Elastin-like polypeptide as well as preparation method and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to elastin-like polypeptide as well as a preparation method and application thereof. The amino acid sequence of the elastin-like polypeptide is (VPGXG) n, X comprises I, A and F, and n is an integer selected from 20-120. The elastin-like polypeptide disclosed by the invention has reversible phase change temperature, a purification method is simple, and the elastin-like polypeptide has good and controllable release capacity. The elastin-like polypeptide disclosed by the invention can serve as a drug storage cavern, can be coupled with various drugs, and can improve the treatment effect of the carried drugs, so that targeted therapy is more accurate.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an elastin-like polypeptide and a preparation method and application thereof. Background Art

[0002] Elastin is an important extracellular matrix protein, mainly found in the connective tissue of arteries, skin, lungs and ligaments. It can combine with microfibrils in the body to form elastic fibers, and together with collagen fibers, it gives tissues elasticity and tensile strength.

[0003] Elastin-like polypeptides (ELPs) are repetitive artificial polypeptides derived from the repetitive amino acid sequence in the hydrophobic domain of tropoelastin. Their structure is mainly composed of pentapeptide repeating units (Val-Pro-Gly-Xaa-Gly, VPGXG) in series, where Xaa is any amino acid except proline. Since different amino acids have different structures and functions, ELPs can exhibit different biological functions with the change of Xaa.

[0004] Elastin-like polypeptides will show reversible phase transition behavior with changes in temperature. When the temperature is lower than its phase transition temperature (transition temperature, Tt), the elastin-like polypeptide is in a soluble state. When the temperature is higher than its Tt, the elastin-like polypeptide is in an insoluble aggregated state. The different amino acids represented by X make the properties of elastin-like polypeptides different. Specifically, if X is a hydrophobic amino acid, the phase transition temperature increases. According to the different properties, proportion composition of X and the number of repetitions of n, elastin-like polypeptides or elastin-like polypeptide fusion proteins with different phase transition temperatures can be designed. When the elastin-like polypeptide shows a reversible transition of the condensed state, the recombinant protein can be separated by centrifugation, thereby achieving simple protein separation. Using this characteristic, the prepared elastin-like polypeptide can be quickly purified.

[0005] Elastin-like polypeptides have special temperature responsiveness. When the temperature is higher than the phase transition temperature, they will show the characteristics of thermodynamically reversible phase transition. The β-turn structure of the repeating unit at the Pro-Gly position changes from an extended and disordered state to a more compact right-handed β-helix. Continuing to increase the temperature will cause the β-helices to gather together. Within the phase transition temperature range, a certain number of amino acids can be observed, forming β-folds within the chain, further promoting the cross-linking and aggregation between elastin-like polypeptide molecular chains. This makes the elastin-like polypeptides become ordered polymers in a water environment. This process is mainly due to the hydrophobic residues, which is conducive to the packaging of hydrophobic drugs.

[0006] Therefore, according to the characteristics and treatment needs of different drugs, the release and targeting of drugs in time and space can be precisely controlled by the sequence and length of elastin-like polypeptides. And the way of combining elastin-like polypeptides with drugs can be to connect the sequences of medicinal proteins and elastin-like polypeptides together by means of genetic engineering to express fusion proteins, design corresponding certain active groups in the elastin-like polypeptide chain, and covalently conjugate the drug to the elastin-like polypeptide chain through chemical reaction with the drug to form ELPs-DOX conjugates. In addition, medicinal proteins or small molecule drugs can be directly encapsulated into elastin-like polypeptides and self-assembled to form microparticles, nanobeams or micelles. Elastin-like polypeptides have excellent pharmacokinetic characteristics when acting as drug carriers. To achieve the slow release of medicinal proteins or small molecule drugs in elastin-like polypeptides, first adjust their phase transition temperature to below the physiological temperature, so that they can undergo phase transition at the injection site after injection to form a drug reservoir, and ensure the slow release of drugs in the form of reservoirs to achieve the long-term effect of drugs or for local delivery of drugs. At the same time, since elastin-like polypeptides are composed of natural amino acids, they have good biocompatibility, are easily biodegradable, have low immunogenicity, and are non-toxic. Using elastin-like polypeptides as drug carriers can effectively prolong the half-life of drugs, increase the solubility of poorly soluble drugs, and reduce the toxic effects of drugs on normal tissues and organs. Based on the above advantages, elastin-like polypeptides can be widely used in biomedical fields such as protein expression and purification, drug delivery, in vitro diagnosis, and tissue engineering.

