Recombinant elastin with repeated amino acid sequence and preparation method thereof

Abstract

The invention belongs to the technical field of protein expression, and particularly relates to recombinant elastin with repeated amino acid sequences and a preparation method thereof.The recombinant elastin is composed of 327 AA sequence units which are repeatedly connected in series twice and a spider silk protein GAAn module, and the AA sequence units are derived from human elastin containing VPGVG peptide fragment repeating units; the invention also discloses a double-repeat tandem sequence protein obtained by serially connecting AA sequence units twice, a coding gene of the double-repeat tandem sequence protein, a recombinant elastin sequence, a coding gene of the recombinant elastin sequence, a high-expression vector and a preparation method, and the problems of low protein expression efficiency and high purification cost at present are solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein expression, and particularly relates to a recombinant elastin with a repetitive amino acid sequence and a preparation method thereof. Background Art

[0002] As a core structural protein in human connective tissues, elastin plays an irreplaceable role in maintaining the biomechanical properties of organs such as skin, blood vessels, and lungs with its unique helical conformation and highly cross-linked elastic fiber network. This three-dimensional dynamic structure composed of repetitive VPGVG peptide units endows tissues with excellent elastic deformation ability and energy dissipation characteristics, enabling them to withstand millions of cycles of stress without structural damage. In the field of biomedicine, the excellent dynamic viscoelasticity and endothelial cell affinity of elastin have shown revolutionary potential in the field of bionic engineering and have become an ideal material for constructing small-diameter artificial blood vessels; when used as a wound dressing, its porous hydrogel form can provide a 3D cell migration scaffold, significantly accelerating the repair process of burns and diabetic ulcers. In addition, with the increasing attention to skin aging problems, the application value of elastin with the effects of firming the skin, reducing wrinkles, and enhancing skin luster has received more and more attention in the cosmetics field.

[0003] However, the industrial application of natural-source elastin still faces many technical barriers: Firstly, the stability of the raw material supply chain is poor, mainly relying on the extraction from animal tissues such as bovine neck ligaments. Limited by factors such as livestock breeding cycles, disease prevention and control, and ethical reviews, the supply is far lower than the market demand; Secondly, the extraction process is complex and cumbersome, requiring cyanidation pretreatment, high-temperature hydrolysis, and multi-stage salting-out purification. The final product protein has a low purity and is difficult to meet the usage standards; At the same time, the production cost remains high, and the preparation cost of per kilogram of animal-derived elastin is relatively high, severely restricting its application expansion in multiple fields.

[0004] Therefore, developing genetic engineering recombinant expression technology to break through the bottleneck of natural extraction and establishing a production system for large-scale preparation of high-purity and high-elastic modulus recombinant elastin has become a key research direction in the field of biomaterials. This technological breakthrough can not only solve the raw material problem but also develop intelligent biomaterials with performance exceeding that of natural proteins through molecular structure customization design. Summary of the Invention

[0005] In view of the above situation, the present invention provides a recombinant elastin with a repetitive amino acid sequence and a preparation method thereof. Through the innovative design of the double-repetitive tandem sequence of 327 AA (amino acid) functional fragments in natural elastin and the directional optimization of the Pichia pastoris expression system, an expression system is constructed by using the design of a simulated growth chain tandem structure. The gene sequence optimized by the codon adaptation index is integrated with the vector to achieve continuous and highly efficient secretion expression without methanol induction. Combined with tangential flow filtration, affinity chromatography and molecular sieve chromatography, a three-step purification process is established to achieve the high-yield and high-purity preparation of recombinant elastin.

[0006] To achieve the above object, the technical solutions adopted by the present invention are as follows:

[0007] The present invention provides a recombinant elastin with a repetitive amino acid sequence. The recombinant elastin is composed of an AA sequence unit tandemly repeated 2 times and a spider silk protein GAAn module. The AA sequence unit is selected from a continuous fragment containing 327 AA residues in human elastin.

