A repetitive structural unit recombinant protein cpE2 and its preparation method and application

Abstract

The present application relates to a repetitive structural unit recombinant protein cpE2 and its preparation method and application. The repetitive structural unit recombinant protein cpE2 has self-assembly performance and is transformed from the E2 subunit of the pyruvate dehydrogenase complex and the dihydrolipoamide transacetylase. Specifically, the present application utilizes the principle of cyclic recombination to place the C-terminus of the E2 protein at the N-terminus through a connecting peptide, thereby constructing the recombinant protein cpE2. The engineered cpE2 protein cage of the present application can self-assemble into a cage-like structure, and the connecting group can be brought into the interior of the protein cage by modifying the N-terminus or C-terminus of cpE2, respectively, thereby overcoming the problem that it is difficult for traditional E2 proteins to place modifiers or drugs into the protein cage. At the same time, the specific particle size of the cpE2 protein cage can be controlled by the length of the connecting peptide to achieve efficient transportation of goods.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a recombinant protein cpE2 having a repeating structural unit, and a preparation method and application thereof. Background Art

[0002] Cancer, cardiovascular and cerebrovascular diseases, and central nervous system diseases are the three most serious diseases. In the field of cancer, the application of nanotechnology has become a research hotspot, among which nanoparticles have shown great potential as drug delivery systems. At present, traditional nano-delivery systems mainly include organic carriers (such as liposomes, polymer nanoparticles), inorganic carriers (such as gold, silica, magnetic nanoparticles) and biomimetic carriers (such as extracellular vesicles, DNA nanostructures, hydrogels).

[0003] Nanoprotein cages have attracted much attention in cargo packaging and delivery due to their unique and ideal properties. Protein nanocages are highly oligomeric structures assembled from single protein components that are rapidly produced by biological expression systems. They can be produced by the precise self-assembly of repeating protein subunits, and the cages usually have a certain amount of space inside to package exogenous molecules. Compared with non-biological nanomaterials, protein cages have higher biocompatibility and biodegradability and are highly safe. As molecular components of nanomedical devices for delivering antigens and therapeutic molecules, nanoprotein cages show many advantages over non-biological nanomaterials, including ultra-high atomic-level precision assembly advantages, geometric surface modification, and biocompatibility.

[0004] Although nanoprotein cages have been used in drug delivery systems, drug delivery still faces severe challenges due to the complex biological systems inside cells, such as endosomal escape and precise positioning of organelles. Problems with existing technologies include: 1. Low delivery efficiency and specificity: Many nanoparticles are difficult to achieve cell-specific targeting, resulting in imprecise drug distribution. 2. Biocompatibility and safety issues: Some nanomaterials may cause immune responses or long-term toxicity. 3. Insufficient stability and controllability: The stability and release behavior of nanoparticles in the body are difficult to control. 4. Complex production process: The synthesis of nanoparticles with specific functions is often complex and difficult to produce on a large scale. 5. Uneven size distribution: Size control and distribution of nanoparticles are the key to achieving efficient delivery, but existing technologies make it difficult to ensure uniformity. Summary of the invention

[0005] In view of this, the present application develops an E2 protein cage delivery system based on a cyclic recombination strategy. The E2 protein cage is derived from the pyruvate dehydrogenase (PDH) complex of Bacillus stearothermophilus. Through cyclic recombination technology, the present application can introduce non-natural functional substances on the surface or inside of the E2 protein cage to enhance drug adsorption or achieve targeted drug delivery, thereby providing a new and efficient drug delivery solution.

[0006] In a first aspect, the present application provides a recombinant protein cpE2 having a repeating structural unit, wherein the recombinant protein cpE2 comprises, from N-terminus to C-terminus, a C-terminal segment of a pyruvate dehydrogenase complex E2 subunit (PDH-E2), a flexible peptide 1, and an N-terminal segment of PDH-E2.

[0007] In some embodiments, the amino acid sequence of the pyruvate dehydrogenase complex E2 subunit (PDH-E2) is as shown in SEQ ID NO: 01.

