Recombinant collagen type vii, preparation method therefor and the use thereof

Abstract

Provided are a recombinant collagen type VII, a preparation method therefor and the use thereof, belonging to the fields of biology, genetic engineering and biologic technologies. Genetic engineering technology is used as a means for increasing the expression level of the recombinant collagen type VII, and an eukaryotic pichia pastoris expression system is used for performing the expression production of the recombinant collagen type VII; the recombinant collagen type VII is expressed in all pichia pastoris strains, can be efficiently secreted and expressed to the outside of cells, and is not prone to degradation during the purification stage, thus reducing the difficulty in purification. The recombinant collagen type VII has physicochemical properties and biological functions similar as that of an original sequence, has a relatively small molecular weight and achieves good absorption effect on tissue, and can be processed into collagen-containing compositions, and therefore can be applied well in each of the fields of drugs, pharmaceutical compositions, medical instruments, biological materials, tissue engineering products, cosmetics or health care products, etc.

Description

Recombinant type VII collagen and its preparation method and application Technical Field

[0001] The present invention belongs to the fields of biology, genetic engineering and biotechnology, and particularly relates to recombinant type VII collagen and a preparation method and application thereof. Background Art

[0002] Anchoring fibers are attachment structures that mediate the adhesion of the human skin to the epidermis, of which type VII collagen (Col7) is the main component of anchoring fibers (AFs). Type VII collagen connects the basal layer to the underlying tissue by interacting with other types of collagen and part of the extracellular matrix. Col7 is a homotrimer composed of three α chains. The Col7 gene encodes a 290kDa α chain, and the three α chains form a 900kDa homotrimer. In Col7, each α chain has a 145kDa central collagen triple helix with a segment sequence characterized by repeating Gly-XY amino acids. The central domain of the helix is ​​flanked by a larger 145kDa non-collagenous domain (NC1) amino-terminal and a 34kDa carboxyl-terminal non-collagenous domain (NC2). In the dermis, Col7 molecules form disulfide-bonded tail-to-tail antiparallel dimers through the carboxyl-terminal NC2 domain. The NC2 domain and its adjacent triple-helical domain initiate the triple-helical assembly of type VII collagen. Subsequently, a portion of the NC2 domain is proteolytically removed, and the antiparallel dimers aggregate laterally to form anchoring fibers, which interact with microfibrils, collagen fibers, and microthread-like fibers within the dermis. These interactions are thought to anchor the epidermis and its underlying basement membrane zone to the underlying dermis.

[0003] Studies have shown that NC1 interacts with multiple extracellular matrix (ECM) components, including fibronectin, laminin 5, collagen type I, and collagen IV. Structural alterations in type VII collagen may disrupt its functional interactions with ECM components, leading to separation of the epidermis from the dermis and dystrophic epidermolysis bullosa (DEB). Col7 mutations can cause the human skin fragility disorder recessive dystrophic epidermolysis bullosa (RDEB), which in turn leads to persistent wound non-healing. Therefore, Col7 is required for the re-epithelialization of laminin 332 at the dermal-epidermal junction. Its absence disrupts the organization of laminin 332 during wound healing, disrupting the strictly polarized expression of integrin α6β4 in basal keratinocytes and negatively affecting laminin 332 / integrin.

[0004] Studies have shown that Col7 promotes skin wound healing through two interrelated mechanisms. First, Col7 enables re-epithelialization by organizing laminin-332 at the dermal-epidermal junction. During wound healing, Col7 loss disrupts laminin-332 organization, which in turn disrupts the strictly polarized expression of integrin α6β4 in basal keratinocytes and negatively affects the laminin-332 / integrin α6β4 signaling axis that directs keratinocyte migration. Col7 then supports dermal fibroblast migration and regulates cytokine production in granulation tissue. Therefore, Col7 plays a key role in physiological wound healing in humans and mice, potentially aiding the development of therapeutic strategies for RDEB and other chronic wounds.

[0005] Collagen VII is a large molecule with a molecular weight exceeding 300,000 Daltons. Synthesis and isolation of large collagen molecules are costly and difficult. Furthermore, large collagen molecules are susceptible to degradation and have poor stability, leading to high protein loss and operational complexity during the production and separation processes. Applications are also limited, hindering large-scale production. Furthermore, currently, there are only a few expression systems for type VII collagen, primarily mammalian cells and Escherichia coli. Mammalian cell culture is expensive, has a slow growth cycle, and produces low expression levels of 15-20 mg / L. Although secreted into the supernatant, this requires extensive purification steps and results in significant product losses, making industrial production impossible. While the E. coli expression system offers improved yields compared to mammalian cells, it has not reached the scale required for commercial production. Consequently, both expression systems suffer from low yields, poor protein stability, and susceptibility to degradation, limiting them to laboratory-scale production. To commercialize type VII collagen products, there is a need for a recombinant type VII protein with high expression levels, as well as an expression system and method for its construction. Summary of the Invention

