Recombinant collagen for targeted activation of Frizzled-8 protein as well as preparation method and application of recombinant collagen

Abstract

The invention relates to recombinant collagen for targeted activation of Frizzled-8 protein as well as a preparation method and application of the recombinant collagen, and belongs to the technical field of recombinant protein. The invention discloses recombinant collagen for targeted activation of Frizzled-8 protein. The amino acid sequence of the recombinant collagen comprises an amino acid sequence shown as any one of SEQ ID No.1-SEQ ID No.4 or an amino acid sequence of which the homology with the amino acid sequence shown as any one of SEQ ID No.1-SEQ ID No.4 is greater than or equal to 80%. And on the basis of realizing intercellular filling, the compound can be combined with Frizzled-8 protein, so that the formation of collagen is remarkably promoted.

Description

Technical Field

[0001] The present application relates to the technical field of recombinant proteins, and in particular to a recombinant collagen protein that targets and activates Frizzled-8 protein, and a preparation method and application thereof. Background Art

[0002] The skin is the largest organ in 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. As we age, the content of collagen in the skin gradually decreases, and the skin shows aging phenomena such as decreased elasticity, sagging, and roughness. As a key component of the skin, collagen is widely used in skin care products. Collagen can not only hydrate and moisturize the skin, but also repair the skin barrier and tighten the skin.

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

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

[0005] In view of the shortcomings of the prior art, the purpose of the embodiments of the present application includes providing a recombinant collagen protein that targets and activates Frizzled-8 protein, and a preparation method and application thereof. On the basis of achieving intercellular filling, it can also bind to Frizzled-8 protein to significantly promote the formation of collagen.

[0006] In the first aspect, the embodiments of the present application provide a recombinant collagen protein for targeted activation of Frizzled-8 protein, wherein 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 having a homology greater than or equal to 80% with any one of SEQ ID NO.1-SEQ ID No.4.

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

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

[0009] The recombinant collagen provided in the present application, whose amino acid sequence is any one of SEQ ID No. 1 to SEQ ID No. 4, can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen.

[0010] In some embodiments of the present application, the amino acid sequence of the recombinant collagen is as shown in SEQ ID No.1 or SEQ ID No.4.

[0011] The recombinant collagen provided in the present application, such as the amino acid sequence shown in SEQ ID No. 1 or SEQ ID No. 4, can better bind to the Frizzled-8 protein, thereby more significantly promoting the formation of collagen.

[0012] In some embodiments of the present application, the amino acid sequence of the recombinant collagen is as shown in SEQ ID No.1.

[0013] The recombinant collagen provided in the present application, whose amino acid sequence is as shown in SEQ ID No. 1, can optimally bind to the Frizzled-8 protein, thereby more significantly promoting the formation of collagen.

[0014] In a second aspect, an embodiment of the present application provides a nucleic acid encoding the recombinant collagen provided in the first aspect.

[0015] The nucleic acid provided in the present application is used to encode the above-mentioned recombinant collagen, which can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen.

[0016] In a third aspect, an embodiment of the present application provides a recombinant plasmid comprising the nucleic acid provided in the second aspect.

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

[0018] In a fourth aspect, an embodiment of the present application provides an engineered bacterium comprising the recombinant plasmid provided in the third aspect.

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

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

[0021] The present application uses Pichia pastoris as a host cell. Pichia pastoris belongs to a eukaryotic expression system. By expressing the above-mentioned recombinant collagen protein through a eukaryotic expression system, the above-mentioned recombinant collagen protein can be closer to the conformation of natural protein and have higher activity.

[0022] In a fifth aspect, the present application embodiment provides a method for preparing a recombinant collagen protein for targeted activation of Frizzled-8 protein as provided in the first aspect, comprising:

[0023] (1) Designing the coding region sequence of the recombinant collagen protein that targets and activates the Frizzled-8 protein, adding a signal peptide and a fusion His tag, synthesizing the expression sequence of the recombinant collagen protein that targets and activates 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) The recombinant plasmid was transfected into Pichia pastoris, induced for expression, and purified to obtain recombinant collagen that targeted and activated Frizzled-8 protein.

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

[0026] In a sixth aspect, an embodiment of the present application provides a use of the recombinant collagen provided in the first aspect in the preparation of cosmetics or drugs that target and activate Frizzled-8 protein.

[0027] Since the recombinant collagen provided in the first aspect can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen, therefore, the cosmetics or medicines prepared using the recombinant collagen can also effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen, and have anti-aging and firming effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is a diagram of the binding model of the protein provided in Experimental Example 1 of the present application and the Frizzled-8 protein; wherein A-Example 1, B-Example 2, C-Example 3, D-Example 4, and E-Control Group. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

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

[0032] To this end, an embodiment of the present application provides a recombinant collagen protein that targets and activates Frizzled-8 protein, wherein 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 having a homology greater than or equal to 80% with any one of SEQ ID NO.1-SEQ ID No.4.

