Recombinant human COLIII collagen, its preparation method and application

Abstract

This invention relates to a recombinant human COLIII collagen, its preparation method, and its applications, belonging to the field of proteins. Its amino acid sequence comprises: n directly tandemly linked or linker-connected amino acid sequences as shown in SEQ ID NO:1, or amino acid sequences with the same or similar properties obtained by substitution and / or deletion of one, two, or three or more amino acid residues as shown in SEQ ID NO:1; n represents the number of repetitions of the amino acid sequence as shown in SEQ ID NO:1, and n is an integer between 2 and 5. This invention, through tandem expression of specific functional sequences, ensures both the stability of the target protein and achieves high expression, while simultaneously verifying its good biological activity and safety. It is expected to be widely used in many fields such as clinical medicine, the cosmetics industry, and tissue engineering, opening up a broader chapter in its applications.

Description

Technical Field

[0001] This invention relates to the field of proteins, and more particularly to recombinant human COLIII collagen, its preparation method, and its applications. Background Technology

[0002] Type III collagen (COLIII), an essential structural protein in the human body, plays a crucial role in physiological functions. It is widely distributed in many important tissues. For example, in the skin—our first line of defense against external aggressors—COLIII acts like a finely woven "elastic net," giving the skin firmness and resilience. In the complex vascular system, it is fundamental to maintaining the integrity of blood vessel walls, ensuring smooth blood circulation. In the lungs, it participates in building the intricate alveolar structure, facilitating efficient gas exchange. In the liver, COLIII maintains the orderly arrangement of liver cells, stabilizing the internal structure of this vital metabolic organ. Furthermore, it bears the responsibility of maintaining the stability of cell and tissue structures. When the body experiences trauma, it responds rapidly, actively promoting wound healing and playing a key role in enhancing tissue elasticity.

[0003] In recent years, with the vigorous development of the life sciences field, researchers have conducted more in-depth and detailed explorations of the biological functions of COLIII. In the field of clinical medicine, it has shown great potential in repairing damaged tissues and assisting in disease treatment. For example, in the treatment plans for some patients with chronic ulcers and burns, it is expected to become a powerful assistant in accelerating wound healing and reducing the risk of infection. In the cosmetics industry, it has become a popular ingredient sought after by many manufacturers due to its excellent moisturizing and anti-wrinkle effects. Consumers favor skin care products containing COLIII, hoping to use them to lock in youthful appearance. In tissue engineering, it provides ideal biomaterial raw materials for constructing various artificial tissues and organs, and is expected to help researchers overcome the shortage of organ transplant donors.

[0004] However, fully realizing the application potential of type III collagen faces a thorny challenge—the extraction and purification of natural type III collagen. Obtaining COLIII from biological tissues is extremely difficult, both in terms of tissue source and content. On the one hand, its content in tissues is relatively limited, and the cost of obtaining raw materials is high; on the other hand, limited by existing extraction processes, ensuring both yield and purity is even more challenging. This predicament directly results in the scarcity of existing natural COLIII and significant variations in purity, far from meeting the growing large-scale application demand in the market, severely restricting its widespread application across various industries.

[0005] Encouragingly, genetic engineering technology has brought us hope, opening up a new path for the production of COLIII. Using gene editing techniques, researchers can introduce the gene encoding type III collagen into a suitable host system, allowing it to function like a precisely operating "biofactory," continuously producing recombinant COLIII. Currently, commonly used recombinant protein expression systems, such as E. coli and yeast, each have their own characteristics and advantages and disadvantages; the specific choice depends on the application scenario of the target protein. While *E. coli*, as a prokaryotic expression system, is rapid in reproduction and easy to culture, it lacks the protein modification mechanisms unique to eukaryotes. Achieving folding and further formation of higher-order structures through recombinant COLIII presents significant challenges. Furthermore, maintaining the stability of the produced proteins requires specific measures, and determining their bioactivity depends on further in-depth validation studies to ultimately establish reliable experience. Although yeast is a eukaryote, its cellular environment differs from human cells, making it difficult for the synthesized proteins to perfectly match human needs in terms of structure and function. Similarly, further in-depth research is needed on key aspects such as protein folding, stability maintenance, and bioactivity assurance. Moreover, there are certain differences between different species, requiring researchers to conduct systematic validation work during the construction of engineered bacteria.

