Recombinant humanized type xvii collagen col1115 and methods of making the same

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a recombinant humanized type XVII collagen COL1115 and a preparation method thereof. The amino acid sequence of the recombinant humanized type XVII collagen COL1115 comprises the amino acid sequence shown in SEQ ID NO:1. SEQ ID NO:5 is cloned into a vector to obtain a recombinant vector; the recombinant vector is transformed into a host bacterium to obtain a recombinant engineering bacterium; and after fermentation culture and induction expression of the recombinant engineering bacterium, the recombinant humanized type XVII collagen COL1115 is obtained. The recombinant humanized type XVII collagen COL1115 prepared by the application has excellent expression performance in a Pichia pastoris expression system, can realize high-efficiency secretory expression, and is confirmed to have significant cell proliferation activity and cell adhesion activity through activity detection, thereby laying a foundation for application of the collagen in the biomedical field.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a recombinant humanized type XVII collagen COL1115 and its preparation method. Background Technology

[0002] Collagen is the most abundant structural protein in the human body, accounting for more than 30% of the total protein in the body. It is widely distributed in tissues such as skin, bones, tendons, blood vessels, and cornea, acting as the "reinforced concrete" of the extracellular matrix. Its typical characteristic is a triple helix formed by Gly-XY repeating sequences. Currently, 28 types have been identified: Type I provides tensile strength to bones and tendons; Types III and V together maintain the elasticity of skin and blood vessels; Type IV constitutes the basement membrane skeleton; and Types X and VII anchor epidermal stem cells and regulate their microenvironment. With age, collagen fibers become thinner and cross-linked, leading to loose skin, brittle bones, and arteriosclerosis. Modern biotechnology uses yeast, E. coli, or plant expression systems to mass-produce recombinant humanized collagen, avoiding the risks of animal-derived viruses and allowing for precise design of functional domains to obtain high-purity, low-immunogenic, and easily cross-linked medical and aesthetic materials. In recent years, Type XVII collagen has attracted much attention due to its transmembrane structure's ability to stabilize hemidesmosomes and activate hair follicle stem cells, becoming a new focus of research in tissue regeneration, wound repair, and anti-aging. In the future, with the help of AI design, organoid models, and gene editing, collagen is expected to be upgraded from a "filling scaffold" to a "smart matrix," enabling spatiotemporally controllable release of cellular instructions and ushering in a new era of precision regenerative medicine.

[0003] Type XVII collagen (BP180) is a transmembrane non-fibrous collagen encoded by the COL17A1 gene. It has a molecular weight of approximately 180 kDa, with its carboxyl terminus located intracellularly and its amino terminus extending extracellularly. It contains a long rod-shaped extracellular region composed of three collagen domains (Gly-XY repeats) and a non-collagenary spacer region. It is anchored to hemidesmosomes via its carboxyl tail and serves as a crucial "molecular rivet" between epidermal stem cells and the basement membrane. Its extracellular segment can be recognized by proteases such as TACE and ADAM9. It sheds during stress or aging, producing a 120 kDa soluble fragment. This process is known as "shedding regulation," which directly affects keratinocyte migration, hair follicle cycle, and epidermal regeneration. Genetic evidence suggests that COL17A1 mutations lead to bullous pemphigoid, borderline epidermolysis bullosa, and premature alopecia. The decline in type XVII collagen expression associated with aging causes epidermal stem cells to lose their anchorage, resulting in "stem cell escape," which has been confirmed as a core mechanism for skin thinning, pigmentation, and poor wound healing. A 2021 report in *Nature* showed that restoring type XVII collagen in mice can reverse skin atrophy and promote hair follicle regeneration, elevating this protein to the status of an "anti-aging star" for the first time. In the industrial sector, high-density expression of recombinant humanized type XVII collagen has been achieved using yeast and human cell lines. By preserving the extracellular collagen region and integrin binding sites, its cell adhesion and basement membrane assembly activity are retained, leading to the development of medical aesthetic products such as hydrogels, microneedles, and periorbital freeze-dried fibers. These products can significantly enhance keratinocyte colony formation rate and shorten the barrier repair time after laser treatment. Chinese patent CN113185604A discloses a recombinant human type XVII collagen, its preparation method, and its application. An attempt was made to express full-length type XVII collagen in Pichia pastoris. The results showed that the target band was not detected in the fermentation supernatant; only a degradation fragment of approximately 120 kDa was observed in the cell lysate. This suggests that the transmembrane protein is anchored to the inner membrane and requires protease cleavage to detach, but cannot be actively secreted extracellularly. This results in a cumbersome extraction process, low yield, and difficulty in scaling up to production. Therefore, screening for gene fragments that can efficiently express type XVII collagen and obtaining collagen with specific functions is of great significance. Summary of the Invention

[0004] The purpose of this invention is to provide a recombinant humanized type XVII collagen COL1115, which can be efficiently expressed in a yeast expression system and exhibits good biological activity; this invention also provides a method for preparing recombinant humanized type XVII collagen COL1115.

[0005] The recombinant humanized type XVII collagen COL1115 of the present invention has an amino acid sequence containing the amino acid sequence shown in SEQ ID NO:1.

[0006] The gene encoding SEQ ID NO:1 is the nucleotide sequence shown in SEQ ID NO:2.

[0007] The recombinant humanized type XVII collagen COL1115 has the amino acid sequence SEQ ID NO:1 or SEQ ID NO:3.

