Oil body protein as well as preparation method and application thereof

Abstract

The invention relates to the technical field of cosmetic functional raw materials, in particular to oleosin as well as a preparation method and application thereof. The amino acid sequence of the oleosin is as shown in SEQ ID No. 1 and / or SEQ ID No. 2. The oleosin has the advantages of being simple in structure, simple in synthesis method and the like, can effectively promote collagen expression, is more suitable for actual production requirements, and can be applied to preparation of cosmetics and / or medical supplies.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetic functional raw materials, and in particular to an oil body protein and a preparation method and application thereof. Background Art

[0002] Oil body membrane protein (OIP) is an inherent component of the surface of plant oil bodies and is usually present in the seeds of various plants. OIP plays a key role in maintaining seed germination, affecting the size of oil bodies in seeds and lipid accumulation during plant growth and development.

[0003] There are many types of plant oil body membrane proteins. To study the effects of a single substance, further purification is needed. The current separation and purification method is to separate it using protein electrophoresis, which cannot be achieved for mass production and industrial application, limiting its further application.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide an oleosomal protein and a preparation method and application thereof. The oleosomal protein has a simple structure and can effectively promote the expression of collagen.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0007] One aspect of the present invention relates to an oleosomal protein, the amino acid sequence of which is shown as SEQ ID No.1 and / or SEQ ID No.2.

[0008] The oil body protein has the advantages of simple structure and simple synthesis method, can effectively promote the expression of collagen, and is more suitable for actual production needs. It can be used to prepare cosmetics and / or medical products.

[0009] Another aspect of the present invention also relates to a method for preparing the oil body protein, comprising the following steps:

[0010] (a) adding piperidine and N,N-dimethylformamide to a carrier resin and then performing a first reaction and a first washing;

[0011] (b) adding the first amino acid raw material, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-dimethylformamide and N,N-diisopropylethylamine to the reaction solution after the first washing to carry out a first stirring reaction;

[0012] (c) performing a ninhydrin colorimetric test on the carrier resin after the first stirring reaction. When the test result is negative, vacuum-evacuating the reaction liquid of the first stirring reaction, adding N,N-dimethylformamide, acetic anhydride and N,N-diisopropylethylamine to perform a second stirring reaction, and vacuum-evacuating the reaction liquid of the second stirring reaction, and performing a second washing;

[0013] (d) adding piperidine and N,N-dimethylformamide to the reaction system after the second washing, and then performing a first deprotection reaction and a third washing;

[0014] (e) adding the second amino acid raw material, ethyl 2-oxime cyanoacetate, N,N'-diisopropylcarbodiimide and N,N-dimethylformamide to the reaction system after the third washing, and then performing a third stirring reaction and a fourth washing; performing a ninhydrin colorimetric detection on the carrier resin, and when the detection result is negative, adding piperidine and N,N-dimethylformamide to the reaction system after the fourth washing, performing a second deprotection reaction and a fifth washing;

[0015] (f) according to the amino acid sequence of the oil body protein, the second amino acid raw material is replaced with the third amino acid raw material and the amino acid raw materials after the third amino acid raw material in sequence, and step (e) is repeated to synthesize the amino acid sequence of the oil body protein;

[0016] (g) performing a capping reaction and a sixth washing on the reaction system after step (f); performing a ninhydrin colorimetric detection on the carrier resin, and when the detection result is negative, performing a shrinkage washing and a first drying on the carrier resin to obtain a peptide resin; cracking and filtering the peptide resin, mixing the filtrate with glacial methyl tert-butyl ether, performing a first centrifugation, and removing the upper layer liquid to obtain a crude polypeptide; mixing the crude polypeptide with glacial methyl tert-butyl ether, performing a second centrifugation, removing the upper layer liquid, and performing a second drying to obtain a crude product;

[0017] (h) purifying and converting the crude product.

[0018] The method for preparing the oil body protein has a simple process and is suitable for industrial mass production. The prepared oil body protein has a simple structure and high purity, has an excellent effect of promoting collagen expression, and can be effectively used in cosmetics and / or medical products.

[0019] Another aspect of the present invention also relates to the use of the oleosomal protein or the oleosomal protein prepared by the preparation method of the oleosomal protein in the preparation of cosmetics and / or pharmaceutical products.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The oil body protein provided by the present invention has the advantages of simple structure and simple synthesis method, can effectively promote the expression of collagen, and is more suitable for actual production needs, and can be used to prepare cosmetics and / or pharmaceutical products.

