Recombinant vector for expressing natural long-sequence recombinant type III collagen, preparation method and application thereof

Abstract

This invention relates to a recombinant vector for expressing natural long-sequence recombinant type III collagen, its preparation method, and its applications. It pertains to the field of proteins and includes a nucleotide sequence encoding recombinant type III collagen. Optionally, the nucleotide sequence encoding recombinant type III collagen may have at least one segment encoding the amino acid sequence shown in SEQ ID NO:1 (optionally, it may have at least one segment of the nucleotide sequence shown in any one of SEQ ID NO:2-4). This invention utilizes genetic engineering technology to construct engineered bacteria that efficiently express the natural long-sequence type III collagen, enabling its large-scale production. This technology can be widely applied in fields such as medicine, clinical skin repair, tissue engineering, and cosmetics.

Description

Technical Field

[0001] This invention relates to the field of proteins, and more particularly to recombinant vectors for expressing natural long-sequence recombinant type III collagen, their preparation methods, and applications. Background Technology

[0002] As a crucial structural protein in the human body, COLIII is widely distributed in various tissues such as skin, blood vessels, and internal organs, acting like a precise "scaffolding" for building life, shouldering the important responsibility of maintaining the integrity of tissue structure. It is not only a "glue" for cell stability, allowing tissues and organs to maintain their proper shape, but also an important matrix for promoting cell proliferation and migration, providing basic raw materials for tissue renewal and repair, and playing an indispensable key role in the complex physiological process of wound healing.

[0003] As scientific research increasingly focuses on type III collagen, its enormous application potential is gradually emerging. In the medical field, COLIII is like an ideal "biological patch" for repairing damaged organs and tissues and treating chronic, difficult-to-heal wounds, promising to bring about entirely new treatment solutions. The beauty and skincare industry also favors it greatly, as the firmness, elasticity, and radiance of human skin largely depend on the content and activity of COLIII, making it a core ingredient for anti-aging and skin rejuvenation. It is also attracting attention in the field of biomaterials, with hopes of integrating it into novel biomedical materials to give implantable medical devices better biocompatibility.

[0004] However, reality is far from the ideal. Extracting natural type III collagen from living organisms is fraught with difficulties. On the one hand, its sources are extremely limited. Animal tissues contain very little collagen, making it difficult to obtain in large quantities. Even with volunteer tissue donations from humans, the amount is extremely limited, and ethically, it is difficult for the public to accept. On the other hand, the purity obtained from extraction is difficult to meet medical-grade standards; impurities are always present, and subsequent purification costs are extremely high. These bottlenecks directly limit its prospects for large-scale commercial application.

[0005] The emergence of gene recombination technology is theoretically a breakthrough. However, the full-length natural sequence of the mature COLIII A1 peptide contains as many as 1068 amino acids and has numerous enzyme cleavage sites, making it extremely susceptible to attack and cleavage by various enzymes in the environment, resulting in very poor molecular stability. During fermentation, the environment in which microorganisms exist is complex and variable, making the target protein chain very easy to break and degrade. The separation and purification stage is even more difficult; the fragile target protein is often largely degraded before reaching high purity through multiple processes, making it a formidable obstacle to obtain sufficient quantity and purity of target protein that meets medical-grade requirements.

[0006] Selecting the most suitable production sequence that balances functionality, expression level, and stability is a research challenge in this field. Summary of the Invention

[0007] Technical issues

[0008] In view of this, the technical problem to be solved by this invention is how to provide a recombinant vector for expressing natural long-sequence recombinant type III collagen, its preparation method, and its applications. The inventors of this invention have focused on the sequence selection and design of type III collagen, and through unremitting efforts and trials, have successfully achieved the efficient expression and production of a relatively long natural COLIII sequence of 507 amino acids. Furthermore, through rigorous scientific safety evaluation and biological efficacy testing, experimental results show that the recombinant natural long-sequence COLIII has good safety and also possesses various biological functions such as promoting human cell proliferation. This achievement provides a good raw material for applications in multiple fields such as skin care, medical dressings, plastic surgery, and even artificial tissues and organs, and is expected to become a new generation of multi-purpose biomedical materials with broad commercial application prospects.

