Recombinant laminin with anti-aging and repairing effects and preparation method thereof

Abstract

The invention discloses recombinant laminin with anti-aging and repairing effects and a preparation method of the recombinant laminin, relates to the technical field of recombinant proteins, and aims to solve the technical problems that existing laminin is relatively low in yield and limited in application. According to the recombinant laminin with the anti-aging and repairing effects, the sequence of the recombinant laminin is shown as SEQ ID No.3, the total number is 363aa, and the predicted molecular weight is 40.4 kD. Compared with the prior art, the recombinant laminin is synthesized by embedding a laminin gene sequence into a pichia pastoris genome for heterologous expression. A cytological detection method for evaluating the efficacy of a skin care product is applied, and a research result shows that the recombinant laminin can remarkably promote cell adhesion and migration activity and can stimulate cells to generate collagen, so that the anti-aging and repairing functions of the recombinant laminin are shown.

Description

Technical Field

[0001] The present application relates to the technical field of recombinant proteins, and in particular to a recombinant laminin with anti-aging and repairing 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 associated with 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) have a cross-shaped structure, consisting of three short arms and a long arm composed of all three chains, and each short arm is composed of different chains. So far, 15 heterotrimers of LN have been known, among which laminin-5 (LN-5), which was once named kalinin, epiligrin and nicein, is the main component protein of the epithelial basement membrane and 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 diffusion. As far as we know, the deformation of the extracellular matrix of the dermis leads to increased skin wrinkles and reduced elasticity, and affects the structural and functional changes of the basement membrane between the epidermis and the dermis or skin aging. Laminin 332 (LN-5), a structural component of the basement membrane, plays an essential role in imparting security between the epidermis and the dermis by firmly fixing the epidermis and the dermis.

[0003] Laminin is currently mainly extracted from natural animals, which limits its production and application, and there are certain biocompatibility risks in its use. Summary of the invention

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

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

[0006] As some optional 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 optional embodiments of the present application, the recombinant laminin has an activity of promoting Hacat cell adhesion of 8490±550 U / mg.

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

[0009] As some optional embodiments of the present application, the recombinant laminin is diluted to 1.0 μg / mL by the maintenance culture medium, and then the recombinant laminin is subjected to a synthesis promotion treatment on type I collagen and type III collagen of 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; After the recombinant laminin was diluted to 5.0 μg / mL by the maintenance culture medium, it was used to promote the synthesis of type I collagen and type III collagen of human fibroblasts. After the synthesis promotion treatment was completed, the content of type I collagen was 21.42±1.55 ng / mL, and the content of type III collagen was 25.78±1.42 ng / mL; After the recombinant laminin was diluted to 25.0 μg / mL in the maintenance culture medium, it was subjected to a synthesis promotion treatment on type I collagen and type III collagen of human fibroblasts. After the synthesis promotion treatment was completed, the content of type I collagen was 63.66±1.24 ng / mL, and the content of type III collagen was 47.26±1.20 ng / mL.

[0010] On the other hand, the present application also provides a method for preparing the recombinant laminin having anti-aging and repairing effects as described above, comprising the following steps: Selecting a target gene sequence; optimizing and designing the target gene sequence to obtain a recombinant laminin protein with an optimized nucleotide sequence as shown in SEQ ID No. 4; synthesizing gene fragments based on the optimized recombinant laminin protein, and inserting the gene fragments into a pPIC9K plasmid through EcoR Ⅰ and Not Ⅰ restriction sites to obtain a recombinant plasmid expressing the recombinant laminin protein; The recombinant plasmid is subjected to Pichia pastoris electroporation treatment and then cultured on MD solid plates until a single transformant colony appears; Screening the transformant single colonies to obtain a high-copy recombinant yeast engineering strain; Inducing the expression of the high-copy recombinant yeast engineering strain to obtain the target protein; The target protein is purified to obtain recombinant laminin with anti-aging and repairing effects.

