A highly hydrophilic and highly transdermal recombinant humanized XVII collagen protein and its synthesis method and application

Abstract

The present invention discloses a recombinant humanized type XVII collagen with high hydrophilicity and high transdermal permeability, and a synthesis method and application thereof. The recombinant humanized collagen comprises functional sequences such as KGD, GER, and GPP, and has a molecular weight of 17 to 30 kDa. At the same time, reasonable design is performed based on information such as isoelectric point, hydrophilicity, and antigenic epitopes to ensure that the recombinant humanized collagen has high hydrophilicity and high transdermal permeability, and has biological activities of promoting cell proliferation, cell migration, cell repair, and anti-wrinkle and firming.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a highly hydrophilic and highly transdermal recombinant humanized XVII collagen protein and a synthesis method and application thereof. Background Art

[0002] Human type XVII collagen is an important transmembrane glycoprotein that mainly acts on the basement membrane zone. By regulating and repairing the basement membrane zone through signals, it opens up the nutrient cycle of the epidermis-basement membrane-dermis, assists in the transmission of nutrients and the elimination of waste, and helps maintain skin health. At the same time, it can promote the production of type I, type III, type IV and type VII collagen, which play a supporting and elastic role in the skin, thereby enhancing the skin's toughness and firmness.

[0003] Type XVII collagen is composed of three identical α1 chains, with a total length of 1497 amino acids, which are roughly divided into an intracellular domain (ICD, 1-467), a transmembrane domain (468-488) and an extracellular domain (ECD, 489-1497). According to the Gly-XY characteristic sequence, it is further divided into 16 non-triple helical regions (NC) and 15 triple helical regions (C), of which the NC region can be divided into NC16C (1-467), NC16B (468-488) and NC16A (489-566). NC16C and NC16B correspond to the intracellular domain and the transmembrane domain, respectively, while NC16A belongs to the extracellular domain and is located between the transmembrane domain and the 15th helical region. The 15th helical region (567-808) is the longest triple helix, including 242 amino acids, and the other 14 helical regions are shorter, about 15-55 amino acids. Among them, C15, C12, C11 and C1 contain integrin binding sequences such as KGD or GER, GEK, and the C14-C11 region is rich in GPP sequences. These functional sequences give collagen specific biological functions.

[0004] In addition to special functional sequences, the biological function of collagen is also closely related to its molecular weight and advanced structure. Collagen with high molecular weight or advanced structure (triple helix structure, fiber structure, hydrogel structure) usually has good stability and support, and is therefore mostly used in tissue filling or medical beauty fields; while collagen peptides with low molecular weight or no advanced structure usually have good transdermal properties and are suitable for the development of cosmetics or non-injectable Class II medical devices.

[0005] As we age, the body's ability to synthesize collagen decreases, resulting in a decrease in the collagen content in the skin, which causes the skin to lose elasticity and produce wrinkles. Supplementing collagen in an appropriate manner can increase the elasticity and firmness of the skin, reduce the formation of wrinkles, and improve nasolabial folds and crow's feet. With the improvement of living standards, daily skin care products have become a must-have skin care method for people of different ages and classes, and collagen-related skin care products are even more dazzling. Most of these collagen raw materials are collagen extracted from animals, with a wide range of molecular weights, ranging from thousands to hundreds of thousands of Daltons, and it is difficult to effectively control the specific molecular weight.

[0006] Recombinant humanized collagen is based on the original sequence of human collagen. The parts with strong water solubility and high biological activity are selected through splicing and recombination or tandem duplication, and the recombinant humanized collagen sequence is obtained after codon optimization. Large-scale production can be achieved by selecting appropriate hosts and fermentation technology. More and more studies have confirmed that recombinant humanized collagen has good water solubility and high biological activity. Its performance is better than that of natural human collagen, and it has broad application prospects in the fields of biomedical materials, beauty cosmetics, food and health care.

[0007] In the application of cosmetics, recombinant collagen with advanced or complex structures has poor solubility and usually requires long-term stirring or the addition of a certain amount of co-solvent to help dissolve; at the same time, they have poor transdermal absorption and it is difficult for them to penetrate the stratum corneum and enter the deep layers of the skin to exert their effects, resulting in most of the recombinant collagen remaining only on the surface of the skin and unable to effectively reach the dermis, which is the main place where collagen exerts its key functions such as promoting cell proliferation and repairing damaged tissues.

[0008] Highly hydrophilic collagen contains a large number of hydrophilic groups, such as hydroxyl and carboxyl groups. The interaction between these residues and water molecules will trigger hydration, forming a hydration film around the collagen molecules. This hydration film can prevent the mutual aggregation of collagen molecules, allowing them to exist more stably in the solution and have higher solubility. However, the stratum corneum of the skin is a lipid bilayer structure. According to the principle of like dissolves like, fat-soluble substances can more easily penetrate the stratum corneum. Highly hydrophilic collagen may have poor transdermal absorption. Therefore, how to design recombinant collagen with high hydrophilicity and high transdermal absorption through genetic engineering is crucial to promote the application of recombinant collagen in cosmetics.

[0009] It should be noted that the above information disclosed in this background technology section is only used to understand the background technology of the present application concept, and therefore, the above description is not considered to constitute information of the prior art. Summary of the invention

[0010] In view of the above problems, the present invention aims to provide a recombinant humanized collagen sequence with high hydrophilicity and high transdermal permeability, which contains multiple integrin binding sequences such as KGD and GER, and rich GPP sequences, giving it biological activity of promoting cell proliferation and cell migration, and having good cell repair and anti-wrinkle and firming effects. At the same time, the molecular weight of the collagen sequence is between 17 and 30 kDa, which belongs to medium and low molecular weight collagen, and the sequence contains a certain proportion of hydrophilic and hydrophobic aliphatic amino acids, which gives the sequence better hydrophilicity and transdermal permeability, thereby improving the skin utilization rate of collagen.

[0011] On the one hand, the present invention provides a recombinant humanized XVII collagen, the amino acid sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2. The molecular weight is 17-30 kDa, and the amino acid sequence is composed of a typical "GXY" characteristic sequence, which contains a certain proportion of hydrophilic and hydrophobic amino acids, has good hydrophilicity and skin permeability; and contains multiple KGD, GER, GPP functional sequences, and has good effects of promoting cell proliferation and migration, promoting skin damage repair and anti-wrinkle and firming.

[0012] Another aspect of the present invention provides a gene encoding the above protein.

[0013] Furthermore, the nucleotide sequence encoding the gene of SEQ ID NO.1 is shown as SEQ ID NO.3, and the nucleotide sequence encoding the gene fragment of SEQ ID NO.2 is shown as SEQ ID NO.4.

[0014] Another aspect of the present invention provides a recombinant plasmid into which the above gene is inserted, and the plasmid comprises a replicon, a screening marker, a promoter, a terminator, a restriction site and a humanized collagen sequence.

[0015] Another aspect of the present invention provides a bioengineering bacterium, wherein the bioengineering bacterium contains or integrates the above-mentioned recombinant plasmid. The bioengineering bacterium can be Pichia pastoris, including any host of Pichia pastoris GS115, KM71H, KM71, SMD1163, SMD1165, SMD1168, SMD1168H and X33, preferably SMD1168H.

[0016] Another aspect of the present invention provides a cosmetic comprising the above-mentioned recombinant humanized XVII collagen.

