Preparation method of recombinant ginseng protein expressed by pichia pastoris

Abstract

The application discloses a preparation method of recombinant ginseng protein expressed by Pichia pastoris. A general 9K carrier skeleton without an a-factor signal peptide element is used to construct a recombinant plasmid with a target gene containing a ginseng protein self signal peptide element, and the recombinant plasmid is transformed into Pichia pastoris, so that the Pichia pastoris can effectively express the recombinant ginseng protein. The obtained recombinant ginseng protein has high biological safety, can promote proliferation of fibroblasts, inhibit ROS activity, and has obvious repair and anti-photoaging effects on skin.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and involves the structural design, efficacy verification, and establishment of production process for recombinant ginseng protein. Background Technology

[0002] Ultraviolet (UV) radiation from land is a major cause of skin aging. Long-wave ultraviolet A (UVA) radiation, with a wavelength of 320–400 nm, accounts for approximately 95% of ultraviolet radiation (UVR). UVA can penetrate the dermis and cause functional damage to dermal fibroblasts. This damage primarily manifests as inhibition of fibroblast proliferation, reduced cell viability and contractility, and increased collagen degradation, leading to photoaging of the skin. To effectively protect the skin from photoaging, it is necessary to develop protective agents that can resist UVA. Furthermore, with the increasing harm to skin health caused by various light sources in the digital age, the development of anti-aging and anti-photoaging functional ingredients has become increasingly urgent.

[0003] Ginseng contains over 300 types of ginseng proteins (GPs), categorized into saponin β-glucosidase, RNase-like proteins, and ribonucleases. Seung Il Kim et al. discovered a ginseng root RNA-like storage protein (characterization of RNase-like major storage protein from the ginseng root by a proteomic approach). Two-dimensional electrophoresis analysis showed that its content varied with the season, and amino acid sequence analysis (AY496964.) showed high homology with plant RNases. Rui Jiang et al. isolated ginseng proteins with molecular weights of 27 kDa and 13 kDa, finding that these proteins could alleviate the inhibitory effect of UVA on cell viability, increase the percentage of NIH-3T3 fibroblasts in the S phase of the cell cycle, prevent UVA-induced photoaging of fibroblasts, and potentially improve fibroblast proliferation and contraction by inhibiting ROS-induced DNA damage and collagen degradation. Previous studies indicated that the coverage of the aforementioned 27 kDa protein with ginseng root RNA-like storage proteins was only 63.03%.

[0004] Although a suitable industrial-scale ginseng protein preparation process has been established through ginseng extraction, yielding ginseng protein with enhanced T-SOD and other antioxidant enzyme activities, the yield is low, the extraction process relies heavily on organic chemical reagents, and production costs are high. Chinese patent CN104829701A utilizes *E. coli* expression to obtain recombinant ginsenoside protein with antibacterial activity, but the expression product is located within the bacterial cell, leading to complex separation and purification processes, affecting yield, and increasing costs and contamination risks. The Pichia pastoris expression system has advantages such as low cost, low contamination, and high yield, and is safer than *E. coli*. However, no reports have been found regarding processes for expressing recombinant ginseng protein using Pichia pastoris. As ginseng is a plant, its protein expression differs from that in yeast; furthermore, codon selection affects protein expression, making the production of recombinant ginseng protein using Pichia pastoris fermentation quite challenging. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing recombinant ginseng protein expressed by Pichia pastoris.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A method for preparing recombinant ginseng protein expressed by Pichia pastoris, the method comprising the following steps:

[0008] The coding sequence of recombinant ginseng protein was inserted into a Pichia pastoris expression vector with the signal peptide element removed in an expression cassette, and then Pichia pastoris was transformed to complete the secretory expression of the recombinant ginseng protein using Pichia pastoris. The recombinant ginseng protein includes a fusion protein composed of ginseng protein signal peptide and mature ginseng protein.

[0009] Preferably, the Pichia pastoris expression vector is selected from any one of the universal 9K vectors. A recombinant plasmid for secretory expression of recombinant ginseng protein is constructed by combining the coding sequence of the recombinant ginseng protein with the backbone of a universal 9K vector with the α-factor signal peptide element removed. The recombinant plasmid is then transformed into Pichia pastoris.

[0010] Preferably, the preparation method of the recombinant ginseng protein specifically includes the following steps:

[0011] 1) Cloning of the target gene

[0012] The coding sequence of the recombinant ginseng protein is artificially designed, compared with the nucleotide sequence of a universal 9K vector (e.g., pPIC9K), and the restriction endonuclease recognition sites and necessary flanking sequences of the restriction endonuclease recognition sites located on both sides of the α-factor signal peptide element are selected (e.g., these sequences are used to ensure that the coding sequence can match the open reading frame of the original expression cassette after insertion). The stop codon is combined with the coding sequence of the recombinant ginseng protein to obtain the target gene sequence with restriction endonuclease recognition sites on both sides (e.g., SEQ.ID.NO.3, where the coding sequence of the recombinant ginseng protein is nucleotides 15 to 728). A cloning plasmid containing the target gene sequence is then constructed or synthesized.

