Recombinant cubilose peptide and application thereof

Abstract

The invention discloses a recombinant cubilose peptide and application thereof, and belongs to the technical field of recombinant peptide preparation. The amino acid sequence of the recombinant cubilose peptide is as shown in SEQ ID NO.1, and the nucleotide sequence for coding the recombinant cubilose peptide is as shown in SEQ ID NO.2. The preparation method of the recombinant cubilose peptide comprises the following steps: (1) cloning a nucleotide sequence for coding the recombinant cubilose peptide into a prokaryotic or eukaryotic expression vector to construct a recombinant expression plasmid; (2) transforming or transfecting the recombinant expression plasmid into a proper expression host cell, wherein the expression host cell is a prokaryotic cell, a eukaryotic cell or a yeast cell; (3) culturing the expression host cell, and performing induced expression or expression under proper conditions; (4) collecting an expression product, crushing or cracking, and centrifuging to obtain a supernatant or precipitate containing the recombinant cubilose peptide; and (5) purifying the recombinant cubilose peptide. The cubilose peptide is prepared by using a recombinant expression technology, the cubilose peptide has remarkable anti-aging, tightening and moisturizing effects, and a foundation is laid for industrial production of the cubilose peptide.

Description

Technical Field

[0001] This invention belongs to the field of recombinant peptide preparation technology, and particularly relates to a recombinant bird's nest peptide and its application. Background Technology

[0002] Bird's nest is made from the saliva secreted by swiftlets (Aerodramus genus). Since ancient times, it has been regarded as a precious tonic in Traditional Chinese Medicine, widely used for strengthening the body and prolonging life. Modern research shows that bird's nest is rich in glycoproteins, sialic acid, amino acids, and various trace elements such as calcium, iron, potassium, and magnesium. It is a natural food with both nutritional and functional properties. Its main components are glycoproteins (approximately 50-60%) and carbohydrates (approximately 10%). Sialic acid (N-acetylneuraminic acid), epidermal growth factor-like substances, and antioxidant peptides are considered key active factors promoting health, possessing various biological functions such as cell repair, immune regulation, and anti-aging.

[0003] Recent in vitro and in vivo studies have shown that bird's nest possesses significant immunomodulatory and anti-inflammatory capabilities. Experiments have demonstrated that bird's nest can promote the proliferation of human peripheral blood mononuclear cells and increase the secretion of various cytokines, thus helping to regulate the human immune system. Sialic acid plays an important role in immune cell signaling and pathogen recognition, enhancing the mucosal immune barrier and improving antiviral defense capabilities. Simultaneously, bird's nest exerts anti-inflammatory effects by inhibiting the production of pro-inflammatory factors such as tumor necrosis factor-alpha, interleukin-6, and nitric oxide, showing potential application value in the intervention of chronic inflammatory diseases and immune disorders.

[0004] Bird's nest shows broad application prospects in the medical field, especially in clinical nutritional support, suitable for postoperative recovery, during tumor radiotherapy and chemotherapy, chronic disease management, and rehabilitation for the elderly. Its proteins and sialic acid help with tissue repair and immune reconstruction, reducing treatment-induced fatigue and immunosuppression. Furthermore, research has found that bird's nest has antioxidant and neuroprotective effects, potentially delaying the onset of neurodegenerative diseases such as Alzheimer's. In respiratory health, the sialic acid in bird's nest can block the binding of viruses to respiratory epithelial cells, exhibiting a certain natural inhibitory effect on influenza viruses. In maternal and child nutrition, bird's nest is widely used for nutritional conditioning during pregnancy and postpartum, not only aiding fetal brain development but also promoting rapid maternal recovery. Currently, the active ingredients in bird's nest are being used in the research and development of natural medicines and immune enhancers, demonstrating its cross-disciplinary potential from traditional tonics to modern medicine.

[0005] In the health and nutrition field, bird's nest is highly favored as a traditional tonic, often used in soups, desserts, and functional beverages. Modern processing techniques have driven the development of various product forms, such as ready-to-drink bird's nest, freeze-dried bird's nest powder, and concentrated capsules, meeting modern consumers' demand for convenient nutrition. Clinical and user feedback indicates that long-term consumption of bird's nest helps improve physical strength, enhance immune function, and improve skin tone and blood circulation. Therefore, it holds an important position in the nutritional supplement market, and is particularly suitable for people with low immunity, physical weakness and fatigue, or those recovering from surgery.

[0006] Furthermore, the value of bird's nest in the beauty and skincare field is becoming increasingly prominent. Its epidermal growth factor-like substances and collagen-boosting active peptides can significantly improve skin hydration and elasticity, reduce wrinkles, and accelerate cell repair and regeneration. Bird's nest extract is widely used in high-end skincare products, such as masks, serums, and anti-aging creams, becoming a popular natural, safe, and multi-functional beauty ingredient, highly favored by consumers seeking natural skincare and delayed skin aging.