[0017] Furthermore, the cosmetics also contain components acceptable to human skin.

[0018] Furthermore, the cosmetic also includes, by weight: 0.1% recombinant humanized collagen, 0.3% hexylene glycol, 0.5% p-hydroxyacetophenone, 0.06% acrylic acid (ester) / C10-30 alkyl acrylate cross-linked polymer, 0.06% arginine, and the balance is made up of water.

[0019] Another aspect of the present invention provides the use of the above-mentioned recombinant humanized XVII collagen for reducing skin wrinkles and / or repairing skin damage.

[0020] Another aspect of the present invention provides the use of the above gene fragments, recombinant plasmids, or bioengineered bacteria in the preparation of skin damage repair and / or anti-wrinkle and firming products.

[0021] Another aspect of the present invention provides a method for synthesizing a recombinant humanized collagen with high hydrophilicity and high skin permeability, comprising the following steps:

[0022] (1) synthesizing the initial sequence of human type XVII collagen, adding Bgl II at the N-terminus of the sequence, BamH I and Xho I restriction sites at the C-terminus, and synthesizing after codon optimization according to the codon preference of the expression host;

[0023] (2) The pPIC9KM plasmid was double-digested with BamH I and Xho I;

[0024] (3) double-digesting the fragment in step (1) with Bgl II and Xho I, and ligating it with the vector in step (2) to obtain a recombinant plasmid;

[0025] (4) linearizing the recombinant plasmid obtained in step (2) with Sal I or Sac I, and then electrotransforming it into Pichia pastoris competent cells, and obtaining positive transformants by screening;

[0026] (5) Plating the positive transformants obtained in step (4) on a high-concentration antibiotic plate for high-copy screening to obtain high-copy transformants;

[0027] (6) The obtained high-copy transformants were inoculated into 96-well plates for culture, induced expression, and high-expression strains were screened by SDS-PAGE;

[0028] (7) fermenting the obtained high-expression strain on a large scale to obtain a large amount of fermentation liquid, and centrifuging and filtering the fermentation liquid to obtain a crude protein liquid;

[0029] (8) The crude protein solution is purified using a phenyl hydrophobic column to obtain a recombinant humanized collagen with a purity of 95% to 99% and high hydrophilicity and skin permeability.

[0030] Another aspect of the present invention provides a method for preparing the above-mentioned human type XVII collagen essence, comprising:

[0031] (1) adding recombinant humanized collagen to deionized water and stirring to completely dissolve to obtain a mixed solution 1;

[0032] (2) adding the thickening suspending agent into deionized water and stirring to dissolve completely to obtain a mixed solution 2;

[0033] (3) adding the pH adjuster into deionized water and stirring to dissolve completely to obtain a mixed solution 3;

[0034] (4) Add the polyol and preservative into deionized water, heat to 80-85° C., stir until completely dissolved, cool to 65° C., add the above-mentioned mixed solution 2 and mixed solution 3 in sequence and stir evenly;

[0035] (5) Cooling the mixture to below 45° C., adding the mixed solution 1, stirring and dispersing the mixture evenly, and obtaining a recombinant humanized collagen essence.

[0036] Beneficial effects:

[0037] We have constructed a recombinant humanized type XVII collagen. Cell and zebrafish experiments have confirmed that the recombinant humanized type XVII collagen has the effects of promoting cell proliferation and cell migration, promoting zebrafish repair and anti-wrinkle and firming; at the same time, it has been proved that the essence containing the recombinant humanized type XVII collagen has excellent anti-wrinkle, firming and repair effects, and with the help of Raman spectroscopy experiments, it has been confirmed that the recombinant humanized type XVII collagen has high transdermal absorption. Please refer to the following for details. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 CIMM-COL17-3 gene retrieval map and transformation map

[0039] Figure 2 CIMM-COL17-5 gene retrieval map and transformation map

[0040] Figure 3 pPIC9KM-CIMM-COL17-3 colony PCR verification image

[0041] Figure 4 Colony PCR verification of pPIC9KM-CIMM-COL17-5

[0042] Figure 5 pPIC9KM-CIMM-COL17-3 plasmid map

[0043] Figure 6 pPIC9KM-CIMM-COL17-5 plasmid map

[0044] Figure 7 Results of pPIC9KM-CIMM-COL17-3 transformation into different Pichia hosts

[0045] Figure 8 Colony PCR images of pPIC9KM-CIMM-COL17-3 transformed into different Pichia hosts

[0046] Fig. 9 High-copy screening of pPIC9KM-CIMM-COL17-3 in different Pichia pastoris hosts

[0047] Fig.10 High expression screening of pPIC9KM-CIMM-COL17-3 in different Pichia pastoris hosts

[0048] Fig.11 Results of pPIC9KM-CIMM-COL17-5 transformation into different Pichia hosts

[0049] Fig.12 Colony PCR images of pPIC9KM-CIMM-COL17-5 transformed into different Pichia hosts

[0050] Fig.13 High-copy screening of pPIC9KM-CIMM-COL17-5 in different Pichia pastoris hosts

[0051] Fig.14 High expression screening of pPIC9KM-CIMM-COL17-5 in different Pichia pastoris hosts

[0052] Fig.15 SDS-PAGE analysis of protein expression in 5L fermenter

[0053] Fig.16 SDS-PAGE analysis of CIMM-COL17-3 purification results

[0054] Fig.17 SDS-PAGE analysis of CIMM-COL17-5 purification results

[0055] Fig.18 Hydrophilic and hydrophobic display of CIMM-COL17-3 and CIMM-COL17-5 structures

[0056] Fig.19 Hydrophilicity and hydrophobicity analysis of CIMM-COL17-3 and CIMM-COL17-5

[0057] Fig. 20 Cell proliferation assay of CIMM-COL17-3 and CIMM-COL17-5

[0058] Fig.21 Cell proliferation assay containing CIMM-COL17-3 and CIMM-COL17-5 essence

[0059] Fig. 22 Cell migration assay of CIMM-COL17-3 and CIMM-COL17-5

[0060] Fig.23 Cell migration assay with CIMM-COL17-3 and CIMM-COL17-5 essences

[0061] Fig.24 Evaluation of the anti-aging efficacy of CIMM-COL17-3 in zebrafish

[0062] Fig.25 Evaluation of the anti-aging efficacy of CIMM-COL17-5 in zebrafish

[0063] Fig.26 Evaluation of the anti-aging effects of CIMM-COL17-3 and CIMM-COL17-5 extracts on zebrafish

[0064] Fig. 27 Evaluation of the repair effect of CIMM-COL17-3 on zebrafish

[0065] Fig.28 Evaluation of the repair effect of CIMM-COL17-5 on zebrafish

[0066] Fig.29 Evaluation of the repairing effect of CIMM-COL17-3 and CIMM-COL17-5 essence on zebrafish

[0067] Fig.30 Raman spectra of CIMM-COL17-3 and CIMM-COL17-5

[0068] Fig.31 Permeability experiments of CIMM-COL17-3 and CIMM-COL17-5 DETAILED DESCRIPTION

[0069] Definition of terms

[0070] Herein, the term "bioengineering bacteria" refers to those cells used for transformation, i.e., cells used to express a target gene. Bioengineering bacteria can be isolated cells or cell lines cultured in culture, or cells present in living tissues or organisms. In the context of the present invention, host cells are preferably cells that can grow in culture. The cells described in the present invention are bacteria or fungi, and the present invention is shown and described in examples with Escherichia coli and Pichia pastoris.