[0007] Many ELPs have been discovered that can be used as drug carriers to treat a variety of diseases, including siEVI1-ELP for the treatment of various cancers (breast cancer, ovarian cancer, pancreatic cancer and lung cancer), VIP-ELP for the treatment of pulmonary hypertension, cardiomyopathy, cystic fibrosis, GLP1-ELP for the treatment of type 2 diabetes, and (VPGVG) as a reservoir for the treatment of neuroinflammation. 120 and (VPGVG) 240 Therefore, ELP development is one of the most promising areas in drug delivery.

[0008] As a suitable candidate for new-era drug delivery and regenerative medicine, elastin-like peptides are particularly concerned about the side effects of small molecule drugs, the persistence of small molecule drugs, and the half-life, as well as the increasing focus on the requirements of high-efficiency and safe and harmless performance. Therefore, it is necessary to develop more elastin-like peptides with good performance to accelerate the application, transformation, and development of elastin-like peptides in drug delivery. Summary of the invention

[0009] In order to solve the deficiencies of the prior art, the object of the present invention is to provide an elastin-like polypeptide and a preparation method thereof, as well as the use of the elastin-like polypeptide as a drug carrier.

[0010] To achieve the above object, the present invention adopts the following technical solutions.

[0011] In one aspect, the present invention provides an elastin-like polypeptide, wherein the amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X includes I, A and F, and n is selected from an integer of 20 to 120.

[0012] In some embodiments, the amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X is I, A and F, and n is selected from an integer of 20-120.

[0013] In some embodiments, the amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X is I, A and F, and n is an integer selected from 50 to 100.

[0014] In some embodiments, the amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X is I, A and F, and n can be selected from 50 or 100.

[0015] In some embodiments, the amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X is I, A and F, and n is 100.

[0016] In some embodiments, the amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X is I, A and F, and the number of amino acids is I:A:F=2:2:1.

[0017] Preferably, the amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X is I, A and F, the number of amino acids is I:A:F=2:2:1, and n is 100.

[0018] Further preferably, the elastin-like polypeptide is 20 A 20 I 20 F 20 A 20 , and has an amino acid sequence as shown in SEQ ID NO:3.

[0019] In some embodiments, the amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X includes I, A, F and L, and n is selected from an integer of 20-120.

[0020] In some embodiments, the amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X is I, A, F and L, and n is an integer selected from 60 to 120.

[0021] In some embodiments, the amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X is I, A, F and L, and n can be selected from an integer of 60 or 120.

[0022] In some embodiments, the amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X is I, A, F and L, and n is 120.

[0023] In some embodiments, the amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X is I, A, F and L, and the number of amino acids is I:A:F:L=2:2:1:1.

[0024] Preferably, the amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X is I, A, F and L, the number of amino acids is I:A:F:L=2:2:1:1, and n is 120.

[0025] Further preferably, the elastin-like polypeptide is 20 A 20 I 20 F 20 A 20 L 20 , and has an amino acid sequence as shown in SEQ ID NO:4.

[0026] In some embodiments, in order to ensure seamless connection of the amino acid sequence of the elastin-like polypeptide of the present invention, I, A, F and L in the sequence are each independently replaced by V.

[0027] In some embodiments, to ensure seamless sequence connection, the first I, A, and F of each monomer ELP module in the amino acid sequence of the elastin-like polypeptide of the present invention is replaced by V.

[0028] In some embodiments, to ensure seamless sequence connection, I, A, F or L in the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4 are each independently replaced by V.

[0029] In some embodiments, to ensure seamless sequence connection, the first I, A, and F of each monomer ELP module in the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:4 are replaced with V.

[0030] In some embodiments, the elastin-like polypeptide further comprises a leader peptide, which can enhance the expression level of the elastin-like polypeptide.

[0031] In some embodiments, the amino acid sequence of the elastin-like polypeptide further comprises a leader peptide, and the leader peptide is selected from MGSSGLVPRGSKGPG and MSKGPG.

[0032] Preferably, the leader peptide is MSKGPG.

[0033] In some embodiments, the elastin-like polypeptide further comprises a trailer peptide, which can control the size, shape and stability of the nanostructure of the elastin-like polypeptide.

[0034] In some embodiments, the tail peptide is (Φ(G)y)z, wherein Φ represents tyrosine (Tyr, Y), phenylalanine (Phe, F) or tryptophan (Trp, W), y represents the number of glycine spacers (y=0, 1 or 2), and z is selected from an integer from 0 to 8.

[0035] Preferably, the tail peptide is (Φ(G)y)z, wherein Φ represents tyrosine (Y), that is, the structure of the tail peptide is [Y(G)y]z.

[0036] In some embodiments, the tail peptide is selected from YGYGYGYGYGYGYG, YGYGYGYGYGYGYG, YGGYGGYGGYGGYGGYGGYGG.

