Recombinant human fibronectin peptide as well as preparation method and application thereof

Abstract

The invention discloses a recombinant human fibronectin peptide as well as a preparation method and application thereof, and relates to the field of protein. The recombinant human fibronectin peptide disclosed by the invention can promote cell adhesion, proliferation or migration, and has a wide application prospect in the fields of cosmetics and medical instruments. The invention further discloses a method for producing the recombinant human fibronectin peptide through genetic engineering and fermentation engineering, and a foundation is laid for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the field of proteins, and in particular to a recombinant human fibronectin peptide and a preparation method and application thereof. Background Art

[0002] Fibronectin (FN) is a high-molecular-weight glycoprotein widely present in blood, body fluids and various tissues. It participates in a wide range of physiological processes such as cell adhesion, migration, growth and differentiation, and helps maintain the homeostasis of the extracellular matrix (Am J Physiol Cell Physiol. 2024 Apr 1; ​​326(4): C1212-C1225). It can bind to many biologically important molecules, including integrins, heparin, collagen / gelatin and fibrin, and play different roles.

[0003] FN is usually connected by a pair of disulfide bonds and exists in the form of a dimer. It consists of two almost identical subunits of about 250 kDa, with variable molecular conformations and splice variants (Adv Drug Deliv Rev. 2021 Mar; 170: 353-368). FN has three different functional domains, consisting of three conservative module types (I, II and III), including 12 type I repeat sequences, two type II repeat sequences and 15-17 type III repeat sequences, which together account for about 90% of the FN sequence. These sequences have six major functional binding domains that can bind to collagen, fibrin, cells, heparin, etc. (J Wound Care. 2005 Jun; 14 (6): 284-7). Fibronectin can promote the formation of collagen fibrils in vivo. Among them, 8-9FNI in the collagen binding domain of fibronectin can bind to α5β1 integrin on the cell surface, and the conformational change induced by α5β1 integrin is necessary for promoting fibrogenesis (Current Opinion in Cell Biology, 2008, 20(5): 495-501). In addition, during embryogenesis, fibronectin is essential for the migration and differentiation of many types of cells. FN is also involved in various stages of wound repair. It can serve as a scaffold for cell migration, recruit fibroblasts to migrate to the wound, and restore tissue structure. During the wound healing process, FN participates in the composition of ECM and maintains its stability. It is necessary for the deposition of type I collagen and other ECM proteins. Therefore, fibronectin has broad application prospects in the fields of medicine, beauty, and skin care.

[0004] At present, there are two main ways to produce FN: traditional animal blood extraction. In 2005, Professor Wang Deliang of Zhengzhou Institute of Light Industry and Professor Zhang Junying of Henan Agricultural University pioneered the "Animal Blood FN Industrialized Co-production Process and Series Product Development" project, which broke through the bottleneck of FN industrialization and raised the purification and production of FN from the laboratory level to the industrial level, filling the gap in the relevant field in China. However, the naturally extracted fibronectin has a large molecular weight and is not uniform. The extracted protein pure product has a limited content and is difficult to form a large-scale production scale. In addition, non-human proteins may greatly increase the risk of immunogens. Another method is to produce through microbial fermentation. Since the natural FN has a large molecular weight and the feasibility of full sequence expression is low, the structural domains of different functions of FN can be selected for expression to obtain recombinant FN with different functions, and the active function is similar to that of humanized collagen. Chinese patent CN 117143224 B combines fibronectin fragments FNIII10 and FNIII1214 and studies their role in protecting and promoting stem cell activity. This provides a new idea for the research in this article to express different functional domains separately and combine different functional modules for recombinant expression to achieve specific efficacy. Summary of the invention

[0005] In order to remove redundant sequences that are prone to aggregation, the collagen domain of FN (FNI) is studied separately. The present invention provides a recombinant human fibronectin peptide and a preparation method and application thereof. The specific scheme of the present invention is as follows:

[0006] In a first aspect, the present invention provides a recombinant human fibronectin peptide, wherein the recombinant human fibronectin peptide comprises at least any one of the following:

[0007] A1) the recombinant human fibronectin peptide comprises the amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.4 or SEQ ID NO.6 or SEQ ID NO.8;

[0008] A2) is a protein obtained by replacing and / or deleting and / or adding amino acid residues in the amino acid sequence of the recombinant human fibronectin peptide in A1) and having more than 95% identity with the protein peptide in A1) and having the same function;

[0009] A3) A fusion protein having the same function is obtained by connecting a tag protein to the N-terminus and / or C-terminus of the recombinant human fibronectin peptide described in A1) and / or A2).

[0010] Preferably,

[0011] A2) the amino acid sequence of the recombinant human fibronectin peptide comprises SEQ ID NO.2 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.2; and / or

[0012] The amino acid sequence of the recombinant human fibronectin peptide comprises SEQ ID NO.4 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.4; and / or

[0013] The amino acid sequence of the recombinant human fibronectin peptide comprises SEQ ID NO.6 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.6; and / or

[0014] The amino acid sequence of the recombinant human fibronectin peptide comprises SEQ ID NO.8 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.8.

