Short peptide composition, recombinant polypeptide chain, recombinant nucleic acid molecule and application

Abstract

The invention discloses an oligopeptide composition, a recombinant polypeptide chain, a recombinant nucleic acid molecule and application. The oligopeptide composition comprises at least one oligopeptide, the amino acid sequence of the oligopeptide is the amino acid sequence of an active domain of HAPLN1 protein, and the oligopeptide composition is used for improving the expression quantity of fibroblasts on the HAPLN1 protein and collagen I.

Description

Technical Field

[0001] This application belongs to the field of anti-aging product technology, and particularly relates to a short peptide composition, a recombinant polypeptide chain, a recombinant nucleic acid molecule, and its application. Background Technology

[0002] Hyaluronic acid and proteoglycan link protein 1 (HAPLN1) is a key protein for maintaining extracellular matrix homeostasis, and its serum levels decline with age. HAPLN1 protein can specifically bind to hyaluronic acid and proteoglycan, thereby constructing a highly hydrated network structure in the pericellular matrix that can lock in moisture, which helps maintain skin plumpness and elasticity.

[0003] In related technologies, HAPLN1 protein is used as a biomarker for detecting skin aging. However, because HAPLN1 protein is a large molecule, it is difficult to effectively penetrate the skin's stratum corneum barrier, resulting in low bioavailability and limiting its effectiveness in directly applying it topically to boost collagen production. Summary of the Invention

[0004] This application provides a method for effectively increasing the expression levels of HAPLN1 protein and collagen I in the skin through short peptide compositions, recombinant polypeptide chains, recombinant nucleic acid molecules, and their applications.

[0005] In a first aspect, embodiments of this application provide a short peptide composition comprising at least one short peptide, wherein the amino acid sequence of the short peptide is the amino acid sequence of the active domain of the HAPLN1 protein, and the short peptide composition is used to increase the expression levels of HAPLN1 protein and collagen I in fibroblasts.

[0006] In some embodiments, the carboxyl terminus of the amino acid sequence of the short peptide is lysine or arginine.

[0007] In some embodiments, the number of amino acids in the short peptide is m, where 5 ≤ m ≤ 30.

[0008] In some embodiments, the short peptide composition includes n short peptides, the n short peptides having different amino acid sequences, and the amino acid sequences of the n short peptides being the amino acid sequences of different active domains of the HAPLN1 protein.

[0009] In some embodiments, the short peptide composition comprises n short peptides, the amino acid sequence of which has at least 90% sequence identity with one of the amino acid sequences of SEQ ID NO. 1 to SEQ ID NO. 15, where 1 ≤ n ≤ 15.

[0010] Secondly, embodiments of this application provide a recombinant polypeptide chain, which comprises n short peptides linked by peptide bonds. The amino acid sequences of the n short peptides are different, and the amino acid sequences of the n short peptides are the amino acid sequences of different active domains of the HAPLN1 protein. The recombinant polypeptide chain is used for enzymatic digestion to obtain the above-mentioned short peptide composition.

[0011] In some embodiments, the recombinant polypeptide chain comprises an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO. 16.

[0012] In some embodiments, the recombinant polypeptide chain comprises an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO. 17.

[0013] In some embodiments, a recombinant polypeptide chain is cleaved to form n short peptides.

[0014] Thirdly, embodiments of this application provide a recombinant nucleic acid molecule comprising a nucleotide sequence encoding the recombinant polypeptide chain provided in the second aspect, wherein the recombinant nucleic acid molecule comprises a nucleotide sequence having at least 90% sequence identity with respect to SEQ ID NO. 18.

[0015] In some embodiments, this application provides a recombinant nucleic acid molecule comprising a nucleotide sequence having at least 90% sequence identity with respect to SEQ ID NO. 19.

[0016] Fourthly, embodiments of this application provide a recombinant vector comprising the recombinant nucleic acid molecule provided in the third aspect.

[0017] Fifthly, embodiments of this application provide a recombinant cell comprising the recombinant nucleic acid molecule provided in the third aspect, or the recombinant vector provided in the fourth aspect.

[0018] In a sixth aspect, embodiments of this application provide a formulation product comprising the short peptide composition provided in the first aspect.

