A recombinant laminin with anti-aging and repair effects and its preparation method

Abstract

The present application discloses a recombinant laminin with anti-aging and repair effects and a preparation method thereof, relating to the technical field of recombinant proteins, aiming to solve the technical problems of low yield and limited application of existing laminin. The recombinant laminin with anti-aging and repair effects has a sequence as shown in SEQ ID No.3, with a total of 363 amino acids and a predicted molecular weight of 40.4 kD. Compared with the prior art, the present application embeds the laminin gene sequence into the genome of Pichia pastoris for heterologous expression to synthesize the recombinant laminin. And through the cytological detection method for evaluating the efficacy of skin care products, the research results show that the recombinant laminin can significantly promote cell adhesion and migration activities and can stimulate cells to produce collagen, thereby demonstrating its anti-aging and repair functions.

Description

Technical Field

[0001] The present application relates to the technical field of recombinant protein, and particularly relates to a recombinant laminin with anti-aging and repair effects and a preparation method thereof. Background Art

[0002] Laminin (LN) is a family of extracellular matrix glycoproteins and is the main non-collagen component of the basement membrane. They are involved in a variety of biological processes, including cell adhesion, differentiation, migration, signal transduction, neurite outgrowth, and metastasis. LN is composed of three different chains: laminin α, β, and γ (A, B1, and B2 respectively) with a cross-shaped structure, consisting of three short arms and one long arm composed of all three chains, and each short arm is composed of a different chain. So far, 15 heterotrimers of LN have been known. Among them, laminin-5 (LN-5), formerly named kalinin, epiligrin, and nicein, as the main constituent protein of the epithelial basement membrane, plays an important role in the stable binding between epithelial cells and the basement membrane. Compared with other known ECMs, LN-5 has a stronger ability to promote cell adhesion, migration, and spreading. As is currently known, due to the deformation of the extracellular matrix in the dermis layer of the skin, the number of skin wrinkles increases and the elasticity decreases, and it affects the structural and functional changes of the basement membrane between the epidermis and the dermis or skin aging. Laminin 332 (LN-5) in the structural components of the basement membrane plays a necessary role in conferring security between the epidermis and the dermis by firmly fixing the epidermis and the dermis.

[0003] Currently, laminin is mainly extracted from animals naturally, which limits its yield and application, and there are certain risks of biocompatibility in its use. Summary of the Invention

[0004] The present application provides a recombinant laminin with anti-aging and repair effects and a preparation method thereof, aiming to solve the technical problems of low yield and limited application of existing laminin.

[0005] On the one hand, to solve the above technical problems, an embodiment of the present application provides: a recombinant laminin with anti-aging and repair effects, the sequence of the recombinant laminin is as shown in SEQ ID No.3, with a total of 363 aa, and the predicted molecular weight is 40.4 kD.

[0006] As some alternative embodiments of the present application, the recombinant laminin is obtained by concatenating the sequence shown in SEQ ID No.1 and the sequence shown in SEQ ID No.2.

[0007] As some alternative embodiments of the present application, the cell adhesion promoting activity of the recombinant laminin on Hacat cells is 8490±550 U / mg.

[0008] In some alternative embodiments of the present application, the cell migration promoting rate of the recombinant laminin is 54.60%.

[0009] In some alternative embodiments of the present application, after the recombinant laminin is diluted to 1.0 μg / mL with the maintenance culture medium, it is used to promote the synthesis of type I collagen and type III collagen in human fibroblasts. After the synthesis promotion treatment is completed, the content of type I collagen is 7.38 ± 1.30 ng / mL, and the content of type III collagen is 8.47 ± 1.52 ng / mL;

[0010] After the recombinant laminin is diluted to 5.0 μg / mL with the maintenance culture medium, it is used to promote the synthesis of type I collagen and type III collagen in human fibroblasts. After the synthesis promotion treatment is completed, the content of type I collagen is 21.42 ± 1.55 ng / mL, and the content of type III collagen is 25.78 ± 1.42 ng / mL;

[0011] After the recombinant laminin is diluted to 25.0 μg / mL with the maintenance culture medium, it is used to promote the synthesis of type I collagen and type III collagen in human fibroblasts. After the synthesis promotion treatment is completed, the content of type I collagen is 63.66 ± 1.24 ng / mL, and the content of type III collagen is 47.26 ± 1.20 ng / mL.

[0012] On the other hand, the embodiments of the present application also provide a preparation method of the recombinant laminin having anti-aging and repair effects as described above, including the following steps:

[0013] Select a target gene sequence; optimize and design the target gene sequence to obtain an optimized recombinant laminin with a nucleotide sequence as shown in SEQ ID No. 4; synthesize gene fragments based on the optimized recombinant laminin and insert them into the pPIC9K plasmid through the restriction enzyme cleavage sites of EcoR I and Not I to obtain a recombinant plasmid expressing the recombinant laminin;

[0014] After subjecting the recombinant plasmid to Pichia pastoris electrotransformation treatment, perform MD solid plate culture until transformant single colonies appear;

[0015] Screen the transformant single colonies to obtain a high-copy recombinant yeast engineering strain;

[0016] Induce the expression of the high-copy recombinant yeast engineering strain to obtain the target protein;

[0017] Purify the target protein to obtain a recombinant laminin having anti-aging and repair effects.

