Synthesis of protein polymer self-assembly with multiple environmental stimuli responsiveness and applications thereof

Abstract

The application discloses synthesis of a protein polymer self-assembly with multiple environmental stimulus responsiveness and application thereof. Compared with common electrically neutral ELPs, the application finds that the negative electrically modified polymer carrier ELPs are not easy to be taken up by cells and metabolically removed in vivo, have longer drug half-life and bioavailability, and obviously improve pharmacokinetic characteristics; compared with traditional cationic nanoparticles, the negative electrically modified polymer carrier ELPs reduce cytotoxicity caused by positive charges, increase stability, and are easy to realize selective enrichment at an inflammation site, improve drug bioavailability and the like due to in-situ accumulation of part of inflammatory positive charges. The application also adds an MMP9 enzyme recognition site to the hydrophilic end of the ELPs, increases drug release and distribution at the inflammation site, and improves drug efficacy.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the synthesis and application of a protein polymer self-assembly with multiple environmental stimulus responses. Background Technology

[0002] A well-developed drug delivery system helps improve drug efficacy and expand the range of drug applications. Appropriate drug delivery strategies can enhance drug absorption and bioavailability; control drug release, prolong treatment cycles, and reduce toxic side effects; and create targeted drugs for precision medicine. Advances in genetic engineering have made it possible to explore the structure and function of proteins, and protein-based materials are increasingly being used in various biomedical fields such as tissue engineering, drug delivery, biosensors, and therapeutic diagnostics. Elastin-like polypeptides (ELPs) are biopolymers derived from natural elastin that can be artificially synthesized with stimulus-responsive properties. ELPs consist of repeating units of a Val-Pro-Gly-X-Gly motif derived from the hydrophobic domain of the original elastin, where X represents a guest amino acid residue, which can be any amino acid except proline. ELPs exhibit specific temperature responsiveness and demonstrate low lower critical solution temperature (LCST) phase behavior. Below the characteristic transition temperature (Tt), ELPs are soluble; above Tt, they reversibly form an insoluble, polymer-rich condensed phase. This temperature-sensitive property is well preserved when ELPs are coupled to other small molecules or peptides. Genetic engineering allows for the artificial synthesis of ELPs, enabling precise manipulation of their bioactivity, chemical reactivity, and physical properties, including environmental stimulus responsiveness and self-assembly behavior, making them attractive for tissue engineering and drug delivery applications. ELPs, as novel drug carriers, possess excellent biocompatibility, superior pharmacokinetic behavior, biodegradability, and extremely low immunogenicity. Therefore, ELPs hold great promise for applications in the biopharmaceutical field, particularly in drug delivery.

[0003] Solid tumors have poor vascular wall integrity and contain numerous vascular permeability factors, significantly enhancing vascular permeability. Nanoparticles, due to their size advantage, can penetrate from the vascular endothelium into the tumor tissue. However, because of the lack of lymphatic drainage in the tumor tissue, the nanoparticles remain at the tumor site. This phenomenon is known as the enhanced permeability and retention (EPR) effect in solid tumor tissues. Similar EPR effects can be observed in some inflammatory diseases, such as inflammatory bowel disease (IBD), characterized by vascular structural defects and poor lymphatic drainage. However, due to the non-targeted nature of drug release, most drugs still cannot reach the effective concentration values ​​that utilize the EPR effect alone, nor can they exhibit better therapeutic effects in inflamed areas. Numerous studies have shown that when IBD occurs, inflammation of the colonic mucosa is accompanied by the consumption of the mucus layer and the in situ accumulation of positively charged proteins, including transferrin and bactericidal / permeability-enhancing proteins. This leads to the accumulation of positive charges on the damaged epithelial surface, providing a molecular target and anchor for drug carriers with negative surface charges. Simultaneously, this facilitates better drug release and improves drug distribution and utilization. Therefore, ELP nanoparticles were selected to respond to inflammation by selecting matrix metalloproteinases (MMPs) that are significantly upregulated and released during inflammation, thereby achieving drug release.

[0004] This invention attempts to construct ELPs protein polymer self-assemblies with surface negative charge modification, temperature and enzyme multi-responsiveness to solve the above-mentioned problems in the delivery of therapeutic proteins or small molecule drugs.

[0005] Currently, therapeutic proteins and small molecule drugs are increasingly used in clinical treatment of various diseases. Small molecule drugs, due to their small molecular weight, are rapidly metabolized and eliminated from the body, requiring frequent high-concentration administration to maintain therapeutically effective levels in the blood to achieve therapeutic effects. This is accompanied by dose-related drug toxicity, a heavy economic burden on patients, and unsatisfactory treatment efficiency. Furthermore, hydrophobic small molecule drugs, due to their poor solubility, are difficult to administer at the required dosage, significantly limiting their application. Compared to small molecule drugs, protein drugs are highly specific, have high activity, and fewer side effects. However, protein drugs also face problems such as physicochemical instability, susceptibility to protease hydrolysis, short circulating half-life, and immunogenicity. Currently, polyethylene glycol (PEG) modification is commonly used to increase the molecular size of drugs, reduce glomerular filtration, and prolong the in vivo circulating half-life. However, PEGylation has some significant drawbacks. PEG itself does have some potential safety risks, such as inability to degrade in vivo, formation of antibodies targeting PEG, PEG allergy, and vacuolation. Furthermore, reduced activity and heterogeneity are also negative factors for PEGylated proteins, which may limit their widespread use. Most non-targeted release drugs still cannot achieve effective concentrations utilizing only the EPR effect, resulting in poor therapeutic efficacy in inflamed areas. The purpose of this invention is to provide the preparation and application of ELPs (ethylene glycol monophosphate) polymeric protein self-assemblies with surface negative charge modification and multiple temperature and enzyme responsiveness. This ELP (Elastic Protein Assemblage) is a biomacromolecule based on amino acids, exhibiting excellent biocompatibility and biodegradability into amino acids required by the body. It can improve drug properties through precise and controllable genetic engineering modification, giving it excellent thermosensitive characteristics. It can self-assemble into nanoparticles of approximately 40 nm at both room temperature and 37 °C, significantly prolonging drug half-life and reducing side effects. By selecting combinations of different reactive amino acids, and using hydrophilic alanine and glutamic acid alone or in proportion, nanoparticles with different negative charges can be obtained, increasing the stability of the ELP nanoparticles and improving the selective enrichment of drugs at sites of intestinal inflammation. Simultaneously, by utilizing the high expression and release of MMP9 at inflammatory sites, MMP9 recognition sites can be assembled, responding to MMP9-specific cleavage to release the drug, improving pharmacokinetic characteristics and enhancing drug efficacy. Summary of the Invention

[0006] The present invention aims to provide a method for synthesizing and preparing a protein polymer self-assembled system that is responsive to multiple environmental stimuli, and its application. This protein polymer is prepared by genetic engineering, selecting amino acids with different reactivity and combining them in different proportions to obtain an ELP self-assembled drug loading system. It undergoes sensitive self-assembly in response to temperature, enzymes, charge, pH, ultrasound, or chemical substances, aiming to maintain drug activity, improve drug stability, prolong drug half-life, increase selective drug accumulation and release, and improve pharmacokinetic parameters.

[0007] Addressing the issues of low efficiency and high toxicity encountered by current therapeutic proteins and small molecule drugs in disease treatment and drug delivery, this environmentally responsive protein polymer self-assembly (ELP) cleverly combines electronegative alanine and electronegative glutamic acid in different proportions within its biological structure to form a hydrophilic chain. Simultaneously, an MMP9 enzyme recognition site is added to its hydrophilic end. Therefore, the biosynthesized ELPs with different amino acid ratios exhibit temperature-sensitive self-assembly characteristics, forming nanoparticles at both 25℃ and 37℃, significantly prolonging the drug's half-life and improving pharmacokinetic characteristics. The biosynthesized ELPs with different reactive amino acids also possess a negatively charged surface, enhancing the selective enrichment of drugs at intestinal inflammatory sites and improving drug utilization. Furthermore, the presence of an MMP9 enzyme recognition cleavage site at the hydrophilic end of the biosynthesized ELPs increases drug release and distribution at the inflammatory site, improving drug efficacy.

[0008] The technical solution adopted in this invention is:

[0009] In a first aspect, the present invention provides a drug delivery carrier comprising a negatively charged elastin-like protein.

[0010] In some embodiments of the present invention, the elastin-like protein is capable of sensitive responses to temperature, pH, enzymes, positive and negative charges, light, or chemical substances.

[0011] In some embodiments of the present invention, the elastin-like sequence includes (VPGXG)n, where n = 80 to 160, and n is an integer.