[0006] Given the current challenges, developing stable and efficient expression systems requires continuous exploration and trial-and-error screening. An ideal expression system should not only efficiently express the designed recombinant collagen sequence, but also possess good stability to ensure the protein maintains its activity during storage, transportation, and application, and more importantly, enable large-scale production to reduce costs. Summary of the Invention

[0007] Technical issues

[0008] In view of this, the technical problem to be solved by the present invention is how to provide a recombinant human COLIII collagen protein, its preparation method, and its applications. The present invention, through tandem expression of specific functional sequences, ensures both the stability of the target protein and achieves high expression, while simultaneously verifying its good biological activity and safety. It is expected to be widely used in many fields such as clinical medicine, the cosmetics industry, and tissue engineering, opening up a broader chapter in its applications.

[0009] Solution To solve the above technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a recombinant human COLIII collagen, the amino acid sequence of which comprises: n amino acid sequences as shown in SEQ ID NO:1 directly tandemly or linked by linkers, or amino acid sequences with the same or similar properties obtained by substitution and / or deletion of one, two or more amino acid residues as shown in SEQ ID NO:1; n represents the number of repetitions of the amino acid sequence as shown in SEQ ID NO:1, and n is an integer between 2 and 5.

[0010] The amino acid sequence shown in SEQ ID NO:1 is as follows: GPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGER GEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPP.

[0011] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1 is shown in SEQ ID NO:3 or 4, wherein the optimized codon is shown in SEQ ID NO:3: GGTCCACCAGGTCCACCAGGTACTGCTGGTTTTCCAGGTTCTCCAGGTGCTAAGGGTGAAGTTGGTCCAGCTGGTTCTCCAGGTTCTAACGGTGCTCCAGGTCAAAGAGGTGAACCAGGTCCACAAGGTCATGCTGGTGCTCAAGGTCCACCAGGTCCACCAGGTATTAACGGTTCTCCAGGTGGTAAGGGTGAAATGGGTCCAGCTGGTATTCCAGGTGCTCCAGGTTTGATGGGTGCTAGAGGTCCACCAGGTCCAGCTGGTGCTAATGGTGCTCCAGGTTTGAGAGGTGGTGCTGGTGAACCAGGTAAGAACGGTGCTAAGGGTGAACCAGGTCCAAGAGGTGAAAGAGGTGAAGCTGGTATTCCAGGTGTTCCAGGTGCTAAGGGTGAAGATGGTAAGGATGGTTCTCCAGGTGAACCAGGTGCTAACGGTTTGCCAGGTGCTGCTGGTGAAAGAGGTGCTCCAGGTTTTAGAGGTCCAGCTGGTCCAAACGGTATTCCAGGTGAAAAGGGTCCAGCTGGTGAAAGAGGTGCTCCAGGTCCAGCTGGTCCAAGAGGTGCTGCTGGTGAACCAGGTAGAGATGGTGTTCCAGGTGGTCCAGGTATGAGAGGTATGCCAGGTTCTCCAGGTGGTCCAGGTTCTGATGGTAAGCCAGGTCCACCAGGTTCTCAAGGTGAATCTGGTAGACCAGGTCCACCA。

[0012] Among them, the codons before optimization are shown in SEQ ID NO:4: GGCCCTCCTGGTCCTCCTGGAACTGCCGGATTCCCTGGATCCCCTGGTGCTAAGGGTGAAGTTGGACCTGCAGGGTCTCCTGGTTCAAATGGTGCCCCTGGACAAAGAGGAGAACCTGGACCTCAGGGACACGCTGGTGCTCAAGGTCCTCCTGGCCCTCCTGGGATTAATGGTAGTCCTGGTGGTAAAGGCGAAATGGGTCCCGCTGGCATTCCTGGAGCTCCTGGACTGATGGGAGCCCGGGGTCCTCCAGGACCAGCCGGTGCTAATGGTGCTCCTGGACTGCGAGGTGGTGCAGGTGAGCCTGGTAAGAATGGTGCCAAAGGAGAGCCCGGACCACGTGGTGAACGCGGTGAGGCTGGTATTCCAGGTGTTCCAGGAGCTAAAGGCGAAGATGGCAAGGATGGATCACCTGGAGAACCTGGTGCAAATGGGCTTCCAGGAGCTGCAGGAGAAAGGGGTGCCCCTGGGTTCCGAGGACCTGCTGGACCAAATGGCATCCCAGGAGAAAAGGGTCCTGCTGGAGAGCGTGGTGCTCCAGGCCCTGCAGGGCCCAGAGGAGCTGCTGGAGAACCTGGCAGAGATGGCGTCCCTGGAGGTCCAGGAATGAGGGGCATGCCCGGAAGTCCAGGAGGACCAGGAAGTGATGGGAAACCAGGGCCTCCCGGAAGTCAAGGAGAAAGTGGTCGACCAGGTCCTCCT。

[0013] Optionally, n is 3.

