High-activity recombinant human type iii collagen and application thereof

Abstract

This invention provides a highly active recombinant human type III collagen, the amino acid sequence of which is shown in SEQ ID NO.2. The invention also provides a gene encoding the above-mentioned recombinant human type III collagen, a recombinant expression vector containing this gene, and an engineered Pichia pastoris strain containing the recombinant expression vector. Furthermore, this invention provides a method for preparing the above-mentioned recombinant human type III collagen and its applications. The advantages of this recombinant human type III collagen are: small molecular weight, high expression level, and excellent activity. It not only exhibits excellent proliferation, adhesion, and migration-promoting effects on human fibroblasts and immortalized human epidermal cells, but also possesses good hemostatic activity, making it widely applicable in hemostasis, wound repair, and medical aesthetics.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a highly active recombinant human type III collagen and its applications. Background Technology

[0002] Collagen is the most abundant structural protein in the human body, possessing unique biological functions and providing crucial mechanical properties in tissues, including elasticity and toughness. Therefore, collagen-related biomaterials are frequently used for tissue repair and regeneration. In recent years, the market demand for collagen has also been gradually increasing.

[0003] Traditional collagen extraction primarily involves the hydrolysis of animal tissues using acid or alkali. However, this method has significant drawbacks: it not only generates strong immune rejection reactions but also carries the risk of potential viruses and infectious diseases, making its safety difficult to guarantee. In contrast, human collagen has weak antigenicity and can induce cell proliferation, migration, and promote cell adhesion, making it a safer and more reliable high-quality biomaterial. Among them, type III human collagen, a fibrillary collagen secreted by fibroblasts and mesenchymal cells, is composed of three α1(III) chains forming a homotrimer. It is the second most abundant collagen in the human body, widely distributed in connective tissues such as skin, lungs, liver, intestines, and vascular system. It plays a positive role in hemostasis, oral surgery, and diabetic wound repair, making it a highly promising tissue regeneration biomaterial.

[0004] The bioactivity of natural collagen depends on the binding of its specific amino acid sequences to cellular receptors: integrins (α / β heterodimer transmembrane receptors) recognize the corresponding amino acid sequences exposed by collagen, mediating intracellular and extracellular signal transduction; heat shock protein HSP47, as a collagen-specific molecular chaperone, is crucial for procollagen folding and functional maintenance; furthermore, the interaction between tyrosine kinase active receptors DDRs (DDR1, DDR2) and platelet-activated collagen receptor GPVI and their corresponding binding sites on collagen can significantly enhance cell migration and platelet aggregation, accelerating wound healing. Therefore, by precisely designing fragments associated with the active sites of human collagen and optimizing their binding efficiency to receptors, recombinant collagen with superior bioactivity can be developed.

[0005] Currently, recombinant collagen expression systems have been gradually expanded to hosts such as animals, plants, Escherichia coli, and yeast. Among them, the Pichia pastoris expression system has become the preferred platform for recombinant collagen expression due to its advantages such as high yield, no endotoxin, simple purification process, controllable cost, and post-translational modification.

[0006] Based on this, the present invention designed a human type III collagen protein rich in multiple active binding sites such as integrins, DDR, and GPVI, and recombinantly expressed it in Pichia pastoris chassis cells. The resulting recombinant collagen protein not only has good biological activity on human skin epidermal cells and dermal cells, but also has excellent hemostatic function, meeting the application needs of medical hemostasis, wound repair and medical aesthetics. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a highly active recombinant human type III collagen and its applications. The recombinant human type III collagen has a small molecular weight, high expression level and excellent activity. It not only has excellent proliferation, adhesion and migration promoting effects on human fibroblasts and human immortalized epidermal cells, but also has good hemostatic activity. It can be widely used in hemostasis, wound repair and medical aesthetics.

[0008] The present invention solves the above-mentioned technical problems by adopting the following technical solutions: A highly active recombinant human type III collagen, named COLIII-23, has the amino acid sequence shown in SEQ ID NO.2.

