Cell-expressed self-assembled triple-helix human collagen and preparation process thereof

Abstract

The invention relates to a human recombinant collagen based on cell expression self-assembly, which is closer to a triple helix structure of natural collagen, and successfully realizes efficient expression and correct folding of recombinant III type collagen through a genetic engineering cell expression system and rational amino acid sequence design. Through verification, the obtained product can be spontaneously assembled to form a stable natural triple-helix structure, and the optimized molecular conformation enables the product to have the following remarkable advantages: the product is closer to the mechanical property of a natural tissue; the biological activity is improved; the cell affinity is obviously improved.

Description

Technical Field

[0001] This invention belongs to the field of recombinant protein technology, and specifically relates to a cell-expressed self-assembled human collagen protein and its preparation process. Background Technology

[0002] Collagen is the most abundant structural protein in mammals, playing a crucial role in maintaining tissue integrity, cell adhesion, migration, and tissue repair. Based on its molecular structure and tissue distribution, collagen can be classified into at least 28 types, among which type I, type II, and type III collagen are the most prevalent fibrous collagens and are widely found in connective tissue.

[0003] Type III collagen (COL3) is a fibroblast-forming collagen mainly composed of three α1(III) chains, exhibiting a typical triple helix structure. COL3 is primarily found in tissues rich in elastic fibers, such as skin, blood vessels, lungs, intestines, and the uterus. It is usually co-expressed with type I collagen, working synergistically to maintain the mechanical strength and elasticity of tissues.

[0004] During tissue development and repair, the expression of type III collagen typically precedes that of type I collagen, suggesting its crucial role in wound healing, angiogenesis, and matrix remodeling. Furthermore, COL3 exhibits characteristic changes in various disease states, demonstrating significant biological associations in aneurysm formation, fibrotic diseases (such as liver and pulmonary fibrosis), and connective tissue diseases (such as Ehlers-Danlos syndrome). Therefore, type III collagen holds significant research and application value in regenerative medicine, biomaterials, biomarker development, and related drug development.

[0005] With the development of tissue engineering and biomaterials, the expression, purification, and application of recombinant human type III collagen (rhCOL3) have attracted increasing attention. Obtaining high-purity type III collagen through genetic engineering not only helps in studying its functional mechanisms but also provides a foundation for developing novel tissue scaffolds, medical dressings, cosmetic products, and therapeutic drugs.

[0006] The triple helix structure of natural collagen refers to procollagen molecules arranging themselves in parallel triple helices according to specific rules, cross-linking through hydrogen bonds to form stable collagen microfibers, which further aggregate into bundles to form collagen fibers. This process involves forces such as hydrogen bonds, which play a crucial role in the stability, strength, and bioactivity of collagen. Triple helix collagen molecules are assembled into supramolecular structures, and collagen with specific amino acid sequences is produced through specific expression systems. Recombinant human collagen is a protein obtained through recombinant expression technology after optimization or the introduction of amino acid sequences with specific functions. It significantly reduces the risk of immune responses triggered by exogenous proteins and occupies a pivotal position in the synthetic biology industry chain.

[0007] Currently, most mass-produced recombinant collagen has a single-chain structure, with very few possessing a triple-helix structure. Unlike proteins that self-assemble into triple-helix structures within cells, collagen cannot achieve the same high-order triple-helix structure, primarily due to the lack of hydroxyproline modification, resulting in lower stability and bioactivity. If collagen is not precisely modified post-translationally, it cannot effectively bind to cell surface receptors, weakening its support for cell regeneration and repair. Misfolded, unmodified collagen may be more easily degraded and eliminated by the body's enzymatic systems, and therefore cannot sustainably exist in the body. Summary of the Invention

[0008] To address the aforementioned technical problems, a first aspect of the present invention provides a recombinant collagen protein, wherein the amino acid sequence of the recombinant collagen protein is SEQ ID No. 1.

[0009] A second aspect of the present invention also provides a polynucleotide encoding the aforementioned recombinant collagen, an expression vector comprising the aforementioned polynucleotide, and a host cell comprising the aforementioned expression vector.

[0010] A third aspect of the present invention also provides a process for preparing recombinant collagen, comprising the following steps: obtaining the recombinant collagen by affinity chromatography based on the crude recombinant collagen product.