[0022] (2) The method for preparing oleosomal protein provided by the present invention effectively solves the problem that it is difficult to obtain high-purity natural oleosomal protein in the existing process. The process is simple and suitable for industrial mass production. The prepared oleosomal protein has a simple structure and high purity and has an excellent effect of promoting collagen expression, and can be effectively used in cosmetics and / or pharmaceutical products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 is the HPLC chromatogram of oil body protein SPVIVP;

[0025] Figure 2 It is the HPLC chromatogram of oil body protein SPVLVP. DETAILED DESCRIPTION

[0026] The technical scheme of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments, but it will be understood by those skilled in the art that the following described embodiments are part of embodiments of the present invention, rather than all embodiments, and are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0027] One aspect of the present invention relates to an oleosomal protein, the amino acid sequence of which is shown as SEQ ID No.1 and / or SEQ ID No.2.

[0028] The oil body protein has the advantages of simple structure and simple synthesis method, can effectively promote the expression of collagen, and is more suitable for actual production needs. It can be used to prepare cosmetics and / or medical products.

[0029] Furthermore, the amino acid sequence of the oil body protein is serine (Serine) - proline (Proline) - valine (Valine) - isoleucine (Isoleucine) - valine (Valine) - proline (Proline) and / or serine (Serine) - proline (Proline) - valine (Valine) - leucine (Leucine) - valine (Valine) - proline (Proline).

[0030] Further, SEQ ID No. 1: Ser-Pro-Val-Iso-Val-Pro (abbreviated as SPVIVP).

[0031] Further, SEQ ID No. 2: Ser-Pro-Val-Leu-Val-Pro (abbreviated as SPVLVP).

[0032] Another aspect of the present invention also relates to a method for preparing the oil body protein, comprising the following steps:

[0033] (a) adding piperidine and N,N-dimethylformamide to a carrier resin and then performing a first reaction and a first washing;

[0034] (b) adding the first amino acid raw material, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-dimethylformamide and N,N-diisopropylethylamine to the reaction solution after the first washing to carry out a first stirring reaction;

[0035] (c) performing a ninhydrin colorimetric test on the carrier resin after the first stirring reaction. When the test result is negative, vacuum-evacuating the reaction liquid of the first stirring reaction, adding N,N-dimethylformamide, acetic anhydride and N,N-diisopropylethylamine to perform a second stirring reaction, and vacuum-evacuating the reaction liquid of the second stirring reaction, and performing a second washing;

[0036] (d) adding piperidine and N,N-dimethylformamide to the reaction system after the second washing, and then performing a first deprotection reaction and a third washing;

[0037] (e) adding the second amino acid raw material, ethyl 2-oxime cyanoacetate, N,N'-diisopropylcarbodiimide and N,N-dimethylformamide to the reaction system after the third washing, and then performing a third stirring reaction and a fourth washing; performing a ninhydrin colorimetric detection on the carrier resin, and when the detection result is negative, adding piperidine and N,N-dimethylformamide to the reaction system after the fourth washing, performing a second deprotection reaction and a fifth washing;

[0038] (f) according to the amino acid sequence of the oil body protein, the second amino acid raw material is replaced with the third amino acid raw material and the amino acid raw materials after the third amino acid raw material in sequence, and step (e) is repeated to synthesize the amino acid sequence of the oil body protein;

[0039] (g) performing a capping reaction and a sixth washing on the reaction system after step (f); performing a ninhydrin colorimetric detection on the carrier resin, and when the detection result is negative, performing a shrinkage washing and a first drying on the carrier resin to obtain a peptide resin; cracking and filtering the peptide resin, mixing the filtrate with glacial methyl tert-butyl ether, performing a first centrifugation, and removing the upper layer liquid to obtain a crude polypeptide; mixing the crude polypeptide with glacial methyl tert-butyl ether, performing a second centrifugation, removing the upper layer liquid, and performing a second drying to obtain a crude product;

[0040] (h) purifying and converting the crude product.

[0041] The method for preparing oleosomal protein effectively solves the problem that it is difficult to obtain high-purity natural oleosomal protein in the existing process. The process is simple and suitable for industrial mass production. The prepared oleosomal protein has a simple structure and high purity, has an excellent effect of promoting collagen expression, and can be effectively used in cosmetics and / or medical products.