[0013] 2) Construction of recombinant plasmids

[0014] Based on the selected restriction endonuclease recognition sites, the universal 9K vector is double-digested to obtain a vector backbone with the α-factor signal peptide element removed. The target gene (i.e., the target fragment obtained by double-digesting the cloned plasmid) is then ligated to the vector backbone to obtain a recombinant plasmid.

[0015] 3) Transformation of Pichia pastoris with recombinant plasmids

[0016] The recombinant plasmid was linearized and then transformed into Pichia pastoris competent cells. High-copy transformants were then screened using G418.

[0017] 4) Identification of recombinant transformants

[0018] Genomic DNA of high-copy transformants was extracted using the freeze-thaw method, and then the recombinant transformants were identified by PCR.

[0019] 5) Screening of fermented seeds

[0020] The recombinant transformants were cultured in shake flasks at 26–32°C, and methanol was used to induce expression during the culture process to screen for expression strains.

[0021] 6) Fermentation of recombinant ginseng protein

[0022] After the expression strain was inoculated into the fermentation medium, it was cultured at 26–32°C. The wet bacterial weight was increased to ≥140 g / L by adding glycerol. Then, methanol was used to induce expression until the expression level of the target protein (the recombinant ginseng protein) no longer increased (at which point the wet bacterial weight was generally greater than 200 g / L), and the culture was terminated.

[0023] Preferably, the preparation method of the recombinant ginseng protein further includes the following steps: purifying the recombinant ginseng protein contained in the supernatant of the fermentation broth by ion exchange chromatography, and then sequentially performing dialysis (with a molecular weight cutoff of 5-10 kDa) for desalting and freeze drying to obtain the pure recombinant ginseng protein.

[0024] Preferably, the ion exchange chromatography uses a chromatography column filled with cation exchange packing material.

[0025] Preferably, the ion exchange chromatography uses a mixture of solution A (91%–95% by volume) and solution B (5%–9% by volume) as the washing reagent, and a mixture of solution A (86%–90% by volume) and solution B (10%–14% by volume) as the elution reagent; wherein solution A is a 10–20 mmol / L citrate buffer solution with pH 5.8–6.0, and solution B is solution A containing 1 M NaCl.

[0026] A cosmetic or medical aesthetic product contains recombinant ginseng protein prepared by the above-mentioned method for preparing recombinant ginseng protein expressed by Pichia pastoris.

[0027] Preferably, the recombinant ginseng protein is used as a functional ingredient for anti-photoaging, anti-aging, or skin repair.

[0028] Preferably, the concentration of the recombinant ginseng protein is ≥700 μg / mL.

[0029] A drug for promoting tissue and / or organ regeneration, comprising recombinant ginseng protein prepared by the above-described method for preparing recombinant ginseng protein expressed by Pichia pastoris.

[0030] Preferably, the recombinant ginseng protein is used as a functional raw material for repairing skin, bones, and blood vessels.

[0031] A nucleic acid molecule comprising a coding sequence for recombinant ginseng protein, said recombinant ginseng protein comprising a fusion protein composed of a ginseng protein signal peptide and mature ginseng protein.

[0032] Preferably, the nucleic acid molecule is selected from a recombinant plasmid for expressing the recombinant ginseng protein, which is constructed from the coding sequence of the recombinant ginseng protein and a universal 9K vector backbone with the α-factor signal peptide element removed.

[0033] A recombinant ginseng protein expression system includes Pichia pastoris host cells and a recombinant plasmid transformed into the host cells. The recombinant plasmid is constructed from the coding sequence of recombinant ginseng protein and a universal 9K vector backbone with the α-factor signal peptide element removed. The recombinant ginseng protein includes a fusion protein composed of a ginseng protein signal peptide and mature ginseng protein.

[0034] Preferably, the mature ginseng protein is selected from any one of the ribonucleases derived from ginseng.

[0035] Preferably, the mature ginseng protein is selected from any one of the ginseng root RNA-like storage proteins, and the ginseng protein signal peptide is the signal peptide in the precursor of the ginseng root RNA-like storage protein, that is, the ginseng protein's own signal peptide.

[0036] Preferably, the amino acid sequence of the fusion protein is shown in SEQ.ID.NO.2.

[0037] The beneficial effects of this invention are reflected in:

[0038] This invention uses recombinant ginseng protein, which is expressed by fusion with Pichia pastoris using the signal peptide of ginseng protein (such as ginseng root RNA-like storage protein), as the experimental subject. Experiments have shown that the recombinant ginseng protein has low cytotoxicity, high biosafety, and can effectively promote fibroblast proliferation and inhibit ROS activity, thereby preventing UVA-induced photoaging of fibroblasts and having significant repair and anti-photoaging effects on the skin.

[0039] This invention utilizes Pichia pastoris to express recombinant ginseng protein. Experiments show that by constructing a recombinant plasmid using a Pichia pastoris expression vector with the signal peptide element removed and a target gene containing the signal peptide element of ginseng protein itself, the Pichia pastoris expression system can not only guide the secretion of recombinant ginseng protein using the signal peptide of ginseng protein itself, but also effectively enhance the expression of the recombinant ginseng protein (e.g., reduce the expression of other proteins in the system).

