Extraction and purification method of elastin

Abstract

The invention relates to the technical field of biology, in particular to an elastin extraction and purification method which is characterized by comprising the following steps: (1) constructing a recombinant nucleic acid sequence from an elastin structural unit, a hydrophilic structure and a cell-penetrating peptide; (2) introducing the recombinant nucleic acid sequence into plasmids, transforming the plasmids into pichia pastoris, and culturing the pichia pastoris to proper conditions for induced expression; and (3) separating and purifying to obtain the recombinant elastin. The invention provides the elastin which can remarkably promote cell proliferation and adhesion activity and promote elastin synthesis, and the high-purity recombinant elastin is finally obtained through optimized fermentation culture and purification. Compared with a conventional fermentation means, the expression quantity of the recombinant elastin can be remarkably increased, and the recombinant elastin has potential application prospects in the fields of biomedical materials, cosmetics and the like.

Description

Technical Field

[0001] The 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, the main component of which is elastic fiber, which can provide elasticity and flexibility to tissues. Elastin plays an important role in the vascular system, skin and lungs. It combines with microfibrils in the body to form elastic fibers, and together with collagen fibers, it gives tissues elasticity and tensile strength. Elastin is rich in non-polar amino acids such as glycine, proline, alanine, leucine and valine, and usually contains a large number of 3-9 amino acid repeating sequences to form a flexible and stable structure.

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

[0004] Prior art CN 118005770 B discloses a highly active human recombinant elastin protein, wherein the recombinant human elastin protein is composed of 9 peptides VAPGTIGTG as structural units connected by a hydrophilic structure AAAKSAAKAA. The elastin protein has a structure and properties similar to those of natural elastin protein, and is efficiently expressed through a prokaryotic expression system. After separation and purification, a highly active recombinant elastin protein with high purity is obtained. The highly active human recombinant elastin protein has broad application prospects in skin repair, cell adhesion, and cell proliferation.

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

[0006] Prior art CN 118480554A discloses that the recombinant elastin encoding gene and the recombinant elastin-like encoding gene provided by the present invention can be used to express recombinant elastin and recombinant elastin-like proteins, and the protein expression amount is considerable, easy to purify and has better stability during storage.

[0007] However, the technical solutions of the above-mentioned prior art are limited, and the structures and effects of the recombinant proteins are uneven, which cannot meet the growing demand. Therefore, it is urgent to provide new recombinant elastin and corresponding elastin extraction and purification methods. 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) constructing an elastin structural unit, a hydrophilic structure and a membrane-penetrating peptide into a recombinant nucleic acid sequence;

[0010] (2) introducing the recombinant nucleic acid sequence into the plasmid and transforming it into Pichia pastoris, and culturing the Pichia pastoris to appropriate conditions to induce 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.

[0013] shown.

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

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

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

[0017] In certain embodiments, the nucleotide sequence of the recombinant elastin protein comprises the polypeptides shown in SEQ ID NO. 4-7.

[0018] In certain embodiments, the amino acid sequence of the recombinant elastin is shown in SEQ ID NO. 4-7.

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

[0020] The present invention also provides a biomaterial, which can encode the above-mentioned recombinant elastin or contains a nucleic acid that can encode the above-mentioned recombinant elastin;

[0021] Optionally, the biological material is one or more of nucleic acid, vector, and cell.

[0022] Finally, the present invention provides a use of the above-mentioned recombinant elastin in preparing a material for promoting cell proliferation, cell adhesion or skin repair.

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

[0024] Compared with the prior art, the present invention optimizes the structure, repeating unit and number of repetitions of the recombinant protein by screening and modifying the elastin sequence, thereby obtaining an optimized recombinant elastin with significant cell proliferation promotion, adhesion activity and elastin synthesis promotion. The Pichia pastoris expression gene is constructed into a Pichia pastoris secretory expression plasmid by homologous recombination, and a recombinant Pichia pastoris engineered strain highly expressing the recombinant elastin is obtained by electroporation screening. After optimized fermentation culture and purification, a high-purity recombinant elastin is finally obtained. BRIEF DESCRIPTION OF THE DRAWINGS

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

[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 , QPCR results of recombinant elastin promoting elastin synthesis. DETAILED DESCRIPTION

[0029] In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, a detailed description will be given below 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 structural unit of elastin 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 cell-penetrating peptide, RKKRRQRRRPP (SEQ ID NO. 3);

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

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

[0036] The amino acid sequences of the recombinant elastin in the experimental groups 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] GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA

[0045] AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG

[0046] VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPG

[0047] KGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGV

[0048] PGKGVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVGRVHHHHHH*;

[0049] SEQ ID NO.6:

[0050] RKKRRQRRRPPVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK

[0051] GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA

[0052] AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG

[0053] VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPG

[0054] KGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGV

[0055] PGKGVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK

[0056] GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA

[0057] AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG

[0058] VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVGRVHHHHHH*;

[0059] SEQ ID NO.7:

[0060] RKKRRQRRRPPVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK

[0061] GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA

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

[0082] The recombinant plasmid and the GS115 competent cells are mixed, and after ice bath, they are electroporated, and then a sorbitol solution is added, mixed, and then transferred to a sterile EP tube, incubated at 30°C for 1h-2h, and then spread on an MD solid plate, and then inverted at 30°C for 2d-5d until a single colony grows; His+ transformants on the MD solid plate are scraped off and diluted, and then spread on a YPD plate containing G418, and inverted at 30°C until a single colony appears, and then the single colony of the YPD plate is selected and transferred to a 96-well culture plate of YPD medium, and continued to be cultured at 30°C to screen the recombinant yeast engineering bacteria;

[0083] The recombinant yeast engineering bacteria are selected and inoculated into a BMGY induction medium, and cultured until the OD600nm absorbance reaches 2.0-6.0; after centrifugation at 3000rpm for 10min, the bacteria are collected and re-inoculated into the BMGY induction medium until the OD600nm absorbance reaches 2.0, and the culture is continued at 30°C and 220rpm, and 0.5% methanol is added every 24h. After induction, the induction supernatant is collected by centrifugation;

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

[0085] Example 2: Preparation of recombinant elastin

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

[0087] The recombinant plasmid and the GS115 competent cells are mixed, and after ice bath, they are electroporated, and then a sorbitol solution is added, mixed, and then transferred to a sterile EP tube, incubated at 30°C for 1h-2h, and then spread on an MD solid plate, and then inverted at 30°C for 2d-5d until a single colony grows; His+ transformants on the MD solid plate are scraped off and diluted, and then spread on a YPD plate containing G418, and inverted at 30°C until a single colony appears, and then the single colony of the YPD plate is selected and transferred to a 96-well culture plate of YPD medium, and continued to be cultured at 30°C to screen the recombinant yeast engineering bacteria;

[0088] The recombinant yeast engineering bacteria are selected and inoculated into a BMGY induction medium, and cultured until the OD600nm absorbance reaches 2.0-6.0; after centrifugation at 3000rpm for 10min, the bacteria are collected and re-inoculated into the BMGY induction medium until the OD600nm absorbance reaches 2.0, and the culture is continued at 30°C and 220rpm, and 0.5% methanol is added every 24h. After induction, the induction supernatant is 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 to obtain human recombinant elastin 2 with a purity of 98.73%.

[0090] Example 3: Preparation of recombinant elastin

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

[0092] The recombinant plasmid and the GS115 competent cells are mixed, and after ice bath, they are electroporated, and then a sorbitol solution is added, mixed, and then transferred to a sterile EP tube, incubated at 30°C for 1h-2h, and then spread on an MD solid plate, and then inverted at 30°C for 2d-5d until a single colony grows; His+ transformants on the MD solid plate are scraped off and diluted, and then spread on a YPD plate containing G418, and inverted at 30°C until a single colony appears, and then the single colony of the YPD plate is selected and transferred to a 96-well culture plate of YPD medium, and continued to be cultured at 30°C to screen the recombinant yeast engineering bacteria;

[0093] The recombinant yeast engineering bacteria are selected and inoculated into a BMGY induction medium, and cultured until the OD600nm absorbance reaches 2.0-6.0; after centrifugation at 3000rpm for 10min, the bacteria are collected and re-inoculated into the BMGY induction medium until the OD600nm absorbance reaches 2.0, and the culture is continued at 30°C and 220rpm, and 0.5% methanol is added every 24h. After induction, the induction supernatant is collected by centrifugation;

[0094] The induced supernatant was purified by a cation exchange chromatography column, and the protein corresponding to the elution peak was eluted and collected to obtain human recombinant elastin 3 with a purity of 97.65%.

