Preparation method for recombinant human collagen iii

Abstract

The present invention relates to the technical field of biology, and provides a method for producing human collagen III, comprising co-transfecting CHO cells with a P4H gene and a human collagen III gene to obtain a stably transfected cell line; and fermenting the stably transfected cell line to obtain human collagen III. The cell line obtained with the provided method has the advantages of high expression and applicability to large-scale production.

Description

A method for preparing recombinant human collagen III Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, this invention discloses a method for producing recombinant human collagen. Background Technology

[0002] Collagen is a biological macromolecule and a major component of animal connective tissue. It is also the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of total protein, and even more than 80% in some organisms.

[0003] Collagen is one of the most abundant proteins in the human body. Collagen hydrolysates have been shown to possess significant biological activities, such as antioxidant properties, antihypertensive activity, lipid-lowering activity, and the ability to repair damaged skin. Collagen plays a dual role in the skin: providing the building blocks for the formation of elastin and collagen, and acting as a ligand or binding to fibroblast receptors to stimulate the synthesis of hyaluronic acid.

[0004] There are many types of collagen, with the most common being type I, type II, type III, type V, and type XI. In normal skin tissue, collagen mainly exists in the form of type I and type III collagen fibers. Human collagen III has broad application prospects in the fields of medical aesthetics, skin care, and medical devices, and has become a hot research direction in the biotechnology and medical aesthetics industries in recent years.

[0005] There are two main methods for preparing human collagen III: natural extraction and recombinant expression. Naturally extracted collagen, due to its animal origin, carries the risk of viral transmission and faces difficulties in scaling up production. Recombinant expression through genetic engineering can effectively overcome these drawbacks. The expression systems for recombinant human collagen III are primarily prokaryotic cells and yeast fermentation. While these systems can efficiently express the target protein, the expressed collagen cannot undergo sufficient hydroxylation modification due to the limitations of the expression systems, making it difficult to form a stable triple helix structure. However, using a CHO cell recombinant protein expression system allows for sufficient hydroxylation modification of collagen amino acids, thus enabling the expression of collagen molecules with a natural triple helix structure. However, CHO cell expression suffers from drawbacks such as low triple helix collagen expression levels (20-200 mg / L) and high costs, preventing large-scale collagen production using CHO cells to date. Summary of the Invention

[0006] To address the aforementioned problems, the present invention aims to provide a method for preparing type III human collagen.

[0007] This invention co-transfects the amino acid hydroxylase P4H gene and the human type III collagen gene into mammalian cells (CHO cells). Through stable cell line screening, a stable cell line with high expression of triple helix collagen was obtained. Using this cell line, a 5L reactor fermentation experiment was conducted, which can stably and highly express the triple helix structure of human type III collagen, with an expression level exceeding 2g / L, meeting the requirements for large-scale production.

[0008] More specifically, in the synthesis of human collagen III, the P4H enzyme is an amino acid hydroxylase. Whether this enzyme can function properly is crucial in determining whether expressed human collagen III can form a triple-helix active structure. To achieve high-level expression of triple-helix human collagen III in CHO cells, the P4H gene of CHO cells needs to be modified. However, mammalian cells (CHO) have a complex genetic background and a large genome, making gene editing of CHO host cells challenging. Furthermore, modified host cells are prone to gene recombination or mutation, leading to host cell instability and affecting protein expression levels and product quality. In this invention, CHO host cells were not genetically edited. Instead, human type III collagen gene and P4H gene (virus) from a specific species were co-transfected into CHO cells at different mass ratios (1:2, 1:1, 2:1). It was found that the expression level of triple-helical collagen was highest when the human collagen III gene and P4H gene were transfected at a mass ratio of 1:1. Therefore, this protocol was followed for double plasmid co-transfection during the construction of stable cell lines. Through stable cell line screening, a stable cell line with high expression of triple-helical collagen was obtained. Using this cell line, a 5L reactor fermentation experiment was conducted, which stably and highly expressed human collagen III with a triple-helical structure, with an expression level exceeding 2 g / L, meeting the requirements for large-scale production.

[0009] According to one aspect of the present invention, a method for producing human collagen III includes the following steps:

[0010] 1. The P4H gene and the human collagen III gene were co-transfected into CHO cells to obtain a stable cell line;

[0011] 2. The stabilized cell line was fermented to obtain human collagen III.