[0040] Furthermore, this invention optimizes the codons of recombinant ginseng protein, constructs a recombinant plasmid with the Pichia pastoris universal 9K vector (with the α-factor signal peptide removed), and transforms Pichia pastoris to achieve secretory expression of recombinant ginseng protein (expression level > 0.8 g / L) using Pichia pastoris recombinant transformants. This allows for the mass production of ginseng protein with good hydrophilicity, intact structure, and excellent function, which can then be used as a raw material for cosmetics, medical aesthetics, and other products.

[0041] Furthermore, this invention employs ion exchange chromatography, dialysis, and freeze-drying purification processes to obtain recombinant ginseng protein with a purity of up to 95% for the constructed Pichia pastoris expression system (see Chinese Pharmacopoeia Purity Detection Method, Electrophoresis). Attached Figure Description

[0042] Figure 1 3D structure modeled for recombinant ginseng protein SWISS-MODEL.

[0043] Figure 2A schematic diagram of the restriction site selection for the Pichia pastoris expression plasmid (pPIC9K,9K): In the figure, the sequence in the upper box is the BamH I restriction site, the sequence in the middle box is the Xho I restriction site, and the sequence in the lower box is the EcoR I restriction site.

[0044] Figure 3 The results of PCR identification of GS115 / 9K-GP recombinant transformants are shown in the figure. In the figure, 1# to 12# represent recombinant transformants from different colonies, M represents the marker, and the size of the target band is approximately 800bp.

[0045] Figure 4 The results of shake-flask expression identification of GS115 / 9K-GP strain (recombinant transformant).

[0046] Figure 5 Comparison of shake-flask expression products of two Pichia pastoris (recombinant transformants) transformed with GS115 / 9K-GP and GS115 / 9K-gp.

[0047] Figure 6 The growth curves of HFF-1 cells cultured in vitro after treatment with different concentrations of recombinant ginseng protein samples are shown.

[0048] Figure 7 Results of recombinant ginseng protein promoting HFF-1 cell proliferation.

[0049] Figure 8 The results of scratch assays on HFF-1 cells treated with different concentrations of recombinant ginseng protein (micrographs).

[0050] Figure 9 The results of scratch assays on HFF-1 cells treated with different concentrations of recombinant ginseng protein are shown in bar graphs.

[0051] Figure 10 Phenotypic diagram of the zebrafish repair efficacy experiment of recombinant ginseng protein (dashed line area is the quantitative region).

[0052] Figure 11 Results of the photoaging effect experiment of recombinant ginseng protein in zebrafish (bar chart). Detailed Implementation

[0053] To better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Obviously, the embodiments described below are only for explaining the present invention and are not intended to limit the scope of protection of the present invention.

[0054] 1. Recombinant Ginseng Protein Molecular Design

[0055] 1.1 Obtaining and Modifying the Ginseng Protein (GP) Sequence

[0056] The amino acid sequence of ginseng root RNA-like storage protein (AY496964.) was found on the NCBI website. After modification, the amino acid sequence of the recombinant ginseng protein to be expressed (i.e. SEQ.ID.NO.2) was formed, in which amino acid residues 1 to 23 are the self-signal peptides of the corresponding ginseng protein contained in AY496964. (i.e. SEQ.ID.NO.1).

[0057] 1.2 Theoretical Properties of Recombinant Ginseng Protein

[0058] The protein has a molecular weight of 27.34 kDa and an isoelectric point of 6.24; see the 3D structure of the protein. Figure 1 .

[0059] 1.3 Recombinant Ginseng Protein Gene Synthesis

[0060] Based on the codon preference of Pichia pastoris, the nucleotide sequence encoding recombinant ginseng protein was artificially selected and optimized. Restriction endonucleases BamHI and EcoRI restriction sites were added to both ends of this sequence. At the same time, the sequence "aaacgatg" was added after the BamHI restriction site, and the stop codon "taa" was added before the EcoRI restriction site. The recombinant ginseng protein gene sequence (i.e., SEQ.ID.NO.3) was finally obtained. Chemical synthesis was carried out to obtain the whole gene synthetic cloning plasmid T-GP (which was synthesized by a gene company and was only used to increase the copy number of the recombinant ginseng protein gene sequence).

[0061] 2. Construction of recombinant plasmids

[0062] 2.1 Materials

[0063] 2×Taq PCR Mastermix (Tiangen Biochemical), T4 DNA Ligase (Thermo), E. coli DH5α competent cells (Tiangen Biochemical), restriction endonuclease EcoRI Fast Digest (Thermo), restriction endonuclease BamHI Fast Digest (Thermo), restriction endonuclease XhoI Fast Digest (Thermo), agarose gel DNA recovery kit (Tiangen Biochemical), LB medium.

[0064] 2.2 Equipment

[0065] PCR instrument (Bio-RAD), horizontal electrophoresis apparatus, centrifuge, shaker.