[0014] Optionally, the amino acid sequence of recombinant human COLIII collagen is as shown in SEQ ID NO:2.

[0015] The amino acid sequence shown in SEQ ID NO:2 is as follows: .

[0016] In a second aspect, a polynucleotide encoding the recombinant human COLIII collagen described in the first aspect is provided; optionally, the polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1 is as shown in SEQ ID NO:3 or 4; optionally, the polynucleotide sequence encoding the human COLIII collagen comprises the nucleotide sequence shown in SEQ ID NO:5 or 6.

[0017] The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2 is shown in SEQ ID NO:5 or 6, wherein the optimized codon is shown in SEQ ID NO:5:

[0018] Thirdly, a nucleic acid construct is provided, comprising the polynucleotides described in the second aspect, optionally having a polynucleotide sequence as shown in SEQ ID NO:7.

[0019] Fourthly, a recombinant vector comprising the nucleic acid construct described in the third aspect is provided, wherein optionally, the backbone vector is a pGAPZ A-αA expression vector.

[0020] Fifthly, a microorganism comprising the recombinant vector described in the fourth aspect is provided; preferably, the microorganism is Pichia pastoris, and optionally, the Pichia pastoris is Pichia pastoris GS115.

[0021] Sixthly, a method for preparing recombinant human COLIII collagen is provided, comprising the following steps: Genetically engineered yeast containing a polynucleotide sequence encoding human COLIII collagen as described in the first aspect, or a polynucleotide as described in the second aspect, or a nucleic acid construct as described in the fourth aspect, is added to a culture medium for fermentation. The fermentation broth is collected and purified to obtain COLIII collagen.

[0022] Furthermore, the culture medium includes a basal medium; the basal medium includes YPD medium, and optionally, the basal medium may also contain the following components by weight as needed: 10-40 parts glycerol, 5-10 parts methanol, 15-30 parts glucose, 10-15 parts yeast extract, 10-20 parts plant peptone, 5-15 parts ammonium sulfate, 5-30 parts ammonium nitrate, 10-40 parts potassium dihydrogen phosphate, 10-25 parts dipotassium hydrogen phosphate, 5-15 parts magnesium sulfate, 0.2-1.2 parts calcium chloride, 5-10 parts zinc sulfate, 3-6 parts copper sulfate pentahydrate, 10-30 parts proline, 5-20 parts lysine, 10-50 parts glycine, 2-5 parts citric acid, 2-10 parts disodium hydrogen phosphate, 10-20 parts sorbitol, and 0.01-0.1 parts Tween-80. Optionally, the culture medium may also include trace elements with the following composition: boric acid 0.015~0.025 g / L, copper sulfate pentahydrate 3~5 g / L, manganese sulfate 1~2.2 g / L, ferrous sulfate 35~45 g / L, riboflavin 0.3~45 g / L, pyridoxal phosphate 0.8 g / L, nicotinic acid 0.5 g / L, and thiamine 0.2 g / L; Optionally, the culture medium may also include trace elements with the following composition: boric acid 0.02 g / L, copper sulfate pentahydrate 3 g / L, manganese sulfate 1 g / L, ferrous sulfate 40 g / L, riboflavin 0.3 g / L, pyridoxal phosphate 0.8 g / L, nicotinic acid 0.5 g / L, and thiamine 0.2 g / L.

[0023] Optionally, the purification method includes: taking the fermentation culture broth and removing cell debris by centrifugation, then using a hollow fiber column to further clarify the centrifuged supernatant, then ultrafiltration to desalt and remove small molecule pigments, and then performing ion exchange chromatography using a cation exchange chromatography column to remove most impurities; optionally, Q resin chromatography is used to remove other impurities, so that the purity of the target protein exceeds 99% and meets medical grade standards.