[0008] The gene encoding SEQ ID NO:3 is the nucleotide sequence shown in SEQ ID NO:4.

[0009] The gene encoding SEQ ID NO:3 is the nucleotide sequence shown in SEQ ID NO:5.

[0010] The preparation method of recombinant humanized type XVII collagen COL1115 according to the present invention includes the following steps: (1) SEQ ID NO:5 was cloned into the vector to obtain the recombinant vector; (2) Transform the host bacteria with the recombinant vector obtained in step (1) to obtain recombinant engineered bacteria; (3) The recombinant engineered bacteria obtained in step (2) were fermented and induced to express to obtain recombinant humanized type XVII collagen COL1115.

[0011] In step (1), the carrier is pPIC9K.

[0012] In step (2), the host bacteria are bacteria or yeast.

[0013] The yeast is Pichia pastoris, preferably Pichia pastoris GS115.

[0014] In step (3), the fermentation culture and induced expression process involves picking a single colony of recombinant engineered bacteria, inoculating it into YPD liquid medium for seed culture, and then inoculating it into BMGY liquid medium for seed culture to obtain seed liquid. Subsequently, the seed liquid is centrifuged to collect the bacterial cells, and the bacterial cells are resuspended in BMMY liquid medium for induced expression. During the induction process, methanol is added every 24 hours to maintain the induction concentration. After induction expression, the fermentation supernatant is collected by centrifugation, and the target protein in the fermentation supernatant is purified to obtain recombinant humanized type XVII collagen COL1115.

[0015] To overcome the bottlenecks of existing technologies, this invention constructs a recombinant humanized type XVII collagen COL1115 that is secretible, easily purified, and retains high activity. Its core design features are as follows: The recombinant humanized type XVII collagen COL1115 sequence was selected from a highly active fragment of humanized type XVII collagen, corresponding to the amino acid sequence SEQ ID NO:1. It removes the transmembrane region to avoid membrane anchoring while retaining the integrin binding site to ensure biological activity.

[0016] The gene sequence encoding SEQ ID NO:1 is shown in SEQ ID NO:2.

[0017] A start codon, a 6×His tag, and a TEV restriction site were introduced at the N-terminus of SEQ ID NO:1, and a stop codon was added at the C-terminus to form the amino acid sequence of SEQ ID NO:3; the DNA sequence corresponding to SEQ ID NO:3 is shown in SEQ ID NO:4.

[0018] Sequence optimization of SEQ ID NO:4 was performed based on Pichia pastoris codon preference, GC content, and mRNA secondary structure. The optimized DNA sequence is shown in SEQ ID NO:5.

[0019] Insert SEQ ID NO:5 into a driver vector with an alcohol oxidase promoter such as pPIC9K, preferably pPIC9K, which can be used to achieve high copy integration and stable inheritance by utilizing its strong promoter and Kan resistance.

[0020] The recombinant engineered bacteria prepared by this invention contain the coding gene sequence SEQ ID NO:5 of the above-mentioned recombinant type XVII human collagen COL1115.

[0021] The preparation method of recombinant humanized type XVII collagen COL1115 according to the present invention includes the following specific steps: (1) The gene sequence (SEQ ID NO:5) encoding recombinant humanized type XVII collagen COL1115 was cloned into the EcoRI and NotI double restriction sites of the pPIC9K vector to form the recombinant vector pPIC9K-COL1115; the recombinant vector was transformed into Escherichia coli DH5α competent cells, and positive clones were selected for DNA sequencing verification. Correct sequencing indicates that the recombinant vector pPIC9K-COL1115 has been successfully constructed. (2) The recombinant vector pPIC9K-COL1115 was digested with restriction endonuclease Sal I. After digestion, the linearized vector product was recovered by conventional nucleic acid recovery method and kept for later use. (3) The recovered linearized vector product was introduced into Pichia pastoris GS115 competent cells by electroporation; positive recombinants were screened by a combination of MD plate screening and PCR verification. After verification, the recombinant engineered strain, namely recombinant Pichia pastoris, was obtained and namedShepherd's pie GS115 COL1115; (4) Recombinant engineered bacteria Shepherd's pie GS115 COL1115 was prepared as a recombinant humanized type XVII collagen COL1115 through fermentation culture and induced expression.

[0022] The fermentation and induced expression process in step (4) specifically involves selecting recombinant engineered bacteria. Peach shepherd Single colonies of GS115 COL1115 were inoculated into 20ml LYPD liquid medium and cultured overnight in a constant temperature shaker at 30℃ and 200rpm. A 2% inoculum was then inoculated into BMGY liquid medium and cultured for 24 hours in a constant temperature shaker at 30℃ and 200rpm to obtain the seed culture. The seed culture was then centrifuged to collect the bacterial cells, which were resuspended in BMMY liquid medium and induced to express. Methanol was added every 24 hours during induction to maintain the induction concentration. After 96 hours of induction, the fermentation supernatant was collected by centrifugation. The target protein in the fermentation supernatant was purified to obtain recombinant humanized type XVII collagen COL1115.

[0023] The beneficial effects of this invention are as follows: 1. The recombinant humanized type XVII collagen COL1115 prepared in this invention has excellent expression performance in the Pichia pastoris expression system, and can achieve efficient secretory expression. Moreover, the activity test confirmed that it has significant cell proliferation activity and cell adhesion activity, which lays the foundation for its application in the biomedical field.

