GDF11 truncated body and application thereof

Abstract

The invention particularly relates to a GDF11 truncated body and application thereof. The GDF11 truncated body provided by the invention is obtained by deleting an N-terminal signal peptide of a full-length GDF11 protein and retaining a complete mature structural domain of the full-length GDF11 protein, and the length of the truncated body is at least one part between 26th to 407th amino acids of an amino acid sequence of the full-length GDF11 protein. The GDF11 truncated body provided by the invention is shorter in sequence, smaller in molecular weight and easy to synthesize and modify, and the sequence length and the molecular weight are remarkably reduced on the basis of reserving high-affinity binding of an original aptamer and GDF11, so that the in-vitro synthesis and modification process is greatly simplified, and the production efficiency is improved. Secondly, the GDF11 truncation provided by the invention also has biological activity of inhibiting skin photoaging and inflammatory response caused by ultraviolet radiation, and provides new possibility for application of the GDF11 truncation in the fields of skin protection, anti-aging, anti-inflammatory treatment and the like.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and specifically relates to a GDF11 truncation and its application. Background Art

[0002] Growth differentiation factor 11 (GDF11) is a member of the TGF-βs subfamily, which is mainly located in the region of human chromosome 12q13.2 and encoded by 407 amino acids. The molecular structure of GDF11 contains multiple key regions, including a signal peptide, an RXXR proteolytic processing site, and a conserved carboxyl-terminal region of cysteine residues, etc. These regions are crucial for the exertion of its biological activity. First, the presence of the signal peptide enables GDF11 to be effectively secreted extracellularly, thereby exerting its long-distance regulatory role. Second, the RXXR proteolytic processing site enables GDF11 to be precisely cleaved in specific biological processes, forming fragments with specific biological activities. In addition, the conserved carboxyl-terminal region of cysteine residues is a structural feature shared by GDF11 and other TGF-β family members, and this region is of great significance for maintaining its biological activity and stability.

[0003] As an important member of the transforming growth factor-β (TGF-β) superfamily, the unique biological characteristics and broad application prospects of GDF11 have attracted extensive attention in the scientific community. GDF11 is widely present in various tissues such as skin, heart, liver, and skeletal muscle, highly expressed in young individuals, and plays a key role in the process of growth and development. Recent studies have shown that GDF11 can promote fibroblasts to secrete collagen (such as type I collagen) and elastin, while inhibiting the overexpression of matrix metalloproteinases (MMPs) (MMPs are markers of skin anti-wrinkle activity, such as MMP-1 degrading collagen and MMP-3 degrading elastic fibers, etc.), thereby reducing collagen degradation and maintaining the integrity of the skin structure. In addition, animal models suggest that GDF11 may regulate the function of skin stem cells by activating the Smad2 / 3 signaling pathway and delay the decline of age-related epidermal regeneration ability.

[0004] However, despite the broad application prospects of GDF11, in the actual research and application process, traditional aptamer technology faces a series of challenges. Aptamers, as artificially synthesized oligonucleotides or oligopeptides, can tightly bind to specific target molecules, thereby achieving the recognition and regulation of target molecules. However, the sequences of traditional aptamers are usually long, which not only significantly increases the synthesis cost and time of the molecule itself, but also leads to more by-products in the chemical synthesis process, thus reducing the purity of the final product. In addition, long aptamers are prone to form complex secondary and tertiary structures, such as stem-loop structures or pseudoknot structures, and this kind of higher-order structure hinders the efficiency of modification reactions, especially when performing functional modifications (such as bioluminescence labeling or chemical cross-linking). More importantly, long-sequence aptamers may lead to a decrease in target binding efficiency due to their large molecular weight and the stability of higher-order structures in in vitro applications, thus affecting the function. For example, large molecular aptamers have weak transmembrane ability in biomedical applications and are difficult to maintain efficient functions in the intracellular and extracellular environments, and this characteristic limits their applications in the biomedical field. In addition, aptamers with longer lengths require higher concentrations of synthetic raw materials in large-scale production, which not only increases the production cost, but also complicates the process and is difficult to meet the requirements of actual industrial applications.

[0005] Therefore, in the process of in vitro synthesis and subsequent modification of traditional aptamers, the longer sequences not only significantly increase the technical difficulty, but also limit their operability in large-scale production and applications. To address these problems, developing a truncated form with a shorter sequence and smaller molecular weight has become a key goal for optimizing aptamer technology. Summary of the Invention

[0006] Aiming at the many problems brought about by the longer sequences of traditional aptamers, the present invention aims to provide a truncated form of GDF11 and its applications. The present invention uses the full-length GDF11 protein as the starting template, truncates the sequence of the GDF11 aptamer through genetic engineering technology, reduces its molecular weight, and reduces the formation of its complex higher-order structures, thereby improving the efficiency of modification reactions and target binding ability. At the same time, short-sequence aptamers have higher operability and economy in in vitro synthesis and large-scale production, and can meet the requirements of actual industrial applications.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions:

[0008] The first objective of the present invention is to provide a truncated form of GDF11, which is obtained by deleting the N-terminal signal peptide of the full-length GDF11 protein and retaining the complete mature domain of the full-length GDF11 protein, and is at least a part between the 26th and 407th amino acids of the amino acid sequence of the full-length GDF11 protein.