[0012] If n is too large, the protein is not easily expressed, reducing the yield; if n is too small, the temperature range of the formed nanoparticles is narrow.

[0013] In some preferred embodiments of the present invention, n = 100 to 125.

[0014] In some embodiments of the present invention, X is any amino acid other than proline P.

[0015] In some embodiments of the present invention, X is at least one selected from arginine, lysine, arginine, glutamic acid, aspartic acid, alanine, glycine, isoleucine, phenylalanine, methionine, serine, and valine.

[0016] In some embodiments of the present invention, X is at least one of alanine, isoleucine, and glutamic acid.

[0017] In some embodiments of the present invention, the drug delivery polymer carrier is a multi-environmentally responsive self-assembled drug delivery polymer carrier, wherein the environmental stimuli responsiveness includes the ability to respond sensitively to temperature, pH, enzymes, positive and negative charges, light or chemical substances.

[0018] In some embodiments of the present invention, the response temperature of the drug delivery polymer carrier is 15-60°C.

[0019] In some embodiments of the present invention, the temperature is preferably 18–42°C.

[0020] In some embodiments of the present invention, the elastin-like sequence includes: (VPGAG)18(VPGAGVPGEG)9(VPGA G)18(VPGIG)60 or (VPGAGVPGEG)18(VPGAG)18(VPGIG)60.

[0021] A second aspect of the present invention provides a protein polymer self-assembly comprising the delivery polymer carrier described in the first aspect of the present invention and an active molecule connected thereto.

[0022] In some embodiments of the present invention, the active molecule includes diagnostic agents and / or therapeutic agents and / or molecular switches.

[0023] In some embodiments of the present invention, the diagnostic agent includes fluorescent labeling, radiolabeling, enzyme labeling, etc.

[0024] In some preferred embodiments of the present invention, the fluorescent label includes at least one of mCherry, GFP, YFP, BFP, and CFP.

[0025] In some embodiments of the present invention, the therapeutic agent includes proteins, antibodies, peptides, small molecule chemical drugs, traditional Chinese medicines, etc.

[0026] In some embodiments of the present invention, the drug includes polyclonal antibodies, monoclonal antibodies, microantibodies, domain antibodies, nanobodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, antibody conjugates, antibody mimics, humanized antibodies and their fragments, insulin, growth factors, interferon, interleukins, annexin, erythropoietin, colony-stimulating factors, neutrophil growth factor and transforming growth factor, etanercept, aflibercept, abatacept, and afasicept. Beracide, liraglutide, pramlinide, teriparatide, exenatide, lantolide, enfuviride, glucagon-like peptide-1 and its analogues, relaxin, octreotide, incretin peptide CCK, gastrointestinal peptide, leptin, receptor antagonist IL-1Ra, asparaginase, uricase, cyclosporine, antidiuretic hormone, oxytocin, pancreatin, superoxide dismutase, gastrin, vaccines, viral vectors, affinity molecules, Her2 receptor, G protein-coupled receptor, VEGF receptor, etc.

[0027] In some embodiments of the present invention, the protein polymer self-assembly further includes a protein as a release switch.

[0028] In some preferred embodiments of the present invention, the molecular switch includes recognition sequences of matrix metalloproteinases (MMPs), esterases, histones, furin proteases, caspases, and enzymes that are highly expressed and released from inflamed or tumorous lesions.

[0029] In some embodiments of the present invention, the amino acid sequence of the protein polymer self-assembly is shown in SEQ ID NO. 15 or SEQ ID NO. 16.

[0030] The present invention also provides a method for preparing protein polymer self-assemblies, comprising the following steps: 1) design and sequential insertion of ELP monomer genes; 2) recursive directional ligation (RDL) of ELP monomer genes; 3) coupling of active molecules with ELPs; 4) expression of active molecules-ELPs.

[0031] In some embodiments of the present invention, the method further includes further purification of the expressed protein polymer self-assemblies.

[0032] A third aspect of the invention provides biomaterials related to the drug delivery carriers described in the first aspect of the invention or the protein polymer self-assemblies described in the second aspect of the invention, wherein the biomaterials are any one of (a1) to (d1) below:

[0033] (a1) Nucleic acid molecules encoding the delivery vector described in the first aspect of the present invention or the protein polymer self-assembly described in the second aspect of the present invention;

[0034] (b1) An expression cassette containing the nucleic acid molecule described in (a1);

[0035] (c1) A recombinant vector containing the nucleic acid molecule described in (a1), or a recombinant vector containing the expression cassette described in (b1);

[0036] (d1) A recombinant microorganism containing the nucleic acid molecule described in (a1), or a recombinant microorganism containing the expression cassette described in (b1), or a recombinant cell containing the recombinant vector described in (c1).

[0037] A fourth aspect of the present invention provides the use of the delivery carrier described in the first aspect of the present invention, or the protein polymer self-assembly described in the second aspect of the present invention, or the biomaterial described in the third aspect of the present invention, in the preparation of diagnostic agents and / or pharmaceuticals.

[0038] In some embodiments of the present invention, the drug includes drugs for the prevention and / or treatment of tumors, autoimmune diseases, metabolic diseases, infectious diseases, organ lesions, and tissue damage.

[0039] A fifth aspect of the present invention provides a method for delivering a drug to target cells, comprising the step of administering to the target cells a drug delivery carrier of the first aspect of the present invention, a protein polymer self-assembly of the second aspect of the present invention, or a biomaterial of the third aspect of the present invention.

[0040] A sixth aspect of the present invention provides a medicament comprising a drug delivery carrier according to the first aspect of the present invention, a protein polymer self-assembly according to the second aspect of the present invention, or a biomaterial according to the third aspect of the present invention.

[0041] In some embodiments of the present invention, the medicament further comprises pharmaceutically acceptable excipients.

[0042] The beneficial effects of this invention are:

[0043] This invention utilizes site-directed modification technology in genetic engineering to design different combinations of the number of negatively charged reactive amino acids and the positional substitutions of neutral amino acids. By rationally selecting enzyme-responsive unstable structures, self-assembling protein drug polymer carriers (ELPs) with different protein structures and multiple stimulus responses are obtained. These ELPs undergo sensitive self-assembly in response to temperature, enzymes, charge, pH, ultrasound, or chemical substances. Compared to ordinary electroneutrally neutral ELPs, this invention reveals that negatively charged modified polymer carrier ELPs are less susceptible to cellular uptake and in vivo metabolic clearance, exhibiting longer drug half-life and bioavailability, and significantly improving pharmacokinetic characteristics. Compared to traditional cationic nanoparticles, they reduce cytotoxicity caused by positive charges, increase stability, and, due to the in-situ accumulation of some inflammatory positive charges, facilitate selective enrichment at inflammatory sites, improving drug utilization. Furthermore, this invention incorporates an MMP9 enzyme recognition site at the hydrophilic end of the ELPs, increasing drug release and distribution at the site of inflammation and enhancing therapeutic efficacy.

[0044] The ELPs of this invention are derived from in vivo elastin and are biosynthesized through genetic engineering. They have good biocompatibility, extremely low immunogenicity, flexible site-specific modification, and biodegradability.

[0045] The self-assembled protein drug delivery polymer carriers (ELPs) of this invention are multi-environmentally responsive self-assembling drug delivery polymer carriers. They exhibit thermo-sensitive self-assembly characteristics at 25°C and 37°C, forming nanoparticles. Compared to the multi-step assembly process of other nanoparticles, this simplifies drug and carrier preparation, reducing the risk of exogenous contamination and batch-to-batch errors. Furthermore, the initial raw materials for the protein drug delivery polymer carriers (ELPs) prepared by this invention are common and readily available, extraction and purification conditions are simple, and the genetic engineering modification design is reasonable. Attached Figure Description

[0046] Figure 1 This is a recursive directional synthesis method for ELPs.

[0047] Figure 2 This is an electrophoresis image. Figure 2 a is an illustration of SDS-PAGE electrophoresis of mCherry. Figure 2 b is an illustration of SDS-PAGE electrophoresis of mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-).

[0048] Figure 3 Illustrations of Native-PAGE electrophoresis of mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-).

[0049] Figure 4For particle size and dispersion factor detection, Figure 4 a represents the particle size of mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-). Figure 4 b is the polymer dispersion coefficient of mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-).

[0050] Figure 5 The zeta potentials are mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-).

[0051] Figure 6 The in vivo retention time of mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-) in small animals for IVIS in vivo imaging; Figure 6 a is a live imaging image. Figure 6 b is a statistical chart of the stay time.

[0052] Figure 7 The pharmacokinetic results of mCherry, mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-) in mice are presented.

