Recombinant collagen targeting activated frizzled-8 protein and preparation method and application thereof

Abstract

The application relates to a recombinant collagen protein targeting activated Frizzled-8 protein and a preparation method and application thereof, and belongs to the technical field of recombinant proteins. The recombinant collagen protein targeting activated Frizzled-8 protein has an amino acid sequence which comprises an amino acid sequence shown in any one of SEQ ID No. 1-SEQ ID No. 4 or an amino acid sequence with homology greater than or equal to 80% with any one of SEQ ID No. 1-SEQ ID No. 4. On the basis of realizing cell interstitial filling, the recombinant collagen protein can be combined with Frizzled-8 protein, and formation of the collagen protein is significantly promoted.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of recombinant proteins, and particularly relates to a recombinant collagen protein targeting activation of Frizzled-8 protein and a preparation method and application thereof. BACKGROUND

[0002] Skin is the largest organ of the human body, and the dermis is the largest and most important component of the skin. Collagen fibers are the most critical component of the dermis. With age, the content of collagen in the skin gradually decreases, and the skin appears to have decreased elasticity, sagging, roughness and other signs of aging. As a key component of the skin, collagen is widely used in skin care products. Collagen not only can hydrate and moisturize the skin, but also can repair the skin barrier and tighten the skin.

[0003] Collagen on the market mainly comes from the tissues of animals such as cattle, pigs and fish, which may have virus risks and immunogenicity defects, and the water solubility of animal collagen is poor, which limits its application in cosmetics. Recombinant collagen prepared by genetic engineering technology completely eliminates the risk of animal viruses, and has the advantages of low immunogenicity and uniform molecular weight. More importantly, the water solubility of recombinant collagen is good, which is convenient to add, and has great application prospects in the fields of cosmetics, medical cosmetology, biological medicine and the like.

[0004] At present, the existing recombinant collagen is only used as an intercellular filler, and is degraded by proteases, making it difficult to continuously maintain the content of intercellular collagen. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the embodiments of the present application includes providing a recombinant collagen protein targeting activation of Frizzled-8 protein and a preparation method and application thereof. On the basis of realizing intercellular filling, the recombinant collagen protein can be combined with Frizzled-8 protein, and the formation of collagen protein is significantly promoted.

[0006] In a first aspect, the embodiments of the present application provide a recombinant collagen protein targeting activation of Frizzled-8 protein, and the amino acid sequence of the recombinant collagen protein includes an amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4 or an amino acid sequence with homology greater than or equal to 80% with any one of SEQ ID No. 1-SEQ ID No. 4.

[0007] The recombinant collagen protein provided in the present application has an amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4, or an amino acid sequence with homology of greater than or equal to 80% to any one of SEQ ID No. 1-SEQ ID No. 4, and can effectively bind to Frizzled-8 protein, thereby significantly promoting the formation of collagen protein.

[0008] In some embodiments of the present application, the amino acid sequence of the recombinant collagen protein comprises an amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4.

[0009] The recombinant collagen protein provided in the present application has an amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4, or an amino acid sequence with homology of greater than or equal to 80% to any one of SEQ ID No. 1-SEQ ID No. 4, and can effectively bind to Frizzled-8 protein, thereby significantly promoting the formation of collagen protein.

[0010] In some embodiments of the present application, the amino acid sequence of the recombinant collagen protein comprises an amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4.

[0011] The recombinant collagen protein provided in the present application has an amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4, or an amino acid sequence with homology of greater than or equal to 80% to any one of SEQ ID No. 1-SEQ ID No. 4, and can effectively bind to Frizzled-8 protein, thereby significantly promoting the formation of collagen protein.

[0012] In some embodiments of the present application, the amino acid sequence of the recombinant collagen protein comprises an amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4.

[0013] The recombinant collagen protein provided in the present application has an amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4, or an amino acid sequence with homology of greater than or equal to 80% to any one of SEQ ID No. 1-SEQ ID No. 4, and can effectively bind to Frizzled-8 protein, thereby significantly promoting the formation of collagen protein.

[0014] In some embodiments of the present application, the amino acid sequence of the recombinant collagen protein comprises an amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4.

[0015] The recombinant collagen protein provided in the present application has an amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4, or an amino acid sequence with homology of greater than or equal to 80% to any one of SEQ ID No. 1-SEQ ID No. 4, and can effectively bind to Frizzled-8 protein, thereby significantly promoting the formation of collagen protein.

