6 kDa, and the molecular weight of a single protein subunit of the artificially evolved protein cage is 37.99 kDa.

[0010] Preferably, the nucleic acid drug is selected from OVA mRNA.

[0011] A second aspect of this application provides a method for preparing the artificially evolved protein cage nanomedicine described in the first aspect; the preparation method includes the following steps:

[0012] The steps for preparing the nanoprotein cage fusion gene are as follows: by gene recombination, a gene encoding a biomolecule drug-binding peptide is fused to the 5' end of the protein subunit monomer gene encoding the nanoprotein cage, and a gene encoding a protein purification tag is fused to the 3' end via a flexible linker gene, thus obtaining the nanoprotein cage fusion gene.

[0013] The steps for preparing a biomacromolecule drug fusion gene are as follows: a gene encoding HIV protease is fused to the 5' end of the gene encoding the biomacromolecule drug through gene recombination to obtain the biomacromolecule drug fusion gene.

[0014] The steps for constructing a gene library include: using error-prone PCR (epPCR) technology to mutagenesis of the nanoprotein cage fusion gene to obtain a gene library of the nanoprotein cage fusion gene;

[0015] The steps for constructing a plasmid library are as follows: the purified nanocage fusion gene library and the biomolecular drug fusion gene are subcloned into the downstream of the first promoter and the downstream of the second promoter of the pretreated dual promoter plasmid to obtain a plasmid library containing the mutant protein cage nanodrug gene.

[0016] The steps of artificial evolution are: transforming a plasmid library containing the gene of the mutant protein cage nanomedicine into competent cells, cell culture and expansion, adding nanoprotein cage inducer and tetracycline to induce expression, self-assembly, purification, and collecting surviving cells.

[0017] The steps of encapsulating biopharmaceutical drugs are as follows: the nanoprotein cage fusion gene extracted from surviving cells and the biopharmaceutical drug fusion gene are sequentially subcloned into the downstream of the first promoter and the downstream of the second promoter of a dual promoter plasmid; transformed into competent cells; cell culture and amplification; induced co-expression; self-assembly; and purification to obtain artificially evolved protein cage nanomedicines; the amino acid sequence of its protein subunits is shown in SEQ ID NO.1.

[0018] Preferably, the artificial evolution process specifically includes the following steps:

[0019] Step A1: Transform the plasmid library containing the mutant protein cage nanomedicine gene into competent cells, culture and expand the cells, add salicylate or IPTG inducer and add tetracycline at concentrations of 100, 200, 400, 800 and 1600 ng / mL at 2-hour intervals to induce expression, self-assemble, purify, and collect the surviving cells.

[0020] Step A2: Subclone the nano-protein cage fusion gene and the biomolecular drug fusion gene extracted from surviving cells into the downstream of the first promoter and the downstream of the second promoter of the dual promoter plasmid to obtain a plasmid library containing the mutant protein cage nano-drug gene.

[0021] Step A3: Transform the plasmid library containing the mutant protein cage nanomedicine gene into competent cells, culture and expand the cells, add salicylate or IPTG inducer and add tetracycline at concentrations of 400, 800, 1200, 1600 and 3200 ng / mL at 2-hour intervals to induce expression, self-assemble, purify, and collect the surviving cells;

[0022] Step A4: Subclone the nano-protein cage fusion gene and the biomolecular drug fusion gene extracted from surviving cells into the downstream of the first promoter and the downstream of the second promoter of the dual promoter plasmid to obtain a plasmid library containing the mutant protein cage nano-drug gene.

[0023] Step A5: Transform the plasmid library containing the mutant protein cage nanomedicine gene into competent cells, culture and expand the cells, add salicylate or IPTG inducer and add tetracycline at concentrations of 800, 1200, 1600, 3200 and 6400 ng / mL sequentially at 2-hour intervals to induce expression, self-assemble, purify, collect the surviving cells, the surviving cells express the protein cage nanomedicine, the protein cage nanomedicine includes nano protein cages and the encapsulated biomolecule drugs;

[0024] Step A6: Subclone the nano-protein cage fusion gene and the biomolecular drug fusion gene extracted from the surviving cells into the downstream of the first promoter and the downstream of the second promoter of the dual promoter plasmid to obtain a plasmid library containing the mutant protein cage nano-drug gene.

[0025] Step A7: Transform the plasmid library containing the mutant protein cage nanomedicine gene into competent cells, culture and expand the cells, add salicylate or IPTG inducer, and add tetracycline at concentrations of 800, 1200, 1600, 3200 and 6400 ng / mL sequentially at 2-hour intervals to induce expression, self-assemble, purify, and collect the surviving cells.

[0026] Preferably, after the artificial evolution step and before the encapsulation of the biological macromolecule drug, the following screening step is also included:

[0027] Step B1: Subclone the nanoprotein cage fusion gene extracted from the surviving cells into the vector plasmid, transform it into competent cells, culture and expand the cells to obtain surviving cells that can express 12 nanoprotein cage variants.

[0028] Step B2: The surviving cells that can express 12 nanoprotein cage variants are sequentially induced to express, screened for protein yield and / or screened for antigens to obtain nanoprotein cages with high protein yield and / or low antigenicity.

