Extracellular matrix fusion protein as well as preparation method and application thereof

Abstract

The invention discloses an extracellular matrix fusion protein as well as a preparation method and application thereof, relates to the technical field of recombinant proteins, and aims to solve the technical problems that protein cosmetics in the market are relatively single in effect and difficult to meet application requirements. According to the extracellular matrix fusion protein, the nucleotide sequence of the extracellular matrix fusion protein is as shown in SEQ ID No.7. According to the extracellular matrix fusion protein with the nucleotide sequence shown as SEQ ID No.7, an in-vitro cell test proves that the fusion protein can remarkably promote cell adhesion, proliferation and cell migration, can act on a corium layer in a transdermal mode and remarkably promote collagen secretion, and due to the fact that recombinant protein belongs to biological protein, the fusion protein can be used for preparing the extracellular matrix fusion protein with the nucleotide sequence shown as SEQ ID No.7. Compared with common chemical cosmetics and hormone drugs, the composition has the advantages of easiness in decomposition, no residue and the like, and has a wider application prospect.

Description

Technical Field

[0001] The present application relates to the technical field of recombinant proteins, and particularly relates to an extracellular matrix fusion protein, a preparation method thereof, and an application thereof. Background Art

[0002] Currently, most protein-based cosmetics on the market use single-component protein raw materials, such as extracted collagen, elastin, fibronectin, etc. Very few use extracellular matrix protein fusion products. Moreover, the efficacy of products using a single protein component is relatively single, far less complete than that of fusion-expressed protein products. However, for fusion-expressed proteins, due to their large molecular weight, there are also problems such as being difficult to be absorbed by tissues and cells, resulting in limited scope of use. Summary of the Invention

[0003] The present application provides an extracellular matrix fusion protein, a preparation method thereof, and an application thereof, aiming to solve the technical problem that the efficacy of existing protein-based cosmetics on the market is relatively single and difficult to meet the application requirements.

[0004] On the one hand, to solve the above technical problem, an embodiment of the present application provides: an extracellular matrix fusion protein, and the nucleotide sequence of the extracellular matrix fusion protein is as shown in SEQ ID No.7.

[0005] As some optional embodiments of the present application, the amino acid sequence of the extracellular matrix fusion protein includes a transdermal short peptide sequence, a laminin amino acid sequence, a humanized type I collagen amino acid sequence, a fibronectin amino acid sequence, and an elastin amino acid sequence sequentially connected in series based on a flexible linker (GGGGS) 2 as shown in SEQ ID No.6.

[0006] As some optional embodiments of the present application, the transdermal short peptide sequence is as shown in SEQ ID No.1; The laminin amino acid sequence is derived from the LG4 domain of LAMA5 with UniProt of PO15230, as shown in SEQ IDNo.2; The humanized type I collagen amino acid sequence is derived from an intercepted fragment of the domain of COL1A1 with UniProt of P02452, as shown in SEQ ID No.3; The fibronectin amino acid sequence is derived from the Fibronectin type-III 11 domain fragment of FN1 with UniProt of P02751, as shown in SEQ ID No.4; The elastin amino acid sequence is derived from one repeat fragment of [VPGKG(VPGVG)8VPGFG] in the hydrophobic domain, as shown in SEQ ID No.5.

[0007] On the other hand, the embodiments of the present application also provide: a method for preparing the extracellular matrix fusion protein as described above, comprising the following steps: Insert the nucleotide sequence of the extracellular matrix fusion protein shown in SEQ ID No.7 into the pPIC9K plasmid by the restriction enzyme cleavage sites of EcoRI and NotI to obtain the pPIC9K-TD-LCFE recombinant plasmid; After linearizing the pPIC9K-TD-LCFE recombinant plasmid, electrotransform it into GS115 competent cells for culturing until multiple transformant single colonies appear; Based on the multiple transformant single colonies, screen to obtain positive transformant single colonies; Culture and induce the positive transformant single colonies to obtain the engineered bacteria fermentation broth; Purify the engineered bacteria fermentation broth to obtain the extracellular matrix fusion protein.

[0008] As some alternative embodiments of the present application, the step of linearizing the pPIC9K-TD-LCFE recombinant plasmid includes: Use Quick Cut Sac I to perform linearizing restriction enzyme digestion on the pPIC9K-TD-LCFE recombinant plasmid, and the digestion conditions are 35°C to 40°C for 3h to 7h; the digestion system includes: 10 μg to 15 μg of pPIC9K-TD-LCFE recombinant plasmid; 8 μL to 12 μL of Quick Cut Sac I; 3 μL to 7 μL of 10× Quick Cut buffer.

[0009] As some alternative embodiments of the present application, the step of electrotransforming into GS115 competent cells for culturing until multiple transformant single colonies appear includes: Mix the GS115 competent cells with the linearized recombinant plasmid at a volume ratio of 10:1, transfer them into a pre-cooled electroporation cup for ice bath treatment; adjust the electroporator to the yeast mode for electroporation treatment, add pre-cooled sorbitol to the electroporation cup, mix well and transfer the mixture to a sterile EP tube for culturing to obtain a bacterial solution; spread the bacterial solution on an MD medium for culturing until multiple transformant single colonies appear.

[0010] As some alternative embodiments of the present application, the step of screening based on the multiple transformant single colonies to obtain positive transformant single colonies includes: Pick the single colony of the transformant, transfer it to a 96-well culture plate containing YPD medium for culture to obtain the first bacterial solution; aspirate the first bacterial solution in each well to a new 96-well culture plate containing YPD medium for continuous culture to obtain the second bacterial solution; aspirate the second bacterial solution in each well to a new 96-well culture plate containing YPD medium for continuous culture to obtain the third bacterial solution; aspirate the third bacterial solution in each well and spot it onto YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418 respectively for continuous culture; Screen the single colony of the transformant corresponding to the third bacterial solution that can grow on YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418 simultaneously as the positive transformant single colony.

[0011] As some alternative embodiments of the present application, the step of culturing and inducing the positive transformant single colony to obtain the engineered bacteria fermentation broth includes: Pick a single colony and inoculate it into BMGY yeast growth medium for culture until the measured OD 600 value is 2 - 6; adjust the volume of BMGY yeast growth medium according to the measured OD 600 value, centrifuge to collect the bacterial cells, resuspend the bacterial cells with BMMY yeast induction medium with the same volume as the BMGY yeast growth medium to make the initial OD 600 value 2.0; continue the culture, add 0.5% methanol to the medium every 24 h, take bacterial liquid samples every 24 h after induction, centrifuge to collect the expression supernatant to obtain the engineered bacteria fermentation broth.

[0012] As some alternative embodiments of the present application, the step of purifying the engineered bacteria fermentation broth to obtain the extracellular matrix fusion protein includes: Use a solid-liquid separation system to separate the engineered bacteria fermentation broth into fermentation supernatant and bacterial cells; 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 20 cm / h, detect the ultraviolet A280 absorbance value, and start loading when it rises; After the loading is completed, balance the cation chromatography medium with phosphate buffer until the ultraviolet and conductivity drop to the lowest and no longer change, and stop loading; Then elute and collect the corresponding protein with a NaH 2 PO 4 -NaCl (0.5 M) buffer, and after ultrafiltration, buffer exchange, and concentration, obtain the recombinant TD-LCFE protein stock solution.

[0013] On the other hand, the embodiments of the present application also provide: an application of the extracellular matrix fusion protein as described above for preparing protein-based skin care products with anti-aging effects.

[0014] Compared with the prior art, the extracellular matrix fusion protein with the nucleotide sequence shown in SEQ ID No.7 in the present application has been proven by in vitro cell experiments to significantly promote cell adhesion, proliferation and migration. However, considering the relatively large molecular weight of this fusion protein, a transdermal peptide TD-1 is added to the N-terminus of the fusion protein to significantly enhance its transdermal efficacy. The experimental results show that this fusion protein can act on the dermis layer through the skin. The results of the Sirius red staining method for promoting collagen secretion show that this fusion protein can significantly promote collagen secretion. Moreover, since the recombinant protein belongs to a biological protein and has the advantages of easy decomposition and no residue, it has a broader application prospect compared with ordinary chemical cosmetics and hormonal drugs. Brief Description of the Drawings

[0015] In order 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 are not necessarily drawn to scale.

