Tibetan sheep embryo III type collagen as well as preparation method and application thereof

Abstract

The invention relates to the field of biomedical materials, in particular to Tibetan sheep embryo type III collagen as well as a preparation method and application of the Tibetan sheep embryo type III collagen. Comprising the following steps: sequentially carrying out cation exchange chromatography purification and gel chromatography purification on an extracting solution which is extracted from a Tibetan sheep embryo and contains type III collagen; in the cation exchange chromatography purification step, a sample is loaded in an acetic acid buffer solution with the pH value of 2.5-3.5 and the concentration of 0.4-0.6 M, an eluent is a sodium chloride solution, and gradient elution is carried out within the eluent concentration range of 0-1 mol / L; in the step of gel chromatography purification, an eluent is a phosphate buffer solution with the pH value of 7.2-7.4, and the concentration of the eluent is 0.01-0.1 M; type III collagen with a complete triple helix structure and high biological activity is prepared from Tibetan sheep embryonic tissue, has the properties of good biocompatibility, good thermal stability, promotion of cell proliferation, adhesion and migration and the like, and has high medical value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomaterials, in particular to a Tibetan sheep embryo type III collagen and a preparation method and application thereof. BACKGROUND

[0002] Collagen is a kind of protein existing in all mammals, and is almost distributed in various organs of mammals, which mainly plays a supporting and protective role in the animal body. Among them, type III collagen is the main structural component of hollow organs, and is a type of collagen mainly existing in human skin, fascia and tendon, and the proportion of type III collagen to type I collagen is about 1 / 4. Type III collagen belongs to fibrous collagen, which is composed of a collagen alpha chain, and is a homotrimer containing three alpha 1((III) chains. These chains are supercoiled with each other in a right-handed triple helix, showing a unique triple helix structure, and this structure is the key factor for type III collagen to have good biological function.

[0003] As a kind of "baby collagen", type III collagen is mainly distributed in the form of a network around type I collagen at the junction of epidermis and dermis. It is relatively small in size and has a loose silk screen shape, and the alpha 1((III) 3 forms a triple helix domain, which mainly endows the skin with elasticity and stress resistance. Type III collagen can also regulate the synthesis of type I collagen and accelerate the formation of collagen fibrils, and has excellent elasticity and repair function. In addition, it also plays an important role in promoting wound healing and reducing scar.

[0004] Sheep embryo is a by-product in the production process of sheep placenta, a substitute for traditional Chinese medicine "Ziheche". Sheep placenta is derived from the fetal membranes of sheep embryos in the middle and late stages of pregnancy. After the fetal membranes are stripped, the embryo is usually discarded as waste, which not only causes environmental pollution, but also leads to serious resource waste. Similar to the human body, animal embryos and infants have a high content of type III collagen, which is suitable for extracting and preparing active collagen. Due to the placental barrier, most of the zoonoses cannot be vertically transmitted, so the probability of collagen product contamination by pathogens is very low. Therefore, compared with collagen from other sources, Tibetan sheep embryo collagen has significant advantages.

[0005] The prior art discloses isolation and purification of type III collagen from animal tissues, and the main steps include: degreasing, impurity removal, enzyme cutting, salting-out, desalting and the like. However, due to the differences in species, parts, fiber strength, mechanical strength, content and associated collagen separation problems of type III collagen, the current method for isolating and purifying type III collagen from animal tissues is not suitable for extracting type III collagen from Tibetan sheep embryos. In the extraction process, degradation and denaturation are prone to occur, it is difficult to maintain the natural and non-denatured triple helix structure of collagen, and finally the purity and yield of the active Tibetan sheep embryo type III collagen obtained are low. SUMMARY

[0006] Therefore, the technical problem to be solved by the present application is to provide a Tibetan sheep embryo type III collagen and a preparation method and use thereof. The type III collagen prepared from Tibetan sheep embryo tissues has a complete triple helix structure and high biological activity, has a low extraction cost, has good biocompatibility, good thermal stability, promotes cell proliferation, adhesion and migration, and has high medical value.

