Recombinant humanized elastin, its preparation method and application

Abstract

This invention provides recombinant humanized elastin, its preparation method, and its applications, relating to the field of bioengineering technology. Based on humanized elastin, this invention utilizes a combination design based on the functional domains of elastin to obtain recombinant humanized elastin, discovering novel bioactive functional fragments of humanized elastin. The recombinant humanized elastin of this invention integrates multiple elastin functional sites. Specifically, this recombinant humanized elastin exhibits significant effects in promoting cell proliferation and increasing the expression of collagen and elastin genes, demonstrating high safety and wide applicability in various scenarios, and providing a new active ingredient for anti-aging related products. Furthermore, the theoretical molecular weight of this recombinant humanized elastin is significantly smaller than that of natural full-length elastin, reducing obstacles for elastin to cross the skin barrier and exert its biological functions, thus exhibiting excellent biological efficacy.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, and in particular to recombinant humanized elastin, its preparation method, and its application. Background Technology

[0002] Elastin is the main component of elastic fibers. Nonpolar amino acids (hydrophobic amino acids) account for 95% of the elastin molecule, glycine accounts for 1 / 3 of the total, proline accounts for 10%, and hydroxyproline accounts for 1%, giving it resistance to acid and alkali hydrolysis. Elastic fibers are mainly found in ligaments and blood vessel walls, coexisting with collagen fibers, giving tissues elasticity and tensile strength. Elastin and its degradation products play an important role in the skin aging process, such as in cell interactions and signal transduction. Elastin maintains and supports skin elasticity and a certain degree of resilience, allowing the skin to stretch and curl freely, much like the springs in a mattress. Therefore, adding elastin to cosmetics is significant in maintaining skin elasticity. Currently, the common method for preparing animal-derived elastin is enzymatic hydrolysis of elastin-rich tissues from animals. The resulting elastin product is a soluble polypeptide chain. However, the composition of elastin extracted from animals is complex, and batch-to-batch variations lead to quality differences, making safety and efficacy uncertain.

[0003] In the invention patent CN102241747A, an elastin sequence with (VAPGVG)3S as the basic unit and highly repeated was constructed. However, this elastin sequence is just a simple repetition of VAPGVG and has a single functional site. Summary of the Invention

[0004] To address the aforementioned issues, this application presents a recombinant humanized elastin prepared using bioengineering and synthetic biology methods. This avoids some of the potential problems associated with traditional extraction methods. The protein integrates multiple functional sites according to the elastin sequence itself, resulting in a rich array of functional sites. It exhibits significant effects in promoting cell proliferation and the expression of HSF collagen and elastin genes in human fibroblasts, and can be widely applied in fields such as medical devices and cosmetics.

[0005] On the one hand, this application provides recombinant humanized elastin, wherein the amino acid sequence of said recombinant humanized elastin includes any one or more of the following A1)-A5):

[0006] A1) The amino acid sequence of the recombinant humanized elastin comprises the sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 95% identity with SEQ ID NO.1;

[0007] A2) The amino acid sequence of the recombinant humanized elastin comprises the sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 95% identity with SEQ ID NO.2;

[0008] A3) The amino acid sequence of the recombinant humanized elastin comprises the sequence shown in SEQ ID NO.3 or an amino acid sequence having at least 95% identity with SEQ ID NO.3;

[0009] A4) The amino acid sequence of the recombinant humanized elastin comprises the sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 95% identity with SEQ ID NO.4;

[0010] A5) The amino acid sequence of the recombinant humanized elastin contains the sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 95% identity with SEQ ID NO.5.