[0029] Step B3: Sequencing of high-protein-yield and / or low-antigenicity nanocages yields the selected nanocage fusion gene extracted from surviving cells.

[0030] Preferably, in the artificial evolution step, cell culture and expansion specifically includes the following steps:

[0031] Step C1: Add chloramphenicol at a concentration of 25 μg / mL to the culture medium used for cell culture and amplification, and culture overnight at 30℃ and 230 rpm / min to screen cells containing plasmid libraries of mutant protein cage nanomedicine genes.

[0032] Step C2: Using a culture medium containing chloramphenicol at a concentration of 25 μg / mL, adjust the initial OD600 of the cells containing the plasmid library of mutant protein cage nanomedicine genes to 0.1 to obtain a culture in the early stage of logarithmic growth.

[0033] Step C3: Incubate the culture in the early stage of logarithmic growth at 30℃ and 230 rpm / min for 1.5 hours to obtain the culture to be induced for expression.

[0034] Preferably, in the step of constructing the gene library, the mutagenesis of the nanoprotein cage fusion gene using error-prone PCR (epPCR) technology includes: using error-prone PCR (epPCR) technology, mutagenesis of the nanoprotein cage fusion gene, primers F and R with homologous arms located on both sides of the coding region of the nanoprotein cage fusion gene, in an Accurate Taq random mutagenesis kit to obtain a gene library of the nanoprotein cage fusion gene.

[0035] Preferably, in the step of constructing the plasmid library, purification is performed by agarose gel electrophoresis, and pretreatment is performed by sequential digestion with EcoRI and AflII enzymes followed by agarose gel electrophoresis purification.

[0036] The third aspect of this application provides the application of the artificially evolved protein cage nanomedicine described in the first aspect in the preparation of tumor therapeutic drugs.

[0037] Compared with existing technologies, the artificially evolved protein cage nanomedicine provided in this application has at least the following beneficial effects:

[0038] 1. This application provides an artificially evolved protein cage nanomedicine, which uses an artificial evolution system to artificially evolve a 240-polymer nanoprotein cage C27-A with PDB number 6NJ8. The resulting artificially evolved protein cage has a large particle size, exceeding 42 nm, which can efficiently encapsulate more nucleic acid-based biomolecules and can express them efficiently with a yield close to 25 mg / L. It also has good delivery and translation efficiency and stability, and exerts significant therapeutic effects such as inhibiting tumor growth.

[0039] 2. The artificially evolved protein cage nanomedicine provided in this application does not cause abnormalities in liver and kidney indicators and anti-protein cage nanomedicine antibody levels, and is a protein cage nanomedicine with good safety. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0041] Figure 1 This is a schematic diagram of the process of preparing the nano-protein cage C27-A by engineering the inner and outer surfaces of the protein cage according to Example 1 of this application.

[0042] Figure 2 This is a spatial schematic diagram of the nano-protein cage C27-A prepared by engineering modification of the inner and outer surfaces of the protein cage according to Example 1 of this application.

[0043] Figure 3 The image shows the cell viability results for Example 1 of this application, obtained by co-expression with IPTG and tetracycline at a concentration of 200-1600 ng / mL or by inducing expression with only tetracycline at a concentration of 200-1600 ng / mL.

[0044] Figure 4 The graph shows the yield results of soluble purified protein of the protein cage nanomedicines provided in Examples 1-2 of this application;

[0045] Figure 5 Electron micrographs of the particle size of the protein cage nanomedicine provided in Examples 1-2 of this application;

[0046] Figure 6 Statistical results of the particle size of protein cage nanomedicines provided in Examples 1-2 of this application;

[0047] Figure 7 These are the results of OVA mRNA encapsulated with protein cage nanomedicines provided in Examples 1-2 of this application;

[0048] Figure 8 The graph shows the translation efficiency results of the in vitro delivery of protein cage nanomedicines provided in Examples 1-2 of this application;

[0049] Figure 9 The graph shows the stability test results of the protein cage nanomedicines provided in Examples 1-2 of this application;

[0050] Figure 10 These are the cell immunoassay results of the protein cage nanomedicines provided in Examples 1-2 of this application;

[0051] Figure 11 The graphs show the humoral immunity test results of the protein cage nanomedicines provided in Examples 1-2 of this application.

[0052] Figure 12 These are the tumor growth inhibition results of the protein cage nanomedicines provided in Examples 1-2 of this application;

[0053] Figure 13 The graphs show the results of liver and kidney indicators in mice provided in Examples 1-2 of this application for protein cage nanomedicine.

[0054] Figure 14 The graph shows the antibody levels of the protein cage nanodrug provided in Examples 1-2 of this application. Detailed Implementation

[0055] This application provides an artificially evolved protein cage nanomedicine, its preparation method, and its application, which addresses the technical problem of low drug encapsulation efficiency in existing protein cage nanomedicines.

