A stable expression of type XVII recombinant collagen and its preparation method and application

Abstract

This invention discloses a stable recombinant type XVII collagen, its preparation method, and its applications, relating to the field of genetic engineering technology. The recombinant type XVII humanized collagen comprises a recombinant collagen single chain; wherein the recombinant collagen single chain is formed by repeating a collagen monomer fragment as shown in SEQ ID NO.2 2 to 10 times. This invention selects and combines three highly hydrophilic and stable key functional domains at positions 954-968, 585-614, and 909-920 of the human type XVII collagen α1 chain, eliminating a large number of non-functional or low-activity regions. This results in the recombinant protein enriching the core active epitopes of natural type XVII collagen within a monomer fragment of only 57 amino acids. The obtained recombinant collagen exhibits significant promoting effects on cell proliferation, cell adhesion, and cell migration, demonstrating excellent biological activity.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a stable XVII type recombinant collagen protein, its preparation method, and its applications. Background Technology

[0002] Type XVII collagen is a unique transmembrane collagen that, as a core structural component of hemidesmosomes, plays a crucial role in the adhesion and connection between epidermal and epithelial cells and the basement membrane. Competition among epidermal stem cells driven by Type XVII collagen is key to delaying or reversing skin aging; epidermal stem cells highly expressing Type XVII collagen are firmly anchored to the basement membrane and displace nearby cells with low Type XVII collagen expression through symmetrical division. Furthermore, Type XVII collagen plays an irreplaceable role in maintaining hair follicle stem cell activity and promoting wound healing. However, due to its extremely low abundance in human and animal tissues, and its long amino acid chain and large molecular weight, natural extraction of Type XVII collagen is extremely difficult, hindering large-scale production. Therefore, recombinant expression of Type XVII collagen through genetic engineering has become a research hotspot in this field.

[0003] Pichia pastoris expression systems, as eukaryotic microorganisms, possess complete post-translational modification capabilities, and recombinant proteins can be secreted extracellularly, avoiding contamination from impurity proteins and endotoxins caused by cell lysis, thus offering significant advantages in industrial production. However, exogenous recombinant proteins expressed in Pichia pastoris are easily non-specifically cleaved by host-secreted proteases during fermentation, resulting in numerous degradation fragments. This not only reduces the yield and purity of the target protein but also significantly increases downstream purification costs. Therefore, improving the expression stability of type XVII recombinant collagen in Pichia pastoris while maintaining its human sequence safety and biological activity is a pressing technical problem to be solved in this field.

[0004] Chinese Patent CN117986353A describes a recombinant human type XVII collagen, its preparation method, and its application. The recombinant human type XVII collagen is a truncated protein of human type XVII collagen, selected from the non-GXY structural region of human type XVII collagen, and has a single functional domain composition.

[0005] Therefore, developing a type XVII recombinant collagen that does not rely on host gene knockout, achieves stable expression in Pichia pastoris through a reasonable combination and repetition of functional domains of human GXY structural regions, and simultaneously possesses excellent thermal stability, acid and alkali tolerance, cell adhesion activity, cell proliferation activity, and safety is a pressing problem that needs to be solved. Summary of the Invention

[0006] Therefore, the present invention provides a stable XVII type recombinant collagen, its preparation method and application, to solve the problems in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: According to a first aspect of the present invention, a recombinant type XVII collagen protein with stable expression is provided, wherein the recombinant type XVII collagen protein comprises a recombinant collagen single chain; wherein the recombinant collagen single chain is composed of collagen monomer fragments as shown in SEQ ID NO.2 repeated 2 to 10 times. As an example, preferably, the collagen single chain contains 3 to 7 collagen monomer fragments; more preferably, repeated 4 to 5 times; most preferably, repeated 6 times.

[0008] COL17 recombinant collagen monomer fragment 1 amino acid sequence SEQ ID NO.2: GPPGPQGPKGDKGDPGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGPPGPPGPKGDQ.

[0009] Furthermore, the collagen monomer fragment is obtained by sequentially combining amino acids at positions 954-968, 585-614, and 909-920 of human type XVII collagen, wherein the amino acid sequence of human type XVII collagen is shown in SEQ ID NO.1.

[0010] COL17A1 amino acid sequence SEQ ID NO.1:

[0011] Furthermore, the nucleotide sequence encoding the recombinant collagen single chain is shown in SEQ ID NO.4.

[0012] COL17-6 nucleotide sequence SEQ ID NO.4:

[0013] The amino acid sequence of this invention is entirely derived from the combination of natural human type XVII collagen sequences, without introducing any non-human exogenous amino acid residues. Theoretically, its sequence is completely consistent with the corresponding region in the human body, thus exhibiting extremely low immunogenicity and avoiding the risk of viruses carried by animal-derived collagen and allergic reactions caused by foreign proteins.

[0014] A method for preparing recombinant collagen single chains according to a second aspect of the present invention includes the following steps: Step 1: Design collagen sequences The amino acids at positions 954-968, 585-614, and 909-920 of human type XVII collagen are sequentially combined to obtain a collagen monomer fragment as shown in SEQ ID NO.2. The collagen monomer fragment is repeated 2 to 10 times to obtain a recombinant collagen single chain. Step 2, constructing the carrier Based on the recombinant collagen single-chain, the corresponding nucleotide sequence was synthesized to construct a recombinant plasmid; Step 3: Transformation and Cultivation The recombinant plasmid was transferred into host cells, engineered strains were screened, and fermentation culture was performed to induce the expression of the target protein. Step 4: Harvesting and Purification The fermentation broth was collected, the expressed protein product was harvested, and purified to obtain the target collagen.

