Lotus root source LEA protein and its application in improving skin dry damage resistance

Abstract

This invention discloses lotus-derived LEA protein and its application in improving skin's resistance to dryness damage, belonging to the field of biotechnology and its application in skin care technology. The amino acid sequence of the LEA protein is shown in SEQ ID NO:1. This invention successfully constructed a recombinant expression vector containing a nucleic acid molecule encoding the LEA protein using genetic engineering methods, and transformed it into *E. coli* for efficient expression. After purification, the target protein was obtained. This LEA protein can significantly protect human immortalized keratinocytes against dryness damage. At a concentration of 100-200 μg / mL, it can restore cell viability under dryness stress to over 85%, with effects comparable to hyaluronic acid. This LEA protein has good biocompatibility and significant anti-dryness activity, and can be used to prepare skin care products that protect skin cells against dryness damage, moisturize, repair the skin barrier, or have anti-aging effects, showing broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of biotechnology and its application in skin care, and particularly to lotus-derived LEA protein and its application in improving skin's resistance to dryness and damage. Background Technology

[0002] Skin is the largest organ in the human body, and its barrier function is crucial for resisting external environmental damage and maintaining the body's water balance. However, environmental factors (such as low humidity, ultraviolet radiation, and pollution) and internal physiological processes (such as aging) often lead to dry skin. Dry skin not only causes discomfort but also damages the skin barrier function, accelerates skin aging, and manifests as decreased elasticity and wrinkle formation. Therefore, developing effective anti-dryness, moisturizing, and anti-aging products for the skin has significant market demand and research value.

[0003] Hyaluronic acid (HA), a classic moisturizer, effectively absorbs and retains moisture and is often used as an active ingredient in skin moisturizing products. However, the extraction cost of naturally derived HA is high, and its molecular weight and degree of cross-linking limit its penetration and persistence in the skin. In recent years, the search for novel, highly effective, and biocompatible natural anti-dryness active ingredients has become a research hotspot.

[0004] Late Embryogenesis Abundant Proteins (LEA proteins) are a class of proteins that are highly expressed during late embryonic development in plants and some invertebrates, and during exposure to abiotic stresses such as desiccation, high salinity, and low temperature. Their structures are typically highly hydrophilic, lacking well-defined secondary and tertiary structures. They are believed to protect cells from damage caused by stresses such as desiccation and freeze-thaw cycles by maintaining the structural stability of biomolecules, preventing protein aggregation, and binding water molecules. These properties make LEA proteins bioactive molecules with enormous application potential in fields such as biomedicine, food preservation, and cosmetics.

[0005] Currently, in-depth research on the anti-dryness protective effects of plant-derived LEA proteins in human skin cells, particularly the exploration of their specific effects and application potential, remains relatively limited. Therefore, developing a LEA protein with a clearly defined source, mature preparation process, and the ability to effectively protect human skin cells from dryness damage has significant practical implications and application value. Summary of the Invention

[0006] To address the limitations of existing anti-dryness ingredients in terms of cost, permeability, and durability, this invention proposes lotus-derived LEA protein and its application in improving skin's resistance to dryness damage.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a LEA protein derived from lotus, the amino acid sequence of which is shown in SEQ ID NO:1.

[0008] In a second aspect, the present invention provides a nucleic acid molecule encoding the LEA protein described in the first aspect.

[0009] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2.

[0010] Thirdly, the present invention provides a recombinant expression vector comprising the nucleic acid molecule described in the second aspect.

[0011] Preferably, the recombinant expression vector is constructed by inserting the nucleic acid molecule into the multiple cloning site of the pET-24a(+) vector.

[0012] Fourthly, the present invention provides a host cell comprising the recombinant expression vector described in the third aspect.

[0013] Preferably, the host cell is Escherichia coli BL21(DE3).

[0014] Fifthly, the present invention provides a method for preparing the LEA protein described in the first aspect, comprising the following steps: (1) Construct a recombinant expression vector containing a nucleic acid molecule encoding the LEA protein described in the first aspect; (2) Transform the recombinant expression vector into host cells; (3) Induce the host cells to express LEA protein; (4) Separate, purify and collect the LEA protein.