[0056] The technical solutions of this application will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0057] Given that the efficiency of protein cage encapsulation plays a crucial role in achieving effective drug concentrations and exerting therapeutic effects in vivo, and considering the current limitations of protein cage delivery tools in terms of encapsulation efficiency, this application provides an artificially evolved protein cage nanomedicine. The provided artificially evolved protein cage nanomedicine comprises an artificially evolved protein cage and a biomolecular drug; wherein, the amino acid sequence (SEQ ID) of the protein subunit of the artificially evolved protein cage encapsulating the biomolecular drug provided in this application is... NO.1) As shown below: MGNARTRRRERRAEKRAQWKAANAGGGNKSQLYPDSPLTDQDFNQLDQTVIEAARRQLVGRRFIEPYGPLGRGMQSAFNDILMESHEGGGEALAEALAEALAEALAGGGAEMDFQGSFDTEVESSRRVDYTIPMLYKDFDLYWRDLEQSKALDIPIDLSVAANAAR DVAFLEDQMIFHGSKEFDIPGPMNVKGRLTHLIGDWYGSGNAFQDIVEARNKPLEMNHNGPYALVLSPELYSLLHRVHKDTNVLEIEHVRELISAGVFQSPVLKGKSGVIVNTGRINLDLAISEDFEAAYLGEEGMYRPFRVYETVVLRIKRPAAICTLIDPEEGGGGGGHHHHHH.

[0058] Currently, by mutating key amino acids on the inner surface of protein cages to positively charged amino acids, the encapsulation efficiency of negatively charged nucleic acid molecules can be enhanced. Introducing specific nucleic acid-binding peptides into protein cages can improve the packaging efficiency of nucleic acid molecules. However, these methods are based on the modifiability of protein cages, and modifying them does not fundamentally improve the encapsulation efficiency. The improvement in encapsulation efficiency is still not significant. The artificially evolved protein cage with a specific amino acid sequence provided in this application was obtained by artificially evolving the 240-polymer nanoprotein cage C27-A with PDB number 6NJ8 through an artificial evolution system. This artificially evolved protein cage has a large particle size, and its internal cavity can efficiently encapsulate nucleic acid-based biomolecules. It can also express drugs efficiently, produce high yields, is not easily degraded, has good stability, and achieves effective concentrations, thus exerting significant therapeutic effects such as inhibiting tumor growth.

[0059] As a preferred embodiment, the artificially evolved protein cage nanomedicine provided in this application has a molecular weight of 9117.6 kDa, obtained based on an artificial evolution system. Furthermore, due to the N-terminus modification with a nucleic acid-binding peptide (amino acid sequence MSTAKLVKSKATN), it can effectively encapsulate nucleic acid drugs such as OVA mRNA.

[0060] The following will describe an artificially evolved protein cage nanomedicine provided by this technical solution in conjunction with embodiments and experimental examples.

[0061] Example 1

[0062] This embodiment 1 provides a method for preparing artificially evolved protein cage nanomedicines. The preparation method includes the steps of preparing a nanoprotein cage fusion gene and a biomacromolecule drug fusion gene, constructing a gene and plasmid library, artificial evolution, and encapsulating the biomacromolecule drug.

[0063] The steps for preparing nanoprotein cage fusion genes and biomolecular drug fusion genes include:

[0064] Based on the native 240-mer protein cage C27 (PDB number 6NJ8), the C-terminus of its protein subunit monomer structure is fully exposed on the outer surface of the cage, and the N-terminus is fully exposed on the inner surface. This allows for the engineering modification of the inner and outer surfaces of the protein cage, enabling the attachment of protein purification tags (amino acid sequence HHHHHH) and specific nucleic acid-binding peptides (amino acid sequence MSTAKLVKSKATN), respectively. The engineering process is as follows: Figure 1 As shown, the spatial diagram is as follows Figure 2 As shown, gene recombination was used to fuse a gene encoding a specific nucleic acid binding peptide at the 5' end of the gene encoding the protein subunit monomer of the nanoprotein cage via a flexible linker gene fusion, and a gene encoding a protein purification tag was also fused at the 3' end via a flexible linker gene fusion, resulting in a nanoprotein cage fusion gene. Gene recombination was used to fuse the gene sequence encoding HIV protease (a dimeric retroviral protease that is toxic to E. coli when produced in the cytoplasm of E. coli) to the 5' end of the OVA mRNA to be packaged, resulting in a biomolecular drug fusion gene, named HIV-OVA. After expression of the nanoprotein cage fusion gene, the inner surface of the protein cage is equipped with 240 nucleic acid binding peptides, and the outer surface carries 240 His tags, named C27-A.

[0065] The steps for constructing gene and plasmid libraries include:

[0066] Error-prone PCR (epPCR) was used to mutagenesis of the prepared nanoprotein cage fusion gene. The epPCR reaction was performed using the Accurate Taq random mutagenesis kit, with a target mutation rate of medium to high (4.5 to 16 mutations / kb). The mutagenesis process used primers F (TTGTTTAACTTTAATAAGGAGATATACCATGGAATTC) and R (ATGCTAGCACTTAAGTTAATGGTGATGATGATGATG) with homologous arms located on both sides of the coding region of the cage gene.

