Collagen with low immunogenicity, high purity and high biological activity as well as preparation method and application thereof

Abstract

The invention discloses low-immunogenicity, high-purity and high-bioactivity collagen as well as a preparation method and application thereof, and belongs to the technical field of medical material preparation. According to the preparation method disclosed by the invention, transgenic pigskin is taken as a raw material, enzymolysis is carried out under monitoring of an intelligent system, and collagen with low immunogenicity, high purity and high biological activity is prepared by combining salting-out with an isoelectric point separation technology and a multi-stage purification technology. And low immunogenicity, high purity and high biological activity of the collagen can be realized while the complete triple-helix structure of the collagen is ensured. The prepared collagen can be applied to the fields of medical treatment and medical beauty.

Description

Technical Field

[0001] This invention belongs to the field of medical material preparation technology, specifically relating to a low-immunogenicity, high-purity, and highly bioactive collagen, its preparation method, and its application. Background Technology

[0002] Collagen is a major component of the extracellular matrix, widely distributed in connective tissues such as bones, skin, and tendons in mammals, accounting for 25-30% of total mammalian protein. The molecular structure of collagen consists of a triple helix structure formed by two α1-peptide chains and one α2-peptide chain intertwined. Each peptide chain is composed of a repeating Gly-XY amino acid sequence. Type I collagen has non-helical structures flanking the Gly-XY repeat sequence, formed by two C-terminal and N-terminal propeptides, namely the C-terminal and N-terminal peptides. These C-terminal peptides can be cleaved and removed by proteases, thus preparing determinated type I collagen.

[0003] Collagen possesses excellent bioactivity, biocompatibility, and a certain degree of mechanical strength, making it widely used in the food, medical, and biological fields. However, its immunogenicity limits its application. Despite its superior biocompatibility, its uncertain immunogenicity can still trigger adverse immune responses in the human body. In the 1980s, the immunogenicity of various collagen products was measured, revealing that non-collagen components or collagen degradation products could induce immune responses. The immunogenicity of type I collagen is mainly distributed in the terminal peptide regions of the molecular chain. This can be inactivated during collagen extraction through hydrolysis or removal, producing terminal-de-peptide type I collagen and reducing its immunogenicity. Some potentially immunogenic substances in collagen, such as nucleic acids, extraneous proteins, polysaccharides, lipids, α-Gal antigens, and other small molecules, can not only reduce collagen purity but may also trigger immune responses, posing significant safety risks to human health.

[0004] Therefore, how to provide a collagen with low immunogenicity, high purity, and high bioactivity is an urgent problem to be solved in this field. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, this invention provides a low-immunogenicity, high-purity, and highly bioactive collagen, its preparation method, and its applications. The collagen prepared by the method of this invention achieves low immunogenicity, high purity, and high bioactivity while ensuring the integrity of the collagen's triple helix structure.

[0006] The technical solution of the present invention is as follows: The first aspect of this invention protects a method for preparing low-immunogenic, high-purity, and highly bioactive collagen, comprising the following steps: S1. Defatting and freezing the genetically modified pig skin to remove impurities, resulting in processed skin flakes; S2. Under the monitoring of the enzymatic hydrolysis reaction system, the treated skin sheet is enzymatically hydrolyzed with protease to obtain an enzymatically hydrolyzed collagen solution. S3. After filtering the enzymatically hydrolyzed collagen solution, the filtrate is mixed with a neutral salt solution for salting out, centrifuged, and the precipitate is reconstituted. S4. Adjust the pH of the reconstituted solution until a white turbidity appears and does not disappear. Let the reaction stand, centrifuge to collect the white precipitate and reconstitute it until a clear, transparent and viscous collagen solution is obtained. S5. The collagen solution described in S4 is subjected to microfiltration, primary ultrafiltration, and secondary ultrafiltration in sequence. S6. The collagen solution that has undergone two ultrafiltrations is concentrated and freeze-dried to obtain collagen with low immunogenicity, high purity, and high bioactivity.

[0007] Preferably, in step S1: The method for preparing the transgenic pig includes: removing the pig's heteroglycogen synthesis genes GGTAI, CMHA, and B4GaINT2 through gene editing, and introducing human complement regulatory proteins hCD46, hCDSS, and hCD59, as well as coagulation inhibitory regulatory proteins hTBM and hCD39. And / or, the removal of impurities includes: soaking in a sodium chloride solution and / or a sodium hydroxide solution for 0.5 to 2 hours, wherein the concentration of the sodium chloride solution is 1 to 2 mol / L and the concentration of the sodium hydroxide solution is 0.5 to 2 mol / L.

[0008] Preferably, in step S2: The protease includes pepsin; And / or, the protease is added in multiple portions; And / or, the total amount of the protease added is 1-2 wt% of the weight of the treated skin graft; And / or, the enzymatic hydrolysis is performed at a pH of 1.8 to 2.2, a temperature of 4 to 20°C, and a time of 48 to 72 hours; And / or, during the enzymatic hydrolysis process, the frequency of the ultrasound is 20~40Hz.

[0009] Preferably, in step S3: The filtration is performed using a 20~40μm filter element; And / or, the neutral salt includes at least one of sodium phosphate, sodium chloride, and sodium sulfate; And / or, the concentration of the neutral salt solution is 10-20 wt%; And / or, the final concentration of the neutral salt is 3-4 wt%; And / or, the salting-out time is 8-12 hours; And / or, the centrifugation speed is 5000~6000 r / min and the time is 8~10 min; And / or, dissolve the precipitate using an 8-12 mmol / L hydrochloric acid solution.

[0010] Preferably, in step S4: The pH of the reconstituted solution is adjusted using sodium hydroxide solution with a concentration of 0.5~2 mol / L. And / or, when the pH is adjusted to 6.8-7.5, a white turbidity appears and does not disappear; And / or, the reaction time is 20-30 hours; And / or, the centrifugation speed is 7000~8500 r / min, the time is 15~20 min, and the temperature is 4~10℃.

[0011] Preferably, in step S5: The microfiltration is performed through a filter element with a pore size of 5~10μm; The molecular weight cutoff of the filter membrane used in the primary ultrafiltration is 100~150 kDa; The molecular weight cutoff of the filter membrane used in the secondary ultrafiltration is 200~300 kDa.

[0012] Preferably, in step S5: The first ultrafiltration step includes: diluting the microfiltered collagen solution with a buffer solution to 2-4 times its volume, then eluting by ultrafiltration until the original volume of collagen solution is reached, and repeating the operation 3-4 times; the buffer solution for the first ultrafiltration step includes a 0.03-0.07 mol / L acetic acid solution. And / or, the secondary ultrafiltration includes: diluting the collagen solution after the first ultrafiltration to 2-4 times its volume with a buffer solution, and then eluting by ultrafiltration until the original volume of collagen solution is reached, repeating the operation 3-4 times; the buffer solution for the secondary ultrafiltration includes 0.03-0.07 mol / L citric acid solution.

[0013] Preferably, in step S6: Concentrate the light using ultrafiltration until the absorbance at 280 nm is greater than 0.22. The freeze-drying process includes pre-freezing, deep freezing, primary sublimation, secondary sublimation, and tertiary sublimation performed sequentially.

[0014] The second aspect of this invention protects a low-immunogenic, high-purity, and highly bioactive collagen, which is prepared by the method described in the first aspect.

[0015] The third aspect of this invention protects the application of a low-immunogenicity, high-purity, and highly bioactive collagen in the medical and aesthetic fields, wherein the collagen is the collagen described in the second aspect and / or collagen prepared by the preparation method described in the first aspect.

