Extracellular matrix fusion protein and preparation method and application thereof

Abstract

The application discloses an extracellular matrix fusion protein and a preparation method and application thereof, relates to the technical field of recombinant proteins, and aims to solve the technical problem that the existing protein cosmetic products on the market have relatively single functions and are difficult to meet application requirements. The extracellular matrix fusion protein has a nucleotide sequence as shown in SEQ ID No. 7. The extracellular matrix fusion protein with the nucleotide sequence as shown in SEQ ID No. 7 can significantly promote cell adhesion and proliferation, promote cell migration, and act on the dermis layer through the skin, and significantly promote collagen secretion, and since the recombinant protein belongs to a biological protein, has the advantages of easy decomposition and no residue, and has a more broad application prospect compared with ordinary chemical cosmetic products and hormone drugs.

Description

TECHNICAL FIELD

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

[0002] At present, protein cosmetics on the market mostly use single protein raw materials such as extracted collagen, elastin and fibronectin, and a few use extracellular matrix protein fusion products. Moreover, products using single protein components have relatively single efficacy, which is far from the complete efficacy of fusion-expressed protein products. However, for fusion-expressed proteins, there are problems such as difficulty in being absorbed by tissues and cells due to their large molecular weight, which limits their use range. SUMMARY

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

[0004] In one aspect, to solve the above technical problem, the present application provides an extracellular matrix fusion protein, and the nucleotide sequence of the extracellular matrix fusion protein is 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 comprises, in sequence, 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 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 shown in SEQ ID No. 1.

[0007] The laminin amino acid sequence is derived from the LG4 domain of LAMA5 with UniProt PO15230, as shown in SEQ ID No. 2.

[0008] The humanized type I collagen amino acid sequence is derived from a truncated fragment of the domain of COL1A1 with UniProt P02452, as shown in SEQ ID No. 3.

[0009] The fibronectin amino acid sequence is derived from the Fibronectin type-III 11 domain fragment of FN1 with UniProt P02751, as shown in SEQ ID No. 4.

[0010] The amino acid sequence of elastin is derived from a repeating segment of [VPGKG(VPGVG)8VPGFG]1 in the hydrophobic domain, as shown in SEQ ID No.5.

[0011] On the other hand, the present invention also provides a method for preparing the extracellular matrix fusion protein, comprising the following steps:

[0012] 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;

[0013] After linearization, the pPIC9K-TD-LCFE recombinant plasmid was electroporated into GS115 competent cells and cultured until multiple transformant colonies appeared;

[0014] Screening is performed based on the multiple transformant single colonies to obtain positive transformant single colonies;

[0015] Cultivate and induce the positive transformant single colony to obtain the engineered bacterial fermentation liquid;

[0016] The fermentation liquid of the engineered bacteria is purified to obtain the extracellular matrix fusion protein.

[0017] As some optional embodiments of the present application, the step of linearizing the pPIC9K-TD-LCFE recombinant plasmid includes:

[0018] Using Quick Cut Sac I. Linearize the pPIC9K-TD-LCFE recombinant plasmid using enzyme digestion conditions at 35° C. to 40° C. for 3 h to 7 h. The enzyme digestion system includes:

[0019] 10 μg~15 μg pPIC9K-TD-LCFE recombinant plasmid;

[0020] 8μL~12μL Quick Cut Sac I;

[0021] 3μL~7μL 10× Quick Cut buffer.

[0022] As some optional embodiments of the present application, the step of electrotransforming into GS115 competent cells and culturing until a plurality of transformant single colonies appear includes:

[0023] GS115 competent cells and the linearized recombinant plasmid were mixed in a volume ratio of 10:1 and transferred to a pre-cooled electroporation cuvette for ice bath treatment; the electroporator was adjusted to the yeast mode for electroporation, and pre-cooled sorbitol was added to the electroporation cuvette. 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 MD medium and cultured until multiple transformant colonies appeared.

[0024] As some optional embodiments of the present application, the step of screening the multiple transformant single colonies to obtain positive transformant single colonies includes:

[0025] Pick a single colony of the transformant and transfer it to a 96-well culture plate containing YPD medium for cultivation to obtain a first bacterial liquid; pipette the first bacterial liquid from each well to a new 96-well culture plate containing YPD medium for continued cultivation to obtain a second bacterial liquid; pipette the second bacterial liquid from each well to a new 96-well culture plate containing YPD medium for continued cultivation to obtain a third bacterial liquid; pipette the third bacterial liquid from each well onto YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418 for continued cultivation;

[0026] 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 and taken as positive transformant single colonies.

[0027] As some optional embodiments of the present application, the step of culturing and inducing a single colony of a positive transformant to obtain an engineered bacterial fermentation broth includes:

[0028] 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 bacteria by centrifugation, and resuspend the bacteria 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, and 0.5% methanol was added to the culture medium every 24 hours. After induction, bacterial samples were taken every 24 hours, and the expression supernatant was collected by centrifugation to obtain the engineered bacterial fermentation broth.

[0029] As some optional embodiments of the present application, the step of purifying the fermentation broth of the engineered bacteria to obtain the extracellular matrix fusion protein includes:

[0030] Separating the engineered bacteria fermentation liquid into fermentation supernatant and bacteria bodies by using a solid-liquid separation system;

[0031] Use cation exchange medium and equilibrate the column with phosphate buffer until the conductivity and A280 absorbance remain unchanged. Set the sample loading flow rate to 20 cm / h and detect the UV A280 absorbance. When it rises, start loading the sample.

[0032] After the sample loading is completed, the cationic chromatography medium is equilibrated with phosphate buffer until the UV and conductivity drop to the lowest and no longer change, and then the sample loading is stopped;

[0033] Then, the corresponding protein was eluted with a buffer solution containing NaH2PO4-NaCl (0.5 M) and collected. After ultrafiltration, liquid replacement and concentration, the recombinant TD-LCFE protein stock solution was obtained.

[0034] On the other hand, an embodiment of the present application further provides: an application of the extracellular matrix fusion protein as described above, for preparing a protein skin care product with anti-aging efficacy.

[0035] Compared with the prior art, the extracellular matrix fusion protein described in the present application, such as the nucleotide sequence shown in SEQ ID No. 7, has been shown in in vitro cell experiments to significantly promote cell adhesion and proliferation as well as cell migration. However, considering the large molecular weight of this fusion protein, the transdermal peptide TD-1 is added to the N-terminus of the fusion protein to significantly enhance the transdermal efficacy of the fusion protein. The test results show that the fusion protein can act on the dermis through the skin. The results of the collagen secretion promotion test using the picrosirius red staining method show that this fusion protein can significantly promote collagen secretion. Moreover, since the recombinant protein is a biological protein, it has the advantages of being easily decomposed and leaving no residue. Compared with ordinary chemical cosmetics and hormone drugs, it has a broader application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] To more clearly illustrate the specific embodiments of this application or the technical solutions in the prior art, the following briefly describes the drawings required for the specific embodiments or the description of the prior art. Similar elements or parts are generally identified by similar reference numerals throughout the drawings. Elements or parts in the drawings are not necessarily drawn to scale.