[0019] In a seventh aspect, embodiments of this application provide the use of a short peptide composition (first aspect), a recombinant polypeptide chain (second aspect), a recombinant nucleic acid molecule (third aspect), a recombinant vector (fourth aspect), a recombinant cell (fifth aspect), or a formulation product (sixth aspect) in the preparation of an anti-skin aging product.

[0020] In the short peptide compositions, recombinant polypeptide chains, recombinant nucleic acid molecules, and applications of this application, the short peptides in the short peptide compositions have a smaller molecular weight than the HAPLN1 protein, making it easier for them to penetrate the stratum corneum and enter fibroblasts. Since the amino acid sequence of the short peptides is the same as the active domain of the HAPLN1 protein, after entering fibroblasts, the short peptides may act as transcriptional coactivators or may influence epigenetic modifications, causing fibroblasts to upregulate the transcription of HAPLN1 itself, leading to increased mRNA levels, increased expression of HAPLN1 protein in fibroblasts, and increased expression of collagen I in fibroblasts. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments of this application will be briefly introduced below. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a bar chart showing the relative expression levels of HAPLN1 in one embodiment and a comparative example of this application; Figure 2 This is a bar chart showing the relative expression levels of collagen I in one embodiment and a comparative example of this application. Detailed Implementation

[0023] The features and exemplary embodiments of various aspects of this application will be described in detail below. To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only intended to explain this application and not to limit it. For those skilled in the art, this application can be implemented without some of these specific details. The following description of the embodiments is merely to provide a better understanding of this application by illustrating examples.

[0024] It should be noted that, in this document, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising..." does not exclude the presence of additional identical elements in the process, method, article, or apparatus that includes the element.

[0025] To address the problems of the prior art, this application provides a short peptide composition, a recombinant polypeptide chain, a recombinant nucleic acid molecule, and its applications. The short peptide composition provided in this application is described below.

[0026] The short peptide composition includes at least one short peptide, the amino acid sequence of which is the amino acid sequence of the active domain of the HAPLN1 protein, and the short peptide composition is used to increase the expression levels of HAPLN1 protein and collagen I in fibroblasts.

[0027] The short peptide composition may include multiple separate short peptides. These multiple short peptides may correspond to only one type of short peptide or to multiple types of short peptides. In this embodiment, the similarity of the amino acid sequences of the short peptides can be used to determine whether they belong to the same category.

[0028] The amino acid sequence of the short peptide is derived from the amino acid sequence of the active domain of the HAPLN1 protein, which gives the short peptide the same biological activity as the active domain of the HAPLN1 protein.

[0029] Because short peptides have a smaller molecular weight compared to the HAPLN1 protein, they can easily penetrate the stratum corneum and enter fibroblasts. Once inside fibroblasts, the short peptides may act as transcriptional coactivators or influence epigenetic modifications, causing fibroblasts to upregulate the transcription of HAPLN1 itself, leading to increased mRNA levels, increased HAPLN1 expression, and increased collagen I expression. HAPLN1 protein can also reduce or eliminate cellular damage accumulated during skin aging. HAPLN1 protein protects the transforming growth factor β receptor 2 on the cell surface from endocytic degradation through mechanisms such as regulating viscoelasticity and CD44 clustering. HAPLN1 protein also regulates the levels of nuclear factor erythroid 2-associated factor 2 (Nrf2), phosphorylated nuclear factor kappa (κB), and some cyclin-dependent kinase inhibitors (such as p16 and p21) to prevent or repair aging skin.

[0030] In some embodiments, the number of amino acids in the short peptide is m, where 5 ≤ m ≤ 30, to enhance the ability of the short peptide to penetrate the stratum corneum of the skin and enter fibroblasts.

[0031] In some embodiments, the carboxyl terminus of the amino acid sequence of the short peptide is lysine or arginine.

[0032] When preparing multiple short peptides simultaneously, amino acid sequences of multiple short peptides can be pre-designed, and recombinant polypeptide chains can be synthesized based on these amino acid sequences. Since the carboxyl terminus of each short peptide is lysine or arginine, the recombinant polypeptide chains can be enzymatically cleaved by proteases that specifically recognize lysine or arginine, thus obtaining multiple short peptides at the same time.

[0033] In some embodiments, the short peptide composition includes n short peptides, the amino acid sequences of which are different, and the amino acid sequences of the n short peptides are the amino acid sequences of different active domains of the HAPLN1 protein, where 2 ≤ n.

