A recombinant type XVII collagen with transdermal effect, its preparation method and application

Abstract

The present invention specifically relates to a recombinant type 17 collagen with a transdermal effect, a preparation method thereof, and an application thereof. The amino acid sequence of the recombinant type 17 collagen provided by the present invention is selected from one of SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.26. The present invention analyzes fragments of natural type 17 collagen, screens out fragments of multiple active sites, further splices and modifies them, and finally obtains a recombinant protein with good biological activity and transdermal effect. Moreover, the present invention realizes the expression of soluble target protein, and the subsequent purification is simple and convenient, which meets the requirements of industrial production and lays the foundation for further expanding the application of recombinant collagen.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and in particular relates to a recombinant type 17 collagen with transdermal effect, a preparation method and an application thereof. Background Art

[0002] Type 17 collagen (Collagen XVII, also known as COL17 / BP180 / BPAG2) is a key transmembrane protein. Recent studies have shown that type 17 collagen is not only closely associated with various skin diseases but also plays a key role in regulating various stem cell functions, with significant associations with aging-related phenotypes such as hair loss and graying. During skin wound repair, type 17 collagen plays a crucial role in repair and regeneration by regulating the migration, proliferation, and differentiation of stem cells. The rapid development of synthetic biology has made it possible to produce recombinant type 17 collagen. As a novel therapeutic protein and anti-aging ingredient, recombinant type 17 collagen demonstrates significant potential in skin repair and anti-aging, offering new avenues for the treatment of related diseases and skin health management.

[0003] At present, type 17 collagen is mainly obtained through two methods: recombinant technology and animal-derived extraction. However, there are significant differences between the two in terms of source, production technology, extraction process and safety. For animal-derived type 17 collagen, it usually needs to be extracted from animal tissues such as pig skin and cow hide. This process is easily affected by various factors such as the age, health status, and breeding environment of the animal, resulting in unstable product quality. In addition, pollutants such as viruses and bacteria in the animal body may be introduced during the extraction process, posing certain safety risks. At the same time, due to species differences and possible structural damage during the extraction process, its biocompatibility is often poor, which limits its widespread use in clinical applications.

[0004] In contrast, recombinant type 17 collagen can be precisely synthesized and efficiently purified in a laboratory environment through genetic engineering and biofermentation techniques. This production method is highly controllable and repeatable, ensuring product consistency and stability. At the same time, the recombinant production process is carried out in a sterile environment and does not require the addition of any animal-derived ingredients, fundamentally avoiding the risk of contamination from viruses, bacteria, and other sources, significantly improving safety. In addition, recombinant type 17 collagen has a high degree of similarity and affinity with the body's own collagen, and therefore exhibits superior biocompatibility, which can effectively reduce rejection and immune responses, giving it important advantages in its application in the medical and cosmetic fields.

[0005] Human collagen XVII is a transmembrane collagen protein with an intracellular domain, a transmembrane region, and an extracellular domain. Generally speaking, when expressing transmembrane proteins, they are often anchored to the cell membrane. Furthermore, human collagen XVII has a very long amino acid sequence (1497 amino acids) and a correspondingly large molecular weight (180 kDa), making it theoretically difficult to express effectively and prone to degradation.

[0006] Therefore, constructing recombinant proteins by screening related sequences is crucial for achieving recombinant type 17 collagen expression and its industrial application. Summary of the Invention

[0007] Based on this, the present invention provides a recombinant type 17 collagen protein that achieves soluble expression of the recombinant protein and facilitates simple and convenient subsequent purification, fully meeting the requirements of industrial production. Furthermore, the recombinant protein of the present invention is histidine-tagged, which reduces subsequent production costs and shortens production time, improves product performance, and expands its application range.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0009] A recombinant type 17 collagen with a transdermal effect, wherein the amino acid sequence of the recombinant type 17 collagen is selected from one of SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, and SEQ ID NO.26.

[0010] The present invention also provides a polynucleotide encoding the recombinant type 17 collagen, wherein the polynucleotide sequence is selected from one of SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16.

[0011] In the present invention, the original nucleotide sequences encoding the amino acid sequences of recombinant type 17 collagen are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8. The amino acid sequences corresponding to these nucleotide sequences not only contain multiple spliced natural type 17 collagen active fragments, but also, for subsequent purification, an 8×His histidine tag is added to the recombinant type 17 collagen fragment. The nucleotide sequence corresponding to the 8×His histidine tag is located at the 3' end of the recombinant type 17 collagen nucleotide sequence, and its amino acid sequence is shown in SEQ ID NO.18. Among these sequences, the nucleotide sequence shown in SEQ ID NO.8 also contains a nucleotide sequence corresponding to the cell-penetrating peptide Tat, which is located at the 5' end of the recombinant type 17 collagen nucleotide sequence. A flexible linker was added between the cell-penetrating peptide and recombinant type 17 collagen to prevent interference between the two functional fragments. The amino acid sequence of the cell-penetrating peptide is shown in SEQ ID NO. 17, and the amino acid sequence of the flexible linker is GGGGSGGGGS. Further optimization based on the preferences of E. coli resulted in the nucleotide sequences shown in SEQ ID NOs. 9-16.

[0012] Among them, the amino acid sequence of the membrane-penetrating peptide is shown in SEQ ID NO. 17, YGRKKRRQRRR (SEQ ID NO. 17); the amino acid sequence of the 8×His histidine tag is shown in SEQ ID NO. 18, HHHHHHHH (SEQ ID NO. 18).

[0013] The present invention also provides a recombinant type 17 collagen expression vector comprising the polynucleotide sequence.

[0014] The present invention also provides a recombinant engineered bacterium, comprising the recombinant type 17 collagen expression vector.

[0015] Preferably, the recombinant engineered bacteria uses Escherichia coli as a host.

[0016] The present invention also provides a method for preparing the recombinant type 17 collagen, comprising the following steps:

[0017] S1. Use biological and repeated synthesis of the nucleotide sequences of SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16 to obtain the target gene fragment;

[0018] S2. The target gene fragments obtained in step S1 were double-digested with EcoRI and NotI and then inserted into the corresponding restriction sites of the shuttle vector PET28a to construct recombinant plasmids PET28a-T17-1, PET28a-T17-2, PET28a-T17-3, PET28a-T17-4, PET28a-T17-5, PET28a-T17-6, PET28a-T17-7, and PET28a-T17-8;

[0019] S3. The recombinant plasmids PET28a-T17-1, PET28a-T17-2, PET28a-T17-3, PET28a-T17-4, PET28a-T17-5, PET28a-T17-6, PET28a-T17-7, and PET28a-T17-8 constructed in step S2 are respectively transformed into Escherichia coli to obtain positive bacteria, which are further cultured and induced to express to obtain bacteria containing the recombinant type 17 collagen;

[0020] S4. Resuspend and crush the recombinant collagen type 17 obtained in step S3; collect the supernatant by centrifugation, filter, and then purify the filtered solution by affinity chromatography on a nickel column to obtain the product.