[0011] As some optional embodiments of the present application, the step of subjecting the recombinant plasmid to Pichia pastoris electroporation and then culturing on MD solid plates until a single transformant colony appears comprises: 5 μg to 10 μg of the recombinant plasmid was linearized by restriction endonuclease SacⅠ at 35°C to 40°C for 1h to 3h, and 5 times the volume of anhydrous ethanol was added and placed in a -15°C to -25°C environment for 1h to 3h, and then centrifuged at 11000r / min to 13000r / min for 15min to 25min to obtain a white precipitate; the white precipitate was dissolved with 15μL to 25μL of sterile pure water to obtain a linearized recombinant plasmid; Prepare GS115 Pichia competent cells; mix the GS115 Pichia competent cells and the linearized recombinant plasmid in a volume ratio of 8:2, transfer to an electroporation cup and place in an ice bath for 3 min to 7 min; use a Bio-rad electroporation instrument, select the yeast mode, and perform electroporation; add 0.5 mL to 1.5 mL of pre-cooled sorbitol to the electroporation cup, mix well, transfer the mixture to a sterile EP tube, and incubate in an incubator at 25° C. to 35° C. for 1 h to 2 h; Take 100μL~200μL of bacterial solution and spread it on the MD solid plate, let it stand at room temperature for 8min~12min, and culture it upside down in a 28℃~32℃ incubator for 2d~5d until a single colony appears.

[0012] As some optional embodiments of the present application, the step of screening the transformant single colony to obtain a high-copy recombinant yeast engineering strain includes: The transformant single colony was 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 a sterile white pipette tip; Single transformant colonies that can simultaneously 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 were screened and obtained as high-copy recombinant yeast engineering strains.

[0013] As some optional embodiments of the present application, the step of inducing expression of the high-copy recombinant yeast engineering strain to obtain the target protein includes: A plurality of single colonies of the high-copy recombinant yeast engineering strain were selected and inoculated into BMGY medium, and cultured at 28°C-30°C and 200r / min-240r / min for 20h-26h until the measured OD 600 The value is 2~6; According to the measured OD 600 Adjust the volume of BMGY culture solution to the value, collect the cells by centrifugation at 3000r / min for 10min, and resuspend the cells in BMMY medium to make the starting OD of all strains 600 The value is 1.0; Continue to culture at 28ºC~32℃ and 200r / min~240r / min, add 0.5% methanol to BMMY medium every 24h, take bacterial sample 72h after methanol induction, centrifuge at 12000r / min for 10min, and collect expression supernatant; The expression of the recombinant strain was analyzed by SDS-PAGE electrophoresis to obtain the target protein.

[0014] As some optional embodiments of the present application, the step of purifying the target protein to obtain a recombinant laminin with anti-aging and repairing effects comprises: The culture supernatant was collected by centrifugation, and the cation exchange medium was used to balance the chromatography column with phosphate buffer until the conductivity value and A280 absorbance value remained unchanged. The sample loading flow rate was set to 2mL / min~8mL / min, and the UV A280 absorbance value was detected. When it increased, the sample was loaded; After the sample loading is completed, the cationic chromatography medium is equilibrated with 20 mM PB until the UV and conductivity drop to the minimum and no longer change, and the equilibration is completed; Then, the corresponding proteins were eluted with 20 mM PB containing 500 mM NaCl to obtain recombinant laminin with anti-aging and repair effects.

[0015] Compared with the prior art, this application adopts the method of inserting the laminin gene sequence into the Pichia pastoris genome for heterologous expression to synthesize recombinant laminin. And by applying 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 activity, and can stimulate cells to produce collagen, thereby showing its anti-aging and repair functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the specific implementation of the present application or the technical solution in the prior art, the following is a brief introduction to the drawings required for the specific implementation or the prior art description. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale.

[0017] Figure 1 This is a diagram showing the expression level identification result of the high-copy strain of the GS115 / pPIC9K-LN-5 engineered bacteria involved in the examples of the present application; Figure 2 This is a diagram showing the purification results of the recombinant laminin SP involved in the examples of the present application; Figure 3 This is a graph showing the results of a test on the Hacat cell adhesion promoting activity of the recombinant laminin involved in the examples of the present application; Figure 4This is a graph showing the test results of the recombinant laminin promoting cell migration activity involved in the examples of the present application; Figure 5 The secretion amount of type I collagen involved in the embodiments of the present application; Figure 6 It is the secretion amount of type III collagen involved in the examples of this application.

[0018] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0020] As mentioned above, laminin (LN) is the main 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 component protein of the epithelial basement membrane and is essential for the binding of cells to the basement membrane. LN-5 has a strong ability to promote cell adhesion and migration. Studies have shown that increased skin wrinkles and decreased elasticity are related to structural and functional changes in LN-5. LN-332 plays a role in fixing the epidermis and dermis in the basement membrane.

[0021] Currently, LN is mainly extracted from animal sources, which limits its production and application.

[0022] Based on this, the present application proposes a recombinant laminin with anti-aging and repairing effects and a preparation method thereof.

[0023] The sequence of the recombinant laminin is shown in 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 in SEQ ID No. 1 and the sequence shown in SEQ ID No. 2.