[0009] Solution

[0010] To solve the above technical problems, the present invention provides the following technical solution:

[0011] In a first aspect, the present invention provides a recombinant vector for expressing natural long-sequence recombinant type III collagen, comprising a nucleotide sequence encoding recombinant type III collagen, optionally having at least one segment encoding an amino acid sequence as shown in SEQ ID NO:1.

[0012] The selected 507 amino acid sequences of COLIII are shown in SEQ ID NO:1:

[0013] GPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAG ERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGP PGPQGLQGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRGGGAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPPGKGDKGEPGGPGADGVPGKDGPR GPTGPIGPPGPAGQPGDKGEGGAPGLPGIAGPRGSPGERGETGPPGPAGFPGAPGQNGEPGGKGERGAPGEKGEGGPPGVAGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPP.

[0014] Furthermore, the nucleotide sequence encoding recombinant type III collagen has at least one nucleotide sequence as shown in any one of SEQ ID NO:2 to 4, and optionally has at least one nucleotide sequence of the amino acid sequence shown in the nucleotide sequence shown in SEQ ID NO:2 or 3.

[0015] The nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:1 are shown in SEQ ID NO:2, 3, or 4, wherein the optimized codon is shown in SEQ ID NO:2:

[0016]

[0017] A nucleotide sequence containing an enzyme cleavage site, a start codon, and a stop codon can be as shown in SEQ ID NO:3:

[0018]

[0019] The natural codon is shown in SEQ ID NO:4:

[0020]

[0021] Optionally, the backbone vector is the pGAPZ A-αA expression vector.

[0022] In a second aspect, a microorganism comprising the recombinant vector described in the first aspect is provided.

[0023] Further, the microorganism is Pichia pastoris, and optionally, the Pichia pastoris is Pichia pastoris GS115.

[0024] Thirdly, a method for preparing naturally expressed long-sequence recombinant type III collagen is provided, comprising the following steps:

[0025] The recombinant vector described in the first aspect is transferred into yeast to obtain genetically engineered yeast, or the microorganism described in the second aspect is added to a culture medium for fermentation, the fermentation broth is collected, and COLIII collagen is purified.

[0026] Furthermore, the culture medium used was YPD medium and trace elements;

[0027] Optionally, the trace elements in the culture medium include the following raw materials in parts by weight: boric acid 0.02–0.03 g / L, copper sulfate pentahydrate 2–3.5 g / L, manganese sulfate 1–2 g / L, ferrous sulfate 25–45 g / L, riboflavin 0.3–0.45 g / L, pyridoxal phosphate 0.8–1.2 g / L, nicotinic acid 0.6–0.9 g / L, thiamine 0.18–0.22 g / L, and biotin 0.01–0.015 g / L;

[0028] Optionally, the trace elements in the culture medium include the following raw materials in parts by weight: boric acid 0.03 g / L, copper sulfate pentahydrate 3.0 g / L, manganese sulfate 2.0 g / L, ferrous sulfate 45 g / L, riboflavin 0.45 g / L, pyridoxal phosphate 0.8 g / L, nicotinic acid 0.6 g / L, thiamine 0.18 g / L, and biotin 0.012 g / L.

[0029] The culture medium may also be supplemented with the following ingredients as needed: glycerol, methanol, glucose, yeast extract, plant peptone, ammonium sulfate, ammonium nitrate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, calcium chloride, zinc sulfate, copper sulfate, proline, lysine, glycine, citric acid, disodium hydrogen phosphate, sorbitol, and Tween-80.

[0030] Optionally, the purification method includes: removing cell debris from the fermentation broth by centrifugation and ultrafiltration, and extracting and purifying the target protein using ion exchange chromatography.

[0031] Fourthly, a long-sequence recombinant type III collagen protein obtained using the preparation method described in the third aspect is provided.

[0032] Fifthly, the application of natural long-sequence recombinant type III collagen prepared by the recombinant vector described in the first aspect, the microorganism described in the second aspect, or the preparation method described in the third aspect in the preparation of skin repair dressings, medical aesthetic injection products, implants, biomaterials, and medical devices.

[0033] Furthermore, the biomaterial is used as a hemostatic dressing.

