Recombinant elastin-like protein as well as preparation method and application thereof

Abstract

The invention relates to a recombinant elastin-like protein as well as a preparation method and application thereof, and belongs to the technical field of biomacromolecules. The invention provides a recombinant elastin-like protein. The recombinant elastin-like protein comprises an elastin-like protein motif and a collagen III functional motif, the amino acid sequence of the elastin-like motif is VPGXG, and X in the VPGXG is any one of H, R, K, I, F, L, W, A, M, C, N, V, G, S, Q, Y, D, E and T; the amino acid sequence of the III type collagen functional motif is as shown in SEQ ID NO. 1. The invention provides a recombinant elastin-like protein which is fused with a collagen motif and an elastin motif of a triple helix structural domain with cell adhesion promoting activity, has temperature-sensitive characteristics, pH response characteristics and gel characteristics, and has anti-aging, anti-inflammatory, skin barrier repairing and soothing effects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomacromolecules, and in particular to a recombinant elastin protein and a preparation method and application thereof. Background Art

[0002] Sensitive skin (SS), also known as sensitive skin, was defined as a syndrome by a special interest group of the International Forum for Itch Research in 2016, characterized by unpleasant sensations (tingling, burning, pain, itching and stinging) after stimuli that should not cause a reaction, such as cold, heat or other physical and / or chemical factors. The pathophysiology of sensitive skin is still poorly understood, but its symptoms suggest the presence of inflammation and are controlled by peripheral nerves. Once skin inflammation occurs in people with sensitive skin, the signal transmission of inflammatory factors is faster, and the inflammation spreads from the stratum corneum to the dermis, causing the degradation of extracellular matrix components such as elastin, fibronectin and collagen. Collagen contains a large number of polar functional group structures such as hydroxyproline, glycine and aspartic acid. Elastin is rich in glycine and proline, which can form a β-helix to expose hydrophilic amino acids to the outside. These characteristics give collagen and elastin the moisturizing properties of epidermal and dermal tissues. During the inflammation stage, the decomposition of collagen can trigger the removal of cell debris and bacteria by immune cells, regulating the degree of inflammatory response.

[0003] The biological preparation of recombinant proteins involves a relatively complex purification process. The purification of recombinant proteins mostly uses affinity filler chromatography technology, such as bioaffinity chromatography, immunoaffinity chromatography and metal affinity chromatography, but column fillers have limited efficiency and high costs. Sometimes multiple chromatography columns are needed to purify the target protein, which significantly increases the cost of industrial production.

[0004] Therefore, there is an urgent need to develop a protein that can resist aging, soothe, resist inflammation and repair the skin barrier, and at the same time, a preparation process that can utilize the characteristics of the protein itself to reduce the cost of industrial production and improve production efficiency. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a recombinant elastin-like protein and a preparation method and application thereof.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a recombinant elastin-like protein, wherein the recombinant elastin-like protein comprises an elastin-like motif and a type III collagen functional motif;

[0008] The amino acid sequence of the elastin-like motif is VPGXG, wherein X in VPGXG is any one of H, R, K, I, F, L, W, A, M, C, N, V, G, S, Q, Y, D, E and T;

[0009] The amino acid sequence of the type III collagen functional motif is shown in SEQ ID NO.1.

[0010] The present invention utilizes the phase change characteristics of elastin-like protein to temperature, pH value and salt ion concentration to construct a recombinant elastin-like protein that fuses a collagen motif and an elastin motif with a triple helical domain that promotes cell adhesion activity. The obtained recombinant elastin-like protein not only has the temperature-sensitive characteristics and pH response characteristics of elastin-like protein, but also exhibits the gelatin characteristics of collagen: at 4-37°C, when the temperature rises to about 25°C, the hydrophobicity is enhanced and a loose porous gel is precipitated through phase change, and at 37°C, the hydrophilicity is enhanced and the protein is reversibly dissolved again. The gradual change of pH between 9.0 and 7.0 and the salt ion concentration can also trigger the phase change of the recombinant protein from a soluble state to a gel state.

[0011] As a preferred embodiment of the recombinant elastin-like protein of the present invention, the number of repetitions of the amino acid sequence of the elastin-like motif in the recombinant elastin-like protein is an integer ≥5, and the number of repetitions of the amino acid sequence of the type III collagen functional motif is an integer ≥1.

[0012] As a preferred embodiment of the recombinant elastin-like protein of the present invention, the amino acid sequence of the elastin-like motif in the recombinant elastin-like protein is repeated 5 to 100 times, and the amino acid sequence of the type III collagen functional motif is repeated 1 to 100 times.

[0013] As a preferred embodiment of the recombinant elastin-like protein of the present invention, the amino acid sequence of the elastin-like motif in the recombinant elastin-like protein is repeated 10 to 60 times, and the amino acid sequence of the type III collagen functional motif is repeated 5 to 50 times.

[0014] As a preferred embodiment of the recombinant elastin-like protein of the present invention, the amino acid sequence of the elastin-like motif in the recombinant elastin-like protein is repeated 30 to 50 times, and the amino acid sequence of the type III collagen functional motif is repeated 8 to 16 times.

[0015] As a preferred embodiment of the recombinant elastin-like protein of the present invention, the recombinant elastin-like protein further comprises a histidine tag, and the histidine tag is connected to the amino terminus and / or carboxyl terminus of the recombinant elastin-like protein. The present invention introduces a histidine tag at the amino terminus or carboxyl terminus of the recombinant elastin-like protein, which is beneficial to the purification and identification of the target protein.