[0021] Preferably, the process of affinity chromatography purification by nickel column shown in step S4 is as follows: the filtrate containing recombinant type 17 collagen is passed through the nickel column, unbound impurities are washed away with binding buffer and washing buffer, and the recombinant type 17 collagen is eluted with elution buffer; added to a G25 desalting column, and washed out with desalting buffer.

[0022] Preferably, the components of the binding buffer include 0.4~0.6MNaCl, 45~55mM PB, 15~25mM imidazole, 7~9M urea, pH=8.3; the components of the wash buffer include 0.4~0.6MNaCl, 45~55mM PB, 70~90mM imidazole, 3~5M urea, pH=8.3; the components of the elution buffer include 0.4~0.6MNaCl, 45~55mM PB, 250~350mM imidazole, 2M urea, pH=8.3; the components of the desalting buffer include 0.1~0.2MNaCl, 45~55mM PB, pH=6.5.

[0023] The present invention also provides an application of the recombinant type 17 collagen in the preparation of a biological skin follicle repair agent.

[0024] Preferably, the biological skin follicle repair agent is in the form of a freeze-dried powder, a biological sponge or a dressing.

[0025] Type XVII collagen contains 1-15 collagen domains, which effectively support keratinocyte adhesion. Loss of various collagen domains leads to epidermal adhesion defects and a fragile skin phenotype. Genetic experiments have demonstrated that the collagen domain of type XVII collagen promotes cell adhesion in vitro in an integrin β1-dependent manner. Related studies have also confirmed that recombinantly expressed collagen domains exhibit enhanced cell adhesion activity and cell migration activity compared to fibronectin. Therefore, the KGD site (a specialized type of integrin binding site) within the collagen domain may constitute the molecular basis for its recognition by keratinocyte integrins, making it functionally irreplaceable.

[0026] Based on this, the present invention designs sequences based on the basic elements of the KGD active region and the antioxidant fragment, while taking into account other active sites or fragments, as well as factors such as sequence stability. Based on the research experience accumulated by researchers, different collagen domains of the α1 chain of human type XVII collagen are selected to combine into the sequence of recombinant type XVII collagen. The recombinant sequence contains different KGD region sequences and antioxidant sites, and is compatible with other active site sequences. Such sequence selection enables the functions of each region sequence to be integrated, and they are not expressed separately but as a whole, avoiding the sequence homogeneity of recombinant collagen.

[0027] Human skin acts as a barrier organ, which not only resists the invasion of foreign harmful substances, but also blocks the entry of nutrients needed by cells. Traditional skin care products and drugs have difficulty effectively penetrating the skin barrier and reaching deep cells. The cell-penetrating peptide Tat acts as a carrier for transmembrane transport. Its positive charge and hydrophilicity enable the cell-penetrating peptide to interact with the negative charge of the cell membrane, thereby effectively crossing the membrane structure and carrying active ingredients such as large molecular proteins for successful transdermal transport. During the skin care process, the cell-penetrating peptide Tat can carry the active ingredients in skin care products through the cell membrane directly to the key layers of the skin to exert their effects, thereby achieving ideal skin care effects. Therefore, the present invention adds the cell-penetrating peptide Tat to a fragment of type 17 collagen to obtain a recombinant type 17 collagen that is not only highly biologically active, but also able to penetrate the skin and better realize biological functions; and further constructs a high-expression recombinant strain. The collagen portion of the recombinant type 17 collagen produced by fermentation of the recombinant strain is completely consistent with the sequence of natural type 17 collagen, and is a recombinant type 17 collagen with good safety. It can improve the transdermal effect of recombinant type 17 collagen, so that recombinant type 17 collagen can better play its role in maintaining skin barrier function and skin health.

[0028] Escherichia coli is a Gram-negative bacillus that can grow in simple culture media and typically reproduces a generation every 20 minutes. This makes its cultivation relatively simple, low-cost, and allows for short fermentation cycles. Furthermore, its extensive genetic background and fully sequenced genome facilitate molecular manipulation of its genetic material, making it one of the most commonly used host cells in biotechnology.

[0029] The protein expression system of Escherichia coli has high transfection efficiency and can produce large amounts of recombinant proteins. The level of exogenous gene products expressed is much higher than that of other gene expression systems, and can even reach more than 30% of the total bacterial protein. This makes it widely used in many fields such as medicine, biotechnology, and agriculture, including the production of preventive vaccines, therapeutic recombinant proteins, therapeutic enzymes, medical and aesthetic raw materials, enzymes for scientific research and industrial use, etc. It is an ideal host for large-scale production of recombinant proteins.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] (1) Based on the amino acid sequence of type 17 collagen, the present invention uses bioinformatics and other means to design the amino acid sequence, and combines it with long-term experimental screening to design a series of fragments. These sequences cover multiple active sites and can better exert the biological function of type 17 collagen;

[0032] (2) The present invention also provides a low-cost and efficient protein purification process suitable for large-scale production. The recombinant type 17 collagen prepared by the above technical solution has excellent skin repair ability and can be well applied in the field of cosmetics. At the same time, the present invention can also be used for the research of skin health and disease, and the protein can be used to prepare drugs to treat skin diseases, which has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the recombinant type 17 collagen 17-4, 17-5, 17-6, 17-7, 17-8 gene fragment and plasmid;

[0034] Figure 2 It is the recombinant type 17 collagen 17-1, 17-2, 17-3 gene fragment and plasmid;

[0035] Figure 3 This is the SDS-PAGE and WB profile of the recombinant type 17 collagen 17-1 expressed in the strain;

[0036] Figure 4 This is the SDS-PAGE and WB profile of the recombinant type 17 collagen 17-2 expressed in the strain;

[0037] Figure 5 This is the SDS-PAGE and WB profile of the recombinant type 17 collagen 17-3 expressed in the strain;

[0038] Figure 6 This is the SDS-PAGE and WB profile of the recombinant type 17 collagen 17-4 expressed in the strain;

[0039] Figure 7 This is the SDS-PAGE and WB profile of the recombinant type 17 collagen 17-5 expressed in the strain;