[0024] In a seventh aspect, the application of the human COLIII collagen described in the first aspect, the polynucleotide described in the second aspect, the nucleic acid construct described in the third aspect, the recombinant vector described in the fourth aspect, the expression system cell described in the fifth aspect, or the human COLIII collagen prepared by the preparation method described in the sixth aspect in the preparation of skin repair dressings, medical aesthetic injection products, implants, biomaterials, and medical devices is provided.

[0025] Furthermore, the biomaterial is a medical-grade raw material that can be used in skin repair dressings, medical aesthetic injection products, implants, biomaterials, medical devices, and also in the development of skin care products.

[0026] Beneficial effects (1) This invention provides a method for preparing an engineered bacterium that expresses recombinant COLIII with 234 amino acids repeated three times in tandem. By designing specific amino acid sequences, optimizing codons, selecting suitable host cells and fermentation, isolation and purification conditions, etc., a highly efficient and stable expression system was successfully constructed, enabling recombinant COLIII to be efficiently expressed, stably stored and retain its biological activity in host cells.

[0027] (2) The 234 amino acid fragments selected in this invention are repeated three times in tandem from human COLIII, containing key regions of the collagen triple helix structure. Through sequence design optimization, its folding efficiency and stability in the recombinant expression system are enhanced, while ensuring that its biological function is not affected.

[0028] 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

[0029] 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.

[0030] Figure 1 A schematic diagram of the recombinant COLIII plasmid of Example 1 of the present invention; Figure 2 Electrophoresis diagram of the enzyme digestion results of recombinant COLIII plasmid-2 in Example 2 of the present invention.

[0031] Figure 3 DNA sequencing results of recombinant COLIII plasmid-2 in Example 2 of the present invention.

[0032] Figure 4 An SDS-PAGE 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.

[0033] Figure 5 The results of the recombinant COLIII protein in Test Example 2 of this invention promoting the proliferation of skin fibroblasts.

[0034] 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 were obtained by dissection. Detailed Implementation

[0035] 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.

[0036] 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.

[0037] 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.

[0038] Example 1.1. Gene Synthesis and Vector Construction 1) Design of gene elements: The coding sequence is tandemly arranged as follows: KpnI + ATG + (COLIII tandem unit) × 3 + TAA + Not I, where the COLIII tandem unit is the base sequence encoding the amino acid sequence shown in SEQ ID NO:1, the KpnI restriction site is GGTACC, the Not I restriction site is GCGGCCGC, ATG corresponds to the start codon, and TAA corresponds to the stop codon. The base sequence of the gene element after codon optimization is shown in SEQ ID NO:7. The gene elements are artificially synthesized, for example, by Shenzhen BGI Genomics Co., Ltd.

[0039] The optimized codon of recombinant COLIII collagen is shown in SEQ ID NO:5, and the unoptimized natural codon is shown in SEQ ID NO:6.

[0040] 2) The above gene elements and pGAPZ A-αA vector were digested with KpnI and Not I enzymes, respectively, and then ligated using T4 DNA ligase to obtain recombinant COLIII plasmids (e.g., Figure 1 As shown, the plasmids 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.

[0041] 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.

[0042] The codon optimization of this invention fully considers the triple helix structure of collagen and the Gly-XY repeat sequence.

[0043] Example 2: Protein Expression, Purification and Detection 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 electrophoresis for detection and analysis.

[0044] The enzyme digestion results of COLIII plasmid-2 are as follows: Figure 2 DNA sequencing results are as follows Figure 3 SDS gel electrophoresis detection and analysis, such as Figure 4 This indicates that the recombinant COLIII plasmid-2 was successfully constructed and can express the target protein (COLIII plasmid-1 is not shown in the figure).

[0045] Protein expression: 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 cultured at 28℃ for 48 h until single colonies appeared. Then, single colonies were picked and inoculated into 300 mL shake flasks containing liquid YPD medium, placed on a shaker, and cultured at 28℃ and 250 rpm for 72 h to bring the cells to 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 5 L fermenter. A suitable basal medium (e.g., YPD medium + trace element formulation 2) was used, with the temperature controlled at 28°C and pH 5.0-6.0. Dissolved oxygen was maintained at 40%-50% 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 71 hours, the cell density stopped increasing. After being placed in the fermentation tank, the fermentation broth was processed and the expression level of the target protein was detected.