[0037] In some embodiments, the tail peptide is YGYGYGYGYGYGY.

[0038] Preferably, the amino acid sequence of the elastin-like polypeptide is as shown in SEQ ID NO: 1 or SEQ ID NO: 2: SEQ ID NO: 1

[0039] MSKGPGVGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGV

[0040] PGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGVGVPGAGVPGAGVPGAGVPG

[0041] AGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGV

[0042] PGAGVPGAGVPGAGVPGAGVPGVGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPGIGVPG

[0043] IGVPGIGP

[0044] FGVPGFGVPGFGVPGFGVPGFGVPGFGVPGFGVPGFGVPGFGVPGFG

[0045] GVPGFGVPGFGVPGFGVPGFGVPGFGVPGVGVPGAGVPGAGVPGAGVPGAGVP

[0046] AGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGAGVPGGAVPGWPYGYGYGYGYGY;

[0047] SEQ ID NO:2

[0048] .

[0049] In another aspect, the present invention provides a polynucleotide, the sequence of which comprises a sequence encoding the above-mentioned elastin-like polypeptide.

[0050] In another aspect, the present invention provides a recombinant expression vector comprising the sequence of the polynucleotide described above.

[0051] In another aspect, the present invention provides a genetically engineered bacterium, wherein the genetically engineered bacterium is Escherichia coli containing the above-mentioned recombinant expression vector.

[0052] In another aspect, the present invention provides a method for preparing the above-mentioned elastin-like polypeptide, the preparation method comprising:

[0053] (1) ELP monomer gene amplification: NdeI-PflM restriction site was added to the N-terminus of the ELP monomer gene module, and BglI-HindIII restriction site was added to the C-terminus, and then amplified to the target size by PCR;

[0054] (2) Construction of ELP monomer gene module expression plasmid: The ELP monomer gene was connected into the pET plasmid and verified to be correct by NdeI and HindIII restriction enzyme digestion and sequencing;

[0055] (3) The plasmid expressing the ELP monomer gene module was cut into linearized plasmid I using PflM single enzyme, and was also cut into linearized plasmid II using PflM and BglI double enzymes, and the ELP monomer gene was purified and recovered using a DNA purification kit. The linearized plasmid I obtained by single enzyme cutting and the linearized plasmid II obtained by double enzyme cutting were ligated overnight under the action of T4 ligase to obtain a recombinant plasmid;

[0056] (4) transfecting the obtained recombinant plasmid into Escherichia coli to induce expression;

[0057] (5) crushing the E. coli obtained in step (4) to obtain a soluble protein solution containing the target protein;

[0058] (6) Elastin-like polypeptides were purified by reversible phase change cycle method.

[0059] Furthermore, the plasmid in step (2) is pET15b.

[0060] Furthermore, the specific method of the crushing treatment in step (6) is at least one of ultrasonic crushing, homogenization crushing, ball milling crushing, repeated freeze-thaw crushing and lysozyme enzymatic crushing.

[0061] Furthermore, the reversible phase change cycle method in step (7) comprises the following steps:

[0062] Add NaCl solid to the supernatant to a final concentration of 2 mol / L, incubate in a 42°C water bath for 15 min, centrifuge at 12000 rpm / min for 15 min, and remove the supernatant; add 35 mL of PBS solution to the precipitate to dissolve the precipitate, transfer it to a 50 mL conical bottom centrifuge tube, rotate it in a refrigerator for 1 h, centrifuge it at 4°C and 12000 rpm / min for 15 min, and collect the supernatant; repeat the above steps and add NaCl solid to the supernatant to a final concentration of 2 mol / L, collect the precipitate, add 5 mL of PBS solution at 4°C to dissolve the precipitate, centrifuge it at 4°C and 12000 r / min for 15 min, and collect the supernatant.

[0063] The elastin-like polypeptide in the present invention has a reversible phase transition temperature. According to this characteristic, when the temperature is higher than the phase transition temperature, the elastin-like polypeptide aggregates to form particles; when the temperature is lower than the phase transition temperature, the elastin-like polypeptide is dispersed in the solution and the solution becomes clear. According to this characteristic, the elastin-like polypeptide can be used to separate and purify.

[0064] In yet another aspect, the present invention further provides a use of the above-mentioned elastin-like polypeptide in preparing a drug delivery system.

[0065] In some embodiments, the elastin-like polypeptide of the present invention contains an active group at the end, and the active group can bind to a drug or a marker to couple and release the drug.

[0066] In some embodiments, the elastin-like polypeptide synthesized in the present invention has an optimal phase transition temperature and exists in a solution state at room temperature. When injected into the human body at body temperature, the elastin-like polypeptide solidifies at the lesion site and does not diffuse as a whole.