[0024] The test confirmed that the Hacat cell adhesion promoting activity of the recombinant laminin described in the present application was 8490±550U / mg, and the cell migration promoting rate of the recombinant laminin was 54.60%; After the recombinant laminin was diluted to 1.0 μg / mL by the maintenance culture medium, it was used to promote the synthesis of type I collagen and type III collagen of human fibroblasts. After the synthesis promotion treatment was completed, the content of type I collagen was 7.38±1.30 ng / mL, and the content of type III collagen was 8.47±1.52 ng / mL; After the recombinant laminin was diluted to 5.0 μg / mL by the maintenance culture medium, it was used to promote the synthesis of type I collagen and type III collagen of human fibroblasts. After the synthesis promotion treatment was completed, the content of type I collagen was 21.42±1.55 ng / mL, and the content of type III collagen was 25.78±1.42 ng / mL; After the recombinant laminin was diluted to 25.0 μg / mL in the maintenance culture medium, it was subjected to a synthesis promotion treatment on type I collagen and type III collagen of human fibroblasts. After the synthesis promotion treatment was completed, the content of type I collagen was 63.66±1.24 ng / mL, and the content of type III collagen was 47.26±1.20 ng / mL.

[0025] It can be seen that the present application heterologously expresses recombinant laminin by inserting the laminin gene sequence into the Pichia pastoris genome, and tests the recombinant laminin according to the cytological detection method for evaluating the efficacy of skin care products. The results show that it promotes cell adhesion and migration activity, and also promotes cell production of collagen, showing anti-aging and repair effects.

[0026] In addition, the present application also proposes a method for preparing the above-mentioned recombinant laminin with anti-aging and repairing effects, comprising the following steps: Step 1, select the target gene sequence; optimize the design of the target gene sequence to obtain the 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 sites of EcoR Ⅰ and Not Ⅰ to obtain a recombinant plasmid expressing recombinant laminin.

[0027] Step 2: After the recombinant plasmid is electro-transformed with Pichia pastoris, it is cultured on MD solid plates until a single transformant colony appears.

[0028] In a further technical solution, step 2 may be: 5 μg to 10 μg of the recombinant plasmid was linearized by restriction endonuclease SacⅠ at 35°C to 40°C for 1h to 3h, and 5 times the volume of anhydrous ethanol was added and placed in a -15°C to -25°C environment for 1h to 3h, and then centrifuged at 11000r / min to 13000r / min for 15min to 25min to obtain a white precipitate; the white precipitate was dissolved with 15μL to 25μL of sterile pure water to obtain a linearized recombinant plasmid; Prepare GS115 Pichia competent cells; mix the GS115 Pichia competent cells and the linearized recombinant plasmid in a volume ratio of 8:2, transfer to an electroporation cup and place in an ice bath for 3 min to 7 min; use a Bio-rad electroporation instrument, select the yeast mode, and perform electroporation; add 0.5 mL to 1.5 mL of pre-cooled sorbitol to the electroporation cup, mix well, transfer the mixture to a sterile EP tube, and incubate in an incubator at 25° C. to 35° C. for 1 h to 2 h; Take 100μL~200μL of bacterial solution and spread it on the MD solid plate, let it stand at room temperature for 8min~12min, and culture it upside down in a 28℃~32℃ incubator for 2d~5d until a single colony appears.

[0029] Furthermore, the restriction endonuclease SacⅠ system includes the recombinant plasmid, restriction endonuclease SacⅠ 2μL, 10×QuickCut Buffer 2μL, and ddH2O is added to 20μL.

[0030] Furthermore, the GS115 Pichia pastoris competent cells are prepared by the following steps: After streaking the GS115 plate, a single colony was picked and inoculated into 20 mL YPD and cultured at 30 °C and 225 rpm for 24 h; the colony was transferred to 50 mL YPD liquid medium at a ratio of 1:1000 and cultured at 30 °C and 225 rpm until OD 600 1.3-1.5; Transfer the bacterial solution into a sterile 50mL centrifuge tube, centrifuge at 4℃, 3000 rpm for 5min, discard the supernatant and collect the bacteria; resuspend the bacterial pellet with 50mL pre-cooled sterile ultrapure water, centrifuge at 4℃, 3000 rpm for 5min; discard the supernatant, and resuspend the bacterial pellet with 50mL pre-cooled sterile ultrapure water; centrifuge at 4℃, 3000 rpm for 5min, discard the supernatant, and resuspend the cell pellet with 40mL pre-cooled sterile 1 M sorbitol; centrifuge at 4℃, 3000 rpm for 5min, discard the supernatant, and resuspend the cell pellet with 100-150μL pre-cooled sterile 1 M sorbitol, gently rotate to mix, and place on ice for use.