[0012] More specifically, a method for preparing human collagen III includes the following steps:

[0013] 1) Construct an expression vector containing a codon-optimized gene encoding human collagen III, wherein the gene encoding human collagen III has the nucleotide sequence shown in SEQ ID NO.2;

[0014] 2) Construct an expression vector containing a codon-optimized gene encoding amino acid hydroxylase P4H, wherein the gene encoding amino acid hydroxylase P4H has the nucleotide sequence shown in SEQ ID NO.4;

[0015] 3) Co-transfect the expression vectors from steps 1) and 2) into CHO cells to obtain a stable cell line;

[0016] 4) The stable cell line obtained in step 3) is fermented to obtain human collagen III.

[0017] In the method of the present invention, the expression vector in steps 1) and 2) is the pXC17.4 vector, the gene encoding human collagen III is ligated to the pXC17.4 vector via HindIII and EcoRI restriction sites, T4, and the gene encoding P4H is ligated to the pXC18.4 vector via HindIII and EcoRI restriction sites, T4.

[0018] In step 3) of the method described in this invention, the gene encoding human collagen III and the gene encoding P4H are co-transfected into CHO cells at a mass ratio of 1:1.

[0019] The present invention also provides a recombinant human collagen III prepared according to the method of the present invention. Attached Figure Description

[0020] Figure 1: Plasmid map of recombinant vector pXC17.4-COL3A1;

[0021] Figure 2: Plasmid map of recombinant vector pXC18.4-A085R;

[0022] Figure 3: SDS-PAGE detection results;

[0023] Figure 4A: Cell viability and cell density as measured by Vicell cell counter;

[0024] Figure 4B: Content of glutamine and glucose in fermentation supernatant;

[0025] Figure 4C: Ammonia ion concentration and glutamine content in fermentation supernatant;

[0026] Figure 5A: SDS-PAGE results of fermentation supernatant in a 5L reactor (non-reduction);

[0027] Figure 5B: Reactor expression levels;

[0028] Figure 6: SDS-PAGE results;

[0029] Figure 7A: SEC-HPLC detection results;

[0030] Figures 7B-1 and 7B-2: HPLC-SEC-MALS detection results;

[0031] Figure 7C: Fourier transform infrared spectroscopy detection results;

[0032] Figure 7D: Circular dichroism (CD) detection results. Detailed implementation method:

[0033] Example 1: Determination of Transfection Ratio

[0034] 1.1 The steps for constructing the recombinant human collagen III plasmid are as follows:

[0035] The full-length amino acid sequence of natural human collagen III was codon-optimized (Cricetulus griseus) and synthesized. The optimized gene sequence (COL3A1) was ligated into the pXC17.4 vector (from the commercial LONZA plasmid) via HindIII and EcoRI restriction sites at T4. The specific experimental steps are as follows:

[0036] 1) Enzyme digestion

[0037] CLA3A1_pUC57 plasmid digestion system

[0038] pXC17.4 vector enzyme digestion system

[0039] Mix the enzyme digestion systems separately and place them in a 37°C constant temperature water bath for 30 min.

[0040] 2) Electrophoresis

[0041] The enzyme digestion system was loaded with samples, electrophoresed at 120V for 30 min, and the gel containing the target DNA was excised and recovered using an agarose gel DNA recovery kit.

[0042] 3) T4 connection

[0043] Connection system:

[0044] The mixed connection system was placed in a 25°C constant temperature water bath and connected for 1 hour.

[0045] 4) Transformation

[0046] Add 10 μl of the ligation product to 100 μl of competent cells, gently tap the tube wall to mix, incubate on ice for 30 min, then heat shock at 42°C for 60 s, immediately cool on ice for 2-3 min, add 900 μl of antibiotic-free LB medium, and incubate at 37°C for 1 h (220 rpm). Spread the transformed competent cells evenly onto LB agar plates (ampicillin resistant), invert them in a 37°C incubator, and incubate overnight.

[0047] 5) Picking bacteria

[0048] The following day, single clones were picked on LB plates and sequenced.

[0049] 6) Plasmid extraction

[0050] The plasmid map of pXC17.4-COL3A1 is shown in Figure 1.

[0051] According to the reference sequence (NCBI Reference Sequence: NM_000090.4) provided by the NCBI database, this is the full-length amino acid sequence of natural human type III collagen. Its amino acid sequence is shown in SEQ ID NO.1. GenScript performed codon optimization (Cricetulus griseus) and synthesized the sequence, and the optimized sequence is shown in SEQ ID NO.2.