[0066] 2.3 Methods

[0067] 2.3.1 Double enzyme digestion of target gene and vector

[0068] like Figure 2 As shown, to remove the α-factor signal peptide element from the pPIC9K vector (Miaoling plasmid, P0214) to construct a recombinant plasmid without the α-factor signal peptide element (instead of introducing the ginseng protein's own signal peptide element), one restriction endonuclease (i.e., BamHI and EcoRI) cleavage site was selected upstream and downstream of the α-factor signal peptide element. Simultaneously, in the earlier stages of the laboratory, to construct a recombinant plasmid retaining the α-factor signal peptide element (instead of introducing the ginseng protein's own signal peptide element), [the following was done]. Figure 2 The restriction endonuclease Xho I cleavage site shown replaces the upstream restriction endonuclease (i.e., BamHI) cleavage site selected above, while the downstream restriction endonuclease (i.e., EcoRI) cleavage site remains unchanged.

[0069] The whole-gene synthetic cloning plasmids T-GP and pPIC9K vector were double-digested using restriction endonucleases BamHI and EcoRI, respectively. The digestion products were detected by 1% agarose gel electrophoresis, and the electrophoretic bands were recovered by gel extraction to obtain the target fragment with sticky ends and the vector backbone without the α-factor signal peptide.

[0070] Similarly, in the early stages of the laboratory, the whole-gene synthetic cloning plasmid T-gp (which differs from T-GP in that it does not contain the ginseng protein's own signal peptide element) and the pPIC9K vector were double-digested using restriction endonucleases XhoI and EcoRI, respectively, to obtain the target fragment with sticky ends and the vector backbone containing the α-factor signal peptide.

[0071] 2.3.2 Ligation of enzyme digestion products

[0072] To construct the recombinant expression vector pPIC9K-GP (a recombinant plasmid without the α-factor signal peptide), T4 DNA ligase was used to ligate the enzyme digestion product (target fragment) containing the nucleotide sequence encoding recombinant ginseng protein and the pPIC9K vector digestion product (vector backbone without the α-factor signal peptide).

[0073] Similarly, in the early stages of the laboratory, the corresponding target fragment was linked to the pPIC9K vector digestion product (a vector backbone containing the α-factor signal peptide) in the same manner, and the recombinant expression vector constructed was designated as pPIC9K-gp. The protein product secreted and expressed by pPIC9K-gp in Pichia pastoris does not contain the α-factor signal peptide, and the amino acid sequence of the target protein is amino acid residues 24 to 238 in SEQ.ID.NO.2.

[0074] 2.3.3 Conversion of Linkage Products

[0075] Taking pPIC9K-GP as an example, E. coli DH5α competent cells were used for plasmid transformation. The transformation solution was evenly spread on LB plates containing 60 μg / mL ampicillin antibiotic, and the plates were inverted and placed in a 37°C incubator overnight.

[0076] 2.3.4 Identification of positive clones

[0077] Select the grown colonies, perform bacterial culture PCR identification, and send the correctly identified strains to a gene company for sequencing. Compare the sequencing results with the designed sequence, and preserve the correctly matched strains.

[0078] 3. Transformation of GS115 yeast with recombinant plasmid

[0079] 3.1 Materials

[0080] Plasmid mini-extraction kit (Tiangen Biochemical), general DNA purification and recovery kit (Tiangen Biochemical DP214-02), restriction endonuclease SalI FastDigest (Thermo), MD plate, YPD plate.

[0081] 3.2 Equipment

[0082] PCR instrument (Bio-RAD), horizontal electrophoresis apparatus, centrifuge, shaker.

[0083] 3.3 Methods

[0084] 3.3.1 Medium-scale extraction of recombinant plasmids

[0085] Recombinant plasmids, such as pPIC9K-GP, were extracted from the preserved strain using a kit. The plasmids were then eluted with 300 μL of sterile ddH2O. The strain was stored at -20°C.

[0086] 3.3.2 Linearization of Recombinant Plasmids

[0087] For recombinant plasmids extracted in medium quantities, such as pPIC9K-GP, linearization was performed using SalI. The linearization reaction system is shown in Table 3-1.

[0088] Table 3-1. Linearization reaction system of recombinant plasmids

[0089] Components Volume (μL) Recombinant plasmid 300 10×Fast Digest buffer 200 SaL I 20 <![CDATA[ddH2O]]> 1480 2000μL in total

[0090] After linearization, the linearized plasmid was recovered using a kit. The plasmid solution was then concentrated to 10–20 μL by baking at 45 °C. It was stored at -20 °C.

[0091] 3.3.3 Preparation of Pichia pastoris competent cells GS115

[0092] Single colonies of Pichia pastoris strain GS115 were selected and cultured overnight at 29°C and 200 rpm in a shaker.