[0053] Figure 8 The pharmacokinetic parameters of mCherry, mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-) in mice are given.

[0054] Figure 9 The tissue distribution of mCherry, mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-) in mice.

[0055] Figure 10 The distribution of mCherry, mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-) in mouse tissues was statistically analyzed.

[0056] Figure 11 The results of selective enrichment of mCherry, mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-) in the colonic tissue of mice. Figure 11 a is the image. Figure 11 b is a statistical chart. Detailed Implementation

[0057] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0058] Example 1: Constructing (VPGAG)n

[0059] Alanine A was chosen as the X amino acid of ELPs. The repeating unit of ELPs(A) is VPGAG, with repeat numbers of 3, 6, 9, 12, 18, 36, 54, and 60.

[0060] 1) Design and synthesize oligonucleotide gene fragments containing repeating units and sticky ends of restriction enzyme sites Acu I and BseR I.

[0061] AF: CGCAGGTGTTCCGGGTGCGGGCGTTCCGGGTGCTGGCGTGCCGGG (SEQ ID NO: 1);

[0062] AR: CGGCACGCCAGCACCCGGAACGCCCGCACCCGGAACACCTGCGCC (SEQ ID NO: 2);

[0063] 2) Phosphorylation of AF and AR was performed to facilitate the ligation reaction. The phosphorylation system consisted of: 5 μL (50 μM) AF oligonucleotide gene, 5 μL (50 μM) AR oligonucleotide gene, 1 μL T4 polynucleotide kinase, 2.5 μL T4 DNA ligation buffer, and 11.5 μL ultrapure water. After incubating at 37°C for 1 h, 1 μL of 1M NaCl was added to terminate the reaction. The mixture was then transferred to a PCR instrument for annealing and extension. The annealing program was: 95°C for 5 min, 55°C for 5 min, and 16°C for 5 min. This annealing process resulted in double-stranded DNA fragments with Acu I / BseR I sticky ends.

[0064] 3) pET24b was digested with BseRI. The digestion system consisted of 30 μL pET24b plasmid, 4 μL rCutsmart Buffer, 0.5 μL BseRI, and 5.5 μL ultrapure water. The mixture was incubated at 37°C for 3 hours. Then, 1 μL of thermosensitive calf intestinal alkaline phosphatase was added, and the mixture was incubated at 37°C for 20 minutes to dephosphorylate the 5′ end to prevent vector self-circulation. The linearized vector was purified by agarose gel electrophoresis, and the recovered fragment was 5294 bp.

[0065] 4) T4 DNA ligation process, specific reaction system: 1 μL T4 DNA ligation buffer, 0.02 pM pET24b recovery vector, 0.5 μL A-DNA, 0.5 μL T4 DNA Ligase, and ultrapure water to a final volume of 10 μL. Incubate at 25°C for 2 hours, then immediately place on ice and let stand for 5 minutes.

[0066] 5) Transformation: Add the DNA ligation product to competent E. coli cells and incubate on ice for 30 min; 42°C, 60 s; immediately place on ice for 5 min; add 700 μL of antibiotic-free LB medium, incubate at 37°C on a shaker at 200 rpm for 1 h; centrifuge at 3000 rpm for 5 min, discard 600 μL of supernatant, mix the remaining liquid, spread it on LB plates containing kanamycin, and incubate overnight at 37°C inverted.

[0067] 6) Select single clones and perform colony PCR identification using universal primers. The bacterial clone suspension was prepared by dissolving the single clone in 10 μL of sterile ultrapure water. The PCR reaction system was: 5 μL of 2x Flash PCR MasterMix, 0.5 μL of ELP-T7-F, 0.5 μL of ELP-T7-R, 1 μL of bacterial suspension, and 3 μL of ultrapure water. Agarose gel electrophoresis was performed, and the correct plasmids were sequenced.

[0068] ELP-T7-F: ACGACTCACTATAGGGGA (SEQ ID NO: 3);

[0069] ELP-T7-R:GAAGCACGGCTCATTTTG (SEQ ID NO:4);

[0070] 7) Use Recursive Directioning (RDL) to quickly obtain multiple repeating units VPGAG. An RDL diagram is shown below. Figure 1As shown, pET24b-A9, obtained by the polypeptide monomer gene insertion method, was used as the parental plasmid. Double digestion with restriction endonucleases AcuⅠ and BglⅠ was performed. The digestion reaction system consisted of 20 μL pET24b-A9 plasmid, 4 μL rCutsmart Buffer, 0.7 μL AcuⅠ, 0.4 μL BglⅠ, and 14.9 μL ultrapure water. After incubation at 37℃ for 3 h, three DNA fragments of different sizes were obtained. After detection by 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment, i.e., the linear fragment A9-1 containing the A9 gene, was purified by gel recovery. Double digestion with restriction endonucleases BseRⅠ and BglⅠ was then performed. The digestion reaction system consisted of 20 μL pET24b-A9 plasmid, 4 μL rCutsmart Buffer, 0.7 μL BseRⅠ, 0.4 μL BglⅠ, and 14.9 μL ultrapure water. After incubating at 37°C for 3 hours, two DNA fragments of different sizes were obtained. After detection by 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment, namely the linear fragment A9-2 containing the A9 gene, was purified by gel recovery. The linear fragments A9-1 and A9-2 were ligated using T4 DNA ligase. The ligation system consisted of: 1 μL T4 DNA ligation buffer, 1 μL A9-1 fragment, 2 μL A9-2 fragment, 0.5 μL T4 DNA ligase, and 5.5 μL ultrapure water. The mixture was incubated at 25°C for 2 hours. The ligation product was then added to competent *E. coli* cells and incubated on ice for 30 minutes; then incubated at 42°C for 60 seconds; immediately placed on ice for 5 minutes; 700 μL of antibiotic-free LB medium was added; the mixture was incubated at 37°C on a shaker at 200 rpm for 1 hour; centrifuged at 3000 rpm for 5 minutes; 600 μL of the supernatant was discarded; the remaining liquid was mixed and plated onto LB agar plates containing kanamycin; and incubated overnight at 37°C inverted. Single clones were selected and colony PCR was performed using universal primers. Bacterial clone suspensions were prepared by dissolving single clones in 10 μL of sterile ultrapure water. The PCR reaction system consisted of: 5 μL 2x Flash PCR MasterMix, 0.5 μL ELP-T7-F, 0.5 μL ELP-T7-R, 1 μL bacterial suspension, and 3 μL ultrapure water. Agarose gel electrophoresis was performed to identify and sequence the correct plasmids. 18 repeat units (VPGAG) were obtained.

[0071] 8) Repeated units (VPGAG)36, (VPGAG)54, and (VPGAG)60 were obtained sequentially using the recursive directional connection method in experimental step 7).

[0072] Example 2: Constructing (VPGIG)n

[0073] Isoleucine I was selected as the X amino acid of ELPs. The repeating unit of ELPs(I) is VPGIG, and the repeating numbers are 3, 6, 12, 18, 24, 36, 48, and 60.

[0074] 1) Design and synthesize oligonucleotide gene fragments containing repeating units and sticky ends of restriction enzyme sites Acu I and BseR I.

[0075] IF: CATCGGTGTTCCGGGTATTGGTGTGCCGGGCATCGGCGTACCGGG (SEQ ID NO: 5);

[0076] IR: CGGTACGCCGATGCCCGGCACACCAATACCCGGAACACCGATGCC (SEQ ID NO: 6).

[0077] 2) Phosphorylation of IF and IR was performed to facilitate the ligation reaction. The phosphorylation system was the same as in Example 1. After incubating in a 37°C water bath for 1 hour, 1 μL of 1M NaCl was added to terminate the reaction. The mixture was then transferred to a PCR instrument for annealing and extension. The annealing procedure was the same as in Example 1. This annealing process resulted in the formation of a double-stranded DNA fragment with sticky Acu I / BseR I ends.

[0078] 3) pET24b was digested with BseR I using the same digestion system as in Example 1. The digestion was carried out in a water bath at 37°C for 3 hours, and 1 μL of heat-sensitive calf intestinal alkaline phosphatase was added. The 5′ end was dephosphorylated at 37°C for 20 minutes to prevent the vector from cyclizing. The linearized vector was purified by agarose gel electrophoresis, and the recovered fragment was 5294 bp.

[0079] 4) The T4 DNA ligation process follows the same reaction system as in Example 1. After incubation at 25°C for 2 hours, immediately place on ice and let stand for 5 minutes.

