CN119409803A - Method for extracting and purifying elastin - Google Patents
Method for extracting and purifying elastin Download PDFInfo
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- CN119409803A CN119409803A CN202411785934.0A CN202411785934A CN119409803A CN 119409803 A CN119409803 A CN 119409803A CN 202411785934 A CN202411785934 A CN 202411785934A CN 119409803 A CN119409803 A CN 119409803A
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Abstract
本发明涉及生物技术领域，具体而言，涉及一种弹性蛋白的提取和纯化方法，其特征在于，所述方法包含以下步骤：(1)将弹性蛋白结构单元、亲水结构和穿膜肽构建成重组的核酸序列；(2)将重组的核酸序列导入质粒后转化入毕赤酵母，培养毕赤酵母至合适条件进行诱导表达；(3)分离纯化获得重组弹性蛋白。本发明提供一种具有显著促细胞增殖、粘附活性以及促进弹性蛋白合成的弹性蛋白，同时经优化后的发酵培养及纯化，最终得到高纯度的重组弹性蛋白。本发明相对于常规的发酵手段能够显著提升上述重组弹性蛋白的表达量，在生物医用材料和化妆品等领域具有潜在的应用前景。
The present invention relates to the field of biotechnology, and in particular to 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 a plasmid and transforming it into Pichia pastoris, culturing the Pichia pastoris to suitable conditions for inducing expression; (3) isolating and purifying to obtain recombinant elastin. The present invention provides an elastin that has significant cell proliferation and adhesion activity and promotes elastin synthesis, and finally obtains a high-purity recombinant elastin through optimized fermentation culture and purification. Compared with conventional fermentation methods, the present invention can significantly increase the expression level of the above-mentioned recombinant elastin, and has potential application prospects in the fields of biomedical materials and cosmetics.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an elastin extraction and purification method.
Background
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 vasculature, skin and lungs, combining with microfibrils in the body to form elastic fibers, which, together with collagen fibers, impart elastic and tensile capabilities to tissues. Elastin is rich in nonpolar amino acids such as glycine, proline, alanine, leucine and valine, and usually has a repetitive sequence consisting of a large number of 3-9 amino acids, forming a flexible and stable structure.
The method for extracting elastin is more, and has advantages and disadvantages. In 1969 Bornstein and Ross, 5mol/L guanidine hydrochloride was used to extract, collagenase was used to hydrolyze other nonelastin components, disulfide bonds were reduced, and elastin was isolated. However, the prior art adopts a mode of constructing recombinant elastin to obtain high-efficiency elastin.
The prior art CN 118005770B discloses a high-activity human recombinant elastin, which is formed by connecting 9 peptide VAPGTIGTG serving as a structural unit through a hydrophilic structure AAAKSAAKAA, has a structure and properties similar to those of natural elastin, is subjected to high-efficiency expression through a prokaryotic expression system, and is separated and purified to obtain the high-activity recombinant elastin with higher purity, and the high-activity human recombinant elastin has wide application prospects in skin repair, cell adhesion and cell proliferation.
The prior art CN 118388666B discloses a fusion protein, which combines elastin polypeptide and collagen polypeptide, utilizes an amino acid sequence designed by elastin fragments rich in VAPGXG motif and a core fragment of an alpha 2 chain of type I collagen to play an important role in promoting fibroblast proliferation, inhibiting metalloprotease activity and enhancing collagen and elastin expression, utilizes a genetic engineering technology to realize efficient expression and purification of the fusion protein in an escherichia coli expression system, remarkably improves production efficiency, reduces cost and has strong expandability, and the transdermal peptide TAT sequence added at the N end of the produced fusion protein enhances the penetration capability of the fusion protein in skin, so that the bioavailability and curative effect of the fusion protein in deep skin are improved.
The prior art CN 118480554A discloses that the recombinant elastin coding gene and the recombinant elastin coding gene provided by the invention can be used for expressing recombinant elastin and recombinant elastin, the protein expression quantity is considerable, the purification is easy, and the stability is better in the storage process.