[0009] As one of the preferred embodiments of the present invention, the recombinant human type III collagen contains 2 integrin recognition sites (GER), 1 integrin recognition site (GEK), 2 integrin recognition sites (GEN), 4 glycoprotein VI recognition sites (GPO), 1 discoid domain receptor recognition site (GFO), and 5 heat shock protein binding sites (GXR).

[0010] As one of the preferred embodiments of the present invention, the recombinant human type III collagen has in vitro hemostatic activity and can promote the proliferation, adhesion and migration of human skin fibroblasts and human immortalized epidermal cells.

[0011] A recombinant human type III collagen encoding gene, the nucleotide sequence of which is shown in SEQ ID NO.1, is used to encode the aforementioned recombinant human type III collagen.

[0012] As one of the preferred embodiments of the present invention, the nucleotide sequence shown in SEQ ID NO.1 is a codon-optimized sequence adapted to the Pichia pastoris expression system (the sequence before optimization is shown in SEQ ID NO.3).

[0013] A recombinant expression vector containing the above-mentioned recombinant human type III collagen encoding gene; and the recombinant expression vector is constructed by seamlessly cloning the target gene into the MF-α signal peptide of the pPIC9K expression vector.

[0014] A recombinant Pichia pastoris engineered strain containing the above-mentioned recombinant expression vector.

[0015] As one of the preferred embodiments of the present invention, the recombinant Pichia pastoris engineered strain is obtained by linearizing the recombinant expression vector and then transferring it into Pichia pastoris chassis cells.

[0016] As one of the preferred embodiments of the present invention, the Pichia pastoris chassis cells are selected from at least one of Pichia pastoris GS115, Pichia pastoris X33, Pichia pastoris KM71H, and Pichia pastoris SMD1168.

[0017] A method for preparing the above-mentioned recombinant human type III collagen, comprising the following steps: (1) Synthesize the nucleotide sequence shown in SEQ ID NO.1 encoding a single strand of recombinant human type III collagen; (2) Design PCR primers to amplify the DNA fragment encoding the single strand of recombinant human type III collagen. The primers are shown in SEQ ID NO.4 and SEQ ID NO.5. (3) Construct a recombinant expression vector comprising the DNA fragment encoding the single strand of recombinant human type III collagen, wherein the expression vector is pPIC9K; (4) After linearizing the constructed recombinant expression vector, it was transformed into Pichia pastoris chassis cells to obtain recombinant Pichia pastoris engineered strains; (5) Culture the above-mentioned recombinant Pichia pastoris engineered strain to express recombinant human type III collagen; (6) The recombinant human type III collagen expressed in step (5) is separated and purified, including hollow fiber microfiltration, tangential flow exchange, ion exchange chromatography, hydrophobic chromatography and dialysis steps.

[0018] As one of the preferred embodiments of the present invention, in step (6), the buffer used for ion exchange chromatography is 10mM sodium acetate, pH 4.5; the buffer used for hydrophobic chromatography is 10mM sodium acetate + 1.5M ammonium sulfate, pH 4.5; and the dialysate used for dialysis is PBS, pH 7.0.

[0019] The application of the above-mentioned recombinant human type III collagen in the preparation of hemostatic sponges, wound repair materials, medical aesthetic and plastic surgery materials and cosmetics.

[0020] The advantages of this invention compared to the prior art are: (1) The recombinant human type III collagen COLIII-23 of this invention has a small molecular weight and is designed with precise sequence. It is rich in 5 integrin recognition sites (including 2 GER, 1 GEK, and 2 GEN), 4 glycoprotein VI recognition sites (GPO), 1 discoid domain receptor recognition site (GFO, which can bind to two members, DDR1 and DDR2), and 5 heat shock protein binding sites (GXR). It can specifically and efficiently bind to a variety of key receptors on the cell surface, mediating signal transduction and biological effects. Experiments show that it can not only significantly promote the proliferation, adhesion, and migration of human fibroblasts and human immortalized epidermal cells, but also has excellent in vitro hemostatic activity. It can be widely used in hemostatic sponges, wound repair materials, medical aesthetics, cosmetics and other fields.