[0006] In response to some deficiencies in the prior art, the present invention provides a recombinant type VII collagen protein, a preparation method thereof, and an application thereof. The present invention uses genetic engineering technology as a means to increase the expression level of recombinant type VII collagen protein, and uses a eukaryotic Pichia pastoris expression system to express and produce recombinant type VII collagen protein. The recombinant type VII collagen protein of the present invention is expressed in Pichia pastoris, can be efficiently secreted and expressed extracellularly, is not easily degraded during the purification stage, and reduces the difficulty of purification. The recombinant type VII collagen protein has similar physical and chemical properties and biological functions to the original sequence, has a smaller molecular weight, has a better effect on tissue absorption, can be made into a composition containing collagen protein, and has good applications in the fields of drugs, pharmaceutical compositions, medical devices, biomaterials, tissue engineering products, cosmetics, or health care products.

[0007] The present invention achieves the above technical objectives through the following technical means.

[0008] The present invention first provides a type VII collagen, which includes an amino acid sequence as shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9 or SEQ ID No.11, or an amino acid sequence that is 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 identical to SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9 or SEQ ID No.11, and maintains the biological activity of the collagen.

[0009] The present invention also provides a nucleic acid encoding the above-mentioned type VII collagen.

[0010] The present invention also provides a recombinant vector comprising the above nucleic acid. Preferably, the recombinant vector includes but is not limited to pPICZαB, pFLDα, and pPIC9K, and the linking site is between XhoI and NotI.

[0011] The present invention also provides a method for preparing the above-mentioned type VII collagen, characterized in that the method comprises:

[0012] (1) Selecting the sequence of type VII collagen to construct a collagen tandem sequence, and then constructing a recombinant expression vector containing the collagen tandem sequence;

[0013] (2) The obtained recombinant expression vector was linearized with Sal I to obtain a linearized plasmid;

[0014] (3) The linearized plasmid is electroporated into a host bacterium, and recombinant engineered bacteria containing or expressing type VII collagen are screened and verified to obtain the recombinant engineered bacteria, and the expression is induced by fermentation to obtain the type VII collagen.

[0015] Preferably, the collagen tandem sequence is expressed in tandem repetitions using type VII collagen as a basic unit, and there is a site for Kex2 enzyme recognition and cleavage between every two adjacent basic units in the collagen tandem sequence.

[0016] Preferably, the type VII collagen comprises an amino acid sequence as shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9 or SEQ ID No.11, or an amino acid sequence that is more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identical to SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9 or SEQ ID No.11, and maintains the biological activity of the collagen.

[0017] Preferably, the collagen tandem sequence may further include a recognition and cleavage site for Ste13 enzyme between each two adjacent basic units.

[0018] Preferably, the recognition site of the Kex2 enzyme includes a KR or RR dibasic amino acid residue, followed by EA, EAEA or other amino acid residues that facilitate cleavage by the Kex2 enzyme or Ste 13 enzyme.

[0019] Preferably, the amino acids of the collagen tandem sequence include a sequence as shown in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10 or SEQ ID No. 12, or an amino acid sequence having more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% identity with the above.

[0020] Preferably, the nucleic acid encoding the collagen tandem sequence includes the nucleotide sequence shown as SEQ ID No. 13 to SEQ ID No. 18, or a degenerate sequence thereof.

[0021] Preferably, in step (1), the recombinant expression vector includes but is not limited to pPICZαB, pFLDα, and pPIC9K, and the linking site is between XhoI and NotI.

[0022] Preferably, in step (3), the host bacteria include Pichia pastoris, Saccharomyces cerevisiae, Hansenula, etc., preferably Pichia pastoris.

[0023] Preferably, in step (3), the recombinant engineered bacteria are deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with the deposit numbers being: CGMCC No. 29601, CGMCC No. 29602, CGMCC No. 29603, CGMCC No. 29604, CGMCC No. 29605, or CGMCC No. 29606.

[0024] The present invention also provides type VII collagen prepared according to the above method.

[0025] The present invention also provides a composition comprising the type VII collagen, or the type VII collagen encoded by the nucleic acid, or the recombinant vector, or the type VII collagen obtained by the method; the composition includes drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

[0026] The present invention also provides a product, which comprises the type VII collagen, the type VII collagen encoded by the nucleic acid, the recombinant vector, the type VII collagen obtained by the method, or the composition; the product includes a drug, a medical device, a biomaterial, a tissue engineering product, a cosmetic or a health product.