[0015] Furthermore, in step two, the plasmid is selected for prokaryotic expression, specifically the pET series vector, pGEX series vector, pBAD series vector, or pUC series vector; or the plasmid is selected for eukaryotic expression, specifically the pcDNA series vector, pCMV series vector, pIRES series vector, pEGFP series vector, pYES series vector, or pPIC series vector. As an example, the preferred plasmid is pPICzα.

[0016] Furthermore, in step three, the host cell is a prokaryotic cell, a yeast cell, or a mammalian cell. For example, the prokaryotic cell is *Escherichia coli* or *Bacillus subtilis*; the yeast cell is *Pichia pastoris* or *Saccharomyces cerevisiae*; and the mammalian cell is HEK293 or CHO cell. Preferably, the host cell is *Pichia pastoris* X33.

[0017] The stable type XVII recombinant collagen prepared by the method described above, according to a third aspect of the present invention, is used in the preparation of collagen-containing products.

[0018] Furthermore, the collagen product is a pharmaceutical, medical device, or cosmetic.

[0019] Optionally, the drug includes wound repair materials, cartilage repair materials, and drug delivery carriers; Optionally, the medical device includes tissue engineering materials, surgical aids, and dental prosthetic materials; Optionally, the cosmetics include skin care products, hair care products, and anti-aging products.

[0020] In one embodiment, the pharmaceutical product includes, but is not limited to, wound repair materials, such as collagen sponges, collagen hemostatic dressings, and collagen membranes; cartilage repair materials, such as collagen injections and collagen-based cartilage grafts; and drug delivery carriers, such as collagen microspheres and collagen gel injections.

[0021] In one embodiment, the medical device includes, but is not limited to, tissue engineering materials, such as collagen scaffolds, collagen-based composite materials, and collagen filling materials; surgical aids include, but are not limited to, collagen sutures and collagen hemostatic sponges; and oral repair materials include, but are not limited to, collagen membranes and collagen periodontal regeneration materials.

[0022] In one embodiment, cosmetics include skin care products, such as collagen masks, collagen serums, and collagen lotions; hair care products, such as collagen shampoos and collagen conditioners; and anti-aging products, such as collagen anti-wrinkle creams and collagen freeze-dried powders.

[0023] A fourth objective of this invention is to provide a collagen product containing any of the aforementioned recombinant collagen single chains; optionally, the collagen product is a food, health product, pharmaceutical, medical device, or cosmetic. Optionally, the food includes functional foods and food additives; the health product includes collagen oral liquid, collagen tablets, collagen powder, collagen joint health soft capsules, and bone health drinks; the pharmaceutical includes wound repair materials, cartilage repair materials, and drug delivery carriers; the medical device includes tissue engineering materials, surgical aids, and oral repair materials; and the cosmetic includes skincare products, hair care products, and anti-aging products.

[0024] The present invention has the following advantages: This invention combines three highly hydrophilic and stable key functional domains at positions 954-968, 585-614, and 909-920 of the α1 chain of human type XVII collagen, eliminating a large number of non-functional or low-activity regions. This results in a recombinant protein enriched with the core active epitopes of natural type XVII collagen in a monomeric fragment of only 57 amino acids. The resulting recombinant collagen has significant promoting effects on cell proliferation, cell adhesion, and cell migration, exhibiting excellent biological activity.

[0025] This invention does not rely on host gene knockout and can achieve stable expression in Pichia pastoris. The fermentation supernatant bands are single and there is no obvious degradation, which greatly reduces the degradation of the target protein during fermentation.

[0026] The amino acid sequence of this invention is entirely derived from the cutting and splicing of the natural sequence of human type XVII collagen, without introducing any non-human exogenous amino acid residues or non-natural linker peptides. The 100% human sequence coverage fundamentally eliminates the risk of animal-derived collagen carrying viruses and greatly reduces the risk of immunogenicity and allergic reactions caused by foreign proteins, providing extremely high safety guarantees for high-end medical aesthetics, regenerative medicine, and clinical treatment.

[0027] The recombinant type XVII collagen prepared by this invention has excellent thermal stability, maintaining structural and purity stability even after heating at 60°C for 4 hours; and it also has good acid and alkali tolerance, maintaining strong stability at pH 6-8.

[0028] The recombinant type XVII collagen prepared by this invention has high cell adhesion and cell proliferation activity, and is non-cytotoxic and highly biocompatible. Attached Figure Description

[0029] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0030] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0031] Figure 1 This is a schematic diagram of a recombinant plasmid provided in Embodiment 1 of the present invention; Figure 2 The SDS-PAGE of the recombinant collagen expression supernatant provided in Example 1 of this invention; wherein Figure 2 In the figure, A represents the SDS-PAGE result of the supernatant of recombinant collagen expression in Pichia pastoris genetically engineered bacteria; Figure 2 In the figure, B represents the SDS-PAGE result of the supernatant from a 5L-scale fermentation. The left side was diluted 10 times, and the right side was diluted 20 times before loading. Figure 3 The results of SDS-PAGE detection during the purification process of recombinant collagen provided in Example 1 of this invention; Figure 4 The results show the thermal stability of the recombinant collagen COL17-6 provided in Test Example 1 of this invention. Figure 5 The results of the acid-base tolerance test of recombinant collagen COL17-6 provided in Test Example 1 of this invention; Figure 6 The results of the recombinant collagen cell adhesion activity test provided in Test Example 1 of this invention; Figure 7 The results of the recombinant collagen cell proliferation activity detection provided in Test Example 1 of this invention; Figure 8 The results of the recombinant collagen cytotoxicity test provided in Test Example 1 of the present invention are shown. Detailed Implementation

[0032] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified in the embodiments of this invention, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products; different manufacturers and models of raw materials do not affect the implementation of the technical solution or the achievement of the technical effect of this invention.

