High-stability recombinant XVII type collagen as well as construction method and application thereof

Abstract

The invention provides a high-stability recombinant XVII type collagen as well as a construction method and application thereof, and belongs to the technical field of bioengineering. According to the invention, an amino acid sequence of natural human (XVII) type collagen is selected as a basic unit and repeated for multiple times to obtain recombinant (XVII) type collagen; in the design process of the recombinant XVII type collagen, an unstable GXY triad is avoided, potential matrix metalloproteinase restriction enzyme cutting sites and glycosylation sites are abandoned, a KGD active sequence is reserved, and the probability of containing sites which are easy to degrade can be reduced; a sequence fragment of the recombinant XVII type collagen is derived from a full-length sequence of a natural collagen gene, has excellent solubility, biological activity and high stability, and can be kept stable in a high-temperature storage environment and under the condition of avoiding alkalinity; the product can be used as a raw material in the production process of medicines, medical equipment, biological materials or cosmetics, and has good practicability.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to highly stable recombinant type XVII collagen and a construction method and application thereof. Background Art

[0002] Type ⅩⅦ collagen is abbreviated as COL17. It is the main component of hemidesmosomes and is mainly found in the skin, mucous membranes and eyes. COL17 is a key regulator of skin aging. Intrinsic or extrinsic aging can lead to genomic instability, which in turn leads to decreased expression and increased hydrolysis of COL17, which ultimately leads to thinning and brittle epidermis, as well as graying and shedding of hair. Moreover, COL17 is a key regulator in wound repair. Upregulation of COL17 can regulate the dynamics and migration of stem cell populations. In addition, 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 forced maintenance of COL17 expression can inhibit the aging of hair follicle stem cells and rescue epidermal thinning and hair loss. As a good stem cell marker that can reflect the potential and self-renewal quality of individual cells, COL17 is a potential therapeutic target for skin anti-aging and wound repair.

[0003] At present, the preparation methods of collagen are mainly animal extraction and biological expression and separation. The collagen obtained by animal extraction has batch differences due to individual differences in animals, and COL17 is less distributed in animals. It has the disadvantages of greater difficulty in extraction, lower production capacity, and high price. It is not suitable for industrial large-scale production, which brings resistance to the sustainable production of products. In addition, some zoonotic viruses may also exist in animals, which brings great risks to the application of animal-derived collagen. The biological expression and separation of recombinant proteins is to introduce the gene sequence of natural collagen or the re-optimized gene sequence into the selected host cells. After cultivation, fermentation, separation and purification, recombinant collagen with the characteristics and functions of natural proteins can be obtained. The molecules of recombinant collagen are relatively simple and the structure is relatively clear, so it is easy to obtain target proteins of specific types, sizes and functions. According to different host cells, the expression system is mainly divided into Escherichia coli expression system (prokaryotic), yeast expression system (eukaryotic), insect expression system, plant expression system, mammalian expression system, etc. The yeast protein expression system is represented by Pichia methanolica, which has the advantages of high protein expression level, low cost, easy induction, easy purification of secreted proteins, and easy high-density fermentation. However, its post-translational processing and modification system is not exactly the same as that 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 will also degrade during the fermentation and purification process, which not only increases the production and storage costs, but also affects the performance and final yield of the recombinant type XVII collagen produced by this method. In addition, as a raw material in the field of medical devices, in order to ensure the quality and safety of the products and the safety and effectiveness of the use of medical device products, the recombinant type XVII collagen should have higher purity and stability to reduce safety accidents and adverse reactions caused by raw material problems in medical device products. Therefore, it is necessary to develop a high-purity and high-stability recombinant type XVII collagen. Summary of the invention

[0005] In view of some deficiencies in the prior art, the present invention provides a highly stable recombinant type XVII collagen and a construction method and application thereof; the present invention selects the amino acid sequence of natural human type XVII collagen as a basic unit and repeats it multiple times to obtain recombinant type XVII collagen; in the design process of the recombinant type XVII collagen, unstable GXY triplet is avoided, potential matrix metalloproteinase cleavage sites and glycosylation sites are abandoned, and KGD active sequence is retained, which can reduce the probability of containing sites that are prone to degradation; the sequence fragment of the recombinant type XVII collagen is derived from the full-length sequence of the natural collagen gene, has excellent solubility, biological activity and high stability, can remain stable in a high-temperature storage environment and under conditions avoiding alkalinity, can be used as a raw material in the production process of drugs, medical devices, biomaterials or cosmetics, and has good practicality.

[0006] In order to achieve the above technical objectives, the present invention adopts 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 includes a plurality of sequence fragments that avoid unstable GXY triplet, abandon potential matrix metalloproteinase cleavage sites and glycosylation sites, and retain KGD activity;

[0008] The sequence fragments include one or more of 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 in human type XVII collagen.

[0009] Preferably, the recombinant type XVII collagen monomer comprises any one of the following sequence fragment combinations:

[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] Among them, one or more monomer fragments in each of the above groups are tightly connected.

[0023] Preferably, the recombinant type XVII collagen monomer is combination 1), and its 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 tandemly repeated with the above recombinant type XVII collagen monomers as basic units, and the recombinant type XVII collagen monomers are the same or different.

[0025] Preferably, the highly stable recombinant type XVII collagen has a size of 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 in which the amino acid sequence in i is substituted, deleted or added with one or more amino acids and has human collagen activity, 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 99% or more identity with the sequence shown in i.

[0029] Preferably, the amino acid sequence of the highly stable recombinant type XVII collagen is as 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 comprises SEQ ID NO: 26 or a degenerate sequence thereof.

[0032] The present invention also provides a recombinant expression vector, which contains nucleotides encoding the above-mentioned highly stable recombinant type XVII collagen.

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

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

[0035] Preferably, the recombinant engineered bacteria is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with the deposit number of CGMCC No.32750.

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

[0037] (1) selecting and designing the sequence of a recombinant type XVII collagen monomer, and then constructing a recombinant type XVII collagen by serially recombining the recombinant type XVII collagen monomer as a basic unit;

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

[0039] (3) electroporating the obtained recombinant expression vector expressing recombinant type XVII collagen into a host bacterium, screening and verifying it, and obtaining a recombinant engineered bacterium;

[0040] (4) Fermentation-induced expression of the recombinant engineered bacteria is performed to obtain the highly stable recombinant type XVII collagen.

[0041] Preferably, in step (1), the recombinant type XVII collagen monomer includes a plurality of sequence fragments that avoid unstable GXY triplet, abandon potential matrix metalloproteinase cleavage sites and glycosylation sites, and retain KGD activity;

[0042] The sequence fragments include one or more of 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.

[0043] Preferably, the recombinant type XVII collagen monomer comprises any one of the following sequence fragment combinations:

[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] Among them, one or more monomer fragments in each of the above groups are tightly connected.