[0034] Beneficial effects

[0035] (1) This invention constructs engineered bacteria that efficiently express the natural long sequence of type III collagen through genetic engineering technology, and realizes its large-scale production. It can be widely used in the fields of medicine, clinical skin repair, tissue engineering and beauty, and can also be used in the development of skin care products and functional foods.

[0036] (2) This invention achieves efficient expression and large-scale production of recombinant COLIII by means of selecting amino acid sequences, codon optimization, vector selection and fermentation conditions, so as to meet the needs of different application fields.

[0037] (3) The inventors of this invention tackled the challenge of sequence selection and design for type III collagen. Through tireless efforts and trials, they successfully achieved the efficient expression and production of a relatively long natural COLIII sequence of 507 amino acids. Furthermore, through rigorous scientific safety evaluation and biological efficacy testing, the experimental results showed that the recombinant natural long-sequence COLIII exhibited good safety and multiple biological effects, including promoting human cell proliferation. This achievement provides a valuable raw material for applications in skincare, medical dressings, plastic surgery, and even artificial tissues and organs, and is expected to become a new generation of multi-purpose biomedical materials with a broad prospect for commercial application.

[0038] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it according to the contents of the specification, and to make the above and other objects, technical features and advantages of the present invention easier to understand, one or more preferred embodiments are listed below and described in detail with reference to the accompanying drawings. Attached Figure Description

[0039] One or more embodiments are illustrated by way of example with reference to the accompanying drawings, and these illustrative examples are not intended to limit the embodiments. The term "illustrative" as used herein means "serving as an example, embodiment, or illustration." Any embodiment illustrated herein as "illustrative" is not necessarily to be construed as superior to or better than other embodiments.

[0040] Figure 1 A schematic diagram of the recombinant COLIII plasmid in Example 1 of the present invention;

[0041] Figure 2 Electrophoresis diagram of the enzyme digestion results of recombinant COLIII plasmid-2 in Example 2 of the present invention.

[0042] Figure 3 DNA sequencing results of recombinant COLIII plasmid-2 in Example 2 of the present invention.

[0043] Figure 4 SDS gel electrophoresis image of the fermentation broth of recombinant COLIII plasmid-2 in Example 2 of the present invention, wherein 234*3aa represents recombinant COLIII plasmid-2.

[0044] Figure 5 The results of the recombinant COLIII protein in Test Example 2 of this invention promoting the proliferation of skin fibroblasts.

[0045] Figure 6 The results of gross observation of the injection site after 4 weeks of subcutaneous injection of recombinant COLIII collagen gel in mice in Test Example 3 of this invention.

[0046] Figure 7 Photographs of the liver lobe wound and the pressure wound of the New Zealand white rabbit in Test Example 5 of this invention, wherein A is the wound cut with a blade and B is the wound pressure wound applied with collagen sponge. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. 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.

[0048] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, methods, means, etc., well-known to those skilled in the art, are not described in detail in order to highlight the spirit of the present invention.

[0049] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0050] Example 1. Gene Synthesis and Vector Construction

[0051] 1) Construction of nucleotide elements: The coding sequence was tandemly constructed in the following order: KpnI + ATG + nucleotide sequence encoding COLIII507AA (SEQ ID NO:1) + TAA + NotI, wherein the KpnI restriction site is GGTACC, the NotI restriction site is GCGGCCGC, ATG corresponds to the start codon, and TAA corresponds to the stop codon. The base sequence encoding the amino acid sequence shown in SEQ ID NO:1 uses optimized codons as shown in SEQ ID NO:2. The constructed nucleotide element is shown in SEQ ID NO:3; simultaneously, nucleotide elements containing natural codons as shown in SEQ ID NO:4 were also synthesized. The nucleotide elements were artificially synthesized, for example, by Shenzhen BGI Genomics Co., Ltd.

[0052] 2) The above nucleotide elements and pGAPZ A-αA vector were digested with KpnI and NotI enzymes, respectively, and then ligated using T4 DNA ligase to obtain the recombinant COLIII plasmid (e.g., Figure 1 As shown), they are named recombinant COLIII plasmid-1 (natural codon before optimization) and recombinant COLIII plasmid-2 (natural codon after optimization) according to whether the codons are optimized.

[0053] The pGAPZ A-αA vector selected in this invention contains the GAP promoter and is suitable for Pichia pastoris expression systems, allowing for expression without inducers.