[0024] 2. The recombinant humanized type XVII collagen COL1115 provided by this invention has 100% homology with the amino acid sequence of the corresponding functional region of natural human type XVII collagen, which can effectively avoid the risk of immune rejection caused by foreign proteins. Moreover, the Pichia pastoris expression system and subsequent purification process can ensure that the product has no endotoxin residue and has high safety. It shows broad application prospects in multiple fields such as tissue engineering, wound repair, and medical aesthetics. Attached Figure Description

[0025] Figure 1 The image shows the SDS-PAGE results of protein expression in COL1115 shake-flask fermentation. In the image, lane M represents the protein marker; lane 1 represents the target protein band detected after 24 hours of induction culture; lane 2 represents the target protein band detected after 48 hours of induction culture; lane 3 represents the target protein band detected after 72 hours of induction culture; and lane 4 represents the target protein band detected after 96 hours of induction culture.

[0026] Figure 2 The image shows the SDS-PAGE results of protein expression in the COL1115 fermenter. In the image, lane M represents the protein marker; lane 1 represents the result of sampling and detection at 0 h of induction (no target protein band); lane 2 represents the target protein band detected at 24 h of induction; lane 3 represents the target protein band detected at 48 h of induction; lane 4 represents the target protein band detected at 72 h of induction; and lane 5 represents the target protein band detected at 96 h of induction. Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments.

[0028] Operational steps not explicitly described in the embodiments shall be performed in accordance with conventional technical specifications in the field; experimental materials, reagents and consumables not specifically described are all commercially available products that can be purchased through commercial channels.

[0029] Example 1: 1. Sequence screening and codon optimization of recombinant humanized type XVII collagen COL1115 This embodiment screened the core sequence of recombinant humanized type XVII collagen COL1115, and performed codon optimization and sequence modification based on the characteristics of the Pichia pastoris expression system. The specific operations are as follows: (1) Screening and determination of core functional sequence The full-length sequence of human type XVII collagen was retrieved from the Genbank database (Genbank accession number: Q9UMD9.3). Bioinformatics analysis was performed on its domain functions, and the extracellular domains 11-15 (collagen region) were extracted as the core functional fragment, named COL1115. This core fragment contains 372 amino acid residues, and its specific amino acid sequence is shown in SEQ ID NO:1. The DNA sequence encoding this core fragment is shown in SEQ ID NO:2.

[0030] (2) Sequence modification and optimization design To achieve efficient expression and subsequent purification of COL1115 in Pichia pastoris, the core sequence was modified at both ends and optimized using codons. A start codon, a 6×His tag coding sequence, and a TEV protease recognition sequence were sequentially introduced upstream of the N-terminal coding region of COL1115. A stop codon was introduced downstream of the C-terminal coding region to terminate protein translation. The modified COL1115 contains 385 amino acid residues, and its complete amino acid sequence is shown in SEQ ID NO:3. The corresponding DNA sequence of SEQ ID NO:3 is shown in SEQ ID NO:4. Based on the sequence shown in SEQ ID NO:4, codon optimization was performed considering the codon bias of Pichia pastoris. The optimized complete DNA sequence is shown in SEQ ID NO:5, with a total length of 1158 bp.

[0031] 2. Construction and screening of recombinant humanized type XVII collagen strain COL1115 (1) Nanjing Genscript Biotech Co., Ltd. was commissioned to synthesize the whole gene of the optimized recombinant humanized type XVII collagen COL1115 encoding gene sequence (SEQ ID NO:5). After synthesis, the gene fragment was directionally cloned into the EcoRI and NotI double restriction sites of the pPIC9K vector to form a recombinant vector. The recombinant vector was transformed into Escherichia coli DH5α competent cells, single colonies were picked for expansion culture and plasmids were extracted. The correctness of the inserted fragment was verified by DNA sequencing. The sequencing results showed that the target gene sequence was correct, indicating that the recombinant vector pPIC9K-COL1115 was successfully constructed.

[0032] (2) Take 10 μg of the recombinant vector pPIC9K-COL1115 that has been verified by sequencing, add Sal I fast digestion enzyme (purchased from TaKaRa), prepare the enzyme digestion mixture according to the reaction system recommended by the kit, and place it in a constant temperature water bath at 37℃ for 30 minutes for enzyme digestion; after the enzyme digestion reaction, use the PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to recover and purify the linearized vector product, and finally elute the product with 10 μL of elution buffer and store it at -20℃ for later use.

[0033] (3) Pick a single colony of Pichia pastoris strain GS115 (purchased from Shanghai Beyotime Biotechnology Co., Ltd.), inoculate it into 20 mL of YPD liquid medium, and incubate it in a constant temperature shaker at 30℃ and 200 rpm until OD. 600The concentration was approximately 1.0. Take 10 mL of the above bacterial culture and place it in an ice bath for 10 minutes to pre-cool. Then, centrifuge at 4℃ and 4000 rpm for 5 minutes, discard the supernatant and collect the bacterial cells. Resuspend the bacterial cells in 10 mL of pre-cooled 1M D-sorbitol solution and repeat the centrifugation and washing twice. Finally, add 5 mL of pre-cooled 1M D-sorbitol solution to resuspend the bacterial cells. Aliquot the bacterial culture into 100 μL / vial and store it in an ultra-low temperature freezer at -80℃ for later use.

