A recombinant human collagen type XVII COL-1012-A, COL-1012-B and preparation method thereof

Abstract

This invention provides recombinant type XVII human collagen COL-1012-A and COL-1012-B and their preparation method, belonging to the fields of genetic engineering and synthetic biology. Specifically, it relates to a recombinant type XVII human collagen COL-1012-A, the amino acid sequence of which is shown in SEQ ID NO.1; a recombinant strain of Pichia pastoris GS115COL-1012-A; and a recombinant type XVII human collagen COL-1012-B, the amino acid sequence of which is shown in SEQ ID NO.12. The recombinant type XVII human collagen COL-1012-A and COL-1012-B provided by this invention can be effectively expressed using Pichia pastoris and exhibit cell adhesion activity and cell proliferation-promoting activity.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and synthetic biology, specifically relating to a recombinant type XVII human collagen COL-1012-A and COL-1012-B and their preparation method. Background Technology

[0002] Collagen, also known as collagen peptide, exists in a triple-helix configuration within body tissues. It is a key structural component of the extracellular matrix, and at least 28 different types are known to exist in all tissues and organs, including skin, bone, tendons, ligaments, cartilage, and other specific tissues. It not only provides tissue strength, durability, and flexibility but also participates extensively in specific biological interactions. As a natural protein macromolecule, collagen is an important biodegradable material. The main functions of collagen in the human body include low immunogenicity, biodegradability, biocompatibility, promotion of cell growth, and hemostasis. Due to its crucial role in human tissues and its significant advantages as a biomaterial, research on collagen mainly includes its synthesis and preparation, the design and development of active collagen peptides, the exploration of new functions of collagen-based materials, and the development and manufacturing of various types of collagen-based materials and medical materials.

[0003] Collagen type XVII (COL17 / BP180 / BPAG2), also known as COL17, is a transmembrane protein containing 1497 amino acids. It includes intracellular, transmembrane, and extracellular domains. Its most prominent structural feature is its intracellular N-terminus and its long extracellular C-terminus, which connects to anchor fibers in the lamina pellucida of the skin's basement membrane. Human COL17 contains 15 collagen domains located in the extracellular domain. These extracellular collagen domains are likely the structural sites where it interacts with extracellular matrix components to stabilize the connection between the epidermis and dermis. These collagen domains are separated from each other by short non-collagenous domains (NC, non-helical domains). Collagen type XVII is expressed in various tissues, with the highest expression level in the skin, accounting for nearly 70%. Type XVII collagen mediates the interaction between stem cells and surrounding cells and the matrix, regulating stem cell migration and proliferation to regulate skin homeostasis, aging, and wound repair. It also promotes hair follicle repair and regeneration, slows hair loss, increases local hair density, and enhances hair diameter. However, the extraction of type XVII collagen from animal sources presents significant challenges due to immunological and viral issues, low expression levels, and difficulties in large-scale production. Therefore, recombinant collagen extraction using genetic engineering techniques is now widely employed. This technology carries a lower potential pathogen risk, enables large-scale, high-purity production, and offers greater sustainability and economic benefits.

[0004] Currently, there are five main expression systems for recombinant collagen: prokaryotic bacterial (Escherichia coli) protein expression systems, eukaryotic yeast protein expression systems (Pichia pastoris / Saccharomyces cerevisiae, etc.), eukaryotic insect cell protein expression systems (insect cells infected with baculovirus, etc.), eukaryotic mammalian cell protein expression systems (CHO cells, HEK293 cells, etc.), and plant expression systems (tobacco, tomato, etc.). Prokaryotic collagen expression requires extremely high purification standards, and prokaryotic expression systems naturally contain endotoxins and peptidoglycans, requiring complex purification processes for removal. Insect cell and mammalian cell expression of human type XVII collagen are generally not used for large-scale collagen production due to their high cost and low yield. Yeast expression systems are theoretically advantageous due to their ease of genetic modification and the ability to synthesize enzymes required for post-translational modifications and protein folding, offering advantages such as easy high-density fermentation and low cost. To date, there has been considerable research on the expression of human collagen using yeast, such as Pichia pastoris and Saccharomyces cerevisiae, with recombinant human collagen obtained using engineered Pichia pastoris showing the highest expression level and hydroxylation efficiency. Pichia pastoris possesses a systematic gene editing system and a complete protein secretion and expression mechanism, making it widely used in the food and pharmaceutical fields. Therefore, utilizing Pichia pastoris to secrete and express recombinant type XVII human collagen has demonstrated significant application value in biomedicine, drug development, skincare products, and hair care.