[0007] Currently, the main methods for obtaining bird's nest and its active peptides include hot water extraction, enzymatic hydrolysis, and ultrasound-assisted extraction. Among these, hot water extraction is the most traditional and widely used method. It typically involves extracting the soluble glycoproteins and peptides from the soaked bird's nest by boiling it in hot water or steaming it under high pressure. While this method is simple to operate, it has significant technical limitations. The main issue is that the high temperature environment can easily destroy heat-sensitive active ingredients (such as epidermal growth factor-like substances), thus reducing the product's functionality. Furthermore, the extraction efficiency is limited, protein recovery is low, and the high-temperature processing can easily cause component denaturation and aggregation, affecting the quality stability of the final product.

[0008] Enzymatic hydrolysis is a modern biotechnology method that has been widely used in the development of functional foods in recent years. This method utilizes specific proteases (such as trypsin, pepsin, or complex enzymes) to target and hydrolyze glycoproteins in bird's nest, obtaining bird's nest peptides with smaller molecular weights and specific physiological activities. Although enzymatic hydrolysis offers high bioavailability and functional retention, this technology requires very strict conditions for the enzymatic hydrolysis reaction. Parameters such as temperature, pH, and enzyme concentration must be precisely controlled; otherwise, over-hydrolysis or loss of activity may occur. Furthermore, enzyme sources are costly, the process system is complex, and stability is poor during scale-up production. Additionally, there is currently a lack of efficient methods for separating and purifying specific functional peptides, making it difficult to ensure product consistency and standardization.

[0009] Ultrasonic-assisted extraction (ALE) is an emerging auxiliary technology, often used in conjunction with hot water extraction or enzymatic hydrolysis. This method utilizes the cavitation and mechanical vibration generated by high-frequency ultrasound to break down the structure of bird's nest tissue, significantly increasing the release rate of active ingredients. Although ALE has demonstrated good extraction efficiency and energy-saving advantages in laboratory studies, its industrial application still faces many challenges. This technology requires extremely precise control of ultrasonic power, frequency, and processing time; even slight errors can damage the spatial structure of target peptides, affecting their functional activity. Furthermore, the high investment cost of large-scale equipment, coupled with the risk of localized overheating or the release of metal impurities during the ultrasound process, also poses a potential impact on product quality and safety.

[0010] The preparation of bird's nest peptides using recombinant expression technology has significant advantages over traditional extraction and enzymatic hydrolysis methods in terms of efficiency, standardization, cost control, and functional preservation, and can largely solve the key technical bottlenecks of the aforementioned methods.

[0011] First, recombinant expression technology enables the efficient and targeted production of target functional peptides, avoiding the problems of low product purity and inconsistent composition caused by factors such as differences in raw materials and unstable extraction conditions in traditional extraction methods. For example, in natural bird's nest, the content of epidermal growth factor-like peptides or specific active peptides is extremely low, making them difficult to separate and purify using hot water or enzymatic hydrolysis. However, through recombinant DNA technology, expression vectors can be constructed in E. coli, yeast, or mammalian cells to achieve quantitative production of specific functional peptides, greatly improving the yield and purity of active ingredients and facilitating product standardization and industrial-scale production.

[0012] Secondly, recombinant technology allows for better preservation of structure and function. Traditional high-temperature extraction often causes denaturation and inactivation of heat-sensitive proteins (such as sialic acid-binding proteins and EGF-like peptides), while recombinant technology allows for the expression and purification of functional peptides under milder conditions, thereby better preserving their native conformation and biological activity. This is of great significance for ensuring the immunomodulatory, antioxidant, or anti-inflammatory effects of the product.

[0013] Third, the preparation of recombinant bird's nest peptides offers better safety and traceability. Natural bird's nests are subject to issues such as origin, batch, and environmental pollution (e.g., nitrites, heavy metals), while recombinant peptides have a clear and controllable source, do not rely on animal resources, and avoid the risks of microbial contamination or allergies. This better meets the requirements of the modern pharmaceutical, cosmetic, and functional food industries for "high purity and low risk."

[0014] Furthermore, in the long term, recombination technology can also help reduce costs and enhance sustainability. Natural bird's nest resources are scarce and expensive, and are subject to significant environmental and ecological policy restrictions. Microbial expression systems, such as E. coli or yeast, have advantages such as rapid growth and low fermentation costs, which can significantly reduce raw material costs and increase production capacity, thus promoting industrialization. Summary of the Invention

[0015] This invention provides a recombinant bird's nest peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0016] In one embodiment of the present invention, the nucleotide sequence encoding the recombinant bird's nest peptide is shown in SEQ ID NO.2.

[0017] The present invention also provides a recombinant DNA expression vector comprising a gene sequence encoding the above-mentioned recombinant bird's nest peptide.

[0018] The present invention also provides a recombinant DNA expression vector comprising a nucleotide sequence encoding the above-mentioned recombinant bird's nest peptide; the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

[0019] The present invention also provides an expression system for expressing the above-mentioned recombinant bird's nest peptide, the expression system including a prokaryotic expression system, a eukaryotic expression system or a yeast expression system.