[0008] Furthermore, the amino acid sequence of the recombinant elastin is as shown in SEQ ID No.1.

[0009] Furthermore, the amino acid sequence of the AA sequence unit is as shown in SEQ ID No.2.

[0010] Furthermore, the amino acid sequence of the spider silk protein GAAn module is GPGGAQGGY.

[0011] Furthermore, the amino acid sequence of the human elastin containing the AA sequence unit is as shown in SEQ ID No.3.

[0012] Furthermore, the base sequence of the coding gene of the human elastin is as shown in SEQ ID No.4.

[0013] Furthermore, the recombinant elastin is expressed using an engineered bacterium.

[0014] Preferably, the engineered bacterium is Pichia pastoris GS115, and the expression vector is the pGAPZαA vector.

[0015] The present invention also provides a preparation method of a recombinant elastin with a repetitive amino acid sequence, which specifically includes the following steps:

[0016] Step 1: Use protein sequencing software to select the amino acid sequence SEQ ID No.2 containing 327 AA fragments from the sequence SEQ ID No.3, design the double-repeat tandem sequence protein SEQ ID No.5, insert the spider silk protein GAAn module into the SEQ ID No.5 sequence, design to obtain recombinant elastin (amino acid sequence SEQ ID No.1), and fuse the His6-FUS LC bifunctional tag at the C-terminus of the recombinant elastin to obtain the recombinant elastin to be expressed (amino acid sequence SEQ ID No.6);

[0017] Step 2: Use gene coding tools to obtain the base sequence SEQ ID No.7 encoding the recombinant elastin to be expressed, replace and optimize the low-frequency codons therein with high-frequency synonymous codons, avoid restriction enzyme cleavage sites, and then perform total gene synthesis and sequencing. The optimized base sequence of the gene encoding the recombinant elastin to be expressed is as shown in SEQ ID No.8 to obtain the target gene;

[0018] Step 3: Clone the target gene into the multiple cloning site of the pGAPZαA vector, use SfiⅠ / XbaⅠ

[0019] and NotⅠ / XbaⅠ double digestion of the vector, replace the original α-factor signal peptide with the SED1 signal peptide, and construct a recombinant plasmid;

[0020] Step 4: Transform the recombinant plasmid into Pichia pastoris GS115 by electroporation, coat the cells on a YPD plate containing antibiotics for culture, pick a single colony and inoculate it into BMGY medium, and sort the high fluorescence intensity strains by flow cytometry after induction expression to obtain positive bacteria;

[0021] Step 5: Inoculate the positive bacteria into a fermenter. The fermentation conditions from 0 to 24 h are 30 °C, pH 5.5, and DO (dissolved oxygen concentration) 40%. The fermentation conditions from 24 to 72 h are 28 °C, pH 6.0, and DO 20%. Centrifuge to collect the fermentation supernatant;

[0022] Step 6: Add 0.1M TEOS (tetraethyl orthosilicate) + 1mM Fe 3+ mixed solution to the fermentation supernatant, shake at 37 °C for 1 h, centrifuge to collect the precipitate, dissolve the precipitate with 0.1M citric acid, filter, and cut the His6-FUS LC bifunctional tag by 365 nm light irradiation to obtain recombinant elastin with the amino acid sequence SEQ ID No.1.

[0023] Furthermore, the amino acid sequence of the His6-FUS LC bifunctional tag is GSHHHHHHGSGGGSSGYSGQSSFSSG.