[0011] A fourth aspect of the present invention also provides a pharmaceutical, cosmetic, medical aesthetic product, biomedical material, or tissue engineering product of recombinant collagen.

[0012] The fifth aspect of the present invention also provides the use of recombinant collagen in the preparation of pharmaceuticals, cosmetics, medical aesthetic products, biomedical materials or tissue engineering products.

[0013] The beneficial effects of this invention lie in its successful achievement of efficient expression and correct folding of recombinant type III collagen through a genetically engineered cell expression system and rationally designed amino acid sequences. Verification has shown that the resulting product can spontaneously assemble into a stable natural triple helix structure. This optimized molecular conformation gives it the following significant advantages: mechanical properties closer to natural tissues; improved bioactivity; and significantly enhanced cell affinity. Triple helix collagen will play a more important biological role in the fields of biomedicine, tissue engineering, medical aesthetics, and orthopedic repair. This invention's recombinant collagen utilizes molecular adaptive technology to spontaneously assemble into a triple helix structure, significantly enhancing its performance in various applications. Attached Figure Description

[0014] Figure 1 Results of cell proliferation assay;

[0015] Figure 2 Adhesion experiment results;

[0016] Figure 3 TIC image of Trypsin digestion in 24S70 sample (sample batch number 20240308AC);

[0017] Figure 4 Trypsin peptide sequence coverage of 24S70 sample (sample batch number 20240308AC);

[0018] Figure 5 The results of circular dichroism spectroscopy of recombinant collagen in this embodiment of the invention are shown.

[0019] Figure 6 To represent the spectrum of the carrier IP292-SNTs 1.0;

[0020] Figure 7 The results are from SDS-PAGE analysis of collagen.

[0021] Figure 8 SEC assay results for IP292A affinity-purified protein;

[0022] Figure 9 SEC assay results for IP292A affinity-purified protein;

[0023] Figure 10 This is the result of the SEC test. Detailed Implementation

[0024] Sources of reagents and materials:

[0025] HindIII enzyme (Thermo); EcoRI enzyme (Thermo); PvuI (Thermo); CD CHO medium (Thermo); CBM-S9 medium (Merck); L-methionine sulfoxide imine (Merck); expression vector SNTs 1.0 (Scissofac); CHO cells (Lonza); pTT5 (Scissofac); CFM-1 medium (Merck); CFM-2 medium (Merck).

[0026] Example 1: Preparation of recombinant human collagen (IP292A)

[0027] 1. Synthesis of target gene and construction of expression vector

[0028] 1.1 Target gene synthesis

[0029] The amino acid sequence (SEQ ID No. 1) of the cell-expressed self-assembled human collagen is: GRPGRPGERGLPGPPGIKGPAGIPGFPGMKGHRGFDGRNGEKGETGAPGLKGENGLPGENGAPGPMGPRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAGSPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARGPPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSPGEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRDGVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGPSGPRGQPGVMGFPGPKGNDGAPGKNGERGGPGGP

[0030] The DNA sequence (SEQ ID No. 2) for cell expression of self-assembled human collagen is as follows:

[0031]

[0032] HindIII restriction site, Kozak sequence, and signal peptide sequence were sequentially added to the 5' end of the nucleotide sequence encoding the target molecule's amino acid. A stop codon and EcoRI restriction site were sequentially added to the 3' end. Codon optimization (CHO) of the nucleotide sequence encoding the target molecule's amino acid and the synthesis of the target gene were commissioned to a third-party gene synthesis company, and plasmid pTT5 was cloned.

[0033] 1.2 Construction of expression vector

[0034] Gene plasmids were synthesized by double digestion with HindIII and EcoRI. The target gene fragment was recovered by gel electrophoresis and cloned into the mammalian expression vector SNTs 1.0 to obtain the expression vector IP292-SNTs 1.0 (e.g., Figure 6 (As shown). The target gene was verified by colony PCR and enzyme digestion. The correctness of the nucleic acid sequence in the constructed expression vector IP292-SNTs 1.0 was verified by Sanger sequencing (first-generation sequencing). The expression vector plasmid was extracted using an endotoxin-free kit and linearized using PvuI. The linearization of the plasmid was confirmed by agarose gel electrophoresis. The concentration of the linearized plasmid was adjusted to 950 ng / μl to 1100 ng / μl. 2≥A 260 / A 280 ≥1.8, the expression vector IP292-SNTs 1.0 was used for cell transfection.