[0071] The following is an explanation of the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the process equipment or devices not specifically noted in the following embodiments all adopt conventional equipment or devices in the art. In addition, it should be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps, unless otherwise specified; it should also be understood that the combined connection relationship between one or more devices / devices mentioned in the present invention does not exclude the presence of other devices / devices before and after the combination device / device or the insertion of other devices / devices between these explicitly mentioned two devices / devices, unless otherwise specified. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, rather than limiting the order of arrangement of each method step or limiting the scope of the present invention. The change or adjustment of its relative relationship, without substantial change in the technical content, should also be regarded as the scope of the present invention.

[0072] Example 1 Construction of recombinant plasmids pPIC9KM-CIMM-COL17-3 and pPIC9KM-CIMM-COL17-5

[0073] The C15, C12, C11, C8 and C1 triple helical regions of human type XVII collagen (GenBanK accession number: NP_000485.3) were selected, and two small molecule collagen sequences were designed based on parameters such as isoelectric point, hydrophilicity, stability, integrin recognition sequence, and antigen epitope sequence. They were named CIMM-COL17-3 and CIMM-COL17-5 (as shown in Table 1). As can be seen from the table, CIMM-COL17-3 and CIMM-COL17-5 contain 185 and 324 amino acid residues, respectively, with molecular weights of 17.26 and 30.19 kDa, respectively; isoelectric points are 9.62 and 9.63, respectively, and instability indexes are 15.54 and 19.38, respectively. Usually, the instability Index≤40 is considered to be a protein with good stability, which indicates that CIMM-COL17-3 and CIMM-COL17-5 have good stability; their overall average hydrophilicity (GRAVY) is -1.045 and -1.051 respectively, and the GRAVY value is negative, indicating that the protein is hydrophilic as a whole. In addition, the sequence contains multiple KGD and GER integrin recognition sequences, as well as a large number of GPP sequences, which will affect the stability of the protein structure and thus affect cell signal transduction and binding to platelets.

[0074] Type XVII collagen, also known as BP180, is an autoantigen of bullous pemphigoid. In patients with bullous pemphigoid, autoantibodies against BP180 are produced. These autoantibodies bind to BP180, triggering a series of immune responses, destroying the normal structure and function of the skin and mucous membranes, and leading to the occurrence of the disease. Giovanni Di Zenzo et al. identified 15 antigenic epitopes distributed throughout the BP180 molecule through phage surface display technology, of which eight epitope sequences, 120-160, 151-170, 176-203, 266-343, 282-341, 299-354, 292-367 and 353-399, were located in the ICD, and seven epitopes, 508-541, 567-622, 773-798, 915-985, 1080-1107, 1331-1404 and 1354-1406, were located in the ECD. Among these 15 antigen epitopes, the two dominant antigen epitopes that are prone to cause allergic reactions are 528-441 (NC16A) and 1331-1404. CIMM-COL17-5 avoided these two dominant antigen epitopes during sequence design, and CIMM-COL17-3 avoided these 15 antigen epitopes. Then, the above sequence was codon optimized according to the codon preference of Pichia pastoris, avoiding the removal of BamH I, Xho I, Bgl II, Sac I and Sal I restriction sites, and Bgl II and Xho I restriction sites were added at both ends of the optimized sequence and handed over to Nanjing GenScript Biotechnology Co., Ltd. for synthesis.

[0075] Table 1 Parameter information of CIMM-COL17-3 and CIMM-COL17-5

[0076]

[0077] By designing primers, the synthesized CIMM-COL17-3 and CIMM-COL17-5 sequences were amplified, and their amino acid sequences were shown in SEQ ID NO.1 and SEQ ID NO.2, and their nucleotide sequences were shown in SEQ ID NO.3 and SEQ ID NO.4. PCR, reaction system (50 μL): 2×Phanta Master Mix 25.0 μL, template 1.0 μL, upstream and downstream primers 1.0 μL each, ddH2O was supplemented to 50 μL.

[0078] CIMM-COL17-3 amino acid sequence, SEQ ID NO.1:

[0079] RSRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGP

[0080] VGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKG

[0081] AMGPAGPDGHQGPRGEQGLTGMPGIRGPPGPSGPRGPPGPPGASGDGSLGPPGPRGHKGEK

[0082] GDKGDQGS

[0083] Amino acid sequence of CIMM - COL17 - 5, SEQ ID NO.2:

[0084] RSGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRG

[0085] REGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQ

[0086] GLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGP

[0087] AGPDGHQGPRGEQGLTGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAP

[0088] GKIGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFSGPPGPPGPQGPKGDKGDPGVPGALGI

[0089] PSGPSGPPGPRGHKGEKGDKGDQGS

[0090] Nucleotide sequence of CIMM - COL17 - 3, SEQ ID NO.3 (579bp):

[0091] AGATCTAGAGGGCGAGAAGGCCCCATGGGACCTCGTGGTGAGGCAGGGCCTCCTGGAT

[0092] CTGGAGAGAAAGGGGAAAGAGGGGCTGCTGGTGAACCAGGTCCTCATGGCCCACCTG

[0093] GTGTCCCAGGTTCTGTGGGTCCCAAAGGTTCCAGCGGCTCTCCTGGCCCACAGGGCCCT

[0094] CCAGGTCCTGTAGGTCTCCAAGGGCTCCGAGGTGAAGTAGGACTTCCTGGTGTCAAAG

[0095] GTGACAAAGGACCAATGGGACCACCAGGACCCAAAGGTGACCAGGGTGAGAAAGGAC

[0096] CTCGAGGCCTCACAGGCGAGCCTGGCATGAGAGGTTTGCCTGGTGCTGTTGGTGAGCC

[0097] CGGGGCTAAAGGAGCAATGGGTCCTGCTGGCCCAGACGGACACCAAGGCCCAAGAGGT

[0098] GAACAAGGTCTTACTGGGATGCCTGGAATCCGTGGCCCACCAGGACCTTCTGGCCCTCG

[0099] GGGTCCGCCAGGGCCACCAGGAGCCAGTGGAGATGGGTCCCTCGGGCCACCTGGCCCT

[0100] CGAGGACACAAGGGAGAAAAAGGAGACAAAGGTGACCAAGGATCCTAATGACTCGAGCIMM - COL17 - 5 nucleotide sequence, SEQ ID NO.4 (996bp):