[0024] The beneficial effects achieved by the present invention are as follows:

[0025] A recombinant elastin with a repetitive amino acid sequence provided by the present invention achieves a huge improvement in the properties of recombinant elastin through structural design and process optimization. At the protein molecular level, existing technologies mostly rely on the linear amplification of a single repetitive unit, resulting in insufficient mechanical properties of the material. In contrast, the present invention performs double-repeat tandem of 327 amino acid functional fragments of human elastin to simulate the periodic network structure of natural elastic fibers, significantly enhancing the mechanical strength and ductility of the material. Further, a breakthrough is made by introducing the spider silk protein GAAn module, and using its cross-species synergistic effect between the β-sheet structure and the α-helix of elastin to endow the material with dynamic response characteristics, thus solving the technical bottleneck of the single function of traditional elastin. In terms of the preparation process, the existing Pichia pastoris expression system is generally limited by the toxicity risk and low-efficiency secretion induced by methanol. However, the present invention reconstructs the gene sequence through global codon optimization, combines with the SED1 signal peptide replacement strategy, and for the first time constructs a methanol-independent high-efficiency secretion expression system. At the same time, a dynamic fermentation parameter regulation strategy is designed to achieve continuous and high-efficiency secretion of the target protein. Aiming at the industry pain points of complex purification process and high cost, the present invention develops a "dual-functional tag-TEOS precipitation-photocleavage" triple purification technology. Through the synergistic effect of the His6-FUS LC tag, affinity capture and phase separation enrichment are combined, and photocleavage with light responsiveness is used to replace the traditional enzymatic cleavage process, which not only simplifies the purification steps but also avoids the risk of protease residue, achieving a leap-forward improvement in efficiency and purity.

[0026] A recombinant elastin with a repetitive amino acid sequence of the present invention forms a complete technical system from structural bionic design to green and efficient preparation, breaking through multiple barriers such as protein performance limitations, low expression efficiency, and high purification cost in the existing technology, and providing a new solution for biomaterials and industrial applications. Brief Description of the Drawings

[0027] Figure 1 Plasmid map of the double-repeat tandem sequence protein SEQ ID No.5 in Example 2;

[0028] Figure 2 Protein structure diagram of the double-repeat tandem sequence protein SEQ ID No.5 in Example 2;

[0029] Figure 3 Single enzyme digestion plasmid detection result of the double-repeat tandem sequence protein SEQ ID No.5 in Example 2;

[0030] Figure 4 Protein structure diagram of the recombinant elastin SEQ ID No.1 in Example 1;

[0031] Figure 5Secondary structure analysis results of the recombinant elastin SEQ ID No.1 in Example 1;

[0032] Figure 6 Gel electrophoresis detection results of the recombinant elastin SEQ ID No.1 in Example 1;

[0033] Figure 7 GC content ratios in SEQ ID No.7 before optimization and SEQ ID No.8 after optimization of the base sequence encoding the recombinant elastin SEQ ID No.1 in Example 1;

[0034] Figure 8 Cell adhesion detection results of the recombinant elastin SEQ ID No.1 in Example 1;

[0035] Figure 9 Human efficacy detection results of the recombinant elastin SEQ ID No.1 in Example 1. Detailed implementation manners

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments; based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0037] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the described content can be applied to the present invention. The preferred implementation methods and materials described herein are only for illustrative purposes and do not limit the content of this application.

[0038] In the following embodiments, unless otherwise specified, all are conventional methods; in the following embodiments, the materials used, unless otherwise specified, the raw materials are all newly purchased materials in the market.

[0039] Example 1: This example provides a recombinant elastin with a repetitive amino acid sequence, and the amino acid sequence of the recombinant elastin is as shown in SEQ ID No.1.

[0040] This example also provides a preparation method of a recombinant elastin with a repetitive amino acid sequence, which specifically includes the following steps:

[0041] Step 1: Design of the recombinant elastin sequence

[0042] Using SnapGene software, a 327-AA fragment (SEQ ID No.2) was intercepted from SEQ ID No.3. A double-repeat tandem sequence protein (SEQ ID No.5) was designed through the Repeat Finder tool, and the spider silk protein GAAn module (GPGGAQGGY) was inserted into its β-sheet region. Subsequently, a His6-FUS LC tag (the sequence contains 6×His and the FUS LC domain) was fused at the C-terminus to generate the target amino acid sequence SEQ ID No.6. The protein structure was predicted using AlphaFold2, and the PSIPRED was used to verify that there was no secondary structure conflict in the linker region, obtaining the target recombinant elastin with the amino acid sequence SEQ ID No.1.