[0034] The amino acid sequences of various short peptides are derived from the active domains of the HAPLN1 protein. These short peptides have different amino acid sequences, which can vary in the number of amino acids or their order.

[0035] The amino acid sequences of various short peptides are derived from the active domains of the HAPLN1 protein, resulting in different biological activities. The short peptide composition includes at least two short peptides, allowing them to synergistically enhance the expression levels of HAPLN1 and collagen I in fibroblasts.

[0036] In some embodiments, the short peptide composition comprises n short peptides, the amino acid sequence of which has at least 90% sequence identity with one of the amino acid sequences of SEQ ID NO. 1 to SEQ ID NO. 15, where 1 ≤ n ≤ 15.

[0037] 1 ≤ n ≤ 15, meaning that 1 to 15 short peptides can be selected as needed to construct a short peptide composition. The short peptide corresponds to one of the amino acid sequences in SEQ ID NO. 1 to SEQ ID NO. 15, and the short peptide has at least 90% sequence identity with that corresponding amino acid sequence. For example, the short peptide corresponds to the amino acid sequence of SEQ ID NO. 1, and the amino acid sequence of the short peptide has at least 90% sequence identity with the amino acid sequence of SEQ ID NO. 1: CEVIEGLEDDTVVVALDLQGVVFPYFPR.

[0038] For example, when n is 1, the short peptide composition comprises a short peptide that corresponds to one of the amino acid sequences of SEQ ID NO. 1 to SEQ ID NO. 15; When n is 5, the short peptide composition includes 5 short peptides, which correspond one-to-one with the 5 amino acid sequences in SEQ ID NO. 1 to SEQ ID NO. 15; When n is 15, the short peptide composition includes 15 short peptides, the amino acid sequences of which correspond to the amino acid sequences of SEQ ID NO. 1 to SEQ ID NO. 15, and have at least 90% sequence identity with the corresponding amino acid sequences of SEQ ID NO. 1 to SEQ ID NO. 15.

[0039] The short peptide composition includes peptides that correspond one-to-one with the amino acid sequences of SEQ ID NO. 1 to SEQ ID NO. 15. The 15 peptides work synergistically to increase the expression of HAPLN1 protein and collagen I in fibroblasts.

[0040] Secondly, this application also provides a recombinant polypeptide chain, which comprises n short peptides linked by peptide bonds, the amino acid sequences of the n short peptides being different, and the amino acid sequences of the n short peptides being the amino acid sequences of different active domains of the HAPLN1 protein, and the recombinant polypeptide chain being used for enzymatic digestion to obtain the short peptide composition as described above.

[0041] The amino acid sequence of the HAPLN1 protein can be analyzed beforehand, and fragments corresponding to multiple active domains can be selected. A sequence of amino acid sequences corresponding to these active domains can be designed, with the order of these fragments not necessarily matching their order in the HAPLN1 protein's amino acid sequence. A protein with this amino acid sequence can then be prepared through synthesis, microbial expression, or other methods. This protein can then be digested with enzymes to obtain short peptides corresponding to each active domain.

[0042] Optionally, by screening for polypeptide fragments ending in lysine and arginine, the recombinant polypeptide chain can be easily digested after expression. By adjusting the amino acid sequence order corresponding to each short peptide in the recombinant polypeptide chain and selecting short peptides without self-complementary tendency, the recombinant polypeptide chain becomes a disordered, linear amino acid chain, exposing protease recognition sites to improve digestion efficiency and specificity. Optionally, the recombinant polypeptide chain has only a primary structure. Optionally, the recombinant polypeptide chain has a primary structure but does not form stable, regular secondary or tertiary structures.

[0043] In some embodiments, the recombinant polypeptide chain comprises an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO. 16.

[0044] In some embodiments, the recombinant polypeptide chain comprises an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO. 17.

[0045] In some embodiments, a recombinant polypeptide chain is cleaved to form n short peptides.

[0046] Optionally, the recombinant polypeptide chain can be digested with trypsin to obtain n short peptides.

[0047] The recombinant polypeptide chain provided in this application embodiment can synthesize the short peptide composition provided in any of the above embodiments, and therefore has the same technical effect, which will not be repeated here.