[0011] Preferred,

[0012] a1) The amino acid sequence of the recombinant humanized elastin comprises the sequence shown in SEQ ID NO.1 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%, or 99.9% identity with SEQ ID NO.1; more preferably, the amino acid sequence of the recombinant humanized elastin is as shown in SEQ ID NO.1; more preferably, the nucleotide sequence encoding the recombinant humanized elastin is as shown in SEQ ID NO.6;

[0013] a2) The amino acid sequence of the recombinant humanized elastin comprises the sequence shown in 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%, or 99.9% identity with SEQ ID NO.2; more preferably, the amino acid sequence of the recombinant humanized elastin is as shown in SEQ ID NO.2; more preferably, the nucleotide sequence encoding the recombinant humanized elastin is as shown in SEQ ID NO.7;

[0014] a3) The amino acid sequence of the recombinant humanized elastin comprises the sequence shown in SEQ ID NO.3 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%, or 99.9% identity with SEQ ID NO.3; more preferably, the amino acid sequence of the recombinant humanized elastin is as shown in SEQ ID NO.3; more preferably, the nucleotide sequence encoding the recombinant humanized elastin is as shown in SEQ ID NO.8;

[0015] a4) The amino acid sequence of the recombinant humanized elastin comprises the 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%, or 99.9% identity with SEQ ID NO.4; more preferably, the amino acid sequence of the recombinant humanized elastin is as shown in SEQ ID NO.4; more preferably, the nucleotide sequence encoding the recombinant humanized elastin is as shown in SEQ ID NO.9;

[0016] a5) The amino acid sequence of the recombinant humanized elastin comprises the sequence shown in SEQ ID NO. 5 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%, or 99.9% identity with SEQ ID NO. 5; more preferably, the amino acid sequence of the recombinant humanized elastin is as shown in SEQ ID NO. 5; more preferably, the nucleotide sequence encoding the recombinant humanized elastin is as shown in SEQ ID NO. 10.

[0017] It is understood that those skilled in the art can select a suitable gene editing system and gene editing method to complete the construction of the above-mentioned recombinant humanized elastin according to the actual situation, or they can choose to use a general method to prepare the recombinant humanized elastin.

[0018] Furthermore, the N-terminus of the recombinant humanized elastin contains an amino acid sequence DDDDK that can be cleaved by enterokinase protease. Preferably, the DDDDK sequence is directly linked to the N-terminus of the protein sequence.

[0019] Those skilled in the art will understand that known modifications and optimizations can be made without affecting the function of the recombinant humanized elastin, and this application does not impose any mandatory limitations in this regard.

[0020] On the other hand, this application also provides biological materials, said biological materials comprising any one of the following B1)-B5):

[0021] B1) A nucleic acid molecule, said nucleic acid molecule containing a nucleic acid molecule encoding said recombinant humanized elastin;

[0022] B2) Expression cassette, wherein the expression cassette contains the nucleic acid molecule described in B1);

[0023] B3) A recombinant vector containing the nucleic acid molecule described in B1) and / or the expression cassette described in B2);

[0024] B4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3);

[0025] B5) Recombinant cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3).

[0026] Further, the nucleic acid molecule comprises: a nucleotide sequence as shown in SEQ ID NO. 6 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 6, and / or a nucleotide sequence as shown in SEQ ID NO. 7 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 7, and / or a nucleotide sequence as shown in SEQ ID NO. 8 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 8, and / or a nucleotide sequence as shown in SEQ ID NO. 9 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 9, and / or a nucleotide sequence as shown in SEQ ID NO. 10 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 10.

[0027] Preferably, the nucleic acid molecule comprises: a nucleotide sequence as shown in SEQ ID NO. 6 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%, or 99.9% identity with SEQ ID NO. 6, and / or 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%, 99.8%, or 99.9% identity with SEQ ID NO. 6, and / or 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%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 6, and / or a nucleotide sequence having 95%, 96%, 97%, 98%, 98.1%, 98.2%, 98.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO. 6, and / or a nucleotide sequence having 95%, 98.1%, 98.2%, 98.3%, 99.4%, 99.5%, 99. NO.7 has a nucleotide sequence with 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%, and 99.9% identity, and / or a nucleotide sequence as shown in SEQ ID NO.8 or the same as SEQ ID NO. NO.8 has a nucleotide sequence with 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%, and 99.9% identity, and / or a nucleotide sequence as shown in SEQ ID NO.9 or the same as SEQ ID NO. NO.9 has a nucleotide sequence with 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%, and 99.9% identity, and / or a nucleotide sequence as shown in SEQ ID NO.10 or the same as SEQ ID NO. NO.10 has nucleotide sequences with 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%, and 99.9% identity.