[0037] Figure 1 This is the plasmid map of pPIC9K-TD-LCFE involved in the examples of this application;

[0038] Figure 2 This is the GS115 / pPIC9K / TD-LCFE PCR electrophoresis pattern involved in the examples of this application;

[0039] Figure 3 This is a graph showing the SDS-PAGE results of the GS115 / pPIC9K / TD-LCFE supernatant involved in the examples of this application;

[0040] Figure 4 SDS-PAGE result of supernatant of GS115 / pPIC9K / TD-LCFE purification involved in the embodiments of the present application;

[0041] Figure 5 Cell state of different treatment groups at 0h and 24h involved in the embodiments of the present application;

[0042] Figure 6 Result diagram of cell adhesion promotion by TD-LCFE fusion protein involved in the embodiments of the present application;

[0043] Figure 7 Working diagram of Franz cell diffusion pool for mouse skin-Franz cell involved in the embodiments of the present application; wherein, 1-place of raw material drug (transdermal area), 2-upper cover of receiving pool, 3-elastic clip, 4-skin or skin substitute, 5-receiving pool, 6-sampling point, 7-shark glass, 8-sampling needle, 9-sampling tube, 10-magnetic stirrer;

[0044] Figure 8 Immunohistochemical result of mouse skin of each group in transdermal test involved in the embodiments of the present application; wherein, Fig. A: result diagram of blank control treatment; Fig. B: result diagram of recombinant type III collagen treatment; Fig. C: result diagram of TD-LCFE fusion protein treatment;

[0045] Figure 9 Scharlach staining result involved in the embodiments of the present application;

[0046] The implementation, functional features and advantages of the present application will be further described with reference to the embodiments and the accompanying drawings. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0048] Extracellular matrix (ECM) is a complex network of proteins and polysaccharides secreted by cells and surrounding them in tissues, which can provide attachment sites and structural support for cells, and also provide important biological information for regulating cell activity and organ / tissue function. According to its composition and structure, it is divided into two types: gel matrix components and fibrous protein components, in which the gel matrix includes glycosaminoglycans (GAG) and proteoglycans (PG). The fibrous proteins mainly include collagen (COL) and elastin (ELP) which play a structural support role, and fibronectin (FN) and laminin (LN) which play a cementing role. The composition and structure of ECM affect the overall structure and biomechanical properties of the body. More and more studies have found that ECM can regulate cell adhesion, drive cell polarization, enhance cell migration, regulate cell proliferation, and guide cell differentiation. How to finely adjust the physical parameters of these ECMs has become one of the current research hotspots.

[0049] Collagen is a family of proteins 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 function. Type I collagen, which is the most abundant fibrous collagen, exists in various connective tissues. Collagen is widely used in the fields of tissue engineering and regenerative medicine due to its excellent biocompatibility, biodegradability, and bioabsorbability.

[0050] Fibronectin was first discovered by Morrison in 1948. Due to its property of precipitating when cold, it was initially called cold insoluble globulin and is now commonly known as fibronectin, with the English abbreviation FN. There are more than 20 types in the human body, widely existing in blood, body fluids, and various tissues. Fibronectin is an important component of the extracellular matrix and can act as a mediator for the interaction between various cells and the extracellular matrix, playing a significant role in cell adhesion, migration, growth, and differentiation. Fibronectin participates in the composition and maintenance of the extracellular matrix, and is essential for the deposition of type I collagen and other extracellular matrix proteins.

[0051] Laminin (LN) is a large molecular glycoprotein (500-800 KDa) in the form of a trimer, which is the main component of the basement membrane (BM) and the earliest extracellular matrix component in embryonic development. It has multiple functions such as enhancing cell adhesion, migration, neurite growth, and angiogenesis. The laminin molecule is composed of an alpha chain, a beta chain, and a gamma chain, which are combined by forming a spiral-shaped coiled coil to form a heterotrimer with three short arms and a rod-shaped long arm. Since the discovery of laminin, five alpha chains (α1-α5), three beta chains (β1-β3), and three gamma chains (γ1-γ3) have been isolated. Like many other extracellular matrix proteins, laminin is composed of many independent structural and functional domains, including domain I, domain II, domain III (domain IIIa, IIIb), domain IV (IVa, IVb), domain V, domain VI, alpha domain, and G domain.

[0052] Elastin is an important extracellular matrix protein in animals, and its content in the body is second only to collagen. It plays an irreplaceable role in tissue stretching and anti-external force deformation. The biggest feature is to give the tissue elasticity. The human elastin gene 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, of which exon 24 is the typical hydrophobic functional domain of elastin. Elastin is composed of a unit of five amino acids (VPGXG), where X is any amino acid except 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 the risk of zoonosis, insufficient supply, complex purification process, and low final yield. Using genetic engineering technology, artificially designed and synthesized elastin-like proteins with similar properties to natural elastin amino acid sequences can solve the problems of natural extraction of elastin.

[0053] There is another class of biological polypeptides that can quickly penetrate into mammalian cells and still retain their original structure and function, but they do not rely on endocytosis to enter cells. This class of polypeptides is called cell-penetrating peptides, which not only penetrate cells themselves, but also load other substances and promote the cell penetration of such substances, such as proteins, DNA, siRNA, liposomes, and nanomaterials. Chen Yongping et al. first applied in vivo spitting body display technology to the field of transdermal enhancer development and discovered a short peptide that can promote protein transdermal penetration. The short peptide contains 11 amino acids and is named "TD1". Its amino acid sequence is ACSSSPSKHCG. This short peptide can also help other proteins or hormones penetrate the skin.

[0054] Currently, most protein cosmetics on the market use single-ingredient protein raw materials, such as extracted collagen, elastin, and fibronectin. Very few use extracellular matrix protein fusion products. Moreover, products using single protein ingredients have relatively limited efficacy and are far less comprehensive than fusion protein products. However, fusion proteins, due to their large molecular weight, also have the problem of being difficult to be absorbed by tissues and cells, which limits their scope of use.

[0055] To solve the above technical problems, the present application provides an extracellular matrix fusion protein and its preparation method and application. The human laminin gene, humanized type I collagen gene, fibronectin gene, and elastin gene are sequentially connected through 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. The gene is then cloned into the pPIC9K expression vector and transformed into Pichia pastoris cells. The in vitro biological activity results of the extracellular matrix fusion protein show that this fusion protein has good effects in promoting cell adhesion, cell proliferation, and cell migration, and has a certain effect in promoting collagen secretion. Each effect is better than that of single humanized type I collagen, fibronectin, and elastin.

[0056] Specifically, an embodiment of the present application provides an extracellular matrix fusion protein, the nucleotide sequence of the extracellular matrix fusion protein is shown in SEQ ID No.7.

[0057] 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.

[0058] Wherein, the transdermal short peptide sequence is shown as SEQ ID No.1;

[0059] The laminin amino acid sequence is derived from the LG4 domain of LAMA5 with UniProt PO15230, as shown in SEQ ID No. 2;

[0060] The amino acid sequence of humanized type I collagen is derived from a fragment of the COL1A1 domain with UniProt P02452, as shown in SEQ ID No. 3;

[0061] 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;

[0062] The amino acid sequence of elastin is derived from a repeating segment of [VPGKG(VPGVG)8VPGFG]1 in the hydrophobic domain, as shown in SEQ ID No.5.