[0021] (2) Based on the expression characteristics of Pichia pastoris, the present invention optimized the nucleotide sequence encoding COLIII-23 protein by codon optimization, making the gene more suitable for the Pichia pastoris expression system; combined with the MF-α signal peptide-mediated secretion expression of the pPIC9K expression vector and the selected Pichia pastoris chassis cells, the efficient synthesis and secretion of recombinant protein were verified by fermentation, and the engineered strain with high efficiency expression was obtained through large-scale fermentation screening, laying a solid foundation for large-scale industrial production.

[0022] (3) The present invention adopts a core purification strategy combining "ion exchange chromatography and hydrophobic chromatography", and further optimizes the step-by-step separation and purification process of "hollow fiber microfiltration → tangential flow liquid exchange → ion exchange chromatography → hydrophobic chromatography → dialysis", which can effectively remove impurities and proteins, and finally obtain COLIII-23 protein with high recovery rate and high purity. Moreover, the entire preparation process does not require complex equipment, and the purification steps are simple and efficient, which reduces the technical threshold and cost of industrial production. At the same time, the recombinant expression vector is constructed through seamless cloning technology, with no enzyme cleavage site residue, 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.

[0023] (4) The recombinant collagen of the present invention is designed with human sequence and has no animal-derived components. It avoids the risk of immune rejection, viral and infectious disease transmission that may exist with animal-derived collagen. It has weak antigenicity and excellent biocompatibility. At the same time, its molecular weight is small and does not contain excess amino acid sequence, making it easy to be absorbed and utilized by the body. It can meet the safe use needs of multiple fields such as medical treatment, medical aesthetics, and cosmetics.

[0024] (5) The present invention selects Pichia pastoris as the host to produce COLIII-23. This expression system not only has the advantages of high yield, no endotoxin, simple purification process and controllable cost, but also can perform necessary post-translational modifications on recombinant proteins, fully support the biological function of collagen products produced by this collagen, and ensure that the activity of recombinant collagen is highly consistent with that of natural human type III collagen, further enhancing the application value of the product. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the active site in the COLIII-23 amino acid sequence in Example 1; Figure 2 This is an agarose gel electrophoresis image of the COLIII-23 DNA fragment from Example 2 (in the image, M is the marker, lanes 1, 2, and 3 are samples; the target band size is 753 bp). Figure 3 This is an electrophoresis image of the protein obtained from the separation and purification of COLIII-23 in Example 4 (in the image, lane 1 is the molecular weight marker, lane 2 is the fermentation supernatant diluted 7 times, lane 3 is the flow-through buffer of the cation exchange column, lane 4 is the contaminating protein eluted from the cation exchange column using 4% NaCl, lane 5 is the target protein eluted from the cation exchange column using 50% NaCl, lane 6 is the flow-through buffer after using a hydrophobic chromatography column, lane 7 is the washed contaminating protein eluted from the hydrophobic chromatography column using 65% NaCl, and lanes 8 and 9 are the target protein eluted from the hydrophobic chromatography column; the molecular weight of the COLIII-23 protein, as determined by electrophoresis, is approximately 30 kDa). Figure 4 This is a graph showing the effect of COLIII-23 protein on the proliferation of human skin fibroblasts and human immortalized epidermal cells in Example 5 (Figure A shows the effect on human fibroblast HSF, and Figure B shows the effect on human immortalized epidermal cells HaCaT; ** indicates P<0.01, *** indicates P<0.001). Figure 5 This is a graph showing the effect of COLIII-23 protein on the adhesion of human skin fibroblasts and human immortalized epidermal cells in Example 5 (Figure A shows the effect on human immortalized epidermal cells HaCaT, and Figure B shows the effect on human fibroblast HSF; ** indicates P<0.01). Figure 6 This is a graph showing the effect of COLIII-23 protein on the migration rate of human skin fibroblasts and human immortalized epidermal cells in Example 5 (Figure A shows the effect on human immortalized epidermal cells HaCaT, and Figure B shows the effect on human fibroblast HSF; ** indicates P<0.01, *** indicates P<0.001). Figure 7The figure shows the results of the in vitro hemostatic activity test of COLIII-23 protein in Example 5 (Figure A shows the actual hemostatic effect of different concentrations of COLIII-23; Figure B shows the comparison of the hemostatic effect of COLIII-23 with other collagen; Figure C is a bar chart of the coagulation rate of different samples; *** indicates P<0.001). Figure 8 This is a graph showing the growth of recombinant Pichia pastoris engineered strains in a 5L fermenter and the expression level of COLIII-23 protein in Example 6. Detailed Implementation

[0026] 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.