[0016] Figure 1 It is the plasmid map of pPIC9K-TD-LCFE involved in the embodiment of the present application; Figure 2 It is the PCR electrophoresis map of GS115 / pPIC9K / TD-LCFE involved in the embodiment of the present application; Figure 3 It is the SDS-PAGE result map of the supernatant of GS115 / pPIC9K / TD-LCFE involved in the embodiment of the present application; Figure 4 It is the SDS-PAGE result map of the purified supernatant protein of GS115 / pPIC9K / TD-LCFE involved in the embodiment of the present application; Figure 5 It is the cell state of different treatment groups at 0h and 24h involved in the embodiment of the present application; Figure 6 It is the result map of the TD-LCFE fusion protein promoting cell adhesion involved in the embodiment of the present application; Figure 7 It is the working schematic diagram of the mouse skin-Franz cell diffusion cell involved in the embodiment of the present application; wherein, 1 - the location of the active ingredient (transdermal area), 2 - the upper cover of the receiving pool, 3 - the elastic clip, 4 - the skin or skin substitute, 5 - the receiving pool, 6 - the sampling point, 7 - the shark glass slide, 8 - the sampling needle, 9 - the sampling tube, 10 - the magnetic stirrer; Figure 8Immunohistochemical results of the skin of each group in the transdermal experiment involved in the embodiments of the present application; among them, Figure A: the result diagram of the blank control treatment; Figure B: the result diagram of the treatment with recombinant type III collagen; Figure C: the result diagram of the treatment with TD-LCFE fusion protein; Figure 9 The Sirius red staining results involved in the embodiments of the present application; The realization, functional characteristics and advantages of the purpose of the present application will be further described with reference to the accompanying drawings in combination with the embodiments. Detailed implementation manners

[0017] Next, the technical solutions in the embodiments of the present application will be clearly and completely described with reference to the accompanying 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. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present application without making creative efforts shall fall within the protection scope of the present application.

[0018] The extracellular matrix (ECM) is secreted by cells and surrounds them in tissues. Its complex network structure of proteins and polysaccharides can not only provide attachment sites and structural support for cells, but also provide important biological information for regulating cell activity and organ / tissue function. According to its different components and structures, it is divided into two types: gel matrix components and fibrous protein components, among which the gel matrix includes glycosaminoglycans (GAGs) and proteoglycans (PGs). Fibrous proteins mainly include collagen (COL) and elastin (ELP) that play a structural support role, as well as fibronectin (FN) and laminin (LN) that play an adhesive role. Changes in the composition and structure of ECM affect the overall structure and biomechanical properties of the body. More and more studies on ECM regulating cell adhesion, driving cell polarization, enhancing cell migration, regulating cell proliferation and guiding cell differentiation have been found. How to finely regulate these physical parameters of ECM has become one of the current research hotspots.

[0019] Collagen is a protein family with a unique triple helix structure in the extracellular matrix and is the most abundant structural and functional protein in the human body. It includes 19 types, and the structural characteristics of each type of collagen are adapted to its specific functions. Among them, type I collagen belongs to the most abundant fibrotic collagen and exists in various connective tissues. Due to its excellent biocompatibility, biodegradability, bioabsorbability and other properties, collagen is widely used in the fields of tissue engineering and regenerative medicine.

[0020] Fibronectin was first discovered by Morrison in 1948. Due to its property of precipitating when cooled, fibronectin was initially called Cold insoluble globulin and is now generally known as fibronectin, with the English abbreviation FN. There are more than 20 types in the human body, widely present in blood, body fluids and various tissues. Fibronectin is an important component commonly present in the extracellular matrix. It can serve as a medium for the interaction between various cells and the extracellular matrix, playing a huge role in cell adhesion, migration, growth and differentiation. Fibronectin participates in the composition of the extracellular matrix and maintains its stability. It is necessary for the deposition of type I collagen and other extracellular matrix proteins.

[0021] Laminin (laminin LN) is a large glycoprotein in the form of a trimer (500 - 800KDa). It is the main component in the basement membrane (BM) and is the earliest extracellular matrix component to appear during embryonic development. It has various functions such as enhancing cell adhesion, migration, neurite growth, and angiogenesis. The laminin molecule is composed of an α chain, a β chain, and a γ chain. The three chains are combined with each other by forming a helical coiled coil to form a heterotrimer with three short arms and a long rod-shaped structure arm. Since the discovery of laminin, 5 types of α chains (α1 - α5), 3 types of β chains (β1 - β3), and 3 types of γ chains (γ1 - γ3) have been isolated. Like many other extracellular matrix proteins, laminin is composed of many independent structural domains and independent functional domains. The main domains include domain I, domain II, domain III (domain IIIa, IIIb), domain IV (IVa, IVb), domain V, domain VI, domain α, and domain G, etc.

[0022] Elastin is an important extracellular matrix protein in animals. Its content in the body is second only to collagen, playing an irreplaceable role in tissue stretching movement, anti-extrinsic deformation, etc. The biggest feature is to endow tissues with elasticity. The elastin gene in the human body has a homology of nearly 80% compared with other mammals, and there is no significant difference in amino acid composition. It is encoded by 36 exons, and exon 24 is the typical hydrophobic functional domain of elastin. Elastin is composed of a unit in which five amino acids (VPGXG) appear repeatedly, where X is any amino acid other than proline. Elastin has a wide range of applications in tissue engineering, such as cartilage and intervertebral disc tissue engineering, eye tissue engineering, liver tissue engineering, etc. However, natural extraction of elastin often faces problems such as zoonosis risk, insufficient source supply, complex purification process, and low final yield. Using genetic engineering technology to artificially design and synthesize elastin-like proteins with similar characteristics to the amino acid sequence of natural elastin is expected to solve the problems existing in the natural extraction of elastin.

[0023] There is also a class of bioactive polypeptides that can rapidly penetrate into mammalian cells and still retain their original structure and function. However, they do not rely on endocytosis to enter cells. Such polypeptides are called cell-penetrating peptides, which can not only penetrate cells by themselves but also load other substances and promote the cellular penetration of these substances, such as proteins, DNA, siRNA, liposomes, and nanomaterials. Chen Yongping et al. first applied in vivo phage display technology to the field of transdermal enhancer development and discovered a short peptide that can promote protein transdermal penetration. This short peptide contains 11 amino acids and is named "TD1", and its amino acid sequence is ACSSSPSKHCG. This short peptide can also help other various proteins or hormones penetrate through the skin.

[0024] Currently, most protein-based cosmetics on the market use single-component protein raw materials, such as extracted collagen, elastin, fibronectin, etc., and very few use extracellular matrix protein fusion products. Moreover, the efficacy of products using single protein components is relatively single, far less comprehensive than that of fusion-expressed protein products. However, for fusion-expressed proteins, due to their large molecular weight, there are also problems such as being difficult to be absorbed by tissues and cells, resulting in limited scope of use.

[0025] To solve the above technical problems, the present application provides an extracellular matrix fusion protein, its preparation method and application. The human laminin gene, humanized type I collagen gene, fibronectin gene, and elastin gene are sequentially connected by a flexible Linker. To promote the absorption of the fusion protein expression product, a transdermal short peptide "TD1" is introduced at the N-terminus of the human laminin gene, and then cloned into the pPIC9K expression vector and transformed into Pichia pastoris cells. The in vitro biological activity results of this extracellular matrix fusion protein show that this fusion protein has good effects on promoting cell adhesion, cell proliferation, cell migration, and has a certain effect on promoting collagen secretion. Each of these effects is superior to single humanized type I collagen, fibronectin, and elastin.