[0027] The present invention also provides the use of the type VII collagen, or the type VII collagen encoded by the nucleic acid, or the recombinant vector, or the type VII collagen obtained by the method, or the composition, or the product in the preparation of drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) Among the type VII collagen monomers described in the present invention, SEQ ID No. 1 contains M and H and has antioxidant activity. SEQ ID No. 3, SEQ ID No. 5, and SEQ ID No. 7 contain RGD, which can specifically bind to 11 types of integrins and effectively promote cell adhesion to biomaterials. SEQ ID No. 9 contains charged amino acids, which can ionize and release charges, and has a high isoelectric point. The six groups of sequences obtained by the present invention based on the series connection of the above six groups of type VII collagen monomers are all GXY triplets. The six groups of recombinant collagen sequences involved are all expressed in Pichia pastoris, can be efficiently secreted and expressed outside the cell, are not easily degraded during the purification stage, and reduce the difficulty of purification.

[0030] (2) The sequence of the collagen protein described in the present invention is derived from human type VI I collagen (Q02388·CO7A1_HUMAN), and the sequence is derived from the triple helical region: a sequence of about 50 amino acids is selected, KR is added to the C-terminus, EA is added to the N-terminus, and 6 copies are connected in series to make the target sequence size about 40KD to adapt to the optimal expression range of Pichia pastoris. After expression in Pichia pastoris, kex2 contained in Pichia pastoris itself cuts at the carboxyl terminus of KR, ste13 cuts the nitrogen-terminal EA, and Kex1 cuts the remaining KR residues, and secreted into the extracellular space via the secretory signal peptide.

[0031] The technical solution of the present invention greatly improves the yield of type VII collagen expression; the technical solution of the present invention does not introduce exogenous proteins and does not use any in vitro enzymatic cleavage methods. During the process of intracellular secretion of type VII collagen, the residual amino acid residues at the enzymatic cleavage sites during the repeated tandem design will be removed, thereby obtaining type VII collagen without non-collagen sequences or with 100% homology to the corresponding region of natural collagen; the entire expression system avoids the cost and risk of exogenous protein residues caused by the use of protease enzymatic cleavage in vitro, and can also shorten the time and cost of the subsequent purification process.

[0032] (3) The recombinant type VII collagen obtained by the method of the present invention has similar physicochemical properties and biological functions to the original sequence, a smaller molecular weight, and better tissue absorption. The present invention conducted cell adhesion experiments using in vitro cultured NIH / 3T3 cells with type VII collagen and commercial collagen. The experiments showed that there was no significant difference in the adhesion activity between the recombinant type VII collagen of the present invention and commercial type I collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a picture of agarose gel verification of the recombinant engineering strain.

[0034] Figure 2 shows the supernatant of recombinant collagen expression in shake flasks after 48h and 72h of induction.

[0035] FIG3 shows the results of protein spectrum detection of recombinant collagen.

[0036] Figure 4 shows the results of mass spectrometry analysis.

[0037] Figure 5 is the electrophoresis of the supernatant of each recombinant collagen 5L tank fermentation

[0038] Figure 6 is the electrophoresis diagram of the purified recombinant collagen

[0039] FIG7 is a graph showing the results of cell adhesion activity detection.

[0040] FIG8 is a graph showing the detection results of cell migration area ratio.

[0041] Figure 9 is a picture of cell migration. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the technical solutions of the present invention, preferred embodiments of the present invention are described in detail below. However, the following embodiments do not limit the scope of protection of the present invention.

[0043] In the embodiments of the present invention, those that are not described in detail are all completed using conventional experimental methods. Those processes involved in the embodiments that are not described in detail are all understandable and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore are not described in detail.

[0044] Example 1:

[0045] S1. Design of amino acid sequence:

[0046] A sequence of about 50 amino acids from the triple helix region of human type VII collagen (Q02388·CO7A1_HUMAN) was selected, KR was added to the C-terminus, EA was added to the N-terminus, and 6 copies were connected in series to make the target sequence size about 40KD to adapt to the optimal expression range of Pichia pastoris. After expression in Pichia pastoris, kex2 contained in Pichia pastoris itself cuts at the carboxyl terminus of KR, ste13 cuts the nitrogen-terminal EA, and Kex1 cuts the remaining KR residues, which are then secreted into the extracellular space via the secretory signal peptide.

[0047] (1) 2156-2207AA of Q02388.CO7A1-HUMAN was selected, LE was added to the C-terminus, and the obtained sequence was named 705, which is shown in SEQ ID No. 1. On the basis of SEQ ID No. 1, EA was added to the N-terminus and KR was added to the C-terminus to form a monomer. The monomer was repeated 6 times to form the amino acid sequence shown in SEQ ID No. 2, thus obtaining the recombinant type VII collagen tandem sequence.