[0046] The expression levels of the target protein before and after codon optimization are shown in Table 1: Table 1. Comparison of protein expression levels before and after codon optimization of recombinant COLIII (5L fermenter)

[0047] As shown in Table 1, the optimized codons used in this invention can effectively increase the expression level of the target protein by 2.2 times.

[0048] In this embodiment, the composition of the culture medium and the formulation of trace elements were also optimized. The results are shown in Table 2. Among them, formulations 2, 3 and 5 showed the best results.

[0049] Table 2. Effects of trace elements on the expression level of recombinant COLIII (5L fermenter)

[0050] The trace element composition of each formula in Table 2 is as follows: Formula 1: Boric acid 0.01 g / L, copper sulfate pentahydrate 2 g / L, manganese sulfate 1.5 g / L, ferrous sulfate 30 g / L, riboflavin 0.35 g / L, pyridoxal phosphate 1.1 g / L, nicotinic acid 0.8 g / L, thiamine 0.15 g / L and biotin 0.01 g / L.

[0051] Formula 2: Boric acid 0.02 g / L, copper sulfate pentahydrate 3 g / L, manganese sulfate 1 g / L, ferrous sulfate 40 g / L, riboflavin 0.3 g / L, pyridoxal phosphate 0.8 g / L, nicotinic acid 0.5 g / L, thiamine 0.2 g / L, biotin 0.01 g / L.

[0052] Formula 3: Boric acid 0.015 g / L, copper sulfate pentahydrate 4 g / L, manganese sulfate 1.5 g / L, ferrous sulfate 35 g / L, riboflavin 0.4 g / L, pyridoxal phosphate 1 g / L, nicotinic acid 0.6 g / L, thiamine 0.15 g / L, biotin 0.015 g / L.

[0053] Formula 4: Boric acid 0.03 g / L, copper sulfate pentahydrate 2.5 g / L, manganese sulfate 2 g / L, ferrous sulfate 50 g / L, riboflavin 0.25 g / L, pyridoxal phosphate 0.6 g / L, nicotinic acid 0.4 g / L, thiamine 0.25 g / L, biotin 0.008 g / L.

[0054] Formula 5: Boric acid 0.025 g / L, copper sulfate pentahydrate 5 g / L, manganese sulfate 2.2 g / L, ferrous sulfate 45 g / L, riboflavin 0.45 g / L, pyridoxal phosphate 1.1 g / L, nicotinic acid 0.7 g / L, thiamine 0.22 g / L, biotin 0.012 g / L.

[0055] Protein purification process: The fermentation broth was centrifuged to remove cell debris. The supernatant was then further clarified using a hollow fiber column (0.45 μm pore size filter membrane), followed by ultrafiltration to desalt and remove small molecule pigments. A cation exchange chromatography column was then used to remove most impurities. Finally, Q-resin chromatography was used to remove any remaining impurities, such as endotoxins and host proteins, ensuring the target protein purity exceeded 99% and met medical-grade standards.

[0056] Test Example 1: Evaluation of the oral safety of recombinant COLIII protein (oral acute toxicity test) Twenty-four healthy adult 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 low, medium, and high dose experimental groups of recombinant COLIII protein expressed on codon-optimized COLIII plasmid-2 (each group was diluted with purified water), at doses of 1000 mg / kg, 5000 mg / kg, and 15000 mg / kg, respectively. Acute toxicity tests were conducted before... First, the experimental animals are housed in the animal husbandry room for 1-2 days to allow them to fully adapt to the laboratory environment. After observation confirming their health, they are randomly assigned to groups. Before the experiment, the experimental animals are fasted overnight, with a fasting period of approximately 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 further mortality occurs 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 Horn's method. 50 value.

[0057] 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.

[0058] Table 3 Acute toxicity (LD50) dose classification

[0059] Table 4. Results of acute oral toxicity test in mice

[0060] The results in Table 4 show that the recombinant COLIII protein prepared in this invention has high safety and is non-toxic.

[0061] Test Example 2: Functional Validation (Experiment to Promote the Proliferation of Human Skin Fibroblasts (HSF)) Recombinant COLIII protein was dissolved in DMEM cell culture medium, with two groups: a blank control group (without recombinant COLIII protein) and experimental groups with low, medium, and high recombinant COLIII protein concentrations (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.