[0018] In some alternative embodiments of the present application, the step of performing MD solid plate culture after electrotransforming the recombinant plasmid with Pichia pastoris until transformant single colonies appear includes:

[0019] Linearize 5 μg to 10 μg of the recombinant plasmid with restriction endonuclease SacⅠ at 35°C to 40°C for 1 h to 3 h, add 5 volumes of absolute ethanol, place it in an environment of -15°C to -25°C for 1 h to 3 h, and then centrifuge at 11000 r / min to 13000 r / min for 15 min to 25 min to obtain a white precipitate; dissolve the white precipitate with 15 μL to 25 μL of sterile pure water to obtain the linearized recombinant plasmid;

[0020] Prepare GS115 Pichia pastoris competent cells; mix the GS115 Pichia pastoris competent cells with the linearized recombinant plasmid at a volume ratio of 8:2, transfer them into an electroporation cuvette, and ice-bath for 3 min to 7 min; use a Bio-rad electroporator, select the yeast mode, and perform electroporation; add 0.5 mL to 1.5 mL of pre-cooled sorbitol to the electroporation cuvette, mix well, transfer the mixture to a sterile EP tube, and statically incubate in an incubator at 25°C to 35°C for 1 h to 2 h;

[0021] Take 100 μL to 200 μL of the bacterial solution and spread it on an MD solid plate, let it stand at room temperature for 8 min to 12 min, and incubate it upside down in an incubator at 28°C to 32°C for 2 d to 5 d until single colonies appear.

[0022] In some alternative embodiments of the present application, the step of screening the transformant single colonies to obtain a high-copy recombinant yeast engineering strain includes:

[0023] Inoculate the transformant single colonies with sterile white pipette tips onto YPD solid plates with G418 concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL respectively;

[0024] Screen and obtain transformant single colonies that can grow on YPD solid plates with G418 concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL respectively as the high-copy recombinant yeast engineering strain.

[0025] In some alternative embodiments of the present application, the step of inducing the high-copy recombinant yeast engineering strain to express and obtain the target protein includes:

[0026] Pick multiple single colonies of the high-copy recombinant yeast engineering strain and inoculate them into BMGY medium. Culture at 28°C - 30°C and 200 r / min - 240 r / min for 20 h - 26 h until the measured OD 600 value is 2 - 6;

[0027] Adjust the volume of the BMGY culture broth according to the measured OD 600 value. Centrifuge at 3000 r / min for 10 min to collect the cells, and resuspend the cells with BMMY medium to make the initial OD 600 value of all strains 1.0;

[0028] Continue to culture at 28°C - 32°C and 200 r / min - 240 r / min. Add 0.5% methanol to the BMMY medium every 24 h. After 72 h of methanol induction, take the bacterial liquid sample, centrifuge at 12000 r / min for 10 min, and collect the expression supernatant;

[0029] Analyze the expression of the recombinant strain by SDS-PAGE electrophoresis to obtain the target protein.

[0030] As some alternative embodiments of the present application, the steps of purifying the target protein to obtain recombinant laminin with anti-aging and repair effects include:

[0031] Centrifuge to collect the supernatant of the culture broth. Use a cation exchange medium to balance the chromatography column with phosphate buffer until the conductivity value and A280 absorbance value remain unchanged. Set the sample loading flow rate at 2 mL / min - 8 mL / min, detect the ultraviolet A280 absorbance value, and start sampling when it rises;

[0032] After the sample loading is completed, balance the cation chromatography medium with 20 mM PB until the ultraviolet and conductivity drop to the lowest and no longer change, and the balancing is completed;

[0033] Then elute and collect the corresponding protein with 20 mM PB containing 500 mM NaCl to obtain recombinant laminin with anti-aging and repair effects.

[0034] Compared with the prior art, the present application embeds the laminin gene sequence into the Pichia pastoris genome for heterologous expression to synthesize recombinant laminin. And through the application of cytological detection methods for evaluating the efficacy of skin care products, the research results show that this recombinant laminin can significantly promote cell adhesion and migration activity, and can stimulate cells to produce collagen, thus demonstrating its anti-aging and repair functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] To more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts do not necessarily draw according to the actual scale.

[0036] Figure 1 It is a diagram showing the identification results of the expression level of the high-copy strain of the GS115 / pPIC9K-LN-5 engineering bacteria involved in the embodiments of the present application;

[0037] Figure 2 It is a diagram showing the purification results of the recombinant laminin SP involved in the embodiments of the present application;

[0038] Figure 3 It is a diagram showing the test results of the activity of the recombinant laminin promoting Hacat cell adhesion involved in the embodiments of the present application;

[0039] Figure 4 It is a diagram showing the test results of the activity of the recombinant laminin promoting cell migration involved in the embodiments of the present application;

[0040] Figure 5 It is the secretion amount of type I collagen involved in the embodiments of the present application;

[0041] Figure 6 It is the secretion amount of type III collagen involved in the embodiments of the present application.