[0035] 2. Cell line construction

[0036] 2.1 Transfection

[0037] The expression vector IP292-SNTs 1.0 was transfected into host cells using electroporation transfection. The specific steps are as follows:

[0038] Resuscitate host CHO cells and passage them using CBM-S9 cells containing 6 mM glutamine. Culture conditions were: 37.0℃, 8% CO2, 110 rpm.

[0039] Take 2.52×10 7Centrifuge CHO cells at 1000 rpm for 5 min (actual cell volume = theoretical requirement × 105%, the 5% increase is centrifugation loss), discard the supernatant; add 1520 μl of CD CHO medium (Thermo) and 120 μl of expression vector IP292-SNTs 1.0 to the centrifuged cells, resuspend and mix; take 800 μl of the mixture into two electroporation cuvettes, place the cuvettes into the electroporator and electroporate (electroporation program: 300V, 900μF, exponential pulse, resistance (∞)); after electroporation, transfer the cells from the two electroporation cuvettes to a shake flask containing 30 ml of preheated CBM-S9 to obtain a pool.

[0040] After 48 hours of pressurized recovery electroporation, the culture medium was centrifuged and replaced with CBM-S9 containing 50 μM L-methionine sulfoxide imide. Cell density and viability were measured 48 hours after electroporation, on day 6, and every two days thereafter, and the cell density was adjusted to (0.3–0.7) × 10⁻⁶. 6 Cells / ml were cultured in suspension on a CO2 shaker with the following parameters: 37.0℃, 8.0% CO2, 110 rpm. When the cell density was ≥1.0 × 10⁻⁶ cells / ml, the cells were cultured in suspension. 6 When the cell count is ≥90% and the cell viability is ≥90%, pressure screening is completed. Five cell lines are cryopreserved per pool, with a cryopreservation density of 1.0 × 10⁶ cells / ml. 7 cells / ml, 1ml / vial, with the remaining cell solution used for fed-batch evaluation.

[0041] 2.2 Monoclonal Plating

[0042] Pool 1 cells obtained from pressure screening were single-cloned using a cloning screening single-cell printing system (c.sight), and seeded onto 10 96-well plates. c.sight parameters were set to cell diameter (10–25) μm and cell roundness (0.5–1.0). The viable cell density was adjusted to 0.8 × 10⁻⁶ cells using CD CHO. 6 cells / ml, plate-forming medium was EX- Add 120 μl of Cloning Medium per well to a 96-well plate. Photographs of the 96-well plates were taken at 2–3 h, 24 h, 48 h, D7 (day 7), and D17 (day 17) after plating. After photographing on D7, 100 μl of medium was added to each well.

[0043] 2.3 Single-clone plate screening

[0044] After D17 plate formation, the confluence rate of most positive wells was greater than 30%. The monoclonal nature of the positive wells was initially assessed, and wells that met the monoclonal criteria (cell images at 2h-3h, 1 cell; 24h, 1 or 2 cells; 48h, 2, 3, 4 or 5 cells) were numbered with 96-well plate serial numbers.

[0045] When the confluence of cells in each well plate exceeded 30%, protein expression levels were detected using a Gator label-free analyzer. The top 10 clones with the highest expression levels were selected and progressively expanded to shake-tube culture. After the high-expression clones in the well plates were expanded to a certain quantity, they were cryopreserved and cultured in fed-batch culture. Five cells were cryopreserved for each clone, with a cryopreservation density of 1.0 × 10⁻⁶ cells / well. 7 cells / ml, 1ml / vial, define the cryopreservation passage as P0, the fed-batch culture protocol is the same as the pool fed-batch culture protocol, see Table 4.

[0046] Table 1. Top 10 Clonal Flow Culture Protocols

[0047]

[0048]

[0049] Based on expression levels and SEC results ( Figure 10 Clone 1 was selected as the final clone.

[0050] 3. Purification steps

[0051] As shown in Tables 2 and 3.

[0052] Table 2. Basic Information on His Affinity Chromatography

[0053]

[0054] Table 3. Specific steps of His affinity chromatography

[0055]

[0056]

[0057] Example 2

[0058] IP292A-20240308S; Quantitative expression level 1g.

