CN119371518A - A highly stable recombinant humanized elastin, a genetically engineered bacterium expressing the protein and its application - Google Patents
A highly stable recombinant humanized elastin, a genetically engineered bacterium expressing the protein and its application Download PDFInfo
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Abstract
The invention provides a high-stability recombinant humanized elastin, a genetically engineered bacterium expressing the protein and application thereof, and belongs to the technical field of combined tissue peptide. After the nucleotide sequence of the humanized elastin is subjected to codon optimization, the humanized elastin is transferred into escherichia coli, and then the genetically engineered bacterium for expressing the recombinant humanized elastin is constructed, so that the expression quantity of the recombinant humanized elastin can be improved, the stability of the recombinant humanized elastin is improved, the mobility of the recombinant humanized elastin to HSF cells is improved, and the h-ELN, h-COL1A1 and h-COL3A1 gene expression quantity of the HSF cells is improved.
Description
Technical Field
The invention relates to a high-stability recombinant humanized elastin, a genetically engineered bacterium expressing the protein and application thereof, and belongs to the technical field of combined tissue peptide.
Background
Elastin (ELN) is an important extracellular matrix protein, the major component of which is Elastin fiber, providing elasticity and flexibility to tissues. There are also differences in elastin content in different tissues, for example in ligaments subjected to the motor load between bones, elastin content of 80%, elastin content in the aorta of 30% -50% of the dry weight, elastin content in the lungs of 10% -25% and elastin content in the skin of 3%.
Elastin plays an important role in promoting various functions such as cell adhesion, proliferation, differentiation, chemotaxis, and migration. Therefore, elastin is widely used in the fields of biology, medicine, medical materials, cosmetology and the like.
CN118005770a discloses a high bioactivity human recombinant elastin, a preparation method and application thereof, the elastin is expressed with high efficiency through a prokaryotic expression system, and the high activity recombinant elastin with high purity is obtained after separation and purification.
CN118480554a discloses recombinant elastin, expression system and application thereof, and the obtained recombinant elastin has the functions of promoting cell proliferation, promoting cell adhesion and promoting cell synthesis of collagen.
However, the recombinant humanized elastin synthesized by the prokaryotic expression system has the problems of poor stability, easy degradation and obvious reduction of efficacy in subsequent product use.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention provides the high-stability recombinant humanized elastin, the genetically engineered bacterium for expressing the protein and the application thereof, the stability of the recombinant humanized elastin is improved, the recombinant humanized elastin is not easy to degrade, and the migration effect on HSF cells and the expression quantity of genes related to anti-aging and anti-wrinkle are improved.
In order to solve the technical problems, the invention adopts the following technical scheme:
a high-stability recombinant humanized elastin has a nucleotide sequence shown in SEQ ID NO. 1.
The amino acid sequence of the recombinant humanized elastin is shown as SEQ ID NO. 4.
The preparation method of the recombinant humanized elastin comprises the steps of constructing recombinant plasmids by using nucleotide sequences of the recombinant humanized elastin, transferring the recombinant plasmids into competent cells of escherichia coli, and inducing expression to obtain the recombinant humanized elastin.
And expressing the recombinant humanized elastin.
The application of the recombinant humanized elastin in preparing skin care products.
Compared with the prior art, the invention has the following beneficial effects:
After the nucleotide sequence of the humanized elastin is subjected to codon optimization, the humanized elastin is transferred into escherichia coli, and then the genetically engineered bacterium for expressing the recombinant humanized elastin is constructed, so that the expression quantity of the recombinant humanized elastin can be improved, the stability of the recombinant humanized elastin is improved, the mobility of the recombinant humanized elastin to HSF cells is improved, and the h-ELN, h-COL1A1 and h-COL3A1 gene expression quantity of the HSF cells is improved.