[0063] To facilitate understanding by those skilled in the art, the present application also provides a method for preparing the extracellular matrix fusion protein as described above, comprising the following steps:

[0064] Step 1: Insert the synthesized gene fragment of the nucleotide sequence of the extracellular matrix fusion protein shown in SEQ ID No. 7 into the pPIC9K plasmid through the EcoRI and NotI restriction sites to obtain the pPIC9K-TD-LCFE recombinant plasmid.

[0065] Step 2: After linearization, the pPIC9K-TD-LCFE recombinant plasmid was electroporated into GS115 competent cells and cultured until multiple transformant colonies appeared.

[0066] More specifically, the step of linearizing the pPIC9K-TD-LCFE recombinant plasmid comprises: using Quick Cut Sac I. Linearize the pPIC9K-TD-LCFE recombinant plasmid using enzyme digestion conditions of 35°C to 40°C for 3h to 7h. The enzyme digestion system includes: 10μg to 15μg pPIC9K-TD-LCFE recombinant plasmid; 8μL to 12μL QuickCut Sac I; 3μL~7μL 10× Quick Cut buffer.

[0067] More specifically, the step of electrotransforming into GS115 competent cells and culturing until multiple transformant single colonies appear includes: mixing GS115 competent cells and the linearized recombinant plasmid in a volume ratio of 10:1, and transferring the mixture into a pre-cooled electrotransformation cuvette for ice bath treatment; adjusting the electroporator to yeast mode for electroshock treatment, adding pre-cooled sorbitol to the electrotransformation cuvette, mixing evenly, and transferring the mixture into a sterile EP tube for culturing to obtain a bacterial solution; and spreading the bacterial solution on MD culture medium for culturing until multiple transformant single colonies appear.

[0068] Step 3: Screening is performed based on the multiple transformant single colonies to obtain positive transformant single colonies.

[0069] 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 and transferring them to a 96-well culture plate containing YPD medium for culturing to obtain a first bacterial liquid; aspirating the first bacterial liquid from each well to a new 96-well culture plate containing YPD medium for continued culturing to obtain a second bacterial liquid; aspirating the second bacterial liquid from each well to a new 96-well culture plate containing YPD medium for continued culturing to obtain a third bacterial liquid; aspirating the third bacterial liquid from each well and spotting them onto YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418 for continued culturing; and screening the transformant single colonies corresponding to the third bacterial liquid that can grow on the YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418 at the same time as the positive transformant single colonies.

[0070] Step 4: Cultivate and induce the positive transformant single colony to obtain the engineered bacterial fermentation liquid.

[0071] More specifically, the step of culturing and inducing a positive transformant single colony to obtain an engineered bacterial fermentation broth comprises: picking a single colony and inoculating it into a BMGY yeast growth medium and culturing 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 bacteria by centrifugation, and resuspend the bacteria 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, and 0.5% methanol was added to the culture medium every 24 hours. After induction, bacterial samples were taken every 24 hours, and the expression supernatant was collected by centrifugation to obtain the engineered bacterial fermentation broth.

[0072] Step 5: purifying the fermentation broth of the engineered bacteria to obtain the extracellular matrix fusion protein.

[0073] More specifically, the step of purifying the engineered bacteria fermentation broth to obtain the extracellular matrix fusion protein includes: using a solid-liquid separation system to separate the engineered bacteria fermentation broth into a fermentation supernatant and bacteria; using a cation exchange medium, balancing the chromatography column with a phosphate buffer until the conductivity value and the A280 absorbance value remain unchanged, setting the sample loading flow rate to 20 cm / h, detecting the ultraviolet A280 absorbance value, and starting to load the sample when it rises; after the loading is completed, balancing the cationic chromatography medium with a phosphate buffer until the ultraviolet and conductivity drop to the lowest and no longer change, and stopping the loading; then eluting and collecting the corresponding protein with a buffer containing NaH2PO4-NaCl (0.5M), ultrafiltration, liquid replacement, and concentration to obtain a recombinant TD-LCFE protein stock solution.

[0074] To facilitate understanding by those skilled in the art, the technical solutions described in this application will be further described below with reference to specific embodiments:

[0075] Example 1

[0076] Step 1:

[0077] The transdermal short peptide sequence described in this application is cited (Wang Shanshan, Research on the Function of Transdermal Enhancement Peptide TD1 and Epidermal Growth Factor Fusion Protein, University of Science and Technology of China, 2011) (11aa, SEQ ID No.1), the adhesion protein amino acid sequence is derived from the LG4 domain of LAMA5 (UniProt: PO15230) (175aa, SEQ ID No.2), the humanized type I collagen amino acid sequence is derived from the truncated fragment of the COL1A1 (UniProt: P02452) domain (150aa, SEQ ID No.3), and the fibronectin amino acid sequence is derived from the Fibronectin type-III 11 domain fragment of FN1 (UniProt: P02751) (94aa, SEQID No.4). The amino acid sequence of elastin selected a repeat of [VPGKG(VPGVG)8VPGFG]1 (100aa, SEQ ID No.5) in the hydrophobic domain, and the polypeptide fragments of different proteins were connected in series through a flexible linker (GGGGS)2 to construct the recombinant protein TD-1-LN-Col-FN-ELA amino acid sequence (560aa, SEQ ID No.6), named TD-LCFE, with a predicted molecular weight of approximately 54.03kDa.

[0078] The DNA sequence encoding the protein was optimized for Pichia pastoris preference to obtain a recombinant protein with nucleotide sequence as shown in SEQ ID No. 7, so that the recombinant protein is more suitable for expression in Pichia pastoris.

[0079] Among them, SEQ ID No.1-SEQ ID No.7 are as follows:

[0080] SEQ ID No.1:

[0081] ACSSSPSKHCG.

[0082] SEQ ID No.2:

[0083] TDGSYLDGTGFARISFDSQISTTKRFEQELRLVSYSGVLFFLKQQSQFLCLAVQEGSLVLLYDFGAGLKKAVPLQPPPPLTSASKAIQVFLLGGSRKRVLVRVERATVYSVEQDNDLELADAYYLGGVPPDQLPPSLRRLFPTGGSVRGCVKGIKALGKYVDLKRLNTTGVSAGC。

[0084] SEQ ID No. 3:

[0085] GPMGPSGPRGLPGPPGAPGPQGFQGPPGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAGDGAPGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGAT。

[0086] SEQ ID No.4:

[0087] VPRDLEVVAATPTSLLISWDAPAVTVRYYRYTYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEID。

[0088] SEQ ID No. 5:

[0089] VPGKGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG.