[0043] Step 2: Gene sequence optimization and synthesis

[0044] The amino acid sequence of SEQ ID No.6 was input into the GenScript Codon Optimization Tool to replace low-frequency codons (Arg from AGG→CGT, Pro from CCC→CCG), and restriction enzyme cleavage sites such as BglⅡ (AGATCT) and EcoRI (GAATTC) were avoided to generate the optimized base sequence SEQ ID No.8. Total gene synthesis was performed, and the sequence accuracy was verified by Sanger sequencing (coverage rate reached 100%), obtaining the target gene fragment.

[0045] Step 3: Expression vector construction and signal peptide replacement

[0046] The pGAPZαA vector was double-digested with SfiⅠ / XbaⅠ. The digestion conditions were 1 μg pGAPZαA vector + 1 μL SfiⅠ + 1 μL XbaⅠ + 2 μL NEBuffer 3.1 + 16 μL H2O, incubated at 37°C for 4 h to excise the original α-factor signal peptide, and the SED1 signal peptide was inserted through Gibson Assembly. Forward primer F: 5'-GGCCATTACGGCCATGGCGAGTTTCTCAAC-3', reverse primer R: 5'-GGGGTCTAGATTACTAGTGATGGTGATGGTGTA-3'. The recombinant plasmid was transformed into E. coli competent cells. After heat shock, a recovery medium containing 0.5% glucose was added, and it was shaken and resuscitated at 37°C for 1 h, then spread on an LB plate containing Zeocin (50 μg / mL) and cultured at 37°C for 16 h. Single colonies were picked for colony PCR verification. pGAPZαA-F: 5'-GACTGGTTCCAATTGACAAGC-3' and SED1-R: 5'-GTCGACCTGCAGCGTA-3' were used to amplify the signal peptide region to obtain the recombinant plasmid.

[0047] Step 4: Pichia pastoris transformation and screening of highly expressing strains

[0048] The linearized recombinant plasmid was transformed into competent cells of Pichia pastoris GS115 by electroporation (1.5 kV, 5 ms), and spread on YPD + Zeocin (100 μg / mL) plates. After culturing at 30 °C for 48 h, single colonies were picked and inoculated into BMGY medium containing 1% glycerol. After culturing at 30 °C until OD600 = 6, it was transferred to BMMY medium for induction for 72 h. The cells were labeled with Anti-His-FITC antibody, and the top 1% of the strains with fluorescence intensity were sorted by flow cytometry to obtain highly expressing positive engineering bacteria;

[0049] Step 5: Fermentation process and supernatant collection

[0050] The positive engineering bacteria were inoculated into a 5 L fermenter and fermented at 30 °C, pH 5.5, and 40% DO for 24 h, then adjusted to 28 °C, pH 6.0, and 20% DO, and continuously fermented for 72 h. After centrifugation at 4 °C and 8000 g for 20 min, the supernatant was collected, filtered through a 0.22 μm filter membrane to remove bacteria, and a sterile supernatant was obtained, which was stored at -80 °C for later use. The protein expression level was detected by BCA protein assay kit and was 11.43 g / L;

[0051] Step 6: Protein purification and tag cleavage

[0052] 0.1 M TEOS and 1 mM FeCl3 were added to the sterile supernatant, and it was shaken at 37 °C for 1 h. After centrifugation at 12000 g for 15 min, the precipitate was collected and dissolved in 0.1 M citric acid (pH 4.0). After filtration through a 0.22 μm filter membrane, it was irradiated with 365 nm ultraviolet light at an intensity of 10 mW / cm 2 for 30 min to cleave the His6-FUS LC tag. The removal of the tag was verified by SDS-PAGE and Western Blot (Anti-His antibody), and the purity was determined to be 97.4%.