Drawings
FIG. 1 is an SDS-PAGE electrophoresis diagram of recombinant humanized elastin induced by each genetically engineered bacterium, wherein M is Marker, 1 is a protein band induced by empty genetically engineered bacterium BL21-pET28a (+), 2 is a protein band induced by genetically engineered bacterium BL21-pET28a (+) -rhELN-before optimization, 3 is a protein band induced by genetically engineered bacterium BL21-pET28a (+) -rhELN-optimization 2, and 4 is a protein band induced by genetically engineered bacterium BL21-pET28a (+) -rhELN-optimization 1;
FIG. 2 is a microscopic image of the cell migration situation of example 5;
FIG. 3 is a bar graph of the relative mobility of cells of example 5;
FIG. 4 is a bar graph showing the expression level of the genes involved in anti-aging and anti-wrinkle in example 6.
Detailed Description
Example 1 preparation method of highly stable recombinant humanized elastin
(1) Sequence design of target Gene
The invention optimizes the codon of the nucleotide sequence of the humanized elastin;
The optimized nucleotide sequence of the humanized elastin is shown as SEQ ID NO.1 or SEQ ID NO.2, and the nucleotide sequence before optimization is shown as SEQ ID NO. 3;
the amino acid sequence of the humanized elastin is shown as SEQ ID NO. 4.
In the nucleotide sequences shown in SEQ ID No.1-SEQ ID No.3, CCATGG is an NcoI cleavage site, and AAGCTT is a HindIII cleavage site.
(2) Construction of recombinant plasmids
The nucleotide sequences of the humanized elastin shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 are entrusted to the gene synthesis of Suzhou Jinzhi biotechnology limited company to obtain plasmids before PUC 57-rhELN-optimization 1, PUC 57-rhELN-optimization 2 and PUC 57-rhELN-optimization;
Plasmid pET28a (+) is subjected to NcoI+HindIII double digestion with plasmids before PUC 57-rhELN-optimizing 1, PUC 57-rhELN-optimizing 2 and PUC 57-rhELN-optimizing respectively, double digestion products are recovered and connected, the double digestion products are transformed into E.coli DH5 alpha competent cells, positive transformants are selected for culturing after resistance screening, sequencing is carried out after PCR identification, and the sequencing results are correct, and recombinant plasmids pET28a (+) -rhELN-optimizing 1, pET28a (+) -rhELN-optimizing 2 and pET28a (+) -rhELN-optimizing are obtained by extracting with a plasmid small extraction kit.
(3) Construction of genetically engineered bacterium expressing recombinant humanized elastin
Mixing 1 μL of recombinant plasmid (with concentration of 1 μg/μL) with 100 μL of competent cell suspension of Escherichia coli BL21 (DE 3) after thawing, ice-bathing for 30min, placing in a 42 ℃ water bath, heat-shocking for 90s, ice-bathing for 5min again, adding 400 μL of non-resistant LB culture medium, shaking at 37 ℃ and 180rpm for 40min, sucking 200 μL of bacterial liquid, uniformly coating on LB agar plate containing 50 μg/mL kanamycin, and culturing overnight at 37 ℃ upside down. The single colony transformed was picked the next day and inoculated into a test tube containing 5mL of LB liquid medium (Kana's final concentration is 50. Mu.g/mL), after overnight culture at 37℃and 180rpm, the plasmid of the bacterial liquid was extracted, and the genetically engineered bacterium expressing the recombinant humanized elastin was obtained by nucleotide sequencing inspection.
The recombinant plasmids pET28a (+) -rhELN-optimizing 1, pET28a (+) -rhELN-optimizing 2 and pET28a (+) -rhELN-are respectively constructed according to the method before optimization to obtain the genetically engineered bacteria BL21-pET28a (+) -rhELN-optimizing 1 and BL21-pET28a (+) -rhELN-optimizing 2 and BL21-pET28a (+) -rhELN-before optimization.
And constructing pET28a (+) according to the method to obtain the empty genetic engineering bacterium BL21-pET28a (+).