[0016] The beneficial technical effects of this invention are as follows: This invention uses transgenic pig skin as raw material, performs enzymatic hydrolysis under intelligent system monitoring, and then prepares low-immunogenic, high-purity, and highly bioactive collagen through salting out combined with isoelectric point separation technology and multi-stage purification technology. It effectively removes terminal peptide structures, DNA, α-Gal antigens, polysaccharides, and other substances that easily induce immunogenicity in humans, achieving low immunogenicity in the collagen. Simultaneously, the purity of the prepared collagen is ≥99.5%, and the content of active collagen (collagen with a complete triple helix structure) is ≥99%, achieving high purity and high bioactivity. Attached Figure Description

[0017] Figure 1 Example 1 ( Figure 1 (Left) and Comparative Example 1 ( Figure 1 (Right) Molecular weight distribution of collagen GPC obtained.

[0018] Figure 2 This is an SDS-PAGE electrophoresis image of BSA gradient concentration solutions.

[0019] Figure 3 Example 1 ( Figure 3 (Left) and Example 2 ( Figure 3 (Right) SDS-PAGE electrophoresis image of the collagen obtained.

[0020] Figure 4 Example 1 ( Figure 4 Above), Example 2 ( Figure 4 The circular dichroism chromatograms of collagen prepared below are shown in the figure. From top to bottom, they are the circular dichroism chromatograms of collagen from Example 1 and Example 2.

[0021] Figure 5 The image shows a circular dichroism chromatogram of the collagen prepared in Comparative Example 1.

[0022] Figure 6 This is a standard curve for L-hydroxyproline reference standard.

[0023] Figure 7 This is a standard curve for C-terminal peptide standards.

[0024] Figure 8 This is a standard curve for N-terminal peptide standards.

[0025] Figure 9 A standard curve for determining the number of HaCat cells using the CCK-8 reagent.

[0026] Figure 10 The cell adhesion rates are those of collagen in Example 1, collagen in Example 2, standard, and blank control.

[0027] Figure 11 The images show the cell fluorescence before and after centrifugation of collagen from Example 1, collagen from Example 2, standard, and blank control.

[0028] Figure 12 The migration of cells in the scratch area under the action of collagen in Example 1, collagen in Example 2, and blank control group.

[0029] Figure 13 Cell migration rates for collagen in Example 1, collagen in Example 2, and the blank control group. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0031] A method for preparing low-immunogenic, high-purity, and highly bioactive collagen includes the following steps: S1. Defatting and freezing the genetically modified pig skin to remove impurities, resulting in processed skin flakes; S2. Under the monitoring of the enzymatic hydrolysis reaction system, the treated skin sheet is enzymatically hydrolyzed with protease to obtain an enzymatically hydrolyzed collagen solution. S3. After filtering the enzymatically hydrolyzed collagen solution, the filtrate is mixed with a neutral salt solution for salting out, centrifuged, and the precipitate is reconstituted. S4. Adjust the pH of the reconstituted solution until a white turbidity appears and does not disappear. Let the reaction stand, centrifuge to collect the white precipitate and reconstitute it until a clear, transparent and viscous collagen solution is obtained. S5. The collagen solution described in S4 is subjected to microfiltration, primary ultrafiltration, and secondary ultrafiltration in sequence. S6. The collagen solution that has undergone two ultrafiltrations is concentrated and freeze-dried to obtain collagen with low immunogenicity, high purity, and high bioactivity.

[0032] In some implementations, the preparation of transgenic pigs includes: removing the pig's heteroglycoantigen synthesis genes GGTAI, CMHA, and B4GaINT2 through gene editing to eliminate hyperacute rejection; introducing human complement regulatory proteins hCD46, hCDSS, and hCD59 to effectively alleviate acute rejection caused by activation of the human complement system; and introducing coagulation-inhibiting regulatory proteins hTBM and hCD39 to effectively avoid rejection caused by coagulation disorders. This controls the introduction of immunogens at the source and reduces the body's immune rejection response.

[0033] It is understood that, compared to ordinary pigs, the transgenic pigs only control the introduction of immunogenicity. Using them as raw materials, combined with the preparation process of this invention, can further reduce the immunogenicity of collagen. Simultaneously, the preparation process of this invention can effectively improve the purity and content of active collagen. The two work synergistically to produce a collagen with low immunogenicity, high purity, and high bioactivity.

[0034] In the following embodiments and comparative examples of this invention, both the transgenic pig skin and the ordinary pig skin used were purchased; the transgenic pig skin was purchased from Chengdu Zhongke Aoge Biotechnology Co., Ltd.; the ordinary pig skin (conventional pig skin) was purchased from COFCO Group Co., Ltd. In the test examples, the porcine type I collagen was purchased from Aladdin Company.

[0035] In some embodiments, in step S2, the protease includes pepsin.

[0036] In some embodiments, in step S2, the total amount of protease added is 1 to 2 wt% of the weight of the treated skin graft.

[0037] In step S2, the pepsin is added in two steps to maintain its enzymatic activity and prevent it from weakening due to prolonged exposure, thereby improving the hydrolysis effect. The first addition is made at the beginning of the hydrolysis, and the second addition is made 24 hours after the hydrolysis reaction.

[0038] In some embodiments, in step S2, the pH of the enzymatic hydrolysis is 1.8 to 2.2, the temperature is 4 to 20°C, and the time is 48 to 72 hours.

[0039] Preferably, in step S2, the enzymatic hydrolysis temperature is 15~20℃; and the time is 56~66h.

[0040] In some embodiments, during step S2, the frequency of the ultrasound during the enzymatic hydrolysis is 20-40 Hz.

[0041] Understandably, in step S2, during the enzymatic hydrolysis reaction, it is necessary to monitor parameters such as pH, temperature, and frequency of ultrasound during the reaction process to ensure that the obtained collagen has a complete triple helix active structure while completely removing its terminal peptide structure.

[0042] If the pH is too low, collagen will undergo acid degradation, which will destroy the triple helix structure and increase the content of small molecule proteins and peptides in collagen, thus affecting the content of active collagen. If the pH is too high, it will also affect the enzymatic hydrolysis effect, resulting in incomplete removal of telopeptides and affecting the immunogenicity of collagen.

[0043] If the temperature is too low, it will affect the enzymatic hydrolysis effect, resulting in incomplete removal of telopeptides and affecting the immunogenicity of collagen; if the temperature is too high, it will also destroy the triple helix structure of collagen, increasing the content of small molecule protein peptides in collagen and affecting the content of active collagen.

[0044] Introducing ultrasound into the enzymatic hydrolysis process allows the hydrolysate to fully penetrate into the skin graft, promoting the enzymatic hydrolysis reaction and thus improving the hydrolysis effect and collagen extraction yield.

[0045] In step S2 of this invention, the enzymatic hydrolysis process is carried out under the monitoring of the enzymatic hydrolysis reaction system. Through an intelligent monitoring system, the enzymatic hydrolysis process achieves controllable ultrasonic-directed enzymatic digestion. During the enzymatic hydrolysis process, the system monitors indicators such as pH value, temperature, and ultrasonic frequency in real time. If any indicator exceeds the set range, the reaction equipment will automatically control the process by adding a pH adjuster (NaOH or HCl), adjusting the hot and cold water circulation, and adjusting the ultrasonic equipment, ensuring that the monitored indicators remain within the set range.

[0046] In step S5, the collagen solution described in S4 is subjected to microfiltration, primary ultrafiltration, and secondary ultrafiltration in sequence.

[0047] In some embodiments, the microfiltration is performed through a filter element with a pore size of 5-10 μm; the molecular weight cutoff of the filter membrane used for the primary ultrafiltration is 100-150 kDa; and the molecular weight cutoff of the filter membrane used for the secondary ultrafiltration is 200-300 kDa.

[0048] Through a multi-stage purification process, after microfiltration, small molecule impurities and cleaved telopeptides are first removed by primary ultrafiltration, and then incomplete triple helix collagen is removed by secondary ultrafiltration, effectively preserving collagen with complete triple helix structure.

[0049] In some embodiments, in steps S3 and S4, the precipitate resolution includes the following method: dissolving the precipitate with an 8-12 mmol / L hydrochloric acid solution.