[0028] Those skilled in the art will recognize that other commonly used expression elements can be adaptively added to the expression cassette described in this application to assist in the expression of the target gene, such as adding tags, fluorescent protein markers, and resistance selection markers.

[0029] Optionally, the recombinant vector may include a nucleic acid molecule encoding the aforementioned protein, a promoter, and transcription and translation termination signals. The promoters or terminators used to regulate expression in this application may be general promoters or terminators, or they may be host-specific promoters or terminators. Those skilled in the art can make adaptive selections according to the actual situation. This application does not impose mandatory limitations on the sequences of promoters or terminators.

[0030] The recombinant vector can be any vector (e.g., plasmid or virus) that facilitates recombinant DNA manipulation and the expression of nucleic acid sequences. The choice of vector typically depends on its compatibility with the host cell to which it will be introduced. The vector can be a linear or closed circular plasmid. The vector can be a self-replicating vector (i.e., a complete structure existing outside the chromosome that can replicate independently of the chromosome), such as plasmids, extrachromosomal elements, minichromosomes, or artificial chromosomes. The vector can contain any mechanism that ensures self-replication. Alternatively, the vector is a vector that, when introduced into a host cell, will integrate into the genome and replicate along with the integrated chromosome. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, can be used, collectively containing the entire DNA to be introduced into the host cell's genome. Preferably, the recombinant vector is pET32a(+).

[0031] Furthermore, the recombinant microorganism is selected from one or more of Escherichia coli, Streptococcus, Bacillus, Saccharomyces cerevisiae, and Pichia pastoris.

[0032] Preferably, the recombinant microorganism is Escherichia coli.

[0033] In a preferred embodiment, the recombinant microorganism is *Escherichia coli* BL21(DE3). Those skilled in the art will understand that conventional fermentation strains or any known industrial strain can be used as the starting strain, as long as they can complete the expression of the recombinant humanized elastin described in this application; no specific strain is limited here.

[0034] In one 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, as will be apparent to those skilled in the art, the nucleic acid molecule can be selectively inserted into the genome of the starting strain or can exist on a free plasmid, as long as the expression of the nucleic acid molecule or the synthesis of recombinant humanized elastin can be achieved.

[0035] In one optional embodiment, the recombinant vector and recombinant microorganism contain an resistance selection marker gene. Those skilled in the art can choose according to the actual situation. This application does not impose mandatory limitations on the resistance selection marker gene.

[0036] A resistance selection marker gene is a gene whose product confers resistance to biocides or viruses, resistance to heavy metals, or a protrophic auxotrophic phenotype. Examples of bacterial selection markers include the dal gene in Bacillus subtilis or Bacillus licheniformis, or resistance markers for antibiotics such as ampicillin, kanamycin, chloramphenicol, or tetracycline.

[0037] On the other hand, this application also provides a method for preparing recombinant humanized elastin, the method comprising: constructing a recombinant microorganism expressing the recombinant humanized elastin, and culturing the recombinant microorganism.

[0038] Preferably, the amino acids of the recombinant humanized elastin comprise the amino acid sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 95% identity with SEQ ID NO.1, and / or the amino acid sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 95% identity with SEQ ID NO.2, and / or the amino acid sequence shown in SEQ ID NO.3 or an amino acid sequence having at least 95% identity with SEQ ID NO.3, and / or the amino acid sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 95% identity with SEQ ID NO.4, and / or the amino acid sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 95% identity with SEQ ID NO.5.

[0039] More preferably, the nucleic acid molecule encoding the recombinant humanized elastin comprises: a nucleotide sequence as shown in SEQ ID NO. 6 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 6, and / or a nucleotide sequence as shown in SEQ ID NO. 7 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 7, and / or a nucleotide sequence as shown in SEQ ID NO. 8 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 8, and / or a nucleotide sequence as shown in SEQ ID NO. 9 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 9, and / or a nucleotide sequence as shown in SEQ ID NO. 10 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 10.