[0034] Yeast extract: Oxoid, LP0021B; Peptone: Oxoid, LP0042B; YNB: Shanghai Yuanye Biotechnology Co., Ltd., S24483; Glycerin: Ruikangda, GY20250113; Biotin: Aladdin, D2317484; Potassium phosphate buffer: Beijing Solarbio Science & Technology Co., Ltd., P1030.

[0035] Seed culture medium YPD (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose); BMGY medium (1% yeast extract + 2% peptone + 1.34% YNB + 1% glycerol + 0.0004% biotin + 100 mM potassium phosphate buffer, pH 6.0); BMMY medium (1% yeast extract + 2% peptone + 1.34% YNB + 0.0004% biotin + 100 mM potassium phosphate buffer, pH 6.0); Fermentation medium (46 g / L glycerol, 2.02 g / L magnesium sulfate heptahydrate, 0.46 g / L calcium sulfate dihydrate, 9.1 g / L potassium sulfate, 11.34 mL / L phosphate, 2.06 g / L potassium hydroxide, 4 mL / L PTM1 trace element); Feeding medium (50% w / v glycerol, 12 mL PTM1 trace element per liter); Induction medium (100% methanol, 12 mL PTM1 trace element per liter). PTM1 (trace elements); PTM1: sterilized by filtration through a 0.22μm filter membrane and stored at 4℃. After the fermentation medium is sterilized at high temperature, PTM1 is added after the temperature drops to room temperature, and the pH is adjusted to 5.0 with ammonia.

[0036] pPICzα empty vector was purchased from Thermo Fisher Scientific; SacI was purchased from Thermo Fisher Scientific.

[0037] In this invention, buffer A comprises citric acid and sodium citrate, and is a 20 mM citrate buffer with pH 3.5. Buffer B comprises citric acid, sodium citrate, and NaCl, with a citrate concentration of 20 mM, a NaCl concentration of 1 M, and a pH of 3.5.

[0038] Example 1 1. Design and expression of recombinant collagen Recombinant collagen was designed based on the sequence of human type XVII collagen (COL17A1, amino acid sequence as shown in SEQ ID NO.1). Amino acids 954-968, 585-614, and 909-920 of human type XVII collagen were sequentially combined to obtain recombinant collagen monomer fragment 1, amino acid sequence as shown in SEQ ID NO.2. The monomer fragment shown in SEQ ID NO.2 was repeated 6 times to obtain a recombinant collagen single chain, named COL17-6, amino acid sequence as shown in SEQ ID NO.3.

[0039] COL17 recombinant collagen monomer fragment 1 amino acid sequence SEQ ID NO.2: GPPGPQGPKGDKGDPGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGPPGPPGPKGDQ.

[0040] COL17-6 recombinant collagen single-chain amino acid sequence SEQ ID NO.3: GPPGPQGPKGDKGDPGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGPPGPPGPKGDQGPPGQGPKGDKGDPGTPGIPGPLGHPGPQGPKGQKGSVGDPGPPGPPGPKGDQGPPGPQGPKGDKGDPGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGPPGPPGPKGDQ GPPGPQGPKGDKGDPGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGPPGPPGPKGDQGPPGQGPKGDKGDPGTPGIPGPLGHPGPQGPKGQKGSVGDPGPPGPPGPKGDQGPPGPQGPKGDKGDPGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGPPGPPGPKGDQ.

[0041] 2. Construction of recombinant plasmids and recombinant bacteria Based on the amino acid sequence SEQ ID NO.3 designed in step 1, Nanjing Genscript Biotech Co., Ltd. was commissioned to optimize and synthesize the corresponding nucleotide sequence, as shown in SEQ ID NO.4. This sequence was then inserted into the α-factor reading frame of the pPICzα empty vector (purchased from Thermo Fisher Scientific) to obtain the recombinant plasmid pPICzα-COL17-6 (COL-XVII-1_pPICZalphaA). A schematic diagram of the plasmid is shown below. Figure 1 As shown.

[0042] The above recombinant plasmid (2-3 μg) was digested with SacI (purchased from Thermo Fisher Scientific) at 37°C for 2 h, linearized, recovered, and electroporated into Pichia pastoris X33 competent cells; the electroporated bacterial culture was plated on YPD / Zeocin plates and incubated upside down at 30°C for 2-5 days. Single colonies were selected to verify that the Pichia pastoris genetically engineered strain was obtained and named P. pastoris-COL17-6.

[0043] 3. Induced expression and identification of recombinant collagen Take the Pichia pastoris genetically engineered bacteria obtained in step 2, inoculate it into a 50 mL centrifuge tube containing 5 mL of BMGY medium, and incubate at 28-30℃ and 220 rpm until OD reaches 100%. 600 The result was 10 (approximately 48 hours). The cells were centrifuged at 3500g for 5 minutes at room temperature, collected, and resuspended in BMMY medium to adjust the OD value. 600The culture medium was 10, and the culture was placed on a shaker at 28℃ and 220rpm for 3 days. Anhydrous methanol was added to the culture medium every 24 hours until the final concentration was 1.0%. Bacterial culture samples were collected at 48h and 72h after methanol induction. The sample volume was 1mL and placed in a 1.5mL EP tube. The tube was centrifuged at 12000g for 5min at 4℃ and the expression supernatant was collected. The samples to be tested were stored at -80℃ for later use.