[0015] In a second aspect, the present invention provides a biomaterial, wherein the biomaterial is at least selected from any one of the following:

[0016] C1) containing a nucleic acid molecule encoding the recombinant human fibronectin peptide according to claim 1;

[0017] C2) an expression cassette containing the nucleic acid molecule;

[0018] C3) a recombinant vector containing the nucleic acid molecule, or a recombinant vector containing the expression cassette described in C2);

[0019] C4) a recombinant microorganism containing the nucleic acid molecule, or a recombinant microorganism containing the recombinant vector of the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3);

[0020] C5) A recombinant cell, wherein the recombinant cell contains the expression cassette described in C2) and the recombinant vector described in C3).

[0021] Furthermore, the nucleic acid molecule is at least selected from any one of the following:

[0022] B1) encoding the amino acid sequence shown in SEQ ID NO.2 includes the nucleotide sequence shown in SEQ ID NO.1 or has a homology of more than 95% with the nucleotide sequence shown in SEQ ID NO.1;

[0023] B2) encoding the amino acid sequence shown in SEQ ID NO.4 includes the nucleotide sequence shown in SEQ ID NO.3 or has a homology of more than 95% with the nucleotide sequence shown in SEQ ID NO.3;

[0024] B3) encoding the amino acid sequence shown in SEQ ID NO.6 includes the nucleotide sequence shown in SEQ ID NO.5 or has a homology of more than 95% with the nucleotide sequence shown in SEQ ID NO.5;

[0025] B4) The amino acid sequence encoding SEQ ID NO.8 includes the nucleotide sequence shown in SEQ ID NO.7 or has a homology of more than 95% with the nucleotide sequence shown in SEQ ID NO.7.

[0026] Preferably, the nucleic acid molecule comprises: a nucleotide sequence as shown in SEQ ID NO.1 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.1, and / or

[0027] The nucleic acid molecule comprises: a nucleotide sequence as shown in SEQ ID NO.3 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.3, and / or

[0028] The nucleic acid molecule comprises: a nucleotide sequence as shown in SEQ ID NO.5 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.5, and / or

[0029] The nucleic acid molecule includes: a nucleotide sequence as shown in SEQ ID NO.7 or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.7.

[0030] Those skilled in the art will appreciate that other commonly used expression elements can be adaptively added to the expression cassette described in the present application to assist in the expression of the target gene, such as adding tags, fluorescent protein markers, resistance screening markers, etc.

[0031] Optionally, the recombinant vector may include a nucleic acid molecule encoding the above protein, a promoter, and transcription and translation termination signals. The promoter or terminator for regulating expression in the present application may be a universal promoter or terminator, or a specific promoter or terminator in the host bacteria. Those skilled in the art may make adaptive selections according to actual conditions, and the present application does not impose mandatory restrictions on the sequence of the promoter or terminator.

[0032] The recombinant vector can be any vector (such as a plasmid or virus) that is convenient for recombinant DNA manipulation and expression of nucleic acid sequences. The selection of the vector usually depends on the compatibility of the vector with the host cell into which it will be introduced. The vector can be a linear or closed-loop plasmid. The vector can be an autonomous replicating vector (i.e., a complete structure present outside the chromosome that can be replicated independently of the chromosome), such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can include any mechanism that ensures self-replication. Alternatively, the vector is a vector that will be integrated into the genome and replicated together with the chromosome into which it is integrated when it is introduced into the host cell. In addition, a single vector or plasmid can be used, or two or more vectors or plasmids, or a transposon, that generally contain the entire DNA that will be introduced into the host cell genome. Preferably, the recombinant vector can be pET30a (+).

[0033] Furthermore, the recombinant microorganism or recombinant cell is selected from one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris, Hansenula, Candida, and mammalian cells.

[0034] Preferably, the recombinant microorganism is Escherichia coli.

[0035] In a preferred embodiment, the recombinant microorganism is Escherichia coli BL21 (DE3). It will be appreciated by those skilled in the art that conventional fermentation strains or any known industrial strains can be used as starting strains, as long as they can complete the expression of the recombinant fibronectin described in the present application, and the specific strains are not limited in detail herein.

[0036] In an optional embodiment, the expression cassette of the nucleic acid molecule is located on a recombinant vector or introduced into a recombinant microorganism using a recombinant vector. It should be noted that it is known to those skilled in the art that the nucleic acid molecule can be inserted into the genome of the starting strain or can exist on a plasmid in a free state, as long as the expression of the nucleic acid molecule or the synthesis of the recombinant human fibronectin peptide can be achieved.

[0037] In an optional embodiment, the recombinant vector and the recombinant microorganism contain a resistance screening marker gene, and those skilled in the art can select it according to actual conditions. The present application does not impose mandatory limitations on the resistance screening marker gene.

[0038] A resistance selection marker gene is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or confers prototrophy to auxotrophs, etc. Examples of bacterial selection markers are the dal genes of Bacillus subtilis or Bacillus licheniformis, or resistance markers to antibiotics such as ampicillin, kanamycin, chloramphenicol or tetracycline.