[0048] SEQ ID No. 1:

[0049]

[0050] SEQ ID No. 2:

[0051]

[0052] (2) 2527-2568AA of Q02388.CO7A1-HUMAN was selected, and LE was added to the C-terminus. The resulting sequence was named 706, and the sequence is shown in SEQ ID No. 3. SEQ ID No. 3 contains an RGD motif, can specifically bind to 11 integrins, can effectively promote cell adhesion to biomaterials, and contains a large number of ionizable polar amino acids.

[0053] Based on SEQ ID No. 3, EA was added to the N-terminus and KR was added to the C-terminus to form a monomer, and this was repeated 6 times to form the amino acid sequence shown in SEQ ID No. 4, thereby obtaining recombinant type VII collagen.

[0054] SEQ ID No.3: GPPGPRGAKGDMGERGPRGLDGDKGPRGDNGDPGDKGSKGEPGLE; SEQ ID No.4:

[0055]

[0056] (3) 1988-2020AA of Q02388.CO7A1-HUMAN was selected and named 707, and the sequence was shown in SEQ ID No. 5. SEQ ID No. 5 contains an RGD motif, can specifically bind to 11 integrins, can effectively promote cell adhesion to biomaterials, and contains a large number of ionizable polar amino acids.

[0057] Based on SEQ ID No. 5, EA was added to the N-terminus and KR was added to the C-terminus to form a monomer, and this was repeated 6 times to form the amino acid sequence shown in SEQ ID No. 6, thereby obtaining recombinant type VII collagen.

[0058] SEQ ID No.5:GPPGKEGPIGFPGERGLKGDRGDPGPQGPPGLA;

[0059] SEQ ID No.6:

[0060]

[0061] (4) 1314-1352AA of Q02388.CO7A1-HUMAN was selected, and LE was added to the C-terminus. The resulting sequence was named 708, and the sequence is shown in SEQ ID No. 7. SEQ ID No. 7 contains an RGD motif, can specifically bind to 11 integrins, can effectively promote cell adhesion to biomaterials, and contains a large number of ionizable polar amino acids.

[0062] Based on SEQ ID No. 7, EA was added to the N-terminus and KR was added to the C-terminus to form a monomer, and this was repeated 6 times to form the amino acid sequence shown in SEQ ID No. 8, thereby obtaining recombinant type VII collagen.

[0063] SEQ ID No.7:GNPGTPGAPGLKGSPGLPGPRGDPGERGPRGPKGEPGAPGLE;

[0064] SEQ ID No.8:

[0065]

[0066] (5) 2617-2655AA of Q02388.CO7A1-HUMAN was selected and the obtained sequence was named 709, which is shown in SEQ ID No. 9. Based on SEQ ID No. 9, EA was added to the N-terminus and KR was added to the C-terminus to form a monomer, and this was repeated 6 times to form the amino acid sequence shown in SEQ ID No. 10, thereby obtaining recombinant type VII collagen.

[0067] SEQ ID No.9:GFMGPRGLKGERGVKGACGLDGEKGDKGEAGPPGRPGLA;

[0068] SEQ ID No.10:

[0069]

[0070] (6) 1483-1527AA of Q02388.CO7A1-HUMAN was selected, and LE was added to the C-terminus. The resulting sequence was named 710 and is shown in SEQ ID No. 11. Based on SEQ ID No. 11, EA was added to the N-terminus and KR was added to the C-terminus to form a monomer. This was repeated 6 times to form the amino acid sequence shown in SEQ ID No. 12, thereby obtaining recombinant type VII collagen.

[0071] SEQ ID No.11:

[0072]

[0073] SEQ ID No.12:

[0074]

[0075] S2. Synthesis of DNA sequences and construction of recombinant expression vectors:

[0076] Nanjing GenScript Biotech Co., Ltd. was commissioned to synthesize the nucleic acid sequences encoding the above-mentioned amino acid sequences SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, and SEQ ID No. 12, and the corresponding nucleic acid sequences are shown in SEQ ID No. 13 to SEQ ID No. 18, respectively.

[0077] The synthesized gene fragments were cloned into pPICZαB respectively, and the exogenous target protein sequences were cloned using XhoI and NotI as restriction sites, so that the target fragments were accurately inserted into the secretory vector reading frame containing the secretion signal α-factor, and recombinant expression vectors expressing SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, and SEQ ID No. 12 were obtained and named pPICZαB-705 to pPICZαB-710.

[0078] SEQ ID No. 13 to SEQ ID No. 18 are shown below, wherein "CTCGAG" is an XhoⅠ restriction site and "GCGGCCGC" is a NotⅠ restriction site.

