A method for extracting and purifying elastin

Abstract

The present invention relates to the field of biotechnology. Specifically, it relates to a method for extracting and purifying elastin, which is characterized in that the method comprises the following steps: (1) constructing a recombinant nucleic acid sequence from an elastin structural unit, a hydrophilic structure, and a transmembrane peptide; (2) introducing the recombinant nucleic acid sequence into a plasmid and then transforming it into Pichia pastoris, culturing Pichia pastoris to appropriate conditions for induced expression; (3) separating and purifying to obtain recombinant elastin. The present invention provides an elastin with significant cell proliferation-promoting, adhesion activity, and promoting elastin synthesis. At the same time, through optimized fermentation culture and purification, finally, highly pure recombinant elastin is obtained. Compared with conventional fermentation means, the present invention can significantly increase the expression level of the above recombinant elastin and has potential application prospects in the fields of biomedical materials and cosmetics, etc.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular, to a method for extracting and purifying elastin. Background Art

[0002] Elastin is an important extracellular matrix protein, mainly composed of elastic fibers, which can provide elasticity and flexibility to tissues. Elastin plays an important role in the vascular system, skin and lungs. In vivo, it combines with microfibrils to form elastic fibers, and together with collagen fibers, endows tissues with elasticity and tensile strength. Elastin is rich in non-polar amino acids such as glycine, proline, alanine, leucine and valine, and usually forms a flexible and stable structure with a large number of repeated sequences composed of 3-9 amino acids.

[0003] There are many methods for extracting elastin, each with its own advantages and disadvantages. In 1969, Bornstein and Ross first extracted with 5mol / L guanidine hydrochloride, then hydrolyzed other non-elastin components with collagenase, and finally reduced disulfide bonds to isolate and extract elastin. However, most of the existing technologies obtain highly efficient elastin by constructing recombinant elastin.

[0004] The prior art CN 118005770 B discloses a highly active human recombinant elastin. The recombinant human elastin is composed of 9-peptide VAPGTIGTG as a structural unit connected by a hydrophilic structure AAAKSAAKAA. This elastin has a structure and properties similar to those of natural elastin, and is highly expressed through a prokaryotic expression system. After separation and purification, a highly pure and highly active recombinant elastin is obtained. This highly active human recombinant elastin has broad application prospects in skin repair, cell adhesion and cell proliferation.

[0005] The prior art CN 118388666 B discloses a fusion protein. By combining an elastin polypeptide with a collagen polypeptide, and using an amino acid sequence designed with an elastin fragment rich in the VAPGXG motif and the core fragment of the α2 chain of type I collagen, it plays an important role in promoting fibroblast proliferation, inhibiting metalloproteinase activity, and enhancing the expression of collagen and elastin; using genetic engineering technology, the efficient expression and purification of the fusion protein are achieved in an Escherichia coli expression system, significantly improving production efficiency, reducing costs and having strong scalability; the addition of a transdermal peptide TAT sequence at the N-terminus of the produced fusion protein enhances its penetration ability in the skin, thereby improving the bioavailability and efficacy of the present invention in the deep layer of the skin.

[0006] The prior art CN 118480554A discloses that the recombinant elastin-encoding gene and recombinant tropoelastin-encoding gene provided by the present invention can be used to express recombinant elastin and recombinant tropoelastin, with considerable protein expression levels, easy purification, and better stability during storage.

[0007] However, the technical solutions of the above prior art are limited, and the structures and effects of its recombinant proteins vary, unable to meet the growing demands. Therefore, there is an urgent need to provide new recombinant elastin and corresponding methods for extracting and purifying elastin. Summary of the Invention

[0008] The present invention first provides a method for extracting and purifying elastin, the method comprising the following steps:

[0009] (1) Construct a recombinant nucleic acid sequence from an elastin structural unit, a hydrophilic structure, and a transmembrane peptide;

[0010] (2) Introduce the recombinant nucleic acid sequence into a plasmid and then transform it into Pichia pastoris, and culture Pichia pastoris under appropriate conditions for induced expression;

[0011] (3) Isolate and purify to obtain recombinant elastin.