[0054] Example 2: Protein Expression, Purification and Detection

[0055] Recombinant COLIII plasmid-1 and recombinant COLIII plasmid-2 were transformed into Pichia pastoris GS115 host cells via electroporation. The transformed cells were then screened for antibiotics (using G418 resistance in YPD medium) to obtain positive clones. Positive clones were selected and their gene integration was confirmed by PCR and restriction enzyme digestion analysis. Plasmid DNA was extracted, and DNA sequencing was used to further verify the insertion of the target gene. The fermentation supernatant was then subjected to SDS-PAGE gel electrophoresis for detection and analysis.

[0056] Confirmation was performed using Xba I restriction enzyme digestion, with the digestion results of COLIII plasmid-2 as follows: Figure 2 DNA sequencing results are as follows Figure 3 SDS gel electrophoresis detection and analysis, such as Figure 4 The results showed that recombinant COLIII plasmid-1 and recombinant COLIII plasmid-2 were successfully constructed and could express the target protein (the figure of recombinant COLIII-1 is not shown).

[0057] Protein expression:

[0058] The fermentation process for producing recombinant collagen in a 5L fermenter (brand and model: XAN-Ferment-5L): Two strains of Pichia pastoris (before and after codon optimization) were picked from glycerol tubes and inoculated onto slant agar plates containing suitable culture medium. They were incubated at 28℃ for 48 hours until single colonies appeared. Then, single colonies were picked and inoculated into 300mL shake flasks containing liquid culture medium (YPD medium + trace elements), placed on a shaker, and incubated at 28℃ and 250rpm for 72 hours to bring the cells into the logarithmic growth phase and achieve a cell density OD0.05. 600 At 3 PM, inoculation was carried out by transferring 8% of the seed culture from the seed tank into a 5L fermenter. A suitable basal medium (e.g., YPD medium + micronutrient formulation 4) was used, with the temperature controlled at 28℃ and pH 5.0-6.0. Dissolved oxygen was maintained at 30%-40% by adjusting the stirring speed and aeration rate. Fed culture phase: When the carbon source and other nutrients in the basal medium were depleted to a certain extent, fed culture medium was started. The feeding rate was adjusted according to cell growth and metabolism to maintain cell growth and product synthesis. Simultaneously, parameters such as dissolved oxygen and pH were closely monitored and adjusted as needed. Cell density (OD) was monitored throughout the culture process. 600 After approximately 73 hours, the cell density stopped increasing. After being placed in a container, the fermentation broth was processed and the expression level of the target protein was detected.

[0059] The expression levels of the target protein before and after codon optimization are shown in Table 1:

[0060] Table 1. Comparison of protein expression levels before and after codon optimization of the 507-amino acid natural long sequence recombinant COLIII (5L fermenter)

[0061]

[0062] As shown in Table 1, when using liquid culture medium containing YPD medium and trace elements (formula 4), the optimized codons used in this invention can effectively increase the expression level of the target protein, and the expression level is increased by nearly 3 times.

[0063] In this embodiment, the composition of the culture medium and the formulation of trace elements were also optimized, and the results are shown in Table 2.

[0064] Table 2. Effects of trace elements on the expression level of 507 amino acid natural long recombinant COLIII sequences (5L fermenter)

[0065]

[0066] The trace element composition of each formula in Table 2 is as follows:

[0067] Formula 1: Boric acid 0.02g / L, copper sulfate pentahydrate 2.0g / L, manganese sulfate 1.0g / L, ferrous sulfate 25g / L, riboflavin 0.30g / L, pyridoxal phosphate 1.0g / L, nicotinic acid 0.9g / L, thiamine 0.20g / L and biotin 0.015g / L.

[0068] Formula 2: Boric acid 0.015g / L, copper sulfate pentahydrate 2.5g / L, manganese sulfate 1.5g / L, ferrous sulfate 30g / L, riboflavin 0.35g / L, pyridoxal phosphate 0.9g / L, nicotinic acid 0.5g / L, thiamine 0.25g / L, biotin 0.020g / L.

[0069] Formula 3: Boric acid 0.025g / L, copper sulfate pentahydrate 3.5g / L, manganese sulfate 1.8g / L, ferrous sulfate 40g / L, riboflavin 0.40g / L, pyridoxal phosphate 1.2g / L, nicotinic acid 0.7g / L, thiamine 0.22g / L, biotin 0.010g / L.