[0059]

[0060] The difference from Example 1 is:

[0061] Table 4. Basic Information on His Affinity Chromatography

[0062]

[0063]

[0064] Table 5. Specific steps of His affinity chromatography

[0065]

[0066] Figure 7 SDS-PAGE of IP292A affinity-purified protein.

[0067] Results analysis:

[0068] After one affinity assay, SDS-PAGE results showed that the purity of the IP292A protein molecule was >95%. The SEC assay result for the IP292A protein was 96%.

[0069] By comparing the intensity of control protein bands with different protein levels in SDS-PAGE, it is estimated that the protein concentration range of IP292A-20240308AC is 2.5–5 mg / ml (BCA platform detection method is not applicable).

[0070] Experimental Example 1: Cell Proliferation Assay

[0071] 5. Reagents, standards and instruments

[0072] 5.1 Cell Counting Kit-8 (CCK-8) is a rapid, highly sensitive assay kit based on WST-8, widely used for the detection of cell proliferation and cytotoxicity. CCK-8 contains WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt), which, in the presence of the electron coupling reagent 1-methoxy-5-methylphenazineonium sulfate dimethyl ester (1-Methoxy PMS), can be reduced by mitochondrial dehydrogenases to the highly water-soluble orange-yellow formazan product, Formazan. The amount of Formazan generated is directly proportional to the number of viable cells.

[0073] 5.2 Complete culture medium for cell culture and sample dilution (DMEM, containing 10% FBS and antibiotics);

[0074] 5.3 96-well cell culture plate;

[0075] 5.4 The microplate reader needs to be preheated for 15 minutes in advance;

[0076] 5.5 37℃, 5% CO2 incubator.

[0077] 6. Testing Procedures

[0078] 6.1 Cell Preparation

[0079] Prepare a suitable concentration of cell suspension using healthy 3T3 cells, and add 100 μL to each well of a 96-well cell culture plate. Typically, add 5 × 10⁶ cells per well. 3 Cells / 100µL (the exact number of cells used per well depends on factors such as cell size and cell proliferation rate). Incubate at 37°C in a 5% CO2 incubator until cells adhere.

[0080] 6.2 Sample Preparation

[0081] Dilute the sample to the highest analyte concentration using complete culture medium (the specific dilution factor depends on the characteristics of the chemical components).

[0082] 6.3 Sample Addition

[0083] Arrange the samples in a 96-well plate and perform triple parallel tests for each concentration. Starting with the second concentration well, pre-add 100 μL of complete culture medium to each well. Using a pipette, pipette 300 mL / well of the highest diluted sample to be tested, three times, and add it to the first concentration well of the 96-well plate; this is the first concentration. Adjust the pipette to 200 mL, pipette 200 mL / well of the first concentration well, three times, and add it to the second concentration well. Gently pipette 10 times to mix; this is the second concentration. Take 200 μL / well of the second concentration from each of the three wells and add it to the third concentration well, gently pipetting 10 times to mix; this is the third concentration. Repeat this process for approximately 10 concentrations. For the last concentration, pipette 200 μL / well from each of the three wells to ensure the same culture volume in each well. Incubate the 96-well plate overnight in a 37°C, 5% CO2 incubator.

[0084] 6.4 CCK-8 reaction

[0085] Prepare a 10% concentration of CCK-8 solution using complete culture medium. Dry the solution in the 96-well plate and add CCK-8 by changing the medium. Incubate the 96-well plate in a 37°C, 5% CO2 incubator for 0.5-4 hours (first measurement within 30 minutes). The absorbance of the negative control should be between 1 and 1.5.

[0086] 6.5 Measurement of absorbance

[0087] Detection was performed using a single wavelength of 450nm.

[0088] 7. Result Calculation

[0089] 7.1 Calculation formula:

[0090] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0091] Cell inhibition rate = [(Ac-As) / (Ac-Ab)] × 100%

[0092] As: Absorbance of experimental wells (including cells, complete culture medium, CCK-8 solution and sample);

[0093] Ac: Absorbance of control wells (containing cells, culture medium, and CCK-8 solution, but excluding sample);

[0094] Ab: Absorbance of blank wells (including culture medium and CCK-8 solution, excluding cells and samples).

[0095] We typically use 450nm baseline data.

[0096] Cell viability = As / Ac × 100%

[0097] Cell inhibition rate = Ac / As × 100%

[0098] 8. Precautions

[0099] 8.1 Set up 3 replicates for each drug concentration well, and finally take the average value to make a curve.