[0067] In some embodiments, the elastin-like polypeptide described above is degradable and its injection point is controllable.

[0068] In some embodiments, the elastin-like polypeptides have good release capacity and good release effect within a certain period of time.

[0069] In some embodiments, the elastin-like polypeptide of the present invention has a good drug release capacity, so the packaged drugs are diverse, including therapeutic proteins (interleukins, tumor necrosis factors, corticosteroids and enzymes, monoclonal antibodies, therapeutic vaccines, insulin, interferons, etc.), radionuclide drugs. And the elastin-like polypeptide is developed as a degradable, injectable, customizable close-range continuous elastin-like polypeptide reservoir. Its treatment method is to use the ELP thermosensitive agglutination and cross-linking properties to form a reservoir, so that the carried drug is retained at the injection position by direct or interventional injection, and in this way, the drug release is accurately controlled for distribution, and the speed and release time of the drug release are controlled, and the target area and safety margin can be set, so that harmful cells can be killed at close range without affecting the surrounding healthy tissues, so that the lesions are reduced or disappeared, or the organ function is improved, or the auxiliary organs are retained, so as to improve the quality of life of patients and prolong the survival period.

[0070] The elastin-like polypeptide of the present invention exists in the form of a reservoir, can be coupled with a variety of drugs, and has the following advantages:

[0071] (1) The release capacity of this type of elastin polypeptide is controllable, which can improve the therapeutic effect of the drug it carries;

[0072] (2) It can make targeted therapy more accurate and improve the enrichment of drugs at the tumor site. The phase transition temperature of the two elastin-like polypeptides of the present invention is lower than the human body temperature and can aggregate and solidify when injected into the human body;

[0073] (3) It can control the dosage of drugs, thereby reducing the toxic and side effects of drugs.

[0074] the term

[0075] In order to make the present invention more easily understood, some technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0076] In the context of the present disclosure, the term "amino acid" is defined as having at least one primary, secondary, tertiary or quaternary amino group and at least one acid group, wherein the acid group can be a carboxylic acid, a sulfonic acid or a phosphoric acid or a mixture thereof. The amino group can be "α", "β", "γ" to "ω" relative to the acid group. Suitable amino acids include, but are not limited to, the D- and L-isomers of the 20 common naturally occurring amino acids (e.g., alanine, arginine, asparagine, cysteine, glutamine, aspartic acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine) found in peptides, as well as naturally occurring and non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways.

[0077] The amino acids mentioned in the present invention are abbreviated as follows according to the nomenclature of IUPAC-IUB:

[0078] Alanine (Ala, A); Arginine (Arg, R); Asparagine (Asn, N); Aspartic acid (Asp, D); Cysteine ​​(Cys, C); Glutamic acid (Glu, E); Glutamine (Gln, Q); 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); Valine (Val, V).

[0079] The terms "polypeptide", "peptide" and "amino acid sequence" are used interchangeably herein to refer to a polymer of amino acid residues of any length. The polymer may be linear or branched, it may contain modified amino acids or amino acid analogs, and may be interrupted by non-amino acid chemical moieties. The term also includes amino acid polymers that have been naturally or artificially modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a label or biologically active component). The term "peptide" includes two or more naturally occurring or synthetic amino acids linked by covalent bonds (e.g., amide bonds).

[0080] The term "including" is used to mean "including but not limited to".

[0081] The term "buffer" refers to a buffered solution that resists changes in pH through the action of its acid-base conjugate components.

[0082] Compared with the prior art, the present invention has at least the following beneficial effects:

[0083] (1) The method for purifying elastin-like polypeptides of the present invention is simple and reduces the preparation cost, and can achieve large-scale separation and purification of elastin-like polypeptides.

[0084] (2) The present invention provides a novel elastin-like polypeptide, provides a new structure for elastin-like polypeptides, enriches the types of ELPs, and provides a new type for drug carriers.

[0085] (3) The elastin-like polypeptide of the present invention has good release capacity both in vivo and in vitro, and the release capacity is controllable, which can improve the therapeutic effect of the carried drug. The phase transition temperature fluctuates around 15 to 20°C, which is lower than the human body temperature, and is suitable as a drug carrier for drug delivery.

