High-stability recombinant humanized elastin, genetically engineered bacterium for expressing protein and application of high-stability recombinant humanized elastin

Abstract

The invention provides a high-stability recombinant humanized elastin, a genetically engineered bacterium for expressing the protein and application of the high-stability recombinant humanized elastin, and belongs to the technical field of binding tissue peptides. The nucleotide sequence of the recombinant humanized elastin is shown as SEQ ID NO.1; and the amino acid sequence is as shown in SEQ ID NO.4. According to the invention, the nucleotide sequence of the humanized elastin is subjected to codon optimization and transferred into escherichia coli to construct the genetically engineered bacterium for expressing the recombinant humanized elastin, so that the expression quantity of the recombinant humanized elastin can be increased, the stability of the recombinant humanized elastin can be improved, and the recombinant humanized elastin can be used for preparing the recombinant humanized elastin. The migration rate of the recombinant humanized elastin to the HSF cell is improved, and the expression quantity of the h-ELN gene, the h-COL1A1 gene and the h-COL3A1 gene of the HSF cell is improved.

Description

Technical Field

[0001] The invention relates to a highly stable recombinant humanized elastin, a genetic engineering bacterium expressing the protein and application thereof, and belongs to the technical field of tissue-binding peptides. Background Art

[0002] Elastin (ELN) is an important extracellular matrix protein, the main component of which is elastic fiber, which provides elasticity and flexibility to tissues. The content of elastin varies in different tissues. For example, in the ligaments that bear the load of inter-bone movement, the content of elastin is 80%, in the aorta, the content of elastin accounts for 30%-50% of the dry weight, in the lungs, the content of elastin is 10%-25%, and in the skin, the content of elastin is 3%.

[0003] Elastin plays an important role in promoting cell adhesion, proliferation, differentiation, chemotaxis, migration, etc. Therefore, elastin is widely used in biology, medicine, medical materials, beauty and other fields.

[0004] CN118005770A discloses a human recombinant elastin with high biological activity and a preparation method and application thereof. The elastin is efficiently expressed through a prokaryotic expression system, and a highly pure and highly active recombinant elastin is obtained after separation and purification.

[0005] CN118480554A discloses recombinant elastin and its expression system and application. The obtained recombinant elastin has the functions of promoting cell proliferation, cell adhesion and cell synthesis of collagen.

[0006] However, the above patent uses a prokaryotic expression system to synthesize recombinant humanized elastin, which has poor stability and is easily degraded, resulting in a significant reduction in efficacy in subsequent product use. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a highly stable recombinant humanized elastin, a genetically engineered bacterium expressing the protein and its application, which improves the stability of the recombinant humanized elastin, is not easy to degrade, and improves the migration effect on HSF cells and the expression level of anti-aging and anti-wrinkle related genes.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions: A highly stable recombinant humanized elastin protein, the nucleotide sequence of the recombinant humanized elastin protein is shown as SEQ ID NO.1.

[0009] The amino acid sequence of the recombinant humanized elastin is shown in SEQ ID NO.4.

[0010] The preparation method of the recombinant humanized elastin is to construct a recombinant plasmid with the nucleotide sequence of the recombinant humanized elastin, transfer the recombinant plasmid into competent Escherichia coli cells, and induce expression to obtain the recombinant humanized elastin.

[0011] A genetically engineered bacterium expressing the recombinant humanized elastin.

[0012] The application of the recombinant humanized elastin in the preparation of skin care products.

