Recombinant human-derived II-type collagen COLII-23k with skin repairing and hemostasis effects and application of recombinant human-derived II-type collagen COLII-23k

Abstract

The invention provides a recombinant human collagen type II COLII-23k. The amino acid sequence of the recombinant human collagen type II COLII-23k is as shown in SEQ ID NO. 1. The invention also provides a pichia pastoris engineering bacterium for expressing the collagen COLII-23k, the engineering bacterium comprises a gene for coding the COLII-23k, and the coding gene is connected to a pichia pastoris expression vector in a seamless cloning manner. Meanwhile, the invention also provides an application of the collagen COLII-23k. The invention has the advantages that the active fragment COLII-23k of the human collagen type II provided by the invention is a complete human sequence, does not have redundant amino acid, and has excellent hemostatic activity and skin cell proliferation, adhesion and migration promotion functions; pichia pastoris engineering bacteria for expressing the protein are constructed through seamless cloning, the expression quantity is high, and industrial production is easy; and the collagen is wide in application scene, and can meet the requirements of multiple fields such as hemostasis, wound repair and medical beauty.

Description

Recombinant human type II collagen COLII-23k with skin repair and hemostatic effects and its applications Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a recombinant human type II collagen COLII-23k with skin repair and hemostasis effects and its applications. Background Technology

[0002] Collagen, the most abundant structural protein in the human body, is a core biomaterial in the field of tissue repair and regeneration, and its market demand has been rising steadily in recent years. Traditional collagen is mainly extracted through acid / alkali hydrolysis of animal tissues, which poses safety risks such as high risk of immune rejection and potential viral and infectious disease contamination, limiting its clinical application. In contrast, human-derived collagen, due to its weak antigenicity and ability to induce cell proliferation, migration, and adhesion, has become a safer alternative material; however, breakthroughs are still needed in its functional development and efficient preparation.

[0003] Type II collagen is a fibrous protein synthesized by chondrocytes, mainly found in cartilage, vitreous humor, and intervertebral discs. It possesses excellent tensile and compressive strength, promoting bone healing, relieving joint pain, and enhancing immunity. Type II collagen interacts with proteoglycans and glycosaminoglycans in the extracellular matrix to form collagen networks, ultimately providing tissue with tensile strength. It plays a positive role in cartilage scaffolds and osteoarthritis. However, current research on the function of type II collagen focuses primarily on cartilage-related applications, with limited exploration in skin repair. Prior art (patent number ZL20240717286.9) reported a type II collagen named COLII-23, which promotes the migration of immortalized human epidermal cells (with limited effect) and only has a weak adhesion effect on mouse fibroblasts, but does not involve the regulation of human fibroblasts (key cells for dermal skin repair), and lacks core functions such as in vitro hemostasis, failing to meet the practical needs of skin wound repair and hemostatic materials.

[0004] Therefore, there is an urgent need to develop a recombinant human type II collagen that combines external hemostatic activity with full-thickness skin repair capabilities and can be prepared efficiently, in order to fill the gap in demand for multifunctional and highly adaptable collagen raw materials in the fields of medical hemostasis, wound repair and medical aesthetics, and at the same time break through the technical bottlenecks of the current single function and insufficient industrialization support of human type II collagen. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a recombinant human type II collagen COLII-23k with skin repair and hemostasis effects and its application.

[0006] The present invention adopts the following technical solution to solve the above technical problems: a recombinant human type II collagen COLII-23k with skin repair and hemostasis effects, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] As one of the preferred embodiments of the present invention, the COLII-23k has in vitro hemostatic activity and can promote the proliferation, adhesion and migration of human immortalized epidermal cells and human fibroblasts.

[0008] A Pichia pastoris engineered strain expressing COLII-23k, wherein the Pichia pastoris engineered strain contains a gene encoding COLII-23k, the gene sequence of which is shown in SEQ ID NO.2, and the encoding gene is ligated into a Pichia pastoris expression vector via seamless cloning; after the encoding gene is ligated into the Pichia pastoris expression vector, a recombinant expression vector is obtained, which is then linearized and transformed into Pichia pastoris chassis cells.

[0009] As one of the preferred embodiments of the present invention, the Pichia pastoris chassis strain includes at least one of Pichia pastoris GS115, Pichia pastoris X33, Pichia pastoris KM71H, and Pichia pastoris SMD1168.

[0010] As one of the preferred embodiments of the present invention, a pPIC9K-based expression vector is used, and the encoding gene is inserted downstream of the signal peptide of pPIC9K through seamless cloning.

[0011] As one of the preferred methods of the present invention, the specific construction method of the Pichia pastoris engineered strain is as follows: (1) Primer design and fragment amplification: Design specific primers according to the coding gene sequence shown in SEQ ID NO.2, and obtain the coding gene fragment by PCR amplification; at the same time, use pPIC9K as a template to obtain the linearized vector fragment by reverse PCR amplification; the linearized vector fragment and the coding gene fragment have homologous arms at both ends; (2) Recombinant vector construction: Mix the coding gene fragment obtained in step (1) with the linearized vector fragment, add homologous recombinase, and connect the fragment and the vector by seamless cloning to construct a recombinant expression vector containing the coding gene; (3) Vector linearization and transformation: Linearize the recombinant expression vector with restriction endonuclease, and transform the linearized recombinant expression vector into Pichia pastoris chassis cells by electroporation, and after static recovery, spread it on YPD solid medium; (4) Strain screening: After single colonies are formed on the medium, select single colonies and transfer them to YPD screening medium containing genimycin, and perform resistance screening by increasing the concentration of genimycin in a gradient, and finally obtain the target Pichia pastoris engineered strain.

