Highly stable recombinant type XVII collagen, its construction method and applications

Abstract

This invention provides a highly stable recombinant type XVII collagen, its construction method, and its applications, belonging to the field of bioengineering technology. The invention selects the amino acid sequence of natural human type XVII collagen as the basic unit and repeatedly replicates it to obtain recombinant type XVII collagen. The design process of the recombinant type XVII collagen avoids unstable GXY triplets, eliminates potential matrix metalloproteinase cleavage sites and glycosylation sites, and retains the KGD active sequence, thus reducing the probability of containing easily degraded sites. The sequence fragment of the recombinant type XVII collagen is derived from the full-length sequence of the natural collagen gene, exhibiting excellent solubility, bioactivity, and high stability. It remains stable under high-temperature storage conditions and without alkaline conditions, making it suitable as a raw material for the production of pharmaceuticals, medical devices, biomaterials, or cosmetics, demonstrating significant practicality.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to highly stable recombinant type XVII collagen, its construction method, and its applications. Background Technology

[0002] Type XVII collagen, abbreviated as COL17, is a major component of hemidesmosomes and is primarily found in the skin, mucous membranes, and eyes. COL17 is a key regulator of skin aging; intrinsic or extrinsic aging leads to genomic instability, resulting in decreased COL17 expression and increased hydrolysis, ultimately causing thinning and fragility of the epidermis, as well as graying and hair loss. Furthermore, COL17 is a crucial regulator of wound repair; upregulation of COL17 can modulate stem cell population dynamics and migration. In addition, the upregulation and shedding of COL17 can regulate the migration speed and direction of stem cells through interactions with other intracellular or extracellular matrix components. Studies have shown that forcibly maintaining COL17 expression can inhibit the senescence of hair follicle stem cells, rescuing epidermal thinning and hair loss. As a good marker of stem cells reflecting individual cellular potential and self-renewal quality, COL17 is a potential therapeutic target for skin anti-aging and wound repair.

[0003] Currently, collagen is mainly prepared through animal extraction and biological expression separation. Collagen obtained through animal extraction suffers from batch-to-batch variations due to individual animal differences, and COL17 is scarce in animals, leading to difficulties in extraction, low production capacity, and high prices. This makes it unsuitable for large-scale industrial production and hinders sustainable manufacturing. Furthermore, some zoonotic viruses may exist in animals, posing significant risks to the application of animal-derived collagen. Biological expression separation of recombinant proteins involves introducing the gene sequence of natural collagen or a redesigned gene sequence into selected host cells. Through culturing, fermentation, separation, and purification, recombinant collagen with the characteristics and functions of the natural protein can be obtained. Recombinant collagen molecules are relatively simple and structurally clear, making it easier to obtain specific types, sizes, and functions of target proteins. Depending on the host cell, expression systems are mainly classified into E. coli expression systems (prokaryotic), yeast expression systems (eukaryotic), insect expression systems, plant expression systems, and mammalian expression systems. Yeast protein expression systems, represented by Pichia pastoris, have advantages such as high protein expression levels, low cost, easy induction, easy purification of secreted proteins, and easy implementation of high-density fermentation. However, their post-translational processing and modification systems are not entirely the same as those of mammals, some protein products are easily degraded, and the expression level is relatively difficult to control.

[0004] In recent years, researchers have developed some recombinant type XVII collagen. However, when using genetic engineering methods to produce and express recombinant type XVII collagen, it can degrade during fermentation and purification processes. This not only increases production and storage costs but also affects the performance and final yield of the recombinant type XVII collagen produced by this method. Furthermore, as a raw material for medical devices, recombinant type XVII collagen requires higher purity and stability to ensure product quality and safety, as well as the safety and effectiveness of medical devices. This is to reduce safety incidents and adverse reactions caused by raw material issues in medical devices. Therefore, there is a need to develop a high-purity, high-stability recombinant type XVII collagen. Summary of the Invention

[0005] To address some shortcomings in existing technologies, this invention provides a highly stable recombinant type XVII collagen, its construction method, and its applications. This invention selects the amino acid sequence of natural human type XVII collagen as the basic unit and repeatedly replicates it to obtain recombinant type XVII collagen. The design process of the recombinant type XVII collagen avoids unstable GXY triplets, eliminates potential matrix metalloproteinase cleavage sites and glycosylation sites, and retains the KGD active sequence, thus reducing the probability of containing easily degraded sites. The sequence fragment of the recombinant type XVII collagen is derived from the full-length sequence of the natural collagen gene, exhibiting excellent solubility, bioactivity, and high stability. It remains stable under high-temperature storage conditions and without alkaline conditions, making it suitable as a raw material for the production of pharmaceuticals, medical devices, biomaterials, or cosmetics, demonstrating significant practicality.

[0006] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0007] The present invention first provides a highly stable recombinant type XVII collagen monomer, wherein the recombinant type XVII collagen monomer comprises multiple sequence fragments that avoid unstable GXY triplets, discard potential matrix metalloproteinase cleavage sites and glycosylation sites, and retain KGD activity.

[0008] The sequence fragments include one or more of the following sequences from human type XVII collagen: p597-p611, p597-p619, p641-p670, p674-p700, p674-p715, p686-p706, p692-p715, p906-p929, p906-p938, p951-p977, p1438-p1461, p1438-p1464, and p1438-p1467.

[0009] Preferably, the recombinant type XVII collagen monomer comprises any combination of the following sequence fragments:

[0010] a) p597-p611, p674-p715, p906-p929;

[0011] b) p692-p715, p906-p938, p951-p977, p1438-p1467;

[0012] c) p597-p619, p641-p670, p674-p715, p906-p929;

[0013] d)p674-p715;

[0014] e)p674-p715, p906-p929;

[0015] f) p597-p611, p674-p715, p906-p929, p951-p977;

[0016] g)p597-p611, p674-p715, p906-p929, p1438-p1464;

[0017] h)p695-p715;

[0018] i)p597-p611, p695-p715;

[0019] j)p686-p706;

[0020] k)p674-p700;

[0021] l)p1438-p1461;

[0022] In each of the above groups, one or more individual fragments are closely connected.

[0023] Preferably, the recombinant type XVII collagen monomer is combination l), whose amino acid sequence is shown in SEQ ID NO:24.

[0024] The present invention also provides a highly stable recombinant type XVII collagen, wherein the highly stable recombinant type XVII collagen is based on the above-mentioned recombinant type XVII collagen monomers repeated in series as basic units, and the recombinant type XVII collagen monomers may be the same or different.

[0025] Preferably, the size of the highly stable recombinant type XVII collagen is 20-25 kDa.

[0026] Preferably, the amino acid sequence of the highly stable recombinant type XVII collagen includes i or ii:

[0027] i. Amino acid sequence such as SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25;

[0028] ii. A protein derived from i that has human collagen activity after substitution, deletion or addition of one or more amino acids in the amino acid sequence shown in i, or an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or more, the same as the sequence shown in i.

[0029] Preferably, the amino acid sequence of the highly stable recombinant type XVII collagen is shown in SEQ ID NO:25.

[0030] The present invention also provides a polynucleotide encoding the above-mentioned highly stable recombinant type XVII collagen.

[0031] Preferably, the sequence of the polynucleotide includes SEQ ID NO:26 or its degenerate sequence.

[0032] The present invention also provides a recombinant expression vector comprising nucleotides encoding the aforementioned highly stable recombinant type XVII collagen.