[0057] Preferably, the recombinant type XVII collagen monomer is combination 1), and its 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 highly stable recombinant type XVII collagen has a size of 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 in which the amino acid sequence in i is substituted, deleted or added with one or more amino acids and has human collagen activity, 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 99% or more identity with the sequence shown in i.

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

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

[0065] Preferably, in step (3), the recombinant engineered bacteria is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with the deposit number of CGMCC No.32750.

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

[0067] The present invention also provides a product, which comprises 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 use of the highly stable recombinant type XVII collagen monomer, the highly stable recombinant type XVII collagen, the composition or the product in the preparation of medical equipment, biomaterials, tissue engineering products and cosmetics.

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] The present invention optimizes several short amino acid sequences of natural human type XVII collagen based on the sequence of natural human type XVII collagen, avoids unstable GXY triplet, abandons potential matrix metalloproteinase cleavage sites and glycosylation sites, retains a KGD active sequence, and uses these amino acid fragments as repeating units to form recombinant type XVII collagen monomers in series, and then recombines the recombinant type XVII collagen monomers in series to obtain the highly stable recombinant type XVII collagen. The amino acid sequence of the highly stable recombinant type XVII collagen of the present invention is 100% homologous to the sequence of the corresponding part of natural type XVII collagen, and has the potential to be applied to the fields of medical devices, cosmetic raw materials, and biomaterials.

[0071] The recombinant type XVII collagen of the present invention has high stability. After performance evaluation, it is found that it has high stability under conventional storage conditions, and it can remain stable in a high-temperature storage environment and avoid alkaline conditions, without degradation, and maintains the original molecular weight and structure. The recombinant type XVII collagen of the present invention can maintain the stability and consistency of the product when used as a raw material in the production process of drugs, medical devices, biomaterials or cosmetics, and has good practicality.

[0072] The recombinant type XVII collagen of the present invention can be efficiently secreted and expressed in a Pichia pastoris expression system and is easy to purify. The protein expression amount reaches 6.51 g / L during high-density fermentation, and the conditions for industrial mass production are met. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1SDS-PAGE diagram of the first batch of designed recombinant human type XVII protein expression.

[0074] Figure 2 SDS-PAGE diagram of the expression of recombinant human type XVII protein designed for the second batch.

[0075] Figure 3 SDS-PAGE diagram of the expression of recombinant human type XVII protein designed for the third batch.

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

[0077] Figure 5 This is the SDS-PAGE picture of the purified highly stable recombinant type XVII collagen.

[0078] Figure 6 SDS-PAGE image (a) and degradation rate analysis results (b) of the stability test of XVII10K106 at different temperatures.

[0079] Figure 7 The SDS-PAGE image (a) and degradation rate analysis result image (b) of stability test of ⅩⅦ10K106 solution samples with different pH values.

[0080] Figure 8 This is a statistical graph of the cytotoxicity test results of recombinant type ⅩⅦ collagen. DETAILED DESCRIPTION

[0081] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto. The technical scheme of the embodiment of the present invention is clearly and completely described below. Obviously, the described embodiment is a part of the embodiment of the present invention, not all of the embodiments. Based on the described embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

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

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

[0084] This example is based on the human type XVII collagen sequence, with the aim of avoiding unstable GXY triplet, discarding potential matrix metalloproteinase cleavage sites and glycosylation sites, retaining KGD active sequence, etc., and selecting one or several amino acid sequence fragments of natural human type XVII collagen, which are used for the subsequent design and screening of recombinant type XVII collagen monomer and recombinant type XVII collagen.

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

[0086] SEQ ID NO: 1:

[0087] GPPGQKGEMGTPGPKGDRGPAGPP GHPGPPGPRGHKGEKGDKGDQVYAGRRRRRSIAVKP.

[0088] The sequence fragments that avoid the unstable GXY triplet, abandon the potential matrix metalloproteinase cleavage sites and glycosylation sites, and retain the 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 as follows:

[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 monomer and recombinant type ⅩⅦ collagen:

[0105] In this example, one or more of the sequence fragments obtained in Example 1 are recombined in series to form the first batch of recombinant type XVII collagen monomers, and then the recombinant type XVII collagen monomers are used as basic units for series recombining to obtain the first batch of recombinant type XVII collagen, the size of which is 20-25 kDa. This example also examines the distribution of the selected sequence fragments and the stability of their expression, which are used as the design basis for the next batch of recombinant type XVII collagen, until a type XVII collagen sequence with high stability is screened out.

[0106] The specific steps are as follows:

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

[0108] Three sequences of human type XVII collagen sequence p597-p611, p674-p715, and p906-p929 were selected and concatenated 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 repeatedly concatenated three times to obtain a recombinant type XVII collagen, denoted as XVII9K32, whose amino acid sequence is 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 sequence p692-p715, p906-p938, p951-p977, and p1438-p1467 were selected and concatenated to obtain a recombinant type XVII collagen monomer with an amino acid sequence as shown in SEQ ID NO:4. The recombinant type XVII collagen monomer was repeatedly concatenated twice to obtain a recombinant type XVII collagen, denoted as XVII12K23, whose amino acid sequence is 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 sequence p597-p619, p641-p670, p674-p715, and p906-p929 were selected and concatenated to obtain a recombinant type XVII collagen monomer with an amino acid sequence as shown in SEQ ID NO:6. The recombinant type XVII collagen monomer was repeatedly concatenated twice to obtain a recombinant type XVII collagen, denoted as XVII6K25, whose amino acid sequence is 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. Synthesis of DNA sequences and construction of recombinant expression vectors:

[0124] Nanjing GenScript Biotech Co., Ltd. was commissioned to synthesize DNA fragments encoding XVII9K32, XVII12K23, and XVII6K25, and the synthesized gene fragments were cloned into the pPIC9K empty vector so that the target fragments were accurately inserted into the secretory vector reading frame containing the secretion signal α factor to obtain recombinant expression vectors expressing XVII9K32, XVII12K23, and XVII6K25.

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

[0126] 10 μg of the recombinant expression vectors expressing XVII9K32, XVII12K23, and XVII6K25 obtained in step S2 were respectively digested with Sal I fast cutting enzyme (Dalian TaKaRa Company) at 37°C for 30 min for linearization, and then the linearized plasmids were recovered using a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.), and the recovery volume was about 20 μL.

[0127] Take 10 μL of linearized plasmid respectively, and electrotransform it into the competent cells of host strain Pichia pastoris GS115 (purchased from Thermo Fisher Scientific). After the electroporation, add 800 μL of pre-cooled YPD medium + 1M sorbitol 1:1 mixture immediately, mix the bacteria and transfer them to a 1.5mL EP tube, incubate at 30°C for 2h, after the incubation, centrifuge the bacterial solution at 3000×g for 5min, discard 600 μL of supernatant, mix well, take 50-80 μL of the bacterial solution and spread it on the MD plate, and culture it upside down at 30°C for 2-5 days until a single colony (positive transformant) appears.