[0090] SEQ ID No. 6:

[0091] ACSSSPSKHCGTDGSYLDGTGFARISFDSQISTTKRFEQELRLVSYSGVLFFLKQQSQFLCLAVQEGSLVLLYDFGAGLKKAVPLQPPPPLTSASKAIQVFLLGGSRKRVLVRVERATVYSVEQDNDLELADAYYLGGVPPDQLPPSLRRLFPTGGSVRGCVKGIKALGKYVDLKRLNTTGVSAGCGGGGSGGGGSGPMGPSGPRGLPGPPGAPGPQGFQGPPGEPGEPGASGPMGPRGPPGPPGKNGDDGEAGKPGRPGERGPPGPQGARGLPGTAGLPGMKGHRGFSGLDGAKGDAGPAGPKGEPGSPGENGAPGQMGPRGLPGERGRPGAPGPAGARGNDGATGGGGSGGGGSVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDGGGGSGGGGSVPGKGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGFGVPGKGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGFG.

[0092] SEQ ID No. 7:

[0093]

[0094] The optimized base sequence of the recombinant protein TD-1-LN-Col-FN-ELA as shown in SEQ ID No. 7 was entrusted to Jinsheng Biotechnology Co., Ltd. for gene fragment synthesis. The synthesized gene fragment was inserted into pPIC9K plasmid through EcoRI and NotI enzyme cutting sites to obtain pPIC9K-TD-LCFE recombinant plasmid as shown in SEQ ID No. 7. Figure 1

[0095] Step 2:

[0096] 2-1) Obtaining of recombinant plasmid: the optimized base sequence as shown in SEQ ID No. 7 was entrusted to Jinsheng Biotechnology Co., Ltd. for gene fragment synthesis, and the corresponding plasmid and strain were provided after sequencing verification, and high-concentration pPIC9K-TD-LCFE plasmid was extracted after expansion culture for standby use.

[0097] 2-2) Linearization treatment of pPIC9K-TD-LCFE plasmid:

[0098] The extracted pPIC9K-TD-LCFE plasmid was linearized and cut by Quick Cut Sac I, and the enzyme cutting condition was 37°C, 5h, and the enzyme cutting system was as shown in Table 1:

[0099] Table 1:

[0100]

[0101] After electrophoresis verification, 0.1 volume of 3M NaAc (pH 5.2) and 2.5 volume of anhydrous ethanol were added, and it was placed at -20°C overnight. 4°C, 13000rpm centrifugation for 20min, discard the supernatant; add 700μL 75% ethanol rinse, 13000rpm centrifugation for 20min, discard the supernatant, repeat once; EP tube is inverted on the super clean table water paper for about 10min, try to remove water and residual ethanol, 20μL ddH2O redissolve the plasmid, take 1μL dilution 10 times, and use one-drop to detect the nucleic acid concentration.

[0102] 2-3) Preparation of GS115 competent cells

[0103] After the GS115 strain plate streaking, a single colony was inoculated in 20mL YPD liquid medium, and it was cultured at 30°C, 225rpm for 24h; it was inoculated in 50mL YPD liquid medium with a 1:1000 inoculation ratio, and it was cultured at 30°C, 225rpm until OD 600 ​The value is 1.3-1.5; transfer the bacterial solution into a sterile 50mL centrifuge tube, centrifuge at 4℃, 3000rpm for 5min, discard the supernatant and collect the bacteria; resuspend the bacterial pellet with 50mL pre-cooled sterile ultrapure water, centrifuge at 4℃, 3000rpm for 5min; discard the supernatant and resuspend the bacterial pellet with 50mL pre-cooled sterile ultrapure water; centrifuge at 4℃, 3000rpm for 5min, discard the supernatant, and resuspend the cell pellet with 40mL pre-cooled sterile 1M sorbitol; centrifuge at 4℃, 3000rpm for 5min, discard the supernatant, and resuspend the cell pellet with 100μL~150μL pre-cooled sterile 1M sorbitol, mix gently with rotation, and place on ice for use.

[0104] 2-4) Electrotransformation into GS115 competent cells

[0105] Mix 100 μL of GS115 competent cells with 10 μL of linearized pPIC9K-TD-LCFE plasmid, transfer to a pre-chilled electroporation cuvette, and immediately place on ice for 5 minutes. Select the yeast mode on the electroporator and perform electroporation. Immediately add 1 mL of pre-chilled 1 M sorbitol to the cuvette, mix thoroughly, and transfer the mixture to a sterile EP tube. Incubate in a 30°C incubator for 1-2 hours. Spread 100-200 μL of the bacterial solution onto MD medium, let it stand at room temperature for 10 minutes, and then incubate inverted in a 30°C incubator for approximately 2-5 days until a single colony appears.

[0106] Step 3:

[0107] Pick a single colony from the YPD plate and transfer it to a 96-well plate containing 200 μL of YPD medium (containing 0.5 mg / mL G418). Continue culturing at 30°C. After 48 hours, homogenize the bacterial solution by airflow and transfer 10 μL per well to a new 96-well plate containing 190 μL YPD. Continue culturing for 24 hours, then repeat the above process. After 24 hours, homogenize the bacterial solution from a third 96-well plate by airflow and transfer 1 μL to YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418, respectively. Transformants that grow simultaneously on YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418 indicate that they contain multiple copies of the target gene and are considered positive transformants.

[0108] In order to verify whether the above-mentioned positive transformant single colony is correct, the present application also designed the following steps to verify it:

[0109] Take the remaining 50 μL of the first block 96-well plate liquid in the above step, boiling in boiling water bath for 10 min, frozen in liquid nitrogen for 30 min, boiling in boiling water bath for 10 min, repeated freeze-thaw, 12000 rpm centrifugation for 5 min, take supernatant as PCR template, identify whether the target gene is integrated into the yeast chromosome by PCR amplification.

[0110] Upstream specific primer: 5'-TTACGTAGAATTCGCATGTTC-3'

[0111] Downstream specific primer: 5'-ATTAATTCGCGGCCGCACCAAA-3'

[0112] PCR conditions: 98℃ pre-denaturation, 5min, 98℃ hot denaturation 50s, 60℃ annealing 30s, 72℃ extension 30s, 35 cycles; 72℃ reannealing 10min.

[0113] The amplified product was subjected to 1.0% agarose gel electrophoresis to identify whether the expected size gene fragment was amplified; the identification result is shown in Figure 2 (about 1700bp).

[0114] Step 4:

[0115] 4-1) Expression of recombinant TD-LCFE protein

[0116] The positive transformant single colony verified in step 3 was inoculated in BMGY yeast growth medium, 30 ºC, 220 rpm for 24 h to OD 600 value of 2~6. According to the measured OD 600 value, adjust the volume of BMGY yeast growth medium, 3000 rpm, 10 min to collect the bacteria, resuspend the bacteria with the same volume of BMMY yeast induction medium as the BMGY yeast growth medium, so that the initial OD 600 value is 2.0. 30 ºC, 220 rpm continued to culture, every 24 h added 0.5% methanol to the culture medium, 24h, 48h, 72h, 96h after induction, respectively, take the bacterial liquid sample, centrifugal collection of expression supernatant, as the engineering bacteria fermentation broth, for use. As shown in Figure 3 , SDS-PAGE electrophoresis analysis of the molecular weight of the target protein is about 54kDa and the expression amount.