[0052] The amino acid sequence of human type III collagen (SEQ ID NO.1):

[0053] Signal peptides are indicated by a single underscore.

[0054] Nucleotide sequence of human type III collagen (SEQ ID NO.2):

[0055] 1.2 Steps for constructing the P4H gene plasmid

[0056] The amino acid sequence of A085R was codon-optimized (Cricetulusgriseus) and synthesized.

[0057] The optimized gene sequence (A085R) was ligated into the pXC18.4 vector (derived from the commercial LONZA plasmid) via HindIII and EcoRI restriction sites at T4. The specific experimental steps are as follows:

[0058] 1) Enzyme digestion

[0059] A085R_pUC57 plasmid digestion system

[0060] pXC18.4 vector enzyme digestion system

[0061] Mix the enzyme digestion systems separately and place them in a 37°C constant temperature water bath for 30 min.

[0062] 2) Electrophoresis

[0063] The enzyme digestion system was loaded with samples, electrophoresed at 120V for 30 min, and the gel containing the target DNA was excised and recovered using an agarose gel DNA recovery kit.

[0064] 3) T4 connection

[0065] Connection system:

[0066] The mixed connection system was placed in a 25°C constant temperature water bath and connected for 1 hour.

[0067] 4) Transformation

[0068] Add 10 μl of the ligation product to 100 μl of competent cells, gently tap the tube wall to mix, incubate on ice for 30 min, then heat shock at 42°C for 60 s, immediately cool on ice for 2-3 min, add 900 μl of antibiotic-free LB medium, and incubate at 37°C for 1 h (220 rpm). Spread the transformed competent cells evenly onto LB agar plates (ampicillin resistant), invert them in a 37°C incubator, and incubate overnight.

[0069] 5) Picking bacteria

[0070] The following day, single clones were picked on LB plates and sequenced.

[0071] 6) Plasmid extraction

[0072] The plasmid map of the recombinant vector pXC18.4-A085R is shown in Figure 2.

[0073] A085R amino acid sequence (SEQ ID NO.3):

[0074] A085R nucleotide sequence (SEQ ID NO.4):

[0075] 1.3 Host cell transfection

[0076] The plasmids containing the human collagen III gene expression vector and the plasmid containing the P4H gene expression vector were linearly purified, and the specific steps are as follows:

[0077] 1) Plasmid linearization and enzyme digestion

[0078] The pXC17.4-CLA3A1 enzyme digestion system is as follows:

[0079] The pXC18.4-A085R enzyme digestion system is as follows:

[0080] Mix the enzyme digestion systems separately and place them in a 37°C constant temperature water bath for 3 hours.

[0081] 2) Recovery of enzyme digestion products

[0082] (1) Add phenol:chloroform:isoamyl alcohol in a 1:1 ratio, shake to mix, and centrifuge at 12000 rpm for 2 min.

[0083] (2) Transfer the supernatant to a new 1.5ml centrifuge tube, add chloroform at a ratio of 1:1, shake to mix, and centrifuge at 12000rpm for 2min.

[0084] (3) Transfer the supernatant to a new 1.5ml centrifuge tube, add one-tenth of the volume of 3M sodium acetate and 2.5 times the volume of anhydrous ethanol, and shake to mix.

[0085] (4) Centrifuge at 12000 rpm for 2 min and discard the supernatant.

[0086] (5) Rinse once with 100 μl of 70% ethanol, centrifuge at 12000 rpm for 2 min, discard the supernatant and remove the residual liquid.

[0087] (6) Air dry at room temperature for 5 minutes.

[0088] (7) Dissolve the plasmid in 100 μl of sterile water and measure the concentration using an enzyme-linked immunosorbent assay (ELISA) reader.

[0089] CHO host cell lines were electroporated at different mass ratios (2:1, 1:1, 1:2). After cell growth recovered, different cell lines were subjected to fed-batch culture experiments in 125 ml shake flasks. The experimental steps are as follows:

[0090] The shaker speed was 110 rpm, the carbon dioxide concentration was 7%, and the temperature was reduced from 37℃ to 33℃ on day 6. Cells were seeded at a density of 0.6E6 / ml in Dynamis (Gibco) basal medium with an initial culture volume of 40 ml. On days 2, 4, 6, 8, 10, and 12 post-seeding, 5% CellBoost7a (2 ml) + 0.5% CellBoost7b (0.2 ml) (Cytiva) supplemental medium was added. On day 5 post-seeding, the sugar concentration in the culture supernatant was measured using a Cedex biochemical analyzer, and if the sugar concentration was below 8 g / L, it was supplemented to 8 g / L. On day 14 post-seeding, the cell culture supernatant was collected for SDS-PAGE analysis. The results are shown in Figure 3.

