Recombinant elastin, preparation method of recombinant elastin, recombinant elastin raw material and application of recombinant elastin raw material

Abstract

The invention relates to a recombinant elastin and a preparation method thereof as well as a recombinant elastin raw material and application thereof, and belongs to the technical field of recombinant proteins. The amino acid sequence of the recombinant elastin is an amino acid sequence as shown in SEQ ID No.1 or a derivative amino acid sequence of which the sequence identity with the SEQ ID No.1 is more than 90%. The protein is high in expression level, high in purity and relatively high in biological activity, and has the effects of supplementing skin collagen, resisting aging and repairing skin barriers.

Description

Technical Field

[0001] This application relates to the field of recombinant protein technology, and in particular to a recombinant elastin, its preparation method, recombinant elastin raw materials, and their applications. Background Technology

[0002] Elastin is an important structural protein in the dermis of the skin, mainly composed of alternating hydrophobic and hydrophilic domains. It plays a crucial role in maintaining skin morphology. Elastin imparts elasticity to the skin, provides an ideal scaffold and site for substance exchange for cell adhesion and tissue growth, and participates in regulating basic physiological processes such as cell proliferation and differentiation. Therefore, the application of elastin in dermatology has attracted much attention.

[0003] With the development of biotechnology, recombinant elastin obtained by gene recombination technology has achieved great success. Compared with natural elastin, recombinant elastin produced by this technology has solved the defects of viral risks in traditional extraction methods, and at the same time significantly improved the stability, hydrophilicity and biocompatibility of elastin. Therefore, recombinant elastin has attracted more and more attention.

[0004] However, the current recombinant elastin expression level is low, the product is difficult to purify, and the biological activity of the expressed product is low. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the objectives of this application include providing a recombinant elastin, its preparation method, raw materials, and applications. This recombinant elastin exhibits high expression levels, high purity, and high biological activity, possessing the effects of replenishing skin collagen, anti-aging, and repairing the skin barrier.

[0006] In a first aspect, embodiments of this application provide a recombinant elastin, the amino acid sequence of which comprises the amino acid sequence shown in SEQ ID No. 1 or a derived amino acid sequence having more than 90% identity with the sequence of SEQ ID No. 1.

[0007] The recombinant elastin provided in this application contains an amino acid sequence as shown in SEQ ID No. 1 or a derived amino acid sequence with more than 90% identity to the sequence in SEQ ID No. 1. It can be expressed efficiently, with a high expression level, is easy to purify to a high purity, and has high biological activity. Even at a low concentration, it has the effects of supplementing skin collagen, anti-aging, and repairing the skin barrier.

[0008] In some embodiments of this application, the amino acid sequence of recombinant elastin is shown in SEQ ID No. 1.

[0009] The recombinant elastin with the amino acid sequence shown in SEQ ID No. 1 provided in this application has a higher expression level, is easier to purify to achieve higher purity, and has higher biological activity. It can replenish skin collagen, resist aging, and repair the skin barrier even at a lower concentration.

[0010] Secondly, embodiments of this application provide a nucleic acid encoding the recombinant elastin provided in the first aspect.

[0011] The nucleic acid provided in this application is used to encode the recombinant elastin containing the amino acid sequence shown in SEQ ID No. 1 or a derived amino acid sequence with more than 90% identity to the sequence of SEQ ID No. 1. The recombinant elastin has a high expression level, is easy to purify to a high purity, and has high biological activity. It has the effects of supplementing skin collagen, anti-aging, and repairing the skin barrier even at a low concentration.

[0012] In some embodiments of this application, the nucleotide sequence of the nucleic acid is shown in SEQ ID No. 2.

[0013] This application provides a recombinant elastin encoded by the nucleotide sequence shown in SEQ ID No. 2, which has a higher expression level, is easier to purify to achieve higher purity, and has higher biological activity. It can replenish skin collagen, resist aging, and repair the skin barrier even at a lower concentration.

[0014] Thirdly, embodiments of this application provide a recombinant expression vector comprising the nucleic acid provided in the second aspect.

[0015] The recombinant expression vector provided in this application contains nucleic acid encoding the aforementioned recombinant elastin. This recombinant expression vector containing nucleic acid encoding the aforementioned recombinant elastin can be transfected into host cells for expression. It can induce the expression of the aforementioned recombinant elastin containing the amino acid sequence shown in SEQ ID No. 1 or a derived amino acid sequence with more than 90% sequence identity with SEQ ID No. 1. The recombinant elastin has a high expression level, is easy to purify to achieve high purity, and has high biological activity. Even at a low concentration, it has the effects of supplementing skin collagen, anti-aging, and repairing the skin barrier.

[0016] Fourthly, embodiments of this application provide a method for preparing the recombinant elastin provided in the first aspect, comprising: (1) Synthesize the nucleotide sequence encoding the recombinant elastin provided in the first aspect, and construct a recombinant expression vector containing the nucleotide sequence; (2) Construct recombinant engineered cells containing the recombinant expression vector of step (1); (3) The recombinant engineered cells from step (2) are cultured and fermented, and the recombinant elastin is isolated and purified from the fermentation product.