[0012] As one of the preferred embodiments of the present invention, in step (1), the specific primers for amplifying the coding gene fragment include an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4. The 5' ends of both the upstream and downstream primers have 20 bp homologous arms, and the homologous arm sequences are complementary to the upstream and downstream sequences of the linearized pPIC9K vector signal peptide, respectively.

[0013] As one of the preferred embodiments of the present invention, in step (2), the coding gene is seamlessly connected to the downstream sequence of the pPIC9K vector signal peptide, and the constructed recombinant expression vector contains only the coding gene sequence shown in SEQ ID NO.2 and the necessary functional sequence of the pPIC9K vector.

[0014] As one of the preferred embodiments of the present invention, the essential functional sequences of the pPIC9K vector include a promoter, a selection marker gene, and a replication origin.

[0015] As one of the preferred embodiments of the present invention, in step (4), when the target Pichia pastoris engineered strain is fermented and cultured, under the conditions of proliferation with glycerol as carbon source and methanol induction expression for 60h in a 5L fermenter, the expression level of COLII-23k is not less than 3.9g / L; when induced and cultured in a 30L fermenter with the same process for 108h, the expression level of collagen is not less than 2.9g / L.

[0016] Application of the above-mentioned recombinant human type II collagen COLII-23k in the preparation of hemostatic sponges, wound repair materials, medical aesthetic and plastic surgery materials and cosmetics.

[0017] The advantages of this invention compared with the prior art are: (1) Filling the application gap of existing type II collagen. This invention breaks through the limitation of traditional type II collagen focusing only on the cartilage field, and endows it with significant in vitro hemostatic activity + full-thickness skin repair ability; compared with the COLII-23 collagen disclosed in patent number ZL20240717286.9, the COLII-23k of this invention adds the core functions of skin repair and hemostasis; the collagen of this invention can not only efficiently promote the proliferation, adhesion and migration of human immortalized epidermal cells (epidermal repair), but also significantly regulate the physiological activity of human fibroblasts (core cells of dermal repair), realize the full-thickness repair of epidermis and dermis, and show excellent healing effect in mouse wound healing model, and is suitable for medical scenarios such as hemostatic sponge and wound repair materials.

[0018] (2) Stronger activity and higher adaptability Compared with the prior art COLII-23 collagen, the COLII-23k of this invention has achieved a qualitative leap in the regulatory effect on human cells: the adhesion ability of human fibroblasts has increased from 110% to 144%; the migration ability of human fibroblasts at 48h has increased from 31% to 57.4%, with a significant migration promotion effect; at the same time, the in vitro procoagulant ability of the collagen of this invention has increased from 77.5% to 99%, with outstanding hemostatic function, and its practicality in trauma emergency rescue, surgical hemostasis and other scenarios far exceeds that of similar products; in addition, due to the use of human sequence, the safety risks such as immune rejection and viral contamination of animal collagen are completely avoided.

[0019] (3) High-efficiency preparation and controllable cost: This invention achieves high-density and high-yield fermentation of recombinant human type II collagen through the construction strategy of "seamless cloning + Pichia pastoris engineered strain": the expression level is ≥3.9g / L after 60h induction in a 5L fermenter and the expression level is still stable at ≥2.9g / L after 108h induction in a 30L fermenter; and the vector constructed by seamless cloning has no residual enzyme cleavage sites, so that the expressed collagen does not contain excess amino acids brought by enzyme cleavage sites, ensuring the complete humanization of the protein and avoiding the problems of reduced protein activity and increased immunogenicity caused by the introduction of non-target amino acids in traditional enzyme cleavage and ligation processes; at the same time, the Pichia pastoris expression system has the ability to modify eukaryotic proteins after translation, which can ensure the natural conformation and biological activity of collagen, reduce the quality control difficulty of large-scale production, and lay the core foundation for industrial application.

[0020] (4) Covering the needs of multiple fields, this invention covers the entire scenario of "medical-grade function + consumer-grade application" from medical (hemostatic sponge, wound repair material) to medical aesthetics (medical beauty and plastic surgery material) and daily chemical (cosmetics), solving the problem of the single application of existing type II collagen, with wider market adaptability and higher commercial value.