[0040] Furthermore, the recombinant microorganism is selected from one or more of Escherichia coli, Streptococcus, Bacillus, Saccharomyces cerevisiae, and Pichia pastoris. Preferably, it is Escherichia coli.

[0041] In a preferred embodiment, a method for preparing recombinant humanized elastin includes:

[0042] Step 1: Construct recombinant microorganisms expressing the recombinant humanized elastin described above;

[0043] Step 2: Culture the recombinant microbial cells at 25℃-37℃ until OD. 600 The concentration of the fermentation broth was approximately 0.8-1.0. IPTG was added to a final concentration of 0.5 mM, and the mixture was cultured at 25℃-37℃ with shaking for 3-4 hours. The supernatant after centrifugation was then purified to obtain the recombinant humanized protein.

[0044] The above purification process can be carried out using general methods.

[0045] In a preferred embodiment, the purification includes: using nickel column affinity chromatography and ion exchange chromatography to elute and collect the protein corresponding to the elution peak.

[0046] On the other hand, this application also provides compositions containing the recombinant humanized elastin or the biomaterial or the recombinant humanized elastin prepared by the method.

[0047] On the other hand, experiments in this application have demonstrated that the recombinant humanized elastin is non-cytotoxic and has a certain degree of promoting effect on cell proliferation.

[0048] In a preferred embodiment, the concentration of the recombinant humanized elastin is 0.001 mg / mL to 10 mg / mL; preferably, 0.05 mg / mL to 0.8 mg / mL.

[0049] The concentration of the recombinant humanized elastin 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.

[0050] Those skilled in the art can adjust the concentration of recombinant humanized elastin according to the actual situation, and no specific restrictions are imposed in this application.

[0051] On the other hand, this application also provides the application of the recombinant humanized elastin or the biomaterial or the recombinant humanized elastin prepared by the method or the composition thereof in wound repair or the preparation of wound repair products; preferably, the wound repair is achieved by promoting cell proliferation.

[0052] On the other hand, this application also provides the application of the recombinant humanized elastin or the biomaterial or the recombinant humanized elastin prepared by the method or the composition thereof in tissue regeneration or the preparation of tissue regeneration products; preferably, the tissue regeneration is achieved by promoting cell proliferation.

[0053] On the other hand, this application also provides the use of the recombinant humanized elastin or the biomaterial or the recombinant humanized elastin prepared by the method or the composition thereof in anti-aging or in the preparation of anti-aging products.

[0054] Preferably, the anti-aging effect is achieved by promoting the expression of collagen and / or elastin, and preferably, the collagen includes type I collagen and / or type III collagen.

[0055] More preferably, the promotion of collagen and / or elastin expression includes promoting the relative expression levels of the type I collagen COL1A1 gene, the type III collagen COL3A1 gene, and the elastin eln gene.

[0056] On the other hand, this application also provides the use of the recombinant humanized elastin or the biomaterial or the recombinant humanized elastin prepared by the method or the composition thereof in promoting collagen and / or elastin expression; preferably, the collagen includes type I collagen and / or type III collagen.

[0057] More preferably, the promotion of collagen and / or elastin expression includes promoting the relative expression levels of the type I collagen COL1A1 gene, the type III collagen COL3A1 gene, and the elastin eln gene.

[0058] The present invention has the following beneficial effects:

[0059] In this invention, based on humanized elastin (GeneID:2006), five new recombinant humanized elastins were obtained by combining and designing according to the functional domains of elastin, and new biologically active functional fragments of humanized elastin were discovered.

[0060] The recombinant humanized elastin in this invention integrates multiple elastin functional sites, which is superior to currently disclosed single-site elastin products. Specifically, this recombinant humanized elastin has significant effects in promoting cell proliferation and increasing the expression of collagen and elastin genes, demonstrating high safety and wide applicability in various scenarios, and providing a new active ingredient for anti-aging related products. Furthermore, the theoretical molecular weight of this recombinant humanized elastin is significantly smaller than that of natural full-length elastin, reducing obstacles for elastin to cross the skin barrier and exert its biological functions, thus exhibiting excellent biological efficacy.