[0050] In some embodiments, step S6 includes freeze-drying, which consists of pre-freezing, deep freezing, first sublimation, second sublimation, and third sublimation performed sequentially.

[0051] In some embodiments, in step S6: the freeze-drying conditions are: pre-freezing temperature -3~-6℃, time 2~4h; deep freezing temperature -35~-45℃, time 3~5h; first sublimation temperature 0~5℃, time 2~4h, vacuum degree 10~30Pa; second sublimation temperature 10~20℃, time 10~15h, vacuum degree 10~30Pa; third sublimation temperature 35~40℃, time 1~2h, vacuum degree 10~30Pa.

[0052] Example 1 A method for preparing low-immunogenic, high-purity, and highly bioactive collagen, comprising the following steps: S1. Use a hair remover to remove the long hairs from the surface of the transgenic pig wet skin slices, use a splitting machine to remove the epidermis and fat layer, leaving only the dermis, clean it, cut it into pieces with a slicer, and freeze it for later use. Take 1000g of frozen genetically modified pig skin, soak it in 1 mol / L NaOH solution for 1 hour, wash it several times with purified water, and spin dry. After the leather pieces have been treated with alkali, they are soaked in a 1.5 mol / L sodium chloride solution for 1 hour, then rinsed three times with purified water and dried to obtain the treated leather pieces.

[0053] S2. Under system monitoring, the treated leather pieces were immersed in 40L of 10 mmol / L hydrochloric acid solution, and 10g of pepsin was added. After 24 hours, another 5g of pepsin was added, and the pH of the solution was adjusted to 2.0. The enzymatic hydrolysis reaction was carried out at 20℃ for 60 hours to obtain an enzymatically hydrolyzed collagen solution. The ultrasonic frequency was 30 Hz. The system will monitor the pH value, temperature, ultrasonic frequency, and other indicators in real time during the enzymatic hydrolysis process. If the indicators exceed the set range, the reaction equipment will automatically control the process by adding pH adjuster (NaOH or HCl), adjusting the hot and cold water circulation, and adjusting the ultrasonic equipment to ensure that the monitored indicators are always within the set range.

[0054] S3. Filter the enzymatically hydrolyzed collagen solution through a 30 μm filter cartridge. While stirring, slowly add a 20 wt% sodium chloride solution to the filtrate, until the final sodium chloride concentration is 3.5 wt%. Allow the salting-out reaction to proceed for 10 hours. After the reaction is complete, centrifuge the collagen solution at 5500 rpm for 10 minutes, remove the upper clear solution, and collect the lower white precipitate. Then, dissolve the white precipitate in 10 mmol / L hydrochloric acid solution until a clear, transparent, and viscous collagen solution is obtained.

[0055] S4. While stirring, add 1 mol / L sodium hydroxide solution to the reconstituted collagen solution until a white turbidity appears and does not disappear. Maintain the pH of the solution at 7.0 and allow it to stand at room temperature for 25 hours. After the reaction is complete, centrifuge at low temperature: 8000 r / min for 20 min, with the temperature controlled at 8℃. Dissolve the white precipitate in 10 mmol / L hydrochloric acid solution until a clear, transparent, viscous collagen solution is obtained.

[0056] S5. Microfiltration: The completely dissolved collagen solution is filtered using a filter cartridge with a pore size of 5μm. First-stage ultrafiltration: Take the collagen solution after microfiltration, use 0.05 mol / L acetic acid as buffer, dilute it to twice the volume, and then elute it by ultrafiltration (the molecular weight cutoff of the ultrafiltration membrane is 100 kDa). When the collagen solution is eluted to the original volume, the first ultrafiltration is completed. Repeat 3 times. Secondary ultrafiltration: Take the collagen solution after the first ultrafiltration, use 0.05 mol / L citric acid as buffer, dilute it to twice the volume, and then elute it by ultrafiltration (the molecular weight cutoff of the ultrafiltration membrane is 300 kDa). When the collagen solution is eluted to its original volume, the first ultrafiltration is completed. Repeat 4 times.

[0057] S6. Take the collagen solution after two-stage ultrafiltration and concentrate it using ultrafiltration. Continuously concentrate and sample. Concentration is complete when the absorbance at 280 nm measured by a UV-Vis spectrophotometer is greater than 0.22. Freeze-dry the concentrated collagen solution using a freeze dryer under the following conditions: pre-freezing temperature -6℃ for 3 hours; deep freezing temperature -40℃ for 4 hours; first sublimation temperature 5℃ for 3 hours under a vacuum of 30 Pa; second sublimation temperature 15℃ for 15 hours under a vacuum of 20 Pa; and third sublimation temperature 38℃ for 2 hours under a vacuum of 10 Pa. After freeze-drying, collagen with low immunogenicity, high purity, and high biological activity is obtained.

[0058] Example 2 A method for preparing low-immunogenic, high-purity, and highly bioactive collagen, comprising the following steps: S1. Use a hair remover to remove the long hairs from the surface of the transgenic pig wet skin slices, use a splitting machine to remove the epidermis and fat layer, leaving only the dermis, clean it, cut it into pieces with a slicer, and freeze it for later use. Take 1000g of frozen genetically modified pig skin, soak it in 1.5 mol / L NaOH solution for 0.5h, wash it several times with purified water, and spin dry; After the leather pieces have been treated with alkali, they are soaked in a 2 mol / L sodium chloride solution for 1 hour, then rinsed three times with purified water and dried to obtain the treated leather pieces.

[0059] S2. Under system monitoring, the treated leather pieces were immersed in 40L of 10 mmol / L hydrochloric acid solution, and 10g of pepsin was added. After 24 hours, another 10g of pepsin was added, and the pH of the solution was adjusted to 1.8. The enzymatic hydrolysis reaction was carried out at 15℃ for 72 hours to obtain an enzymatically hydrolyzed collagen solution. The ultrasonic frequency was 20 Hz. The system will monitor the pH value, temperature, ultrasonic frequency, and other indicators in real time during the enzymatic hydrolysis process. If the indicators exceed the set range, the reaction equipment will automatically control the process by adding pH adjuster (NaOH or HCl), adjusting the hot and cold water circulation, and adjusting the ultrasonic equipment to ensure that the monitored indicators are always within the set range.

[0060] S3. Filter the enzymatically hydrolyzed collagen solution through a 30 μm filter cartridge. While stirring, slowly add a 20 wt% sodium chloride solution to the filtrate, until the final sodium chloride concentration is 3.5 wt%. Allow the salting-out reaction to proceed for 12 hours. After the reaction is complete, centrifuge the collagen solution at 6000 rpm for 8 minutes, remove the upper clear solution, and collect the lower white precipitate. Then, dissolve the white precipitate in 10 mmol / L hydrochloric acid solution until a clear, transparent, and viscous collagen solution is obtained.

[0061] S4. While stirring, add 1 mol / L sodium hydroxide solution to the reconstituted collagen solution until a white turbidity appears and does not disappear. Maintain the pH of the solution at 7.0 and allow it to stand at room temperature for 20-30 hours. After the reaction is complete, centrifuge at a low temperature of 7500 rpm for 15 minutes at 4°C. Dissolve the white precipitate in 10 mmol / L hydrochloric acid solution until a clear, transparent, viscous collagen solution is obtained.

[0062] S5. Microfiltration: The completely dissolved collagen solution is filtered using a filter cartridge with a pore size of 10μm. First-stage ultrafiltration: Take the collagen solution after microfiltration, use 0.05 mol / L acetic acid as buffer, dilute it to twice the volume, and then elute it by ultrafiltration (the molecular weight cutoff of the ultrafiltration membrane is 150 kDa). When the collagen solution is eluted to the original volume, the first ultrafiltration is completed. Repeat 3 times. Secondary ultrafiltration: Take the collagen solution after the first ultrafiltration, use 0.05 mol / L citric acid as buffer, dilute it to twice the volume, and then elute it by ultrafiltration (the molecular weight cutoff of the ultrafiltration membrane is 250 kDa). When the collagen solution is eluted to its original volume, the first ultrafiltration is completed. Repeat 4 times.