[0117] 4-2) Chinese-style fermentation of yeast engineering bacteria

[0118] Seed solution preparation: Pichia pastoris engineered strain (GS115 / pPIC9K / TD-LCFE) was inoculated into 50 mL of YPD liquid medium (10 g / L yeast powder, 20 g / L peptone, 20 g / L glucose) containing 0.25 mg / mL geneticin. The culture was shaken at 30°C and 220 rpm for 18–20 h to obtain the primary seed solution. The primary seed solution was then inoculated into 200 mL of YPD liquid medium at a 5% inoculum size and shaken at 30°C and 220 rpm for 18–20 h to obtain the secondary seed solution.

[0119] Preparation for inoculation: Prepared BSM medium was transferred to a 5-L glass fermenter (Shanghai Bailun Biotechnology Co., Ltd.) and sterilized in an autoclave at 121°C for 15 minutes. After autoclaving, the medium was cooled to below 30°C. Then, under a flame ring, 2 mL of defoamer and 8.7 mL of PTM1 solution were added.

[0120] Yeast Inoculation: Inoculate 2 L of the secondary seed solution into the aforementioned 5 L glass fermenter. Maintain a culture temperature of 30°C and a stirring speed of 800 r / min. Automatically add 50% (w / w) ammonia to maintain a pH of approximately 5.0. Aerate with air or pure oxygen to maintain a dissolved oxygen (DO) value above 50%.

[0121] Between 18 and 20 hours of culture, the glycerol in the BSM medium is depleted, and the DO value rises sharply, indicating that the glycerol in the medium is being consumed by the cells. At this point, approximately 400 mL of feed medium (50% w / v glycerol, 12 mL of PTM1 trace element per liter) is added. Once the cell wet weight reaches 180 g / L–220 g / L, glycerol addition is stopped and starvation is continued for approximately 1–2 hours. Once the DO in the tank reaches its maximum value again, induction medium (100% methanol, 12 mL of PTM1 trace element per liter) is added to induce the engineered Pichia pastoris to express recombinant human thioredoxin. The feed rate is adjusted regularly to maintain DO within a cyclical range of 20%–50%. At 24, 48, 72, 96, and 120 hours of induction, the supernatant is centrifuged, diluted 5-fold, and 10 μL is loaded onto the sample for SDS-PAGE and concentration analysis.

[0122] Step 5:

[0123] The fermentation broth of the engineered bacteria obtained in step 4 was collected by centrifugation, and the fermentation supernatant and the bacteria were separated using a solid-liquid separation system. Using a cation exchange medium (the chromatography filler is SP Purose 6 High Performance produced by Qianchun, loaded on a GE Akta chromatography system), the chromatography column was equilibrated with phosphate buffer (20mM NaH2PO4, pH 5.0) until the conductivity value and A280 absorbance value remained unchanged. The sample loading flow rate was set to 20cm / h, and the UV A280 absorbance value was detected. When it rose, the sample was started. After the loading was completed, the cationic chromatography medium was equilibrated with phosphate buffer until the UV and conductivity dropped to the lowest and no longer changed, and the sample was stopped. The corresponding protein was then eluted and collected with a buffer containing NaH2PO4-NaCl (0.5M). After ultrafiltration, liquid replacement, and concentration, the recombinant TD-LCFE protein stock solution was obtained. The molecular weight and purity were detected by SDS-PAGE, and the results are as follows. Figure 4 shown.

[0124] It should be noted that the culture medium and its formula involved in the examples are as follows:

[0125] YPD liquid medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L (if solid medium is used, add 2% agar);

[0126] MD medium: YNB without amino acids, nitrogen source 13.4 g / L; 0.4 mg / L biotin; 20 g / L glucose (solid medium containing 2% agar);

[0127] BMGY Yeast Growth Medium:

[0128] Yeast extract 10g / L, peptone 20g / L, 3g / L K2HPO4, 11.8g / L K2HPO4, add water to 890mL, sterilize at 121℃ for 20 minutes, then add 10×YNB 100mL (13.4g / L), 500×biotin 1mL (4×10 -4 g / L), glycerol 10mL.

[0129] BMMY yeast induction medium:

[0130] Yeast extract 10g / L, peptone 20g / L, 3g / L K2HPO4, 11.8g / L K2HPO4, add water to 895mL, sterilize at 121℃ for 20 minutes, then add 100×YNB 100mL (13.4 g / L), 500×biotin 1mL (4×10 -4 g / L), methanol 5mL.

[0131] Fermentation medium: 85% H3PO4 26.7 mL / L; CaSO4·2H2O 1.175 g / L; K2SO4 18.2 g / L; MgSO4·7H2O 14.9 g / L; KOH 4.13 g / L; glycerol 40.0 g / L; PTM 14.35 mL / L;

[0132] Feed medium: 50% w / v glycerol, plus 12 mL of PTM1 trace elements per liter;

[0133] Induction medium: 100% methanol, add 12 mL of PTM1 trace elements per liter; add PTM1 trace elements, sterilize the fermentation medium at high temperature, add PTM1 when the temperature drops to room temperature, and adjust the pH to 5.0 with ammonia water.

[0134] The PTM1 solution was prepared according to the Invitrogen manual. The specific formula was as follows: CuSO4·5H2O 6.0 g / L; NaI 0.08 g / L; MnSO4·H2O 3.0 g / L; NaMoO4·2H2O 0.2 g / L; H3BO3 0.02 g / L; CoCl2 0.5 g / L; ZnCl2 20.0 g / L; FeSO4·7H2O 65.0 g / L; biotin 0.2 g / L; H2SO4 5.0 mL / L. Sterilize by filtration through a 0.22 μm filter membrane and store at 4°C.

[0135] In order to verify the efficacy of the recombinant TD-LCFE protein solution obtained in Example 1, the following experimental examples were set up:

[0136] Experimental Example 1: Effect of TD-LCFE fusion protein on promoting cell proliferation

[0137] 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 test of the fusion protein prepared in this application was performed by promoting cell proliferation.

[0138] 1. Experimental Methods

[0139] 1.1 Experimental Materials

[0140] Test sample: TD-LCFE, prepared in Example 1, batch number 20241008.

[0141] Reference substance: recombinant type III collagen (rhCOLⅢ), prepared according to application number CN202411045813.2, batch number 20240807.

[0142] Standard: EGF, titer 500,000 U / mg, purchased from Sigma.

[0143] Cell line: Mouse embryonic fibroblasts (3T3), passage 8, cryopreservation batch number 20221215, purchased from ATCC, USA.

[0144] Other reagents (complete culture medium HyClone / AJ30742864, PBS, 0.25% trypsin) and other items (96-well cell culture plates, TIP tips, and micropipettes) were routine laboratory items and were sterilized or filter-sterilized before the experiment and passed the sterility test.

[0145] 2. Experimental Procedure

[0146] 2.1 Cell culture and passaging

[0147] The cells were cultured normally in a medium containing 10% calf serum. When the cells grew to a confluence rate of more than 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 rounded, the trypsin was removed and a medium containing 10% calf serum was added and gently pipetted. The cells were collected and centrifuged. The cells were counted and the cell concentration was adjusted to an appropriate concentration and inoculated into 1 mL of 1.0×10 5 ~5.0×10 5 When the cell fusion rate reaches more than 90%, the next passage is performed.