[0070] Formula 4: Boric acid 0.03g / L, copper sulfate pentahydrate 3.0g / L, manganese sulfate 2.0g / L, ferrous sulfate 45g / L, riboflavin 0.45g / L, pyridoxal phosphate 0.8g / L, nicotinic acid 0.6g / L, thiamine 0.18g / L, biotin 0.012g / L.

[0071] Formula 5: Boric acid 0.01g / L, copper sulfate pentahydrate 4.5g / L, manganese sulfate 1.8g / L, ferrous sulfate 35g / L, riboflavin 0.25g / L, pyridoxal phosphate 1.3g / L, nicotinic acid 0.8g / L, thiamine 0.30g / L, biotin 0.018g / L.

[0072] Protein purification process:

[0073] Cell debris was removed from the fermentation broth by centrifugation and ultrafiltration. The target protein was then extracted and purified using ion-exchange chromatography. Purity was confirmed by SDS-PAGE, and the purity of the target protein needed to reach 99% or higher.

[0074] Test Example 1: Evaluation of the oral safety of 507 amino acid natural long-sequence recombinant COLIII (oral acute toxicity test).

[0075] Twenty-four adult healthy Kunming mice (6-8 weeks old, weighing 20-25g, half male and half female) were purchased from Beijing Jinmuyang Experimental Animal Breeding Co., Ltd. They were randomly divided into four groups of six mice each: a blank control group and three experimental groups (diluted with purified water) containing low, medium, and high doses of the 507-amino acid natural long-sequence recombinant COLIII protein expressed by the recombinant COLIII-2 expression system, at doses of 1000 mg / kg, 5000 mg / kg, and 15000 mg / kg, respectively.

[0076] Before conducting acute toxicity tests, experimental animals are housed in the animal husbandry room for 1-2 days to allow them to fully acclimatize to the laboratory environment. After confirming their health, they are randomly assigned to groups. Before the test, experimental animals are fasted overnight, generally for about 16 hours, but water intake is not restricted. The test substance is administered orally by gavage, and observations are conducted at 7 and 14 days after oral administration. If experimental animals continue to die after 4 days, the observation period needs to be extended to 14 days, and if necessary, to 28 days. The number of deaths, time of death, and symptoms of poisoning are recorded. If any animals die, the LD50 of the test substance is determined using the Horn method. 50 value.

[0077] Oral LD50 in mice 50 The corresponding lethal dose classifications for humans are shown in Table 3. The experimental results are shown in Table 4.

[0078] Table 3 Acute toxicity (LD50) dose classification

[0079]

[0080] Table 4. Results of acute oral toxicity test in mice

[0081]

[0082] The results in Table 4 show that the 507-amino acid natural long-sequence recombinant COLIII collagen prepared in this invention has high safety and is non-toxic.

[0083] Test Example 2: Functional Validation (Assay to Promote the Proliferation of Human Skin Fibroblasts (HSF))

[0084] 507 amino acid-rich natural long-sequence recombinant COLIII was dissolved in cell culture medium, serving as a blank control group (without 507 amino acid-rich natural long-sequence recombinant COLIII) and low, medium, and high concentrations of 507 amino acid-rich natural long-sequence recombinant COLIII, at doses of 1 mg / mL, 5 mg / mL, and 10 mg / mL, respectively. Skin fibroblast cryopreservation tubes (purchased from the Cell Bank of the Chinese Academy of Sciences) were removed from liquid nitrogen and quickly placed in a 37°C water bath with gentle agitation until completely thawed. The cell suspension was transferred to centrifuge tubes, an appropriate amount of culture medium was added, and the cells were centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in fresh culture medium, transferred to culture flasks, and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 80%-90%, the cells were passaged. Skin fibroblasts in the logarithmic growth phase were digested with trypsin, and a single-cell suspension was prepared with culture medium. The cells were counted and the cell density was adjusted to 1 × 10⁶ cells / mL. 4 Cells / mL. 100 μL of cell suspension was seeded into each well of a 96-well plate and incubated for 24 h to allow cell adhesion. After cell adhesion, the original culture medium was discarded, and 100 μL of culture medium containing different concentrations of recombinant COLIII was added to each well, with 5-6 replicates for each concentration. Subsequent operations were performed at 24 h and 48 h after drug addition. MTT assay: At the corresponding time point, 20 μL of MTT solution was added to each well, and incubation continued for 4 h. The supernatant was carefully aspirated, and 150 μL of DMSO was added to each well, shaken for 10-15 min to fully dissolve the crystals. The absorbance (OD value) of each well was measured at 490 nm using a microplate reader. The average OD values ​​of different concentration groups at 24 h and 48 h were calculated, and the cell proliferation rate was calculated using the control group as a reference.