[0033] The present invention also provides a recombinant engineered bacterium, which contains nucleotides encoding the above-mentioned highly stable recombinant type XVII collagen or the above-mentioned recombinant expression vector.

[0034] Preferably, the host bacteria include Pichia pastoris, and more preferably Pichia pastoris strain GS115, Pichia pastoris strain X33, Pichia pastoris strain KM71H, or Pichia pastoris strain SMD116.

[0035] Preferably, the recombinant engineered bacteria are deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 32750.

[0036] This invention also provides a method for preparing the above-mentioned highly stable recombinant type XVII collagen, the method comprising:

[0037] (1) Select and design the sequence of recombinant type XVII collagen monomers, and then construct recombinant type XVII collagen by tandem recombination based on recombinant type XVII collagen monomers;

[0038] (2) Construct a recombinant expression vector for recombinant type XVII collagen;

[0039] (3) Electroporate the obtained recombinant expression vector for recombinant type XVII collagen into the host bacteria, screen and verify, and obtain recombinant engineered bacteria;

[0040] (4) The recombinant engineered bacteria were fermented and induced to express the high-stability recombinant type XVII collagen.

[0041] Preferably, in step (1), the recombinant type XVII collagen monomer includes multiple sequence fragments that avoid unstable GXY triplets, discard potential matrix metalloproteinase cleavage sites and glycosylation sites, and retain KGD activity.

[0042] The sequence fragments include one or more of the following sequences from human type XVII collagen: p597-p611, p597-p619, p641-p670, p674-p700, p674-p715, p686-p706, p692-p715, p695-p715, p906-p929, p906-p938, p951-p977, p1438-p1461, p1438-p1464, and p1438-p1467.

[0043] Preferably, the recombinant type XVII collagen monomer comprises any combination of the following sequence fragments:

[0044] a) p597-p611, p674-p715, p906-p929;

[0045] b) p692-p715, p906-p938, p951-p977, p1438-p1467;

[0046] c) p597-p619, p641-p670, p674-p715, p906-p929;

[0047] d)p674-p715;

[0048] e)p674-p715, p906-p929;

[0049] f) p597-p611, p674-p715, p906-p929, p951-p977;

[0050] g)p597-p611, p674-p715, p906-p929, p1438-p1464;

[0051] h)p695-p715;

[0052] i)p597-p611, p695-p715;

[0053] j)p686-p706;

[0054] k)p674-p700;

[0055] l)p1438-p1461;

[0056] In each of the above groups, one or more individual fragments are closely connected.

[0057] Preferably, the recombinant type XVII collagen monomer is combination l), whose amino acid sequence is shown in SEQ ID NO:24.

[0058] Preferably, in step (1), the recombinant type XVII collagen monomers are the same or different.

[0059] Preferably, in step (1), the size of the highly stable recombinant type XVII collagen is 20-25 kDa.

[0060] Preferably, in step (1), the amino acid sequence of the highly stable recombinant type XVII collagen includes i or ii:

[0061] i. Amino acid sequence such as SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25;

[0062] ii. A protein derived from i that has human collagen activity after substitution, deletion or addition of one or more amino acids in the amino acid sequence shown in i, or an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more or more, the same as the sequence shown in i.

[0063] Preferably, in step (1), the amino acid sequence of the highly stable recombinant type XVII collagen is shown in SEQ ID NO:25.

[0064] Preferably, in step (3), the host bacteria include Pichia pastoris, and more preferably Pichia pastoris strain GS115, Pichia pastoris strain X33, Pichia pastoris strain KM71H, or Pichia pastoris strain SMD116.

[0065] Preferably, in step (3), the recombinant engineered bacteria are deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 32750.

[0066] The present invention also provides a composition comprising the above-mentioned highly stable recombinant type XVII collagen monomer or highly stable recombinant type XVII collagen.

[0067] The present invention also provides an article comprising the above-mentioned highly stable recombinant type XVII collagen monomer, highly stable recombinant type XVII collagen, or a combination thereof.

[0068] The present invention also provides the application of the above-mentioned highly stable recombinant XVII type collagen monomer, highly stable recombinant XVII type collagen, composition or product in the preparation of medical devices, biomaterials, tissue engineering products and cosmetics.

[0069] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0070] This invention optimizes several short amino acid sequences of natural human type XVII collagen based on its sequence, avoiding unstable GXY triplets, eliminating potential matrix metalloproteinase cleavage sites and glycosylation sites, and retaining a KGD active sequence. These amino acid fragments are then tandemly linked as repeating units to form recombinant type XVII collagen monomers. These monomers are then recombined to obtain the highly stable recombinant type XVII collagen. The amino acid sequence of the highly stable recombinant type XVII collagen of this invention shows 100% homology with the corresponding portion of the natural type XVII collagen sequence, and it has potential applications in medical devices, cosmetic raw materials, and biomaterials.

[0071] The recombinant type XVII collagen described in this invention exhibits high stability. Performance evaluation revealed that it maintains high stability under normal storage conditions, and remains stable without degradation in high-temperature storage environments and under conditions avoiding alkalinity, preserving its original molecular weight and structure. When used as a raw material in the production of pharmaceuticals, medical devices, biomaterials, or cosmetics, the recombinant type XVII collagen described in this invention can maintain product stability and consistency, demonstrating excellent practicality.

[0072] The recombinant type XVII collagen described in this invention can be efficiently secreted and expressed in a Pichia pastoris expression system and is easy to purify. It achieves a protein expression level of 6.51 g / L during high-density fermentation, making it suitable for large-scale industrial production. Attached Figure Description

[0073] Figure 1This is an SDS-PAGE image of the first batch of recombinant human XVII protein designed for expression.

[0074] Figure 2 SDS-PAGE image of the second batch of recombinant human XVII protein expression.

[0075] Figure 3 SDS-PAGE image of the third batch of recombinant human XVII protein expression.

[0076] Figure 4 SDS-PAGE image of recombinant type XVII collagen with high stability expressed in a small-batch, high-density fermentation experiment.

[0077] Figure 5 This is an SDS-PAGE image of purified, highly stable recombinant type XVII collagen.

[0078] Figure 6 The stability test results of XVII 10K106 at different temperatures are shown in SDS-PAGE (a) and degradation rate analysis results (b).

[0079] Figure 7 The stability test results of XVII 10K 106 solution samples at different pH values ​​are shown in SDS-PAGE (a) and degradation rate analysis results (b).

[0080] Figure 8 This is a graph showing the statistical results of cytotoxicity testing for recombinant type XVII collagen. Detailed Implementation

[0081] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. The technical solutions of the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0082] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0083] Example 1. Screening of recombinant type XVII collagen monomer fragments:

[0084] This embodiment is based on the human type XVII collagen sequence. With the aim of avoiding unstable GXY triplets, discarding potential matrix metalloproteinase cleavage sites and glycosylation sites, and retaining KGD active sequences, one or several amino acid sequence fragments of natural human type XVII collagen are selected and used for subsequent design and screening of recombinant type XVII collagen monomers and recombinant type XVII collagen.

[0085] The human type XVII collagen sequence references the Uniprot Q9UMD9 sequence (https: / / www.uniprot.org / uniprot / Q9UMD9) and the NCBI reference sequence Q9UMD9 (https: / / www.ncbi.nlm.nih.gov / protein / Q9UMD9), both of which are identical and are shown in SEQ ID NO:1.