[0053] Example 2: This example provides a protein with a double repeat tandem sequence, and its amino acid sequence is SEQ ID No.5. The specific preparation method is as follows:

[0054] Step 1: Use SnapGene software to design and obtain the double repeat tandem sequence protein SEQ ID No.5;

[0055] Step 2: Use the GenScript Codon Optimization Tool to design the base sequence of SEQ ID No.5, and after optimization, obtain the base sequence of the double repeat tandem sequence protein SEQ ID No.9. The whole gene was synthesized, and the sequence accuracy was verified by Sanger sequencing to obtain the target gene fragment;

[0056] Step 3: Clone the target gene fragment onto the pGAPZαA vector to construct a recombinant plasmid. Transform the recombinant plasmid into E. coli competent cells. After heat shock, add recovery medium containing 0.5% glucose, incubate at 37 °C with shaking for 1 h, spread on an LB plate containing Zeocin (50 μg / mL), culture at 37 °C for 16 h, pick single colonies for colony PCR verification. Use pGAPZαA-F: 5'-GACTGGTTCCAATTGACAAGC-3' and SED1-R: 5'-GTCGACCTGCAGCGTA-3' to amplify the signal peptide region to obtain the recombinant plasmid;

[0057] Step 4: Transform the recombinant plasmid into Pichia pastoris GS115 by electroporation (1.5 kV, 5 ms), spread on a YPD + Zeocin (100 μg / mL) plate, culture at 30 °C for 48 h, pick single colonies and inoculate them into BMGY medium containing 1% glycerol, culture at 30 °C until OD600 = 6, transfer to BMMY medium for induction for 72 h, label the bacterial cells with Anti-His-FITC antibody, and sort the top 1% of the strains with the highest fluorescence intensity by flow cytometry to obtain highly expressed positive engineering bacteria;

[0058] Step 5: Inoculate the positive engineering bacteria into a 5 L fermenter, ferment at 30 °C, pH 5.5, 40% DO for 24 h, then adjust to 28 °C, pH 6.0, 20% DO, continue fermentation for 72 h, centrifuge at 4 °C, 8000 g for 20 min, collect the supernatant, filter and sterilize it through a 0.22 μm filter membrane to obtain a sterile supernatant, store it at -80 °C for later use. The protein expression level detected by the BCA protein expression kit is 8.15 g / L;

[0059] Step 6: Add 2.5 M NaCl to the sterile supernatant and precipitate it on ice bath for 30 min. After centrifugation, resuspend the precipitate with 20 mM Tris-HCl (pH 8.0), concentrate and desalt it through a 10 kDa ultrafiltration membrane until the conductivity < 5 mS / cm. Subsequently, perform HiTrap SPHP cation exchange chromatography (20 mM sodium citrate, pH 4.5; 0 - 1 M NaCl gradient elution), collect the main peak fraction, add 5% trehalose and 1% glycine as lyophilization protectants, pre-freeze and then vacuum lyophilize. The purity of the final product is verified to be 92.6% by SDS-PAGE, HPLC and MALDI-TOF.

[0060] Physicochemical property analysis

[0061] Use protein bioanalysis software to analyze the molecular weight, stability index and hydrophilicity of the recombinant elastin and double repeated tandem sequence proteins obtained in Examples 1 - 2, and use a Malvern nano laser particle size analyzer to measure the isoelectric point. The results are shown in Table 1.

[0062] The plasmid map of the designed double-repeat tandem sequence protein is shown in Figure 1 , and the protein structure is shown in Figure 2 . The single enzyme digestion result of the plasmid is shown in Figure 3 .

[0063] The recombinant elastin structure is shown in Figure 4 , Two . The analysis result of the secondary structure is shown in Figure 5 , and the SDS-PAGE gel electrophoresis result is shown in Figure 6 .