However, the prior art has limited technical solutions, and the structure and effect of the recombinant protein are uneven, so that the increasing demands cannot be met, and therefore, it is needed to provide new recombinant elastin and to match with the corresponding extraction and purification methods of elastin.
Disclosure of Invention
The invention firstly provides an elastin extraction and purification method, which 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) The recombinant nucleic acid sequence is transferred into pichia pastoris after being led into plasmid, and the pichia pastoris is cultivated to proper condition for induced expression;
(3) Separating and purifying to obtain recombinant elastin.
In certain embodiments, the elastin building block has an amino acid sequence as set forth in SEQ ID NO.1
As shown.
In certain embodiments, the elastin building blocks are repeated 3-10 times.
In certain embodiments, the amino acid sequence of the hydrophilic structure is set forth in SEQ ID NO. 2.
In certain embodiments, the amino acid sequence of the transmembrane peptide is shown in SEQ ID NO. 3.
In certain embodiments, the nucleotide sequence of the recombinant elastin comprises a polypeptide as set forth in SEQ ID NO. 4-7.
In certain embodiments, the amino acid sequence of the recombinant elastin is shown in SEQ ID NO. 4-7.
The invention also provides a recombinant elastin, which is prepared by the method.
The present invention also provides a biological material capable of encoding the recombinant elastin described above or comprising a nucleic acid capable of encoding the recombinant elastin described above;
Optionally, the biological material is one or more of a nucleic acid, a vector, a cell.
The invention finally provides an application of the recombinant elastin in preparing materials for promoting cell proliferation or cell adhesion or skin repair.
Compared with the prior art, the invention has at least the following beneficial effects:
compared with the prior art, the structure, the repeating units and the repeating times of the recombinant protein are optimized through screening and transformation from the elastin sequence, the optimized recombinant elastin with obvious cell proliferation promoting and adhesion activity and elastin synthesis promoting effects is obtained, pichia pastoris expression genes are utilized, the recombinant pichia pastoris expression genes are constructed into pichia pastoris secretion expression plasmids through homologous recombination, recombinant pichia pastoris engineering strains with high expression recombinant elastin are obtained through electric transfer screening, and the recombinant elastin with high purity is finally obtained through fermentation culture and purification after optimization.
Drawings
FIG. 1, SWISS-MODEL, predicts the tertiary structure MODEL of recombinant elastin 1;
FIG. 2, cell proliferation promoting activity of recombinant elastin;
FIG. 3, cell adhesion promoting activity of recombinant elastin;
FIG. 4 is a graph showing the result of QPCR of recombinant elastin to promote elastin synthesis.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
The nucleotide sequence of the elastin building block, (VPGKG) 5 (SEQ ID No. 1);
repeating the elastin structural unit n times, wherein n is 3-10;
amino acid sequence of hydrophilic structure AAAKSAAKAA (SEQ ID NO. 2);
amino acid sequence of the transmembrane peptide RKKRRQRRRPP (SEQ ID NO. 3);
the recombinant elastin structure of the experimental group is:
RKKRRQRRRPP-[(VPGKG)5-AAAKSAAKAA-(VPGKG)5]n-VGRVHHHHHH*。
The amino acid sequence of the recombinant elastin in the experimental group is shown as SEQ ID NO.4-7, and the corresponding n is 3, 5, 7 and 9 respectively.
SEQ ID NO.4:
RKKRRQRRRPPVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK
GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA
AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG
VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVGRVHHHHHH*;
SEQ ID NO.5:
RKKRRQRRRPPVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK
GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA
AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG
VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPG
KGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGV
PGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVGRVHHHHHH*;
SEQ ID NO.6:
RKKRRQRRRPPVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK
GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA
AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG
VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPG
KGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGV
PGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK
GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA
AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG
VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVGRVHHHHHH*;
SEQ ID NO.7:
RKKRRQRRRPPVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK
GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA
AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG
VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPG
KGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGV
PGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGK
GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAA
AKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG
VPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVPG
KGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGV
PGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGAAAKSAAKAAVPGKGVPGKGVPGKGVPGKGVPGKGVGRVHHHHHH*;
The recombinant elastin structure of comparative example 1 is:
RKKRRQRRRPP-[(VPGKG)5-(VPGKG)5]n-VGRVHHHHHH*。
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.