[0086] (5) The elastin-like polypeptide of the present invention can act as a drug reservoir, can be coupled with a variety of drugs, is degradable, injectable, integrally solidified, non-migrating, and has a controllable release capacity within a certain range. It can be used as a package for drugs that require targeted treatment, such as radionuclide drugs, and can accurately control the radiation dose and the precise injection location. It has a safety margin, so that it can kill diseased tissues at close range without damaging surrounding normal tissues and organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 The ITC purification result diagram of ELPs shown in SEQ ID NO: 1 and SEQ ID NO: 2, wherein: Figure 1 A is the purification result of ELPs shown in SEQ ID NO: 1, Figure 1 B is the purification result of ELPs shown in SEQ ID NO: 2, Figure 1 C is the purity verification of ELPs shown in SEQ ID NO: 1 (1) and SEQ ID NO: 2 (2), the concentration is 15 μM, and the sample volume is 10 μL;

[0088] Figure 2 OD of ELPs shown in SEQ ID NO: 1 and SEQ ID NO: 2 in PBS buffer 350 The turbidity curve of Figure 2 A shows the change in turbidity of the ELPs shown in SEQ ID NO: 1 as the temperature rises. Figure 2 B shows the change in turbidity of the ELPs represented by SEQ ID NO: 2 as the temperature increases;

[0089] Figure 3 The ELPs repetitive phase transition diagram and OD shown in SEQ ID NO: 1 and SEQ ID NO: 2 350 Detection phase change temperature change diagram, where: Figure 3 A is the ELPs repetitive phase transition diagram shown in SEQ ID NO: 1 and SEQ ID NO: 2, Figure 3 B is the ELPsOD shown in SEQ ID NO: 1 350 Detection of phase change temperature change diagram, Figure 3 C is the ELPs OD shown in SEQ ID NO: 2 350 Detect phase change temperature change diagram;

[0090] Figure 4 The graph is a relationship between the fluorescence amount released in vitro and time of the ELPs shown in SEQ ID NO:1 and SEQ ID NO:2 labeled with FITC and simulated blood circulation, wherein: Figure 4 A is a graph showing the relationship between the fluorescence released in vitro and time of the ELPs simulated blood circulation shown in SEQ ID NO: 1, Figure 4 B is a graph showing the relationship between the fluorescence released in vitro and time of the ELPs simulated blood circulation shown in SEQ ID NO: 2;

[0091] Figure 5 The graph is a graph showing the relationship between the release capacity of ELPs shown in SEQ ID NO:1 and SEQ ID NO:2 at different concentrations in mice and the time, wherein: Figure 5 A is a graph showing the relationship between the release capacity of ELPs shown in SEQ ID NO: 1 in mice and the change in time, Figure 5 B is a graph showing the relationship between the release capacity of the ELPs shown in SEQ ID NO: 2 in mice and the change in time. Specific embodiments

[0092] The present invention is further described in detail below in conjunction with specific embodiments. The embodiments of the present invention are only for describing the technical ideas and features of the present invention, rather than for limiting the protection scope of the present invention.

[0093] Example 1 Purification of ELPs shown in SEQ ID NO: 1 and SEQ ID NO: 2

[0094] 1. Construction of SEQ ID NO: 1 and SEQ ID NO: 2 monomer modules

[0095] (1) Construction of leader peptide and tail peptide vector pET-V: The leader peptide and tail peptide DNA sequences were connected, NcoⅠ-MSKGPGWPYGYGYGYGYGYGY-XhoⅠ, the sequence was shown in SEQ ID No. 5, and the sequence was synthesized and connected into the pET15b plasmid, and verified by NcoⅠ and XhoⅠ restriction enzyme digestion and sequencing.

[0096] (2) Monomer ELP module plasmid construction: 20 pentapeptide linker modules such as (VPGVG)1(VPGIG) 19 、(VPGVG)1(VPGAG) 19 、(VPGVG)1(VPGFG) 19 、(VPGLG) 20 As shown, NdeI-PflM I and BglI-HindIII restriction sites were added to the N-terminus and C-terminus respectively during construction. The DNA sequences of the connecting modules of the above 20 pentapeptides are shown in SEQ ID No.6 to SEQ ID No.9, wherein SEQ ID No.6 is (VPGVG)1(VPGIG) 19 The DNA sequence of SEQ ID No.7 is (VPGVG)1(VPGAG) 19 The DNA sequence of SEQ ID No. 8 is (VPGVG)1(VPGFG) 19 The DNA sequence of SEQ ID No.9 is (VPGLG) 20 The DNA sequence was annealed and connected to the pUC18 plasmid, which was verified by NdeⅠ and HindⅢ restriction enzyme digestion and sequencing, and the pUC18-ELP (V1I 19 )、pUC18-ELP(V1A 19 )、pUC18-ELP(V1F 19 )、pUC18-ELP(L 20 ) plasmids, etc.