[0128] Add 2 mL of sterile ultrapure water to the surface of the MD plate, cover and incubate for a few seconds, then use a sterile 1 mL pipette tip to gently blow and mix the His+ transformants on the surface of the plate, and transfer the bacterial suspension to a 1.5 mL centrifuge tube. Dilute the bacterial suspension with sterile double distilled water and measure the OD 600 After the value, take 10 5 The cells were spread on a YPD plate containing 0.5 mg / mL G418 and inverted cultured at 30°C for 2-5 days until single colonies appeared, thereby obtaining 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 mix it in the medium, and culture it overnight at 180 rpm at 30°C. The next day, mix the bacterial solution in the well and take 80 μL of the bacterial solution for Pichia colony PCR. Take 5 μL of the PCR reaction product to run a DNA gel, and select a single colony with a clear band, size and position for shake flask verification.

[0130] The Pichia pastoris strains expressing XVII9K32, XVII12K23, and XVII6K25 with clear bands and reasonable size and position were inoculated into 100 mL conical flasks containing 15 mL of 15% BMGY medium, and cultured at 220 rpm for 24 h at 30 ° C. After calculating the OD value, the final OD 600 The bacterial solution with a final OD value of 150 was centrifuged at 3000×g for 5 min, and the supernatant was discarded. The bacteria were resuspended in 15 mL of BMM medium to a final OD value of 150. 600 The value is 10, and the culture medium is placed in a 30°C incubator with shaking at 220 rpm. After culturing for 24 hours, 200 μL of 100% methanol is added to the culture medium to a final methanol concentration of about 1%, and then induced for 24 hours. After the culture is completed, a bacterial sample is taken, centrifuged at 12000×g for 5 minutes at 4°C, and the supernatant is collected. The supernatant is prepared into a 1× SDS-PAGE electrophoresis sample with 5× loading buffer, and the sample is prepared after 100°C metal bath for 5 minutes, and SDS-PAGE detection is performed.

[0131] Test results such as Figure 1 As shown in the figure, it can be seen that the expression level of ⅩⅦ12K23 is extremely low, and degradation bands are faintly visible; ⅩⅦ6K25 has protein expression, but the main band accounts for a low proportion, there is serious degradation, and it is unstable; ⅩⅦ9K32 has the highest expression level, but degradation bands can also be seen. Since the sequences of ⅩⅦ9K32 and ⅩⅦ6K25 have overlapping regions, which are p597-p611 and p674-p715 fragments, these two fragments are relatively stable fragments, and the possibility of the expressed peptide fragments breaking and degrading is low;

[0132] Compared with XVII9K32, XVII6K25 has additional segments p612-p619 and p641-p670, which greatly reduce its stability and show obvious degradation bands. This segment should be avoided in the next batch of sequence design.

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

[0134] In this example, based on the distribution of sequence fragments selected in Example 2 and the stability of their expression, one or more sequence fragments obtained in Example 1 are recombined in series to form a second batch of recombinant type XVII collagen monomers, and then the recombinant type XVII collagen monomers are used as basic units for series recombining to obtain a second batch of recombinant type XVII collagen, and further screening of highly stable type XVII collagen is performed.

[0135] The specific steps are as follows:

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

[0137] When designing the recombinant type XVII collagen monomer, the fragments that can increase the degradation rate during protein expression determined in Example 2 are avoided, and in the sequence with low expression level, shorter fragments are intercepted for construction and expression screening. The specific steps are as follows:

[0138] The human type XVII collagen sequence p674-p715 was selected to obtain a recombinant type XVII collagen monomer with an amino acid sequence as shown in SEQ ID NO:8. The recombinant type XVII collagen monomer was repeatedly connected in series 6 times to obtain a recombinant type XVII collagen, which was recorded as XVII12K61 and has an amino acid sequence as shown in SEQ ID NO:9.

[0139] SEQ ID NO:8:

[0140] GPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPPGPKGDQGEK;

[0141] SEQ ID NO:9:

[0142] GPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEK GPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEK.

[0143] Two sequences of human type XVII collagen sequence p674-p715 and p906-p929 were selected and concatenated to obtain a recombinant type XVII collagen monomer with an amino acid sequence as shown in SEQ ID NO:10. The recombinant type XVII collagen monomer was repeatedly concatenated 4 times to obtain recombinant type XVII collagen, denoted as XVII12K42, whose amino acid sequence is shown in SEQ ID NO:11.

[0144] SEQ ID NO:10:

[0145] GPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQ GPPGPRGHQ;

[0146] SEQ ID NO:11:

[0147] GPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQ GPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQ.

[0148] Four sequences of human type XVII collagen sequence p597-p611, p674-p715, p906-p929, and p951-p977 were selected and concatenated to obtain a recombinant type XVII collagen monomer with an amino acid sequence as shown in SEQ ID NO:12. The recombinant type XVII collagen monomer was repeatedly concatenated twice to obtain a recombinant type XVII collagen, recorded as XVII10K25, whose amino acid sequence is shown in SEQ ID NO:13.

[0149] SEQ ID NO:12:

[0150] GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGE KGPPGPPGPPGPKGDQGPPGPRGHQGPPGPPGPQGPKGDKGDPGVPGALGIP;

[0151] SEQ ID NO:13:

[0152] GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGPPGPPGPQGPKGDKGDPGVPGALGIP GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGPPGPPGPQGPKGDKGDPGVPGALGIP.

[0153] Four sequences of human type XVII collagen sequence p597-p611, p674-p715, p906-p929, and p1438-p1464 were selected and concatenated to obtain a recombinant type XVII collagen monomer with an amino acid sequence as shown in SEQ ID NO: 14. The recombinant type XVII collagen monomer was repeatedly concatenated twice to obtain a recombinant type XVII collagen, denoted as XVII8K26, whose amino acid sequence is shown in SEQ ID NO: 15.

[0154] SEQ ID NO:14:

[0155] GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGE KGPPGPPGPPGPKGDQGPPGPRGHQGPPGQKGEMGTPPGKGDRGPAGPPGHP;

[0156] SEQ ID NO:15:

[0157] GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGPPGQKGEMGTPPGKGDRGPAGPPGHP GHPGPQGPKGQKGSVGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGPPGPPGPKGDQGPPGPRGHQGPPGQKGEMGTPPGKGDRGPAGPPGHP.

[0158] The steps of S2 and S3 in Example 2 were used to construct the recombinant expression vector, construct the recombinant engineering strain, express the recombinant protein, and screen the recombinant protein. The SDS-PAGE test was performed. The test results were as follows: Figure 2 shown.