[0079] SEQ ID No.13:

[0080]

[0081] SEQ ID No.14:

[0082]

[0083] SEQ ID No.15:

[0084]

[0085] SEQ ID No.16:

[0086]

[0087] SEQ ID No.17:

[0088]

[0089] SEQ ID No.18:

[0090]

[0091] S3. Construction of recombinant engineering strains and screening of strains:

[0092] 10 μg of the recombinant expression vectors with different sequences obtained in step 2 were linearized with Sal I (Dalian TaKaRa) enzyme, digested in a metal bath at 37°C for 30 min, and purified with a PCR product purification kit (Shanghai Sangon, according to the kit instructions) to recover the linearized plasmids.

[0093] The linearized plasmid was electroporated into the competent cells of the host strain Pichia pastoris X33 (purchased from Thermo Fisher Scientific), and then 200 μL of the electroporated bacterial solution was spread on a YPDZ (100 μg / mL) plate and cultured in an inverted manner in a 30°C biochemical incubator for 2-3 days until a single colony (positive transformant) appeared.

[0094] Use a sterile pipette tip to pick a single colony and streak it onto a YPDZ (300 μg / mL) plate. Incubate it upside down at 30°C for 2-3 days until a single colony (positive transformant) appears. For each sequence, pick 16 single colonies in 200 mL of YPD medium and place them in a 96-well plate. Incubate them in a 30°C incubator for 2 days. Take 50 μL of the bacterial solution to extract the template. Take 2 μL of the extracted template and add it to a PCR tube. Perform polymerase chain reaction by PCR. After the reaction, verify the target band by nucleic acid electrophoresis.

[0095] The primers used in the above PCR reaction are:

[0096] 5'AOX:GACTGGTTCCAATTGACAAGC (SEQ ID No. 19);

[0097] 3'AOX:GCAAATGGCATTCTGACATCC (SEQ ID No. 20);

[0098] The PCR reaction conditions were as follows: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min, for 30 cycles.

[0099] The results of nucleic acid electrophoresis verification are shown in Figure 1. As can be seen from Figure 1, the target band is about 1200bp-1500bp. Compared with the marker size band, the band around 1200bp-1500bp is the positive bacteria. The selected positive strain contains the nucleic acid sequence expressing the target protein. The positive strain is selected for subsequent strain screening experiments.

[0100] Positive strains are inoculated onto plates containing high-concentration Zeocin. The number of positive strains that grow on the plates with high Zeocin concentrations is used to determine whether the strain contains multiple copies of the target gene. If a positive strain can grow on the plate with high Zeocin concentrations, it indicates that the strain contains multiple copies of the target gene, meaning that multiple recombinant fragments have entered the yeast and integrated into the yeast chromosome through homologous recombination. Through screening, recombinant engineered bacteria with high copy numbers and high expression efficiency can be obtained.

[0101] The obtained high-copy, high-efficiency expression recombinant engineered bacteria were respectively sent to the General Microbiology Center of China Culture Collection Administration for Microorganisms for preservation, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit numbers: CGMCC No. 29601, CGMCC No. 29602, CGMCC No. 29603, CGMCC No. 29604, CGMCC No. 29605, CGMCC No. 29606, and the deposit date was January 12, 2024. The classification name is Komagataella phaffii.

[0102] S4. Inducible expression and identification of recombinant collagen:

[0103] Select the strain with the target band and inoculate it into 15 mL of BMGY medium. Cultivate it at 28-30°C and 220 rpm until the OD600 is 2-15 (16-24 hours). Centrifuge it at 1500-3000g for 5 minutes at room temperature, collect the bacteria, and resuspend them in BMMY medium to adjust the OD600 to 2-15. 600 The cells were incubated for 3 days in a biochemical incubator at 28-30°C and 220 rpm, with 1% of the final volume of methanol added every 12 hours. Samples were collected after 48 and 72 hours of induction and the supernatant was collected by centrifugation at 1500-3000g for 5 minutes at room temperature. The collected expression supernatant was added with 2× loading buffer (Coomassie Brilliant Blue, denaturant DTT, SDS buffer), heated in a 100°C metal bath for 5 minutes, and then subjected to GLASS Gel precast gel Tricine protein gel analysis. The results are shown in Figure 2. As can be seen, all target bands were secreted in the culture supernatant.

[0104] The expected target bands 705, 706, 707, 708, 709, and 710 on the GLASS gel Tricine were cut out and digested with trypsin. The trypsin-digested peptides of the recombinant collagen were detected by Nano-HPLC-MS / MS mass spectrometry (completed by Suzhou Putai Biotechnology Co., Ltd.), and the detected peptides were aligned with the theoretical sequence. The alignment results are shown in Figures 3 and 4.