[0080] 5) Transformation: Add the DNA ligation product to competent E. coli cells and incubate on ice for 30 min; 42°C, 60 s; immediately place on ice for 5 min; add 700 μL of antibiotic-free LB medium, incubate at 37°C on a shaker at 200 rpm for 1 h; centrifuge at 3000 rpm for 5 min, discard 600 μL of supernatant, mix the remaining liquid, spread it on LB plates containing kanamycin, and incubate overnight at 37°C inverted.

[0081] 6) Select single clones and perform colony PCR identification using universal primers. The bacterial clone suspension was prepared by dissolving the single clone in 10 μL of sterile ultrapure water. The PCR reaction system was the same as in Example 1. Agarose gel electrophoresis was performed, and the correctly identified plasmids were sequenced.

[0082] 7) Utilize recursive directed fast ( Figure 1 Multiple repeating units VPGIG were obtained. Using pET24b-I6, obtained via polypeptide monomer gene insertion, as the parent plasmid, double digestion with restriction endonucleases AcuⅠ and BglⅠ was performed, with the digestion reaction system identical to that in Example 1. After incubation at 37°C for 3 hours, three DNA fragments of different sizes were obtained. After detection by 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment, i.e., the linear fragment I6-1 containing the I6 gene, was recovered and purified from the gel. Double digestion with restriction endonucleases BseRⅠ and BglⅠ was performed, with the digestion reaction system identical to that in Example 1. After incubation at 37°C for 3 hours, two DNA fragments of different sizes were obtained. After detection by 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment, i.e., the linear fragment I6-2 containing the I6 gene, was recovered and purified from the gel. Using T4... DNA ligase was used to ligate linear fragments I6-1 and I6-2 using the same ligation system as in Example 1. The mixture was incubated at 25°C for 2 hours. The ligation product was added to competent *E. coli* cells and incubated on ice for 30 minutes; then at 42°C for 60 seconds; immediately placed on ice for 5 minutes; 700 μL of antibiotic-free LB medium was added, and the mixture was incubated at 37°C for 1 hour at 200 rpm using a shaker; then centrifuged at 3000 rpm for 5 minutes, discarding 600 μL of the supernatant. The remaining liquid was mixed and plated onto LB agar plates containing kanamycin, and incubated overnight at 37°C inverted mode. Single clones were selected, and colony PCR was performed using universal primers. The bacterial clone suspension was prepared by dissolving a single clone in 10 μL of sterile ultrapure water. The PCR reaction system consisted of: 5 μL of 2x Flash PCR MasterMix, 0.5 μL of ELP-T7-F, 0.5 μL of ELP-T7-R, 1 μL of bacterial suspension, and 3 μL of ultrapure water. Agarose gel electrophoresis was used to identify the correct plasmids for sequencing. Repeat units (VPGIG)12 were obtained.

[0083] 8) Repeated units (VPGIG)18, (VPGIG)24, (VPGIG)36, (VPGIG)48, and (VPGIG)60 were obtained sequentially using the recursive directional connection method in experimental step 7).

[0084] Example 3: Constructing (VPGAGVPGEG)n

[0085] Alanine A and glutamic acid E were selected as the X amino acids of ELPs. The repeating unit of ELPs (AE) is VPGAGVPGEG, with repeat numbers of 2, 3, 4, 6, 9, 18, 24, and 36, respectively.

[0086] 1) Design and synthesize oligonucleotide gene fragments containing repeating units and sticky ends of restriction enzyme sites Acu I and BseR I.

[0087] AE-F: CGCGGGTGTTCCGGGTGAAGGCGTTCCGGG (SEQ ID NO: 7);

[0088] AE-R: CGGAACGCCTTCACCCGGAACACCCGCGCC (SEQ ID NO: 8);

[0089] 2) Phosphorylation of AE-F and AE-R was performed to facilitate the ligation reaction. The phosphorylation system was the same as in Example 1. After incubating in a 37°C water bath for 1 hour, 1 μL of 1M NaCl was added to terminate the reaction. The mixture was then transferred to a PCR instrument for annealing and extension. The annealing procedure was the same as in Example 1. IF / IR annealing was then performed to form double-stranded DNA fragments with Acu I / BseR I sticky ends.

[0090] 3) pET24b was digested with BseR I using the same digestion system as in Example 1. The digestion was carried out in a water bath at 37°C for 3 hours, and 1 μL of heat-sensitive calf intestinal alkaline phosphatase was added. The 5′ end was dephosphorylated at 37°C for 20 minutes to prevent the vector from cyclizing. The linearized vector was purified by agarose gel electrophoresis, and the recovered fragment was 5294 bp.

[0091] 4) The T4 DNA ligation process follows the same reaction system as in Example 1. After incubation at 25°C for 2 hours, immediately place on ice and let stand for 5 minutes.

[0092] 5) Transformation: Add the DNA ligation product to competent E. coli cells and incubate on ice for 30 min; 42°C, 60 s; immediately place on ice for 5 min; add 700 μL of antibiotic-free LB medium, incubate at 37°C on a shaker at 200 rpm for 1 h; centrifuge at 3000 rpm for 5 min, discard 600 μL of supernatant, mix the remaining liquid, spread it on LB plates containing kanamycin, and incubate overnight at 37°C inverted.

[0093] 6) Select single clones and perform colony PCR identification using universal primers. The bacterial clone suspension was prepared by dissolving the single clone in 10 μL of sterile ultrapure water. The PCR reaction system was the same as in Example 1. Agarose gel electrophoresis was performed, and the correctly identified plasmids were sequenced.

[0094] 7) Multiple repeating units VPGAGVPGEG were rapidly obtained using recursive orientation. pET24b-(AE)3 and pET24b-(AE)6, obtained by polypeptide monomer gene insertion, were used as parental plasmids. pET24b-(AE)3 was double-digested with restriction endonucleases AcuⅠ and BglⅠ, using the same digestion system as in Example 1. After incubation at 37℃ for 3 hours, three DNA fragments of different sizes were obtained. After detection by 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment, i.e., the linear fragment (AE)3-1 containing the (AE)3 gene, was recovered and purified from the gel. pET24b-(AE)6 was double-digested with restriction endonucleases BseRⅠ and BglⅠ, using the same digestion system as in Example 1. After incubation at 37℃ for 3 hours, two DNA fragments of different sizes were obtained. After detection by 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment, i.e., the linear fragment (AE)6-2 containing the (AE)6 gene, was recovered and purified from the gel. Using T4... DNA ligase was used to ligate linear fragments (AE)3-1 and (AE)6-2 using the same ligation system as in Example 1. The mixture was incubated at 25°C for 2 hours. The ligation product was added to competent *E. coli* cells and incubated on ice for 30 minutes; then at 42°C for 60 seconds; immediately placed on ice for 5 minutes; 700 μL of antibiotic-free LB medium was added, and the mixture was incubated at 37°C for 1 hour at 200 rpm using a shaker; then centrifuged at 3000 rpm for 5 minutes, discarding 600 μL of the supernatant. The remaining liquid was mixed and plated onto LB agar plates containing kanamycin, and incubated overnight at 37°C with the plates inverted. Single clones were selected, and colony PCR was performed using universal primers. The bacterial clone suspension was prepared by dissolving a single clone in 10 μL of sterile ultrapure water. The PCR reaction system consisted of: 5 μL of 2xFlash PCR MasterMix, 0.5 μL of ELP-T7-F, 0.5 μL of ELP-T7-R, 1 μL of bacterial suspension, and 3 μL of ultrapure water. Agarose gel electrophoresis was used to identify and sequence the correct plasmids. Repeat units (VPGAGVPGEG)9 were obtained.

[0095] 8) Repeated units (VPGAGVPGEG)12, (VPGAGVPGEG)18, (VPGAGVPGEG)24, and (VPGAGVPGEG)36 were obtained sequentially using the recursive directional connection method in experimental step 7).

[0096] Example 4: Constructing (VPGAG)60 (VPGIG)60

[0097] 1) Recursive orientation was used to obtain (VPGAG)60 (VPGIG)60 (also known as E0). pET24b-(A)60 and pET24b-(I)60, obtained by the RDL method, were used as parental plasmids. pET24b-(A)60 was double-digested with restriction endonucleases AcuⅠ and BglⅠ, using the same digestion system as in Example 1. After incubation at 37℃ for 3 hours, three DNA fragments of different sizes were obtained. These fragments were then detected by 1.2% agarose gel electrophoresis. The larger molecular weight DNA fragment, namely the linear fragment (A)60-1 containing the (A)60 gene, was recovered and purified. pET24b-(I)60 was double-digested with restriction endonucleases BseRI and BglRI, using the same digestion system as in Example 1. After incubation at 37°C for 3 hours, two DNA fragments of different sizes were obtained. After detection by 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment, namely the linear fragment (I)60-2 containing the I60 gene, was recovered and purified from the gel.