[0086] SEQ ID NO:1:

[0087] GPPGQKGEMGTPGPKGDRGPAGPP GHPGPPGPRGHKGEKGDKGDQVYAGRRRRRSIAVKP.

[0088] The sequence fragments that avoid unstable GXY triplets, eliminate potential matrix metalloproteinase cleavage sites and glycosylation sites, and retain KGD activity are p597-p611, p597-p619, p641-p670, p674-p700, p674-p715, p686-p706, p692-p715, p695-p715, p906-p929, p906-p938, p951-p977, p1438-p1461, p1438-p1464, and p1438-p1467 in human type XVII collagen.

[0089] The amino acid sequence of the sequence fragment is shown below:

[0090] p597-p611: GHPGPQGPKGQKGSV (SEQ ID NO: 27);

[0091] p597-p619: GHPGPQGPKGQKGSVGDPGMEGP (SEQ ID NO: 28);

[0092] p641-p670: GEKGERGAAGEPGPHGPPGVPGSVGPKGSS (SEQ ID NO: 29);

[0093] p674-p700: GPQGPPGPVGLQGLRGEVGLPGVKGDK (SEQ ID NO: 22);

[0094] p674-p715: GPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPPGPKGDQGEK (SEQ ID NO: 8);

[0095] p686-p706: GLRGEVGLPGVKGDKGPMGPP (SEQ ID NO: 20);

[0096] p692-p715: GLPGVKGDKGPMGPPPGPKGDQGEK (SEQ ID NO: 30);

[0097] p695-p715: GVKGDKGPMGPPGPKGDQGEK (SEQ ID NO: 16);

[0098] p906-p929:GPPGPPGPPGPKGDQGPPGPRGHQ (SEQ ID NO: 31);

[0099] p906-p938:GPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGFS (SEQ ID NO: 32);

[0100] p951-p977:GPPGPPGPQGPKGDKGDPGVPGALGIP (SEQ ID NO:33);

[0101] p1438-p1461: GPPGQKGEMGTPPGPKGDRGPAGPP (SEQ ID NO: 24);

[0102] p1438-p1464: GPPGQKGEMGTPPGPKGDRGPAGPPGHP (SEQ ID NO: 34);

[0103] p1438-p1467: GPPGQKGEMGTPPGPKGDRGPAGPPGHPGPP (SEQ ID NO: 35).

[0104] Example 2. Design and screening of recombinant type XVII collagen monomers and recombinant type XVII collagen:

[0105] In this embodiment, one or more sequence fragments obtained in Example 1 are tandemly recombined to form the first batch of recombinant XVII type collagen monomers. Then, these recombinant XVII type collagen monomers are used as base units for tandem recombination to obtain the first batch of recombinant XVII type collagen, which has a size of 20-25 kDa. This embodiment also examines the distribution of the selected sequence fragments and their expression stability, using this as the design basis for the next batch of recombinant XVII type collagen until highly stable XVII type collagen sequences are screened out.

[0106] The specific steps are as follows:

[0107] S1. Design of recombinant type XVII collagen monomers and recombinant type XVII collagen:

[0108] Three sequences of human type XVII collagen, p597-p611, p674-p715, and p906-p929, were selected and tandemly to obtain a recombinant type XVII collagen monomer with an amino acid sequence as shown in SEQ ID NO:2. The recombinant type XVII collagen monomer was tandemly repeated three times to obtain recombinant type XVII collagen, denoted as XVII9K32, with an amino acid sequence as shown in SEQ ID NO:3.

[0109] SEQ ID NO:2:

[0110] GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQ;

[0111] SEQ ID NO:3:

[0112] GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGD KGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQ.

[0113] Four sequences of human type XVII collagen, p692-p715, p906-p938, p951-p977, and p1438-p1467, were selected and tandemly to obtain recombinant type XVII collagen monomers with amino acid sequences as shown in SEQ ID NO:4. The recombinant type XVII collagen monomers were tandemly repeated twice to obtain recombinant type XVII collagen, denoted as XVII12K23, with an amino acid sequence as shown in SEQ ID NO:5.

[0114] SEQ ID NO:4:

[0115] GLPGVKGDKGPMGPPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGEQGLPGPP GPPGPQGPKGDKGDPGVPGALGIPGPPGQKGEMGTPPGKGDRGPAGPPGHPGPP;

[0116] SEQ ID NO:5:

[0117] GLPGVKGDKGPMGPPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPPRGHQGEQGLPGPPGPPGPQGPKGDKGDPGVPGALGIPGPPGQKGEMGTPPGKGDRGPAGPPGHPGPP GLPGVKGDKGPMGPPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPPRGHQGEQGLPGPPGPPGPQGPKGDKGDPGVPGALGIPGPPGQKGEMGTPPGPKGDRGPAGPPGHPGPP.

[0118] Four sequences of human type XVII collagen, p597-p619, p641-p670, p674-p715, and p906-p929, were selected and tandemly to obtain recombinant type XVII collagen monomers with amino acid sequences as shown in SEQ ID NO:6. The recombinant type XVII collagen monomers were tandemly repeated twice to obtain recombinant type XVII collagen, denoted as XVII6K25, with an amino acid sequence as shown in SEQ ID NO:7.

[0119] SEQ ID NO:6:

[0120] GHPGPQGPKGQKGSVGDPGMEGPGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGPQ GPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPR GHQ;

[0121] SEQ ID NO:7:

[0122] GHPGPQGPKGQKGSVGDPGMEGPGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQ GHPGPQGPKGQKGSVGDPGMEGPGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQ.

[0123] S2. DNA sequence synthesis and construction of recombinant expression vectors:

[0124] We commissioned Nanjing Genscript Biotech Co., Ltd. to synthesize DNA fragments encoding XVII9K32, XVII12K23, and XVII6K25. The synthesized gene fragments were cloned into the pPIC9K empty vector, so that the target fragments were accurately inserted into the reading frame of the secretory vector containing the secretion signal α factor, in order to obtain a recombinant expression vector that expresses XVII9K32, XVII12K23, and XVII6K25.

[0125] S3. Construction of recombinant engineered strains and expression of recombinant type XVII collagen:

[0126] 10 μg of the recombinant expression vectors for XVII9K32, XVII12K23, and XVII6K25 obtained in step S2 were linearized by digesting them with Sal I rapid digestion enzyme (Dalian TaKaRa Company) at 37℃ for 30 min. The linearized plasmids were then recovered using a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.), with a recovery volume of approximately 20 μL.

[0127] Take 10 μL of linearized plasmid and electroporate it into competent Pichia pastoris GS115 cells (purchased from Thermo Fisher Scientific). Immediately after electroporation, add 800 μL of pre-cooled YPD medium + 1M sorbitol 1:1 solution, mix the cells well, transfer to 1.5 mL EP tubes, and incubate at 30°C for 2 h. After incubation, centrifuge the bacterial solution at 3000×g for 5 min, discard 600 μL of supernatant, mix well, and spread 50-80 μL of the bacterial solution onto MD plates. Incubate at 30°C upside down for 2-5 days until single colonies (positive transformants) appear.