[0016] As a preferred embodiment of the recombinant elastin-like protein of the present invention, the recombinant elastin-like protein comprises a linker sequence, and the linker sequence connects the elastin-like protein motif and the type III collagen functional motif; the amino acid sequence of the linker sequence is shown in SEQ ID NO.2.

[0017] In a second aspect, the present invention provides a method for preparing the above-mentioned recombinant elastin-like protein, which comprises the following steps:

[0018] S1, transferring the constructed vector containing elastin-like motif, type III collagen functional motif, linker sequence and histidine tag into Escherichia coli to obtain an engineered bacterium expressing recombinant elastin-like protein;

[0019] S2, subjecting the engineered bacteria expressing recombinant elastin-like protein obtained in step S1 to high-density fermentation to obtain a fermentation product;

[0020] S3, subjecting the fermentation product obtained in step S2 to ultrasonic crushing and centrifugation, and subjecting the obtained supernatant to reversible phase change purification to obtain a crude product;

[0021] S4, resuspending the crude product obtained in step S3 in a buffer, incubating at 35-37° C. until the crude product is dissolved, and centrifuging to obtain a supernatant;

[0022] S5, subjecting the supernatant obtained in step S4 to reversible phase change purification to obtain a purified product;

[0023] S6. Repeat the operation of step S4 with the purified product obtained in step S5, and the obtained supernatant is the recombinant elastin-like protein.

[0024] The present invention has developed a method for efficiently expressing recombinant elastin-like proteins using recombinant Escherichia coli. The proportion of the expressed target protein can reach more than 40%, the protein is highly expressed and the fermentation cycle is short. Based on the phase change characteristics of the target protein, the fermented bacteria undergo two rounds of precipitation-dissolution reversible phase change cycle (ITC) purification to obtain the target protein with a purity of more than 90%. On the other hand, based on the reversible phase change characteristics of the recombinant elastin-like proteins, the protein can be preserved without the addition of a protein protective agent, which reduces the purification cost and simplifies the purification steps.

[0025] As a preferred embodiment of the preparation method of the present invention, in step S1, the number of elastin-like motifs in the carrier is an integer ≥5, the number of type III collagen functional motifs in the carrier is an integer ≥1, the linker sequence in the carrier connects the elastin-like motif and the type III collagen functional motif, and the histidine tag is connected to the amino terminus and / or carboxyl terminus of the recombinant elastin.

[0026] As a preferred embodiment of the preparation method of the present invention, in step S2, the high-density fermentation operation includes the following steps:

[0027] (1) preparing the engineered bacteria expressing recombinant elastin-like protein obtained in step S1 into a secondary seed solution, inoculating the secondary seed solution into a bioreactor, adding glucose with an initial concentration of 5-6 g / L and magnesium sulfate of 0.6-0.7 g / L, stirring at a speed of 180-200 rpm, and maintaining the dissolved oxygen content above 30%;

[0028] (2) After culturing at 35-37°C for 3.5-4 hours, feed was added until the bacterial weight was ≥40 g / L, the dissolved oxygen content was maintained at 80-85%, IPTG was added at a final concentration of 0.1-1 M, and the culture was continued for 4 hours to obtain the fermentation product.

[0029] As a preferred embodiment of the preparation method of the present invention, in steps S3 and S5, the reversible phase change purification operation mainly comprises adjusting the pH value of the supernatant to 7.0-7.5, standing at 23-28°C until a precipitate is precipitated, and collecting the precipitate by centrifugation. The obtained precipitate is the crude product or the purified product.

[0030] In a third aspect, the present invention provides the use of the above-mentioned recombinant elastin-like protein in the preparation of anti-aging, anti-inflammatory, skin barrier repair and / or soothing products.

[0031] The recombinant elastin provided by the present invention has the activities of repairing skin barrier, inhibiting skin inflammatory response, soothing and anti-aging, and is expected to be developed as an active raw material for application in the fields of biomedicine and cosmetics; at the same time, based on its gel properties, active biomaterials can be developed as matrix materials for tissue engineering.

[0032] As a preferred embodiment of the application of the present invention, the products include medicines, foods, health products, cosmetics and biological products.

[0033] In a fourth aspect, the present invention provides an anti-aging, anti-inflammatory, skin barrier repair and soothing cosmetic comprising the above-mentioned recombinant elastin-like protein.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The present invention provides a recombinant elastin-like protein that combines a collagen motif and an elastin motif with a triple helical domain having cell adhesion promoting activity, and has temperature-sensitive properties, pH-responsive characteristics and gel properties: it is in a soluble state at pH = 9.0, and in a gel state at pH = 7.0; when the temperature rises to about 25°C at a temperature of 4 to 37°C, the hydrophobicity is enhanced and a loose porous gel is precipitated through a phase transition; at 37°C, the hydrophilicity is enhanced and the gel can be reversibly dissolved again.

[0036] 2. The present invention provides a simple protein purification process based on the reversible phase change characteristics of recombinant elastin. The temperature and pH value are used to mediate the transformation of protein hydrophobicity and hydrophilicity. Through two rounds of reversible phase change cycles, recombinant elastin with a purity of more than 90% can be obtained; compared with affinity chromatography purification, the process is simple and efficient, the cost is significantly reduced, and it is suitable for industrial production.

