A method for preparing human tropoelastin based on sequence optimization and use thereof

Abstract

This invention belongs to the field of biomedicine, specifically relating to a method for the expression and purification of recombinant human tropoelastin. More specifically, it relates to a method for efficiently expressing human tropoelastin using *E. coli* as a host by constructing an expression plasmid containing a T7 promoter, and obtaining a high-purity product through a series of purification processes. The technical problem to be solved by this invention is to provide a human tropoelastin with intact structure and high biological activity that can be efficiently expressed in a prokaryotic expression system, as well as its preparation method and applications.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine. Specifically, it relates to a method for the expression and purification of recombinant human elastin. More specifically, it relates to a method for efficiently expressing human elastin by constructing expression plasmids using Escherichia coli as a host, and obtaining a high-purity product through a series of purification processes. Background Technology

[0002] Elastin is a key extracellular matrix protein that endows mammalian tissues such as skin, blood vessels, and lungs with elasticity. Tropoelastin is a soluble monomer of elastin. In the human body, tropoelastin is mainly synthesized by smooth muscle cells and fibroblasts. It is encoded by a single gene and undergoes selective splicing to form tropoelastin with a molecular weight of 60-72 kDa, which is then secreted into the extracellular space. Subsequently, it forms an insoluble elastin network structure through cross-linking of lysine residues. As a core functional protein of the human extracellular matrix, elastin is mainly composed of repetitive amino acid sequences (such as GPVGX). Its unique hydrophobic domains and lysine-rich hydrophilic regions work synergistically to endow tissues with excellent elastic recovery and mechanical stability, giving it irreplaceable application value in fields such as skin anti-aging, tissue repair, and biomedical materials. However, the human body's ability to synthesize elastin decreases significantly with age, and the content of elastin in natural tissues is extremely low (e.g., only 2%-4% of the dry weight of skin), making it difficult to meet the needs of large-scale applications through direct extraction. This has driven the development of artificial preparation technologies.

[0003] Traditional elastin preparation techniques mainly rely on two pathways: animal-derived extraction and chemical synthesis, but both have significant technical bottlenecks.

[0004] Animal-derived extraction method: using ligaments and aortas of mammals such as cattle and pigs as raw materials, elastin or its fragments are obtained through enzymatic hydrolysis, chemical extraction and other processes. However, this method has the following risks or defects: (1) Biosafety: there may be pathogens in the animal body that can infect humans. If the inactivation is not thorough, the pathogens can infect humans; (2) Immunogenicity: animal-derived proteins have low homology with proteins from different human sources, which can easily trigger immune reactions. Repeated injections can easily produce antibodies, leading to aggravated local injection reactions, which can easily cause local redness, swelling, pain, itching and other symptoms, thus limiting its application in high-requirement fields such as medicine and medical aesthetics; (3) Destruction of structural integrity: the enzymatic hydrolysis, high temperature and strong chemical treatment commonly used in the extraction process of animal-derived proteins can easily lead to the degradation of the natural secondary structure of elastin, causing it to lose its unique elasticity and biological activity; (4) Poor uniformity of animal tissue raw material quality: each batch of raw materials to be processed comes from many different animals. The age of the animals, the freshness of the tissues, the storage status and other factors are inconsistent, which makes it difficult to achieve controllability of raw material quality.

[0005] Chemical synthesis: Solid-phase or liquid-phase synthesis strategies can be used to obtain elastin peptides with certain biological functions. Although this method avoids the safety risks of animal-derived products to some extent, it is limited by the efficiency of chemical synthesis, the length of the synthesized peptide chain is limited, and it usually cannot simulate the complete spatial structure and complex function of natural full-length elastin (about 60-72 kDa). Moreover, the synthesis cost is high, making it difficult to meet market demand.

[0006] The development of genetic engineering technology has made it possible to produce recombinant elastin on a large scale and at low cost. However, in existing technologies, due to the large molecular weight of elastin and the highly repetitive nature of its gene sequence, direct heterologous expression often faces the challenge of extremely low expression levels or even no expression, resulting in high production costs. Currently, most elastin products on the market are elastin-like peptides (ELPs), which are artificial peptides that mainly mimic short repetitive sequences of elastin (such as VPGVG). Although ELPs retain some of the phase transition properties and cell adhesion sites of elastin, their generally short peptide chain length (molecular weight typically between 10-30 kDa) results in insufficient biological activity and weak mechanical properties compared to full-length proteins, making it difficult to meet the needs of regenerative medicine, tissue engineering, and other applications that require high integrity of protein structure and function. Therefore, the market urgently needs to develop a method for efficiently expressing structurally intact, highly biologically active, and low-immunogenic human full-length elastin. Summary of the Invention

[0007] The present invention aims to solve the above-mentioned problems existing in the prior art. Specifically, the technical problem to be solved by the present invention is to provide a human tropoelastin that can be efficiently expressed in a prokaryotic expression system, has an intact structure and high biological activity, as well as its preparation method and application.

[0008] In a first aspect, the present invention provides an optimized human elastin, the amino acid sequence of which is shown in SEQ ID NO.3.

[0009] Secondly, the nucleotide sequence corresponding to the human elastin amino acid sequence described in this invention can be designed according to conventional methods in the art, with a preferred embodiment shown in SEQ ID NO.4.

[0010] Preferably, its nucleotide sequence is shown in SEQ ID NO.4.

[0011] Thirdly, the present invention provides a recombinant expression vector containing the aforementioned nucleic acid, such as the pET or pGEX series. Preferably, the recombinant expression vector is a pET series vector, and more preferably, a pET-30a(+) vector.

[0012] Fourthly, the present invention provides a genetically engineered bacterium containing the recombinant expression vector, such as BL21 or Rosetta series. Preferably, the genetically engineered bacterium is Escherichia coli, and more preferably, it is BL21(DE3) strain.