[0063] S6. Take the collagen solution after two-stage ultrafiltration and concentrate it using ultrafiltration. Continuously concentrate and sample. Concentration is complete when the absorbance at 280 nm measured by a UV-Vis spectrophotometer is greater than 0.22. Freeze-dry the concentrated collagen solution using a freeze dryer under the following conditions: pre-freezing temperature -4℃ for 3 hours; deep freezing temperature -35℃ for 3 hours; first sublimation temperature 0℃ for 4 hours under a vacuum of 30 Pa; second sublimation temperature 20℃ for 15 hours under a vacuum of 20 Pa; and third sublimation temperature 40℃ for 1 hour under a vacuum of 10 Pa. After freeze-drying, collagen with low immunogenicity, high purity, and high biological activity is obtained.

[0064] Comparative Example 1 A method for preparing collagen, comprising the following steps: S1. Remove the long hair from the surface of the wet pig skin slices with a hair remover, and use a splitting machine to remove the epidermis and fat layer, leaving only the dermis. After cleaning, cut the skin slices into pieces with a slicer and freeze. Take 1000g of frozen pigskin, soak it in 1 mol / L NaOH solution for 1 hour, then wash it several times with purified water and spin dry. After the leather pieces have been treated with alkali, they are soaked in a 1.5 mol / L sodium chloride solution for 1 hour, then rinsed three times with purified water and dried to obtain the treated leather pieces.

[0065] S2. The treated leather pieces were placed in 40L of 10 mmol / L hydrochloric acid solution, 15g of pepsin was added, the pH of the solution was adjusted to 2.0, and the enzymatic hydrolysis reaction was carried out at 20℃ for 60h.

[0066] S3. Filter the enzymatically hydrolyzed collagen solution through a 30μm filter cartridge. While stirring, slowly add a 20wt% sodium chloride solution to the filtrate, until the final sodium chloride concentration is 3.5wt%. Allow the salting-out reaction to proceed for 10 hours. After the reaction is complete, centrifuge the collagen solution at 6000 rpm for 8 minutes, remove the upper clear solution, and collect the lower white precipitate. Then, dissolve the white precipitate in 10 mmol / L hydrochloric acid solution until a clear, transparent, and viscous collagen solution is obtained.

[0067] S4. Microfilter the completely dissolved collagen solution using a filter cartridge with a pore size of 5μm. Take the microfiltered collagen solution and dilute it to twice its volume using 0.05mol / L acetic acid as a buffer. Then elute it by ultrafiltration (the ultrafiltration membrane has a molecular weight cutoff of 100 kDa). When the collagen solution is eluted to its original volume, one ultrafiltration is complete. Repeat 6 times.

[0068] S5. Take the collagen solution after ultrafiltration and concentrate it using ultrafiltration. Continuously concentrate and sample. Concentration is complete when the absorbance at 280 nm measured by a UV-Vis spectrophotometer is greater than 0.22. Freeze-dry the concentrated collagen solution using a freeze dryer under the following conditions: pre-freezing temperature -6℃ for 3 hours; deep freezing temperature -40℃ for 4 hours; first sublimation temperature 5℃ for 3 hours under a vacuum of 30 Pa; second sublimation temperature 15℃ for 15 hours under a vacuum of 20 Pa; and third sublimation temperature 38℃ for 2 hours under a vacuum of 10 Pa. The resulting collagen is obtained after freeze-drying.

[0069] Test case 1. Determination of collagen purity and impurities (1) Size exclusion chromatography (GPC) Mobile phase: 0.1 M phosphate buffer (pH 6.8), filtered through a 0.45 μm filter membrane and degassed before use.

[0070] Preparation of sample solution: Accurately weigh 10 mg of sample, dissolve and dilute with 10 mL of 0.5% acetic acid solution to achieve a collagen concentration of 0.5~1 mg / mL, place at 4℃ for 24 h to allow complete dissolution, filter through a 0.45 μm filter membrane, and keep the filtrate for later use.

[0071] GPC procedure: After stabilizing the baseline with the mobile phase, inject an appropriate amount of sample filtrate and analyze the sample at a flow rate of 0.5 mL / min and a column temperature of 30℃. Using thyroglobulin as a standard, determine the purity of the collagen sample according to the "size exclusion chromatography" method in the "Chromatography" section of the Pharmacopoeia of the People's Republic of China, and calculate the purity of the collagen sample using the area normalization method.

[0072] Figure 1 Example 1 ( Figure 1 (Left) and Comparative Example 1 ( Figure 1 (Right) Molecular weight distribution of collagen GPC obtained. According to... Figure 1 As can be seen, the collagen obtained in Example 1 has only one main peak (elution time approximately 10.7 min), which is collagen protein, while the remaining smaller peaks are impurities or other proteins. The purity of the collagen, calculated using the area normalization method, is 99.5%. The collagen obtained in Comparative Example 1, in addition to the main peak, also has several small impurity peaks, which are either impurities or other proteins. The purity of the collagen obtained using the area normalization method is 98.88%, lower than that of the collagen in Example 1. This demonstrates that the collagen prepared using the method of this invention can achieve higher purity.

[0073] (2) SDS-polyacrylamide gel electrophoresis The method was performed in Appendix A of YY / T 1453-2016 "Characteristics of Type I Collagen in Tissue-Engineered Medical Devices".

[0074] (a) Staining limits analysis of Coomassie Brilliant Blue for BSA Solution preparation: Dilute BSA to a gradient concentration of 0.001~0.025 mg / mL with PBS solution (pH 7.4, 0.01M).

[0075] SDS-PAGE analysis: 10 μL of each BSA solution was loaded for SDS-PAGE electrophoresis. After electrophoresis, the destained films were analyzed using a gel imaging system to determine the staining limit of Coomassie Brilliant Blue for BSA.

[0076] (b) Analysis of extraneous proteins in the sample Sample ①: Dissolve the collagen sample in 0.5% acetic acid to make the protein concentration 1 mg / mL.

[0077] Sample ②: Prepared with collagenase digestion solution (enzyme concentration of 1 mg / mL) to achieve the same protein concentration as Sample ①, and incubated in a water bath at 37 ℃ for 2-4 h.

[0078] Sample ③: Take ultrapure water and add collagenase digestion solution to make the concentration of collagenase the same as that in Sample ②.

[0079] Sample loading and analysis: Mix the above samples ①②③ with 5X SDS-PAGE protein loading buffer at a volume ratio of 4:1 (if the solution turns slightly yellow, adjust to blue with NaOH), then place in hot water at 90–100 °C for 2 min before performing SDS-PAGE electrophoresis (8% precast gel). The loading volumes of samples, BSA (using the staining limit), and molecular weight standards are all 10 μL, and the marker loading volume is 5 μL. The electrophoresis voltage is 80–110 V. After electrophoresis, remove the gel and stain it with Coomassie Brilliant Blue rapid staining solution under gentle shaking for 1–2 h. When clear blue bands are visible, remove the gel and then destain it with pure water until the gel background is transparent. Finally, analyze the destaining gel using a gel imaging analysis system and record the band optical density.

[0080] (c) Calculation of collagen purity When B-C≠0, the purity of collagen in the sample is calculated according to formula (1).

[0081] Purity (%) = {A - (B - C)} / A × 100% (1) In the formula: A—The sum of the optical densities of all bands in sample ①; B—The sum of the optical densities of all bands in sample ②; C—The sum of the optical densities of all stripes in sample ③.

[0082] When B-C=0, the purity of collagen in the sample is calculated according to formula (2).

[0083] Purity (%) = (10000 - BSA limit) / 10000 × 100% (2) (d) Calculation of miscellaneous protein content The content of extraneous proteins in the sample is calculated according to formula (3).