[0101] AGATCTGGAAGCCCTGGCCCTAAAGGTGACATGGGAAGTCCAGGCCCTAAAGGAGATC

[0102] GAGGGTTCCCTGGGACTCCAGGTATCCCTGGGCCCTTGGGCCACCCAGGTCCACAAGG

[0103] ACCAAAGGGTCAAAAAGGCAGCGTGGGAGATCCTGGCATGGAAGGCCCCATGGGCCAG

[0104] AGAGGGCGAGAAGGCCCCATGGGACCTCGTGGTGAGGCAGGGCCTCCTGGATCTGGAG

[0105] AGAAAGGGGAAAGAGGGGCTGCTGGTGAACCAGGTCCTCATGGCCCACCTGGTGTCCC

[0106] AGGTTCTGTGGGTCCCAAAGGTTCCAGCGGCTCTCCTGGCCACAGGGCCCTCCAGGTC

[0107] CTGTAGGTCTCCAAGGGCTCCGAGGTGAAGTAGGACTTCCTGGTGTCCAAAGGTGACAA

[0108] AGGACCAATGGGACCACCAGGACCCAAAGGTGACCAGGGTGAGAAAGGACCTCGAGG

[0109] CCTCACAGGCGAGCCTGGCATGAGAGGTTTGCCTGGTGCTGTTGGTGAGCCCGGGGCTA

[0110] AAGGAGCAATGGGTCCTGCTGCGCCAGACGGACACCAAGGCCCAAGAGGTGAACAAG

[0111] GTCTTACTGGGATGCCTGGAATCCGTGGCCCACCAGGACCTTCTGGAGACCCAGGAAAG

[0112] CCAGGTCTCACAGGACCCCAGGGACCTCAGGGACTTCCCGGTACCCCTGGCCGACCAG

[0113] GAATAAAAGGTGAACCAGGGCTCCAGGCAAGATCGGCCCCCCAGGCCCCACCTGGCCC

[0114] CCCAGGTCCCAAGGGAGACCAAGGTCCCCCAGGCCCCAGAGGACACCAAGGCGAGCA

[0115] AGGCCTCCCAGGTTTCTCAGGACCACCAGGCCCACCTGGCCCCCAGGGACCCAAAGGT

[0116] GACAAAGGTGATCCAGGTGTTCCAGGGGCTCTTGGCATTCCTAGTGGTCCTTCTGGGCC

[0117] ACCTGGCCCTCGAGGACACAAGGGAGAAAAAGGAGACAAAGGTGACCAAGGATCCTA

[0118] ATGACTCGAG

[0119] The PCR amplification program was as follows: (1) denaturation at 95°C for 10 min, (2) denaturation at 94°C for 10 s, (3) annealing at 55°C for 30 s, and (4) extension at 72°C for 3 min 30 s. Steps (2) to (4) were repeated for 35 cycles, and finally extension was performed at 72°C for 10 min. The PCR amplification product was stored at 4°C.

[0120] PCR results are as follows Figure 1 and Figure 2 As shown in the figure, CIMM-COL17-3 and CIMM-COL17-5 bands with lengths of 579 bp and 996 bp were successfully obtained by PCR. After purification and recovery using a gel recovery purification kit, they were ligated with the pPIC9KM plasmid double-digested with BamH I and Xho I by T4 DNA ligase for 2 h. After that, the ligation solution was transformed into E. coli Top10 competent cells and coated on a plate containing 200 μg mL -1 After the transformants grow on the ampicillin-resistant plate ( Figure 3 and Figure 4 The results of colony PCR are shown in Figure 5 and 6 As shown in the figure, PCR successfully amplified the bands of corresponding sizes, proving that the recombinant plasmids pPIC9KM-CIMM-COL17-3 and pPIC9KM-CIMM-COL17-5 were successfully constructed. The plasmid maps are shown in Figure 5 and Figure 6 After the plasmids were extracted from the corresponding bacteria, they were sent to General Biotechnology (Anhui) Co., Ltd. for sequencing, and the strains were preserved after the sequencing was correct.

[0121] Example 2 Construction of different Pichia pastoris recombinant engineering bacteria containing pPIC9KM-CIMM-COL17-3 and comparison of protein expression

[0122] The Tiangen medium extraction kit was used to extract the recombinant plasmid pPIC9KM-CIMM-COL17-3 that was successfully sequenced above, and the extracted plasmid was linearized by single enzyme digestion using Sac I or Sal I. The enzyme digestion system (50 μL) was as follows: 10×Q-cut buffer 5 μL, Sac I or Sal 1 μL, plasmid 1-2.0 μg, ddH2O was added to 50 μL, and the enzyme digestion reaction was carried out at 37°C for 2-4 hours. After the enzyme digestion was completed, the Tiangen large-scale PCR product recovery kit was used to purify and recover the enzyme digestion product for later use.

[0123] Pick a single colony and inoculate it into a sterile shaking tube containing 3 mL of YPD medium. Incubate it at 30°C, 250 rpm overnight. The next day, transfer 1 mL of seed solution to a 500 mL conical flask containing 50 mL of YPD medium and incubate it at 30°C, 250 rpm until OD 600 The pH value is 1.0, then collect the bacterial solution in a 50mL round-bottom inner spiral centrifuge tube, centrifuge at 4℃, 1500g for 5min, and discard the supernatant. Resuspend the bacteria with 40mL pre-cooled sterile water, centrifuge at 4℃, 1500g for 5min, and discard the supernatant. Repeat the above sterile water washing step once. Resuspend the bacteria with 10mL pre-cooled 1M sorbitol, centrifuge at 4℃, 1500g for 5min, and discard the supernatant. Then add 300μL pre-cooled 1M sorbitol, gently pipette and mix with a pipette tip, and dispense the competent cells into 1.5mL centrifuge tubes at 100μL per tube for later use.

[0124] 10 μL of the purified and recovered products were added to Pichia pastoris KM71H, SMD1168H, SMD1168, SMD1163 and GS115 competent cells respectively, and then placed on ice for 5 min. The mixture was then transferred to a 0.2 cm electric shock cup, and the electroporator was set to the SC2 program for electric shock. After the electric shock, 1 mL of pre-cooled 1 M sorbitol was added, and then placed in a shaker, cultured at 30 ° C, 150 rpm for 1 h, and then smeared to contain 0.3 mg·mL -1 After the transformants grow on the YPD plate of G418, colony PCR verification is performed. Figure 7 As shown in the figure, it can be seen that the growth of CIMM-COL17-3 is different in different hosts: for example, the nutrient-deficient hosts SMD1163 and SMD1168 obtained fewer transformants, while SMD1168H obtained more transformants, which may be related to the fact that the former two are histidine dehydrogenase-deficient hosts; for the fast-utilizing methanol host GS115, fewer transformants were obtained, which may be related to the integration efficiency of different restriction endonucleases after linearization into different positions of the host. Then a certain number of transformants were selected for colony PCR verification, and the PCR results are shown in Figure 8As shown in the figure, it can be seen that PCR successfully amplified a CIMM-COL17-3 band of about 1200 bp, indicating that the linearized product was successfully integrated into the yeast genome.

[0125] Since homologous recombination between yeast genome and linearized plasmid is a random integration event, recombinant bacteria with multiple copies of target gene integration will appear. Therefore, in order to screen recombinant bacteria with high copy integration, the positive transformants obtained above were inoculated in 0.5 mg mL -1 and 1.0 mg mL -1 G418 was screened on a high-resistance YPD plate. The screening results of the high-resistance plate are shown in Fig. 9 As shown by Fig. 9 It can be seen that all transformants were able to grow at 0.5 mg mL -1 G418 can grow on YPD plates. For plates containing higher concentrations of antibiotics, only a small number of bacteria can grow, and these strains may be high-copy strains.

[0126] In order to screen and obtain high-expression strains, the high-copy strains obtained above were sequentially inoculated into 96-well plates for culture and induced expression. The specific steps are as follows:

[0127] (1) The colonies that can grow on the high-concentration G418 plate were picked up and placed in a 96-well deep-well plate containing 300 μL YPD medium and cultured at 30° C. and 250 rpm for 12 to 24 h.