[0034] (4) Take one (100 μL) of Pichia pastoris GS115 competent cells, thaw them on ice, add 10 μL of the recovered linearized vector product, gently pipette and mix, and transfer to a pre-cooled electroporation cup; place the electroporation cup in an electroporator (purchased from Bio-Rad Biomedical Products (Shanghai) Co., Ltd.), select the "PIC" special program for electroporation; after electroporation, quickly add 1 mL of pre-cooled 1M D-sorbitol solution to the electroporation cup and gently mix; transfer the above mixture to a sterile centrifuge tube and place it in a 30℃ constant temperature incubator for static recovery culture for 1 h; after recovery, centrifuge at 4℃ and 4000 rpm for 5 minutes, discard part of the supernatant, and retain about 100 μL of supernatant to resuspend the cells; spread the resuspended bacterial solution evenly on MD solid medium and place it in a 30℃ constant temperature incubator for about 3 days until single colonies grow on the plate.

[0035] (5) Prepare YPD solid screening medium containing 4 g / L G418, draw 1 cm × 1 cm grids on the surface of the medium and number them; use a sterile toothpick to pick up a single colony from the MD plate, spot it onto the G418 screening medium according to the corresponding number, and incubate it in a constant temperature incubator at 30℃ for about 3 days. Screen for high copy recombinants according to the size and plumpness of the colony growth (usually high copy recombinants have larger colony diameters) to obtain recombinant engineered bacteria, and name them Shepherd's pie GS115 COL1115.

[0036] 3. Induced expression of recombinant humanized type XVII collagen by recombinant strain COL1115 With the above-mentioned recombinant engineered bacteria Shepherd's pie Using GS115 and COL1115 as the starting strain, the recombinant humanized type XVII collagen COL1115 was efficiently expressed through seed culture, scale-up culture, and methanol-induced expression.

[0037] Select high-copy recombinant engineered bacteria obtained through screening Shepherd's pieSingle colonies of GS115 and COL1115 were inoculated into 20 mL of YPD liquid medium and cultured overnight in a shaker at 30°C and 200 rpm to obtain a seed culture. At a volume ratio of 2%, the seed culture was transferred to a 250 mL Erlenmeyer flask containing 50 mL of BMGY medium and cultured for 24 h in a shaker at 30°C and 200 rpm. The expanded seed culture was transferred to sterile centrifuge tubes and centrifuged at 4°C and 12000 rpm for 10 min. The supernatant was discarded, and the bacterial cells were collected. The bacterial cells were resuspended in 10 mL of sterile physiological saline and washed once by centrifugation to thoroughly remove residual glycerol and culture medium components. The washed bacterial cells were resuspended in 10 mL of BMMY medium and then transferred to a 500 mL Erlenmeyer flask containing 100 mL of BMMY medium. The Erlenmeyer flasks were placed in a constant-temperature shaker at 25℃ and 200rpm for induction expression. To maintain induction efficiency, methanol was added to the culture medium every 24 hours, at a volume of 1% of the culture medium (i.e., 1 mL of methanol per 100 mL of culture medium). Simultaneously, 1 mL of methanol was taken after each addition and frozen at -20℃ for subsequent protein expression level detection. Induction was terminated after 96 hours.

[0038] SDS-PAGE electrophoresis was used to detect the expression of recombinant humanized type XVII collagen COL1115 in fermentation supernatants at different induction times to verify their expression. Fermentation samples taken from shake flasks at different induction time points were thawed at room temperature and centrifuged at 12000 rpm for 1 min. The supernatants were then analyzed by SDS-PAGE. The SDS-PAGE electrophoresis results are shown below. Figure 1 As shown, specific protein bands appeared in the fermentation supernatant samples at a relative molecular mass of approximately 35 kDa, which is consistent with the theoretical molecular mass of recombinant humanized type XVII collagen COL1115, indicating that the target protein has been correctly expressed in Pichia pastoris and secreted into the fermentation broth.

[0039] 4. Fermentation preparation of recombinant humanized type XVII collagen COL1115 With recombinant engineered bacteria Shepherd's pie GS115 and COL1115 are the production strains. Through seed activation, fermenter-scale culture, and methanol-induced expression, the target protein is prepared on a large scale through fermentation.

[0040] (1) Activation culture of seed liquid Select recombinant engineered bacteria Shepherd's pieSingle colonies of GS115 and COL1115 were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of YPD liquid medium and cultured overnight in a constant temperature shaker at 30°C and 200 rpm to obtain primary seed culture. The primary seed culture was then transferred to 1000 mL Erlenmeyer flasks containing 200 mL of YPD liquid medium at a volume ratio of 2% and cultured at 30°C and 200 rpm. The OD value of the bacterial culture was monitored in real time using a UV spectrophotometer. When the OD value reached approximately 6.0, the secondary seed culture was terminated, and the seed activation was completed to obtain the secondary seed culture.

[0041] (2) Preparation of fermentation culture medium and feed solution The formulations and preparation methods of the fermentation medium, trace element stock solution, and feed solution used are as follows: BSM fermentation medium: 26.7 mL / L phosphate (85%, volume fraction), 0.93 g / L calcium sulfate, 18.2 g / L potassium sulfate, 14.9 g / L magnesium sulfate, 4.13 g / L potassium hydroxide, and 40 g / L glycerol; weigh each component according to the above formula, dissolve in deionized water and make up to volume; place the prepared medium in a fermenter and autoclave at 121℃ for 20 min; after the medium temperature drops to room temperature, add sterilized trace element stock solution PTM1 at a rate of 4.35 mL / L, stir and mix well, and adjust the pH of the medium to 5.0 with 50% (volume fraction) ammonia water, and set aside.

