Method for heterologous expression of snail-derived mucoprotein and application of snail-derived mucoprotein

Abstract

The invention discloses a method for heterologous expression of snail-derived mucoprotein and application of the snail-derived mucoprotein. The snail-source mucoprotein is derived from African large snails, and the amino acid sequence of the snail-source mucoprotein is shown as SEQ ID NO.1. The protein is expressed and fermented by pichia pastoris, and the yield is obviously improved by optimizing a signal peptide sequence and a purification method and can reach 5g / L or above; and the mucoprotein with the purity of more than 95% is obtained. The snail-derived mucoprotein is subjected to efficacy characterization in vitro and on the cellular level, and the result shows that the protein has a remarkable effect in the aspect of repairing and further has the effects of resisting oxidation, relieving, moisturizing and whitening. The method can be used as a substitute of existing snail mucus, and solves the problems that snail mucus extraction is difficult in large-scale production, large in batch difference, high in pathogen pollution risk, inconsistent with the animal welfare principle and the like. Therefore, the invention has great application potential in the aspects of cosmetics, medicines and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to snail-derived mucin and a preparation method thereof and applications in moisturizing, anti-oxidation, whitening, soothing and repairing. Background Art

[0002] When a snail crawls, its foot glands can secrete a kind of mucus, which can reduce friction and assist its walking, and can resist the invasion of ants, bacteria, etc. The research on snail mucus can be traced back to the ancient Greek period. It is mainly used in the medical field and has moisturizing, anti-inflammatory and anti-aging effects. With the deepening of the research on snail mucus, snail mucus also has the effects of repairing and regenerating, eliminating acne scars and anti-oxidation. It is currently used by many companies in skin care. It is a growing market, and its value is expected to increase to US$770 million by 2025.

[0003] At present, typical methods for extracting snail mucus include electric shock of snail feet and salt stimulation. However, it is extremely challenging to purify large amounts of high-quality mucus from natural sources. On the one hand, the amount of mucus secreted from animal organs is limited, and the production process relies on extraction procedures with poor scalability, which greatly limits large-scale production. On the other hand, differences between product batches are almost inevitable, especially when the animal is sick or the organ is inflamed, the composition of the mucus will be different, and the animal source significantly increases the risk of pathogen contamination. And because the obtained snail mucus cannot be used directly on human skin, its effective ingredients need to be extracted, which greatly increases the cost of the product. Therefore, it has become necessary to seek a sustainable, batch-parallel and easy-to-purify method for snail mucus, among which artificial snail stock solution is a more feasible method.

[0004] It is known that snail mucus is a multi-component mixture, and its main components are allantoin, elastin, collagen, mucin, antimicrobial peptides, vitamin C, vitamin E, hyaluronic acid and glucuronic acid. Among them, allantoin, elastin, collagen, vitamin C, vitamin E, hyaluronic acid and glucuronic acid have clear effects in skin care, are widely used in skin care products, and the raw materials are easy to obtain. However, there is no snail mucin raw material on the market, and its efficacy is unknown. Therefore, snail mucin is the key to artificial snail mucus. It is known that mucin is a copolymer of protein and sugar, and is a family of high molecular weight, heavily glycosylated proteins. Its key feature is the ability to form gels, so they are the key components of most gel-like secretions, providing lubrication, cell signaling pathways and chemical barriers. At present, mucin on the market is mainly extracted from bovine submandibular glands, which are used in scientific research and artificial tears. Snail mucin mainly exists in the form of snail secretion filtrate, so the preparation method of snail mucin becomes the first problem to be solved. The literature currently discloses the sequences of a variety of snail mucins, making heterologous expression of snail mucins possible.

[0005] Secondly, the application form of snail mucin is snail concentrate. Due to the nature of the mixture, little is known about the efficacy of snail mucin itself. At present, the application of snail mucin is limited to skin care, which greatly limits its application scenarios. Therefore, it is necessary to clarify the efficacy of snail mucin. Summary of the invention

[0006] The purpose of the present invention is to provide a heterologously expressed snail mucin and a preparation method thereof, as well as applications in whitening, anti-oxidation, soothing, cell repair and the like.

[0007] The invention provides a method for preparing snail mucin, which comprises the following steps: connecting a nucleic acid encoding a mucin with an amino acid sequence as shown in SEQ ID NO.1 or a mucin with an identity of more than 90%, more than 95%, more than 98%, more than 99% and having the same function as the mucin from the giant African snail (Achatina fulica) to a nucleic acid encoding an alpha signal peptide, an OST1 signal peptide, an SA signal peptide or an SP23 signal peptide, introducing the nucleic acid into Pichia pastoris through a recombinant expression vector to obtain a recombinant Pichia pastoris, and fermenting and purifying the mucin to obtain the recombinant Pichia pastoris.