[0033] The amino acid sequence provided in the present application is as shown in any one of SEQ ID No.1-SEQ ID No.4, or a recombinant collagen having 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, which can effectively bind to Frizzled-8 protein, which is an extracellular receptor of the Wnt / β-catenin pathway. When the recombinant collagen binds to the Frizzled-8 protein, the Wnt / β-catenin pathway can be activated, regulating the cell growth cycle, thereby significantly promoting the formation of collagen. Therefore, the amino acid sequence provided in the present application is as shown in any one of SEQ ID No.1-SEQ ID No.4, or a recombinant collagen having 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, which can significantly promote the formation of collagen, and is used to prepare cosmetics or drugs, and has anti-aging and firming effects.

[0034] As an example, the amino acid sequence of the recombinant collagen may have a homology with the amino acid sequence shown in any of SEQ ID No.1-SEQ ID No.4, but is not limited to 80%, 82%, 85%, 88%, 90%, 95%, 99% and 99.9%, or any range between two of them.

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

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

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

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

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

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

[0041] In some embodiments of the present application, the host cell of the engineered bacteria is Pichia pastoris. Pichia pastoris belongs to a eukaryotic expression system. By expressing the above-mentioned recombinant collagen protein through a eukaryotic expression system, the above-mentioned recombinant collagen protein can be closer to the conformation of the natural protein and have higher activity. It should be noted that the host cell refers to any cell type that is susceptible to transformation, transfection, transduction, etc. of the recombinant plasmid containing the present application. The host cell can be any cell useful in the production of the recombinant collagen protein of the present application.

[0042] The present application embodiment provides a method for preparing the recombinant collagen for targeted activation of Frizzled-8 protein as described above, comprising:

[0043] (1) Designing the coding region sequence of the recombinant collagen protein that targets and activates the Frizzled-8 protein, adding a signal peptide and a fusion His tag, synthesizing the expression sequence of the recombinant collagen protein that targets and activates 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) The recombinant plasmid was transfected into Pichia pastoris, induced for expression, and purified to obtain recombinant collagen that targeted and activated Frizzled-8 protein.

[0045] The expression sequence of the above-mentioned recombinant collagen containing a signal peptide and a 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 an amino acid sequence as shown in any one of SEQ ID No. 1 to SEQ ID No. 4. The recombinant collagen can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen.

[0046] It should be noted that methods for transfecting recombinant plasmids into host cells are well known, and the method may also be introduction, microinjection technology, gene gun technology, liposome-mediated method, etc.

[0047] The embodiments of the present application provide a use of the recombinant collagen provided in the first aspect in preparing cosmetics or drugs for targeted activation of Frizzled-8 protein.

[0048] Since the above-mentioned recombinant collagen protein including the amino acid sequence shown in any one of SEQ ID No.1-SEQ ID No.4 can effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen, therefore, the cosmetics or medicines prepared using the recombinant collagen protein can also effectively bind to the Frizzled-8 protein, thereby significantly promoting the formation of collagen, and have anti-aging and firming effects.

[0049] The features and performance of the present application are further described in detail below in conjunction with the embodiments.

[0050] The equipment used in the embodiments 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; 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); 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 was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0056] 3.Ni 2+Affinity chromatography columns were purchased from Thermo Fisher Scientific.

[0057] 4. BJ cells were purchased from Shanghai Stemcell Biotechnology Co., Ltd.

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

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

[0060] Example 1

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

[0062] The complete amino acid sequence of human type III collagen α1 subunit (ID: P02461) was obtained from the UniPort protein database for screening and optimization, and a recombinant collagen protein having an amino acid sequence including any one of SEQ ID No.1-SEQ ID No.4 was designed.

[0063] Example 2

[0064] This embodiment provides a method for constructing and expressing recombinant collagen 1 that targets and activates Frizzled-8 protein as follows:

[0065] (1) Designing the coding region sequence of recombinant collagen 1 targeting and activating Frizzled-8 protein according to the amino acid sequence as shown in SEQ ID No. 1, adding a signal peptide and a fusion His tag, synthesizing the expression sequence of recombinant collagen 1 targeting and activating Frizzled-8 protein, and inserting the expression sequence into the MSC cloning site of the pPIC9K plasmid to obtain a recombinant plasmid (commissioned to Suzhou Biotechnology Co., Ltd. for synthesis);

[0066] (2) Transfecting the recombinant plasmid into Pichia pastoris, preparing Pichia pastoris electroporation competent cells and electroporation transformation. The preparation of Pichia pastoris electroporation competent cells was carried out according 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) to construct an engineered bacterium;

[0067] (3) Streaking the bacterial solution of the engineered bacteria on YPD solid medium to activate the strain;

[0068] (4) After a single colony grows on the plate, pick a single colony and place it in BMGY medium for 24 h. Inoculate the bacterial solution into BMMY medium at a 2% inoculum volume and start fermentation 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 and purified by Ni 2+ The affinity chromatography column binds the recombinant collagen in the fermentation supernatant, and then the recombinant collagen is eluted with 400mM imidazole buffer. The eluate is collected and concentrated to 1mg / mL using an ultrafiltration tube. The resulting solution is the recombinant collagen 1 that targets and activates Frizzled-8 protein (commissioned to Binary Hesheng Company for preparation).