[0128] Add 2 mL of sterile ultrapure water to the surface of an MD plate, cover and incubate for a few seconds. Then, gently pipette the His+ transformants on the plate surface using a sterile 1 mL pipette tip, and transfer the bacterial suspension to a 1.5 mL centrifuge tube. Dilute the bacterial suspension with sterile double-distilled water and calculate the OD. 600 After taking the value, take 10. 5 Cells were spread on YPD plates containing 0.5 mg / mL G418 and incubated upside down at 30°C for 2-5 days until single colonies appeared, yielding Pichia pastoris engineered strains expressing XVII9K32, XVII12K23, and XVII6K25, respectively.

[0129] Add 200 μL / well of YPD medium to a sterile 96-well plate. Pick a single colony from the YPD plate and add it to the medium, mix well, and incubate overnight at 30°C and 180 rpm. The next day, mix the bacterial culture in the wells and take 80 μL of the culture for Pichia pastoris colony PCR. Take 5 μL of the PCR product and run it on a DNA gel. Select single colonies with clear bands and reasonable size and position for shake-flask verification.

[0130] Pichia pastoris engineered strains expressing XVII9K32, XVII12K23, and XVII6K25, with clear bands of appropriate size and position, were inoculated into 100mL Erlenmeyer flasks containing 15mL of 15% BMGY medium. The flasks were incubated at 30℃ with shaking at 220rpm for 24h. After calculating the OD value, the final OD was collected. 600 The bacterial suspension with a final OD value of 150 was centrifuged at 3000×g for 5 min, the supernatant was discarded, and the bacterial cells were resuspended in 15 mL of BMM medium to achieve the final OD value. 600 The culture medium was set to a value of 10 and incubated at 30°C with shaking at 220 rpm for 24 hours. Then, 200 μL of 100% methanol was added to the medium until the final methanol concentration was approximately 1%. The medium was then induced for another 24 hours. After incubation, bacterial samples were collected and centrifuged at 12000×g for 5 minutes at 4°C. The supernatant was collected. The supernatant was then prepared into a 1× SDS-PAGE electrophoresis sample using 5× loading buffer. The sample was prepared by incubating in a 100°C metal bath for 5 minutes and then analyzed by SDS-PAGE.

[0131] Test results as follows Figure 1 As shown in the figure, XVII12K23 expression is extremely low, and degradation bands are faintly visible; XVII6K25 shows protein expression, but the main band percentage is low, indicating severe degradation and instability; XVII9K32 has the highest expression level, but degradation bands are also visible. Since XVII9K32 and XVII6K25 have overlapping sequences (p597-p611 and p674-p715 fragments), these are relatively stable fragments, and the likelihood of peptide fragmentation and degradation is low.

[0132] XVII6K25 has additional segments p612-p619 and p641-p670 compared to XVII9K32, resulting in significantly reduced stability and the appearance of obvious degradation bands. These segments should be avoided in the design of the next batch of sequences.

[0133] Example 3. Design and screening of recombinant type XVII collagen monomers and recombinant type XVII collagen:

[0134] Based on the sequence fragment distribution and expression stability selected in Example 2, this embodiment recombines one or more sequence fragments obtained in Example 1 in series to form a second batch of recombinant type XVII collagen monomers. Then, the recombinant type XVII collagen monomers are used as basic units for tandem recombination to obtain a second batch of recombinant type XVII collagen, and further screening for highly stable type XVII collagen is performed.

[0135] The specific steps are as follows:

[0136] S1. Design of recombinant type XVII collagen monomers and recombinant type XVII collagen:

[0137] When designing recombinant type XVII collagen monomers, the fragments that could increase the degradation rate during protein expression, as identified in Example 2, were avoided. Furthermore, shorter fragments were extracted from sequences with low expression levels for construction and expression screening. The specific steps are as follows:

[0138] Human type XVII collagen sequence p674-p715 was selected to obtain recombinant type XVII collagen monomers with amino acid sequences as shown in SEQ ID NO:8. The recombinant type XVII collagen monomers were tandemly repeated 6 times to obtain recombinant type XVII collagen, denoted as XVII12K61, whose amino acid sequence is shown in SEQ ID NO:9.

[0139] SEQ ID NO:8:

[0140] GPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPPGPKGDQGEK;

[0141] SEQ ID NO:9:

[0142] GPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEK GPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEK.

[0143] Two sequences, p674-p715 and p906-p929, of human type XVII collagen were selected and tandemly synthesized to obtain a recombinant type XVII collagen monomer with the amino acid sequence shown in SEQ ID NO:10. This recombinant type XVII collagen monomer was then tandemly repeated four times to obtain recombinant type XVII collagen, denoted as XVII12K42, with the amino acid sequence shown in SEQ ID NO:11. SEQ ID NO:10:

[0144] GPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQ GPPGPRGHQ;

[0145] SEQ ID NO:11:

[0146] GPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQ GPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQ.

[0147] Four sequences of human type XVII collagen, p597-p611, p674-p715, p906-p929, and p951-p977, were selected and tandemly synthesized to obtain recombinant type XVII collagen monomers with amino acid sequences as shown in SEQ ID NO:12. The recombinant type XVII collagen monomers were tandemly synthesized twice to obtain recombinant type XVII collagen, denoted as XVII10K25, with an amino acid sequence as shown in SEQ ID NO:13.

[0148] SEQ ID NO:12:

[0149] GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGE KGPPGPPGPPGPKGDQGPPGPRGHQGPPGPPGPQGPKGDKGDPGVPGALGIP;

[0150] SEQ ID NO:13:

[0151] GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGPPGPPGPQGPKGDKGDPGVPGALGIP GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGPPGPPGPQGPKGDKGDPGVPGALGIP.

[0152] Four sequences of human type XVII collagen, p597-p611, p674-p715, p906-p929, and p1438-p1464, were selected and tandemly to obtain recombinant type XVII collagen monomers with amino acid sequences as shown in SEQ ID NO:14. The recombinant type XVII collagen monomers were tandemly repeated twice to obtain recombinant type XVII collagen, denoted as XVII8K26, with an amino acid sequence as shown in SEQ ID NO:15.

[0153] SEQ ID NO:14:

[0154] GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGE KGPPGPPGPPGPKGDQGPPGPRGHQGPPGQKGEMGTPPGKGDRGPAGPPGHP;

[0155] SEQ ID NO:15:

[0156] GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGPPGQKGEMGTPPGKGDRGPAGPPGHP GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGPPGQKGEMGTPPGKGDRGPAGPPGHP.

[0157] The recombinant expression vector was constructed, the recombinant engineered strain was constructed, and the recombinant protein was expressed and screened, following the steps S2 and S3 in Example 2. SDS-PAGE analysis was then performed, and the results are as follows: Figure 2 As shown.

[0158] from Figure 2It can be seen that XVII12K61 and XVII8K26 have high expression levels, while XVII12K42 and XVII10K25 have low expression levels, and XVII10K25 shows a clearly visible degradation band. The XVII12K42 sequence differs from the XVII12K61 sequence only by the addition of a p906-p929 segment, yet its expression level is significantly reduced. This may be because this segment affects the expression efficiency of the recombinant protein, and it should be avoided in subsequent sequence design. Furthermore, sequences VII10K25 and XVII8K26 share the p597-p611, p674-p715, and p906-p929 segments. The difference lies in that the XVII10K25 unit ends with the p951-p977 segment, while the XVII8K26 sequence units end with p1438-p1464. The expression level of XVII8K26 is significantly higher than that of XVII10K25. Therefore, the p1438-p1464 segment is considered the superior sequence.