[0040] Figure 8 This is the SDS-PAGE and WB profile of the recombinant type 17 collagen 17-6 expressed in the strain;

[0041] Figure 9 This is the SDS-PAGE and WB profile of the recombinant type 17 collagen 17-7 expressed in the strain;

[0042] Figure 10 This is the SDS-PAGE and WB profile of the recombinant type 17 collagen 17-8 expressed in the strain;

[0043] Figure 11 It is recombinant type 17 collagen 17-5, 17-6, and 17-8 expressed by high-density fermentation of bacteria;

[0044] Figure 12is the SDS-PAGE profile of purified recombinant type 17 collagen 17-6;

[0045] Figure 13 is the SDS-PAGE profile of purified recombinant type 17 collagen 17-5;

[0046] Figure 14 is the SDS-PAGE profile of purified recombinant type 17 collagen 17-8;

[0047] Figure 15 This is the result of the recombinant type 17 collagen of the present invention on cell proliferation;

[0048] Figure 16 This is the result of the recombinant type 17 collagen of the present invention adhering to cells;

[0049] Figure 17 This is the result of the recombinant type 17 collagen transdermal experiment.

[0050] Figure 18 This is a lyophilized sample of purified recombinant type 17 collagen 17-8. DETAILED DESCRIPTION

[0051] The present invention will be further explained below in conjunction with specific examples. However, it should be noted that the following examples are only used to explain the present invention and are not intended to limit the present invention. All technical solutions that are the same or similar to the present invention are within the scope of protection of the present invention. Where specific techniques or conditions are not specified in the present examples, the operations were carried out in accordance with conventional technical methods and instrument specifications in the art; where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0052] The buffers involved in the experiment are as follows:

[0053] The components of 50mM PB (i.e., PB mentioned below) include 2.65mM sodium dihydrogen phosphate dihydrate + 47.35mM sodium dihydrogen phosphate dodecahydrate, pH=8.3; the components of the binding buffer (equilibrium buffer) include 0.5MNaCl, 50mM PB, 20mM imidazole, 8M urea, pH=8.3; the components of the wash buffer include 0.5MNaCl, 50mM PB, 80mM imidazole, 4M urea, pH=8.3; the components of the elution buffer include 0.5MNaCl, 50mM PB, 300mM imidazole, 2M urea, pH=8.3; the components of the desalting buffer include 0.15MNaCl, 50mM PB, pH=6.5.

[0054] Example 1 Design of recombinant type 17 collagen sequence

[0055] The amino acid sequence of natural human type 17 collagen (entry: Q9UMD9) was obtained from the Uniprot database. Bioinformatics methods were used to analyze the sequence, identifying the triple-helical fragment region within the original sequence and the corresponding active site within the sequence. The corresponding protein sequence was then designed based on the amino acid characteristics and the predicted spatial structure of the protein sequence. The recombinant type 17 collagen of the present invention is characterized by splicing sequences of different active sites. The specific amino acid sequences are shown in SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, and SEQ ID NO. 26. These sequences contain a GXXGER integrin binding site, a proliferation or adhesion motif, antioxidant amino acids GF, GP, and QP, chemotactic amino acids PGP, PG, and RGD, and anti-aging active fragments, ensuring the biological function of the recombinant type 17 collagen. Furthermore, the fragments are predicted to have good water solubility and stability, enabling efficient expression. Each sequence contains a histidine tag, making subsequent purification convenient and simple.

[0056] Example 2 Construction of recombinant E. coli BL21(DE3)pLysS / PET28a(+)-recombinant type 17 collagen

[0057] According to the codon preference of Escherichia coli, the nucleotide sequence encoding the amino acid sequence of Example 1 (specific sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8) was codon optimized. The optimized coding gene nucleotide sequences are shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44, SEQ ID NO.45, SEQ ID NO.46, SEQ ID NO.47, SEQ ID As shown in NO.16, the codon-optimized nucleotide sequence was commissioned to Qingke Biotechnology for synthesis, and then the synthesized target gene was cloned into the Escherichia coli expression vector PET28a(+), and further transformed into competent E. coli (DH5α). Positive clones were obtained by screening, and then plasmids were extracted to obtain recombinant expression vectors PET28a(+)-T17-1, PET28a(+)-T17-2, PET28a(+)-T17-3, PET28a(+)-T17-4, PET28a(+)-T17-5, and P ET28a(+)-T17-6, PET28a(+)-T17-7, PET28a(+)-T17-8, and the recombinant expression vectors PET28a(+)-T17-1, PET28a(+)-T17-2, PET28a(+)-T17-3, PET28a(+)-T17-4, PET28a(+)-T17-5, PET28a(+)-T17-6, PET28a(+)-T17-7, PET28a(+)-T17-8 were successfully constructed and verified by DNA sequencing.

[0058] The successfully sequenced plasmids PET28a(+)-T17-1, PET28a(+)-T17-2, PET28a(+)-T17-3, PET28a(+)-T17-4, PET28a(+)-T17-5, PET28a(+)-T17-6, PET28a(+)-T17-7, and PET28a(+)-T17-8 were then transformed into Escherichia coli expression bacteria BL21(DE3)pLysS and screened on kanamycin-resistant LB plates to obtain recombinant E. coli BL21(DE3). pLysS / PET28a(+)-T17-1, PET28a(+)-T17-2, PET28a(+)-T17-3, PET28a(+)-T17 -4, PET28a(+)-T17-5, PET28a(+)-T17-6, PET28a(+)-T17-7, PET28a(+)-T17-8.

[0059] The results are as follows Figure 1 as well as Figure 2 As shown, Figure 1 Band 1 is PET28a(+)-T17-4, band 2 is gene fragment T17-4, band 3 is PET28a(+)-T17-5, band 4 is gene fragment T17-5, band 5 is PET28a(+)-T17-6, band 6 is gene fragment T17-6, band 7 is PET28a(+)-T17-7, band 8 is gene fragment T17-7, band 9 is PET28a(+)-T17-8, and band 10 is gene fragment T17-8. Figure 2 Among them, band 11 is PET28a(+)-T17-1, band 12 is gene fragment T17-1, band 13 is PET28a(+)-T17-2, band 14 is gene fragment T17-2, band 15 is PET28a(+)-T17-3, and band 16 is gene fragment T17-3. It can be seen that the recombinant expression vectors PET28a(+)-T17-1, PET28a(+)-T17-2, PET28a(+)-T17-3, PET28a(+)-T17-4, PET28a(+)-T17-5, PET28a(+)-T17-6, PET28a(+)-T17-7, PET28a(+)-T17-8 and the target genes contained therein meet the requirements.