[0061] This invention utilizes bioengineering technology to achieve efficient expression and purification of recombinant humanized elastin in an Escherichia coli expression system. Compared with traditional protein extraction and purification methods, the quality is more stable, more economical, and suitable for widespread application. Attached Figure Description

[0062] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings:

[0063] Figure 1 The images are SDS-PAGE electrophoresis images of purified recombinant humanized elastin, where A) is the electrophoresis image of purified SE1 protein, B) is the electrophoresis image of purified SE2 protein, C) is the electrophoresis image of purified SE3 protein, D) is the electrophoresis image of purified SE4 protein, and E) is the electrophoresis image of purified SE5 protein.

[0064] Figure 2 This is a diagram showing the proliferation of recombinant humanized elastin cells.

[0065] Figure 3 Diagram showing the gene expression of type I collagen promoted by recombinant humanized elastin;

[0066] Figure 4 Diagram showing the expression of the type III collagen-promoting gene by recombinant humanized elastin;

[0067] Figure 5 This is a diagram illustrating the expression of the elastin-proelastin gene in recombinant humanized elastin. Detailed Implementation

[0068] Identity: refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules in molecular evolution studies.

[0069] Recombination: In a broad sense, any gene exchange process that causes a change in genotype is called recombination.

[0070] Expression cassette: An expression cassette is a set of DNA sequences that consists of promoters, target genes, and reporter genes, and can be expressed in specific tissues and is easily detected.

[0071] Recombinant vectors: Recombinant vectors are vectors into which the target gene is transferred based on the basic framework of a cloning vector, thereby enabling the target gene to be expressed.

[0072] Recombinant microorganisms: bacterial cell lines in which foreign genes are expressed efficiently using genetic engineering methods.

[0073] Recombinant cells: The term "recombinant cell" refers to any cell type that is readily transformed, transfected, transduced, etc., using nucleic acid constructs or expression vectors containing the polynucleotides of the present invention. The term "recombinant cell" also encompasses any parental cell progeny that is not entirely identical to the parental cell due to mutations that occur during replication.

[0074] To more clearly illustrate the overall concept of this application, a detailed description is provided below with reference to the accompanying drawings and embodiments. Numerous specific details are set forth in the following description to provide a more thorough understanding of the invention. However, it will be apparent to those skilled in the art that the invention can be practiced without one or more of these details. In other instances, certain technical features well-known in the art have not been described to avoid confusion with the invention.

[0075] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. Where specific conditions are not specified in the embodiments, they are performed under standard conditions or conditions recommended by the manufacturer.

[0076] The plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products. The specific operations were performed according to the kit instructions.

[0077] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Specifically, they can be performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0078] Example 1: Construction of Recombinant Humanized Elastin Expression Engineered Bacteria

[0079] 1. Obtaining the Target Gene: Based on humanized elastin (GeneID: 2006), recombinant humanized elastin was designed by combining its functional domains. An enterokinase restriction site (DDDDK) was added to the N-terminus of the recombinant humanized elastin. Recombinant humanized elastin SE1-4 were designed using the SE5 sequence as the core. The specific amino acid sequences of the recombinant humanized elastin are shown in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5. The target sequence encoding the recombinant humanized elastin was optimized according to the codon preferences of *E. coli*. The optimized nucleotide sequences are shown in SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, and SEQ ID No. 10. The recombinant humanized elastin gene was artificially synthesized by GenScript. A recombinant vector was constructed using pET32a(+) as the backbone, with restriction endonuclease sites BglⅡ (AGATCT) and XhoⅠ (CTCGAG).