[0105] As can be seen from Figures 3 and 4, the peptides detected after enzymatic hydrolysis of 705, 706, 707, 708, 709, and 710 all belong to the theoretical sequence of the corresponding collagen, indicating that the collagen was successfully expressed, and further indicating that the recombinant 705, 706,

[0106] Collagen 707, 708, 709, and 710. Figure 3 shows the comparison results of the characteristic peptides of 705, 706, 707, 708, 709, and 710 with the sequence of human type VII collagen α1 chain in the database and the theoretical sequence. The red color in Figure 4 is the compared theoretical sequence. The results in Figure 3 show that the band of about 5KD contains 1-2 major proteins and matches the characteristic peptide of human type VII collagen α1 chain in the recorded peptide coverage. The relative abundance of human type VII collagen α1 chain in this band is more than 75%, confirming that it is human type VII collagen α1 chain. Trypsin is used for protein spectrum identification, and the cleavage sites are K and R. The peptides after cleavage generally end with K or R. During database retrieval and software analysis, the peptide between K and R is also the detection peptide. If the N-terminal sequence of the sequence is in the middle part in the database sequence, the theoretical cleavage peptide during software analysis has one more amino acid than the one in the database, so it cannot be matched. Sequences 708 and 709 belong to this situation, but they can be compared with the theoretical sequence, and the relative abundance is 86.6% and 97.1%, confirming that the target protein sequence has been synthesized.

[0107] S5. High-density fermentation and purification test:

[0108] (1) The recombinant engineered bacteria selected in step 3 are subjected to a high-density fermentation test to express and produce the recombinant 705, 706, and 710 collagens on a large scale to obtain a fermentation broth containing the recombinant collagens.

[0109] Among them, seed culture medium YPG: yeast powder 10g / L, peptone 20g / L, glycerol 10g / L;

[0110] 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; after high-temperature sterilization of the fermentation medium, add PTM1 when the temperature drops to room temperature, and adjust the pH to 5.0 with ammonia water.

[0111] Feed medium: 50% w / v glycerol, plus 12 mL PTM1 trace elements per liter;

[0112] Induction medium: 100% methanol, 12 mL of PTM1 trace elements per liter;

[0113] PTM1: Sterilize by filtration using a 0.22 μm filter and store at 4°C.

[0114] The batch culture conditions and induced expression conditions of the engineered strains are as follows: adopt the batch fed culture method, and the culture temperature is 30°C. The engineered bacteria are inoculated into a 1L shake flask containing 200ml of seed culture medium YPG, and cultured at 220rpm and 30°C for 18-20h to OD600=2~10. A 5L fermenter (Baoxing Biology) is used, with a liquid volume of 2L of fermentation medium and 2% glycerol sterilized separately. Before inoculation, the speed is adjusted to 300rpm, the ventilation volume is 4L / min, and the temperature is 30°C. The pH is adjusted with an alkaline solution prepared with concentrated ammonia water, and the pH is set to 4.5. Then 0.9ml PTM1 is first inoculated, and then the prepared 200ml seed liquid is inoculated into the tank (flame circle inoculation), and then the dissolved oxygen electrode is clicked for calibration, and fermentation is started after calibration. When the dissolved oxygen drops to 30% for the first time during growth, the dissolved oxygen cascade speed function is used to maintain 30%; wait for the glycerol to be consumed, the dissolved oxygen rebounds, and the dissolved oxygen is greater than 70% (OD 600 Value is about 20), cancel the dissolved oxygen cascade speed, increase the stirring to 650rpm, use 30% linkage feeding of glycerol, and feed 80ml. Stop feeding glycerol, after the dissolved oxygen rebounds to more than 70%, set pH4 and temperature to 29℃, and induce culture with a mixed carbon source of methanol and glycerol (methanol: 50% glycerol = 7:3). Manually add 5ml, after the dissolved oxygen rebounds to more than 70%, set the feeding rate to 8ml / h, increase to 10ml / h after one hour, and increase 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 feed linkage after the dissolved oxygen returns to 30%. After 40-60h of induction, the protein concentration measured by UV does not increase significantly or decreases, then the tank can be released.

[0115] UV protein quantification formula: C (mg / mL) = 0.144*(A215-A225), A215<1.5.

[0116] The fermentation supernatant was collected for protein electrophoresis detection, which showed that as shown in Figure 5, under high-density fermentation conditions, each sequence collagen had almost only the target band within 24 hours of induction, and the main band ratio in optical density analysis was more than 85%.

[0117] (2) Purification:

[0118] Buffer A: 20 mM KH2PO4, pH 4.0;

[0119] Buffer B: 20 ​​mM KH2PO4, 1 M NaCl, pH 4.0.