[0159] from Figure 2It can be seen that the expression levels of XVII12K61 and XVII8K26 are higher, while those of XVII12K42 and XVII10K25 are lower, and XVII10K25 has a clearly visible degradation band. The sequence of XVII12K42 has only one more segment p906-p929 than that of XVII12K61, and the expression level is greatly reduced. This may be because this segment will affect the expression efficiency of the recombinant protein, and this segment should be avoided in subsequent sequence design. In addition, VII10K25 and XVII8K26 sequences share p597-p611, p674-p715, and p906-p929 segments, but the difference is that the XVII10K25 unit ends with p951-p977, while the XVII8K26 sequence unit ends with p1438-p1464. The expression level of XVII8K26 is much higher than that of XVII10K25. Therefore, the p1438-p1464 segment is considered to be the better sequence.

[0160] Example 4. Design and screening of recombinant type ⅩⅦ collagen monomer and recombinant type ⅩⅦ collagen:

[0161] In this example, based on the screening in Examples 2 and 3, it was found that the p597-p611, p674-p715, and p1438-p1464 segments had higher expression levels and better stability. Because some sequences still had slightly degraded bands, a third batch of units with shorter fragment lengths was designed in order to screen for amino acid sequences of recombinant type XVII collagen with high expression levels and high stability. The specific steps are as follows:

[0162] S1. Design of recombinant type ⅩⅦ collagen monomer and recombinant type ⅩⅦ collagen:

[0163] When designing the recombinant type XVII collagen monomer, the fragments that can increase the degradation rate during protein expression determined in Example 2 are avoided, and in the sequence with low expression level, shorter fragments are intercepted for construction and expression screening. The specific steps are as follows:

[0164] The human type XVII collagen sequence p695-p715 was selected to obtain a recombinant type XVII collagen monomer with an amino acid sequence as shown in SEQ ID NO:16. The recombinant type XVII collagen monomer was repeatedly connected in series 11 times to obtain a recombinant type XVII collagen, which was recorded as XVII22K111 and has an amino acid sequence as shown in SEQ ID NO:17.

[0165] SEQ ID NO:16:

[0166] GVKGDKGPMGPPGPKGDQGEK;

[0167] SEQ ID NO:17:

[0168] GVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGP PGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEKGVKGDKGPMGPPGPKGDQGEK.

[0169] Select two segments of human type XVII collagen sequence p597-p611 and p695-p715, connect them in series to obtain a recombinant type XVII collagen monomer with an amino acid sequence as shown in SEQ ID NO: 18, and repeat the recombinant type XVII collagen monomer in series 6 times to obtain a recombinant type XVII collagen, denoted as XVII12K62, whose amino acid sequence is shown in SEQ ID NO: 19. SEQ ID NO: 18:

[0170] GHPGPQGPKGQKGSVGVKGDKGPMGPPGPKGDQGEK;

[0171] SEQ ID NO: 19:

[0172] GHPGPQGPKGQKGSVGVKGDKGPMGPPGPKGDQGEKGHPGPQGPKGQKGSVGVKGDKGPMGPPGPKGDQGEKGHPGPQGPKGQKGSVGVK GDKGPMGPPGPKGDQGEKGHPGPQGPKGQKGSVGVKGDKGPMGPPGPKGDQGEKGHPGPQGPKGQKGSVGVKGDKGPMGPPGPKGDQGEK GHPGPQGPKGQKGSVGVKGDKGPMGPPGPKGDQGEK.

[0173] The human type XVII collagen sequence p686-p706 was selected to obtain a recombinant type XVII collagen monomer with an amino acid sequence as shown in SEQ ID NO:20. The recombinant type XVII collagen monomer was repeatedly connected in series 11 times to obtain a recombinant type XVII collagen, which was recorded as XVII11K113 and has an amino acid sequence as shown in SEQ ID NO:21.

[0174] SEQ ID NO:20:

[0175] GLRGEVGLPGVKGDKGPMGPP;

[0176] SEQ ID NO:21:

[0177] GLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGV KGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPPGLRGEVGLPGVKGDKGPMGPP.

[0178] The human type XVII collagen sequence p674-p700 was selected to obtain a recombinant type XVII collagen monomer with an amino acid sequence as shown in SEQ ID NO:22. The recombinant type XVII collagen monomer was repeated in series 9 times to obtain a recombinant type XVII collagen, which was recorded as XVII9K95 and has an amino acid sequence as shown in SEQ ID NO:23.

[0179] SEQ ID NO:22:

[0180] GPQGPPGPVGLQGLRGEVGLPGVKGDK;

[0181] SEQ ID NO:23:

[0182] GPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGL RGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDKGPQGPPGPVGLQGLRGEVGLPGVKGDK.

[0183] The human type XVII collagen sequence p1438-p1461 was selected to obtain a recombinant type XVII collagen monomer with an amino acid sequence as shown in SEQ ID NO:24. The recombinant type XVII collagen monomer was repeatedly connected in series 10 times to obtain a recombinant type XVII collagen, which was recorded as XVII10K106 and has an amino acid sequence as shown in SEQ ID NO:25.

[0184] SEQ ID NO:24:

[0185] GPPGQKGEMGTPGPKGDRGPAGPP;

[0186] SEQ ID NO:25:

[0187] GPPGQKGEMGTPPGPKGDRGPAGPPGPPGQKGEMGTPPGKGDRGPAGPPGPPGQKGEMGTPPGKGDRGPAGPPGPPGQKGEMGTPPGKGDRGPAGPPGPPGQKGEMGTPPGKGDRGPAGPP GPPGQKGEMGTPPGPKGDRGPAGPPGPPGQKGEMGTPPGKGDRGPAGPPGPPGQKGEMGTPPGKGDRGPAGPPGPPGQKGEMGTPPGPKGDRGPAGPPGPPGQKGEMGTPPGPKGDRGPAGPP.

[0188] The steps of S2 and S3 in Example 2 were used to construct the recombinant expression vector, construct the recombinant engineering strain, express the recombinant protein, and screen the recombinant protein. The SDS-PAGE test was performed. The test results were as follows: Figure 3 shown.

[0189] from Figure 3 It can be seen that the ⅩⅦ12K62 sequence has an additional 15-amino acid p597-p611 fragment compared to the ⅩⅦ22K111 sequence, and its expression level is much lower than that of ⅩⅦ22K111. In addition, there are more short peptide sequences that are not the target bands in its expression product. It is suspected that there is a break site in the p597-p611 segment. After repeated multiple times, the unit sequence breaks at the same place, resulting in multiple uniformly distributed bands below the target band. In comparison, the possibility of breaking in the p695-p715 segment is much smaller. Similarly, the ⅩⅦ11K113 sequence located in the p674-p715 segment was selected to select the p686-p706 segment of human type ⅩⅦ collagen. The expression level is also very high, but the generation of degradation bands can also be observed. From the perspective of stability, it is not preferred. ⅩⅦ9K95 selected the p674-p700 segment of the human type ⅩⅦ collagen sequence. From the gel image, its expression level is not particularly high, and almost no degradation bands are visible. Similarly, ⅩⅦ10K106, located in the p1438-p1461 segment of the first helical region of human type ⅩⅦ collagen, was selected. The expression level is relatively low, but it has considerable expression stability.