[0037] 3. The recombinant elastin provided by the present invention has the activity of repairing skin barrier, inhibiting skin inflammatory response, soothing and anti-aging, and is expected to be developed as an active raw material for application in the fields of biomedicine and cosmetics; at the same time, based on its gel properties, active biomaterials can be developed as matrix materials for tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a spectrum of the recombinant elastin-like protein expression vector constructed in Example 1 of the present invention;

[0039] Figure 2 This is the SDS-PAGE electrophoresis diagram of the engineered bacteria expressing recombinant elastin-like protein in Example 1 of the present invention;

[0040] Figure 3 The SDS-PAGE detection for optimizing the expression conditions of recombinant elastin-like protein rhELT, Figure A is the SDS-PAGE detection induced by different temperatures; Figure B is the SDS-PAGE detection induced by different concentrations of IPTG;

[0041] Figure 4 The SDS-PAGE electrophoresis diagram of the recombinant elastin-like protein expressed by high-density fermentation at different induction times in Effect Example 1 of the present invention, wherein the band indicated by the arrow in the diagram is the recombinant elastin-like protein;

[0042] Figure 5 The SDS-PAGE and WB results of the recombinant elastin-like protein after purification in Example 1 of the present invention are shown in Figure A. Figure A is an SDS-PAGE electrophoresis diagram, in which lane 1 is a round of ITC product, lane 2 is a pH 7.0 centrifugal supernatant, lane 3 is a pH 7.0 centrifugal precipitate re-dissolved solution, lane 4 is a second round of ITC centrifugal supernatant product, and Figure B is a WB result;

[0043] Figure 6 The phase transition observation results of the recombinant elastin-like protein in Example 1 of the present invention are shown in Figure A, which shows the appearance of the protein sample during the process of adjusting the pH from 9.0 to 7.0, Figure B shows the cross-section electron microscopy scanning result of the precipitate obtained at pH = 7.0, and Figure C shows the longitudinal section electron microscopy scanning result of the precipitate obtained at pH = 7.0;

[0044] Figure 7The results of the detection of the degranulation of P815 mast cells by recombinant elastin in Example 2 of the present invention, Figure A is the result of neutral red staining of cells, Figure B is the statistics of cell degranulation rate, *P<0.05, **P<0.01, ***P<0.001 vs. model group;

[0045] Figure 8 The results of the test of the effect of recombinant elastin on the expression of matrix metalloproteinases (MMPs) in fibroblasts in Example 3 of the present invention, *P<0.05, **P<0.01, ***P<0.001 vs. control group (-UV group);

[0046] Fig. 9 The results of the test of the effect of recombinant elastin on the expression of collagen and fibronectin in the extracellular matrix of fibroblasts in Example 3 of the present invention, *P<0.05, **P<0.01, ***P<0.001 vs. control group (-UV group);

[0047] Fig.10 The results are the test results of the effect of recombinant elastin on the expression of barrier proteins in HNEK cells in Example 3 of the present invention. *P<0.05, **P<0.01, ***P<0.001 vs. blank group.

[0048] Fig.11 The results are the test results of the effect of recombinant elastin on the transcription level of capsaicin receptor TRPV1 gene in Effect Example 3 of the present invention. *P<0.05, **P<0.01, ***P<0.001 vs. model group.

[0049] Fig.12 Figure 3 is the detection result of the effect of recombinant elastin on the release of inflammatory factor TNF-α from RAW264.7 cells. Figure A is the determination of the content of inflammatory factor TNF-α, and Figure B is the statistical inhibition rate of recombinant elastin rhELT on TNF-α production, *P<0.05, ***P<0.001 vs. model group, ns indicates no significant difference.

[0050] Fig.13 The results of the test on the effect of recombinant elastin on the ROS content of fibroblasts in Example 3 of the present invention. *P<0.05, **P<0.01, ***P<0.001 vs. model group; ### P<0.001vs. model group.

[0051] In the above figure, "rhELT" refers to recombinant elastin. DETAILED DESCRIPTION

[0052] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0053] Unless otherwise specified, other materials, reagents, etc. used in the examples can be obtained from commercial sources.

[0054] In the following examples, comparative examples and effect examples, unless otherwise specified, the feed is 5 g / L sterile glucose solution and 0.65 g / L sterile magnesium sulfate solution.

[0055] Mouse mast cell tumor cell P815 was provided by ATCC with the catalog number TIB-64.

[0056] Human immortalized keratinocytes HaCat were provided by Beina Biotechnology with the catalog number BNCC101683.

[0057] Mouse mononuclear macrophage RAW264.7 was provided by ATCC with the catalog number TIB-71.

[0058] The techniques not described in detail in the following embodiments and effect examples are all commonly used techniques in the art, and reference may be made to "Handbook of Molecular Biology Experiments" (Ma Wenli, People's Military Medical Publishing House), "Molecular Biology Experiments (Second Edition)" (Zhejiang University Press), and "Cell Biology Experiments" (Yang Hongbing, Hou Lixia, Zhang Yuxi, Higher Education Press).