[0009] Preferably, the truncated body is obtained by retaining at least a part of the amino acids between the 363rd and 407th amino acids in the amino acid sequence of the full-length GDF11 protein, including the highly conserved cysteine knot Cys391 / Cys407 and the TGF-β characteristic CCTPTK motif in the C-terminal domain.

[0010] Preferably, the amino acid sequence of the GDF11 truncated body is as shown in SEQ ID NO.3.

[0011] Preferably, the sequence encoding the amino acid is a polynucleotide sequence, and the polynucleotide sequence is as shown in SEQ ID NO.7.

[0012] The second object of the present invention is to provide a recombinant expression vector containing the polynucleotide sequence encoding the above-mentioned GDF11 truncated body.

[0013] Another object of the present invention is to provide a recombinant bacterium containing the above-mentioned recombinant expression vector.

[0014] Preferably, the recombinant bacterium uses a Pichia pastoris engineering bacterium as the host.

[0015] Preferably, the Pichia pastoris engineering bacterium includes Pichia pastoris GS115.

[0016] Another object of the present invention is to provide an application of the above-mentioned GDF11 truncated body in the preparation of skin care products for skin photoaging caused by ultraviolet radiation.

[0017] Another object of the present invention is to provide an application of the above-mentioned GDF11 truncated body in the preparation of skin care products for inhibiting inflammatory reactions caused by ultraviolet radiation.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a GDF11 truncated body with a shorter sequence, smaller molecular weight, and easier to synthesize and modify. On the basis of retaining the high-affinity binding of the original aptamer to GDF11, the sequence length and molecular weight are significantly reduced, thus greatly simplifying the in vitro synthesis and modification process and improving the production efficiency. In addition, due to the smaller molecular weight of the truncated body, its transportation and distribution in the body are more efficient, and it can more effectively target diseased tissues or cells, further enhancing the therapeutic effect.

[0020] Secondly, through in vitro experiments, it is proved that the GDF11 truncated body provided by the present invention has significant biological activity, and it has the effect of inhibiting skin photoaging and inflammatory reactions caused by ultraviolet radiation. This discovery provides new possibilities for the application of the GDF11 truncated body in the fields of skin protection, anti-aging, and anti-inflammatory treatment. Brief Description of the Drawings

[0021] Figure 1 It is a graph for hydrophilicity-hydrophobicity analysis and binding site prediction;

[0022] Figure 2 It is a two-dimensional prediction graph of protein;

[0023] Figure 3 It is a three-dimensional prediction graph of protein;

[0024] Figure 4 It is a gel graph for detecting high-copy transformants;

[0025] Figure 5 It is a Page graph of induction expression conditions;

[0026] Figure 6 It is a graph for identifying the purity of truncated body C1 by HPLC;

[0027] Figure 7 It is an MS graph for qualitative identification of C1 truncated body;

[0028] Figure 8 It is a total ion current graph - C1 truncated body;

[0029] Figure 9 It is a graph of quantitative ion pairs;

[0030] Figure 10 It is a graph of extracted ion current of quantitative ion 1039.75;

[0031] Figure 11 It is a graph of extracted ion current of quantitative ion 819.73;

[0032] Figure 12 It is a graph of the result that C1 has low toxicity to human epidermal cells and can protect against damage caused by ultraviolet rays;

[0033] Figure 13 It is a graph of the result of photoaging skin inflammation;

[0034] Figure 14 It is a graph of the anti-wrinkle effect of truncated body C1;

[0035] Figure 15 It is a graph of the moisturizing effect of truncated body C1. Specific implementation mode

[0036] In the following examples of the present invention, the experimental methods without specific conditions are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. All kinds of commonly used chemical reagents used in the examples are commercially available products.

[0037] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by those skilled in the technical field to which this invention belongs. The terms used in the description of this invention are only for the purpose of describing specific embodiments and are not intended to limit this invention.

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the following further details this invention in conjunction with specific embodiments. It should be understood that these descriptions are exemplary and not intended to limit the scope of this invention. In addition, in the following descriptions, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of this invention.

[0039] The following embodiments further describe this invention, but these embodiments are not intended to limit the protection scope of this invention.