[0042] The realization, functional characteristics and advantages of the purpose of the present application will be further described in combination with the embodiments with reference to the drawings. Specific Embodiments

[0043] The following will clearly and completely describe the technical solutions in the embodiments of the present application in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope protected by the present application.

[0044] As described above, laminin (LN) is a major component of the extracellular matrix and is related to biological processes such as cell adhesion, differentiation, and migration. LN is composed of three chains, α, β, and γ, forming a cross-shaped structure. There are 15 known LN heterotrimers, among which LN-5 is the main constituent protein of the epithelial basement membrane and is crucial for the binding of cells to the basement membrane. LN-5 has strong abilities in promoting cell adhesion and migration. Research shows that the increase in skin wrinkles and the decrease in elasticity are related to the structural and functional changes of LN-5. LN-332 plays a role in fixing the epidermis and dermis in the basement membrane.

[0045] Currently, LN is mainly extracted from animal sources, which limits its production and application.

[0046] Based on this, the present application proposes a recombinant laminin with anti-aging and repair effects and its preparation method.

[0047] Among them, the sequence of the recombinant laminin is shown as SEQ ID No.3, with a total of 363 aa and a predicted molecular weight of 40.4 kD; the recombinant laminin is obtained by concatenating the sequence shown as SEQ ID No.1 and the sequence shown as SEQ ID No.2.

[0048] It has been tested and confirmed that the cell adhesion-promoting activity of the recombinant laminin of the present application on Hacat cells is 8490±550 U / mg, and the cell migration rate of the recombinant laminin is 54.60%;

[0049] After the recombinant laminin is diluted to 1.0 μg / mL with the maintenance culture medium and its synthesis of type I collagen and type III collagen in human fibroblasts is promoted, after the synthesis promotion treatment is completed, the content of type I collagen is 7.38±1.30 ng / mL, and the content of type III collagen is 8.47±1.52 ng / mL;

[0050] After the recombinant laminin is diluted to 5.0 μg / mL with the maintenance culture medium and its synthesis of type I collagen and type III collagen in human fibroblasts is promoted, after the synthesis promotion treatment is completed, the content of type I collagen is 21.42±1.55 ng / mL, and the content of type III collagen is 25.78±1.42 ng / mL;

[0051] After the recombinant laminin is diluted to 25.0 μg / mL with the maintenance culture medium and its synthesis of type I collagen and type III collagen in human fibroblasts is promoted, after the synthesis promotion treatment is completed, the content of type I collagen is 63.66±1.24 ng / mL, and the content of type III collagen is 47.26±1.20 ng / mL.

[0052] It can be seen that in the present application, the laminin gene sequence is inserted into the Pichia pastoris genome for heterologous expression of recombinant laminin, and the recombinant laminin is tested according to the cytological detection method for evaluating the efficacy of skin care products. The results show that it promotes cell adhesion and migration activities, and also promotes cells to produce collagen, indicating that it has anti-aging and repair effects.

[0053] In addition, the present application also proposes a preparation method for the above-mentioned recombinant laminin with anti-aging and repair effects, including the following steps:

[0054] Step 1: Select the target gene sequence; optimize and design the target gene sequence to obtain the optimized recombinant laminin with the nucleotide sequence shown in SEQ ID No. 4; synthesize gene fragments based on the optimized recombinant laminin, and insert them into the pPIC9K plasmid through the restriction enzyme cleavage sites of EcoR Ⅰ and Not Ⅰ to obtain a recombinant plasmid expressing recombinant laminin.

[0055] Step 2: After subjecting the recombinant plasmid to electrotransformation treatment with Pichia pastoris, perform MD solid plate culture until transformant single colonies appear.

[0056] In a further technical solution, Step 2 can be:

[0057] Linearize 5 μg - 10 μg of the recombinant plasmid with the restriction enzyme SacⅠ at 35°C - 40°C for 1 h - 3 h, add 5 volumes of absolute ethanol, place it in an environment of -15°C - 25°C for 1 h - 3 h, and then centrifuge at 11000 r / min - 13000 r / min for 15 min - 25 min to obtain a white precipitate; dissolve the white precipitate with 15 μL - 25 μL of sterile pure water to obtain a linearized recombinant plasmid;

[0058] Prepare GS115 Pichia pastoris competent cells; mix the GS115 Pichia pastoris competent cells with the linearized recombinant plasmid at a volume ratio of 8:2, transfer them into an electroporation cup and ice-bath for 3 min - 7 min; use a Bio-rad electroporator, select the yeast mode, and perform electroporation; add 0.5 mL - 1.5 mL of pre-cooled sorbitol to the electroporation cup, mix well, transfer the mixture to a sterile EP tube, and statically incubate it in an incubator at 25°C - 35°C for 1 h - 2 h;

[0059] Take 100 μL - 200 μL of the bacterial solution and spread it on an MD solid plate, let it stand at room temperature for 8 min - 12 min, and incubate it upside down in an incubator at 28°C - 32°C for 2 d - 5 d until single colonies appear.