[0059] In the following embodiments and effect examples, "V" refers to valine (Val), "P" refers to proline (Pro), "G" refers to glycine (Gly), "H" refers to histidine (His), "R" refers to arginine (Arg), "K" refers to lysine (Lys), "I" refers to isoleucine (Ile), "F" refers to phenylalanine (Phe), "L" refers to leucine (Leu), "W" refers to tryptophan (Trp), "A" refers to alanine (Ala), "M" refers to methionine (Met), "C" refers to cysteine ​​(Cys), "N" refers to asparagine (Asn), "S" refers to serine (Ser), "Q" refers to glutamine (Gln), "Y" refers to tyrosine (Tyr), "D" refers to aspartic acid (Asp), "E" refers to glutamic acid (Glu), and "T" refers to threonine (Thr).

[0060] Example 1

[0061] This embodiment provides a recombinant elastin-like protein and a preparation method thereof, the preparation method comprising the following steps:

[0062] 1. Construction of engineered bacteria expressing recombinant elastin-like proteins

[0063] The "X" in the 1.1-type elastin motif VPGXG is I, and the number of repetitions of the amino acid sequence of VPGIG is set to 50 times, which is recorded as (VPGIG)50; the number of repetitions of the amino acid sequence of the type III collagen functional motif is set to 16, and the amino acid sequence of the type III collagen functional motif is shown in SEQ ID NO.1, which is recorded as (aa)16 and named rhELT;

[0064] 1.2 The (VPGIG)50 and (aa)16 obtained in step 1.1 are connected through a linker sequence, and a histidine tag (HHHHHH) is introduced at the carboxyl terminus of the target protein. The amino acid sequence of the linker is shown in SEQ ID NO.2. The amino acid sequence of the obtained recombinant elastin-like protein is shown in SEQ ID NO.3. The amino acid sequence of the recombinant elastin-like protein is codon-optimized, and NdeⅠ and HindⅢ restriction site sequences are introduced at the amino terminus and the carboxyl terminus, and the obtained optimized nucleic acid sequence with restriction site introduction is shown in SEQ ID NO.4;

[0065] 1.3 Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the recombinant elastin gene fragment (SEQ ID NO.4) obtained in step 1.2 with optimized and introduced restriction sites, and the synthesized recombinant elastin gene fragment was connected to the vector pET20b through the NdeⅠ and HindⅢ restriction sites, and transformed into Escherichia coli DH5α, and positive clones were screened by LB resistance plate, and the positive clones were verified by colony PCR and sequencing to obtain the recombinant plasmid rhELT / pET20b (plasmid map see Figure 1 );

[0066] 1.4 The recombinant plasmid rhELT / pET20b obtained in step 1.3 was transferred into Escherichia coli BL21 (DE3), and positive clones were screened and identified according to the method in step 1.3. After identification, it was determined that the Escherichia coli carried the recombinant vector rhELT / pET20b, that is, the obtained recombinant Escherichia coli was an engineered bacterium expressing recombinant elastin-like protein.

[0067] 2. Expression and identification of recombinant elastin-like proteins

[0068] Pick up the engineered bacteria expressing recombinant elastin obtained in step 1.4 (hereinafter referred to as engineered bacteria) and add them to 5 mL of resistant LB medium. Culture at 37°C until OD 600 =0.6-0.8, add IPTG with a final concentration of 1mM, induce culture for 4h, collect bacteria, and detect the expression level of target protein by SDS-PAGE. The SDS-PAGE results are shown in Figure 2 .

[0069] like Figure 2As shown, the 7 selected recombinant engineering bacteria rhELT / pET20b-BL21 all expressed the target protein, and the apparent molecular weight thereof was consistent with the theoretical molecular weight, which was about 80.8 kDa. The grayscale analysis results showed that the background protein content was low, and the target proteins No. 1-7 accounted for 40%, 45%, 47%, 40%, 45%, 42% and 45%, respectively, all of which were highly expressed, indicating that the present invention successfully and efficiently expressed recombinant elastin-like protein through the Escherichia coli system.

[0070] 3. Optimization of inducible expression conditions

[0071] In order to determine the most suitable induction conditions for high expression of the target protein, the above-mentioned engineering bacteria with high expression levels were selected for the three-factor test of induction temperature, time and inducer concentration. The induction temperature was set at 20℃, 30℃ and 37℃, and the corresponding induction time was 4h, 6h and 18h ​​respectively; after determining the optimal induction temperature and time, the optimal inducer concentration was screened, and the inducer was IPTG, and the concentration was set at 0.1, 0.25, 0.5, 1.0mM, and the expression level of the target protein under different induction conditions was detected by SDS-PAGE electrophoresis.

[0072] like Figure 3 As shown, the expression levels of the target protein under different induction temperatures and times are 20°C, 4h>37°C, 18h>30°C, 6h, and there is no obvious difference in the expression level of the target protein under different IPTG concentrations and induction times.