[0008] Specifically, the sequence of the α signal peptide is SEQ ID NO: 3: MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEG DFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA, the sequence of the OST1 signal peptide is SEQ ID NO: 4: MRQVWFSWIVGLFL CFFNVSSA, the sequence of the SA signal peptide is SEQ ID NO: 5: MKWVTFISLLFLFSSAYS, and the sequence of the SP23 signal peptide is SEQ ID NO: 6: MKI LSALLLLFTLAFA;

[0009] Preferably, the nucleotide sequence of the nucleic acid encoding the mucin is shown in SEQ ID NO: 2: the nucleotide sequence of the α signal peptide is shown in SEQ ID NO: 7: ATGAGATTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAG AAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCAT TTTCCAACAGCACAAATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCGA GAAAAGAGGCTGAAGCT; the nucleotide sequence of the OST1 signal peptide is shown in SEQ ID NO: 8: ATGCGACAGGTGTGGTTTAGTTGGA TAGTAGGACTTTTTCTATGCTTCTTCAATGTCTCCAGCGCT; the nucleotide sequence of the SA signal peptide is shown in SEQ ID NO: 9: ATGAAGTG GGTTACCTTTATCTCACTCCTATTCTTGTTCTCGTCTGCTTATAGT; the nucleotide sequence of the SP23 signal peptide is shown in SEQ ID NO: 10: ATGAAGATTCTTAGCGCTCTACTCTTATTGTTCACCCTGGCCTTTGCG.

[0010] Preferably, the starting vector of the recombinant expression vector is pPIC9K.

[0011] In a specific embodiment, the fermentation is to inoculate the recombinant Pichia pastoris into BMGY medium and culture at 30° C. for 3 days, and add 1% methanol every 24 hours; after the fermentation is completed, the fermentation supernatant is taken to obtain a crude product containing the mucin.

[0012] Preferably, the purification is performed by a cation exchange column SP column, specifically, the fermentation supernatant is dialyzed overnight in 50mM acetic acid-sodium acetate buffer, pH 4.5, and the SnMS protein is purified in the following manner: equilibrium mobile phase: 50mM acetic acid-sodium acetate buffer, pH 4.5; elution mobile phase: 50mM acetic acid-sodium acetate buffer, 1M NaCl, pH 4.5, gradient elution 20 column volumes.

[0013] The present invention also provides a use of snail mucin in preparing soothing and repairing, cell repairing, skin whitening and anti-oxidation preparations, wherein the amino acid sequence of the snail mucin is as shown in SEQ ID NO.1, or a mucin with the same function as that of the snail mucin having more than 90%, more than 95%, more than 98%, or more than 99% identity therewith and coming from the giant African snail (Achatina fulica).

[0014] Preferably, the mucin is obtained by the preparation method of the present invention.

[0015] The present invention also provides a snail mucin, whose amino acid sequence is a mucin as shown in SEQ ID NO.1 or a mucin with the same function as that of Achatinafulica and having more than 90%, more than 95%, more than 98%, or more than 99% identity thereto, and optionally further comprising an α signal peptide, an OST1 signal peptide, an SA signal peptide, or an SP23 signal peptide.

[0016] Furthermore, a gene encoding the snail mucin is also provided. Preferably, the nucleotide sequence thereof is shown in SEQ ID NO:2.

[0017] Beneficial effects: The present invention realizes the expression of snail-derived mucin in Pichia pastoris for the first time, and establishes a purification method with a purity of more than 90%. Although the efficacy of snail mucus has been disclosed in the prior art, it contains a variety of active ingredients, and the efficacy of snail mucin is known. Therefore, the present invention also characterizes the efficacy of pure snail mucin in vitro and at the cellular level for the first time. The heterologously expressed snail mucin SnMS has significant cell repair effects, in addition to whitening, moisturizing, soothing and antioxidant effects. It can be used as a substitute for existing snail mucus to solve the problems of difficult large-scale production of snail mucus extraction, large batch differences, high risk of pathogen contamination, and non-compliance with animal welfare principles. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :SDS-PAGE of the induced expression of SnMS protein in yeast strains. M: maker; Lanes 1-3: SnMS protein.

[0019] Figure 2 :Detection of fermentation level of SnSM strains with different signal peptides. Wherein, M: marker; 1 and 2: represent different transformant numbers.

[0020] Figure 3 : Purification of SnMS protein by anion exchange column. M: maker; lane 1: sample before purification; lane 2: flow-through; lanes 3-7: gradient elution samples.

[0021] Figure 4 : Purification of SnMS protein by Ni column. M: marker; Lane 1: fermentation supernatant; Lane 2: flow-through; Lanes 3-7: gradient eluted proteins.