[0148] 2.2 Cell seeding

[0149] The cells were selected for determination 24h~36h after passage. The culture medium in the culture flask was discarded, the cells were digested and collected, and PRIM1640 medium containing 10% fetal bovine serum was used to prepare 5.0×10 4 ~8.0×10 4 The cell suspension of 100 cells was inoculated into a 96-well cell culture plate, 100 μL per well, and cultured at 37°C and 5% carbon dioxide.

[0150] 2.3 Cell synchronization

[0151] After 24 h, the cells were replaced with maintenance medium (PRIM1640 medium containing 2% fetal bovine serum) and cultured at 37°C and 5% carbon dioxide for another 24 h.

[0152] 2.4 Administration

[0153] After 24 hours, the sample was serially diluted 4-fold using maintenance medium and 100 μL of the diluted sample was added to each well of the cells. A blank control group was also set up with 2 replicates per group and the culture was continued.

[0154] 2.5 MTT assay

[0155] After the treated cells were cultured for 72 hours, 20 μl of MTT solution (0.5 mg / ml) was added to each well, incubated at 37°C for 4 hours with 5% CO2, and then the solution was discarded. DMSO was added for lysis and shaking, and the absorbance was measured at 570 nm.

[0156] 2.6 Data Processing

[0157] The experimental data were processed using computer programs or four-parameter regression calculation method.

[0158] 3. Experimental Results

[0159] The test results are shown in Table 2 below:

[0160] Table 2 Results of cell proliferation-promoting activity assay of TD-LCFE fusion protein

[0161]

[0162] 4. Conclusion

[0163] From the results in Table 2, it can be seen that the cell proliferation promoting activity of the fusion protein TD-LCFE is at the same order of magnitude as that of the patented reference substance recombinant type III collagen, and the cell proliferation promoting activity reaches 1000 U / mg.

[0164] Experimental Example 2: TD-LCFE fusion protein promotes cell scratch test

[0165] 1. Experimental Principle

[0166] When cells grow to a confluent monolayer, a blank area, called a "scratch," is artificially created on the confluent monolayer. Cells at the edge of the scratch gradually migrate into the blank area, healing the scratch. Images are captured at the beginning and at regular intervals during cell migration, and the images are compared to determine the cell migration rate.

[0167] 2. Experimental Materials

[0168] Test sample: TD-LCFE, prepared in Example 1, batch number 20241008.

[0169] Reference substance: recombinant elastin, prepared according to application number CN202411012422.0, batch number 20240824.

[0170] Cell line: Balb / c 3T3 cells, purchased from ATCC, USA.

[0171] Other reagents (cell nutrient solution, serum-free culture medium, PBS, 0.25% trypsin) and other items (96-well cell culture plates, TIP tips, and micropipettes) were routine laboratory items and were sterilized or filtered before the experiment, and passed the sterility test.

[0172] 3. Experimental Procedure

[0173] (1) First, use a marker pen to align the ruler on the back of the 6-well plate and draw horizontal lines evenly, approximately every 0.5 cm to 1 cm, across the holes.

[0174] (2) Add 5×10 5 2mL of cell suspension with a cell count of 1 cell / mL.

[0175] (3) On the second day, observe that all cells in the 6-well plate have grown into a monolayer. Use the tip of the gun to measure the ruler and make two scratches perpendicular to the horizontal line on the back of the plate.

[0176] (4) Wash the cells three times with PBS to remove the suspended cells.

[0177] (5) Add 1.8 mL of serum-free culture medium to the wells according to the grouping, then add 200 μL of sample, and add an equal amount of PBS solution to the cell control wells.

[0178] (6) Place in a 37°C, 5% CO2 incubator and incubate. Take a photo at 0:00 and record the photo location in each well. Observe and photograph the fixed location during subsequent observations.

[0179] (7) Data processing: Area detection method (scratch distance measurement is equivalent measurement)

[0180] Average scratch width = scratch gap area / length

[0181] Cell migration rate = (0h scratch width - 24h scratch width) / 0h scratch width × 100%

[0182] 4. Experimental Results

[0183] The observation results of the cell control well and each sample well at 0h and 24h are as follows Figure 5 and as shown in Table 3.

[0184] Table 3 Results of TD-LCFE fusion protein promoting 3T3 cell migration

[0185]

[0186] 5. Conclusion

[0187] The results of in vitro cell migration rate calculated according to the formula show that TD-LCFE fusion protein has a significant effect in promoting cell migration.

[0188] Experimental Example 3: Effect of TD-LCFE fusion protein on promoting cell adhesion

[0189] 1. Experimental Principle

[0190] Cell adhesion and retention are essential for cell repair and growth. Fibronectin has a strong ability to promote cell adhesion. This study tested whether the TD-LCFE fusion protein has the same adhesion-promoting activity as fibronectin through a cell adhesion (adhesion) assay.

[0191] 2. Test Materials

[0192] 2.1 Main instruments: clean bench, cell culture incubator.

[0193] 2.2 Reagent preparation

[0194] 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.

[0195] Serum-free medium: Measure 1 mL of double antibody, add 99 mL of 1640 culture medium, and store at 4°C.

[0196] Digestion solution: 0.25% trypsin.

[0197] 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 them in water and make up to 1000 mL. Sterilize by autoclaving at 121°C for 15 min.

[0198] 2.3 Cells

[0199] MDBK cells grew as monolayers and adherently in complete cell culture medium. They were passaged every 4 to 5 days, digested and passaged at a ratio of 1:2, and grown and propagated in complete cell culture medium.

[0200] 2.4 Test sample: TD-LCFE, prepared as in Example 1, batch number 20241008.

[0201] 2.5 Positive control: recombinant fibronectin (rhFN), prepared according to application number CN202110357340.X, batch number 20240716.

[0202] 3. Test operation

[0203] 3.1 Sample dilution and incubation

[0204] The TD-LCFE fusion protein was pre-diluted to 0.5 μg / mL with PBS. After pre-dilution, a 2-fold serial dilution was performed in a 96-well plate for a total of 10 dilutions, with 50 μL of TD-LCFE fusion protein samples of different dilutions per well. Positive control wells for the serial dilutions and a negative control well (50 μL of PBS was added as a control) were set up and incubated overnight at 4°C.

[0205] 3.2 Cell adhesion assay

[0206] After incubation, 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 MDBK cell suspension (resuspended in serum-free medium) was added. The cell seeding density was 1.0 × 10 5 Each well was inoculated with 100 μL of the culture medium and incubated in a thermostat for 5 h.

[0207] 4. Experimental Results

[0208] The incubated cell plate was washed three times with PBS and the cell adhesion was observed under a microscope. The results of TD-LCFE fusion protein promoting cell adhesion were as follows: Figure 6 shown.