[0085] Cell proliferation rate (%) = (OD value of experimental group - OD value of control group) / OD value of control group × 100%.

[0086] The significant differences between different concentration groups and the control group were compared using statistical methods such as one-way ANOVA to determine the promoting effect of the 507-amino acid natural long sequence recombinant COLIII on the proliferation of skin fibroblasts. A p-value < 0.05 was considered statistically significant.

[0087] The results are as follows Figure 5 The results showed that the 507-amino acid natural long-sequence recombinant COLIII protein of the present invention significantly promoted the proliferation of skin fibroblasts, and the effect increased with increasing concentration.

[0088] Test Example 3: Preliminary evaluation of the safety and in vivo anti-degradation ability of a 507-amino acid natural long-sequence recombinant COLIII collagen cross-linked gel.

[0089] Experimental Procedure: Five healthy New Zealand white rabbits of similar weight (Beijing Jinmuyang Experimental Animal Breeding Co., Ltd.), weighing 2.0-2.5 kg, were selected and acclimatized for one week. A 3.0% (w / w) solution of a natural long-sequence recombinant COLIII containing 507 amino acids was cross-linked by 2 kGy of gamma rays to form a translucent gel, which was then filled into a glass syringe and injected using a 27# needle. First, following standard operating procedures, rabbits were anesthetized via intravenous injection of a 10% chloral hydrate solution at a dose of 7.5 mL / kg body weight via the marginal ear vein. After anesthesia, hair removal cream was applied to the rabbits' backs, covering an area of ​​200 mm × 80 mm, symmetrically arranged around the spine. Residual cream was rinsed off with water, and the treated area was disinfected with iodine solution followed by 75% alcohol to remove the iodine. Then, 0.5 mL of 507 amino acid-based natural long-sequence recombinant COLIII cross-linked gel was subcutaneously injected at three points (approximately 50 mm apart) on each side of the spine on the rabbits' backs. The injection sites were photographed and recorded. For weeks 1-4 post-injection, the rabbits' general condition was observed daily, including their mental state, appetite, and activity. Their weight was measured weekly, and any local reactions such as redness, swelling, induration, or exudation were recorded. Four weeks later, the patient was euthanized after excessive anesthesia following hair removal on the back. The skin at the injection site on the back was quickly dissected and photographed to observe changes in the morphology and volume of the gel, and to preliminarily assess the degree of degradation of the injected gel and whether there was an inflammatory reaction. If necessary, further pathological analysis will be conducted.

[0090] The experimental results showed that at 1, 2, 3, and 4 weeks after injection, the experimental rabbits exhibited no abnormalities in mental state, appetite, or activity, and no local reactions such as redness, swelling, induration, or exudation occurred at the injection site. After 4 weeks, an average of approximately 37% of the recombinant collagen at the injection site (approximately 331 mg remaining after 4 weeks from each injection of approximately 525 mg) had degraded (dissection results are shown in the figure). Figure 6 Furthermore, the implanted material showed intact subcutaneous structure after dissection, with no obvious inflammatory reaction observed in the surrounding tissues. This indicates that the 507-amino acid natural long-sequence recombinant COLIII cross-linked gel has good biocompatibility. At 4 weeks, more than 35% of the material remained unexplained, demonstrating good anti-degradation ability. It is expected to be developed into a medical aesthetic injection product and other implantable medical device products.

[0091] Test Example 4: Evaluation of the efficacy of 507-amino acid natural long-sequence recombinant COLIII collagen in promoting collagen secretion at the cellular level.