SEQ ID NO.8:
RKKRRQRRRPPVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGK
GVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVP
GKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKG
VPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPG
KGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVPGKGVGRVHHHHHH*;
EXAMPLE 1 preparation of recombinant elastin
Synthesizing a gene fragment according to an amino acid sequence shown in Seq ID No.4, and inserting the synthesized gene fragment between enzyme cutting sites EcoRI and NotI on pPIC9K plasmid to obtain a recombinant plasmid;
Mixing the recombinant plasmid with the GS115 competent cells, performing electric shock after ice bath, adding sorbitol solution, transferring to an aseptic EP tube after uniform mixing, standing at 30 ℃ for incubation for 1-2 h, coating on an MD solid plate, and performing inversion culture at 30 ℃ for 2d-5d until single bacterial colony grows; scraping His+ transformants on the MD solid plates, diluting, coating the transformants on YPD plates containing G418, culturing the transformants in an inverted manner at 30 ℃ until single colonies appear, and then selecting the single colonies on the YPD plates, transferring the single colonies into a 96-well culture plate of a YPD culture medium, and culturing the single colonies continuously at 30 ℃ to screen recombinant yeast engineering bacteria;
Inoculating the recombinant yeast engineering bacteria into a BMGY induction culture medium, culturing until the OD600nm absorbance reaches 2.0-6.0, centrifuging at 3000rpm for 10min, collecting thalli, inoculating again into the BMGY induction culture medium to enable the OD600nm absorbance to reach 2.0, continuously culturing at 30 ℃ and 220rpm, adding 0.5% methanol every 24h, and centrifuging to collect an induction supernatant after induction;
Purifying the induced supernatant by a cation exchange chromatographic column, eluting and collecting proteins corresponding to elution peaks to obtain the human recombinant elastin 1 with the purity of 98.45%, predicting a three-level structure MODEL of the recombinant elastin 1 by SWISS-MODEL, wherein the result in FIG. 1 shows that the recombinant elastin 1 has dynamic balance of Random Coil and ordered structure, mimics the characteristics of natural elastin, and in theory, the recombinant elastin 2-4 can be expected to have the characteristics of similar natural elastin, and can be subjected to subsequent experiments.
EXAMPLE 2 preparation of recombinant elastin
Synthesizing a gene fragment according to an amino acid sequence shown in Seq ID No.5, and inserting the synthesized gene fragment between enzyme cutting sites EcoRI and NotI on pPIC9K plasmid to obtain a recombinant plasmid;
Mixing the recombinant plasmid with the GS115 competent cells, performing electric shock after ice bath, adding sorbitol solution, transferring to an aseptic EP tube after uniform mixing, standing at 30 ℃ for incubation for 1-2 h, coating on an MD solid plate, and performing inversion culture at 30 ℃ for 2d-5d until single bacterial colony grows; scraping His+ transformants on the MD solid plates, diluting, coating the transformants on YPD plates containing G418, culturing the transformants in an inverted manner at 30 ℃ until single colonies appear, and then selecting the single colonies on the YPD plates, transferring the single colonies into a 96-well culture plate of a YPD culture medium, and culturing the single colonies continuously at 30 ℃ to screen recombinant yeast engineering bacteria;
Inoculating the recombinant yeast engineering bacteria into a BMGY induction culture medium, culturing until the OD600nm absorbance reaches 2.0-6.0, centrifuging at 3000rpm for 10min, collecting thalli, inoculating again into the BMGY induction culture medium to enable the OD600nm absorbance to reach 2.0, continuously culturing at 30 ℃ and 220rpm, adding 0.5% methanol every 24h, and centrifuging to collect an induction supernatant after induction;
Purifying the induced supernatant by a cation exchange chromatographic column, eluting and collecting the protein corresponding to the eluting peak to obtain the human recombinant elastin 2 with the purity of 98.73%.