[0209] The calculation results are shown in Table 4-Table 5:

[0210] Table 4 Cell counts at five points in each well of the plate

[0211]

[0212] Table 5 Statistical results of cell adhesion promotion by TD-LCFE fusion protein

[0213]

[0214] 5. Conclusion

[0215] According to the statistical results, both TD-LCFE fusion protein and recombinant fibronectin have in vitro cell adhesion promoting activity.

[0216] Experimental Example 4: Transdermal absorption test of TD-LCFE fusion protein

[0217] 1. Test Principle

[0218] The protein solution was tested for transdermal absorption in an upright Franz diffusion cell placed in a drug transdermal diffusion tester and maintained in a constant temperature circulating water bath at 37°C. The in vitro transdermal performance of the TD-LCFE fusion protein was compared and evaluated.

[0219] 2. Test methods

[0220] 2. Test materials:

[0221] 2.1 Ex vivo skin: BALB / c mice aged 6-8 weeks were selected for ex vivo skin preparation.

[0222] 2.2 Main test reagents

[0223] Test sample: TD-LCFE fusion protein produced in Example 1, batch number 20241008;

[0224] Reference substance: recombinant type III collagen, prepared according to application number CN202411045813.2, batch number 20240807;

[0225] Normal saline; type III collagen monoclonal antibody (purchased from Abcam, UK).

[0226] 2.3 Experimental Instruments

[0227] Honghua ZTY intelligent transdermal tester, with a maximum receiving cell capacity of 20mL and an effective transdermal area of ​​1.36cm 2 .

[0228] 3. Experimental steps

[0229] 3.1 In vitro skin permeation test

[0230] 3.1.1 Mouse Skin Installation

[0231] Transdermal absorption studies were conducted in an upright Franz diffusion cell placed in a transdermal drug diffusion tester. The treated excised skin was positioned between the donor and receiver cells, with the stratum corneum facing the donor cell and the dermis facing the receiver. The water bath temperature was adjusted to 37°C, with a stirring rate of 100 rpm. Preheated 37°C physiological saline was added to the receiver cell, and air bubbles were removed. To minimize interference, the inner surface of the mouse skin was first exposed to the receiver solution without drug administration. Then, 5 mL of solutions of varying concentrations were injected into the donor cell, placed against the skin.

[0232] 3.1.2 Sampling

[0233] At 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, and 24 hours after the start of the test, approximately 1 mL of the receiving fluid was drawn with a syringe as the sample solution. At the same time, the receiving chamber was filled with an equal amount of physiological saline solution. Finally, the sample solutions collected at each time period were tested. Figure 7 shown.

[0234] 3.1.3 Trial Grouping

[0235] Set up three groups, namely blank group (normal saline), test group, and control group, and dilute the sample to a concentration of 0.1 mg / mL with normal saline.

[0236] 4. Result detection

[0237] 4.1 Protein content detection

[0238] The protein content in the receiving fluid samples taken at each time point was determined using the Coomassie Brilliant Blue method. The cumulative permeation amount (Q) was calculated as follows:

[0239]

[0240] Among them, C n Indicates the concentration of the sample taken at the nth time, C i represents the concentration of the sample taken at the i-th time, V represents the volume of the receiving pool, and V i represents the sampling volume, and S represents the transdermal area.

[0241] 4.2 Immunohistochemical Slice Preparation and Observation

[0242] After 24 hours of treatment, the transdermal sections of the rat skin (including the hair follicle and non-follicle areas) were fixed and immunohistochemical sections were prepared. The prepared immunohistochemical sections were placed under a microscope to observe the location of proteins in the skin tissue of each group.

[0243] 4.3 Immunohistochemistry Slide Preparation Process

[0244] (1) Place the slices in a drying oven at 66°C for 20 to 30 minutes.

[0245] (2) Pass through xylene three times in sequence, each time for 5 minutes.

[0246] (3) Pass through three layers of ethanol (100%, 95% and 80%) in sequence, each layer for 3 minutes.

[0247] (4) Place the slices in a beaker and rinse slowly with running water to remove the ethanol until the slices are clean and transparent.

[0248] (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 slices in, cover the pot, spray the air and time for 2 minutes, then stop heating and slowly rinse the pressure cooker lid with running water until it cools down.

[0249] (6) Block endogenous peroxidase: incubate the slices in 3% H2O2 at room temperature, wash with distilled water three times, draw a hydrophobic circle, and rinse with PBS-T three times.

[0250] (7) Shake off excess liquid from the slices, add primary antibody (type III collagen monoclonal antibody), cover and incubate in a 37°C incubator for 60 minutes. Remove the slices and rinse with PBS-T three times.

[0251] (8) Shake off excess liquid from the slices, add secondary antibody, cover and incubate in a 37°C incubator for 30 minutes. Remove the slices and rinse with PBS-T three times.

[0252] (9) Shake off the excess liquid on the slice, add DAB color developer, control the color development time under a microscope, stop the color development when there is a positive result, and rinse with distilled water.

[0253] (10) Stain with hematoxylin for 2 to 5 minutes, then rinse with water; differentiate with 1% hydrochloric acid and alcohol for a few seconds, then rinse with water.

[0254] (11) Lithium carbonate solution was bluing for 30 seconds and then washed with water.

[0255] (12) Conventional dehydration, xylene transparency.

[0256] (13) Seal the slide with neutral gum and observe the results under a microscope. A brown color indicates a positive reaction.

[0257] 5. Experimental Results

[0258] 5.1 Protein concentration of samples at different time periods

[0259] The cumulative permeability per unit area (Q) at each time point was calculated according to the formula as shown in Table 6:

[0260] Table 6 Protein concentration of samples at each time point and cumulative permeation per unit area Q

[0261]

[0262] Note: (1) Sample concentration is in μg / mL; (2) Q value is in μg / cm 2

[0263] From the results in Table 6, it can be seen that TD-LCFE fusion protein has transdermal performance, while recombinant type III collagen cannot penetrate the skin.

[0264] 5.2 Immunohistochemistry Results

[0265] Immunohistochemical detection was performed on the isolated mouse skin 24 hours after the experiment. The results were as follows: Figure 8 As shown, it can be seen that the TD-LCFE fusion protein in the test group penetrates into the dermis ( Figure 8 C red arrow), showing an obvious brown positive reaction; while the control group recombinant type III collagen had basically no protein penetration, the positive reactions were all outside the epidermis, there was no obvious concentrated positive reaction in the dermis, and only the collagen structure of the skin itself had scattered positive reactions ( Figure 8 B); Figure 8 There was no obvious concentrated positive reaction in the blank group A.

[0266] According to the above test results, it can be seen that TD-LCFE fusion protein has obvious transdermal absorption effect.

[0267] Experimental Example 5: TD-LCFE fusion protein promotes collagen secretion test

[0268] 1. Test Principle

[0269] Collagen content is measured using staining and enzyme-linked immunosorbent assay (ELISA). ELISA can also visualize type I and III collagen fibrils, but the antibodies used are expensive and the procedure is time-consuming. Sirius red staining, on the other hand, is inexpensive, specifically binds to the Gly-XY helical structure of fibrillar collagen (types I to V), and is simple to perform. Sirius red is a strongly acidic anionic dye that reacts with alkaline collagen. Collagen content is positively correlated with the intensity of the stain. Optical density (OD) is measured at 450 nm using a microplate reader to calculate collagen content.