[0044] The results of SDS-PAGE analysis of the supernatant are as follows: Figure 2 As shown in A, the results indicate that the designed type XVII collagen sequence can be secreted into the culture supernatant, and the bands are clear and uniform.

[0045] 4. Fermentation preparation of recombinant collagen The Pichia pastoris-COL17-6 genetically engineered yeast obtained in step 3 was used for high-density fermentation to express recombinant collagen on a large scale. A fed-batch culture method was used, and the culture temperature was 28℃. The steps are as follows: (1) Pichia pastoris genetic engineering was inoculated into 1L shake flasks containing 100mL of seed culture medium YPD and cultured at 220rpm and 28℃ for 22-24h until OD was reached. 600 Reaching 8~12; (2) Use a 5L fermenter, fill with 2L of fermentation medium, adjust the rotation speed to 600rpm, the aeration rate to 3L / min, and the temperature to 28℃ before inoculation. Adjust the pH with the alkaline solution prepared with concentrated ammonia water and set the pH to 5. Inoculate 100mL of the prepared seed liquid into the tank (inoculate with a flame ring), click the dissolved oxygen electrode to calibrate to 100%, and start fermentation after calibration. (3) When the dissolved oxygen level drops to 30% for the first time during growth, use the dissolved oxygen cascade speed function to maintain it at 30%; wait for the glycerol to be depleted, the dissolved oxygen to rebound, and the dissolved oxygen level to be greater than 60% (OD). 600 With a protein concentration of approximately 100, methanol induction was initiated, with a feed rate set at 6 mL / h. After two hours, this was increased to 10 mL / h, and finally to 16 mL / h. During induction, the temperature was maintained at 28℃, the rotation speed was adjusted to 800 rpm, dissolved oxygen was controlled at 20%, and pH was maintained at 5 by adding ammonia. After approximately 96 hours of induction, the protein was discharged from the tank when SDS-PAGE analysis showed that protein degradation had begun or when no significant increase in protein concentration was observed.

[0046] Collect the fermentation supernatant and perform SDS-PAGE electrophoresis for analysis. The results are as follows: Figure 2 As shown in B, under high-density fermentation conditions, after 72 hours of induction, collagen COL17-6 showed almost only the target band, and the proportion of the main band in the optical density analysis exceeded 90%.

[0047] 5. Purification of recombinant collagen Collect the fermentation supernatant prepared in step 1, and dilute the fermentation broth with purified water until the conductivity is 5.0 mS / cm. Add 1 M citric acid solution to adjust the pH of the fermentation broth to 3.50. Equilibrate the cation exchange medium (chromatographic packing material: Changzhou Tiandi Renhe SP Big Beads, loaded in Jiaxing Dongfulong Qianchun Biotechnology QCXKC-16 / 20) with buffer A until the absorbance and conductivity at 220 nm wavelength are stable. Start loading the sample at a flow rate of 5 mL / min and a loading volume of 0.3 L / time. Measure the absorbance at 220 nm wavelength. When the absorbance is higher than 100 mAu, collect the flow-through fermentation supernatant.

[0048] After sample loading, the cationic chromatography medium was equilibrated with buffer A. When the absorbance at 220 nm dropped to 100 mAu, collection of the flow-through fermentation supernatant was stopped. After equilibration with buffer A until the UV absorbance and conductivity stabilized, the proportion of buffer B was adjusted to 5% for washing, and then adjusted to 50% for elution of the target protein. Protein solution collection began when the 220 nm UV absorbance exceeded 50 mAu. The protein composition of the washing and elution buffers was analyzed. The target protein solution was concentrated by ultrafiltration until the conductivity was below 0.4 mS / cm. The ultrafiltration concentrate was then filtered through a 0.22 μm sterile filter and freeze-dried to obtain collagen sponge.

[0049] The SDS-PAGE electrophoresis results of the COL17-6 purification process are as follows: Figure 3 As shown.

[0050] Example 2 To screen for the optimal number of repetitions, under the same conditions as in Example 1, recombinant collagen single chains with 1, 2, 3, 4, 6, 8, and 10 repetitions were constructed and named COL17-1, COL17-2, COL17-3, COL17-4, COL17-6, COL17-8, and COL17-10, respectively. They were prepared using the same expression and purification conditions (as in Example 1) and their performance was tested according to the methods in Test Examples 1-3. The results are shown in Table 1.

[0051] Table 1 Performance comparison at different number of repetitions

[0052] Table 1 shows that the best overall performance was achieved with 6 repetitions, with the main band accounting for 93%. It remained stable after heating at 60℃ for 4 hours, forming a typical triple helix structure, and both cell adhesion and proliferation rates reached their peak. Performance was similar to 6 repetitions with 4 repetitions, exhibiting good stability and biological activity. Therefore, the preferred number of repetitions in this invention is 3-7, more preferably 4-5, and most preferably 6. With fewer repetitions, although expression stability is highest, biological activity is significantly lower. Because the monomer fragment contains only one set of functional domains and has a small molecular weight, it cannot form a stable triple helix structure, resulting in cell adhesion and proliferation rates far lower than with 6 repetitions. This indicates that the "stability" of the sequence alone is insufficient to endow collagen with its due biological function. With too many repetitions, the proportion of the main band continuously decreases with increasing repetitions, and the stability after heating at 60℃ for 4 hours gradually deteriorates from "basically stable" to "slightly degraded." This is because excessively high repetition counts amplify the protease recognition sites and unstable local secondary structures hidden in the sequence, leading to non-specific degradation of the target protein during fermentation. Meanwhile, biological activity also tends to decrease with excessive repetition.