[0091] Lane 1 contains host cell culture supernatant without transfection with plasmid; lane 2 contains supernatant transfected with collagen protein particles; lane 3 contains cell culture supernatant transfected with collagen protein particles: P4H plasmid 2:1; lane 4 contains cell culture supernatant transfected with collagen protein particles: P4H plasmid 1:1; lane 5 contains cell culture supernatant transfected with collagen protein particles: P4H plasmid 1:2; and lane 6 contains Native Human Collagen III protein (1047178-1, abcam, ab7535).

[0092] As shown in Figure 3, in the supernatant of host cells without plasmid transfection, only a small amount of host cell protein is present, and no collagen is expressed (lane 1); in the supernatant transfected only with collagen protein particles, most of the protein consists of fragments, with only a small amount of monomers and dimers, and a very small amount of trimers (lane 2); in the supernatant of collagen protein particles: P4H plasmid 2:1 cell culture, in addition to fragments, dimers, and monomers, obvious trimer bands appear (lane 3); in the supernatant of collagen protein particles: P4H plasmid 1:1 cell culture, most of the protein is trimer, and because trimers are more stable, the number of fragments is significantly reduced (lane 4); in the supernatant of collagen protein particles: P4H plasmid 1:2 cell culture, in addition to trimers, multimers with a molecular weight larger than trimers also appear.

[0093] The results show that without P4H co-transfection, collagen cannot form a large amount of stable triple helix structure, and single-chain collagen is easily degraded. Therefore, the supernatant contains only a small amount of collagen monomers and a large amount of collagen degradation fragments. As the number of P4H genes increases, the number of trimers increases significantly. However, when there is too much P4H, it is easy to form multimers. Therefore, co-transfection of collagen protein plasmid and P4H plasmid at a ratio of 1:1 is the optimal condition.

[0094] Example 2 Construction of stable cell lines and identification of protein expression

[0095] 2.1 Construction of stable cell lines and protein expression

[0096] The expression vector containing the target gene prepared in Example 1 and the expression vector containing the P4H gene were mixed at a 1:1 mass ratio (20ug:20ug) and then co-electroporated (GenePulserXcell). TM1E7 CHO cells (1 ml) were added to the culture medium. After the cells recovered, they were diluted with CD CHO medium (Gibco) and seeded into 96-well polyclonal plates at a density of 3000 cells / well. A total of 10 96-well plates were seeded (culture volume 200 μl / well). Based on cell growth, after 2 weeks of plating, 20 μL of cell supernatant was collected, and the concentration of the target protein in the supernatant was determined by ELISA. 120 polyclonal clones with high expression levels were selected and passaged into 24-well plates (1 ml / well). After 10 days, 20 μL of cell supernatant was collected, and the concentration of the target protein in the supernatant was determined by ELISA. 60 polyclonal clones with high expression levels were selected and passaged into 6-well plates (4 ml / well). After 10 days, 20 μL of cell supernatant was collected, and the concentration of the target protein in the supernatant was determined by ELISA. 12 polyclonal clones with high expression levels were selected and passaged into 10 cm dishes (10 ml). After 10 days, 20 μL of cell supernatant was collected, and the concentration of the target protein in the supernatant was determined by ELISA. 3 polyclonal clones with high expression levels were selected and passaged into 125 ml (40 ml). Once the cell density reached 2-3 E6 / ml, the cells were cultured using CD45. Cells were cryopreserved using CHO + 10% DMSO (Sigma) cryopreservation medium at a density of 1E7 / ml. A polyclonal cell line was selected, and using a limiting dilution method, cells were seeded at a density of 0.5 cells / well in 96-well plates. After 3 weeks, when the confluence of individual cells reached 60-70%, 20 μL of supernatant was aspirated for ELISA concentration analysis. Based on the ELISA results, 120 clones with high expression levels were selected and passaged into 24-well plates (1 ml / well). After 10 days, 20 μL of cell supernatant was aspirated, and the concentration of the target protein in the supernatant was determined by ELISA. 60 polyclonal clones with high expression levels were selected and passaged into 6-well plates (4 ml / well). After 10 days, 20 μL of cell supernatant was aspirated, and the concentration of the target protein in the supernatant was determined by ELISA. 12 polyclonal clones with high expression levels were selected and passaged into 10 cm⁻¹ plates. Place the cells in a culture dish (10 ml culture volume); after 10 days, aspirate 20 μl of cell supernatant and determine the concentration of the target protein in the supernatant using ELISA. Select 10 single clones with high expression levels and passage them into 125 ml (40 ml culture volume). Once the cell density reaches 2-3 E6 / ml, cryopreserve the cells using CD CHO + 10% DMSO (Sigma) cell cryopreservation solution, with a cryopreservation density of 1 E7 / ml.