[0080] 2. Recombinant Vector Transformation: Mix 2 μL of the recombinant vector obtained in step 1 with *E. coli* BL21(DE3) competent cells and incubate on ice for 30 min. Heat shock at 42℃ for 90 s, then incubate on ice for 2 min. Add 1 mL of antibiotic-free LB medium and incubate at 37℃ and 220 rpm for 1 h with shaking. Centrifuge for 1 min, discard 800 μL of supernatant, resuspend the remaining precipitate, and evenly spread it on LB agar plates containing ampicillin (Amp). Incubate overnight at 37℃. The next day, pick transformed colonies for inoculation and culture to obtain recombinant human-like elastin-expressing engineered bacteria.

[0081] 3. Shake-flask expression: The recombinant human-like elastin expression engineered bacteria obtained in step 2 were revitalized with LB medium containing Amp resistance, inoculated into liquid LB medium, and cultured at 37°C until OD500. 600 When the protein content is approximately 0.8-1.0, add IPTG to a final concentration of 0.5 mM and incubate at 37°C with shaking for 4 h to induce cell growth. After harvesting the cells, resuspend them in 20 mM PB phosphate buffer (pH = 7.4), sonicate, centrifuge (10000 rpm, 15 min), and collect the supernatant after centrifugation, which is the crude protein sample.

[0082] 4. Protein Purification: The cell lysis supernatant was purified using a Cytiva AKTApure purification system. First, a column was packed using BorgLone nickel affinity chromatography packing material, and the Ni was washed with 5 column volumes of purified water. 2+Chelate the affinity chromatography column and then equilibrate it with 2-3 column volumes of Binding Buffer. After the absorbance at UV 280 nm stabilizes, start loading the crude protein sample, setting the flow rate through the pump and column to 1.5 mL / min. Then, pass the sample through the column with Binding Buffer (20 mM phosphate buffer, 250 mM NaCl, 10 mM imidazole, pH 7.4) to wash away any unbound proteins until UV 280 nm stabilizes. Perform linear elution of the target protein using 20 column volumes of Elution Buffer (20 mM phosphate buffer, 250 mM NaCl, 500 mM imidazole, pH 7.4).

[0083] After nickel column purification, the protein solution was purified using different ion chromatography methods based on the predicted isoelectric point. The pH and conductivity of the protein solution were adjusted to match the buffer solution before loading. Linear elution was performed using 20 column volumes to elute the target protein. Following nickel column affinity chromatography and ion chromatography, the protein samples, namely recombinant humanized elastin samples SE1, SE2, SE3, SE4, and SE5, were obtained. Specific protein information is shown in Table 1. SDS-PAGE analysis of the purified proteins is as follows: Figure 1 As shown.

[0084] Table 1 Protein Information

[0085]

[0086] Note: The T1 sequence is shown in SEQ ID NO.11, the T2 sequence is shown in SEQ ID NO.12, and the T3 sequence is shown in SEQ ID NO.13.

[0087] Example 2: Cellular Experiments with Recombinant Humanized Elastin

[0088] 1. Cytotoxicity assay

[0089] a)Planning

[0090] HaCaT cells in the logarithmic growth phase were harvested at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL in 96-well plates. The culture medium was DMEM high glucose medium (Bloomage Biotechnology (Hainan) Co., Ltd.) with 10% fetal bovine serum (Gibco) added. The seeded cells were incubated overnight in a CO2 incubator at 37°C with 5% CO2.

[0091] b) Sample solution preparation

[0092] Sample solutions were prepared using serum-free culture medium (DMEM high glucose medium, Bloomage Biotechnology (Hainan) Co., Ltd.), and sterilized by filtration through a 0.22μm filter membrane. The solutions were prepared and used immediately.

[0093] c) Adding medicine

[0094] After 24 hours of routine culture, the old culture medium was discarded, and the experimental group was replaced with 100 μL samples of different concentrations (SE1, SE2, SE3, SE4, and SE5 proteins obtained in Example 1). The negative control group was given an equal amount of serum-free culture medium, with 6 parallel wells for each level.

[0095] d) Detection

[0096] After culturing for another 24 hours, the relative cell proliferation rate was detected using the CCK-8 assay. The culture medium was discarded, and 100 μL of CCK-8 diluted 10-fold with serum-free medium was added to each well. The cells were then incubated in a cell culture incubator for another 2 hours, and the absorbance was measured at 450 nm using a microplate reader. The relative proliferation rate (RGR) was the ratio of the absorbance of the experimental group to that of the negative control group. The experimental results are shown in Table 2. A graph was plotted from Table 2 to obtain the desired results. Figure 2 .