[0021] In summary, this invention not only solves the technical pain points of existing type II collagen, such as "single function and high safety risks", but also breaks through the bottleneck of "difficult industrial preparation". It combines technological innovation and industrial practicality and can effectively fill the gap in demand for multifunctional human collagen raw materials in the fields of medical treatment and medical aesthetics. Attached Figure Description

[0022] Figure 1 is an electrophoresis diagram of the protein obtained by separation and purification of recombinant human type II collagen COLII-23k of the present invention (in the figure, lane 1 is the fermentation supernatant, lane 2 is the flow-through medium, and lane 3 is the purified COLII-23k protein); Figure 2 is the in vitro hemostatic activity detection result of recombinant human type II collagen COLII-23k of the present invention (in the figure, Figure A is a visual photograph of the in vitro hemostatic effect of different collagen samples, and Figure B is a bar chart of the in vitro coagulation rate of different collagen samples; **** indicates P<0.0001); Figure 3 is the effect of recombinant human type II collagen COLII-23k of the present invention on the proliferation, adhesion and migration activity of human immortalized epidermal cells HaCaT (in the figure, Figure A is the proliferation rate result, Figure B is the adhesion rate result, Figure C is the migration rate result at 24 h, and Figure D is the migration rate result at 48 h). Migration rate results, Figure E is a representative photograph of the scratch test; * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001); Figure 4 shows the effects of the recombinant human type II collagen COLII-23k of the present invention on the proliferation, adhesion and migration activities of human skin fibroblasts (HSF) (Figure A shows the proliferation rate, Figure B shows the adhesion rate, Figure C shows the migration rate at 24h, and Figure D shows the migration rate at 48h). The migration rate results are shown in Figure E, which is a representative photograph of the scratch test; * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001). Figure 5 shows the effect of recombinant human type II collagen COLII-23k on the repair of mouse skin wounds (Figure A shows representative photographs of wounds at different time points, and Figure B shows the quantitative analysis curve of wound healing rate). Figure 6 shows the cell growth and COLII-23k expression results of the recombinant Pichia pastoris engineered strain fermented in a fermenter (Figure A shows the wet weight and protein expression level of the 5L fermenter, Figure B shows the SDS-PAGE analysis results of the 5L fermenter, and Figure C shows the wet weight and protein expression level of the 30L fermenter). Figure 7 is a schematic diagram of the expression plasmid structure of the recombinant human type II collagen COLII-23k of the present invention; Figure 8 is a schematic diagram of the expression vector structure of the prior patent COLII-23; Figure 9 is a comparison of the differences in in vitro hemostatic activity between COLII-23k of the present invention and the prior patent COLII-23 (in the figures, A and B show the hemostatic effect of different additions of the prior patent COLII-23, and C and D show the hemostatic effect of different additions of the present invention COLII-23k; ** indicates P<0.01, *** indicates P<0.001, **** indicates P<0.0001). Detailed Implementation

[0023] The embodiments of the present invention are described in detail below. These embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0024] The Pichia pastoris GS115 strain used in the following examples was obtained from Shanghai Angyu Company, and the expression vector pPIC9K was obtained from Shanghai Sangon Biotech Co., Ltd. Both strains and vectors are readily available for purchase. The culture medium formulations used are as follows: 1) YPD medium (1L): Add 10g / L yeast extract and 20g / L peptone to 900mL of water, autoclave, and then add 100mL of sterile 10× glucose solution. The solid culture medium contains 1.5% agar.

[0025] 2) MD medium (1L): First, autoclave 800mL of water, then cool it to 60℃, and then add 100mL of 10X YNB, 2mL of 500X Biotin, and 100mL of 10X glucose. The solid medium contains 2% agar.

[0026] 3) BMGY medium (1L): Add 10g / L yeast extract and 20g / L peptone to 700mL of water, autoclave, cool to room temperature, and then add 100mL of 1M potassium phosphate buffer (pH 6.0), 100mL of 10X YNB, 2mL of 500X biotin and 100mL of 10X glycerol.

[0027] 4) BMMY medium (1L): Add 10g / L yeast extract and 20g / L peptone to 700mL of water, autoclave, cool to room temperature, and then add 100mL of 1M potassium phosphate buffer (pH 6.0), 100mL of 10X YNB, 2mL of 500X biotin, and 100mL of 10X methanol.

[0028] 5) BSM medium: CaSO4 0.93g / L, K2SO4 18.2g / L, MgSO4·7H2O 14.9g / L, KOH 4.13g / L, glycerol 40g / L, defoamer 1g / L; after sterilization, add 26.7mL of 85% phosphoric acid.

[0029] 6) PTM1 Trace Elements: CuSO4·5H2O 6g / L, NaI 0.08g / L, MnSO4·H2O 3g / L, NaMoO4·2H2O 0.2g / L, H3BO3 0.02g / L, CoCl2 0.5g / L, ZnCl2 20g / L, FeSO4·7H2O 65g / L, Biotin 0.2g / L, H2SO4 5mL.

[0030] Meanwhile, the DMEM basal culture medium, PBS, fetal bovine serum (FBS), and trypsin used in the following examples were from Vivacell, the human immortalized epidermal cell line HaCaT was from Oricell, and the human skin fibroblast cell line HSF was from Fudancell.

[0031] Example 1: Recombinant human type II collagen COLII-23k: The recombinant human type II collagen COLII-23k in this example is a completely human type II collagen with the amino acid sequence shown in SEQ ID NO.1. It has in vitro hemostatic activity and can promote the proliferation, adhesion and migration of human immortalized epidermal cells and human fibroblasts.