[0013] Fifthly, the present invention provides a method for preparing the human elastin, comprising the following steps:

[0014] 1) Synthesize a nucleotide sequence (preferably SEQ ID NO.4) encoding the amino acid sequence shown in SEQ ID NO.3, and insert it into a prokaryotic expression vector to construct a recombinant expression plasmid;

[0015] 2) Transform the recombinant expression plasmid constructed in step 1) into competent E. coli cells to obtain genetically engineered bacteria;

[0016] 3) Ferment and culture the genetically engineered bacteria, and induce them to express the target protein;

[0017] 4) Isolate and purify the target protein from the fermented cells to obtain human elastin.

[0018] The nucleotide sequence in step (1) is shown in SEQ ID NO.4, and the prokaryotic expression vector is pET-30a(+).

[0019] Among them, the competent Escherichia coli cells in step (2) are BL21(DE3).

[0020] The fermentation culture in step (3) includes: culturing in a culture medium containing 10-20 g / L peptone, 5-10 g / L yeast extract, 8-15 g / L sodium chloride and 5-10 g / L glucose at 36-38°C and pH 6.8-7.4.

[0021] When the OD600 value of the bacterial culture reaches 20-30, add IPTG to a final concentration of 0.4-1.0 mM for induction, and the induction time is 3-5 hours.

[0022] The purification method described in step (4) includes the following steps:

[0023] a) Collect inclusion bodies after bacterial cell disruption;

[0024] b) Wash the inclusion bodies;

[0025] c) Dissolve the inclusion bodies in a urea-containing buffer solution;

[0026] d) Perform cation exchange chromatography;

[0027] e) Complication;

[0028] f) Purification via phase change cycle.

[0029] In a further preferred embodiment, the phase transition cyclic purification is performed by adding NaCl to the protein solution to a final concentration of 1M, incubating at 35-40°C, and then centrifuging.

[0030] The purification method described in step (4) includes the following steps:

[0031] (1) Cell disruption: Add 10 times (w / w) of 20mM PB buffer (pH 8.0) to the cells and disrupt the cells by ultrasonic or high-pressure homogenizer, disrupting them a total of 3 times; after disruption, centrifuge at 15000×g and 2-8℃ for 20min, discard the supernatant, collect the precipitate, and obtain inclusion bodies;

[0032] (2) Inclusion body washing: Add 20mM PB buffer (pH 8.0) containing 0.5% Triton X-100 to the inclusion body at a ratio of 1:10 (w / w), stir thoroughly with a homogenizer, centrifuge at 15000×g and 2-8℃ for 20min, collect the precipitate, complete one round of washing, and wash for a total of 3 rounds;

[0033] (3) Inclusion body dissolution: Add 20mM PB buffer (pH 8.0) containing 6-8M urea to the inclusion bodies at a ratio of 1:20 (w / w), stir at room temperature for more than 3 hours, then filter with a 0.45μm filter membrane or centrifuge (15000×g, 25℃, 20min) to remove insoluble matter and collect the supernatant;

[0034] (4) Cation exchange chromatography: After equilibrating the NanoGel-50SP column with 20mM PB buffer (pH 8.0) containing 6-8M urea, start loading the sample. After loading the sample, perform gradient elution with the above buffer containing NaCl to harvest the target protein.

[0035] (5) Renaturation: Slowly add 7 times the volume of 20mM PB buffer (pH 7.4) to the protein solution containing urea, and stir at 2-8℃ for 12-16h; the 20mM PB buffer may optionally contain 1-5mM of reduced glutathione and / or dithiothreitol or β-mercaptoethanol; after renaturation, filter or centrifuge at high speed to remove insoluble matter, and collect the filtrate or the supernatant after centrifugation;

[0036] (6) Phase transition cycle purification: Add NaCl to the renatured protein solution to a final concentration of 1M, heat in a water bath until the protein solution temperature reaches 35~40℃, let stand for more than 20 minutes, centrifuge at ≥15000×g for more than 10 minutes, and collect the precipitate; add 20mM PB buffer (pH 7.4) at a temperature not higher than 20℃ to the precipitate, stir to fully dissolve the precipitate; obtain a solution with a recombinant human elastin concentration of 5-100mg / ml;

[0037] (7) The recombinant human elastin solution obtained in step (6) is filtered through a 0.22 μm sterile filter membrane to remove bacteria, thereby obtaining human primordial elastin solution; or human primordial elastin lyophilized powder is obtained by freeze drying.

[0038] For a further preferred preparation method of the present invention, please refer to the examples.

[0039] Sixthly, the present invention also provides the application of the aforementioned human elastin in the preparation of pharmaceuticals, cosmetics, medical devices, or biomedical materials. For example, it can be used to prepare skin anti-aging products, tissue repair materials, drug sustained-release carriers, or artificial blood vessels.

[0040] In one specific implementation, the human elastin can be used to: (1) be injected subcutaneously into the human body via aseptic injection to supplement the elastin in the human skin; (2) be injected into the joint cavity to act as a lubricant to relieve joint pain; and (3) be added to the wound site of the epidermis to promote wound healing.

[0041] Applications of human elastin in the preparation of drugs, health products, or biomaterials that supplement elastin in human skin, lubricate and relieve joint pain, and promote wound healing.

[0042] In a seventh aspect, the present invention also provides a human elastin product prepared by the above method, wherein the product is characterized in that its purity is not less than 99% as detected by SDS-PAGE and SEC-HPLC.