[0016] In some embodiments of the present application, the amino acid sequence of the recombinant collagen protein comprises an amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4.

[0017] The recombinant plasmid provided in the application contains the nucleic acid, the recombinant plasmid is transfected into a host cell, and the expression of the above-mentioned recombinant collagen protein is induced, which can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen protein.

[0018] In a fourth aspect, the embodiments of the present application provide an engineering bacteria containing the recombinant plasmid provided in the third aspect.

[0019] The engineering bacteria provided in the application contains the above-mentioned recombinant plasmid, and the expression of the above-mentioned recombinant collagen protein can be induced, which can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen protein.

[0020] In some embodiments of the present application, the host cell of the engineering bacteria is Pichia pastoris.

[0021] The Pichia pastoris used as the host cell in the present application belongs to the eukaryotic expression system, and the above-mentioned recombinant collagen protein is expressed through the eukaryotic expression system, which can make the above-mentioned recombinant collagen protein closer to the conformation of natural protein and has higher activity.

[0022] In a fifth aspect, the embodiments of the present application provide a preparation method of the recombinant collagen protein for targeting and activating the Frizzled-8 protein as provided in the first aspect, comprising:

[0023] (1) designing a coding region sequence of the recombinant collagen protein for targeting and activating the Frizzled-8 protein, adding a signal peptide and a fusion His tag at the same time, synthesizing an expression sequence of the recombinant collagen protein for targeting and activating the Frizzled-8 protein, and inserting the expression sequence into the MSC cloning site of the pPIC9K plasmid to obtain a recombinant plasmid;

[0024] (2) transfecting the recombinant plasmid into Pichia pastoris, inducing expression and purification to obtain the recombinant collagen protein for targeting and activating the Frizzled-8 protein.

[0025] The preparation method of the recombinant collagen protein for targeting and activating the Frizzled-8 protein provided in the present application inserts the expression sequence of the above-mentioned recombinant collagen protein containing the signal peptide and the His tag into the MSC cloning site of the pPIC9K plasmid to obtain a recombinant plasmid, and then transfecting into Pichia pastoris to express the recombinant collagen protein which can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen protein.

[0026] In a sixth aspect, the embodiments of the present application provide an application of the recombinant collagen protein as provided in the first aspect in the preparation of a cosmetic or a drug for targeting and activating the Frizzled-8 protein.

[0027] Since the recombinant collagen provided by the first aspect can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen, the cosmetic or pharmaceutical prepared by using the recombinant collagen can also effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen, and has the effects of anti-aging and firming. BRIEF DESCRIPTION OF DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings.

[0029] Figure 1 The figure shows the binding model of the protein provided in Test Example 1 of the present application to the Frizzled-8 protein; wherein A-Example 1, B-Example 2, C-Example 3, D-Example 4, E- Control group. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased on the market.

[0031] At present, the existing recombinant collagen is only used as an intercellular filler, and is degraded by proteases, making it difficult to continuously maintain the content of intercellular collagen.

[0032] Therefore, the embodiments of the present application provide a recombinant collagen targeting the activation of Frizzled-8 protein, and the amino acid sequence of the recombinant collagen includes the amino acid sequence shown in any one of SEQ ID No. 1-SEQ ID No. 4 or an amino acid sequence with a homology of greater than or equal to 80% with any one of SEQ ID No. 1-SEQ ID No. 4.

[0033] The recombinant collagen protein having the amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4 or the amino acid sequence having homology of greater than or equal to 80% with any one of SEQ ID No. 1-SEQ ID No. 4 can effectively bind to the Frizzled-8 protein, which is an extracellular receptor of the Wnt / β-catenin pathway. When the recombinant collagen protein binds to the Frizzled-8 protein, the Wnt / β-catenin pathway can be activated, the cell growth cycle can be regulated, and thus the formation of collagen can be significantly promoted. Therefore, the recombinant collagen protein having the amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4 or the amino acid sequence having homology of greater than or equal to 80% with any one of SEQ ID No. 1-SEQ ID No. 4 can significantly promote the formation of collagen, and can be used for preparing cosmetics or drugs, and has the effects of anti-aging and firming.