[0048] Thirdly, a recombinant nucleic acid molecule is provided, comprising a nucleotide sequence encoding the aforementioned recombinant polypeptide chain, wherein the recombinant nucleic acid molecule comprises an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO. 18.

[0049] In another embodiment, the recombinant nucleic acid molecule comprises an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO.19.

[0050] Recombinant nucleic acid molecules can be introduced into hosts such as microorganisms and animal cells as exogenous genes, and then expressed heterologously using the host's transcription and translation systems to obtain recombinant polypeptide chains encoded by the recombinant nucleic acid molecules.

[0051] The recombinant nucleic acid molecules provided in this application have the same technical effects because they use the nucleotide sequences corresponding to the recombinant polypeptide chains provided in any of the above embodiments, and will not be described again here.

[0052] Fourthly, a recombinant vector is provided, which includes the recombinant nucleic acid molecule as described above.

[0053] Recombinant nucleic acid molecules can be cloned into a target vector to obtain a recombinant vector; this vector can then be transformed into a microbial host to achieve heterologous expression and production of recombinant polypeptide chains. The target vector can be a eukaryotic expression system vector or a prokaryotic expression system vector. The eukaryotic expression system vector can be the pET28a vector, and the prokaryotic expression system vector can be the pPIC9K vector.

[0054] The recombinant vector provided in this application embodiment has the same technical effect as the recombinant nucleic acid molecule provided in any of the above embodiments, and will not be described again here.

[0055] Fifthly, a recombinant cell is provided, which includes the aforementioned recombinant nucleic acid molecule or the aforementioned recombinant vector.

[0056] Recombinant cells can be microbial or animal cells. By culturing these cells, recombinant nucleic acid molecules within the cells or in the recombinant vector can be expressed, resulting in the production of recombinant polypeptide chains.

[0057] Optionally, the recombinant cells are Escherichia coli or Pichia pastoris.

[0058] The recombinant cells provided in this application have the same technical effects because they use the recombinant nucleic acid molecules provided in any of the above embodiments, and will not be described again here.

[0059] In a sixth aspect, a formulation product is provided, comprising the short peptide composition as described above.

[0060] The formulation product can be a gel, aqueous solution, cream, ointment, lotion, spray, injection, syringe, or dressing, etc. The formulation product may also include a base solvent, active ingredient, natural extract, stabilizer, emulsifier, etc. The formulation product can also be a skin care product, health product, or pharmaceutical product, etc. The formulation products provided in the embodiments of this application have the same technical effects because they use the short peptide composition provided in any of the above embodiments, and will not be described again here.

[0061] In a seventh aspect, the use of the short peptide composition, recombinant polypeptide chain, recombinant nucleic acid molecule, carrier, recombinant cell, or formulation product as described above in the preparation of anti-skin aging products is provided.

[0062] Anti-aging products include, but are not limited to, toners, serums, lotions, creams, shampoos, shower gels, facial cleansers, masks, capsules, tablets, oral liquids, powders, and granules. These anti-aging products have moisturizing and anti-aging effects. The applications provided in this application, due to the use of the short peptide compositions provided in any of the above embodiments, have the same technical effects, and will not be elaborated further here.

[0063] This application also provides the following verification experiments to illustrate the effects of the short peptide composition, recombinant polypeptide chain, recombinant nucleic acid molecule and application provided in this application.

[0064] Experimental Example 1: Multiple short peptide amino acid sequences were selected from the amino acid sequence of the active domain of the HAPRN1 protein. The multiple short peptide amino acid sequences are shown as SEQ ID NO. 1-SEQ ID NO. 15, and the active domain was obtained from https: / / www.ebi.ac.uk / interpro / . Fifteen amino acid sequences were spliced ​​together to form the amino acid sequence shown in SEQ ID NO. 16, and then converted into the nucleotide sequence shown in SEQ ID NO. 18 based on the amino acid sequence shown in SEQ ID NO. 16. A recombinant nucleic acid molecule was synthesized according to the nucleotide sequence shown in SEQ ID NO. 18, and the recombinant nucleic acid molecule was cloned into the pET28a vector to obtain the recombinant vector.

[0065] The recombinant vector was transformed into Escherichia coli expression strain BL21(DE3) to obtain recombinant cells. The recombinant cells were placed in LB liquid medium and cultured in a shaker at 37°C and 220 rpm until the OD600 reached 0.6.