[0060] Example 3 Induced expression of recombinant type 17 collagen

[0061] The recombinant E. coli BL21 (DE3) pLysS / PET28a (+) - recombinant type 17 (i.e., T17-1 to T17-8) collagen single colony obtained by screening in Example 2 was selected and cultured in LB liquid medium (containing 50 μg / mL kanamycin) at 37 ° C and 250 rpm. When the cell density reached OD600 = 0.6, a sample (the sampling amount was half of the total cell) was collected by centrifugation and marked as the pre-induction sample. Then, IPTG with a final concentration of 0.5 mmol was added to the remaining cell liquid for induction. The induction conditions were 25 ° C and 250 rpm for 4 hours, and then the cells were collected by centrifugation at 12000 rpm for 5 minutes, and marked as the post-induction sample. The samples before and after induction were resuspended in lysozyme solution, digested at 37°C for 10 min to break the bacteria, and then subjected to SDS-PAGE and WB detection. The theoretical sizes of recombinant type 17 collagen T17-1, T17-2, T17-3, T17-4, T17-5, T17-6, T17-7, and T17-8 were 20.13 kDa, 10.29 kDa, 13.17 kDa, 30.48 kDa, 25.11 kDa, 15.51 kDa, 52.85 kDa, and 17.96 kDa, respectively.

[0062] The results of SDS-PAGE and WB are as follows Figure 3-10 As shown, Figure 3In the figure, 1-14 represent SDS-PAGE gel images, and 15-28 represent WB images. BL21(DE3)plysS-1 bacteria - before induction; bands 2 and 27 are 17-1-BL21(DE3)plysS-1 bacteria - after induction; bands 3 and 26 are 17-1-BL21(DE3)plysS-2 bacteria - before induction; bands 4 and 25 are 17-1-BL21(DE3)plysS-2 bacteria - after induction; bands 5 and 24 are 17-1-BL21(DE3)plysS-3 bacteria - before induction; bands 6 and 23 are 17-1-BL21(DE3)plysS-3 bacteria - after induction; bands 7 and 22 are 17-1-BL21(DE3)plysS-4 bacteria - before induction; bands 8 and 21 are 1 7-1-BL21(DE3)plysS-4 - after induction; bands 9 and 20 are 17-1-BL21(DE3)plysS-5 - before induction; bands 10 and 19 are 17-1-BL21(DE3)plysS-5 - after induction; bands 11 and 18 are 17-1-BL21(DE3)plysS-6 - before induction; bands 12 and 17 are 17-1-BL21(DE3)plysS-6 - after induction; bands 13 and 16 are 17-1-BL21(DE3)plysS-7 - before induction; bands 14 and 15 are 17-1-BL21(DE3)plysS-7 - after induction. This shows that the 17-1 fragment is not expressed.

[0063] Figure 4Bands 1 and 15 represent 17-2-BL21(DE3)plysS-1 bacteria before induction; bands 2 and 16 represent 17-2-BL21(DE3)plysS-1 bacteria after induction; bands 3 and 17 represent 17-2-BL21(DE3)plysS-2 bacteria before induction; bands 4 and 18 represent 17-2-BL21(DE3)plysS-2 bacteria after induction; bands 5 and 19 represent 17-2-BL21(DE3)plysS-3 bacteria before induction; bands 6 and 20 represent 17-2-BL21(DE3)plysS-3 bacteria after induction; bands 7 and 21 represent 17-2-BL21(DE3)plysS-4 bacteria before induction; bands Bands 8 and 22 represent 17-2-BL21(DE3)plysS-4 strain after induction; bands 9 and 23 represent 17-2-BL21(DE3)plysS-5 strain before induction; bands 10 and 24 represent 17-2-BL21(DE3)plysS-5 strain after induction; bands 11 and 25 represent 17-2-BL21(DE3)plysS-6 strain before induction; bands 12 and 26 represent 17-2-BL21(DE3)plysS-6 strain after induction; bands 13 and 27 represent 17-2-BL21(DE3)plysS-7 strain before induction; and bands 14 and 28 represent 17-2-BL21(DE3)plysS-7 strain after induction. This indicates that the 17-2 fragment is not expressed.

[0064] Similarly, Figure 5 The induced expression of 17-3 is shown in the figure. 1 and 11 are 17-3-BL21 (DE3) plysS-1 bacteria before induction; 2 and 12 are 17-3-BL21 (DE3) plysS-1 bacteria after induction; 3 and 13 are 17-3-BL21 (DE3) plysS-2 bacteria before induction; 4 and 14 are 17-3-BL21 (DE3) plysS-2 bacteria after induction; 5 and 15 are 17-3-BL21 (DE3) plysS- 3 before induction; 6 and 16 are 17-3-BL21(DE3)plysS-3 after induction; 7 and 17 are 17-3-BL21(DE3)plysS-4 before induction; 8 and 18 are 17-3-BL21(DE3)plysS-4 after induction; 9 and 19 are 17-3-BL21(DE3)plysS-5 before induction; 10 and 20 are 17-3-BL21(DE3)plysS-5 after induction. This indicates that the 17-3 protein fragment is 13.3 kDa and is not significantly expressed in E. coli.

[0065] Figure 6The induced expression of 17-4 is shown in the figure. 1 and 15 are 17-4-BL21 (DE3) plysS-1 bacteria before induction; 2 and 16 are 17-4-BL21 (DE3) plysS-1 bacteria after induction; 3 and 17 are 17-4-BL21 (DE3) plysS-2 bacteria before induction; 4 and 18 are 17-4-BL21 (DE3) plysS-2 bacteria after induction; 5 and 19 are 17-4-BL21(DE3)plysS-3 bacteria before induction; 6 and 20 are 17-4-BL21(DE3)plysS-3 bacteria after induction; 7 and 21 are 17-4-BL21(DE3)plysS-4 bacteria before induction; 8 and 22 are 17-4-BL21(DE3)plysS-4 bacteria after induction; 9 and 23 are 17-4-BL21(DE3)plysS 17-4-BL21(DE3)plysS-No. 5 strain - before induction; 10 and 24 are 17-3-BL21(DE3)plysS-No. 5 strain - after induction; 11 and 25 are 17-4-BL21(DE3)plysS-No. 3 strain - before induction; 12 and 26 are 17-4-BL21(DE3)plysS-No. 4 strain - after induction; 13 and 27 are 17-4-BL21(DE3)plysS-No. 4 strain - before induction; 14 and 28 are 17-4-BL21(DE3)plysS-No. 5 strain - after induction. It can be seen that the size of the 17-4 protein fragment is 30.5KD. There is no obvious difference before and after induction, and WB also does not show color, indicating that the target protein is not expressed.