[0008] In some embodiments, the C-terminal segment of the above-mentioned pyruvate dehydrogenase complex E2 subunit (PDH-E2) comprises positions 240-242 of SEQ ID NO: 01; preferably, comprises positions 238-242 of SEQ ID NO: 01; more preferably, comprises positions 235-242 of SEQ ID NO: 01; and / or, the N-terminal segment of the above-mentioned pyruvate dehydrogenase complex E2 subunit (PDH-E2) comprises positions 1-200 of SEQ ID NO: 01; preferably, comprises positions 1-230 of SEQ ID NO: 01; more preferably, comprises positions 1-234 of SEQ ID NO: 01.

[0009] In some embodiments, the C-terminal segment of the pyruvate dehydrogenase complex E2 subunit (PDH-E2) comprises positions 240-242 of SEQ ID NO: 01; preferably, comprises positions 238-242 of SEQ ID NO: 01; more preferably, comprises positions 235-242 of SEQ ID NO: 01.

[0010] In a preferred embodiment, the C-terminal segment of the pyruvate dehydrogenase complex E2 subunit (PDH-E2) comprises positions 235-242 of SEQ ID NO:01.

[0011] In a preferred embodiment, the C-terminal segment of the pyruvate dehydrogenase complex E2 subunit (PDH-E2) is positions 235-242 of SEQ ID NO: 01.

[0012] In some embodiments, the N-terminal segment of the pyruvate dehydrogenase complex E2 subunit (PDH-E2) comprises positions 1-200 of SEQ ID NO: 01; preferably, comprises positions 1-230 of SEQ ID NO: 01; more preferably, comprises positions 1-234 of SEQ ID NO: 01.

[0013] In a preferred embodiment, the N-terminal segment of the pyruvate dehydrogenase complex E2 subunit (PDH-E2) comprises positions 1-234 of SEQ ID NO:01.

[0014] In a preferred embodiment, the N-terminal segment of the pyruvate dehydrogenase complex E2 subunit (PDH-E2) is positions 1 to 234 of SEQ ID NO: 01.

[0015] In some embodiments, the amino acid sequence of the flexible peptide 1 is selected from GGS, GGGS, GGGGS, GGSGSS, and GTGGSGSS.

[0016] In a preferred embodiment, the amino acid sequence of the flexible peptide 1 is selected from GTGGSGSS.

[0017] In a preferred embodiment, the amino acid sequence of the above-mentioned repeating structural unit recombinant protein cpE2 is shown as SEQ ID NO: 02.

[0018] On the other hand, the present application provides a fusion protein, which includes, from N-terminus to C-terminus, a targeting polypeptide, the above-mentioned repeating structural unit recombinant protein cpE2, flexible peptide 2, and cytokine.

[0019] In some embodiments, the above-mentioned targeting polypeptide is selected from tumor homing peptides, cell penetrating peptides, and cell targeting peptides; preferably LinTT1 peptide, LyP-1 peptide, and Ln5-P4 peptide; more preferably LinTT1 peptide; and / or, the above-mentioned cytokine is selected from interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), and colony stimulating factor (CSF); preferably interferon (IFN).

[0020] In some embodiments, the targeting polypeptide is selected from tumor homing peptides, cell penetrating peptides, and cell targeting peptides; preferably, LinTT1 peptide, LyP-1 peptide, and Ln5-P4 peptide; more preferably, LinTT1 peptide.

[0021] In a preferred embodiment, the above-mentioned targeting polypeptide is selected from LinTT1 peptide.

[0022] In some embodiments, the above-mentioned cytokine is selected from interleukin (IL), interferon (IFN), tumor necrosis factor (TNF), colony stimulating factor (CSF); preferably, interferon (IFN).

[0023] In a preferred embodiment, the above-mentioned cytokine is selected from interferon (IFN).

[0024] In a preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 05.

[0025] The present application provides a biological material, which is a polynucleotide, wherein the polynucleotide encodes the above-mentioned repeating structural unit recombinant protein cpE2, or the above-mentioned fusion protein.

[0026] The present application provides a biological material, which is an expression vector, and the expression vector contains the above-mentioned polynucleotide.

[0027] The present application provides a biological material, which is a cell, wherein the cell comprises the above-mentioned expression vector.