[0040] Example 1 Design of GDF11 Truncated Forms

[0041] 1. Construction of GDF11 Truncated Forms

[0042] Obtain the complete sequence of GDF11 and confirm its protein sequence information using the UniProt database. Among them, the amino acid sequence information of GDF11 is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2. First, use SignalP 5.0 (default parameters, eukaryotic mode) to predict the signal peptide region of GDF11. The results show that the N-terminal 1-25 amino acids are typical signal peptides (D-score = 0.87), and the cleavage site is between Gly25 and Ser26. Through NetCorona 1.0 protease cleavage prediction (threshold > 0.5), it is found that there are three potential Furin protease cleavage sites in the mature peptide segment (26-407): RSKR (positions 300-303, probability 0.82), RKAR (positions 189-192, probability 0.68), and RRER (positions 158-161, probability 0.59). Among them, the RSKR site is consistent with the C-terminal processing site of the reported native mature GDF11 (26-407).

[0043] Based on the above analysis, the truncated form design strategy is as follows: C1 (26-407) retains the complete mature domain; C2 (26-300) is truncated at the RSKR site to remove the C-terminal domain; C3 (26-192) is truncated based on the RKAR site; C4 (26-161) is truncated at the RRER site.

[0044] Based on the above analysis, the truncation design strategy is as follows: C1 (corresponding to positions 363 - 407 of the original sequence), retaining the highly conserved cysteine knot (Cys391 / Cys407) and the TGF-β characteristic CCTPTK motif in the C-terminal domain. C2 (corresponding to positions 137 - 172 of the original sequence), truncated upstream of RSKR (300 - 303), including the β-sheet core region (137 - 200) and part of the α-helix linker region. C3 (corresponding to positions 173 - 212 of the original sequence), truncated based on the RKAR (189 - 192) site, covering the starting segment of the α-helix (173 - 192) and the adjacent loop region. C4 (corresponding to positions 213 - 250 of the original sequence), truncated downstream of RRER (158 - 161), retaining the C-terminal cysteine-rich region (Cys213 / Cys225 / Cys238) and part of the CCTPTK motif. A total of 4 groups of GDF11 truncations, C1, C2, C3, and C4, were constructed, and their amino acid sequence information is shown as SEQ ID NO.3 - 6 below.

[0045] Meanwhile, the original polypeptide (26 - 407) has typical TGF-β family structural characteristics (β-sheet core region: positions 50 - 120, α-helix domain: positions 200 - 280). Truncation structure analysis shows that: C1: retains the complete C-terminal cysteine knot but lacks the N-terminal β-sheet (54% reduction in proportion). C2: completely retains the β-sheet core (100% coverage), but lacks the C-terminal α-helix (positions 200 - 280). C3: only retains the starting segment of the α-helix (positions 173 - 192, 61% coverage), and the loop region (193 - 212) has increased flexibility (B-factor > 80). C4: the cysteine-rich region forms a stable disulfide bond network (Disulfide bonds: Cys213 - Cys238, Cys225 - Cys245), but the CCTPTK motif (positions 243 - 248) shows a conformational shift due to truncation All truncation cleavage sites are located in evolutionarily conserved regions (Clustal Omega multiple sequence alignment, BLOSUM62 matrix), among which the RSKR site has a conservation rate of 98% (15 / 16 species) in vertebrates, supporting the rationality of its functional cleavage.

[0046] The amino acid sequence information of the GDF11 is as follows:

[0047] MVLAAPLLLGFLLLALELRPRGEAAEGPAAAAAAAAAAAAAGVGGERSSRPAPSVAPEPDGCPVCVWRQHSRELRLESIKSQILSKLRLKEAPNISREVVKQLLPKAPPLQQILDLHDFQGDALQPEDFLEEDEYHATTETVISMAQETDPAVQTDGSPLCCHFHFSPKVMFTKVLKAQLWVYLRPVPRPATVYLQILRLKPLTGEGTAGGGGGGRRHIRIRSLKIELHSRSGHWQSIDFKQVLHSWFRQPQSNWGIEINAFDPSGTDLAVTSLGPGAEGLHPFMELRVLENTKRSRR N LGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS(SEQ ID NO.1)

[0048] The nucleotide sequence information of GDF11:

[0049]

[0050] Amino acid sequence information of the GDF11 truncated form C1:

[0051] CCTPTKMSPINMLYFNDKQQIIYGKIPGMVVDRCGCS (SEQ ID NO.3)

[0052] Amino acid sequence information of the GDF11 truncated form C2:

[0053] NLGLDCDEHSSESRCCRYPLTVDFEAFGWDWIIAPK (SEQ ID NO.4)

[0054] Amino acid sequence information of the GDF11 truncated form C3:

[0055] AFGWDWIIAPKRYKANYCSGQCEYMFMQKYPHTHLVQQ (SEQ ID NO.5)