[0042] Trace element stock solution PTM1: Copper sulfate 6g / L, sodium iodide 0.08g / L, manganese sulfate 3g / L, sodium molybdate 14.9g / L, boric acid 0.02g / L, cobalt chloride 0.5g / L, zinc chloride 20g / L, ferrous sulfate heptahydrate 65g / L, biotin 0.2g / L, sulfuric acid 5mL / L; After dissolving each component in deionized water and adjusting the volume, the solution was filtered through a 0.22 μm filter membrane for sterilization. The sterilized mother liquor was then sealed and stored in a refrigerator at 4°C for later use.

[0043] Glycerin replenishment solution: 50% (mass fraction) glycerin aqueous solution; mix glycerin and deionized water at a mass ratio of 1:1 and autoclave at 121℃ for 20 min; after the temperature drops to room temperature, add trace element stock solution PTM1 at a volume of 12 mL / L, stir thoroughly and mix well, and set aside.

[0044] Methanol feed solution: 100% methanol; take analytical grade methanol directly and add it to trace element mother liquor PTM1 at a volume of 12 mL / L, stir magnetically to mix well, and set aside.

[0045] (3) Fermenter culture and induced expression Initial culture stage: Add the prepared BSM fermentation medium to the fermenter and inoculate the activated secondary seed culture at a volume ratio of 10%. Set the initial culture parameters as follows: temperature 30℃, stirring speed 300rpm, aeration rate 2vvm (volume air / volume medium / minute). Maintain the pH value of the fermentation broth at 5.0 by automatically adding 50% ammonia water. At the same time, ensure that the dissolved oxygen (DO) content in the fermenter is maintained above 20% through a dissolved oxygen-related stirring speed feedback mechanism.

[0046] Glycerol fed-batch culture stage: After continuous culture for 18-20 hours, when the glycerol in the BSM medium is depleted, the DO value in the fermentation broth will rapidly rise to over 60%. At this point, glycerol feeding is initiated to promote secondary cell proliferation. The feeding strategy uses dissolved oxygen feedback control. When the DO value is greater than 30%, the glycerol feeding solution is fed at a rate of 1.0 mL / min. The wet cell weight is sampled periodically. When the wet cell weight reaches 300 g / L, glycerol feeding is stopped, and the stirring speed is increased to 900 rpm to enter the starvation culture stage. After the DO value rises to 100% and stabilizes for 30 minutes, the starvation culture is ended, and the induction stage begins.

[0047] Methanol-induced expression stage: The fermenter temperature was lowered to 25℃, and methanol feed solution was started for induction. The first 4 hours were the cell adaptation period, and the methanol feed solution flow rate was controlled at 0.06 mL / min to help the cells adapt to the change of carbon source from glycerol to methanol. After 4 hours, the feed strategy was adjusted, and the flow rate was increased to 0.12 mL / min, while still using dissolved oxygen feedback feed mode (feeding was started when DO value ≥ 30%). After continuous induction culture for 96 hours, fermentation was stopped, and the fermentation broth was collected for subsequent separation and purification of the target protein.

[0048] Fermentation broth containing recombinant humanized type XVII collagen COL1115 was collected at methanol induction times of 0 h, 24 h, 48 h, 72 h, and 96 h, respectively. The broth was frozen at -20℃, thawed at room temperature, and centrifuged at 12000 rpm for 1 min. The supernatant was then analyzed by SDS-PAGE. The results are shown below. Figure 2 As shown.

[0049] from Figure 2It was found that no target protein band appeared after 0 h of methanol induction, indicating that the AOX1 promoter was in a repressed state before methanol induction, and the target gene was not expressed. Specific protein bands with a relative molecular mass of approximately 35 kDa appeared in the fermentation supernatant after 24 h, 48 h, 72 h, and 96 h of methanol induction, consistent with the theoretical molecular mass of recombinant humanized type XVII collagen COL1115. This indicates that the recombinant humanized type XVII collagen COL1115 of this invention can still be stably expressed and secreted into the fermentation broth in the industrial-scale culture system of the fermenter. Furthermore, the brightness of the target protein band gradually increased from 24 h to 96 h of methanol induction, indicating that the expression level of recombinant humanized type XVII collagen COL1115 in the fermenter culture continuously accumulated with the extension of methanol induction time, reaching a high level at 96 h of induction.

[0050] In summary, the recombinant Pichia pastoris containing recombinant humanized type XVII collagen COL1115 of the present invention can be scaled up from shake flask fermentation culture to fermenter fermentation culture, and can stably achieve extracellular secretion and high accumulation of recombinant humanized type XVII collagen, thus realizing the large-scale production of recombinant humanized type XVII collagen COL1115.

[0051] 5. Purification of recombinant humanized type XVII collagen COL1115 Recombinant humanized type XVII collagen COL1115 was purified using a magnetic bead method. The magnetic beads were chelated Ni. 2+ Cross-linked superparamagnetic agarose microspheres were purchased from Nanjing Yuzhulong Biotechnology Co., Ltd.