[0128] (2) 10-100 μL of bacterial solution was transferred to two 48-well deep-well plates containing 500 μL of BMGY medium and cultured at 30°C and 250 rpm for 12-18 h.

[0129] (3) Centrifuge at 4000 rpm for 10 min, discard the supernatant, add 500 μL BMMY medium, and induce at 28°C, 250 rpm for 24 to 48 h. Add 1% methanol at 24 h.

[0130] (4) After induction, centrifuge at 4000 rpm for 10 min, use a pistol to pipette 20 μL of supernatant into a 96-well PCR tube, add 5 μL of 5×Sample Loading Buffer and denature at room temperature for 5 min, centrifuge at 4000 rpm for 10 min, and pipette 10 μL of supernatant for SDS-PAGE analysis. After Coomassie Brilliant Blue staining and decolorization, you can take pictures.

[0131] The SDS-PAGE screening results of all hosts are as follows Fig.10As shown in the figure, CIMM-COL17-3 can be expressed in different Pichia pastoris, but the expression effects are different. The host with the best expression effect is Pichia pastoris SMD1168H, followed by KM71H, SMD1163, GS115 and SMD1168.

[0132] Example 3 Construction of different Pichia pastoris recombinant engineering strains containing pPIC9KM-CIMM-COL17-5 and comparison of protein expression

[0133] The above examples have demonstrated that the expression effect of XVII collagen in Pichia pastoris SMD1168H and KM71H is good. Therefore, for pPIC9KM-CIMM-COL17-5, this example preferentially transforms it into two competent cells of Pichia pastoris SMD1168H and KM71H: 10 μL of the purified and recovered product is added to Pichia pastoris KM71H and SMD1168H competent cells in turn, ice bathed for 5 minutes, and then the mixed solution is transferred to a 0.2 cm electric shock cup, and the electroporator is adjusted to the SC2 program for electric shock. After the electric shock, 1 mL of pre-cooled 1 M sorbitol is added, and then placed in a shaker, 30 ° C, 150 rpm After 1 hour, smear until it contains 0.3 mg·mL -1 After transformants were grown on the YPD plate containing G418, colony PCR was performed for verification.

[0134] The conversion result is as follows Fig.11 As shown in the figure, CIMM-COL17-5 obtained a large number of transformants in both hosts, which is consistent with the transformation results of pPIC9KM-CIMM-COL17-3 mentioned above. Then a certain number of transformants were selected for colony PCR verification. The PCR results are shown in Fig.12 As shown in the figure, it can be seen that PCR successfully amplified a CIMM-COL17-5 band of about 1500 bp, indicating that the linearized product was successfully integrated into the yeast genome.

[0135] The positive transformants obtained above were inoculated in 0.5 mg mL -1 and 1.0 mg mL -1 G418 was screened on a high-resistance YPD plate. The screening results of the high-resistance plate are shown in Fig.13 As shown by Fig.13 It can be seen that the transformants of KM71H host can be grown at 0.5 mg mL -1 and 1.0 mg mL -1 G418 was grown on YPD plates. For SMD1168H host, most transformants could grow at 0.5 mg mL -1G418 grew on YPD plates, but only a small part could grow on 1.0 mg mL -1 G418 grew on YPD plates, which may be related to the fact that SMD 1168 is a proteinase A-deficient host.

[0136] Then, the positive transformants were screened by 96-well plate according to the above example to verify their high expression. The SDS-PAGE screening results were as follows: Fig.14 As shown in the figure, it can be seen that CIMM-COL17-5 can be expressed in both Pichia pastoris hosts, and the expression effect of SMD1168H host is significantly better than that of KM71H, which is consistent with the expression screening of CIMM-COL17-3 mentioned above.

[0137] Example 4 5L fermentation tank fermentation of Pichia pastoris

[0138] Pichia pastoris SMD1168H / pPIC9KM-CIMM-COL17-3 and Pichia pastoris SMD1168H / pPIC9KM-CIMM-COL17-5 were streaked in 0.3 mg mL -1 G418 was cultured on YPD plates at 30°C for 72 h, and a single colony was picked and inoculated into 50 mL of BMG medium and continued to be cultured until OD 600 The inoculum was transferred to 100 mL BMG medium at a rate of 10% and cultured until the OD reached 8.0 to 12.0. The secondary seed solution was transferred to a 5 L fermentation tank and 4.35 mL L -1 PTM 1 trace elements and 4.35mL·L -1 0.02% biotin, start fermentation tank culture, when DO quickly rises to 50-70%, start glycerol feeding, glycerol feeding rate is 10-20mL·h -1 ·L -1 DO was maintained at 25-30% by fine-tuning the glycerol feeding rate. Feeding was stopped after 5-6 hours of feeding. After "starvation" for 30-60 minutes, methanol induction was started. Methanol flow rate was 0.5-7.2 mL·h -1 ·L -1 The induction temperature was 28°C, and samples were taken during the period to detect the wet weight and protein concentration of the bacteria.

[0139] Seed culture medium BMG (1L): 10 g glycerol, 2.3 g K2HPO4, 11.81 g KH2PO4, dilute to 900 mL, sterilize at 121 °C for 15 min, add 100 mL 13.4% YNB and 2 mL 0.02% biotin after cooling;

[0140] Fermentation medium BSM (1L): 80g glycerol, 26.7mL H3PO4, 1.175g CaSO4, 18.2g K2SO4, 7.28g MgSO4, 4.13g KOH, add water to dissolve and make up to 2000mL. After sterilization, add 4.35mL PTM1;

[0141] Feed growth medium: 50% (w / v) glycerol (containing 12 mL·L -1 PTM1)

[0142] Fermentation induction medium: 100% methanol (containing 12 mL·L -1 PTM1).

[0143] PTM1 ingredients (1L): CuSO4·5H2O 6.0g, NaI 0.08g, MnSO4·H2O 3.0g, Na2MoO4·2H2O0.2g, H3BO3 0.02g, CoCl2 0.5g, ZnCl2 20.0g, FeSO4·7H2O 65.0g, H2SO4 5mL·L -1 0.2 g of biotin was sterilized by filtration and stored at 4°C for later use.

[0144] Protein expression during fermentation of Pichia pastoris SMD1168H / pPIC9KM-CIMM-COL17-3 and Pichia pastoris SMD1168H / pPIC9KM-CIMM-COL17-5 is shown in Fig.15 As shown in the figure, with the extension of fermentation time, the expression levels of CIMM-COL17-3 and CIMM-COL17-5 proteins increased significantly, reaching 6.7 and 9.1 g·L at 72 h, respectively. -1 .

[0145] Example 5 Protein Purification

[0146] The fermented liquid after the fermentation is centrifuged to remove the bacterial cells, and the supernatant is obtained. The supernatant is clarified by a 50-200 nm ceramic membrane, and the supernatant is collected. Further, the supernatant is concentrated by a 3-50 kDa hollow fiber membrane (Shandong Bona Biotechnology Group Co., Ltd.), and after the concentration is completed, the pH is adjusted to 4.5 and the conductivity is 100-150 mS / cm for standby use.