[0056] Amino acid sequence information of the GDF11 truncated form C4:

[0057] YCSGQCEYMFMQKYPHTHLVQQANPRGSAGPCCTPTKMS (SEQ ID NO.6)

[0058] 2. Physicochemical properties and activity screening of GDF11 truncated form construction

[0059] Based on the above sequence information, the ProtParam tool (https: / / web.expasy.org / protparam / ) on the ExPASy platform was used to analyze the physicochemical parameters of the peptide sequence, specifically including: molecular weight calculated based on the standard molecular weights of each amino acid (without considering disulfide bond modification), isoelectric point (pI) calculated using the Bjellqvist method, instability index evaluated based on the formula of Guruprasad et al. (1990), grand average of hydropathicity (GRAVY) calculated by the Kyte & Doolittle (1982) algorithm, and net charge calculated based on the dissociation constants (pKa) of each amino acid at pH 7.0; the Hydropath. / Kyte&Doolittle algorithm was selected using ProtScale (https: / / web.expasy.org), with a window size of 9 and row sliding window averaging to predict the hydrophilic and hydrophobic region distribution characteristics of the protein. The EMBOSSPepstats tool (https: / / www.ebi.ac.uk / ) was used for amino acid composition analysis, where the classification criteria for non-polar amino acids (Ala, Val, Leu, Ile, Pro, Phe, Trp, Met), polar amino acids (Gly, Ser, Thr, Cys, Tyr, Asn, Gln), acidic amino acids (Asp, Glu), and basic amino acids (Lys, Arg, His) refer to the amino acid property classification system of the International Union of Biochemistry and Molecular Biology (IUBMB). All calculation results were generated based on the default parameters of the tools, and the original algorithms and calculation formulas were cited from the technical documents of the corresponding tools and relevant original literature.

[0060] Among them, the physicochemical parameters of the four groups of GDF11 truncations C1, C2, C3, and C4 are shown in Table 1 below. The results of the hydrophilic and hydrophobic analysis and binding site prediction of the GDF11 truncations are as Figures 1 - 3 shown. The toxicity prediction results of different GDF11 truncations are shown in Table 2 below.

[0061] Table 1 Physicochemical properties of different truncations

[0062]

[0063]

[0064] Table 2 Toxicity prediction results of different truncations

[0065] Group Bioactivity Probability Predicted Value Prediction Result C1 0.666007 0.09 Non - Toxin C2 0.607631 1.08 Toxin C3 0.412511 0.4 Toxin C4 0.208399 0.48 Toxin

[0066] From the above experimental results, it can be seen that the GDF11 truncated form C1 may have good amphiphilicity and thermal stability, a more reasonable amino acid distribution, a secondary structure and a tertiary structure that are relatively similar to the original full-length GDF11 sequence, and the original biological activity of GDF11 and low toxicity.

[0067] Example 2 Expression of GDF11 truncated form C1

[0068] Select the expression vector: pPIC9K; determine the subcloning site: EcoRI / NotI. The constructed plasmid was linearized with SalI, PmeI, and SacI enzymes respectively, and then electrotransformed into the yeast GS115 host yeast cells, and recombinants were screened on plates. Incubate at 30 °C for 2 - 4 days. After the transformants grew out, the transformants were rinsed, mixed, and collected with sterile water. A small part was taken and spread on YPDA + G418 (125 and 200 μg / mL) plates for resistance screening; the finally grown clones were verified by PCR.

[0069] 1. Construction of the truncated form C1 strain

[0070] According to the sequence of GDF11 truncated form C1, PCR primers containing specific restriction sites were designed, and the sequence information is as follows:

[0071] The nucleotide sequence information of the GDF11 truncated form C1:

[0072] GAATTCAGTGAATCCAGATGTTGCAGATATCCATTGACTGTTGACTTTGAGGCATTCGGATGGGATTGGATTATCGCACCTAAGAGATACAAAGCAAACTATTGCTCCGGACAATGTGAGTACATGTTTATGCAGAAGTATCCACATACACATTTGGTTCAACAAGCTAATCCAAGAGGTAGTGCTGGACCTTGTTGTACGCCAACTAAGATGAGTCCAATCAATATGCTGTACTTTAACGATAAGCAACAAATTATCTACGGTAAGATTCCAGGTATGGTTGTGGATAGATGTGGTTGTTCTCACCACCACCACCACCACTAAGCGGCCGC(SEQ ID NO.7).

[0073] The PCR verification primers are as follows:

[0074] The sequence information of the 5'AOX1 primer: 5'-GACTGGTTCCAATTGACAAGC-3' (SEQ ID NO.8);

[0075] The primer sequence information of 3'AOX1: 5'-GCAAATGGCATTCTGACATCC-3' (SEQ ID NO.9).