[0022] Figure 5 : Color comparison of SnMS protein purified by Ni column. Among them, a: SnMS sample purified by Ni column; b: SnMS protein sample purified by cation exchange column.

[0023] Figure 6 : Purification of SnMS protein by SP column. Lane 1: flow-through; Lanes 2-8: gradient eluted proteins; Lane 9: fermentation supernatant.

[0024] Figure 7 :SDS-PAGE electrophoresis of the fermentation samples of SnMS protein at different time points in 10L tank fermentation. Note: 85 / 2 means the sample loading of 85h is halved; 74 / 2 means the sample loading of 74h is halved.

[0025] Figure 8 : SnMS freeze-dried sample of 10L fermentation broth.

[0026] Fig. 9 :Safety detection of SnMS protein on HaCaT cells.

[0027] Fig.10 : Comparison of images of cells treated with different concentrations of SnMS protein for 24 hours.

[0028] Fig.11 : Typical diagram of soothing effect (the dotted line area is the quantitative area). DETAILED DESCRIPTION

[0029] The present invention is described below through specific embodiments in order to better understand the present invention, but it does not constitute a limitation of the present invention.

[0030] Example 1: Construction of snail-derived mucin recombinant strain

[0031] Through literature search, we found a mucin from the disclosed genome sequence of the giant African snail Achatina fulica, which we named SnMS, and its amino acid sequence is shown in SEQ ID NO.1. The SnMS protein coding sequence was optimized according to the codon preference of Pichia pastoris, and the encoding nucleotide sequence is shown in SEQ ID NO.2. pPIC9K was used as an expression vector, and protein secretion was guided by its own signal peptide for full gene synthesis. The recombinant vector SnMS-pPIC9K was linearized with restriction endonuclease SalI and then electroporated into the competent cell of Pichia pastoris GS115 for recombination, and positive transformants were screened using histidine auxotrophy and geneticin resistance G418.

[0032] Example 2: Shake flask fermentation

[0033] The single colony obtained in Example 1 was picked and spotted onto a YPD (1% yeast extract, 2% peptone, 2% glucose, 1.5% agar) plate containing 2 mg / ml G418 resistance, and the strain that can grow on the resistant plate was selected for fermentation verification. First, the strain was inoculated into YPD liquid culture medium for overnight culture, and then inoculated into BMGY culture medium at a 2% inoculation ratio and cultured at 30°C for 16h. After centrifugation and washing, the strain was suspended in BMMY culture medium and cultured at 30°C for 3d, with 1% methanol added every 24h. After fermentation, the fermentation supernatant was taken for SDS-PAGE electrophoresis.

[0034] The results are as follows Figure 1 As shown in the figure, SnMS is secreted and expressed in a small amount in Pichia pastoris GS115. The molecular size is about 180KDa, which is 3.5 times larger than the molecular weight predicted by its amino acid sequence. The reason may be that the high proportion of glycosylation sites in SnMS leads to a large deviation in protein size.

[0035] Example 3: Modification of SnMS expression strain to improve fermentation level

[0036] It can be seen from the fermentation results of Example 2 that the fermentation level of SnMS is low, and it needs to be optimized to meet the production needs. At present, the signal peptide part of the SnMS protein is a signal peptide carried by itself, and it is speculated that it may not be applicable in the yeast expression system. Therefore, a strategy for optimizing its signal peptide is adopted to increase its secretion level. Four different signal peptides were selected, and their amino acid sequences are shown in Table 1. The signal peptide with a short sequence was inserted in front of the target gene by primer design, and the α signal peptide was introduced into the expression vector by fragment amplification and insertion. The primer design is shown in Table 2 below. Taking the construction of the α signal peptide expression vector as an example, other vectors are constructed in this way. First, pPIC9K is used as a template to linearize the vector with primer pair α-1f / α-1r, and the SnMS coding sequence with the signal peptide part removed is amplified with primer pair α-2f / α-2r. After the linearized vector and SnMS fragment are recovered, they are recombined and transformed according to the ClonExpress IIOne Step Cloning Kit instructions. The successfully constructed recombinant vector was electroporated into GS115 competent cells according to the method of Example 1, and positive transformants were selected using auxotrophy as screening markers and G418 resistance. Using the self-signal peptide strain as a control, shake flask fermentation was performed on strains with different signal peptides according to the method of Example 2. Two strains were selected for each signal peptide, and samples were taken for SDS-PAGE detection after 3 days of fermentation. The results are shown in Figure 2 shown.