[0100] 8.2 After adding CCK-8 solution, pay attention to the incubation time. The length of incubation time depends on the cell type and cell density, etc. For the first experiment, you can use an ELISA reader to detect the absorbance after 0.5, 1, 2 and 4 hours, and then select a time point with a suitable absorbance range for subsequent experiments.

[0101] 8.3 When cells are cultured in an incubator, the outermost wells of the culture plate are prone to drying and evaporation, which increases the error due to inaccurate volume. Therefore, the outermost four wells of the plate are not used for testing, and PBS is added.

[0102] After incubation at 8.4CCK-8, the orange-yellow formazan product may clump together. You can gently tap the side of the well plate to mix it before loading it onto the machine.

[0103] 8.5 Before using an ELISA reader, ensure that there are no air bubbles in each well, otherwise it will interfere with the assay.

[0104] Result: As Figure 1 As shown, compared with bovine serum albumin (BSA) and yeast-expressed collagen, cell-expressed recombinant human collagen is more conducive to cell proliferation, indicating that it has better cell biocompatibility.

[0105] Experiment Example 2 Adhesion Experiment

[0106] 1. Adhesion Experiment

[0107] Experimental procedure:

[0108] NIH / 3T3 cells were cultured normally. The recombinant human collagen purified lyophilized product obtained in Example 1 and the control product were dissolved in bovine serum albumin (BSA) (ultrapure water or 1M HCl solution). The protein concentration was determined using the empirical formula for UV protein quantification: C(mg / mL) = 0.144*(A215-A225), and then diluted to 0.5 mg / mL with PBS (pH 7.4). 100 μL of various protein solutions and blank PBS solution were added to each well of a 96-well cell culture plate and incubated at room temperature for 60 min. Then, 10⁵ well-cultured 3T3 cells were added to each well and incubated at 37°C and 5% CO₂ for 60 min. The cells were washed four times with PBS. The absorbance at OD492 nm was measured using an LDH detection kit (Roche, 04744926001).

[0109] Experimental results:

[0110] The absorbance at OD492nm can characterize the cell adhesion activity of collagen samples: the higher the absorbance value, the more cells the protein adheres to, the higher the adhesion activity, and the more quickly collagen can help cells adhere to the cell wall or the extracellular matrix, which is more conducive to building a better extracellular environment.

[0111] as follows Figure 2 As shown, the recombinant human collagen of Example 1 has better cell adhesion activity compared with BSA.

[0112] Experimental Example 3: Post-translational Modification Analysis Experiment of 24S70

[0113] 1. Experimental Objective

[0114] The proline hydroxylation modification of the 24S70 sample (recombinant human collagen from Example 1) was tested using Trypsin peptide mapping, and compared with the proline sites of the natural protein P02461 reported by Uniprot to compare the similarity between the 24S70 protein and the natural protein P02461 in terms of proline hydroxylation modification.

[0115] 2. Experimental Background

[0116] The P02461 molecule is a type III collagen composed of 1466 amino acids. Hydroxyproline, formed by the hydroxylation of proline, is the most important modification in collagen, playing a crucial role in maintaining its triple helix structure and biological properties. The 24S70 molecule consists of 369 amino acids, and its amino acid sequence is identical to amino acids 234-602 of the natural protein P02461.

[0117] This experiment aims to test the 24S70 proline hydroxylation modification site and its ratio, and to compare it with the proline sites hydroxylated in the range of 234-602 reported by Uniprot in the natural protein P02461. Information such as sample names and batch numbers is shown in Table 4. The sample (batch number 20240308AC) was prepared using the same method as in Example 1.

[0118] Table 4 Sample Information

[0119] Sample Name Sample batch number concentration Other information IP292A-20240308AC 20240308AC 2.5-5mg / ml Buffer solution: 20mM Tris, 5mM EDTA, 50mM NaCl

[0120] 3. Experimental instruments and reagents

[0121] 3.1 Experimental Apparatus

[0122] pipette Eppendorf / 20μl~200μl LK-AS-123 pipette Eppendorf / 100μl~1000μl LK-AS-117

[0123] 3.2 Experimental reagents or consumables

[0124]

[0125] 4. Experiment Overview

[0126] The proline hydroxylation modification of 20240308AC was detected using a SCIEX 7600 Zeno-QTOF mass spectrometer and SCIEX Be software.