[0012] In certain embodiments, the amino acid sequence of the elastin structural unit is as shown in SEQ ID NO.1

[0013] as shown.

[0014] In certain embodiments, the elastin structural unit repeats 3 - 10 times.

[0015] In certain embodiments, the amino acid sequence of the hydrophilic structure is as shown in SEQ ID NO.2.

[0016] In certain embodiments, the amino acid sequence of the transmembrane peptide is as shown in SEQ ID NO.3.

[0017] In certain embodiments, the nucleotide sequence of the recombinant elastin comprises polypeptides as shown in SEQ ID NOs.4 - 7.

[0018] In certain embodiments, the amino acid sequence of the recombinant elastin is as shown in SEQ ID NOs.4 - 7.

[0019] The present invention also provides a recombinant elastin, which is prepared by the above method.

[0020] The present invention also provides a biomaterial, which can encode the above recombinant elastin or comprises a nucleic acid capable of encoding the above recombinant elastin;

[0021] Optionally, the biological material is one or more of nucleic acids, vectors, and cells.

[0022] Finally, the present invention provides an application of the above-mentioned recombinant elastin in the preparation of materials for promoting cell proliferation, promoting cell adhesion, or promoting skin repair.

[0023] Compared with the prior art, the present invention has at least the following beneficial effects:

[0024] Compared with the prior art, in the present invention, through screening and modification from the elastin sequence, the structure, repeat unit, and repeat times of the recombinant protein are optimized, and a recombinant elastin with significantly enhanced cell proliferation, adhesion activity, and promotion of elastin synthesis is obtained. The gene is expressed using Pichia pastoris, constructed into a Pichia pastoris secretion expression plasmid through homologous recombination, and a recombinant Pichia pastoris engineering strain with high expression of recombinant elastin is obtained through electrotransformation screening. After optimized fermentation culture and purification, high-purity recombinant elastin is finally obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The tertiary structure model of recombinant elastin 1 predicted by SWISS-MODEL;

[0026] Figure 2 The cell proliferation-promoting activity of recombinant elastin;

[0027] Figure 3 The cell adhesion-promoting activity of recombinant elastin;

[0028] Figure 4 The QPCR result diagram of recombinant elastin promoting elastin synthesis. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] To make the technical problems, technical solutions, and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0030] The nucleotide sequence of the elastin structural unit, (VPGKG) 5 (SEQ ID NO.1);

[0031] The elastin structural unit is repeated n times, where n is 3 - 10;

[0032] The amino acid sequence of the hydrophilic structure, AAAKSAAKAA (SEQ ID NO.2);

[0033] The amino acid sequence of the transmembrane peptide, RKKRRQRRRPP (SEQ ID NO.3);

[0034] The structure of the recombinant elastin in the experimental group is:

[0035] RKKRRQRRRPP-[(VPGKG) 5 -AAAKSAAKAA-(VPGKG) 5 n-VGRVHHHHHH*。

[0036] The amino acid sequences of the recombinant elastin in the experimental group are shown in SEQ ID NO.4-7, and the corresponding n values are 3, 5, 7, and 9 respectively.

[0037] SEQ ID NO.4:

[0038] RKKRRQRRRPPVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK

[0039] GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA

[0040] AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG

[0041] VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVGRVHHHHHH*;

[0042] SEQ ID NO.5:

[0043] RKKRRQRRRPPVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK

[0044] GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA

[0045] AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG

[0046] VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPG

[0047] KGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGV

[0048] PGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVGRVHHHHHH*;

[0049] SEQ ID NO.6:

[0050] RKKRRQRRRPPVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK

[0051] GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA

[0052] AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG

[0053] VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPG

[0054] KGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGV

[0055] PGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK

[0056] GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA

[0057] AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG

[0058] VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVGRVHHHHHH*;

[0059] SEQ ID NO.7:

[0060] RKKRRQRRRPPVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK

[0061] GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA

[0062] AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG

[0063] VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPG

[0064] KGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGV

[0065] PGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK

[0066] GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA

[0067] AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG

[0068] VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPG

[0069] KGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGV

[0070] PGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVGRVHHHHHH*;

[0071] The recombinant elastin structure of Comparative Example 1 is:

[0072] RKKRRQRRRPP - [(VPGKG) 5 -(VPGKG) 5 n - VGRVHHHHHH*.