[0039] The third aspect of the present invention provides a method for producing the recombinant human fibronectin peptide, the method comprising the following steps:

[0040] (1) introducing the nucleic acid molecule into a target cell or target microorganism for expression;

[0041] (2) Collecting and / or purifying the recombinant human fibronectin peptide expressed in step (1).

[0042] Furthermore, the target cell or target microorganism in step (1) is at least one or more selected from Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris, Hansenula, Candida, and mammalian cells. Preferably, Escherichia coli.

[0043] Furthermore, the method further comprises the step of culturing the target cells or target microorganisms obtained in step (1).

[0044] Preferably, the amino acids of the recombinant human fibronectin peptide comprise the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence that is 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to SEQ ID NO.4; more preferably, the amino acid sequence of the recombinant human fibronectin peptide is as shown in SEQ ID NO.2; more preferably, the nucleotide sequence encoding the recombinant human fibronectin peptide is as shown in SEQ ID NO.1.

[0045] Preferably, the amino acids of the recombinant human fibronectin peptide comprise the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity with SEQ ID NO.4; more preferably, the amino acid sequence of the recombinant human fibronectin peptide is as shown in SEQ ID NO.4; more preferably, the nucleotide sequence encoding the recombinant human fibronectin peptide is as shown in SEQ ID NO.3.

[0046] Preferably, the amino acids of the recombinant human fibronectin peptide comprise the amino acid sequence shown in SEQ ID NO.6 or an amino acid sequence that is 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to SEQ ID NO.4; more preferably, the amino acid sequence of the recombinant human fibronectin peptide is as shown in SEQ ID NO.6; more preferably, the nucleotide sequence encoding the recombinant human fibronectin peptide is as shown in SEQ ID NO.5.

[0047] Preferably, the amino acids of the recombinant human fibronectin peptide comprise the amino acid sequence shown in SEQ ID NO.8 or an amino acid sequence that is 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to SEQ ID NO.4; more preferably, the amino acid sequence of the recombinant human fibronectin peptide is as shown in SEQ ID NO.8; more preferably, the nucleotide sequence encoding the recombinant human fibronectin peptide is as shown in SEQ ID NO.7.

[0048] In a specific embodiment of the present invention, the nucleic acid molecule described in step (1) further includes a solubility-promoting tag.

[0049] In a preferred embodiment, a method for preparing a recombinant human fibronectin peptide comprises:

[0050] Step 1, constructing a recombinant microorganism expressing the recombinant human fibronectin peptide;

[0051] Step 2: Cultivate the recombinant microbial cells at 25°C-37°C until OD 600 The pH value is about 0.8-1.0, and IPTG with a final concentration of 100 μM is added. The culture is induced by shaking at 25°C-37°C for 2-6 hours. The supernatant after centrifugation of the fermentation broth is purified to obtain the recombinant human fibronectin peptide.

[0052] The above purification process can be carried out using general methods.

[0053] In a preferred embodiment, the purification comprises: using a nickel ion affinity chromatography column to elute and collect the protein corresponding to the elution peak.

[0054] In a fourth aspect, the present invention provides a composition, which comprises at least the recombinant human fibronectin peptide or the nucleic acid molecule or the biomaterial or the recombinant human fibronectin peptide prepared by the method described above. Furthermore, the composition also comprises an acceptable auxiliary material.

[0055] In a fifth aspect, the present invention provides the use of the recombinant human fibronectin peptide, the nucleic acid molecule, the biomaterial, or the recombinant human fibronectin peptide prepared by the method, or the composition, wherein the use includes at least any one of the following:

[0056] D1) Application in the preparation of products promoting cell adhesion;

[0057] D2) Application in the preparation of products promoting cell proliferation;

[0058] D3) Application in the preparation of products promoting cell migration;

[0059] D4) Application in the preparation of products for promoting collagen production;

[0060] D5) Application in the preparation of products for repairing skin damage;

[0061] D6) Application in the preparation of tissue regeneration products;

[0062] D7) Use in the preparation of products promoting anti-aging and / or anti-wrinkle;

[0063] D8) Application in the preparation of skin tightening products;

[0064] D9) Application in the field of daily chemicals;

[0065] D10) Application in the field of medicine and equipment;

[0066] D11) Application in engineering tissue materials.

[0067] Regarding D2) above, the experiments in this application prove that the recombinant human fibronectin peptide has no cytotoxicity and promotes cell proliferation to a certain extent.

[0068] In a preferred embodiment, the concentration of the recombinant human fibronectin peptide is 0.001 mg / mL-10 mg / mL; preferably, 0.05 mg / mL-0.8 mg / mL.

[0069] The concentration of the recombinant human fibronectin peptide can be 0.001 mg / mL, 0.002 mg / mL, 0.003 mg / mL, 0.004 mg / mL, 0.005 mg / mL, 0.006 mg / mL, 0.007 mg / mL, 0.008 mg / mL, 0.009 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.0 7mg / mL, 0.08mg / mL, 0.09mg / mL, 0.1mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, 0.5mg / mL, 0.6mg / mL, 0.7mg / mL , 0.8mg / mL, 0.9mg / mL, 1mg / mL, 2mg / mL, 3mg / mL, 4mg / mL, 5mg / mL, 6mg / mL, 7mg / mL, 8mg / mL, 9mg / mL, 10mg / mL.