[0037] Table 1 Signal peptide and amino acid sequence

[0038]

[0039] Table 2 Primer sequences

[0040]

[0041] The results are as follows Figure 2 As shown, compared with the SnMS protein's own signal peptide (control), the α signal peptide, OST1, SA and SP23 all showed higher fermentation levels, among which the SP23-2 strain had the highest protein production, with an estimated production increase of 2-3 times, and could be used as a subsequent large-scale fermentation verification strain.

[0042] Example 3: Protein purification methods and conditions

[0043] 1. Purification of SnMS protein using anion exchange column Q

[0044] The isoelectric point of SnMS protein was inferred from its amino acid sequence as pI=6.37, which is alkaline in neutral buffer. Therefore, anion exchange resin can be used for purification. Since the fermentation broth is large, it needs to be ultrafiltered and concentrated before purification and dialyzed overnight in the equilibrium phase. The purification conditions are as follows: equilibrium mobile phase: 20mMTris-HClpH 8.5; elution mobile phase: 20mMTris-HCl, 1M NaCl, pH 8.5; gradient elution. The purification results are shown in Figure 3 shown.

[0045] The results are analyzed as Figure 3 As shown, when an anion exchange column is used for protein purification, both the target protein and the impurity protein are bound to the column filler, and the target protein and the impurity protein cannot be effectively separated during elution.

[0046] 2. Purification of SnMS protein by Ni affinity chromatography

[0047] Since the SnMS protein sequence carries a 6*His tag at the C-terminus, the target protein can be purified using a Ni affinity column. The purification conditions are as follows: equilibrium mobile phase: 20mM PB, 150mM NaCl, pH7.5; elution mobile phase: 20mM PB, 150mM NaCl, 500mM imidazole, gradient elution. The purification results are shown in Figure 2. Figure 4 shown.

[0048] The results are as follows Figure 4 As shown in the figure, the target protein can be purified by Ni column purification, but it is still not well separated from the impurities. And most importantly, the purified protein is darker in color and contains some residues of fermentation medium components (such as Figure 5 Therefore, the purification method needs to be further optimized.

[0049] 3. Cation exchange column SP column purification

[0050] The fermentation broth was dialyzed overnight in 50 mM acetic acid-sodium acetate buffer, pH 4.5, and the SnMS protein was purified in the following manner: equilibrium mobile phase: 50 mM acetic acid-sodium acetate buffer, pH 4.5; elution mobile phase: 50 mM acetic acid-sodium acetate buffer, 1 M NaCl, pH 4.5, gradient elution 20 column volumes. The purification results are shown in Figure 6 shown.

[0051] Result analysis: Figure 6 It can be seen that the target protein is almost completely bound to the column filler and the purity of the protein is above 95%.

[0052] Example 5: Snail mucin SnMS protein 10L tank fermentation

[0053] The SnMS expression strain verified by the shake flask was reactivated on the YPD plate, transferred to the YPD-G418 (2mg / ml) plate, and the colony was picked and activated to the YPD liquid, cultured at 30℃ and 220rpm for 24 hours, and transferred to 1% of the BMGY liquid medium, cultured at 30℃ and 220rpm for 24 hours, and inoculated into the fermenter at 10%. The fermenter used BSM basal salt medium, and 4.4ml of PTM1 trace element mother solution was added per liter. The fermenter was controlled at 30℃, pH 5.5, and the speed was linked to control the dissolved oxygen>30%. The culture was continued until the glycerol was exhausted for 18h, at which time the OD 600 About 100, glycerol was added to control the dissolved oxygen to about 30%, cultured to OD300, the temperature was lowered to 25°C, alkali was added to adjust the pH to 6.0, methanol was added to induce and control the dissolved oxygen to about 25%, and cultured continuously until the yield no longer increased. SDS-PAGE gel electrophoresis was used to detect the protein yield at different induction times, such as Figure 7 As shown. The collected fermentation supernatant was replaced with 50mM, pH5.0 acetic acid-sodium acetate buffer, and the target protein was purified using a strong cation exchange column, phase A: 50mM, pH5.0 acetic acid-sodium acetate buffer, phase B: 50mM acetic acid-sodium acetate buffer and 1M NaCl solution, pH4.5. The SnMS protein-rich eluate was collected, and then the eluate was desalted and concentrated using a hollow fiber ultrafiltration system with a molecular weight cutoff of 3.0KDa, and the retentate was collected and freeze-dried (the final sample obtained is shown in Figure 8 shown).

[0054] The results are as follows Figure 7 As shown, with the extension of fermentation time, the yield of target protein SnMS gradually increased, and the yield after 85h of fermentation was not less than 5mg / ml.