[0013] Compared with the prior art, the present invention achieves the following beneficial effects: The present invention performs codon optimization on the nucleotide sequence of humanized elastin and transfers it into Escherichia coli to construct a genetically engineered bacterium expressing recombinant humanized elastin, which can improve the expression amount of recombinant humanized elastin, improve the stability of recombinant humanized elastin, improve the mobility of recombinant humanized elastin to HSF cells, and improve the expression amounts of h-ELN, h-COL1A1 and h-COL3A1 genes of HSF cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The SDS-PAGE electrophoresis diagrams of the recombinant humanized elastin expressed by each genetically engineered bacterium after induction; wherein M is a marker; 1 is a protein band expressed by the genetically engineered bacterium BL21-pET28a (+) after induction; 2 is a protein band expressed by the genetically engineered bacterium BL21-pET28a (+)-rhELN-before optimization after induction; 3 is a protein band expressed by the genetically engineered bacterium BL21-pET28a (+)-rhELN-optimization 2 after induction; 4 is a protein band expressed by the genetically engineered bacterium BL21-pET28a (+)-rhELN-optimization 1 after induction; Figure 2 This is a microscope image of cell migration in Example 5; Figure 3 is a bar graph of the relative migration rate of cells in Example 5; Figure 4 This is a bar graph of the expression levels of anti-aging and anti-wrinkle related genes in Example 6. DETAILED DESCRIPTION

[0015] Example 1 A method for preparing highly stable recombinant humanized elastin (1) Sequence design of target gene The present invention performs codon optimization on the nucleotide sequence of humanized elastin; The humanized elastin, the nucleotide sequence after optimization is shown in SEQ ID NO.1 or SEQ ID NO.2, and the nucleotide sequence before optimization is shown in SEQ ID NO.3; The amino acid sequence of the humanized elastin is shown in SEQ ID NO.4.

[0016] In the nucleotide sequences shown in SEQ ID NO.1 to SEQ ID NO.3, CCATGG is an NcoI restriction site, and AAGCTT is a HindIII restriction site.

[0017] (2) Construction of recombinant plasmid The nucleotide sequences of humanized elastin as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3 were entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. for gene synthesis to obtain PUC57-rhELN-optimized 1, PUC57-rhELN-optimized 2 and PUC57-rhELN-optimized pre-plasmids; The plasmid pET28a (+) was double-digested with NcoI + HindIII with the plasmids of PUC57-rhELN-optimized 1, PUC57-rhELN-optimized 2 and PUC57-rhELN-before optimization, respectively. The double-digestion products were recovered and ligated, and then transformed into Escherichia coli DH5α competent cells. After resistance screening, the positive transformants were picked out for culture, and sequencing was performed after PCR identification. The sequencing results were correct, and the recombinant plasmids pET28a (+) -rhELN-optimized 1, pET28a (+) -rhELN-optimized 2 and pET28a (+) -rhELN-before optimization were extracted using a plasmid mini-extraction kit.

[0018] (3) Construction of genetically engineered bacteria expressing recombinant humanized elastin Take 1μL of recombinant plasmid (concentration of 1μg / μL) and mix with 100μL of thawed E. coli BL21 (DE3) competent cell suspension, put it in an ice bath for 30min, heat shock it in a 42℃ water bath for 90s, and then put it in an ice bath for 5min. Then add 400μL of LB medium without resistance, and culture it in a shaking incubator at 37℃ and 180rpm for 40min. Take 200μL of bacterial solution and spread it evenly on LB agar plate containing 50μg / mL kanamycin, and invert it to culture it overnight at 37℃. The next day, pick a single colony of transformation and inoculate it into a test tube containing 5mL LB liquid medium (the final concentration of Kana is 50μg / mL). After overnight culture at 37℃ and 180rpm, extract the plasmid of the bacterial solution, and if it is correct by nucleotide sequencing, the genetically engineered bacteria expressing recombinant humanized elastin are obtained.

[0019] The recombinant plasmids pET28a(+)-rhELN-optimized 1, pET28a(+)-rhELN-optimized 2 and pET28a(+)-rhELN-optimized before were constructed according to the above method to obtain genetically engineered bacteria BL21-pET28a(+)-rhELN-optimized 1, BL21-pET28a(+)-rhELN-optimized 2 and BL21-pET28a(+)-rhELN-optimized before.

[0020] pET28a(+) was constructed according to the above method to obtain the empty genetic engineering bacteria BL21-pET28a(+).