[0005] Type XVII human collagen is a transmembrane protein. When expressed in eukaryotic cells, it is mostly not secreted extracellularly but rather fixed to the cell membrane. Furthermore, Type XVII human collagen has a very long amino acid sequence (1497 amino acids) and a large molecular weight (180 kDa), theoretically making effective extracellular secretion difficult and prone to degradation. Successful expression requires careful selection of relevant sequences. Current technologies for recombinant Type XVII human collagen often employ either selecting a single collagen region-related amino acid sequence and repeating it tandemly as the basic unit, or selecting multiple collagen region-related amino acid sequences and repeating them tandemly.

[0006] There are numerous reports on recombinant forms of human collagen type XVII in existing technologies. However, the effects of different recombination methods on collagen are currently unpredictable, and it is impossible to determine whether the recombinant forms can be effectively expressed in host bacteria or whether they possess biological activity. Given the wide range of applications for human collagen type XVII, developing and researching recombinant forms of human collagen type XVII that can be effectively expressed and possess certain biological activities is of great significance. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides recombinant type XVII human collagen COL-1012-A and COL-1012-B and their preparation method.

[0008] The technical solution of the present invention is as follows:

[0009] A recombinant type XVII human collagen COL-1012-A, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] The coding gene sequence of the above-mentioned recombinant type XVII human collagen COL-1012-A is SEQ ID NO.4, which contains 6 HIS tag coding genes.

[0011] A recombinant vector comprising the gene sequence encoding the above-mentioned recombinant type XVII human collagen COL-1012-A, SEQ ID NO.4.

[0012] According to a preferred embodiment of the present invention, the expression vector of the recombinant vector is pPIC9K.

[0013] A recombinant bacterium containing the coding gene sequence SEQ ID NO.4 of the above-mentioned recombinant type XVII human collagen COL-1012-A.

[0014] According to a preferred embodiment of the present invention, the host strain of the recombinant bacteria is Pichia pastoris.

[0015] A method for constructing recombinant Pichia pastoris includes the following steps: cloning the above gene sequence SEQ ID NO.4 into the EcoRI and Not I restriction sites of pPIC9K to obtain the recombinant expression plasmid pPIC9K-COL-1012-A, and then transforming the recombinant expression plasmid pPIC9K-COL-1012-A into Pichia pastoris to obtain recombinant Pichia pastoris.

[0016] According to a preferred embodiment of the present invention, the Pichia pastoris in the construction method is Pichia pastoris GS115.

[0017] A Pichia pastoris GS115 COL-1012-A specimen was deposited on October 14, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20242195.

[0018] The application of the above-mentioned Pichia pastoris GS115 COL-1012-A strain in the preparation of the above-mentioned recombinant type XVII human collagen COL-1012-A.

[0019] The preparation method of the above-mentioned recombinant type XVII human collagen COL-1012-A includes the following steps:

[0020] The recombinant Pichia pastoris strain constructed using the above method or the above-mentioned Pichia pastoris GS115 COL-1012-A strain were fermented in shake flasks and fermenters, and the fermentation culture was obtained by induction with methanol. The bacterial cells were separated and the liquid was retained. Recombinant type XVII human collagen COL-1012-A was extracted from the liquid.

[0021] According to a preferred embodiment of the present invention, in the preparation method, recombinant Pichia pastoris or Pichia pastoris GS115COL-1012-A strain is inoculated into BSM medium for fermentation, and methanol is used to induce expression for 24-96 hours to obtain fermentation culture.

[0022] A recombinant type XVII human collagen COL-1012-B, the amino acid sequence of which is shown in SEQ ID NO.12.

[0023] The gene sequence encoding the recombinant type XVII human collagen COL-1012-B is SEQ ID NO.15, which contains 6 HIS tag encoding genes.

[0024] A recombinant vector comprising the encoding gene sequence SEQ ID NO.15 of the above-mentioned recombinant type XVII human collagen COL-1012-B.

[0025] According to a preferred embodiment of the present invention, the expression vector of the recombinant vector is pPIC9K.

[0026] A recombinant bacterium containing the coding gene sequence SEQ ID NO.15 of the above-mentioned recombinant type XVII human collagen COL-1012-B.

[0027] According to a preferred embodiment of the present invention, the host strain of the recombinant bacteria is Pichia pastoris.

[0028] A method for constructing recombinant Pichia pastoris includes the following steps: cloning the above gene sequence SEQ ID NO.15 into the EcoRI and NotI restriction sites of pPIC9K to obtain the recombinant expression plasmid pPIC9K-COL-1012-B, and then transforming the recombinant expression plasmid pPIC9K-COL-1012-B into Pichia pastoris to obtain recombinant Pichia pastoris.

[0029] The preparation method of the above-mentioned recombinant type XVII human collagen COL-1012-B includes the following steps:

[0030] The recombinant Pichia pastoris constructed using the above method was fermented in shake flasks and fermenters, and the fermentation culture was obtained by induction with methanol. The cells were separated and the liquid was retained. Recombinant type XVII human collagen COL-1012-B was extracted from the liquid.