[0067] The amplified epPCR product (1078 bp) was first purified by 1% agarose gel electrophoresis to obtain a purified gene library of the nanocage fusion gene. Then, the pACYCDuet 1 dual T7 promoter plasmid was digested and purified with EcoRI and AflII to obtain a pretreated dual promoter plasmid. The purified nanocage fusion gene library and the biomolecular drug fusion gene were then subcloned into the downstream of the first and second promoters of the pretreated dual promoter plasmid using the QuarCam seamless cloning kit and purified to obtain a plasmid library containing the gene encoding the mutant protein cage nanodrug.

[0068] The steps of artificial evolution include:

[0069] The quantity and quality of the library were evaluated. The plasmid library was transformed into electrocompetent E. coli DH10B cells via electroporation and then incubated in 30 mL of antibiotic-free LB medium at 30 °C and 230 rpm for 1 hour. The library size was estimated by plating serially diluted cells onto LB agar plates containing chloramphenicol (25 μg / mL). The library showed sufficient mutation coverage. Sequencing of representative clones was performed to verify the library quality, and the library showed sufficient effective mutations. The library size confirmed by each round of directed evolution was determined to be 5 × 10⁻⁶. 5 ;

[0070] The cell culture amplification steps are as follows: First, the initial culture of the plasmid library is cultured overnight in LB medium containing chloramphenicol (25 μg / mL) at 30°C and 230 rpm / min to ensure that all surviving cells in the library contain the target plasmid, avoiding interference from empty or low-expression clones in subsequent screening. Then, the initial OD600 is adjusted to 0.1 using fresh LB medium containing chloramphenicol (25 μg / mL) to ensure that all cells are in the early logarithmic growth phase, eliminating interference from differences in growth rate, and this is recorded as the second batch of culture. The second batch of culture is then incubated at 30°C and 230 rpm / min for 1.5 hours to ensure that the culture is in the optimal physiological state for subsequent induction, yielding the culture to be induced for expression.

[0071] The process involved adding a nano-protein cage inducer and tetracycline to induce expression. Salicylate (100 μM) (Sigma; St. Louis, USA) was added to the culture to induce expression to induce the formation of protein cages, which was designated as the third batch of cultures. The third batch of cultures was then induced to express expression by adding tetracycline at concentrations of 100, 200, 400, 800 and 1600 ng / mL at 2-hour intervals. The cells were then self-assembled, purified, and the surviving cells were collected and the protein cage nanomedicine was isolated, thus completing the first round of artificial evolution.

[0072] Total RNA, encoding the nanocage fusion gene and the biomolecule drug fusion gene, was extracted from the protein cage nanomedicine using the Trizol method. This RNA was subcloned downstream of the first and second promoters of a dual-promoter plasmid, yielding a plasmid library containing the gene encoding the mutant protein cage nanomedicine. The cell culture amplification steps were then repeated. Salicylate (100 μM) (Sigma; St. Louis, USA) was added to the culture to induce expression to generate protein cages, resulting in the third batch of cultures. The third batch of cultures was then induced with tetracycline at concentrations of 400, 800, 1200, 1600, and 3200 ng / mL at 2-hour intervals for expression, self-assembly, and purification. Surviving cells were collected, and the protein cage nanomedicine was isolated, marking the second round of artificial evolution.

[0073] Total RNA, encoding the protein cage nanomedicine fusion gene and the biomolecule drug fusion gene, was extracted from the protein cage nanomedicine using the Trizol method. This RNA was then subcloned downstream of the first and second promoters of a dual promoter plasmid to obtain a plasmid library containing the gene encoding the mutant protein cage nanomedicine. The cell culture amplification steps were then repeated. Salicylate (100 μM) (Sigma; St. Louis, USA) was added to the culture to induce expression to induce the generation of protein cages, and this was recorded as the third batch of culture. The third batch of culture was then induced by adding tetracycline at concentrations of 800, 1200, 1600, 3200, and 6400 ng / mL at 2-hour intervals. The cells were then self-assembled, purified, and the surviving cells were collected and the protein cage nanomedicine was isolated. This was the third round of artificial evolution.

[0074] Total RNA, encoding the nanocage fusion gene and the biomolecule drug fusion gene, was extracted from the protein cage nanomedicine using the Trizol method. This RNA was then subcloned downstream of the first and second promoters of a dual-promoter plasmid, yielding a plasmid library containing the gene encoding the mutant protein cage nanomedicine. The cell culture amplification process was repeated, and salicylate (100 μM) (Sigma; St. Louis, USA) was added to the culture to induce expression, resulting in the third batch of cultures. Tetracycline at concentrations of 800, 1200, 1600, 3200, and 6400 ng / mL was added to the third batch of cultures at 2-hour intervals to induce expression, self-assembly, and purification. Viable cells were collected, and the protein cage nanomedicine was isolated, marking the fourth round of artificial evolution. Finally, total RNA, encoding the nanocage fusion gene and the biomolecule drug fusion gene, was extracted from the protein cage nanomedicine again using the Trizol method and subcloned downstream of the first and second promoters of a dual-promoter plasmid, yielding a plasmid library containing the gene encoding the mutant protein cage nanomedicine.