[0064] The changes in the C and G contents in the base sequence SEQ ID No.7 of the recombinant elastin coding gene to be expressed before optimization and the base sequence SEQ ID No.8 of the target gene after optimization are shown in Figure 7 .

[0065] Cell adhesion experiment

[0066] Take human fibroblast cell lines in the logarithmic growth phase. The prepared recombinant elastin samples are respectively formulated into three different concentrations of high, medium, and low (10 mg / mL, 5 mg / mL, 1 mg / mL) with PBS buffer. Then, set up high-concentration group, medium-concentration group, and low-concentration group in a 96-well plate. The blank control group adds 100 μL of PBS buffer. Each group is set with 6 replicate wells. Incubate at 37 °C and 5% CO2 for 2 h to allow the protein to fully adsorb on the bottom of the well plate. After the incubation ends, carefully aspirate the liquid in each well, avoiding damaging the adsorbed protein layer; digest the cells and adjust the cell concentration to 5×10 4 cells / mL, inoculate into the above 96-well plate and continue to culture for 2 h. Slowly rinse each well 3 times with pre-cooled PBS buffer to remove the unadhered cells. Add 100 μL of cell fixative to each well and fix at room temperature for 20 min. After the fixation is completed, aspirate the fixative and rinse; stain with 10 min of crystal violet staining solution, wash with distilled water until colorless, remove the excess staining solution and dry, then add 100 μL of eluent, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance A of each well at 492 nm. The results are shown in Figure 8 .

[0067] Human efficacy test

[0068] Recruit 48 healthy volunteers aged 20 - 50 years old with neutral, dry or combination skin types who have not used recombinant protein-based skin care products recently; set three dose concentrations of 0.1%, 0.5%, and 1.0%, which are marked as the low-dose group, medium-dose group, and high-dose group respectively, and the purified water group is the blank group, with 12 people in each group; the volunteers fill in the basic information questionnaire and skin type test, and sign the informed consent form. One week before the experiment, all volunteers stop using any skin care products or beauty means that may affect skin elasticity, and only use mild cleansing products. After cleansing the face morning and evening every day, each group of volunteers takes an appropriate amount of recombinant elastin solution and evenly applies it on the face, gently massaging until absorbed; the blank control group uses placebo (purified water) in the same way. Use the German CK MPA 580 multi-probe skin tester to measure the skin elasticity of the left and right cheeks, the center of the forehead, and the chin at the time points before use, 14 days after use, and 28 days after use, and evaluate the functional effect of recombinant elastin in improving skin elasticity. The results are shown in Figure 9 .

[0069] Table 1 Physicochemical properties of recombinant elastin and double-repeat tandem sequence protein

[0070]

[0071] Table 1 results show that the recombinant elastin and double-repeat tandem sequence protein have similar physicochemical properties, strong protein stability, good lipid solubility, and are conducive to cell absorption and utilization.

[0072] Figure 1 The designed double-repeat tandem sequence plasmid is reasonable, with a single restriction enzyme and a small cutting point error.

[0073] Figure 2 In the double-repeat tandem sequence protein, there are more α-helix and β-sheet secondary structures, fewer folding structures at both ends of the protein, and there is a certain steric hindrance.

[0074] Figure 3 The single enzyme digestion result of the double-repeat tandem sequence plasmid shows that the plasmid is linearized by single enzyme digestion, which is Figure 1 consistent with the designed plasmid result and contains the target sequence protein band.

[0075] Figure 4 In the structure of recombinant elastin, due to the addition of the spider silk protein GAAn module at the tandem of the repeat sequence units, the folding effect of its tertiary structure is stronger and the secondary structure is more abundant.

[0076] Figure 5 The analysis result of the secondary structure of recombinant elastin corresponds to Figure 4 the result, and the α-helix structure is obvious.