[0055] Example 6: In vitro efficacy test - moisturizing, anti-oxidation and whitening

[0056] 1. Moisture determination - weighing method experiment

[0057] The weighing method is used to calculate the water absorption rate and moisturizing rate by utilizing the interaction between the sample and the water molecules in the environment under constant temperature and humidity conditions, so as to measure the moisturizing effect of the sample. The specific steps are as follows: 1. Place the saturated potassium carbonate solution in a closed condition overnight to keep the drying box at a suitable and stable humidity; 2. Take 0.4 ml of the sample and cover it in a culture dish with 3M medical breathable tape (2 sections of 4 cm), weigh the initial weight with an analytical balance, and place the culture dish in a drying box; 3. Take out the sample at 2h, 4h, 6h, and 8h, weigh the mass, and immediately put it in a dryer with constant humidity after weighing; 4. Calculate the water loss rate according to the following formula: Water loss rate = (m1-m2) / m1*100% (m1 is the mass of the sample before the experiment; m2 is the mass of the sample after the experiment). According to the above steps, the moisturizing rate of protein SnM with concentrations of 0.25%, 0.5% and 1% was measured, and the results are shown in Table 3:

[0058] Table 3: Determination of moisture retention rate of three different snail mucins

[0059]

[0060] Result analysis: With the increase of SnMS protein concentration, the water loss rate gradually decreased, indicating that SnMS protein has the effect of moisturizing.

[0061] 2. Antioxidant assay-DPPH free radical scavenging experiment

[0062] 1,1-Diphenyl-2-trinitrophenylhydrazine (DPPH) is a stable free radical that appears purple in ethanol solution and has a strong absorption peak at 517nM. Samples with antioxidant efficacy can weaken their absorption through electron pairing. Based on this principle, the antioxidant efficacy of 0.5% SnMS protein was determined.

[0063] The specific steps are as follows: 1. Test substance treatment: water-soluble test substances are diluted with water to form multi-level concentration samples, and oil-soluble test substances are diluted with 95% ethanol to form multi-level concentration samples. 2. According to Table 4, use 10mL test tubes to set up sample tubes (T), sample background (T0), DPPH tubes (C) and solvent background (C0). Three parallel tubes need to be set up for each sample tube (T), and three parallel tubes need to be set up for DPPH (C). 3. According to Table 1, add sample solution, water or 95% ethanol solvent, 95% ethanol and DPPH ethanol solution in order, mix well, and stand at room temperature away from light for 5 minutes; 4. Transfer each reaction solution into a 1cm cuvette or well plate, measure the absorbance at 517nm, and use 0.3mg / ml vitamin C as a positive control. Process and calculate the data as follows:

[0064] Calculate the DPPH free radical scavenging rate:

[0065]

[0066] (1) Where:

[0067] T-sample tube absorbance, that is, the absorbance of the solution after the sample reacts with DPPH:

[0068] T 0 -Sample background absorbance:

[0069] The average value of the absorbance of the C-DPPH tube for 3 times, that is, the absorbance of the DPPH solution without adding the sample:

[0070] C 0 -Solvent background absorbance.

[0071] Table 4: Sample loading requirements

[0072] T-sample tube <![CDATA[T 0 -Sample Background]]> C-DPPH tube <![CDATA[C 0 -Solvent Background]]> Sample solution (mL) 1 1 - - Water or 95% ethanol solvent (mL) 2 2 3 3 DPPH ethanol solution (mL) 1 - 1 - 95% ethanol (mL) - 1 - 1 Parallel times 3 / Sample 1 / sample 3 / Experimental 1 / Experiment

[0073] Result analysis: The results of the free radical scavenging experiment are shown in Table 5. The vitamin C free radical scavenging rate of 98.26% indicates that the control is normal and the experiment is credible. All three snail moisturizing proteins can scavenge the free radical DPPH and have antioxidant effects, among which Smuc2 has the worst antioxidant effect and Smuc4 has the best antioxidant effect.

[0074] Table 5: DPPH free radical scavenging experiments of three different snail mucins

[0075] Test sample (0.5%) DPPH free radical scavenging rate SnMS 21.71％ Vitamin C 98.26％

[0076] 3. Whitening efficacy determination-tyrosinase inhibition test

[0077] Tyrosinase is a key enzyme in the synthesis of melanin, which can catalyze L-tyrosine to form dopaquinone, which is the precursor of melanin. The test substance with tyrosinase activity inhibition can slow down the tyrosinase-catalyzed conversion of L-tyrosine into dopaquinone. The absorbance of dopaquinone at 475nm is measured, and the inhibitory effect of the test substance on tyrosinase activity is evaluated according to the change of absorbance.