[0075] The steps involved in encapsulating biological macromolecular drugs include:

[0076] The steps for screening nanocage variants included: extracting nucleic acids from surviving cells collected after the fourth round of artificial evolution of the nanocage nanodrug, subcloning it into dual promoter plasmids, and sequencing the constructed plasmid library to obtain a set of representative genes encoding nanocage fusion genes. Then, a set of representative single clones were induced to express the nanocages by adding IPTG, and purified using Ni-NTA and size exclusion chromatography to obtain a set of 12 multimeric nanocage variants of C27, named C27-A1 to C27-A12. Subsequently, the protein purification yield of the 12 variants was analyzed, and antigenicity was assessed. Variants with high yield and low antigenicity were selected for subsequent experiments. The nanocage variant C27-A5 was selected, exhibiting both high yield and low antigenicity.

[0077] The co-expression process involved subcloning the gene encoding the nanocage variant C27-A5 and the OVA mRNA into the downstream of the first and second promoters of the dual promoter plasmid pACYCDuet 1, respectively. The OVA mRNA was then transformed into competent BL21 (DE3) cells, cultured in LB medium for amplification, induced with IPTG and tetracycline for co-expression, self-assembly, and purified by metal affinity chromatography and size exclusion chromatography. This one-step specific encapsulation of OVA mRNA was achieved during the self-assembly of the nanocage variant C27-A5, and the encapsulation system was named C27-A5_OVA.

[0078] The amino acid sequence (SEQ ID NO.1) of the protein subunit in the nanoprotein cage variant C27-A5 in this embodiment is shown below:

[0079] C27-A5 (mutant; not documented in PDB; molecular weight: 9117.6 kDa)

[0080] MGNARTRRRERRAEKRAQWKAANAGGGNKSQLYPDSPLTDQDFNQLDQTVIEAARRQLVGRRFIEPYGPLGRGMQSAFNDILMESHEGGGEALAEALAEALAEALAGGGAEMDFQGSFDTEVESSRRVDYTIPMLYKDFDLYWRDLEQSKALDIPIDLSVAANAARDVAFLEDQMIFHGSKEFDIPGPMNVKGRLTHLIGDWYGSGNAFQDIVEARNKPLEMNHNGPYALVLSPELYSLLHRVHKDTNVLEIEHVRELISAGVFQSPVLKGKSGVIVNTGRINLDLAISEDFEAAYLGEEGMYRPFRVYETVVLRIKRPAAICTLIDPEEGGGGGGHHHHHH (This sequence is the monomeric amino acid sequence of a protein cage, which consists of 240 identical subunits.)

[0081] In this embodiment, after the steps of preparing the nanoprotein cage fusion gene and the biomolecular drug fusion gene, and before the steps of constructing the gene and plasmid library and artificial evolution; to investigate the reliability of artificial evolution, the nanoprotein cage fusion gene and the biomolecular drug fusion gene HIV-OVA were subcloned into the downstream of the first promoter and the downstream of the second promoter of the dual promoter plasmid pACYCDuet 1, respectively. These were then transformed into competent BL21(DE3) cells, cultured and expanded in LB medium, and induced with co-expression of IPTG and tetracycline at concentrations of 200–1600 ng / mL, or with only tetracycline at concentrations of 200–1600 ng / mL. Cell viability was then tested, and the results are as follows: Figure 3 As shown, from Figure 3 It can be seen that when tetracycline is added alone to induce expression, cell viability is poor, and as the concentration of tetracycline increases, cell viability decreases further, making it difficult for cells to survive. However, when tetracycline and IPTG are added simultaneously, cell survival rate is significantly improved. Cells encapsulating HIV-OVA nanocage drugs can survive, while cells that do not express nanocage drugs cannot encapsulate HIV-OVA, resulting in cell death. This indicates that using tetracycline and IPTG at progressively increasing concentrations can serve as a screening condition for artificial evolution.

[0082] Example 2

[0083] This Example 2 provides a method for preparing a protein cage nanomedicine, which serves as a comparative example of Example 1. The preparation method includes the steps of preparing a nanoprotein cage fusion gene and encapsulating a biomacromolecule drug.

[0084] The steps for preparing the nanoprotein cage fusion gene include: Based on the 240-polymer natural protein cage C27 (PDB number 6NJ8), utilizing the property that its protein subunit monomer structure has the C-terminus fully exposed on the outer surface and the N-terminus fully exposed on the inner surface, which can be used to install protein purification tags (amino acid sequence HHHHHH) and specific nucleic acid binding peptides (amino acid sequence MSTAKLVKSKATN), respectively, the inner and outer surfaces of the protein cage are engineered. The engineering process is as follows: Figure 1 As shown, the spatial diagram is as follows Figure 2 As shown, gene recombination was used to fuse a gene encoding a specific nucleic acid binding peptide at the 5' end of the gene encoding the protein subunit monomer of the nanoprotein cage through a flexible linker gene, and a gene encoding a protein purification tag was also fused at the 3' end through a flexible linker gene, resulting in a nanoprotein cage fusion gene. After expression of the nanoprotein cage fusion gene, the inner surface of the protein cage is equipped with 240 nucleic acid binding peptides and the outer surface carries 240 His tags, and it is named C27-A.