[0066] Figure 7The induced expression of 17-5, among which, bands 1 and 20 are 17-5-BL21 (DE3) plysS-1 bacteria before induction; bands 2 and 19 are 17-5-BL21 (DE3) plysS-1 bacteria after induction; bands 3 and 18 are 17-5-BL21 (DE3) plysS-2 bacteria before induction; bands 4 and 17 are 17-5-BL21 (DE3) plysS-2 bacteria after induction; bands 5 and 16 are 17-5-BL21 (DE3) plysS- Bands 6 and 15 represent 17-5-BL21(DE3)plysS from strain 3 before induction; bands 7 and 14 represent 17-5-BL21(DE3)plysS from strain 4 before induction; bands 8 and 13 represent 17-5-BL21(DE3)plysS from strain 4 after induction; bands 9 and 12 represent 17-5-BL21(DE3)plysS from strain 5 before induction; and bands 10 and 11 represent 17-5-BL21(DE3)plysS from strain 5 after induction. The 17-5 protein fragment size is 25.3 kD, and the expressed protein is consistent with the theoretical value, indicating that 17-5-BL21(DE3)plysS is significantly expressed. Strain 4 was selected for the next fermentation.

[0067] Figure 8The results of 17-6 induced expression are shown in the figure. 1 and 15 are 17-6-BL21 (DE3) plysS-1 bacteria before induction; 2 and 16 are 17-6-BL21 (DE3) plysS-1 bacteria after induction; 3 and 17 are 17-6-BL21 (DE3) plysS-2 bacteria before induction; 4 and 18 are 17-6-BL21 (DE3) plysS-2 bacteria after induction; 5 and 19 are 17-6-BL21(DE3)plysS-3 bacteria before induction; 6 and 20 are 17-6-BL21(DE3)plysS-3 bacteria after induction; 7 and 21 are 17-6-BL21(DE3)plysS-4 bacteria before induction; 8 and 22 are 17-6-BL21(DE3)plysS-4 bacteria after induction; 9 and 23 are 17-6-BL21(DE3)plysS 17-6-BL21(DE3)plysS-No. 5 strain - before induction; 10 and 24 are 17-6-BL21(DE3)plysS-No. 5 strain - after induction; 11 and 25 are 17-6-BL21(DE3)plysS-No. 4 strain - before induction; 12 and 26 are 17-6-BL21(DE3)plysS-No. 4 strain - after induction; 13 and 27 are 17-6-BL21(DE3)plysS-No. 5 strain - before induction; 14 and 28 are 17-6-BL21(DE3)plysS-No. 5 strain - after induction. It can be seen that the size of the 17-6 protein fragment is 15.5KD, and the expressed protein is consistent with the theoretical value, indicating that 17-6-BL21(DE3)plysS has obvious expression, and strain 4 was selected for the next fermentation.

[0068] Figure 9 The results of induced expression of 17-7 are shown in the figure. 1 and 11 are before induction of 17-7-BL21(DE3)plysS-1 bacteria; 2 and 12 are after induction of 17-7-BL21(DE3)plysS-1 bacteria; 3 and 13 are before induction of 17-7-BL21(DE3)plysS-2 bacteria; 4 and 14 are after induction of 17-7-BL21(DE3)plysS-2 bacteria; 5 and 15 are before induction of 17-7-BL21(DE3)plysS-2 bacteria; Figure 6 shows 17-7-BL21(DE3)plysS-3 before induction; Figures 6 and 16 show 17-7-BL21(DE3)plysS-3 after induction; Figures 7 and 17 show 17-7-BL21(DE3)plysS-4 before induction; Figures 8 and 18 show 17-7-BL21(DE3)plysS-4 after induction; Figures 9 and 19 show 17-7-BL21(DE3)plysS-5 before induction; and Figures 10 and 20 show 17-7-BL21(DE3)plysS-5 after induction. As can be seen, the 17-7 protein fragment is 53.5 kD in size. There is no significant difference before and after induction, and no color development is observed by Western blotting, indicating that the target protein is not expressed.

[0069] Figure 10 The induced expression results of 17-8 are shown in the figure. Bands 1 and 11 are 17-8-BL21(DE3)plysS-1 before induction; 2 and 12 are 17-8-BL21(DE3)plysS-1 after induction; 3 and 13 are 17-8-BL21(DE3)plysS-2 before induction; 4 and 14 are 17-8-BL21(DE3)plysS-2 after induction; 5 and 15 are 17-8-BL21(DE3)plysS-3 before induction; 6 and 16 are 17-8-BL21(DE3) plysS-3 strain - after induction; 7 and 17 are 17-8-BL21(DE3)plysS-4 strain - before induction; 8 and 18 are 17-8-BL21(DE3)plysS-4 strain - after induction; 9 and 19 are 17-8-BL21(DE3)plysS-5 strain - before induction; 10 and 20 are 17-8-BL21(DE3)plysS-5 strain - after induction. It can be seen that the size of the 17-8 protein fragment is 17.96KD, and the expressed protein is consistent with the theoretical value. WB shows that 17-8-BL21(DE3)plysS has obvious expression. Later, strain 1 was selected as the seed for subsequent experiments. Figure 14 Figure 17-8 purification results. 1: 17-8-BL21(DE3) plysS supernatant (sample loading); 2: 17-8-BL21(DE3) plysS pellet; 3: nickel column purification - flow-through; 4: nickel column purification - equilibration solution rinse; 5: nickel column purification - 50mM imidazole rinse; 6: nickel column purification - 100mM imidazole rinse; 7: nickel column purification - 150mM imidazole rinse; 8: nickel column purification - 250mM imidazole rinse; 9: nickel column purification - 500mM imidazole rinse; 10: G25 - desalted sample. This shows that after nickel column purification, the protein can be purified to a single band.

[0070] In summary, after actual induction, only T17-5, T17-6 and T17-8 had obvious bands at 25KD-35KD, 15KD-20KD and 18.4KD-25KD respectively, and WB experiments showed that obvious bands could be detected. According to the above experimental results, the recombinant type 17 collagen T17-5, T17-6 and T17-8 of the present invention can be successfully induced to express. Figure 7 , Figure 8 , Figure 10 As shown, after the constructed strains were induced, obvious bands appeared. The results showed that the strains producing recombinant type 17 collagen T17-5, T17-6 and T17-8 were successfully constructed.