[0073] 4. Production and purification of recombinant elastin

[0074] 4.1 The engineered bacteria highly expressing recombinant elastin-like protein obtained in step 2 were inoculated into 50 mL of resistant LB medium, and cultured at 37°C with shaking for 12-16 hours to obtain a primary seed solution;

[0075] 4.2 Inoculate 50 mL of the first-level seed solution obtained in step 4.1 into 2 L of resistant LB medium and culture at 37°C with shaking for 12-16 hours to obtain the second-level seed solution;

[0076] 4.3 Inoculate 2L of the secondary seed solution obtained in step 4.3 into a 20L bioreactor, add a sterile glucose solution with an initial concentration of 5g / L and a sterile magnesium sulfate solution of 0.65g / L, stir at 200rpm, aerate at 5L / min, maintain dissolved oxygen above 30%, and culture for 3.5h;

[0077] 4.4 Feed until the bacterial weight is ≥40g / L, the dissolved oxygen content is maintained at 80%, add 7mL 1M IPTG, induce at 30℃ for 4h, and obtain the fermentation product;

[0078] 4.5 The fermentation product obtained in step 4.4 was centrifuged at 4°C and 30,000 g for 10 min to collect the cells, and the cells were resuspended in 50 mM Tris buffer (pH = 9.0) (cells: Tris buffer = 1:20), and the cells were broken once at 4°C and normal pressure with a high-pressure homogenizer, and broken twice at 4°C and 600 Pa, and the supernatant was obtained by centrifugation;

[0079] 4.6 The pH value of the supernatant obtained in step 4.5 was adjusted to 7.5, and the mixture was allowed to stand at 23-28°C for 0.5 h to precipitate, and the precipitate was collected by centrifugation at 4°C and 18000 rpm for 20 min. The precipitate obtained was the crude product;

[0080] 4.7 Add an equal volume of 50 mM Tris buffer to the precipitate obtained in step 4.6, incubate in a 37°C water bath until the precipitate is dissolved, and centrifuge at 25°C and 18,000 rpm for 20 min to collect the supernatant;

[0081] 4.8 Repeat the operation of step 4.6 with the supernatant obtained in step 4.7 to obtain a precipitate, which is the purified product;

[0082] 4.9 Repeat the operation of step 4.7 with the precipitate obtained in step 4.8 to obtain a supernatant, which is the recombinant elastin-like protein solution.

[0083] Example 2

[0084] The present embodiment provides a recombinant elastin-like protein and a preparation method thereof. The preparation method is similar to that of Example 1, except that in step 1.1, "X" in the elastin-like protein motif VPGXG is V, and the number of repetitions of the amino acid sequence of VPGVG is set to 30 times, recorded as (VPGVG)30; the number of repetitions of the amino acid sequence of the type III collagen functional motif is set to 1, and the amino acid sequence of the type III collagen functional motif is shown in SEQ ID NO.1, recorded as (aa)1, and the remaining steps and parameter conditions remain unchanged, and the product is named rhELT-1.

[0085] Example 3

[0086] The present embodiment provides a recombinant elastin-like protein and a preparation method thereof. The preparation method is similar to that of Example 1, except that in step 1.1, "X" in the elastin-like protein motif VPGXG is 1, the number of repetitions of the amino acid sequence of VPGIG is set to 30 times, recorded as (VPGIG)30; the number of repetitions of the amino acid sequence of the type III collagen functional motif is set to 1, the amino acid sequence of the type III collagen functional motif is shown in SEQ ID NO.1, recorded as (aa)8, and the remaining steps and parameter conditions remain unchanged, and the product is named rhELT-2.

[0087] Example 4

[0088] The present embodiment provides a recombinant elastin-like protein and a preparation method thereof. The preparation method is similar to that of Example 1, except that in step 1.1, "X" in the elastin-like protein motif VPGXG is Y, and the number of repetitions of the amino acid sequence of VPGYG is set to 30 times, recorded as (VPGYG)30; the number of repetitions of the amino acid sequence of the type III collagen functional motif is set to 1, and the amino acid sequence of the type III collagen functional motif is shown in SEQ ID NO.1, recorded as (aa)8, and the remaining steps and parameter conditions remain unchanged, and the product is named rhELT-3.

[0089] Example 5

[0090] The present embodiment provides a recombinant elastin-like protein and a preparation method thereof. The preparation method is similar to that of Example 1, except that in step 1.1, "X" in the elastin-like protein motif VPGXG is Y, and the number of repetitions of the amino acid sequence of VPGYG is set to 50 times, recorded as (VPGYG)50; the number of repetitions of the amino acid sequence of the type III collagen functional motif is set to 1, and the amino acid sequence of the type III collagen functional motif is shown in SEQ ID NO.1, recorded as (aa)16, and the remaining steps and parameter conditions remain unchanged, and the product is named rhELT-4.

[0091] Effect Example 1

[0092] The recombinant elastin obtained in Example 1 was characterized, and the specific scheme is as follows:

[0093] 1. The fermentation product obtained in step 4.4 was subjected to SDS-PAGE detection, and the recombinant elastin-like proteins in steps 4.7 and 4.9 were subjected to SDS-PAGE and Western Blot detection (the primary antibody was histidine antibody, and the secondary antibody was mouse anti-histidine antibody). The results are shown in Figure 4-5 .

[0094] like Figure 4 As shown, the expression level of recombinant elastin gradually increased after induction. The grayscale analysis results showed that the proportion of target protein reached 27% at 3.5h, and the bacterial density reached 52g / L after the fermentation.

[0095] like Figure 5 As shown in A, the effect of pH and temperature on the phase transition of recombinant elastin-like protein is utilized to purify it. After two rounds of ITC purification, a protein with higher purity can be obtained; Figure 5 As shown in B, the immunoblot band is single and consistent with the molecular weight in SDS-PAGE. The above results show that the preparation method of the present invention can effectively prepare and purify recombinant elastin-like protein.