[0043] Beneficial effects

[0044] Compared with existing technologies, this invention has the following beneficial effects: ① Structural integrity: The protein prepared by this invention is full-length human tropoelastin, and its sequence has been optimized (as shown in SEQ ID NO.3), overcoming the defect of incomplete structure of existing ELP peptides, and possessing a more complete spatial structure and function closer to that of natural proteins. ② High yield and low cost: Through targeted codon optimization and N-terminal truncation modification of the coding sequence of human tropoelastin, it can achieve efficient soluble expression or inclusion body expression in the E. coli system. Measurements show that under optimal culture conditions, its fermentation yield can reach 0.5~1.0 g / L, significantly higher than the expression level of full-length protein in existing technologies, greatly reducing production costs. ③ High purity and low impurities: Through the purification method provided by this invention, the purity of the final obtained human tropoelastin can reach over 99% (detected by SDS-PAGE and SEC-HPLC), and the content of host protein residues, exogenous DNA residues, and endotoxins all meet the relevant standards of the Chinese Pharmacopoeia for biological raw materials, laying the foundation for its application in high-requirement fields such as pharmaceuticals and medical aesthetics. ④ Compared to existing technologies that directly express full-length genes (such as the sequence encoded by SEQ ID NO.1) with no expression or extremely low expression (see...) Figure 1 This invention, through specific N-terminal truncation and codon optimization, unexpectedly achieved high-level expression of the target protein in *E. coli*. This modification not only overcomes the technical bottleneck of expressing long, highly repetitive protein fragments, but also preserves the core functional domains of the obtained protein, demonstrating biological activity superior to the expected technical results. Attached Figure Description

[0045] Figure 1 SDS-PAGE expression profile of engineered bacteria containing the unoptimized target gene sequence (SEQ ID NO.2). Lane M: Protein molecular weight marker; Lane 1: Whole bacterial protein before induction; Lane 2: Whole bacterial protein after induction. Results showed no obvious target protein band after induction.

[0046] Figure 2 SDS-PAGE expression profile of engineered bacteria containing the optimized target gene sequence (SEQ ID NO.4). Lane M: Protein molecular weight marker; Lane 1: Whole-cell protein before induction; Lane 2: Whole-cell protein after induction. Results showed a clear induction band at approximately 60.6 kDa, consistent with the expected molecular weight.

[0047] Figure 3SDS-PAGE purity assays of human elastin stock solutions purified from different batches. Lane M: Protein molecular weight marker; Lanes 1, 3, 5, and 7: Non-reducing electrophoresis of purified products from different production batches (e.g., batches 20251231, 20260106, 20260107, and 20260108). Lanes 2, 4, 6, and 8: Reducing electrophoresis of purified products from different production batches (e.g., batches 20251231, 20260106, 20260107, and 20260108). The results show that the target protein bands in each batch are single, indicating high purity.

[0048] Figure 4 SEC-HPLC purity determination chromatogram of human elastin stock solution. The results show that the main peak of the target protein is symmetrical, and the purity is above 99%.

[0049] Figure 5 Comparison of growth viability of L929 cells and Vero cells in culture media containing different concentrations of elastin. The results showed that the cell viability was higher after the addition of elastin than that of the control group.

[0050] Figure 6 The growth of L929 cells cultured for 72 hours with different concentrations of elastin was observed. Results showed that after adding elastin, L929 cells exhibited normal morphology and higher cell density compared to the control group.

[0051] Figure 7 The growth of Vero cells cultured for 72 hours with different concentrations of elastin was observed. The results showed that after the addition of elastin, Vero cells exhibited normal morphology and higher cell density compared to the control group.

[0052] Terminology Explanation:

[0053] To facilitate understanding of this invention, some of the terms and abbreviations used in the specification are explained below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0054] NCBI: National Center for Biotechnology Information.

[0055] SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis.

[0056] SEC-HPLC: Size Exclusion Chromatography-High Performance Liquid Chromatography.

[0057] IPTG: Isopropyl β-D-Thiogalactoside.

[0058] PB: Phosphate Buffer.

[0059] ELP: Elastin-like Peptide.

[0060] BSA: Bovine Serum Albumin.

[0061] CCK-8: Cell Counting Kit-8.

[0062] Triton X-100: Polyethylene glycol octylphenyl ether.

[0063] PBS (Phosphate Buffered Saline)

[0064] DTT (dithiothreitol) Detailed Implementation

[0065] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. The present invention will be further described below in conjunction with specific embodiments, but should not be construed as limiting the present invention. Unless otherwise specified, the methods used in the embodiments are conventional methods in the art, and the reagents used are commercially available conventional biochemical reagents in the art.

[0066] Example 1: Sequence Optimization and Expression Testing

[0067] (1) Sequence design and synthesis

[0068] First, the amino acid sequence of human elastin was obtained from the NCBI database (referred to as the unoptimized sequence, SEQ ID NO.1). Codon optimization was performed on this sequence to address the codon usage bias of the *E. coli* expression system, resulting in the corresponding nucleotide sequence (referred to as the unoptimized nucleic acid sequence, SEQ ID NO.2). To further improve expression efficiency and protein stability, the amino acid sequence shown in SEQ ID NO.1 was modified by deleting 27 amino acids from its N-terminus, resulting in the optimized amino acid sequence (SEQ ID NO.3). Then, based on SEQ ID NO.3, codon optimization was performed again specifically for *E. coli*, resulting in the optimized nucleotide sequence (SEQ ID NO.4). The nucleic acid sequences shown in SEQ ID NO.2 and SEQ ID NO.4 were respectively commissioned to a third-party company for whole-genome synthesis.

[0069] (2) Construction of engineered bacteria

[0070] The synthesized gene fragments SEQ ID NO.2 and SEQ ID NO.4 were inserted into pET-30a(+) expression vectors linearized with restriction endonucleases (such as NdeI and XhoI), respectively, to construct recombinant expression plasmids pET-30a-SEQ ID NO.2 and pET-30a-SEQ ID NO.4. After verification by sequencing, the two recombinant plasmids were transformed into E. coli BL21(DE3) competent cells, as follows:

[0071] ① Take 2 μl of plasmid solution (about 10-100 ng), add it to 100 μl of BL21 (DE3) competent cells thawed on ice, gently tap to mix, and incubate on ice for 30 min.