[0034] For example, the amino acid sequence of the recombinant collagen protein can have homology of any one of 80%, 82%, 85%, 88%, 90%, 95%, 99%, and 99.9% or a range value between any two of them with the amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4.

[0035] In some embodiments of the present application, the amino acid sequence of the recombinant collagen protein includes the amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4. The recombinant collagen protein having the amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4 can effectively bind to the Frizzled-8 protein, and thus can significantly promote the formation of collagen.

[0036] In some embodiments of the present application, the amino acid sequence of the recombinant collagen protein is as shown in SEQ ID No. 1 or SEQ ID No. 4. The recombinant collagen protein having the amino acid sequence as shown in SEQ ID No. 1 or SEQ ID No. 4 can more optimally bind to the Frizzled-8 protein, and thus can more significantly promote the formation of collagen.

[0037] In some embodiments of the present application, the amino acid sequence of the recombinant collagen protein is as shown in SEQ ID No. 1. The recombinant collagen protein having the amino acid sequence as shown in SEQ ID No. 1 can most optimally bind to the Frizzled-8 protein, and thus can more significantly promote the formation of collagen.

[0038] The embodiment of the present application provides a nucleic acid for encoding the recombinant collagen protein provided in the above. The nucleic acid provided in the present application is used for encoding the recombinant collagen protein, and the recombinant collagen protein can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen protein.

[0039] The embodiment of the present application provides a recombinant plasmid comprising the nucleic acid. The recombinant plasmid comprising the nucleic acid is transfected into a host cell, and the expression of the recombinant collagen protein with the amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4 is induced, so that the recombinant collagen protein can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen protein.

[0040] The embodiment of the present application provides an engineering bacterium comprising the recombinant plasmid. The engineering bacterium comprising the recombinant plasmid can induce the expression of the recombinant collagen protein with the amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4, so that the recombinant collagen protein can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen protein.

[0041] In some embodiments of the present application, the host cell of the engineering bacterium is Pichia pastoris. Pichia pastoris belongs to a eukaryotic expression system, and the expression of the recombinant collagen protein through the eukaryotic expression system can make the recombinant collagen protein closer to the conformation of natural protein, and the activity is higher. It should be noted that the host cell refers to any cell type, which is easy to be transformed, transfected, transduced and the like by the recombinant plasmid comprising the present application. The host cell can be any cell which is useful in the production of the recombinant collagen protein of the present application.

[0042] The embodiment of the present application provides a preparation method of the recombinant collagen protein for targeting and activating the Frizzled-8 protein as described above, comprising:

[0043] (1) designing a coding region sequence of the recombinant collagen protein for targeting and activating the Frizzled-8 protein, adding a signal peptide and a fusion His tag at the same time, synthesizing an expression sequence of the recombinant collagen protein for targeting and activating the Frizzled-8 protein, and inserting the expression sequence into the MSC cloning site of the pPIC9K plasmid to obtain a recombinant plasmid;

[0044] (2) transfecting the recombinant plasmid into Pichia pastoris, inducing expression and purification, and obtaining the recombinant collagen protein for targeting and activating the Frizzled-8 protein.

[0045] The expression sequence of the recombinant collagen containing the signal peptide and the His tag is inserted into the MSC cloning site of the pPIC9K plasmid to obtain a recombinant plasmid, which is then transfected into Pichia pastoris to express a recombinant collagen having an amino acid sequence including any one of the amino acid sequences shown in SEQ ID No. 1-SEQ ID No. 4, which can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen.

[0046] It should be noted that the method of transfecting the recombinant plasmid into the host cell is well known, and can also be introduction, microinjection technology, gene gun technology, liposome-mediated method, etc.

[0047] The application provides an application of the recombinant collagen provided in the first aspect in the preparation of a cosmetic or a drug for targeted activation of the Frizzled-8 protein.

[0048] Since the recombinant collagen having an amino acid sequence including any one of the amino acid sequences shown in SEQ ID No. 1-SEQ ID No. 4 can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen, the cosmetic or the drug prepared by using the recombinant collagen can also effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen, and has the effects of anti-aging and firming.

[0049] The features and performances of the application are further described in detail below in combination with the examples.

[0050] The apparatuses used in the examples and test examples are as follows:

[0051] 1. Culture medium:

[0052] (1) YPD solid medium: 1% yeast extract; 2% peptone; 2% glucose; 1.5% agar; and the rest is distilled water.