[0093] The following day, the OD600 was measured using a spectrophotometer. When the OD600 value was between 1.1 and 1.7, it could be used for competent cell preparation, as detailed below:

[0094] Centrifuge at 3000 rpm for 5 min at 4℃ and discard the supernatant; pre-cool the cells with 30 mL of sterile ice-cold ddH2O, suspend the cells, centrifuge at 3000 rpm for 5 min at 4℃ and discard the supernatant (repeat the steps twice).

[0095] Resuspend the cells in 30 mL of sterile, ice-cold 1 M D-sorbitol, centrifuge at 3000 rpm for 5 min at 4 °C, and discard the supernatant (repeat this step twice). Resuspend the cells in 1 M D-sorbitol to obtain competent cells. Aliquot the competent cells for later use.

[0096] 3.3.4 Electroconversion and Screening of Linearized Plasmids

[0097] Electroporation: Taking pPIC9K-GP as an example, add 10 μL of linearized plasmid to 90 μL of competent cells, mix well, and then transfer 100 μL of the mixture to a 2 mm electroporation cuvette and pre-chill on ice for 10 min. Electroporate according to the set parameters (voltage 2000V, capacitance 25uF, resistance 200Ω). Immediately after electroporation, add 1.5 mL of 1M pre-chilled D-sorbitol on ice, mix well, and then aspirate the bacterial culture into a 1.5 mL sterile centrifuge tube.

[0098] Screening: Taking pPIC9K-GP as an example, spread 200 μL of bacterial suspension on each MD plate using a sterile spreader and incubate upside down at 29°C for 3–4 days. Prepare YPD plates containing 4 mg / mL G418. Transfer all single colonies growing on the MD plates to the YPD plates containing 4 mg / mL G418 using a sterile toothpick and incubate upside down at 29°C for 4–5 days. Prepare YPD plates by transferring all single colonies growing on the G418 plates containing 4 mg / mL G418 to the YPD plates using a sterile toothpick and incubate upside down at 29°C for 1–2 days.

[0099] 4. Identification of Pichia pastoris recombinant transformants

[0100] 4.1 Materials

[0101] 2×Taq PCR Mastermix (Tiangen Biotech).

[0102] 4.2 Equipment

[0103] PCR instrument (Bio-RAD), centrifuge, microwave oven, -80℃ refrigerator.

[0104] 4.3 Methods

[0105] 4.3.1 Genomic DNA extraction from transformants

[0106] Use a toothpick to pick up colonies grown on a YPD plate and transfer them to an EP tube. Mix well and centrifuge at 4000 rpm for 1 min at room temperature. Discard the supernatant. Microwave on medium heat for 5 min, then freeze at -80°C for 20 min; repeat this process twice. Add 50 μL of double-distilled water, mix well, and centrifuge at 4000 rpm for 1 min at room temperature. Transfer the supernatant (genomic DNA) to a sterile centrifuge tube and store at -20°C.

[0107] 4.3.2 Transformant Genomic PCR

[0108] Using extracted genomic DNA as a template, PCR was performed with primers containing the inserted target gene fragment. Strains capable of amplifying the target fragment were designated as recombinant transformants (e.g., GS115 / pPIC9K-GP, or GS115 / 9K-GP). The PCR reaction system is shown in Table 4-1.

[0109] Table 4-1. GS115 / pPIC9K-GP Genomic DNA PCR Reaction System

[0110] Components Volume (μL) Genomic DNA 4.0 GP-XF 2.0 GP-ER 2.0 2×Taq PCR Mastermix 20.0 <![CDATA[ddH2O]]> 12.0 40μL in total

[0111] The primer design is as follows:

[0112] GP-XF: 5'-ATGGATCCAAACGATGATGAGAGCCATT-3'

[0113] GP-ER: 5'-TAGAATTCTTAAATAATACTATTCGTTCT-3'

[0114] The specific PCR procedure is as follows:

[0115] (1) Take a sterilized EP tube, prepare the reaction system according to Table 4-1, mix well, and centrifuge briefly.

[0116] (2) Perform the reaction in the PCR instrument according to the set program:

[0117] After pre-denaturation at 94℃ for 5 min, repeat the following steps for 30 cycles: 94℃, 30 s (de-chaining), 60℃, 30 s (annealing), 72℃, 1 min (extension).

[0118] Extend the heat at 72°C for another 5 minutes. Then maintain at 4°C.

[0119] (3) Based on the PCR results, the recombinant transformants were preliminarily identified, and the corresponding plates were stored at -4℃.

[0120] 4.4 Results

[0121] Ultimately, multiple strains carrying the target vector (e.g., pPIC9K-GP) were successfully identified. Figure 3 The recombinant transformants can be used for shake-flask expression screening.

[0122] 5. Recombinant transformant expression in shake flasks

[0123] 5.1 Materials

[0124] BMGY medium, BMMY medium.

[0125] 5.2 Equipment

[0126] Shaking incubator, clean bench, sterilizer.

[0127] 5.3 Methods

[0128] (1) Pick GS115 / 9K-gp (containing α-factor signal peptide, which was prepared in our laboratory in the previous stage), GS115 / 9K-GP (without α-factor signal peptide) and blank strain (GS115 single colony) respectively, and inoculate them into 250mL Erlenmeyer flasks containing 30mL BMGY medium and incubate at 29℃ and 225rpm for 60 hours.