[0027] The Pichia pastoris strains and expression vectors used in the following examples are all commercially available strains and vectors. The culture media used are the commonly used formulations for Pichia pastoris fermentation. Additionally, the DMEM basal culture medium, PBS, fetal bovine serum (FBS), and trypsin used in the following examples are from Vivacell, while calf serum (BCS), human skin fibroblast cell line HSF, and human immortalized epidermal cell line HaCaT are from Oricell.

[0028] Example 1: Recombinant human type III collagen COLIII-23 encoding gene: The final optimized recombinant human type III collagen COLIII-23 encoding gene of this invention is shown in SEQ ID NO.1, and its corresponding amino acid sequence is shown in SEQ ID NO.2 (the active sites in the amino acid sequence of recombinant human type III collagen COLIII-23 are shown in [link to relevant documentation]). Figure 1 ).

[0029] Gene screening and optimization process: (1) Based on the human type III collagen gene sequence in GeneBank, a fragment containing 5 integrin recognition sites (2 GER, 1 GEK, 2 GEN), 4 glycoprotein VI recognition sites (GPO), 1 discoid domain recognition site (GFO), and 5 heat shock protein binding sites (GXR) was found, as shown in SEQ ID NO.3.

[0030] (2) In response to the codon preference of Pichia pastoris and the AT content of the DNA sequence, online codon optimization software was used to change the corresponding original recombinant human type III collagen codon bases to the codon-preferred bases of Pichia pastoris without changing the protein coding sequence. The optimized gene sequence is shown in SEQ ID NO.1. The optimized gene sequence was artificially synthesized by Shanghai Sangon Biotech Co., Ltd.

[0031] Example 2: Recombinant expression vector pPIC9K- COLIII-23 Construction: 1. Amplification of the target gene fragment Specific primers were designed based on the coding gene sequence shown in SEQ ID NO.1, and the DNA fragment encoding COLIII-23 protein was amplified by PCR. The PCR system is shown in Table 1.

[0032] Table 1. PCR amplification reaction system and reaction conditions for COLIII-23

[0033] The target fragment was detected by 1% agarose gel electrophoresis, and the results are as follows: Figure 2 As shown (target strip size is 753bp).

[0034] 2. Carrier linearization processing To achieve seamless cloning of the target gene and the vector, specific primers (upstream primer: SEQ ID NO. 6; downstream primer: SEQ ID NO. 7) were designed for PCR linearization amplification of the circular pPIC9K vector. Homologous arm sequences complementary to the ends of the target gene were introduced at the 5' ends of the primers. The PCR amplification reaction system and conditions are shown in Table 2.

[0035] Table 2. PCR amplification reaction system and reaction conditions for pPIC9K expression vector

[0036] After amplification, the linearized pPIC9K vector fragment was recovered by 1% agarose gel electrophoresis.

[0037] 3. Seamless cloning to construct recombinant expression plasmids The recovered COLIII-23 encoding gene fragment and the linearized pPIC9K vector fragment were taken, homologous recombinase was added, and the reaction was carried out in the system shown in Table 3. After the sequence fragment was inserted downstream of the pPIC9K signal peptide, the corresponding recombinant expression vector pPIC9K-COLIII-23 was obtained.

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

[0039] Example 3: Construction of recombinant Pichia pastoris engineered strain: (1) The successfully constructed recombinant expression vector pPIC9K- COLIII-23 After linearization, the plasmid was transferred into Pichia pastoris GS115 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 (0.5 mg / mL G418) medium. Incubate at 30°C and 240 rpm for 18-24 h. Then transfer them sequentially to YPD liquid medium containing 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL G418 for further culture.