[0070] The amino acid sequence of the recombinant collagen 1 that targets and activates the Frizzled-8 protein is shown in SEQ ID No. 1: GGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGP KGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGD TGPPGPQGLQGLPGTGGPPGENGKPGEPGPK.

[0071] Example 3

[0072] This embodiment provides a method for constructing and expressing recombinant collagen 2 that targets and activates Frizzled-8 protein as follows:

[0073] (1) Designing the coding region sequence of recombinant collagen 2 that targets and activates Frizzled-8 protein according to the amino acid sequence shown in SEQ ID No. 2, adding a signal peptide and a fusion His tag, synthesizing the expression sequence of recombinant collagen 2 that targets and activates Frizzled-8 protein, and inserting the expression sequence into the MSC cloning site of the pPIC9K plasmid to obtain a recombinant plasmid (commissioned to Suzhou Biotechnology Co., Ltd. for synthesis);

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

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

[0076] Example 4

[0077] This embodiment provides a method for constructing and expressing recombinant collagen 3 that targets and activates Frizzled-8 protein as follows:

[0078] (1) Designing the coding region sequence of recombinant collagen 3 that targets and activates Frizzled-8 protein according to the amino acid sequence as shown in SEQ ID No. 3, adding a signal peptide and a fusion His tag, synthesizing the expression sequence of recombinant collagen 3 that targets and activates Frizzled-8 protein, and inserting the expression sequence into the MSC cloning site of the pPIC9K plasmid to obtain a recombinant plasmid (commissioned to Suzhou Biotechnology Co., Ltd. for synthesis);

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

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

[0081] Example 5

[0082] This embodiment provides a method for constructing and expressing recombinant collagen 4 that targets and activates Frizzled-8 protein as follows:

[0083] (1) Designing the coding region sequence of recombinant collagen 4 targeting and activating Frizzled-8 protein according to the amino acid sequence as shown in SEQ ID No. 4, adding a signal peptide and a fusion His tag, synthesizing the expression sequence of recombinant collagen 4 targeting and activating Frizzled-8 protein, and inserting the expression sequence into the MSC cloning site of the pPIC9K plasmid to obtain a recombinant plasmid (commissioned to Suzhou Biotechnology Co., Ltd. for synthesis);

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

[0085] The amino acid sequence of the recombinant collagen 4 that targets and activates Frizzled-8 protein is shown in SEQ ID No.4: GPAGEEGKRGARGEPGPTGLPGPPGERGGPGSRGFPGADGVAGPKGPAGERGSPGPAGPKGSPGEAGRPGEAGLPGAKGLTGSPGSPGPDGKTGPPGPAGQDGRPGPPGPPGARGQAGVMGFPGPKGAAGEPGKAGERGVPGPPGAVGPAGKDGEAGAQGPPGPAGPAGERGEQGPAGSPGFQGLPGPAGPPGEAGKPGEQGVPGDLGAPGPSGARGERGFPGERGVQGPPGPAGPRGANGAPGNDGAKGDAGAPGAPGSQGAPGLQGMPGERGAAGLPGPKGDRGDAGPKGADGSPGKD.

[0086] Test Example 1

[0087] In this test example, the recombinant collagen targeted to activate Frizzled-8 protein provided in Examples 2-5 was used to calculate the best docking score and confidence score of the best binding model between the recombinant collagen targeted to activate Frizzled-8 protein and the Frizzled-8 protein using the molecular mechanics / generalized Born surface area method of the HDOCK SERVER platform. 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 best docking score and confidence score of the best binding model with Frizzled-8 protein were calculated by the same method as above as a control group.

[0089] Each combination model is as follows Figure 1 The best docking scores and confidence scores are shown in Table 1.

[0090] Table 1 The best docking scores and confidence scores of recombinant collagen and Frizzled-8 protein

[0091]

[0092] Figure 1In the examples, the yellow models in A are the recombinant collagen provided in Example 2, the yellow models in B are the recombinant collagen provided in Example 3, the yellow models in C are the recombinant collagen provided in Example 4, the yellow models in D are the recombinant collagen provided in Example 5, and the yellow models in E are the Wnt1 protein in the control group. Figure 1 It can be seen that the recombinant collagen provided in Examples 2-5 and the Wnt1 protein in the control group both bind to the Frizzled-8 protein.