[0127] Conduct data analysis.

[0128] 5. Experimental Methods

[0129] 5.1 Sample preparation

[0130] 5.1.1 Transmutation and Reduction

[0131] Take a 1.5 mL centrifuge tube, add 300 μL of 8M guanidine hydrochloride denaturing buffer, add 15 μL of 1M NH4HCO3 (pH 7.8), then add 15 μL of 0.5M DTT, vortex to mix for a few seconds, and finally take 200 μg of sample (sample concentration is 2.5 mg / mL, take 80 μL) and add it to the above 1.5 mL centrifuge tube, vortex to mix for a few seconds, place in a metal bath and incubate at 50 °C for 30 min, remove the sample and cool to room temperature.

[0132] 5.1.2 Closure

[0133] Add 30 μL of 0.5 M IAM solution to the above sample, vortex mix for a few seconds, and then incubate at room temperature in the dark for 1 h.

[0134] 5.1.3 Fluid Change

[0135] Add 500 μL of 50 mM NH4HCO3 (pH 7.8) to the ultrafiltration tube, centrifuge at 12000 rpm for 5 min to rinse, discard the buffer solution in the ultrafiltration tube, and transfer the entire sample to the rinsed ultrafiltration tube. Centrifuge at 12000 rpm for 5 min. Add 50 mM NH4HCO3 (pH 7.8) to a final volume of 500 μL, centrifuge at 12000 rpm for 5 min, and repeat this step 4 times. Transfer the sample after buffer replacement to a new 1.5 mL centrifuge tube, rinse the ultrafiltration tube with an appropriate amount of 50 mM NH4HCO3 (pH 7.8), and then transfer the entire sample to the centrifuge tube, with a total volume of approximately 200 μL. Store at -80℃ for one month.

[0136] 5.1.4 Trypsin digestion

[0137] Take 50 μL of the sample obtained in 5.1.3, add 1.25 μL of PNGase F (40 μg protein: 1 μL glycosidase), vortex mix for a few seconds, incubate in a 50℃ water bath for 30 min, remove and cool to room temperature, then add 4 μL of 0.5 mg / mL Trypsin (protein: endonuclease w / w = 25:1), vortex mix for a few seconds, and incubate in a 37℃ metal bath for 7 h.

[0138] 5.1.5 Termination

[0139] Add 1.25 μL of 20% FA (final concentration approximately 0.5%) to the sample obtained in 5.1.4 to terminate the reaction, vortex mix for a few seconds, centrifuge at 12000 rpm for 5 min, and then transfer the supernatant into the insertion tube for analysis.

[0140] 5.2 Instrument Conditions

[0141] 5.2.1UPLC conditions

[0142]

[0143]

[0144]

[0145] 6. Results and Discussion

[0146] 6.1 Data Analysis Parameters

[0147]

[0148]

[0149]

[0150] Note: * indicates Oxidation(P) modification, which means Hydroxy-P modification.

[0151] 6.2 Mass spectrometry TIC chromatogram

[0152] like Figure 3 As shown.

[0153] 6.3 Sequence Coverage

[0154] The peptide mapping, performed using Trypsin desaccharification enzyme, showed a sequence coverage of 98.4% after secondary confirmation. See details... Figure 4 .

[0155] 6.4 Results of proline hydroxylation modification

[0156] The trypsin digestion peptide mapping was used to characterize the proline hydroxylation sites and proportions in the 24S70 sample (sample batch number 20240308AC). When proline undergoes hydroxylation, the peptide mass spectrometry molecular weight increases by the mass number of one oxygen atom. The hydroxylation sites were identified by the shift in the primary molecular weight of the peptide mass spectrometry and by the secondary mass spectrometry. The proportion of hydroxylation at a single site was then calculated using the response value ratio, as shown in the following formula:

[0157] The proline hydroxylation modification rate at a certain site = (Response value of the hydroxylated peptide at that site / Response value of the hydroxylated peptide containing that site + Response value of the non-hydroxylated peptide containing that site) × 100%

[0158] The results show that the proline hydroxylation modification sites in 20240308AC are similar to those reported by Uniprot (molecule P02461). Mass spectrometry data of hydroxyproline used in this application indicate that hydroxyproline modification occurred at 52 out of 369 uninterrupted amino acid sites, and proline accounts for approximately 2 / 9 of this sequence; that is, the present invention states that "the hydroxyproline content reaches more than 60% of the total proline."