[0031] Take 80 μL of Pichia pastoris competent cells and 20 μL of the linearized recombinant plasmid, mix them, and transfer them to an electroporation cup and place them in an ice bath for 3 min to 7 min; use a Bio-rad electroporation instrument, select the yeast mode, and perform electroporation; add 0.5 mL to 1.5 mL of pre-cooled sorbitol to the electroporation cup, mix well, transfer the mixture to a sterile EP tube, and incubate it in an incubator at 25°C to 35°C for 1 h to 2 h; Take 100μL~200μL of bacterial solution and spread it on the MD solid plate, let it stand at room temperature for 8min~12min, and culture it upside down in a 28℃~32℃ incubator for 2d~5d until a single colony appears.

[0032] Step 3: Screen the transformant single colonies to obtain high-copy recombinant yeast engineering strains.

[0033] In a further technical solution, step 3 may be: The transformant single colony was 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 a sterile white pipette tip; Single transformant colonies that can simultaneously 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 were screened and obtained as high-copy recombinant yeast engineering strains.

[0034] Step 4: Induce the expression of the high-copy recombinant yeast engineering strain to obtain the target protein.

[0035] In a further technical solution, step 4 may be: A plurality of single colonies of the high-copy recombinant yeast engineering strain were selected and inoculated into BMGY medium, and cultured at 28°C-32°C and 200r / min-240r / min for 20h-26h until the measured OD 600 The value is 2~6; According to the measured OD 600 Adjust the volume of BMGY culture solution to the value, collect the cells by centrifugation at 3000r / min for 10min, and resuspend the cells with an appropriate amount of BMMY medium to make the starting OD of all strains 600 The value is 1.0; Continue to culture at 28ºC~32℃ and 200r / min~240r / min, add 0.5% methanol to BMMY medium every 24h, take bacterial sample 72h after methanol induction, centrifuge at 12000r / min for 10min, and collect expression supernatant; The expression of the recombinant strain was analyzed by SDS-PAGE electrophoresis to obtain the target protein.

[0036] Step 5: purify the target protein to obtain recombinant laminin with anti-aging and repair effects.

[0037] In a further technical solution, step 5 may be: The culture supernatant was collected by centrifugation, and the cation exchange medium was used to balance the chromatography column with phosphate buffer until the conductivity value and A280 absorbance value remained unchanged. The sample loading flow rate was set to 2mL / min~8mL / min, and the UV A280 absorbance value was detected. When it increased, the sample was loaded; After the sample loading is completed, the cationic chromatography medium is equilibrated with 20 mM PB until the UV and conductivity drop to the minimum and no longer change, and the equilibration is completed; Then, the corresponding proteins were eluted with 20 mM PB containing 500 mM NaCl to obtain recombinant laminin with anti-aging and repair effects.

[0038] To facilitate understanding by those skilled in the art, the sequences involved in the technical solution described in this application are listed below: SEQ ID No.1 NERALGAIQRQVKEINSLQSDFTKYLTTADSSLLQTNIALQLKEKSQKEYEKLAASLNEARQELSDKVRELSRSAGKTSLVEEAEKHARSLQELAKQLEEIKRNASGDELV RCAVDAATAYENILNAIKAAEDAANRAASASESALQTVIKEDLPRKAKTLSSSNSDKLLNEAKMTQKKLKQEVSPALNNLQQTLNIVTVQKEVIDTNLTTLRDGLHGIQRGD SEQ ID No.2 YFEGTGYARVPTQPHAPIPTFGQTIQTTVDRGLLFFAENGDRFISLNIEDGKLMVRYKLNSELPKERGVGAAINNGRDHSIQIKIGKLQKRMWINVDVQNTIIDGEVFDFSTYYLGGIPIAIRERFNISTPAFRGCMKNLK SEQ ID No.3 NERALGAIQRQVKEINSLQSDFTKYLTTADSSLLQTNIALQLKEKSQKEYEKLAASLNEARQELSDKVRELSRSAGKTSLVEEAEKHARSLQELAKQLEEIKRNASGDELVRCAVDAATAYENILNAIKAAEDAANRAASASESALQTVIKEDLPRKAKTLSSNSDKLLNEAKMTQKKLKQEVSPALNNLQQTLNIVTVQKEVIDTNLTTLRDGLHGIQRGDYFEGTGYARVPTQPHAPIPTFGQTIQTTVDRGLLFFAENGDRFISLNIEDGKLMVRYKLNSELPKERGVGAAINNGRDHSIQIKIGKLQKRMWINVDVQNTIIDGEVFDFSTYYLGGIPIAIRERFNISTPAFRGCMKNLK- SEQ ID No.4 The technical solution described in this application is described in detail below in conjunction with specific implementation methods: Example 1 Construction, identification and preparation of engineered bacteria 1. Gene sequence selection The recombinant laminin described in the present application is the α3 chain of human laminin-5, and its Genbank gene accession number is CAA59428.1. Two sequences are selected, and the sequences are 439-660 bp (SEQ ID No. 1) and 982-1122 bp (SEQ ID No. 2), respectively. 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.