[0060] Furthermore, the restriction enzyme SacⅠ system includes the recombinant plasmid, 2 μL of the restriction enzyme SacⅠ, 2 μL of 10×QuickCut Buffer, and add ddH2O to 20 μL.

[0061] Furthermore, the GS115 Pichia pastoris competent cells are prepared and obtained through the following steps:

[0062] The single colonies were picked after streaking the GS115 strain on the plate and inoculated into 20 mL of YPD, and cultured at 30 °C and 225 rpm for 24 h; then transferred to 50 mL of YPD liquid medium at an inoculation ratio of 1:1000 and cultured at 30 °C and 225 rpm until OD 600 reached 1.3 - 1.5; the bacterial liquid was transferred into a sterile 50 mL centrifuge tube, centrifuged at 4 °C and 3000 rpm for 5 min, and the supernatant was discarded to collect the cells; the cell pellet was resuspended with 50 mL of pre-cooled sterile ultrapure water, centrifuged at 4 °C and 3000 rpm for 5 min; after discarding the supernatant, the cell pellet was resuspended with 50 mL of pre-cooled sterile ultrapure water; centrifuged at 4 °C and 3000 rpm for 5 min, after discarding the supernatant, the cell pellet was resuspended with 40 mL of pre-cooled sterile 1 M sorbitol; centrifuged at 4 °C and 3000 rpm for 5 min, after discarding the supernatant, the cell pellet was resuspended with 100 - 150 μL of pre-cooled sterile 1 M sorbitol, gently rotated and mixed, and placed on ice for later use.

[0063] Take 80 μL of GS115 Pichia pastoris competent cells and mix them with 20 μL of the linearized recombinant plasmid, then transfer them into an electroporation cuvette and ice-bath for 3 min - 7 min; use a Bio-rad electroporator, select the yeast mode, and perform electroporation; add 0.5 mL - 1.5 mL of pre-cooled sorbitol to the electroporation cuvette, mix well and transfer the mixture to a sterile EP tube, and incubate statically in an incubator at 25 °C - 35 °C for 1 h - 2 h;

[0064] Take 100 μL - 200 μL of the bacterial liquid and spread it on an MD solid plate, let it stand at room temperature for 8 min - 12 min, and incubate it upside down in an incubator at 28 °C - 32 °C for 2 d - 5 d until single colonies appear.

[0065] Step 3: Screen the single colonies of the transformants to obtain a high-copy recombinant yeast engineering strain.

[0066] In a further technical solution, step 3 can be:

[0067] Inoculate the single colonies of the transformants onto YPD solid plates with G418 concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL respectively using sterile white pipette tips;

[0068] Screen and obtain the single colonies of transformants that can grow on YPD solid plates with G418 concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL respectively as the high-copy recombinant yeast engineering strain.

[0069] Step 4: Induce the expression of the high-copy recombinant yeast engineering strain to obtain the target protein.

[0070] In a further technical solution, step 4 may be as follows:

[0071] Pick multiple single colonies of the high-copy recombinant yeast engineering strain and inoculate them into BMGY medium, and culture them at 28°C to 32°C and 200 r / min to 240 r / min for 20 h to 26 h until the measured OD 600 value is 2 to 6;

[0072] According to the measured OD 600 value, adjust the volume of the BMGY culture broth, centrifuge at 3000 r / min for 10 min to collect the cells, and resuspend the cells with an appropriate amount of BMMY medium to make the initial OD of all strains 600 value be 1.0;

[0073] Continue to culture at 28°C to 32°C and 200 r / min to 240 r / min, add 0.5% methanol to the BMMY medium every 24 h. After 72 h of methanol induction, take a sample of the bacterial liquid, centrifuge at 12000 r / min for 10 min, and collect the expression supernatant;

[0074] Analyze the expression of the recombinant strain by SDS-PAGE electrophoresis to obtain the target protein.

[0075] Step 5: Purify the target protein to obtain recombinant laminin with anti-aging and repair effects.

[0076] In a further technical solution, step 5 may be as follows:

[0077] Centrifuge to collect the supernatant of the culture broth, use a cation exchange medium, balance the chromatography column with phosphate buffer until the conductivity value and A280 absorbance value remain unchanged, set the sample loading flow rate at 2 mL / min to 8 mL / min, detect the ultraviolet A280 absorbance value, and start sampling when it rises;

[0078] After the sample loading is completed, balance the cation chromatography medium with 20 mM PB until the ultraviolet and conductivity drop to the lowest and no longer change, and the balance is completed;

[0079] Then elute with 20 mM PB containing 500 mM NaCl and collect the corresponding protein to obtain recombinant laminin with anti-aging and repair effects.