(4) Inducible expression of genetically engineered bacteria
Selecting single colony of genetically engineered bacteria, inoculating into 5mL of LB (Kana final concentration is 50 mug/mL) liquid culture medium, shaking and culturing overnight at 37 ℃ and 180rpm, absorbing 300 mug of overnight bacterial liquid, inoculating into a triangular flask containing 30mL of LB (Kana final concentration is 50 mug/mL) liquid culture medium, culturing for 2.5h until OD 600 reaches 0.68, adding inducer IPTG to make the final concentration be 0.2mM, inducing temperature be 37 ℃ and inducing time be 5h, taking 1mL of bacterial liquid after the induction is finished, centrifuging at 12000rpm and 4 ℃ for 2min, discarding supernatant, adding 500 mug of ddH 2 O to resuspension, taking 40 mug of heavy suspension, adding 10 mug of 5X loading buffer solution, uniformly mixing, boiling water for 5 min, instantaneous separating, taking 20 mug of liquid, and carrying out 12% SDS-PAGE protein electrophoresis to detect the expression.
The genetic engineering bacteria BL21-pET28a (+) -rhELN-optimized 1, BL21-pET28a (+) -rhELN-optimized 2 and BL21-pET28a (+) -rhELN-pre-optimized empty genetic engineering bacteria BL21-pET28a (+) are respectively induced and expressed according to the method, and the result of protein electrophoresis is shown in figure 1.
As can be seen from FIG. 1, the empty genetically engineered bacterium BL21-pET28a (+) is induced, the supernatant obtained by cell disruption has no specific band, and the expression level of the genetically engineered bacterium BL21-pET28a (+) -rhELN-optimization 1 on the recombinant humanized collagen is far higher than that of BL21-pET28a (+) -rhELN-optimization 2 and BL21-pET28a (+) -rhELN-before optimization.
(5) Fermentation culture of genetically engineered bacteria
And (3) identifying the bacterial liquid of the correct genetic engineering bacteria, absorbing 200 mu L of the bacterial liquid, inoculating the bacterial liquid into a triangular flask containing 65mL of LB culture medium, culturing for 12 hours at 200rpm and 37 ℃ to obtain first-stage seeds, absorbing 6mL of the first-stage seeds, inoculating the first-stage seeds into a triangular flask containing 130mL of LB culture medium, culturing for 8 hours at 200rpm and 37 ℃, fully inoculating 130mL of bacterial liquid into a fermentation tank of 2.5L of TB culture medium, culturing at 37 ℃ and 1000rpm until the OD 600 reaches 30, adding IPTG to the final concentration of 0.2mM, and inducing for 5 hours to finish fermentation to obtain the fermentation bacterial liquid of the genetic engineering bacteria.
(6) Protein purification
Centrifuging the fermentation broth of each genetic engineering bacterium in the step (5) at 8000rpm for 30min, taking precipitated thalli, adding 7mL of 0.2M NaCl aqueous solution into 1g of thalli to dissolve thalli, homogenizing at low pressure once (400 bar) by using a Natong high-pressure homogenizer, homogenizing at high pressure three times (800 bar), centrifuging at 8000rpm for 30min, taking supernatant, slowly adding citric acid monohydrate to control pH to a specific condition (pH=4.2+/-0.3), centrifuging again under the same condition, taking supernatant after centrifuging, slowly adding 20% saturated ammonium sulfate to completely dissolve, centrifuging at 8000rpm for 30min, and taking supernatant for the next experiment.
Sample purification was performed using a Bio-Lab laboratory chromatography system instrument, tofflon, rigose MMC Hires (High Sub) cationic mixed mode High-intensity agarose medium self-loading column, to obtain a purified protein stock solution.
The recombinant humanized elastin expressed before the optimization of the genetically engineered bacteria BL21-pET28a (+) -rhELN-optimized 1 and BL21-pET28a (+) -rhELN-optimized 2 and BL21-pET28a (+) -rhELN-are subjected to protein purification according to the method to obtain purified protein stock solution, and the purity of the purified protein stock solution is higher than 90 percent before the optimization of rhELN-optimized 1, rhELN-optimized 2 and rhELN-marked.
(7) Freeze-drying
And (3) carrying out freeze-drying treatment on the purified protein stock solution by adopting a vacuum freeze dryer of the sea creature to obtain recombinant humanized elastin freeze-dried powder, wherein the freeze-drying procedure is shown in table 1.