[0031] According to a preferred embodiment of the present invention, in the preparation method, recombinant Pichia pastoris is inoculated into BSM medium for fermentation, and methanol is used to induce expression for 24-96 hours to obtain a fermentation culture.

[0032] The beneficial effects of the present invention include at least the following:

[0033] The recombinant human collagen COL-1012-A and COL-1012-B of type XVII provided by this invention can be effectively expressed using Pichia pastoris. Furthermore, the recombinant human collagen COL-1012-A exhibits superior cell adhesion activity compared to natural human collagen and bovine serum albumin, and also demonstrates cell proliferation-promoting activity. Compared to COL-1012-B, recombinant human collagen COL-1012-A shows superior cell adhesion and cell proliferation-promoting effects. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the recombinant plasmid pPIC9K-COL-1012-A for recombinant type XVII human collagen constructed in this invention.

[0035] Figure 2 This is a schematic diagram of the recombinant plasmid pPIC9K-COL-1012-B of recombinant type XVII human collagen constructed in this invention.

[0036] Figure 3 This is a schematic diagram of the recombinant plasmid pPIC9K-COL-24-A for recombinant type XVII human collagen constructed in this invention.

[0037] Figure 4 Nucleic acid gel electrophoresis image for verifying the recombinant plasmid constructed in this invention;

[0038] In the figure: lane M is the protein marker; lanes 1-2 are for verifying the gene fragment COL-1012-A transformed into the pPIC9K plasmid; lanes 3-4 are for verifying the gene fragment COL-1012-B transformed into the pPIC9K plasmid; lanes 5-6 are for verifying the gene fragment COL-24-A transformed into the pPIC9K plasmid.

[0039] Figure 5 The image shows the SDS-PAGE results of the supernatant of the fermentation broth obtained after methanol induction for 24 h for the fermentation culture of recombinant Pichia pastoris strain.

[0040] In the figure: Lane M is the protein marker; Lane 1 is the target protein band of recombinant strain Pichia pastoris GS115 COL-1012-A, Lane 2 is the target protein band of recombinant strain Pichia pastoris GS115 COL-1012-B, and Lane 3 is the target protein band of recombinant strain Pichia pastoris GS115 COL-24-A.

[0041] Figure 6 The image shows the Westonblot results of the supernatant of the fermentation broth obtained after methanol induction for 24 h for the fermentation culture of recombinant Pichia pastoris strain.

[0042] In the figure: Lane M is the protein marker; Lane 1 is the target protein band of recombinant strain Pichia pastoris GS115 COL-1012-A, and Lane 2 is the target protein band of recombinant strain Pichia pastoris GS115 COL-1012-B.

[0043] Figure 7 The HPLC chromatogram shows the protein purity of recombinant type XVII human collagen COL-1012-A.

[0044] Figure 8 The chromatogram for HPLC determination of protein purity of recombinant type XVII human collagen COL-1012-B.

[0045] Figure 9 This figure shows the comparison of cell adhesion activities between recombinant type XVII human collagen, natural human collagen, and bovine serum albumin.

[0046] Figure 10 The figure shows the verification results of the cell proliferation-promoting effect of recombinant type XVII human collagen. Detailed Implementation

[0047] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto.

[0048] In the embodiments of the present invention, unless otherwise described, conventional experimental methods were used. The processes involved in the embodiments, unless otherwise described, can be understood and implemented by those skilled in the art based on the product manual or basic knowledge in the field, and therefore will not be described in detail.

[0049] In the following examples, Pichia pastoris GS115 was used as the starting strain. Recombinant human type XVII collagen was expressed using pPIC9K as the integration vector. The nucleotide sequence was synthesized, PCR amplified, and purified by Nanjing Genscript Biotech Co., Ltd., and then seamlessly cloned into pPIC9K.

[0050] Example 1: Construction of recombinant Pichia pastoris strain

[0051] (1) Design of the amino acid sequence and expression of the DNA sequence of recombinant type XVII human collagen

[0052] In this invention, the sequence optimization is based on type XVII human collagen. The specific sequence of type XVII human collagen is referenced from NCBI reference sequence Q9UMD9.3 (https: / / www.ncbi.nlm.nih.gov / protein / Q9UMD9.3).

[0053] The sequence selected in this invention is the 9th to 12th collagen regions of the extracellular domain of type XVII human collagen. Amino acids 906-1047 of the full-length sequence of type XVII human collagen, totaling 142 amino acids, were selected, including 4 collagen regions and 3 non-collagen regions. The inventors discovered that this not only enables highly efficient secretory and soluble expression of type XVII human collagen in eukaryotic host cells such as Pichia pastoris, but also exhibits superior cell adhesion and cell proliferation activities compared to commercially available natural human collagen. This is the preferred sequence for type XVII human collagen, and the specific amino acid sequence is shown in SEQ ID NO.1. The encoded DNA sequence is SEQ ID NO.2. Based on SEQ ID NO.2, 6 HIS tags were added to the carboxyl terminus of the sequence to obtain sequence SEQ ID NO.3. The codons of the encoding sequence SEQ ID NO.3 were optimized using Pichia pastoris as the host, and the optimized sequence is shown in SEQ ID NO.4, named COL-1012-A.