[0080] For the convenience of those skilled in the art to understand, the sequences involved in the technical solution described in this application are listed below:

[0081] SEQ ID No.1

[0082] NERALGAIQRQVKEINSLQSDFTKYLTTADSSLLQTNIALQLKEKSQKEYEKLAASLNEARQELSDKVRELSRSAGKTSLVEEAEKHARSLQELAKQLEEIKRNASGDELVRCAVDAATAYENILNAIKAAEDAANRAASASESALQTVIKEDLPRKAKTLSSNSDKLLNEAKMTQKKLKQEVSPALNNLQQTLNIVTVQKEVIDTNLTTLRDGLHGIQRGD

[0083] SEQ ID No.2

[0084] YFEGTGYARVPTQPHAPIPTFGQTIQTTVDRGLLFFAENGDRFISLNIEDGKLMVRYKLNSELPKERGVGAAINNGRDHSIQIKIGKLQKRMWINVDVQNTIIDGEVFDFSTYYLGGIPIAIRERFNISTPAFRGCMKNLK

[0085] SEQ ID No.3

[0086] NERALGAIQRQVKEINSLQSDFTKYLTTADSSLLQTNIALQLKEKSQKEYEKLAASLNEARQELSDKVRELSRSAGKTSLVEEAEKHARSLQELAKQLEEIKRNASGDELVRCAVDAATAYENILNAIKAAEDAANRAASASESALQTVIKEDLPRKAKTLSSNSDKLLNEAKMTQKKLKQEVSPALNNLQQTLNIVTVQKEVIDTNLTTLRDGLHGIQRGDYFEGTGYARVPTQPHAPIPTFGQTIQTTVDRGLLFFAENGDRFISLNIEDGKLMVRYKLNSELPKERGVGAAINNGRDHSIQIKIGKLQKRMWINVDVQNTIIDGEVFDFSTYYLGGIPIAIRERFNISTPAFRGCMKNLK-

[0087] SEQ ID No.4

[0088]

[0089] The technical solution of the present application will be described in detail below in conjunction with specific embodiments:

[0090] Example 1 Construction, Identification and Preparation of Engineering Bacteria

[0091] 1. Gene Sequence Selection

[0092] The recombinant laminin described in the present application is the α3 chain of human laminin-5, with the Genbank gene accession number CAA59428.1. Two sequences were selected, namely 439-660 bp (SEQ ID No.1) and 982-1122 bp (SEQ ID No.2). The sequence SEQ ID No.3 obtained by concatenating the two sequences has a total of 363 aa and a predicted molecular weight of 40.4 kD.

[0093] A terminator (TGA) was added at the C-terminus. Considering the differences in gene transcription and translation processes in different hosts, the DNA sequence encoding recombinant laminin was optimized and designed, and its corresponding nucleotide sequence is SEQ ID No.4, so that recombinant laminin is more likely to be expressed in Pichia pastoris.

[0094] The optimized recombinant laminin gene sequence SEQ ID No.4 was entrusted to GenScript Biotech Corporation for gene fragment synthesis. The synthesized gene fragment was inserted into the pPIC9K plasmid through the restriction enzyme cleavage sites of Eco R Ⅰ and Not Ⅰ to obtain a recombinant plasmid expressing recombinant laminin, named recombinant plasmid pPIC9K-LN-5.

[0095] 2. Electroporation of Pichia pastoris

[0096] 5 μg - 10 μg of the recombinant plasmid pPIC9K-LN-5 was digested with the restriction endonuclease SacⅠ to linearize it. After recovering the linearized recombinant plasmid with absolute ethanol, 100 μL of GS115 Pichia pastoris competent cells were mixed with the linearized plasmid, transferred into a pre-cooled electroporation cuvette, and immediately ice-bathed for 5 min. Using a Bio-rad electroporator, select the yeast mode for electroporation. Then immediately add 1 mL of pre-cooled 1 M sorbitol to the electroporation cuvette, mix well, and transfer the mixture to a sterile EP tube. Incubate it in a 30 °C incubator for 1 h - 2 h. Take 100 μL - 200 μL of the bacterial solution and spread it on an MD solid plate. Let it stand at room temperature for 10 min, and then incubate it upside down in a 30 °C incubator for about 2 d - 5 d until single colonies appear.

[0097] 3. Screening of High-copy Recombinant Yeast Engineering Strains

[0098] The single colonies on the MD plate were inoculated onto YPD solid plates with G418 concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL respectively using sterile white pipette tips. If a single colony can grow on YPD solid plates with G418 concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL simultaneously, it indicates that the single colony contains multiple copies of the target gene, namely a high-copy recombinant yeast engineering strain that can be highly expressed.