[0066] LB liquid medium was cultured in a shaker at 22 ℃ and 220 rpm for 10 h, and 1 mM isopropyl-β-D-thiogalactoside (IPTG) was added to the culture for induction to improve the solubility of recombinant polypeptide chains produced by recombinant cells. Centrifuge the culture for 3 minutes to collect the bacterial cells, resuspend the bacterial cells in LB medium and sonicate to disrupt them, filter to remove cell debris and insoluble impurities, and obtain a crude protein extract containing recombinant polypeptide chains. Trypsin was added to the crude protein extract to enzymatically hydrolyze the recombinant polypeptide chain, and the mixture was filtered and freeze-dried to obtain a freeze-dried powder containing a short peptide composition.

[0067] Experimental Example 2: Multiple short peptide amino acid sequences were selected from the amino acid sequence of the active domain of the HAPRN1 protein, as shown in SEQ ID NO. 1-SEQ ID NO. 15; the active domain was obtained from https: / / www.ebi.ac.uk / interpro / . Fifteen amino acid sequences were spliced ​​together to form the amino acid sequence shown in SEQ ID NO. 17, and then converted into the nucleotide sequence shown in SEQ ID NO. 19 based on the amino acid sequence shown in SEQ ID NO. 17. A recombinant nucleic acid molecule was synthesized according to the nucleotide sequence shown in SEQ ID NO. 19, and the recombinant nucleic acid molecule was cloned into the pPIC9K vector to obtain the recombinant vector.

[0068] The SacI-digested linearized plasmid was electroporated into Pichia pastoris GS115 (purchased from Beijing Qingke Biotechnology Co., Ltd.), cultured on a shaker at 220 rpm for 1-2 h, and the cells were collected by centrifugation at 5000 rpm for 5 min.

[0069] Spread the bacterial cells on His-deficient plates and incubate for 2-3 days, then select single colonies. Recombinant cells containing selection markers were screened using 3-5 mg / mL G418 plates; The recombinant cells were transferred into 50 mL centrifuge tubes containing 10-15 mL of BMGY medium with a breathable plug, and cultured on a shaker at 28°C and 220 rpm until the OD600 reached 4-5. The cells were then collected by centrifugation at 5000 rpm for 5 min. The bacterial cells were suspended in BMM medium and cultured in a shaker at 220 rpm. 0.5% methanol was added every 24 hours for induction, and the culture was carried out for 4-5 days. Centrifuge the culture for 3 minutes to collect the bacterial cells, resuspend the bacterial cells in BMM medium and sonicate to disrupt them, filter to remove cell debris and insoluble impurities, and obtain a crude protein extract containing recombinant polypeptide chains. Trypsin was added to the crude protein extract to enzymatically hydrolyze the recombinant polypeptide chain, and the mixture was filtered and freeze-dried to obtain a freeze-dried powder containing a short peptide composition.

[0070] Comparative Example 1: The amino acid sequence of the HAPLN1 protein shown in SEQ ID NO. 20 was converted into the nucleotide sequence shown in SEQ ID NO. 21; A recombinant nucleic acid molecule was synthesized according to the nucleotide sequence shown in SEQ ID NO. 21. The recombinant nucleic acid molecule was cloned into the pET28a vector to obtain the recombinant vector.

[0071] The recombinant vector was transformed into Escherichia coli expression strain BL21(DE3) to obtain recombinant cells. The recombinant cells were placed in LB liquid medium and cultured in a shaker at 37°C and 220 rpm until the OD600 reached 0.6.

[0072] LB liquid medium was cultured in a shaker at 22 ℃ and 220 rpm for 10 h, and 1 mM isopropyl-β-D-thiogalactoside (IPTG) was added to the culture for induction to improve the solubility of recombinant protein produced by recombinant cells. Centrifuge the culture for 3 minutes to collect the bacterial cells, resuspend the bacterial cells in LB medium and sonicate them, filter to remove cell debris and insoluble impurities, and obtain a crude protein extract containing recombinant protein. The crude protein extract was microfiltered and ultrafiltered to obtain pure recombinant HAPRN1 protein.