[0097] Table 2 Results of relative cell proliferation rate

[0098]

[0099] As shown in Table 2 and Figure 2 The results show that, according to the standard of a relative proliferation rate of over 70% being considered non-cytotoxic, samples SE1, SE2, SE3, SE4, and SE5 showed no potential cytotoxicity within the experimental concentration range and had a certain degree of promoting effect on cell proliferation.

[0100] 2. Experiments on the efficacy of promoting type I and III collagen genes and elastin genes.

[0101] a)Planning

[0102] HSF cells in logarithmic growth phase were digested with trypsin until they became rounded and suspended. Digestion was terminated by adding an appropriate amount of serum-containing DMEM cell culture medium, and the cell density was adjusted to 1 x 10⁻⁶ cells / year. 5 Cells were seeded at a density of 1 mL / well in 24-well plates, ensuring even distribution at the bottom of each well. The seeded cells were then incubated at 37°C with 5% CO2 for 24 hours.

[0103] b) Adding medication

[0104] After 24 hours of routine culture, the culture medium in the well plate was discarded. The experimental group was given sample solution (SE1, SE2, SE3, SE4, and SE5 proteins obtained in Example 1) with a protein concentration of 0.2 mg / mL. The negative control group was given an equal volume of serum-free culture medium and cultured in a carbon dioxide incubator for another 24 hours.

[0105] c) Cell collection and RNA extraction

[0106] After incubation and culture, cells were collected for total RNA extraction. Total RNA extraction was performed according to the instructions of the Novizan FastPure Cell / Tissue Total RNA Isolation Kit V2. RNA concentration was then measured after extraction.

[0107] d) Reverse transcription

[0108] 1 μg of total RNA extracted from cells was used to remove genomic DNA and then reverse transcribed. The experimental procedure was performed according to the instructions of the Novizan HiScript III RT SuperMix for qPCR (+gDNAwiper) kit.

[0109] e) Quantitative fluorescence experiment

[0110] The quantitative fluorescent primers are shown in Table 3 for the quantitative detection of the COL1A1 gene promoting type I collagen, the COL3A1 gene promoting type III collagen, and the eln gene promoting elastin.

[0111] Table 3 Primer sequences

[0112]

[0113] Quantitative PCR was performed using the SYBR Green I chimeric fluorescence method. The kit used was the Novizan ChamQ Universal SYBR qPCR Master Mix kit. The mixture was prepared in the qPCR tubes as shown in Table 4.

[0114] Table 4

[0115]

[0116]

[0117] Perform qPCR reactions according to the conditions in Table 5.

[0118] Table 5

[0119]

[0120] The experimental results were obtained using 2 -△△CT The method was used for calculation. The experimental results of the COL1A1 gene promoting type I collagen are shown in Table 6. Based on Table 6, a graph was plotted... Figure 3 The experimental results of promoting type III collagen COL3A1 gene therapy are shown in Table 7. Based on Table 7, a graph was plotted... Figure 4 The experimental results of the elastin eln gene are shown in Table 8. Based on Table 8, a graph was plotted... Figure 5 .

[0121] Table 6

[0122]

[0123] Table 7

[0124]

[0125] Table 8

[0126]

[0127] From Table 6 and Figure 3 It is evident that, compared to the negative control, the SE1, SE2, SE3, SE4, and SE5 proteins all had a promoting effect, with SE4 showing the best gene expression-promoting effect.

[0128] From Table 7 and Figure 4 It is evident that, compared to the negative control, the SE1, SE2, SE3, SE4, and SE5 proteins all exhibited a promoting effect, with SE1 showing the best gene expression-promoting effect.

[0129] From Table 8 and Figure 5 It is evident that, compared to the negative control, SE1, SE2, SE3, SE4, and SE5 proteins can all promote the expression of the elastin eln gene, with SE4 showing the best gene expression-promoting effect.