[0041] (3) Ferment the well-growing strains as follows: The bacterial culture was inoculated into 10 mL / 50 mL BMGY at a 2% inoculum and cultured at 30 °C and 240 rpm for 18–24 h. After centrifugation, the supernatant was discarded, and the culture was resuspended in 10 mL of BMGY. The culture was then incubated at 28 °C and 240 rpm for 48 h, with 100% methanol added every 24 h until the final concentration reached 2.5%. After centrifugation, the supernatant was collected for SDS-PAGE analysis to obtain recombinant Pichia pastoris engineered bacteria with high expression levels.

[0042] Example 4: Production and purification of COLIII-23: (1) Take the recombinant Pichia pastoris engineered strain (glycerol strain) successfully constructed above and activate it in YPD (G418 containing the corresponding resistance) medium at 30℃, 240 rpm, for 18 h; (2) 2% inoculum was transferred to 200 mL / L of BMGY and cultured at 30°C and 240 rpm for 26-28 h to completely consume the glycerol in the culture medium; (3) Take 200 mL of BMMY to resuspend the bacterial cells, and incubate at 28℃ and 240 rpm for 48 h; add 100% methanol every 24 h until the final concentration is 2.5%.

[0043] (4) After fermentation, the supernatant was filtered through hollow fiber microfiltration to obtain a clear fermentation supernatant. The supernatant was then separated and purified by tangential flow exchange, ion exchange chromatography (buffer: 10mM sodium acetate, pH 4.5), hydrophobic chromatography (buffer: 10mM sodium acetate + 1.5M ammonium sulfate, pH 4.5), and dialysis (dialysis solution: PBS, pH 7.0).

[0044] Figure 3Electrophoresis image of the protein obtained from the isolation and purification of COLIII-23. The purified COLIII-23 protein solution was analyzed by gel filtration chromatography and non-reducing SDS-PAGE, and the purity was >95% and the recovery rate was >85%.

[0045] Example 5: Bioactivity assay of COLIII-23: 1. MTT assay showed that recombinant human type III collagen promotes the proliferation of human skin fibroblasts (HSF) and immortalized human epidermal cells (HaCaT): (1) Take HSF or HaCaT cells, digest, centrifuge and resuspend them, and then dilute to 4×10⁻⁶. 4 Cells / mL, 100 μL per well into a 96-well cell culture plate; simultaneously, cell-free culture medium was added as a blank control (culture medium only, no cells), and cultured at 37°C, 5% CO2, 95% humidity for 24 h.

[0046] (2) Prepare COLIII-23 protein suspensions of 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, and 0.015625 mg / mL.

[0047] (3) Discard the culture medium and add it to the corresponding system according to the group: The blank control was supplemented with an equal volume of culture medium (to maintain a cell-free state). Negative control (NC): Add to a system containing cells + culture medium + 5% DMSO; Positive control (PC): A system containing cells, culture medium, and bovine type I collagen standard; Experimental group: The system containing cells, culture medium, and different concentrations of COLIII-23 protein solution was added.

[0048] All groups were incubated at 37℃, 5% CO2, and 95% humidity for 24 hours.

[0049] (4) Add 20 μL of MTT to each well and incubate in an incubator for 4 h at 37 °C, 5% CO2, and 95% humidity. Add 150 μL of DMSO and incubate on a shaker for 10 min to fully dissolve the crystals. Detect the absorbance at 490 nm using a microplate reader.

[0050] Test results as follows Figure 4 As shown. By Figure 4 A and B show that, with the negative control group as 100%, the survival rate of the HSF experimental group with added COLIII-23 was 133%; and the survival rate of the HaCaT experimental group with added COLIII-23 was 130%.

[0051] 2. COLIII-23 promotes the adhesion of human skin fibroblasts and human immortalized epidermal cells: (1) Human skin fibroblasts (HSF) or immortalized human epidermal cells (HaCaT) were taken, digested, centrifuged, resuspended, and diluted to 5 × 10⁻⁶. 4 100 μL of COLII-23 protein solution at a concentration of 0.0156 mg / mL was seeded into each well of a 96-well cell culture plate. Then, 100 μL of COLII-23 protein solution at a concentration of 0.0156 mg / mL 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.

[0052] (2) 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.