[0099] 4. Induced expression of recombinant yeast engineering strain

[0100] Pick 14 single colonies of the high-copy recombinant yeast engineering strain pPIC9K-LN-5 and inoculate them into BMGY medium. Culture at 30 ºC and 220 rpm for 24 h until the measured OD 600 value is 2 - 6. Adjust the volume of the BMGY culture broth according to the measured OD 600 value; and centrifuge at 3000 rpm for 10 min to collect the bacterial cells, resuspend the bacterial cells with BMMY medium of the same volume as BMGY to make the initial measured OD 600 value 1.0. Continue to culture at 30 ºC and 220 rpm, add 0.5% methanol to the medium every 24 h. After 72 h of methanol induction, take the bacterial liquid samples respectively and centrifuge to collect the expression supernatant. Analyze the expression of the recombinant strain by SDS-PAGE electrophoresis. The expressed target protein is named rhLN-5 (molecular weight is 40.4 kD). As a result, obvious target bands can be seen in the expression of seven single colonies in the shake flask at the 72nd h of induction. The results of the expression level identification are shown in Figure 1 ; among them, Figure 1 M represents marker in

[0101] 5. Purification of target protein

[0102] Centrifuge to collect the supernatant of the culture broth. Use a cation exchange medium (the chromatography packing material is SPSanarose 6 HP produced by Xiamen Sanji, loaded on the GE Akta chromatography system), balance the chromatography column with phosphate buffer (20 mM, pH 5.0) until the conductivity value and A280 absorbance value remain unchanged. Set the sample loading flow rate at 5 mL / min, detect the ultraviolet A280 absorbance value, and start sampling when it rises. After the sample loading is completed, balance the cation chromatography medium with 20 mM PB until the ultraviolet and conductivity drop to the lowest and no longer change, and the balance is completed. Then elute with 20 mM PB containing NaCl (500 mM) and collect the corresponding protein, that is, obtain the purified recombinant laminin. The SP purification result of the recombinant laminin is asFigure 2 as shown Figure 2 In it, M represents marker; 1 represents supernatant; 2 represents flow-through; 3 represents elution with 500 mM NaCl.

[0103] Example 2 Testing the activity of recombinant laminin in promoting Hacat cell adhesion

[0104] The recombinant laminin prepared in Example 1 was diluted to 1.0 μg / mL with 20 mM PBS solution at pH 7.4, and 2-fold serial dilutions were performed in a 96-well plate, with a total of 10 dilution degrees. Each well contained 50 μL of different dilutions, and each sample was tested in parallel three times. At the same time, 20 mM PBS was used as a negative control, and commercially available natural extracted laminin (purchased from Shanghai Maokang Biotechnology Co., Ltd., product number MX0929, the same below) was set as a positive control, and incubated overnight at 4°C. After incubation, the liquid in the plate was discarded, and 100 μL of 30 g / L BSA was added to each well for blocking, and incubated in an incubator at 37°C for 1 h; the liquid in the plate was taken out and discarded, and human immortalized keratinocytes Hacat in the logarithmic growth phase were added, 1.0×10 5 / mL, 100 μL per well, and incubated in an incubator at 37°C for 5 h. The cell plate after incubation was washed 3 times with 20 mM PBS, and the cell adhesion was observed under a microscope. Five points were selected at 200-fold magnification except at the edge to count the number of adherent cells, and the titer was obtained by fitting a four-parameter curve according to the counting results.

[0105] The results are shown in Table 1 and Figure 3 as shown, the recombinant laminin prepared in Example 1 of this application has a significantly higher effect on promoting Hacat cell adhesion than the commercially available laminin.

[0106] Table 1: Activity of recombinant laminin in promoting Hacat cell adhesion

[0107]

[0108] Example 3 Activity of recombinant laminin in promoting Hacat cell migration

[0109] The recombinant laminin prepared in Example 1 of this application was used for the experiment of promoting Hacat cells. The specific method is as follows:

[0110] 1. Use a black marker to draw horizontal lines evenly on the back of a 6-well cell culture plate with a ruler aligned, about one line every 0.5 - 1 cm, crossing the wells;

[0111] 2. Collect Hacat cells in the logarithmic growth phase and adjust the cell density to 5×10 5 / mL, 2 mL per well, add it to the above 6-well cell culture plate, and place it in an incubator at 37°C with 5% carbon dioxide for 24 h. When the cells grow into a monolayer, use a pipette tip against a ruler to make scratches perpendicular to the horizontal line on the back. Make 2 horizontal scratches in each well. Wash the cells 3 times with PBS to wash away the suspended cells that have been scratched off, and then add 1.8 mL of serum-free DMEM medium to the wells.

[0112] 3. Add the recombinant laminin prepared in Example 1 to the wells, and use commercially available natural extract laminin (the same as above) as the positive control group. The above protein concentrations are all 50 μg / mL, and PBS is used as the blank control group.

[0113] 4. Place the 6-well cell culture plate with the test samples added into an incubator at 37°C with 5% carbon dioxide for 24 h. Take pictures at the 0 h of culture to record the positions for taking pictures in each well. At the 24 h of culture, observe and take pictures at the fixed positions. Use Image J software to process the pictures of cell migration to obtain the data of the initial scratch area and the cell-free blank area, and calculate the migration rate. Migration rate = cell-free blank area / initial scratch area × 100%.