Table 1 vacuum freeze drying process
EXAMPLE 2 stability experiment of recombinant humanized elastin lyophilized powder
The recombinant humanized elastin lyophilized powder before rhELN-optimization 1, rhELN-optimization 2 and rhELN-optimization was placed at 25 ℃, 37 ℃ and 50 ℃ for 0h, 8h, 24h, 48h and 30d respectively, then prepared into a solution of 10mg/mL with purified water, and the purity was measured by reverse phase high performance liquid chromatography, and the results are shown in Table 2.
Table 2 results of purity measurement of protein lyophilized powder
As can be seen from Table 2, the purity of the protein lyophilized powder rhELN-optimized 2, rhELN-before-optimization drops faster and the purity of rhELN-optimized 1 remains almost unchanged when placed at 25 ℃, 37 ℃ and 50 ℃ for 0h, 8h,24h,48h and 30d, indicating that the stability is better and the degradation is not easy.
Example 3 stability experiment of recombinant humanized elastin lyophilized powder solution
The recombinant humanized elastin lyophilized powder rhELN-optimization 1, rhELN-optimization 2, rhELN-before optimization, 10mg/mL of solution was prepared with purified water, and then placed at 25 ℃, 37 ℃ and 50 ℃ for 0h,8h,24h,48h and 30d, and the purity was measured by reverse phase high performance liquid chromatography, and the results are shown in Table 3.
TABLE 3 determination of the purity of protein lyophilized powder solutions
As can be seen from Table 3, the purity of the protein lyophilized powder solution before rhELN-optimization 1, rhELN-optimization 2, rhELN-optimization was rapidly decreased when the protein lyophilized powder solution was left at 25 ℃,37 ℃ and 50 ℃ for 0h, 8h,24h,48h and 30d, while the purity of the protein lyophilized powder solution before rhELN-optimization 2, rhELN-optimization was almost unchanged, indicating that the stability was good and the protein lyophilized powder solution was not easily degraded.
Example 4 cytotoxicity assay
Cytotoxicity was detected by MTT method, and human skin immortalized fibroblasts (HSF) in logarithmic growth phase were inoculated into 96-well plates at 2.0X10 5/mL, 100. Mu.L/well, and cultured at 37℃in 5% CO 2 for 24 hours. Administration was performed when the cell plating rate in 96-well plates reached 50%.
A. Sample group 100. Mu.L of FM medium containing different concentrations (10 mg/mL, 5mg/mL, 2.5mg/mL, 1.25mg/mL, 0.625mg/mL, 0.313mg/mL, 0.156mg/mL, 0.078 mg/mL) of protein lyophilized powder was added per well;
B. control (NC) 100. Mu.L of FM medium was added;
C. Blank (BC) containing no cells, 100. Mu.L of FM medium was added;
After 24 hours of cultivation, the waste liquid was changed to a culture liquid containing MTT (0.5 mg/mL, final concentration), 100. Mu.L/well, and cultivation was continued for 4 hours. Adding 100 mu L/hole DMSO into the waste liquid, fully oscillating and developing, detecting OD 490 by using an enzyme-labeling instrument, and calculating the relative activity of the cells according to the formula (1):
Cytotoxicity criterion, that is, if the cell viability is greater than 70%, then no cytotoxicity reaction is considered, otherwise, cytotoxicity reaction is considered.
The results of the relative viability of the cells are shown in Table 4.
TABLE 4 results of relative cell viability
As can be seen, the cell survival rate is more than 70% after the rhELN-optimizing 1, rhELN-optimizing 2 and rhELN-pre-optimizing protein freeze-dried powder solution is reacted with HSF cells for 24 hours at the concentration of less than or equal to 10.0mg/mL, and the protein freeze-dried powder solution has no toxicity to the cells.