[0062] Cell proliferation rate (%) = (OD value of experimental group - OD value of control group) / OD value of control group × 100%.

[0063] The statistical methods, including one-way ANOVA, were used to compare the significant differences between different concentration groups and the control group to determine the promoting effect of recombinant type III collagen on the proliferation of skin fibroblasts. P A value <0.05 is considered statistically significant.

[0064] As shown in Figure 5, the results indicate that the recombinant COLIII protein of the present invention has a significant promoting effect on the proliferation of skin fibroblasts, and this effect increases with increasing concentration.

[0065] Test Example 3: Subcutaneous Injection Experiment of Recombinant COLIII Protein Crosslinking Gel in Mice Experimental objective: To preliminarily evaluate the safety and in vivo anti-degradation ability of recombinant COLIII protein cross-linked gel.

[0066] Experimental Procedure: Six healthy Kunming mice (6-8 weeks old, 20-25 g) with similar weights were selected and acclimatized for one week. A 3.0% recombinant COLIII protein solution was cross-linked with 2 kGY of gamma rays to form a translucent gel, which was then filled into a glass syringe and injected using a #27 needle. Mice were first anesthetized by intraperitoneal injection of 0.3 mL / 100 g body weight of 10% chloral hydrate solution according to standard operating procedures. After anesthesia with chloral hydrate, hair was removed from the back of the mice using depilatory cream, covering an area of ​​40 mm × 30 mm, symmetrically arranged around the spine. Residual depilatory cream was rinsed off with water, and the area was disinfected with iodine and then deiodized with 75% alcohol. Finally, 0.2 mL of recombinant COLIII cross-linked gel was injected subcutaneously on each side of the spine in the hair-removed area on the mouse's back, and the injection sites were photographed and recorded. During weeks 1, 2, 3, and 4 post-injection, the animals' general condition was observed daily, including their mental state, appetite, and activity. Weight was measured weekly, and any local reactions such as redness, swelling, induration, or exudation were recorded. Four weeks later, the mice were euthanized after excessive anesthesia following hair removal on their backs. The skin at the injection site on the back was quickly dissected and photographed to observe changes in the gel's morphology and volume. Any remaining gel was peeled off and accurately weighed to preliminarily assess the degree of gel degradation and the presence of inflammatory reactions. If necessary, further pathological analysis will be conducted.

[0067] Experimental results showed that at 1, 2, 3, and 4 weeks post-injection, the mice 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 31% of the recombinant collagen at the injection site (approximately 145 mg remaining after 4 weeks from an injection of approximately 210 mg per point) had degraded (dissection results are shown in the figure). Figure 6 Furthermore, no visible inflammatory reaction was observed in the surrounding tissues, indicating that the recombinant COLIII protein cross-linked gel possesses certain anti-degradation capabilities and good biocompatibility, and is expected to be developed into a medical aesthetic injection product and other implantable medical device products.

[0068] 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. Recombinant human COLIII collagen, the amino acid sequence of which is shown in SEQ ID NO:

2.

2. A polynucleotide encoding the recombinant human COLIII collagen of claim 1; the polynucleotide sequence encoding the recombinant human COLIII collagen is shown in SEQ ID NO:5 or 6.

3. A nucleic acid construct comprising the polynucleotide of claim 2.

4. The nucleic acid construct according to claim 3, characterized in that, It contains a polynucleotide sequence as shown in SEQ ID NO:

7.

5. A recombinant vector comprising the nucleic acid construct of claim 3 or 4.

6. The recombinant vector according to claim 5, characterized in that, The backbone vector is the pGAPZ A-αA expression vector.

7. A microorganism comprising the recombinant vector of claim 5 or 6.

8. The microorganism according to claim 7, characterized in that, The microorganism in question is Pichia pastoris.

9. The microorganism according to claim 8, characterized in that, The Pichia pastoris is Pichia pastoris GS115.

10. A method for preparing recombinant human COLIII collagen, characterized in that, Includes the following steps: Genetically engineered yeast containing a polynucleotide sequence encoding the recombinant human COLIII collagen of claim 1, or the polynucleotide of claim 2, or the nucleic acid construct of claim 3, is added to a culture medium for fermentation. The fermentation broth is collected and purified to obtain recombinant human COLIII collagen.