[0026] Specifically, the embodiment of the present application provides an extracellular matrix fusion protein, and the nucleotide sequence of the extracellular matrix fusion protein is as shown in SEQ ID No.7.

[0027] Among them, the amino acid sequence of the extracellular matrix fusion protein includes a transdermal short peptide sequence, a laminin amino acid sequence, a humanized type I collagen amino acid sequence, a fibronectin amino acid sequence, and an elastin amino acid sequence sequentially connected based on a flexible linker (GGGGS) 2 as shown in SEQ ID No.6.

[0028] Among them, the transdermal short peptide sequence is as shown in SEQ ID No.1; The amino acid sequence of laminin is derived from the LG4 domain of LAMA5 with UniProt ID PO15230, as shown in SEQ ID No. 2; The amino acid sequence of humanized type I collagen is derived from a truncated fragment of the domain of COL1A1 with UniProt ID P02452, as shown in SEQ ID No. 3; The amino acid sequence of fibronectin is derived from the Fibronectin type-III 11 domain fragment of FN1 with UniProt ID P02751, as shown in SEQ ID No. 4; The amino acid sequence of elastin is derived from one repeat fragment of [VPGKG(VPGVG)8VPGFG] in the hydrophobic domain, as shown in SEQ ID No. 5.

[0029] For the convenience of those skilled in the art to understand, the embodiments of the present application also provide: a method for preparing the extracellular matrix fusion protein as described above, comprising the following steps: Step 1: Insert the synthesized gene fragment into the pPIC9K plasmid through the restriction enzyme sites of EcoRI and NotI for the nucleotide sequence of the extracellular matrix fusion protein as shown in SEQ ID No. 7 to obtain the pPIC9K-TD-LCFE recombinant plasmid.

[0030] Step 2: After linearizing the pPIC9K-TD-LCFE recombinant plasmid, electrotransform it into GS115 competent cells for culture until multiple transformant single colonies appear.

[0031] More specifically, the step of linearizing the pPIC9K-TD-LCFE recombinant plasmid includes: using Quick Cut Sac I to perform linearizing restriction enzyme digestion on the pPIC9K-TD-LCFE recombinant plasmid, and the restriction enzyme digestion conditions are 35°C to 40°C for 3h to 7h; the system for the restriction enzyme digestion includes: 10 μg to 15 μg of the pPIC9K-TD-LCFE recombinant plasmid; 8 μL to 12 μL of QuickCut Sac I; 3 μL to 7 μL of 10× Quick Cut buffer.

[0032] More specifically, the step of electrotransforming into GS115 competent cells and culturing them until multiple transformant single colonies appear includes: mixing GS115 competent cells with the linearized recombinant plasmid in a volume ratio of 10:1, transferring them into a pre-cooled electroporation cuvette for ice bath treatment; adjusting the electroporator to the yeast mode for electroporation treatment, adding pre-cooled sorbitol to the electroporation cuvette, mixing evenly, and transferring the mixture to a sterile EP tube for culturing to obtain a bacterial solution; spreading the bacterial solution on an MD medium for culturing until multiple transformant single colonies appear.

[0033] Step 3: Screen based on the multiple transformant single colonies to obtain positive transformant single colonies.

[0034] More specifically, the step of screening based on the multiple transformant single colonies to obtain positive transformant single colonies includes: picking the transformant single colonies, transferring them to a 96-well culture plate containing YPD medium for culturing to obtain a first bacterial solution; aspirating the first bacterial solution in each well to a new 96-well culture plate containing YPD medium for continuous culturing to obtain a second bacterial solution; aspirating the second bacterial solution in each well to a new 96-well culture plate containing YPD medium for continuous culturing to obtain a third bacterial solution; aspirating the third bacterial solution in each well and spotting them onto YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418 for continuous culturing; screening the transformant single colonies corresponding to the third bacterial solution that can grow on YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418 simultaneously as positive transformant single colonies.

[0035] Step 4: Culture and induce the positive transformant single colonies to obtain an engineered bacteria fermentation broth.

[0036] More specifically, the step of culturing and inducing the positive transformant single colonies to obtain an engineered bacteria fermentation broth includes: picking single colonies and inoculating them into BMGY yeast growth medium for culturing until the measured OD 600 value is 2 - 6; adjusting the volume of BMGY yeast growth medium according to the measured OD 600 value, centrifuging to collect the bacterial cells, resuspending the bacterial cells with BMMY yeast induction medium with the same volume as BMGY yeast growth medium to make the starting OD 600 value 2.0; continuing the culture, adding 0.5% methanol to the medium every 24 h, taking bacterial solution samples every 24 h after induction, centrifuging to collect the expression supernatant to obtain an engineered bacteria fermentation broth.

[0037] Step 5: Purify the engineered bacteria fermentation broth to obtain an extracellular matrix fusion protein.

[0038] More specifically, the step of purifying the engineered bacteria fermentation broth to obtain the extracellular matrix fusion protein includes: separating the engineered bacteria fermentation broth into a fermentation supernatant and bacterial cells using a solid-liquid separation system; using a cation exchange medium, equilibrating the chromatography column with a phosphate buffer until the conductivity value and A280 absorbance value remain unchanged, setting the sample loading flow rate at 20 cm / h, detecting the ultraviolet A280 absorbance value, and starting to load the sample when it rises; after the sample loading is completed, equilibrating the cation chromatography medium with a phosphate buffer until the ultraviolet and conductivity drop to the lowest and no longer change, and stopping the sample loading; then eluting and collecting the corresponding protein with a NaH 2 PO 4 -NaCl (0.5 M) buffer, and after ultrafiltration, buffer exchange, and concentration, obtaining the recombinant TD-LCFE protein stock solution.

[0039] For the convenience of understanding by those skilled in the art, the following will further elaborate on the technical solutions of the present application in combination with specific embodiments: Example 1 Step 1: The transdermal short peptide sequence described in the present application is cited (Wang Shanshan, Research on the Function of the Transdermal Enhancement Peptide TD1 and the Epidermal Growth Factor Fusion Protein, University of Science and Technology of China, 2011) (11 aa, SEQ ID No.1). The amino acid sequence of laminin is derived from the LG4 domain (175 aa, SEQ ID No.2) of LAMA5 (UniProt: PO15230). The amino acid sequence of humanized type I collagen is derived from the truncated fragment (150 aa, SEQ ID No.3) of the domain of COL1A1 (UniProt: P02452). The amino acid sequence of fibronectin is derived from the Fibronectin type-III 11 domain fragment (94 aa, SEQID No.4) of FN1 (UniProt: P02751). The amino acid sequence of elastin selects [VPGKG(VPGVG) 8 VPGFG] 1 one repeat (100 aa, SEQ ID No.5) of the hydrophobic domain, and connects the polypeptide fragments of different proteins through a flexible linker (GGGGS) 2 to construct the recombinant protein TD-1-LN-Col-FN-ELA amino acid sequence (560 aa, SEQ ID No.6), named TD-LCFE, with a predicted molecular weight of approximately 54.03 kDa.

[0040] The DNA sequence encoding the above protein is optimized and designed according to the preference of Pichia pastoris to obtain the recombinant protein with the nucleotide shown in SEQ IDNo.7, so that the recombinant protein is more suitable for expression in Pichia pastoris.