[0190] 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.

[0191] SEQ ID No:26:

[0192] ggaccaccgggtcaaaagggggaaatgggcacccccggccctaagggcgaccggggcccggctggacccccaggtccgcctgggcaaaagggcgagatgggaactccaggtcccaaaggggacagagggccggcagggccccccgggccgcctgggcagaaaggggagatgggcactccggggccgaagggtgaccgagggcctgctggaccccccggtcccccaggtcaaaagggtgaaatgggtactcccggcccgaaaggcgatcgtggcccagcgggaccgccgggaccgcccggtcaaaaaggtgaaatgggtacccctggacccaaaggcgaccgcggacctgcgggaccgccaggaccccccggccagaagggagagatggggacacctggtccgaagggtgatagagggccagccgggccccctggtcctcctggacagaaaggagagatggggacacctggcccaaaaggcgacaggggaccggctggtcccccaggcccacctggccagaagggtgaaatgggtacgcctgggcctaagggagatcgagggccggcaggcccgccagggccccctggccaaaaaggggaaatgggtacccccggcccaaaaggagatcgtggcccagcgggacctcccggcccaccgggtcagaagggagagatggggacgcctggtccaaaaggagatcgcggaccagccgggccaccgtgataa.

[0193] The recombinant engineered bacteria expressing XVII12K62 were sent to the General Microbiological Center of China Microbiological Culture Collection Administration, and the culture collection number is: CGMCC NO.32750. Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; Deposit date: November 22, 2024, classification name: Pichia pastoris Komagataella phaffii.

[0194] Example 5. Small batch high density fermentation test

[0195] In this example, some of the recombinant type XVII collagen in Examples 2 to 4 were selected for small batch high-density fermentation experiments to evaluate the expression level and stability of the recombinant type XVII collagen.

[0196] (1) High-density fermentation of genetically engineered bacteria:

[0197] Expression of recombinant type ⅩVII collagen ⅩVII9K32, ⅩVII12K61, ⅩVII22K111, ⅩVII11K113,

[0198] The genetically engineered bacteria of XVII9K95 and XVII10K106 are used for large-scale expression production to obtain a fermentation liquid containing recombinant collagen. The specific steps are as follows:

[0199] S1. Preparation of culture medium:

[0200] Seed culture medium YPG: yeast powder 10g / L, peptone 20g / L, glycerol 10g / L;

[0201] Fermentation medium: NH 4 H 2 PO 4 190.4g / L, KH 2 PO 4 10.06g / L, CaSO 4 ·2H 2 O 1.18g / L, K 2 SO 4 18.2g / L, MgSO 4 7H 2 O 14.9g / L, glycerol 40g / L;

[0202] Feed medium: 50% glycerol (W / V) and PTM1, with 12 mL of PTM1 trace elements added per liter of 50% glycerol;

[0203] Induction medium: 100% methanol and PTM1, with 12 mL PTM1 trace elements added per liter of methanol;

[0204] PTM1: CuSO 4 ·5H2 O 6.0g / L, NaI 0.08g / L, MnSO 4 ·H2O 3.0g / L, NaMoO 4 ·2H 2 O0.2g / L, H 3 BO 3 0.02g / L, CoCl 2 0.5g / L, ZnCl 2 20.0g / L, FeSO 4 7H 2 O 65.0g / L, biotin 0.2g / L, H 2 SO 4 5.0mL / L, sterilize by filtration with a 0.22μm filter membrane, and store at 4℃.

[0205] S2. Batch culture conditions and induced expression of engineering strains:

[0206] The genetically engineered bacteria expressing recombinant type ⅩVII collagen ⅩVII9K32, ⅩVII12K61, ⅩVII22K111, ⅩVII11K113, ⅩVII9K95, and ⅩVII10K106 were inoculated into 1 L shake flasks containing 200 mL of seed medium YPG and cultured at 220 rpm and 30 °C for 18 to 24 h until the OD 600 =2~10, and obtain seed solution.

[0207] Add 2L of fermentation medium to a 5L fermenter (Baoxing Bio). Before inoculation, adjust the speed in the tank to 300rpm, the ventilation volume to 4L / min, and the temperature to 30°C. Then adjust the pH value in the tank to 4.5 with alkali solution prepared with concentrated ammonia water. Then, add 0.9mL of PTM1 to the fermenter, and then add 200mL of the prepared seed solution to the tank (flame circle inoculation). Then click the dissolved oxygen electrode to calibrate, and start fermentation after calibration.

[0208] When the dissolved oxygen drops to 30% for the first time during growth, use the dissolved oxygen cascade speed function to maintain 30% and wait for the glycerol to be consumed, the dissolved oxygen rebounds and is greater than 70% (OD 600When the value is about 20), cancel the dissolved oxygen cascade speed, increase the stirring to 650rpm, use 30% dissolved oxygen linkage feeding, and add 80mL of feeding medium. After the feeding is completed, wait for the dissolved oxygen to rebound to more than 70%, set the pH to 5.0 and the temperature to 30℃, and flow-feed induction medium for induction culture. Manually add 5mL of induction medium, and after the dissolved oxygen rebounds to more than 70%, set the feeding rate to 8mL / h, increase it to 10mL / h after one hour, and increase it again to 20mL / h after one hour. When the dissolved oxygen value is lower than 30%, stop feeding, wait for the dissolved oxygen to rebound, and link feeding after the dissolved oxygen rises to 30%. After 40-60h of induction culture, the tank can be released if the protein concentration measured by UV does not increase significantly or decreases.

[0209] The UV values ​​of the protein samples were measured by photometer at a wavelength of 215 nanometers (A215) and 225 nanometers (A225), and the quantitative formula for protein concentration was: C (mg / mL) = 0.144*(A215-A225), A215 <1.5.

[0210] The fermentation supernatant after tanking was collected and tested by SDS-PAGE electrophoresis. The measurement results were as follows: Figure 4 As shown in the figure, under high-density fermentation conditions, recombinant collagens XVII9K32 and XVII11K113 showed serious degradation after 48 hours of induction, which did not occur in small-batch shake flasks, and it was considered that they did not meet the conditions for large-scale production. The remaining four recombinant XVII collagens XVII12K61, XVII22K111, XVII9K95, and XVII10K106 were purified to obtain high-purity recombinant collagen sponges.

[0211] (2) Purification:

[0212] Buffer A: 20 mM sodium dihydrogen phosphate, pH 4.4;

[0213] The chromatographic filler of the cation exchange medium is UniGel-80sp produced by Suzhou Nano-Micro, which is loaded on the Hanbang Bio-Lab100 chromatograph;

[0214] Buffer B: 20 ​​mM sodium dihydrogen phosphate + 1 M sodium chloride, pH 4.4.