[0020] The present invention also provides a method for preparing the above-mentioned recombinant bird's nest peptide, which includes the following steps: (1) cloning the nucleotide sequence encoding the above-mentioned recombinant bird's nest peptide into a prokaryotic or eukaryotic expression vector to construct a recombinant expression plasmid; (2) transforming or transfecting the recombinant expression plasmid into a suitable expression host cell, wherein the expression host cell is a prokaryotic cell, a eukaryotic cell or a yeast cell; (3) culturing the expression host cell and inducing expression or expressing under suitable conditions; (4) collecting the expression product and treating it by crushing or lysing, and centrifuging to obtain a supernatant or precipitate containing the recombinant bird's nest peptide.

[0021] In one embodiment of the present invention, the expression host cell is selected from Escherichia coli BL21, Rosetta strain, Pichia pastoris, Saccharomyces cerevisiae, CHO cells, HEK293 cells or Sf9 cells.

[0022] In one embodiment of the present invention, the fermentation medium used to culture the expression host cells in step (3) is composed of: glucose 30.0 g / L, yeast extract 50.0 g / L, dipotassium hydrogen phosphate 8.7 g / L, sodium dihydrogen phosphate 4.2 g / L, ammonium sulfate 5.5 g / L, magnesium sulfate 2.5 g / L, ethylenediaminetetraacetic acid 1.0 g / L, trace element solution 1.0 g / L, and defoamer 0.1 g / L; the trace element solution is composed of: manganese sulfate (MnSO4·H2O) 3.0 g, ferric sulfate (FeSO4·7H2O) 2.0 g, zinc sulfate (ZnSO4·7H2O) 0.5 g, copper sulfate (CuSO4·5H2O) 0.1 g, potassium iodide (KI) 0.05 g, boric acid (H3BO3) 0.3 g, and sodium molybdate (Na2MoO4·2H2O). 0.05 g, diluted to 1 L with deionized water.

[0023] In one embodiment of the present invention, a protein purification step is further included after step (4).

[0024] This invention also provides the application of the above-mentioned recombinant bird's nest peptide in the preparation of medical nutritional preparations, immunomodulators, neuroprotective agents, anti-inflammatory or antioxidant preparations, skin care products, beverages, health products and other products with cell repair, anti-aging or enhanced health functions.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] This invention utilizes recombinant expression technology to prepare bird's nest peptides, laying the foundation for the industrial production of bird's nest peptides. Attached Figure Description

[0027] Figure 1 The results of SDS-PAGE identification of BN01 protein expression in Example 1 are shown, where M: protein molecular weight standard, 1: uninduced, 2: induced, 3: supernatant after induction and lysis, and 4: precipitate after induction and lysis.

[0028] Figure 2 The SDS-PAGE results for identifying the purification of BN01 protein in Example 1 are shown below. In the SDS-PAGE results, M represents the molecular weight standard of the protein, 1 represents the sample after cleavage, 2 represents the runoff, 3 represents DEAE elution, and 4 represents the elution from secondary molecular purification.

[0029] Figure 3The results show the statistical results of HSF cell viability in the blank control group (0% sample) and the experimental groups (0.05%, 0.1%, 0.25%, 0.5%, and 1% samples) in Example 2. Statistical analysis was performed using GraghPad 8.3.0. ns represents no significant difference, * represents p < 0.05, and ** represents p < 0.01.

[0030] Figure 4 This is a bar chart comparing β-galactosidase activity (OD value) in Example 3. Statistical analysis was performed using GraghPad 8.3.0. ** represents P < 0.01, and *** represents P < 0.001.

[0031] Figure 5 This is a bar chart comparing the absorbance (OD value) of elastase activity in Example 4. Statistical analysis was performed using GraghPad 8.3.0. ** represents P < 0.01, and *** represents P < 0.001.

[0032] Figure 6 This is a bar chart comparing the relative expression levels of the AQP-3 gene in Example 6. Statistical analysis was performed using GraghPad 8.3.0. *** represents P < 0.001. Detailed Implementation

[0033] Example 1: Construction, expression, and purification of BN01 vector

[0034] 1. Carrier Construction

[0035] The amino acid sequence of the recombinant bird's nest peptide is (SEQ ID NO.1):

[0036] MGGGDLAYLDQGHRLFWSPSVYLKNPPADLHKGGGGSGGSLMITAAVAAGLSNIQAGYQIAELGKGGGSGGGGSYLDYFHVMTYGWPHLEDNYLDWFRAFDSNVIETEQSHVEEVR.

[0037] The corresponding nucleotide sequence (SEQ ID NO.2) is:

[0038] ATGGGTGGTGGCGATCTGGCATACCTGGACCAAGGTCATCGTCTGTTCTGGTCTCCTTCTGTCTACCTGAAGAACCCGCCGGCAGATCTGCATAAAGGTGGTGGTTCTGGCGGCGGTAGCCTGATGATCACTGCAGCTGTCGCTGCCGGCCTGTCTAACATTCAGGCGGGCTAT CAGATTGCAGAGCTGGGTAAAGGTGGTGGCTCTGGTGGTGGTGGCTCCTACCTGGACTACTTCCACGTTATGACTTACGGCTGGCCGCACCTGGAAGACAACTACCTGGATTGGTTCCGTGCCTTCGACTCCAACGTGATCGAAACCGAACAGTCTCATGTCGAGGAAGTCCGC.