[0052] (1) First, pretreat the magnetic beads. Each milliliter of magnetic bead solid volume can adsorb approximately 30 mg of the target protein. Estimate the content of the target protein in the sample to ensure that the amount of magnetic beads added is sufficient. Equilibrate the magnetic beads using equilibration buffer. Take a certain amount of magnetic bead suspension in an Erlenmeyer flask, add a magnetic rod, and the magnetic beads will immediately adsorb onto the surface of the magnetic rod. Remove the supernatant. Add an appropriate amount of equilibration buffer, remove the magnetic rod, resuspend the magnetic beads, mix well, add the magnetic rod again, and remove the supernatant. Repeat this magnetic bead equilibration step once.

[0053] (2) The fermentation broth obtained above was centrifuged at 4℃ and 8000rpm for 20min, and the fermentation supernatant was collected.

[0054] (3) Resuspend the balanced magnetic beads in the fermentation supernatant and incubate at 4°C and 80 rpm for 30 min to ensure that the magnetic beads are fully and evenly mixed with the fermentation supernatant, so as to ensure that the magnetic beads bind to the target protein to the greatest extent.

[0055] (4) After incubation, place the magnetic rod into the above incubation system. After the magnetic beads are adsorbed onto the surface of the magnetic rod, transfer the magnetic rod to another Erlenmeyer flask, add 5 times the volume of the magnetic beads in washing buffer, remove the magnetic rod, resuspend the magnetic beads, mix well, and place the magnetic rod again. After the magnetic beads are adsorbed onto the surface of the magnetic rod, remove the supernatant and retain a small sample for subsequent detection. Repeat this washing step twice.

[0056] (5) Add 5 times the volume of the magnetic beads to the washed magnetic beads and incubate at 4°C and 80 rpm for 5 min. Place the magnetic rod in the incubator, and collect the eluted target protein after the magnetic beads are adsorbed onto the surface of the magnetic rod. To obtain a higher recovery rate, repeat the elution step twice and combine the eluents to obtain the purified collagen solution.

[0057] (6) Add 5 times the volume of the magnetic beads in regeneration buffer to the Erlenmeyer flask containing the eluted magnetic beads, resuspend the magnetic beads, and incubate on a shaker at 4°C for 10 min. Place the magnetic rod into the Erlenmeyer flask, and discard the supernatant after the magnetic beads are adsorbed onto the surface of the magnetic rod. Repeat this alkaline washing step once. Wash the regenerated magnetic beads twice with 5 times the volume of the magnetic beads in distilled water.

[0058] (7) Add 25% ethanol solution with a volume of 3 times that of the magnetic beads to completely immerse the magnetic beads and store them in a refrigerator at 4°C.

[0059] The above buffer solution formulation is as follows: Equilibration buffer: 20mM phosphate buffer, 300mM sodium chloride, 10mM imidazole, pH 6.5.

[0060] Wash buffer: 20 mM phosphate buffer, 300 mM sodium chloride, 20 mM imidazole, pH 6.5.

[0061] Elution buffer: 20mM phosphate buffer, 300mM sodium chloride, 250mM imidazole, pH 6.5.

[0062] Regeneration buffer: 8M urea.

[0063] (8) The purified recombinant humanized type XVII collagen COL1115 was subjected to dialysis desalting and freeze-drying, and the purity of the product was detected by high performance liquid chromatography (HPLC). The recombinant humanized type XVII collagen COL1115 solution obtained by the above purification process was placed into a dialysis bag with a molecular weight cutoff of 10 kDa. The two ends of the dialysis bag were sealed with dialysis clips to ensure no leakage. The sealed dialysis bag was placed in a dialysis container containing sufficient pure water and dialyzed at 4°C. To ensure the desalting effect, the dialysis solution was changed every 4 hours, and dialysis was continued for 24 hours to completely remove residual imidazole and inorganic salt impurities in the protein solution.

[0064] After dialysis, the collagen solution in the dialysis bag was collected and transferred to a sterile lyophilization bottle, which was then placed in a freeze dryer. The lyophilization parameters were set as follows: pre-freezing at -40℃ for 4 hours; after the solution was completely frozen, the vacuum pump was turned on, maintaining a vacuum below 10 Pa, and the temperature was gradually increased to 25℃ for sublimation drying for 24 hours. After lyophilization, the lyophilization bottle was removed, quickly sealed, and recombinant humanized type XVII collagen COL1115 lyophilized powder was obtained and stored at -20℃ for later use.

[0065] 6. Cell proliferation activity assay of recombinant humanized type XVII collagen COL1115 Using human immortalized epidermal cells (HaCaT) as the research object, the CCK-8 assay was used to detect the promoting effect of recombinant humanized type XVII collagen COL1115 on cell proliferation, clarify its biological activity, and provide experimental basis for the application of this protein in tissue repair and other fields.

[0066] (1) Experimental materials and reagents Immortalized human epidermal cells (HaCaT, purchased from the Cell Bank of the Chinese Academy of Sciences) can simulate the cell proliferation scenario during skin tissue repair. The recombinant humanized type XVII collagen COL1115 lyophilized powder prepared in this invention (purity ≥93% as determined by purity testing) was dissolved and diluted to a series of concentrations in DMEM medium containing 10% fetal bovine serum. Control samples: ① Positive control: natural human type XVII collagen (purchased from ProSpec, Israel, catalog number: COL-795); ② Negative control: bovine serum albumin (BSA, purchased from Sigma, purity ≥98%); ③ Blank control: DMEM medium containing 10% fetal bovine serum. Core reagents: DMEM high-glucose medium (Gibco), fetal bovine serum (FBS, Ausbian), 0.25% trypsin-EDTA digestion solution (Shanghai Beyotime Biotechnology Co., Ltd.), PBS buffer (pH 7.4, Solarbio), CCK-8 assay kit (Tongren Chemical Research Institute, catalog number: CK04). (2) Experimental methods Frozen HaCaT cells were removed from the -80°C freezer and rapidly thawed in a 37°C water bath. After centrifugation at 1000 rpm for 5 min, the cryopreservation solution was discarded. The cells were resuspended in DMEM medium containing 10% fetal bovine serum and seeded into T25 cell culture flasks, which were then incubated at 37°C, 5% CO2, and saturated humidity. When the cell confluence reached 80-90%, the cells were digested and passaged using 0.25% trypsin-EDTA digestion solution. Cells in the logarithmic growth phase were used for subsequent experiments.