[0084] Impurity protein content (%) = 100% - Collagen purity (3) SDS-PAGE electrophoresis images of BSA gradient concentration solutions are shown below. Figure 2 As shown. When B-C≠0, collagen purity (%) = A-(B-C); when B-C=0, collagen purity = (10000-BSA limit) / 10000×100%, and the content of impurities in the sample (%) = 100%-collagen purity. Figure 3 Example 1 ( Figure 3 (Left) and Example 2 ( Figure 3 (Right) SDS-PAGE electrophoresis image of the collagen prepared, B-C=0, Coomassie Brilliant Blue staining limit for BSA is 5 μg / mL, BSA limit value is 50 ng. The purity of collagen in Example 1 and Example 2 was calculated to be 99.5%, and the content of impurities was 0.5%.

[0085] Purity (%) = ((10000 - 50) / 10000) × 100% = 99.5% Contrast protein content (%) = 100% - 99.5% = 0.5% 2. Determination of the content of active collagen (collagen with a complete triple helix structure) in collagen. (1) Characterization by circular dichroism Take 5 mL of acetic acid, dilute it with water to 1000 mL, and prepare a 0.5% acetic acid solution for later use.

[0086] Weigh an appropriate amount of collagen sample and dissolve it in a 0.5% (v / v) acetic acid solution to prepare a collagen solution with a concentration of 0.5 mg / mL. Place the dissolved collagen solution into a quartz cuvette and use the 0.5% acetic acid solution as a reference solution for detection on a circular dichroism spectrometer. Detection temperature: room temperature (25℃); sample cell optical path: 1 mm; scanning range: 180 ~ 260 nm; scanning interval: 0.5 nm; scanning speed: 50 nm / min.

[0087] The collagen from Examples 1, 2, and Comparative Example 1 was characterized by circular dichroism spectroscopy according to the above method.

[0088] Type I collagen's unique right-handed superhelical structure gives it a distinctive circular dichroism (CD) spectrum. Normally, a negative absorption peak around 195 nm and a positive absorption peak around 221 nm are characteristic of collagen's CD spectrum. Studies have shown that when collagen denatures or its triple-helix structure is disrupted, its CD spectrum exhibits a red shift of the negative peak and the disappearance of the positive peak. Furthermore, the intensity of the positive absorption peak can indicate the extent of the triple-helix structure.

[0089] Figure 4 , Figure 5 The images show circular dichroism chromatograms of collagen from Examples 1, 2, and 1, respectively. Figure 4 , 5 It can be seen that Examples 1, 2 and Comparative Example 1 all exhibit a typical collagen helical conformation, namely a negative absorption peak at around 195 nm and a positive absorption peak at 221 nm; indicating that the collagen prepared in the examples and comparative examples all have a stable triple helix structure.

[0090] Table 1 shows the ratio of the absolute values ​​of the positive and negative peaks of collagen in Examples 1, 2, and Comparative Example 1.

[0091] As can be seen from Table 1, the ratio of the absolute values ​​of the 221 nm positive peak and the 195 nm negative peak of collagen in Examples 1, 2, and Comparative Example 1 all meet the industry standard requirement of a positive to negative peak ratio of "0.09~0.15" in the circular dichroism chromatogram of triple-helix collagen. However, the ratio of the examples is greater than that of the comparative example and is closer to the upper limit of the industry standard requirement of "0.09~0.15". This indicates that the collagen prepared by the method of the present invention is almost entirely active collagen with a complete triple-helix structure.

[0092] (2) Content of active collagen (collagen with a complete triple helix structure) in collagen (a) Preparation of pepsin digestive solution Prepare a 0.1 mol / L acetic acid solution for later use; dissolve pepsin in the 0.1 mol / L acetic acid solution to prepare a pepsin digestion solution with an enzyme activity of 100~150 U / mL.

[0093] (b) Sample and reference preparation ① Collagen sample test solution: Weigh 10 mg of sample, add 0.1 mol / L acetic acid solution to dissolve until the collagen concentration is 1 mg / mL, and set aside.

[0094] ② Non-denatured collagen reference solution: Weigh 10 mg of porcine type I collagen reference standard, add an appropriate amount of 0.1 mol / L acetic acid solution to dissolve the collagen, and then make up the volume to 10 mL with 0.1 mol / L acetic acid solution.

[0095] ③ Denatured collagen reference solution: Take 5 mL of the non-denatured collagen reference solution prepared in ② into a capped test tube or centrifuge tube, tighten the cap, heat at 60℃ for 1 h, and immediately place on ice for 10 min for later use.

[0096] (c) Pepsin digestion Accurately pipette 500 μL each of the sample solution and the control solution into 1.5 mL capped conical centrifuge tubes. Add 100 μL of pepsin digestion solution, tighten the cap, and mix thoroughly. Set up two replicates for each sample group. Place the tubes in a constant-temperature shaker at 30 °C and shake at 100 rpm for 24 h. Remove from the shaker and immediately place in an ice bath.

[0097] (d) NaCl precipitate Accurately measure 300 μL each of the sample digest and control digest obtained in step (c) above using a pipette, and transfer them to 1.5 mL capped conical centrifuge tubes. Add 200 μL of 5 mol / L sodium chloride solution. Cap the centrifuge tubes, invert them to mix thoroughly, and shake at 200 rpm for 30 min on a shaker. Centrifuge at 20,000 × g for 20 min, and carefully remove all supernatant using a pipette, reserving the precipitate for later use. Note that during the removal of supernatant, care should be taken to avoid aspirating the precipitate as much as possible. Add 500 μL of 2 mol / L NaCl solution to the precipitate, cap the centrifuge tube, and invert it to mix thoroughly. Centrifuge at 20,000 × g for 20 min, and carefully remove all supernatant using a pipette, reserving the precipitate. Wash the precipitate once more with 2 mol / L NaCl, centrifuge, and reserve the precipitate for later use. (e) Determination of hydroxyproline content The test was performed according to Method 1 in Appendix B of YY / T 1453-2016, "Characteristics of Type I Collagen in Tissue-Engineered Medical Devices".

[0098] Accurately measure 0 mL, 1.25 mL, 2.5 mL, 5 mL, and 10 mL of 20 μg / mL L-hydroxyproline reference stock solution and place them in 10 mL volumetric flasks. Dilute to volume with water to prepare a series of L-hydroxyproline reference solutions and construct a hydroxyproline standard curve.

[0099] Add 0.5 mL of 6 mol / L hydrochloric acid to the precipitate obtained in (d), dissolve it completely, and transfer the entire solution to a hydrolysis tube. Rinse the centrifuge tube once with 0.5 mL of 6 mol / L hydrochloric acid solution, and transfer the entire washing solution to the hydrolysis tube. Seal the tube with nitrogen. Hydrolyze at 105℃ for 24 h, cool, and transfer the hydrolysis product to a volumetric flask. Wash the hydrolysis tube with water, combine the washing solutions, and transfer them to a volumetric flask. Add 2 drops of phenolphthalein indicator, and add 6 mol / L NaOH dropwise until the solution turns pink. Dilute to 5 mL with water, shake well, and the test solution is prepared.

[0100] Accurately measure 250 μL each of solutions ①②③ from (b) using a pipette, and place them separately in hydrolysis tubes. Add 0.75 mL of 8 mol / L hydrochloric acid, and seal the tubes with nitrogen. Hydrolyze at 105 ℃ for 24 h, cool, and transfer the hydrolysis products to a volumetric flask. Wash the hydrolysis tubes with water, combine the washings, and transfer them to the volumetric flask. Add 2 drops of phenolphthalein indicator, and add 6 mol / L NaOH dropwise until the solution turns pink. Dilute to 5 mL with water, shake well, and the test solution is obtained.