[0021] (4) Induced expression of genetically engineered bacteria A single colony of the genetically engineered bacteria was selected and inoculated into 5 mL of LB (the final concentration of Kana was 50 μg / mL) liquid medium. The culture was shaken at 37°C and 180 rpm overnight. 300 μL of the overnight bacterial solution was aspirated and inoculated into a triangular flask containing 30 mL of LB (the final concentration of Kana was 50 μg / ml) liquid medium. The culture was continued for 2.5 h until the OD 600 Reach 0.68, add inducer IPTG to make its final concentration 0.2mM, the induction temperature is 37℃, the induction time is 5h, after the induction, take 1mL of bacterial solution, centrifuge at 12000rpm, 4℃ for 2min, discard the supernatant, add 500μL ddH2O to resuspend, take 40μL of the resuspension, add 10μL of 5× loading buffer, mix well, boil in boiling water for 5 minutes, centrifuge instantly, take 20μL of the liquid for 12% separation gel SDS-PAGE protein electrophoresis to detect the expression level.

[0022] The genetically engineered bacteria BL21-pET28a (+)-rhELN-optimized 1, BL21-pET28a (+)-rhELN-optimized 2, BL21-pET28a (+)-rhELN-optimized before, and the genetically engineered bacteria BL21-pET28a (+) without loading were induced to express according to the above method, and the protein electrophoresis results were shown in Figure 1 shown.

[0023] Recombinant humanized elastin has a specific band at 35-40KD (the theoretical molecular weight is 31KD, and the apparent molecular weight is larger than the theoretical molecular weight). Figure 1 It can be seen that after the empty genetically engineered bacteria BL21-pET28a (+) were induced, the supernatant obtained by breaking the bacteria had no specific bands, and the expression level of recombinant humanized collagen by the genetically engineered bacteria BL21-pET28a (+)-rhELN-optimized 1 was much higher than that of BL21-pET28a (+)-rhELN-optimized 2 and BL21- pET28a (+)-rhELN-optimized before.

[0024] (5) Fermentation culture of genetically engineered bacteria 200 μL of the bacterial solution of the genetically engineered bacteria identified correctly in step (3) was inoculated into a flask containing 65 mL of LB medium and cultured at 200 rpm and 37°C for 12 h to obtain primary seeds; 6 mL of the primary seeds were inoculated into a flask containing 130 mL of LB medium and cultured at 200 rpm and 37°C for 8 h; all 130 mL of the bacterial solution was inoculated into a fermenter containing 2.5 L of TB medium and cultured at 37°C and 1000 rpm until the OD 600 When the pH value reached 30, IPTG was added to a final concentration of 0.2 mM and induced for 5 h to end the fermentation and obtain the fermentation broth of the genetically engineered bacteria.

[0025] (6) Protein purification The fermentation broth of each genetically engineered bacterium in the above step (5) was centrifuged at 8000 rpm for 30 min, and then the precipitated bacteria were collected. 7 mL of 0.2 M NaCl aqueous solution was added to 1 g of bacteria to dissolve the bacteria. A Naton high-pressure homogenizer was used to perform low-pressure homogenization once (400 bar) and high-pressure homogenization three times (800 bar). After centrifugation at 8000 rpm for 30 min, the supernatant was collected, monohydrated citric acid was slowly added to control the pH to a specific condition (pH = 4.2 ± 0.3), and then centrifuged again under the same conditions. After centrifugation, 20% saturated ammonium sulfate was slowly added to the supernatant to completely dissolve it. After centrifugation at 8000 rpm for 30 min, the supernatant was collected for the next experiment.

[0026] The sample was purified using the Bio-Lab laboratory chromatography system instrument of Hanbang Technology Company and the Rigose MMC Hires (High Sub) cationic mixed mode high-strength agarose medium self-packed column of Tofflon Company to obtain the purified protein stock solution.