[0032] Example 2: Construction of a fully human type II collagen COLII-23k expression plasmid: 1. Amplification of the target gene fragment: Specific primers were designed based on the coding gene sequence shown in SEQ ID NO.2; using the coding gene sequence shown in SEQ ID NO.2 as a template, PCR amplification was performed using the designed and synthesized upstream primer (SEQ ID NO.3) and downstream primer (SEQ ID NO.4) to obtain the COLII-23k coding gene fragment. Both the upstream and downstream primers have a 20bp homologous arm at their 5' ends. The PCR amplification reaction system and reaction conditions are shown in Table 1.

[0033] Table 1. PCR amplification reaction system and reaction conditions

[0034] 2. Vector Linearization: Using the pPIC9K vector as a template, reverse PCR amplification was employed to linearize the vector (the amplified region covers the downstream multiple cloning site of the signal peptide). The specific primers used for reverse PCR must be designed to match the homologous arm characteristics of the target gene fragment. Specifically, the 5' 20bp homologous arm sequence of the reverse PCR primer sequence is complementary to the 5' 20bp homologous arm sequence of the specific primers (SEQ ID NO. 3, SEQ ID NO. 4) used to amplify the COLII-23k encoding gene in the previous steps, ensuring seamless and efficient ligation of the linearized vector and the target gene fragment. The reverse PCR amplification reaction system and conditions are shown in Table 2.

[0035] Table 2. Reverse PCR amplification reaction system and reaction conditions

[0036] After amplification, the linearized pPIC9K vector fragment was recovered by 1% agarose gel electrophoresis (ensuring that the linearized vector had 20bp sequences homologous to the target gene fragment at both ends).

[0037] 3. Seamless cloning and construction of recombinant expression plasmids: The recovered COLII-23k encoding gene fragment and the linearized pPIC9K vector fragment were combined with homologous recombinase and reacted according to the system in Table 3. After inserting the sequence fragment downstream of the pPIC9K signal peptide, the fully human type II collagen COLII-23k expression plasmid pPIC9K-II-23k was obtained.

[0038] Table 3. Homologous recombination system and reaction conditions

[0039] Example 3, Construction of recombinant Pichia pastoris engineered strain: (1) The pPIC9K-II-23k plasmid constructed in Example 2 was transferred into Pichia pastoris competent cells.

[0040] (2) Pick single colonies from the transformed MD plate and place them in a 48-well culture plate containing 500 μL YPD (0.75 mg / mL G418) medium. Incubate at 30°C and 240 rpm for 18-24 h. Then transfer them to YPD liquid medium with gradually increasing G418 content for further culture.

[0041] (3) Ferment the strain with good growth as follows: Inoculate the bacterial solution into 10 mL / 50 mL BMGY and culture at 30℃ and 240 rpm for 18-24 h; centrifuge to remove the supernatant, add 10 mL BMMY to resuspend, culture at 28℃ and 240 rpm for 48 h, and add 100% methanol every 24 h until the final concentration is 1.5%; centrifuge, take the supernatant for SDS-PAGE analysis, and obtain the recombinant Pichia pastoris engineered strain with high expression level.

[0042] Example 4, Production and purification of type II collagen COLII-23k: (1) Take the recombinant Pichia pastoris engineered strain successfully constructed in Example 3 and activate it in YPD (containing the corresponding resistant G418) medium at 30℃, 240rpm, for 18h; (2) Transfer 2% inoculum to 200mL / 1L BMGY and culture at 30℃, 240rpm for 26~28h to completely consume the glycerol in the medium; (3) Take 200mL BMMY to resuspend the bacterial cells and culture at 25℃, 240rpm for 48h; add 100% methanol every 24h to the final concentration of 1.5%.

[0043] (4) After fermentation, the supernatant was filtered through a 0.22 μm aqueous filter membrane to obtain a clear fermentation supernatant. The fully human type II collagen fragment COLII-23k was separated and purified by dialysis (dialysis solution: 20 mM Tris-HCl, pH 8), ion exchange chromatography, and secondary dialysis (dialysis solution: PBS, pH 7.0).

[0044] Figure 1 shows the electrophoresis diagram of the COLII-23k protein obtained after separation and purification. The purified COLII-23k protein solution was analyzed by gel filtration chromatography and non-reducing SDS-PAGE, and the purity was >90% and the recovery rate was >85%.

[0045] Example 5: Validation of the in vitro hemostatic activity of COLII-23k: This experiment uses COLII-23k purified in Example 4 as the experimental material to verify its in vitro coagulation efficacy. The core indicator is to determine the ability of the biomaterial to induce the formation of blood clots on the surface of the material after contact with calcified whole blood. Specifically, the procoagulant activity of different samples is evaluated by the blood clotting index (BCI) test.

[0046] I. Experimental Materials Sample Group: COLII-23k protein solution purified in Example 4; Control Group: physiological saline (negative control, NC), bovine type I collagen (COLI (Bovine)), and chicken type II collagen (COLII (Chick)).