[0097] 2. Construction of SEQ ID NO: 1 and SEQ ID NO: 2

[0098] (1) pUC18-ELP(V1I 19 V1A 19 V1I 19 V1F 19 V1A 19 ) and pUC18-ELP(V1I 19 V1A 19 V1I 19 V1F 19 V1A 19 L 20 )Plasmid construction: pUC18-ELP(V1A19 ) plasmid was linearized by PflM I single restriction enzyme digestion and recovered after dephosphorylation; pUC18-ELP(V1F 19 ) plasmid was double digested with PflM and BglI to recover the monomer fragment, and the two were connected by T4 DNA ligase. After transformation of Top10 competent cells, the plasmid was extracted and verified by NdeⅠ / / HindⅢ digestion or sequencing. pUC18-ELP(V1F 19 V1A 19 Repeat the above steps until pUC18-ELP(V1I 19 V1A 19 V1I 19 V1F 19 V1A 19 ) and pUC18-ELP(V1I 19 V1A 19 V1I 19 V1F 19 V1A 19 L 20 ). pUC18-ELP(V1I 19 V1A 19 V1I 19 V1F 19 V1A 19 ) in which the DNA sequence between NdeI and HindIII is shown in SEQ ID No.10, and pUC18-ELP(V1I 19 V1A 19 V1I 19 V1F 19 V1A 19 L 20 ) The DNA sequence between NdeI and HindIII is shown as SEQ ID No.11.

[0099] (2) pET15b-ELP(V1I 19 V1A 19 V1I 19 V1F 19 V1A 19 ) and pET15b-ELP

[0100] (V1I 19 V1A 19 V1I 19 V1F 19 V1A 19 L 20 ) Construction of expression plasmid: pUC18-ELP(V1I 19 V1A 19 V1I 19 V1F19 V1A 19 ) and pUC18-ELP(V1I 19 V1A 19 V1I 19 V1F 19 V1A 19 L 20 ) was double-digested with PflM and BglI to recover the target fragment; the pET-V plasmid was linearized with SfiⅠ and recovered after dephosphorylation. The linearized vector and the target fragment were connected by T4 DNA ligase and transformed into Arctic Express competent cells. After the plasmid was extracted, it was verified by NcoⅠ and XhoⅠ digestion. pET15b-ELP(V1I 19 V1A 19 V1I 19 V1F 19 V1A 19 ) and pET15b-ELP

[0101] (V1I 19 V1A 19 V1I 19 V1F 19 V1A 19 L 20 ) plasmid.

[0102] 3. Protein Expression and Purification

[0103] (1) Activation of inoculation: The successfully constructed expression vector was inoculated and activated, and then expanded into 300 mL of TB liquid culture medium. At the same time, 1 / 1000 of ampicillin and 1 / 10 of phosphate buffer were added, and cultured at 37°C for 5-6 hours. IPTG was added to a final concentration of 1 mM (1 / 1000), and after induction at 37°C for 3 hours, the bacterial solution was centrifuged at 4°C, 4000 rpm for 5 minutes, and the bacterial cells were collected and the supernatant was discarded.

[0104] (2) Ultrasonic disruption: Add 1×PBS solution to each bottle of bacteria at a volume of 50 mL to resuspend, disrupt with ultrasound (power 60%) for 10 min, centrifuge on ice and collect the supernatant.

[0105] (3) ITC (Inverse transition cycling) purification: Add NaCl solid to the supernatant to a final concentration of 2 mol / L, incubate at 42°C in a water bath for 15 min, centrifuge at 12000 rpm / min for 15 min, and remove the supernatant; add 35 mL of PBS solution to the precipitate to dissolve the precipitate, transfer it to a 50 mL conical bottom centrifuge tube, rotate it in the refrigerator for 1 hour, centrifuge at 4°C and 12000 rpm / min for 15 min, and collect the supernatant; repeat the above steps and add NaCl solid to the supernatant to a final concentration of 2 mol / L, collect the precipitate, add 5 mL of PBS solution at 4°C to dissolve the precipitate, centrifuge at 4°C and 12000 r / min for 15 min, and collect the supernatant. Use SDS-PAGE to detect the protein size and purity. Figure 1 A and 1B show that the ELPs shown in SEQ ID NO: 1 and SEQ ID NO: 2 were purified successfully. Figure 1 C shows that when the concentration is 15 μM and the sample volume is 10 μL, the protein band is clean and free of impurities, and the purity is >95%.