[0053] (2) BMGY medium: 1% yeast extract; 2% peptone; 1.34% YNB; 1% glycerol; 10% 1M PBS (PH = 6.0); and the rest is distilled water.

[0054] (3) BMMY medium: 1% yeast extract; 2% peptone; 1.34% YNB; 1% methanol; 10% 1M PBS (PH = 6.0); and the rest is distilled water.

[0055] 2. Pichia pastoris GS115 is purchased from Shanghai Blue Sky Bio-Technology Co., Ltd.

[0056] 3. Ni 2+Affinity chromatography column, purchased from Thermo Fisher Scientific.

[0057] 4. BJ cells, purchased from Sartomer (Shanghai) Biotech Co., Ltd.

[0058] 5. Wnt1 protein, purchased from Sigma-Aldrich, purity ≥98%.

[0059] 6. Nucleic acid extraction kit, purchased from Yixing Biotech (Shanghai) Co., Ltd.

[0060] Example 1

[0061] This example provides the amino acid sequence design of the recombinant collagen targeting the activation of Frizzled-8 protein, the method is as follows:

[0062] The full amino acid sequence of human type III collagen alpha 1 subunit (ID: P02461) is obtained from UniPort protein database for screening and optimization, and the recombinant collagen with the amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4 is designed.

[0063] Example 2

[0064] This example provides the construction and expression method of the recombinant collagen 1 targeting the activation of Frizzled-8 protein, the method is as follows:

[0065] (1) The coding region sequence of the recombinant collagen 1 targeting the activation of Frizzled-8 protein is designed according to the amino acid sequence as shown in SEQ ID No. 1, and a signal peptide and a fusion His tag are added, the expression sequence of the recombinant collagen 1 targeting the activation of Frizzled-8 protein is synthesized, and the expression sequence is inserted into the MSC cloning site of pPIC9K plasmid to obtain a recombinant plasmid (synthesized by Suzhou Biotech Co., Ltd. on commission);

[0066] (2) The recombinant plasmid is transfected into Pichia pastoris, and Pichia pastoris electrotransformation competent cells are prepared and electroporation transformed, the preparation of Pichia pastoris electrotransformation competent cells refers to the method of (Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris. Biotechniques. 2005 January. 38(1): 44-48. Lin-Cereghino J, 2005), and the engineering bacteria are constructed;

[0067] (3) The bacterial liquid of the engineering bacteria was streaked on YPD solid medium to activate the bacterial strain;

[0068] (4) After single colonies were grown on the plate, a single colony was picked into BMGY medium and cultured for 24 h. The bacterial liquid was inoculated into BMMY medium at a 2% inoculation amount, and fermentation was started at 30°C and 250 rpm for 5 days to obtain a fermentation broth;

[0069] (5) The fermentation broth was centrifuged at 4°C and 8000 rpm for 15 min, and the fermentation supernatant was collected. The recombinant collagen in the fermentation supernatant was bound using a Ni 2+ affinity column, and then eluted with 400 mM imidazole buffer. The eluate was collected, and the recombinant collagen was concentrated to 1 mg / mL using an ultrafiltration tube. The obtained solution was the recombinant collagen 1 targeting the activated Frizzled-8 protein (prepared by the company Biogems).

[0070] The amino acid sequence of the recombinant collagen 1 targeting the activated Frizzled-8 protein is shown in SEQ ID No. 1: GGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGLQGLPGTGGPPGENGKPGEPGPK.

[0071] Example 3

[0072] This example provides a construction and expression method of the recombinant collagen 2 targeting the activated Frizzled-8 protein as follows:

[0073] (1) The coding region sequence of the recombinant collagen 2 targeting the activated Frizzled-8 protein was designed according to the amino acid sequence shown in SEQ ID No. 2, and a signal peptide and a fusion His tag were added. The expression sequence of the recombinant collagen 2 targeting the activated Frizzled-8 protein was synthesized, and the expression sequence was inserted into the MSC cloning site of the pPIC9K plasmid to obtain a recombinant plasmid (synthesized by Suzhou Bio-Technology Co., Ltd.).

[0074] The rest is the same as the method of Example 2.