[0147] The prepared samples were purified using buffer A (20 mM KH2PO4, 2 M NaCl, pH 4.5) and buffer B (20 mM KH2PO4, pH 4.5) in combination with a protein purifier (SCG-P100 series from Suzhou Saipu Instrument Co., Ltd., loaded with Phenyl Purose 6Fast Flow chromatographic column, and phenyl hydrophobic filler purchased from Boglon (Zhejiang) Biotechnology Co., Ltd.). The specific steps were as follows: first, the chromatographic column was equilibrated with 5 CV buffer A, and the sample was loaded after the equilibration (the loading amount was 200 mg). After the loading, the column was rinsed with buffer A until A 210 The protein purity was detected by SDS-PAGE. Fig.16 and Fig.17 As shown in the figure, it can be seen that after purification, the protein purity of CIMM-COL17-3 and CIMM-COL17-5 was significantly improved, and the band was single. After the purified proteins were concentrated, the purity was measured, and the purities were 96% and 99% respectively after SEC measurement.

[0148] Example 6 Protein structure and hydrophilicity analysis

[0149] The AlphaFold 3.0 software was used to model the structures of CIMM-COL17-3 and CIMM-COL17-5, and the hydrophilicity distribution of the amino acid sequences of the two proteins was displayed and calculated on the surface. The results are as follows: Fig.18 and 19 As shown, blue represents hydrophilicity, brown represents hydrophobicity, Score value <0 represents hydrophilicity, Score value >0 represents hydrophobicity, Fig.18 It can be seen that most of the regions of CIMM-COL17-3 and CIMM-COL17-5 proteins are displayed in blue. Fig.19 It can be seen that the score values ​​of most areas of CIMM-COL17-3 and CIMM-COL17-5 proteins are <0, which indicates that both collagens are highly hydrophilic proteins.

[0150] Example 7 Preparation of anti-wrinkle repair essence containing recombinant humanized type XVII collagen

[0151] This embodiment provides a method for preparing a recombinant humanized type XVII collagen anti-wrinkle repair essence, and the specific operations are as follows:

[0152] (1) According to the mass percentage, 0.1% of the above recombinant humanized collagen was added into 5% deionized water, and stirred at 300 rpm for 5 min. After the solution was completely dissolved, a mixed solution 1 was obtained;

[0153] (2) adding 0.06% of acrylic acid (ester) / C10-30 alkyl acrylate cross-linked polymer into 5% deionized water, stirring at 500 rpm for 1 hour, and obtaining a mixed solution 2 after the mixture is completely dissolved;

[0154] (3) Add 0.06% arginine into 5% deionized water and stir at 300 rpm for 5 min until the solution is completely dissolved to obtain a mixed solution 3;

[0155] (4) 0.3% hexylene glycol and 0.5% p-hydroxyacetophenone were added to 70% deionized water in sequence, and heated to 80-85° C., 300 rpm, and stirred for 10 min. After complete dissolution and cooling to 65° C., the above-mentioned mixed solution 2 and mixed solution 3 were added in sequence while stirring, 300 rpm, and stirred for 10 min to obtain mixed solution 4;

[0156] (5) After the temperature of the mixed solution 4 drops below 45° C., the mixed solution 1 is added while stirring, and the remaining amount of deionized water is added. The mixture is stirred at 300 rpm for 10 min to finally obtain a clear and transparent recombinant humanized type XVII collagen essence.

[0157] Example 8 Cell proliferation experiment of recombinant humanized type XVII collagen and anti-wrinkle firming repair essence

[0158] The cell colony formation assay was used to compare the cell proliferation effects of two types of recombinant humanized type XVII collagen.

[0159] L-929 fibroblasts in good growth state and in the logarithmic growth phase were taken, digested with 0.25% trypsin, neutralized with complete medium (10% FBS, 1% penicillin-streptomycin, RPMI 1640 medium), counted, and gradiently diluted to prepare a final cell density of 3.0×10 4 / mL cell suspension; after mixing the cell suspension, place it in a sterile sample tank, use a spray gun to seed a 96-well plate, the inoculation volume is 100μL / well, the inoculation amount of cells is 3000 / well, every 6 duplicate wells are regarded as a parallel, PBS solution is added to the edge wells of the well plate (to prevent drying), after the well plate is inoculated, it is placed on the table and left to stand for 10 minutes, and then placed in a cell culture incubator with a culture environment of 37°C, 5% CO2, 95% air and saturated humidity, and cultured continuously for 24 hours; aspirate the complete culture medium and divide the 96-well plate into a blank control group and an experimental treatment group: the experimental treatment groups were added with different concentrations of CIMM-COL17-3 (1, 5, 10, 20, 40, 60, 80, 100μg·mL -1 ), CIMM-COL17-5 collagen (1, 5, 10, 20, 40, 60, 80, 100 μg mL -1 ) and RPMI 1640 complete medium with essence base A and essence B and C with CIMM-COL17-3 and CIMM-COL17-5 collagen added; the blank control group was added with the corresponding volume of RPMI 1640 complete medium; then, the cells were placed in a cell culture incubator with a culture environment of 37°C, 5% CO2, 95% air and saturated humidity for 24 hours; the old culture medium was removed, and a CCK-8 solution with a final concentration of 10% in RPMI 1640 complete medium was added to each well; after incubation at 37°C in the dark for 1 to 3 hours, the absorbance value (OD value) of each well was measured at 450nm using a full-band microplate reader. The cell survival rate was calculated according to the mean absorbance of each group according to the following formula:

[0160] Cell viability (%) = [(OD of experimental group) - (OD of blank group)] / [(OD of control group) - (OD of blank group)] × 100%

[0161] The results of cell viability assay were as follows Fig. 20 As shown in the figure, it can be seen that within the tested concentration range (1~100μg·mL -1 ), CIMM-COL17-3 and CIMM-COL17-5 collagens showed good cell proliferation activity on L-929 fibroblasts.

[0162] At the same time, the effects of CIMM-COL17-3 and CIMM-COL17-5 collagen essences on cell proliferation were investigated. Group A was an essence base formula without the above two collagens, and Group B and Group C were essence group formulas with corresponding concentrations of CIMM-COL17-3 and CIMM-COL17-5, respectively. Fig.21It can be seen that under high concentration conditions, Essence Base A had a certain effect on cell proliferation, but the experimental groups adding CIMM-COL17-3 and CIMM-COL17-5 could restore cell viability to 80% to 90% of the blank group compared with the control group, which once again showed that these two collagens can promote cell proliferation.