[0096] 2. The recombinant elastin obtained in Example 1 was subjected to phase change observation and scanning electron microscopy observation. The phase change observation includes the following operations: the pH value of the recombinant elastin solution is adjusted from 9.0 to 7.0, and the state of the recombinant elastin is observed when the pH changes. A small amount of the protein precipitated at pH = 7.0 is taken for freeze drying, and the freeze-dried protein sample is adhered to a conductive tape, and after gold spraying, it is placed under a scanning electron microscope for observation. The results are shown in FIG. Figure 6 .

[0097] like Figure 6 As shown, the changes in the hydrophobicity and hydrophilicity of the recombinant elastin-like protein are directly related to the temperature and the pH value of the buffer. When the pH of the buffer is adjusted from 9.0 to 7.0, the target protein gradually precipitates from a soluble state to a loose and porous gel.

[0098] Effect Example 2

[0099] The soothing efficacy comparison test of the recombinant elastin obtained in Examples 1-5 was conducted, and the specific scheme is as follows:

[0100] P815 mouse mast cell tumor cells in logarithmic growth phase were counted and inoculated into 24-well plates. After incubation for 24 hours, the original culture medium was discarded and replaced with a medium containing drugs for 24 hours. The positive control group was 1 mg / mL sodium cromoglycate, and the recombinant elastin rhELT, rhELT-1, rhELT-2, rhELP-3, and rhELT-4 groups were set to 200 nM. The original culture medium was discarded and replaced with a medium containing C48 / 80 stimulant (20 μg / mL) to stimulate P815 cells for 1 hour. The drugs were present at the same time, and an equal volume of basal culture medium was added to the blank control group. The supernatant was discarded, 200 μL of 0.33% neutral red dye was added to cover the cells, and the cells were stained for 5 minutes. An equal volume of PBS was added for dilution, and the degranulation rate was observed under a microscope and photographed, and the results were statistically analyzed. Figure 7 .

[0101] like Figure 7 As shown, the results of neutral red staining show that at the same concentration, rhELT alleviates the degranulation of P815 allergic cells better than rhELT-1, rhELT-2, rhELT-3, and rhELT-4, indicating that the recombinant elastin obtained in the present invention has a good soothing effect.

[0102] Effect Example 3

[0103] The function of the recombinant elastin obtained in Example 1 was tested, and the specific scheme is as follows:

[0104] 1. Analysis of gene expression of matrix metalloproteinases (MMP1, MMP3, MMP9) in fibroblasts

[0105] Human fibroblasts that are growing well were seeded into 24-well plates and incubated in 5% CO 2 The cells were cultured in an incubator for 24 h. The original culture medium was discarded and replaced with pre-cooled PBS. The cells were placed in a UV curing box for UVA irradiation (30 min, with the dose set to 3.33 mW / cm 2 , i.e. 6J / cm 2 After irradiation, PBS was aspirated, and 2 mL of basal culture medium was added to each well of the blank group and model group, and the drug group was added to the culture medium containing 250 nM recombinant elastin. Each group had 3 replicates and was cultured for 72 h. Using GAPDH as a reference gene, the transcription levels of MMP1, MMP3, and MMP9 protein-related genes were detected by qRT-PCR (CFX384 Touch). The results are shown in Figure 8 .

[0106] Matrix metalloproteinases (MMPs) play an important role in the aging process. MMPs are a group of enzymes that can degrade the extracellular matrix (ECM). They play a role in tissue remodeling, repair, cell migration and other processes. MMP1, MMP3 and MMP9 are members of the MMPs family, and their role in aging and longevity has attracted much attention. Studies have shown that reducing the expression of MMPs or inhibiting their activity may help reduce the destruction of the extracellular matrix, thereby maintaining tissue structure and function and delaying aging. Figure 8 As shown, the recombinant elastin of the present invention can significantly inhibit the expression of MMPs under the condition of 250 nM administration (P≤0.05), indicating that the recombinant elastin of the present invention can reduce the degradation of the extracellular matrix induced by ultraviolet light, thereby achieving the anti-aging effect.

[0107] 2. Gene expression analysis of fibroblast extracellular matrix proteins (COL1A1, COL3A1, COL4A1, COL17A1 and FN1)

[0108] Human fibroblasts that are growing well were seeded into 24-well plates and incubated in 5% CO 2 The cells were cultured in an incubator for 24 h. The original culture medium was discarded and replaced with pre-cooled PBS. The cells were placed in a UV curing box for UVA irradiation (30 min, with the dose set to 3.33 mW / cm 2 , i.e. 6J / cm 2 After irradiation, PBS was aspirated, and 2 mL of basal culture medium was added to each well of the blank group and model group, and the drug group was added with culture medium containing 250 nM recombinant elastin. Each group had 3 replicates and was cultured for 72 h. Using GAPDH as a reference gene, qRT-PCR (CFX384 Touch) was used to detect the transcription levels of COL1A1, COL3A1, COL4A1, COL17A1, and FN1 protein-related genes. The results are shown in Fig. 9 .

[0109] The extracellular matrix (ECM) plays an important role in the anti-aging process. ECM is a complex network composed of a variety of macromolecules, including structural proteins (such as collagen and elastin), connecting proteins (such as fibronectin and laminin), glycosaminoglycans, proteoglycans, and various growth factors. With age, the composition, structure, and function of ECM change. These changes are related to the decline in cell functional activity, which ultimately leads to the aging of tissue structure and function. In anti-aging strategies, reducing the damage to ECM by external factors (such as ultraviolet radiation) and directly supplementing ECM or stimulating its secretion are two main methods. Collagen and fibronectin are key proteins in ECM, and they play a vital role in maintaining skin elasticity, toughness, and overall appearance. With age, the content of collagen and fibronectin in the skin will gradually decrease, leading to aging phenomena such as loose skin, sagging, and wrinkles. Such as Fig. 9 As shown, the recombinant elastin of the present invention can significantly promote the expression of type I, type III, type IV and type XVII collagen and fibronectin under the condition of administration of 250 nM (P≤0.05), reduce the damage of ultraviolet radiation to ECM, and has anti-aging effect.