[0098] 2) Linear fragments (A)60-1 and (I)60-2 were ligated using T4 DNA ligase, with the ligation system the same as in Example 1. The cells were incubated at 25°C for 2 hours. The DNA ligation product was added to competent E. coli cells and incubated on ice for 30 minutes. The cells were then incubated at 42°C for 60 seconds and then quickly placed on ice for 5 minutes. 700 μL of antibiotic-free LB medium was added and the cells were incubated at 37°C on a shaker at 200 rpm for 1 hour. The cells were then centrifuged at 3000 rpm for 5 minutes. 600 μL of the supernatant was discarded, and the remaining liquid was mixed and spread onto LB plates containing kanamycin. The plates were then incubated overnight at 37°C with the plates inverted. Single clones were selected and colony PCR was performed using universal primers. Bacterial clone suspensions were prepared by dissolving single clones in 10 μL of sterile ultrapure water. The PCR reaction system consisted of: 5 μL 2x Flash PCR MasterMix, 0.5 μL ELP-T7-F, 0.5 μL ELP-T7-R, 1 μL bacterial suspension, and 3 μL ultrapure water. Agarose gel electrophoresis was performed, and correctly identified plasmids were sequenced. (VPGAG)60 (VPGIG)60 was obtained.

[0099] Example 5: Constructing (VPGAG)18(VPGAGVPGEG)9(VPGAG)18(VPGIG)60

[0100] 1) Using recursive orientation, (VPGAG)18(VPGAGVPGEG)9(VPGAG)18(VPGIG)60 (also known as ELP(E9-)) was obtained. pET24b-(A)18 and pET24b-(AE)9, obtained by the RDL method, were used as parental plasmids. pET24b-(A)18 was double-digested with restriction endonucleases AcuⅠ and BglⅠ, using the same digestion system as in Example 1. After incubating at 37℃ for 3 hours, three DNA fragments of different sizes were obtained. 1.2 After 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment, namely the linear fragment (A)18-1 containing the (A)18 gene, was recovered and purified from the gel. pET24b-(AE)9 was then double-digested with restriction endonucleases BseRI and BglRI, using the same digestion system as in Example 1. After incubation at 37°C for 3 hours, two DNA fragments of different sizes were obtained. After 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment, namely the linear fragment (AE)9-2 containing the (AE)9 gene, was recovered and purified from the gel. Linear fragments (A)18-1 and (AE)9-2 were ligated using T4 DNA ligase, following the same ligation system as in Example 1. The mixture was incubated at 25°C for 2 hours. The ligation product was then added to competent *E. coli* cells and incubated on ice for 30 minutes; then at 42°C for 60 seconds; immediately placed on ice for 5 minutes; 700 μL of antibiotic-free LB medium was added, and the mixture was incubated at 37°C for 1 hour at 200 rpm using a shaker; then centrifuged at 3000 rpm for 5 minutes, discarding 600 μL of the supernatant. The remaining liquid was mixed and plated onto LB agar plates containing kanamycin, and incubated overnight at 37°C inverted mode. Single clones were selected, and colony PCR was performed using universal primers. The bacterial clone suspension was prepared by dissolving the single clone in 10 μL of sterile ultrapure water. The PCR reaction system consisted of: 5 μL of 2x Flash PCR MasterMix, 0.5 μL of ELP-T7-F, 0.5 μL of ELP-T7-R, 1 μL of bacterial suspension, and 3 μL of ultrapure water. Agarose gel electrophoresis was used to identify the correct plasmids for sequencing. (VPGAG)18(VPGAGVPGEG)9 was obtained.

[0101] 2) Using pET24b-(A)18(AE)9 and pET24b-(A)18 obtained by the RDL method as parental plasmids, pET24b-(A)18(AE)9 was double-digested with restriction endonucleases AcuⅠ and BglⅠ, in the same digestion system as in Example 1. After incubation at 37℃ for 3 hours, three DNA fragments of different sizes were obtained. After detection by 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment was recovered and purified from the gel. The linear fragment (A)18(AE)9-1 containing the (A)18(AE)9 gene was obtained. pET24b-(A)18 was double-digested with restriction endonucleases BseRI and BglRI, using the same digestion system as in Example 1. After incubation at 37°C for 3 hours, two DNA fragments of different sizes were obtained. After detection by 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment, i.e., the linear fragment (A)18-2 containing the (A)18 gene, was recovered and purified from the gel. Linear fragments (A)18(AE)9-1 and (A)18-2 were ligated using T4 DNA ligase, following the same ligation system as in Example 1. The cells were incubated at 25°C for 2 hours. The ligation product was then added to competent E. coli cells and incubated on ice for 30 minutes; then at 42°C for 60 seconds; immediately placed on ice for 5 minutes; 700 μL of antibiotic-free LB medium was added, and the cells were incubated at 37°C on a shaker at 200 rpm for 1 hour; centrifuged at 3000 rpm for 5 minutes, discarding 600 μL of the supernatant. The remaining liquid was mixed and plated onto LB agar plates containing kanamycin, and incubated overnight at 37°C inverted. Single clones were selected and colony PCR was performed using universal primers. Bacterial clone suspensions were prepared by dissolving single clones in 10 μL of sterile ultrapure water. The PCR reaction system consisted of: 5 μL 2x Flash PCR MasterMix, 0.5 μL ELP-T7-F, 0.5 μL ELP-T7-R, 1 μL bacterial suspension, and 3 μL ultrapure water. Agarose gel electrophoresis was performed to identify and sequence the correct plasmids. The desired plasmids were (VPGAG)18(VPGAGVPGEG)9(VPGAG)18.

[0102] 3) Using pET24b-(A)18(AE)9(A)18 and pET24b-(I)60 obtained by the RDL method as parental plasmids, pET24b-(A)18(AE)9(A)18 was double-digested with restriction endonucleases AcuⅠ and BglⅠ, in the same digestion system as in Example 1. After incubation at 37℃ for 3 hours, three DNA fragments of different sizes were obtained. After detection by 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment was recovered and purified from the gel. The linear fragment (A)18(AE)9(A)18-1 containing the (A)18(AE)9(A)18 gene was obtained. pET24b-(I)60 was double-digested with restriction endonucleases BseRI and BglRI, using the same digestion system as in Example 1. After incubation at 37°C for 3 hours, two DNA fragments of different sizes were obtained. After detection by 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment, i.e., the linear fragment (I)60-2 containing the (I)60 gene, was recovered and purified from the gel. Linear fragments (A)18(AE)9(A)18-1 and (I)60-2 were ligated using T4 DNA ligase, following the same ligation system as in Example 1. The cells were incubated at 25°C for 2 hours. The ligation product was then added to competent E. coli cells and incubated on ice for 30 minutes; then at 42°C for 60 seconds; immediately placed on ice for 5 minutes; 700 μL of antibiotic-free LB medium was added; the cells were incubated at 37°C on a shaker at 200 rpm for 1 hour; centrifuged at 3000 rpm for 5 minutes, discarding 600 μL of the supernatant. The remaining liquid was mixed and plated onto LB agar plates containing kanamycin and incubated overnight at 37°C inverted. Single clones were selected and colony PCR was performed using universal primers. Bacterial clone suspensions were prepared by dissolving single clones in 10 μL of sterile ultrapure water. The PCR reaction system consisted of: 5 μL 2xFlash PCR MasterMix, 0.5 μL ELP-T7-F, 0.5 μL ELP-T7-R, 1 μL bacterial suspension, and 3 μL ultrapure water. Agarose gel electrophoresis was performed to identify and sequence the correct plasmids. The desired plasmid composition was (VPGAG)18(VPGAGVPGEG)9(VPGAG)18(VPGIG)60.