[0215] The fermentation broth containing recombinant type XVII collagen XVII12K61, XVII22K111, XVII9K95, and XVII10K106 was collected respectively, and the bacterial cells and the fermentation supernatant were separated by centrifugation at 2000×g, 30 min, and 4°C. The cation exchange medium was balanced with buffer A, and the fermentation supernatant was diluted with pure water to a conductivity of less than 8ms / cm. The pH value was adjusted to 4.4 and then chromatography was started. The chromatography process was as follows:

[0216] a) Column equilibration: Manual mode, flow rate set to 28 mL / min, inlet A4 (buffer A), column valve forward flushing, and continuous monitoring of the Cond conductivity curve and pH curve on the spectrum interface until the conductivity curve drops to the lowest point and flattens out (about 3 column volumes). When the pH curve flattens out, it indicates that the column has been equilibrated. Click Pause.

[0217] b) Sample loading: Manual mode, flow rate set to 28mL / min, inlet A1 (diluted fermentation supernatant, pH 4.4, 5L), click Pause when the injection volume reaches the required volume.

[0218] c) Rebalance: Manual mode, flow rate set to 28 mL / min, inlet A4 (buffer A), click Continue, run the program to continuously monitor the spectrum curve, and continue balancing after the UV curve, conductivity curve, and pH curve are stable, and click Pause.

[0219] d) Elution: Manual mode, flow rate set to 28mL / min, inlet B1 (buffer B), click Continue, when the A215 spectrum curve rises, click Outlet1, click Continue, start eluting and collecting the target component, until UV215 drops to the lowest point, and the collection is completed. Collect the eluate, detect and determine the components separately, and then dialyze (dialysis fluid is ultrapure water), then concentrate and freeze-dry, and collect the purified freeze-dried collagen sponge.

[0220] The purified freeze-dried sponge was dissolved in ultrapure water and subjected to SDS-PAGE electrophoresis. The results were as follows: Figure 5 As shown in the figure, the purified recombinant collagen ⅩⅦ12K61, ⅩⅦ22K111, and ⅩⅦ9K95 showed obvious degradation bands in gel images, indicating poor stability, while ⅩⅦ10K106 showed a relatively pure single band in SDS-PAGE electrophoresis, indicating better expression and stability.

[0221] Example 6. Characterization of the properties of recombinant collagen

[0222] In this example, high performance liquid chromatography-mass spectrometry (LC-MSMS) was used to analyze the amino acid sequence coverage of the ⅩVII10K106 freeze-dried sponge sample. The specific steps are as follows:

[0223] Weigh 10 μg of the purified freeze-dried sponge into 5 1.5 mL centrifuge tubes, add ultrapure water to dissolve, and then dilute to 100 μL with the corresponding enzyme buffer, with a final concentration of 0.1 mg / mL, recorded as samples 1 to 5.

[0224] The buffer for sample 1 was 50 mM NH 4 HCO 3;

[0225] The buffer for sample 2 was ultrapure water;

[0226] The buffer for sample 3 was 50 mM Tris-HCl;

[0227] The buffer for sample 4 was 25 mM Tris-HCl, 1 mM EDTA (pH 8.5)

[0228] The buffer for sample 5 was 50 mM Tris-HCl (pH 8.0), 10 mM CaCl 2 .

[0229] 1 μL of 1M dithiothreitol (DTT) solution was added to samples 1 to 5 respectively, so that the final concentration of DTT was 10 mM, and then the reduction reaction was carried out in a 56°C water bath for 1 h. After the reaction, 2 μL of 1M iodoacetamide (IAM) solution was added to the reaction solution respectively, so that the final concentration of IAM was 20 mM. The reaction was carried out in the dark at room temperature for 40 min. After the reaction, 1 μL of 1M dithiothreitol (DTT) solution was added to the sample respectively, so that the final concentration of DTT was 10 mM to neutralize the unreacted IAM, and reaction solutions 1 to 5 were obtained.

[0230] The corresponding enzymes were added to reaction solutions 1 to 5, respectively, wherein 2 μL of 0.25 μg / μL Trypsin enzyme was added to reaction solution 1, 2 μL of 0.5 μg / μL Glu-c enzyme was added to reaction solution 2, 2 μL of 0.1 μg / μL Elastase enzyme was added to reaction solution 3, 2 μL of 0.2 μg / μL Lys-C enzyme was added to reaction solution 4, and 1 μL of 0.1 μg / μL Protease K enzyme was added to reaction solution 5, and then incubated at 37°C for 16 h. After the incubation, a C18 desalting column was used for desalting, and then vacuum centrifugation and drying were performed at 45°C to re-dissolve the peptides. The data were collected using a Vanquish Neo-Oribitrap Fusion Lumos high-resolution liquid chromatography-mass spectrometry system, and the data were analyzed using the Peptide Mapping analysis function of the BioPharma Finder software.

[0231] After combined analysis of the coverage information of 5 enzymes, the coverage of XVII10K106 reached 100%, completely matching the theoretical sequence SEQ ID NO:25. Therefore, the recombinant type XVII collagen XVII10K106 of the present invention is indeed obtained by repeating the monomer selected from the human type XVII collagen sequence p1438-p1461 10 times.

[0232] Example 7. Stability test of recombinant type ⅩⅦ collagen

[0233] Based on Examples 4 and 5, it can be preliminarily determined that recombinant collagen XVII10K106 has high stability. Since the subsequent production and application process involves short-term storage of raw materials, it is required to maintain the initial molecular weight and not degrade or degrade less. Therefore, in this example, the storage stability of recombinant type XVII collagen XVII10K106 at different ambient temperatures and the accelerated stability of recombinant collagen solutions with different pH values ​​under high temperature conditions were tested.

[0234] (1) Stability test of recombinant type XVII collagen under different temperature conditions:

[0235] Weigh an appropriate amount of recombinant ⅩVII collagen ⅩVII10K106 freeze-dried 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 an ultra-clean workbench. After filtration, take the 0-day sample and prepare it as an electrophoresis sample for the control sample of the subsequent temperature stability experiment.

[0236] The sterile recombinant collagen solution was divided into 3 sterile centrifuge tubes, which were placed at 40°C, 25°C, and 4°C respectively to accelerate the thermal stability experiment process. At the same time, it can simulate the normal room temperature and normal sample storage temperature to explore the effect of storage temperature conditions on the samples. Each group took samples on the 1st, 4th, and 8th day after the start of the experiment, and prepared them into electrophoresis samples for SDS-PAGE detection. The results are as follows: Figure 6 shown.