[0039] The nucleotide sequence corresponding to the above amino acid sequence was inserted between NdeI and XhoI in the pET41a vector, named BN01. In a biosafety cabinet, 0.5 μL of the successfully constructed plasmid was transformed into competent cells (e.g., BL21, Rosetta, etc.). A 1.5 mL centrifuge tube containing competent cells and plasmid was incubated on ice for 5 min, then heat-shocked at 42°C for 45 s, followed by an ice incubation for 10 min. After the ice incubation, 900 μL of LB liquid medium (10 g / L, yeast extract 5 g / L, NaCl 10 g / L) was added to the 1.5 mL centrifuge tube in a biosafety cabinet. The tube was then placed in a shaker at 37°C, 100 rpm for 2 h. After 2 h, the tube was centrifuged at 900 Xg at room temperature for 3 min. The supernatant was discarded in a biosafety cabinet, and 100 μL of supernatant was gently mixed by pipetting. Using a spreader, evenly spread kanamycin at a final concentration of 50 μg / mL onto LB agar plates (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L Agar, brought to a final volume of 100 mL, and sterilized at high temperature). Take 20 μL of the mixed product and spread it onto the plates. After spreading, incubate overnight at 37 °C.

[0040] 2. Fermentation expression of BN01

[0041] Single colonies were picked from LB solid medium and inoculated into 50 mL Erlenmeyer flasks containing 10 mL of seed medium. The flasks were incubated at 32°C and 200 rpm for 10–12 hours to obtain the first-generation seed culture. Subsequently, the resulting seed cultures were transferred to 1000 mL Erlenmeyer flasks containing 300 mL of LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) and incubated under the same conditions for another 10–12 hours to prepare the second-generation seed culture.

[0042] The cultured second-generation seed culture was inoculated into 3.5 L of fermentation medium (glucose 30.0 g / L, yeast extract 50.0 g / L, dipotassium hydrogen phosphate 8.7 g / L, sodium dihydrogen phosphate 4.2 g / L, ammonium sulfate 5.5 g / L, magnesium sulfate 2.5 g / L, ethylenediaminetetraacetic acid 1.0 g / L, and trace element solution (trace element solution (1 L): manganese sulfate (MnSO4·H2O) 3.0 g, ferric sulfate (FeSO4·7H2O) 2.0 g, zinc sulfate (ZnSO4·7H2O) 0.5 g, copper sulfate (CuSO4·5H2O) 0.1 g, potassium iodide (KI) 0.05 g, boric acid (H3BO3) 0.3 g, sodium molybdate (Na2MoO4·2H2O) 0.05 g, diluted to 1 L with deionized water) 1.0 g / L. In a 6 L fermenter containing 0.1 g / L of antifoam 204 (Sigma-Aldrich), the primary fermentation stage was controlled at 37°C. During the batch feeding and feed-by-feed stages, dissolved oxygen (DO) levels were maintained at 20% of air saturation by adjusting air flow, increasing stirring speed, and applying appropriate pressure. The pH of the fermentation broth was automatically adjusted to 6.8 using 25% ammonia (w / w) (this pH was maintained throughout the fermentation process). Once the glucose in the tank was completely consumed, a near-exponential feed-by-feed strategy (1000.0 g / L glucose, 400.0 g / L yeast extract, 26 g / L dipotassium hydrogen phosphate, 12.6 g / L sodium dihydrogen phosphate, 16.5 g / L ammonium sulfate, 20 g / L magnesium sulfate, 3.0 g / L EDTA, and 3.0 g / L trace element solution) was employed to maintain a steady specific growth rate.

[0043] When the bacterial cell optical density (OD) 600When the concentration reaches 20, add 1 mM IPTG, then lower the temperature to 25℃ to initiate induction expression, and continue induction for 16 hours to complete the expression of the target protein. Take 20 μL of bacterial culture and centrifuge at 4℃, 13000 rpm, 5 min. After centrifugation, discard the supernatant, add 20 μL of protein loading buffer, mix well by pipetting, and then boil at 98℃ for 10 min using a constant temperature mixer (heating type). Take 5 μL of sample for loading and perform SDS-PAGE detection and observation.

[0044] 3. Cell disruption

[0045] Before cell lysis, the fermented cells were first recovered by centrifugation at 4°C and 6,000g for 20 minutes. Then, the cells were thoroughly resuspended in pre-chilled lysis buffer (50mM Tris-HCl, 150mM NaCl, 5% glycerol, pH 7.5, with 1mM PMSF protease inhibitor added) at a ratio of 1g wet weight cells to 5mL buffer. The entire process must be performed on ice to maintain a low temperature and prevent protein degradation.