[0110] 3. Analysis of barrier protein expression in NHEK cells

[0111] Normal human epidermal keratinocytes (NHEK) (PCS-200-010, ATCC) in good growth condition were seeded into 12-well plates and incubated in 5% CO 2 Cultured in an incubator until the cell plating rate reached 50% to 60%; discarded the original culture medium, added 2 mL of basal culture medium to each well of the blank control group, and added equal volumes of culture medium containing 50 nM and 200 nM recombinant elastin to each well of the drug group, with 3 replicates per group. After continuing to culture for 24 hours, discarded the original culture medium, washed twice with PBS, and collected the cells by centrifugation. Using GAPDH as a reference gene, the transcription levels of FLG and LOR protein-related genes were detected by qRT-PCR. The results are shown in Fig.10 .

[0112] like Fig.10 As shown, the recombinant elastin of the present invention can significantly promote the transcription levels of FLG and LOR protein related genes under the condition of 200 nM administration, indicating that the recombinant elastin of the present invention can promote the expression of HNEK cell barrier related proteins to improve the skin barrier.

[0113] 4. Analysis of TRPV1 gene expression in keratinocytes

[0114] Human immortalized keratinocytes HaCat were stimulated with capsaicin to construct an in vitro efficacy evaluation model for relieving nerve pain. A total of 5 groups were designed in the experiment, namely blank control group, model group, positive group, low-dose group and high-dose group, and they were treated as follows:

[0115] The blank control group received no treatment;

[0116] Model group, 10 μM capsaicin was added;

[0117] positive group, 10 μM capsaicin and 50 μM 4-tert-butylcyclohexanol were added;

[0118] The low-dose group was supplemented with 10 μM capsaicin and 250 nM recombinant elastin;

[0119] In the high-dose group, 10 μM capsaicin and 750 nM recombinant elastin were added.

[0120] The specific operation is as follows: HaCat cells with good growth status were inoculated into 12-well plates and incubated in 5% CO 2 The cells were cultured in an incubator until the cell plating rate reached 50% to 60%. The original culture medium was discarded, and 300 μL of basal culture medium, basal culture medium containing 4-tert-butylcyclohexanol, or basal culture medium containing recombinant elastin were added according to the above treatment. After culturing for 24 hours, the culture medium was discarded.

[0121] According to the above treatment, 300 μL of basal medium, basal medium containing capsaicin, basal medium containing recombinant elastin and capsaicin, or basal medium containing 4-tert-butyl cyclohexanol and capsaicin were added respectively, and the culture was continued for 24 h. The cells were collected by centrifugation, and the mRNA levels of TRPV1 receptor on the cell membrane of the blank control group, model group, positive group and recombinant elastin group were detected by fluorescent quantitative PCR. The results are shown in Fig.11 .

[0122] like Fig.11 As shown, at a dosage concentration of 750 nM, the recombinant elastin-like protein can effectively reduce the mRNA expression level of TRPV1, indicating that the recombinant elastin-like protein of the present invention can relieve nerve tingling and has a certain effect of soothing and calming the skin.

[0123] 5. Expression analysis of inflammatory factors released by RAW264.7 macrophages

[0124] Mouse mononuclear macrophages RAW264.7 cells that are in good growth condition were seeded into 24-well plates and incubated in 5% CO 2The cells were cultured in an incubator for 24 h; the original culture medium was discarded, 2 mL of basal culture medium was added to each well of the blank group, the model group was cultured with 1 μg / mL LPS, the positive group was cultured with 1 μg / mL LPS and 100 μg / mL dexamethasone, and the drug group was cultured with 1 μg / mL LPS and 100 nM or 250 nM recombinant elastin. Each group had 3 replicates and was cultured for 24 h. The cells were collected and the content of inflammatory factor TNF-α was detected by Mouse TNF-α (Tumor Necrosis Factor Alpha) ELISA Kit (E-EL-M3063, Elabscience). The results are shown in Table 1. Fig.12 .

[0125] like Fig.12 As shown, the recombinant elastin of the present invention has a significant inhibitory effect on the release of inflammatory factor TNF-α by RAW264.7 cells stimulated by LPS under the condition of 250 nM administration.

[0126] 6. Analysis of ROS content generated by fibroblasts

[0127] Select human fibroblasts with good growth status and inoculate them into 6-well plates, with 2 mL of cell suspension per well, and incubate in 5% CO 2 The cells were cultured in an incubator for 24 h. The original culture medium was discarded and replaced with pre-cooled PBS. The cells were placed in a UV curing box for UVA irradiation (30 min, with the dose set to 3.33 mW / cm 2 , i.e. 6J / cm 2 After irradiation, PBS was aspirated, and 2 mL of basal culture medium was added to each well of the blank group and model group, the positive group was culture medium containing 100 μg / mL ascorbic acid, and the drug group was culture medium containing 0.2%, 0.5% or 1% recombinant elastin-like protein, with 3 replicates per group, and culture was continued for 1 h.