[0053] Example 3 To screen for the optimal fragment combination, under the same conditions as in Example 1 (all repeated 6 times), recombinant collagen single chains with the following different fragment combinations were constructed: (1) COL17-6: 954-968 + 585-614 + 909-920 (i.e., the SEQ ID NO.2 fragment in Example 1); (2) COL17-Nat: The natural continuous amino acid sequence of human type XVII collagen from position 909 to 968 was selected as SEQ ID NO. 5: PGPPGPRGHQGEQGLPGFSTSGSSSFGLNLQGPPGPPGPPGPGKGDKGDPGVPGALGIPS; Repeat 6 times; (3) COL17-Rev: 909-920 + 585-614 + 954-968 (reverse the splicing order) repeat 6 times; (4) COL17-A: only amino acids at positions 909-920 are repeated 6 times; (5) COL17-B: only amino acids at positions 954-968 are repeated 6 times; (6) COL17-C: only amino acids at positions 585-614 are repeated 6 times.

[0054] The same expression and purification conditions were used for preparation (same as in Example 1), and the performance was tested according to the methods of Test Examples 1-3. The results are shown in Table 2.

[0055] Table 2 Performance Comparison of Different Fragment Combinations

[0056] Table 2 shows that while COL17-A, COL17-B, and COL17-C, containing only single functional fragments, exhibit extremely high expression stability due to their short sequences, their cell adhesion and proliferation rates are significantly lower than those of COL17-3F from this invention due to the lack of a complete functional domain combination of type XVII collagen. This indicates that retaining only a single functional fragment cannot form a complete biological functional conformation, and a single fragment cannot replace the synergistic effect of multiple fragments. Although COL17-Nat retains the complete native sequence, the main band accounts for only 55%, and significant degradation occurs after heating at 60°C. This is because the native continuous sequence contains multiple protease recognition sites, and these sites are further amplified after repetition, leading to a large amount of non-specific degradation of the target protein during fermentation, making industrial production difficult. COL17-Rev has a main band accounting for 83%, and slight degradation occurs after heating at 60°C. Its stability and activity are both lower than those of COL17-3F from this invention, indicating that the order of fragment splicing has a significant impact on protein folding efficiency and exposure to protease attack sites.

[0057] Example 4 Collagen microneedle patches were prepared using the recombinant collagen obtained in Example 1: Microneedle patch formulation: 5.0%~10.0% (w / v) recombinant type XVII humanized collagen lyophilized sponge, 1.0%~3.0% (w / v) sodium hyaluronate, 5.0%~10.0% (w / v) polyvinylpyrrolidone (PVP K90), 1.0%~3.0% (v / v) glycerin, and water for injection to bring the total to 100%.

[0058] Preparation method: (1) Dissolve the recombinant XVII type humanized collagen freeze-dried sponge prepared in Example 1 with water for injection to prepare a 10% (w / v) collagen solution. Stir magnetically or at low speed (4°C) to dissolve it fully and avoid generating bubbles.

[0059] (2) Weigh sodium hyaluronate and PVP K90, add water for injection to dissolve them, prepare a mixed matrix solution, and filter to remove insoluble matter.

[0060] (3) Mix the collagen solution and the matrix solution at a volume ratio of 6:4, add glycerol, and stir evenly to obtain the microneedle precursor solution.

[0061] (4) The microneedle precursor solution was injected into the polydimethylsiloxane (PDMS) microneedle mold, and after vacuum degassing, it was dried at room temperature for 24~48h. The recombinant type XVII collagen microneedle patch was obtained by demolding.

[0062] How to use: After cleansing the skin, apply the microneedle patch to the target area, press gently to allow the microneedles to penetrate the epidermis, and remove after 15-30 minutes.

[0063] Example 5 Collagen compound gel dressing was prepared using the recombinant collagen obtained in Example 1: It is divided into two parts, A and B. Part A is a sterile freeze-dried sponge containing 4 mg of recombinant type XVII humanized collagen. Part B consists of the following components by mass percentage: sodium hyaluronate 0.25%, carbomer 1%, propylene glycol 2.5%, glycerin 2%, methylparaben 0.05%, propylparaben 0.05%, and the remainder is purified water. When using, mix parts A and B thoroughly.

[0064] Preparation method: (1) Accurately weigh carbomer, sodium hyaluronate, glycerin, propylene glycol, methylparaben, and propylparaben, stir for 1 hour, add purified water, and dissolve completely. (2) Accurately weigh recombinant type XVII humanized collagen freeze-dried sponge. (3) Fill under aseptic conditions, seal and package, and sterilize by irradiation to obtain the recombinant collagen compound gel dressing product.

[0065] Instructions for use: Bottles A and B can be aseptically and quickly mixed before use.