[0092] Experimental procedure:

[0093] (1) Cell seeding: Human skin fibroblasts (HSF cells, purchased from the Cell Bank of the Chinese Academy of Sciences) were thawed and expanded according to standard cell culture procedures and then seeded into 96-well plates at a density of 5 × 10⁶ cells / well. 3 -1×10 4Cells / wells were cultured in basal medium containing 10% fetal bovine serum to 60%–70% confluence.

[0094] (2) Experimental grouping: The experiment was divided into 4 groups, namely blank control group and 3 experimental groups, with 6 wells in each group. The blank control group used normal serum-containing culture medium, and the experimental groups were supplemented with 507 amino acid natural long sequence recombinant COLIII on the basis of normal serum-containing culture medium, so that the final concentrations were 1 mg / mL, 2 mg / mL and 5 mg / mL, respectively.

[0095] (3) Cell processing and sample collection: The experimental group was replaced with the corresponding concentration of culture medium, while the blank control group remained unchanged. After culturing for 48 hours, the cell culture supernatant was collected from each well and centrifuged to remove impurities for later use.

[0096] (4) Collagen secretion detection: Hydroxyproline content detection kit (Shanghai Yaji Biotechnology Co., Ltd.) was used to determine hydroxyproline content. Each well of the blank control group and each experimental group had approximately 100 μL of sample solution. 900 μL of 6.7 mol / L hydrochloric acid was added to each sample and hydrolyzed at 110℃ for 24 h under sealed conditions. After hydrolysis, the solution was cooled to room temperature, filtered, and the filtrate was used for later use.

[0097] (5) Standard curve preparation: Prepare hydroxyproline standard solutions of 0, 5, 10, 20, 40, 60, and 80 μg / mL. Take 1 mL of each concentration standard solution into a stoppered test tube, add 0.5 mL of chloramine-T solution, mix thoroughly, and let stand at room temperature for 20 min. Then add 0.5 mL of perchloric acid solution, shake well, and let stand for 10 min. Finally, add 1 mL of p-dimethylaminobenzaldehyde solution, shake well, heat in a 60℃ water bath for 15 min, and cool to room temperature. Using a blank tube as a control, measure the absorbance of each tube at a wavelength of 558 nm and plot the standard curve.

[0098] (6) Sample determination: 1.0 mL of the treated sample solution is processed according to the determination method in the standard curve plotting step. The absorbance of the sample solution is measured, and the content of hydroxyproline in the sample is calculated according to the standard curve.

[0099] (7) Result Calculation: Based on the hydroxyproline content in the sample and the average hydroxyproline content in collagen (calculated at 13.4%, collagen (μg / mL) = hydroxyproline content (μg) × 7.46), the collagen content in the sample was calculated using the formula (Note: the sample concentration was diluted to 25% of the original concentration before testing, and the final result needs to be converted). Statistical Analysis: One-way ANOVA was used to compare the differences between each experimental group and the blank control group. The significance criteria were *P<0.05, **P<0.01, and ***P<0.001.

[0100] The test results are shown in Table 5 below.

[0101] Table 5. Results of collagen secretion promotion by recombinant COLIII with 507 amino acid natural long sequences (after conversion based on hydroxyproline content).

[0102]

[0103] The results showed that the 507-amino acid natural long sequence recombinant COLIII of the present invention had a good effect on promoting collagen secretion.

[0104] Test Example 5: Evaluation of the hemostatic effect of collagen sponge prepared from 507 amino acid natural long-sequence recombinant COLIII collagen.

[0105] Preparation process of recombinant collagen sponge: Take 507 amino acid natural long-sequence recombinant type III collagen freeze-dried raw material, dissolve it, and prepare a solution with a concentration of 1.5 wt%; pour the prepared 1.5 wt% recombinant collagen solution into a polytetrafluoroethylene mold, and control the liquid surface thickness to (2±0.2) mm; place the mold filled with solution in a refrigerator and let it stand overnight at 2-8℃ to allow the solution to form a gel; take the formed gel out of the refrigerator and irradiate it with a cobalt-60 or electron accelerator irradiation source at an irradiation dose of 2 kGy; put the irradiated gel into a freeze dryer, pre-freeze it to -40℃, and then start the freeze-drying program and freeze-dry for 48 hours to finally obtain the recombinant collagen sponge (referred to as 507AA recombinant collagen sponge).