[0270] 2. Experimental Materials

[0271] Test sample: TD-LCFE fusion protein produced in Example 1, batch number 20241008;

[0272] Reference substance: recombinant type III collagen (rhCOLⅢ), prepared according to application number CN202411045813.2, batch number 20240807.

[0273] Reference substance 2: recombinant fibronectin (rhFN), prepared according to application number CN202110357340.X, batch number 20240716.

[0274] Cell line: Balb / c 3T3 cells were purchased from ATCC, USA.

[0275] Other reagents (RPMI 1640 medium, fetal bovine serum, trypsin solution, PBS, picrosirius red staining solution, acetic acid, sodium hydroxide) and other items (96-well cell culture plates, TIP tips, and micropipettes) were routine laboratory materials and were sterilized or filter-sterilized before the experiment and passed the sterility test.

[0276] 3. Experimental Procedure

[0277] 3.1 Detection of cell collagen content by staining

[0278] 3.1.1 Cell culture and passaging

[0279] The cells were cultured normally in a medium containing 10% calf serum. When the cells grew to a confluence rate of more than 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 rounded, the trypsin was removed and a medium containing 10% calf serum was added and gently pipetted. The cells were collected and centrifuged. The cells were counted and the cell concentration was adjusted to an appropriate concentration and inoculated into 1 mL of 1.0×10 5 ~5.0×10 5 When the cell fusion rate reaches more than 90%, the next passage is performed.

[0280] 3.1.2 Cell seeding

[0281] The cells were selected for determination 24h~36h after passage. The culture medium in the culture flask was discarded, the cells were digested and collected, and the complete culture medium was used to prepare a mixture containing 5.0×10 4 ~8.0×10 4 The cell suspension of 100 cells was inoculated into a 96-well cell culture plate, 100 μL per well, and cultured at 37°C and 5% carbon dioxide.

[0282] 3.1.3 Cell synchronization

[0283] After 24 h, the culture medium was changed to maintenance medium and cultured for another 24 h at 37°C and 5% carbon dioxide.

[0284] 3.1.4 Drug administration

[0285] After 24 hours, dilute the sample and positive control with maintenance medium and add 0.2 mL of the diluted sample to each well of the cells. Simultaneously, set up a cell control group, add only 0.2 mL of maintenance medium, and continue culturing in triplicate wells per group.

[0286] 3.1.5 Collection of cell supernatant

[0287] After the incubation, 200 μL of cell culture supernatant was collected from each well into a 1.5 mL sterile centrifuge tube and stored in a -80°C ultra-low temperature refrigerator.

[0288] 3.1.6 Fixation of cell cultures

[0289] The adherent cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes, the fixative was discarded, and 0.2 mL PBS was added to each well and washed twice.

[0290] 3.1.7 Sirius red staining

[0291] Add 0.15 mL of 0.1% picric acid-staining solution to each well and stain at room temperature for 1 hour. Aspirate the staining solution and add 0.2 mL of 0.1% glacial acetic acid to each well. Wash three times, 5 minutes each time.

[0292] 3.1.8 Detection

[0293] Aspirate 0.1% glacial acetic acid, add 0.2 mL of 0.1 mol / L sodium hydroxide to each well, shake thoroughly at room temperature for 1 h, and place in a microplate reader to detect the absorbance A value at a wavelength of 540 nm to detect changes in collagen.

[0294] 4. Experimental Results

[0295] The test results are as follows Figure 9 And as shown in Table 7:

[0296] Table 7 Collagen content upregulation rate

[0297]

[0298] The results showed that TD-LCFE and recombinant type III collagen had a significant effect in promoting collagen secretion, and TD-LCFE had the best effect, with an increase rate of 35.7% in collagen content.

[0299] The above are only preferred embodiments of the present application and do not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the 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. 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. A method for preparing the extracellular matrix fusion protein according to claim 1, 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; After linearization, the pPIC9K-TD-LCFE recombinant plasmid was electroporated into GS115 competent cells and cultured until multiple transformant colonies appeared; Screening is performed based on the multiple transformant single colonies to obtain positive transformant single colonies; Cultivate and induce the positive transformant single colony to obtain the engineered bacterial fermentation liquid; The fermentation liquid of the engineered bacteria is purified to obtain the extracellular matrix fusion protein.

3. The method for preparing the extracellular matrix fusion protein according to claim 2, 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 using enzyme digestion conditions at 35° C. to 40° C. for 3 h to 7 h. The enzyme digestion system includes: 10 μg~15 μg pPIC9K-TD-LCFE recombinant plasmid; 8μL~12μL Quick Cut Sac I; 3μL~7μL 10× Quick Cut buffer.

4. The method for preparing the extracellular matrix fusion protein according to claim 2, 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: GS115 competent cells and the linearized recombinant plasmid were mixed in a volume ratio of 10:1 and transferred to a pre-cooled electroporation cuvette for ice bath treatment; the electroporator was adjusted to the yeast mode for electroporation, and pre-cooled sorbitol was added to the electroporation cuvette. 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 MD medium and cultured until multiple transformant colonies appeared.

5. The method for preparing the extracellular matrix fusion protein according to claim 2, characterized in that: The step of screening the multiple transformant single colonies to obtain positive transformant single colonies comprises: Pick a single colony of the transformant and transfer it to a 96-well culture plate containing YPD medium for cultivation to obtain a first bacterial liquid; pipette the first bacterial liquid from each well to a new 96-well culture plate containing YPD medium for continued cultivation to obtain a second bacterial liquid; pipette the second bacterial liquid from each well to a new 96-well culture plate containing YPD medium for continued cultivation to obtain a third bacterial liquid; pipette the third bacterial liquid from each well onto YPD plates containing 1 mg / mL, 2 mg / mL, and 3 mg / mL G418 for continued cultivation; 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 and taken as positive transformant single colonies.

6. The method for preparing the extracellular matrix fusion protein according to claim 2, characterized in that: The step of culturing and inducing a positive transformant single colony 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 bacteria by centrifugation, and resuspend the bacteria 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, and 0.5% methanol was added to the culture medium every 24 hours. After induction, bacterial samples were taken every 24 hours, and the expression supernatant was collected by centrifugation to obtain the engineered bacterial fermentation broth.

7. The method for preparing the extracellular matrix fusion protein according to claim 2, characterized in that: The step of purifying the fermentation broth of the engineered bacteria to obtain the extracellular matrix fusion protein comprises: Separating the engineered bacteria fermentation liquid into fermentation supernatant and bacteria bodies by using a solid-liquid separation system; Use cation exchange medium and equilibrate the column with phosphate buffer until the conductivity and A280 absorbance remain unchanged. Set the sample loading flow rate to 20 cm / h and detect the UV A280 absorbance. When it rises, start loading the sample. After the sample loading is completed, the cationic chromatography medium is equilibrated with phosphate buffer until the UV and conductivity drop to the lowest and no longer change, and then the sample loading is stopped; The corresponding protein was then 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.

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