[0073] Comparative Example 2: Multiple short peptide amino acid sequences were selected from the amino acid sequence of the inactive domain of the HAPRN1 protein, as shown in SEQ ID NO. 22-SEQ ID NO. 31. The above 10 amino acid sequences are spliced ​​together to form the amino acid sequence shown in SEQ ID NO. 32, and then converted into the nucleotide sequence shown in SEQ ID NO. 33 based on the amino acid sequence shown in SEQ ID NO. 32. A recombinant nucleic acid molecule was synthesized according to the nucleotide sequence shown in SEQ ID NO. 33, and the recombinant nucleic acid molecule was cloned into the pET28a vector to obtain the recombinant vector.

[0074] The recombinant vector was transformed into Escherichia coli expression strain BL21(DE3) to obtain recombinant cells. The recombinant cells were placed in LB liquid medium and cultured in a shaker at 37°C and 220 rpm until the OD600 reached 0.6.

[0075] LB liquid medium was cultured in a shaker at 22 ℃ and 220 rpm for 10 h, and 1 mM isopropyl-β-D-thiogalactoside (IPTG) was added to the culture for induction to improve the solubility of recombinant polypeptide chains produced by recombinant cells. Centrifuge the culture for 3 minutes to collect the bacterial cells, resuspend the bacterial cells in LB medium and sonicate to disrupt them, filter to remove cell debris and insoluble impurities, and obtain a crude protein extract containing recombinant polypeptide chains. Trypsin was added to the crude protein extract to enzymatically hydrolyze the recombinant polypeptide chain, and the mixture was filtered and freeze-dried to obtain a freeze-dried powder containing a short peptide composition.

[0076] Experimental Examples 3-17, Comparative Example 3: The components shown in Table 1 were added to DMEM (Dulbecco's Modified Eagle Medium) to prepare DMEM medium containing the short peptide compositions of Experimental Examples 1-2, DMEM medium containing the short peptides of Experimental Examples 3-17, DMEM medium containing the protein of Comparative Example 1 and the short peptide compositions of Comparative Example 2, respectively, and DMEM medium containing 0.1% DMSO in Comparative Example 3. Normal human skin fibroblasts (HSF) were placed in the culture medium of Experimental Examples 1-17 and Comparative Examples 1-3, and incubated at 37°C with 5% CO2. Incubate in an incubator for 24 hours; Total RNA was extracted from the culture products of Experimental Examples 1-17 and Comparative Examples 1-3, and cDNA was synthesized by reverse transcription. Real-time quantitative PCR amplification was performed using specific primers for the HAPLN1 and collagen I genes. The relative expression levels of the corresponding mRNAs of the HAPLN1 and collagen I genes were calculated using GAPDH as an internal reference gene. Each group was divided into three replicates.

[0077] The specific primers for the HAPLN1 gene are: 5'-CCTATGGAGGCTACCAGGGT-3'; 5'-GTCAGTTTGGTGGGGTGGAT-3'.

[0078] The specific primers for the collagen I gene are: 5'-CCACGACATCAAGGAGAAGA-3'; 5'-TCCTTGATGGTGGTGTTGTC-3'.

[0079] The specific primers for the GAPDH gene are: 5'-CAATGACCCCTTCATTGACC-3'; 5'-GACAAGCTTCCCGTTCTCAG-3'.

[0080] Table 1 Please see Figure 1 and Figure 2 The bar chart shows the relative expression levels of HAPLN1 and collagen I mRNA in Experimental Examples 1-2 and Comparative Examples 1-3. Figure 1 and Figure 2 As can be seen, compared with Comparative Examples 1-3, culturing fibroblasts in the culture medium corresponding to Experimental Examples 1 and 2 can increase the expression level of mRNA encoding HAPRN1 and collagen I in fibroblasts, thus indicating that the relative expression levels of HAPRN1 and collagen I in fibroblasts in Experimental Examples 1-2 are significantly higher than those in Comparative Examples 1-3.

[0081] Please refer to Table 1. As can be seen from Table 1, compared with Comparative Examples 1-3, culturing fibroblasts in the culture medium corresponding to Experimental Examples 1-17 can increase the expression level of mRNA encoding HAPLN1 and collagen I in fibroblasts.