[0027] The recombinant humanized elastin expressed by the genetically engineered bacteria BL21-pET28a (+) -rhELN-optimized 1, BL21-pET28a (+) -rhELN-optimized 2, and BL21-pET28a (+) -rhELN-optimized before was purified according to the above method to obtain purified protein stock solutions, which were labeled as rhELN-optimized 1, rhELN-optimized 2, and rhELN-optimized before, and the purity was higher than 90%.

[0028] (7) Freeze-drying The purified protein stock solution was freeze-dried using a vacuum freeze dryer from Haier Bio to obtain recombinant humanized elastin freeze-dried powder. The freeze-drying procedure is shown in Table 1.

[0029] Table 1 Vacuum freeze drying process

[0030] Example 2 Stability test of recombinant humanized elastin freeze-dried powder The recombinant humanized elastin freeze-dried powders of rhELN-optimized 1, rhELN-optimized 2 and rhELN-pre-optimized were placed at 25°C, 37°C and 50°C for 0 h, 8 h, 24 h, 48 h and 30 d, respectively, and then prepared into a 10 mg / mL solution with purified water. The purity was determined by reversed-phase high performance liquid chromatography. The results are shown in Table 2.

[0031] Table 2 Purity determination results of protein freeze-dried powder

[0032] As can be seen from Table 2, when placed at 25°C, 37°C and 50°C for 0h, 8h, 24h, 48h and 30d, the purity of protein freeze-dried powder rhELN-optimized 2 and rhELN-pre-optimized decreased rapidly, while the purity of rhELN-optimized 1 remained almost unchanged, indicating that it has good stability and is not easy to degrade.

[0033] Example 3 Stability test of recombinant humanized elastin freeze-dried powder solution Recombinant humanized elastin freeze-dried powder rhELN-optimized 1, rhELN-optimized 2, and rhELN-optimized before were prepared with purified water to prepare 10 mg / mL solutions, and then placed at 25°C, 37°C, and 50°C for 0 h, 8 h, 24 h, 48 h, and 30 d, and the purity was determined by reversed-phase high performance liquid chromatography. The results are shown in Table 3.

[0034] Table 3 Purity determination results of protein freeze-dried powder solution

[0035] As can be seen from Table 3, when the protein lyophilized powder solutions of rhELN-optimized 1, rhELN-optimized 2 and rhELN-before optimization were placed at 25°C, 37°C and 50°C for 0h, 8h, 24h, 48h and 30d, the purity of the protein lyophilized powder solutions of rhELN-optimized 2 and rhELN-before optimization decreased rapidly, while the purity of the protein lyophilized powder solution of rhELN-optimized 1 remained almost unchanged, indicating that it has good stability and is not easy to degrade.

[0036] Example 4 Cytotoxicity Experiment The cytotoxicity was detected by MTT method. Human skin immortalized fibroblasts (HSF) in logarithmic growth phase were cultured at 2.0×10 5 100 μL / well was inoculated into a 96-well plate and cultured at 37°C, 5% CO2 for 24 h. The drug was administered when the cell plating rate in the 96-well plate reached 50%.

[0037] A. Sample group: 100 μL of FM medium containing lyophilized protein powder of different concentrations (10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.313 mg / mL, 0.156 mg / mL, 0.078 mg / mL) was added to each well; B, control group (NC): add 100 μL of FM medium; C, blank group (BC): no cells, only 100 μL FM medium was added; After 24 hours of culture, discard the solution and replace it with a culture medium containing MTT (0.5 mg / mL, final concentration), 100 μL / well, and continue to culture for 4 hours. Discard the solution and add 100 μL / well of DMSO, shake thoroughly, develop color, and detect OD with an enzyme reader. 490 , the relative cell viability was calculated according to formula (1):

[0038] Cytotoxicity criteria: If the cell survival rate is greater than 70%, it is considered to be free of cytotoxicity; otherwise, it is considered to be cytotoxic.

[0039] The results of relative cell viability are shown in Table 4.

[0040] Table 4 Results of relative cell viability

[0041] It can be seen that when the protein lyophilized powder solutions of rhELN-optimization 1, rhELN-optimization 2, and rhELN-before optimization were at a concentration of ≤10.0 mg / mL, the cell survival rate was greater than 70% after acting on HSF cells for 24 hours, and there was no toxicity to the cells.