[0129] (2) Number the 50mL centrifuge tubes, pour the BMGY culture medium obtained in the previous step into the centrifuge tubes, centrifuge at room temperature and 3000rpm for 5min, and collect the bacterial cells in the shake flask.

[0130] (3) Add 30 mL of sterile double-distilled water to resuspend the bacterial cells. Centrifuge at 3000 rpm for 5 min at room temperature and collect the bacterial cells.

[0131] (4) Repeat step 3 twice.

[0132] (5) Add 30 mL of BMMY medium to resuspend the bacterial cells, pour 30 mL of the mixed BMMY culture solution into a 250 mL Erlenmeyer flask, and culture on a shaker at 29 °C and 225 rpm.

[0133] (6) When cultured for 24, 48 and 72 hours, methanol with a final concentration of 1% (v / v) was added for induction.

[0134] (7) Incubate for 96 hours, collect the supernatant from the shake flask, and store at -20℃.

[0135] (8) SDS-PAGE electrophoresis was used to detect the supernatant of the shake flask to confirm the expression results of recombinant ginseng protein.

[0136] 5.4 Results

[0137] Depend on Figure 4It can be seen that the experimental results of recombinant transformants with different numbers (e.g., GS115 / 9K-GP) demonstrate that multiple expression strains can be obtained, indicating that the above procedure for preparing recombinant transformants is not a random experiment and is effective and reproducible. Figure 5 It can be seen that, compared with the blank strain, both GS115 / 9K-GP (without α-factor signal peptide) and GS115 / 9K-gp (with α-factor signal peptide) expressed the target protein. However, among the two transformants constructed with different recombinant plasmids, the recombinant ginseng protein expression level of GS115 / 9K-GP was significantly higher than that of GS115 / 9K-gp. Moreover, GS115 / 9K-gp expressed more impurities, which increased the difficulty of purification and was not conducive to subsequent applications. Furthermore, when the molecular size of the target protein after expression was compared with the theoretical value, it was found that the recombinant ginseng protein expressed by GS115 / 9K-GP was slightly higher than the theoretical value, indicating that the ginseng protein's own signal peptide was not cleaved, that is, the expressed recombinant ginseng protein retained the ginseng protein's own signal peptide.

[0138] 6. Fermentation process

[0139] 6.1 Shake-flask seed culture

[0140] The prepared culture medium was dispensed into 200 mL Erlenmeyer flasks (500 mL each). The GS115 / 9K-GP expression strain, as determined in the shake-flask experiments above, was inoculated and cultured at 29±0.5℃ on a shaker (220±10 rpm) for 20–28 hours. Under flame protection, 500–800 mL of the shake-flask seed culture was combined into a sterile inoculation bottle and inoculated into a 10 L fermenter.

[0141] 6.2 Fermentation

[0142] The fermentation tank pressure is 0.030–0.050 MPa, and the fermentation tank air flow rate is 2–5 m³ / s. 3 The culture was carried out at a stirring speed of 100 rpm for 1 hour. After the dissolved oxygen recovered, the glycerol batch growth stage was started, and 50% (volume fraction) glycerol was added. When the wet cell weight reached 140 g / L, methanol induction was performed. After induction to a wet cell weight of 220 g / L, the increase in the target protein no longer increased significantly, the cells aged, and the fermentation was terminated.

[0143] 6.3 Fermentation Results

[0144] GS115 / 9K-GP (without α-factor signal peptide) can effectively express recombinant ginseng protein via methanol induction, with an expression level greater than 0.8 mg / mL.

[0145] 7. Recombinant Ginseng Protein Purification Process

[0146] 7.1 Method

[0147] After fermentation was terminated, the fermentation broth was released. After centrifugation, 7 L of supernatant containing recombinant ginseng protein was collected. The supernatant was purified using a CM-Sepharose FF (CM-Sepharose FF) ion exchange chromatography column. For purification, the column was first equilibrated with 3 column volumes of phase A before loading the sample. Then, the column was washed with 95% phase A + 5% phase B (volume fraction) to remove impurities. Next, 10 column volumes of phase A + 10% phase B (volume fraction) were used for elution. The eluent was collected, where phase A was 10 mmol / L citrate buffer at pH 6.0 and phase B was phase A containing 1 M NaCl. Finally, the eluent was dialyzed (dialysis bag molecular weight cutoff 10 kDa) to desalt and lyophilized to obtain pure recombinant ginseng protein.

[0148] 7.2 Results

[0149] Finally, 20g of recombinant ginseng protein lyophilized powder was prepared using GS115 / 9K-GP (without α-factor signal peptide) and used for the following experiments.

[0150] 8. Experiment on the proliferation of HFF-1 cells promoted by recombinant ginseng protein

[0151] 8.1 Materials

[0152] DMEM medium (containing 5% fetal bovine serum), PBS, 75% alcohol, cell culture flasks, 96-well plates, pipettes, centrifuge tubes, alcohol lamp, 1mL syringe, 0.22μL filter membrane, steel ruler, marker pen.