[0072] ② Heat shock competent cells in a 42℃ water bath for 90 seconds, then quickly transfer them to ice and incubate on ice for 2-3 minutes.

[0073] ③ Under aseptic conditions, add 1 ml of sterile LB liquid medium to competent cells and incubate at 37°C and 220 rpm for 60 min with shaking to allow the cells to recover and express the resistance gene.

[0074] ④ Centrifuge the revived bacterial culture at 5000g for 1 min, discard part of the supernatant, and keep about 100μl to resuspend the bacterial cells. Spread the resuspended cells on LB solid medium plates containing 50μg / ml kanamycin and invert them in a 37℃ constant temperature incubator overnight.

[0075] ⑤ Pick a single colony from the plate and inoculate it into a test tube containing 5 ml of LB liquid medium (containing 50 μg / ml kanamycin), and incubate overnight at 37°C with shaking at 220 rpm.

[0076] ⑥ Preservation of bacterial cultures: Take an appropriate amount of bacterial culture and mix it with sterile 80% glycerol at a ratio of 9:1 to make the final glycerol concentration 8%. Aliquot the mixture into cryovials and store at -70℃ to obtain two engineered Escherichia coli strains containing SEQ ID NO.2 and SEQ ID NO.4 genes.

[0077] (3) Expression test

[0078] Take glycerol culture from each of the two engineered bacteria mentioned above and inoculate them at 1% (v / v) into Erlenmeyer flasks containing 120 ml LB medium (containing 50 μg / ml kanamycin), and incubate at 37°C with shaking at 220 rpm. When the OD of the bacterial culture... 600 When the pH value reaches 0.6-0.8, add IPTG to a final concentration of 0.4 mM and continue induction culture at 37℃ for 4 h. After induction, collect the bacterial cells by centrifugation and perform SDS-PAGE analysis.

[0079] (4) Results

[0080] SDS-PAGE test results are as follows: Figure 1 and Figure 2 As shown. The results indicate that the engineered bacteria containing the SEQ ID NO.2 gene did not show significant expression of the target protein after induction. Figure 1 The engineered bacteria containing the SEQ ID NO.4 gene, after induction, showed a distinct protein band at approximately 60.6 kDa, consistent with the expected molecular weight of human elastin. Figure 2 This indicates that the optimized sequence shown in SEQ ID NO.4 was efficiently expressed in Escherichia coli. This result demonstrates that by truncating the N-terminus and optimizing the codons, the technical challenge of expressing full-length human tropoelastin in prokaryotic systems was successfully overcome.

[0081] Example 2: Fermentation culture of engineered bacteria

[0082] This embodiment uses a 10L fermenter as an example to describe in detail the high-density fermentation culture method of engineered bacteria containing the SEQ ID NO.4 gene.

[0083] (1) Strain recovery: Strain recovery: Take one strain of engineered bacteria and inoculate it into 120ml LB medium (containing 50 μg / ml kanamycin) at 0.1% (volume ratio). Set the constant temperature incubator to 220rpm and incubate overnight at 37℃ to obtain the recovered engineered bacteria seed.

[0084] (2) The revived engineered bacteria seed was inoculated into a 10L fermenter (containing 6L of fermentation medium). The fermentation medium formula is shown in Table 1:

[0085] Table 1. Fermentation medium formulation

[0086] Serial Number Components Amount added (g) 1 peptone 10 2 Yeast extract 5 3 Sodium chloride 10 4 glucose 5 5 Potassium dihydrogen phosphate 2 6 dipotassium hydrogen phosphate 3 7 Magnesium sulfate 2 8 Purified water Adjust the volume to 1L

[0087] (3) Control the fermentation temperature to 36-38℃, pH to 6.8-7.4, dissolved oxygen ≥30%, feed with 50% (w / v) glucose solution, and adjust the pH with ammonia. OD should be measured every 1 hour. 600 Value, pending OD 600 When the concentration reaches 20-30, add IPTG to a final concentration of 0.4-1.0 mmol / L to begin induction. After 3-5 hours of induction, stop fermentation and collect the cells by high-speed centrifugation at 15000×g.

[0088] Example 3: Optimization of fermentation medium for engineered bacteria

[0089] This embodiment optimizes the fermentation medium based on Example 2 to obtain higher protein yield.

[0090] (1) Strain recovery: Strain recovery: Take one strain of engineered bacteria and inoculate it into 120ml LB medium at 0.1% (volume ratio). Set the constant temperature incubator to 220rpm and shake at 37℃ overnight to obtain the recovered engineered bacteria seed.

[0091] (2) The revived engineered bacteria seed was inoculated into a 20L fermenter (containing 12L of fermentation medium). The fermentation medium formula is shown in Table 2:

[0092] Table 2 Comparison of fermentation medium formulations

[0093] Serial Number Components Dosage of Formula 1 (g) Dosage of Formula 2 (g) Addition amount of formula 3 (g) 1 peptone 8 10 15 2 Yeast extract 4 5 7.5 3 Sodium chloride 10 10 10 4 glucose 5 5 5 5 Potassium dihydrogen phosphate 2 2 2 6 dipotassium hydrogen phosphate 3 3 3 7 Magnesium sulfate 1 1 1 8 Purified water Adjust the volume to 1L Adjust the volume to 1L Adjust the volume to 1L

[0094] (3) Control the fermentation temperature to 36-38℃, pH to 6.8-7.4, dissolved oxygen ≥30%, feed with 50% (w / v) glucose solution, and adjust the pH with ammonia. OD should be measured every 1 hour. 600 Value, pending OD 600 When the concentration reaches 20, add IPTG to a final concentration of 0.4 mmol / L to begin induction. After 4 hours of induction, stop fermentation and collect the cells by high-speed centrifugation at 15000×g.