[0120] The fermentation broth in step (1) was collected and centrifuged at 2000g, 30min, and 4°C to separate the bacterial cells and the fermentation supernatant. The cation exchange medium was balanced with buffer A (the chromatography filler was UniGel-80sp produced by Suzhou Nano Micro, loaded on an XK50 / 30 chromatography column produced by Lisui Technology, and a GE AKTAPure protein separation chromatography purification system was used) until the A215 absorbance and conductivity values ​​remained unchanged. The flow rate was set to 100us / cm for sample loading, and the sample volume was 0.5L / time. The ultraviolet A215 absorbance value was detected. When it rose, the sample was started. When the A215 absorbance value dropped, the sample was stopped until the ultraviolet and conductivity dropped to the lowest and no longer changed. After the sample loading was completed, the sample was closed and the cationic chromatography medium was balanced with buffer A. The eluate was collected, and after the components were detected and determined, dialyzed (the dialysate was ultrapure water), then concentrated and freeze-dried to collect the freeze-dried collagen sponge.

[0121] The purified freeze-dried sponge was dissolved in ultrapure water and subjected to protein electrophoresis as shown in Figure 6. As can be seen from the figure, bands 705, 706 and 710 are clear and the target band has a high content.

[0122] Example 2: Recombinant collagen cell adhesion activity experiment

[0123] In this example, natural collagen was used as a control, and recombinant 705, 706, and 710 collagens (recombinant type VII collagens with amino acid sequences as shown in SEQ ID No. 2, SEQ ID No. 4, and SEQ ID No. 12) were used as examples to investigate the cell adhesion activity of the recombinant type VII collagen of the present invention.

[0124] The specific steps are as follows:

[0125] (1) Material preparation: Normally culture NIH / 3T3 cells (purchased from the Chinese Academy of Sciences Cell Bank, catalog number GNM6, culture and passaging methods are carried out according to the cell instructions). Recombinant 705, 706, and 710 collagen freeze-dried sponges (respectively designated as sample 1, sample 2, and sample 3) and positive control human collagen (Sigma, catalog number C7774-5MG) were obtained.

[0126] The positive control, human collagen, was prepared as follows: a 5 mL sterile centrifuge tube was weighed, human collagen was added in a clean bench, and the sample was weighed. The sample was then dissolved in ultrapure water and acetic acid was added to a pH of 3.0 (concentration of 5 mg / mL) to obtain a milky white solution. The protein concentration was determined using the UV protein quantification formula: C (mg / mL) = 0.144 * (A215 - A225). Upon use, the solution was diluted to a concentration of 0.5 mg / mL with serum-free DMEM medium and sterilized by filtration through a 0.22 μm sterile filter.

[0127] (2) Coating preparation:

[0128] Add 100 μL of sample (standard / sample / blank control) to each well of a 96-well plate. Standard: Positive control human collagen 0.5 mg / mL (Sigma, Cat. No. C7774-5MG); Samples: 705, 706, and 710 (representing sample 1, sample 2, and sample 3, respectively) collagen 0.5 mg / mL; Blank: D-PBS phosphate buffer.

[0129] Four wells were prepared for each sample coating, and the cells were incubated in a 37°C, 5% CO2 (v / v) incubator for 1-4 hours. Excess coating solution was removed from the wells, and 100 μL of 1% BSA-PBS solution was added. The cells were incubated in a 37°C, 5% CO2 incubator for 1 hour. After removing the liquid from the wells, the cells were washed three times with D-PBS, and the washing solution was discarded. Hoechst-33342 fluorescent dye (10%) was premixed with complete culture medium, and the NIH / 3T3 cells were diluted to 5×10 4 Cells were added to the wells at 100 μL, covered with aluminum foil, and incubated at 37°C, 5% CO₂ for 1 hour. Three replicates were measured; the fourth well was used to adjust microscope parameters and its measurement was not used. The experiment showed that the coated samples exhibited stronger adhesion and were superior to the positive control group (Figure 7), demonstrating that the recombinant type VII collagen described herein exhibits superior cell adhesion compared to native collagen.

[0130] Example 3: Recombinant collagen cell migration experiment

[0131] In this example, NIH / 3T3 cells consistent with those in Example 2 were used to examine the cell migration ability of recombinant 705 (SEQ ID No. 2), 706 (SEQ ID No. 4), and 710 (SEQ ID No. 12) collagen freeze-dried sponges (respectively designated as sample 1, sample 2, and sample 3) and a positive control human collagen (Sigma, catalog number C7774-5MG).

[0132] (1) Experimental preparation: First, use a marker pen to draw horizontal lines evenly on the back of a 6-well plate, using a ruler. Draw lines every 0.5 cm to 1 cm across the holes, with 3 lines drawn across each hole. Add about 5 × 10 5 cells.

[0133] (2) Scratch test: On the second day of cell culture, use the tip of a pipette to scratch the horizontal line on the back of the pipette perpendicular to the ruler. The pipette tip should be vertical and not tilted. Then, rinse the cells three times with PBS to remove the scratched cells. Serum-free medium containing the test sample is added as the experimental group at a concentration of 0.05% (mass to volume ratio). Incubate the cells at 37°C, 5% CO2 in an incubator. Samples are taken at 0, 6, and 24 hours after incubation and photographed.