[0071] Example 4 High-density culture of recombinant bacteria

[0072] The recombinant bacteria BL21(DE3) pLysS / PET28a(+)-T17-5, No. 4 of BL21(DE3) pLysS / PET28a(+)-T17-6, and No. 1 of BL21(DE3) pLysS / PET28a(+)-T17-8 in Example 3 were placed in a glycerol tube and inoculated into 50 mL of a primary seed culture medium (1% peptone, 1% sodium chloride, 0.5% yeast powder) containing 50 μg / mL kanamycin at an inoculum size of 1‰. The cells were cultured at 37°C and 250 rpm for about 16 h, and then transferred to 1000 mL of a secondary seed culture medium (1% peptone, 1% yeast extract powder, 0.4% sodium chloride, 0.5% diphosphate) containing 50 μg / mL kanamycin. The seed solution was incubated at 37°C and 250 rpm for approximately 10 hours. The cultured seed solution was then transferred to a 20-L fermentor containing 10 L of fermentation medium (1.6% peptone, 2.3% yeast extract powder, 0.4% sodium chloride, 0.25% potassium dihydrogen phosphate, 0.133% dipotassium hydrogen phosphate, 0.07% magnesium sulfate heptahydrate, 0.25% glucose, pH adjusted to 7.0 with sodium hydroxide). Initial fermentation parameters were: temperature 37°C, rotation speed 350 rpm, aeration 1000 L / h, pressure 0.02 MPa, uncontrolled pH (pH range 6.0-7.3), and DO ≥ 30%. After the dissolved oxygen (DO) returned to 60%, feed was initiated (feed medium formulation: peptone 1.6%, yeast extract powder 2.3%, sodium chloride 0.4%, potassium dihydrogen phosphate 0.25%, dipotassium hydrogen phosphate 0.133%, magnesium sulfate heptahydrate 3.25%, glycerol 50%). The initial feed rate was primarily controlled by pH to control flow acceleration. The pH drop was no more than 1 / hour. When the pH dropped below 6.0, sodium hydroxide was used to adjust the pH to 7.0. Feeding was then continued. The dissolved oxygen (DO) was maintained at 20% by increasing the aeration rate. When the OD600 reached 100, 1 mM IPTG was added. The temperature was maintained at 37°C, and the DO was maintained at around 30%. The induction time was 4-5 hours. Samples were taken periodically after the induction period, and the fermentation broth was analyzed by SDS-PAGE.

[0073] SDS-PAGE results are as follows Figure 11 As shown, Figure 11Band 1 represents 17-6-BL21(DE3)plysS-4 before induction; Band 2 represents 17-6-BL21(DE3)plysS-4 after induction; Band 3 represents 17-5-BL21(DE3)plysS-4 before induction; Band 4 represents 17-5-BL21(DE3)plysS-4 after induction; Band 5 represents 17-8-BL21(DE3)plysS-1 before induction; Band 2 represents 17-8-BL21(DE3)plysS-1 after induction. This indicates that the target proteins can be effectively expressed in high-density fermentations of the above strains, indicating successful production and fermentation with these strains.

[0074] Example 5 Purification of recombinant type 17 collagen

[0075] The fermentation broth of the recombinant bacteria BL21(DE3)pLysS / PET28a(+)-T17-6, BL21(DE3)pLysS / PET28a(+)-T17-5 and BL21(DE3)pLysS / PET28a(+)-T17-8 detected and correct in Example 4 after high-density fermentation was centrifuged at 10,000 rpm in a tubular centrifuge to obtain bacterial sludge. The bacterial sludge was subjected to the following purification steps to obtain pure recombinant type 17 collagen:

[0076] ① Bacterial cell disruption: The bacterial sludge was resuspended in a solution with a mass-to-volume ratio of 1:6 (50 mM Pb + 500 mM NaCl + 20 mM imidazole + 8 M urea, pH = 8.3), and then the bacterial cells were disrupted using a homogenizer at a pressure of 800 bar. The bacterial cells were disrupted three times to obtain a bacterial solution containing the target protein;

[0077] ② Collection of bacterial solution: Centrifuge the bacterial solution containing the target protein at 4°C, 20,000 rpm for 20 min, remove the precipitate, and collect the supernatant, which is the bacterial solution containing the target protein;

[0078] ③ Nickel column purification: The collected bacterial solution was purified by nickel column. The purification method is as follows:

[0079] 1) Nickel column equilibration: First, rinse the column with 3 CV of deionized water to remove the protective solution (20% ethanol) in the column, then rinse the column with 3 CV of equilibration buffer (i.e., binding buffer);

[0080] 2) Loading the bacterial solution: Pass the bacterial solution containing the target protein collected in step ② through a 0.45 μm membrane and then load the sample. The loading volume is 2 CV.

[0081] 3) Protein removal: Rinse the column with washing buffer until the UV280 curve flattens.

[0082] 4) Elution of the target protein: Use elution buffer for elution. When a new peak appears at UV280, start collecting the sample until the peak flattens.

[0083] 5) Gel (SDS gel) detection: Take the above target protein elution sample and run the gel test. If the test is qualified (protein band size is 15KD-20KD, 25KD-35KD, 18.4KD-25KD), proceed to the next step;

[0084] ④ Molecular sieve purification (G25 filler): The target protein that has passed the nickel column purification test in step 5) is further purified by the following purification method:

[0085] 1) Equilibrate the molecular sieve column: First, rinse the column with 3CV of deionized water to remove the protective solution (20% ethanol) in the column, and then rinse the column with desalting buffer for 3CV;

[0086] 2) Sample loading: The target protein that has passed the above test is loaded into the sample with a sample volume of 0.2CV;

[0087] 3) Desalting: Continue to flush the column with desalting buffer until the protein flows out. Collect the protein as the desired sample.