EXAMPLE 3 preparation of recombinant elastin
Synthesizing a gene fragment according to an amino acid sequence shown in Seq ID No.6, and inserting the synthesized gene fragment between enzyme cutting sites EcoRI and NotI on pPIC9K plasmid to obtain a recombinant plasmid;
Mixing the recombinant plasmid with the GS115 competent cells, performing electric shock after ice bath, adding sorbitol solution, transferring to an aseptic EP tube after uniform mixing, standing at 30 ℃ for incubation for 1-2 h, coating on an MD solid plate, and performing inversion culture at 30 ℃ for 2d-5d until single bacterial colony grows; scraping His+ transformants on the MD solid plates, diluting, coating the transformants on YPD plates containing G418, culturing the transformants in an inverted manner at 30 ℃ until single colonies appear, and then selecting the single colonies on the YPD plates, transferring the single colonies into a 96-well culture plate of a YPD culture medium, and culturing the single colonies continuously at 30 ℃ to screen recombinant yeast engineering bacteria;
Inoculating the recombinant yeast engineering bacteria into a BMGY induction culture medium, culturing until the OD600nm absorbance reaches 2.0-6.0, centrifuging at 3000rpm for 10min, collecting thalli, inoculating again into the BMGY induction culture medium to enable the OD600nm absorbance to reach 2.0, continuously culturing at 30 ℃ and 220rpm, adding 0.5% methanol every 24h, and centrifuging to collect an induction supernatant after induction;
purifying the induced supernatant by a cation exchange chromatographic column, eluting and collecting the protein corresponding to the eluting peak to obtain the human recombinant elastin 3 with the purity of 97.65%.
EXAMPLE 4 preparation of recombinant elastin
Synthesizing a gene fragment according to an amino acid sequence shown in Seq ID No.7, and inserting the synthesized gene fragment between enzyme cutting sites EcoRI and NotI on pPIC9K plasmid to obtain a recombinant plasmid;
Mixing the recombinant plasmid with the GS115 competent cells, performing electric shock after ice bath, adding sorbitol solution, transferring to an aseptic EP tube after uniform mixing, standing at 30 ℃ for incubation for 1-2 h, coating on an MD solid plate, and performing inversion culture at 30 ℃ for 2d-5d until single bacterial colony grows; scraping His+ transformants on the MD solid plates, diluting, coating the transformants on YPD plates containing G418, culturing the transformants in an inverted manner at 30 ℃ until single colonies appear, and then selecting the single colonies on the YPD plates, transferring the single colonies into a 96-well culture plate of a YPD culture medium, and culturing the single colonies continuously at 30 ℃ to screen recombinant yeast engineering bacteria;
Inoculating the recombinant yeast engineering bacteria into a BMGY induction culture medium, culturing until the OD600nm absorbance reaches 2.0-6.0, centrifuging at 3000rpm for 10min, collecting thalli, inoculating again into the BMGY induction culture medium to enable the OD600nm absorbance to reach 2.0, continuously culturing at 30 ℃ and 220rpm, adding 0.5% methanol every 24h, and centrifuging to collect an induction supernatant after induction;
purifying the induced supernatant by a cation exchange chromatographic column, eluting and collecting the protein corresponding to the eluting peak to obtain the human recombinant elastin 4 with the purity of 98.16%.