[0066] Example 6 Collagen injectable photocrosslinked hydrogels were prepared using the recombinant collagen obtained in Example 1. Hydrogel formulation: 15% (w / v) methacrylamide recombinant type XVII collagen (CMA), 0.25% (w / v) photoinitiator LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphonate), and PBS buffer to 100%. Optionally, bone marrow mesenchymal stem cells (BMSCs) are loaded at a cell density of 1×10⁻⁶ cells / mL. 7 per mL.

[0067] Preparation method: (1) The recombinant XVII type humanized collagen lyophilized sponge prepared in Example 1 was dissolved in PBS to prepare a collagen solution of 10 mg / mL.

[0068] (2) The recombinant XVII type collagen was modified by methacrylamide according to conventional methods to obtain methacrylamide recombinant XVII type collagen (CMA), which was then lyophilized and stored.

[0069] (3) Before use, dissolve CMA in PBS to prepare a 15% (w / v) solution, add photoinitiator LAP to a final concentration of 0.25% (w / v), and dissolve thoroughly.

[0070] (4) If cell loading is required, resuspend BMSCs in CMA precursor solution and adjust the cell density to 1×10⁻⁶. 7 per mL.

[0071] (5) Inject the precursor solution into the target site and use blue light (wavelength 405nm, light intensity 10mW / cm²). 2 Irradiate for 30-60 seconds to achieve in-situ photocrosslinking and curing.

[0072] Application: Suitable for tissue repair scenarios requiring minimally invasive injection and in-situ gelation, such as articular cartilage defects and osteochondral defects.

[0073] Example 7 Collagen liquid dressing was prepared using the recombinant collagen obtained in Example 1: The recombinant XVII type humanized collagen lyophilized sponge contains 5 mg / mL of collagen. The remaining components, by mass percentage, include: 0.05% sodium hyaluronate, 10% glycerol, 2% carbomer, and the remainder is purified water.

[0074] Preparation method: (1) Accurately weigh carbomer, sodium hyaluronate, and glycerin, stir for 1 hour to fully dissolve. (2) Accurately weigh recombinant type XVII humanized collagen freeze-dried sponge, add purified water, and dissolve fully. (3) After mixing, fill under aseptic conditions, seal and package, and sterilize by irradiation to obtain the recombinant collagen liquid dressing product.

[0075] How to use: After cleansing, apply evenly to the face and gently massage or pat until fully absorbed.

[0076] Comparative Example 1 Collagen microneedle patches were prepared using collagen obtained from COL17-Nat (same as Example 4).

[0077] Comparative Example 2 Collagen compound gel dressings were prepared using collagen obtained from COL17-Nat (same as Example 5).

[0078] Comparative Example 3 Collagen microneedle patches were prepared using collagen obtained from COL17-Rev (same as Example 4).

[0079] Comparative Example 4 Collagen compound gel dressings were prepared using collagen obtained from COL17-Rev (same as Example 5).

[0080] Comparative Example 5 Collagen microneedle patches were prepared using collagen containing only a single functional fragment COL17-B (positions 954-968, repeated 6 times) (same as Example 4).

[0081] Test Example 1 The properties of the recombinant collagen freeze-dried collagen sponge obtained in Example 1 were tested: 1. Thermal stability Recombinant type XVII humanized collagen lyophilized sponge was prepared into a 10 mg / mL solution with ultrapure water, filtered through a 0.22 μm filter membrane, and then aliquoted into sterile centrifuge tubes. The tubes were heated at 60 °C for 1 h, 2 h, 3 h, and 4 h, and samples were taken for SDS-PAGE analysis.

[0082] The stability results of the recombinant XVII type humanized collagen freeze-dried sponge are as follows: Figure 4 As shown, the results indicate that the treated recombinant XVII type humanized collagen freeze-dried sponge still maintains structural and purity stability, with no significant difference from 0h; the results also show that the protein structure was not destroyed at 60℃, and no serious degradation or diffusion occurred, proving that the recombinant XVII type humanized collagen freeze-dried sponge has extremely high thermal stability.

[0083] 2. Acid and alkali tolerance stability test The stability of recombinant XVII type humanized collagen lyophilized sponge was tested under different pH conditions, and the results are as follows: Figure 5 As shown, the results indicate that the recombinant XVII type humanized collagen freeze-dried sponge has good pH suitability, maintains strong stability at pH 6-8, and has good clinical pH suitability.

[0084] 3. Cell proliferation experiment Recombinant humanized collagen COL17 solution and BSA solution of the same concentration were prepared as experimental groups. PBS solution was added to the control group. Then, 100 μL of solution was added to each well of a 96-well plate. Each sample was coated to prepare 3 wells. The plates were incubated at 37°C for 2 h in a 5% CO2 incubator. The excess coating solution was removed and the 96-well plates were refrigerated at 2-8°C overnight.

[0085] NIH / 3T3 cells were digested, counted, and the suspension was diluted with growth medium. The cells were then seeded at a density of 2000 cells / well (100 μL per well) in 96-well plates, and the seeding time was recorded as 0 h. The plates were incubated at 37°C for 11 h in a 5% CO2 incubator. After incubation, 10 μL of CCK-8 solution was added to each well, and the plates were incubated again at 37°C for 1 h. The absorbance was then measured at 450 nm.

[0086] 4. Cell adhesion experiment Coating: Prepare recombinant type XVII humanized collagen COL17 solution and BSA solution with the same concentration as the experimental group. Add PBS solution to the control group. Add 100 μL of solution to each well of a 96-well plate. Coat 3 wells for each sample and incubate at 37°C for 2 h in a 5% CO2 incubator.