[0095] (4) Results

[0096] Table 3 Comparison of Fermentation Medium Formulations

[0097] Group Formula 1 Formula 2 Formula 3 Cell yield (g) 580 679 463 Bacterial cell unit production capacity g / L 48 57 39

[0098] The results of the comparison of culture medium formulations are shown in Table 3. The cell productivity of formulations 1, 2, and 3 were 48, 57, and 39 g / L, respectively, with formulation 2 showing the highest productivity. This indicates that the comparison of culture medium formulations improved cell productivity.

[0099] Example 4: Protein Purification

[0100] The cells obtained from fermentation in Example 2 were purified using the following specific steps:

[0101] (1) Cell disruption: Add 10 times (w / w) of 20mM PB (pH 8.0) buffer to the cells, mix thoroughly, and then disrupt the cells using a high-pressure homogenizer at a pressure of 600-800 bar for a total of 3 cycles. After disruption, centrifuge at 15000×g and 2-8℃ for 20 min. Discard the supernatant and collect the precipitate to obtain inclusion bodies.

[0102] (2) Inclusion body washing: Add 20 mM PB (containing 0.5% Triton X-100, pH 8.0) buffer at a ratio of 1:10 (w / w) to the inclusion bodies, mix thoroughly with a homogenizer, centrifuge at 15000×g and 2~8℃ for 20 min, collect the precipitate, and complete one round of washing. A total of 3 rounds of washing are performed.

[0103] (3) Inclusion body dissolution: Add 20 mM PB (containing 8 M urea, pH 8.0) buffer at a ratio of 1:10 to 1:20 (w / w) to the inclusion bodies and dissolve at room temperature for 3 h. Centrifuge at 15000×g at 25℃ for 20 min and collect the supernatant.

[0104] (4) Cation exchange chromatography: After equilibrating the NanoGel-50SP column with 20mM PB (containing 6-8M urea, pH 8.0), the sample was loaded. After loading, the sample was washed with 20mM PB (containing 6-8M urea, pH 8.0) buffer to the baseline level, and then gradient eluted with buffer containing 500 mM NaCl to harvest the target protein.

[0105] (5) Refolding: Slowly add 7 times the amount of 20mM PB, pH 7.4 buffer solution containing 6-8M urea to the protein solution and stir at 2-8℃ for 12-16h.

[0106] (6) Phase transition: Add NaCl to the renatured protein solution to a final concentration of 1M, incubate in a water bath until the protein solution reaches 35-40℃, centrifuge at 15000×g for 20min, discard the supernatant and collect the precipitate. Add 20mM PB (pH 7.4) buffer to the precipitate and stir at low speed to fully dissolve the precipitate. Filter through a 0.22μm sterile filter membrane to obtain the target protein stock solution.

[0107] (7) Purity detection: The final protein stock solution was analyzed by SDS-PAGE and SEC-HPLC, and the results are as follows. Figure 3 and Figure 4 As shown in the figure. SDS-PAGE showed that the target protein band was single, and SEC-HPLC results showed that the purity of the main peak of the target protein was greater than 99%, indicating that the target protein had high purity after purification. The results of host cell protein residue, exogenous DNA residue, and endotoxin detection are shown in Table 4, indicating that the purification process of the present invention can effectively remove impurities such as host proteins, nucleic acids, and endotoxins to obtain high-purity human elastin.

[0108]

[0109] Example 5: Cell proliferation assay

[0110] Elastin solution was diluted with MEM complete medium containing 5% FBS to concentrations of 4, 2, 1, 0.5, 0.2, 0.1, and 0.05 mg / ml. Vero and L929 cells in logarithmic growth phase were then taken, digested, and their cell densities adjusted to 5-8 × 10⁻⁶ cells / ml using MEM complete medium containing 5% FBS. 4 Cells / mL). Add 50 μl of diluted elastin solution to a 96-well cell culture plate, then add 50 μl of diluted cells and mix well. Incubate at 37°C with 5% CO2. After incubation, remove the 96-well plate and add 10 μL of CCK-8 solution to each well according to the group. Gently tap the edge of the plate to mix, then return to the incubator for another 1–4 hours. Measure the OD value at 450 nm using a microplate reader. Calculate the cell viability (%) for each group using the formula: Cell viability (%) = (OD of test group) / (OD of control group) × 100%. Results ( Figure 5 , Figure 6 , Figure 7 The results showed that after 48 h and 72 h of culture, the cell viability of cells with added elastin was significantly increased compared with the control group, and the cell viability was highest when the elastin concentration was 2 mg / ml. These results indicate that the human elastin prepared in this invention can promote cell proliferation.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0112] sequence list

[0113] <110> Green Bamboo Biological Products (Beijing) Co., Ltd.; Beijing Rumeng Biotechnology Co., Ltd.

[0114] <120> A method for preparing human oxyelastin based on sequence optimization and its applications