[0017] The method for preparing recombinant elastin provided in this application first synthesizes a nucleotide sequence encoding a recombinant elastin whose amino acid sequence includes the amino acid sequence shown in SEQ ID No. 1 or a derived amino acid sequence with more than 90% sequence identity with SEQ ID No. 1. Then, a recombinant expression vector encoding the nucleotide sequence of the recombinant elastin is constructed. This recombinant expression vector is then transfected into host cells to construct recombinant engineered cells. These recombinant engineered cells are cultured and fermented to express a recombinant elastin with high expression levels, easy purification to achieve high purity, and high biological activity. Even at low concentrations, it exhibits the effects of supplementing skin collagen, anti-aging, and repairing the skin barrier. The amino acid sequence includes the amino acid sequence shown in SEQ ID No. 1 or a derived amino acid sequence with more than 90% sequence identity with SEQ ID No. 1.

[0018] In some embodiments of this application, the host cell for the recombinant engineered cells is Escherichia coli.

[0019] This application uses Escherichia coli as the host cell, which is beneficial to improve expression efficiency, thereby increasing the expression level of recombinant elastin, and also helps to make the recombinant elastin more closely resemble the conformation of the natural protein, resulting in higher activity.

[0020] In some embodiments of this application, purification includes: resuspending and lysing the recombinant engineered cells isolated from the fermentation product using a buffer containing 4-10 M urea, and collecting the supernatant; purifying the supernatant using an affinity chromatography column; and desalting the purified solution by chromatography using a buffer containing 100-150 mM sodium chloride.

[0021] This application uses a buffer solution containing an appropriate concentration of urea to resuspend and lyse the recombinant engineered cells isolated from the fermentation product, thereby effectively improving the purification effect and increasing the yield and purity of recombinant elastin. At the same time, a buffer solution containing an appropriate concentration of sodium chloride is used to desalt the purified solution, effectively improving the desalting recovery rate, thereby further improving the yield and purity of recombinant elastin.

[0022] Fifthly, embodiments of this application provide a recombinant elastin raw material, comprising recombinant elastin provided in the first aspect at a concentration of 0.2~0.5 mg / mL, glycerol at a mass percentage of 30%~40%, pentylene glycol at a mass percentage of 4.5%~5%, and the balance being water.

[0023] This application uses a suitable concentration of the above-mentioned recombinant elastin and a suitable mass percentage of glycerol, and adapts it with pentylene glycol to form a recombinant elastin raw material, which has good storage stability and can be stored stably at 4°C.

[0024] In some embodiments of this application, the pH value of the recombinant elastin raw material is 5 to 5.5.

[0025] This application can further improve the storage stability of recombinant elastin raw materials by adjusting the pH value of the recombinant elastin raw materials to a suitable range.

[0026] Sixthly, embodiments of this application provide the application of the recombinant elastin provided in the first aspect or the recombinant elastin raw material provided in the fifth aspect in the preparation of cosmetics. The recombinant elastin provided in this application, containing the amino acid sequence shown in SEQ ID No. 1 or a derived amino acid sequence with more than 90% identity to the sequence in SEQ ID No. 1, can be expressed efficiently, has high purity, and exhibits high biological activity. Even at low concentrations, it has the effects of replenishing skin collagen, anti-aging, and repairing the skin barrier. When the above-mentioned recombinant elastin or recombinant elastin raw material containing the above-mentioned recombinant elastin is used in the preparation of cosmetics, it has the effects of replenishing skin collagen, anti-aging, and repairing the skin barrier. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 The image shows the detection results of recombinant elastin before and after purification, as provided in Example 4 of this application.

[0029] Figure 2 The image shows the detection results of recombinant elastin before and after desalting, as provided in Example 4 of this application.

[0030] Figure 3 This is a graph showing the cytotoxicity results in Test Example 1 of this application.

[0031] Figure 4 This is a diagram showing the results of recombinant elastin promoting collagen synthesis in Experiment Example 2 of this application.

[0032] Figure 5 This is the survival curve of wild-type N2 elegans nematode in Experiment Example 3 of this application.

[0033] Figure 6 For example 3 of this application, BE93 ( dpy-2 (Image of stained fluorescent nematodes)

[0034] Figure 7 The graph shows the stability test results of the recombinant elastin raw materials provided in Examples 9-10 and Examples 13-14 of Test Example 6 of this application.

[0035] Figure 8 The graph shows the stability test results of the recombinant elastin raw materials provided in Examples 11-12 and Examples 17-18 of Test Example 6 of this application.

[0036] Figure 9 The graph shows the stability test results of the recombinant elastin raw materials provided in Examples 15-16 and Examples 19-20 of Test Example 6 of this application.

[0037] Figure 10 This is a graph showing the stability test results of the recombinant elastin raw material provided in Comparative Example 3 of Test Example 6 of this application. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0039] Currently, recombinant elastin expression levels are low, product purification is difficult, and the expressed product has low biological activity.

[0040] Therefore, embodiments of this application provide a recombinant elastin, wherein the amino acid sequence of the recombinant elastin comprises the amino acid sequence shown in SEQ ID No. 1 or a derived amino acid sequence having more than 90% identity with the sequence of SEQ ID No. 1.

[0041] The recombinant elastin provided in this application contains an amino acid sequence as shown in SEQ ID No. 1 or a derived amino acid sequence with more than 90% identity to the sequence in SEQ ID No. 1. It can be expressed efficiently, with a high expression level, is easy to purify to a high purity, and has high biological activity. Even at a low concentration, it has the effects of supplementing skin collagen, anti-aging, and repairing the skin barrier.