[0067] Sample dilution: Recombinant humanized type XVII collagen COL1115 was diluted in DMEM medium containing 10% fetal bovine serum to five concentration gradients: 1 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, and 40 μg / mL; the positive control and negative control were both diluted to 20 μg / mL for later use. Cell seeding: After digesting HaCaT cells in logarithmic growth phase, the cell concentration was adjusted to 5 × 10⁶ cells / year using DMEM medium containing 10% fetal bovine serum. 3 Add 100 μL of cell suspension to each well of a 96-well cell culture plate and incubate for 24 h to allow the cells to adhere and grow.

[0068] Sample intervention and proliferation detection: ① Sample intervention: After 24 hours of culture, the old culture medium in each well of the 96-well plate was discarded. 100 μL of DMEM culture medium containing 10% fetal bovine serum was added to the blank control well. 100 μL of the corresponding concentration of sample solution was added to the negative control well, positive control well, and each concentration group of COL1115. Six replicates were set for each concentration to avoid experimental error. ② Proliferation Detection: After 24h, 48h, and 72h of sample intervention, 10μL of CCK-8 reagent was added to each well. The culture plate was gently shaken to thoroughly mix the reagent with the culture medium, and then incubated in an incubator for another 1h. After incubation, the absorbance (OD) of each well was immediately measured at 450nm using a microplate reader. 450 (Value), record experimental data.

[0069] Data processing and analysis: Cell proliferation rate (%) = (OD value of experimental group - OD value of blank control group) / (OD value of blank control group) × 100%. Statistical analysis was performed using GraphPad Prism 9.0 software. Data are expressed as mean ± standard deviation (x ± s). Differences between groups were analyzed using one-way ANOVA, with P < 0.05 considered statistically significant.

[0070] (3) Experimental results and analysis The effects of different concentrations of recombinant humanized type XVII collagen COL1115 on the proliferation rate of HaCaT cells are shown in Table 1.

[0071] Table 1. Effects of different samples on HaCaT cell proliferation rate (n=6, x±s) , As shown in Table 1, the cell proliferation rate of the negative control BSA group was less than 6% at all time points, and it had no significant promoting effect on HaCaT cell proliferation. In contrast, all concentrations of recombinant humanized type XVII collagen COL1115 significantly promoted HaCaT cell proliferation, exhibiting a dual concentration-time dependence. (1) Time-dependent: Under the same concentration of COL1115, the cell proliferation rate increased significantly with the extension of intervention time. For example, in the 20 μg / mL COL1115 group, the proliferation rate was 27.2% at 24h, increased to 54.8% at 48h, and reached 76.3% at 72h, indicating that its proliferation-promoting effect is sustained. (2) Concentration dependence: Under the same intervention time, the cell proliferation rate increased with the increase of COL1115 concentration. When the concentration was increased from 1 μg / mL to 20 μg / mL, the proliferation rate increased from 38.9% to 76.3% after 72 h. When it was further increased to 40 μg / mL, the proliferation rate did not change significantly (P>0.05), indicating that 20 μg / mL was close to the optimal concentration. (3) Activity comparison: There was no significant difference between the proliferation rate of the 20 μg / mL COL1115 group and the positive control (natural collagen) group at each time point (P>0.05). For example, at 72 h, the proliferation rate of the COL1115 group was 76.3% and that of the natural collagen group was 78.6%, and the two groups had comparable activity.

[0072] The above experimental results confirm that the recombinant humanized type XVII collagen COL1115 prepared in this invention has significant cell proliferation promoting activity, and its activity is similar to that of natural human type XVII collagen, providing important functional support for the application of this protein in the fields of skin wound repair, medical aesthetics and anti-aging.

[0073] 7. Cell adhesion activity assay of recombinant humanized type XVII collagen COL1115 In vitro cell adhesion experiments were conducted to verify the adhesion-promoting activity of recombinant humanized type XVII collagen COL1115 on mammalian cells, providing data support for its biological functional applications.

[0074] (1) Experimental materials and reagents The recombinant humanized type XVII collagen COL1115 lyophilized powder prepared above was dissolved and diluted to different concentration gradients in serum-free DMEM medium; human immortalized epidermal cells HaCaT (purchased from the Cell Bank of the Chinese Academy of Sciences) were used to simulate skin tissue cell adhesion scenarios; bovine serum albumin (BSA, purity ≥98%, purchased from Sigma) served as a negative control; natural human type XVII collagen (purchased from ProSpec) served as a positive control; main reagents: DMEM high glucose medium (containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptomycin), serum-free DMEM medium, 0.25% trypsin-EDTA digestion solution, CCK-8 assay kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.), PBS buffer (pH 7.4). (2) Experimental methods Sample Coating: Recombinant humanized type XVII collagen COL1115, positive control, and negative control were diluted with serum-free DMEM medium. The concentration gradient of COL1115 was set at 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL, while the concentration of positive control and negative control was 40 μg / mL. 100 μL of each sample was added to a 96-well cell culture plate, with 3 replicates for each concentration. A blank control group containing only serum-free medium was also set up. After coating overnight at 4°C, the liquid in the wells was discarded, and the cells were washed twice with PBS buffer for 3 min each time to remove unbound protein. Cell treatment: HaCaT cells in logarithmic growth phase were digested with 0.25% trypsin-EDTA digestion solution. After collecting the cells, they were resuspended in serum-containing DMEM medium, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in serum-free DMEM medium and counted. The cell concentration was adjusted to 1×10⁶ cells / year. 5 per mL.