[0073] The amino acid sequence of the recombinant elastin of Comparative Example 1 is shown in SEQ ID NO.8, and the corresponding n is 5.

[0074] SEQ ID NO.8:

[0075] RKKRRQRRRPPVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGK

[0076] GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVP

[0077] GKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG

[0078] VPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPG

[0079] KGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVGRVHHHHHH*;

[0080] Example 1: Preparation of Recombinant Elastin

[0081] Gene fragments were synthesized according to the amino acid sequence shown in Seq ID NO.4, and the synthesized gene fragments were inserted between the restriction enzyme sites EcoRⅠ and NotⅠ on the pPIC9K plasmid to obtain a recombinant plasmid;

[0082] The recombinant plasmid was mixed with the GS115 competent cells. After ice-bathing, electrotransformation was carried out, and then sorbitol solution was added. After mixing, it was transferred to a sterile EP tube. After static incubation at 30 °C for 1 h - 2 h, it was spread on an MD solid plate and then incubated in an inverted manner at 30 °C for 2 d - 5 d until single colonies grew; The His+ transformants on the MD solid plate were scraped off, diluted, and then spread on a YPD plate containing G418. After incubation in an inverted manner at 30 °C until single colonies appeared, the single colonies on the YPD plate were selected and transferred to a 96-well culture plate containing YPD medium and continued to be cultured at 30 °C to screen for recombinant yeast engineering bacteria;

[0083] The selected recombinant yeast engineering bacteria were inoculated into BMGY induction medium and cultured until the OD600nm absorbance value reached 2.0 - 6.0; After centrifugation at 3000 rpm for 10 min, the cells were collected, re-inoculated into BMGY induction medium to make the OD600nm absorbance value reach 2.0, and continued to be cultured at 30 °C and 220 rpm, and 0.5% methanol was added every 24 h. After induction, the induced supernatant was collected by centrifugation;

[0084] The induced supernatant was purified through a cation exchange chromatography column, and the protein corresponding to the elution peak was eluted and collected, thus obtaining human recombinant elastin 1 with a purity of 98.45%. The three-dimensional structure model of recombinant elastin 1 was predicted by SWISS-MODEL,Figure 1 The results showed that recombinant elastin 1 had a dynamic balance between random coil and ordered structure, mimicking the properties of natural elastin. Theoretically, it could be expected that recombinant elastins 2-4 would also have properties similar to natural elastin, and subsequent experiments could be carried out.

[0085] Example 2: Preparation of Recombinant Elastin

[0086] Gene fragments were synthesized according to the amino acid sequence shown in Seq ID NO.5, and the synthesized gene fragments were inserted between the restriction enzyme sites EcoRⅠ and NotⅠ on the pPIC9K plasmid to obtain a recombinant plasmid;

[0087] The recombinant plasmid was mixed with the GS115 competent cells. After ice bath, electrotransformation was carried out, and then sorbitol solution was added. After mixing, it was transferred to a sterile EP tube. After static incubation at 30 °C for 1 h - 2 h, it was spread on an MD solid plate and then inverted and cultured at 30 °C for 2 d - 5 d until single colonies grew; The His+ transformants on the MD solid plate were scraped off, diluted, and then spread on a YPD plate containing G418. After inverted culture at 30 °C until single colonies appeared, the single colonies on the YPD plate were selected and transferred to a 96-well culture plate containing YPD medium and continued to be cultured at 30 °C to screen for recombinant yeast engineering bacteria;

[0088] The selected recombinant yeast engineering bacteria were inoculated into BMGY induction medium and cultured until the OD600nm absorbance value reached 2.0 - 6.0; After centrifugation at 3000 rpm for 10 min, the cells were collected, re-inoculated into BMGY induction medium to make the OD600nm absorbance value reach 2.0, and continued to be cultured at 30 °C and 220 rpm, and 0.5% methanol was added every 24 h. After induction, the induced supernatant was collected by centrifugation;

[0089] The induced supernatant was purified by a cation exchange chromatography column, and the protein corresponding to the elution peak was eluted and collected, and human recombinant elastin 2 with a purity of 98.73% was obtained.