[0078] Based on the above principle, the whitening efficacy of SnMS was tested. The specific steps are as follows: 1. The positive control kojic acid was diluted with PBS buffer to form a series of mass concentration gradient solutions: 0.080g / L, 0.040g / L, 0.020g / L, 0.010g / L, 0.008g / L, 0.005g / L, 0.002g / L, 0.001g / L; 2. The solvent background well (Ta), solvent reaction well (Tb), sample background well (Tc), and sample reaction well (Td) were set in the 96-well ELISA plate. Among them, the Ta group is the solvent background group, without adding the substrate L-tyrosine solution and the sample solution; the Tb group is the solvent reaction group, with the substrate L-tyrosine solution but without the sample solution; the Tc group is the sample background group, without adding the substrate L-tyrosine solution and adding the sample solution; the Td group is the sample reaction group, with both the substrate L-tyrosine solution and the sample solution. Three replicate wells were made for each group; 3. Referring to the reagent addition amount in Table 4, L-tyrosine solution, sample solution / solvent, and PBS buffer were added to each well in turn, mixed thoroughly, incubated at 37°C for 10 min, and then 20 μL of tyrosinase solution was added to each well in turn. After mixing at 37°C for 5 min±5 s, the well was immediately placed in a microplate reader and measured at a wavelength of 475 nm (Note: the time from the addition of tyrosinase solution to the measurement of absorbance in each well was kept consistent (5 min±5 s). SnMS, Smuc2, and Smuc4 with a content of 0.5% were measured by referring to the above steps. The tyrosinase activity inhibition rate of each test sample was calculated according to the following formula.

[0079] Calculate according to formula (1):

[0080]

[0081] Where:

[0082] Y-tyrosinase activity inhibition rate;

[0083] A d - Absorbance of sample reaction wells;

[0084] A c -Sample background well absorbance;

[0085] A b - Average absorbance of solvent reaction wells;

[0086] A a - Average absorbance of solvent background wells.

[0087] Table 6: Sample loading table for tyrosinase activity inhibition experiment

[0088]

[0089] Result analysis: It can be seen from Table 7 that SnMS can inhibit tyrosinase activity, and its effect is better than 0.008 mg / ml kojic acid.

[0090] Table 7: Experimental study on the inhibition rate of tyrosinase activity of three different snail mucins

[0091] Test samples Tyrosinase inhibition rate 0.5% SnMS 16.74％ 0.008mg / ml Kojic acid 8.04％ 0.04mg / ml kojic acid 43.62％ 0.2mg / ml kojic acid 91.38％ 1mg / ml kojic acid (positive control) 98.62％

[0092] Example 7: SnMS-Cellular Level Toxicity Testing

[0093] Based on the principle that WST-8 in the CCK8 kit can be oxidized and reduced by NAD+ to a water-soluble yellow formazan product in the presence of living cells, the cytotoxicity of the samples and their effects on cell proliferation were evaluated. The specific steps are as follows: The CCK-8 method was used to detect the effects of the test substances on the survival rate of the HaCaT cell line. Cells in the exponential growth phase were taken, digested with 0.25% trypsin-EDTA and blown evenly, and the cells were plated at 2×10 4 The cells were inoculated into a 96-well plate at a density of 100 μL per well and incubated at 37°C with 5% CO 2 Cultured overnight in an incubator; discarded the supernatant, added 100 μL of culture medium containing different concentrations of the test substance, and made 3 replicates for each concentration; incubated for 48 hours. The cell viability was detected using a CCK-8 kit, and the absorbance (OD value) at 450 nm was measured using an enzyme marker, and the cell survival rate was calculated according to formula (1).

[0094] Cell survival rate = (OD value of each well - OD value of blank group) / (OD value of control group - OD value of blank group) * 100%

[0095] Results: When the survival rate is low, the potential cytotoxicity of the test sample is high. If the survival rate drops to <70% of the blank, it is potentially cytotoxic. Fig. 9 From the effect of SnMS protein on the cell survival rate of HaCaT cell line shown in the figure, the cell survival rate at the concentrations of 0.5% and 1% of SnMS protein was above 90%, indicating that the concentration of 0.5% was within its safe applicable range and had no cytotoxicity, and the cell survival rate at the concentration of 1% was greater than 100%, indicating that SnMS protein may have cell proliferation activity.