[0046] Transfer the resuspended cell solution to a pre-cooled stainless steel storage tank, ensuring that the feed temperature of the high-pressure homogenizer is below 8°C. Set the pressure to 800 bar during homogenization and perform 2 to 3 cycles. After each cycle, take a small sample and observe it under a microscope to determine the degree of cell disruption until more than 90% of the cells are completely lysed.

[0047] After homogenization, the lysis buffer was immediately centrifuged at 4°C and 13,000g for 30 minutes, and the soluble protein supernatant was collected for subsequent purification experiments.

[0048] In addition, take 20 μL of homogenized sample, centrifuge at 13,000 rpm for 5 minutes at 4℃, and transfer the supernatant to a new centrifuge tube. Add 20 μL of protein loading buffer to each supernatant, mix thoroughly by pipetting, and then place in a heated mixer (heated type) at 98℃ for 10 minutes to boil the sample. Take 5 μL of the treated sample for SDS-PAGE electrophoresis to observe the expression of soluble proteins. The results are shown in [link to results]. Figure 1 .

[0049] 4. Protein purification

[0050] In the protein purification experiment, the completely lysed soluble protein solution was first centrifuged at 4°C (13,000 rpm, 30 minutes) to remove cell debris and insoluble impurities. The supernatant after centrifugation was then filtered through a 0.45 μm filter membrane to further remove particulate matter that was not completely removed after high-speed centrifugation. After obtaining a clear protein solution, the entire purification process was carried out at low temperature to ensure that the protein activity was not destroyed.

[0051] Protein purification was performed using a protein chromatography system equipped with a DEAE weak anion exchange column (10 mL column volume). First, the column was pre-equilibrated with equilibration buffer (Buffer A: 25 mM Tris, 10% glycerol, pH 8.0) at a flow rate of 1.5 mL / min, for a total equilibration volume of five column volumes, to ensure a stable and consistent column environment. Subsequently, the filtered protein sample was loaded into the column with sample buffer (same as Buffer A) at the same flow rate.

[0052] The elution stage employed a linear gradient elution mode, achieving protein separation and elution by gradually increasing the NaCl concentration. Specifically, Buffer B (25 mM Tris, 10% glycerol, 1 M NaCl, pH 8.0) containing 1 M NaCl was used in addition to Buffer A, with the concentration linearly increased from 0% to 100% within 20 column volumes, maintaining a flow rate of 1.5 mL / min throughout the elution process. The appearance and changes of protein elution peaks were monitored using UV absorption at 280 nm. The peak corresponding to the target protein was identified based on the spectrum, and the eluted fractions were collected in 3 mL increments. Subsequent SDS-PAGE electrophoresis was used to analyze the protein composition of the eluted fractions, determining the elution location and purity of the target protein.

[0053] The collected BN01 sample required further dialysis buffer replacement (MWCO 3, 500 Da dialysis bag). The protein solution was placed in a 25 mM Tris, 100 mM NaCl, 10% glycerol, pH 8.0 buffer solution and dialyzed three times at 4°C, changing the dialysate each time and maintaining the process for 4 hours to thoroughly remove small molecule components such as NaCl and glycerol from the protein solution. After dialysis, the sample was centrifuged at low speed and filtered through a 0.22 μm filter membrane to remove particulate impurities. Next, the BN01 was concentrated using an ultrafiltration concentrator with a molecular weight cutoff of 3,500 Da, ultimately concentrating the BN01 concentration to 10 mg / mL.

[0054] 5. Secondary purification using molecular sieves

[0055] In the secondary purification stage of BN01, a Sephadex G-200 molecular sieve chromatography column was used to further separate the proteins according to their molecular size. The dialyzed and concentrated protein sample was centrifuged at 12,000 rpm for 30 minutes at 4°C to remove any possible precipitates or aggregates, and then filtered through a 0.22 μm filter to obtain a clear supernatant. The processed BN01 was then loaded into a column fully equilibrated with Buffer C (25 mM Tris, 100 mM NaCl, 10% glycerol, pH 8.0).

[0056] The elution process employed isocratic elution mode, using the same Buffer C buffer throughout, and separation was performed at a flow rate of 1.0 mL / min. Protein elution peaks were monitored online using UV absorption (280 nm), and elution was performed sequentially according to molecular size. Protein fractions at different peak positions were collected during elution and analyzed by SDS-PAGE electrophoresis (see [link to SDS-PAGE analysis]). Figure 2 To confirm the molecular weight and purity of the target protein, the components containing the target protein were concentrated using an ultrafiltration concentrator with a molecular weight cutoff of 3,500 Da, ultimately concentrating the protein to a concentration of 10 mg / mL for subsequent functional studies.

[0057] Example 2 BN01 Cytotoxicity (MTT) Test

[0058] First, HSF cells were seeded in culture dishes and incubated in a constant temperature incubator at 37°C and 5% CO2. After the cells entered the logarithmic growth phase, they were digested and then seeded into 96-well plates to prepare a homogeneous cell suspension.