[0015] Preferably, the preparation method of the LEA protein includes the following steps: The genome sequence file of *Nelumbo nucifera* (Chinese ancient lotus) is obtained from the Nelumbo Genome Database. After gene alignment analysis using MEGA software, it is imported into the NBCI database for BLAST gene alignment analysis. A gene fragment encoding a late embryonic development protein is synthesized using gene synthesis methods and ligated into the *E. coli* expression vector pET-24a(+). This vector is then transformed into the engineered bacterium *E. coli* BL21(DE3) for induced expression. The expressed late embryonic development protein is purified to finally obtain the late embryonic development protein derived from lotus. The specific steps are as follows: (1) Obtain the genome sequence file of Nelumbonucifera from the Nelumbo Genome Database, perform sequence analysis, import it into the NBCI database for BLAST gene alignment analysis, and number the LEA protein as NunLEA-002 (species origin-protein type-experiment number). (2) The above DNA sequences were synthesized by gene synthesis and Nde I and Xho I restriction endonuclease sites were added to the 5' and 3' ends, respectively. The LEA gene fragment with Nde I and Xho I restriction endonuclease sites was obtained by PCR amplification. The PCR product and pET-24a(+) vector were double digested with restriction endonucleases Nde I and Xho I to ensure directional ligation. The reaction conditions were set at 37℃ for 1-2 h.

[0016] (3) Electrophoresis and gel recovery of the double digestion products: The single LEA gene and the linearized pET-24a(+) fragment after double digestion were recovered using a gel recovery kit. The digested vector products and the LEA fragment were ligated with the linearized pET-24a(+) fragment using T4 ligase to form the pET-24a(+)-LEA recombinant plasmid. After the system was prepared, it was incubated at 22℃ for 0.5-1h. After the ligation reaction was completed, it was inactivated by heat at 65℃ for 10-20min.

[0017] (4) Take 5-10 μL of the ligation product and place it in 50-100 μL of E. coli BL21(DE3) competent cells. The transformation process is as follows, and single clones are picked the next day: ① Take the E. coli BL21(DE3) competent cells out of the -80℃ ultra-low temperature freezer and place them on ice to thaw; ② In a clean bench, take 300-500 ng of plasmid and add it to 50-100 μL of E. coli. ① Gently tumble the BL21(DE3) competent cells to ensure even distribution, and place them on ice for 30-60 min. (All the following operations are performed in a clean bench.) ② Place the competent cells with the added plasmid in a pre-opened 42℃ water bath and heat shock for 90 s. ③ Remove the competent cells and place them on ice for 3-5 min. ④ Add 750-900 μL of antibiotic-free LB medium to the tube containing the competent cells and incubate on a shaker at 200-220 rpm / min for 30-60 min. ⑤ Centrifuge at 2000-3000 rpm / min for 3-5 min at room temperature. ⑥ After centrifugation, discard most of the medium, leaving approximately 100-150 μL of liquid medium, and mix it with the bacterial pellet by pipetting. Add the suspension dropwise to a solid culture medium containing kanamycin resistance, spread it evenly with a spreader until it feels rubbed against the surface of the medium; ⑧ Seal the culture dish with sealing film, incubate it upside down at 37℃ overnight, and observe the growth status of the plaques the next day; ⑨ The next day, pick a single colony and add it to 5-10 mL of LB liquid medium, and add an appropriate proportion of 100 mg / mL kanamycin stock solution according to the ratio of 50-100 μg / mL kanamycin resistance, and incubate at 37℃ and 200-220 rpm / min; ⑩ Store the bacterial culture at a ratio of 300 μL bacterial culture to 300 μL 50% glycerol, label it with the name and date, and store it in an ultra-low temperature freezer at -80℃; Perform plasmid mini-extraction according to the Tiangen plasmid mini-extraction kit, and measure the plasmid concentration using a Nanodrop 2000 nucleic acid quantification instrument after plasmid extraction.