[0153] 8.2 Equipment

[0154] Liquid nitrogen tank, clean bench, cell culture incubator, inverted microscope, centrifuge, enzyme-linked immunosorbent assay (ELISA) reader.

[0155] 8.3 Methods

[0156] 8.3.1 Laying out and processing

[0157] HFF-1 cells were divided into 10 3 Each sample was inoculated into a 96-well plate (7 plates). After 24 hours of incubation, different concentration gradients of recombinant ginseng protein diluted with 5% serum were added (120 μg / mL, 160 μg / mL, 200 μg / mL, 240 μg / mL, 280 μg / mL, 320 μg / mL). The blank control group was inoculated with only 5% serum. The plates were then incubated, and one plate was taken every 24 hours for MTT assay.

[0158] 8.3.2 Detection

[0159] Cell status was recorded using an inverted imaging microscope. The culture medium was then removed, and 50 μL of MTT solution was added to each well. The cells were incubated at 37°C with 5% CO2 for 2 h. The MTT solution was then removed, and 100 μL of isopropanol was added to each well. The cells were shaken at room temperature for 30 min. The absorbance of each well was measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0160] 8.8.3 Data Recording and Calculation

[0161] The absorbance values ​​of recombinant ginseng protein after treatment were analyzed using Prism 8 (the average OD value for each concentration was calculated), and line graphs of OD values ​​and time were plotted for each treatment concentration. A higher OD value indicates a greater number of viable cells, meaning that the recombinant ginseng protein treatment at that concentration has a more significant promoting effect on cell proliferation.

[0162] 8.8.4 Results

[0163] Depend on Figure 6 It was observed that the cells were in the logarithmic growth phase on days 4-5, reaching maximum viable cell density on day 6. With decreasing nutrients and living space, the cells underwent apoptosis due to nutrient depletion on days 7-8. The results showed that recombinant ginseng protein was non-toxic to human fibroblast growth and effectively promoted the growth and proliferation of human fibroblasts. Furthermore, the proliferative effect of different concentrations of recombinant ginseng protein was superior to that of 5% fetal bovine serum, indicating that it played a positive role in cell growth.

[0164] Depend on Figure 7 It can be seen that after 6 days of culture, the number of cells in the sample group containing 240 μg / mL recombinant ginseng protein was greater than that in the blank control group, which means that recombinant ginseng protein can significantly promote the adhesion and growth of human fibroblasts.

[0165] 9. Recombinant ginseng protein cell scratch assay

[0166] 9.1 Materials

[0167] DMEM medium (containing 10% fetal bovine serum), PBS, 75% alcohol, low-glucose DMEM medium (containing 1% fetal bovine serum), cell culture flasks, 6-well plates, pipettes, centrifuge tubes, alcohol lamp, 1mL syringe, 0.22μL filter membrane, steel ruler, marker pen.

[0168] 9.2 Equipment

[0169] Liquid nitrogen tank, clean bench, cell culture incubator, inverted microscope, centrifuge, enzyme-linked immunosorbent assay (ELISA) reader.

[0170] 9.3 Methods

[0171] (1) HFF-1 cells were prepared at a concentration of 2.5 × 10⁻⁶. 5 / The cells were inoculated into 24-well plates and cultured in medium containing 10% serum for 24 hours to achieve a cell confluence of 85%–95%.

[0172] (2) After the cells have grown to full size, use a marker to mark three equal parts of each well at the bottom of the well plate with a steel ruler.

[0173] (3) Draw a vertical line with the pipette tip, add PBS to wash the cells 3 times, and remove the drawn and suspended cells.

[0174] (4) Add 2 mL of recombinant ginseng protein samples with concentrations of 50 μg / mL and 25 μg / mL, respectively. A blank control was set up in low-glucose DMEM medium containing 1% serum. Take pictures under a microscope after 0, 6, 20 and 28 hours of culture.

[0175] (5) Data analysis: The scratch area was measured using Image-J software and the cell migration rate of each group was calculated.

[0176] Cell migration rate = (Area of ​​migrating cells in the fixed scratch zone / Initial scratch zone area) × 100%

[0177] 9.4 Results

[0178] Depend on Figure 8 , Figure 9 It was found that low concentrations (25 μg / mL and 50 μg / mL) of recombinant ginseng protein promoted cell migration from 0 h to 28 h, and both concentrations showed highly significant differences compared with the blank control within 6 h to 28 h (P < 0.01). Among them, 50 μg / mL showed a better effect in promoting migration, indicating that recombinant ginseng protein has a strong skin repair effect at the cellular level.

[0179] 10. Repair Efficacy Experiment

[0180] 10.1 Experimental Principle

[0181] The zebrafish caudal fin has a simple structure, is easy to operate on surgically, does not affect postoperative survival, and is easy to observe, making it an important model for studying tissue regeneration processes. Zebrafish caudal fin regeneration consists of three processes: wound healing, bud formation, and regeneration outcome, with bud formation being the most crucial. Cells involved in fin regeneration originate from multiple sources, including various cell types such as epidermal cells, fibroblasts, and osteoblasts, and these cells exhibit high lineage restriction. The regeneration mechanism of the zebrafish caudal fin is similar to that of human skin, bones, blood vessels, and other tissues and organs.