[0054] The nucleotide sequence SEQ ID NO.4 of recombinant human collagen type XVII COL-1012-A was synthesized by Nanjing Genscript Biotech Co., Ltd., and then cloned into the EcoRI and Not I restriction sites of the Pichia pastoris expression vector pPIC9K (purchased from Thermo Fisher Scientific) to obtain the recombinant expression plasmid pPIC9K-COL-1012-A.

[0055] The present invention also selects the collagen regions of regions 9 to 12 and some non-collagen regions of regions 2 to 5 of the extracellular domain of type XVII human collagen. The recombinant sequence is composed of seven sequences, that is, the non-collagen regions of regions 9 to 12 are replaced with some non-collagen regions of regions 2 to 5. The recombinant amino acid sequence is composed of SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, and SEQ ID NO.11 in sequence to obtain the amino acid sequence SEQ ID NO.12.

[0056] The above sequence contains 142 amino acids, including 4 collagen regions and 3 non-collagen regions. The encoded DNA sequence is shown in SEQ ID NO.13. Based on SEQ ID NO.13, 6 HIS tags were added to the carboxyl terminus of the sequence to obtain the sequence SEQ ID NO.14, as shown below. The codons of the sequence encoding SEQ ID NO.14 were optimized using Pichia pastoris as the host, and the optimized sequence was named COL-1012-B, as shown in SEQ ID NO.15.

[0057] The nucleotide sequence SEQ ID NO.15 of recombinant human type XVII collagen COL-1012-B was synthesized by Nanjing Genscript Biotech Co., Ltd. and cloned into the EcoRI and NotI restriction sites of pPIC9K to obtain the recombinant expression plasmid pPIC9K-COL-1012-B.

[0058] This invention also selected the sequences of extracellular domains 2 to 5 of type XVII human collagen, and amino acids 1215-1413 of the full-length sequence of type XVII human collagen, totaling 199 amino acids, including 4 collagen regions and 3 non-collagen regions, named COL-24, with the specific amino acid sequence shown in SEQ ID NO.16. The DNA sequence encoding COL-24 is SEQ ID NO.17. Based on SEQ ID NO.17, 6 HIS tags were added to the carboxyl terminus of the sequence to obtain sequence SEQ ID NO.18. The codons of the sequence encoding SEQ ID NO.18 were optimized using Pichia pastoris as the host, and the optimized sequence was named COL-24-A, with the specific sequence shown in SEQ ID NO.19.

[0059] The nucleotide sequence SEQ ID NO.19 of recombinant human type XVII collagen COL-24-A was synthesized by Nanjing Genscript Biotech Co., Ltd. and cloned into the EcoRI and NotI restriction sites of pPIC9K to obtain the recombinant expression plasmid pPIC9K-COL-24-A.

[0060] (2) Construction and screening of recombinant Pichia pastoris strains

[0061] The target sequence in the recombinant expression plasmid was amplified using universal primers (AOX-F / R). After verification by colony PCR and sequencing, the recombinant expression plasmids pPIC9K-COL-1012-A, pPIC9K-COL-1012-B, and pPIC9K-COL-24-A were digested with Sal I (purchased from Takara Bio Inc., specific operation according to the kit instructions) at 37℃ for half an hour to obtain linearized plasmids, which were then recovered using a product purification kit (purchased from Nanjing Novizan Biotechnology Co., Ltd.).

[0062] Competent cells of Pichia pastoris GS115 (purchased from Beyotime Biotechnology Co., Ltd.) with empty host strain were prepared. Single colonies were picked from antibiotic-free YPD plates and inoculated into YPD (1% yeast extract, 2% peptone, and 2% glucose) liquid shake flasks. The cells were incubated overnight at 30°C and 220 rpm on a shaker until OD was reached. 600 =1.3-1.6, centrifuged at 3000r for 3 min at 4℃, collected the cells and resuspended them in pre-cooled sterile water, repeated twice. Centrifuged again and collected the cells, resuspended them in pre-cooled 1M sorbitol, repeated twice. Centrifuged again and collected the cells, resuspended them in 100 μL of pre-cooled 1M sorbitol to obtain the prepared competent Pichia pastoris GS115. 10 μL of the product (the linearized recovery product above) was mixed with competent Pichia pastoris GS115 and transferred to a pre-cooled electroporation vessel, and placed on ice for 10 min. After electroporation with PIC parameters, 1M sorbitol solution was added immediately, and incubated in a 30℃ incubator for 2 h. 100 μL of the transformed bacterial solution was spread on MD (2% glucose, 1.34% YNB, 4×10⁻⁶ mcg) plates. -4Incubate the culture medium in biotin and 1.5% agar plates in an incubator at 30°C, inverted for 2-4 days, until single colonies appear. Single colonies were spotted onto YPD plates containing 4 mg / mL G418 and incubated upside down at 30°C for 3-4 days. Transformants with good growth on the G418 plates were selected for purification and secondary screening. Genome extraction was performed on the transformed strains, and after PCR verification, they were preserved with 50% glycerol to obtain recombinant Pichia pastoris strains expressing recombinant plasmids pPIC9K-COL-1012-A, pPIC9K-COL-1012-B, and pPIC9K-COL-24-A. The constructed recombinant strain Pichia pastoris GS115 COL-1012-A, Pichia pastoris GS115 COL-1012-B, and Pichia pastoris GS115 COL-24-A was then obtained. Sample COL-1012-A was sent to the China Center for Type Culture Collection for preservation. The preservation information for the strain is as follows:

[0063] A Pichia pastoris GS115 COL-1012-A specimen was deposited on October 14, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20242195.

[0064] Figure 1 , Figure 2 , Figure 3 The diagrams show the recombinant plasmids pPIC9K-COL-1012-A, pPIC9K-COL-1012-B, and pPIC9K-COL-24-A constructed according to this invention. Figure 4 The image shows a nucleic acid gel electrophoresis diagram for verifying the recombinant plasmid constructed in this invention. Lane M is a protein marker; lanes 1-2 are for verifying the gene fragment COL-1012-A transformed into the pPIC9K plasmid; lanes 3-4 are for verifying the gene fragment COL-1012-B transformed into the pPIC9K plasmid; and lanes 5-6 are for verifying the gene fragment COL-24-A transformed into the pPIC9K plasmid.

[0065] Example 2: Fermentation of recombinant Pichia pastoris strain and purification of recombinant human collagen type XVII

[0066] (1) Fermentation of recombinant Pichia pastoris strain

[0067] The recombinant Pichia pastoris strain was inoculated into BMGY (1% yeast extract, 2% peptone, 100 mmol / L potassium phosphate buffer (pH 6.0), 1.34% YNB, 4 × 10⁻⁶ ppm). -4In a culture medium containing biotin and 1% glycerol, incubate at 30°C and 220 rpm for 20 hours until OD500 reaches zero. 600 =2-6, collect bacterial cells by centrifugation at 12000r for 10min, and use BMMY (1% yeast extract, 2% peptone, 100mmol / L potassium phosphate buffer (pH 6.0), 1.34% YNB, 4×10 -4 The bacterial cells were resuspended in a culture medium containing biotin and 1% methanol and incubated at 25°C and 220 rpm for 96 h. Every 24 h, samples were taken and 100% methanol was added to bring the final concentration to 1.0%. After sampling, the cells were centrifuged at 12000 rpm for 1 min, and the supernatant was collected and added to 5× loading buffer. The cells were then heated in a 100°C metal bath for 10 min before SDS-PAGE and Western Blot analysis. Figure 5 The results of SDS-PAGE analysis of the supernatant of the fermentation broth obtained after methanol induction for 24 h from the fermentation culture of recombinant Pichia pastoris strain are shown. Lane M is the protein marker; lane 1 is the target protein band of recombinant Pichia pastoris GS115 COL-1012-A, lane 2 is the target protein band of recombinant Pichia pastoris GS115 COL-1012-B, and lane 3 is the target protein band of recombinant Pichia pastoris GS115 COL-24-A. SDS-PAGE analysis concluded that recombinant type XVII human collagen COL-1012-A and COL-1012-B of the corresponding sizes were successfully and effectively expressed, effectively secreted in the supernatant, and exhibited single bands on electrophoresis. The theoretically predicted molecular weights of COL-1012-A, COL-1012-B, and COL-24-A collagen were 15.28 kDa, 13.16 kDa, and 19.24 kDa, respectively, while the apparent molecular weights detected on SDS-PAGE were larger than the theoretical predictions. Figure 5 The target protein expressed by recombinant strain Pichia pastoris GS115 COL-1012-A was higher than that expressed by recombinant strain Pichia pastoris GS115 COL-1012-B, while recombinant strain Pichia pastoris GS115 COL-24-A did not express any protein. The expressed protein was detected by Western blotting. Figure 6 The results of Western blotting analysis of the supernatant of the fermentation broth obtained after methanol induction for 24 h in the fermentation culture of recombinant Pichia pastoris strain are shown. Lane M is the protein marker; lane 1 is the target protein band of recombinant Pichia pastoris GS115 COL-1012-A; and lane 2 is the target protein band of recombinant Pichia pastoris GS115 COL-1012-B.