Comparative example 1 preparation of recombinant elastin
Synthesizing a gene fragment according to an amino acid sequence shown in Seq ID No.8, and inserting the synthesized gene fragment between enzyme cutting sites EcoRI and NotI on pPIC9K plasmid to obtain a recombinant plasmid;
Mixing the recombinant plasmid with the GS115 competent cells, performing electric shock after ice bath, adding sorbitol solution, transferring to an aseptic EP tube after uniform mixing, standing at 30 ℃ for incubation for 1-2 h, coating on an MD solid plate, and performing inversion culture at 30 ℃ for 2d-5d until single bacterial colony grows; scraping His+ transformants on the MD solid plates, diluting, coating the transformants on YPD plates containing G418, culturing the transformants in an inverted manner at 30 ℃ until single colonies appear, and then selecting the single colonies on the YPD plates, transferring the single colonies into a 96-well culture plate of a YPD culture medium, and culturing the single colonies continuously at 30 ℃ to screen recombinant yeast engineering bacteria;
Inoculating the recombinant yeast engineering bacteria into a BMGY induction culture medium, culturing until the OD600nm absorbance reaches 2.0-6.0, centrifuging at 3000rpm for 10min, collecting thalli, inoculating again into the BMGY induction culture medium to enable the OD600nm absorbance to reach 2.0, continuously culturing at 30 ℃ and 220rpm, adding 0.5% methanol every 24h, and centrifuging to collect an induction supernatant after induction;
purifying the induced supernatant by a cation exchange chromatographic column, eluting and collecting the protein corresponding to the eluting peak to obtain the human recombinant elastin 5 with the purity of 86.75%.
Comparative example 2 preparation of recombinant elastin
Human recombinant elastin encoding high bioactivity was prepared according to the method of example 1 described in CN118005770 a.
Comparative example 3 preparation of recombinant elastin
A polypeptide for promoting regeneration of skin collagen and elastin is prepared according to the method described in CN 118388666A, and its amino acid sequence is as follows ：MYGRKKRRQRRRVAPGVGVAPGVGVAPGVGS VAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSVAPGVGVAPGVGVAPGVGSGLMGPRGLPGSPGNIGPAGKEGPVGLPGIDGRPGPIGPAGARGEPGNIGFPGPKGPTGDPGKNGDKGHAGLAGARGAPGPDGNNGAQGPPGPQGVQGGKGEQGPPGPPGFQGLPGPSGPAGEVGKPGERGLHGEFRHHHHHH(SEQ ID NO.9).
Example 5 cell proliferation-promoting experiment of recombinant elastin
Skin primary fibroblasts were inoculated into DMEM medium, cultured at 37 ℃ to logarithmic phase, and then inoculated into 96-well cell culture plates (cell concentration 1.0x10 5cells/ml-5.0×105 cells/ml, 100 μl per well). The protein solution was diluted to 1mg/mL with PBS buffer and then added to 96-well cell culture plates in proportion to give a final concentration of 50ppm of recombinant elastin of examples 1-4 and comparative examples 1-3, taking the sample added with PBS buffer as a blank. 3 replicate wells were treated for each concentration. After 48 hours of incubation at 37 ℃, a final concentration of 5 mg/ml of thiazole blue solution (MTT, 10 μl/well) was added and incubation was continued for 4 hours at 37 ℃. The medium was discarded, 100. Mu.L of dimethyl sulfoxide (DMSO) was added to each well, and the mixture was placed on a shaker for slow shaking for 10 minutes. Finally, the absorbance measurement wavelength of each well was 490nm by using an enzyme-labeled instrument, and the results are shown in FIG. 3.
As shown in FIG. 2, 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 most remarkable (p < 0.001), which indicates that example 2 has excellent effect of promoting cell proliferation, has good anti-aging repair ability, and is an effective biomedical material.
Example 6 cell adhesion promotion experiment of recombinant elastin
Cells in log phase (HaCaT cells) were plated at 1×10 6 cells/well in 6-well plates and incubated for 24h in an incubator at 37 ℃ with 5% co 2. The recombinant elastin solution was diluted with DMEM, and then, the sample group was added to the recombinant protein sample solutions of examples 1 to 4 and comparative examples 1 to 3 (final concentration of recombinant elastin was 50 ppm), and the blank group was added to a serum-free medium, 37 ℃ and 5% co 2 incubator for culturing for 24 hours. According to the cell adhesion kit (Shanghai Bei Bo Bio Inc.) instructions, 100. Mu.L of coating solution was added to each well of the new 96-well plate, and the plate was placed in a refrigerator at 4℃for 24 hours. After the cells to be measured are treated, they are digested with pancreatin, washed with PBS and resuspended in the corresponding medium to give a cell suspension. The cells/well were plated in coated 96-well plates at 5 x 10 4 cells/well, 6 multiplex wells were set and incubated in a 37 ℃ 5% CO 2 incubator for 1h. After 3 washes, 100 μl fresh medium was added to each well. mu.L of cell stain B was added to each well and incubated at 37℃for 1h. OD at 450nm was measured. The cell adhesion rate calculation formula is shown below:
Wherein, V (%) -cell adhesion rate,%), OD sample-absorbance of the reaction system containing the sample to be detected, OD blank-blank absorbance without any substance, OD cell control-absorbance of the reaction system without the sample to be detected.