[0087] Washing: Remove excess coating solution from the wells and wash twice with PBS solution.

[0088] Cell counting: Cells were resuspended in culture medium and counted. The cell count was diluted to a final concentration of 2 × 10⁻⁶. 5 per mL.

[0089] Assay: The cell suspension was aspirated and added to each well of a 96-well plate, 100 μL per well. After incubation at 37°C for 4 h in a 5% CO2 incubator, the culture medium was discarded. The cells were gently washed three times with PBS to remove any unadhered cells. Fresh culture medium was then added and cultured for 2 h. After that, 10 μL of CCK-8 solution was added to each well and the plate was incubated for another 1 h. The absorbance was then measured at 450 nm.

[0090] Observation of cell adhesion quantity and morphology: (1) The number and morphology of NIH / 3T3 cells were observed under a 100x light microscope when they were first seeded into the well plates of the three samples; (2) After incubation for 4 hours, the changes in cell morphology and the number of adherent cells were observed and compared under the same magnification conditions.

[0091] 5. Cell migration experiment Coating: Prepare recombinant type XVII humanized collagen solution and BSA solution of the same concentration as the experimental group. Add PBS solution to the control group. Add 1.5 mL of solution to each well of a marked 6-well plate. Coat two wells for each sample. Incubate at 37°C for 2 h in a 5% CO2 incubator.

[0092] Washing: Remove excess coating solution from the wells and wash twice with PBS solution.

[0093] Cell counting: Cells were resuspended in culture medium and counted. The cell count was diluted to a final concentration of 3 × 10⁻⁶. 5 per mL.

[0094] Detection: Aspirate the above cell suspension and add 1 mL to each well of a 6-well plate. Incubate at 37°C for 20-24 hours in a 5% CO2 incubator until 95%-100% confluence is achieved. Using a 1 mL pipette tip, mark the horizontal lines vertically and gently push downwards to create a scratch (approximately 1.0-1.5 mm wide). Gently wash twice with PBS to remove the scratched cells, then add 2 mL of fresh culture medium and incubate at 37°C in a 5% CO2 incubator. Observe the healing of the cell scratches under a microscope at 0 h and 16 h, using the intersection of the horizontal and vertical scratches as the core, and photograph the cells under a 40x microscope for analysis.

[0095] Experimental results are as follows Figure 6 and Figure 7 The results showed that recombinant type XVII humanized collagen COL17 promoted cell proliferation, cell adhesion, and cell migration.

[0096] 6. Safety The testing should be conducted according to GB / T16886.5-2017, and the steps are as follows: The potential cytotoxic effects of recombinant humanized type XVII collagen were tested using in vitro cultured mammalian L-929 cells. Recombinant collagen (1 mg / mL, 2 mg / mL, 4 mg / mL) and control samples were placed in MEM medium containing 10% fetal bovine serum and incubated at 37°C for 24 hours. After extraction, 96-well plates (103 wells) cultured for 24 hours were... 4 Remove the culture medium from each cell (well), replace it with the appropriate extraction solution, and incubate in a cell culture incubator (37℃, 5% CO2, >90% humidity) for 24 hours.

[0097] After culture, cell morphology and cell lysis were observed under a microscope, and the cytotoxicity value of the test sample was determined using the CCK8 assay. Results are as follows: Figure 8 As shown, the results indicate that the blank control group and cells maintained normal morphology throughout the experiment and showed no cytotoxic reaction. After incubating the cells at a concentration of 4 mg / mL for 24 hours, the cell morphology remained basically intact and the cell viability was close to 100%. This shows that recombinant type XVII humanized collagen had no cytotoxic effect under the CCK8 cytotoxicity assay conditions.

[0098] Test Example 2 An evaluation experiment was conducted using the products from Example 4 and Comparative Examples 1, 3, and 5 to assess androgenetic alopecia (AGA): Sixty healthy SPF-grade C57BL / 6 mice, 6-8 weeks old and weighing 18-22g, were used and acclimatized for one week before the experiment. Before modeling, the backs of the mice were dehaired with hair removal cream, and an AGA mouse model was established by inducing hair regrowth on the backs of successfully modeled mice. Successfully modeled mice showed significantly delayed hair regrowth on the backs. The successfully modeled mice were divided into 5 groups of 12 mice each.

[0099] The microneedle patches obtained in Example 4 and Comparative Examples 1, 3, and 5 were applied to the hair-removed areas on the backs of mice every two days, while the control group was treated with PBS, for a total of 21 days. Hair regeneration was recorded by photographing every 7 days, and the area of ​​hair regeneration was measured. After 21 days, skin tissue was harvested for H&E staining and immunohistochemical analysis to detect the number of hair follicles and angiogenesis. The results are shown in Table 3.

[0100] Table 3. Effects of different microneedle patches on hair regeneration in AGA mice.

[0101] Note: This indicates that the value of P < 0.05 compared to the control group.

[0102] As shown in Table 3, the microneedle patch prepared in Example 4 of this invention has a good effect on promoting hair regeneration, with a hair regeneration rate of over 90% after 21 days. Compared with the control group, the microneedle patches of Comparative Examples 1, 3, and 5 were all effective compared with the blank control, with Comparative Example 1 showing better results than Comparative Examples 3 and 5; however, their effects were worse than those of Example 4. The recombinant type XVII collagen microneedle patch of this invention can promote hair regeneration by enhancing angiogenesis and reducing tissue inflammation.