[0082] The lyophilized powders prepared in Experimental Examples 1 and 2, and Comparative Examples 1 and 2, were prepared into 0.1% (w / v) aqueous solutions. Comparative Example 4 was water. 0.1 ml of each aqueous solution was applied to the left side of the test subject's face, and Comparative Example 4 was applied to the right side of the face. This was done twice daily, morning and evening, for 30 consecutive days, with 10 test subjects in each group. After 30 days, the skin's moisture content, skin elasticity, fine line depth, and average area of ​​fine lines around the eyes were measured and averaged. The results are shown in Table 2 below, where the measurement results for Comparative Example 4 are the average values ​​obtained from 40 test subjects.

[0083] Table 2 Please refer to Table 2. As can be seen from Table 2, applying the aqueous solutions of the peptide compositions provided in Examples 1 and 2 has a better anti-aging effect on the skin than applying the aqueous solutions of the compositions provided in Comparative Examples 1 and 2. Applying the aqueous solution of the composition provided in Comparative Example 1 is not significantly different from applying the aqueous solution of Comparative Example 4.

[0084] Furthermore, the term "and / or" in this document is merely a description of the association relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship. It should be understood that in the embodiments of this application, "B corresponding to A" means that B is associated with A, and B can be determined based on A. However, it should also be understood that determining B based on A does not mean determining B solely based on A; B can also be determined based on A and / or other information.

[0085] The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A short peptide composition, characterized in that, The composition includes at least one short peptide, the amino acid sequence of which is the amino acid sequence of the active domain of the HAPLN1 protein, and the short peptide composition is used to increase the expression levels of the HAPLN1 protein and collagen I in fibroblasts.

2. The short peptide composition according to claim 1, characterized in that, The number of amino acids in the short peptide is m, where 5 ≤ m ≤ 30.

3. The short peptide composition according to claim 1, characterized in that, The carboxyl terminus of the amino acid sequence of the short peptide is lysine or arginine.

4. The short peptide composition according to any one of claims 1 to 3, characterized in that, The short peptide composition comprises n short peptides, wherein the amino acid sequences of the n short peptides are different, and the amino acid sequences of the n short peptides are respectively the amino acid sequences of different active domains of the HAPLN1 protein, where 2≤n.

5. The short peptide composition according to any one of claims 1 to 3, characterized in that, The short peptide composition comprises n short peptides, wherein the amino acid sequence of the short peptides has at least 90% sequence identity with one of the amino acid sequences of SEQ ID NO. 1 to SEQ ID NO. 15, and 1 ≤ n ≤ 15.

6. A recombinant polypeptide chain, characterized in that, The recombinant polypeptide chain comprises n short peptides linked by peptide bonds, wherein the amino acid sequences of the n short peptides are different, and the amino acid sequences of the n short peptides are respectively the amino acid sequences of different active domains of the HAPLN1 protein. The recombinant polypeptide chain is used for enzymatic digestion to obtain the short peptide composition as described in any one of claims 1 to 5.

7. The recombinant polypeptide chain according to claim 6, characterized in that, The recombinant polypeptide chain comprises an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO.

16. Alternatively, the recombinant polypeptide chain comprises an amino acid sequence having at least 90% sequence identity with respect to SEQ ID NO.

17.

8. A recombinant nucleic acid molecule, characterized in that, It includes a nucleotide sequence encoding the recombinant polypeptide chain of any one of claims 6 or 7, wherein the recombinant nucleic acid molecule comprises a nucleotide sequence having at least 90% sequence identity with respect to SEQ ID NO.

18. Alternatively, the recombinant nucleic acid molecule comprises a nucleotide sequence having at least 90% sequence identity with respect to SEQ ID NO.

19.

9. A recombinant vector, characterized in that, It includes the recombinant nucleic acid molecules as described in claim 8.

10. A recombinant cell, characterized in that, It includes the recombinant nucleic acid molecule as described in claim 8, or the recombinant vector as described in claim 9.

11. A pharmaceutical preparation, characterized in that, It includes the short peptide composition as described in any one of claims 1 to 5.

12. The use of a short peptide composition as described in any one of claims 1 to 5, a recombinant polypeptide chain as described in any one of claims 6 to 7, a recombinant nucleic acid molecule as described in claim 8, a recombinant vector as described in claim 9, a recombinant cell as described in claim 10, or a formulation product as described in claim 11 in the preparation of an anti-skin aging product.

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