[0068] Recombinant bacteria Pichia pastoris GS115 COL-24-A and Pichia pastoris GS115 COL-1012-B were inoculated into YPD liquid medium and cultured overnight at 30°C and 220 rpm to obtain primary seed culture. These were then transferred to secondary seed culture flasks at a volume ratio of 2% and cultured at 30°C and 220 rpm until OD... 600 The pH range was 6.0-10.0. Fermentation was conducted using BSM fermentation medium, with successfully activated secondary seed culture inoculated into the fermenter at a 10% (v / v) inoculum. The initial culture temperature was 30℃, the agitator speed was 200 rpm, and aeration was 1 vvm. The pH was adjusted to 5.0 using 50% ammonia. Dissolved oxygen (DO) in the fermenter was maintained above 20% by adjusting the dissolved oxygen-correlated agitation speed. After 18-20 hours of fermentation, the glycerol in the medium was depleted, and the DO began to rise. Dissolved oxygen feedback agitation was used to feed glycerol at a rate of 1.0 mL / min when the DO exceeded 30%. Feeding was stopped when the wet cell mass reached 250-400 g / L, and the agitator speed was increased to 900 rpm. Once the DO stabilized, methanol was added for induction. The induction temperature was 25℃. For the first 4 hours of induction, the methanol feed rate was 0.06 mL / min to promote cell adaptation to the carbon source conversion to methanol. After 4 hours of induction, feeding was stopped. Once the dissolved oxygen (DO) value began to rise, it indicated that the methanol in the fermenter had been consumed. At this point, dissolved oxygen feedback was set to start adding the remaining methanol feed solution at a feeding rate of 0.12 mL / min. Simultaneously, dissolved oxygen was monitored, and feeding was started when the DO value was above 30%. Induction continued for 72 hours to obtain the fermentation broth. Samples were taken every 8 hours during the induction period for subsequent analysis and detection.

[0069] The above BSM fermentation medium formula is as follows: 26.7 mL / L phosphate (85%), 0.93 g / L calcium sulfate, 18.2 g / L potassium sulfate, 14.9 g / L magnesium sulfate, 4.13 g / L potassium hydroxide, 40 g / L glycerol, and water as solvent; after sterilization at 121℃ for 20 min, PTM1 trace element stock solution is added at 4.35 mL / L after the temperature drops to room temperature, and the pH is adjusted to 5.0 with 50% (v / v) ammonia water.

[0070] The above glycerol feed solution formula is as follows: 50% (mass ratio) glycerol, sterilized at 121℃ for 20 min, and after the temperature drops to room temperature, add PTM1 trace element stock solution at 12 mL / L and mix well;

[0071] The above methanol feed solution formula is: 100% methanol, added to PTM1 trace element mother liquor at a rate of 12 mL / L, and mixed well;

[0072] The formula for the above-mentioned trace element mother liquor PTM1 is as follows: copper sulfate 6 g / L, sodium iodide 0.08 g / L, manganese sulfate 3 g / L, sodium molybdate 14.9 g / L, boric acid 0.02 g / L, cobalt chloride 0.5 g / L, zinc chloride 20 g / L, ferrous sulfate heptahydrate 65 g / L, biotin 0.2 g / L, sulfuric acid 5 mL / L, and water as solvent; it is filtered through a 0.22 μm filter membrane for sterilization and stored at 4℃ for later use.

[0073] The protein concentration in the supernatant after fermentation was determined using BCA protein quantification technology. A BCA kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) was prepared. 1.2 ml of protein standard preparation solution was added to one tube of protein standard (30 mg BSA) and thoroughly dissolved to prepare a 25 mg / ml protein standard solution. An appropriate amount of the 25 mg / ml protein standard was taken and diluted to a final concentration of 0.5 mg / ml. Based on the sample quantity, a BCA working solution was prepared by mixing BCA reagent A and BCA reagent B at a volume ratio of 50:1. The mixture was thoroughly mixed. Protein concentrations were determined by adding 0, 1, 2, 4, 8, 12, 16, and 20 μL of standard to the wells of a 96-well plate, and then adding standard diluent to bring the total volume to 20 μL. These concentrations correspond to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml, respectively. Add an appropriate volume of sample to the wells of a 96-well plate. Add 200 μL of BCA working solution to each well and mix thoroughly by pipetting or shaking the plate. Incubate at 37°C for 20-30 minutes. Measure the absorbance at 562 nm using a microplate reader. Calculate the protein concentration of the sample based on the standard curve and the sample volume used. The total protein content in the supernatant of recombinant strains Pichia pastoris GS115 COL-1012-A and Pichia pastoris GS115 COL-1012-B after fermentation was 11.37 g / L and 4.60 g / L, respectively.