[0059] Subsequently, the test samples were diluted to five different concentration gradients with volume fractions of 1%, 0.5%, 0.25%, 0.1%, and 0.05%. When the cell deposition rate in the 96-well plates reached approximately 40%, the above-mentioned samples at different concentrations were added to the experimental group, and the cells were cultured at 37°C and 5% CO2.

[0060] After 24 hours of cell treatment, 10 μL of MTT reagent was added to each well, and the cells were incubated for another 4 hours in the dark. Then, 100 μL of formazan solvent was added to each well to dissolve the formed purple crystals, and incubation continued for another 2 to 4 hours. Finally, the absorbance (OD value) of each well was measured at 570 nm using a microplate reader.

[0061] Cell viability is calculated based on the measured OD value using the following formula: Cell viability = (OD value of experimental group - OD value of reagent control group) / (OD value of blank control group - OD value of reagent control group) × 100%.

[0062] By analyzing cell viability, plotting activity curves corresponding to sample toxicity, and simultaneously observing and recording cell morphological changes through image capture, this method can be used to assess the potential toxic effects of samples on cells.

[0063] In the experiment, the relative survival rates of cells in the 0.05%, 0.1%, and 0.25% concentration groups were all within the range of 80%-99% (referring to the cytotoxicity evaluation criteria in the United States Pharmacopeia), all meeting the acceptable standards. Significant differences in HSF cytotoxicity among the samples were compared (see...). Figure 3 The selected sample treatment concentrations were 0.05%, 0.1%, and 0.25%.

[0064] Table 1

[0065]

[0066] Example 3

[0067] BN01 Anti-aging Efficacy Test (Spectrophotometer Method)

[0068] (1) Cell seeding: Human fibroblasts (HSF) were seeded at a rate of 2 × 10⁶ cells / year. 5 Cells were seeded at a density of [number] cells / well in 6-well culture plates. The experimental setup included a normal control group, a model group, and a sample treatment group. Three concentrations were selected for the sample group based on MTT assay results. After seeding, cells were cultured at 37°C and 5% CO2 for 24 hours to ensure cell adhesion and growth.

[0069] (2) Construction of senescence model: After the cells adhered stably, medium containing 200 mM D-galactose was added to each well of the model group and the sample group, and the cells were cultured for 7 days to induce the senescence phenotype. The normal control group was cultured in the conventional medium.

[0070] (3) Sample intervention: After modeling, the sample groups were added to serum-free culture medium containing different concentrations of BNO1 solution to evaluate the anti-aging effect of different doses. The model group and the normal control group were added with an equal amount of serum-free culture medium as a reference. All groups were incubated in an incubator for 48 hours.

[0071] (4) Sample Collection and Detection: After culture, the cell culture supernatant from each well was collected for subsequent detection. Following the instructions of the BCA protein quantification kit, appropriate amounts of substrate buffer, test supernatant, and elastase solution were added sequentially to the 96-well plate. After thorough mixing, the plate was incubated at 37°C for 30 minutes. The reaction was then terminated by adding stop solution, and the absorbance (OD value) of each well was measured using a microplate reader at 405 nm. Finally, the β-galactosidase activity of each treatment group was calculated based on the standard curve (see [reference missing]). Figure 4 This allows for the evaluation of the anti-aging efficacy of the samples.

[0072] Anti-aging efficacy index *100%

[0073] Table 2 Statistical results of β-galactosidase activity (OD value)

[0074]

[0075] Table 3. Statistical analysis of the efficacy of BN01 on β-galactosidase activity in HSF cells.

[0076]

[0077] Example 4: BN01 Firming Efficacy Test (Spectrophotometer Method)

[0078] (1) Cell seeding: Human fibroblasts (HSF) were seeded at a rate of 2 × 10⁶ cells / year. 5 Cells were seeded at a density of [number] cells / well in 6-well plates, with a normal control group, a model group, and a sample treatment group. Three concentrations were selected for the sample groups based on the MTT cytotoxicity test results. After seeding, the cells were incubated at 37°C with 5% CO2 for 24 hours to ensure cell adhesion and adaptation to the environment.

[0079] (2) Model establishment: Serum-free culture medium containing elastase (final concentration of 50 μg / mL) was added to each well in the model group and sample group to simulate the skin relaxation caused by the degradation of elastic fibers; the normal control group was given an equal amount of serum-free culture medium as a baseline control. Each group was incubated in an incubator for 24 hours to complete the model establishment.

[0080] (3) Sample treatment: After modeling, the sample groups were added to serum-free culture medium containing different concentrations of BNO1 solution to evaluate its effect on improving cell tightening-related functions. The model group and the control group were added to the same amount of serum-free culture medium. All groups were cultured at 37℃ and 5% CO2 for 48 hours.

[0081] (4) Sample collection and detection: After culture, the cell culture supernatant of each well was collected. According to the instructions of the elastase assay kit, an appropriate amount of substrate buffer, test supernatant, and elastase solution were added to the 96-well plate in sequence, mixed well, and incubated at 37°C for 30 minutes. Then, the reaction was terminated by adding stop solution, and the absorbance (OD value) of each well was measured at 405 nm using a microplate reader to reflect the changes in cell elastase activity, thereby evaluating the firming effect of the sample.