[0101] Accurately measure 0.5 mL of blank (water), hydroxyproline reference solutions, and the test solution, add 1 mL of isopropanol and 0.5 mL of oxidant solution respectively, mix, and let stand at room temperature for 4 min; then add 6.5 mL of colorimetric reagent to each, mix, and heat each tube in a 60 ℃ water bath for 15 min, then cool. Detect the absorbance at 560 nm using an ELISA reader. Perform linear regression analysis on the absorbance of the hydroxyproline reference solutions at various concentrations to obtain the linear regression equation, and calculate the hydroxyproline content in the test solution.

[0102] (f) Preparation of oxidizing agent pH=6.0 buffer: Weigh 57 g sodium acetate trihydrate, 37.5 g trisodium citrate, 5.5 g citric acid monohydrate, 385 mL isopropanol, add 500 mL water, adjust the pH to 6.0 with citric acid monohydrate, and dilute with water to 1000 mL; Chloramine T solution: Weigh 3.5 g chloramine T, dilute with water to 50 mL, and prepare fresh before use; Oxidizing agent solution: Mix chloramine T solution and pH=6.0 buffer solution at a ratio of 1:4.

[0103] (g) Preparation of colorimetric reagent 60% perchloric acid solution: Measure 43 mL of perchloric acid and dilute with water to 50 mL; p-Dimethylaminobenzaldehyde solution: Weigh 10 g of p-dimethylaminobenzaldehyde and dissolve it in 15 mL of 60% perchloric acid solution; Colorimetric reagent: Measure 15 mL of p-dimethylaminobenzaldehyde solution and dissolve it in 65 mL of isopropanol.

[0104] (h) Results and Calculations The total mass of hydroxyproline is calculated according to formula (1): The mass of hydroxyproline in the sample (μg) = C × V················ (1) Where: C—concentration of L-hydroxyproline in the test solution obtained from the standard curve, μg / mL; V—dilution volume, mL, which is 5 mL in this experiment. The denatured collagen content in the sample is calculated according to formula (2), and the non-denatured collagen content in the sample is calculated according to formula (3): The percentage of denatured collagen in the sample (%) = (m2-m1) / m2 × 100% (2) The content of non-denatured collagen in the sample (%) = m1 / m2 × 100% (3) Where: m1—mass of hydroxyproline in the precipitate, μg; m2—mass of hydroxyproline in 250 μL of sample test solution or reference solution, μg.

[0105] The above method is based on the following idea: Collagen with a triple helix structure can resist digestion by most proteases, while denatured collagen is easily digested by proteases. After the collagen sample is treated with proteases, the non-denatured collagen in the digestion solution is recovered by salting out. By detecting the hydroxyproline content in the sample and the recovered non-denatured collagen, the content of non-denatured and denatured collagen in the sample can be quantitatively analyzed. Figure 6 The standard curve for L-hydroxyproline was used to obtain the denatured collagen content of the collagen sample through detection and calculation.

[0106] The contents of non-denatured collagen and denatured collagen in Example 1, Example 2 and Comparative Example 1 were tested according to the above scheme, and the test results are shown in Table 2.

[0107] Table 2: Content of Undenatured and Denatured Collagen in Collagen

[0108] As shown in Table 2, the content of non-denatured collagen (active collagen) in the collagen prepared in Examples 1 and 2 is much higher than that in Comparative Example 1, while the content of deformed collagen in the collagen prepared in Examples 1 and 2 is much lower than that in Comparative Example 1. This indicates that the collagen prepared by the method of the present invention has a very high content of non-denatured collagen (active collagen), with an active collagen content greater than 99%.

[0109] 3. Evaluation of collagen immunogenicity (1) DNA residue The residual DNA content was determined according to the method specified in YY / T 0606.25-2014 "Tissue-engineered medical products - Part 25: Determination of residual DNA in animal-derived biological materials - Fluorescent staining method" using the Quant-iT™ PicoGreends DNA Assay Kit.

[0110] Protease pretreatment: 1) Take approximately 20 mg of sample for DNA extraction. Prepare 3 parallel samples.

[0111] 2) Prepare a series of recovery samples using DNA standards.

[0112] 3) As needed, mix an appropriate amount of protease with protease buffer at a volume ratio of 1:6.

[0113] 4) Add 140 μL of a protease and buffer mixture to the prepared samples and recovery samples, respectively. Incubate in a 55°C water bath until the solid matter in the reactants disappears. If the sample digestion is incomplete, increase the amount of protease and protease buffer accordingly until complete digestion.

[0114] DNA purification: Use the PrepSEQ™ Nucleic Acid Extraction Kit. 1) Add 360 μL of lysis buffer to each of the above samples, shake for 10 seconds to mix the samples completely, and let stand at room temperature for 1 hour to allow the samples to dissolve completely.

[0115] 2) Add 30 μL of magnetic beads and mix by inverting the bowl.

[0116] 3) Add 300 μL of binding solution, invert and mix well, then vortex for 5 min.

[0117] 4) Centrifuge for 15 seconds, let stand on a magnetic rack for 10 minutes, and remove the supernatant.

[0118] 5) Add 300 μL of rinsing solution, invert the container to mix, and vortex for 5 seconds.

[0119] 6) Centrifuge for 15 seconds, let stand on a magnetic rack for 5 minutes, and remove the supernatant.

[0120] 7) Repeat steps 5) and 6) once. Completely remove the supernatant.

[0121] 8) Open the lid and let it dry at room temperature for 5 minutes.

[0122] 9) Add 180 μL of elution buffer and vortex at high speed for 10 seconds.

[0123] 10) Incubate in a 70℃ bath for 7 minutes, shaking 3 times during the process.

[0124] 11) Centrifuge for 15 seconds, then let stand on a magnetic rack for 2 minutes.

[0125] 12) Transfer the supernatant to a new DNase-free centrifuge tube.

[0126] Using the above method, the amount of DNA residue in the collagen prepared in Example 1, Example 2, and Comparative Example 1 was detected, and the detection results are shown in the table below.

[0127] Table 3: DNA Residue in Collagen

[0128] As shown in Table 3, the residual DNA levels in the collagen prepared in Examples 1 and 2 were 6.6 ng / mg and 5.8 ng / mg, respectively, which were lower than the residual DNA level of 12.3 ng / mg in the collagen prepared in Comparative Example 1, and far lower than the 50-100 ng / mg in conventional decellularized matrix. This indicates that the collagen prepared using the method of the present invention can control the residual DNA level to an extremely low level.

[0129] (2) Residual amount of terminal peptides Take appropriate amounts of collagen samples from Examples 1, 2, and Comparative Example 1, and porcine type I collagen control (Aladdin), respectively. Dissolve the samples in 3% acetic acid to achieve a final collagen concentration of 1 mg / mL. Incubate at 56°C for 24 h before use. Prepare collagen C-terminal peptide standards at concentrations of 7.5, 15, 30, 60, and 90 nmol / L, and N-terminal peptide standards at concentrations of 0.75, 1.5, 3, 6, and 9 nmol / L. Detect the concentration of terminal peptides in the samples according to the ELISA kit method. The detection range is 0.3–12 nmol / L.

[0130] C-terminal peptide concentration in collagen: Based on the test results, a standard curve was plotted with the concentration of the C-terminal peptide standard on the x-axis and the absorbance at 450 nm on the y-axis (e.g., Figure 7 As shown), its equation is y = 0.0308x - 0.0194 (R²). 2 =0.9914), and then the absorbance values ​​of the collagen sample and the porcine type I collagen control were substituted into the regression equation to obtain the concentrations of the C-terminal peptides of both, as shown in Table 4.

[0131] Table 4: Results of Collagen C-terminal Peptide Concentration Detection

[0132] As shown in Table 4, the concentration of C-terminal peptides in the collagen prepared in Examples 1 and 2 of this invention is very low, below the limit of detection (3 nmol / L) of the kit, meaning that C-terminal peptides were not detected. This is significantly lower than the C-terminal peptide concentration of 4.97 ± 0.28 nmol / L in Comparative Example 1, and also lower than the C-terminal peptide concentration in commercially available porcine type I collagen. This indicates that the collagen prepared using the method of this invention has extremely low C-terminal peptide content. After enzymatic hydrolysis and the multi-stage purification process of this invention, the C-terminal peptides in the collagen are almost completely removed, and the prepared collagen exhibits obvious C-terminal peptide-free characteristics.