[0070] Those skilled in the art can adjust the concentration of the recombinant human fibronectin peptide according to actual conditions, and this application does not impose any specific limitation on this.

[0071] With respect to the above D5) and D6), the repair of skin damage and / or tissue regeneration is achieved by promoting cell proliferation and / or epidermal cell migration.

[0072] Preferably, the amino acid sequence of the recombinant human fibronectin peptide is as shown in SEQ ID NO.2; more preferably, the nucleotide sequence encoding the recombinant human fibronectin peptide is as shown in SEQ ID NO.1.

[0073] Preferably, the amino acid sequence of the recombinant human fibronectin peptide is as shown in SEQ ID NO.4; more preferably, the nucleotide sequence encoding the recombinant human fibronectin peptide is as shown in SEQ ID NO.3.

[0074] Preferably, the amino acid sequence of the recombinant human fibronectin peptide is as shown in SEQ ID NO.6; more preferably, the nucleotide sequence encoding the recombinant human fibronectin peptide is as shown in SEQ ID NO.5.

[0075] Preferably, the amino acid sequence of the recombinant human fibronectin peptide is as shown in SEQ ID NO.8; more preferably, the nucleotide sequence encoding the recombinant human fibronectin peptide is as shown in SEQ ID NO.7.

[0076] With respect to D4) and D7) above, the anti-aging and / or anti-wrinkle effect is achieved by promoting the expression of collagen. Preferably, the collagen includes type I collagen and / or type III collagen.

[0077] Preferably, the amino acid sequence of the recombinant human fibronectin peptide is as shown in SEQ ID NO.8; more preferably, the nucleotide sequence encoding the recombinant human fibronectin peptide is as shown in SEQ ID NO.7.

[0078] The beneficial effects of the present invention include but are not limited to:

[0079] 1. The present invention provides a highly active recombinant human fibronectin peptide, which has a lower molecular weight than traditional fibronectin (FN), removes redundant sequences that are prone to aggregation, and has the characteristics of low immunogenicity risk and high safety;

[0080] 2. The recombinant human fibronectin peptide of the present invention can promote cell adhesion and / or proliferation and / or migration. At the same time, the recombinant human fibronectin peptide FN4 has good thermal stability and can be widely used in the preparation of cosmetics, medical devices, tissue engineering materials or their raw materials, and has broad application prospects;

[0081] 3. The present invention screens and obtains recombinant human fibronectin peptides through genetic engineering and fermentation engineering as well as adding solubility-promoting tags, and realizes soluble intracellular expression in an engineered strain of Escherichia coli, and then obtains highly active recombinant human fibronectin peptides through a purification process. The preparation process is simple, the stability is high, and it can be applied to large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0083] Figure 1 This is a schematic diagram of the expression effect of recombinant human fibronectin peptides FN1, FN2, FN3 and FN4 detected by SDS-PAGE in an embodiment of the present invention;

[0084] Figure 2 This is a schematic diagram of the purification effect of recombinant human fibronectin peptides FN1, FN2, FN3 and FN4 detected by SDS-PAGE in an embodiment of the present invention;

[0085] Figure 3 Schematic diagram of the adhesion rate of recombinant human fibronectin peptides FN1, FN2, FN3 and FN4 to mouse fibroblasts L929 in the embodiment of the present invention

[0086] Figure 4 This is a schematic diagram of the results of promoting the proliferation rate of mouse fibroblast L929 by recombinant human fibronectin peptides FN1, FN2, FN3 and FN4 in the embodiments of the present invention;

[0087] Figure 5 This is a schematic diagram of the results of photographing the migration rate of mouse fibroblast L929 cells by recombinant human fibronectin peptides FN1, FN2, FN3 and FN4 in a 24-well plate in an embodiment of the present invention;

[0088] Figure 6 Schematic diagram of the results of promoting the migration rate of mouse fibroblast L929 by recombinant human fibronectin peptides FN1, FN2, FN3 and FN4 in the embodiments of the present invention;

[0089] Figure 7 This is a schematic diagram of the results of the recombinant human fibronectin peptide FN4 promoting the expression of type I collagen in mouse fibroblasts L929 in an embodiment of the present invention;

[0090] Figure 8 This is a schematic diagram of the results of the recombinant human fibronectin peptide FN4 promoting the expression of type III collagen in mouse fibroblasts L929 in an embodiment of the present invention;

[0091] Fig. 9 Schematic diagram of the results of the thermal stability study of the recombinant human fibronectin peptide FN4 in the examples of the present invention. DETAILED DESCRIPTION

[0092] The present invention is described in detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present invention are purchased through commercial channels.

[0093] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or between the amino acid sequences of two protein molecules in molecular evolution research.

[0094] Recombination: Broadly speaking, any gene exchange process that causes genotype changes is called recombination.