[0163] Example 9 Cell migration experiment of recombinant humanized type XVII collagen and anti-wrinkle firming repair essence

[0164] Keratinocytes HaCaT were used to simulate the keratinocyte layer of the skin to explore the skin damage repair effect of collagen solution. Use a ruler and a marker to evenly draw horizontal lines on the back of a 6-well plate, approximately every 0.5 cm (at least 5 lines per well); take keratinocytes HaCaT cells in good growth state and in the logarithmic growth phase, digest them with 0.25% trypsin, neutralize them with full culture medium (10% FBS, 1% penicillin-streptomycin, high-glucose DMEM cell culture medium), count them, and dilute them in a gradient to prepare a cell density of 1.5×10 5 2 mL of the above cell suspension was inoculated in 6-well culture plates, with an inoculation volume of 3.0 × 10 cells / mL per well. 5 After that, the cells were placed in a cell culture incubator with a culture environment of 37°C, 5% CO2, 95% air and saturated humidity. The cells were observed regularly during the culture process. When the cell confluence rate was ≥90%, the serum-free medium was replaced and starvation culture was continued for 24 hours. After starvation, a 200μL pipette tip was used to gently push down the horizontal line on the back; the cells were rinsed with PBS 2 to 3 times, and after confirming that the cells were clean under a microscope, 50 and 100μg·mL of DMEM high-glucose basal medium were added to the experimental groups respectively. -1 CIMM-COL17-3, CIMM-COL17-5 collagen solution and 50 μg mL -1 Essence base A and essence B and C added with CIMM-COL17-3 and CIMM-COL17-5 collagen were cultured (three replicate wells were set), and the same volume of DMEM high-glucose basal medium was added to the blank control group; after culturing in a cell culture incubator with a culture environment of 37°C, 5% CO2, 95% air and saturated humidity for 0h, 24h and 48h, the horizontal scratches on the back of the 6-well plate were wiped off, and photos were taken under a 10x objective lens to ensure that the scratches were centered and vertical, and pay attention to the consistency of the background; samples were taken at 0h and 48h, and the images were opened using Image J software after taking photos and recording, and 6 to 8 horizontal lines were randomly drawn to calculate the mean of the distance between cells or the mean of the scratch area, and the cell migration rate was calculated according to the following formula.

[0165] Cell migration rate = (t 0hInitial scratch area-t 48h Time scratch area) / t 0h Initial scratch area.

[0166] The experimental results are as follows Fig. 22 As shown in the figure, there are no obvious cells in the central scratch area at 0h. After culturing in the culture medium containing CIMM-COL17-3 and CIMM-COL17-5 collagen solutions for 48h, HaCaT cells gradually migrate to the central scratch area. At the tested concentration, the migration rates of HaCaT cells in the experimental group with added collagen were 39.6% and 45.8%, respectively, which were significantly higher than those in the blank group (18.8%), indicating that CIMM-COL17-3 and CIMM-COL17-5 collagens have good ability to promote skin damage repair.

[0167] At the same time, the effects of CIMM-COL17-3 and CIMM-COL17-5 collagen essence on the migration activity of HaCaT cells were investigated. Fig.23 As shown in the figure, it can be seen that at the same concentration, the cell migration rate of essence A without adding collagen is the same as that of the blank group. In contrast, the cell migration rates of essences B and C with added CIMM-COL17-3 and CIMM-COL17-5 collagen reached 42.7% and 45.6%, respectively, which are 2.0 and 2.1 times that of the control group A, respectively, significantly promoting the migration of HaCaT cells, indicating that the essence with added recombinant collagen has a better effect in promoting skin damage repair.

[0168] Example 10 Evaluation of the anti-wrinkle efficacy of recombinant humanized type XVII collagen and anti-wrinkle firming repair essence

[0169] Collagen, as a structural protein, is widely present in animal skin, tendons and other connective tissues, and is the main extracellular matrix in human skin. The appearance of skin wrinkles is closely related to the normal synthesis and expression of collagen. The loss of collagen leads to decreased skin elasticity and relaxation, and increased wrinkles. In addition, when exposed to external ultraviolet rays, the protein content on the surface of zebrafish decreases. At this time, it can be seen through a microscope that the skin of the tail fin shrinks, which is specifically manifested as a decrease in the area of ​​the tail fin. Therefore, the anti-wrinkle effect of the test sample can also be evaluated by analyzing the degree of deformation of the zebrafish tail fin.

[0170] The embryos were divided into blank group, model group and sample group 72 hours after fertilization, with 10 embryos in each group. The embryos in the blank group and model group were not treated in any way and were directly added to the culture water for cultivation. The sample group was added according to the following instructions: different concentrations of CIMM-COL17-3 and CIMM-COL17-5 collagen (concentrations of 1, 10 and 100 μg mL-1 ) raw material group, essence base A group without collagen, and CIMM-COL17-3 with different concentrations (1, 10, and 100 μg mL -1 ) and CIMM-COL17-5 (at concentrations of 1, 10, and 100 μg mL -1 ) was placed in a 28.5°C incubator for 2 hours. After the incubation, the model group and the sample group were placed under a UVC irradiation lamp for irradiation to simulate the situation when the embryos were irradiated. The irradiation time was 6 minutes and 30 seconds. After the irradiation, the zebrafish embryos in the model group and the sample group were cultured for 18 hours under the above conditions. After the incubation, each zebrafish was placed under a microscope for photographing, and the area of ​​the zebrafish tail fin was calculated using ImageJ, and the data was processed according to the following formula:

[0171]

[0172] The experimental results are as follows Fig.24 and 25 As shown in the figure, after UV irradiation, the tail of zebrafish embryos showed obvious shrinkage, while the tail of zebrafish embryos exposed to CIMM-COL17-3 and CIMM-COL17-5 collagen experimental groups showed significantly alleviated shrinkage after UV irradiation, indicating that CIMM-COL17-3 and CIMM-COL17-5 collagens can effectively resist wrinkles caused by photoaging caused by UV irradiation, and the anti-wrinkle effect gradually improved with the increase of collagen concentration. -1 At the concentration of 1.34, the anti-wrinkle ability reached 50%, which shows that CIMM-COL17-3 and CIMM-COL17-5 collagens have good anti-wrinkle effects, and the anti-wrinkle effect of CIMM-COL17-5 collagen is significantly better than that of CIMM-COL17-3 collagen ( Fig.26 ).

[0173] Furthermore, the experiment compared the addition of 1 μg mL -1 CIMM-COL17-3, CIMM-COL17-5 collagen raw material group and essence base A group without collagen, add 1μg·mL -1 CIMM-COL17-3 extract group B and added 1μg·mL -1 The anti-wrinkle ability of CIMM-COL17-5 Essence C Group is shown in the following results: Fig.26As shown in the figure, the anti-wrinkle abilities of the experimental groups with only CIMM-COL17-3 and CIMM-COL17-5 were 18.0% and 26.5% respectively, and the anti-wrinkle ability of the essence of group A without collagen was only 5.3%. The anti-wrinkle abilities of the essences of groups B and C with CIMM-COL17-3 and CIMM-COL17-5 were 32.7% and 47.4% respectively, which were 6.2 and 8.9 times higher than those of the base formula group A, showing excellent anti-wrinkle efficacy, even though the concentration of the added recombinant humanized collagen was only 1 μg mL -1 , but the anti-wrinkle effect is particularly significant, which once again proves the anti-wrinkle effect of the recombinant humanized collagen of the invention patent.

[0174] Example 11 Evaluation of the repair efficacy of recombinant humanized type XVII collagen

[0175] The zebrafish embryo tail amputation inflammation model is a traumatic inflammation model. After the tail of the zebrafish is amputated, local damage to the zebrafish tail fin will be induced, prompting the zebrafish to have an autoimmune response. After the tail fin is cut off, the wound will immediately shrink and be covered with epithelial cells. An inflammatory reaction will occur within a few hours, and the number of neutrophils in the wound will increase. The neutrophils of the transgenic neutrophil fluorescent zebrafish can be clearly observed under a fluorescent microscope, and the AB type wild zebrafish can be seen to regenerate the damaged part of the tail fin under a microscope. Therefore, the repair efficacy of the sample can be evaluated by the regeneration speed of the tail fin or the number of neutrophils near the wound.