[0041] Among them, SEQ ID No.1 - SEQ ID No.7 are as follows: SEQ ID No.1: ACSSSPSKHCG。

[0042] SEQ ID No.2: TDGSYLDGTGFARISFDSQISTTKRFEQELRLVSYSGVLFFLKQQSQFLCLAVQEGSLVLLYDFGAGLKKAVPLQPPPPLTSASKAIQVFLLGGSRKRVLVRVERATVYSVEQDNDLELADAYYLGGVPPDQLPPSLRRLFPTGGSVRGCVKGIKALGKYVDLKRLNTTGVSAGC。

[0043] SEQ ID No.3: GPMGPSGPRGLPGPPGAPGPQGFQGPPGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGAT。

[0044] SEQ ID No.4: VPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEID。

[0045] SEQ ID No.5: VPGKGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGFGVPGKGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGFG。

[0046] SEQ ID No.6: ACSSSPSKHCGTDGSYLDGTGFARISFDSQISTTKRFEQELRLVSYSGVLFFLKQQSQFLCLAVQEGSLVLLYDFGAGLKKAVPLQPPPPLTSASKAIQVFLLGGSRKRVLVRVERATVYSVEQDNDLELADAYYLGGVPPDQLPPSLRRLFPTGGSVRGCVKGIKALGKYVDLKRLNTTGVSAGCGGGGSGGGGSGPMGPSGPRGLPGPPGAPGPQGFQGPPGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGATGGGGSGGGGSVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDGGGGSGGGGSVPGKGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGFGVPGKGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGFG。

[0047] SEQ ID No.7:

[0048] The optimized base sequence of the recombinant protein TD-1-LN-Col-FN-ELA shown in SEQ ID No.7 was entrusted to GenScript Biotech Co., Ltd. to synthesize the gene fragment. The synthesized gene fragment was inserted into the pPIC9K plasmid through the restriction sites of EcoRI and NotI to obtain Figure 1 The pPIC9K-TD-LCFE recombinant plasmid is shown.

[0049] Step 2: 2-1) Acquisition of recombinant plasmid: The optimized base sequence shown in SEQ ID No.7 was entrusted to GenScript Biotech Co., Ltd. for gene fragment synthesis. After sequencing verification, the corresponding plasmid and bacterial strain were provided. After expanded culture, high-concentration pPIC9K-TD-LCFE plasmid was extracted for use.

[0050] 2-2) Linearization of pPIC9K-TD-LCFE plasmid: The pPIC9K-TD-LCFE plasmid extracted above was linearized using Quick Cut Sac I. The enzyme digestion conditions were 37°C for 5 h. The enzyme digestion system is shown in Table 1: Table 1:

[0051] After electrophoresis verification, add 0.1 times the volume of 3 M NaAc (pH 5.2) and 2.5 times the volume of anhydrous ethanol, and place at -20°C overnight. Centrifuge at 4°C, 13000rpm for 20min, discard the supernatant; add 700μL 75% ethanol for rinsing, centrifuge at 13000rpm for 20min, discard the supernatant, repeat; invert the EP tube on the clean bench absorbent paper for about 10min to remove as much water and residual ethanol as possible, and 20μL ddH 2 Redissolve the plasmid in 5% CO, take 1 μL and dilute it 10 times, and use one-drop to detect the nucleic acid concentration.

[0052] 2-3) Preparation of GS115 competent cells After streaking the GS115 strain on the plate, a single colony was picked and inoculated into 20 mL YPD liquid medium 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 600The value is 1.3 - 1.5; Transfer the bacterial solution into a sterile 50 mL centrifuge tube, centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant and collect the bacterial cells; Resuspend the bacterial cell pellet with 50 mL of pre-cooled sterile ultrapure water, centrifuge at 4°C and 3000 rpm for 5 min; After discarding the supernatant, resuspend the bacterial cell pellet with 50 mL of pre-cooled sterile ultrapure water; Centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant, and resuspend the cell pellet with 40 mL of pre-cooled sterile 1 M sorbitol; Centrifuge at 4°C and 3000 rpm for 5 min, discard the supernatant, and resuspend the cell pellet with 100 μL - 150 μL of pre-cooled sterile 1 M sorbitol, gently rotate and mix evenly, place on ice for later use.

[0053] 2 - 4) Electrotransform into GS115 competent cells Take 100 μL of GS115 competent cells and mix with 10 μL of linearized pPIC9K - TD - LCFE plasmid, transfer into a pre-cooled electroporation cuvette, and immediately ice-bath for 5 min. Select the yeast mode of the electroporator and perform electroporation. Then immediately add 1 mL of pre-cooled 1 M sorbitol to the electroporation cuvette, mix evenly and transfer the mixture to a sterile EP tube, incubate statically in a 30°C incubator for 1 h - 2 h. Take 100 μL - 200 μL of the bacterial solution and spread it on the MD medium, let it stand at room temperature for 10 min, and incubate it upside down in a 30°C incubator for about 2 d - 5 d until single colonies appear.

[0054] Step 3: Pick single colonies from the YPD plate, transfer them into a 96-well culture plate containing 200 μL of YPD medium (containing 0.5 mg / mL G418), and continue to culture at 30°C. After 48 h, blow the bacterial solution evenly, aspirate 10 μL from each well and transfer it to a new 96-well plate (containing 190 μL of YPD), repeat the above steps after continuing to culture for 24 h. After 24 h, blow the bacterial solution in the third 96-well plate evenly, and aspirate 1 μL and spot it on the YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418 respectively and continue to culture. If the transformants can grow on the YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418 at the same time, it indicates that the transformants contain multiple copies of the target gene, that is, positive transformant single colonies.

[0055] To verify whether the above positive transformant single colonies are correct, this application also designs the following steps to verify them: Take 50 μL of the remaining bacterial solution in the first 96-well plate in the above steps, boil it in a boiling water bath for 10 min, freeze it in liquid nitrogen for 30 min, boil it in a boiling water bath for 10 min, repeat freeze-thawing, centrifuge at 12000 rpm for 5 min, take the supernatant as the PCR template, and identify whether the target gene is integrated into the yeast chromosome by PCR amplification.

[0056] Upstream specific primer: 5’-TTACGTAGAATTCGCATGTTC-3’ Downstream specific primer: 5’-ATTAATTCGCGGCCGCACCAAA-3’ PCR conditions: Pre-denaturation at 98°C for 5 min, hot denaturation at 98°C for 50 s, annealing at 60°C for 30 s, extension at 72°C for 30 s, 35 cycles; final extension at 72°C for 10 min.

[0057] Perform 1.0% agarose gel electrophoresis on the amplified product to identify whether the gene fragment of the expected size is amplified; the identification result diagram is as Figure 2 shown (about 1700 bp).

[0058] Step 4: 4-1) Expression of recombinant TD-LCFE protein Pick a single colony of the positive transformant verified in Step 3 and inoculate it into BMGY yeast growth medium, culture at 30 ºC, 220 rpm for 24 h until the OD 600 value is 2 - 6. Adjust the volume of BMGY yeast growth medium according to the measured OD 600 value, collect the cells at 3000 rpm for 10 min, resuspend the cells with an equal volume of BMMY yeast induction medium as the BMGY yeast growth medium to make the initial OD 600 value 2.0. Continue to culture at 30 ºC, 220 rpm, add 0.5% methanol to the medium every 24 h, take the bacterial liquid samples at 24 h, 48 h, 72 h, and 96 h after induction respectively, centrifuge to collect the expressed supernatant as the fermentation broth of the engineered bacteria for standby. As Figure 3 shown, analyze the molecular weight and expression level of the target protein by SDS-PAGE electrophoresis.

[0059] 4-2) Medium-scale fermentation of yeast engineered bacteria Seed liquid preparation: Select the Pichia pastoris engineered bacteria (GS115 / pPIC9K / TD-LCFE) and inoculate it into 50 mL of YPD liquid medium containing 0.25 mg / mL geneticin (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L), culture at 30°C, 220 r / min with shaking for 18 - 20 h to obtain the primary seed liquid; inoculate the primary seed liquid into 200 mL of YPD liquid medium at an inoculation amount of 5%, and culture at 30°C, 220 r / min with shaking for 18 - 20 h to obtain the secondary seed liquid.

[0060] Preparation before inoculation: Transfer the prepared BSM medium into a 5L glass fermenter (Shanghai Bailun Biotech Co., Ltd.). Place the fermenter in a high-pressure steam sterilizer and sterilize it at 121°C for 15 min. After sterilization, wait until it cools down to below 30°C, and then add 2 mL of antifoaming agent and 8.7 mL of PTM1 solution under the protection of a flame circle.