[0159] Example 4. Design and screening of recombinant type XVII collagen monomers and recombinant type XVII collagen:

[0160] Based on the screening in Examples 2 and 3, this embodiment found that the p597-p611, p674-p715, and p1438-p1464 segments showed high expression levels and good stability. However, because some sequences still showed slight degradation bands, a third batch of shorter units was designed to screen for amino acid sequences of recombinant type XVII collagen with high expression levels and high stability. The specific steps are as follows:

[0161] S1. Design of recombinant type XVII collagen monomers and recombinant type XVII collagen:

[0162] When designing recombinant type XVII collagen monomers, the fragments that could increase the degradation rate during protein expression, as identified in Example 2, were avoided. Furthermore, shorter fragments were extracted from sequences with low expression levels for construction and expression screening. The specific steps are as follows:

[0163] Human type XVII collagen sequence p695-p715 was selected to obtain recombinant type XVII collagen monomers with amino acid sequences as shown in SEQ ID NO:16. The recombinant type XVII collagen monomers were tandemly repeated 11 times to obtain recombinant type XVII collagen, denoted as XVII22K111, whose amino acid sequence is shown in SEQ ID NO:17.

[0164] SEQ ID NO:16:

[0165] GVKGDKGPMGPPGPKGDQGEK;

[0166] SEQ ID NO:17:

[0167] GVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGP PGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEK.

[0168] Two sequences, p597-p611 and p695-p715, of human type XVII collagen were selected and tandemly synthesized to obtain a recombinant type XVII collagen monomer with the amino acid sequence shown in SEQ ID NO:18. This recombinant type XVII collagen monomer was then tandemly repeated six times to obtain recombinant type XVII collagen, denoted as XVII12K62, with the amino acid sequence shown in SEQ ID NO:19. SEQ ID NO:18:

[0169] GHPGPQGPKGQKGSVGVKGDKGPMGPPGPKGDQGEK;

[0170] SEQ ID NO:19:

[0171] GHPGPQGPKGQKGSVGVKGDKGPMGPPGPKGDQGEKGHPGPQGPKGQKGSVGVKGDKGPMGPPGPKGDQGEKGHPGPQGPKGQKGSVGVK GDKGPMGPPGPKGDQGEKGHPGPQGPKGQKGSVGVKGDKGPMGPPGPKGDQGEKGHPGPQGPKGQKGSVGVKGDKGPMGPPGPKGDQGEK GHPGPQGPKGQKGSVGVKGDKGPMGPPGPKGDQGEK.

[0172] Human type XVII collagen sequence p686-p706 was selected to obtain recombinant type XVII collagen monomers with amino acid sequences as shown in SEQ ID NO:20. The recombinant type XVII collagen monomers were tandemly repeated 11 times to obtain recombinant type XVII collagen, denoted as XVII11K113, whose amino acid sequence is shown in SEQ ID NO:21.

[0173] SEQ ID NO:20:

[0174] GLRGEVGLPGVKGDKGPMGPP;

[0175] SEQ ID NO:21:

[0176] GLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGV KGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPP.

[0177] Human type XVII collagen sequence p674-p700 was selected to obtain recombinant type XVII collagen monomers with amino acid sequences as shown in SEQ ID NO:22. The recombinant type XVII collagen monomers were tandemly repeated 9 times to obtain recombinant type XVII collagen, denoted as XVII9K95, whose amino acid sequence is shown in SEQ ID NO:23.

[0178] SEQ ID NO:22:

[0179] GPQGPPGPVGLQGLRGEVGLPGVKGDK;

[0180] SEQ ID NO:23:

[0181] GPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGL RGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDK.

[0182] Human type XVII collagen sequence p1438-p1461 was selected to obtain recombinant type XVII collagen monomers with amino acid sequences as shown in SEQ ID NO:24. The recombinant type XVII collagen monomers were tandemly repeated 10 times to obtain recombinant type XVII collagen, denoted as XVII10K106, whose amino acid sequence is shown in SEQ ID NO:25.

[0183] SEQ ID NO:24:

[0184] GPPGQKGEMGTPGPKGDRGPAGPP;

[0185] SEQ ID NO:25:

[0186] GPPGQKGEMGTPPGPKGDRGPAGPPGPPGQKGEMGTPPGKGDRGPAGPPGPPGQKGEMGTPPGKGDRGPAGPPGPPGQKGEMGTPPGKGDRGPAGPPGPPGQKGEMGTPPGKGDRGPAGPP GPPGQKGEMGTPPGPKGDRGPAGPPGPPGQKGEMGTPPGKGDRGPAGPPGPPGQKGEMGTPPGKGDRGPAGPPGPPGQKGEMGTPPGKGDRGPAGPPGPPGQKGEMGTPPGPKGDRGPAGPP.

[0187] The recombinant expression vector was constructed, the recombinant engineered strain was constructed, and the recombinant protein was expressed and screened, following the steps S2 and S3 in Example 2. SDS-PAGE analysis was then performed, and the results are as follows: Figure 3 As shown.

[0188] from Figure 3 It can be seen that the XVII12K62 sequence has an additional 15 amino acid segment, p597-p611, compared to the XVII22K111 sequence. Its expression level is significantly lower than that of XVII22K111, and its expression product contains many short peptide sequences that are not the target band. This suggests a possible break point in the p597-p611 segment. Repeated sequences breaking at the same location can result in multiple uniformly distributed bands below the target band. In contrast, the likelihood of a break in the p695-p715 segment is much lower. Similarly, the XVII11K113 sequence, located at p674-p715, was used to select the p686-p706 segment of human XVII type collagen. While the expression level was also high, degradation bands were observed, making it a less desirable option from a stability perspective. XVII9K95 was selected from the p674-p700 segment of the human XVII collagen sequence. The gel electrophoresis results showed that its expression level was not particularly high, with almost no visible degradation bands. Similarly, XVII10K106, located in the p1438-p1461 segment of the first helix region of human XVII collagen, showed relatively low expression levels but considerable expression stability.

[0189] Therefore, the XVII 10K106 is a recombinant type XVII collagen with high stability, and the polynucleotide sequence encoding XVII 10K106 is shown as SEQ ID No:26.

[0190] SEQ ID No:26:

[0191] ggaccaccgggtcaaaagggggaaatgggcacccccggccctaagggcgaccggggcccggctggacccccaggtccgcctgggcaaaagggcgagatgggaactccaggtcccaaaggggacagagggccggcagggccccccgggccgcctgggcagaaaggggagatgggcactccggggccgaagggtgaccgagggcctgctggaccccccggtcccccaggtcaaaagggtgaaatgggtactcccggcccgaaaggcgatcgtggcccagcgggaccgccgggaccgcccggtcaaaaaggtgaaatgggtacccctggacccaaaggcgaccgcggacctgcgggaccgccaggaccccccggccagaagggagagatggggacacctggtccgaagggtgatagagggccagccgggccccctggtcctcctggacagaaaggagagatggggacacctggcccaaaaggcgacaggggaccggctggtcccccaggcccacctggccagaagggtgaaatgggtacgcctgggcctaagggagatcgagggccggcaggcccgccagggccccctggccaaaaaggggaaatgggtacccccggcccaaaaggagatcgtggcccagcgggacctcccggcccaccgggtcagaagggagagatggggacgcctggtccaaaaggagatcgcggaccagccgggccaccgtgataa。

[0192] The recombinant engineered strain expressing XVII10K106 was sent to the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.32750. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession date: November 22, 2024; classification and name: *Pichia pastoris* Komagataella phaffii.