[0114] Observation results of the cell control wells and each sample well at 0 h and 18 h Figure 4 As shown, and calculate the promoting migration effect of each treatment group on HaCaT keratinized cells.

[0115] The results show that compared with the commercially available natural extract laminin, the recombinant laminin of the present application has a better effect on promoting the migration of HaCat cells, and there is a significant difference compared with the negative control. It shows that the recombinant laminin prepared in Example 1 has a good effect on promoting cell migration. It shows that the recombinant adhesin prepared in Example 1 can promote the migration of human skin fibroblasts and has an anti-aging effect.

[0116] Example 4 Recombinant adhesin promotes the production of collagen by fibroblasts

[0117] Experimental principle: Collagen is one of the main components of the extracellular matrix of dermal cells. Procollagen is synthesized intracellularly by fibroblasts and secreted extracellularly. After the terminal procollagen peptidase acts and the terminal peptides are separated, they polymerize to form collagen fibers. Fibroblasts can be used as a cell model to study the improvement of collagen content in cosmetics. By measuring the upregulation rate of collagen (type I collagen and type III collagen) content after blank control and administration of the test substance, evaluate whether the test substance has an effect in promoting collagen synthesis.

[0118] The determination of the content of collagen produced by fibroblasts by the recombinant laminin of this application adopts the enzyme-linked immunosorbent assay (ELISA). In the ELISA method, after type I or type III collagen specifically binds to the collagen antibody coated on the enzyme-labeled plate, it binds to the anti-type I or type III collagen antibody with a substrate label. After the substrate is catalyzed by the enzyme, a colored product is generated, and the content of type I or type III collagen is positively correlated with the depth of the color of the colored product. The optical density (OD value) is measured at a wavelength of 450 nm with an enzyme-labeled instrument, and the content of type I or type III collagen is calculated.

[0119] Main experimental materials: BALB / C 3T3 mouse embryo fibroblasts (purchased from ATCC), RPMI 1640 culture medium (purchased from gibco), 0.01M PBS, fetal bovine serum (purchased from gibco); human type I collagen ELISA detection kit (purchased from abcam, ab285250); human type III collagen α1 (COL3α1) ELISA kit (purchased from Shanghai Yubo Biotechnology Co., Ltd., product number YB-COL3a1-Hu).

[0120] Experimental steps: The cells are normally cultured in a complete culture medium (RPMI1640 containing 10% fetal bovine serum). When the cell growth reaches a confluence rate of more than 90%, the culture medium is removed, and the cells are washed twice with PBS. Then, 0.25% trypsin is added for digestion. When the cells shrink and become round, the trypsin is poured off, and a culture medium containing 10% calf serum is added and gently pipetted. The cells are collected and centrifuged, and the cells are counted. The cell concentration is adjusted to an appropriate concentration and inoculated into 1 mL containing 1.0×10 5 ~5.0×10 5 cells. Cells at 24 - 36 hours after passage are selected for determination. Discard the culture medium in the culture flask, digest and collect the cells, and prepare them into a solution containing 1.0×10 4 / mL cell suspension was inoculated into a 96-well cell culture plate, 100 μL per well, and cultured at 37 °C under 5% carbon dioxide. After 24 hours of culture, the medium was replaced with maintenance medium and continued to be cultured at 37 °C under 5% carbon dioxide for another 24 hours. After 24 hours, the recombinant laminin prepared in Example 1 was diluted to 1.0 μg / mL, 5.0 μg / mL and 25 μg / mL with maintenance medium, and the commercial natural laminin positive control (the same as above) was diluted to 25 μg / mL. The diluted samples were added to the cells, 200 μL per well. At the same time, a cell control group was set up, and only 200 μL of maintenance medium was added to each well. There were 3 replicates in each group. The cells were continued to be cultured at 37 °C under 5% carbon dioxide for 36 h. Cell supernatant samples were taken, and the content of collagen was determined using a human type I collagen ELISA kit and a human type III collagen α1 (COL3α1) ELISA kit to evaluate the promoting effect on the synthesis of type I and type III collagen in human fibroblasts. The test results of the content of type I and type III collagen are shown in Table 2 and Figure 5 and Figure 6 shown

[0121] Table 2 Recombinant laminin promotes the production of collagen by fibroblasts

[0122]

[0123] As can be seen from Table 2 and Figure 5 、 Figure 6 shown, the content of type I and type III collagen secreted by fibroblasts promoted by the recombinant laminin prepared in Example 1 was significantly higher than that of the commercial natural extracted laminin and the negative control, indicating that the recombinant laminin prepared in Example 1 can promote the production of collagen by human skin fibroblasts and has an anti-aging effect.

[0124] It can be seen that the recombinant laminin described in the present application can promote fibroblast adhesion, migration and collagen secretion, achieving anti-aging and repair effects in skin care products.

[0125] The above are only the preferred embodiments of the present application, and do not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the specification and drawings of the present application, or directly or indirectly applied in other related technical fields, shall be included in the patent protection scope of the present application by the same token.