[0115] <160> 4

[0116] <210> 1

[0117] <212> PRT

[0118] <213> Artificial sequence

[0119] <400> 1

[0120] 1 MAGLTAAAPR PGVLLLLLSI LHPSRPGGVP GAIPGGVPGG VFYPGAGLGA LGGGALGPGGKPLKPVPGGL

[0121] 71 AGAGLGAGLG AFPAVTFPGA LVPGGVADAA AAYKAAKAGA GLGGVPGVGG LGVSAGAVVPQPGAGVKPGK

[0122] 141 VPGVGLPGVY PGGVLPGARF PGVGVLPGVP TGAGVKPKAP GVGGAFAGIP GVGPFGGPQPGVPLGYPIKA

[0123] 211 PKLPGGYGLP YTTGKLPYGY GPGGVAGAAG KAGYPTGTGV GPQAAAAAAA KAAAKFGAGAAGVLPGVGGA

[0124] 281 GVPGVPGAIP GIGGIAGVGT PAAAAAAAAA AKAAKYGAAA GLVPGPGFG PGVVGVPGAGVPGVGVPGAG

[0125] 351 IPVVPGAGIP GAAVPGVVSP EAAAKAAAKA AKYGARPGVG VGGIPTYGVG AGGFPGFGVGVGGIPGVAGV

[0126] 421 PSVGGVPGVG GVPGVGISPE AQAAAAKAA KYGVGTPAAA AAKAAAKAAQ FALLNLAGLVPGVGVAPGVG

[0127] 491 VAPGVGVAPG VGLAPGVGVA PGVGVAPGVG VAPGIGPGGV AAAAKSAAKV AAKAQLRAAAGLGAGIPGLG

[0128] 561 VGVGVPGLGV GAGVPGLGVG AGVPGFGAVP GALAAAKAAK YGAAVPGVLG GLGALGGVGIPGGVVGAGPA

[0129] 631 AAAAAAKAAA KAAQFGLVGA AGLGGLGVGG LGVPGVGGLG GIPPAAAAKA AKYGAAGLGGVLGGAGQFPL

[0130] 701 GGVAARPGFG LSPIFPGGAC LGKACGRKRK

[0131] <210>2

[0132] <212>DNA

[0133] <213>Artificial Sequence

[0134] <400>2

[0135] 1 ATGGCCGGTC TGACTGCTGC TGCTCCGCGT CCGGGTGTTC TGCTGCTGCT GCTGAGCATCCTGCACCCGT

[0136] 71 CCCGCCCTGG CGGTGTTCCG GGCGCGATCC CGGGCGGCGT ACCTGGCGGC GTCTTTTACCCGGGCGCTGG

[0137] 141 CCTGGGTGCT CTGGGCGGTG GTGCTCTGGG CCCGGGTGGT AAGCCGCTGA AACCGGTACCTGGCGGCCTG

[0138] 211 GCTGGCGCCG GTCTGGGCGC GGGCCTGGGT GCTTTCCCGG CAGTTACCTT CCCGGGCGCACTGGTTCCAG

[0139] 281 GCGGTGTAGC CGACGCAGCA GCTGCGTATA AGGCTGCAAA AGCTGGTGCC GGTCTGGGCGGCGTTCCGGG

[0140] 351 CGTTGGTGGT CTGGGCGTAT CCGCAGGCGC GGTAGTGCCG CAGCCGGGTG CTGGTGTTAAACCGGGCAAA

[0141] 421 GTTCCTGGTG TTGGCCTGCC GGGTGTTTAT CCGGGCGGCG TTCTGCCGGG CGCGCGTTTCCCGGGTGTCG

[0142] 491 GTGTTCTGCC GGGCGTGCCG ACTGGTGCCG GTGTGAAACC GAAAGCGCCG GGTGTCGGCGGTGCGTTCGC

[0143] 561 GGGTATCCCG GGCGTAGGTC CGTTCGGTGG CCCGCAGCCG GGTGTTCCGC TGGGTTACCCGATCAAGGCT

[0144] 631 CCGAAACTGC CGGGCGGTTA CGGTCTGCCG TATACCACCG GTAAACTGCC GTACGGTTATGGCCCGGCG

[0145] 701 GTGTTGCAGG TGCGGCGGGC AAAGCAGGTT ATCCTACTGG CACCGGCGTG GGCCCGCAGGCCGCGGCGGC

[0146] 771 TGCGGCGGCT AAAGCCGCTG CAAATTCGG CGCTGGCGCG GCAGGCGTTC TGCCAGGTGTTGGTGGCGCA

[0147] 841 GGTGTACCGG GTGTGCCGGG TGGCATCCCG GGTATCGGTG GCATCGCTGG TGTCGGTACTCCGGCAGCAG

[0148] 911 CGGCAGCGGC TGCCGCGGCG GCGAAAGCGG CCAAATATGG TGCAGCGGCT GGTCTGGTTCCGGGTGGTCC

[0149] 981 TGGTTTCGGT CCGGGTGTCG TCGGTGTTCC GGGCGCTGGC GTACCGGGCG TAGGTGTACCGGGCGCAGGC

[0150] 1051 ATTCCGGTGG TACCAGGTGC TGGCATCCCG GGCGCAGCTG TGCCGGGTGTTGTCTCCCCG GAGGCGGCGG

[0151] 1121 CCAAAGCGGC GGCGAAAGCT GCAAAGTACG GCGCTCGCCC TGGTGTGGGTGTCGGCGGTA TCCCGACTTA

[0152] 1191 TGGTGTTGGT GCGGGTGGTT TTCCGGGTTT CGGCGTTGGC GTGGGTGGTATTCCGGGCGT TGCGGGCGTG

[0153] 1261 CCGAGCGTTG GTGGTGTTCC GGGCGTTGGT GGCGTGCCGG GTGTGGGCATTTCCCCGGAA GCTCAGGCAG

[0154] 1331 CAGCTGCGGC GAAAGCGGCC AAGTATGGTG TTGGTACCCC GGCGGCGGCTGCAGCTAAAG CGGCGGCGAA

[0155] 1401 AGCAGCACAG TTCGCCCTGC TGAACCTGGC TGGCCTGGTG CCGGGTGTCGGTGTCGCGCC GGGCGTAGGC

[0156] 1471 GTTGCTCCGG GCGTTGGTGT CGCCCCGGGC GTTGGCCTGG CGCCGGGCGTGGGTGTAGCA CCAGGCGTTG

[0157] 1541 GTGTCGCACC AGGTGTAGGC GTTGCTCCAG GTATCGGCCC GGGTGGCGTTGCTGCAGCCG CAAAAAGCGC

[0158] 1611 AGCCAAAGTG GCCGCCAAGG CTCAGCTGCG TGCCGCGGCA GGCCTGGGTGCTGGCATCCC GGGTCTGGGT

[0159] 1681 GTAGGCGTTG GTGTACCGGG CCTGGGTGTT GGCGCTGGTG TCCCGGGCCTGGGTGTGGGC GCAGGCGTAC

[0160] 1751 CGGGTTTCGG TGCGGTGCCA GGCGCCCTGG CTGCAGCGAA AGCGGCAAAATATGGTGCTG CTGTTCCGGG

[0161] 1821 TGTGCTGGGT GGTCTGGGTG CTCTGGGTGG TGTTGGCATC CCGGGTGGCGTCGTTGGCGC GGGCCCGGCA