[0076] Use the designed primers and template DNA for PCR amplification to obtain the DNA fragment of GDF11 truncated form C1. The PCR system is as follows:

[0077] Table 3 Reaction system

[0078] Reaction System Addition Amount (μL) 5'AOX1 Primer 2.0 3'AOX1 Primer 2.0 KOD Enzyme 0.2 dNTP (2mM) 2.0 KOD Buffer 5.0 Colony Template 2.0 <![CDATA[ddH2O]]> Add to 50

[0079] Reaction conditions: 98°C for 3 min; 98°C for 15 s (cycles 20 - 25); 55°C for 15 s (cycles 20 - 25); 72°C for 60 s (cycles 20 - 25); 72°C for 1 min; 10°C forever.

[0080] Verify the size of the target fragment by 1.5% agarose gel electrophoresis (120 V, 20 min) of the PCR product, purify the DNA fragment using a gel extraction kit (Jinsha Biology GE706), and determine the concentration and purity by Nanodrop.

[0081] 2. Electroporation experiment

[0082] Activation and cultivation of the strain: Streak and activate the preserved Pichia pastoris (GS115) strain on a YPD plate (containing 1% yeast extract, 2% peptone, 2% glucose, 2% agar) at 30°C for 48 h, pick a single colony and inoculate it into 5 mL of YPD liquid medium, and culture it with shaking at 30°C and 250 rpm until OD600 ≈ 2.0.

[0083] Preparation of competent cells: Take 1 mL of the bacterial solution and inoculate it into 50 mL of YPD medium, culture it at 30°C until OD600 ≈ 1.3 - 1.5. Centrifuge at 4°C and 1500×g for 5 min to collect the bacterial cells, wash them once with pre-cooled sterile water, and then resuspend them in 30 mL of pre-cooled 1 M sorbitol solution, and incubate on ice for 30 min. After centrifugation, resuspend the bacterial cells in 1 mL of pre-cooled 1 M sorbitol, aliquot (80 μL / tube) and immediately use them for electroporation.

[0084] Electroporation operation: Mix 10 μg of linearized recombinant plasmid with 80 μL of competent cells and incubate on ice for 5 min. Transfer them to a pre-cooled electroporation cuvette (2 mm gap), set the electroporation parameters: voltage 1500 V, capacitance 25 μF, resistance 200 Ω, pulse time 5 ms. Immediately add 1 mL of pre-cooled 1 M sorbitol after electroporation, let it stand and recover at 30°C for 1 h, and resuspend it in 100 μL of sorbitol solution after centrifugation.

[0085] 3. Screening and verification

[0086] Plate coating and culture: Spread the electrotransformation products on YPDS plates (containing 100 - 500 μg / mL Zeocin), and culture them upside down at 30 °C for 3 - 5 days until single colonies appear.

[0087] Select monoclonal colonies: Randomly pick 5 - 10 colonies and inoculate them into 2 mL of BMGY medium (containing 100 μg / mL Zeocin) respectively. Culture at 30 °C and 250 rpm for 24 h. Centrifuge to collect the cells before induction and transfer them to BMMY medium (containing 0.5% methanol) for continued induction expression.

[0088] Verify the transformants: Amplify the inserted fragment with AOX1 universal primers and verify the target band by electrophoresis.

[0089] The experimental results are as Figure 4 shown, and it can be seen from Figure 4 that the position of the target band is consistent with the theoretical band.

[0090] 4. Expression identification and purification of the GDF11 truncation:

[0091] Select high-copy cell lines with positive colony PCR for expression. The process is as follows:

[0092] A. Pre-culture: Inoculate the positive clone into 10 mL of BMGY medium (containing 1% glycerol, 100 μg / mL Zeocin), and culture it with shaking at 28 °C and 250 rpm until OD600 ≈ 2 - 6.

[0093] B. Induced expression: After culturing in BMGY, centrifuge to collect the cells (3000×g, 5 min), transfer them to BMMY medium for continued induction culture for 3 days at 28 °C, and the methanol concentration is 1.0%.

[0094] C. Sampling analysis and centrifugation, collect the supernatant of the medium, and detect it by Page after purification.

[0095] The experimental process of the Page detection includes: SDS-PAGE gel preparation: Prepare 12% separating gel and 5% stacking gel, and add pre-stained protein Marker (such as 10 - 180 kDa). Sample treatment: Mix 20 μL of the supernatant with 5×SDS loading buffer (containing β-mercaptoethanol), boil at 95 °C for 10 min, and load 10 μL after centrifugation. Electrophoresis conditions: Constant voltage 80 V (stacking gel) → 120 V (separating gel), and electrophorese until the bromophenol blue migrates to the bottom of the gel. Staining and decolorization: Stain with Coomassie Brilliant Blue for 30 min, and decolorize with decolorizing solution (10% acetic acid + 40% methanol) with shaking until the background is clear. Result interpretation: Confirm the expression of the GDF11 truncation C1 by comparing with the Marker.