[0028] The present application provides a protein cage for drug delivery, which is prepared from the above-mentioned repeating structural unit recombinant protein cpE2.

[0029] The present application provides the use of the above-mentioned protein cage in the preparation of a drug delivery carrier.

[0030] The present application has the following advantages: 1. High stability: stable at high temperature, able to resist protein degradation in serum in vivo and able to penetrate cells, suitable for the in vivo environment, and an ideal drug delivery container. 2. Uniform size: The dodecahedral E2 protein cage is composed of 60 identical subunits, which self-assemble to form a regular hollow structure with a diameter of 25 nm. 3. Biocompatibility: As a natural protein, it has good biocompatibility and biodegradability. 4. Potential for genetic engineering: New functions such as targeting ligands or pH-responsive polypeptides can be introduced through genetic engineering. Through cyclic recombination technology, the present invention uses the protein cage E2 protein as a drug carrier, which can improve the targeting of treatment and reduce side effects, meeting the current urgent need for precision treatment in the medical field. The present invention can introduce non-natural functional substances on the surface or inside of the E2 protein cage, enhance drug adsorption or achieve targeted drug delivery, thereby providing a new and efficient drug delivery solution. The purpose of the present invention is to improve the efficiency and specificity of drug delivery through this innovative protein cage delivery system, while ensuring biocompatibility and safety, and providing new possibilities for medical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Mechanism diagram of this application.

[0032] Figure 2 : Schematic diagram of the protein expression results of Example 1.

[0033] Figure 3 : Schematic diagram of the protein expression results of Example 1.

[0034] Figure 4 : Schematic diagram of the characterization of cpE2 and LinTT1-cpE2@IFN in Example 2.

[0035] Figure 5 : Transmission electron microscopy schematic diagram of cpE2 and LinTT1-cpE2@IFN in Example 2.

[0036] Figure 6 : Schematic diagram of the LinTT1-cpE2@eGFP Transwell experiment in Example 3.

[0037] Figure 7 : Schematic diagram of the results of nanoparticles killing GL261 cell line CCK8 in Example 4.

[0038] Figure 8 : Schematic diagram of the results of nanoparticles killing U87 cell line CCK8 in Example 4.

[0039] Figure 9 : Schematic diagram of the results of nanoparticles killing GL261 and U87 cell lines by cell scratching in Example 5.

[0040] Figure 10 : Schematic diagram of the results of the effect of nanoparticles on the migration ability of GL261 cells in Example 5.

[0041] Figure 11 : Schematic diagram of the results of the effect of nanoparticles on the ability of U87 cells to kill migration in Example 5.

[0042] Detailed description of the invention

[0043] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0044] the term

[0045] Unless otherwise stated, each of the following terms shall have the meaning set forth below.

[0046] In this article, the terms "comprises", "comprising" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of more restrictions, an element defined by the sentence "comprising a ..." does not exclude the presence of other identical elements in the process, method, article or device that includes the element.

[0047] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0048] It should be noted that, as used herein and in the claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. For example, a nucleic acid molecule refers to one or more nucleic acid molecules. Thus, the terms "a", "a", "one or more", and "at least one" can be used interchangeably. Similarly, the terms "comprising", "including", and "having" can be used interchangeably and should generally be understood to be open-ended and non-restrictive, e.g., not excluding other unlisted elements or steps.

[0049] The term "amino acid" refers to an organic compound containing both an amino group and a carboxyl group, such as an α-amino acid, which can be encoded by a nucleic acid directly or in the form of a precursor. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called codons or base triplets). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code". Amino acids include natural amino acids and unnatural amino acids. Natural amino acids include alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

[0050] The term "polypeptide" is intended to encompass the singular "polypeptide" as well as the plural "polypeptide", and refers to a molecule composed of amino acid monomers linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any single chain or multiple chains of two or more amino acids, and does not refer to the specific length of the product. Therefore, the definition of "polypeptide" includes peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to two or more amino acid chains, and the term "polypeptide" can be used to replace any of the above terms, or to be used interchangeably with any of the above terms. The term "polypeptide" is also intended to refer to products modified after the expression of the polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protection / blocking groups, proteolytic cleavage or non-natural amino acid modifications. The polypeptide can be derived from a natural biological source or produced by recombinant technology, but it is not necessarily translated from a specified nucleic acid sequence, and it may be produced in any manner including chemical synthesis.