[0082] *100%

[0083] Compared with the control group, the elastase activity of HSF cells in the model group was significantly increased, indicating the successful induction of the skin fibroblast damage model and a decrease in skin elasticity. Compared with the model group, the elastase activity of each treatment concentration group was significantly reduced, and the inhibitory effect of elastase increased with increasing concentration, thus revealing that BN01 solution has a significant firming effect by significantly inhibiting elastase activity. The elastase inhibition rate was 34.62% at a treatment concentration of 0.05%, 57.69% at a treatment concentration of 0.1%, and 73.08% at a treatment concentration of 0.25% (see [reference missing]). Figure 5 ).

[0084] Table 4. Statistical results of elastase activity (OD value)

[0085]

[0086] Table 5. Statistical analysis of the inhibitory efficacy of samples on HSF cell proteases.

[0087]

[0088] Example 5: Preparation of Facial Mask

[0089] Table 6

[0090]

[0091] Prepare the face mask according to the ingredients listed in the table above:

[0092] 1. Solution preparation

[0093] Place injection-grade purified water in a stainless steel mixing container and start the magnetic stirrer, maintaining a medium speed of 80 rpm / min. Slowly add glycerol while stirring, ensuring it is fully dissolved in the aqueous phase to form a homogeneous, transparent liquid. Then slowly sprinkle in hydroxyethyl cellulose while stirring continuously to prevent clumping. Stir for at least 30 minutes until a transparent colloidal substrate without noticeable bubbles or particles is formed. The viscosity of this substrate system is 300 mPa·s. After preparation, allow it to stand for 30 minutes to remove bubbles.

[0094] 2. Addition of functional ingredients

[0095] Add the various active ingredients sequentially. First, add the aseptically filtered BN01 and gently stir for 10 minutes to ensure even dispersion. Then, add β-glucan and sodium hyaluronate sequentially, maintaining stirring for 30 minutes to promote full dissolution and uniform distribution of each component. Avoid vigorous stirring during the process to prevent the introduction of air bubbles that could affect the transparency and stability of the final product. Note: The control group does not add BN01; any volume deficiency is made up with pure water.

[0096] 3. Osmotic pressure regulation and pH adjustment

[0097] After the functional components have dissolved evenly, add sodium chloride to adjust the osmotic pressure of the system to approximately 0.9%. Monitor the pH value of the system in real time using a precision pH meter, and fine-tune using 0.1 M NaOH or HCl solution to maintain the final pH around 6.5. After adjustment, continue stirring for 10 minutes, and finally filter to remove impurities (using a 0.22 μm membrane) before use.

[0098] 4. Filling and Aseptic Processing

[0099] Transfer the prepared membrane solution to a quantitative filling machine. Medical-grade facial mask sheets (silk), pre-sterilized with an electron beam of 15 kGy, should be placed into similarly sterilized mask bags. Fill each bag with 25 mL of membrane solution and seal using a heat-sealing device.

[0100] The packaged masks are placed in an isolation box and uniformly subjected to irradiation sterilization treatment (electron beam 15 kGy) to become sterile masks.

[0101] Example 6: Moisturizing Efficacy Test of BN01 Facial Mask (qRT-PCR Method)

[0102] (1) Cell resuscitation and culture: The HaCaT cell cryopreservation tubes were removed from liquid nitrogen and rapidly revived in a 37°C water bath. The cell suspension was then transferred to centrifuge tubes containing complete culture medium and centrifuged at 1,000 rpm for 5 minutes. After discarding the supernatant, the cells were resuspended in fresh complete culture medium and seeded into cell culture flasks. The cells were then cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%–90%, the cells were passaged.

[0103] (2) Modeling and drug administration: Cells in the logarithmic growth phase were digested with trypsin-EDTA and the cell density was adjusted to 5 × 10⁻⁶. 5 Cells were seeded at a density of 2 mL / well in 6-well plates and incubated for 24 hours to allow for full cell adhesion. Subsequently, the cells were divided into groups: the control group received fresh complete culture medium; the model group received medium containing 400 mM mannitol; and the sample group received medium containing different concentrations of BN01 compound mask solutions along with 400 mM mannitol. All groups were incubated for another 24 hours to establish a hyperosmolar injury model and to allow for sample intervention.

[0104] (3) RNA extraction: After culture, discard the culture medium and wash the cells 2–3 times with PBS buffer. Add 1 mL of Trizol reagent to each well to lyse the cells, mix thoroughly by pipetting, and transfer to a centrifuge tube. Add chloroform, vortex vigorously, and centrifuge to separate the layers. Take the upper aqueous phase and add an equal volume of isopropanol to precipitate RNA. After centrifugation, discard the supernatant, wash the RNA precipitate with 75% ethanol, air dry, and dissolve the RNA in DEPC-treated water. After determining the concentration and purity of the RNA, store it at -80℃ for later use.