[0095] Example 4: Preparation of recombinant elastin

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

[0097] The recombinant plasmid and the GS115 competent cells are mixed, and after ice bath, they are electroporated, and then a sorbitol solution is added, mixed, and then transferred to a sterile EP tube, incubated at 30°C for 1h-2h, and then spread on an MD solid plate, and then inverted at 30°C for 2d-5d until a single colony grows; His+ transformants on the MD solid plate are scraped off and diluted, and then spread on a YPD plate containing G418, and inverted at 30°C until a single colony appears, and then the single colony of the YPD plate is selected and transferred to a 96-well culture plate of YPD medium, and continued to be cultured at 30°C to screen the recombinant yeast engineering bacteria;

[0098] The recombinant yeast engineering bacteria are selected and inoculated into a BMGY induction medium, and cultured until the OD600nm absorbance reaches 2.0-6.0; after centrifugation at 3000rpm for 10min, the bacteria are collected and re-inoculated into the BMGY induction medium until the OD600nm absorbance reaches 2.0, and the culture is continued at 30°C and 220rpm, and 0.5% methanol is added every 24h. After induction, the induction supernatant is collected by centrifugation;

[0099] The induced supernatant was purified by a cation exchange chromatography column, and the protein corresponding to the elution peak was eluted and collected to obtain human recombinant elastin 4 with a purity of 98.16%.

[0100] Comparative Example 1: Preparation of recombinant elastin

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

[0102] The recombinant plasmid and the GS115 competent cells are mixed, and after ice bath, they are electroporated, and then a sorbitol solution is added, mixed, and then transferred to a sterile EP tube, incubated at 30°C for 1h-2h, and then spread on an MD solid plate, and then inverted at 30°C for 2d-5d until a single colony grows; His+ transformants on the MD solid plate are scraped off and diluted, and then spread on a YPD plate containing G418, and inverted at 30°C until a single colony appears, and then the single colony of the YPD plate is selected and transferred to a 96-well culture plate of YPD medium, and continued to be cultured at 30°C to screen the recombinant yeast engineering bacteria;

[0103] The recombinant yeast engineering bacteria are selected and inoculated into a BMGY induction medium, and cultured until the OD600nm absorbance reaches 2.0-6.0; after centrifugation at 3000rpm for 10min, the bacteria are collected and re-inoculated into the BMGY induction medium until the OD600nm absorbance reaches 2.0, and the culture is continued at 30°C and 220rpm, and 0.5% methanol is added every 24h. After induction, the induction supernatant is collected by centrifugation;

[0104] The induced supernatant was purified by a cation exchange chromatography column, and the protein corresponding to the elution peak was eluted and collected to obtain human recombinant elastin 5 with a purity of 86.75%.

[0105] Comparative Example 2: Preparation of recombinant elastin

[0106] According to the method of Example 1 described in CN118005770A, human recombinant elastin encoding high biological activity was prepared.

[0107] Comparative Example 3: Preparation of recombinant elastin

[0108] According to the method described in CN 118388666A, a polypeptide for promoting the regeneration of skin collagen and elastin was prepared, and its amino acid sequence was as follows: MYGRKKRRQRRRVAPGVGVAPGVGVAPGVGS VAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSGLMGPRGLPGSPGNIGPAGKEGPVGLPGIDGRPGPIGPAGARGEPGNIGFPGPKGPTGDPGKNGDKGHAGLAGARGAPGPDGNNGAQGPPGPQGVQGGKGEQGPPGPPGFQGLPGPSGPAGEVGKPGERGLHGEFRHHHHHH (SEQ ID NO.9).

[0109] Example 5: Experiment on promoting cell proliferation by recombinant elastin

[0110] Primary skin fibroblasts were inoculated into DMEM medium and cultured at 37°C until the logarithmic growth phase, and then inoculated into 96-well cell culture plates (cell concentration was 1.0×10 5 cells / ml-5.0×10 5 cells / ml, 100 μL per well). The protein solution was diluted to 1 mg / mL with PBS buffer, and then added to a 96-well cell culture plate in proportion so that the final concentration of the recombinant elastin in Examples 1 to 4 and Comparative Examples 1-3 was 50 ppm, and the sample added with PBS buffer was used as the blank control group. Three replicate wells were treated with each concentration. After culturing at 37°C for 48 hours, a thiazolyl blue solution (MTT, 10 μL / well) with a final concentration of 5 mg / mL was added, and the culture was continued at 37°C for 4 hours. The culture medium was discarded, 100 μL of dimethyl sulfoxide (DMSO) was added to each well, and then the well was placed on a shaker and slowly shaken for 10 minutes. Finally, the absorbance of each well was measured using an enzyme reader at a wavelength of 490 nm. The results are shown in FIG. Figure 3 .