[0061] Inoculate yeast: Inoculate the secondary seed liquid into the above-mentioned 5L glass fermenter with a liquid volume of 2L. The culture temperature is 30°C, the stirring speed is 800 r / min, and 50% (w / w) ammonia water is automatically added to maintain the pH of the medium at about 5.0. Air or pure oxygen is introduced to control the dissolved oxygen (DO) value above 50%.

[0062] At the 18th - 20th hour of cultivation, the glycerol in the BSM medium is exhausted, and the DO value suddenly increases, indicating that the glycerol in the medium has been depleted by the cells. At this time, start to supplement about 400 mL of feeding medium (50% W / V glycerol, 12 mL of PTM 1 trace elements per liter). When the wet weight of the cell thallus reaches 180 g / L - 220 g / L, stop supplementing glycerol and perform starvation treatment for about 1 - 2 h. Wait until the DO in the fermenter rises to the maximum value again, and then start to supplement the induction medium (100% methanol, 12 mL of PTM 1 trace elements per liter) to induce the expression of recombinant human thioredoxin by the Pichia pastoris engineering bacteria. Regularly adjust the feeding rate to maintain the DO fluctuating periodically within the range of 20% - 50%. At 24h, 48h, 72h, 96h, and 120h of induction respectively, centrifuge the supernatant, dilute the supernatant 5 times, load 10 μL, and perform SDS-PAGE and concentration tests.

[0063] Step 5: Centrifuge and collect the engineered bacteria fermentation broth obtained in Step 4, and use a solid-liquid separation system to separate the fermentation supernatant and the thallus. Use a cation exchange medium (the chromatography packing is SP Purose 6 High Performance produced by Qianchun, loaded on a GE Akta chromatography system), and equilibrate the chromatography column with phosphate buffer (20 mM NaH 2 PO 4 , pH 5.0) until the conductivity value and the A280 absorbance value remain unchanged. Set the sample loading flow rate at 20 cm / h, detect the ultraviolet A280 absorbance value, and start sampling when it rises. After the sample loading is completed, equilibrate the cation exchange medium with phosphate buffer again until the ultraviolet and conductivity drop to the lowest and no longer change, and then stop sampling. Then use the solution containing NaH 2 PO 4-Elute and collect the corresponding protein with NaCl (0.5M) buffer, and obtain the recombinant TD-LCFE protein stock solution after ultrafiltration, buffer exchange, and concentration. Detect the molecular weight and purity by SDS-PAGE, and the results are as Figure 4 shown.

[0064] It should be noted that the media and their formulations involved in the examples are as follows: YPD liquid medium: 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose (if it is a solid medium, add 2% agar); MD medium: 13.4 g / L YNB without amino acid nitrogen source; 0.4 mg / L biotin; 20 g / L glucose (solid medium contains 2% agar); BMGY yeast growth medium: 10 g / L yeast extract, 20 g / L peptone, 3 g / L K 2 HPO 4 4, 11.8 g / L K 2 HPO 4 4, add water to 890 mL, sterilize at 121 °C for 20 minutes, and then add 100 mL of 10×YNB (13.4 g / L), 1 mL of 500×biotin (4×10 -4 g / L), and 10 mL of glycerol on the laminar flow bench after the temperature drops to 60 °C.

[0065] BMMY yeast induction medium: 10 g / L yeast extract, 20 g / L peptone, 3 g / L K 2 HPO 4 4, 11.8 g / L K 2 HPO 4 4, add water to 895 mL, sterilize at 121 °C for 20 minutes, and then add 100 mL of 100×YNB (13.4 g / L), 1 mL of 500×biotin (4×10 -4 g / L), and 5 mL of methanol on the laminar flow bench after the temperature drops to 60 °C.

[0066] Fermentation medium: 85% H 3 PO 4 4 26.7 mL / L; CaSO 4 4·2H 2 2O 1.175 g / L; K 2 2SO 4 4 18.2 g / L; MgSO 4 4·7H 2 2O 14.9 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM 14.35 mL / L; Feed medium: 50% W / V glycerol, add 12 mL of PTM1 trace elements per liter; Induction medium: 100% methanol, add 12 mL of PTM1 trace elements per liter; among which PTM1 trace elements are added, and after the fermentation medium is autoclaved, PTM1 is added when the temperature drops to room temperature, and the pH value is adjusted to 5.0 with ammonia water. The PTM1 solution refers to the operation manual of Invitrogen Corporation, and the specific formula is: CuSO 4 ·5H 2 O 6.0 g / L; NaI 0.08 g / L; MnSO 4 ·H 2 O 3.0 g / L; NaMoO 4 ·2H 2 O 0.2 g / L; H 3 BO 3 0.02 g / L; CoCl 2 0.5 g / L; ZnCl 2 2 0.0 g / L; FeSO 4 ·7H 2 O 65.0 g / L; biotin 0.2 g / L; H 2 SO 4 5.0 mL / L, filter and sterilize with a 0.22 μm filter membrane, and store at 4°C.

[0067] To verify the efficacy of all aspects of the recombinant TD-LCFE protein stock solution obtained in Example 1, the following experimental examples are specifically set: Experimental Example 1 Effect Test of TD-LCFE Fusion Protein on Promoting Cell Proliferation The fusion protein prepared in this application contains collagen. Therefore, according to the requirements of "YY / T 1849-2022 Recombinant Collagen", the in vitro biological activity detection of the fusion protein prepared in this application is detected by promoting cell proliferation.

[0068] 1. Experimental method 1.1 Experimental materials Test article: TD-LCFE, prepared in Example 1, batch number 20241008.

[0069] Control article: Recombinant type III collagen (rhCOLⅢ), prepared according to application number CN202411045813.2, batch number 20240807.

[0070] Standard article: EGF, titer 500,000 U / mg, purchased from sigma.

[0071] Cell line: Mouse embryonic fibroblasts (3T3), passage 8, cryopreservation batch number 20221215, purchased from ATCC, USA.

[0072] Other reagents (complete medium HyClone / AJ30742864, PBS, 0.25% trypsin) and other items (96-well cell culture plates, TIP heads and micropipettes) are routine in the laboratory. They are sterilized or filter-sterilized before the experiment, and the sterility test is qualified.

[0073] 2. Experimental procedures 2.1 Cell culture and subculture The cells were cultured normally in a medium containing 10% calf serum. When the cell confluence reached over 90%, the medium was removed, and the cells were washed twice with PBS. Then, 0.25% trypsin was added for digestion. When the cells shrank and became round, the trypsin was poured off, and a medium containing 10% calf serum was added and gently pipetted to collect the cells, which were then centrifuged and counted. The cell concentration was adjusted to an appropriate concentration and inoculated into 1 mL of medium containing 1.0×10 5 ~5.0×10 5 cells. When the confluence reached over 90% again, the next subculture was carried out.

[0074] 2.2 Cell seeding Cells at 24 h - 36 h after subculture were selected for determination. The culture medium in the culture flask was discarded, and the cells were digested and collected. They were resuspended in PRIM1640 medium containing 10% fetal bovine serum to prepare a cell suspension with 5.0×10 4 ~8.0×10 4 cells per 1 mL. The cell suspension was inoculated into 96-well cell culture plates, 100 μL per well, and cultured at 37°C and 5% carbon dioxide.

[0075] 2.3 Cell synchronization After 24 h, the medium was changed to maintenance medium (PRIM1640 medium containing 2% fetal bovine serum), and the cells were continued to be cultured at 37°C and 5% carbon dioxide for another 24 h.

[0076] 2.4 Drug administration After 24 h, the sample was serially diluted 4-fold with maintenance medium, and 100 μL of the diluted sample was added to each well of the cells. At the same time, a blank control group with 2 replicates per group was set up, and the cells were continued to be cultured.