[0042] The first amino acid raw material mentioned in the present invention refers to the amino acid raw material required for synthesizing the first amino acid of oleosomal protein, and the subsequent amino acid raw materials are analogous. For example, the first amino acid of oleosomal protein is proline, and the first amino acid raw material is Fmoc-Pro-OH, and the second amino acid is valine, and the second amino acid raw material is Fmoc-Val-OH.

[0043] The present invention does not specifically limit the type of carrier resin, and any conventional carrier resin in the art can be used to implement the technical solution of the present invention. In some specific embodiments, the carrier resin includes but is not limited to: Rink Amide-AM resin.

[0044] Furthermore, in step (a), the carrier resin, piperidine and N,N-dimethylformamide are added in a ratio of 50 g: 60 mL: 240 mL. Adding the carrier resin, piperidine and N,N-dimethylformamide in a certain amount can ensure that the carrier resin, piperidine and N,N-dimethylformamide react fully.

[0045] Furthermore, the time of the first reaction is 25 minutes. The time of the first reaction is limited to a certain range, so that the reaction raw materials can fully react.

[0046] Furthermore, the first washing is performed 7 times, using 500 mL of washing solution each time.

[0047] Furthermore, in step (b), the addition ratio of the first amino acid raw material, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-dimethylformamide and N,N-diisopropylethylamine is 100mmol:40.7g:210mL:35mL. The feeding amount of step (b) is limited to ensure that the first stirring reaction is fully carried out.

[0048] Furthermore, the first stirring reaction time is 2 hours.

[0049] Furthermore, the carrier resin is subjected to a ninhydrin colorimetric test. If the carrier resin does not develop color, the test result is negative.

[0050] Furthermore, in step (c), the addition ratio of N,N-dimethylformamide, acetic anhydride and N,N-diisopropylethylamine is 280 mL: 10 mL: 10 mL. The amount of raw materials added in step (c) is limited to ensure that the second stirring reaction is fully carried out.

[0051] Furthermore, the second stirring reaction time is 0.5 h.

[0052] Furthermore, the second washing is repeated 3 times, and the volume ratio of the washing liquid to the carrier resin used each time is 1:10.

[0053] Further, in step (d), the amount of piperidine and N,N-dimethylformamide added to the reaction system after the second washing is 120 mL of piperidine and 480 mL of N,N-dimethylformamide. The amount of piperidine and N,N-dimethylformamide added is limited to ensure that the first deprotection reaction is fully carried out.

[0054] Furthermore, the first deprotection reaction time is 25 min.

[0055] Furthermore, the third washing is repeated 7 times, and the volume ratio of the washing liquid to the carrier resin used each time is 1:10.

[0056] Furthermore, in step (e), the addition ratio of the second amino acid raw material, ethyl 2-oxime cyanoacetate, N,N'-diisopropylcarbodiimide and N,N-dimethylformamide is 100mmol:100mmol:100mmol:280mL. Limiting the feeding amount of the third stirring reaction can ensure that the third stirring reaction is fully carried out and avoid the generation of by-products.

[0057] Furthermore, the third stirring reaction time is 1.5 hours.

[0058] Furthermore, the fourth washing is repeated 3 times, and the volume ratio of the washing liquid to the carrier resin used each time is 1:10.

[0059] Further, in step (e), the amount of piperidine and N,N-dimethylformamide added to the reaction system after the fourth washing is 120 mL piperidine and 480 mL N,N-dimethylformamide. The amount of piperidine and N,N-dimethylformamide added during the second deprotection reaction is limited to ensure that the second deprotection reaction is carried out thoroughly.

[0060] Furthermore, the second deprotection reaction time is 25 min.

[0061] Furthermore, the fifth washing is repeated 5 times, and the volume ratio of the washing liquid to the carrier resin used each time is 1:10.

[0062] Furthermore, 320 mL of end-capping liquid was added to the reaction system after step (f) to carry out an end-capping reaction.

[0063] Furthermore, the capping liquid for the capping reaction includes N,N-dimethylformamide, N,N-diisopropylethylamine (DIEA) and acetic anhydride (Ace) in a volume ratio of 28:1:1.

[0064] Furthermore, the end-capping reaction time is 0.5 h.