[0053] (3) Discard the culture medium, add 150 μL of DMSO, shake and incubate 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 light absorption value of each well on the microplate reader.

[0054] (4) Data analysis: SPSS 11.0 was used for statistical analysis. P < 0.05 was considered statistically significant, and P < 0.01 was considered significantly significant.

[0055] Test results as follows Figure 5 As shown. From Figure 5 It can be seen that COLII-23 has a certain cell adhesion-promoting effect on both HSF and HaCaT cells. With the negative control group as 100%, the adhesion rate of the HSF experimental group with COLII-23 was 124%; the adhesion rate of the HaCaT experimental group with COLII-23 was 128%.

[0056] 3. COLIII-23 promotes the migration of human skin fibroblasts and immortalized human epidermal cells: (1) Cell culture: Using a marker pen, draw 5 horizontal lines at approximately 0.5 cm intervals in each well of a 6-well plate as markers. HaCaT cells were selected, and blank, control, and experimental groups were set up. After digestion, centrifugation, and resuspending, the cells were diluted to 2×10⁻⁶. 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.

[0057] (2) Scratch assay: 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 COLIII-23 protein was added to each of the six wells, with final concentrations of 0.5 mg / mL, 0.05 mg / mL, 0.005 mg / mL, 0.0005 mg / mL, and 0.00005 mg / mL. The blank group only received culture medium, while the control group received bovine type I collagen standard, commercially available recombinant collagen (COLIII), and fish collagen.

[0058] (3) Incubate in an incubator at 37℃, 5% CO2, and 95% humidity, and take microscopic photographs at fixed positions after 0h, 24h, and 48h.

[0059] (4) Use Image J to measure the area of ​​the scratched area: The scratch images (0h, 24h) were processed using software to obtain the cell migration rate. ×100%.

[0060] Test results as follows Figure 6 As shown. For HaCaT cells, the 24-hour migration rate was 13% in the blank control group (NC); 20% in commercially available recombinant collagen; 14% in fish skin collagen; and 38% in the experimental group supplemented with 0.0005 mg / mL COLIII-23. For HSF cells, the 24-hour migration rate was 30% in the blank control group (NC); 38% in commercially available recombinant collagen; 42% in fish skin collagen; and 61% in the experimental group supplemented with 0.0005 mg / mL COLIII-23.

[0061] 4. In vitro hemostasis test: COLIII-23 promotes in vitro coagulation. The core indicator for evaluating in vitro coagulation efficacy is to determine the ability of biomaterials to induce blood clot formation on the surface of the material after contact with calcified whole blood. Specifically, the procoagulant activity of different samples is evaluated through the blood clotting index (BCI) test. ×100%.

[0062] Take an appropriate amount of COLIII-23 sample and add it to a 10 mL centrifuge tube. Incubate at 37 °C for 5 min. Add 200 μL of preheated sodium citrate anticoagulated whole blood to the sample. Immediately add 20 μL of preheated 0.2 M CaCl2 solution. Incubate at 37 °C for 2 min. Slowly add 10 mL of preheated deionized water along the wall. Incubate at 37 °C for 2 min. Centrifuge at 2000 rpm for 5 min. Transfer 100 μL of the supernatant to a 96-well plate and measure the OD value at a wavelength of 540 nm.

[0063] Test results as follows Figure 7 As shown, COLIII-23 exhibits a significant procoagulant effect at concentrations of 0.6 mg / mL to 0.8 mg / mL, with a maximum BCI index of 34.3%.

[0064] Example 6: Fermentation of COLIII-23 engineered bacteria in a 5L fermenter: (1) Seed culture: Pick fresh single colonies from YPD plates and inoculate them into 50mL centrifuge tubes containing 5mL BMGY medium. Incubate at 30℃ and 240rpm for 18-24h. Transfer the inoculum to 1L shake flasks containing 200mL BMGY seed medium at a 2% inoculation rate. Incubate at 30℃ and 240rpm for 14-18h until OD. 600 =5~6. Inoculate the seed culture medium into a 5L fermenter containing basal salt medium at a 10% inoculation rate.