[0077] 2.5 MTT assay After the cells were treated with drugs and continued to be cultured for 72 h, 20 μL of MTT solution (0.5 mg / mL) was added to each well. The cells were incubated at 37°C in 5% CO 2 , and after 4 h of incubation, the supernatant was discarded. DMSO was added for lysis and shaking, and the absorbance was measured at 570 nm.

[0078] 2.6 Data processing The test data is processed using a computer program or a four-parameter regression calculation method.

[0079] 3. Experimental results The test results are shown in Table 2 below: Table 2 Detection results of the cell proliferation-promoting activity of TD-LCFE fusion protein

[0080] 4. Conclusion From the results in Table 2, the cell proliferation-promoting activity of the fusion protein TD-LCFE is at the same order of magnitude as that of the control recombinant type III collagen with a patent, and the cell proliferation-promoting activity reaches 1000 U / mg.

[0081] Experimental Example 2 Cell scratch test of TD-LCFE fusion protein 1. Experimental principle When the cells grow to a confluent monolayer state, an artificial blank area is created on the confluent monolayer cells, called a "scratch". The cells at the scratch edge will gradually enter the blank area to heal the "scratch". Images are captured at the beginning and regularly during the cell migration process, and the cell migration rate is determined by comparing the images.

[0082] 2. Experimental materials Test sample: TD-LCFE, prepared in Example 1, batch number 20241008.

[0083] Control: Recombinant elastin, prepared according to Application No. CN202411012422.0, batch number 20240824.

[0084] Cell line: Balb / c 3T3 cells, purchased from ATCC, USA.

[0085] Other reagents (cell nutrient solution, serum-free medium, PBS, 0.25% trypsin) and other items (96-well cell culture plates, TIP heads and micropipettes) are routine in the laboratory and are sterilized or filter-sterilized before the test, and the sterility test is qualified.

[0086] 3. Experimental steps (1) First, use a marker pen to draw horizontal lines on the back of the 6-well plate, align with a ruler, and draw evenly, about one line every 0.5 cm to 1 cm, crossing the wells horizontally.

[0087] (2) Add 2 mL of a cell suspension with a concentration of 5×10 5 cells / mL to the wells of the 6-well plate.

[0088] (3) On the second day, it was observed that the cells in the 6-well plate had all grown into a monolayer. Using a pipette tip against a ruler, make two scratches perpendicular to the horizontal line on the back in each well.

[0089] (4) Wash the cells 3 times with PBS to wash away the detached suspended cells.

[0090] (5) According to the grouping, add 1.8 mL of serum-free culture medium into the wells, and then add 200 μL of the sample. Add an equal volume of PBS solution to the cell control wells.

[0091] (6) Place it in an incubator at 37 °C and 5% CO 2 incubate. Take pictures at 0 h and record the positions for taking pictures in each well. Observe and take pictures at the fixed positions during subsequent observations.

[0092] (7) Data processing: Area detection method (scratch distance measurement is equivalent measurement) Average scratch width = scratch gap area / length Cell migration rate = (scratch width at 0 h - scratch width at 24 h) / scratch width at 0 h × 100% 4. Experimental results The observation results of the cell control wells and each sample well at 0 h and 24 h are as Figure 5 shown in Table 3.

[0093] Table 3 Results of TD-LCFE fusion protein promoting 3T3 cell migration

[0094] 5. Conclusion According to the results of calculating the in vitro cell migration rate by the formula, it can be seen that the TD-LCFE fusion protein has an obvious effect on promoting cell migration.

[0095] Experimental Example 3 Effect test of TD-LCFE fusion protein on promoting cell adhesion 1. Experimental principle The adhesion and attachment of cells are necessary conditions for cell repair and cell growth completion. Fibronectin has a good effect on promoting cell adhesion. In this experiment, through the test of promoting cell adhesion (adhesion to the wall), it is detected whether the TD-LCFE fusion protein has the same adhesion-promoting activity as fibronectin.

[0096] 2. Test materials 2.1 Main instruments: Laminar flow hood, cell incubator.

[0097] 2.2 Reagent preparation Complete cell culture medium: Measure 10 mL of fetal bovine serum and 1 mL of double antibody, add 90 mL of DMEM culture medium, and store at 4 °C.

[0098] Serum-free medium: Measure 1 mL of double antibody, add it to 99 mL of 1640 culture medium, and store at 4°C.

[0099] Digestive solution: 0.25% trypsin.

[0100] PBS buffer: Weigh 8.0 g of sodium chloride, 0.20 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of potassium dihydrogen phosphate, dissolve in water and make up the volume to 1000 mL, and sterilize by autoclaving at 121°C for 15 minutes.

[0101] 2.3 Cells MDBK cells grow in a monolayer and adhere to the wall in complete cell culture medium, and are passaged every 4 to 5 days, digested and passaged at a ratio of 1:2, and grow and reproduce in complete cell culture medium.

[0102] 2.4 Test article: TD-LCFE, prepared in Example 1, batch number 20241008.

[0103] 2.5 Positive control: Recombinant fibronectin (rhFN), prepared according to application number CN202110357340.X, batch number 20240716.

[0104] 3. Test operation 3.1 Sample dilution and incubation Pre-dilute the TD-LCFE fusion protein with PBS to 0.5 μg / mL. After pre-dilution, perform 2-fold serial dilution in a 96-well plate, with a total of 10 dilution degrees. Add 50 μL of TD-LCFE fusion protein samples at different dilution degrees to each well, set up serial dilution positive control wells and negative control (add 50 μL of PBS as a control), and incubate overnight at 4°C.

[0105] 3.2 Cell adhesion promotion test After incubation, discard the liquid in the plate, add 100 μL of 30 g / L BSA to each well for blocking, and incubate in an incubator at 37°C for 1 h; take out and discard the liquid in the plate, add MDBK cell suspension (resuspended with serum-free medium), the cell seeding density is 1.0×10 5 cells / mL, add 100 μL to each well, and incubate in an incubator for 5 h.

[0106] 4. Experimental results Wash the cell plate after incubation 3 times with PBS, observe the cell adhesion under the microscope, and the results of the TD-LCFE fusion protein promoting cell adhesion are as Figure 6 shown.

[0107] The result calculation is shown in Table 4 - Table 5: Table 4 The number of five-point cells in each well of the well plate

[0108] Table 5 Statistical Results of the Potency of TD-LCFE Fusion Protein in Promoting Cell Adhesion

[0109] 5. Conclusion From the statistical results, both TD-LCFE fusion protein and recombinant fibronectin have the activity of promoting cell adhesion in vitro.

[0110] Experimental Example 4 Transdermal Absorption Test of TD-LCFE Fusion Protein 1. Test Principle The transdermal absorption test of the protein solution was carried out in an upright Franz diffusion cell. The diffusion cell was placed in a drug transdermal diffusion tester and incubated in a 37 °C constant temperature circulating water bath. The in vitro transdermal performance of TD-LCFE fusion protein was compared and evaluated.

[0111] 2. Test Method 2. Test Materials 2.1 Ex vivo skin: Ex vivo skin was prepared from 6-8 week-old BALB / c mice. 2.2 Main Test Reagents Test sample: TD-LCFE fusion protein produced in Example 1, batch number 20241008; Control: Recombinant type III collagen, prepared according to application number CN202411045813.2, batch number 20240807; Normal saline; type III collagen monoclonal antibody (purchased from abcam, UK).

[0112] 2.3 Experimental Instruments Honghua ZTY intelligent transdermal tester, maximum capacity of the receiving cell is 20 mL, effective transdermal area is 1.36 cm 2 .