[0133] N-terminal peptide concentration in collagen: Based on the test results, a standard curve was plotted with the concentration of the N-terminal peptide standard on the x-axis and the absorbance at 450 nm on the y-axis (e.g., ...). Figure 8As shown in the figure, its equation is y = 0.2589x + 0.025 (R). 2 =0.9955), and then the absorbance values ​​of the collagen sample and the porcine type I collagen control were substituted into the regression equation to obtain the concentration of the N-terminal peptide of both, and the results are shown in Table 5.

[0134] Table 5: Results of N-terminal peptide concentration detection in collagen

[0135] As shown in Table 5, the N-terminal peptide concentrations of collagen in Examples 1 and 2 were very low, at 0.09 ± 0.04 nmol / L and 0.06 ± 0.03 nmol / L, respectively, significantly lower than the limit of detection (0.3 nmol / L) of the kit, meaning that N-terminal peptides were not detected. These concentrations were far lower than those in the collagen of Comparative Example 1, and also far lower than those in commercially available porcine type I collagen. This indicates that the collagen prepared using the method of this invention contains almost no N-terminal peptides, suggesting that after enzymatic hydrolysis and multi-stage purification during preparation, the N-terminal peptides of the collagen were almost completely removed, and the prepared collagen exhibited obvious N-terminal peptide-free characteristics.

[0136] (3) α-Gal antigen residue The residual α-Gal antigen in conventional pig skin, transgenic pig skin, collagen from Example 1, collagen from Example 2, and collagen from Comparative Example 1 were determined according to the test methods in YY / T1465.5-2016 "Methods for Immunogenicity Evaluation of Medical Devices Part 5: Determination of α-Gal Antigen Clearance Rate in Animal-Derived Medical Devices Using M86 Antibody" and YY / T1561-2017 "Detection of Residual α-Gal Antigen in Animal-Derived Scaffold Materials of Tissue-Engineered Medical Devices". The test results are shown in Table 6.

[0137] Table 6: Number of α-Gal antigen epitopes in collagen from conventional pigskin, transgenic pigskin, examples, and comparative examples

[0138] As shown in Table 6, the number of α-Gal antigen epitopes in regular pig skin is 3.63 × 10⁻⁶. 14 The number of α-Gal antigen epitopes in transgenic pig skin was 2.95 × 10⁻⁶. 13 Compared to conventional pig skin, the expression level of α-Gal antigen in transgenic pig skin decreased by 92%, indicating that the transgenic pig skin of this invention can effectively control the introduction of α-Gal antigen.

[0139] It can also be seen that the number of α-Gal antigen epitopes in Examples 1 and 2 are 0.27 × 10⁻⁶, respectively. 13 0.31×10 13This indicates that the present invention further removes α-Gal antigen during the collagen preparation process, resulting in a significant reduction in the number of α-Gal antigen epitopes in the prepared collagen compared to transgenic pig skin. In Example 1, the α-Gal removal rate was 90.8%. This is in contrast to the α-Gal antigen epitope number of 3.38 × 10⁻⁶ in Comparative Example 1. 13 In this invention, the α-Gal antigen in the collagen prepared has been basically removed, and the residual amount can be controlled at an extremely low level.

[0140] 4. Bioactivity assay of collagen (1) Cell adhesion (centrifugation method) The detection was performed according to the method in Appendix B of YY / T 1849-2022 "Recombinant Collagen".

[0141] a) Weigh an appropriate amount of collagen solid and dissolve it in 0.1 mol / L acetic acid solution to 1 mg / mL. After the solid is completely dissolved, dilute the solution with pure water to 10 μg / mL to obtain the sample test solution for later use.

[0142] b) In a 96-well adherent cell culture plate, add 100 μL of pure water (blank control), standard (positive control), and sample test solution, respectively, and incubate at room temperature for 1 h. Set up six replicates for each sample. Discard the supernatant and wash with 200 μL of PBS. After discarding the supernatant, block with 100 μL of 1% BSA in PBS solution and incubate at room temperature for 1 h. Discard the supernatant, wash twice with 200 μL of PBS, discard the supernatant again, and set aside for use. Store the coated cell culture plate at room temperature until use, and use it on the same day.

[0143] c) Harvest HaCaT cells that have been stably passaged more than twice, when the cell confluence is approximately 80-95%, using 0.25% trypsin-EDTA digestion. Resuspend the cells in DMEM containing 2% fetal porcine serum, count them, and adjust the cell density to 102. 5 Cells / mL were mixed with CCK8 reagent at a ratio of 10:1, and 110 μL of cell suspension was added to each well. The 96-well plate was placed in a cell culture incubator and incubated for 1 h. The readings were taken at 450 nm using a microplate reader.

[0144] d) After 1 h of incubation, add approximately 310 μL of DMEM medium to each well until the liquid level rises. Seal the plate using a sealing film (ensure no air bubbles are generated after sealing; otherwise, add DMEM medium and reseal). Invert the plate and centrifuge for 5 min at 600 rpm. The appropriate centrifugation force can be determined by the ratio of the adhesion percentage of the positive control to that of the blank control; generally, the ratio should be around 2. After centrifugation, remove the sealing film, discard the original medium, wash with 200 μL of PBS, add fresh medium mixed with CCK8 reagent, and incubate at 37°C for 1 h. Then, read the values ​​at 450 nm using a microplate reader.

[0145] e) Cell fluorescence images can be obtained by adding Hoechst 33342 at a final concentration of 1 μg / mL. During the operation, care should be taken to avoid light to obtain cell fluorescence images covering the entire well area.

[0146] f) Data Calculation Calculate the adhesion percentage using formula (1) and the relative cell adhesion ratio using formula (2).

[0147] V = NB / NA×100%·················(1) In the formula: V—Percentage of adhesion, % NB—Number of cells after centrifugation; NA—Number of cells before centrifugation, in units.

[0148] P = V1 / V2···············(2) In the formula: P—Relative cell adhesion ratio; V1—The average adhesion percentage of each replicate well in the sample or positive control, % V2—Average adhesion percentage of each duplicate well in the blank control, %.

[0149] HaCat cells were cultured in collagen-coated culture dishes for 1 h. Unattached cells were eluted by centrifugation at 600 rpm. The number of adherent cells before and after centrifugation was determined using CCK-8 reagent (standard curve shown). Figure 9 As shown, R 2 It reached 0.9999.

[0150] Figure 10 The cell adhesion rates were measured in collagen from Example 1, collagen from Example 2, porcine type I collagen (standard), and a blank control (pure water). Based on... Figure 10As can be seen, the cell adhesion percentages of collagen in Examples 1 and 2 reached 67.3% and 57.9%, respectively, which were significantly higher than 8.5% of the blank control group and 48.0% of porcine type I collagen (standard), indicating that the collagen prepared by this invention has excellent cell adhesion-promoting properties.

[0151] Figure 11 Cell fluorescence images before and after centrifugation of collagen from Example 1, collagen from Example 2, standard (porcine type I collagen), and pure water (blank control). Based on... Figure 11 It can be seen that, compared with before centrifugation, the number of cells in the blank control group was significantly reduced after centrifugation, and the number of cells in the standard group was also reduced; while the number of cells in the collagen groups of Example 1 and Example 2 was not significantly reduced, further demonstrating that the collagen prepared by the present invention can promote cell adhesion.

[0152] (2) Cell migration (cell scratch assay) The detection was performed according to the method in Appendix C of YY / T 1849-2022 "Recombinant Collagen".

[0153] a) Cell culture: Place sterilized ibidi scratch inhibitors into 24-well plates, seeding approximately 5 × 10⁶ cells per inhibitor. 5 Cells. After overnight culture, remove the insert and observe whether the cells have reached 95-100% confluence. Each group should have at least 3 replicate wells.