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

[0112] Example 6: Experiment on promoting cell adhesion by recombinant elastin

[0113] Take cells in logarithmic growth phase (HaCaT cells) and calculate 1*10 6 Each well was inoculated in a 6-well plate and incubated in an incubator at 37°C and 5% CO2 for 24 hours. The recombinant elastin solution was diluted with DMEM. Subsequently, the recombinant protein sample solutions of Examples 1 to 4 and Comparative Examples 1-3 were added to the sample group (the final concentration of the recombinant elastin was 50 ppm), and serum-free culture medium was added to the blank control group. The plates were cultured in an incubator at 37°C and 5% CO2 for 24 hours. According to the instructions of the cell adhesion kit (Shanghai Beibo Biotechnology Co., Ltd.), 100 μL of coating solution was added to each well of a new 96-well plate and placed in a refrigerator at 4°C for 24 hours. After the cells to be tested were treated, they were digested with trypsin, washed with PBS, and then resuspended with the corresponding culture medium to make a cell suspension. Press 5*10 4 Cells / well were inoculated in a coated 96-well plate, 6 replicates were set up, and cultured in a 37°C 5% CO2 incubator for 1 hour. The culture medium was aspirated, washed 3 times, and 100 μL of fresh culture medium was added to each well. 10 μL of cell staining solution B was added to each well and incubated at 37°C for 1 hour. The OD value at 450nm was measured. The formula for calculating the cell adhesion rate is as follows:

[0114]

[0115] In the formula: V (%) - cell adhesion rate, %; OD sample - absorbance of the reaction system containing the sample to be tested; OD blank control - absorbance of an empty plate without any substance; OD cell control - absorbance of the reaction system without the sample to be tested.

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

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

[0118] HaCaT cells in logarithmic growth phase were cultured at 1*10 610 cells / well were inoculated in a 6-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours. Subsequently, the 6-well plate was taken out, and the recombinant protein sample solutions of Examples 1-4 and Comparative Examples 1-3 were added to the sample group (the final concentration of recombinant elastin was 20 ppm), serum-free culture medium was added to the blank control group, and the corresponding positive drug solution was added to the positive control group, and cultured in a 37°C, 5% CO2 incubator for 24 hours. Subsequently, the supernatant was discarded, and the cells were collected after washing twice with PBS. RNA was extracted from the collected cells according to the RNA extraction kit (Beijing Quanshijin Biological Company). After determining the concentration, 1 μg was used for reverse transcription using a reverse transcription kit (Beijing Quanshijin Biological Company) to obtain cDNA. Subsequently, the cDNA was subjected to RT-qPCR (Nanjing Novozyme Biotechnology Company) according to the kit steps. The 2^(-ΔΔCt) value of the test sample, that is, the relative expression level, and the standard error (SD) of each test sample well were calculated to obtain a data table. The results are as follows Figure 4 .

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

[0120] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting and purifying elastin, characterized in that: The method comprises the following steps: (1) constructing an elastin structural unit, a hydrophilic structure and a membrane-penetrating peptide into a recombinant nucleic acid sequence; (2) introducing the recombinant nucleic acid sequence into the plasmid and transforming it into Pichia pastoris, and culturing the Pichia pastoris to appropriate conditions to induce expression; (3) Isolate and purify elastin.

2. The extraction and purification method according to claim 1, characterized in that The amino acid sequence of the elastin structural unit is shown in SEQ ID NO.

1.

3. The extraction and purification method according to claim 1, characterized in that The elastin structural unit is repeated 3-10 times.

4. The extraction and purification method according to claim 1, characterized in that The amino acid sequence of the hydrophilic structure is shown in SEQ ID NO.

2.

5. The extraction and purification method according to claim 1, characterized in that The amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO.

3.

6. The extraction and purification method according to claim 1, characterized in that The nucleotide sequence of the recombinant elastin contains polypeptides as shown in SEQ ID NO. 4-7.

7. The extraction and purification method according to claim 1, characterized in that The amino acid sequence of the recombinant elastin is shown in SEQ ID NO. 4-7.

8. A recombinant elastin protein, characterized in that: The recombinant elastin is prepared by the method according to any one of claims 1-7.

9. A biomaterial, characterized in that: It can encode the recombinant elastin protein according to claim 8 or contains a nucleic acid that can encode the recombinant elastin protein according to claim 8; Optionally, the biological material is one or more of nucleic acid, vector, and cell.

10. Use of the recombinant elastin as claimed in claim 8 in preparing a material for promoting cell proliferation, cell adhesion or skin repair.

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