[0039] A terminator (TGA) was added to the C-terminus, and the DNA sequence encoding the recombinant laminin was optimized and designed in view of the differences in the transcription and translation processes of genes in different hosts. The corresponding nucleotide sequence is SEQ ID No. 4, so that the recombinant laminin can be more easily expressed in Pichia pastoris.

[0040] The optimized recombinant laminin gene sequence SEQ ID No.4 was commissioned to GenScript Biotech Co., Ltd. to synthesize the gene fragment. Eco R Ⅰ and Not The restriction site of Ⅰ was inserted into the pPIC9K plasmid to obtain a recombinant plasmid expressing recombinant laminin, which was named recombinant plasmid pPIC9K-LN-5.

[0041] 2. Pichia pastoris electroporation 5μg~10μg of the recombinant plasmid pPIC9K-LN-5 was linearized by restriction endonuclease SacⅠ. After the linearized recombinant plasmid was recovered by anhydrous ethanol, 100μL of GS115 Pichia competent cells were mixed with the linearized plasmid, and then transferred to a pre-cooled electroporation cup and immediately placed in an ice bath for 5min. Using the Bio-rad electroporation instrument, select the yeast mode and perform electroporation. Then immediately add 1mL of pre-cooled 1 M sorbitol to the electroporation cup, mix well, transfer the mixture to a sterile EP tube, and incubate it in a 30 ℃ incubator for 1h-2h. Take 100μL~200μL of bacterial solution and spread it on the MD solid plate, let it stand at room temperature for 10min, and invert it in a 30℃ incubator for about 2d~5d until a single colony appears.

[0042] 3. Screening of high-copy recombinant yeast engineering strains Use a sterile white pipette tip to inoculate the single colonies on the MD plate 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. 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, it means that the single colony contains multiple copies of the target gene, that is, a high-copy recombinant yeast engineering strain with efficient expression.

[0043] 4. Induced expression of recombinant yeast engineering strains Fourteen single colonies of the high-copy recombinant yeast strain pPIC9K-LN-5 were selected and inoculated into BMGY medium and cultured at 30 ºC and 220 rpm for 24 h until the measured OD 600 The value is 2-6. According to the measured OD 600 The volume of BMGY culture solution was adjusted to the value; the cells were collected by centrifugation at 3000 rpm for 10 min, and the cells were resuspended in BMMY medium with an equal volume of BMGY to make the initial measured OD 600 The value is 1.0. Continue to culture at 30 ºC and 220 rpm, add 0.5% methanol to the culture medium every 24 hours, and take bacterial samples 72 hours after methanol induction, and collect the expression supernatant by centrifugation. The expression of the recombinant strain was analyzed by SDS-PAGE electrophoresis. The expressed target protein was named rhLN-5 (molecular weight 40.4kD). The results showed that seven single colonies were expressed in the 72th hour of induction in the shake flask and obvious target bands were visible. The expression identification results are shown in Figure 1 ;in, Figure 1 M in the middle indicates marker; Lanes 1 to 14 indicate the expression of recombinant laminin after 14 single colonies were picked and induced with methanol for 72 hours.

[0044] 5. Purification of target protein The culture supernatant was collected by centrifugation, and the cation exchange medium (chromatographic filler is SPSanarose 6 HP produced by Xiamen Sanji, loaded on the GE Akta chromatography system) was used to balance the chromatography column with phosphate buffer (20mM, pH 5.0) until the conductivity value and A280 absorbance value remained unchanged, and the sample loading flow rate was set to 5mL / min. The ultraviolet A280 absorbance value was detected, and when it rose, the sample was started. After the loading was completed, the cationic chromatography medium was balanced with 20mM PB until the ultraviolet and conductivity dropped to the minimum and no longer changed, and the balance was completed. Then, 20mM PB containing NaCl (500mM) was used to elute and collect the corresponding protein to obtain pure recombinant laminin. The purification results of the recombinant laminin SP are as follows. Figure 2 As shown; Figure 2In the figure, M indicates marker; 1 indicates supernatant; 2 indicates flow-through; and 3 indicates elution with 500 mM NaCl.