[0042] As an example, the sequence identity of the derived amino acid sequence with SEQ ID NO.1 may be, but is not limited to, any one of the values ​​of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, and 100%, or any range between two. It should be noted that the derived amino acid sequence is an amino acid sequence formed by substituting, inserting, and / or knocking out one or more amino acids based on the amino acid sequence shown in SEQ ID NO.1.

[0043] In some embodiments of this application, the amino acid sequence of recombinant elastin is shown in SEQ ID No. 1.

[0044] The recombinant elastin with the amino acid sequence shown in SEQ ID No. 1 provided in this application has a higher expression level, is easier to purify to achieve higher purity, and has higher biological activity. It can replenish skin collagen, resist aging, and repair the skin barrier even at a lower concentration.

[0045] This application provides a nucleic acid encoding the aforementioned recombinant elastin.

[0046] The nucleic acid provided in this application is used to encode the recombinant elastin containing the amino acid sequence shown in SEQ ID No. 1 or a derived amino acid sequence with more than 90% identity to the sequence of SEQ ID No. 1. The recombinant elastin has a high expression level, is easy to purify to a high purity, and has high biological activity. It has the effects of supplementing skin collagen, anti-aging, and repairing the skin barrier even at a low concentration.

[0047] In some embodiments of this application, the nucleotide sequence of the nucleic acid is shown in SEQ ID No. 2. The recombinant elastin encoded by the nucleotide sequence shown in SEQ ID No. 2 has a higher expression level, is easier to purify to achieve higher purity, and has higher biological activity, exhibiting the effects of replenishing skin collagen, anti-aging, and repairing the skin barrier even at lower concentrations.

[0048] This application provides a recombinant expression vector containing the aforementioned nucleic acid. The recombinant expression vector contains the nucleic acid encoding the aforementioned recombinant elastin. This recombinant expression vector containing the nucleic acid encoding the aforementioned recombinant elastin can be transfected into host cells for expression. It can induce the expression of the aforementioned recombinant elastin containing the amino acid sequence shown in SEQ ID No. 1 or a derived amino acid sequence with more than 90% sequence identity to SEQ ID No. 1. The recombinant elastin exhibits high expression levels, is easily purified to achieve high purity, and possesses high biological activity, demonstrating the effects of supplementing skin collagen, anti-aging, and repairing the skin barrier even at low concentrations.

[0049] This application provides a method for preparing the above-mentioned recombinant elastin, comprising: (1) Synthesize the nucleotide sequence encoding the above-mentioned recombinant elastin and construct a recombinant expression vector containing the nucleotide sequence; (2) Construct recombinant engineered cells containing the recombinant expression vector of step (1); (3) The recombinant engineered cells from step (2) are cultured and fermented, and the recombinant elastin is isolated and purified from the fermentation product.

[0050] The method for preparing recombinant elastin provided in this application first synthesizes a nucleotide sequence encoding a recombinant elastin whose amino acid sequence includes the amino acid sequence shown in SEQ ID No. 1 or a derived amino acid sequence with more than 90% sequence identity with SEQ ID No. 1. Then, a recombinant expression vector encoding the nucleotide sequence of the recombinant elastin is constructed. This recombinant expression vector is then transfected into host cells to construct recombinant engineered cells. These recombinant engineered cells are cultured and fermented to express a recombinant elastin with high expression levels, easy purification to achieve high purity, and high biological activity. Even at low concentrations, it exhibits the effects of supplementing skin collagen, anti-aging, and repairing the skin barrier. The amino acid sequence includes the amino acid sequence shown in SEQ ID No. 1 or a derived amino acid sequence with more than 90% sequence identity with SEQ ID No. 1.

[0051] In some embodiments of this application, the host cell for the recombinant engineered cells is *Escherichia coli*. Using *E. coli* as the host cell helps to improve expression efficiency, thereby increasing the expression level of recombinant elastin, and also helps to make the recombinant elastin more closely resemble the conformation of the natural protein, resulting in higher activity.

[0052] In some embodiments of this application, purification includes: resuspending and lysing the recombinant engineered cells isolated from the fermentation product using a buffer containing 4-10 M urea, and collecting the supernatant; purifying the supernatant using an affinity chromatography column; and desalting the purified solution by chromatography using a buffer containing 100-150 mM sodium chloride. Using a buffer containing an appropriate concentration of urea to resuspend, lyse, and purify the recombinant engineered cells isolated from the fermentation product effectively improves the purification effect and increases the yield and purity of recombinant elastin; simultaneously, using a buffer containing an appropriate concentration of sodium chloride to desalt the purified solution effectively improves the desalting recovery rate, thereby further improving the yield and purity of recombinant elastin.

[0053] This application provides a recombinant elastin raw material, comprising the aforementioned recombinant elastin at a concentration of 0.2~0.5 mg / mL, 30%~40% glycerol by mass, 4.5%~5% pentanediol by mass, and the balance being water. Using a suitable concentration of the aforementioned recombinant elastin and a suitable mass percentage of glycerol, combined with pentanediol, forms a recombinant elastin raw material with good storage stability, allowing for stable storage at 4°C.

[0054] In some embodiments of this application, the pH value of the recombinant elastin raw material is 5-5.5. Adjusting the pH value of the recombinant elastin raw material to a suitable range can further improve its storage stability.