[0103] Example 6: Constructing (VPGAGVPGEG)18(VPGAG)18(VPGIG)60

[0104] 1) Using recursive orientation, (VPGAGVPGEG)18(VPGAG)18(VPGIG)60 (also known as ELP(E18-)) was obtained. pET24b-(AE)18 and pET24b-(A)18, obtained by the RDL method, were used as parental plasmids. pET24b-(AE)18 was double-digested with restriction endonucleases AcuⅠ and BglⅠ, using the same digestion system as in Example 1. After incubating at 37℃ for 3 hours, three DNA fragments of different sizes were obtained and subjected to 1.2% agarose gel permeation. After gel electrophoresis, the larger molecular weight DNA fragment, namely the linear fragment (AE)18-1 containing the (AE)18 gene, was recovered and purified from the gel. pET24b-(A)18 was then double-digested with restriction endonucleases BseRI and BglRI, using the same digestion system as in Example 1. After incubation at 37°C for 3 hours, two DNA fragments of different sizes were obtained. After 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment, namely the linear fragment (A)18-2 containing the (A)18 gene, was recovered and purified from the gel. Linear fragments (AE)18-1 and (A)18-2 were ligated using T4 DNA ligase, following the same ligation system as in Example 1. The mixture was incubated at 25°C for 2 hours. The ligation product was then added to competent E. coli cells and incubated on ice for 30 minutes; then at 42°C for 60 seconds; immediately placed on ice for 5 minutes; 700 μL of antibiotic-free LB medium was added; the mixture was incubated at 37°C on a shaker at 200 rpm for 1 hour; centrifuged at 3000 rpm for 5 minutes, discarding 600 μL of the supernatant. The remaining liquid was mixed and plated onto LB agar plates containing kanamycin, and incubated overnight at 37°C inverted. Single clones were selected and colony PCR was performed using universal primers. Bacterial clone suspensions were prepared by dissolving single clones in 10 μL of sterile ultrapure water. The PCR reaction system consisted of: 5 μL 2x Flash PCR MasterMix, 0.5 μL ELP-T7-F, 0.5 μL ELP-T7-R, 1 μL bacterial suspension, and 3 μL ultrapure water. Agarose gel electrophoresis was performed to identify and sequence the correct plasmids. (VPGAGVPGEG)18(VPGAG)18 was obtained.

[0105] 2) Using pET24b-(AE)18(A)18 and pET24b-(I)60 obtained by the RDL method as parental plasmids, pET24b-(AE)18(A)18 was double-digested with restriction endonucleases AcuⅠ and BglⅠ, in the same digestion system as in Example 1. After incubation at 37℃ for 3 hours, three DNA fragments of different sizes were obtained. After detection by 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment was recovered and purified. The linear fragment (AE)18(A)18-1 containing the (AE)18(A)18 gene was obtained. pET24b-(I)60 was double-digested with restriction endonucleases BseRI and BglRI, using the same digestion system as in Example 1. After incubation at 37°C for 3 hours, two DNA fragments of different sizes were obtained. After detection by 1.2% agarose gel electrophoresis, the larger molecular weight DNA fragment, i.e., the linear fragment (I)60-2 containing the (I)60 gene, was purified from the gel. Linear fragments (AE)18(A)18-1 and (I)60-2 were ligated using T4 DNA ligase, following the same ligation system as in Example 1. The cells were incubated at 25°C for 2 hours. The ligation product was then added to competent E. coli cells and incubated on ice for 30 minutes; then at 42°C for 60 seconds; immediately placed on ice for 5 minutes; 700 μL of antibiotic-free LB medium was added, and the cells were incubated at 37°C on a shaker at 200 rpm for 1 hour; centrifuged at 3000 rpm for 5 minutes, discarding 600 μL of the supernatant. The remaining liquid was mixed and plated onto LB agar plates containing kanamycin, and incubated overnight at 37°C inverted. Single clones were selected and colony PCR was performed using universal primers. Bacterial clone suspensions were prepared by dissolving single clones in 10 μL of sterile ultrapure water. The PCR reaction system consisted of: 5 μL 2x Flash PCR MasterMix, 0.5 μL ELP-T7-F, 0.5 μL ELP-T7-R, 1 μL bacterial suspension, and 3 μL ultrapure water. Agarose gel electrophoresis was performed to identify and sequence the correct plasmids. The desired plasmid composition was (VPGAGVPGEG)18(VPGAG)18(VPGIG)60.

[0106] Example 7: Construction of mCherry, mCherry-(VPGAG)60(VPGIG)60

[0107] 1) Design and synthesize gene fragments for expressing reporter fluorescent protein mCherry primers.

[0108] mCherry-F: AACTTTAAGAAGGAGATATACATATGGTGAGCAAGGGCGAGGAGGA (SEQ IDNO: 9);

[0109] mCherry-R: CAGTGGGTGGTGGTGGTGGTGCTCGAGCTTGTACAGCTCGTCCATGCC (SEQ ID NO: 10);

[0110] Design and synthesize gene fragments for coupling the reporter fluorescent protein mCherry to (VPGAG)60 (VPGIG)60 primers.

[0111] 24b-mCherry-F:TTTAAGAAGGAGATATACATATGCACCACCACCACCACGTGAGCAAGGGCGAGGAG (SEQ ID NO: 11);

[0112] mCherry-M9-R: atcatccaggccgcaaaagaagaaaaaCTTGTACAGCTCGTCCATGCCGCC (SEQID NO: 12);

[0113] M9-AE-R: CCTGCCTTCGCTCCTCCGCTAGCGCTGCCACCGCCACCatcatccaggccgcaaaaga SEQ ID NO: 13);

[0114] PCR amplification system: 12.5 μL of 2x Phanta Max Master Mix, 1 μL of primer F, 1 μL of primer R, 10 μL of ultrapure water, and 0.5 μL of pcDNA3.1-mCherry plasmid;

[0115] Amplification program: 95℃ for 30s; 95℃ for 15s, 55℃ for 10s, 72℃ for 30s / kb, 34 cycles; 72℃ for 8min.

[0116] Agarose gel electrophoresis, gel recovery, the recovered fragment was mCherry, size 757bp.

[0117] Using pcDNA3.1-mCherry as a template, mCherry-1 was amplified by PCR using primers 24b-mCherry-F and mCherry-M9-R. The agarose gel electrophoresis and gel recovery yielded the mCherry-1 fragment, which was 773 bp in size. Using the recovered mCherry-1 as a template, mCherry-2 was amplified by PCR using primers 24b-mCherry-F and M9-AE-R. The agarose gel electrophoresis and gel recovery yielded the mCherry-1 fragment, which was 811 bp in size.

[0118] 2) Digest the pET24b plasmid vector with NdeI and XhoI. The digestion system is as follows: 3 μL 10x buffer, 24 μL pET24b plasmid, 1 μL NdeI, and 1 μL XhoI.

[0119] The pET24b-ELP(E0) plasmid vector was digested with Nde I and Nhe I. The digestion system consisted of 3 μL of 10x buffer, 24 μL of pET24b-ELP(E0) plasmid, 1 μL of Nde I, and 1 μL of Xhe I.

[0120] After 3 hours at 37°C, agarose gel electrophoresis was performed, and pET24b and pET24b-ELP(E0) fragments with sizes of 5370bp and 7094bp were recovered from the gel.

[0121] 3) Homologous recombination process, specific reaction system: 0.02 x 757 bp (or 811 bp) of mCherry (or mCherry-2) fragment = 15.1 (or 16.2) ng, 0.01 x 5370 bp (or 7094 bp) of pET24b (or pET24b-ELP(E0)) plasmid vector = 53.7 (or 70.9) ng, 2 x clonExpression Mix (the sum of the volumes of the above DNA fragment and plasmid vector).

[0122] At 50℃, after 15 minutes, immediately place on ice and let stand for 5 minutes.

[0123] 4) Transformation: Add the recombinant product to competent E. coli cells and incubate on ice for 30 min; 42℃ for 60 s; immediately place on ice for 5 min; add 700 μL of antibiotic-free LB medium, incubate at 37℃ on a shaker at 200 rpm for 1 h; centrifuge at 3000 rpm for 5 min, discard 600 μL of supernatant, mix the remaining liquid, spread it on LB plates containing kanamycin, and incubate in an inverted incubator at 37℃ overnight.

[0124] 5) Select single clones and perform colony PCR identification using universal primers. The bacterial clone suspension was prepared by dissolving the single clone in 10 μL of sterile ultrapure water. The PCR reaction system was: 5 μL 2x Flash PCR MasterMix, 0.5 μL ELP-T7-F, 0.5 μL 22b-mCherry-R, 1 μL bacterial suspension, and 3 μL ultrapure water. Agarose gel electrophoresis was performed, and the correct plasmid was sequenced. The amino acid sequence of mCherry-(VPGAG)60(VPGIG)60 is shown in SEQ ID NO:14.

[0125] Where M is the start codon; HHHHHH is the his tag; a single underscore represents the mCherry amino acid sequence; a double underscore represents the MMP9 enzyme recognition site; GGGGS is the linker; and italics represent a portion of the vector's own sequence, which is the enzyme cleavage site.

[0126] Example 8: Constructing mCherry-(VPGAG)18(VPGAGVPGEG)9(VPGAG)18(VPGIG)60 and mCherry-(VPGAGVPGEG)18(VPGAG)18(VPGIG)60

[0127] 1) Prepare the recycled gum product mCherry-2 obtained in Example 7.