[0130] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. Recombinant humanized elastin, characterized in that, The amino acid sequence of the recombinant humanized elastin includes any one or more of the following: A1)-A5): A1) The amino acid sequence of the recombinant humanized elastin comprises the sequence shown in SEQ ID NO.1 or an amino acid sequence having at least 95% identity with SEQ ID NO.1; A2) The amino acid sequence of the recombinant humanized elastin comprises the sequence shown in SEQ ID NO.2 or an amino acid sequence having at least 95% identity with SEQ ID NO.2; A3) The amino acid sequence of the recombinant humanized elastin comprises the sequence shown in SEQ ID NO.3 or an amino acid sequence having at least 95% identity with SEQ ID NO.3; A4) The amino acid sequence of the recombinant humanized elastin comprises the sequence shown in SEQ ID NO.4 or an amino acid sequence having at least 95% identity with SEQ ID NO.4; A5) The amino acid sequence of the recombinant humanized elastin contains the sequence shown in SEQ ID NO.5 or an amino acid sequence having at least 95% identity with SEQ ID NO.

5.

2. A biomaterial, characterized in that, The biomaterial includes any one of the following B1)-B5): B1) A nucleic acid molecule, said nucleic acid molecule containing a nucleic acid molecule encoding the recombinant humanized elastin of claim 1; B2) Expression cassette, wherein the expression cassette contains the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1) and / or the expression cassette described in B2); B4) Recombinant microorganisms, wherein the recombinant microorganisms contain the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3); B5) Recombinant cells containing the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3).

3. The biomaterial according to claim 2, characterized in that, The nucleic acid molecule comprises: a nucleotide sequence as shown in SEQ ID NO. 6 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 6, and / or a nucleotide sequence as shown in SEQ ID NO. 7 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 7, and / or a nucleotide sequence as shown in SEQ ID NO. 8 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 8, and / or a nucleotide sequence as shown in SEQ ID NO. 9 or a nucleotide sequence having at least 95% identity with SEQ ID NO. 9, and / or a nucleotide sequence as shown in SEQ ID NO. 10 or a nucleotide sequence having at least 95% identity with SEQ ID NO.

10.

4. The biomaterial according to claim 2, characterized in that, The recombinant microorganisms are selected from one or more of Escherichia coli, Streptococcus, Bacillus, Saccharomyces cerevisiae, and Pichia pastoris.

5. A method for preparing recombinant humanized elastin, characterized in that, The method includes: constructing a recombinant microorganism expressing the recombinant humanized elastin as described in claim 1, and culturing the recombinant microorganism.

6. A composition, characterized in that, It contains the recombinant humanized elastin as described in claim 1, or the biomaterial as described in any one of claims 2-4, or the recombinant humanized elastin prepared using the method described in claim 5.

7. The application of the recombinant humanized elastin as described in claim 1, or the biomaterial as described in any one of claims 2-4, or the recombinant humanized elastin prepared using the method as described in claim 5, or the composition as described in claim 6, in wound repair or the preparation of wound repair products; preferably, the wound repair is achieved by promoting cell proliferation.

8. The use of the recombinant humanized elastin as described in claim 1, or the biomaterial as described in any one of claims 2-4, or the recombinant humanized elastin prepared by the method as described in claim 5, or the composition as described in claim 6, in tissue regeneration or the preparation of tissue regeneration products; preferably, the tissue regeneration is achieved by promoting cell proliferation.

9. The use of the recombinant humanized elastin as described in claim 1, or the biomaterial as described in any one of claims 2-4, or the recombinant humanized elastin prepared by the method as described in claim 5, or the composition as described in claim 6, in anti-aging or the preparation of anti-aging products.

10. The use of the recombinant humanized elastin of claim 1, or the biomaterial of any one of claims 2-4, or the recombinant humanized elastin prepared by the method of claim 5, or the composition of claim 6, in promoting collagen and / or elastin expression; preferably, the collagen includes type I collagen and / or type III collagen.

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