[0162] 1891 GCGGCAGCGG CCGCCGCGAA AGCTGCGGCA AAGGCTGCGC AATTTGGTCTGGTGGGTGCG GCTGGTCTGG

[0163] 1961 GTGGTCTGGG TGTGGGTGGT CTGGGTGTCC CGGGTGTGGG CGGCCTGGGTGGCATCCCGC CGGCAGCGGC

[0164] 2031 CGCGAAGGCC GCAAAATATG GTGCAGCGGG TCTGGGTGGC GTGCTGGGCGGCGCCGGTCA GTTCCCGCTG

[0165] 2101 GGTGGCGTTG CGGCCCGTCC GGGTTTCGGC CTGTCCCCGA TCTTCCCTGGCGGTGCATGC CTGGGCAAAG

[0166] 2171 CATGTGGTCG TAAGCGTAAA

[0167] <210> 3

[0168] <212> PRT

[0169] <213> artificial sequence

[0170] <400> 3

[0171] 1 MGVPGAIPGG VPGGVFYPGA GLGALGGGAL GPGGKPLKPV PGGLAGAGLG AGLGAFPAVTFPGALVPGGV

[0172] 71 ADAAAAYKAA KAGAGLGGVP GVGGLGVSAG AVVPQPGAGV KPGKVPGVGL PGVYPGGVLPGARFPGVGVL

[0173] 141 PGVPTGAGVK PKAPGVGGAF AGIPGVGPFG GPQPGVPLGY PIKAPKLPGG YGLPYTTGKLPYGYGPGGVA

[0174] 211 GAAGKAGYPT GTGVGPQAAA AAAAKAAAKF GAGAAGVLPG VGGAGVPGVP GAIPGIGGIAGVGTPAAAAA

[0175] 281 AAAAAKAAKY GAAAGLVPGG PFGPGVVGV PGAGVPGVGV PGAGIPVVPG AGIPGAAVPGVVSPEAAAKA

[0176] 351 AAKAAKYGAR PGVGVGGIPT YGVGAGGFPG FGVGVGGIPG VAGVPSVGGV PGVGGVPGVGVGISPEAQAAAA

[0177] 421 AKAAKYGVGT PAAAAAKAAA KAAQFALLNL AGLVPGVGVA PGVGVAPGVG VAPGVGLAPGVGVAPGVGVA

[0178] 491 PGVGVAPGIG PGGVAAAAKS AAKVAAKAQL RAAAGLGAGI PGLGVGVGVP GLGVGAGVPGLGVGAGVPGF

[0179] 561 GAVPGALAAA KAAKYGAAVP GVLGGLGALG GVGIPGGVVG AGPAAAAAAA KAAAKAAQFGLVGAAGLGGL

[0180] 631 GVGGLGVPGV GGLGGIPPAA AAKAAKYGAA GLGGVLGGAG QFPLGGVAAR PGFGLSPIFPGGACLGKACG

[0181] 701 RKRK

[0182] <210>4

[0183] <212>DNA

[0184] <213>Artificial sequence

[0185] <400>4

[0186] 1 ATGGGTGTGC CAGGTGCAAT TCCGGGAGGT GTACCTGGAG GCGTTTTTTA TCCGGGTGCGGGCCTGGGCG

[0187] 71 CGCTGGGTGG TGGCGCGCTG GGGCCGGGTG GTAAACCGCT GAAACCGGTT CCAGGCGGTCTGGCAGGTGC

[0188] 141 GGGCCTGGGT GCTGGCTTGG GTGCGTTTCC TGCGGTTACC TTTCCGGGCG CGCTGGTTCCGGGCGGTGTC

[0189] 211 GCGGACGCAG CGGCGGCATA TAAGGCGGCT AAGGCGGGCG CCGGTTTGGG CGGAGTGCCGGGTGTGGGAG

[0190] 281 GTTTGGGCGT GTCTGCTGGC GCTGTTGTTC CGCAACCGGG GGCGGGCGTA AAACCGGGTAAGGTCCCGGG

[0191] 351 CGTTGGCCTC CCGGGAGTGT ATCCGGGTGG CGTGCTGCCT GGTGCACGCT TTCCGGGCGTGGGCGTTTTG

[0192] 421 CCGGGCGTGC CGACCGGTGC TGGAGTGAAA CCGAAAGCTC CGGGCGTTGG CGGTGCGTTTGCAGGGATTC

[0193] 491 CAGGCGTGGG CCCTTTTGGT GGTCCGCAGC CGGGTGTTCC GCTGGGGTAC CCGATTAAAGCGCCAAAACT

[0194] 561 GCCGGGTGGC TATGGTTTAC CGTATACCAC CGGCAAACTG CCTTATGGTT ACGGTCCGGGTGGTGTGGCG

[0195] 631 GGTGCTGCGG GCAAAGCCGG CTACCCGACC GGAACGGGTG TTGGTCCTCA AGCAGCGGCCGCGGCCGCGG

[0196] 701 CTAAGGCTGC GGCGAAGTTC GGTGCTGGTG CGGCGGGAGT ATTGCCGGGC GTTGGTGGGGCTGGTGTGCC

[0197] 771 GGGCGTCCCG GGCGCGATTC CGGGCATCGG CGGTATTGCA GGTGTGGGTA CTCCGGCAGCGGCAGCTGCG

[0198] 841 GCAGCAGCGG CTGCGAAGGC CGCGAAGTAC GGTGCCGCAG CTGGTTTGGT TCCGGGTGGCCCTGGTTTCG

[0199] 911 GTCCAGGTGT GGTTGGTGTT CCGGGTGCTG GTGTTCCGGG CGTGGGTGTT CCGGGAGCCGGTATCCCGGT

[0200] 981 TGTCCCAGGC GCGGGTATCC CGGGTGCGGC CGTTCCGGGG GTCGTGTCGC CGGAGGCAGCGGCGAAGGCA

[0201] 1051 GCTGCTAAGG CAGCGAAGTA CGGCGCGCGC CCTGGCGTGG GTGTTGGTGGCATCCCAACG TACGGCGTCG

[0202] 1121 GTGCGGGTGG CTTCCCAGGA TTCGGTGTCG GTGTAGGAGG CATCCCGGGTGTTGCCGGTG TCCCAAGCGT

[0203] 1191 TGGCGGTGTT CCGGGTGTTG GTGGTGTTCC GGGCGTAGGT ATTAGCCCGGAAGCACAGGC TGCGGCGGCT

[0204] 1261 GCGAAAGCAG CCAAGTATGG CGTTGGCACC CCGGCAGCGG CGGCAGCCAAGGCAGCGGCA AAGGCGGCGC

[0205] 1331 AATTCGCCTT GCTGAACCTG GCAGGCCTGG TGCCGGGCGT GGGTGTGGCCCCTGGCGTAG GCGTGGCCCC

[0206] 1401 GGGTGTGGGA GTGGCGCCTG GCGTTGGTCT GGCACCGGGC GTGGGTGTGGCCCCAGGTGT CGGTGTTGCA

[0207] 1471 CCGGGAGTGG GCGTCGCACC GGGGATCGGT CCGGGCGGTG TTGCAGCGGCCGCGAAAAGC GCAGCTAAAG

[0208] 1541 TTGCAGCGAA GGCTCAACTG CGTGCAGCGG CGGGTCTGGG TGCTGGCATCCCGGGTTTGG GCGTTGGCGT