[0106] Example 2 ELPs Thermal Characterization Study

[0107] The OD values ​​of the ELPs shown in SEQ ID NO: 1 and SEQ ID NO: 2 at concentrations of 25 μM, 125 μM, 250 μM, 500 μM, 750 μM and 1000 μM were measured using a carry 3500 UV-Vis spectrometer. 350 , set the temperature range to 5~30℃, measure OD every 0.5℃ / min 350 . Figure 2 The turbidity curves of the ELPs shown in SEQ ID NO: 1 and SEQ ID NO: 2 at various concentrations are shown. As can be seen from the figure, the phase transition temperature of the ELPs shown in SEQ ID NO: 1 and SEQ ID NO: 2 decreases with increasing concentration, and it is found that the phase transition temperature of the ELPs fluctuates around 15-20°C, which is lower than the human body temperature, indicating that the ELPs shown in SEQ ID NO: 1 and SEQ ID NO: 2 are suitable for use as human drug carriers.

[0108] Example 3 ELPs stability study - repeated phase transition study

[0109] According to the phase transition temperature measured at each concentration above, a constant temperature water bath was set at 40°C, 1 mL of a sample with a concentration of 25 μM was taken, and the phase transition was repeated 6 times between 40°C and 4°C, and the change in phase transition temperature after repeated phase transition was measured. The results are as follows Figure 3 As shown in A, after repeated phase transitions, ELPs can still return to a clear state at low temperatures, and Figure 3 B shows that the phase transition temperature of the ELPs shown in SEQ ID NO: 1 and SEQ ID NO: 2 remains basically unchanged after 6 repeated phase transitions. The phase transition temperature of the ELPs shown in SEQ ID NO: 1 is maintained at about 18.6°C, and the phase transition temperature of the ELPs shown in SEQ ID NO: 2 is maintained at about 19.8°C.

[0110] Example 4 In vitro release study of ELPs

[0111] The ELPs shown in SEQ ID NO:1 and SEQ ID NO:2 were replaced with NaHCO3 buffer solution (pH=8.5) by ITC to make the protein solution 5 mg / mL (rotate at 4°C until completely dissolved). FITC was dissolved in 100% DMSO at a concentration of 20 mg / mL. At 4°C, the FITC solution:ELP solution was mixed at a molar ratio of 10:1 and incubated overnight with rotation. Two rounds of ITC were performed using the phase change characteristics to separate the ELP-fluorophore conjugate from the unreacted fluorophore. After one round of ITC, the tube wall was briefly purged with PBS solution (37°C), and the original volume of PBS solution was added and incubated at 4°C with rotation until completely dissolved. After two rounds of ITC, 2 mL of PBS solution was added and it was rotated at 4°C to dissolve, and unlabeled samples were prepared at the same time.

[0112] The above FITC-labeled ELP was half-doped into the unlabeled sample to make the final concentration of 1mM, and the total fluorescence of the 1mM sample was calculated. Three concentrations of 250μM, 500μM and 750μM were set, with 3 groups for each concentration and 500μL for each group. The treated samples and the control group were placed in a 37°C constant temperature incubator, centrifuged at 12000rpm for 2min after natural sedimentation, and rotated and incubated to simulate human blood flow. 500μL was taken at 0h, 24h, 4d, 12d, 20d, 30d, 40d, 50d, 60d, 72d, 82d and 100d, and 500μL of PBS solution was replenished. The samples and standard samples were measured using a multifunctional microplate reader, and the fluorescence values ​​of the samples were measured according to the standard curve. After data processing, the release capacity of the ELPs shown in SEQ ID NO:1 and SEQ ID NO:2 was confirmed. The results are shown in Figure 2. Figure 4 As shown, when the ELP concentration is high, the release ability is weak, on the contrary, when the concentration is low, the release ability is strong, and the results show that the cumulative release of ELPs shown in SEQ ID NO:1 and SEQ ID NO:2 at concentrations of 250 μM, 500 μM and 750 μM within 100 days is less than 50%, indicating that the ELPs shown in SEQ ID NO:1 and SEQ ID NO:2 have a good half-life and can ensure the release of the drug to the greatest extent.

[0113] Example 5 Endotoxin Level Assessment

[0114] In order to ensure that endotoxin does not affect the in vivo stability experiment, causing endotoxin to exceed the standard and cause hemorrhagic fever and death of mice, this experiment used Endotoxin Removal Beads to remove endotoxin and used Limulus amebocyte lysate to detect endotoxin limit.

[0115] Mix the Endotoxin Removal Beads thoroughly, draw 1 mL of slurry into the chromatogram with a non-pyrogenic pipette tip, open the lower outlet and remove the protective solution. Wash with 3 mL of regeneration solution, control the flow rate at 0.25 m / min, or less than 10 drops per minute, and control the temperature at 2-8°C. Repeat at least twice to ensure that there is no endotoxin in the column.

[0116] Use 3 mL of equilibration solution to equilibrate the inner wall of the column tube and the filler, drain until dry, with a flow rate of about 0.5 mL / min and the temperature controlled at 2-8°C. Repeat at least twice.