[0090] Example 3: Preparation of Recombinant Elastin

[0091] Gene fragments were synthesized according to the amino acid sequence shown in Seq ID NO.6, and the synthesized gene fragments were inserted between the restriction enzyme sites EcoRⅠ and NotⅠ on the pPIC9K plasmid to obtain a recombinant plasmid;

[0092] Mix the recombinant plasmid with the GS115 competent cells. After ice-bathing, perform electroporation, then add sorbitol solution, mix well, transfer to a sterile EP tube, incubate statically at 30°C for 1 h - 2 h, coat on an MD solid plate, and then incubate in an inverted position at 30°C for 2 d - 5 d until single colonies grow; scrape the His+ transformants on the MD solid plate, dilute them, coat on a YPD plate containing G418, incubate in an inverted position at 30°C until single colonies appear, and then select the single colonies on the YPD plate and transfer them to a 96-well culture plate with YPD medium, and continue to culture at 30°C to screen for recombinant yeast engineering bacteria;

[0093] Select the recombinant yeast engineering bacteria and inoculate them into BMGY induction medium, culture until the absorbance value at OD600nm reaches 2.0 - 6.0; centrifuge at 3000 rpm for 10 min, collect the thalli, re-inoculate them into BMGY induction medium to make the absorbance value at OD600nm reach 2.0, and continue to culture under the conditions of 30°C and 220 rpm, and add 0.5% methanol every 24 h. After induction, centrifuge to collect the induced supernatant;

[0094] Purify the induced supernatant through a cation exchange chromatography column, elute and collect the protein corresponding to the elution peak, and thus obtain human recombinant elastin 3 with a purity of 97.65%.

[0095] Example 4: Preparation of recombinant elastin

[0096] Synthesize a gene fragment according to the amino acid sequence shown in Seq ID NO.7, and insert the synthesized gene fragment between the restriction enzyme sites EcoRⅠ and NotⅠ on the pPIC9K plasmid to obtain a recombinant plasmid;

[0097] Mix the recombinant plasmid with the GS115 competent cells. After ice-bathing, perform electroporation, then add sorbitol solution, mix well, transfer to a sterile EP tube, incubate statically at 30°C for 1 h - 2 h, coat on an MD solid plate, and then incubate in an inverted position at 30°C for 2 d - 5 d until single colonies grow; scrape the His+ transformants on the MD solid plate, dilute them, coat on a YPD plate containing G418, incubate in an inverted position at 30°C until single colonies appear, and then select the single colonies on the YPD plate and transfer them to a 96-well culture plate with YPD medium, and continue to culture at 30°C to screen for recombinant yeast engineering bacteria;

[0098] Select the recombinant yeast engineering bacteria and inoculate them into the BMGY induction medium, and culture until the absorbance value at OD600nm reaches 2.0 - 6.0; after centrifuging at 3000 rpm for 10 min, collect the bacterial cells, re-inoculate them into the BMGY induction medium to make the absorbance value at OD600nm reach 2.0, and continue to culture under the conditions of 30 °C and 220 rpm, and add 0.5% methanol every 24 h. After induction, centrifuge to collect the induced supernatant;

[0099] Purify the induced supernatant through a cation exchange chromatography column, elute and collect the protein corresponding to the elution peak, and thus obtain human recombinant elastin 4 with a purity of 98.16%.