[0095] Expression cassette: An expression cassette is a group of DNA sequences consisting of a promoter, a target gene, and a reporter gene, which can be expressed in specific tissues and easily detected.

[0096] Recombinant vector: A recombinant vector is a vector that transfers the target gene into the basic skeleton of a cloning vector, thereby enabling the target gene to be expressed.

[0097] Recombinant microorganism: A fungal cell strain that uses genetic engineering methods to efficiently express exogenous genes.

[0098] Recombinant cell: The term "recombinant cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "recombinant cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0099] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0100] Unless otherwise specified, in the following embodiments, reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market. If no specific conditions are indicated in the examples, conventional conditions or conditions recommended by the manufacturer are followed.

[0101] The plasmids, endonucleases, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products, and the specific operations are performed in accordance with the kit instructions.

[0102] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields, and can be specifically performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0103] In addition, the "water" mentioned in the present invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion exchange water, double distilled water, high-purity water, and purified water.

[0104] Example 1: Construction of E. coli expression vector of recombinant human fibronectin peptide

[0105] According to the amino acid sequence information of human fibronectin in the NCBI genebank database, a series of recombinant human fibronectin peptides were screened and recombined by analyzing the spatial structure of human fibronectin using bioinformatics methods, and were named FN1, FN2, FN3 and FN4, respectively. The amino acid sequences were shown in SEQ ID NO.2 or SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8, respectively. The obtained recombinant human fibronectin peptides were optimized according to the codon preference of Escherichia coli to obtain the corresponding nucleotide sequence, and the specific information is shown in Table 1. The whole gene was synthesized according to the codon preference of Escherichia coli (the gene synthesis work was completed by Beijing Qingke Biotechnology Co., Ltd.). The HIS purification tag and MBP solubility-promoting tag were fused to the N-terminus of the target gene, and the fusion gene was synthesized on the expression vector pET30a, and the recombinant vectors were named pET30a-FN1, pET30a-FN2, pET30a-FN3 and pET30a-FN4.

[0106] Table 1

[0107] name Amino acid sequence Nucleotide sequence FN1 As shown in SEQ ID NO.2 As shown in SEQ ID NO.1 FN2 As shown in SEQ ID NO.4 As shown in SEQ ID NO.3 FN3 As shown in SEQ ID NO.6 As shown in SEQ ID NO.5 FN4 As shown in SEQ ID NO.8 As shown in SEQ ID NO.7

[0108] Example 2: Expression of recombinant human fibronectin peptide in Escherichia coli:

[0109] The recombinant vectors pET30a-FN1, pET30a-FN2, pET30a-FN3 and pET30a-FN4 were transformed into Escherichia coli BL21 (DE3) by the heat shock method, respectively, and spread on LB (containing 50 μg / μL kanamycin) plates containing 1.5% agar to obtain recombinant Escherichia coli BL21 strains expressing FN1, FN2, FN3 and FN4 recombinant fibronectin.

[0110] A single clone of the expression strain constructed above was selected and cultured overnight in LB liquid medium (containing 50 μg / mL kanamycin resistance), and then transferred to fresh LB liquid medium (containing 50 μg / mL kanamycin resistance) and cultured at OD 600 =0.8, add IPTG with a final concentration of 100 μM, and induce the expression of the target protein at 37°C. After culturing for 4 hours, collect the bacterial cells and use ddH 2 After resuspending the cells at OD, centrifuge again to wash the cells, and resuspend the cells again in lysis buffer (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole) to adjust the OD 600 =100, use ultrasonic crusher to break for 10 minutes, centrifuge to remove the precipitate, and keep the supernatant as the crude protein solution. The crude protein solution was analyzed by SDS-PAGE electrophoresis.

[0111] The experimental results are as follows Figure 1As shown, the results of SDS PAGE experiments showed that recombinant human fibronectin peptides FN1, FN2, FN3 and FN4 were successfully expressed in BL21 (DE3), the positions of the target bands were consistent with the expected results, and good soluble expression was achieved.

[0112] Example 3: Recombinant human fibronectin peptide affinity chromatography purification

[0113] The crude protein solution obtained above was purified by nickel ion affinity chromatography, and the purification process was as follows:

[0114] Rinse the column with 20 mL of water;

[0115] Equilibrate the column with 20 mL binding buffer;

[0116] After the broken bacterial solution is centrifuged, the supernatant is passed through the column;

[0117] Rinse with 20 mL binding buffer and collect the eluate;

[0118] Rinse with Tris-HCl buffer containing 50 mM imidazole to clean the impurities;

[0119] The column was washed with Tris-HCl buffer containing 200 mM imidazole, and the eluate was collected, which was the purified sucrose phosphorylase;

[0120] The column was washed with Tris-HCl buffer containing 500 mM imidazole to remove all proteins from the column;

[0121] The column was rinsed with water and stored in 20% ethanol.

[0122] The purified sucrose phosphorylase obtained by the above method was detected by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). Figure 2 The purified recombinant human fibronectin peptide was stored at -20°C for subsequent determination.