[0055] This application provides an example of the use of the recombinant elastin described above or the recombinant elastin raw material provided in the fifth aspect in the preparation of cosmetics. The recombinant elastin provided in this application, containing the amino acid sequence shown in SEQ ID No. 1 or a derived amino acid sequence with more than 90% identity to the sequence in SEQ ID No. 1, can be expressed efficiently, has high purity, and exhibits high biological activity. Even at low concentrations, it has the effects of replenishing skin collagen, anti-aging, and repairing the skin barrier. When the above-mentioned recombinant elastin or recombinant elastin raw material containing the above-mentioned recombinant elastin is used in the preparation of cosmetics, it has the effects of replenishing skin collagen, anti-aging, and repairing the skin barrier. The features and performance of this application will be further described in detail below with reference to the embodiments.

[0056] The specific equipment used in the embodiments and test examples is as follows: 1. Uracil synthesis-deficient Escherichia coli OP50 ( E. coli OP50), wild-type Caenorhabditis elegans (the Bridtol strain N2), BE93 ( dpy-2 ) Caenorhabditis strain: purchased from the Caenorhabditis Genetics Center (USA).

[0057] 2. RAW 264.7 cells: purchased from iCell.

[0058] 3. Human skin fibroblasts (BJ cells): purchased from the Cell Bank of the Chinese Academy of Sciences.

[0059] Example 1 This embodiment provides the design of the amino acid sequence of recombinant elastin, using the following method: The complete amino acid sequence of human elastin was obtained from the NCBI protein database and screened and optimized to design a recombinant elastin with the amino acid sequence shown in SEQ ID No. 1.

[0060] Example 2 This embodiment provides the construction of a recombinant expression vector for recombinant elastin, as follows: The gene sequence encoding recombinant elastin, as shown in SEQ ID NO.1, was optimized to synthesize a nucleotide sequence as shown in SEQ ID NO.2; and cloned into the pET-28a(+) vector, and fused with a His tag to construct a recombinant expression vector containing the nucleotide sequence shown in SEQ ID NO.2.

[0061] Example 3 This embodiment provides the construction of recombinant engineered cells containing a recombinant expression vector, as follows: The recombinant expression vector containing the nucleotide sequence shown in SEQ ID NO.2, constructed in Example 2, was transfected into Escherichia coli (BL21(DE3)) competent cells using chemical transformation. Positive clones in the transformed LB solid plate colonies were screened by agarose gel electrophoresis. The screened positive clones were transferred to 2 mL of LB liquid medium and cultured at 37°C and 200 rpm for 16 h to obtain recombinant engineered cells containing the recombinant expression vector containing the nucleotide sequence shown in SEQ ID NO.2.

[0062] Example 4 This embodiment provides the fermentation purification of the recombinant engineered cells provided in Example 3, as follows: (1) Fermentation culture of recombinant engineered cells: 10 μL of recombinant engineered cell culture from Example 3 was spread on LB solid plates and cultured in an incubator at 37 ℃ for 12 h. Single clones were selected and inoculated into 50 mL of LB liquid medium containing 50 µg / mL kanamycin. The medium was placed in a shaker and cultured at 37 ℃ and 200 rpm for 14 h. Then, it was inoculated into 200 mL of TB liquid medium containing 50 µg / mL kanamycin at a ratio of 1:100 and cultured at 37 ℃ until the OD of the bacterial culture reached 0.8. Isopropyl-β-D-thiogalactoside (IPTG, Huaxiang Kejie) was added to make the final concentration of IPTG reach 1 mM. The culture was continued for 4 h. Then, the cells were collected by centrifugation at 4 ℃ and 4500 rpm for 15 min. (2) Purification: The bacterial cells collected in step (1) were resuspended at a ratio of 1:9 (v / v) in a buffer containing 8 M urea (including 50 mM Tris-HCl, 50 mM NaCl, and 5% glycerol, adjusted to pH 7.9), homogenized at 800 bar, and then centrifuged at 14000×g, 4 ºC for 20 min. The supernatant was collected. After being filtered through a 0.45 μm filter, the supernatant was loaded onto a 60 mL nickel affinity chromatography column (NW Rose Ni FF) at a flow rate of 1 mL / min. The baseline was washed back with a buffer containing 5 mM imidazole and 20 mM Tris-HCl + 150 mM NaCl. Impurities were washed with a buffer containing 100 mM imidazole and 20 mM Tris-HCl + 150 mM NaCl. The target protein was eluted with 400 mM imidazole buffer to obtain a purified recombinant elastin solution. The supernatant before purification and the purified recombinant elastin solution were then analyzed by SDS-PAGE electrophoresis. The results are shown below. Figure 1 As shown in the image, the sixth protein band indicates that the purity of the recombinant elastin after purification is >90%. The purified recombinant elastin solution was deimidazoleized using a Sephadex G-25 molecular sieve (NW Dex G-25 F) in a buffer solution of 50 mM PB + 150 mM NaCl to obtain a recombinant elastin solution. The undesalted and desalted recombinant elastin solutions were analyzed by SDS-PAGE electrophoresis. The results are shown below. Figure 2 As shown, during the desalting process, five tubes of recombinant elastin solution were collected according to the peak area of ​​the purifier and tested separately. As shown in the 2nd to 6th protein bands, the purity of recombinant elastin after desalting is >90%.

[0063] Example 5 This embodiment provides the fermentation purification of recombinant engineered cells provided in Example 3. The difference between this embodiment and Example 4 is that the concentration of NaCl in the buffer solution used for desalting after purification is 100 mM.