[0018] (5) Single colony selection and culture after transformation: After overnight culture in a 37℃ incubator, remove the plate and observe the number and morphology of colonies. Select single colonies and prepare them for inoculation with liquid culture medium. Take out a bottle of sterilized liquid culture medium, add 50-100 μg / mL kanamycin, mix well, take out a sterilized clean test tube, use a sterile pipette tip to aliquot 5 mL of liquid culture medium into the test tube, use a sterile pipette tip to pick a single colony and inoculate it into the test tube, write a mark on the test tube wall, tilt the test tube and place it in a shaker, 37℃, 200-220 rpm / min, shake and culture overnight. After the culture is completed, take out the test tube and check the cell concentration. When the cell OD reaches 0.7-1.0, add 0.25-0.5 mM IPTG and continue to induce at 37℃ for 10-15 hours. After induction, centrifuge to collect the cells and perform ultrasonic lysis (ultrasonic volume is 3-5 mL, ultrasonic conditions are 2-5 seconds on, 2-5 seconds off, total ultrasonic time is 5-10 minutes). After centrifugation at 10000-12000 rpm / min, the supernatant was filtered through a 0.45 μm pore size filter membrane to collect the total bacterial protein. The total protein was then purified using Ni-NTA agarose gel purification resin to purify the recombinant histidine-tagged LEA protein. The purified protein was concentrated and desalted using ultrafiltration to obtain concentrated LEA protein. SDS-PAGE was used for electrophoresis, and the total protein concentration was determined using the BCA method. (6) HaCaT cell drying model and LEA protein administration anti-drying activity assay: A cell drying damage model was constructed by controlling the relative humidity in a closed culture environment using saturated BaCl2 solution, and the protective effect of LEA protein on HaCaT cell drying damage was evaluated by combining CCK-8 cell activity assay.

[0019] In a sixth aspect, the present invention provides the use of the LEA protein described in the first aspect in the preparation of products for protecting skin cells against dryness damage.

[0020] Preferably, the skin cells are human immortalized keratinocytes (HaCaT).

[0021] In a seventh aspect, the present invention provides a skin care composition for protecting skin cells against dryness damage, comprising an effective amount of the LEA protein described in the first aspect.

[0022] Preferably, the skin care composition is a serum, lotion, cream, mask, or freeze-dried powder.

[0023] In an eighth aspect, the present invention provides a method for protecting skin cells against dryness damage, comprising applying to skin cells an effective amount of the LEA protein described in the first aspect or the composition described in the seventh aspect.

[0024] Compared with the prior art, the beneficial effects of the present invention are: 1. The lotus LEA protein (NunLEA-002) disclosed in this invention exhibits significant protective activity against desiccation damage in HaCaT cells in an in vitro cell model. [For example, at a concentration of 100-200 μg / mL, it can restore cell viability to more than 85%, significantly higher than the desiccation control group (about 50%)], and its protective effect is comparable to that of the classic moisturizing agent HA.

[0025] 2. Naturally derived and highly biocompatible: LEA protein is derived from the natural plant lotus and has excellent biocompatibility. Compared with chemically synthesized ingredients, it has higher safety and consumer acceptance when used in skin care products, reducing potential immunogenicity risks.

[0026] 3. Potential for developing novel moisturizing and anti-aging ingredients: The anti-drying properties of LEA proteins indicate that they have broad application prospects in the fields of skin moisturizing, skin barrier repair, anti-oxidation, and anti-aging, providing new solutions for developing novel, high-performance daily chemical and cosmetic raw materials. Attached Figure Description

[0027] Figure 1 This is an image showing the agarose gel electrophoresis results of the DNA of the target gene NunLEA-002 in this invention; Figure 2 The results of purity identification of the purified NunLEA-002 protein sample by SDS-PAGE gel electrophoresis are shown. Figure 3 This is a schematic diagram illustrating the effect of different concentrations of NunLEA-002 protein on the cell viability of HaCaT cells under in vitro drying (hyperosmolarity) modeling conditions. Detailed Implementation

[0028] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with existing known technologies. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0029] Example 1: Obtain the LEA protein DNA sequence from ancient Chinese lotus and construct an expression vector containing the LEA gene.