[0123] The results are as follows Figure 2 As shown, the SDS PAGE results showed that the recombinant human fibronectin peptides FN1-FN4 with a purity greater than 90% were obtained by the purification method.

[0124] Example 3: Cellular efficacy assay

[0125] 3.1 Determination of cell adhesion efficacy

[0126] Protein preparation: FN1-FN4 and commercially available protein (QD-B-240620-01) were uniformly diluted to 0.3 g / L with PBS solution and filtered with a 0.22 μm filter membrane for later use. The protein concentration was set at 0.005 g / L for adhesion experiments.

[0127] Protein coating: add 100 μL PBS solution (0.22 μm filtered) to the outer circle of the ELISA plate, add 100 μL prepared protein solution to the inner circle, and coat the ELISA plate at 37°C for 2 h.

[0128] BSA blocking: prepare 5% BSA-V protein solution (0.22 μm filtered), add 100 μL directly to the ELISA plate without washing, and block at 37°C for 1 h. At the same time, add 100 μL BSA solution to the PBS solution as a blank control.

[0129] Process and count cells: Remove the serum culture medium of mouse fibroblasts (L929) (purchased from Wuhan Pronocell Life Science Co., Ltd.) grown to 80%-90%, collect the cells after trypsin digestion, and resuspend them in 2 mL MEM. At this time, the number of cells is about 10 6 Take 100 μL of trypan blue, add 80 μL of PBS solution, and then add 20 μL of cell solution. Take 20 μL of the cell solution diluted 10 times and add it to the cell counting plate for counting. Perform a second dilution based on the average count value to dilute the cell concentration to 1×10 6 / mL, for future use.

[0130] Washing to remove protein: Aspirate the protein from the coated ELISA plate and wash twice with MEM culture medium.

[0131] Cell incubation: Add 100 μL of treated cell solution to the washed ELISA plate (add slowly and mix in time), and add 100 μL (record the total number of cells) to the blank ELISA plate and incubate for 1 hour.

[0132] Incubate in dark for color development: Aspirate the cell solution in the ELISA plate, wash four times with 100 μL PBS solution, prepare a color development solution with a ratio of 1:10 of MEM and Cell Counting Kit 8, add 100 μL of color development solution in dark, wrap with tin foil and incubate at 37°C for 1 hour.

[0133] A450 detection: Detect the absorbance of the ELISA plate at 450nm.

[0134] Calculation of cell adhesion rate:

[0135] Relative adhesion rate (%) = (Mean value of sample group - Mean value of blank group) / (Mean value of control group - Mean value of blank group) × 100%

[0136] The mean value is the absorbance value of the ELISA plate detected at 450 nm.

[0137] like Figure 3The results showed that after 24 hours of action of recombinant human fibronectin peptide, the relative adhesion rate of FN2 to L929 cells was 768.97%, the relative adhesion rate of FN1 to L929 cells also reached 625.67%, the relative adhesion rate of FN3 to L929 cells reached 749.71%, and FN4 had the highest adhesion promoting effect on L929 cells, with a relative adhesion rate of 777.54%. Overall, the cell adhesion promoting effects of the four recombinant human fibronectin peptide sequences in this experiment were much higher than the 241% of the positive control bovine collagen, while the adhesion promoting rate of other commercially available products was 376.73%, which was also better than the positive control, but only half of the effect of the recombinant human fibronectin peptide in this experiment.

[0138] 3.2 Determination of cell proliferation efficacy

[0139] 96-well plate culture cells (plating): L929 cells in logarithmic growth phase were obtained, digested with trypsin, resuspended in MEM high-glucose culture medium (added with 10% fetal bovine serum) and the cell density was adjusted to 1×10 5 The cells were inoculated at a density of 100 μL / mL in a 96-well plate. After inoculation, the plate was placed in a carbon dioxide incubator at 37°C and 5% CO 2 Routine culture for 24 hours.

[0140] Preparation of protein sample culture medium: Dilute FN1, FN2, FN3 and FN4 proteins and commercially available proteins to 0.3 g / L with PBS solution, and filter with a 0.22 μm filter membrane for later use. Prepare 0.05 g / L sample solution with serum-free culture medium.

[0141] Dosing: After culturing cells in 96-well plates for 24 hours, discard the old culture medium, add 100 μL protein sample culture medium containing different concentrations to the experimental group, add culture medium without sample to the negative control group, and add culture medium supplemented with 10% fetal bovine serum to the positive control group. Set up 5 parallels for each sample.

[0142] Detection: After culturing for 24 hours, discard the culture medium, add 100 μL of CCK-8 diluted 10 times with serum-free culture medium to each well, place in the cell culture incubator and continue incubation for 1 hour, and measure the absorbance at a wavelength of 450 nm with an enzyme marker.

[0143] Relative proliferation rate (RGR) = [As / Ac] × 100% where As: absorbance of the experimental group; Ac: absorbance of the negative control group.