[0047] II. Experimental Procedure: Take four clean 10mL centrifuge tubes and add 1mL of solution to each of the sample group, negative control group, bovine type I collagen group, and chicken type II collagen group, respectively. Incubate in a 37℃ constant temperature water bath for 5 minutes to ensure temperature stability. Quickly add 200μL of preheated (37℃) sodium citrate anticoagulated whole blood to each centrifuge tube, and immediately add 20μL of preheated 0.2M sodium citrate solution. CaCl2 solution (triggers coagulation reaction), gently invert the centrifuge tube 3 times to mix, and continue incubation at 37℃ for 2 min; slowly add 10 mL of preheated 37℃ deionized water along the wall of the centrifuge tube, and incubate at 37℃ for 2 min; centrifuge at 2000 rpm for 5 min, and transfer 100 μL of supernatant to a 96-well plate (3 replicates per group), and measure the absorbance (Abs) at 540 nm using an ELISA reader; calculate the coagulation index (BCI) according to the following formula: BCI = (Abs of sample group / Abs of negative control group) × 100%, the lower the BCI value, the stronger the procoagulant activity.

[0048] III. Experimental Results The results are shown in Figure 2.

[0049] The results above show that the BCI index of COLII-23k is only 1.0%, which is significantly lower than that of the negative control group (100%) and other collagen control groups, proving that it has excellent in vitro hemostatic activity.

[0050] Example 6: Verification of skin cell proliferation activity of COLII-23k: In this example, COLII-23k purified in Example 4 was used as material, and the MTT assay was used to detect its proliferation-promoting effect on human immortalized epidermal cells (HaCaT) and human skin fibroblasts (HSF).

[0051] I. Experimental Materials Sample Group: COLII-23k protein solution purified in Example 4 (concentration gradient: 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL, 0.00625 mg / mL, diluted with DMEM medium containing 10% fetal bovine serum); Control Group: DMEM medium containing 10% fetal bovine serum (negative control, NC), DMEM medium containing 5% DMSO (positive control, used to verify the cytotoxicity boundary), and cell-free DMEM medium (blank control).

[0052] II. Experimental Procedure: HaCaT cells and HSF cells were digested, centrifuged, and resuspended (in DMEM medium containing 10% fetal bovine serum), and then diluted to 4 × 10⁻⁶. 4 Cells / mL; 100 μL was seeded into each well of a 96-well cell culture plate and incubated at 37°C, 5% CO2, and 95% humidity for 24 h; the original culture medium was discarded, and DMEM medium was added to the blank control wells (culture medium only) and negative control wells, and DMEM medium containing 5% DMSO was added to the positive control wells. Experimental groups were added to DMEM medium containing different concentrations of COLII-23k. Each group was divided into 3 replicates, and the cells were incubated for another 24 h; 20 μL of MTT reagent was added to each well, and the cells were incubated at 37°C, 5% CO2, and 95% humidity for 4 h; 150 μL of LDMSO was added, and the cells were shaken for 10 min to fully dissolve the crystals. The absorbance at 490 nm was measured using a microplate reader. Cell viability was calculated using the formula: Cell viability = (Experimental group Abs - Blank control group Abs) / (Negative control group Abs - Blank control group Abs) * 100%.

[0053] III. Experimental Results The results are shown in Figure 3A and Figure 4A.

[0054] The results above show that, with the negative control group as 100%, the survival rate of the HaCaT experimental group with COLII-23k and the survival rate of the HSF experimental group with COLII-23k were both 123%, demonstrating that they can effectively promote the proliferation of epidermal and dermal core cells.

[0055] Example 7: Verification of skin cell adhesion and migration activity of COLII-23k: In this example, the MTT assay (adhesion test) and scratch assay (migration test) were used to detect the adhesion and migration promoting effects of COLII-23k on HaCaT cells and HSF cells, respectively.

[0056] I. Experimental Materials and Samples: COLII-23k protein solution purified in Example 4 (adhesion test concentrations: 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL, 0.00625 mg / mL; migration test concentrations: 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL); Control Groups: physiological saline (negative control for adhesion test), DMEM medium containing 10% fetal bovine serum (negative control for migration test, NC), bovine type I collagen (positive control), and cell-free DMEM medium (blank control for migration test).

[0057] II. Cell Adhesion Activity Assay (MTT Assay): HaCaT cells and HSF cells were digested, centrifuged, resuspended, and diluted to 5 × 10⁻⁶ cells / mL. 4 Cells / mL were seeded at 100 μL per well into a 96-well cell culture plate, and then 100 μL of COLII-23k protein solution of different concentrations was added to each well. Physiological saline was added as a negative control. The cells were cultured at 37°C, 5% CO2, and 95% humidity for 6-8 hours.

[0058] Discard the supernatant, wash once with PBS, add 100 μL of fresh culture medium and 20 μL of MTT solution to each well, and incubate in an incubator for 4 h at 37°C, 5% CO2, and 95% humidity.

[0059] Discard the culture medium, add 150 μL of DMSO, and incubate on a shaker for 10 min to fully dissolve the crystals. Select 570 nm (490 nm) as the detection wavelength and 630 nm as the reference wavelength, and measure the absorbance of each well on a microplate reader. Calculate the adhesion rate using the formula: Adhesion rate = (Experimental group Abs / Negative control group Abs) × 100%.