[0096] The experimental results are as Figure 5As shown by Figure 5 It can be seen that after the proteins inserted into the yeast genome at different sites are induced to express and detected, the red arrow should be the target protein.

[0097] Example 3 Synthesis and Identification of GDF11 Truncated Form C1

[0098] 1. Synthesis of GDF11 truncated form C1

[0099] Based on the above sequence information, the GDF11 truncated form C1 was synthesized using the solid phase peptide synthesis (SPPS) method. The specific steps are as follows:

[0100] (1) Resin preparation: 3 g of RINK resin (100-200mesh, Sigma, 855130) was weighed into a 150 mL reactor and soaked with 50 mL of dichloromethane (Sigma-Aldrich, D84309). After 2 hours, the resin was washed with 3 times the volume of the resin in nitrogen-dimethylformamide (Sigma-Aldrich, D4551) and then dried. This was repeated four times and the resin was dried for later use.

[0101] (2) Deprotection (Fmoc): Add 20% piperidine (piperidine / DMF, Sigma-Aldrich, P4501) in a volume of 5 to 10 times the volume of the resin to the reactor and shake on a decolorizing shaker for 20 min to remove the Fmoc protecting group on the resin. After deprotection, wash four times with 3 to 5 times the volume of the resin in DMF and then drain.

[0102] (3) Deprotection detection: Take a small amount of resin and detect it using the ninhydrin (nine-well hydrated ninhydrin) method (react at 80-100°C for 1-3 minutes). If the resin has color, it means that the deprotection is successful.

[0103] (4) Coupling the first amino acid: Weigh an appropriate amount of the first amino acid at the C-terminus and an appropriate amount of 1-hydroxy-benzotriazole (Sigma-Aldrich, H9750) into a 50-mL centrifuge tube, add 20 mL of DMF to dissolve them, then add 3 mL of N,N-diisopropylcarbodiimide (DIC) and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30°C for reaction.

[0104] (5) Capping and re-deprotection: After 2 hours, cap the resin with a certain amount of acetic anhydride (Sigma-Aldrich, A1536) (acetic anhydride: DIEA: DCM = 1:1:2) for half an hour, then wash it four times with DMF 3 times the volume of the resin, and drain it for later use. Add 20% piperidine (piperidine / DMF = 1:4) 5 to 10 times the volume of the resin to the reactor, and shake it on a decolorization shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF and then drain it.

[0105] (6) Take a small amount of resin and test it using the ninhydrin (nine-well hydrated ninhydrin) method (two drops each of test A and test B, react at 100°C for 1 min). If the resin has color, it means that the deprotection is successful.

[0106] (7) Weigh an appropriate amount of the second amino acid and an appropriate amount of HOBT into a 50-mL centrifuge tube, add 25 mL of DMF to dissolve them, then add 2.5 mL of DIC and shake for 1 min. After the solution is clarified, add it to the reactor, and then place the reactor in a shaker at 30°C for reaction.

[0107] (8) After 1 hour, take a small amount of resin for testing using the ninhydrin method (two drops of test A and test B, react at 100°C for 1 min). If the resin is colorless, the reaction is complete; if the resin has color, the condensation is incomplete and the reaction should be continued.

[0108] (9) After the reaction is complete, wash the resin four times with DMF and then drain it. Add a certain amount of 20% piperidine (piperidine / DMF = 1:4) to the reactor and shake it on a decolorizing shaker for 20 minutes to remove the Fmoc protecting group on the resin. After deprotection, wash it four times with DMF and then drain it to check whether the protection is removed.

[0109] (10) Follow steps 9 to 11 to connect the following amino acids.

[0110] (11) After the last amino acid is attached, the protection is removed, the resin is washed four times with DMF, and then the resin is drained with methanol. The peptide is then cut from the resin with a cutting solution (trifluoroacetic acid: 1,2-ethanedithiol: 3, isopropylsilane: water = 95:2:2:1) (Sigma-Aldrich, T6508, D9122, I9491) (1-20 mL of cutting solution per gram of resin) and centrifuged four times with ice ether (cutting solution: ether = 1:9, Sigma-Aldrich, 470406). Finally, it is separated and purified by HPLC and then freeze-dried to obtain a peptide of a certain purity.

[0111] (12) HPLC separation and purification conditions: stationary phase: C18; mobile phase configuration: Pump A: V(TFA) / V(water) = 1 / 1000; Pump B: V(TFA) / V(acetonitrile) = 1 / 1000; flow rate: 1 - 10 mL / min; retention time: between 20 - 30 min.