[0088] Samples are taken at each step of the purification process to show that the protein purification process is feasible. Figures 12 to 14 As shown, Figure 12 The following are the purification results of the 17-6 sample. Bands 1-7 are SDS-PAGE run results: Band 1 represents the 17-6-BL21(DE3)plysS lysis sample; Band 2 represents the load sample (lysis sample, adjusted to pH 8.0); Band 3 represents the flow-through (FT) sample; Band 4 represents the equilibration solution wash (25mM imidazole); Band 5 represents the 50mM imidazole wash; Band 6 represents the 250mM imidazole wash; and Band 7 represents the G25-desalted sample. This demonstrates that the protein was purified to a single band after nickel column and desalting column purification. Figure 13 The following are the protein purification results for 17-5. Figure 1 shows the supernatant from 17-5-BL21(DE3) plysS lysis (sample loading); 2 shows the flow-through (FT) sample from nickel column purification; 3 shows the equilibration rinse from nickel column purification; 4 shows the 50mM imidazole rinse from nickel column purification; 5 shows the 250mM imidazole rinse from nickel column purification; 6 shows the 500mM imidazole rinse from nickel column purification; and 7 shows the G25-desalted sample. This demonstrates that after purification on both the nickel and desalting columns, the protein was purified to a single band. Figure 14Figure 17-8 purification results. 1: 17-8-BL21(DE3)plysS supernatant (sample loading); 2: 17-8-BL21(DE3)plysS pellet; 3: nickel column purification - flow-through; 4: nickel column purification - equilibration solution rinse; 5: nickel column purification - 50mM imidazole rinse; 6: nickel column purification - 100mM imidazole rinse; 7: nickel column purification - 150mM imidazole rinse; 8: nickel column purification - 250mM imidazole rinse; 9: nickel column purification - 500mM imidazole rinse; 10: G25 - desalted sample. This shows that after purification on both the nickel column and desalting column, the sample was purified to a single band.

[0089] The results showed that the purity of the protein purified through the above steps was greater than 90%.

[0090] Example 6 Recombinant type 17 collagen promotes cell proliferation experiment

[0091] HaCaT cells in logarithmic growth phase were routinely cultured in DMEM medium (Gibco, USA) containing 10% FBS to 80%-90% of the wells, and then digested with 0.25% trypsin (m / v) and seeded into 96-well plates, with 7.0×103 cells per well. After culturing at 37°C and 5% CO2 for 8 h, serum-free DMEM medium was changed to continue culturing for 16 h. The medium was aspirated and the purified 17-5, 17-6, and 17-8 recombinant collagens were added, respectively. At the same time, animal collagen (bovine collagen) and commercially available recombinant collagen were added. Type 17 collagen (Shanghai Boersen Biotechnology Co., Ltd., catalog number: BES21424RP, same below) was added as a control. Final concentrations of recombinant type 17 collagen were 147.06 ng / mL, 36.76 ng / mL, 9.19 ng / mL, 2.30 ng / mL, 0.57 ng / mL, 0.14 ng / mL, 0.04 ng / mL, and 0.01 ng / mL, respectively (all samples were diluted with PBS). Three wells were prepared for each concentration, and 100 μL of collagen was added to each well. Cells were then cultured in a cell culture incubator (37°C, 5% CO2, Thermo Fisher Scientific, USA). After 24 hours of culture, the culture medium was aspirated and serum-free culture medium containing 10% CCK8 (purchased from MCE) was added. The cells were then incubated in a cell culture incubator (37°C, 5% CO2) for 1 hour. After shaking, the absorbance was detected at a wavelength of 450 nm.

[0092] The results are as follows Figure 15 As shown, it can be seen that recombinant type 17 collagen 17-6, 17-5 and 17-8 all have good cell proliferation promoting activity, among which the activity of 17-8 is significantly better than that of other proteins.

[0093] Example 7 Recombinant Type 17 Collagen Cell Adhesion Assay

[0094] 1. Test sample preparation: Positive control (bovine collagen) solution preparation: Dilute 6.18 mg / mL bovine collagen to 1 μg / mL with 0.4% glacial acetic acid, sterilize through a 0.22 μm filter, and store at -20°C until ready for use. During testing, dilute the control with ultrapure water and perform three replicate wells. All operations are performed under sterile conditions. The preparation process for commercially available collagen is the same as above.

[0095] Sample solution preparation: Dilute the recombinant collagen samples 17-5, 17-6, and 17-8 of Example 6 to 1 μg / mL with ultrapure water and perform three replicate wells. All the above operations were performed under sterile conditions.

[0096] Preparation of PBS solution: weigh 8 g of sodium chloride, 0.2 g of potassium chloride, 3.63 g of disodium hydrogen phosphate dodecahydrate, and 0.24 g of potassium dihydrogen phosphate, add 800 mL of deionized water to fully dissolve, dilute to 1000 mL in a volumetric flask, sterilize, and store in a sealed container at 4°C.

[0097] Complete culture medium preparation: Take 10 mL of fetal bovine serum, 1 mL of penicillin-streptomycin double antibody, and add DMEM basal culture medium to make up to 100 mL.

[0098] 2. Test process: The specific operation process is as follows:

[0099] (1) Add 100 μL of the positive control, negative control (ultrapure water), and sample solution to a 96-well plate, seal, and incubate at 4°C overnight. During the experiment, discard the liquid, wash once with PBS, and set aside.

[0100] (2) Digest the HaCaT cells in the logarithmic phase with trypsin, resuspend them in 10% FBS culture medium, and add 100 μL of the diluted cell suspension to the above 96-well plate to a cell density of 15,000 cells / well;

[0101] (3) After 3 hours of inoculation (the time can be adjusted according to the cell status), observe the cell adhesion status under a microscope;

[0102] (4) Add 10 μL of CCK8 to each well, mix well, and continue to incubate for 2 hours. Then take it out of the incubator, place it on a microplate reader, detect the absorbance at a wavelength of 450 nm, and record the measurement results.

[0103] (5) Use Excel software to process the data and express the results as adhesion rate. The calculation formula is:

[0104]

[0105] The test results are as follows Figure 16 As shown, the results showed that recombinant type 17 collagen 17-5, 17-6 and 17-8 all had good cell adhesion promoting activity, among which the activity of 17-8 was significantly better than that of other proteins (Note: based on experimental results).

[0106] Example 8 Transdermal performance test of recombinant type 17 collagen

[0107] The skin is composed of three parts: the epidermis, dermis, and subcutaneous tissue. Transdermal absorption primarily refers to the process by which substances penetrate and are absorbed into the body through the epidermis. Cosmetic ingredients produce their effects by penetrating the epidermis and being absorbed into the dermis.

[0108] This test used pig skin tissue to test the permeability of the sample. Fluorescence microscopy was used to observe the accumulation of fluorescently labeled substances in the ex vivo skin tissue at different time points to evaluate the skin permeation behavior of the sample. The test was conducted according to the experimental plan in Table 1.