[0030] (1) The genome sequence file of Chinese lotus (Nelumbonucifera) was obtained from the Nelumbo Genome Database. After sequence analysis, it was imported into the NBCI database for BLAST gene alignment analysis. The nucleotide sequence of the LEA protein obtained is shown in SEQ ID NO:2; the corresponding amino acid sequence is shown in SEQ ID NO:1.

[0031] The LEA protein was designated as NunLEA-002 (species origin-protein type-experiment number).

[0032] (2) Synthesize the above sequence and ligate it into the pET-24a(+) vector: The Nun-LEA002 DNA sequence was synthesized using gene synthesis techniques, and Nde I and Xho I restriction endonuclease sites were added to the 5' and 3' ends, respectively. The LEA gene fragment containing Nde I and Xho I restriction sites was obtained by PCR amplification. The PCR product and pET-24a(+) vector were double-digested with Nde I and Xho I restriction endonucleases to ensure directional ligation. The reaction conditions were set at 37℃ for 1-2 hours. The double digestion system is shown in Table 1 below. Table 1. Enzyme digestion system formulation (3) Electrophoresis and gel recovery of double-digested products: The double-digested products were subjected to 2.5% agarose gel electrophoresis. Weigh 1g of agarose into an Erlenmeyer flask, add 40mL of 1×TAE, heat in a microwave oven, shake after boiling, repeat this operation until the solution becomes clear and transparent without particles, add 2 drops of nucleic acid dye and pour into the tank until solidification. Spot the double-digested pUC57-LEA plasmid into the well and spot a marker on one side. Connect the apparatus correctly and electrophoresis for 20min at 120V / 30mA. Place the gel under a gel cutter and use a blade to recover the single gel carrier fragment, removing as much of the excess as possible, and place it in a pre-weighed 1.5mL EP tube. Weigh the gel. Use a gel recovery kit to recover the single SOD gene and linearized pET-24a(+) fragment after digestion. The vector digestion product and LEA fragment were ligated with linearized pET-24a(+) using T4 ligase to construct the pET-24a(+)-LEA recombinant plasmid. After preparing the system, it was incubated at 22℃ for 30 min. The ligation system is shown below. After the ligation reaction was completed, the plasmid was inactivated by heat at 65℃ for 10 min to obtain the recombinant expression vector pET-24a(+)-LEA002.

[0033] Table 2. Electrophoresis System Formulation Figure 1 DNA agarose gel electrophoresis results confirmed that the target gene NunLEA-002 had been successfully inserted into the pET-24a(+) vector through Nde I and Xho I restriction sites, and the correct recombinant expression plasmid pET-24a-LEA002 was constructed.

[0034] Lane 1 contains the recombinant plasmid pET-24a-LEA002 DNA after double digestion with Nde I and Xho I restriction endonucleases. This lane shows two clear DNA bands: one around 3500 bp, corresponding to the linearized fragment of the pET-24a(+) vector (3385 bp in size); and the other around 250 bp, corresponding to the inserted target gene fragment NunLEA-002 (285 bp in length). The two fragments obtained after digestion are highly consistent with the expected size, and the clear bands indicate complete digestion.

[0035] Lane 2 contains the undigested recombinant plasmid pET-24a-LEA002 DNA. A bright and diffuse band with a molecular weight greater than 3000 bp is visible above the gel, consistent with the characteristics of supercoiled and linear isomers of undigested plasmids in agarose gel.

[0036] like Figure 2 As shown, on a Tris-Glycine 4-20% gradient gel, the Nun-LEA002 concentrated sample (lane labeled 'NunLEA-002 (concentrated)') exhibits a significant and clear main band between approximately 16 kDa and 30 kDa, with a molecular weight consistent with the theoretical molecular weight of the LEA protein of this invention.

[0037] The main band is dark in color and bright, indicating that the Nun-LEA002 protein has high purity and a high concentration. Although a small number of extraneous protein bands are visible in molecular weight regions above the main band (such as at approximately 37 kDa and 52 kDa), their abundance is low and does not affect the subsequent application of the target protein.