[0074] (2) Purification of recombinant type XVII human collagen

[0075] ① Centrifuge the bacterial solution after it has been placed in the tank at 9000 rpm for 15 min, collect the supernatant, and microfilter it with a 0.22 μm hollow fiber to obtain the permeate. The original solution is 100 mL. Wash it with three times the volume of ultrapure water to obtain the clear solution.

[0076] ② The clarified solution obtained above was concentrated by ultrafiltration using a 5kDa hollow fiber filter, followed by single-volume washing, i.e., adding an equal volume of buffer solution (30mM NaAC, 2M NaCl, pH=4.8) once for washing, repeated three times, finally obtaining a concentrated solution with pH 5.0 and conductivity of 4-5ms / cm.

[0077] ③ After ultrafiltration concentration of the pretreatment sample, ion exchange chromatography was performed. The column (TH-S ion exchange chromatography column produced by Chutian Microsphere Biotechnology Co., Ltd.) was equilibrated with buffer A (30mM NaAC, pH=4.8) before loading the sample. The UV setting was 220nm. 800mL of 3% (buffer B to water ratio) buffer B (30mM NaAC, 2M NaCl, pH=4.8) was used for elution, and the eluent was collected. Protein was collected by eluting with 35% buffer B. Protein collection began when the peak was displayed on the screen. Multiple loadings were performed, and the final collected solution was the purified target protein solution. Product purity was determined by HPLC using a BioCoreSEC-150 column. The mobile phase was 50mM phosphate buffer (pH 6.8) containing 300mM sodium chloride, with isocratic elution, a flow rate of 0.5mL / min, an injection volume of 5μL, and a column temperature of 30℃. The purified protein was filtered and injected, and detected at 220nm. The chromatogram is shown below. Figure 7 and Figure 8 As shown, the purities of recombinant proteins COL-1012-A and COL-1012-B were 98.79% and 97.91%, respectively. After purification, the concentrate was obtained by desalting ultrafiltration using a 5 kDa hollow fiber, followed by freeze-drying to obtain lyophilized powder.

[0078] Example 3: Bioactivity experiment of recombinant type XVII human collagen

[0079] 1. Cell adhesion experiment

[0080] Specific implementation method: Recombinant type XVII human collagen COL-1012-A and COL-1012-B lyophilized products, and control natural human collagen (Sigma, catalog number C7774) and bovine serum albumin (BSA, purchased from Sangon Biotech (Shanghai) Co., Ltd.) were diluted to 0.5 mg / mL with PBS (pH 7.4). 100 μL of each protein solution and blank PBS solution were added to a 96-well cell culture plate and incubated at room temperature for 60 min. Then, 1 × 10⁻⁶ ppm of PBS was added to each well. 5 One well-cultured NIH / 3T3 cell line (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GMNM6; culture and passage methods were performed according to the cell instructions) was incubated at 37°C and 5% CO2 for 60 min. Cells in the wells were washed four times with PBS. OD was measured using a cell adhesion assay kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.). 492 nm The absorbance value was used to calculate the cell adhesion rate based on the blank control.

[0081] Cell adhesion rate = [(Experimental group cell OD - blank OD) / (Control group cell OD - blank OD)] × 100%

[0082] like Figure 9 As shown, the cell adhesion activities of recombinant type XVII human collagen COL-1012-A and COL-1012-B were no less than or better than those of natural human collagen (purchased from Sigma, catalog number C7774) and bovine serum albumin; at the same concentration, the cell adhesion activity of recombinant type XVII human collagen COL-1012-A was significantly better than that of COL-1012-B.

[0083] 2. Cell proliferation experiment

[0084] Specific implementation method: Recombinant type XVII human collagen COL-1012-A and COL-1012-B lyophilized products were dissolved in pure water, the pH was adjusted to 7.4, and sterilized by filtration through a 0.22μm filter membrane. The maintenance culture medium (0.5% FBS + 95.5% DMEM(H)) was diluted to 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL as negative controls. hEGF (purchased from Lonza) was used as a positive control, with concentrations of 1 ng / mL, 0.5 ng / mL, 0.25 ng / mL, and 0.125 ng / mL. HaCat cell line (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number SCSP-5091) was cultured in complete culture medium (88% DMEM + 10% FBS + 1% glutamine + 1% sodium pyruvate) at 37℃ and 5% carbon dioxide, with the cell concentration controlled at (1.0-5.0) × 10⁻⁶ cells per mL. 5 Cells were passaged and used for biological activity assays 24 hours later. Cells were collected and reconstituted with complete culture medium to a concentration of 5.0 × 10⁶ cells per mL. 4 A cell suspension of 100 μL was added to a 96-well cell culture plate and cultured at 37°C and 5% CO2. After 24 hours, the medium was replaced with maintenance medium. The maintenance medium was discarded, and control and test solutions were added to the prepared cell culture plate. The plate was then cultured at 37°C and 5% CO2 for 36 hours. The 96-well plate was removed, and cell morphology was observed under a microscope. The liquid was removed, and 50 μL of LTT solution (purchased from Shanghai Beyotime Biotechnology Co., Ltd., concentration 1 mg / mL) was added to each well. The plate was then incubated at 37°C and 5% CO2. After 4 hours, the supernatant was removed, and 100 μL of isopropanol was added to each well to dissolve the crystals. The absorbance was measured at 570 nm. Figure 10As shown, hEGF, as a positive control, promoted HaCat cell proliferation. Recombinant type XVII human collagen COL-1012-A and COL-1012-B also promoted HaCat cell proliferation at concentrations of 0.125-1 mg / mL, especially COL-1012-A, whose HaCat cell proliferation activity increased with increasing sample concentration. Compared with COL-1012-B, recombinant type XVII human collagen COL-1012-A showed superior cell adhesion and cell proliferation activity.