[0112] 2. LC-MS identification of GDF11 truncated form C1

[0113] The truncated form C1 was dissolved in water to prepare a solution with a concentration of 1 mg / mL, and the solution to be tested was obtained by filtering through a 0.45 μm filter membrane. Use Thermo Scientific TM MAbPac TM Protein A chromatographic column, and the analytical conditions were set as follows: mobile phase of 0.10 - 0.25% phosphoric acid water - methanol, elution conditions from 0 min to 5 - 10 min, methanol ratio from 3 - 10% to 10 - 18%, from 5 - 10 min to 20 - 38 min, methanol ratio from 12% to 48%, from 20 - 38 min to 45 min, methanol ratio from 48% to 75%, from 45 min to 55 min, methanol ratio from 75% to 90%.

[0114] The experimental result graph is as Figure 6 shown. It can be seen from Figure 6 that under the above elution conditions, the retention time of the truncated form C1 is 9.166 min. The chromatographic peak shape of the truncated form C1 is good, without obvious impurity peak interference, indicating that the truncated form C1 has good resolution and purity in LC-MS analysis.

[0115] In Auto MS / MS mode, ESI ion source was used to collect primary and secondary mass spectrometry data. The ion source drying gas temperature was set at 320 °C, nitrogen flow rate was 8 L / min, sheath gas flow rate was 12 L / min, sheath gas temperature was 50 °C; the MS acquisition rate was set at 6 spectrum / s; MS2 acquisition rate: 12 spectrum / s. Positive ion mode was used for collection respectively. The capillary voltage in positive ion mode was set at 4000 V, and 12 ions in the primary spectrum were selected for secondary scanning. The secondary collision energies were set at 10 V and 40 V respectively. The primary mass scanning range was set at 50 - 1600 m / z, and the secondary mass scanning range was set at 20 - 1600 m / z.

[0116] The experimental results are as Figures 7 - 11As shown, it can be seen that the molecular weight of the truncated body C1 was determined to be 4155.92, which is consistent with the theoretically calculated molecular weight, further verifying the accurate synthesis of the truncated body C1. At the same time, through mass spectrometry analysis, the qualitative and quantitative ion pairs of the truncated body C1 peptide segments were determined to be 1039.75 and 819.73, and these ion pairs can be used for subsequent quantitative analysis.

[0117] In summary, through the above experiments, the polypeptide was successfully synthesized, separated and purified, and the polypeptide product - truncated body C1 with a purity of 91.69% was obtained. The truncated body C1 showed good resolution and purity in LC-MS analysis, and its molecular weight was 4155.92, which is the same as the calculated molecular weight. The qualitative and quantitative ion pairs of the truncated body C1 peptide segments were 1039.75 and 819.73, providing reliable data support for subsequent research.

[0118] Example 4 Anti-ultraviolet damage ability of truncated body C1

[0119] HaCaT cells (purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, catalog number: GNHu64) were cultured in DMEM medium containing 10% fetal bovine serum at 37 °C and 5% CO2 until they adhered to the wall; the HaCaT cells were inoculated into a 96-well cell culture plate at a cell density of 1×10 4 cells / mL with DMEM medium containing 10% fetal bovine serum and cultured overnight in a 37 °C, 5% CO2 incubator.

[0120] The medium was discarded. Except for the control group, the HaCaT cells in other groups were given a 5 - 15 min mixed ultraviolet radiation of 100 mL - 200 mL J UBV + 50 - 150 mJ UVA, and then the PBS was aspirated; a blank control group and a model group were set up, and 200 μl of DMEM was given to each well. Each experimental group was given 200 μL per well of DMEM containing 6.25, 12.5, 25, 50, and 100 ng / mL of the truncated body respectively, and cultured for another 48 h; the original solution was discarded, and 100 μL of working solution containing 5% CCK8 was given to each well and incubated at 37 °C for 30 min, and the absorbance at 450 nm was measured.

[0121] The experimental results are as Figure 12 shown. The truncated body C1 had no obvious toxicity to epidermal cells at a concentration of 100 ng / mL. At the same time, the truncated body could effectively protect human epidermal cells from ultraviolet-induced damage.

[0122] Example 5 Detection of the expression levels of inflammatory factors, MMPs and AQP-3 genes by RT-qPCR

[0123] By means of RT-qPCR detection method, analyze the effects of truncated C1 and full-length GDF11 at different concentrations (purchased from Shanghai Kanglang Biotechnology Co., Ltd., product number KL11365RP) on the gene expression levels of inflammatory factors (IL-6, IL-1β, COX-2), matrix metalloproteinases (MMP-1, MMP-3, MMP-9) and aquaporin-3 (AQP-3) in a photoaging skin model.