[0097] One monoclonal cell line was selected from the 10 selected monoclonal cell lines and inoculated into two 5L reactors for fed-batch culture to express recombinant human collagen III. After cell resuscitation, the cells were scaled up stepwise (30ml→300ml→1L) in Dynamis basal medium at 37℃, 110rpm, and 7% CO2. When the cell seed density reached 3-4E6 / ml, the cells were inoculated into 5L reactors at a density of 0.6*106 / ml. The reactor control parameters were: pH: 6.7-7.2, stirring speed: 180rpm, and temperature: cooled from 37℃ to 33℃ on day 6. The initial inoculation volume was 2.8L (560ml seed culture + 2.24L Dynamis medium). On days 2, 4, 6, 8, 10 and 12 after inoculation, 5% Cellboost7a (140ml) + 0.5% Cellboost7b (14ml) supplemental medium was added. On day 5 after inoculation, the sugar concentration in the supernatant of the medium was detected by a Cedex biochemical analyzer. If the sugar concentration was lower than 8g / L, it was supplemented to 8g / L.

[0098] The direct ELISA method was used. The test sample and the standard curve sample (TS precipitate (source) was serially diluted 2-fold to a standard curve concentration range of 15.625 ng / mL to 1000 ng / mL) were directly coated onto an ELISA plate. Type III collagen (TS-1) rabbit polyclonal antibody (Junshi) bound to the sample immobilized on the ELISA plate. Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP (Invitrogen, 31460), bound to the type III collagen (TS-1) rabbit polyclonal antibody bound to the solid-phase sample. HRP catalyzes the TMB to turn blue under hydrogen peroxide, and the intensity of the blue color is positively correlated with the amount of Goat anti-Rabbit IgG (H+L) Secondary Antibody bound. The color turned yellow after termination with 2M hydrochloric acid. The absorbance (OD value) was measured at 450 nm / 650 nm using a SpectraMax M5 ELISA reader, and the sample concentration was calculated from the standard curve. The specific method parameters are as follows: TS precipitate was used as the standard and serially diluted 2-fold with 2mM acetic acid coating buffer to a standard curve concentration range of 15.625 ng / mL to 1000 ng / mL. The test samples and standard curve samples were incubated overnight at 2–8℃. The plates were washed with PBST (1×PBS solution containing 0.05% Tween 20), blocked with 2% BSA, and incubated at 37℃ for 1.5–2 h. TS-1 rabbit polyclonal antibody was diluted to 10 μg / mL with 1% BSA, washed, and added to 100 μL / well of the plate, and incubated at 37℃ for 1.5 h. Anti-Rabbit IgG (H+L) Secondary antibody was diluted 2000–5000 times with 1% BSA, washed, and added to 100 μL / well of the plate, and incubated at 37℃ for 1 h. After washing, TMB was developed and incubated at 37℃ in the dark for 10 ± 2 minutes. The reaction was terminated with 2M HCl, and the plate was read using a microplate reader within 10 minutes at OD 450nm / 650nm wavelength. The results are shown in Figure 4. Figure 4A shows the cell viability and cell density in the fermenters; line 1 represents the cell viability in fermenter 1; line 2 represents the cell viability in fermenter 2; line 3 represents the cell density in fermenter 1; and line 4 represents the cell density in fermenter 2.

[0099] In Figure 4B, line 1 represents the glutamine content in the supernatant of fermenter 1; line 2 represents the glutamine content in the supernatant of fermenter 2; line 3 represents the glucose content in the supernatant of fermenter 1; and line 4 represents the glucose content in the supernatant of fermenter 2.