[0065] Furthermore, the sixth washing is repeated 3 times, and the volume ratio of the washing liquid to the carrier resin used each time is 1:10.

[0066] Furthermore, the shrinkage washing uses 350 mL of methanol as a washing liquid.

[0067] Furthermore, the lysis solution comprises trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) and water in a volume ratio of 90:5:5.

[0068] Furthermore, the addition ratio of the peptide resin to the lysis solution is 1 g: 8 mL. The peptide resin can be fully lysed by controlling the usage ratio of the peptide resin to the lysis solution within a reasonable range.

[0069] Furthermore, the lysis time is 3h.

[0070] Furthermore, when performing the first centrifugation, the volume of glacial methyl tert-butyl ether added is 8 times the volume of the filtrate.

[0071] Furthermore, during the second centrifugation, the volume of glacial methyl tert-butyl ether added is 8 times the volume of the filtrate.

[0072] Furthermore, the speed of the first centrifugation is 3000 r / min, and the time of the first centrifugation is 2 min.

[0073] Furthermore, the speed of the second centrifugation is 3000 r / min, and the time of the second centrifugation is 2 min.

[0074] Furthermore, the purification is carried out by liquid chromatography.

[0075] Furthermore, the mobile phase A for purification is 0.1 vol% acetic acid aqueous solution, the mobile phase B is 0.1 vol% acetic acid acetonitrile solution, and the detection wavelength is 220 nm or 254 nm.

[0076] Furthermore, the salt conversion is carried out using a 0.3 vol% acetic acid aqueous solution.

[0077] Furthermore, the washing liquids of the first washing, the second washing, the third washing, the fourth washing, the fifth washing and the sixth washing include, but are not limited to: N,N-dimethylformamide.

[0078] Another aspect of the present invention also relates to the use of the oleosomal protein or the oleosomal protein prepared by the preparation method of the oleosomal protein in the preparation of cosmetics and / or pharmaceutical products.

[0079] The raw material information for preparing oil body protein in the specific implementation mode of the present application is shown in Table 1.

[0080] Table 1

[0081] name Abbreviation Packing Specifications factory Fmoc-Pro-OH P 500g / bag Chengdu Zhengyuan Biochemical Technology Co., Ltd. Fmoc-Val-OH V 500g / bag Shanghai Zhengji Biotechnology Co., Ltd. Fmoc-Ile-OH I 500g / bottle Gill Biochemical (Shanghai) Co., Ltd. Fmoc-Ser(tBu)-OH S 500g / bottle Gill Biochemical (Shanghai) Co., Ltd. Fmoc-Leu-OH L 500g / bottle Gill Biochemical (Shanghai) Co., Ltd. Acetic anhydride Ace 500ml / bottle Sinopharm Chemical Reagent Co., Ltd.

[0082] The reagent information for preparing oil body protein in the specific implementation mode of the present application is shown in Table 2.

[0083] Table 2

[0084]

[0085] The resin information for preparing oil body protein in the specific implementation mode of the present application is shown in Table 3.

[0086] Table 3

[0087] name Theoretical degree of substitution batch number factory Rink Amide-AM Resin 0.93mmol / g 08-240107 Xi'an Lanxiao Technology New Materials Co., Ltd.

[0088] The present invention adopts HPLC method to carry out purity and content test.

[0089] The present invention uses HPLC-MS / MS (high performance liquid chromatography-mass spectrometry) to identify components.

[0090] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0091] Example 1

[0092] The method for preparing the oil body protein with the amino acid sequence of SPVIVP provided in this embodiment comprises the following steps:

[0093] a. Weigh 50 g of Rink Amide-AM resin and place it in a polypeptide reactor. Add 60 mL of piperidine and 240 mL of industrial grade N,N-dimethylformamide and react for 25 min. Then, add DMF and wash 7 times, each time using 500 mL.