[0128] DCFH-DA was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μM. After the cells were collected, they were suspended in the diluted DCFH-DA at a cell concentration of 1 million to 20 million / ml and incubated in a 37°C cell culture incubator for 20 minutes. During the incubation period, the cells were inverted and mixed every 3-5 minutes to allow the probe and cells to fully contact. The cells were washed three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells, and then detected by flow cytometry (Beckman, CytoFlex), and images were collected and analyzed (Beckman, CytExpert). The results are shown in Fig.13 .

[0129] The free radicals produced by mitochondria during electron transfer will attack cell membranes, proteins and even DNA, leading to cell aging and even death, and are closely related to a variety of skin diseases. In daily life, the skin is often exposed to ultraviolet radiation, which causes living cells to produce a large number of free radicals. Free radicals further damage living cells, leading to excessive ROS in skin cells, which are toxic and cause oxidative stress damage to cells. Fig.13 As shown, the recombinant elastin of the present invention can significantly reduce the ROS content of the UV-induced fibroblast aging model under the administration conditions of 0.2% and 0.5%, indicating that the recombinant elastin may enhance the ability of the skin to resist UV radiation damage by reducing the ROS content, and has anti-photoaging and anti-aging effects.

[0130] In summary, the recombinant elastin-like protein of the present invention has the effects of improving and repairing the skin barrier, soothing, anti-inflammatory and anti-aging, and has good prospects for medical and cosmetic applications.

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A recombinant elastin-like protein, characterized in that: The recombinant elastin-like protein comprises an elastin-like motif and a type III collagen functional motif; The amino acid sequence of the elastin-like motif is VPGXG, wherein X in VPGXG is any one of H, R, K, I, F, L, W, A, M, C, N, V, G, S, Q, Y, D, E and T; The amino acid sequence of the type III collagen functional motif is shown in SEQ ID NO.

1.

2. The recombinant elastin-like protein according to claim 1, characterized in that: The number of repetitions of the amino acid sequence of the elastin-like motif in the recombinant elastin-like protein is an integer ≥5, and the number of repetitions of the amino acid sequence of the type III collagen functional motif is an integer ≥1.

3. The recombinant elastin-like protein according to claim 1, characterized in that: The recombinant elastin-like protein further comprises a histidine tag, and the histidine tag is connected to the amino end and / or carboxyl end of the recombinant elastin-like protein.

4. The recombinant elastin-like protein according to claim 1, characterized in that: The recombinant elastin-like protein includes a linker sequence, and the linker sequence connects the elastin-like protein motif and the type III collagen functional motif; the amino acid sequence of the linker sequence is shown in SEQ ID NO.

2.

5. The method for preparing the recombinant elastin-like protein according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1, transferring the constructed vector containing elastin-like motif, type III collagen functional motif, linker sequence and histidine tag into Escherichia coli to obtain an engineered bacterium expressing recombinant elastin-like protein; S2, subjecting the engineered bacteria expressing recombinant elastin-like protein obtained in step S1 to high-density fermentation to obtain a fermentation product; S3, crushing and centrifuging the fermentation product obtained in step S2, and performing reversible phase change purification on the obtained supernatant to obtain a crude product; S4, resuspending the crude product obtained in step S3 in a buffer, incubating at 35-37° C. until the crude product is dissolved, and centrifuging to obtain a supernatant; S5, subjecting the supernatant obtained in step S4 to reversible phase change purification to obtain a purified product; S6. Repeat the operation of step S4 with the purified product obtained in step S5, and the obtained supernatant is the recombinant elastin-like protein.

6. The preparation method according to claim 5, characterized in that: In step S1, the number of elastin-like motifs in the vector is an integer ≥5, the number of type III collagen functional motifs in the vector is an integer ≥1, the linker sequence in the vector connects the elastin-like motif and the type III collagen functional motif, and the histidine tag is connected to the amino terminus and / or carboxyl terminus of the recombinant elastin.

7. The preparation method according to claim 5, characterized in that: In step S2, the high-density fermentation operation includes the following steps: (1) preparing the engineered bacteria expressing recombinant elastin-like protein obtained in step S1 into a secondary seed solution, inoculating the secondary seed solution into a bioreactor, adding glucose with an initial concentration of 5-6 g / L and magnesium sulfate of 0.6-0.7 g / L, stirring at a speed of 180-200 rpm, and maintaining the dissolved oxygen content above 30%; (2) After culturing at 30-37°C for 3.5-4 hours, feed was added until the bacterial weight was ≥40 g / L, the dissolved oxygen content was maintained at 80-85%, IPTG was added at a final concentration of 0.1-1 M, and cultured for 4 hours to obtain the fermentation product.

8. The preparation method according to claim 5, characterized in that: In steps S3 and S5, the reversible phase change purification operation mainly comprises adjusting the pH value of the supernatant to 7.0-7.5, standing at 23-28°C until a precipitate is precipitated, and collecting the precipitate by centrifugation. The obtained precipitate is the crude product or the purified product.

9. Use of the recombinant elastin-like protein according to any one of claims 1 to 4 in the preparation of anti-aging, anti-inflammatory, skin barrier repairing and / or soothing products.

10. The use according to claim 9, characterized in that The products include medicines, foods, health products, cosmetics and biological products.

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