[0193] Example 5. Small-batch high-density fermentation experiment

[0194] In this embodiment, some recombinant type XVII collagen from Examples 2 to 4 were selected for small-batch high-density fermentation experiments to evaluate the expression level and stability of recombinant type XVII collagen.

[0195] (1) High-density fermentation with genetically engineered bacteria:

[0196] For expression of recombinant type XVII collagen XVII9K32, XVII12K61, XVII22K111, XVII11K113,

[0197] The genetically engineered bacteria XVII9K95 and XVII10K106 were used for large-scale expression and production to obtain fermentation broth containing recombinant collagen. The specific steps are as follows:

[0198] S1. Preparation of culture medium:

[0199] YPG seed culture medium: yeast extract 10 g / L, peptone 20 g / L, glycerol 10 g / L;

[0200] Fermentation medium: NH4H2PO4 190.4 g / L, KH2PO4 10.06 g / L, CaSO4·2H2O 1.18 g / L, K2SO4 18.2 g / L, MgSO4·7H2O 14.9 g / L, glycerol 40 g / L;

[0201] Feeding medium: 50% glycerol (w / v) and PTM1, with 12 mL of PTM1 micronutrients added per liter of 50% glycerol;

[0202] Induction medium: 100% methanol and PTM1, with 12 mL of PTM1 trace elements added per liter of methanol;

[0203] PTM1: CuSO4·5H2O 6.0g / L, NaI 0.08g / L, MnSO4·H2O 3.0g / L, NaMoO4·2H2O 0.2g / L, H3BO3 0.02g / L, CoCl2 0.5g / L, ZnCl2 20.0g / L, FeSO4·7H2O 65.0g / L, Biotin 0.2g / L, H2SO4 5.0mL / L, sterilized by filtration through a 0.22μm filter membrane, stored at 4℃.

[0204] S2. Batch culture conditions and induced expression of engineered strains:

[0205] A fed-batch culture method was used, with a culture temperature of 30℃. Genetically engineered bacteria expressing recombinant type XVII collagen (XVII9K32, XVII12K61, XVII22K111, XVII11K113, XVII9K95, and XVII10K106) were inoculated into 1L shake flasks containing 200mL of seed culture medium YPG and cultured at 220rpm and 30℃ for 18–24 hours until OD (dose retardation) was achieved. 600 =2~10, to obtain seed liquid.

[0206] Add 2L of fermentation medium to a 5L fermenter (Baoxing Biotechnology). Before inoculation, adjust the rotation speed inside the tank to 300rpm, the aeration rate to 4L / min, and the temperature to 30℃. Then, adjust the pH of the tank to 4.5 with an alkaline solution prepared with concentrated ammonia. Then, add 0.9mL of PTM1 to the fermenter, and then add 200mL of the prepared seed liquid to the tank (inoculation with a flame ring). Then, click the dissolved oxygen electrode to calibrate. After calibration, fermentation begins.

[0207] Once the dissolved oxygen level drops to 30% for the first time during growth, use the dissolved oxygen cascade rotation function to maintain it at 30%, waiting for the glycerol to be depleted and the dissolved oxygen to rebound and exceed 70% (OD). 600 When the dissolved oxygen level is approximately 20%, cancel the dissolved oxygen cascade speed, increase the stirring speed to 650 rpm, and use 30% dissolved oxygen-linked feeding, adding 80 mL of feeding medium. After feeding, wait for the dissolved oxygen to rebound to above 70%, set the pH to 5.0 and the temperature to 30℃, and feed induction medium for induction culture. Manually add 5 mL of induction medium. After the dissolved oxygen rebounds to above 70%, set the feeding rate to 8 mL / h, increase it to 10 mL / h after one hour, and then increase it again to 20 mL / h after another hour. When the dissolved oxygen level drops below 30%, stop feeding and wait for the dissolved oxygen to rebound. Resume feeding when the dissolved oxygen level rises back to 30%. Induction culture for 40-60 hours. Once the protein concentration increase is not significant or decreases according to UV measurement, the culture can be transferred to the container.

[0208] The UV values ​​of the protein samples were measured by a photometer at wavelengths of 215 nm (A215) and 225 nm (A225), respectively. The protein concentration quantification formula was: C (mg / mL) = 0.144 * (A215 - A225), where A215 < 1.5.

[0209] The fermentation supernatant after being removed from the tank was collected and analyzed by SDS-PAGE electrophoresis. The measurement results are as follows: Figure 4 As shown in the figure, under high-density fermentation conditions, recombinant collagen XVII9K32 and XVII11K113 exhibited severe degradation after 48 hours of induction, a phenomenon not seen in small-batch shake flasks, indicating they are not suitable for large-scale production. The remaining four recombinant XVII type collagen proteins—XVII12K61, XVII22K111, XVII9K95, and XVII10K106—were purified to obtain high-purity recombinant collagen sponges.

[0210] (2) Purification:

[0211] Buffer A: 20mM sodium dihydrogen phosphate, pH 4.4;

[0212] The cation exchange medium used was UniGel-80sp produced by Suzhou Nanomicro, which was loaded into a Hanbang Bio-Lab100 chromatography instrument.

[0213] Buffer B: 20mM sodium dihydrogen phosphate + 1M sodium chloride, pH 4.4.

[0214] Fermentation broths containing recombinant type XVII collagen (XVII12K61, XVII22K111, XVII9K95, and XVII10K106) were collected separately. Cells and fermentation supernatants were separated by centrifugation at 2000×g, 30 min, and 4℃. The cation exchange medium was equilibrated with buffer A. The fermentation supernatant was diluted with pure water until the conductivity was below 8 mS / cm. After adjusting the pH to 4.4, chromatography was initiated. The chromatography process is as follows:

[0215] a) Column equilibration: Manual mode, flow rate set to 28 mL / min, inlet A4 (buffer A), column position valve positive flush, continuously monitor the Cond conductivity curve and pH curve on the spectrum interface until the conductivity curve drops to its lowest point and flattens out (about 3 column volumes). After the pH curve flattens out, it means that the column has been equilibrated. Click pause.

[0216] b) Sample loading: Manual mode, flow rate set to 28 mL / min, inlet A1 (diluted fermentation supernatant, pH 4.4, 5 L), click pause when the injection volume reaches the required volume.

[0217] c) Reequilibration: Manual mode, flow rate set to 28 mL / min, inlet A4 (buffer A), click continue, run the program to continuously monitor the chromatogram curves, and continue equilibration after the UV curve, conductivity curve and pH curve have stabilized, then click pause.

[0218] d) Elution: Manual mode, flow rate set to 28 mL / min, inlet B1 (buffer B), click Continue. When the A215 spectrum curve rises, click Outlet1, click Continue to start eluting and collecting the target component until UV215 drops to its lowest point, at which point collection ends. Collect the eluent, analyze and confirm the components, then dialyze (dialysis fluid is ultrapure water), followed by concentration, freeze-drying, and collection of the purified freeze-dried collagen sponge.

[0219] The purified lyophilized sponge was dissolved in ultrapure water and subjected to SDS-PAGE electrophoresis. The results are as follows: Figure 5 As shown in the figure, the purified recombinant collagen XVII12K61, XVII22K111, and XVII9K95 showed obvious degradation bands in gel electrophoresis, indicating poor stability. XVII10K106 showed a relatively pure single band in SDS-PAGE electrophoresis, with better expression levels and stability.