[0113] 3. Test Procedures 3.1 In vitro Transdermal Test 3.1.1 Installation of Mouse Skin The transdermal absorption test was carried out in an upright Franz diffusion cell. The diffusion cell was placed in a drug transdermal diffusion tester. The processed ex vivo skin was fixed between the supply cell and the receiving cell, with the stratum corneum facing the supply cell and the dermal layer facing the receiving chamber. The temperature of the water bath system was adjusted to 37 °C, the stirring speed was 100 r / min, and pre-warmed normal saline at 37 °C was added to the receiving chamber to exhaust air bubbles. To reduce interference, without administration, the inner surface of the mouse skin was first brought into contact with the receiving solution, and then 5 mL of solutions with different concentrations were injected into the supply chamber and closely attached to the mouse skin.

[0114] 3.1.2 Sampling At 1 h, 2 h, 4 h, 6 h, 8 h, 12 h and 24 h after the start of the experiment, about 1 mL of the receiving solution was drawn with a syringe as the sample solution. At the same time, the receiving chamber was filled with an equal amount of normal saline solution. Finally, the sample solutions collected at each time period were tested. The working schematic diagram of the mouse skin-Franz cell diffusion cell is as Figure 7 shown.

[0115] 3.1.3 Experimental grouping A total of 3 groups were set up, namely the blank group (normal saline), the test article group, and the reference standard group. The samples were diluted to a concentration of 0.1 mg / mL with normal saline.

[0116] 4. Result detection 4.1 Protein content detection The protein content in the receiving solution samples taken at each time point was determined by the Coomassie brilliant blue method. The formula for calculating the cumulative permeation amount (Q) is as follows:

[0117] where C n represents the sample concentration at the nth sampling, C i represents the sample concentration at the ith sampling, V represents the volume of the receiving cell, V i represents the sampling volume, and S represents the transdermal area.

[0118] 4.2 Preparation and observation of immunohistochemical sections The transdermal parts of the mouse skin (including the comparison between the hair follicle part and the non-hair follicle part) after 24 h of the 5 groups of experiments were taken for fixation to prepare immunohistochemical sections. The prepared immunohistochemical sections were placed under a microscope to observe the location of the protein in each group of skin tissues.

[0119] 4.3 Preparation process of immunohistochemical sections (1) The sections were placed in an oven at 66 °C for baking for 20 min to 30 min.

[0120] (2) Pass through 3 baths of xylene in sequence, 5 min for each bath.

[0121] (3) Pass through 3 baths of ethanol (100%, 95% and 80%) in sequence, 3 min for each bath.

[0122] (4) The sections were placed in a beaker and rinsed slowly with running water to wash away the ethanol until the sections were clean and transparent.

[0123] (5) Antigen high-pressure repair: Prepare 2000 mL of citrate repair solution with a pH of 6.0 in a pressure cooker, heat it to boiling on an induction cooker, put the sections in, cover the lid of the pressure cooker, start timing for 2 min after the steam jets out, then stop heating, and rinse the lid of the pressure cooker slowly with running water until it cools down.

[0124] (6) Block endogenous peroxidase: Place the sections in 3% H 2 O 2 and incubate at room temperature for a certain time. Wash 3 times with distilled water, draw a hydrophobic circle, and rinse 3 times with PBS-T.

[0125] (7) Drain the excess liquid on the sections, add the primary antibody (monoclonal antibody against type III collagen), cover, and incubate in an incubator at 37°C for 60 min. Take out the sections and rinse 3 times with PBS-T.

[0126] (8) Drain the excess liquid on the sections, add the secondary antibody, cover, and incubate in an incubator at 37°C for 30 min. Take out the sections and rinse 3 times with PBS-T.

[0127] (9) Drain the excess liquid on the sections, add the DAB chromogenic agent, control the chromogenic time under the microscope, stop chromogenesis when positive, and rinse thoroughly with distilled water.

[0128] (10) Counterstain with hematoxylin for 2 min - 5 min, wash clean; differentiate with 1% hydrochloric acid alcohol for a few seconds, wash clean.

[0129] (11) Blue with lithium carbonate solution for 30 s, wash clean.

[0130] (12) Dehydrate routinely and clear with xylene.

[0131] (13) Mount with neutral balsam and observe the results under the microscope. Brown indicates a positive reaction.

[0132] 5. Experimental Results 5.1 Protein Concentrations of Samples at Each Time Period The cumulative permeation amount per unit area (Q) at each time point was calculated according to the formula, and the results are shown in Table 6: Table 6 Protein Concentrations of Samples and Cumulative Permeation Amount Q per Unit Area at Each Time Point

[0133] Note: (1) The unit of sample concentration is μg / mL; (2) The unit of Q value is μg / cm 2 Judging from the results in Table 6, the TD-LCFE fusion protein has transdermal performance, while recombinant type III collagen cannot penetrate the skin.

[0134] 5.2 Immunohistochemical Results Immunohistochemical detection was performed on the excised mouse skin after 24 h of the experiment. The results are as Figure 8 shown. It can be seen that: in the test article group, the TD-LCFE fusion protein penetrated through the skin into the dermis layer ( Figure 8At the red arrow in C), an obvious brown positive reaction was presented; while for the recombinant type III collagen in the control group, there was basically no protein penetration, and the positive reaction was outside the epidermal layer, with no obvious concentrated positive reaction in the dermal layer. Only scattered positive reactions were observed in the collagen structure of the skin itself ( Figure 8 B); Figure 8 There was also no obvious concentrated positive reaction in the blank group of A.

[0135] According to the above test results, it can be seen that the TD-LCFE fusion protein has an obvious transdermal absorption effect.

[0136] Experimental Example 5 TD-LCFE Fusion Protein Promoting Collagen Secretion Test 1. Test Principle The determination of collagen content adopts the staining method and the enzyme-linked immunosorbent assay (ELISA). The enzyme-linked immunosorbent assay can also show type I and type III collagen fibers, but the antibodies used are expensive and the operation is time-consuming. While the Sirius red staining method uses inexpensive reagents and can specifically bind to the Gly-X-Y helical structure of fibrous collagen (type I to type V). The operation is simple. Sirius red is a strong acidic anionic dye that can react with alkaline collagen. The collagen content is positively correlated with the depth of the staining color. The optical density (OD value) is measured at a wavelength of 450 nm with an enzyme-labeled instrument to calculate the collagen content. 2. Experimental Materials Test sample: TD-LCFE fusion protein produced in Example 1, batch number 20241008; Control: Recombinant type III collagen (rhCOLⅢ), prepared according to application number CN202411045813.2, batch number 20240807.

[0137] Control 2: Recombinant fibronectin (rhFN), prepared according to application number CN202110357340.X, batch number 20240716.

[0138] Cell line: Balb / c 3T3 cells, purchased from ATCC, USA.

[0139] Other reagents (RPMI 1640 medium, fetal bovine serum, trypsin solution, PBS, Sirius red staining solution, acetic acid, sodium hydroxide) and other items (96-well cell culture plates, TIP heads and micropipettes) are routine in the laboratory and are sterilized or filtered and sterilized before the test, and the sterility test is qualified.

[0140] 3. Experimental Procedures 3.1 Detection of Cell Collagen Content by Staining Method 3.1.1 Cell Culture and Subculture Cells were normally cultured in a medium containing 10% calf serum. When the cell confluence reached over 90%, the medium was removed, and the cells were washed twice with PBS. Then, 0.25% trypsin was added for digestion. When the cells shrank and became round, the trypsin was poured off, and a medium containing 10% calf serum was added and gently pipetted to collect the cells, which were then centrifuged, counted, and adjusted to an appropriate concentration and seeded into 1 mL containing 1.0×10 5 ~5.0×10 5 cells. When the confluence reached over 90% again, the next passage was carried out.

[0141] 3.1.2 Cell seeding Cells at 24 h - 36 h after passage were selected for determination. The culture medium in the culture flask was discarded, and the cells were digested and collected. They were made into a cell suspension containing 5.0×10 4 ~8.0×10 4 cells per 1 mL with complete culture medium and seeded into a 96 - well cell culture plate, 100 μL per well, and cultured at 37℃ and 5% carbon dioxide.