[0051] Unless otherwise indicated, fusion protein is a recombinant protein comprising the amino acid sequence from at least two unrelated proteins, which have been linked together to form a single protein by a peptide bond. The amino acid sequences of unrelated proteins can be directly connected to each other, or can be connected using a joint. As used herein, if the amino acid sequence of an albumen is usually not linked together via a peptide bond in its natural environment (e.g., in a cell), then they are unrelated. For example, usually the amino acid sequence of bacterial enzymes such as Bacillus stearothermophilus pyruvate dehydrogenase (PDH) complex E2 and the amino acid sequence of interferon (IFN) protein are not linked together by a peptide bond.

[0052] The term "encoding" when applied to a polynucleotide refers to a polynucleotide that is said to "encode" a polypeptide, which, in its native state or when manipulated by methods well known to those skilled in the art, can produce the polypeptide and / or its fragments via transcription and / or translation.

[0053] A polynucleotide is composed of a specific sequence of four bases: adenine (A), cytosine (C), guanine (G), thymine (T), or uracil (U) when the polynucleotide is RNA. A "polynucleotide sequence" can be represented by the letters of the polynucleotide molecule. This letter representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, such as functional genomics and homology searches.

[0054] The terms "polynucleotide," "polynucleotide," and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.

[0055] The term "recombinant" in relation to a polypeptide or polynucleotide refers to a form of the polypeptide or polynucleotide that does not occur in nature, and by way of non-limiting example, can be produced by combination of polynucleotides or polypeptides that do not normally occur.

[0056] The abbreviations of the present invention have the following meanings: E2-WT represents the wild-type ketoacid dehydrogenase (PDH) complex E2 subunit; cpE2 represents the modified ketoacid dehydrogenase (PDH) complex E2 subunit; E2-3NC represents the modified ketoacid dehydrogenase (PDH) complex E2 subunit; E2-NC represents the modified ketoacid dehydrogenase (PDH) complex E2 subunit; cpE2@IFN represents cpE2 connected to interferon IFN, and the link symbols "@" and "-" in the names have no special meanings.

[0057] The experimental reagents not specifically described in the present invention are all conventional reagents in the art, and can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers; the experimental methods not specifically described are all conventional methods in the art, and reference can be made to relevant experimental manuals, such as molecular cloning experimental manuals or instructions of relevant reagent manufacturers. DETAILED DESCRIPTION

[0058] Example 1. Gene design, expression and purification of the repeating structural unit recombinant protein cpE2 and LinTT1-cpE2@IFN

[0059] Based on the protein crystal structure of the E2 subunit of the pyruvate dehydrogenase (PDH) complex of Bacillus stearothermophilus, the sequence from 235 to 242 of the C-terminus of E2-WT was placed at the N-terminus and connected through a flexible GTGGSGSS octapeptide to obtain cpE2. At the same time, the sequence from 60 to 242 of the C-terminus of E2-WT was placed at the N-terminus to obtain E2-3NC. The sequence from 48 to 242 of the C-terminus of E2-WT was placed at the N-terminus to obtain E2-NC.

[0060] Based on the morphological plasticity of E2, we added the glioma targeting peptide LinTT1 to the N-terminus (outer surface) of cpE2, and connected IFN-α to the C-terminus (inner surface) through a flexible GGSGG pentapeptide to obtain LinTT1-cpE2@IFN. At the same time, we designed cpE2@IFN, which was only connected to IFN-α at the C-terminus (inner surface) through a flexible GGSGG pentapeptide as a control, and its N-terminus had no targeting ligand.

[0061] The sequences involved in this embodiment are shown in Table 1, and the design scheme is as follows Figure 1 shown.