[0096] Example 8: Toxicological testing - acute eye irritation test

[0097] Experimental method: 3 male New Zealand rabbits weighing 2.0-2.5kg were selected and adapted to the laboratory animal room environment for 3 days. Both eyes of the New Zealand rabbits were examined within 24 hours before the start of the experiment (including the use of sodium fluorescein for examination), and no obvious eye irritation symptoms, corneal defects and conjunctival damage were found. Gently pull open the left lower eyelid, drip 0.1mL of the test substance into the conjunctival sac, and passively close the upper and lower eyelids for 1 second to prevent the loss of the test substance. The eyes were not rinsed within 24 hours after the test substance was administered, and the right eye was not treated as a self-control. The damage to the conjunctiva, cornea and iris was examined and recorded 1, 24, 48 and 72 hours after the test substance was administered. The scoring was carried out according to the "Table 1 Scoring Standards for Eye Damage" in Chapter 6, 5 of the "Safety Technical Specifications for Cosmetics" (2015 Edition), and the eyes of all animals were further examined with sodium fluorescein 24 hours later. No irritation reaction occurred 72 hours after poisoning, and the test was terminated. The intensity of eye irritation reaction is determined by evaluating the mean value of the irritation response scores of the cornea, iris and conjunctiva of the animals at 24h, 48h and 72h after administration of the test substance and the recovery time, and judging the intensity of eye irritation reaction in accordance with “Table 2 Classification of Eye Irritation Reaction of Raw Materials” in Chapter 6.5 of “Technical Specifications for Safety of Cosmetics” (2015 edition).

[0098] The test report results showed that 0.5% SnMS protein had no acute eye irritation to New Zealand rabbits.

[0099] Results of acute eye irritation test on New Zealand rabbits

[0100] (Do not rinse)

[0101]

[0102] Note: The mean integral value is rounded to 2 decimal places.

[0103] Example 9: SnMS-Cellular Level Repair Efficacy Test

[0104] 1. Oxidative damage repair

[0105] Malondialdehyde (MDA) is an important product of lipid peroxidation. Reactive oxygen species (ROS) are also produced in large quantities when damaged. The accumulation of these substances causes degradation of the extracellular matrix, and inflammation leads to skin aging. AAPH (azobisisobutylamidine hydrochloride) can induce the production and accumulation of cellular ROS. The effect of raw materials on the proliferation activity of AAPH-treated skin keratinocytes (HaCaT) can be detected by CCK8, which can determine the safety of the raw materials and evaluate the repair efficacy of the raw materials on damaged skin keratinocytes. Based on the above principles, the cell damage repair experiment was carried out according to the following steps: First, cells in the logarithmic growth phase were taken, and the cell suspension was inoculated in a 96-well plate with 100 μL per well, so that the cell density was 7000 cells / well. After 24 days of culture, the drug addition operation (AAPH) was performed, and the cells were placed in a 5% CO2 incubator at 37°C for 2 hours; then, after the culture was completed, the culture medium was discarded, and complete culture medium containing samples of different concentrations was added to continue the culture for 24 hours; finally, 10 μL of CCK8 reagent was added to each well after the culture was completed, and a blank group was set to exclude the influence of the color of the working solution. The 96-well plate was incubated in an incubator for about 45-90 minutes, and the absorbance was measured at 450 nm by a microplate reader. The cell proliferation rate and repair rate were calculated according to the following formula: cell activity = (OD value of the experimental group - blank group) / (control group - blank group) × 100; repair rate = (cell activity of the experimental group - cell activity of the model group) / (cell activity of the blank group - cell activity of the model group) * 100.

[0106] Results: As shown in Table 8, the cell activity of 0.5% SnMS protein was significantly better than that of the model group, and the cell repair rate was 39.2%, indicating that SnMS protein has a repairing effect on cell oxidative damage.

[0107] Table 8: SnMS repair test on oxidative damage

[0108]

[0109]

[0110] 2. Cell Migration Assay

[0111] The wound healing assay is one of the earliest methods developed to study directional cell migration in vitro. This method simulates the migration process of cells during the healing process in vivo. The basic steps include creating a "wound" in the cell monolayer, capturing images at the beginning and periodically during cell migration to close the wound, and comparing images to determine the rate of cell migration. Based on the above principles, the following steps were used to test SnMS proteins at different concentrations, with 0.1ug / ml bFGF as a positive control: 1) 35mm*10mm cell culture dishes were plated, a half-dividing line was drawn on the back of the dish, and the Culture-Insert was placed in the middle of the line with a 10*105 1) Take 70ul of the culture medium at a density of 100 μl / ml and add it to the three wells of the Culture-Insert. Stably culture it in a 5% CO2 incubator for 24 hours; 2) Gently remove the Culture-Insert with sterile tweezers and check whether the cell layer is still attached to the surface of the μ-Dish; 3) Wash the cell layer with PBS to remove cell debris and non-attached cells; 4) Use the recommended drug preparation system to fill the μ-Dish with 2ml; 5) Take pictures at 0h, 4h, 6h, 24h, and 48h (observation at 24h is obvious and no pictures can be taken at 48h), analyze and process the pictures with image, and process the data. Mobility calculation formula: Mobility % = (area 0h -area Nh ) / area 0h *100;

[0112] Result: Combined Fig.10 As shown in the results of Table 9, SnMS proteins of different concentrations all showed better migration rates than the control group, and the scratches basically disappeared after 24 hours of treatment, indicating that SnMS protein showed better repair efficacy than the positive control, and within a certain range, the cell migration rate was proportional to the SnMS concentration.