[0100] In Figure 4C, line 1 represents the ammonium ion concentration in the supernatant of fermenter 1; line 2 represents the ammonium ion concentration in the supernatant of fermenter 2; line 3 represents the glutamine content in the supernatant of fermenter 1; and line 4 represents the glutamine content in the supernatant of fermenter 2.

[0101] In Figure 5A, lane 1 is the positive control; lanes 2 and 3 are the supernatants from fermenters 1 and 2 on day 10; lanes 4 and 5 are the supernatants from fermenters 1 and 2 on day 12; lanes 6 and 7 are the supernatants from fermenters 1 and 2 on day 13; and lanes 8 and 9 are the supernatants from fermenters 1 and 2 on day 14.

[0102] In Figure 5B, 1, 2, 3, and 4 represent the supernatant expression levels in fermenter No. 1 on days 10, 12, 13, and 14, respectively; 5, 6, 7, and 8 represent the supernatant expression levels in fermenter No. 2 on days 10, 12, 13, and 14, respectively.

[0103] The results showed that cell growth and metabolism were similar in both reactors, with a peak cell density of approximately 30E6 / ml. At the end of day 14, cell viability was around 80% (1A). Throughout the fermentation process, sugar concentration was maintained between 2-10 g / L. Ammonium ions reached their peak on days 7-8 after inoculation and then began to decline. Glutamine and lactic acid showed an accumulation trend in the later stages of culture (1B, 1C). SDS-PAGE protein analysis of the supernatants from days 10, 12, 13, and 14 of culture in both reactors revealed that the target protein was primarily present in the supernatant as a trimer in all samples.

[0104] The results above show that a stable cell line can be constructed by co-transfection of collagen protein particles and P4H plasmid. Preliminary experiments in a 5L reactor show that the cell line is metabolically stable and the expression level can reach 2g / L, which can be used for large-scale production.

[0105] 2.2 Protein purification

[0106] Take 1 L of cell culture supernatant, add 25% ammonium sulfate, stir at 4℃ for 3 h to precipitate, centrifuge to remove supernatant, resuspend the precipitate in 500 mL of 0.5 M acetic acid (pH 2.5), add 0.1 g of pepsin (Aladdin, P736658) and digest overnight. After digestion, purify using a cation exchange chromatography column (Borglon, Diamond SP Mustang), following the Diamond SP Mustang instruction manual for chromatographic conditions. After cation exchange chromatography, replace the ultrafiltration buffer with an ultrafiltration tube (Cobati, HFELP00301011P), following the product instruction manual for ultrafiltration conditions. Dilute the protein with LDS Sample Buffer (4X) and ultrapure water to a 1 mg / mL test solution, incubate at 70℃ for 1 min, and add 4 μL to the loading channels of a 4-20% precast gel. Then start electrophoresis. After electrophoresis, remove the gel, wash, and then perform staining and destaining.

[0107] The results are shown in Figure 6. As can be seen from the results in Figure 6, the purified PD105 protein has higher purity than the naturally extracted Native Human Collagen III protein (1047178-1, abcam, ab7535). PD105 contains only a triple helix structure, while Native Human Collagen III protein contains not only triple helix but also a large number of dimers and monomers.

[0108] 2.3 Protein Identification

[0109] SEC-HPLC

[0110] Impurities related to recombinant human type III collagen were separated using size exclusion chromatography (MSC). Neutral pH buffer was used as the mobile phase for elution, and the molecular weight fractions were eluted sequentially in descending order of molecular weight. Detection was performed at 214 nm. Peak area normalization was used to determine the content of polymers, monomers, and low molecular weight impurities; the monomer content was used to determine the purity of the monoclonal antibody. Specifically, the sample was diluted to 1 mg / mL, and XBridge Protein BEH SEC Column chromatography was used. A 3.5μm, 7.8mm x 300mm column (catalog number: 186007640) was used. The column temperature was set to 25℃. The mobile phase consisted of 50mM phosphate buffer, 300mM sodium chloride, and pH 7.0±0.2. The injection volume was 20μL. Isocratic elution was performed at a flow rate of 1.0mL / min for 20min. Detection was performed at a wavelength of 214nm, and the monomer content was obtained using the peak area normalization method. The results are shown in Figure 7A.