[0094] b. Weigh 100 mmol of amino acid (Fmoc-Pro-OH) and 40.7 g of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, add 210 mL of AR-grade N,N-dimethylformamide and 35 mL of N,N-diisopropylethylamine, stir evenly and add to the polypeptide reactor, start the polypeptide reactor and stir for 2 hours;

[0095] c. The resin was tested with ninhydrin and the result was negative, indicating that the degree of substitution of Rink Amide-AM resin was 0.93 mmol / g;

[0096] d. After vacuuming the reaction solution, add AR grade N,N-dimethylformamide 280mL, acetic anhydride 10mL, N,N-diisopropylethylamine 10mL and stir the reaction for 0.5h, vacuum dry and wash 3 times with industrial grade N,N-dimethylformamide, each washing amount is 10 times the volume of the resin;

[0097] e. Add 600 mL of 20% piperidine / N,N-dimethylformamide solution (i.e., 120 mL piperidine and 480 mL N,N-dimethylformamide) for deprotection reaction for 25 min, and then add industrial grade N,N-dimethylformamide for washing 7 times, with each washing amount being about 10 times the resin volume;

[0098] f. Using ethyl 2-oxime cyanoacetate and N,N'-diisopropylcarbodiimide (Oxyma, DIC) system for condensation, Fmoc-Val-OH, ethyl 2-oxime cyanoacetate, N,N'-diisopropylcarbodiimide were added in an amount of 100 mmol, AR grade N,N-dimethylformamide 280 mL, stirred for reaction for 1.5 h, after the condensation was completed, industrial grade N,N-dimethylformamide was added for washing 3 times, each washing amount was 10 times the resin volume; the carrier resin was negative when tested by ninhydrin; 600 mL of 20% piperidine / N,N-dimethylformamide solution (i.e., 120 mL piperidine and 480 mL N,N-dimethylformamide) was added, deprotection was performed for 25 min, after the deprotection was completed, industrial grade N,N-dimethylformamide was added for washing 5 times, each washing amount was 10 times the resin volume;

[0099] g. According to the amino acid sequence (Pro-Val-Ile-Val-Pro-Ser), repeat the condensation reaction - washing - detection - deprotection - washing process of step (f) until the sequence is condensed;

[0100] h. Capping solution configuration: N,N-dimethylformamide:DIEA:Ace=28:1:1(v / v / v), add 320mL of capping solution, capping reaction for 0.5h, add N,N-dimethylformamide to wash 3 times after the reaction, the amount of each wash is about 10 times the amount of resin; the carrier resin is negative when tested by ninhydrin; add 350mL of methanol to shrink and wash the carrier resin, and then vacuum dry the resin for 5h to obtain 77.5g of peptide resin;

[0101] i. Lysis buffer configuration: trifluoroacetic acid: 1,2-ethanedithiol: water = 90:5:5 (v / v / v);

[0102] j. Add lysate at a ratio of peptide resin: lysate = 1g: 8mL, stir at room temperature for 3h, filter (remove the resin), add 8 times the volume of icy methyl tert-butyl ether to the filtrate, stir for 5min, centrifuge at 3000r / min for 2min, remove the upper liquid, and obtain a crude polypeptide; add the same volume of icy methyl tert-butyl ether as the first time, disperse evenly, re-centrifuge once, and obtain a white solid. Vacuum freeze-dry for 8h to obtain a crude product; take a sample and send it to MS and HPLC for inspection to confirm that it is the target;

[0103] k. Purify the crude product using a preparative liquid chromatograph, mobile phase: A: 0.1 vol% acetic acid aqueous solution; B: 0.1 vol% acetic acid acetonitrile solution, detection wavelength: λ = 220 nm, purity ≥ 95%, collect the target; salt-transfer the purified polypeptide using 0.3 vol% acetic acid aqueous solution, collect the salt-transfer sample, purity ≥ 95% ( Figure 1 ), the yield of pure peptide was 66.3%;

[0104] 1. Place the sample solution obtained after salt conversion into a freeze dryer, freeze-dry at <20pa for 76h to obtain a white powder; dissolve the obtained white powder in a 5 vol% acetonitrile aqueous solution and place it into a freeze dryer again for freeze drying for 65-80h.

[0105] Example 2

[0106] The method for preparing the oil body protein with the amino acid sequence of SPVLVP provided in this embodiment comprises the following steps:

[0107] a~f. Same as Example 1;

[0108] g. According to the amino acid sequence (Pro-Val-Leu-Val-Pro-Ser), repeat the condensation reaction - washing - detection - deprotection - washing process of step (f) until the sequence is condensed;

[0109] h~j. Same as Example 1;

[0110] k. Purify the crude product using a preparative liquid chromatograph, mobile phase: A: 0.1 vol% acetic acid aqueous solution; B: 0.1 vol% acetic acid acetonitrile solution, detection wavelength: λ = 220 nm, purity ≥ 95%, collect the target; salt-transfer the purified polypeptide using 0.3 vol% acetic acid aqueous solution, collect the salt-transfer sample, purity ≥ 95% ( Figure 2 ), the yield of pure peptide was 62.9%;

[0111] 1. Same as Example 1.