[0064] Example 6 This embodiment provides the fermentation purification of recombinant engineered cells provided in Example 3. The difference between this embodiment and Example 4 is that the concentration of urea in the buffer solution used for purification and resuspension is 4 M.

[0065] Example 7 This embodiment provides the fermentation purification of recombinant engineered cells provided in Example 3. The difference between this embodiment and Example 4 is that the concentration of urea in the buffer solution used for purification and resuspension is 6 M.

[0066] Example 8 This embodiment provides the fermentation purification of recombinant engineered cells provided in Example 3. The difference between this embodiment and Example 4 is that the concentration of urea in the buffer solution used for purification and resuspension is 10 M.

[0067] Example 9 This embodiment provides a recombinant elastin raw material, comprising the recombinant elastin provided in Example 4 at a concentration of 0.2 mg / mL, 30% by mass of glycerol, 5% by mass of pentanediol, and the balance being water.

[0068] Example 10 This embodiment provides a recombinant elastin raw material, which differs from Example 9 in that sodium dihydrogen phosphate is used to adjust the pH value of the recombinant elastin raw material to 5.5.

[0069] Example 11 This embodiment provides a recombinant elastin raw material, which differs from Example 9 in that the concentration of the recombinant elastin provided in Example 4 is 0.5 mg / mL.

[0070] Example 12 This embodiment provides a recombinant elastin raw material, which differs from Example 11 in that sodium dihydrogen phosphate is used to adjust the pH value of the recombinant elastin raw material to 5.5.

[0071] Example 13 This embodiment provides a recombinant elastin raw material, which differs from Example 9 in that the mass percentage of glycerol is 35%.

[0072] Example 14 This embodiment provides a recombinant elastin raw material, which differs from Example 13 in that sodium dihydrogen phosphate is used to adjust the pH value of the recombinant elastin raw material to 5.5.

[0073] Example 15 This embodiment provides a recombinant elastin raw material, which differs from Example 13 in that the concentration of the recombinant elastin provided in Example 4 is 0.5 mg / mL.

[0074] Example 16 This embodiment provides a recombinant elastin raw material, which differs from Example 15 in that sodium dihydrogen phosphate is used to adjust the pH value of the recombinant elastin raw material to 5.5.

[0075] Example 17 This embodiment provides a recombinant elastin raw material, which differs from Example 9 in that the mass percentage of glycerol is 40%.

[0076] Example 18 This embodiment provides a recombinant elastin raw material, which differs from Example 17 in that sodium dihydrogen phosphate is used to adjust the pH value of the recombinant elastin raw material to 5.5.

[0077] Example 19 This embodiment provides a recombinant elastin raw material, which differs from Example 17 in that the concentration of the recombinant elastin provided in Example 4 is 0.5 mg / mL.

[0078] Example 20 This embodiment provides a recombinant elastin raw material, which differs from Example 19 in that sodium dihydrogen phosphate is used to adjust the pH value of the recombinant elastin raw material to 5.5.

[0079] Comparative Example 1 This comparative example provides the fermentation purification of recombinant engineered cells provided in Example 3. The difference between Example 3 and Example 4 is that the concentration of NaCl in the buffer solution used for desalting after purification is 50 mM.

[0080] Comparative Example 2 This comparative example provides the fermentation purification of recombinant engineered cells provided in Example 3. The difference between Example 3 and Example 4 is that the buffer solution used for purification and resuspension does not contain urea.

[0081] Comparative Example 3 This comparative example provides a recombinant elastin raw material, which differs from Example 9 in that the mass percentage of glycerol is 20%.

[0082] Comparative Example 4 This comparative example provides a recombinant elastin raw material, which differs from Comparative Example 3 in that sodium dihydrogen phosphate is used to adjust the pH value of the recombinant elastin raw material to 5.5.

[0083] The fermentation and purification methods for recombinant engineered cells provided in Examples 4-8 and Comparative Examples 1-2 are shown in Table 1: Table 1. Fermentation purification methods for recombinant engineered cells

[0084] The recombinant elastin raw materials provided in Examples 9-20 and Comparative Examples 3-4 are shown in Table 2: Table 2 Recombinant Elastin Raw Materials

[0085] Note: In Table 2, " / " indicates that the pH value is not tested or adjusted.

[0086] Experimental Example 1 This experimental example uses the recombinant elastin provided in Example 4 to conduct cell experiments. The specific method is as follows: RAW 264.7 cells in logarithmic growth phase were seeded into 96-well plates at a density of 2.5 x 10⁻⁶ cells / well. 5 Cells were cultured in 96-well plates for 24 h. Recombinant elastin samples were prepared in serum-free 2% FBS medium to concentrations of 18.75 μg / mL, 37.5 μg / mL, 75 μg / mL, 150 μg / mL, and 300 μg / mL, respectively, and added to each well (3 replicates). A positive control group (10% FBS) and a blank control group (2% FBS) were also included. Cells were cultured for another 24 h, and cell morphology and number were observed periodically under a microscope. After 48 h, CCK-8 was diluted 10-fold with serum-free 2% FBS medium, and 100 µL of the CCK-8 dilution was added to each well. The plates were then incubated at 37 ℃ for 0.5 h, and the absorbance was measured using a microplate reader at a wavelength of 450 nm. Cell viability (%) was calculated as OD0. 试验 / OD 空白 ×100%; where OD 试验 The absorbance value of the sample group or positive control group, OD 空白 The absorbance values ​​are for the blank control group. Results are as follows: Figure 3 As shown.