[0065] (2) Fermentation culture: During the glycerol culture stage, maintain the temperature at 30℃, pH 5.0, and adjust the rotation speed to 400~800 rpm to maintain DO > 20%. When the glycerol in the BSM medium is depleted, at which point DO = 70~80, enter the glycerol feeding stage, starting with the addition of 50% glycerol to further increase the cell density. When the cell wet weight is approximately 180~220 g / L, enter the starvation period. After starvation for 1 hour, enter the methanol feeding stage, adjusting the induction temperature to 28℃ and starting the addition of methanol to maintain DO at around 20%. The pH is maintained 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.

[0066] The growth curves of COLIII-23 cells and the expression level of the target protein in a 5L fermenter are shown below. Figure 8 As shown. By Figure 8 It can be seen that the recombinant Pichia pastoris engineered strain of the present invention has the highest COLIII-23 yield of 4.06 g / L after 84 h of induction.

[0067] In summary, the recombinant human type III collagen COLIII-23 designed in this invention has high expression levels and multiple active sites in Pichia pastoris. It not only has good in vitro hemostatic effects, but also has excellent effects on promoting cell proliferation, migration and adhesion in human fibroblasts and human immortalized epidermal cells. It can be widely used in hemostatic sponges, wound repair materials, medical aesthetics and plastic surgery, cosmetics and other fields.

[0068] 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 highly active recombinant human type III collagen, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

2.

2. The recombinant human type III collagen according to claim 1, characterized in that, The recombinant human type III collagen contains 2 integrin recognition sites (GER), 1 integrin recognition site (GEK), 2 integrin recognition sites (GEN), 4 glycoprotein VI recognition sites (GPO), 1 discoid domain receptor recognition site (GFO), and 5 heat shock protein binding sites (GXR).

3. The recombinant human type III collagen according to claim 1, characterized in that, The recombinant human type III collagen has in vitro hemostatic activity and can promote the proliferation, adhesion and migration of human skin fibroblasts and human immortalized epidermal cells.

4. A recombinant human type III collagen encoding gene, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID NO.1, and it is used to encode the recombinant human type III collagen as described in any one of claims 1 to 3.

5. The recombinant human type III collagen encoding gene according to claim 4, characterized in that, The nucleotide sequence shown in SEQ ID NO.1 is a codon-optimized sequence adapted to the Pichia pastoris expression system.

6. A recombinant expression vector, characterized in that, The recombinant human type III collagen encoding gene as described in claim 4 or 5 is contained in the recombinant expression vector, which is constructed by seamlessly cloning the target gene into the MF-α signal peptide of the pPIC9K expression vector.

7. A recombinant Pichia pastoris engineered strain, characterized in that, The recombinant expression vector as described in claim 6.

8. The recombinant Pichia pastoris engineered strain according to claim 7, characterized in that, The recombinant Pichia pastoris engineered strain was obtained by linearizing the recombinant expression vector and then transferring it into Pichia pastoris chassis cells.

9. A method for preparing recombinant human type III collagen as described in any one of claims 1 to 3, characterized in that, The steps are as follows: (1) Synthesize the nucleotide sequence shown in SEQ ID NO.1 encoding a single strand of recombinant human type III collagen; (2) Design PCR primers to amplify the DNA fragment encoding the single strand of recombinant human type III collagen. The primers are shown in SEQ ID NO.4 and SEQ ID NO.

5. (3) Construct a recombinant expression vector comprising the DNA fragment encoding the single strand of recombinant human type III collagen, wherein the expression vector is pPIC9K; (4) After linearizing the constructed recombinant expression vector, it was transformed into Pichia pastoris chassis cells to obtain recombinant Pichia pastoris engineered strains; (5) Cultivate the above-mentioned recombinant Pichia pastoris engineered strain to express recombinant human type III collagen; (6) The recombinant human type III collagen expressed in step (5) is separated and purified, including hollow fiber microfiltration, tangential flow exchange, ion exchange chromatography, hydrophobic chromatography and dialysis steps.

10. The application of recombinant human type III collagen as described in any one of claims 1 to 3 in the preparation of hemostatic sponges, wound repair materials, medical aesthetic and plastic surgery materials, and cosmetics.

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