[0220] Example 6. Characterization of the properties of recombinant collagen

[0221] In this embodiment, high-performance liquid chromatography-mass spectrometry (LC-MSMS) was used to analyze the full amino acid sequence coverage of the XVII 10K 106 lyophilized sponge sample. The specific steps are as follows:

[0222] Weigh 10 μg of the purified lyophilized sponge into five 1.5 mL centrifuge tubes, add ultrapure water to dissolve, and then adjust the volume to 100 μL with the corresponding enzyme buffer. The final concentration is 0.1 mg / mL, and these are designated as samples 1 to 5.

[0223] The buffer solution for sample 1 was 50 mM NH4HCO3 with a pH of 8.0;

[0224] The buffer solution for sample 2 was ultrapure water;

[0225] The buffer for sample 3 was 50 mM Tris-HCl;

[0226] The buffer for sample 4 was 25 mM Tris-HCl and 1 mM EDTA (pH 8.5).

[0227] The buffer solution for sample 5 was 50 mM Tris-HCl (pH 8.0) and 10 mM CaCl2.

[0228] Add 1 μL of 1M dithiothreitol (DTT) solution to samples 1–5 to make the final DTT concentration 10 mM. Then, reduce the samples for 1 h in a 56℃ water bath. After the reaction, add 2 μL of 1M iodoacetamide (IAM) solution to each sample to make the final IAM concentration 20 mM. React the samples in the dark at room temperature for 40 min. After the reaction, add 1 μL of 1M dithiothreitol (DTT) solution to each sample to make the final DTT concentration 10 mM to neutralize the unreacted IAM, thus obtaining reaction solutions 1–5.

[0229] The corresponding enzymes were added to reaction solutions 1 through 5 respectively. Specifically, 2 μL of 0.25 μg / μL Trypsin was added to reaction solution 1, 2 μL of 0.5 μg / μL Glu-c was added to reaction solution 2, 2 μL of 0.1 μg / μL Elastase was added to reaction solution 3, 2 μL of 0.2 μg / μL Lys-C was added to reaction solution 4, and 1 μL of 0.1 μg / μL Protease K was added to reaction solution 5. The mixture was then incubated at 37°C for 16 h. After incubation, the peptides were desalted using a C18 desalting column, then vacuum-centrifuged at 45°C to dry, and reconstituted. Data were acquired using a Vanquish Neo-Oribitrap Fusion Lumos high-resolution liquid chromatography-mass spectrometry system, and analyzed using the Peptide Mapping function of BioPharma Finder software.

[0230] After combining and analyzing the coverage information of five enzymes, the coverage of XVII10K106 reached 100%, completely matching the theoretical sequence SEQ ID NO:25. Therefore, the recombinant XVII type collagen XVII10K106 described in this invention is indeed obtained by repeating the monomers of human XVII type collagen sequence p1438-p1461 10 times.

[0231] Example 7. Stability test of recombinant type XVII collagen

[0232] Based on Examples 4 and 5, it can be preliminarily concluded that recombinant collagen XVII10K106 has high stability. Since subsequent production and application processes involve short-term raw material storage, it is required to maintain its initial molecular weight and minimize or eliminate degradation. Therefore, this example tested the storage stability of recombinant XVII type collagen XVII10K106 at different ambient temperatures and the accelerated stability of recombinant collagen solutions at different pH values ​​under high-temperature conditions.

[0233] (1) Stability test of recombinant type XVII collagen under different temperature conditions:

[0234] Weigh an appropriate amount of recombinant XVII collagen XVII 10K106 lyophilized sponge and dissolve it in ultrapure water to prepare a 1 g / L recombinant collagen solution. Filter the recombinant collagen solution using a 0.45 μm filter membrane in a clean bench. After filtration, take the sample from day 0 and prepare an electrophoresis sample for use as a control sample in subsequent temperature stability experiments.

[0235] The sterile recombinant collagen solution was aliquoted into sterile centrifuge tubes (3 tubes in total) and placed at ambient temperatures of 40℃, 25℃, and 4℃ respectively to accelerate the thermal stability experiment and simulate normal room temperature and standard sample storage temperature, thus investigating the effect of storage temperature on the samples. Samples from each group were collected on days 1, 4, and 8 of the experiment and prepared for electrophoresis and SDS-PAGE analysis. The results are as follows: Figure 6 As shown.

[0236] from Figure 6 It can be seen that the recombinant XVII collagen XVII10K106 solution remained stable without degradation after 8 days at 4℃, exhibiting high stability. Degradation was accelerated at 25℃ and 40℃, but no significant degradation band was observed below the target band; only the protein content of the target band decreased. After 8 days, the retention rate of samples stored at 25℃ exceeded 60%, and samples stored at 40℃ also had a retention rate of nearly 50%. This indicates that the recombinant XVII collagen XVII10K106 described in this invention has high stability, remaining stable without degradation in refrigerated cabinets or cold storage rooms, maintaining high purity and stability. Although degradation occurs during storage at room temperature or higher temperatures, a high concentration of the target protein remains, making it suitable for large-scale industrial production.

[0237] (2) Stability test of recombinant type XVII collagen at different pH values ​​at room temperature:

[0238] Weigh an appropriate amount of recombinant XVII collagen XVII 10K106 lyophilized sponge and dissolve it in ultrapure water to prepare a 1 g / L recombinant collagen solution. Filter the recombinant collagen solution using a 0.45 μm filter membrane in a clean bench. After filtration, divide the solution into three equal portions and prepare recombinant XVII collagen solutions with pH values ​​of 5, 7, and 11, respectively. Take the sample from day 0 to prepare an electrophoresis sample, which will serve as a control sample for subsequent stability experiments of the recombinant collagen solutions at different pH values.

[0239] Recombinant collagen solutions with different pH values ​​were placed together at 40℃ for accelerated testing to investigate the stability of the samples. Samples from each group were collected on days 1, 4, and 8 of the experiment, prepared for electrophoresis, and analyzed by SDS-PAGE. The results are shown below. Figure 7 As shown.

[0240] The results show that XVII10K106 is relatively stable under both the initial slightly acidic pH and neutral conditions. After 8 days at 40℃, the sample at pH=5 showed only about 15% degradation, exhibiting high stability, while the sample at pH=7 also had a high retention rate of 60%. Alkaline conditions are detrimental to the preservation of recombinant XVII collagen XVII10K106, which is essentially degraded by day 4. Therefore, avoiding alkaline conditions can effectively improve the stability of recombinant proteins.

[0241] In summary, in a real production environment, the recombinant type VII collagen XVII10K106 described in this invention can remain highly stable for at least 8 days at 4°C, which fully meets the production requirements. Adjusting its pH value to neutral can further improve its stability.

[0242] Example 8. Cytotoxicity test of recombinant type XVII collagen

[0243] In this embodiment, MEM medium containing 10% (v / v) fetal bovine serum (FBS) was used as a blank control, Hatano Research's Negative RM-C was used as a negative control, and Sangon Biotech's DMSO was used as a positive control. The recombinant type VII collagen XVII10K106 lyophilized sponge prepared according to this invention was used as the experimental sample to investigate the cytotoxicity of recombinant type VII collagen. The specific steps are as follows:

[0244] Samples were extracted according to the conditions in Table 1. After extraction, changes in the extract were checked. No filtering, centrifugation, dilution, or pH adjustment was required to obtain blank, negative, and positive control samples, which were directly used in subsequent experiments. The extract of the XVII 10K106 lyophilized sponge obtained from Table 1 was used as the final concentration of 100% and serially diluted to 0.75, 2, and 4 times, respectively, to obtain extracts with final concentrations of 75%, 50%, and 25%.