[0106] Twelve healthy New Zealand white rabbits (weighing 4.0-4.5 kg, male and female) were randomly divided into two groups of six each. One group received 507AA recombinant collagen sponge, and the other received commercially available animal collagen sponge (purchased from Wuxi Bedy Biotechnology Co., Ltd.). After ear hair removal, the rabbits were anesthetized by intravenous injection of 2 mL of 2.5% sodium pentobarbital solution. The abdomen was simply shaved, and the abdomen was opened layer by layer to expose the liver. A 1.0 cm × 1.0 cm incision was made in the center of the liver lobe (e.g., ...). Figure 7 A) During wound formation, any oozing blood should be quickly absorbed with gauze, and then the prepared 1.5cm × 1.5cm recombinant collagen sponge of 507AA and the control animal collagen sponge should be quickly applied to the wound (e.g., ...). Figure 7B) Record the time to complete hemostasis (observation of no more visible bleeding from the wound), observe the adhesion of the two types of sponges to the wound, and photograph the hemostasis site on the liver. At the end of the experiment, the patient was euthanized under deep anesthesia according to ethical requirements. Experimental data are expressed as ±SD. Statistical analysis was performed using SPSS 27.0 software. One-way ANOVA was used to analyze differences between groups, with P < 0.05 considered statistically significant. The results showed highly significant differences between groups (P < 0.001).

[0107] The results are shown in Table 6.

[0108] Table 6. Statistics on the hemostasis time of different collagen sponges in liver trauma

[0109]

[0110] The results show that, compared with commercially available animal collagen sponge, the present invention has a shorter hemostasis time, a tighter adhesion between the material and the wound, and a lower expected postoperative risk, which is expected to improve the success rate of clinical surgical procedures.

[0111] The foregoing description of specific exemplary embodiments of the present invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. Any simple modifications, equivalent changes, and alterations made to the foregoing exemplary embodiments should fall within the scope of protection of the present invention.

Claims

1. The application of a recombinant vector expressing a natural long-sequence recombinant type III collagen in the preparation of hemostatic dressings, wherein the recombinant vector comprises a nucleotide sequence encoding a recombinant type III collagen, and the amino acid sequence of the recombinant type III collagen is as shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The nucleotide sequence encoding recombinant type III collagen is as shown in SEQ ID NO:

2.

3. The application according to claim 1, characterized in that, In the recombinant vector, the backbone vector is the pGAPZ A-αA expression vector.

4. The application according to claim 1, characterized in that, The recombinant vector expressed the target protein in the Pichia pastoris host.

5. The application according to claim 4, characterized in that, The Pichia pastoris is Pichia pastoris GS115.

6. The application according to claim 1, characterized in that, The preparation method for expressing natural long-sequence recombinant type III collagen includes the following steps: The recombinant vector was transferred into yeast to obtain genetically engineered yeast, which was then added to a culture medium for fermentation. The fermentation broth was collected and purified to obtain recombinant type III collagen.

7. The application according to claim 6, characterized in that, The culture medium consists of basal medium and trace elements.

8. The application according to claim 6 or 7, characterized in that, The trace elements in the culture medium include the following raw materials in parts by weight: boric acid 0.02~0.03 g / L, copper sulfate pentahydrate 2~3.5 g / L, manganese sulfate 1~2 g / L, ferrous sulfate 25~45 g / L, riboflavin 0.3~0.45 g / L, pyridoxal phosphate 0.8~1.2 g / L, nicotinic acid 0.6~0.9 g / L, thiamine 0.18~0.22 g / L and biotin 0.01~0.015 g / L.

9. The application according to claim 6 or 7, characterized in that, The trace elements in the culture medium include the following raw materials in parts by weight: boric acid 0.03 g / L, copper sulfate pentahydrate 3.0 g / L, manganese sulfate 2.0 g / L, ferrous sulfate 45 g / L, riboflavin 0.45 g / L, pyridoxal phosphate 0.8 g / L, nicotinic acid 0.6 g / L, thiamine 0.18 g / L, and biotin 0.012 g / L.

10. The application according to claim 6 or 7, characterized in that, Purification methods include: removing cell debris from the fermentation broth by centrifugation and ultrafiltration, and extracting and purifying the target protein using ion exchange chromatography.

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