[0042] Example 5 Cell migration promotion experiment HSF cells in the logarithmic growth phase were cultured at 1×10 6 The cells were inoculated into 6-well plates at a density of cells / well and incubated overnight in an incubator (37°C, 5% CO2). The scratch test was performed when the plating rate reached more than 70%. Use a 10μL pipette tip to make two vertical scratches in the six-well plate. The vertical scratches were used as the baseline (the pipette tip was perpendicular to the edge of the ruler), and the distance between the scratches was kept at 2cm; after making the vertical scratches, scratch horizontally near the center axis of the six-well plate perpendicular to the baseline. After the scratches, the cells were washed three times with PBS. The dosing concentration of rhELN-optimized-rhLOX, rhELN-pre-optimized-rhLOX, and rhELN-optimized protein lyophilized powder was 0.156mg / mL, and the dosage per well was 2mL. Two replicate wells were set up for each group. The culture was continued in an incubator (37°C, 5% CO2) for 24h. After taking pictures of each migration group using an inverted microscope (see Figure 2 ), the migration rate of cells in the negative control group was normalized, and the relative migration rate of each group was calculated. The results are shown in Table 5 and Figure 3 .

[0043] Table 5 Cell migration results

[0044] As shown in Table 5, rhELN-optimized 1 can significantly improve the relative migration rate of cells compared with rhELN-optimized 2 and rhELN-before optimization.

[0045] Example 6 Anti-aging and anti-wrinkle experiment HSF cells in the logarithmic growth phase were cultured at 5×10 5 The cells were inoculated into 6-well plates at a density of 100 cells / well and incubated overnight in an incubator (37°C, 5% CO2). When the cell plating rate in the 6-well plate reached 45%, rhELN-optimized 1, rhELN-optimized 2, and rhELN-optimized pre-lyophilized powder were administered at a concentration of 0.156 mg / mL, with a dosage of 2 mL per well and 1 well per group. After the administration, the 6-well plate was placed in an incubator (37°C, 5% CO2) and incubated for 24 hours.

[0046] Increase the total RNA of cells and use ultra-micro spectrophotometer to detect the purity of extracted RNA. 260 / OD 280 The values ​​are all between 1.8 and 2.0, and the next step of reverse transcription experiment can be carried out.

[0047] The RNA was reverse transcribed to obtain reverse transcription products, and real-time quantitative PCR experiments were performed to detect the expression levels of genes h-ELN, h-COL1A1 and h-COL3A1 related to cell anti-aging and anti-wrinkle.

[0048] The results of fluorescent quantitative PCR gene detection are shown in Table 6 and Figure 4 The results showed that the highly stable humanized elastin rhELN-optimized 1 of the present invention can significantly increase the expression levels of h-ELN, h-COL1A1 and h-COL3A1 genes in HSF cells compared with rhELN-optimized 2 and rhELN-before optimization, indicating that the highly stable humanized elastin rhELN-optimized 1 of the present invention has good anti-aging and anti-wrinkle effects.

[0049] Table 6 Results of fluorescent quantitative PCR gene detection

Claims

1. A highly stable recombinant humanized elastin, characterized in that: The nucleotide sequence of the recombinant humanized elastin is shown in SEQ ID NO.

1.

2. The highly stable recombinant humanized elastin according to claim 1, characterized in that: The amino acid sequence of the recombinant humanized elastin is shown in SEQ ID NO.

4.

3. The method for preparing the recombinant humanized elastin according to claim 1 or 2, characterized in that: The nucleotide sequence of the recombinant humanized elastin is used to construct a recombinant plasmid, which is then transferred into competent Escherichia coli cells and induced to express to obtain the recombinant humanized elastin.

4. A genetically engineered bacterium expressing the recombinant humanized elastin according to claim 1.

5. Use of the recombinant humanized elastin according to claim 1 or 2 in the preparation of skin care products.

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