[0237] from Figure 6 It can be seen that the recombinant ⅩVII collagen ⅩVII10K106 solution was stable and not degraded when placed at 4°C for 8 days, and had high stability; placing it at room temperature of 25°C and 40°C would accelerate its degradation, but no obvious degradation band was seen below the target band, and only the protein content of the target band decreased. After 8 days, the retention rate of the sample placed at 25°C exceeded 60%, and the sample placed at 40°C also had a retention rate of nearly 50%. This shows that the recombinant ⅩVII collagen ⅩVII10K106 described in the present invention has high stability, can be stable in a refrigerator or cold room without degradation, and maintain high purity and high stability. Although degradation will occur when stored at room temperature or higher temperatures, a relatively high concentration of the target protein is still retained, which can be used for large-scale factory production.

[0238] (2) Stability test of recombinant type XVII collagen at different pH values ​​at room temperature:

[0239] Weigh an appropriate amount of recombinant ⅩVII collagen ⅩVII10K106 freeze-dried sponge and dissolve it in ultrapure water to prepare a 1g / L recombinant collagen solution. Filter the recombinant collagen solution using a 0.45μm filter membrane in an ultra-clean workbench, divide it into three equal parts after filtration, and prepare recombinant ⅩVII collagen solutions with pH values ​​of 5, 7, and 11 respectively. Take the 0-day sample to prepare the electrophoresis sample, which is used as the control sample for the subsequent stability experiment of the recombinant collagen solution with different pH values.

[0240] The recombinant collagen solutions with different pH values ​​were placed together at 40°C for accelerated experiments to explore the stability of the recombinant collagen solution samples with different pH values. Each group was sampled on the 1st, 4th, and 8th day after the start of the experiment, and the samples were prepared for SDS-PAGE detection. The test results are shown in the figure. Figure 7 shown.

[0241] From the results, it can be seen that XVII10K106 is relatively stable under the original pH (acidic) and neutral conditions. When placed at 40°C for 8 days, the sample with pH = 5 only degraded by about 15%, which has high stability, and the retention rate of the sample with pH = 7 is as high as 60%. Alkaline conditions are not conducive to the preservation of recombinant XVII collagen XVII10K106, which is basically degraded on the 4th day. Therefore, avoiding alkaline conditions can effectively improve the stability of recombinant proteins.

[0242] In summary, in an actual production environment, the recombinant type XVII collagen XVII10K106 described in the present invention can remain highly stable at 4°C for at least 8 days, which is fully capable of meeting production requirements. Adjusting its pH value to neutral can further improve its stability.

[0243] Example 8. Cytotoxicity test of recombinant type XVII collagen

[0244] In this example, MEM medium containing 10% (v / v) fetal bovine serum (FBS) was used as a blank control, Negative RM-C from HatanoResearch was used as a negative control, and DMSO from Bioengineering was used as a positive control. The recombinant type XVII collagen XVII10K106 freeze-dried sponge prepared by the present invention was used as an experimental sample to investigate the cytotoxicity of the recombinant type XVII collagen. The specific steps are as follows:

[0245] The sample was extracted according to the conditions in Table 1. After the extraction, the changes in the extract were checked. The blank control sample, negative control sample and positive control sample were obtained without filtering, centrifuging, diluting, adjusting the pH and other operations on the extract, and they were directly used in subsequent experiments. The extract of the test sample XVII10K106 freeze-dried sponge obtained according to Table 1 was used as the final concentration of 100%, and gradient dilution was performed to 0.75 times, 2 times and 4 times, respectively, to obtain extracts with final concentrations of 75%, 50% and 25%.

[0246] Table 1. Extraction conditions and state of extracts of different groups of samples

[0247]

[0248] L929 cells (cell bank of Chinese Academy of Sciences, mouse fibroblast NCTC cLone 929 [L ceLL, L-929, derivative of Strain L]) were cultured in MEM medium (containing 10% FBS, 1% double antibody (v / v, penicillin-streptomycin mixture) at 37°C and 5% CO 2 The cells grown to the logarithmic growth phase were cultured under the conditions of 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 and counted to obtain 1×10 5 / mL of cell suspension.

[0249] The obtained cell suspension was inoculated in a 96-well plate, 100 μL per well, and then incubated in a cell culture incubator at 37°C and 5% CO 2 , >90% humidity for 24 h, and observe the cell morphology under a microscope. When the cells adhered to the wall and grew to about 70%, the MEM medium in the 96-well plate was discarded, and 100 μL of the extract (final concentration of 100%, 75%, 50% and 25%), blank control sample, negative control sample and positive control sample were added to the corresponding wells of the 96-well plate, and then the 96-well plate was placed in a cell culture incubator at 37°C and 5% CO 2 The cells were cultured for 24 h at a humidity of >90%, and 6 replicate wells were set up in each group.

[0250] After the culture was completed, the 96-well plate was taken out and the cell morphology was observed under a microscope. The upper layer of liquid was then discarded, 50 μL of MTT (1 mg / mL) was added to each well, and the plate was placed in a carbon dioxide incubator for 2 h. The supernatant was then discarded, 100 μL of isopropanol solution was added to each well, the plate was shaken, and the absorbance was measured at 570 nm on a microplate reader (reference wavelength 650 nm).

[0251] The statistical method was to calculate the mean ± standard deviation of each group. The survival rate calculation formula is: Survival rate (%)

[0252] =100%*OD570 e / OD570 b , where: OD570 e is the absorbance of each test group (sample group, negative control group, positive control group); OD570 b is the absorbance of the medium control group (blank). If the average value of the blank control OD570 is ≥0.2, and the difference between the average values ​​of the medium controls in the left and right columns and the average value of all medium controls is not greater than 15%, the test meets the acceptance criteria. When the survival rate is low, the test sample has a 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 the survival rate of the 100% extract, otherwise the test should be repeated; the survival rate of the 100% test sample extract is the final result.

[0253] The description of the microscopic morphology of the cells is shown in Table 2.

[0254] Table 2. Cell morphological characteristics under microscope

[0255]

[0256]

[0257] The statistical results of cytotoxicity detection are shown in Table 3.

[0258] Table 3. Cytotoxicity test results

[0259]

[0260] Combining Table 3 and Figure 8 It can be seen that the recombinant type XVII collagen XVII10K106 of the present invention has no potential cytotoxicity to L929 cells under the condition of 100% of 10 mg / mL extract, which shows that the recombinant type XVII collagen of the present invention has good biosafety and can be used as a raw material in the production process of drugs, medical devices, biomaterials or cosmetics.

[0261] In summary, the present invention selects the amino acid sequence of natural human type XVII collagen as the basic unit and repeats it multiple times to obtain recombinant type XVII collagen; in the design process of the recombinant type XVII collagen, the unstable GXY triplet is avoided, the potential matrix metalloproteinase cleavage site and glycosylation site are abandoned, and the KGD active sequence is retained, which can reduce the probability of containing sites that are prone to degradation; the sequence fragment of the recombinant type XVII collagen is derived from the full-length sequence of the natural collagen gene, has excellent solubility, biological activity and high stability, can remain stable in a high-temperature storage environment and avoid alkaline conditions, can be used as a raw material in the production process of drugs, medical devices, biomaterials or cosmetics, and has good practicality.