[0245] Table 1. Extraction conditions and state of extract for different groups of samples

[0246]

[0247] L929 cells (Chinese Academy of Sciences Cell Bank, mouse fibroblast NCTC cLone 929 [L ceLL, L-929, derivative of Strain L]) were cultured in MEM medium (containing 10% FBS and 1% penicillin-streptomycin mixture, v / v) at 37°C and 5% CO2. Cells that reached the logarithmic growth phase were digested with 0.25% trypsin (containing EDTA, Gibco, catalog number 25200072). After digestion, the cell suspension was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in MEM medium. Cells were counted to obtain 1×10⁶ cells. 5 Cell suspension of cells / mL.

[0248] The obtained cell suspension was seeded into 100 μL per well in a 96-well plate, and then cultured in a cell culture incubator at 37°C, 5% CO2, and >90% humidity for 24 h. Cell morphology was observed under a microscope. When the cells adhered and grew to about 70%, the MEM medium in the 96-well plate was discarded, and 100 μL of extraction solution (final concentrations of 100%, 75%, 50%, and 25%) was added to the corresponding wells of the 96-well plate. Blank control, negative control, and positive control samples were then added. The 96-well plate was then placed in a cell culture incubator and cultured at 37°C, 5% CO2, and >90% humidity for 24 h. Six replicates were set up for each group.

[0249] After the culture was completed, the 96-well plate was removed and the cells were observed under a microscope. The supernatant was then discarded, and 50 μL of MTT (1 mg / mL) was added to each well. The plate was then incubated in a CO2 incubator for 2 h. The supernatant was then discarded, and 100 μL of isopropanol solution was added to each well. The plate was shaken and the absorbance was measured at 570 nm using a microplate reader (reference wavelength 650 nm).

[0250] The statistical method is to calculate the mean ± standard deviation of each group. The formula for calculating survival rate is: Survival Rate (%)

[0251] =100% * OD570 e / OD570 b In the formula: OD570 e The absorbance of each test group (sample group, negative control group, positive control group); OD570 bThis is the absorbance of the control group (blank). If the average OD570 of the blank control is ≥0.2, and the difference between the average values ​​of the left and right columns of the medium control and the average value of all medium controls is no greater than 15%, then the test meets the acceptance criteria. When the survival rate is low, the test sample has high potential cytotoxicity; if the survival rate drops to <70% of the blank, it has potential cytotoxicity; the survival rate of the 50% extract of the test sample should be at least the same as or higher than that of the 100% extract; otherwise, the test should be repeated; the survival rate of the 100% test sample extract is the final result.

[0252] The microscopic morphology of the cells is described in Table 2.

[0253] Table 2. Microscopic morphological characteristics of cells

[0254]

[0255]

[0256] The statistical results of cytotoxicity detection are shown in Table 3.

[0257] Table 3. Cytotoxicity test results

[0258]

[0259] Combined with Table 3 and Figure 8 It can be seen that the recombinant type XVII collagen XVII10K106 described in this invention has no potential cytotoxicity to L929 cells under 100% conditions of 10 mg / mL extract. This indicates that the recombinant type XVII collagen described in this invention has good biosafety and can be used as a raw material in the production process of pharmaceuticals, medical devices, biomaterials or cosmetics.

[0260] In summary, this invention selects the amino acid sequence of natural human type XVII collagen as the basic unit and repeatedly repeats it to obtain recombinant type XVII collagen. The design of this recombinant type XVII collagen avoids unstable GXY triplets, eliminates potential matrix metalloproteinase cleavage sites and glycosylation sites, and retains the KGD active sequence, thus reducing the probability of containing easily degradable sites. The sequence fragment of this recombinant type XVII collagen is derived from the full-length sequence of the natural collagen gene, exhibiting excellent solubility, bioactivity, and high stability. It remains stable under high-temperature storage conditions and without alkaline conditions, making it suitable as a raw material for the production of pharmaceuticals, medical devices, biomaterials, or cosmetics, demonstrating significant practicality.

[0261] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A highly stable recombinant type XVII collagen, characterized in that, The highly stable recombinant type XVII collagen is based on the tandem repetition of recombinant type XVII collagen monomers as basic units; The amino acid sequence of the highly stable recombinant type XVII collagen is shown in SEQ ID NO:

25.

2. A polynucleotide encoding the highly stable recombinant type XVII collagen of claim 1.

3. The polynucleotide according to claim 2, characterized in that, The sequence of the polynucleotide includes SEQ ID NO:26 or its degenerate sequence.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains a polynucleotide encoding the highly stable recombinant type XVII collagen as described in any one of claims 2 or 3.

5. A recombinant engineered bacterium, characterized in that, The recombinant engineered bacteria contain a polynucleotide encoding a highly stable recombinant type XVII collagen as described in claim 2 or 3, or the recombinant expression vector as described in claim 4.

6. The recombinant engineered bacteria according to claim 5, characterized in that, The host bacteria of the recombinant engineered bacteria include Pichia pastoris.

7. The recombinant engineered bacteria according to claim 6, characterized in that, The host bacteria are Pichia pastoris strain GS115, Pichia pastoris strain X33, Pichia pastoris strain KM71H, and Pichia pastoris strain SMD116.

8. The recombinant engineered bacteria according to claim 5, characterized in that, The recombinant engineered bacteria are deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 32750.

9. The method for preparing the highly stable recombinant type XVII collagen according to claim 1, characterized in that, The preparation method includes: (1) Select and design the sequence of recombinant type XVII collagen monomers, and then construct recombinant type XVII collagen by tandem recombination based on recombinant type XVII collagen monomers; (2) Construct a recombinant expression vector for recombinant type XVII collagen; (3) The recombinant expression vector expressing recombinant type XVII collagen was electroporated into the host bacteria, screened and verified to obtain recombinant engineered bacteria; (4) The recombinant engineered bacteria were fermented and induced to express the high-stability recombinant type XVII collagen.

10. The method according to claim 9, characterized in that, In step (1), the amino acid sequence of the recombinant type XVII collagen monomer is shown in SEQ ID NO:

24.

11. The method according to claim 9, characterized in that, In step (1), the amino acid sequence of the highly stable recombinant type XVII collagen is shown in SEQ ID NO:

25.

12. The method according to claim 9, characterized in that, In step (3), the host bacteria include Pichia pastoris.

13. The method according to claim 12, characterized in that, The host bacteria are Pichia pastoris strain GS115, Pichia pastoris strain X33, Pichia pastoris strain KM71H, and Pichia pastoris strain SMD116.

14. The method according to claim 10, characterized in that, In step (3), the recombinant engineered bacteria are deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 32750.

15. A composition, characterized in that, The composition comprises the highly stable recombinant type XVII collagen of claim 1.

16. An article, characterized in that, The product comprises the highly stable recombinant type XVII collagen of claim 1, or the composition of claim 15; the product is selected from medical devices, biomaterials, tissue engineering products, and cosmetics.

17. The use of the highly stable recombinant type XVII collagen of claim 1, or the composition of claim 15, or the article of claim 16 in the preparation of medical devices, biomaterials, tissue-engineered products, and cosmetics.

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