[0182] 10.2 Detection

[0183] Recombinant ginseng protein samples were sent to a third-party testing institution for efficacy testing using wild-type AB strain zebrafish, in accordance with the "Standard Operating Procedures for Evaluation of Zebrafish Repair Efficacy".

[0184] During testing, the caudal fin was severed perpendicular to the torso using a scalpel. The regenerated caudal fin area was used to quantitatively evaluate the repair efficacy of the samples. Zebrafish were divided into three groups: a normal control group, a model group, and a recombinant ginseng protein group. The normal control group received no treatment. The model group and the recombinant ginseng protein group underwent caudal fin amputation. Subsequently, the recombinant ginseng protein group was supplemented with solutions at final concentrations of 0.024% (0.024 g / 100 mL, i.e., 240 μg / mL), 0.048% (0.048 g / 100 mL, i.e., 480 μg / mL), and 0.096% (0.096 g / 100 mL, i.e., 960 μg / mL). After incubation for a period of time, the caudal fins of the three groups of zebrafish were photographed, and the caudal fin area was analyzed to evaluate the repair efficacy.

[0185] 10.3 Results

[0186] Depend on Figure 10 As shown in Table 10-1, the efficacy value of recombinant ginseng protein was 16% at a detection concentration of 0.096%, which was significantly different from that of the model group (P < 0.001). The regenerated tail fin area of ​​zebrafish treated with recombinant ginseng protein was significantly increased compared with that of the model group, proving that recombinant ginseng protein has a good repair effect.

[0187] Table 10-1. Evaluation of Repair Effect

[0188]

[0189] 11. Anti-photoaging efficacy experiment

[0190] 11.1 Experimental Principle

[0191] Ultraviolet (UV) radiation is the primary exogenous environmental factor causing skin damage and aging. It easily induces physiological effects such as inflammation, oxidative stress (which occurs when the production of free radicals exceeds the body's ability to eliminate them, potentially causing severe damage to cell structure), and DNA damage. It can even disrupt the active structures of collagen and elastin, manifesting as skin redness, pigmentation, sagging, and fine lines. Zebrafish fins resemble human limbs, and their skin structure is similar to that of humans. UV damage to zebrafish fins leads to a series of harmful physiological effects, manifesting as atrophy and reduction in the area of ​​the tail fin. The effective absorption of UV radiation by the active ingredients in skincare products on the fin surface prevents UV damage. Therefore, the anti-photoaging efficacy of recombinant ginseng protein samples was detected by measuring changes in ROS fluorescence values ​​in zebrafish after UV radiation.

[0192] 11.2 Detection

[0193] Recombinant ginseng protein samples were sent to a third-party testing institution for efficacy testing using wild-type AB strain zebrafish, in accordance with the "Standard Operating Procedures for Evaluation of Zebrafish Repair Efficacy".

[0194] During testing, zebrafish were divided into three groups: a normal control group, a model group, and a recombinant ginseng protein group. The normal control group received no treatment, while the model group and the recombinant ginseng protein group underwent multiple irradiations with ultraviolet light. Subsequently, the recombinant ginseng protein group was incubated with a final concentration of 0.096% (0.096 g / 100 mL, i.e., 960 μg / mL). After a period of incubation, the ROS fluorescence values ​​of the three groups of zebrafish were measured. The ROS fluorescence value results of each group were analyzed to evaluate the anti-photoaging efficacy.

[0195] 11.3 Results

[0196] Depend on Figure 11 As shown in Table 11-1, the efficacy value of recombinant ginseng protein was 85% at a detection concentration of 0.096%, which was significantly different from that of the model group (P < 0.05). The ROS fluorescence value of the recombinant ginseng protein sample was significantly lower than that of the model group, indicating that recombinant ginseng protein has a good anti-photoaging effect.

[0197] Table 11-1. Sample Effect Evaluation

[0198] Testing items Detected concentration (%) effect(%) p-value Test results Anti-photoaging effects 0.096 85 P＜0.05 Significant

Claims

1. Use of a recombinant ginseng protein in the preparation of a cosmetic or medical cosmesis product, characterized in that: The recombinant ginseng protein is a fusion protein of a ginseng protein signal peptide and a mature ginseng protein, and the amino acid sequence of the fusion protein is shown as SEQ.ID.NO.

2.

2. Use according to claim 1, characterized in that: The concentration of the recombinant ginseng protein is greater than or equal to 700 µg / mL.

3. Use of a recombinant ginseng protein in the preparation of a medicament for promoting regeneration of a tissue and / or an organ, characterized in that: The recombinant ginseng protein is a fusion protein of a ginseng protein signal peptide and a mature ginseng protein, and the amino acid sequence of the fusion protein is shown as SEQ.ID.NO.2.

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