[0093] From the results in Table 1, it can be seen that the confidence scores of the recombinant collagens provided in Examples 2-5 binding to the Frizzled-8 protein are all greater than 0.65, and all can bind to the Frizzled-8 protein. Among them, the confidence score of the recombinant collagen provided in Example 2 binding to the Frizzled-8 protein is greater than 0.8, and is similar to the confidence score of the Wnt1 protein binding to the Frizzled-8 protein in the control group, and its binding effect with the Frizzled-8 protein is the best. It shows that the recombinant collagen provided in the present application, whose amino acid sequence includes the amino acid sequence shown in any one of SEQID No.1-SEQ ID No.4, can significantly bind to the Frizzled-8 protein, activate the Wnt / β-catenin pathway, regulate the cell growth cycle, and significantly promote the formation of collagen.

[0094] Test Example 2

[0095] This test example tests the performance of the recombinant collagen protein that targets and activates Frizzled-8 protein provided in Example 2-5 in promoting collagen formation. The specific method is as follows:

[0096] (1) BJ cells were placed in DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody, and cultured at 37°C and 5% carbon dioxide. When the cells were in good growth condition, 1×10 cells were added to each well. 6 The BJ cells were seeded in a 6-well plate at a density of 10 cells / well, 2 mL of culture medium was added to each well, and the plates were incubated at 37°C for 24 h. The recombinant collagen protein for targeted activation of Frizzled-8 protein provided in Example 1-4 and the Wnt1 protein of the control group were added to the culture medium, respectively, and the final concentration was set to 50 ng / mL. The cells were collected after further culturing for 24 h.

[0097] (2) Total RNA of cells in each experimental group was extracted using a nucleic acid extraction kit and reverse transcribed into cDNA. The sequence of human type III collagen and related information were searched, primers were synthesized, and RT-PCR was performed according to the kit steps to detect the mRNA expression level of cells in each experimental group. The relative expression of inflammatory factor type III collagen was calculated by the method. The results are shown in Table 2.

[0098] Table 2 Relative expression of type III collagen

[0099] sample Relative expression of type III collagen 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 in Table 2, the recombinant collagens provided in Examples 2-5 can significantly increase the relative expression of type III collagen in BJ cells. Among them, the recombinant collagen provided in Example 2 has the best effect in promoting the relative expression of type III collagen in BJ cells, and there is no significant difference in the relative expression of type III collagen in BJ cells compared with the control group Wnt1 protein. This indicates that the recombinant collagen provided in the present application, whose amino acid sequence includes any one of SEQ ID No.1-SEQ ID No.4, can significantly bind to Frizzled-8 protein, activate the Wnt / β-catenin pathway, regulate the cell growth cycle, and significantly promote the formation of collagen.

[0101] The embodiments described above are part of the embodiments of the present application, rather than all of the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application for protection, but merely represents the selected embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present application.

Claims

1. A recombinant collagen protein that targets and activates Frizzled-8 protein, characterized in that: The amino acid sequence of the recombinant collagen includes an amino acid sequence as shown in any one of SEQ ID No.1-SEQ ID No.4 or an amino acid sequence having a homology greater than or equal to 80% with any one of SEQ ID NO.1-SEQ ID No.

4.

2. The recombinant collagen according to claim 1, characterized in that 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.

3. The recombinant collagen according to claim 2, characterized in that The amino acid sequence of the recombinant collagen is the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.

4.

4. The recombinant collagen according to claim 3, characterized in that The amino acid sequence of the recombinant collagen is the amino acid sequence shown in SEQ ID No.

1.

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

6. A recombinant plasmid, characterized in that: Comprising the nucleic acid of claim 5.

7. An engineered bacterium, characterized in that: Comprising the recombinant plasmid according to claim 6.

8. The engineered bacteria according to claim 7, characterized in that: The host cell of the engineering bacteria is Pichia pastoris.

9. A method for preparing recombinant collagen for targeted activation of Frizzled-8 protein according to any one of claims 1 to 4, characterized in that: include: (1) designing the coding region sequence of the recombinant collagen protein that targets and activates the Frizzled-8 protein, adding a signal peptide and a fusion His tag, synthesizing the expression sequence of the recombinant collagen protein that targets and activates the Frizzled-8 protein, and inserting the expression sequence into the MSC cloning site of the pPIC9K plasmid to obtain a recombinant plasmid; (2) Transfecting the recombinant plasmid into Pichia pastoris, inducing expression, and purifying to obtain recombinant collagen that targets and activates Frizzled-8 protein.

10. Use of the recombinant collagen according to any one of claims 1 to 4 in the preparation of cosmetics or medicines for targeted activation of Frizzled-8 protein.

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