[0060] II. Cell Migration Activity Assay (Scratch Test): Five horizontal lines were drawn on the back of each well of a 6-well plate using a marker pen, with each line approximately 0.5 cm apart. HaCaT cells or HSF cells were used, and control, experimental, and blank control groups were established. After digestion, centrifugation, and resuspending, the cells were diluted to 2 × 10⁻⁶ cells / well. 6 Cells / mL, 2mL of cell suspension per well, to ensure that the cell density of each group is the same and that 95-100% confluence can be achieved after 24 hours of culture.

[0061] After culturing cells for 24 hours, a 200 μL pipette tip was used to vertically and closely scrape across the cell layer to form a scratch. The cells were washed three times with PBS to remove the scraped cells. 2 mL of culture medium containing COLII-23k protein was added to each of the six wells.

[0062] The cells were incubated in an incubator at 37°C, 5% CO2, and 95% humidity, and then photographed at fixed locations after 0h, 24h, and 48h.

[0063] ImageJ was used to measure the area of ​​the scratched region: the scratch images (0h, 24h / 48h) were processed using the software to obtain the cell migration rate. ×100%.

[0064] IV. Experimental Results The results of cell adhesion activity detection are shown in Figures 3B and 4B. These results indicate that, with the negative control group as 100%, the adhesion rate of the HaCaT experimental group supplemented with COLII-23k was 144%, and the adhesion rate of the HSF experimental group supplemented with COLII-23k was also 144%, significantly better than the negative control and bovine type I collagen, thus promoting skin cell colonization in the wound matrix.

[0065] The results of cell migration activity assays are shown in Figures 3C-E and 4C-E. These results indicate that COLII-23k significantly accelerated the migration of HaCaT cells and HSF cells to the center of the wound at 48 hours, reaching 61% and 57.4% respectively, providing crucial impetus for epidermal coverage and dermal repair.

[0066] Example 8: Verification of COLII-23k's wound healing activity in mice: This example uses COLII-23k purified in Example 4 as the intervention material to verify its function of promoting skin wound repair in vivo, strictly following the ethical guidelines for animal experiments.

[0067] I. Experimental Materials and Samples: COLII-23k protein solution purified in Example 4 (1.2 mg / cm³) 2**Wound Treatment Dosage:** Control group: Physiological saline (negative control, used for routine wound treatment); **Experimental Procedure:** Six male Kunming rats (8-10 weeks old) were housed under controlled conditions: relative humidity 50%, light / dark cycle 12 hours, and temperature 22 ± 1 ℃. All animals had free access to water and standard feed. This animal experiment was approved by the Animal Care and Use Committee of Anhui University (Animal Use Protocol Approval Number: IACUC(AHU)-2025-103; Approval Date: November 15, 2025). After a one-week acclimatization period, bilateral symmetrical modeling was performed on each mouse. Mice were anesthetized with ether, and two 4 mm biopsy perforations were made on the skin of each dorsal side. The wounds were treated with a specified concentration of COLII-23k, and the dressings were changed every 2 days, while the control group used physiological saline. Wound area was measured daily by digital radiography. On the sixth day post-surgery, the animals were euthanized with an ether overdose.

[0068] III. Experimental Results The results are shown in Figure 5.

[0069] The results show that during the experiment, COLII-23k significantly accelerated skin wound healing compared to untreated wounds. Untreated wounds exhibited an inflammatory response with a healing rate of 40%. Conversely, skin tissue in COLII-23k-treated areas showed significant central migration, reduced inflammation, and a wound healing rate reaching 68.5% by the second day. By day 4, the wound areas in mice were covered with scabs; by day 6, the wounds showed significant contraction, with a healing rate of 95% for COLII-23k-treated wounds and 88% for untreated wounds. These results demonstrate that COLII-23k can effectively accelerate skin wound healing in mice.

[0070] Example 9. Fermentation of COLII-23k recombinant Pichia pastoris engineered strain in a fermenter: I. Fermentation in a 5L fermenter: 1. Pick a fresh single colony from a YPD plate and inoculate it into a 50mL centrifuge tube containing 5mL of BMGY medium. Incubate at 30℃ and 240rpm for 18-24h. Transfer the inoculum at a rate of 2% to a 1L shake flask containing 200mL of BMGY seed medium. Incubate at 30℃ and 240rpm for 14-18h until OD (dose retardation). 600 =5~6. Inoculate the seed culture medium into a 5L fermenter containing basal salt medium at a 10% inoculation rate.

[0071] 2. During the glycerol culture phase, maintain a temperature of 30℃, pH 5.0, and a rotation speed of 400-800 rpm to maintain DO > 20%. When the glycerol in the BSM medium is depleted (DO = 70-80%), enter the glycerol feeding phase, starting with the addition of 50% glycerol to further increase cell density. When the cell wet weight is approximately 180-220 g / L, enter the starvation phase. After 1 hour of starvation, enter the methanol feeding phase, adjusting the induction temperature to 28℃ and starting the methanol feeding to maintain DO at around 20%. Maintain the pH throughout the fermentation process using ammonia and phosphoric acid. After 84 hours of induction, remove the culture from the tank and collect the supernatant by centrifugation.