[0124] 1. Detection method:

[0125] (1) Cell treatment and RNA extraction: First, establish a photoaging model for hacat cells, that is, irradiate with a mixture of 200 mJ of UBV and 150 mJ of UVA to establish a model of skin photoaging. Then, divide the cells into different experimental groups (control group (non-model group), model group, positive control group, C1 group), and add truncated C1 at different concentrations (12.5 ng / mL, 25 ng / mL, 50 ng / mL) to the C1 group and full-length GDF11 (50 ng / mL) to the positive control group. After treatment for a certain time, extract the total RNA of each group of cells by the Trizol method, and reverse transcribe to obtain cDNA using the Evo M-MLV RT Rremix for qPCR kit (Aikery Biotech, AG11706).

[0126] (2) Real-time fluorescence quantitative PCR: Use real-time fluorescence quantitative PCR (Real-Time PCR) to detect the mRNA expression of IL-6, IL-1β, COX-2, MMP-1, MMP-3, MMP-9, AQP-3, and GAPDH in each experimental group. Reaction conditions: 95°C for 30 s; 95°C for 5 s (cycles 40); 60°C for 30 s (cycles 40); 95°C for 10 s (cycles 40); 72°C for 1 min; 10°C forever. The PCR primers involved are shown in Table 4 below, where m is a murine primer and h is a human primer.

[0127] Table 4 Primer sequence information involved in RT-qPCR

[0128]

[0129]

[0130] (3) Detection results:

[0131] The experimental results are as Figures 13 - 15 shown, among which, by Figure 13It can be seen that compared with the control group (non-model group), after administration of truncated C1 at different concentrations and full-length GDF11 at 50 ng / mL, the results showed that IL-6, IL-1β and COX-2 were all significantly down-regulated, indicating that truncated C1 can inhibit skin inflammation caused by photoaging.

[0132] It can be seen from Figure 14 that as the concentration of truncated C1 increased, MMP-1, MMP-3 and MMP-9 were all significantly down-regulated, and full-length GDF11 also showed a similar inhibitory effect. This indicates that truncated C1 can inhibit the formation of skin wrinkles caused by photoaging.

[0133] It can be seen from Figure 15 that in the experimental groups given truncated C1 and full-length GDF11, the results showed that aquaporin AQP-3 was significantly up-regulated and increased with the increase in the concentration of truncated C1. This shows that truncated C1 can promote the expression of aquaporin AQP-3, thus having a moisturizing effect after photoaging.

[0134] In summary, the present invention successfully detected the gene expression levels of inflammatory factors, MMPs and AQP-3 in a photoaged skin model by RT-qPCR technology, and found that truncated C1 and full-length GDF11 can significantly inhibit skin inflammation and wrinkle formation caused by photoaging, while promoting the expression of aquaporin AQP-3, having potential anti-skin photoaging and moisturizing effects. These findings provide a scientific basis for the application of truncated C1 in the field of skin anti-aging.

[0135] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A GDF11 truncation, characterized in that, The truncated body is obtained by deleting the N-terminal signal peptide of the full-length GDF11 protein and retaining at least a part of the full-length GDF11 protein within the range of amino acids 26 to 407 in the amino acid sequence of the full-length GDF11 protein.

2. The GDF11 truncation according to claim 1, wherein The truncated body is obtained by retaining the highly conserved cysteines Cys391 / Cys407 and the TGF-β characteristic CCTPTK motif in the C-terminal domain, and retaining at least a part of the truncated body within the range of amino acids 363 to 407 in the amino acid sequence of the full-length GDF11 protein.

3. The truncated GDF11 according to claim 1, wherein, The amino acid sequence of the GDF11 truncated body is shown in SEQ ID NO.

3.

4. The truncated GDF11 according to claim 1, wherein The sequence encoding the amino acid is a polynucleotide sequence, and the polynucleotide sequence is shown in SEQ ID NO.

7.

5. A recombinant expression vector, characterized in that, It comprises a polynucleotide sequence encoding the GDF11 truncated body as claimed in claim 4.

6. A recombinant bacterium, characterized in that, It comprises a recombinant expression vector as claimed in claim 5.

7. The recombinant bacterium according to claim 6, characterized in that, The recombinant bacterium uses Pichia pastoris engineering bacteria as the host.

8. The recombinant bacterium according to claim 7, characterized in that, The Pichia pastoris engineering bacteria include Pichia pastoris GS115.

9. Use of the GDF11 truncated body as claimed in claim 1 for preparing skin care products for skin photoaging caused by ultraviolet radiation.

10. Use of the GDF11 truncated body as claimed in claim 1 for preparing skin care products for inhibiting inflammatory responses caused by ultraviolet radiation.

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