[0062] Table 1. Amino acid sequences of E2-WT, cpE2, E2-3NC, E2-NC, and LinTT1-cpE2@IFN

[0063] Name Amino acid sequence Number E2-WT AAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNTSIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLM SEQ ID NO: 01 cpE2 SDPELLLMGTGGSGSSAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNTSIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLL SEQ ID NO: 02 E2-3NC AIAAEKGIKLTFLPYVVKALVSALREYPVLNTSIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLMAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFK SEQ ID NO: 03 E2-NC VTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNTSIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLSDPELLLMAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEAD SEQ ID NO:04 LinTT1-cpE2@IFN AKRGARSTAEFSDPELLLMGTGGSGSSAAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNTSIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLGGSGGCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE SEQ ID NO: 05 cpE2@IFN SDPELLLMGTGGSGSSAAAKPATTEGEFPETREKMSGIRRAIAKAMVHSKHTAPHVTLMDEADVTKLVAHRKKFKAIAAEKGIKLTFLPYVVKALVSALREYPVLNTSIDDETEEIIQKHYYNIGIAADTDRGLLVPVIKHADRKPIFALAQEINELAEKARDGKLTPGEMKGASCTITNIGSAGGQWFTPVINHPEVAILGIGRIAEKPIVRDGEIVAAPMLALSLSFDHRMIDGATAQKALNHIKRLLGGSGGCDLPQTHSLGSRRTLMLLAQMRRISLFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSSAAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRKYFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE SEQ ID NO: 06

[0064] In a 10μl system, use 1μl BamH Ⅰ and 1μl Hind Ⅲ restriction endonucleases to cut 6μl of the target DNA fragment, and use the same system to cut the plasmid vector (PET-28A) to produce compatible sticky ends or blunt ends. Mix the cut target DNA fragment and plasmid vector, add T4 DNA ligase and ligase buffer. The ligation reaction is usually carried out at room temperature for 2-3h to allow a stable connection between the DNA fragment and the vector.

[0065] The ligation product (recombinant plasmid) was mixed with competent cells (BL21) and incubated on ice for 30 minutes. The mixed plasmid was heat-shocked at 42°C for 90 seconds and then quickly cooled on ice for 5 minutes to promote DNA absorption. 200-300μl of antibiotic-free culture medium was added to the transformed cells and cultured at 37°C and 200rpm for 45 minutes to express the antibiotic resistance gene and restore the bacteria to normal growth. The cultured bacteria were spread on a plate containing kan +On the antibiotic agar plate, invert and culture overnight in a 37℃ incubator for 12-16h. Identify the recombinant plasmid by double digestion with BamH Ⅰ and Hind Ⅲ. Preserve the correctly identified bacteria, take 300μl 50% glycerol and mix with 700μl bacterial solution, and store in a -80℃ refrigerator.

[0066] Take 100μl glycerol bacteria containing the above protein expression vectors and inoculate them into 5ml LB liquid medium containing kanamycin (100μg / ml), and culture them overnight at 37℃ and 200rpm. Transfer 5ml of overnight cultured bacteria into 1.5L LB liquid medium containing kanamycin (100μg / ml, 1:1000), and culture them at 37℃ and 200rpm until OD600 is between 0.6-0.8, add 1M IPTG450μl, and induce protein expression at 16℃ and 200rpm for 36-48h. Collect the bacterial solution by centrifugation at 4000rpm and 4℃ for 20min, add 100ml binding buffer to resuspend the bacterial solution, and crush it with low temperature ultrasonic for 4-5 times. After crushing, centrifuge the bacterial solution at 8000rpm for 100min and collect it. Binding buffer was used to balance the Ni-NTA affinity column, and the collected supernatant was passed through the Ni-NTA affinity column. Wash buffer was used to wash away non-specifically bound impurities, and elution buffer was used to elute the target protein. The eluate was collected. 20 μl of the effluent from each step was added to 5 μl of 5× SDS-PAGE loading buffer, heated in a 100°C metal bath for 10 min, and then tested by SDS-PAGE. Figures 2 - 3 As shown, the protein was successfully expressed and purified.