[0144] like Figure 4The results showed that FN1 had a good effect on L929 cell proliferation, with a relative proliferation rate of 124%, and FN4 had a relative proliferation rate of 122%, which was close to the positive control, which had a cell proliferation effect of 131% with the addition of 10% fetal bovine serum. FN2 also had a certain cell proliferation effect, with a relative proliferation rate of 115%, and FN3 also had a certain proliferation effect, which was slightly better than the commercially available products. However, other commercially available products FN (QD-B-240620-01) at the same concentration had no obvious effect on promoting proliferation of L929.

[0145] 3.3 Determination of cell migration efficacy

[0146] Plating: Place the ibidi chamber in a new 24-well culture plate (place it as straight as possible for easy photography, with the bottom of the chamber just touching the bottom of the 24-well plate). Take L929 cells in the logarithmic growth phase, digest them and incubate at 3×10 5 The cells were inoculated at a density of 100 / mL in the ibidi chamber in a 24-well plate. 70 μL of cell suspension was added to each of the left and right chambers. Add as slowly as possible, otherwise the cells will be unevenly dispersed. 400 μL of MEM culture medium was added outside the chamber and incubated at 37°C and 5% CO 2 Cultured under the conditions for 24 h.

[0147] Sample solution preparation: Prepare FN1-FN4 and commercially available protein sample solutions at a concentration of 0.05 g / L in serum-free culture medium, filter through a 0.22 μm filter membrane for sterilization, and use immediately after preparation.

[0148] Dosing: Gently use tweezers to remove the chamber, take pictures to observe whether the cells are full (if the cells are not full, the cell plating concentration can be appropriately increased in the next experiment), aspirate and discard the old culture medium, wash the cells 3 times with preheated 1× PBS, add 1mL of protein sample solution, replace the negative control group with culture medium without sample, and the positive control is EGF (10mg / L).

[0149] Photographing: After continuing to culture for a certain period of time, about 24 hours, observe the 24-well plate and take photos. The area of ​​the scratched area in the photo was analyzed using Image J software.

[0150] Cell migration rate (%) = (1-scratch area after drug addition / initial scratch area) × 100%;

[0151] Relative migration rate (%) = migration rate of cells in the experimental group / migration rate of cells in the negative control group × 100%.

[0152] The results are as follows Figure 5 and Figure 6As shown in the figure, after the protein was incubated with L929 cells for 24 hours, FN1 had a good effect on the migration of L929 cells, and the relative migration rate after 24 hours of culture reached 165.91%, which was higher than the positive control EGF's cell migration effect of 121.88%; FN2 and FN3 had a certain cell migration effect on L929 cells, which was basically the same as the effect of commercially available products. The cell migration effect of FN4 was the most prominent. Compared with the negative control, FN4 promoted the migration rate of L929 cells by 278%, which was much better than the positive control EGF's 122%, while other commercially available products FN (QD-B-240620-01) did not detect obvious L929 cell migration effects.

[0153] 3.4 Determination of the efficacy of promoting collagen production

[0154] (1) Plating: After L929 cells were cultured to the logarithmic phase, a new 6-well plate was taken and the digestion count was 2.2×10 5 Cells / ml were inoculated into 6-well plates, and 2 mL was inoculated into each well. 1 mL was added each time, and then 1 mL was added after shaking to make the cells evenly distributed. Incubate at 37°C and 5% CO 2 Cultured for 24 h under the same conditions;

[0155] (2) Dosing: After culturing for 24 h, use serum-free MEM medium to prepare the protein sample solution. Based on the above experimental results, only 0.05 g / L FN4 protein solution was prepared this time, and the solution was sterilized by filtration with a filter membrane. The cultured cells were slowly aspirated without washing, and 2 mL of protein was added to each well (two parallel wells) to treat the cells.

[0156] (3) Sampling: After 6 h, 12 h, and 24 h, the residual protein solution was washed with 1xPBS solution for 3 times, and the cells were collected using RNA-easyTM Isolation Reagent (500 μL) in the RNA extraction kit of FastPure / Tissue Total RNA Isolation Kit V2 of Nanjing Novazon Biotechnology Co., Ltd. The cells were viscous during collection and were transferred to a 1.5 mL centrifuge tube and frozen at -80°C as soon as possible;

[0157] (4) RNA extraction: The cell samples frozen at -80°C were taken out, and the total RNA of each cell sample was extracted using the FastPure / Tissue Total RNA Isolation Kit V2 kit (Nanjing Novezan Biotechnology Co., Ltd.), and the concentration of the extracted RNA was determined.

[0158] (5) Reverse transcription: using the Novagen HiScript III RT SuperMix for qPCR (+gDNA wiper) kit.

[0159] (6) qPCR reaction: The reaction was performed using the ChamQ Universal SYBR qPCR Master Mix kit from Norvegic. GAPDH was used as the internal reference gene, and type I and type III collagen were selected for expression measurement. The specific primer sequences are shown in Table 2.

[0160] Table 2

[0161]

[0162] The results are as follows Figure 7 and Figure 8 Compared with the negative control, FN1 and FN4 had significant effects on promoting the production of type I collagen and type III collagen after being co-incubated with cells for 12 hours. Among them, the relative expression of type I collagen was 138% and the relative expression of type III collagen was 137% after being co-incubated with cells for 12 hours.