[0128] 2) Digest pET24b-ELP(E9-) and pET24b-ELP(E18-) plasmid vectors with Nde I and Nhe I. The digestion system is: 3 μL 10x buffer, 24 μL plasmid, 1 μL Nde I, and 1 μL Xhe I.

[0129] After 3 hours at 37°C, agarose gel electrophoresis was performed, and pET24b-ELP(E9-) and pET24b-ELP(E18-) fragments, both 7004 bp in size, were recovered from the gel.

[0130] 3) Homologous recombination process, specific reaction system: mCherry-2 fragment 0.02 x 811 bp = 16.2 ng, pET24b-ELP(E9-) and pET24b-ELP(E18-) plasmid vectors 0.01 x 7004 bp = 70 ng, 2 x clonExpressionMix (the sum of the volumes of the above DNA fragments and plasmid vectors).

[0131] At 50℃, after 15 minutes, immediately place on ice and let stand for 5 minutes.

[0132] 4) Transformation: Add the recombinant product to competent E. coli cells and incubate on ice for 30 min; 42℃ for 60 s; immediately place on ice for 5 min; add 700 μL of antibiotic-free LB medium, incubate at 37℃ on a shaker at 200 rpm for 1 h; centrifuge at 3000 rpm for 5 min, discard 600 μL of supernatant, mix the remaining liquid, spread it on LB plates containing kanamycin, and incubate in an inverted incubator at 37℃ overnight.

[0133] 5) Select single clones and perform colony PCR identification using universal primers. The bacterial clone suspension was prepared by dissolving the single clone in 10 μL of sterile ultrapure water. The PCR reaction system was: 5 μL of 2x Flash PCR MasterMix, 0.5 μL of ELP-T7-F, 0.5 μL of 22b-mCherry-R, 1 μL of bacterial suspension, and 3 μL of ultrapure water. Agarose gel electrophoresis was performed, and the correct plasmids were sequenced. The amino acid sequences of mCherry-(VPGAG)18(VPGAGVPGEG)9(VPGAG)18(VPGIG)60 are shown in SEQ ID NO:15, and the amino acid sequence of mCherry-(VPGAGVPGEG)18(VPGAG)18(VPGIG)60 is shown in SEQ ID NO:16.

[0134] (SEQ ID NO:15);

[0135] MHHHHHHVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKFFFFCGLDDGGGGSASGGAKAGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGEGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGKMSRASGIPKLLEHHHHHH(SEQ ID NO:16).

[0136] Example 9: Expression and purification of mCherry, mCherry-ELP (E0) in Example 6, and mCherry-ELP (E9-), mCherry-ELP (E18-) in Example 7

[0137] 1) The recombinant plasmid was transformed into BL21(DE3) Escherichia coli, plated on LB agar plates containing kanamycin, and cultured overnight. Single colonies were picked from the LB plates and placed in LB liquid medium containing 50 μg / ml kanamycin for overnight shaking culture. Subsequently, the culture was scaled up in LB liquid medium containing 50 μg / ml kanamycin for 2-3 h. When the OD600 value was detected to be 0.6-0.8, IPTG was added to a final concentration of 0.4 mM, and expression was induced at 16℃ for 20 h. The cells were collected by centrifugation at 6000 rpm for 5 min, and the cells were resuspended in 200 ml of PBS.

[0138] 2) Reagent preparation and sample preparation: All buffers must be prepared with high-purity water. Lysis equilibration buffer (LEBuffer): 100mM Na₂HPO₄, 10mM Tris·Cl, 8M Urea, pH 8.0; Wash buffer: 100mM Na₂HPO₄, 10mM Tris·Cl, 10mM imidazole, 8M Urea, pH 8.0; Elution buffer: 100mM Na₂HPO₄, 10mM Tris·Cl, 250mM imidazole, 8M Urea, pH 8.0. Sonicate the cell suspension on ice for 2 seconds, then cool for 3 seconds, for a total time of 1 hour. Centrifuge (12000 rpm, 4℃, 10 min), discard the precipitate, and retain the supernatant. Centrifuge the supernatant again (12000 rpm, 4℃, 10 min), discard the precipitate, and retain the supernatant.

[0139] 3) Column packing and purification: Gently invert the bottle several times to ensure thorough mixing of the medium. Add a certain amount of medium to the column, allow it to settle freely, and drain the stock solution. Add 8 column volumes of equilibration buffer to equilibrate the chromatography medium or the eluent to a minimum and stable UV absorbance (A280). Add the protein sample to the pre-equilibrated Ni-NTA resin and allow it to flow out slowly at a rate of approximately 0.5-1 ml / min. Add 15 column volumes of wash buffer to remove non-specifically bound proteins from the Ni-NTA resin at a rate of approximately 0.25-0.5 ml / min. Slowly add elution buffer containing 250 mM imidazole (pre-cooled to 4°C), collect the eluent, and analyze the protein purity using sodium dodecyl sulfate-polyacrylamide SDS-PAGE. The results are shown below. Figure 2As shown in a and 2b, due to the partial hydrolysis of the acylamide bond in the mCherry chromophore during high-temperature denaturation, we performed non-deformation PAGE electrophoresis on mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-). Due to the temperature-sensitive self-assembly properties of mCherry-ELPs, the entire electrophoresis process was conducted on ice to maintain low-temperature electrophoresis. Figure 3 The results show that the purity of the purified target protein is over 95%.

[0140] 4) Protein dialysis, quantification, and storage: The purified mCherry, mCherry-ELP conjugates were placed in a 14000 Da dialysis bag and dialyzed in pre-chilled PBS buffer. The PBS buffer was changed twice, every 4 hours. After dialysis, the protein solution was centrifuged at 9000 rpm for 20 min at 4°C, and the supernatant was collected. The supernatant was filtered through a 0.22 μm filter for sterilization, and the protein concentration was determined by the BCA method. The obtained protein concentrations were mCherry (2.2 mg / mL), mCherry-ELP(E0) (3.3 mg / mL), mCherry-ELP(E9-) (3.2 mg / mL), and mCherry-ELP(E9-) (3.0 mg / L). The protein solutions were aliquoted and stored at -80°C.

[0141] Example 10: Physicochemical characterization of mCherry-ELP (E0), mCherry-ELP (E9-) and mCherry-ELP (E18-)

[0142] The particle size, polymer dispersion index (PDI), and zeta potential at pH 7.4 of mCherry-ELP (E0), mCherry-ELP (E9-), and mCherry-ELP (E18-) at 37℃ were determined using a Malvern particle size analyzer. Samples were prepared by adding 20 μL of sample to an 800-1000 μL detection cell. The test results are shown below. Figure 4 a,mCherry, after coupling with ELPs, retains the thermosensitive behavior of ELPs and self-assembles into nanoparticles at 37℃, with a particle size between 45-60 nm and a polymer dispersion index (PDI) between 0.1 and 0.3. Figure 4 b). When testing the zeta potential, a sample solution is drawn for testing, and the detection results are as follows: Figure 5mCherry-ELP(E0) is electroneutrally neutral, while mCherry-ELP(E9-) and mCherry-ELP(E18-) are electronegative, with potentials of approximately -23.94±0.21 V and -12.9±0.75 V, respectively. This indicates that the ELPs were successfully modified with negative charges and exhibit temperature-responsive self-assembly behavior.

[0143] Example 11: Retention time of mCherry-ELP (E0), mCherry-ELP (E9-) and mCherry-ELP (E18-) in vivo

[0144] Fluorescent protein mCherry was conjugated with ELPs in the form of mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-), which were injected into mice via the tail vein. In vivo imaging (IVIS) was used to observe the retention time of the nanoparticles in the abdominal region of the mice. Three male C57BL / 6 mice were used in each group.

[0145] Specific steps:

[0146] 1. Sample preparation

[0147] Remove mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-) stored at -80℃, add sterile PBS buffer, and quantify 0.4 μM, 250 μL volume of fluorescent protein ELP conjugate per mouse.

[0148] 2. Tail vein injection in mice

[0149] Negative control group: PBS buffer was injected via tail vein.

[0150] 3. In vivo retention time analysis

[0151] Mice were photographed using a small animal in vivo imaging system at 4h, 6h, 12h, 24h, 48h, and 72h after tail vein injection. Imaging was performed using the mCherry fluorescence channel with excitation of 580 nm and emission of 620 nm. The experimental results are as follows: Figure 6 The negative control group showed no fluorescence, while mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-) all exhibited fluorescence in the mouse abdomen. Among them, mCherry-ELP(E9-) showed the highest fluorescence intensity and the longest retention time, reaching 72 hours, followed by mCherry-ELP(E18-). This indicates that ELPs modified with negative surface charges are not easily cleared by internal organs, demonstrating favorable pharmacokinetic characteristics.