[0103] Test Example 3 An evaluation experiment was conducted on blue light-induced skin photoaging using the products of Example 5 and Comparative Examples 2 and 4: Forty healthy SPF-grade SD rats, weighing 200-250g, were used and acclimatized for one week before the experiment. After hair removal from the rats' backs, blue light (wavelength 450nm, intensity 30mW / cm²) was applied. 2 A blue light photoaging model was established by irradiating rats for 2 hours daily for 4 weeks. After successful modeling, the rats were divided into 4 groups of 10 each.

[0104] The gel dressings obtained in Example 5 and Comparative Examples 2 and 4 were applied twice daily to the photo-aged areas on the backs of rats, while the control group was treated with PBS, for a period of 4 weeks. After the experiment, transepidermal water loss (TEWL), skin elasticity, epidermal thickness, expression levels of key basement membrane components (COL4A1, COL7A1, COL17A1), and MMP activity were measured. The results are shown in Table 4.

[0105] Table 4. Effects of different gel dressings on the skin of rats aged by blue light.

[0106] As shown in Table 4, the gel dressing prepared in Example 5 of this invention can effectively reduce blue light-induced oxidative stress and inflammatory response, restore the expression of key components of the basement membrane, reduce MMP activity, and maintain the integrity of the epidermal-dermal structure. Compared with the control group, the gel dressings of Comparative Example 2 and Comparative Example 4 have certain effects, but the effect of Example 5 is significantly better than that of Comparative Example 2 and Comparative Example 4.

[0107] Test Example 4 The product from Example 5 was used to evaluate the repair of bullous pemphigoid (BP) skin wounds: Thirty healthy SPF-grade SD rats, weighing 230-250g, were used and acclimatized for one week before the experiment. A BP-like skin vesicle model was induced by intradermal injection of BP180 (type XVII collagen) antibody. Rats with successful modeling developed subepidermal vesicles and erosions on their backs. The successfully modeled rats were divided into three groups of 10 each.

[0108] The gel dressing obtained in Example 5 was applied to the skin lesions twice daily for 14 days; the control group was treated with PBS; and the positive control group was treated with conventional corticosteroid ointment. The time for blister resolution and wound healing rate were measured, and keratinocyte proliferation and adhesion were assessed. The results are shown in Table 5.

[0109] Table 5. Effects of different gel dressings on the repair of BP-like skin lesions

[0110] As shown in Table 5, the gel dressing prepared in Example 5 of this invention can promote the proliferation and adhesion of primary human keratinocytes, accelerate the healing of BP skin wounds, shorten the blister resolution time, and reduce the risk of secondary infection. Compared with the control group and the positive control group, the gel dressing of Example 5 has advantages in both wound healing speed and blister resolution time.

[0111] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A recombinant collagen protein of type XVII that is stably expressed, characterized in that, The stable XVII type recombinant collagen expressed includes a recombinant collagen single chain; wherein the recombinant collagen single chain is a collagen monomer fragment as shown in SEQ ID NO.2 repeated 2 to 10 times.

2. The recombinant collagen of type XVII with stable expression according to claim 1, characterized in that, The recombinant collagen single chain is obtained by cutting and splicing the human type XVII collagen α1 chain; wherein the amino acid sequence of the human type XVII collagen is shown in SEQ ID NO.

1.

3. The recombinant collagen of type XVII with stable expression according to claim 2, characterized in that, The collagen monomer fragment is obtained by sequentially combining amino acids at positions 954-968, 585-614, and 909-920 of human type XVII collagen, wherein the amino acid sequence of human type XVII collagen is shown in SEQ ID NO.

1.

4. The recombinant collagen of type XVII with stable expression according to claim 3, characterized in that, The amino acid sequence of the recombinant collagen single chain is shown in SEQ ID NO.

3.

5. The recombinant collagen of type XVII with stable expression according to claim 3, characterized in that, The nucleotide sequence encoding the recombinant collagen single chain is shown in SEQ ID NO.

4.

6. A method for preparing recombinant type XVII collagen with stable expression as described in any one of claims 1-5, characterized in that, Includes the following steps: Step 1: Design collagen sequences The amino acids at positions 954-968, 585-614, and 909-920 of human type XVII collagen are sequentially combined to obtain a collagen monomer fragment as shown in SEQ ID NO.

2. The collagen monomer fragment is repeated 2 to 10 times to obtain a recombinant collagen single chain. Step 2, constructing the carrier Based on the recombinant collagen single-chain, the corresponding nucleotide sequence was synthesized to construct a recombinant plasmid; Step 3: Transformation and Cultivation The recombinant plasmid was transferred into host cells, engineered strains were screened, and fermentation culture was performed to induce the expression of the target protein. Step 4: Harvesting and Purification The fermentation broth was collected, the expressed protein product was harvested, and purified to obtain the target collagen.

7. The preparation method according to claim 6, characterized in that, In step two, the plasmids used are prokaryotic expression plasmids, specifically pET series vectors, pGEX series vectors, pBAD series vectors, or pUC series vectors.

8. The preparation method according to claim 6, characterized in that, In step three, the host cell is a prokaryotic cell, a yeast cell, or a mammalian cell.

9. The use of a recombinant type XVII collagen with stable expression as described in any one of claims 1-5 in the preparation of collagen-containing products.

10. The application according to claim 9, characterized in that, The collagen products mentioned are pharmaceuticals, medical devices, or cosmetics.

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