[0176] After hatching, AB wild zebrafish embryos were placed under a stereo microscope. Tricaine was used to anesthetize the zebrafish embryos in advance, or the zebrafish embryos were placed on an agar plate and the caudal fin of the embryos was cut. After cutting the tail, a zebrafish tail-cut inflammation model was constructed. Each embryo was photographed and numbered. The zebrafish without tail-cutting were taken as the blank group, and the tail-cut zebrafish were divided into a model group and a sample group. The blank group and the model group were cultured with aquaculture water, and the sample group was added with different concentrations of CIMM-COL17-3 and CIMM-COL17-5 (concentrations of 1, 10, and 100 μg mL -1 ) and essence base A without collagen, and essence group B with different concentrations of CIMM-COL17-3 and essence group C with different concentrations of CIMM-COL17-5 were added for cultivation, and placed in a 28.5℃ incubator for 2 days. After the cultivation, the corresponding numbered zebrafish tail fin part of each group was photographed, and the area of ​​the tail fin part was statistically compared with that of the tail just cut using ImageJ. The repair rate of the tail fin of each group of zebrafish embryos was calculated according to the following formula, and the repair rate of each group was compared to screen and judge the strength of the repair effect of the test samples.

[0177]

[0178] The test results are as follows Fig. 27 , 28 As shown in the figure, after 48 hours of exposure to different concentrations of collagen, the resected part of the zebrafish embryo tail fin gradually recovered, indicating that the two collagens can repair the damaged part of the zebrafish tail fin and promote the regeneration of the zebrafish tail fin. It shows that both CIMM-COL17-3 and CIMM-COL17-5 collagens have good repair ability, and the repair ability increases significantly with the increase of collagen concentration. At 100 μg mL -1 At the same concentration, the repair rates of the two proteins reached 155.6% and 176.1% respectively. In comparison, the repair effect of CIMM-COL17-5 collagen was significantly better than that of CIMM-COL17-3 collagen.

[0179] Furthermore, the experiment compared the addition of 1 μg mL -1 CIMM-COL17-3, CIMM-COL17-5 collagen raw material group and essence base A group without collagen, add 1μg·mL -1 CIMM-COL17-3 extract group B and added 1μg·mL -1 The repairing ability of CIMM-COL17-5 essence C group, the results are as follows Fig.29 As shown, the repair rate of the essence in Group A without added collagen was only 28.8%, while the repair rates of the essence in Groups B and C with added CIMM-COL17-3 and CIMM-COL17-5 reached 90.9% and 132.1%, respectively, which are 3.25 and 4.58 times that of Group A, indicating that both CIMM-COL17-3 and CIMM-COL17-5 collagens have very good repair effects.

[0180] Example 12 Permeability test of recombinant humanized type XVII collagen

[0181] Selection of subjects: According to the inclusion and exclusion criteria, 5 healthy subjects aged 18 to 60 years old were selected to participate in this test. Test requirements: Before the test, the subjects washed their faces with clean water, exposed their faces in a constant temperature and humidity environment (temperature (22±1)℃, relative humidity: 55%±5%) and balanced for 30 minutes. During the equilibrium process, the two sides of the face of the subjects were marked with an area with a diameter of 2cm as the test area. Confocal Raman spectroscopy was used to select an excitation laser source of 671nm to detect the thickness of the stratum corneum of the skin in the test area of ​​the subjects. 0.1% recombinant humanized type XVII collagen (CIMM-COL17-3 and CIMM-COL17-5) aqueous solution was applied to the test area of ​​the subjects, and the measurement was performed before, 30min, 1h, 3h and 5h after use. Using confocal Raman technology, an excitation light source of 785nm was selected to detect the penetration depth and amount of recombinant humanized type XVII collagen in the stratum corneum of the skin in the test area of ​​the subjects. Test parameters: Sample application amount: 4mg / cm 2 , detection exposure time: 2s, scanning depth: from 0μm on the skin surface to 30μm under the skin layer, scanning step length 2μm, and 3 non-repeated sites were randomly selected for testing in each test area. The instrument-matched data analysis software Skin Tools (2.0) was used to process the raw Raman spectroscopy data, and the exported results were analyzed by heat map drawing using the software Origin 8.0.

[0182] The results of Raman mapping are as follows Fig.30 As shown in the figure, the characteristic peaks of recombinant humanized type XVII collagen are consistent with the characteristic peaks of standard type XVII collagen; the permeation analysis results of CIMM-COL17-3 and CIMM-COL17-5 are shown in Fig.31 As shown in the figure, it can be seen that with the extension of time, the penetration amount of the two recombinant humanized type XVII collagens is getting higher and higher, and the penetration depth is getting deeper and deeper, which can reach 30μm, passing through the entire stratum corneum (0-10μm), reaching the granular layer (4-30μm), spinous layer (18-50μm) and basal layer (30-74μm), close to the dermis (≥30μm), which indicates that CIMM-COL17-3 and CIMM-COL17-5 have higher skin permeability and can maximize the biological activity of XVII collagen.

[0183] In summary, the present invention provides a highly hydrophilic and highly transdermal recombinant humanized type XVII collagen and its use, wherein the sequence is selected from the triple helical structure region of human type XVII collagen, comprises a variety of functional sequences such as GER, KED and GPP, and does not comprise dominant or all reported antigenic epitope sequences, and the sequence is made to have high hydrophilicity and high transdermal transability through reasonable design; cell and zebrafish experiments confirm that the recombinant humanized type XVII collagen has the effects of promoting cell proliferation activity, cell migration activity, promoting zebrafish repair and anti-wrinkle and firming; at the same time, it is proved that the essence containing the recombinant humanized type XVII collagen has excellent anti-wrinkle, firming and repairing effects, and with the help of Raman spectroscopy experiments, it is confirmed that the recombinant humanized type XVII collagen has high transdermal absorption, and is very suitable for the development of cosmetics or Class II medical device products.

[0184] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limitations of the present invention. In addition, the various modifications listed herein and the variations of methods and compositions in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications obvious to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A recombinant humanized XVII collagen, characterized in that: The amino acid sequence of the recombinant humanized XVII collagen is shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. A gene, characterized in that Encoding the recombinant humanized XVII collagen as described in claim 1.

3. The gene according to claim 2, characterized in that The nucleotide sequence encoding the gene of SEQ ID NO.1 is shown in SEQ ID NO.3; the nucleotide sequence encoding the gene of SEQ ID NO.2 is shown in SEQ ID NO.

4.

4. A recombinant plasmid, characterized in that: The gene according to claim 2 or 3 is inserted into the recombinant plasmid.

5. A bioengineering strain, characterized in that: The bioengineered bacterial strain contains or integrates the recombinant plasmid as claimed in claim 4.

6. The bioengineering strain according to claim 5, characterized in that: The bioengineering bacteria is Pichia pastoris, preferably SMD1168H.

7. A cosmetic, characterized in that: The cosmetic contains the recombinant humanized XVII collagen as claimed in claim 1.

8. The cosmetic according to claim 7, characterized in that: The cosmetics also contain components acceptable to human skin.

9. Use of the recombinant humanized XVII collagen as claimed in claim 1 for reducing skin wrinkles and / or repairing skin damage.

10. Use of the gene according to claim 2 or 3, the recombinant plasmid according to claim 4, or the bioengineered bacteria according to claim 5 or 6 in the preparation of skin damage repair and / or anti-wrinkle and firming products.

Images