[0087] Depend on Figure 3 The results showed that the cell survival rate was greater than 70% under different concentrations of recombinant elastin, indicating that the recombinant elastin provided in this application is non-cytotoxic and safe and mild.

[0088] Experimental Example 2 This experimental example uses the recombinant elastin provided in Example 4 to conduct cell experiments. The specific method is as follows: Recombinant elastin samples were prepared in serum-free 2% FBS medium at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL. Human skin fibroblast BJ cell lines in 6-well plates were grown to approximately 70% abundance, then the medium was replaced with 2% FBS medium and cultured for 24 h. After starvation, the medium was replaced with the aforementioned medium containing different concentrations of recombinant elastin, and the treatment was repeated for 24 h, serving as the experimental group. The 2% FBS medium served as the control group. RNA was extracted from the cells using TRIzol reagent, and after determining the RNA concentration, the mRNA was reverse transcribed into cDNA. This cDNA was used as a template for quantitative real-time PCR to detect the gene expression levels of recombinant elastin, specifically detecting the expression levels of type I collagen (COL1 gene) and type III collagen (COL3 gene). The following calculations were made: Type I collagen synthesis = COL1 gene expression level in the experimental group / COL1 gene expression level in the control group; Type III collagen synthesis = COL3 gene expression level in the experimental group / COL3 gene expression level in the control group. Results are as follows: Figure 4 As shown.

[0089] Depend on Figure 4 The results showed that recombinant elastin at different concentrations could promote the synthesis of both type I and type III collagen, and even at a low concentration of 50 μg / mL, it could promote the synthesis of both type I and type III collagen. This indicates that the recombinant elastin provided in this application can promote collagen synthesis, and its effective concentration is low, meaning it has high biological activity. Even at a low concentration, it has the effects of replenishing skin collagen, anti-aging, and repairing the skin barrier.

[0090] Experimental Example 3 This experimental example tested the recombinant elastin provided in Example 4 for its anti-aging effects and skin barrier repair efficacy. The specific method was as follows: (1) Preparation of nematode culture medium and reagents: ① 1 M potassium phosphate buffer: Add 108.39 g of KH2PO4 and 35.69 g of K2HPO4 to water to 1 L, and adjust the pH to 6.0; ②M9 buffer: 6 g Na2HPO4, 3 g KH2PO4, 5 g NaCl and 0.25 g MgSO4·7H2O, add water to 1 L, sterilize at 121℃ for 15 min, and prepare fresh before use; ③LB liquid medium: Prepare a final concentration of 10 g / L tryptone, 10 g / L sodium chloride, and 5 g / L yeast extract with distilled water, adjust the pH to 7.0 with 1 M sodium hydroxide solution, and sterilize at 121℃ for 15 min; ④ 1 L Nematode Growth Medium (NGM): Dissolve 3 g NaCl, 17 g Agar, 2.5 g Tryptone, and 0.2 g Streptomycin in 975 mL of distilled water. Shake well, sterilize at 121℃ for 30 min, and keep warm at 80℃ for 15 min. Add the following sterilized solutions (cholesterol is filtered for sterilization, and the remaining solutions are sterilized at high temperature): 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, 1 mL of 5 mg / mL cholesterol (solvent is ethanol), and 25 mL of 1 M potassium phosphate buffer. ⑤ Liquefaction buffer: Dissolve 0.1 g of NaOH and 1.3 mL of NaClO in 4 mL of distilled water and mix thoroughly (prepare fresh before use); (2) Basic operation methods of nematodes ① Uracil synthesis defective Escherichia coli OP50 ( E. coli Cultivation of OP50 Pick E. coli The OP50 strain was streaked on LB agar plates, and a single colony was picked and placed in 10 mL of LB liquid medium. The culture was carried out at 37°C and 200 rpm for 12 h with shaking until OD600=0.4. The culture was then used to inoculate NGM-fed normal nematodes. ② E. coli OP50 coating Add an appropriate amount to each NGM plate E. coli OP50 bacterial suspension (generally 100 μL for 60 mm diameter plates) is evenly spread on NGM plates using a sterile spreader or the bottom of a glass test tube. Note that the edge of the bacterial suspension should be about 0.5 cm away from the edge of the plate. The NGM plates with bacteria spread should be left overnight at room temperature (21-25℃) and then placed in a cold room or a 4℃ refrigerator for later use. ③ Nematode synchronization Wash young adult wild-type Caenorhabditis elegans (the Bridtol strain N2) on NGM plates twice with 1 mL of M9 buffer, then transfer to a sterile 2 mL centrifuge tube. Add 1 mL of freshly prepared lysis buffer, vortex thoroughly for 3-5 min, centrifuge at 3000 rpm for 1 min, discard the supernatant, wash the nematodes again with 1 mL of M9 buffer, and centrifuge twice under the same conditions. Discard the supernatant, leaving 0.3-0.4 mL of buffer containing nematode eggs. Gently pipette to mix the eggs, and drop approximately 100 μL of the buffer containing eggs onto the NGM plate with bacteria spread close to the surface. E. coli In the sterile area of ​​OP50, after approximately 48 hours, the fertilized nematode eggs basically developed into L4 stage larvae, completing synchronization and yielding L4 stage larvae of wild-type N2 nematodes; BE93 ( dpy-2 The same procedure was performed on young adult Nematode elegans strains to obtain BE93 ( dpy-2 L4 stage larvae of *Nematodea elegans*; (3) Preparation of NGM containing recombinant elastin: Will E. coli The OP50 bacterial suspension and the recombinant elastin provided in Example 4 were mixed thoroughly (the concentrations of the recombinant elastin were 50 μg / mL, 100 μg / mL, and 200 μg / mL). Using a pipette, 150 μL of the mixed bacterial suspension was gently pipetted into the center of each NGM plate as the sample group. For the blank group, 150 μL of the blank group was pipetted... E. coli The OP50 bacterial suspension was evenly spread, and the NGM plates with bacteria were dried in the dark and then sealed and stored at 4°C for later use. (4) Anti-aging lifespan testing: L4 stage larvae of wild-type N2 *C. elegans* were selected and transferred to the surface of the culture media in the control and sample groups. Approximately 55 larvae were placed in each plate, with three replicates per group. The survival time of the nematodes was calculated from the time of transfer, and the day of transfer was recorded as the actual lifespan. During the experiment, in the early stage (D4-D10), nematodes were transferred daily to new culture media in the control and sample groups to differentiate parent nematodes and ensure sufficient bacterial suspension. In the middle and late stages, nematodes were transferred every one to two days to ensure sufficient bacterial suspension and prevent contamination. The number of surviving and dying nematodes was recorded throughout the experiment. Nematodes that crawled out of the petri dish wall and naturally disappeared, or burrowed into the agar, were recorded as escapes. Survival curves were plotted using Graph Pad Prism 8 software (0 represents nematode escape, 1 represents nematode death). Figure 5 As shown, the log-rank test was used to analyze the significance of the survival curve. p <0.05 indicates a significant difference. Mean lifespan data for each group are expressed as mean ± standard deviation (X ± SD). t-test analysis was used to analyze differences between groups. p <0.05 was considered statistically significant. The results are shown in Table 3. Life extension rate (%) = (average lifespan of the experimental group - average lifespan of the control group) / average lifespan of the control group × 100%.