[0045] Example 2 Testing the activity of recombinant laminin in promoting Hacat cell adhesion The recombinant laminin prepared in Example 1 was diluted to 1.0 μg / mL with 20 mM PBS solution at pH 7.4, and then diluted 2-fold in a 96-well plate, with a total of 10 dilutions, 50 μL of different dilutions in each well, and each sample was tested in parallel three times. At the same time, 20 mM PBS was used as a negative control, and commercial natural extracted laminin (purchased from Shanghai Maokang Biotechnology Co., Ltd., item number MX0929, the same below) was set as a positive control, and incubated at 4°C overnight. After the incubation was completed, the liquid in the plate was discarded, 100 μL of 30 g / L BSA was added to each well for blocking, and the plate was incubated at 37°C for 1 hour; the liquid in the plate was removed and discarded, and human skin immortalized keratinocytes Hacat in the logarithmic growth phase were added, and 1.0×10 5 / mL, 100μL per well, incubate at 37℃ for 5h. Wash the incubated cell plate 3 times with 20mM PBS, observe the cell adhesion under a microscope, select five points except the edge under a 200x microscope to count the number of adherent cells, and calculate the titer based on the four-parameter fitting curve of the counting results.

[0046] The results are shown in Table 1 and Figure 3 As shown, the recombinant laminin prepared in Example 1 of the present application has a significantly higher effect on promoting Hacat cell adhesion than commercial laminin.

[0047] Table 1: Recombinant laminin promotes Hacat cell adhesion activity

[0048] Example 3 Activity of recombinant laminin in promoting Hacat cell migration The recombinant laminin prepared in Example 1 of the present application was used to carry out a Hacat cell promotion test. The specific method is as follows: 1. Use a black marker pen to align the ruler on the back of the 6-well cell culture plate and draw horizontal lines evenly, approximately every 0.5-1 cm, across the holes; 2. Collect Hacat cells in the logarithmic growth phase and adjust the cell density to 5×10 5 / mL, 2mL / well, added to the above 6-well cell plate, placed in a 37℃ 5% carbon dioxide incubator for 24h, until the cells grew into a full monolayer, use the gun tip to compare with the ruler, and scratch perpendicular to the horizontal line on the back, draw 2 horizontal lines in each well, wash the cells 3 times with PBS, wash off the scratched suspended cells, and then add 1.8mL of serum-free DMEM medium to the wells.

[0049] 3. The recombinant laminin prepared in Example 1 was added into the wells, and commercial natural extracted laminin (same as above) was used as a positive control group. The above protein concentrations were all 50 μg / mL, and PBS was used as a blank control group.

[0050] 4. Place the 6-well cell plate with the sample to be tested in a 37°C 5% carbon dioxide incubator for 24 hours. Take a photo at the 0th hour of incubation and record the photo position in each well. At the 24th hour of incubation, observe and take photos of the fixed position. Use Image J software to process the pictures of cell migration, obtain the initial scratch area and the area of ​​the cell-free blank area, and calculate the migration rate. Migration rate = cell-free blank area / initial scratch area × 100%.

[0051] Observation results of cell control wells and each sample well at 0h and 18h Figure 4 As shown, the migration promoting effect of each treatment group on HaCaT keratinocytes was calculated.

[0052] The results show that the recombinant laminin of the present application has a better effect on promoting the migration of HaCat cells than the commercial natural extracted laminin, and the difference is significant compared with the negative control. This shows that the recombinant laminin prepared in Example 1 has a good effect on promoting cell migration. This shows that the recombinant laminin prepared in Example 1 can promote the migration of human skin fibroblasts and has an anti-aging effect.

[0053] Example 4 Recombinant adhesion protein promotes fibroblasts to produce collagen Experimental principle: Collagen is one of the main components of the extracellular matrix of dermis. Procollagen is synthesized by fibroblasts in cells and secreted outside the cells. Under the action of terminal procollagen peptidase, the terminal peptides are separated and polymerized to form collagen fibers. Fibroblasts can be used as a cell model to study the increase of collagen content in cosmetics. By measuring the increase rate of collagen (type I collagen and type III collagen) content after administration of blank control and test substance, the efficacy of the test substance in promoting collagen synthesis is evaluated.