[0085] The co-expression process involved subcloning the gene encoding the nanocage C27-A and the OVA mRNA into the downstream of the first and second promoters of the dual promoter plasmid pACYCDuet 1, respectively. The OVA mRNA was then transformed into competent BL21 (DE3) cells, cultured and expanded in LB medium, induced with IPTG and tetracycline, self-assembled, purified by metal affinity chromatography and size exclusion chromatography. This one-step specific encapsulation of OVA mRNA was achieved during the self-assembly of the nanocage variant C27-A, and the encapsulation system was named C27-A_OVA.

[0086] Experimental Example 1

[0087] Example 1 of this experiment tests the performance of the protein cage nanomedicines provided in Examples 1-2. The performance tests include protein purification yield, particle size and encapsulation efficiency.

[0088] The yield results of soluble purified proteins of the protein cage nanomedicines provided in Examples 1-2 are as follows: Figure 4 As shown, from Figure 4 As can be seen, compared with the protein cage nanomedicine provided in Example 2, the protein cage nanomedicine provided in Example 1 has significantly improved protein purification yield due to artificial evolution, with the yield approaching 25 mg / L.

[0089] Electron micrographs of the particle size of the protein cage nanomedicines provided in Examples 1-2 are shown below. Figure 5 As shown, the results are as follows Figure 6 As shown, from Figure 5-6As can be seen, compared with the protein cage nanomedicine provided in Example 2, the particle size of the protein cage nanomedicine provided in Example 1 exceeds 42 nm, while the particle size of the protein cage nanomedicine provided in Example 2 is only about 40 nm. Therefore, the protein cage nanomedicine provided in Example 1 is expected to encapsulate higher capacity nucleic acid molecules OVA mRNA.

[0090] The encapsulation efficiency test of the protein cage nanomedicines provided in Examples 1-2 first used the Trizol method to extract total RNA from the protein cages, and then used next-generation sequencing technology to detect the proportion of OVA mRNA in the total RNA. The results are as follows: Figure 7 As shown, from Figure 7 As can be seen, compared with the protein cage nanomedicine provided in Example 2, the protein cage nanomedicine provided in Example 1 can encapsulate up to 85% of OVA mRNA, which is significantly better than the protein cage nanomedicine provided in Example 2.

[0091] Experiment Example 2

[0092] Example 2 of this experiment tests the performance of the protein cage nanomedicines provided in Examples 1-2. The performance tests include in vitro delivery translation efficiency and stability tests.

[0093] In vitro delivery translation efficiency assays included: fusing OVA mRNA genes encapsulated in nanocage variants C27-A5 and C27-A nanocages into eGFP, named C27-A5_OVA. eGFP and C27-A_OVA eGFP Subsequently, the DC2.4 cells were divided into three groups, one of which served as a negative control group, while the other two groups were treated with the same molar amount of C27-A5_OVA. eGFP and C27-A_OVA eGFP After 24 hours of incubation, the expression of eGFP-labeled antigen was detected by flow cytometry. The results are as follows: Figure 8 As shown, from Figure 8 It can be seen that C27-A5_OVA eGFP The stronger fluorescence intensity indicates that the nanocage variant C27-A5 can deliver and translate the encapsulated eGFP OVA mRNA more efficiently.

[0094] In vitro stability tests included: Benzonase and RNase nucleases were used to co-incubate the protein cage nanomedicines C27-A5_OVA and C27-A_OVA provided in Examples 1-2 at 37°C for 2 hours, respectively. The integrity of the protein cage nanomedicines provided in Examples 1-2 and the degradation of guest mRNA within the protein cages were then assessed. The results are as follows: Figure 9 As shown; from Figure 9It can be seen that, compared with before treatment, the integrity of the protein cage nanomedicine and the integrity of the guest mRNA within the protein cage provided in Example 2 decreased significantly after treatment.

[0095] Experimental Example 3

[0096] Example 3 of this experiment tested the efficacy of the protein cage nanomedicines provided in Examples 1-2. The efficacy tests included cellular immunity tests, humoral immunity tests, and tumor growth inhibition tests.

[0097] The performance testing process included: C57BL / 6 mice were divided into 3 groups, all of which were subcutaneously inoculated with 1×10⁻⁶ mice. 6 Three mouse tumor models were constructed using E.G7-OVA tumor cells. Seven days after inoculation, 50 μg of the protein cage nanomedicines C27-A5_OVA and C27-A_OVA provided in Examples 1-2 were injected intramuscularly into the three mouse tumor models, with injections every 7 days for a total of 3 doses. The negative control group was treated with the same injection volume of PBS. After day 23, the spleen, serum, and tumor tissue of the three groups of mice were collected for evaluation of the efficacy of the vaccine via different routes.

[0098] Cellular immunoassay involved preparing single-cell suspensions from collected mouse spleens and detecting antigen-specific CD8 in each group of mice using flow cytometry. + The activation ratios of T cells, central memory T cells (TCM), and effector memory T cells (TEM) were as follows: Figure 10 As shown, from Figure 10 It can be seen that the negative control group activated CD8 + The proportions of T cells, central memory T cells, and effector memory T cells were all at their lowest levels, while the activated CD8+ T cells in mice injected with the protein cage nanomedicine C27-A5_OVA provided in Example 1 were significantly reduced. + The proportions of T cells, central memory T cells, and effector memory T cells were all the highest.