[0113] Table 9 Cell migration rate of SnMS protein at different concentrations

[0114]

[0115] Example 10: SnMS-zebrafish soothing efficacy test

[0116] Sodium dodecyl sulfate (SLS) may cause irritation in the body after acting on the body. The irritant enters the body of zebrafish, induces an inflammatory response, and neutrophils have an immune response, migrate to the skin epidermis and aggregate. The changes in the number of skin neutrophils before and after treatment with transgenic neutrophil green fluorescent strain zebrafish (MPX) were used to detect whether the sample has a soothing effect. Based on the above principles, the SnMS protein was tested in the following steps: 1) Randomly select zebrafish in a 6-well plate, 15 per well, and give SLS in water to establish a zebrafish skin inflammation model. At the same time, a normal control group and a model control group were set up, with a volume of 3mL per well, and incubated in the dark at 28°C for 18h; 2) 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Advanced image processing software was used to analyze and collect data, and the number of zebrafish skin neutrophils (N) was analyzed. The soothing effect of the sample was calculated according to the formula to determine whether it has a soothing effect:

[0117]

[0118] Results: Fig.11As shown, the number of neutrophils in the SnMS group was significantly reduced compared with the model control group, revealing that the sample has a soothing effect.

Claims

1. A method for preparing snail mucin, It is characterized in that The nucleic acid encoding the mucin with an amino acid sequence as shown in SEQ ID NO. 1 or a snail mucin with an identity of more than 90%, more than 95%, more than 98%, or more than 99% and having the same function as the snail mucin from the giant African snail (Achatinafulica) is connected to the nucleic acid encoding the α signal peptide, the OST1 signal peptide, the SA signal peptide, or the SP23 signal peptide, and introduced into Pichia pastoris through a recombinant expression vector to obtain recombinant Pichia pastoris, which is then fermented and purified to obtain the result.

2. The method according to claim 1, It is characterized in that The amino acid sequence of the α signal peptide is shown in SEQ ID NO: 3, the amino acid sequence of the OST1 signal peptide r is shown in SEQ ID NO: 4, the amino acid sequence of the SA signal peptide is shown in SEQ ID NO: 5, and the amino acid sequence of the SP23 signal peptide is shown in SEQ ID NO: 6; The nucleotide sequence of the snail mucin encoding nucleic acid is shown in SEQ ID NO: 2; the nucleotide sequence of the α signal peptide is shown in SEQ ID NO: 2; the nucleotide sequence of the OST1 signal peptide is shown in SEQ ID NO: 7; the nucleotide sequence of the SA signal peptide is shown in SEQ ID NO: 8; and the nucleotide sequence of the SP23 signal peptide is shown in SEQ ID NO:

9.

3. The method according to claim 1, It is characterized in that The starting vector of the recombinant expression vector is pPIC9K.

4. The method according to claim 1, It is characterized in that The fermentation is carried out by inoculating the recombinant Pichia pastoris into a BMGY medium and culturing the medium at 30° C. for 3 days, supplementing 1% methanol every 24 hours; after the fermentation is completed, the fermentation supernatant is taken to obtain a crude product containing the mucin.

5. The method according to claim 4, It is characterized in that The purification is carried out by cation exchange column SP column, specifically, the fermentation supernatant is dialyzed overnight in 50mM acetic acid-sodium acetate buffer at pH4.5, and the SnMS protein is purified in the following manner: equilibrium mobile phase: 50mM acetic acid-sodium acetate buffer, pH4.5; elution mobile phase: 50mM acetic acid-sodium acetate buffer, 1M NaCl, pH4.5, gradient elution 20 column volumes.

6. A use of snail mucin in the preparation of soothing, cell repair, skin whitening and anti-oxidation preparations, wherein the amino acid sequence of the snail mucin is as shown in SEQ ID NO. 1, or a mucin with the same function as that of the giant African snail (Achatina fulica) and having more than 90%, more than 95%, more than 98%, or more than 99% identity thereto.

7. The use according to claim 6, It is characterized in that The mucin is obtained by the method according to any one of claims 1 to 5.

8. A snail mucin, whose amino acid sequence is as shown in SEQ ID NO. 1 or a mucin with more than 90%, more than 95%, more than 98%, more than 99% identity thereto and having the same function from the giant African snail (Achatina fulica), optionally further comprising an α signal peptide, an OST1 signal peptide, an SA signal peptide or an SP23 signal peptide.

9. A gene encoding the snail mucin as claimed in claim 8.

10. The gene according to claim 9, It is characterized in that The nucleotide sequence thereof is shown in SEQ ID NO:2.

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