[0054] The recombinant laminin in this application promotes the determination of collagen content in fibroblasts by enzyme-linked immunosorbent assay (ELISA). The ELISA method is to specifically bind type I or type III collagen to the collagen antibody coated on the enzyme-labeled plate, and then to the anti-type I or type III collagen antibody labeled with a substrate. After the substrate is catalyzed by the enzyme, a colored product is generated. The content of type I or type III collagen is positively correlated with the color depth of the colored product. The optical density (OD value) is measured at a wavelength of 450 nm using an enzyme-labeled instrument to calculate the content of type I or type III collagen. Main experimental materials: BALB / C 3T3 mouse embryonic 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).

[0055] Experimental steps: The cells were cultured normally with complete culture medium (RPMI1640 containing 10% fetal bovine serum). When the cells grew to a fusion rate of more than 90%, the culture medium was removed, the cells were washed twice with PBS, and then 0.25% trypsin was added for digestion. When the cells shrank and rounded, the trypsin was removed, and the culture medium containing 10% calf serum was added and gently blown. The cells were collected and centrifuged, and the cells were counted. The cell concentration was adjusted to an appropriate concentration and inoculated into 1 mL containing 1.0×10 5 ~5.0×10 5 10 cells. 24-36 hours after passage, the cells were selected for determination. The culture medium in the culture flask was discarded, the cells were digested and collected, and a complete culture medium containing 1.0×10 4 / mL cell suspension, inoculated in a 96-well cell culture plate, 100 μL per well, and cultured at 37°C and 5% carbon dioxide. After 24 hours of culture, the maintenance medium was replaced and cultured for another 24 hours at 37°C and 5% carbon dioxide. 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 using the maintenance medium, and the commercial natural laminin positive control (same as above) was diluted to 25 μg / mL, and 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, with 3 replicates per group, and cultured for 36 hours at 37°C and 5% carbon dioxide. The cell supernatant sample was taken, and the collagen content was determined using a human type I collagen ELISA detection kit and a human type III collagen α1 (COL3α1) ELISA kit to evaluate the promotion of type I collagen and type III collagen synthesis in human fibroblasts. The test results of type I collagen and type III collagen content are shown in Table 2. Figure 5 and Figure 6 shown.

[0056] Table 2 Recombinant laminin promotes collagen production by fibroblasts

[0057] From Table 2 and Figure 5 , Figure 6As shown, the recombinant laminin prepared in Example 1 promotes fibroblasts to secrete type I and type III collagen at levels significantly higher than those of commercial natural extracted laminin and negative control, indicating that the recombinant laminin prepared in Example 1 can promote human skin fibroblasts to produce collagen and has an anti-aging effect.

[0058] It can be seen that the recombinant laminin described in the present application can promote fibroblast adhesion, migration and collagen secretion, and achieve anti-aging and repair effects in skin care products.

[0059] The above are only preferred embodiments of the present application, and are not intended to limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the present application specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A recombinant laminin with anti-aging and repairing effects, characterized in that: The sequence of the recombinant laminin is shown in SEQ ID No. 3, which is 363aa in total and has a predicted molecular weight of 40.4kD.

2. The recombinant laminin with anti-aging and repairing effects according to claim 1, characterized in that: The recombinant laminin is obtained by concatenating the sequence shown in SEQ ID No. 1 and the sequence shown in SEQ ID No.

2.

3. The recombinant laminin with anti-aging and repairing effects according to any one of claims 1 to 2, characterized in that: The recombinant laminin has an activity of promoting Hacat cell adhesion of 8490±550 U / mg.

4. The recombinant laminin with anti-aging and repairing effects according to any one of claims 1 to 2, characterized in that: The cell migration promoting rate of the recombinant laminin is 54.60%.

5. The recombinant laminin with anti-aging and repairing effects according to any one of claims 1-2, characterized in that: After the recombinant laminin was diluted to 1.0 μg / mL by the maintenance culture medium, it was used to promote the synthesis of type I collagen and type III collagen of human fibroblasts. After the synthesis promotion treatment was completed, the content of type I collagen was 7.38±1.30 ng / mL, and the content of type III collagen was 8.47±1.52 ng / mL; After the recombinant laminin was diluted to 5.0 μg / mL by the maintenance culture medium, it was used to promote the synthesis of type I collagen and type III collagen of human fibroblasts. After the synthesis promotion treatment was completed, the content of type I collagen was 21.42±1.55 ng / mL, and the content of type III collagen was 25.78±1.42 ng / mL; After the recombinant laminin was diluted to 25.0 μg / mL in the maintenance culture medium, it was subjected to a synthesis promotion treatment on type I collagen and type III collagen of human fibroblasts. After the synthesis promotion treatment was completed, the content of type I collagen was 63.66±1.24 ng / mL, and the content of type III collagen was 47.26±1.20 ng / mL.