[0159] Experimental Example 4: Circular Dichroism Detection

[0160] The circular dichroism signals of the peptides were measured using a Chirascan V100 (Plus) instrument from Applied Photophysics, UK. The instrument was purged with nitrogen for 15 minutes and preheated before the xenon lamp was turned on to ensure test stability. The hydrothermal circulation and testing software were then activated.

[0161] The test temperature was set at 4-65℃, with intervals of 3 degrees Celsius; the test wavelength was 190-260 nm; the scan rate was 100 nm / min; 100 μL of sample was added to a cuvette with a 0.1 mm optical path for testing; the UV absorbance of the sample was ensured to be below 2.5 before testing, and the buffer was used as background subtraction. The instrument's built-in Pro-Data Viewer software was used to store and retrieve data, and CDNN software was used to analyze the secondary structure changes of the peptides.

[0162] Test results: such as Figure 5 As shown, the typical triple helix structure of collagen exhibits a positive absorption peak around 220 nm and a negative absorption peak around 195 nm; both conditions are indispensable. The positive absorption peak near 220 nm: This peak is usually associated with the triple helix structure of collagen, indicating the difference in absorption of left-handed and right-handed circularly polarized light at this wavelength. The negative absorption peak near 195 nm: This peak is also associated with the triple helix structure, further confirming the characteristic structure of collagen.

[0163] Experimental Example 5: Multimer Detection

[0164] Experimental method: Column: Biocore SEC-300 7.8x300mm 5um; Mobile phase: 100mM phosphate containing 100mM anhydrous sodium sulfate, pH 6.8; Flow rate: 1ml / min; Temperature: 25℃; Detection wavelength: 280nm; Injection volume: 50ul; Time: 20min.

[0165] Test results: such as Figure 8 As shown, the theoretical molecular weight of a single collagen molecule is about 45KD, while the results from SEC show that the molecular weight of the purified collagen reaches 150KD, indicating that the protein molecule we expressed is a trimer collagen molecule, further proving that it is a collagen molecule with a triple helix structure.

[0166] like Figure 9 As shown, the product peak is at 15 min, proving that the recombinant collagen of this invention is a self-assembled triple helix.

[0167] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A recombinant collagen protein, characterized in that: The amino acid sequence of the recombinant collagen is SEQ ID No.

1.

2. A polynucleotide encoding the recombinant collagen of claim 1, or an expression vector comprising the polynucleotide, or a host cell comprising the expression vector.

3. The preparation process of recombinant collagen as described in claim 1, characterized in that... The process includes the following steps: obtaining the recombinant collagen from the crude recombinant collagen product through affinity chromatography; preferably, the affinity chromatography column is a HisTrap. TM Excel.

4. The manufacturing method as described in claim 3, characterized in that, Recombinant crude collagen is obtained based on the host cell described in claim 2; preferably, the host cell is a CHO cell.

5. The manufacturing method as described in claim 4, characterized in that, The method for constructing the host cell is as follows: the expression vector is transferred into the host cell using electroporation transfection.

6. The manufacturing method as described in claim 5, characterized in that, The method for constructing the expression vector is as follows: digesting the gene plasmid with enzymes, recovering the target gene fragment by gel extraction, and cloning the target gene into the expression vector; preferably, the expression vector is SNTs 1.

0.

7. The manufacturing method as described in claim 6, characterized in that, The method for constructing the target gene is as follows: add HindIII restriction site, Kozak sequence and signal peptide sequence to the polynucleotide described in claim 2, and add stop codon and EcoRI restriction site to the C-terminus in sequence.

8. The manufacturing method as described in claim 7, characterized in that, The method for constructing the gene plasmid is as follows: inserting the target gene into the plasmid; preferably, the plasmid is pTT5.

9. A recombinant collagen protein, characterized in that: The DNA sequence of the recombinant collagen is SEQ ID No.

2.

10. A pharmaceutical product, cosmetic product, medical aesthetic product, biomedical material, or tissue engineering product prepared from recombinant collagen as described in any one of claims 1 or 2, and the use of recombinant collagen as described in claim 1 in the preparation of pharmaceutical products, cosmetic products, medical aesthetic products, biomedical materials, or tissue engineering products.

Images