11. The preparation method according to claim 10, characterized in that, The culture medium used is a basal medium.

12. The preparation method according to claim 11, characterized in that, The basic culture medium includes YPD medium.

13. The preparation method according to claim 11, characterized in that, The basal culture medium includes: 10-40 parts glycerol, 5-10 parts methanol, 15-30 parts glucose, 10-15 parts yeast extract, 10-20 parts plant peptone, 5-15 parts ammonium sulfate, 5-30 parts ammonium nitrate, 10-40 parts potassium dihydrogen phosphate, 10-25 parts dipotassium hydrogen phosphate, 5-15 parts magnesium sulfate, 0.2-1.2 parts calcium chloride, 5-10 parts zinc sulfate, 3-6 parts copper sulfate pentahydrate, 10-30 parts proline, 5-20 parts lysine, 10-50 parts glycine, 2-5 parts citric acid, 2-10 parts disodium hydrogen phosphate, 10-20 parts sorbitol, and 0.01-01 parts Tween-800.

14. The preparation method according to claim 12, characterized in that, The culture medium also includes the following trace elements: boric acid 0.015~0.025 g / L, copper sulfate pentahydrate 3~5 g / L, manganese sulfate 1~2.2 g / L, ferrous sulfate 35~45 g / L, riboflavin 0.3~45 g / L, pyridoxal phosphate 0.8 g / L, nicotinic acid 0.5 g / L, and thiamine 0.2 g / L.

15. The preparation method according to claim 12, characterized in that, The culture medium also contains the following trace elements: boric acid 0.02 g / L, copper sulfate pentahydrate 3 g / L, manganese sulfate 1 g / L, ferrous sulfate 40 g / L, riboflavin 0.3 g / L, pyridoxal phosphate 0.8 g / L, nicotinic acid 0.5 g / L, and thiamine 0.2 g / L.

16. The preparation method according to any one of claims 10 to 15, characterized in that, The purification method includes: taking the fermentation culture broth and removing cell debris by centrifugation, then using a hollow fiber column to further clarify the centrifuged supernatant, followed by ultrafiltration to desalt and remove small molecule pigments, and then performing ion exchange chromatography using a cation exchange chromatography column to remove most of the impurities; optionally, Q resin chromatography is used to remove other impurities, so that the purity of the target protein exceeds 99% and meets medical grade standards.

17. The use of recombinant human COLIII collagen as described in claim 1, the polynucleotide as described in claim 2, the nucleic acid construct as described in claim 3 or 4, the recombinant vector as described in claim 5 or 6, the microorganism as described in any one of claims 7 to 9, or the recombinant human COLIII collagen prepared by any one of the preparation methods described in claims 10 to 16 in the preparation of skin repair dressings.

18. The use of recombinant human COLIII collagen as described in claim 1, the polynucleotide as described in claim 2, the nucleic acid construct as described in claim 3 or 4, the recombinant vector as described in claim 5 or 6, the microorganism as described in any one of claims 7 to 9, or the recombinant human COLIII collagen prepared by any one of the preparation methods described in claims 10 to 16 in the preparation of medical aesthetic injection products.

19. The use of a recombinant human COLIII collagen as described in claim 1, a polynucleotide as described in claim 2, a nucleic acid construct as described in claim 3 or 4, a recombinant vector as described in claim 5 or 6, a microorganism as described in any one of claims 7 to 9, or a recombinant human COLIII collagen prepared by any one of the preparation methods described in claims 10 to 16 in the preparation of an implant.

20. The use of recombinant human COLIII collagen as described in claim 1, the polynucleotide as described in claim 2, the nucleic acid construct as described in claim 3 or 4, the recombinant vector as described in claim 5 or 6, the microorganism as described in any one of claims 7 to 9, or the recombinant human COLIII collagen prepared by any one of the preparation methods described in claims 10 to 16 in the preparation of skin care products.

21. The use of recombinant human COLIII collagen as described in claim 1, the polynucleotide as described in claim 2, the nucleic acid construct as described in claim 3 or 4, the recombinant vector as described in claim 5 or 6, the microorganism as described in any one of claims 7 to 9, or the recombinant human COLIII collagen prepared by any one of the preparation methods described in claims 10 to 16 in the preparation of medical devices.

Images