[0091] Table 3 Anti-aging lifespan

[0092] As shown in Table 3, the average lifespan of nematodes in the recombinant elastin sample group was longer than that in the control group, and this lifespan increased with increasing recombinant elastin concentration. This indicates that the recombinant elastin provided in this application has high biological activity and an effective concentration, exhibiting anti-aging effects even at low concentrations.

[0093] (5) Skin barrier repair test: Pick BE93( dpy-2 L4 stage larvae of *C. elegans* were placed on the surface of the culture medium in the sample group and cultured for 72 h. The nematodes were then washed into centrifuge tubes with M9 buffer and allowed to settle naturally. The larvae were washed twice with M9 buffer to remove as much supernatant as possible, yielding a nematode pellet. Hoechst 33258 stock solution (Sigma 94403) was diluted with M9 buffer to a working solution of 1 μg / mL. 0.8 mL of the Hoechst 33258 working solution was added to the nematode pellet, and the mixture was incubated at room temperature in the dark for 30 minutes by rotation. The nematodes settled naturally and were washed twice rapidly with M9 buffer, retaining a small amount of M9. A small amount of the nematode suspension was dropped onto a clean glass slide, covered with a coverslip, and immediately observed under a fluorescence microscope to examine the nematode heads. Fluorescence images were captured under a DAPY filter. Figure 6As shown; nematodes with ≥15 cell nuclei stained in their head were considered to have osmotic defects; BE93 (not cultured on the surface of the sample group's culture medium) was used. dpy-2 L4 stage larvae of *C. elegans* were used as the control group; each treatment group contained at least 60 worms, and the proportion of nematodes exhibiting "permeability defects," i.e., the staining positivity rate, was calculated for each group. The results are shown in Table 4.

[0094] Table 4. Staining Positive Rate

[0095] As shown in Table 4, the staining positivity rate of nematodes in the recombinant elastin sample group was low, and decreased with increasing recombinant elastin concentration. This indicates that the recombinant elastin provided in this application has high biological activity and an effective concentration, demonstrating its skin barrier repair efficacy even at low concentrations.

[0096] Test Example 4 This experimental example examines the effect of the recombinant elastin provided in Example 4 on skin elasticity using the following specific method: The elastin essence was prepared as shown in Table 5: Table 5 Elastin Serum

[0097] Fifteen people were randomly selected. After cleansing and applying toner every morning and evening, they applied an appropriate amount of elastin serum to their entire face, paying special attention to the eye area and nasolabial folds, and massaged until absorbed. Before application and 15 minutes after application, the skin elasticity R2 value and skin firmness F4 value of the skin where elastin serum No. 1 was applied were tested using a Cutometer MPA580 (Courage & Khazaka, Germany). The results are shown in Tables 6 and 7. Furthermore, before application and 14 days after application, the dermal density of the skin where elastin serum No. 1 was applied was tested using an Ultrasound Standard Probe (Cortex, Denmark). The results are shown in Table 8.

[0098] Table 6 Skin elasticity R2 value

[0099] As shown in Table 6, 15 minutes after applying the elastin serum, the subjects' skin elasticity R2 value increased by 21.42%, indicating that skin elasticity was enhanced 15 minutes after application. This demonstrates that the recombinant elastin provided in this application can replenish skin collagen and improve skin elasticity.