[0099] The humoral immunity test involves collecting mouse serum and using ELISA to detect antigen-specific antibody levels. The results are as follows: Figure 11 As shown, from Figure 11 It can be seen that mice injected with the protein cage nanodrug C27-A5_OVA provided in Example 1 can induce a higher titer of antigen-specific antibody IgG, which is higher than that induced in mice injected with the protein cage nanodrug C27-A_OVA provided in Example 2.

[0100] The tumor growth inhibition test involved measuring the wet weight of collected mouse tumor tissues and calculating the inhibitory effect of each drug group on tumor growth. The results are as follows: Figure 12 As shown, from Figure 12It can be seen that the negative control group had no inhibitory effect. The mice injected with the protein cage nanodrug C27-A_OVA provided in Example 2 had a heavier tumor wet weight, ranging from 1.5 to 2 g, and a lower inhibition rate. In contrast, the mice injected with the protein cage nanodrug C27-A5_OVA provided in Example 1 showed significant tumor inhibition, with a tumor weight of less than 0.5 g, and the best inhibitory effect.

[0101] Experiment Example 4

[0102] Example 4 of this experiment tested the safety of the protein cage nanomedicines provided in Examples 1-2. The safety tests included mouse liver and kidney marker tests and detection of anti-protein cage nanomedicine antibody levels.

[0103] The safety testing process included: C57BL / 6 mice were divided into 3 groups. Two groups of mice were intramuscularly injected with 100 μg of the protein cage nanomedicines C27-A5_OVA and C27-A_OVA provided in Examples 1-2 on days 1, 14, and 35, respectively. The remaining group of mice was a negative control group treated with the same injection volume of PBS. On day 56, serum from each group of mice was collected for safety assessment.

[0104] Mouse liver and kidney marker tests involved collecting mouse serum and using commercially available kits to measure aspartate aminotransferase (AST), alanine aminotransferase (ALT), uric acid (UA), and creatinine (CRE). The test results are as follows: Figure 13 As shown, from Figure 13 It can be seen that the liver and kidney function indicators of the mice injected with the protein cage nanomedicines C27-A5_OVA and C27-A_OVA provided in Examples 1-2 and the mice injected with PBS did not change significantly, indicating that high-dose, multiple vaccinations did not produce toxic side effects in the mice.

[0105] Antibody levels in anti-protein cage nanomedicine were detected using ELISA in collected mouse serum. The results are as follows: Figure 14 As shown, from Figure 14 It can be seen that the antibody level in the serum of mice injected with C27-A_OVA was higher than that in mice injected with C27-A5_OVA. This indicates that the protein cage nanodrug C27-A5_OVA provided in Example 1 is safer than the protein cage nanodrug C27-A_OVA provided in Example 2. The immune response of the body to the protein cage nanodrug C27-A5_OVA itself is weaker. The protein cage nanodrug C27-A5_OVA can be repeatedly injected over a long period of time and has the potential to be used as a universal nucleic acid delivery platform.

[0106] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application 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 therein. Such 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 this application.

Claims

1. An artificially evolved protein cage nanomedicine, characterized in that, Including artificially evolved protein cages and biological macromolecular drugs; The amino acid sequence of the protein subunit of the artificially evolved protein cage is shown in SEQ ID NO.1; The biological macromolecular drugs are selected from nucleic acid drugs; The nucleic acid drug is selected from OVA mRNA.

2. The artificially evolved protein cage nanomedicine according to claim 1, characterized in that, The molecular weight of the artificially evolved protein cage is 9117.6 kDa.

3. The method for preparing an artificially evolved protein cage nanomedicine according to claim 1 or 2, characterized in that, Includes the following steps: The steps for preparing the nanoprotein cage fusion gene are as follows: by gene recombination, a gene encoding a biomolecule drug-binding peptide is fused to the 5' end of the protein subunit monomer gene encoding the nanoprotein cage, and a gene encoding a protein purification tag is fused to the 3' end via a flexible linker gene, thus obtaining the nanoprotein cage fusion gene. The steps for preparing a biomacromolecule drug fusion gene are as follows: a gene encoding HIV protease is fused to the 5' end of the gene encoding the biomacromolecule drug through gene recombination to obtain the biomacromolecule drug fusion gene. The steps for constructing a gene library include: using error-prone PCR (epPCR) technology to mutagenesis of the nanoprotein cage fusion gene to obtain a gene library of the nanoprotein cage fusion gene; The steps for constructing a plasmid library are as follows: the purified nanocage fusion gene library and the biomolecular drug fusion gene are subcloned into the downstream of the first promoter and the downstream of the second promoter of the pretreated dual promoter plasmid to obtain a plasmid library containing the mutant protein cage nanodrug gene. The steps of artificial evolution are: transforming a plasmid library containing the gene of the mutant protein cage nanomedicine into competent cells, cell culture and expansion, adding nanoprotein cage inducer and tetracycline to induce expression, self-assembly, purification, and collecting surviving cells. The steps for encapsulating biopharmaceutical drugs are as follows: the nanoprotein cage fusion gene extracted from surviving cells and the biopharmaceutical drug fusion gene are sequentially subcloned into the downstream of the first promoter and the downstream of the second promoter of a dual promoter plasmid; the cells are transformed into competent cells; the cells are cultured and expanded; co-expression is induced; self-assembly is performed; and the cells are purified to obtain artificially evolved protein cage nanomedicines.