[0075] The amino acid sequence of the recombinant collagen 2 targeting the activated Frizzled-8 protein is shown in SEQ ID No. 2: KDGPAGKDGESGRPGRPGERGLP.

[0076] Example 4

[0077] The present embodiment provides a method for constructing and expressing recombinant collagen 3 targeting and activating Frizzled-8 protein as follows:

[0078] (1) The coding region sequence of recombinant collagen 3 targeting and activating Frizzled-8 protein was designed according to the amino acid sequence as shown in SEQ ID No. 3, and a signal peptide and a fusion His tag were added. The expression sequence of recombinant collagen 3 targeting and activating Frizzled-8 protein was synthesized, and the expression sequence was inserted into the MSC cloning site of pPIC9K plasmid to obtain a recombinant plasmid (synthesized by Suzhou Biotechnology Co., Ltd. on commission).

[0079] The rest of the method is the same as that of Example 2.

[0080] The amino acid sequence of the recombinant collagen 3 targeting and activating Frizzled-8 protein is shown in SEQ ID No. 3: GEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGE PGAN.

[0081] Example 5

[0082] The present embodiment provides a method for constructing and expressing recombinant collagen 4 targeting and activating Frizzled-8 protein as follows:

[0083] (1) The coding region sequence of recombinant collagen 4 targeting and activating Frizzled-8 protein was designed according to the amino acid sequence as shown in SEQ ID No. 4, and a signal peptide and a fusion His tag were added. The expression sequence of recombinant collagen 4 targeting and activating Frizzled-8 protein was synthesized, and the expression sequence was inserted into the MSC cloning site of pPIC9K plasmid to obtain a recombinant plasmid (synthesized by Suzhou Biotechnology Co., Ltd. on commission).

[0084] The rest of the method is the same as that of Example 2.

[0085] The amino acid sequence of the recombinant collagen 4 targeting and activating Frizzled-8 protein is shown as SEQ ID No. 4: GPAGEEGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGV AGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKD.

[0086] Test Example 1

[0087] In this test example, the molecular mechanics / generalized Born surface area method of the HDOCK SERVER platform was used to calculate the optimal docking score and confidence score of each recombinant collagen targeting and activating Frizzled-8 protein and the optimal binding model of Frizzled-8 protein for the recombinant collagens targeting and activating Frizzled-8 protein provided in Examples 2-5. The human Frizzled-8 protein structure file (ID: 5UN6) was obtained from the PDB protein database.

[0088] In addition, the Wnt1 protein structure file (ID: 6L8M) was obtained from the PDB protein database, and the optimal docking score and confidence score of the optimal binding model thereof and Frizzled-8 protein were calculated as a control group according to the same method as described above.

[0089] Each binding model is shown in Figure 1 The optimal docking score and confidence score results are shown in Table 1.

[0090] Table 1 Optimal docking score and confidence score of recombinant collagen and Frizzled-8 protein

[0091]

[0092] Figure 1In the table, the yellow model in A is the recombinant collagen provided by Example 2 in turn, the yellow model in B is the recombinant collagen provided by Example 3 in turn, the yellow model in C is the recombinant collagen provided by Example 4 in turn, the yellow model in D is the recombinant collagen provided by Example 5 in turn, and the yellow model in E is the Wnt1 protein in the control group. It can be seen from Figure 1 In the table, the yellow model in A is the recombinant collagen provided by Example 2 in turn, the yellow model in B is the recombinant collagen provided by Example 3 in turn, the yellow model in C is the recombinant collagen provided by Example 4 in turn, the yellow model in D is the recombinant collagen provided by Example 5 in turn, and the yellow model in E is the Wnt1 protein in the control group. It can be seen from

[0093] It can be seen from the results in Table 1 that the confidence scores of the recombinant collagens provided by Examples 2-5 and the Wnt1 protein in the control group in combination with the Frizzled-8 protein are all greater than 0.65, and all can be combined with the Frizzled-8 protein. Among them, the confidence score of the recombinant collagen provided by Example 2 in combination with the Frizzled-8 protein is greater than 0.8, and is close to the confidence score of the Wnt1 protein in the control group in combination with the Frizzled-8 protein, and the combination effect with the Frizzled-8 protein is the best. It is illustrated that the recombinant collagen provided by the amino acid sequence including the amino acid sequence as shown in any one of SEQ ID No. 1-SEQ ID No. 4 can be significantly combined with the Frizzled-8 protein, activate the Wnt / β-catenin pathway, can regulate the cell growth cycle, and significantly promote the formation of collagen.