Claims

1. A recombinant type XVII human collagen COL-1012-A, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. The encoding gene sequence of the recombinant type XVII human collagen COL-1012-A according to claim 1 is SEQ ID NO.4, which contains 6 HIS tag encoding genes.

3. A recombinant vector, characterized in that, The gene sequence encoding the recombinant type XVII human collagen COL-1012-A as described in claim 2 is SEQ ID NO.4; Preferably, the expression vector of the recombinant vector is pPIC9K; Preferably, a recombinant bacterium contains the gene sequence encoding the recombinant type XVII human collagen COL-1012-A as described in claim 2, SEQ ID NO.4; Preferably, the host strain of the recombinant bacteria is Pichia pastoris.

4. A method for constructing recombinant Pichia pastoris, characterized in that, The process includes the following steps: cloning the gene sequence SEQ ID NO.4 described in claim 2 into the EcoRI and NotI restriction sites of pPIC9K to obtain the recombinant expression plasmid pPIC9K-COL-1012-A, and then transforming the recombinant expression plasmid pPIC9K-COL-1012-A into Pichia pastoris to obtain recombinant Pichia pastoris. Preferably, the Pichia pastoris in the construction method is Pichia pastoris GS115.

5. A strain of Pichia pastoris GS115 COL-1012-A was deposited on October 14, 2024, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO:M 20242195.

6. The use of the Pichia pastoris GS115 COL-1012-A strain of claim 5 in the preparation of the recombinant type XVII human collagen COL-1012-A of claim 1.

7. The method for preparing recombinant type XVII human collagen COL-1012-A according to claim 1, characterized in that, Includes the following steps: The recombinant Pichia pastoris strain constructed by the method described in claim 4 or the Pichia pastoris GS115COL-1012-A strain described in claim 5 was used for shake flask and fermenter fermentation, and methanol was used to induce the fermentation culture. The cell cells were separated and the liquid was retained. Recombinant type XVII human collagen COL-1012-A was extracted from the liquid. Preferably, in the preparation method, recombinant Pichia pastoris or Pichia pastoris GS115 COL-1012-A is inoculated into BSM medium for fermentation, and methanol is used to induce expression for 24-96 hours to obtain the fermentation culture.

8. A recombinant type XVII human collagen COL-1012-B, characterized in that, The amino acid sequence is shown in SEQ ID NO. 12; Preferably, the gene sequence encoding the recombinant type XVII human collagen COL-1012-B is SEQ ID NO.15, which contains 6 HIS tag encoding genes.

9. A recombinant vector, characterized in that, The gene sequence encoding the recombinant type XVII human collagen COL-1012-B as described in claim 8 is SEQ ID NO.15; Preferably, the expression vector of the recombinant vector is pPIC9K; Preferably, a recombinant bacterium comprises the gene sequence encoding the recombinant type XVII human collagen COL-1012-B as described in claim 8, SEQ ID NO.15; Preferably, the host strain of the recombinant bacteria is Pichia pastoris.

10. A method for constructing recombinant Pichia pastoris, characterized in that, The process includes the following steps: cloning the gene sequence SEQ ID NO.15 described in claim 8 into the EcoRI and NotI restriction sites of pPIC9K to obtain the recombinant expression plasmid pPIC9K-COL-1012-B, and then transforming the recombinant expression plasmid pPIC9K-COL-1012-B into Pichia pastoris to obtain recombinant Pichia pastoris. Preferably, the preparation method of recombinant type XVII human collagen COL-1012-B according to claim 8 includes the following steps: The recombinant Pichia pastoris constructed using the method was fermented in shake flasks and fermenters, and the fermentation culture was obtained by induction with methanol. The cell cells were separated and the liquid was retained. Recombinant type XVII human collagen COL-1012-B was extracted from the liquid. Preferably, in the preparation method, recombinant Pichia pastoris is inoculated into BSM medium for fermentation, and methanol is used to induce expression for 24-96 hours to obtain the fermentation culture.

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