[0134] (3) Data Processing: ImageJ image processing software was used to calculate the scratch area of ​​each image. The migration rate of each group of cells was calculated by dividing the total area of ​​migrating cells in the fixed scratch area by the initial area of ​​the fixed scratch area. Graphs were plotted with time as the horizontal axis and the migration area ratio as the vertical axis (unit: %). Photos of the experimental and control groups at time 0 and at the end of the experiment were compared. One-way analysis of variance was used to analyze the differences in data between the experimental groups, using the chi-square test. The experimental results are shown in Figures 8 and 9.

[0135] Combining Figures 8 and 9, we can see that at 6 hours into the 24-hour experiment, there was no significant difference between the experimental and blank control groups. However, 24 hours later, the cell migration rate in the sample group was higher. This suggests that as cells continue to grow over time, the experimental group samples have a more pronounced effect on promoting cell migration under physiological conditions, effectively promoting cell adhesion to biomaterials.

[0136] In summary, the recombinant type VII collagen obtained by the method of the present invention has similar physicochemical properties and biological functions to the original sequence, a smaller molecular weight, and improved tissue absorption. Cell adhesion experiments using in vitro cultured NIH / 3T3 cells with type VII collagen and commercial collagen demonstrated no significant difference in adhesion activity between the recombinant type VII collagen of the present invention and commercial type I collagen.

[0137] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above implementations. 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 are within the scope of protection of the present invention.

Claims

1. A type VII collagen, characterized in that The type VII collagen has an amino acid sequence as shown in SEQ ID No.1, SEQ ID No.3, SEQ ID No.5, SEQ ID No.7, SEQ ID No.9 or SEQ ID No.

11.

2. A nucleic acid encoding the type VII collagen according to claim 1.

3. A recombinant vector, characterized in that The recombinant vector comprises the nucleic acid according to claim 2.

4. The recombinant vector according to claim 3, characterized in that The recombinant vector includes pPICZαB, pFLDα, and pPIC9K, and the connection site is between XhoI and NotI.

5. The method for preparing type VII collagen according to claim 1, wherein The method comprises: (1) Selecting a type VII collagen sequence to construct a collagen tandem sequence, and then constructing a recombinant expression vector comprising the collagen tandem sequence; the amino acid sequence of the collagen tandem sequence is a sequence as shown in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, or SEQ ID No. 12; (2) The obtained recombinant expression vector was linearized with Sal I to obtain a linearized plasmid; (3) The linearized plasmid is electroporated into a host bacterium, and recombinant engineered bacteria containing or expressing type VII collagen are screened and verified to obtain the recombinant engineered bacteria, and the expression is induced by fermentation to obtain the type VII collagen.

6. The method for preparing type VII collagen according to claim 5, wherein: The nucleic acid encoding the collagen tandem sequence is a nucleotide sequence as shown in SEQ ID No. 13 to SEQ ID No. 18 or a degenerate sequence thereof.

7. The method for preparing type VII collagen according to claim 5, characterized in that: In step (1), the recombinant expression vector includes pPICZαB, pFLDα, and pPIC9K, and the connection site is between XhoI and NotI.

8. The method for preparing type VII collagen according to claim 5, wherein: In step (3), the host bacteria include Pichia pastoris, Saccharomyces cerevisiae, and Hansenula.

9. The method for preparing type VII collagen according to claim 8, characterized in that: The host bacteria is Pichia pastoris.

10. The method for preparing type VII collagen according to claim 5, characterized in that: In step (3), the recombinant engineered bacteria are deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms, with the deposit numbers being: CGMCC No. 29601, CGMCC No. 29602, CGMCC No. 29603, CGMCC No. 29604, CGMCC No. 29605, and CGMCC No. 29606.

11. A composition characterized in that The composition comprises the type VII collagen according to claim 1, or the type VII collagen encoded by the nucleic acid according to claim 2, or the recombinant vector according to any one of claims 3 to 4; the composition is a biomaterial, a tissue engineering product, or a cosmetic.

12. A product, characterized in that The product comprises the type VII collagen described in claim 1, or the type VII collagen encoded by the nucleic acid described in claim 2, or the recombinant vector described in any one of claims 3 to 4, or the composition described in claim 11; the product is a biomaterial, a tissue engineering product, or a cosmetic.

13. Use of the type VII collagen according to claim 1, or the type VII collagen encoded by the nucleic acid according to claim 2, or the recombinant vector according to any one of claims 3 to 4, or the composition according to claim 11, or the product according to claim 12 in the preparation of biomaterials, tissue engineering products, and cosmetics that promote cell adhesion.

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