[0100] Table 7 Skin Firmness F4 Value

[0101] As shown in Table 7, 15 minutes after applying the elastin serum, the subjects' skin firmness F4 value decreased by 12.84%, indicating that skin firmness improved 15 minutes after application. This demonstrates that the recombinant elastin provided in this application can replenish skin collagen, combat aging, and improve skin firmness.

[0102] Table 8. Dermal Density of Skin

[0103] As shown in Table 8, after 14 days of applying the elastin essence, the dermal density of the subjects increased by 4.11%, indicating that the collagen content in the skin increased after 14 days of application. This demonstrates that the recombinant elastin provided in this application can replenish skin collagen and combat aging.

[0104] Experimental Example 5 This experiment tested the yield of recombinant elastin after desalting provided in Examples 4-5 and Comparative Example 1; and tested the yield of recombinant elastin before desalting provided in Examples 6-8 and Comparative Example 2. The results are shown in Table 9.

[0105] Table 9. Yield of recombinant elastin

[0106] As shown in Table 9, using a buffer solution containing 4-10 M urea to resuspend and lyse the recombinant engineered cells isolated from the fermentation product can effectively improve the purification effect and increase the yield and purity of recombinant elastin. At the same time, using a buffer solution containing 100-150 mM sodium chloride to desalt the purified solution can effectively improve the desalting recovery rate, thereby further improving the yield and purity of recombinant elastin.

[0107] Experimental Example 6 In this experiment, the recombinant elastin raw materials provided in Examples 9-20 and Comparative Examples 3-4 were stored at -18°C, 4°C, and 22°C for 3 months, respectively. The stability was then assessed by SDS-PAGE electrophoresis. The results are as follows: Figures 7-10 As shown.

[0108] Depend on Figures 7-10The results showed that in Examples 9-10, 13-14, and 17-18, when the recombinant elastin concentration in the recombinant elastin raw material was 0.2 mg / mL, the recombinant elastin raw material with a glycerol mass percentage of 30%-40% was relatively stable when stored at -18℃ and 4℃. Slight degradation occurred at 22℃, but after adjusting the pH to 5.5, it remained relatively stable at 22℃. In Comparative Example 3, the recombinant elastin raw material with a glycerol mass percentage of 20% degraded after one month of storage at both 4℃ and 22℃, especially at 22℃ where the recombinant elastin was almost completely degraded. Comparative Example 4, based on Comparative Example 3, used sodium dihydrogen phosphate to adjust the pH of the recombinant elastin raw material to 5.5. The recombinant elastin raw material solution became turbid, and protein precipitated. This was because sodium dihydrogen phosphate significantly increased the ionic strength in the solution, causing protein salting out. When the recombinant elastin concentration in the recombinant elastin raw material is 0.5 mg / mL, the recombinant elastin raw material with a glycerol mass percentage of 30%~40% shows slight degradation at 4℃, but after adjusting its pH to 5.5, it is relatively stable during storage.

[0109] This application uses 0.2~0.5 mg / mL of the recombinant elastin provided in this application and 30%~40% by mass of glycerol, and adapts it with pentylene glycol to form a recombinant elastin raw material, which has good storage stability and can be stored stably at 4°C; adjusting its pH value to 5~5.5 can further improve the storage stability of the recombinant elastin raw material.

[0110] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A recombinant elastin, characterized in that, The amino acid sequence of the recombinant elastin comprises the amino acid sequence shown in SEQ ID No. 1 or a derived amino acid sequence that has more than 90% identity with the sequence of SEQ ID NO.

1.

2. The recombinant elastin according to claim 1, characterized in that, The amino acid sequence of the recombinant elastin is shown in SEQ ID No.

1.

3. A nucleic acid encoding the recombinant elastin of claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that, The nucleotide sequence of the nucleic acid is shown in SEQ ID No.

2.

5. A recombinant expression vector, characterized in that, It contains the nucleic acid as described in claim 3 or 4.

6. A method for preparing recombinant elastin as described in claim 1 or 2, characterized in that, include: (1) Synthesize a nucleotide sequence encoding the recombinant elastin of claim 1 or 2, and construct a recombinant expression vector containing the nucleotide sequence; (2) Construct recombinant engineered cells containing the recombinant expression vector described in step (1); (3) The recombinant engineered cells described in step (2) are cultured and fermented, and the fermentation product is separated and purified to obtain recombinant elastin; Preferably, the host cell of the recombinant engineered cells is Escherichia coli.

7. The preparation method according to claim 6, characterized in that, The purification process includes: resuspending and lysing the recombinant engineered cells isolated from the fermentation product using a buffer containing 4-10 M urea, and collecting the supernatant; purifying the supernatant using an affinity chromatography column; and desalting the purified solution by chromatography using a buffer containing 100-150 mM sodium chloride.

8. A recombinant elastin raw material, characterized in that, It comprises the recombinant elastin of claim 1 or 2 at a concentration of 0.2 to 0.5 mg / mL, 30% to 40% by mass of glycerol, 4.5% to 5% by mass of pentylene glycol, and the balance being water.

9. The recombinant elastin raw material according to claim 8, characterized in that, The pH value of the recombinant elastin raw material is 5~5.

5.

10. The use of a recombinant elastin as described in claim 1 or 2, or a recombinant elastin raw material as described in claim 8 or 9, in the preparation of cosmetics.

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