[0105] (4) Reverse transcription reaction: Follow the instructions of the reverse transcription kit. Using total RNA as a template, add random primers, dNTP mixture, reverse transcription buffer, reverse transcriptase and other reaction components to form a 20 μL reaction system. Incubate at 42℃ for 60 minutes, then incubate at 70℃ for 10 minutes to terminate the reverse transcription reaction. Store the synthesized cDNA product at -20℃ for later use.

[0106] (5) Real-time quantitative PCR detection: Using the synthesized cDNA as a template, a 20 μL qPCR reaction system was constructed, including SYBR Green Master Mix, upstream and downstream primers, cDNA template, and ddH2O. Real-time quantitative PCR was performed according to the set PCR amplification program, and the expression level of the target gene AQP-3 was detected. Based on the Ct value, the relative expression level of the AQP-3 gene in each treatment group was calculated using the 2^(-ΔΔCt) method to evaluate the regulatory role of the samples in moisturizing function.

[0107] *100%

[0108] Compared with the control group, the relative expression level of AQP-3 gene in Hacat immortalized keratinocytes in the model group was significantly lower than that in the control group, indicating that the mannitol cell dehydration model was successfully constructed. Compared with the model group, the relative expression level of AQP-3 gene in the three concentration groups of the compound masks (compounds 1, 2, and 3) was significantly increased (see [link to sample group]). Figure 6 Furthermore, the concentration-dependent effect of the treatment concentration was observed, indicating that the sample could significantly increase the expression level of aquaporin genes in cells, restore the cells' water absorption and transport capabilities, and reveal a significant moisturizing effect.

[0109] Table 7. AQP-3 gene expression level test

[0110]

[0111] Table 8. Statistics on the Efficacy Index of BN01 Compound Mask

[0112]

[0113] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A recombinant bird's nest peptide, characterized in that, The amino acid sequence of the recombinant bird's nest peptide is shown in SEQ ID NO.

1.

2. The recombinant bird's nest peptide according to claim 1, characterized in that, The nucleotide sequence encoding the recombinant bird's nest peptide is shown in SEQ ID NO.

2.

3. A recombinant DNA expression vector, characterized in that, It contains a gene sequence encoding the recombinant bird's nest peptide as described in claim 1.

4. A recombinant DNA expression vector, characterized in that, The expression vector contains a nucleotide sequence encoding the recombinant bird's nest peptide of claim 1; the expression vector is a prokaryotic expression vector or a eukaryotic expression vector.

5. An expression system, characterized in that, The expression system is used to express the recombinant bird's nest peptide of claim 1, and the expression system includes a prokaryotic expression system, a eukaryotic expression system, or a yeast expression system.

6. A method for preparing the recombinant bird's nest peptide of claim 1, characterized in that, The process includes the following steps: (1) cloning the nucleotide sequence encoding the recombinant bird's nest peptide into a prokaryotic or eukaryotic expression vector to construct a recombinant expression plasmid; (2) transforming or transfecting the recombinant expression plasmid into a suitable expression host cell, wherein the expression host cell is a prokaryotic cell, eukaryotic cell, or yeast cell; (3) culturing the expression host cell and inducing expression or expressing under suitable conditions; (4) collecting the expression product and, after being broken or lysed, centrifuging to obtain a supernatant or precipitate containing the recombinant bird's nest peptide.

7. The method according to claim 6, characterized in that, The expression host cells are selected from Escherichia coli BL21, Rosetta strain, Pichia pastoris, Saccharomyces cerevisiae, CHO cells, HEK293 cells, or Sf9 cells.

8. The preparation method according to claim 7, characterized in that, In step (3), the host cells for expression were cultured using a fermentation medium, the composition of which was: glucose 30.0 g / L, yeast extract 50.0 g / L, dipotassium hydrogen phosphate 8.7 g / L, sodium dihydrogen phosphate 4.2 g / L, ammonium sulfate 5.5 g / L, magnesium sulfate 2.5 g / L, ethylenediaminetetraacetic acid 1.0 g / L, trace element solution 1.0 g / L, and defoamer 0.1 g / L; the trace element solution consisted of: manganese sulfate (MnSO4·H2O) 3.0 g, ferric sulfate (FeSO4·7H2O) 2.0 g, zinc sulfate (ZnSO4·7H2O) 0.5 g, copper sulfate (CuSO4·5H2O) 0.1 g, potassium iodide (KI) 0.05 g, boric acid (H3BO3) 0.3 g, and sodium molybdate (Na2MoO4·2H2O) 0.05 g. g, dilute to 1 L with deionized water.

9. The preparation method according to claim 8, characterized in that, The step (4) is followed by a protein purification step.

10. The use of the recombinant bird's nest peptide according to claim 1 in the preparation of medical nutritional preparations, immunomodulators, neuroprotective agents, anti-inflammatory or antioxidant preparations, skin care products, beverages, health products and other products with cell repair, anti-aging or enhanced health functions.

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