[0112] Experimental Example 1

[0113] Promoting collagen-I test, based on fibroblast COL-I expression detection test method:

[0114] (1) Inoculation: The cells were inoculated into a 24-well plate and incubated in a 37° C., 5% CO 2 incubator for 18-24 h.

[0115] (2) Solution preparation: Test and control samples were prepared according to Table 4. The sample group was prepared by first dissolving oleosomal protein in DMSO (the mass ratio of oleosomal protein to DMSO was 1:10), and then adding it to the cell culture medium to dilute it to the corresponding mass concentration. The negative control group was added with cell culture medium containing 0.1% DMSO by mass fraction.

[0116] (3) Sampling: According to the experimental grouping and concentration settings in Table 5, after the cells were plated and grown in the 24-well plate for 18-24 hours, they were grouped and sampled, with 3 replicate wells in each group. The cells were cultured in an incubator at 37° C. and 5% CO2 for 48 hours.

[0117] (4) Detection of type I collagen content: Take the supernatant and detect it using an ELISA kit.

[0118] (5) Data processing: All data obtained in the experiment were processed and plotted using Excel software. SPSS17.0 was used for statistical analysis, and one-way analysis of variance (ANOVA) was used for inter-group comparison. When p < 0.05, the difference was considered significant.

[0119] Test groups are shown in Table 4.

[0120] Table 4 Efficacy test groups and test concentrations (col-I)

[0121]

[0122] Table 5 col-I content test data results

[0123]

[0124] According to the test results, it can be seen that the oil body proteins SPVIVP and SPVLVP can effectively promote the increase of type I collagen content in fibroblasts when added in an amount of 0.0005% to 0.01%, and the promoting effect is positively correlated with the content of oil body protein fragments; and compared with the control group, there is a statistically significant difference.

[0125] Experimental Example 2

[0126] Matrix metalloproteinase MMP-1 test, based on the detection method of MMP-1 expression in fibroblasts:

[0127] (1) Inoculation: The cells were inoculated into a 24-well plate and incubated in a 37° C., 5% CO 2 incubator for 18-24 h.

[0128] (2) Solution preparation: The test substance and positive control were prepared according to Table 6. The sample group was prepared by first dissolving oleosomal protein in DMSO (the mass ratio of oleosomal protein to DMSO was 1:10), and then adding it to the cell culture medium to dilute it to the corresponding mass concentration. The negative control group was added with a cell culture medium containing 0.1% DMSO by mass fraction.

[0129] (3) Sampling: According to the experimental grouping and concentration settings in Table 7, after the cells were plated and grown in the 24-well plate for 18-24 hours, they were grouped and sampled, with 3 replicate wells in each group. The cells were cultured in an incubator at 37° C. and 5% CO2 for 48 hours.

[0130] (4) Detection of matrix metalloproteinase-1 content: The supernatant was taken and detected using an ELISA kit.

[0131] (5) Data processing: All data obtained in the experiment were processed and plotted using Excel software. SPSS17.0 was used for statistical analysis, and one-way analysis of variance (ANOVA) was used for inter-group comparison. The difference was considered significant when P < 0.05.

[0132] Table 6 Efficacy test groups and test concentrations (MMP-1)

[0133]

[0134] Table 7 MMP-1 content test data results

[0135]

[0136]

[0137] Note: The addition of 0.01% VA alcohol will reduce the viability of fibroblasts to below 50%, so it was not investigated.

[0138] According to the test results, it can be seen that the oil body protein fragments SPVIVP and SPVLVP can effectively inhibit the activity of type I matrix metalloproteinases in fibroblasts when added in an amount of 0.0005% to 0.01%, avoid excessive MMP-1 activity in the skin that would destroy the structural integrity of the skin, and reduce the rate of collagen decomposition; and compared with the control group, there is a statistically significant difference.