[0209] 1611 CGGAGTACCG GGTCTCGGCG TTGGTGCGGG AGTCCCGGGA CTGGGGGTTGGCGCCGGTGT GCCGGGTTTC

[0210] 1681 GGCGCGGTTC CGGGTGCTTT AGCAGCTGCG AAGGCCGCTA AATACGGCGCGGCGGTGCCG GGTGTCCTGG

[0211] 1751 GTGGTTTAGG GGCCCTGGGC GGCGTGGGCA TCCCGGGCGG CGTGGTTGGTGCCGGTCCGG CAGCGGCGGC

[0212] 1821 TGCGGCCGCG AAAGCAGCAG CGAAGGCGGC CCAGTTTGGA CTGGTTGGGGCGGCTGGCCT TGGCGGACTG

[0213] 1891 GGCGTGGGCG GCCTGGGCGT TCCGGGGGTG GGCGGTCTGG GTGGTATCCCTCCGGCAGCG GCCGCTAAGG

[0214] 1961 CCGCTAAATA CGGCGCTGCG GGTTTGGGTG GAGTCTTAGG CGGGGCCGGTCAGTTCCCCC TGGGTGGCGT

[0215] 2031 CGCTGCTCGT CCGGGCTTCG GTCTCTCCCC GATTTTTCCG GGTGGAGCCTGCCTGGGCAA AGCGTGTGGT

[0216] 2101 CGTAAACGTA AA

Claims

1. A human elastin, characterized in that, The amino acid sequence of the human elastin is shown in SEQ ID NO.

3.

2. A nucleic acid encoding the human tropoelastin of claim 1, characterized in that, The nucleotide sequence of the nucleic acid is shown in SEQ ID NO.

4.

3. A recombinant expression vector containing the nucleic acid of claim 2.

4. A genetically engineered bacterium containing the recombinant expression vector of claim 3, characterized in that, The genetically engineered bacterium is Escherichia coli BL21(DE3).

5. A method for preparing the human elastin of claim 1, characterized in that, Includes the following steps: 1) Synthesize the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.3 and insert it into a prokaryotic expression vector to construct a recombinant expression plasmid; 2) Transform the recombinant expression plasmid constructed in step 1) into competent E. coli cells to obtain genetically engineered bacteria; 3) Ferment and culture the genetically engineered bacteria, and induce them to express the target protein; 4) Isolate and purify the target protein from the fermented cells to obtain human elastin.

6. The method according to claim 5, characterized in that, Step 3) The fermentation culture includes: culturing in a medium containing 10-20 g / L peptone, 5-10 g / L yeast extract, 8-15 g / L sodium chloride, and 5-10 g / L glucose at 36-38°C and pH 6.8-7.4; when the OD of the bacterial culture... 600 When the value reaches 20-30, add IPTG to a final concentration of 0.4-1.0 mM for induction, and the induction time is 3-5 hours.

7. The method according to claim 5, characterized in that, Step 4) The purification method includes the following steps: a) Collect inclusion bodies after bacterial cell disruption; b) Wash the inclusion bodies; c) Dissolve the inclusion bodies in a urea-containing buffer solution; d) Perform cation exchange chromatography; e) Complication; f) Perform phase transition cycle purification.

8. The method according to claim 7, characterized in that, The phase transition cyclic purification was performed by adding NaCl to the protein solution to a final concentration of 1M, incubating at 35-40°C, and then centrifuging.

9. The human elastin product prepared by the method according to any one of claims 5-8, characterized in that, SDS-PAGE analysis showed a purity of no less than 99%.

10. The use of the human elastin of claim 1 or the product of claim 9 in the preparation of pharmaceuticals, cosmetics, medical devices or biomedical materials; preferably, the use includes subcutaneous injection to supplement elastin, injection into the joint cavity for lubrication, or addition to epidermal wounds to promote healing.

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