[0142] 3.1.3 Cell synchronization After 24 h, the medium was replaced with maintenance medium and continued to be cultured at 37℃ and 5% carbon dioxide for another 24 h.

[0143] 3.1.4 Drug administration After 24 h, the samples and positive controls were diluted with maintenance medium, and the diluted samples were added to the cells, 0.2 mL per well. At the same time, a cell control group was set up, and only 0.2 mL of maintenance medium was added to each well. There were 3 replicates in each group, and the culture was continued.

[0144] 3.1.5 Collection of cell supernatant After the incubation culture ended, 200 μL of cell culture supernatant was collected from each well into a 1.5 mL sterile centrifuge tube and stored frozen at -80℃ in an ultra - low temperature freezer.

[0145] 3.1.6 Fixation of cell cultures For adherent cells, 4% paraformaldehyde was added for fixation at room temperature for 20 minutes. The fixing solution was discarded, and 0.2 mL of PBS was added to each well for washing twice.

[0146] 3.1.7 Sirius red staining 0.15 mL of 0.1% Sirius red picric acid staining solution was added to each well and stained at room temperature for 1 h. The staining solution was aspirated, and 0.2 mL of 0.1% glacial acetic acid was added to each well and washed 3 times, 5 min each time.

[0147] 3.1.8 Detection Aspirate 0.1% glacial acetic acid, add 0.2 mL of 0.1 mol / L sodium hydroxide to each well, shake well at room temperature for 1 h, place it in an enzyme-labeled instrument to detect the absorbance A value at a wavelength of 540 nm, and detect the change in collagen.

[0148] 4. Experimental results The test results are as Figure 9 shown in Table 7: Table 7 Up-regulation rate of collagen content

[0149] The results show that TD-LCFE and recombinant type III collagen have obvious effects on promoting collagen secretion, and TD-LCFE has the best effect, with an up-regulation rate of collagen content reaching 35.7%.

[0150] 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 by using the content of the specification and drawings of the present application, or directly or indirectly applied in other related technical fields, shall be similarly included in the patent protection scope of the present application.

Claims

1. An extracellular matrix fusion protein, characterized in that: The nucleotide sequence of the extracellular matrix fusion protein is shown in SEQ ID No.

7.

2. The extracellular matrix fusion protein according to claim 1, characterized in that: The amino acid sequence of the extracellular matrix fusion protein includes a transdermal short peptide sequence, a laminin amino acid sequence, a humanized type I collagen amino acid sequence, a fibronectin amino acid sequence and an elastin amino acid sequence that are sequentially connected in series based on a flexible linker (GGGGS) 2, as shown in SEQ ID No.

6.

3. The extracellular matrix fusion protein according to claim 2, characterized in that: The transdermal short peptide sequence is shown in SEQ ID No. 1; The laminin amino acid sequence is derived from the LG4 domain of LAMA5 with UniProt PO15230, as shown in SEQ ID No. 2; The amino acid sequence of humanized type I collagen is derived from a fragment of the domain of COL1A1 with UniProt P02452, as shown in SEQ ID No. 3; The amino acid sequence of fibronectin is derived from the Fibronectin type-III 11 domain fragment of FN1 with UniProt P02751, as shown in SEQ ID No. 4; The amino acid sequence of elastin is derived from a repeating fragment of [VPGKG(VPGVG)8VPGFG]1 in the hydrophobic domain, as shown in SEQ ID No.

5.

4. A method for preparing the extracellular matrix fusion protein according to any one of claims 1 to 3, characterized in that: The following steps are involved: The nucleotide sequence of the extracellular matrix fusion protein shown in SEQ ID No. 7 was inserted into the pPIC9K plasmid through the EcoRI and NotI restriction sites to obtain the pPIC9K-TD-LCFE recombinant plasmid; The pPIC9K-TD-LCFE recombinant plasmid is linearized and then electroporated into GS115 competent cells for culture until multiple transformant colonies appear; Screening is performed based on the multiple transformant single colonies to obtain positive transformant single colonies; Cultivating and inducing the positive transformant single colony to obtain the engineered bacteria fermentation liquid; The fermentation liquid of the engineered bacteria is purified to obtain the extracellular matrix fusion protein.

5. The method for preparing the extracellular matrix fusion protein according to claim 4, characterized in that: The step of linearizing the pPIC9K-TD-LCFE recombinant plasmid comprises: Using Quick Cut Sac I linearize the pPIC9K-TD-LCFE recombinant plasmid under the conditions of 35°C to 40°C for 3h to 7h; the enzyme digestion system includes: 10μg~15μg pPIC9K-TD-LCFE recombinant plasmid; 8μL~12μL Quick Cut Sac I; 3μL~7μL 10× Quick Cut buffer.

6. The method for preparing the extracellular matrix fusion protein according to claim 4, characterized in that: The step of electrotransforming the cells into GS115 competent cells and culturing the cells until a plurality of transformant colonies appear comprises: The GS115 competent cells and the linearized recombinant plasmid were mixed in a volume ratio of 10:1, and transferred into a pre-cooled electroporation cup for ice bath treatment; the electroporator was adjusted to the yeast mode for electroporation, and pre-cooled sorbitol was added to the electroporation cup. After mixing evenly, the mixture was transferred to a sterile EP tube for culturing to obtain a bacterial solution; the bacterial solution was spread on an MD medium for culturing until a plurality of transformant single colonies appeared.

7. The method for preparing the extracellular matrix fusion protein according to claim 4, characterized in that: The step of screening the multiple transformant single colonies to obtain positive transformant single colonies comprises: Pick up a single colony of the transformant, transfer it to a 96-well culture plate containing YPD medium for culturing, and obtain a first bacterial solution; pipette the first bacterial solution from each well to a new 96-well culture plate containing YPD medium for continued culturing, and obtain a second bacterial solution; pipette the second bacterial solution from each well to a new 96-well culture plate containing YPD medium for continued culturing, and obtain a third bacterial solution; pipette the third bacterial solution from each well and spot it on YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418 for continued culturing; The transformant single colonies corresponding to the third bacterial solution that can grow on YPD plates containing 1 mg / mL, 2 mg / mL and 3 mg / mL G418 at the same time are screened as positive transformant single colonies.

8. The method for preparing the extracellular matrix fusion protein according to claim 4, characterized in that: The step of culturing and inducing a single colony of a positive transformant to obtain an engineered bacterial fermentation broth comprises: Pick a single colony and inoculate it into BMGY yeast growth medium and culture it until the measured OD 600 The value is 2~6; according to the measured OD 600 Adjust the volume of BMGY yeast growth medium to the value, collect the cells by centrifugation, and resuspend the cells with BMMY yeast induction medium of the same volume as BMGY yeast growth medium to make the starting OD 600 The value was 2.0; the culture was continued, 0.5% methanol was added to the culture medium every 24 hours, and bacterial samples were taken every 24 hours after induction, and the expression supernatant was collected by centrifugation to obtain the engineered bacterial fermentation broth.

9. The method for preparing the extracellular matrix fusion protein according to claim 4, characterized in that: The step of purifying the engineered bacteria fermentation broth to obtain the extracellular matrix fusion protein comprises: The engineered bacteria fermentation liquid is separated into fermentation supernatant and bacteria by using a solid-liquid separation system; Use 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 to 20cm / h, detect the ultraviolet A280 absorbance value, and start loading when it rises; After the sample loading is completed, the cationic chromatography medium is equilibrated with phosphate buffer until the UV and conductivity drop to the minimum and no longer change, and then the sample loading is stopped; Then, the corresponding protein was eluted with a buffer solution containing 0.5 M NaH2PO4-NaCl and collected. After ultrafiltration, liquid replacement and concentration, the recombinant TD-LCFE protein stock solution was obtained.

10. A use of the extracellular matrix fusion protein according to any one of claims 1 to 3, characterized in that: Used to prepare protein skin care products with anti-aging effects.

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