[0100] Comparative Example 1: Preparation of recombinant elastin

[0101] Synthesize gene fragments according to the amino acid sequence shown in Seq ID NO.8, and insert the synthesized gene fragments between the restriction enzyme sites EcoRⅠ and NotⅠ on the pPIC9K plasmid to obtain a recombinant plasmid;

[0102] Mix the recombinant plasmid with the GS115 competent cells, after ice bath, perform electrotransformation, then add sorbitol solution, mix well, transfer to a sterile EP tube, after static incubation at 30 °C for 1 h - 2 h, coat on an MD solid plate, and then invert and culture at 30 °C for 2 d - 5 d until single colonies grow; scrape the His+ transformants on the MD solid plate, dilute them, and coat on a YPD plate containing G418, after inverting and culturing at 30 °C until single colonies appear, then select the single colonies on the YPD plate and transfer them to a 96-well culture plate of YPD medium, and continue to culture at 30 °C to screen for recombinant yeast engineering bacteria;

[0103] Select the recombinant yeast engineering bacteria and inoculate them into the BMGY induction medium, and culture until the absorbance value at OD600nm reaches 2.0 - 6.0; after centrifuging at 3000 rpm for 10 min, collect the bacterial cells, re-inoculate them into the BMGY induction medium to make the absorbance value at OD600nm reach 2.0, and continue to culture under the conditions of 30 °C and 220 rpm, and add 0.5% methanol every 24 h. After induction, centrifuge to collect the induced supernatant;

[0104] Purify the induced supernatant through a cation exchange chromatography column, elute and collect the protein corresponding to the elution peak, and thus obtain human recombinant elastin 5 with a purity of 86.75%.

[0105] Comparative Example 2: Preparation of recombinant elastin

[0106] Prepare human recombinant elastin with high biological activity according to the method of Example 1 described in CN118005770A.

[0107] Comparative Example 3: Preparation of Recombinant Elastin

[0108] Prepare a polypeptide that promotes the regeneration of skin collagen and elastin according to the method described in CN 118388666A. Its amino acid sequence is as follows: MYGRKKRRQRRRVAPGVGVAPGVGVAPGVGS VAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSGLMGPRGLPGSPGNIGPAGKEGPVGLPGIDGRPGPIGPAGARGEPGNIGFPGPKGPTGDPGKNGDKGHAGLAGARGAPGPDGNNGAQGPPGPQGVQGGKGEQGPPGPPGFQGLPGPSGPAGEVGKPGERGLHGEFRHHHHHH (SEQ ID NO.9).

[0109] Example 5: Cell Proliferation Promotion Experiment of Recombinant Elastin

[0110] Inoculate primary skin fibroblasts into DMEM medium and culture them at 37°C until the logarithmic growth phase, and then inoculate them into a 96-well cell culture plate (cell concentration is 1.0×10 5 cells / ml - 5.0×10 5 cells / ml, 100 μL per well). Dilute the protein solution to 1 mg / mL with PBS buffer, and then add it to the 96-well cell culture plate in proportion so that the final concentration of the recombinant elastin in Examples 1 - 4 and Comparative Examples 1 - 3 is 50 ppm. The sample added with PBS buffer is used as the blank control group. Each concentration is treated with 3 replicate wells. After culturing at 37°C for 48 hours, add thiazolyl blue solution (MTT) with a final concentration of 5 mg / ml (10 μL / well), and continue to culture at 37°C for 4 hours. Discard the medium, add 100 μL of dimethyl sulfoxide (DMSO) to each well, and then place it on a shaker and shake slowly for 10 minutes. Finally, use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance of each well at a wavelength of 490 nm. The results are shown in Figure 3 .

[0111] The results are as Figure 2 shown. The cell proliferation rates of Examples 1 - 4 and Comparative Examples 1 - 3 are significantly higher than those of the control group, and the effect of Example 2 is the most significant (p < 0.001); it shows that Example 2 has an excellent effect on promoting cell proliferation, has good anti-aging and repair capabilities, and is an effective biomedical material.