As shown in FIG. 3, the cell adhesion rates of examples 1-4 and comparative examples 1-3 were significantly higher than those of the control group, and the effect of example 2 was most remarkable (p < 0.001), indicating that example 2 has excellent cell adhesion promoting effect, has good anti-aging repair ability, and is an effective biomedical material.
EXAMPLE 7 detection of elastin Synthesis ability by recombinant elastin-QPCR
HaCaT cells in log phase were seeded at 1 x 10 6 cells/well in 6-well plates, incubated at 37 ℃ in a 5% co 2 incubator for 24h. Subsequently, the 6-well plate was removed, the sample set was added to the recombinant protein sample solutions of examples 1 to 4 and comparative examples 1 to 3 (final concentration of recombinant elastin was 20 ppm), the blank set was added to the serum-free medium, the positive control set was added to the corresponding positive drug solution, and incubated in a 5% CO 2 incubator at 37℃for 24 hours. The supernatant was then discarded, and after 2 washes with PBS, the cells were collected. The collected cells were subjected to RNA extraction according to the method of RNA extraction kit (Beijing full gold Bio Inc.). After measuring the concentration, reverse transcription was performed with 1. Mu.g of a reverse transcription kit (Beijing full gold Bio Inc.), and cDNA was obtained. The cDNA was then subjected to RT-qPCR (Nanjinouzan Biol) following the kit procedure. The data table is obtained by calculating the 2 (-DeltaDeltaCt) value of the detection sample, namely the relative expression amount, and the standard error (SD) of each detection sample well, and the result is shown in FIG. 4.
As shown in FIG. 4, the relative expression amounts of Elastin (ELN) in examples 1-4 and comparative examples 1-3 were significantly higher than those in the control group, and the effect of example 2 was most remarkable (p < 0.001), indicating that example 2 has excellent Elastin synthesis expression ability, good anti-aging repair ability, and is an effective biomedical material.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A method for the extraction and purification of elastin, said method comprising the steps of:
(1) Constructing an elastin structural unit, a hydrophilic structure and a membrane penetrating peptide into a recombinant nucleic acid sequence;
(2) The recombinant nucleic acid sequence is transferred into pichia pastoris after being led into plasmid, and the pichia pastoris is cultivated to proper condition for induced expression;
(3) Separating and purifying to obtain elastin.
2. The method of extraction and purification according to claim 1, wherein the elastin building block has the amino acid sequence shown in SEQ ID No. 1.
3. The method of extraction and purification according to claim 1, wherein the elastin structural units are repeated 3-10 times.
4. The method for extraction and purification according to claim 1, wherein the amino acid sequence of the hydrophilic structure is shown in SEQ ID No. 2.
5. The method for extracting and purifying the peptide according to claim 1, wherein the amino acid sequence of the peptide is shown in SEQ ID NO. 3.
6. The method of extraction and purification according to claim 1, wherein the nucleotide sequence of the recombinant elastin comprises a polypeptide as shown in SEQ ID No. 4-7.
7. The method of extraction and purification according to claim 1, wherein the amino acid sequence of the recombinant elastin is shown in SEQ ID No. 4-7.
8. A recombinant elastin prepared by the method of any one of claims 1-7.
9. A biological material, characterized in that it is capable of encoding the recombinant elastin of claim 8 or comprises a nucleic acid capable of encoding the recombinant elastin of claim 8;
Optionally, the biological material is one or more of a nucleic acid, a vector, a cell.
10. Use of a recombinant elastin according to claim 8 for the preparation of a material that promotes cell proliferation or promotes cell adhesion or promotes skin repair.
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