[0139] Experimental Example 3

[0140] Collagen-XVII test, based on keratinocyte detection method:

[0141] (1) Inoculation: The cells were inoculated into a 24-well plate and incubated in a 37° C., 5% CO 2 incubator for 18-24 h.

[0142] (2) Solution preparation: The test substances and positive controls were prepared according to Table 8. The sample group was prepared by first dissolving oleosomal protein in DMSO (the mass ratio of oleosomal protein to DMSO was 1:10), and then adding it to the cell culture medium to dilute it to the corresponding mass concentration.

[0143] (3) Sampling: According to the experimental grouping and concentration setting in Table 9, after the cells were plated and grown in the 24-well plate for 18-24 hours, the samples were grouped and sampled, with 3 replicates in each group. The blank control and negative control groups were added with cell culture medium containing 0.1% DMSO, the sample group was added with cell complete culture medium containing samples of corresponding concentrations, the positive control was added with cell culture medium containing 10 ng / mL hEGF, and the sample group was added with cell complete culture medium containing samples of corresponding concentrations, and cultured in an incubator at 37°C with a volume fraction of 5% CO2 for 18-24 hours.

[0144] (4) Detection of COL-17 expression: Immunofluorescence staining was performed, the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, permeabilized with 0.5% Triton X-100 for 20 min, washed three times with PBS, blocked with 5% BSA at room temperature for 60 min, and incubated with primary antibody at 4°C overnight. The next day, the cells were washed three times with PBS, incubated with secondary antibody at room temperature for 1 h, and then washed three times with PBS. The cells were observed and photographed under a fluorescence microscope.

[0145] (5) Data processing: Image J software was used to process the fluorescence photos and calculate the IOD value. The acquired data were processed and plotted using Excel software. SPSS17.0 was used for statistical analysis, and one-way analysis of variance (ANOVA) was used for inter-group comparison. The difference was considered significant when P < 0.05.

[0146] Table 8 Efficacy test groups and test concentrations (col-XVII)

[0147]

[0148] Table 9 col-XVII content test data results

[0149]

[0150] According to the test results, the oil body protein fragments SPVIVP and SPVLVP can effectively promote the increase of type XVII collagen content in fibroblasts. At an addition amount of 0.001%, the promoting effect is 15.5 times that of the control group. The promoting effect is positively correlated with the content of oil body protein fragments, which further indicates that the invention patent has the function of supporting the structural stability of the skin; and compared with the blank group, there is a statistically significant difference.

[0151] Although the present invention has been illustrated and described with specific embodiments, it should be appreciated that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Those skilled in the art should understand that the technical solutions described in the above embodiments may be modified, or some or all of the technical features thereof may be replaced by equivalents without departing from the spirit and scope of the present invention. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. Therefore, this means that all such replacements and modifications within the scope of the present invention are included in the appended claims.

Claims

1. An oil body protein, characterized in that The amino acid sequence of the oil body protein is shown in SEQ ID No.1 and / or SEQ ID No.

2.

2. The method for preparing oleosomal protein according to claim 1, characterized in that: The following steps are involved: (a) adding piperidine and N,N-dimethylformamide to a carrier resin and then performing a first reaction and a first washing; (b) adding the first amino acid raw material, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-dimethylformamide and N,N-diisopropylethylamine to the reaction solution after the first washing to carry out a first stirring reaction; (c) performing a ninhydrin colorimetric test on the carrier resin after the first stirring reaction. When the test result is negative, vacuum-evacuating the reaction liquid of the first stirring reaction, adding N,N-dimethylformamide, acetic anhydride and N,N-diisopropylethylamine to perform a second stirring reaction, and vacuum-evacuating the reaction liquid of the second stirring reaction, and performing a second washing; (d) adding piperidine and N,N-dimethylformamide to the reaction system after the second washing, and then performing a first deprotection reaction and a third washing; (e) adding the second amino acid raw material, ethyl 2-oxime cyanoacetate, N,N'-diisopropylcarbodiimide and N,N-dimethylformamide to the reaction system after the third washing, and then performing a third stirring reaction and a fourth washing; performing a ninhydrin colorimetric detection on the carrier resin, and when the detection result is negative, adding piperidine and N,N-dimethylformamide to the reaction system after the fourth washing, performing a second deprotection reaction and a fifth washing; (f) according to the amino acid sequence of the oil body protein, the second amino acid raw material is replaced with the third amino acid raw material and the amino acid raw materials after the third amino acid raw material in sequence, and step (e) is repeated to synthesize the amino acid sequence of the oil body protein; (g) performing a capping reaction and a sixth washing on the reaction system after step (f); performing a ninhydrin colorimetric detection on the carrier resin, and when the detection result is negative, performing a shrinkage washing and a first drying on the carrier resin to obtain a peptide resin; cracking and filtering the peptide resin, mixing the filtrate with ice methyl tert-butyl ether, and performing a first centrifugation to remove the upper liquid to obtain a crude polypeptide; The crude polypeptide and glacial methyl tert-butyl ether are mixed and then centrifuged for the second time, and the upper liquid is removed and then dried for the second time to obtain a crude product; (h) purifying and converting the crude product.