[0038] Example 2: Construction of a system for expressing LEA protein in Escherichia coli 5 μL of the recombinant expression vector pET-24a(+)-LEA002 was placed in 100 μL of E. coli BL21(DE3) competent cells. The transformation process was as follows: E. coli BL21(DE3) competent cells were removed from a -80℃ cryogenic freezer and thawed on ice; in a clean bench, 300 ng of plasmid was added to 50 μL of E. coli... In BL21(DE3) competent cells, gently tap to mix and distribute evenly among the competent cells, then place on ice for 30 min. (All subsequent operations are performed in a clean bench.) Place the competent cells with added plasmid in a pre-opened 42°C water bath and heat shock for 90 s. Remove the competent cells and place them on ice for 3 min. Add 750 μL of antibiotic-free LB medium to the tube containing the competent cells and incubate on a shaker at 200 rpm / min for 60 min. After centrifuging at 2000 rpm / min for 3 min at room temperature, discard most of the medium, leaving approximately 100 μL of liquid medium. Mix the remaining liquid medium with the bacterial pellet by pipetting. Add the suspension dropwise to a solid culture medium containing kanamycin resistance, spreading it evenly with a spreader until a friction sensation is felt against the surface of the medium. Seal the culture dish with sealing film and incubate inverted at 37°C overnight. The next day, pick a single colony and transfer it to 5 mL of LB liquid medium, adding an appropriate proportion of kanamycin stock solution according to the 50 μg / mL kanamycin resistance ratio. Incubate at 37°C and 220 rpm / min. Take an appropriate amount of bacterial cells for plasmid mini-extraction using a plasmid mini-extraction kit. After plasmid extraction, determine the band size using single enzyme digestion.

[0039] Example 3: Induction of NunLEA-002 protein expression in Escherichia coli Using a sterile pipette tip, pick a single colony from the plate and inoculate it into a 5 mL liquid LB medium (containing 50 μg / mL kanamycin) test tube. Tilt the test tube and place it in a shaker. Incubate overnight at 37°C and 200 rpm. After incubation, remove the test tube and check the bacterial concentration. When the bacterial OD reaches 0.7, add 0.25 mM IPTG and continue induction at 37°C for 10 hours. After induction, collect the bacterial cells by centrifugation and perform ultrasonic lysis (ultrasonic volume: 3 mL, sonication conditions: 2 seconds on, 3 seconds off, total sonication time: 5 minutes). Centrifuge at 12000 rpm and analyze the supernatant using SDS-PAGE.

[0040] Example 4: NunLEA-002 protein expression and purification in E. coli 100 μL of overnight cultured bacterial solution was added to 1 L of LB medium (containing 50 μg / mL kanamycin) and cultured overnight at 37°C in a shaker. When the bacterial OD reached 0.7, 0.25 mM IPTG was added and incubated at 37°C for 10 hours. After induction, the bacterial cells were collected and subjected to sonication lysis. The sonication conditions were 5 seconds on, 5 seconds off, for a total sonication time of 15 minutes. After centrifugation at 12000 rpm / min, the supernatant was collected and filtered through a 0.45 μm pore size filter membrane to obtain total bacterial protein. The total protein was purified using Ni-NTA agarose resin to purify the recombinant histidine-tagged LEA protein. The purified protein was concentrated and desalted by ultrafiltration to obtain concentrated Nun-LEA002 protein. The concentration of concentrated Nun-LEA002 protein was determined by SDS-PAGE electrophoresis and BCA method to be 4 mg / mL, with a total volume of 9 mL.

[0041] Example 5: HaCaT cell drying model and determination of anti-drying activity after NunLEA-002 protein administration Using the method described in Chinese patent CN118895239A, "A Bionic Drying Model for Cells and Its Application in Evaluating Moisturizing Efficacy and Dryness Damage Repair Efficacy," the protective effect of NunLEA-002 protein against dryness damage in HaCaT cells was evaluated through CCK-8 cell viability assay. This model achieves gentle yet continuous dryness stress on cells by precisely controlling relative humidity using a saturated salt solution in a closed incubator environment, thus more closely mimicking the physiological response of skin under natural dry conditions.