[0109] Table 1 Experimental plan for skin permeability test of the present invention

[0110]

[0111] The specific process of transdermal experiment is as follows:

[0112] (1) Sample preparation: The sample to be tested was prepared into a liquid sample according to a certain ratio. The sample concentration was 0.1 μg / mL and contained the recombinant type 17 collagen (17-5, 17-6, 17-8) prepared in this protocol.

[0113] (2) Transdermal cell culture: A skin model of miniature pig skin is used to simulate human skin. The miniature pig skin used in the present invention is the skin of a small Bama miniature pig, standard A1 type, two-month-old, with the following parts: whole body skin (abdomen and back), skin type: full-thickness skin (keratin + epidermis + dermis), item number: HBZ20A1 (Beijing Hebang Xingye Scientific Instrument Co., Ltd.), specifications: diameter: 25 mm, thickness 0.75-1.0 mm, size about 20*30 cm;

[0114] (3) Apply the drug: Apply the drug to be tested evenly on the transdermal model.

[0115] (4) Waiting for absorption: According to the requirements of the experimental design, observe the transdermal effect of the drug in the transdermal model at 0 min and 60 min respectively.

[0116] (5) Sampling and analysis: At the above time points, samples are collected from the transdermal model using a specific method. The accumulation of fluorescent labeled substances in the ex vivo skin tissue at the above time points is observed using a fluorescence microscope and analyzed.

[0117] The results show that if Figure 17 As shown, the cumulative amount of fluorescent substances in all three experimental groups increased over time, and the fluorescence intensity of recombinant type 17 collagen (17-8) was significantly greater than that of recombinant type 17 collagen (17-5) and recombinant type 17 collagen (17-6). These test results indicate that compared with recombinant type 17 collagen (17-5) and recombinant type 17 collagen (17-6), recombinant collagen with a membrane-penetrating peptide (17-8) has the best transdermal performance.

[0118] Example 9 Lyophilization of recombinant type 17 collagen

[0119] The purified type 17 collagen (17-8) with the best cell activity and transdermal ability (i.e., 17-8 in Example 5) was mixed with mannitol and freeze-dried using a vacuum freeze dryer. The specific freeze-drying formula is shown in Table 2 below:

[0120] Table 2 Freeze-dried product formula of the present invention

[0121]

[0122] After the purified product is dissolved according to the above formula, it is freeze-dried according to the following steps:

[0123] Accurately weigh the excipient components of the freeze-dried powder preparation, mannitol and recombinant type 17 collagen (17-8) stock solution, add them to a 20mM PBS buffer system, mix thoroughly, and sterile filter using a 0.22µm filter. Dispense into vials and freeze-dry to obtain the freeze-dried powder. The freeze-drying process is as follows:

[0124] (1) Prefreezing: Place the vial containing the recombinant type 17 collagen (17-8) sample on the freeze dryer partition, set the temperature to 4°C for prefreezing, set the time to 1 hour, and then cool it to 40°C for freezing, and set the time to 5 hours;

[0125] (2) Sublimation drying: Heat the partition system to -35°C for 60 minutes. After reaching the preset temperature, evacuate the system until the vacuum degree reaches 0.1 mBar and perform sublimation drying.

[0126] (3) Re-drying: Continue to heat the partition system to 20 ° C, the preset time is 1 hour, and after reaching the preset temperature, dry it for 10 hours. At the same time, evacuate to a vacuum degree of 0.1 mBar, and the freeze-drying is completed.

[0127] The test results are as follows Figure 18 As shown, the results showed that the purified recombinant type 17 collagen (17-8) had a good appearance after freeze-drying (white block solid).

[0128] It should be noted that the above-described embodiments are to be understood as illustrative and not limiting of the scope of protection of the present invention, which is subject to the claims. It will be apparent to those skilled in the art that non-essential improvements and adjustments to the present invention, without departing from the spirit and scope of the present invention, still fall within the scope of protection of the present invention.

Claims

1. A recombinant type 17 collagen with transdermal effect, characterized in that: The amino acid sequence of the recombinant type 17 collagen is shown in SEQ ID NO.26; the polynucleotide sequence encoding the recombinant type 17 collagen is shown in SEQ ID NO.

16.

2. A recombinant type 17 collagen expression vector, characterized in that: A polynucleotide sequence comprising the recombinant type 17 collagen according to claim 1.

3. A recombinant engineered bacterium, characterized in that: The invention comprises the recombinant type 17 collagen expression vector according to claim 2.

4. The recombinant engineered bacterium according to claim 3, characterized in that Escherichia coli as the host.

5. A method for preparing recombinant type 17 collagen according to claim 1, characterized in that: The steps include: S1. Synthesize the nucleotide sequence of SEQ ID NO. 16 by biosynthesis to obtain the target gene fragment; S2. The target gene fragment obtained in step S1 was double-digested with EcoRI and NotI, and then inserted into the corresponding restriction sites of the shuttle vector PET28a to construct the recombinant plasmid PET28a-T17-8; S3, transferring the recombinant plasmid PET28a-T17-8 constructed in step S2 into Escherichia coli to obtain positive bacteria, further culturing and inducing expression to obtain bacteria containing the recombinant type 17 collagen; S4. Resuspend and crush the recombinant collagen type 17 obtained in step S3; collect the supernatant by centrifugation, filter, and then purify the filtered solution by affinity chromatography on a nickel column to obtain the product.

6. The preparation method according to claim 5, wherein The process of affinity chromatography purification by nickel column shown in step S4 is as follows: the filtrate containing recombinant type 17 collagen is passed through the nickel column, unbound impurities are washed away with binding buffer and washing buffer, and the recombinant type 17 collagen is eluted with elution buffer; the filtrate is added to a G25 desalting column and washed out with desalting buffer.

7. The preparation method according to claim 6, wherein The components of the binding buffer include 0.4~0.6MNaCl, 45~55mM PB, 15~25mM imidazole, 7~9M urea, pH=8.3; the components of the wash buffer include 0.4~0.6MNaCl, 45~55mM PB, 70~90mM imidazole, 3~5M urea, pH=8.3; the components of the elution buffer include 0.4~0.6MNaCl, 45~55mM PB, 250~350mM imidazole, 2M urea, pH=8.3; the components of the desalting buffer include 0.1~0.2MNaCl, 45~55mM PB, pH=6.

5.

8. Use of the recombinant type 17 collagen as claimed in claim 1 in the preparation of a biological skin hair follicle repair agent.

9. The use according to claim 8, characterized in that The dosage form of the biological skin follicle repair agent is one of freeze-dried powder, biological sponge or dressing.

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