[0072] The growth curves of COLII-23k fermentation cells and the expression level of the target protein in a 5L fermenter are shown in Figure 6A. The results of SDS-PAGE analysis of samples taken every 12 hours (diluted 8 times) are shown in Figure 6B.

[0073] The results above show that in a 5L fermenter, the highest yield of COLIII-23k was 3.9 g / L after 60 hours of induction with the recombinant engineered bacteria of this invention.

[0074] II. Fermentation in a 30L fermenter: 1. Pick a fresh single colony from a YPD plate and inoculate it into a 50mL centrifuge tube containing 5mL of BMGY medium. Incubate at 30℃ and 240rpm for 18-24 hours. Transfer the inoculum to a 2L shake flask containing 500mL of BMGY seed medium at a 2% inoculation rate. Incubate at 30℃ and 240rpm until OD reaches 0.50. 600 =5~6. Inoculate the seed culture medium into a 30L fermenter at a 10% inoculation rate.

[0075] 2. During the glycerol culture phase, maintain a temperature of 30℃, pH 5.0, and a rotation speed of 400-800 rpm to maintain DO > 20%. When the glycerol in the BSM medium is depleted (DO = 70-80%), enter the glycerol feeding phase, starting with the addition of 50% glycerol to further increase cell density. When the cell wet weight is approximately 180-220 g / L, enter the starvation phase. After 1 hour of starvation, enter the methanol feeding phase, adjusting the induction temperature to 28℃ and starting the methanol feeding to maintain DO at around 20%. Maintain the pH throughout the fermentation process using ammonia and phosphoric acid. After 108 hours of induction, remove the culture from the tank and collect the supernatant by centrifugation.

[0076] The growth curves of COLII-23k fermentation cells and the expression level of the target protein in a 30L fermenter are shown in Figure 6C. The results of SDS-PAGE analysis of samples taken every 12 hours (diluted 6 times) are shown in Figure 6D.

[0077] The results above show that in a 30L fermenter, the highest yield of COLIII-23k was 2.9g / L when the recombinant engineered bacteria of this invention was induced for 108h.

[0078] Example 10: Performance comparison of COLII-23k of the present invention and COLII-23 of the prior patent: This example uses COLII-23k purified in Example 4 (the present invention) and COLII-23 disclosed in the prior patent (patent number ZL20240717286.9) as the research objects. The analysis is carried out from two dimensions: "difference in carrier construction" and "comparison of core performance" to clarify the technical innovation of the present invention. The experimental methods are the same as those in Examples 5 (hemostasis) and 7 (adhesion / migration).

[0079] I. Experimental Materials 1. Test Samples: Samples of this application: COLII-23k protein solution purified in Example 4 (adhesion test concentration 0.0156 mg / mL, migration test concentration 0.1 mg / mL, hemostasis test concentration same as in Example 5); Samples of prior patents: COLII-23 protein and corresponding expression vector prepared according to the method described in patent number ZL20240717286.9, with protein concentration consistent with the sample of this application (the amino acid sequence of COLII-23 protein is shown in SEQ ID NO.7).

[0080] 2. Control group: Adhesion / migration test: physiological saline (negative control, NC), bovine type I collagen (positive control, same as in Example 7); Hemostasis test: physiological saline (negative control, NC), same as in Example 5.

[0081] II. Differences in Vector Construction The plasmid structures of the expression vectors corresponding to the COLII-23k protein of this invention and the prior patent COLII-23 protein are shown in Figures 7 and 8, respectively. The expression vector corresponding to the COLII-23 protein uses the traditional enzyme digestion and ligation method, resulting in extra amino acid sequences at the gene-vector junction after insertion. In contrast, this invention uses a seamless cloning method (through homologous arm complementarity ligation), seamlessly inserting the target gene downstream of the pPIC9K signal peptide without extra amino acids, resulting in a completely human sequence.

[0082] Based on this, the present invention avoids the "excess amino acid interference" caused by the enzymatic cleavage ligation of prior patents, and keeps COLII-23k in a fully humanized structure. This is one of the core reasons why its adhesion, migration and hemostatic properties are significantly better than COLII-23 (see the experimental results below) - excess amino acids may change the spatial conformation of the protein and affect its ability to bind to cell surface receptors and coagulation factors, while the native conformation of the protein in this application is more likely to exert biological activity.

[0083] III. Core Performance Comparison Experiment 1. Comparison of Adhesion Effect of Human Fibroblasts (HSF) Refer to the procedure of "Cell Adhesion Activity Detection (MTT Method)" in Example 7, only adding "Prior Patent COLII-23 Group".

[0084] The results showed that COLII-23 protein promoted the adhesion function of human fibroblasts by 110%, while the COLII-23k protein of this invention promoted the adhesion function of human fibroblasts by 144%.

[0085] 2. Comparison of human fibroblast (HSF) migration effect after 48 hours: Refer to the procedure of "cell migration activity detection (scratch test)" in Example 7, and add "pre-patent COLII-23 group".