[0042] HaCaT cells were seeded in 96-well plates and cultured for 24 hours to allow them to adhere.

[0043] Grouping and drug administration: Discard the old culture medium and gently wash once with sterile PBS. Set up the following groups (6 replicates per group): Normal control group (Untreated): Add 100 μL of fresh complete culture medium.

[0044] Dry damage group: No liquid added.

[0045] Positive control group (HA): 100 μL of complete culture medium containing 500 µg / mL hyaluronic acid was added.

[0046] LEA protein treatment group: 100 μL of complete culture medium containing different gradient concentrations (25, 50, 100, 200, 300, 400, 500 µg / mL) of NunLEA-002 was added respectively.

[0047] Drying and Modeling: After drug administration, the cell culture plates were pre-incubated in an incubator for 4 hours to allow for full protein interaction. Subsequently, the liquid in all wells (except the normal control group) was carefully aspirated. The sterile water tray at the bottom of the cell culture incubator was replaced with a sterile open container filled with saturated BaCl2 solution to stabilize the relative humidity (RH) in the incubator at approximately 89-90%. The treated 96-well plates (with the lids open but placed on a sterile culture dish base to prevent contamination) were placed in a humidity-equilibrated 37°C, 5% CO2 incubator for drying for 18 hours. The normal control group was cultured normally with the lids on in the same incubator.

[0048] Cell viability assay: After drying, 100 μL of fresh complete culture medium was quickly added to all wells (including the normal control group) to stop drying, followed by 10 μL of CCK-8 solution. After incubation for 2-3 hours, the OD value was measured at 450 nm using a microplate reader.

[0049] All experiments were independently repeated three times. Experimental data are expressed as mean ± standard deviation (Mean ± SD). GraphPad Prism 9.0 software was used for data analysis and graphing. One-way ANOVA was used for comparisons among multiple groups, combined with Tukey's post-hoc test for pairwise comparisons. P < 0.05 was considered statistically significant.

[0050] By measuring the anti-drying activity of Nun-LEA002 protein after administration, it was found that it has moisturizing and anti-drying activities similar to HA in HaCaT cells.

[0051] like Figure 3 The effect of different concentrations of NunLEA-002 protein on the cell viability of HaCaT cells under in vitro drying (hyperosmolarity) modeling conditions is shown. Experimental results are expressed as cell viability percentage (%). The results indicate that lotus NunLEA-002 protein has a significant protective effect on HaCaT cells under in vitro drying (hyperosmolarity) modeling conditions. Its protective effect is dose-dependent, exhibiting optimal activity at moderate concentrations (100-200 μg / mL), effectively reducing drying-induced cell damage and improving cell viability; its protective effect is comparable to that of hyaluronic acid.

[0052] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Lotus-derived LEA protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the lotus-derived LEA protein of claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

2.

3. A recombinant expression vector comprising the nucleic acid molecule of claim 2, characterized in that, The recombinant expression vector is constructed by inserting the nucleic acid molecule into the pET-24a(+) vector.

4. A host cell comprising the recombinant expression vector of claim 3, characterized in that, The host cell was Escherichia coli BL21.

5. A method for preparing the lotus-derived LEA protein of claim 1, characterized in that, Includes the following steps: (1) Construct a recombinant expression vector containing a nucleic acid molecule encoding the lotus-derived LEA protein of claim 1; (2) Transform the recombinant expression vector into host cells; (3) Inducing the host cells to express lotus-derived LEA protein; (4) Separate, purify and collect the lotus-derived LEA protein.

6. The application of the lotus-derived LEA protein of claim 1 in improving skin's resistance to dryness damage, and in the preparation of products for protecting skin cells against dryness damage.

7. The application according to claim 6, characterized in that, The skin cells are human immortalized keratinocytes.

8. A skin care composition for protecting skin cells against dryness damage, characterized in that, It contains an effective amount of the lotus-derived LEA protein of claim 1.

9. The skin care composition according to claim 8, characterized in that, The skin care composition is a serum, lotion, cream, mask, or freeze-dried powder.

Images