[0111] Two-color circle

[0112] 500 μg of recombinant collagen III and Native Human Collagen III protein (1047178-1, abcam, ab7535) were transferred to a 10 kDa ultrafiltration tube and ultrafiltered three times with 50 mM potassium phosphate buffer. The sample was then transferred to a 1.5 mL centrifuge tube, and the volume was brought up to 1 mL with 50 mM potassium phosphate buffer. Data acquisition was performed using a Chirascan Plus V100 circular dichroism chromatograph (CDI). The scanning wavelength range was set to 185-260 nm, with a bandwidth and step size of 1.0 nm. Scans were repeated three times using 0.5 mm quartz cuvettes. After scanning, the sample data were averaged, background subtracted, and smoothed. The results are shown in Figure 7D.

[0113] Fourier transform infrared spectroscopy detection

[0114] The recombinant collagen III (PD105-20240301SP-2-B4) and Native Human Collagen III protein (1047178-1, abcam, ab7535) samples were diluted to 1 mg / mL using formulation buffer. Detection was performed using a Thermo Fisher Fourier Transform Infrared Spectrometer (Nicolet iS20) in attenuated total reflectance (ATR) mode. Other data acquisition parameters were as follows: range 4000–650 cm⁻¹, automatic gain, 64 scans, resolution 8, correction None, and absorbance format. After data acquisition, formulation buffer subtraction, second derivative calculation, smoothing curve analysis, and correlation analysis were performed sequentially. The results are shown in Figure 7C.

[0115] SEC-MALS detection

[0116] Analysis was performed using a Waters 2695 HPLC system and a Wyatt Multi-Angle Laser Scattering (MALS) detector. The recombinant human type III collagen (20241020) sample was diluted to 4 mg / mL. A TSKgel G3000SWXL column was used, with a column temperature of 25℃. The mobile phase consisted of 50 mM phosphate buffer, 300 mM sodium chloride, and pH 7.0 ± 0.2. The injection volume was 10 μL, and isocratic elution was performed at a flow rate of 0.6 mL / min for 25 min. Detection was performed at 214 nm, and the molecular weight of each component was determined using the MALS detector. The results are shown in Figures 7B-1 and 7B-2.

[0117] The purified recombinant human collagen III was analyzed by HPLC-SEC. Figure 7A shows that over 95% of the protein existed in solution as a trimer. Subsequent HPLC-SEC-MALS analysis revealed that the molecular weight of the purified collagen standard recombinant human type III collagen was 364.4 kDa (Figures 7B-1 and 7B-2), which was consistent with expectations. The secondary and tertiary structures of the recombinant human collagen III molecule were then confirmed by Fourier transform infrared spectroscopy (FTIR) and circular dichroism spectroscopy (CD). In FTIR analysis, the recombinant human type III collagen molecule and Native Human Collagen III protein were simultaneously detected. Comparative analysis of the spectra showed a high similarity of 0.9742 (the closer to 1, the higher the similarity) (Figure 7C), indicating that their secondary structures are essentially identical. Circular dichroism spectroscopy (CD) was then performed to examine the presence of a triple helix structure in the recombinant human type III collagen molecule. As shown in Figure 7D, the recombinant human collagen III molecule is highly consistent with the spectrum of the native human collagen III protein. Both have a negative peak at a wavelength of around 195 nm and a positive peak at a wavelength of around 221 nm, suggesting that the recombinant human collagen III molecule, like the native human collagen III protein, has a stable triple helix structure.

Claims

1. A method for preparing human collagen III, comprising the following steps: 1) Construct an expression vector containing a codon-optimized gene encoding human collagen III, wherein the gene encoding human collagen III has the nucleotide sequence shown in SEQ ID NO.2; 2) Construct an expression vector containing a codon-optimized gene encoding amino acid hydroxylase P4H, wherein the gene encoding amino acid hydroxylase P4H has the nucleotide sequence shown in SEQ ID NO.4; 3) Co-transfect the expression vectors from steps 1) and 2) into CHO cells to obtain a stable cell line; 4) The stable cell line obtained in step 3) is fermented to obtain human collagen III.

2. The method of claim 1, wherein the expression vector in steps 1) and 2) is a pXC17.4 vector, the gene encoding human collagen III is ligated to the pXC17.4 vector via HindIII and EcoRI restriction sites, T4, and the gene encoding P4H is ligated to the pXC18.4 vector via HindIII and EcoRI restriction sites, T4.

3. The method of claim 1 or 2, wherein in step 3), the gene encoding human collagen III and the gene encoding P4H are co-transfected into CHO cells at a mass ratio of 1:

1.

4. A recombinant human collagen III prepared according to any one of claims 1-3.

Images