[0094] Test Example 2

[0095] In this test example, the recombinant collagens provided by Examples 2-5 targeting the Frizzled-8 protein are detected for their performance of promoting the formation of collagen. The specific method is as follows:

[0096] (1) Take the BJ cells and place them in a DMEM high-sugar culture medium containing 10% fetal bovine serum and 1% double antibody, and culture them in an environment of 37°C and 5% carbon dioxide. When the cells grow well, inoculate the BJ cells in a 6-well plate at a density of 1x10 6 cells per hole, add 2mL of culture medium to each hole, incubate at 37°C for 24h, and then add the recombinant collagens provided by Examples 1-4 targeting the Frizzled-8 protein and the Wnt1 protein in the control group to the culture medium, respectively, to set the final concentration to be 50ng / mL. Continue to culture for 24h, and then collect the cells;

[0097] (2) Use a nucleic acid extraction kit to extract the total RNA of the cells in each experimental group, and reverse transcribe it into cDNA. Find the human type III collagen protein sequence and related information, synthesize primers, and perform RT-PCR detection of the mRNA expression amount of the cells in each experimental group according to the steps of the kit. Calculate the relative expression amount of the inflammatory factor type III collagen by the method. The results are shown in Table 2.

[0098] Table 2 Relative expression amount of collagen type III

[0099] Sample Relative expression amount of collagen type III Blank group 1.0±0.1 Example 1 8.9±0.3 Example 2 2.4±0.6 Example 3 3.1±0.2 Example 4 5.4±0.6 Control group 9.0±0.2

[0100] As can be seen from the results of Table 2, the recombinant collagen proteins provided in Examples 2-5 can all significantly improve the relative expression amount of collagen type III of BJ cells. Among them, the recombinant collagen protein provided in Example 2 has the best effect of improving the relative expression amount of collagen type III of BJ cells, and has no significant difference with the control group Wnt1 protein in improving the relative expression amount of collagen type III of BJ cells. It is illustrated that the recombinant collagen protein provided in the present application, which has an amino acid sequence comprising any one of SEQ ID No. 1-SEQ ID No. 4, can significantly bind to Frizzled-8 protein, activate Wnt / β-catenin pathway, regulate cell growth cycle, and significantly promote the formation of collagen protein.

[0101] The above-described embodiments are part of the embodiments of the present application, rather than all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but only represents selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

Claims

1. A recombinant collagen targeting activation of Frizzled-8 protein, characterized in that, The amino acid sequence of the recombinant collagen is shown in any one of SEQ ID No. 1-SEQ ID No.

4.

2. The recombinant collagen of claim 1, wherein, The amino acid sequence of the recombinant collagen is shown in any one of SEQ ID No. 1-SEQ ID No.

4.

3. The recombinant collagen of claim 2, wherein, The amino acid sequence of the recombinant collagen is shown in any one of SEQ ID No. 1-SEQ ID No.

4.

4. A nucleic acid encoding the recombinant collagen according to any one of claims 1-3.

5. A recombinant plasmid, characterized by comprising the nucleotide sequence of SEQ ID NO:

1. comprising the nucleic acid according to claim 4.

6. An engineered bacterium, characterized in that, comprising the recombinant plasmid according to claim 5.

7. The engineered bacterium of claim 6, wherein, The host cell of the engineered bacteria is Pichia pastoris.

8. A method of producing a recombinant collagen targeting the activation of a Frizzled-8 protein according to any one of claims 1 to 3, characterized in that, comprising: (1) designing the coding region sequence of the recombinant collagen targeting and activating Frizzled-8 protein, adding a signal peptide and a fusion His tag, synthesizing the expression sequence of the recombinant collagen targeting and activating Frizzled-8 protein, inserting the expression sequence into the MSC cloning site of pPIC9K plasmid to obtain a recombinant plasmid; (2) transfecting the recombinant plasmid into Pichia pastoris, inducing expression and purification to obtain the recombinant collagen targeting and activating Frizzled-8 protein.

9. Use of the recombinant collagen according to any one of claims 1-3 in the preparation of a cosmetic product.

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