[0075] Cell seeding: Add 100 μL of HaCaT cell suspension of adjusted concentration to each well of the coated 96-well plate and incubate at 37℃ and 5% CO2 for 1.5 h to allow the cells to adhere fully; Removal of non-adhered cells: After culture, gently aspirate the culture medium from the wells and slowly wash each well three times with PBS buffer preheated to 37°C. After each wash, slowly add liquid along the well wall to avoid rinsing off the adhered cells. Activity assay: Add 100 μL of serum-free DMEM medium containing 10% CCK-8 reagent to each well, incubate in an incubator for 1 h, and then measure the absorbance (OD value) of each well at a wavelength of 450 nm using a microplate reader.

[0076] The cell adhesion rate was calculated using the following formula: Adhesion rate (%) = (OD value of experimental group - OD value of blank control group) / (OD value of positive control group - OD value of blank control group) × 100%. Statistical analysis was performed using SPSS 22.0 software. Data are expressed as mean ± standard deviation. Differences between groups were analyzed using the t-test, and P < 0.05 was considered statistically significant.

[0077] (3) Experimental results and analysis Table 2 shows the results of HaCaT cell adhesion activity assays in different sample coating wells. Table 2. Effects of different samples on HaCaT cell adhesion rate (n=3) , As shown in Table 2, the cell adhesion rate of the negative control BSA group was only 8.0%, while all concentrations of recombinant humanized type XVII collagen COL1115 significantly promoted HaCaT cell adhesion in a concentration-dependent manner: when the concentration of COL1115 was 10 μg / mL, the adhesion rate reached 39.5%; when the concentration was increased to 40 μg / mL, the adhesion rate reached 96.2%, which was not significantly different from that of natural human type XVII collagen (100%) (P>0.05); when the concentration was further increased to 80 μg / mL, the adhesion rate approached 100%, indicating that its adhesion activity had reached saturation.

[0078] The above results confirm that the recombinant humanized type XVII collagen COL1115 prepared in this invention has excellent cell adhesion activity, which is comparable to that of natural human type XVII collagen, providing important functional basis for its application in skin wound repair, tissue engineering scaffold construction and other fields.

Claims

1. A recombinant humanized type XVII collagen COL1115, characterized in that... Its amino acid sequence contains the amino acid sequence shown in SEQ ID NO:

1.

2. The recombinant humanized type XVII collagen COL1115 according to claim 1, characterized in that... The gene encoding SEQ ID NO:1 is the nucleotide sequence shown in SEQ ID NO:

2.

3. The recombinant humanized type XVII collagen COL1115 according to claim 1, characterized in that... Its amino acid sequence is SEQ ID NO:1 or SEQ ID NO:

3.

4. The recombinant humanized type XVII collagen COL1115 according to claim 3, characterized in that... The gene encoding SEQ ID NO:3 is the nucleotide sequence shown in SEQ ID NO:

4.

5. The recombinant humanized type XVII collagen COL1115 according to claim 3, characterized in that... The gene encoding SEQ ID NO:3 is the nucleotide sequence shown in SEQ ID NO:

5.

6. A method for preparing recombinant humanized type XVII collagen COL1115 as described in claim 5, characterized in that... Includes the following steps: (1) SEQ ID NO:5 was cloned into the vector to obtain the recombinant vector; (2) Transform the host bacteria with the recombinant vector obtained in step (1) to obtain recombinant engineered bacteria; (3) The recombinant engineered bacteria obtained in step (2) were fermented and induced to express to obtain recombinant humanized type XVII collagen COL1115.

7. The method for preparing recombinant humanized type XVII collagen COL1115 according to claim 6, characterized in that... In step (1), the carrier is pPIC9K.

8. The method for preparing recombinant humanized type XVII collagen COL1115 according to claim 6, characterized in that... In step (2), the host bacteria are bacteria or yeast.

9. The method for preparing recombinant humanized type XVII collagen COL1115 according to claim 8, characterized in that... The yeast is Pichia pastoris.

10. The method for preparing recombinant humanized type XVII collagen COL1115 according to claim 6, characterized in that... In step (3), the fermentation culture and induced expression process involves picking a single colony of the recombinant engineered bacteria, inoculating it into YPD liquid medium for seed culture, and then inoculating it into BMGY liquid medium for seed culture to obtain seed liquid. The seed culture was then centrifuged to collect the bacterial cells, which were then resuspended in BMMY liquid medium for induced expression. Methanol was added every 24 hours during the induction process to maintain the induction concentration. After induction, the fermentation supernatant was collected by centrifugation, and the target protein in the fermentation supernatant was purified to obtain recombinant humanized type XVII collagen COL1115.

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