[0112] Example 6: Promoting cell adhesion experiment of recombinant elastin

[0113] Take cells in the logarithmic growth phase (HaCaT cells) and inoculate them into a 6-well plate at 1×10 6 cells / well, and incubate in an incubator at 37°C and 5% CO 2 for 24 h. Dilute the recombinant elastin solution with DMEM. Subsequently, add the recombinant protein sample solutions of Examples 1-4 and Comparative Examples 1-3 to the sample group (the final concentration of recombinant elastin is 50 ppm), add serum-free medium to the blank control group, and culture in an incubator at 37°C and 5% CO 2 for 24 h. According to the instructions of the cell adhesion kit (Shanghai Baibo Biotech Co., Ltd.), add 100 μL of coating solution to each well of a new 96-well plate, place it in the refrigerator at 4°C for 24 h. After the cells to be measured are processed, digest them with trypsin, wash them with PBS, and then resuspend them with the corresponding medium to prepare a cell suspension. Inoculate the coated 96-well plate at 5×10 4 cells / well, set 6 replicates, and culture in an incubator at 37°C and 5% CO 2 for 1 h. Aspirate the medium, wash 3 times, and then add 100 μL of fresh medium to each well. Add 10 μL of cell staining solution B to each well and incubate at 37°C for 1 h. Measure the OD value at 450 nm. The calculation formula for cell adhesion rate is as follows:

[0114]

[0115] Where: V(%)——cell adhesion rate, %; OD sample——absorbance of the reaction system containing the sample to be measured; OD blank control——absorbance of the empty plate without any substance; OD cell control——absorbance of the reaction system without the sample to be measured.

[0116] The results are as Figure 3 shown. The cell adhesion rates of Examples 1-4 and Comparative Examples 1-3 are significantly higher than those of the control group, and the effect of Example 2 is the most significant (p < 0.001); it shows that Example 2 has an excellent effect on promoting cell adhesion, has good anti-aging and repair abilities, and is an effective biomedical material.

[0117] Example 7: Detection of the ability of recombinant elastin to promote elastin synthesis - QPCR

[0118] Inoculate HaCaT cells in the logarithmic growth phase into a 6-well plate at 1×10 6 cells / well, and culture at 37°C and 5% CO 2Incubate in an incubator for 24 h. Subsequently, take out the 6-well plate. Add the recombinant protein sample solutions of Examples 1-4 and Comparative Examples 1-3 (the final concentration of recombinant elastin is 20 ppm) to the sample group, add serum-free medium to the blank control group, and add the corresponding positive drug solution to the positive control group. Incubate at 37°C with 5% CO 2 Incubate in an incubator for 24 h. Subsequently, discard the supernatant, wash twice with PBS, and collect the cells. Extract RNA from the collected cells according to the method of the RNA extraction kit (TransGen Biotech Co., Ltd., Beijing). After measuring the concentration, perform reverse transcription with 1 μg using the reverse transcription kit (TransGen Biotech Co., Ltd., Beijing) to obtain cDNA. Subsequently, perform RT-qPCR on the cDNA according to the kit steps (Vazyme Biotech Co., Ltd., Nanjing). Calculate the 2^(-ΔΔCt) value of the test sample, i.e., the relative expression level, and the standard error (SD) of each test sample well to obtain a data table. The results are as Figure 4 .

[0119] The results are as Figure 4 shown. The relative expression levels of elastin (Elastin, ELN) in Examples 1-4 and Comparative Examples 1-3 are significantly higher than those of the control group, and the effect of Example 2 is the most significant (p < 0.001); it shows that Example 2 has excellent ability to promote the synthesis and expression of elastin, has good anti-aging and repair ability, and is an effective biomedical material.

[0120] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing elastin, characterized in that: The method comprises the following steps: (1) constructing a nucleic acid sequence encoding recombinant elastin from elastin structural units, hydrophilic structures and membrane-penetrating peptides; (2) introducing the nucleic acid sequence encoding the recombinant elastin into a plasmid and then transforming it into Pichia pastoris, and culturing the Pichia pastoris to appropriate conditions to induce expression; (3) Isolation and purification to obtain recombinant elastin; The amino acid sequence of the recombinant elastin is shown in SEQ ID NO. 4-7.

2. A recombinant elastin, characterized in that: The recombinant elastin is prepared by the method according to claim 1.

3. A biomaterial, characterized in that: It can encode the recombinant elastin protein according to claim 2 or contains a nucleic acid that can encode the recombinant elastin protein according to claim 2.

4. The biomaterial according to claim 3, characterized in that The biological material is one or more of nucleic acid, vector and cell.

5. Use of the recombinant elastin as claimed in claim 2 in the preparation of materials for promoting skin anti-aging and repair.

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