3. The method for preparing oleosin according to claim 2, characterized in that: Includes at least one of the following technical features: (1) In step (a), the carrier resin, piperidine and N,N-dimethylformamide are added in a ratio of 50 g: 60 mL: 240 mL; (2) The first reaction time is 25 min; (3) The first washing was performed 7 times, using 500 mL of washing solution each time.

4. The method for preparing oleosin according to claim 2, characterized in that: Includes at least one of the following technical features: (1) Step (b), the addition ratio of the first amino acid raw material, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-dimethylformamide and N,N-diisopropylethylamine is 100 mmol:40.7 g:210 mL:35 mL; (2) The first stirring reaction time is 2 hours.

5. The method for preparing oleosin according to claim 2, characterized in that: Includes at least one of the following technical features: (1) Step (c), the addition ratio of N,N-dimethylformamide, acetic anhydride and N,N-diisopropylethylamine is 280 mL:10 mL:10 mL; (2) The second stirring reaction time is 0.5h; (3) The second washing was repeated 3 times, and the volume ratio of the washing liquid to the carrier resin used each time was 1:

10.

6. The method for preparing oleosin according to claim 2, characterized in that: Includes at least one of the following technical features: (1) Step (d), adding piperidine and N,N-dimethylformamide in an amount of 120 mL piperidine and 480 mL N,N-dimethylformamide to the reaction system after the second washing; (2) The first deprotection reaction time is 25 min; (3) The third washing was repeated 7 times, and the volume ratio of the washing liquid to the carrier resin used each time was 1:

10.

7. The method for preparing oleosin according to claim 2, characterized in that: Includes at least one of the following technical features: (1) Step (e), the ratio of the second amino acid raw material, ethyl 2-oxime cyanoacetate, N,N'-diisopropylcarbodiimide and N,N-dimethylformamide added is 100 mmol: 100 mmol: 100 mmol: 280 mL; (2) The third stirring reaction time is 1.5 h; (3) The fourth washing is repeated 3 times, and the volume ratio of the washing liquid to the carrier resin used each time is 1:10; (4) Step (e), adding piperidine and N,N-dimethylformamide in an amount of 120 mL piperidine and 480 mL N,N-dimethylformamide to the reaction system after the fourth washing; (5) The second deprotection reaction time is 25 min; (6) The fifth washing is repeated 5 times, and the volume ratio of the washing liquid to the carrier resin used each time is 1:

10.

8. The method for preparing oleosin according to claim 2, characterized in that: Includes at least one of the following technical features: (1) adding 320 mL of end-capping liquid to the reaction system after step (f) to carry out an end-capping reaction; (2) the capping solution of the capping reaction comprises N,N-dimethylformamide, N,N-diisopropylethylamine and acetic anhydride in a volume ratio of 28:1:1; (3) The end-capping reaction time is 0.5 h; (4) The sixth washing is repeated three times, and the volume ratio of the washing liquid to the carrier resin used each time is 1:

10.

9. The method for preparing oleosin according to claim 2, characterized in that: Includes at least one of the following technical features: (1) The shrinkage washing uses 350 mL of methanol as a washing liquid; (2) The lysis solution comprises trifluoroacetic acid, 1,2-ethanedithiol and water in a volume ratio of 90:5:5; (3) the addition ratio of the peptide resin and the lysate is 1 g:8 mL; (4) The lysis time is 3 hours.

10. Use of the oleosin according to claim 1 or the oleosin prepared by the method for preparing the oleosin according to any one of claims 2 to 9 in the preparation of cosmetics and / or pharmaceutical products.

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