Example 5 cell migration promotion experiment
HSF cells in the logarithmic growth phase were seeded at a density of 1×10 6 cells/well in 6-well plates and incubated overnight in an incubator (37 ℃, 5% CO 2). And (5) carrying out a scratch experiment when the plating rate reaches more than 70%. The 10 mu L gun head is used for longitudinally scribing two lines in the six-hole plate, vertical marks are used as a base line (the gun head is vertical to the edge of the ruler), the mark distance is kept at 2cm, and after the vertical marks are scribed, the vertical marks are vertical to the base line, and horizontal scratches are formed near the central axis of the six-hole plate. After the scratch is finished, the cells are washed by PBS for 3 times, the administration concentration of rhELN-after-optimization-rhLOX, rhELN-before-optimization-rhLOX, rhELN-after-optimization-protein freeze-dried powder is 0.156mg/mL, the administration amount of each hole is 2mL, and each group is provided with 2 compound holes. The incubator (37 ℃, 5% co 2) continues to incubate for 24h. After photographing each group of migration using an inverted microscope (see fig. 2), the cell mobilities of the negative control group were normalized, and the relative mobilities of each group were calculated, and the results are shown in table 5 and fig. 3.
TABLE 5 cell migration results
Table 5 shows that rhELN-optimization 1 can significantly improve the relative mobility of cells compared to rhELN-optimization 2, rhELN-before optimization.
EXAMPLE 6 anti-aging and anti-wrinkle experiment
HSF cells in the logarithmic growth phase were seeded at an seeding density of 5×10 5 cells/well in 6-well plates and incubated overnight in incubator (37 ℃, 5% co 2). When the cell plating rate in the 6-hole plate reaches 45%, rhELN-optimizing 1, rhELN-optimizing 2 and rhELN-freeze-dried powder before optimization is dosed according to the concentration of 0.156mg/mL, the dosing amount of each hole is 2mL, and each group is provided with 1 hole. After the completion of the administration, the 6-well plate was placed in an incubator (37 ℃ C., 5% CO 2) and incubated for 24 hours.
The total RNA of the cells is improved, the purity of the extracted RNA is detected by using an ultra-micro spectrophotometer, the OD 260/OD280 value is between 1.8 and 2.0, and the next reverse transcription experiment can be carried out.
And (3) carrying out reverse transcription on the RNA to obtain a reverse transcription product, carrying out a real-time quantitative PCR experiment, and detecting the expression levels of genes h-ELN, h-COL1A1 and h-COL3A1 related to cell anti-aging and anti-wrinkle.
The fluorescent quantitative PCR gene detection results are shown in Table 6 and FIG. 4, and the results show that compared with rhELN-optimization 2 and rhELN-optimization, the high-stability humanized elastin rhELN-optimization 1 can obviously improve the gene expression quantity of h-ELN, h-COL1A1 and h-COL3A1 of HSF cells, and the high-stability humanized elastin rhELN-optimization 1 has better anti-aging and anti-wrinkle effects.
TABLE 6 fluorescent quantitative PCR Gene detection results
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111417404A (en) * | 2017-09-28 | 2020-07-14 | 格尔托公司 | Recombinant collagen and elastin molecules and their uses |
| CN112041434A (en) * | 2018-04-27 | 2020-12-04 | 克里斯托生物技术股份有限公司 | Recombinant nucleic acids encoding one or more cosmetic proteins for cosmetic applications |
| CN117886923A (en) * | 2024-03-14 | 2024-04-16 | 山东福瑞达生物股份有限公司 | Recombinant humanized collagen and encoding gene and application thereof |
| CN118064468A (en) * | 2024-04-12 | 2024-05-24 | 山东兴瑞生物科技有限公司 | Recombinant elastin, preparation method and application thereof |
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| CN111417404A (en) * | 2017-09-28 | 2020-07-14 | 格尔托公司 | Recombinant collagen and elastin molecules and their uses |
| CN112041434A (en) * | 2018-04-27 | 2020-12-04 | 克里斯托生物技术股份有限公司 | Recombinant nucleic acids encoding one or more cosmetic proteins for cosmetic applications |
| CN117886923A (en) * | 2024-03-14 | 2024-04-16 | 山东福瑞达生物股份有限公司 | Recombinant humanized collagen and encoding gene and application thereof |
| CN118064468A (en) * | 2024-04-12 | 2024-05-24 | 山东兴瑞生物科技有限公司 | Recombinant elastin, preparation method and application thereof |
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