[0077] Figure 6The results of gel electrophoresis showed that the prepared recombinant elastin had a high purity and its molecular weight was in line with the expected design.

[0078] Figure 7 Before optimization, the GC base content of SEQ ID No.7 was 79.97%, and after optimization, the GC base content of SEQ ID No.8 was 49.44%. The optimized gene sequence was more adaptable to the codon preference of the host engineering bacteria, improving the translation efficiency and protein expression level.

[0079] Figure 8 The results of the cell adhesion experiment showed that all experimental groups could significantly improve the cell adhesion on recombinant elastin. There was no significant difference between the medium and high concentration groups, and a good cell adhesion effect could be achieved at the medium concentration.

[0080] Figure 9 The results showed that as the number of times and duration of using recombinant elastin increased, the viscoelasticity of human skin was significantly improved, demonstrating the advantage of recombinant elastin in improving skin elasticity and firmness.

[0081] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

[0082] The above describes the present invention and its implementation manners. Such a description is not restrictive. What is shown in the drawings is only one of the implementation manners of the present invention, and the actual application is not limited thereto. Generally speaking, if those of ordinary skill in the art are inspired by it and design similar ways and embodiments without creative efforts without departing from the purpose of the present invention, they should fall within the protection scope of the present invention.

Claims

1. A recombinant elastin having a repetitive amino acid sequence, characterized in that, The recombinant elastin is composed of AA sequence units tandemly repeated twice and the spider silk protein GAAn module, and the amino acid sequence is shown in SEQ ID No.1; The AA sequence unit is derived from human elastin, and the amino acid sequence is shown in SEQ ID No.2; The amino acid sequence of the human elastin is shown in SEQ ID No.

3.

2. The recombinant elastin with a repetitive amino acid sequence according to claim 1, wherein, The base sequence of the coding gene of the human elastin is shown in SEQ ID No.

4.

3. The recombinant elastin having a repetitive amino acid sequence according to claim 1, characterized in that, The original coding gene sequence of the recombinant elastin is shown in SEQ ID No.7, and the target gene sequence capable of expressing the recombinant elastin after optimization is shown in SEQ ID No.

8.

4. A recombinant elastin having a repetitive amino acid sequence according to claim 1, characterized in that, The recombinant elastin is expressed using an engineered bacterium, which is Pichia pastoris GS115, and the vector used for the expression is pGAPZαA.

5. A method for preparing a recombinant elastin having a repetitive amino acid sequence according to any one of claims 1-4, characterized in that, Specifically, it includes the following steps: Step 1: Design a double tandem amino acid sequence and insert the spider silk protein GAAn module, and introduce a photosensitive tag to obtain the recombinant elastin to be expressed; Step 2: Design the coding gene of the recombinant elastin to be expressed, and obtain the target gene after optimization; Step 3: Clone the target gene into the vector, use double digestion of the vector, and replace the signal peptide to obtain a recombinant plasmid; Step 4: Transform the recombinant plasmid into the engineered bacterium and sort the positive engineered bacteria; Step 5: Gradient ferment and centrifuge the positive engineered bacteria to collect the fermentation supernatant; Step 6: Precipitate, centrifuge, and photolytically cleave the tag of the fermentation supernatant to obtain the recombinant elastin.

6. The preparation method of a recombinant elastin having a repetitive amino acid sequence according to claim 5, characterized in that, In Step 3, the double digestion of the vector uses SfiⅠ / XbaⅠ and NotⅠ / XbaⅠ endonucleases, and the replacement of the signal peptide is to replace the α-factor signal peptide with the SED1 signal peptide.

7. The preparation method of a recombinant elastin having a repetitive amino acid sequence according to claim 6, characterized in that, The double tandem amino acid sequence is shown in SEQ ID No.5; The photosensitive tag is the His6-FUS LC bifunctional tag, and the amino acid sequence of the recombinant elastin to be expressed is shown in SEQID No.6.

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