[0117] Add the sample to the balanced Endotoxin RemovalBeads, adjust the flow rate to 0.25m / min, or less than 10 drops per minute. When the effluent flows out for about 1m, start collecting the effluent. After it dries up, add 1mL of equilibration solution and continue collecting.

[0118] The endotoxin content in the samples tested by the horseshoe crab reagent (endotoxin content is lower than the international standard (radioinjection K = EU2.5 / gh)f), and the endotoxin detection level is shown in Table 1:

[0119] Table 1 SEQ ID No: 1, SEQ ID No: 2 Endotoxin detection level

[0120]

[0121] The results show that the endotoxin content in the ELPs sample of the present invention is low, which can ensure the safety of clinical medication.

[0122] Example 6 In vivo experimental release study

[0123] In order to study the release ability and stability of the ELP in vivo, the release ability and stability of the ELP were detected by injecting Cy5.5-labeled ELP into mice and observing the decay of fluorescence values.

[0124] The corresponding Cy5.5 (10 mg / mL) was added to ELP (5 mg / mL, NaHCO3 solution, pH 8.4) at a molar ratio of Cy5.5:ELP = 2:1, and the reaction was rotated and labeled overnight at 4°C in the dark. The ELP-fluorophore conjugate was separated from the unreacted fluorophore by ITC and PD-10 chromatography column, and the Cy5.5-ELP concentration was determined using a carry 3500 UV-Vis spectrometer, and the fluorescence value of the labeled sample was verified.

[0125] Cy5.5-ELP was mixed with unlabeled ELP (250 μM, 500 μM and 750 μM) and injected. Nude mice were anesthetized by weighing and inhalation anesthetic (1%, w / v, isoflurane in 2L oxygen) and injected with about 25 μL of ELPs-Cy5.5 solution at fixed points. In vivo fluorescence images and near-infrared fluorescence size were taken at different time intervals (0d, 7d, 14d, 19d, 28d, 35d, 42d, 49d, 56d, 63d, 70d, 77d, 84d, 92d) after anesthesia using a near-infrared imaging instrument. The results are shown in Figure 2. Figure 5 shown.

[0126] Figure 5 The data show that the cumulative fluorescence released by the ELPs shown in SEQ ID NO:1 and SEQ ID NO:2 is within a controllable range. SEQ ID NO:1 can still maintain about 80% of its fluorescence after 70 days in vivo. Compared with the in vitro release experiment, the release ability of SEQ ID NO:2 at the same concentration level in vivo is stronger than that in vitro. The possible reason is that there are multiple enzymes and other substances in the body that can degrade ELP. This shows that ELPs are qualified to act as local drug reservoirs and can control the release effect of drugs.

[0127] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An elastin-like polypeptide, characterized in that: The amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X includes I, A and F, and n is selected from an integer of 20 to 120.

2. The elastin-like polypeptide according to claim 1, characterized in that The amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X is I, A and F, the number of amino acids in the elastin-like polypeptide is I:A:F=2:2:1, and n is 100; preferably, the elastin-like polypeptide is I 20 A 20 I 20 F 20 A 20 , and has an amino acid sequence as shown in SEQ ID NO:

3.

3. The elastin-like polypeptide according to claim 1, characterized in that The amino acid sequence of the elastin-like polypeptide is (VPGXG)n, wherein X is I, A, F and L, the number of amino acids in the elastin-like polypeptide is I:A:F:L=2:2:1:1, and n is 120; preferably, the elastin-like polypeptide is I 20 A 20 I 20 F 20 A 20 L 20 , and has an amino acid sequence as shown in SEQ ID NO:

4.

4. The elastin-like polypeptide according to any one of claims 1 to 3, characterized in that The first I, A, and F of each monomer ELP module in the elastin-like polypeptide is replaced by V.

5. The elastin-like polypeptide according to any one of claims 1 to 4, characterized in that The elastin-like polypeptide further comprises a leader peptide and a tail peptide, wherein the leader peptide is selected from MGSSGLVPRGSKGPG and MSKGPG, and the tail peptide is selected from YGYGYGYGYGYGYG, YGYGYGYGYGYGYG, and YGGYGGYGGYGGYGGYGGYGGG.

6. An elastin-like polypeptide, characterized in that: The amino acid sequence of the elastin-like polypeptide is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

7. A polynucleotide, characterized in that The polynucleotide can encode the elastin-like polypeptide according to any one of claims 1 to 6.

8. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the polynucleotide according to claim 7.

9. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria is Escherichia coli containing the recombinant expression vector according to claim 8.

10. Use of the elastin-like polypeptide according to any one of claims 1 to 6 in the preparation of a drug delivery system.

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