[0152] Example 12: Pharmacokinetic characteristics of mCherry-ELP (E0), mCherry-ELP (E9-) and mCherry-ELP (E18-)

[0153] To further investigate the pharmacokinetic characteristics of the fluorescent protein mCherry conjugates mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-), the nanoparticles were injected into mice via the tail vein, as in Example 11. Blood was collected at 10 min, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h, 72 h, and 96 h, with a blood volume of 30 μl each time. The fluorescence detection channel of an ELISA reader was used for detection. The detection results are as follows: Figure 7 As shown, electronegatively modified nanoparticles significantly prolonged the half-life of the fluorescent protein mCherry. To further analyze its pharmacokinetic parameters, we performed non-compartmental model calculations using Phoenix Winnonlin 8.1 software, as shown... Figure 8 As shown, the standalone fluorescent protein mCherry has an extremely short half-life of only 4.09±0.46 h and is rapidly metabolized in vivo. However, the conjugates of mCherry and ELPs form nanoparticles mCherry-ELP(E0), mCherry-ELP(E9-), and mCherry-ELP(E18-), which effectively increase the in vivo circulation and half-life to 22.15±2.07 h, 37.41±2.20 h, and 35.88±2.57 h, respectively, which are 5.4 times, 9.1 times, and 8.7 times that of the standalone protein. The bioavailability of the drug can be preliminarily understood by calculating the area under the concentration curve (AUC). The AUC of mCherry was (19.58±272) h·mg / L, while that of mCherry-ELP(E0) was (162.46±8.59) h·mg / L, mCherry-ELP(E9-) was (416.73±67.87) h·mg / L, and mCherry-ELP(E18-) was (350.58±58.54) h·mg / L, representing 8.3, 21.3, and 17.9 times that of mCherry monomeric protein, respectively. This data indicates that ELP nanoparticles have a more significant ability to increase the total amount of drug in the blood compared to half-life. Furthermore, compared to electronegative and electronegative nanoparticles, negatively charged nanoparticles showed significant improvements in both half-life and bioavailability, with ELP(E9-) showing the most significant improvement.

[0154] Example 13: In vivo distribution and inflammatory gut enrichment of mCherry-ELP (E0), mCherry-ELP (E9-) and mCherry-ELP (E18-)

[0155] Thermosensitive self-assembled ELPs modified with negative surface charge were used to deliver red fluorescent mCherry reporter protein in C57BL / 6 mice. The mice were injected via the tail vein, and organs were lysed after 4 hours of blood circulation. The distribution characteristics of the nanoparticles in vivo could be detected.

[0156] Specific steps:

[0157] 1. Induction of inflammatory bowel disease in mice by drinking water with 3% sodium dextran sulfate (DSS).

[0158] C57BL / 6 male mice were randomly divided into 4 groups. 3% (m / V) DSS was weighed and dissolved in the mice's drinking water. The mice were induced to drink the water continuously for 6 days, and a mouse model of inflammatory bowel disease was successfully obtained.

[0159] 2. Dissected organs, IVIS imaging and fluorescence assay of nanoparticle distribution.

[0160] The quantitative analysis of mCherry conjugates with ELPs was performed as in Example 10. Four hours after tail vein injection, mice were sacrificed, and organs such as heart, liver, spleen, lung, kidney, brain, small intestine, and colon were harvested. IVIS tissue fluorescence imaging was performed, the tissues were ground, and lysed on ice for 30 min using 0.5% TriTon X-100 lysis buffer. The supernatant was collected by centrifugation, and fluorescence was tested using an ELISA reader.

[0161] 3. Analysis of Experimental Results

[0162] Distribution of ELP nanoparticles in various organs as follows Figure 9 , Figure 10 As shown, the nanoparticles first aggregate in the kidneys, and then in the liver, consistent with the organ distribution characteristics of ELP nanoparticles. Based on the characteristic features of the lesion tissue at the site of IBD inflammation, namely the accumulation of positive charges in situ in the intestine, this invention mainly explores the pharmacokinetic characteristics and selective enrichment in the intestine after negative charge modification. Therefore, IVIS imaging analysis and fluorescence testing were performed on the nanoparticles in colon tissue alone. The experimental results are as follows... Figure 11 As shown, negatively charged ELPs, especially ELP(E9-), exhibited fluorescence intensity 2-3 times higher than normal electrically neutral ELPs, significantly enhancing the accumulation of nanoparticles at sites of colonic inflammation and laying the foundation for subsequent therapeutic drug delivery experiments.

[0163] In summary, this invention creatively proposes a negatively charged modified protein drug delivery polymer carrier (ELP) with multi-environmentally responsive self-assembly. Its structure can be modified in various ways through genetic engineering or chemical synthesis to obtain drug carriers with different environmental stimuli responses. By regulating its properties, it can self-assemble into nanoparticles under certain response conditions, increasing the size and solubility of different drugs. The nanoparticles are tightly packed, making them less susceptible to metabolic clearance in vivo, maintaining the stability of in vivo transport, prolonging the drug's residence time, increasing its half-life, and improving bioavailability. Simultaneously, due to the EPR effect in tumor tissue and inflamed sites, the nanoparticles can be effectively passively enriched, increasing drug efficacy. The negatively charged modified nanoparticles of this invention have the following characteristics: First, they are not easily taken up and cleared, greatly improving pharmacokinetic characteristics; second, they readily accumulate selectively in intestinal tissue at sites of positive charge accumulation in inflamed areas, contacting high-release MMP9 to release the drug and improve efficacy; third, they reduce the toxicity caused by frequent drug administration, improving drug biosafety. This negatively charged, self-assembly protein drug delivery polymer carrier (ELP) system, which is responsive to multiple environmental stimuli, is suitable for many pharmaceutical proteins, enzymes, antibodies, small molecule drugs, and polymers. It can not only reduce the frequency of drug administration but also improve the stability, bioactivity, and therapeutic effect of drugs, reduce toxic side effects, and significantly improve the quality of life of patients.

[0164] The above detailed embodiments have provided a comprehensive description of the present invention. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A drug delivery carrier, characterized in that, The drug delivery carrier contains a negatively charged elastin-like protein; The elastin-like sequence is: (VPGAG)18(VPGAGVPGEG)9(VPGAG)18(VPGIG)60 or (VPGAGVPGEG)18(VPGAG)18(VPGIG)60.

2. The drug delivery carrier according to claim 1, characterized in that, The response temperature of the drug delivery carrier is 15-60℃.

3. A protein polymer self-assembly comprising the drug delivery carrier and active molecule as described in any one of claims 1 to 2.

4. The protein polymer self-assembled assembly according to claim 3, characterized in that, The active molecules include diagnostic agents and / or therapeutic agents and / or molecular switches.

5. The protein polymer self-assembled assembly according to claim 4, characterized in that, The diagnostic agents include fluorescently labeled, radiolabeled, or enzyme-labeled agents.

6. The protein polymer self-assembled assembly according to claim 4, characterized in that, The therapeutic agents include proteins, peptides, small molecule chemicals, or traditional Chinese medicine.

7. The protein polymer self-assembled assembly according to claim 4, characterized in that, The molecular switch includes a recognition sequence for matrix metalloproteinases (MMPs), esterases, histones, furin proteases, or caspases.

8. The protein polymer self-assembled assembly according to claim 5, characterized in that, The fluorescent label includes at least one of mCherry, GFP, YFP, BFP, and CFP.

9. A biomaterial relating to the drug delivery carrier according to any one of claims 1 to 2 or the protein polymer self-assembly according to any one of claims 3 to 8, wherein the biomaterial is any one of (a1) to (d1) below: (a1) A nucleic acid molecule encoding the drug delivery carrier according to any one of claims 1 to 2 or the protein polymer self-assembly according to any one of claims 3 to 8; (b1) An expression cassette containing the nucleic acid molecule described in (a1); (c1) A recombinant vector containing the nucleic acid molecule described in (a1), or a recombinant vector containing the expression cassette described in (b1); (d1) A recombinant microorganism containing the nucleic acid molecule described in (a1), or a recombinant microorganism containing the expression cassette described in (b1), or a recombinant cell containing the recombinant vector described in (c1).

10. The use of the delivery carrier according to any one of claims 1 to 2, the protein polymer self-assembly according to any one of claims 3 to 8, or the biomaterial according to claim 9 in the preparation of diagnostic agents and / or pharmaceuticals.

11. A drug comprising the delivery carrier according to any one of claims 1 to 2, the protein polymer self-assembly according to any one of claims 3 to 8, or the biomaterial according to claim 9.

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