[0163] After 12 hours of cell co-incubation, the relative expression of type Ⅰ collagen was 117%, while the relative expression of type Ⅲ collagen was 144%.

[0164] Example 4: Stability study of recombinant human fibronectin

[0165] The recombinant human fibronectin peptide FN4 obtained above was placed at 80°C and 100°C for 10 min and 20 min, respectively. The protein residue was analyzed using Image J software after SDS-PAGE detection. The results are shown in Fig. 9 . Fig. 9 The results showed that after the recombinant human fibronectin peptide was placed at 80°C for 10 minutes, 93.23% of the protein remained, and after 20 minutes, 78.85% of the protein remained; after 100°C for 10 minutes, 88.73% of the protein remained, and after 20 minutes,

[0166] The residual enzyme activity was 71.45%, which proved that the recombinant human fibronectin FN4 of the present invention had excellent thermal stability.

[0167] The above description is only an embodiment of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent substitution, improvement, etc. made within the technical ideas and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A recombinant human fibronectin peptide, characterized in that: The recombinant human fibronectin peptide comprises at least any one of the following: A1) the recombinant human fibronectin peptide contains the amino acid sequence shown in SEQ ID NO.2 or SEQ ID NO.4 or SEQ ID NO.6 or SEQ ID NO.8; A2) is a protein obtained by replacing and / or deleting and / or adding amino acid residues in the amino acid sequence of the recombinant human fibronectin peptide in A1) and having more than 95% identity with the protein peptide in A1) and having the same function; A3) A fusion protein having the same function is obtained by connecting a tag protein to the N-terminus and / or C-terminus of the recombinant human fibronectin peptide described in A1) and / or A2).

2. Biomaterial, characterized in that The biological material includes at least one of the following: B1) containing a nucleic acid molecule encoding the recombinant human fibronectin peptide according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule of B1), or a recombinant microorganism containing the recombinant vector of the expression cassette of B2), or a recombinant microorganism containing the recombinant vector of B3); B5) a recombinant cell, said recombinant cell comprising the nucleic acid molecule of B1), or a recombinant cell comprising the recombinant vector of the expression cassette of B2), or a recombinant cell comprising the recombinant vector of B3), or a recombinant cell comprising the recombinant microorganism of B4).

3. The biomaterial according to claim 2, characterized in that The B1) nucleic acid molecule includes at least any one of the following: C1) comprising the nucleotide sequence shown in SEQ ID NO.1 or a nucleotide sequence having 95% or more identity with the nucleotide sequence shown in SEQ ID NO.1 and encoding the same protein; C2) comprising the nucleotide sequence shown in SEQ ID NO.3 or a nucleotide sequence having 95% or more identity with the nucleotide sequence shown in SEQ ID NO.3 and encoding the same protein; C3) comprising the nucleotide sequence shown in SEQ ID NO.5 or a nucleotide sequence having 95% or more identity with the nucleotide sequence shown in SEQ ID NO.5 and encoding the same protein; C4) includes the nucleotide sequence shown in SEQ ID NO.7 or a nucleotide sequence that has more than 95% identity with the nucleotide sequence shown in SEQ ID NO.7 and encodes the same protein.

4. A method for producing recombinant human fibronectin peptide, characterized in that: The method at least comprises constructing a recombinant microorganism expressing the recombinant human fibronectin peptide according to claim 1, and culturing the recombinant microorganism.

5. The method according to claim 4, characterized in that The recombinant microorganism is at least selected from one or more of Escherichia coli, Corynebacterium glutamicum, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris, Hansenula, and Candida.

6. A composition, characterized in that The composition at least comprises the recombinant human fibronectin peptide according to claim 1 or the recombinant human fibronectin prepared by the method according to any one of claims 4-5.

7. Use of the recombinant human fibronectin peptide according to claim 1, or the biomaterial according to claims 2-3, or the recombinant human fibronectin prepared by any of the methods of claims 4-5, or the composition according to claim 6, characterized in that: The application includes at least any of the following: D1) Application in the preparation of products promoting cell adhesion; D2) Application in the preparation of products promoting cell proliferation; D3) Application in the preparation of products promoting cell migration; D4) Application in the preparation of products promoting collagen production.

8. Use of the recombinant human fibronectin peptide according to claim 1, or the biomaterial according to claims 2-3, or the recombinant human fibronectin prepared by the method according to any one of claims 4-5, or the composition according to claim 6, characterized in that: The application includes at least any of the following: E1) Application in the preparation of products for repairing skin damage; E2) Application in the preparation of tissue regeneration products; E3) Use in the preparation of products promoting anti-aging and / or anti-wrinkle; E4) in the preparation of skin firming products.

9. Use of the recombinant human fibronectin peptide according to claim 1, or the biomaterial according to claims 2-3, or the recombinant human fibronectin prepared by the method according to any one of claims 4-5, or the composition according to claim 6, characterized in that: The application includes at least any of the following: F1) Application in daily chemical field; F2) Application in the field of medicine and medical devices; F3) Application in engineering tissue materials.

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