[0086] The results showed that the migration ability of the blank control group was 31% after 48 hours, the migration ability of human fibroblasts promoted by COLII-23 protein after 48 hours was 37.4%, and the migration ability of human fibroblasts promoted by COLII-23k protein of the present invention after 48 hours was 57.4%.

[0087] 3. The in vitro hemostatic function comparison follows the procedure of "in vitro hemostatic activity verification (BCI determination)" in Example 5, with the addition of "pre-existing patent COLII-23 group".

[0088] The results are shown in Figure 9.

[0089] The results above show that the prior patent COLII-23 protein has the best hemostatic effect when the addition amount is 4 mg / mL, with a procoagulant index of 22.5%; while the present invention COLII-23k protein (represented as II-23k in the figure) can achieve the best hemostatic effect when the addition amount is only 0.4 mg / mL, with a procoagulant index as high as 99%.

[0090] As can be seen from the comparison, the optimal hemostatic addition amount of COLII-23k protein in this invention is only 1 / 10 of that in the prior patent COLII-23, and the procoagulant index is increased by more than 4 times, proving that it has stronger hemostatic activity and requires a lower dosage.

[0091] In summary, compared to the prior patent COLII-23, this invention COLII-23k achieves a comprehensive upgrade in core performance through optimization of "seamless cloning vector construction + fully humanized sequence": significantly enhanced adhesion ability of human fibroblasts and greatly improved migration promotion effect. At the same time, it adds "high-activity, low-dose" in vitro hemostasis function, solving the problems of single protein function, insufficient activity, and limited application scenarios of the prior patent, and is more suitable for the actual needs of medical and aesthetic fields such as trauma emergency care and skin wound repair.

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant human type II collagen COLII-23k with skin repair and hemostatic effects, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

1.

2. The COLII-23k according to claim 1, characterized in that, The COLII-23k has in vitro hemostatic activity and can promote the proliferation, adhesion and migration of human immortalized epidermal cells and human fibroblasts.

3. An engineered Pichia pastoris strain expressing COLII-23k as described in claim 1 or 2, characterized in that, The engineered Pichia pastoris strain contains a gene encoding COLII-23k, the gene sequence of which is shown in SEQ ID NO.2, and the encoding gene is ligated into the Pichia pastoris expression vector via seamless cloning.

4. The engineered Pichia pastoris strain according to claim 3, characterized in that, The Pichia pastoris expression vector is based on pPIC9K, and the coding gene is inserted downstream of the signal peptide of pPIC9K via seamless cloning.

5. The engineered Pichia pastoris strain according to claim 4, characterized in that, The specific construction method of the Pichia pastoris engineered strain is as follows: (1) Primer design and fragment amplification: Design specific primers according to the coding gene sequence shown in SEQ ID NO.2, and obtain the coding gene fragment by PCR amplification; at the same time, use pPIC9K as a template to obtain the linearized vector fragment by reverse PCR amplification; the linearized vector fragment and the coding gene fragment have homologous arms at both ends; (2) Recombinant vector construction: Mix the coding gene fragment obtained in step (1) with the linearized vector fragment, add homologous recombinase, and connect the fragment and the vector by seamless cloning to construct a recombinant expression vector containing the coding gene; (3) Vector linearization and transformation: Linearize the recombinant expression vector with restriction endonuclease, and transform the linearized recombinant expression vector into Pichia pastoris chassis cells by electroporation, and after static recovery, spread it on YPD solid medium; (4) Strain screening: After single colonies are formed on the medium, select single colonies and transfer them to YPD screening medium containing genimycin, and perform resistance screening by increasing the concentration of genimycin in a gradient, and finally obtain the target Pichia pastoris engineered strain.

6. The engineered Pichia pastoris strain according to claim 5, characterized in that, In step (1), the specific primers for amplifying the coding gene fragment include an upstream primer and a downstream primer. The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

4. The 5' ends of both the upstream and downstream primers have 20 bp homologous arms, and the homologous arm sequences are complementary to the upstream and downstream sequences of the linearized pPIC9K vector signal peptide, respectively.

7. The engineered Pichia pastoris strain according to claim 5, characterized in that, In step (2), the coding gene is seamlessly connected to the downstream sequence of the pPIC9K vector signal peptide, and the constructed recombinant expression vector contains only the coding gene sequence shown in SEQ ID NO.2 and the necessary functional sequence of the pPIC9K vector.

8. The engineered Pichia pastoris strain according to claim 7, characterized in that, The essential functional sequences of the pPIC9K vector include the promoter, selection marker gene, and origin of replication.

9. The engineered Pichia pastoris strain according to claim 5, characterized in that, In step (4), when the target Pichia pastoris engineered strain is fermented and cultured, under the conditions of proliferation with glycerol as carbon source and methanol induction for 60h, the expression level of COLII-23k is not less than 3.9g / L; when induced and cultured in a 30L fermenter with the same process for 108h, the expression level of collagen is not less than 2.9g / L.

10. The application of COLII-23k as described in claim 1 or 2 in the preparation of hemostatic sponges, wound repair materials, medical aesthetic and plastic surgery materials, and cosmetics.

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