[0067] Example 2: Characterization and performance testing of the repeating structural unit recombinant protein cpE2 and LinTT1-cpE2@IFN

[0068] The particle size, ζ potential and morphological characteristics of cpE2 and LinTT1-cpE2@IFN were detected by particle size analysis dynamic light scattering (DLS) and transmission electron microscopy (TEM). The nanoparticles in Example 1 were filtered into a 0.22 microporous membrane to filter the complex. Through particle size analysis dynamic light scattering (DLS), the particle size of cpE2 was increased from 25nm to 38nm compared with the wild-type E2-WT, and the particle size of engineered LinTT1-cpE2@IFN was increased from 38nm to 67nm compared with cpE2, which nearly doubled the drug loading. 10ul of the nanoparticles were added to the copper mesh at a concentration of 0.1mg / ml, soaked for 3min, and washed with ultrapure water 3 times (1min / time). Through transmission electron microscopy, it was observed that cpE2 and LinTT1-cpE2@IFN were uniform and regular spherical. However, E2-3NC and E2-NC could not complete self-assembly to form aggregates. The results are as follows Figure 4 andFigure 5 shown.

[0069] Example 3: Transwell experiment of LinTT1-cpE2@eGFP

[0070] The nanoparticles prepared in Example 1 were subjected to a Transwell experiment, and eGFP was used to simulate anti-tumor drugs or molecules to verify that after cpE2 encapsulated the drugs or molecules, these drug molecules could function.

[0071] Take the Hacat cell suspension and add it to the Transwell chamber, and add the GL261 cell suspension to the lower chamber. Place the 24-well plate at 37°C, 5% CO2, and 90% humidity for 24-48 hours to observe the fluorescence. Figure 6 As shown, the traswell experiment showed that LinTT1-cpE2@eGFP had obvious penetration ability.

[0072] Example 4: Tumor cell killing experiment of LinTT1-cpE2@IFN

[0073] The nanoparticles LinTT1-cpE2@IFN were subjected to CCK8 detection, and the cells used were GL261 and U87. The cell suspension (200 μL / well) was inoculated in a 96-well plate, and the culture plate was placed in an incubator for pre-culture for 24 hours. Then, different concentrations of the drug to be tested were added to the culture plate, and the culture plate was incubated in the incubator for 24 hours. Finally, 20 μL of CCK-8 solution (MeilunBio) was added to each well, and the culture plate was placed in the incubator for incubation for 30 minutes, and the absorbance at 450 nm was measured with an enzyme-labeled instrument. CCK-8 experiment as Figures 7 - 8 As shown, the results showed that 20 μM LinTT1-cpE2@IFN had a maximum killing effect of about 60% on GL261 and U87.

[0074] Example 5: Tumor cell scratch experiment of LinTT1-cpE2@IFN

[0075] The nanoparticles LinTT1-cpE2@IFN were used for cell scratch experiments, and the cells used were GL261 and U87. The cell suspension (2mL / well) was inoculated in a 6-well plate, and the culture plate was placed in an incubator for pre-culture for 24 hours. Use a marker pen to draw a horizontal line on the bottom of the well plate with a ruler. After 24 hours of cell culture, use a 10uL gun tip to align the well plate vertically with a ruler (you can also use a special scratch cell culture dish), gently push down the longitudinal line to form a scratch, rinse the cells 3 times with PBS, and remove the scratched cells. Add different concentrations of the drug to be tested to the culture plate, and incubate the culture plate in an incubator for 24 hours. At 37°C, 5% CO 2After culturing in an incubator for 0, 12, and 24 hours, photographs were taken under an optical microscope. Figures 9 - 11 As shown, the results showed that LinTT1-cpE2@IFN can inhibit the invasion of brain glioma cells.

[0076] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A repetitive structural unit recombinant protein cpE2, characterized in that: The amino acid sequence of the repeating structural unit recombinant protein cpE2 is shown in SEQ ID NO:

02.

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

05.

3. A polynucleotide, characterized in that Encoding the repeating structural unit recombinant protein cpE2 as described in claim 1 or the fusion protein as described in claim 2.

4. An expression vector, characterized in that: Comprising the polynucleotide as claimed in claim 3.

5. A cell, characterized in that Comprising the expression vector as claimed in claim 4.

6. Use of the repeating structural unit recombinant protein cpE2 according to claim 1 in preparing a drug delivery vector.

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