[0262] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A highly stable recombinant type XVII collagen monomer, characterized in that: The recombinant type XVII collagen monomer includes multiple sequence fragments that avoid unstable GXY triplet, abandon potential matrix metalloproteinase cleavage sites and glycosylation sites, and retain KGD activity; The sequence fragments include one or more of 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 in human type XVII collagen.

2. The highly stable recombinant type XVII collagen monomer according to claim 1, characterized in that: The recombinant type XVII collagen monomer comprises any one of the following sequence fragment combinations: a) p597-p611, p674-p715, p906-p929; b) p692-p715, p906-p938, p951-p977, p1438-p1467; c) p597-p619, p641-p670, p674-p715, p906-p929; d) p674-p715; e) p674-p715, p906-p929; f) p597-p611, p674-p715, p906-p929, p951-p977; g)p597-p611, p674-p715, p906-p929, p1438-p1464; h) p695-p715; i) p597-p611, p695-p715; j) p686-p706; k) p674-p700; l) p1438-p1461; Among them, one or more monomer fragments in each of the above groups are tightly connected.

3. The highly stable recombinant type XVII collagen monomer according to claim 2, characterized in that: The recombinant type XVII collagen monomer is combination 1), and its amino acid sequence is shown in SEQ ID NO:

24.

4. A highly stable recombinant type XVII collagen, characterized in that: The highly stable recombinant type XVII collagen is based on the recombinant type XVII collagen monomers according to any one of claims 1 to 3 as a basic unit repeated in tandem, and the recombinant type XVII collagen monomers are the same or different.

5. The highly stable recombinant type XVII collagen according to claim 4, characterized in that: The size of the highly stable recombinant type XVII collagen is 20-25 kDa.

6. The highly stable recombinant type XVII collagen according to claim 4, characterized in that: The amino acid sequence of the highly stable recombinant type XVII collagen includes i or ii: 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; ii. A protein derived from i in which the amino acid sequence in i is substituted, deleted or added with one or more amino acids and has human collagen activity, 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 99% or more identity with the sequence shown in i.

7. The highly stable recombinant type XVII collagen according to claim 6, characterized in that: The amino acid sequence of the highly stable recombinant type XVII collagen is shown in SEQ ID NO:

25.

8. A polynucleotide encoding the highly stable recombinant type XVII collagen according to any one of claims 4 to 7.

9. The polynucleotide according to claim 8, characterized in that The sequence of the polynucleotide includes SEQ ID NO: 26 or a degenerate sequence thereof.

10. A recombinant expression vector, characterized in that: The recombinant expression vector comprises nucleotides encoding the highly stable recombinant type XVII collagen according to any one of claims 4 to 7.

11. A recombinant engineered bacterium, characterized in that: The recombinant engineered bacteria comprises the nucleotide encoding the highly stable recombinant type XVII collagen according to any one of claims 4 to 7 or the recombinant expression vector according to claim 10.

12. The recombinant engineered bacterium according to claim 11, characterized in that: The host bacteria include Pichia pastoris.

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

14. The recombinant engineered bacterium according to claim 11, characterized in that: The recombinant engineered bacteria are deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with the deposit number of CGMCC No.32750.

15. The method for preparing the highly stable recombinant type XVII collagen according to any one of claims 4 to 7, characterized in that: The preparation method comprises: (1) selecting and designing the sequence of a recombinant type XVII collagen monomer, and then constructing a recombinant type XVII collagen by serially recombining the recombinant type XVII collagen monomer as a basic unit; (2) constructing a recombinant expression vector for expressing recombinant type XVII collagen; (3) electroporating the obtained recombinant expression vector expressing recombinant type XVII collagen into a host bacterium, screening and verifying it, and obtaining a recombinant engineered bacterium; (4) Fermentation-induced expression of the recombinant engineered bacteria is performed to obtain the highly stable recombinant type XVII collagen.

16. The method according to claim 15, characterized in that In step (1), the recombinant type XVII collagen monomer includes a plurality of sequence fragments that avoid unstable GXY triplet, abandon potential matrix metalloproteinase cleavage sites and glycosylation sites, and retain KGD activity; The sequence fragments include one or more of 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.

17. The method according to claim 16, characterized in that The recombinant type XVII collagen monomer comprises any one of the following sequence fragment combinations: a) p597-p611, p674-p715, p906-p929; b) p692-p715, p906-p938, p951-p977, p1438-p1467; c) p597-p619, p641-p670, p674-p715, p906-p929; d) p674-p715; e) p674-p715, p906-p929; f) p597-p611, p674-p715, p906-p929, p951-p977; g)p597-p611, p674-p715, p906-p929, p1438-p1464; h) p695-p715; i) p597-p611, p695-p715; j) p686-p706; k) p674-p700; l) p1438-p1461; Among them, one or more monomer fragments in each of the above groups are tightly connected.

18. The method according to claim 17, characterized in that The recombinant type XVII collagen monomer is combination 1), and its amino acid sequence is shown in SEQ ID NO:

24.

19. The method according to claim 15, characterized in that In step (1), the recombinant type XVII collagen monomers are the same or different.

20. The method according to claim 15, characterized in that In step (1), the size of the highly stable recombinant type XVII collagen is 20-25 kDa.

21. The method according to claim 15, characterized in that In step (1), the amino acid sequence of the highly stable recombinant type XVII collagen includes i or ii: 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; ii. A protein derived from i in which the amino acid sequence in i is substituted, deleted or added with one or more amino acids and has human collagen activity, 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 99% or more identity with the sequence shown in i.

22. The method according to claim 15, characterized in that In step (1), the amino acid sequence of the highly stable recombinant type XVII collagen is as shown in SEQ ID NO:

25.

23. The method according to claim 15, characterized in that In step (3), the host bacteria includes Pichia pastoris.

24. The method according to claim 23, characterized in that The host bacteria are Pichia pastoris GS115 strain, Pichia pastoris X33 strain, Pichia pastoris KM71H strain and Pichia pastoris SMD116 strain.

25. The method according to claim 15, characterized in that In step (3), the recombinant engineered bacteria is deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration with the deposit number of CGMCC No.32750.

26. A composition, characterized in that The composition comprises the highly stable recombinant type XVII collagen monomer according to any one of claims 1 to 3 or the highly stable recombinant type XVII collagen according to any one of claims 4 to 7.

27. A product, characterized in that The product comprises the highly stable recombinant type XVII collagen monomer according to any one of claims 1 to 3, or the highly stable recombinant type XVII collagen according to any one of claims 4 to 7, or the composition according to claim 26.

28. Use of the highly stable recombinant type XVII collagen monomer according to any one of claims 1 to 3, or the highly stable recombinant type XVII collagen according to any one of claims 4 to 7, or the composition according to claim 26, or the product according to claim 27 in the preparation of medical devices, biomaterials, tissue engineering products, and cosmetics.

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