4. The method for preparing an artificially evolved protein cage nanomedicine according to claim 3, characterized in that, The steps of artificial evolution specifically include the following: Step A1: Transform the plasmid library containing the mutant protein cage nanomedicine gene into competent cells, culture and expand the cells, add salicylate or IPTG inducer and add tetracycline at concentrations of 100, 200, 400, 800 and 1600 ng / mL at 2-hour intervals to induce expression, self-assemble, purify, and collect the surviving cells. Step A2: Subclone the nano-protein cage fusion gene and the biomolecular drug fusion gene extracted from surviving cells into the downstream of the first promoter and the downstream of the second promoter of the dual promoter plasmid to obtain a plasmid library containing the mutant protein cage nano-drug gene. Step A3: Transform the plasmid library containing the mutant protein cage nanomedicine gene into competent cells, culture and expand the cells, add salicylate or IPTG inducer and add tetracycline at concentrations of 400, 800, 1200, 1600 and 3200 ng / mL at 2-hour intervals to induce expression, self-assemble, purify, and collect the surviving cells. Step A4: Subclone the nano-protein cage fusion gene and the biomolecular drug fusion gene extracted from surviving cells into the downstream of the first promoter and the downstream of the second promoter of the dual promoter plasmid to obtain a plasmid library containing the mutant protein cage nano-drug gene. Step A5: Transform the plasmid library containing the mutant protein cage nanomedicine gene into competent cells, culture and expand the cells, add salicylate or IPTG inducer and add tetracycline at concentrations of 800, 1200, 1600, 3200 and 6400 ng / mL at 2-hour intervals to induce expression, self-assemble, purify, and collect the surviving cells. Step A6: Subclone the nano-protein cage fusion gene and the biomolecular drug fusion gene extracted from the surviving cells into the downstream of the first promoter and the downstream of the second promoter of the dual promoter plasmid to obtain a plasmid library containing the mutant protein cage nano-drug gene. Step A7: Transform the plasmid library containing the mutant protein cage nanomedicine gene into competent cells, culture and expand the cells, add salicylate or IPTG inducer, and add tetracycline at concentrations of 800, 1200, 1600, 3200 and 6400 ng / mL sequentially at 2-hour intervals to induce expression, self-assemble, purify, and collect the surviving cells.

5. The method for preparing an artificially evolved protein cage nanomedicine according to claim 3, characterized in that, Following the artificial evolution step and preceding the encapsulation of biological macromolecules, the following screening steps are also included: Step B1: Extract the nano-protein cage fusion gene from the surviving cells and induce its expression to obtain surviving cells that can express 12 nano-protein cage variants. Step B2: Screen surviving cells that can express 12 nanoprotein cage variants for protein yield and / or antigen to obtain nanoprotein cages with high protein yield and / or low antigenicity. Step B3: Sequencing the high-protein-yield and / or low-antigenicity nanocages to obtain the selected nanocage fusion gene extracted from surviving cells.

6. The method for preparing an artificially evolved protein cage nanomedicine according to claim 3, characterized in that, The artificial evolution process specifically includes the following steps in cell culture and expansion: Step C1: Add chloramphenicol at a concentration of 25 μg / mL to the culture medium used for cell culture and amplification, and culture overnight at 30℃ and 230 rpm / min to screen cells containing plasmid libraries of mutant protein cage nanomedicines. Step C2: Using a culture medium containing chloramphenicol at a concentration of 25 μg / mL, adjust the initial OD600 of the cells containing the plasmid library of mutant protein cage nanomedicine genes to 0.1 to obtain a culture in the early stage of logarithmic growth. Step C3: Incubate the culture in the early stage of logarithmic growth at 30℃ and 230 rpm / min for 1.5 hours to obtain the culture to be induced for expression.

7. The method for preparing an artificially evolved protein cage nanomedicine according to claim 3, characterized in that, The step of constructing the gene library includes mutagenesis of the nanoprotein cage fusion gene using error-prone PCR (epPCR) technology, which involves using error-prone PCR (epPCR) technology to mutagenesis of the nanoprotein cage fusion gene, primers F and R with homologous arms located on both sides of the coding region of the nanoprotein cage fusion gene, in an Accurate Taq random mutagenesis kit to obtain a gene library of the nanoprotein cage fusion gene.

8. The method for preparing an artificially evolved protein cage nanomedicine according to claim 3, characterized in that, In the step of constructing the plasmid library, purification is performed by agarose gel electrophoresis, and pretreatment is performed by sequential digestion with EcoRI and AflII enzymes followed by agarose gel electrophoresis purification.

9. The application of the artificially evolved protein cage nanomedicine according to claim 1 or 2 in the preparation of tumor therapeutic drugs.

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