6. A method for preparing the recombinant laminin with anti-aging and repairing effects as claimed in any one of claims 1 to 5, characterized in that: The following steps are involved: Select a target gene sequence; optimize and design the target gene sequence to obtain a recombinant laminin protein with an optimized nucleotide sequence as shown in SEQ ID No. 4; Based on the optimized recombinant laminin, gene fragments are synthesized, and inserted into pPIC9K plasmid through EcoR Ⅰ and Not Ⅰ restriction sites to obtain a recombinant plasmid expressing recombinant laminin; The recombinant plasmid is subjected to Pichia pastoris electroporation treatment and then cultured on MD solid plates until a single transformant colony appears; Screening the transformant single colonies to obtain a high-copy recombinant yeast engineering strain; Inducing the expression of the high-copy recombinant yeast engineering strain to obtain the target protein; The target protein is purified to obtain recombinant laminin with anti-aging and repairing effects.

7. The method for preparing the recombinant laminin with anti-aging and repairing effects according to claim 6, characterized in that: The step of subjecting the recombinant plasmid to electro-transformation of Pichia pastoris and then culturing on a MD solid plate until a single transformant colony appears comprises: 5 μg to 10 μg of the recombinant plasmid was linearized by restriction endonuclease SacⅠ at 35°C to 40°C for 1h to 3h, and 5 times the volume of anhydrous ethanol was added and placed in a -15°C to -25°C environment for 1h to 3h, and then centrifuged at 11000r / min to 13000r / min for 15min to 25min to obtain a white precipitate; the white precipitate was dissolved with 15μL to 25μL of sterile pure water to obtain a linearized recombinant plasmid; Prepare GS115 Pichia competent cells; mix the GS115 Pichia competent cells and the linearized recombinant plasmid in a volume ratio of 8:2, transfer to an electroporation cup and place in an ice bath for 3 min to 7 min; use a Bio-rad electroporation instrument, select the yeast mode, and perform electroporation; add 0.5 mL to 1.5 mL of pre-cooled sorbitol to the electroporation cup, mix well, transfer the mixture to a sterile EP tube, and incubate in an incubator at 25° C. to 35° C. for 1 h to 2 h; Take 100μL~200μL of bacterial solution and spread it on the MD solid plate, let it stand at room temperature for 8min~12min, and culture it upside down in a 28℃~32℃ incubator for 2d~5d until a single colony appears.

8. The method for preparing the recombinant laminin with anti-aging and repairing effects according to claim 6, characterized in that: The step of screening the transformant single colony to obtain a high-copy recombinant yeast engineering strain comprises: The transformant single colony was 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 a sterile white pipette tip; Single transformant colonies that can simultaneously 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 were screened and obtained as high-copy recombinant yeast engineering strains.

9. The method for preparing the recombinant laminin with anti-aging and repairing effects according to claim 6, characterized in that: The step of inducing the expression of the high-copy recombinant yeast engineering strain to obtain the target protein comprises: A plurality of single colonies of the high-copy recombinant yeast engineering strain were selected and inoculated into BMGY medium, and cultured at 28°C-32°C and 200r / min-240r / min for 20h-26h until the measured OD 600 The value is 2~6; According to the measured OD 600 Adjust the volume of BMGY culture solution to the value, collect the cells by centrifugation at 3000r / min for 10min, and resuspend the cells in BMMY medium to make the starting OD of all strains 600 The value is 1.0; Continue to culture at 28ºC~32℃ and 200r / min~240r / min, add 0.5% methanol to BMMY medium every 24h, take bacterial sample 72h after methanol induction, centrifuge at 12000r / min for 10min, and collect expression supernatant; The expression of the recombinant strain was analyzed by SDS-PAGE electrophoresis to obtain the target protein.

10. The method for preparing the recombinant laminin with anti-aging and repairing effects according to claim 6, characterized in that: The step of purifying the target protein to obtain recombinant laminin with anti-aging and repairing effects comprises: The culture supernatant was collected by centrifugation, and the cation exchange medium was used to balance the chromatography column with phosphate buffer until the conductivity value and A280 absorbance value remained unchanged. The sample loading flow rate was set to 2mL / min~8mL / min, and the UV A280 absorbance value was detected. When it increased, the sample was loaded; After the sample loading is completed, the cationic chromatography medium is equilibrated with 20 mM PB until the UV and conductivity drop to the minimum and no longer change, and the equilibration is completed; Then, the corresponding proteins were eluted with 20 mM PB containing 500 mM NaCl to obtain recombinant laminin with anti-aging and repair effects.

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