Claims

1. A recombinant laminin with anti-aging and repair effects, characterized in that, The sequence of the recombinant laminin is shown in SEQ ID No.3, with a total of 363 amino acids.

2. A preparation method of the recombinant laminin with anti-aging and repair effects as described in claim 1, characterized in that, It includes the following steps: Select the target gene sequence; perform optimized design on the target gene sequence to obtain the optimized recombinant laminin with the nucleotide sequence shown in SEQ ID No.4; Based on the optimized recombinant laminin, conduct gene fragment synthesis and insert it into the pPIC9K plasmid through the restriction enzyme cleavage sites of EcoR Ⅰ and Not Ⅰ to obtain a recombinant plasmid expressing recombinant laminin; After subjecting the recombinant plasmid to electrotransformation treatment with Pichia pastoris, perform MD solid plate culture until transformant single colonies appear; Screen the transformant single colonies to obtain a high-copy recombinant yeast engineering strain; Induce the expression of the high-copy recombinant yeast engineering strain to obtain the target protein; Purify the target protein to obtain recombinant laminin with anti-aging and repair effects.

3. The preparation method of the recombinant laminin with anti-aging and repair effects according to claim 2, wherein, The step of performing MD solid plate culture until transformant single colonies appear after subjecting the recombinant plasmid to electrotransformation treatment with Pichia pastoris includes: Linearize 5 μg - 10 μg of the recombinant plasmid with the restriction enzyme SacⅠ at 35°C - 40°C for 1 h - 3 h, add 5 times the volume of absolute ethanol, place it in an environment of -15°C - 25°C for 1 h - 3 h, then centrifuge at 11000 r / min - 13000 r / min for 15 min - 25 min to obtain a white precipitate; dissolve the white precipitate with 15 μL - 25 μL of sterile pure water to obtain the linearized recombinant plasmid; Prepare GS115 Pichia pastoris competent cells; mix the GS115 Pichia pastoris competent cells with the linearized recombinant plasmid at a volume ratio of 8:2, transfer them into an electroporation cuvette and ice-bath for 3 min - 7 min; use a Bio-rad electroporator, select the yeast mode, and perform electroporation; add 0.5 mL - 1.5 mL of pre-cooled sorbitol to the electroporation cuvette, mix evenly, transfer the mixture to a sterile EP tube, and statically incubate in an incubator at 25°C - 35°C for 1 h - 2 h; Take 100 μL - 200 μL of the bacterial solution and spread it on an MD solid plate, let it stand at room temperature for 8 min - 12 min, and incubate it upside down in an incubator at 28°C - 32°C for 2 d - 5 d until single colonies appear.

4. The preparation method of the recombinant laminin with anti-aging and repair effects according to claim 2, characterized in that, The step of screening the transformant single colonies to obtain a high-copy recombinant yeast engineering strain includes: Inoculate the transformant single colonies with sterile white pipette tips onto YPD solid plates with G418 concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL respectively; Screen and obtain the transformant single colonies that can grow on YPD solid plates with G418 concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL respectively as the high-copy recombinant yeast engineering strain.

5. The preparation method of the recombinant laminin with anti-aging and repair effects according to claim 2, characterized in that, The step of inducing the expression of the high-copy recombinant yeast engineering strain to obtain the target protein includes: Pick multiple single colonies of the high-copy recombinant yeast engineering strain and inoculate them into BMGY medium. Culture at 28°C to 32°C and 200 r / min to 240 r / min for 20 h to 26 h until the measured OD 600 value is 2 to 6; According to the measured OD 600 Adjust the volume of the BMGY culture broth, centrifuge at 3000 r / min for 10 min to collect the cells, and resuspend the cells with BMMY medium so that the initial OD 600 value of all strains is 1.0; Continue culturing under the conditions of 28°C to 32°C and 200 r / min to 240 r / min, add 0.5% methanol to the BMMY medium every 24 h. After 72 h of methanol induction, take the bacterial liquid sample, centrifuge at 12000 r / min for 10 min, and collect the expressed supernatant; Analyze the expression of the recombinant strain by SDS-PAGE electrophoresis to obtain the target protein.

6. The preparation method of the recombinant laminin with anti-aging and repair effects according to claim 2, characterized in that, The steps of purifying the target protein to obtain recombinant laminin with anti-aging and repair effects include: Centrifuge to collect the supernatant of the culture medium. Use a cation exchange medium to equilibrate the chromatography column with phosphate buffer until the conductivity value and A280 absorbance value remain unchanged. Set the sample loading flow rate at 2 mL / min to 8 mL / min, detect the ultraviolet A280 absorbance value, and start sampling when it rises; After the sample loading is completed, equilibrate the cation chromatography medium with 20 mM PB until the ultraviolet and conductivity drop to the lowest and no longer change, and the equilibration is completed; Then elute and collect the corresponding protein with 20 mM PB containing 500 mM NaCl to obtain recombinant laminin with anti-aging and repair effects.

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