[0154] b) Weigh an appropriate amount of collagen solid and dissolve it in 0.1 mol / L acetic acid (HAc) solution to a concentration of 5 mg / mL. After the solid is completely dissolved, dilute the solution to 0.5 mg / mL using serum-free culture medium. This solution will be used as the test solution for later use.

[0155] c) Scratch test: 1 mL of serum-free culture medium was added to the collagen groups of Example 1 and Example 2, respectively, to achieve a final collagen concentration of 0.5 mg / mL. The blank control group (Control) was treated with only serum-free culture medium. The mixtures were incubated at 37 °C in a 5% CO2 incubator, and photographs were taken under a 4× microscope at 0 h, 6 h, and 12 h.

[0156] d) Data processing: Measure the area of ​​the scratched region and calculate the cell migration rate for each group by dividing the total area of ​​migrating cells in the scratched region by the initial area of ​​the fixed scratched region.

[0157] like Figure 12 As shown, the cell scratch region was prepared using the ibidi scratch plugin, photographed under a microscope, and the area of ​​the scratch region in the photograph was measured. The cell migration rate was calculated by dividing the total area of ​​migrating cells in the scratch region by the initial area of ​​the fixed scratch region. Figure 13Cell migration rates were calculated for collagen in Example 1, collagen in Example 2, and the blank control group. Based on... Figure 13 As can be seen, at 6 h, cells cultured with 0.5% collagen solution showed good migration. The cell migration rates of the sample groups in Example 1 and Example 2 reached 32.78% and 28.28%, respectively, which were significantly higher than the 5.49% migration rate of the blank control group (Control group), indicating a significant difference between the sample groups and the Control group. With prolonged culture time, cells gradually grew to cover the scratched area, and the cell migration rate increased. At 12 h, the cell migration rates of the sample groups in Example 1 and Example 2 reached 71.13% and 65.59%, respectively, while the cell migration rate of the Control group reached 42.21%, with a still significant difference between the sample groups and the Control group. That is, at both 6 h and 12 h, the cell migration rates of the sample groups were significantly higher than those of the Control group, indicating that the collagen prepared in this invention has a cell migration-promoting effect.

[0158] In summary, the collagen prepared by this invention using transgenic pig skin as raw material through ultrasonically controlled directional enzymatic digestion, salting out combined with isoelectric point separation, and multi-stage purification techniques achieves a purity of 99.5% and a contaminating protein content of 0.5%. The residual DNA content is 5.8 ng / mg, significantly lower than that of conventionally extracted and purified collagen. The concentrations of C- and N-terminal peptides are far below the detection limits of the kit, meaning that C- and N-terminal peptides were not detected. The α-Gal antigen epitope number is 0.27 × 10⁻⁶. 13 This invention demonstrates that the method can effectively remove terminal peptides, α-Gal antigens, and DNA from collagen, significantly reducing its immunogenicity. Furthermore, the prepared collagen promotes cell adhesion and migration.

[0159] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A method for preparing low-immunogenicity, high-purity, and highly bioactive collagen, characterized in that, Includes the following steps: S1. Defatting and freezing the genetically modified pig skin to remove impurities, resulting in processed skin flakes; S2. Under the monitoring of the enzymatic hydrolysis reaction system, the treated skin sheet is enzymatically hydrolyzed with protease to obtain an enzymatically hydrolyzed collagen solution. S3. After filtering the enzymatically hydrolyzed collagen solution, the filtrate is mixed with a neutral salt solution for salting out, centrifuged, and the precipitate is reconstituted. S4. Adjust the pH of the reconstituted solution until a white turbidity appears and does not disappear. Let the reaction stand, centrifuge to collect the white precipitate and reconstitute it until a clear, transparent and viscous collagen solution is obtained. S5. The collagen solution described in S4 is subjected to microfiltration, primary ultrafiltration, and secondary ultrafiltration in sequence. S6. The collagen solution that has undergone two ultrafiltrations is concentrated and freeze-dried to obtain collagen with low immunogenicity, high purity, and high bioactivity.

2. The preparation method according to claim 1, characterized in that, In step S1: The method for preparing the transgenic pig includes: removing the pig's heteroglycogen synthesis genes GGTAI, CMHA, and B4GaINT2 through gene editing, and introducing human complement regulatory proteins hCD46, hCDSS, and hCD59, as well as coagulation inhibitory regulatory proteins hTBM and hCD39. And / or, the removal of impurities includes: soaking in a sodium chloride solution and / or a sodium hydroxide solution for 0.5 to 2 hours, wherein the concentration of the sodium chloride solution is 1 to 2 mol / L and the concentration of the sodium hydroxide solution is 0.5 to 2 mol / L.

3. The preparation method according to claim 1, characterized in that, In step S2: The protease includes pepsin; And / or, the protease is added in multiple portions; And / or, the total amount of the protease added is 1-2 wt% of the weight of the treated skin graft; And / or, the enzymatic hydrolysis is performed at a pH of 1.8 to 2.2, a temperature of 4 to 20°C, and a time of 48 to 72 hours; And / or, during the enzymatic hydrolysis process, the frequency of the ultrasound is 20~40Hz.

4. The preparation method according to claim 1, characterized in that, In step S3: The filtration is performed using a 20~40μm filter element; And / or, the neutral salt includes at least one of sodium phosphate, sodium chloride, and sodium sulfate; And / or, the concentration of the neutral salt solution is 10-20 wt%; And / or, the final concentration of the neutral salt is 3-4 wt%; And / or, the salting-out time is 8-12 hours; And / or, the centrifugation speed is 5000~6000 r / min and the time is 8~10 min; And / or, dissolve the precipitate using an 8-12 mmol / L hydrochloric acid solution.

5. The preparation method according to claim 1, characterized in that, In step S4: The pH of the reconstituted solution is adjusted using sodium hydroxide solution with a concentration of 0.5~2 mol / L. And / or, when the pH is adjusted to 6.8-7.5, a white turbidity appears and does not disappear; And / or, the reaction time is 20-30 hours; And / or, the centrifugation speed is 7000~8500 r / min, the time is 15~20 min, and the temperature is 4~10℃.

6. The preparation method according to claim 1, characterized in that, In step S5: The microfiltration is performed through a filter element with a pore size of 5~10μm; The molecular weight cutoff of the filter membrane used in the primary ultrafiltration is 100~150 kDa; The molecular weight cutoff of the filter membrane used in the secondary ultrafiltration is 200~300 kDa.

7. The preparation method according to claim 1 or 6, characterized in that, In step S5: The first ultrafiltration step includes: diluting the microfiltered collagen solution with a buffer solution to 2-4 times its volume, then eluting by ultrafiltration until the original volume of collagen solution is reached, and repeating the operation 3-4 times; the buffer solution for the first ultrafiltration step includes a 0.03-0.07 mol / L acetic acid solution. And / or, the secondary ultrafiltration includes: diluting the collagen solution after the first ultrafiltration to 2-4 times its volume with a buffer solution, and then eluting by ultrafiltration until the original volume of collagen solution is reached, repeating the operation 3-4 times; the buffer solution for the secondary ultrafiltration includes 0.03-0.07 mol / L citric acid solution.

8. The preparation method according to claim 1, characterized in that, In step S6: Concentrate the light using ultrafiltration until the absorbance at 280 nm is greater than 0.

22. The freeze-drying process includes pre-freezing, deep freezing, primary sublimation, secondary sublimation, and tertiary sublimation performed sequentially.

9. A low-immunogenic, high-purity, and highly bioactive collagen, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 8.

10. The application of a low-immunogenic, high-purity, and highly bioactive collagen in the medical and aesthetic fields, characterized in that... The collagen is the collagen of claim 9, and / or the collagen prepared by any one of the preparation methods of claims 1 to 8.

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