[0007] To this end, the present application provides the following technical solutions:

[0008] The preparation method of the Tibetan sheep embryo type III collagen provided by the embodiments of the present application comprises: sequentially subjecting an extract containing type III collagen extracted from a Tibetan sheep embryo to cation exchange chromatography purification and gel chromatography purification.

[0009] In the cation exchange chromatography purification step, the sample is equilibrated in an acetic acid buffer solution with a pH of 2.5-3.5 and a concentration of 0.4-0.6 M, and the eluent is a sodium chloride solution, and gradient elution is performed within a concentration range of 0-1 mol / L.

[0010] In the gel chromatography purification step, the eluent is a phosphate buffer solution with a pH of 7.2-7.4 and a concentration of 0.01-0.1 M.

[0011] In some embodiments, in the cation exchange chromatography purification step, the pH value can be any one of 2.5, 2.8, 3.0, 3.3 or 3.5 or a range value between any two of them. The concentration of the acetic acid buffer solution can be any one of 0.4, 0.45, 0.5, 0.55, 0.6 M or a range value between any two of them.

[0012] In some embodiments, in the gel chromatography purification step, the pH of the eluent is 7.2, 7.3 or 7.4, and the concentration of the phosphate buffer solution in the eluent is any one of 0.01, 0.03, 0.05, 0.07, 0.1 M or a range value between any two of them.

[0013] In some embodiments, the cation exchange chromatography purification step comprises at least one of the following:

[0014] (1) the gradient elution condition is gradient elution using sodium chloride solutions with concentrations of 0 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.5 mol / L, and 1 mol / L, in sequence;

[0015] (2) the effluent with a concentration of 0.1 mol / L to 0.3 mol / L is collected;

[0016] (3) the elution rate of the eluent with different concentrations is 1.0 to 2.0 mL / min;

[0017] (4) the cation exchange chromatography uses SP Sepharose HP ion exchange chromatography;

[0018] (5) the pressure is not higher than 0.3 MPa;

[0019] (6) the sample loading amount is 75-150 mg / mL column volume.

[0020] In some embodiments, the elution rate of the eluent with different concentrations can be 1.0, 1.5, or 2.0 mL / min.

[0021] In some embodiments, the sample loading amount is any one of 75, 85, 95, 100, 120, 135, 150 mg / mL column volume, or a range value between any two of the values.

[0022] In some embodiments, the gel chromatography purification step comprises at least one of the following:

[0023] (1) the effluent from 10-20 min is collected;

[0024] (2) the flow rate of the eluent during the elution process is 0.25-0.5 mL / min;

[0025] (3) the filler of the gel chromatography uses Sephadex G-200 gel chromatography;

[0026] (4) the pressure is not higher than 0.15 MPa;

[0027] (5) the sample loading amount is 1%-5% of the column volume.

[0028] In some embodiments, the flow rate of the eluent in the elution process can be any one of 0.25, 0.3, 0.4, 0.5 ml / min or a range value between any two of them.

[0029] In some embodiments, the loading amount is any one of 1%, 2%, 4%, or 5% of the column volume or a range value between any two of them.

[0030] In some embodiments, the preparation step of the collagen type III-containing extract solution extracted from Tibetan sheep embryos includes at least one of pretreatment, degreasing, impurity removal, enzyme cutting, or salting-out.

[0031] In some embodiments, after the gel chromatography purification, a desalination and concentration step is further included.

[0032] In some embodiments, in the salting-out step, a mixed solution of sodium chloride and ammonium sulfate is added to the sample for salting-out, wherein the final concentration of ammonium sulfate is 0.5-1 mol / L, and the final concentration of sodium chloride is 0-1 mol / L. In some embodiments, the final concentration of ammonium sulfate can be any one of 0.5, 0.7, 0.9, 1.0 mol / L or a range value between any two of them. The final concentration of sodium chloride is any one of 0, 0.3, 0.6, 1.0 mol / L or a range value between any two of them.

[0033] In some embodiments, in the salting-out step, the salting-out time is 12-24 hours, such as any one of 12, 15, 18, 20, 22, 24 h or a range value between any two of them.

[0034] In some embodiments, in the salting-out step, after the salting-out is completed, a step of resolubilizing and dialyzing the obtained precipitate is further included.

[0035] In some embodiments, the resolubilizing solution is an acetic acid solution with a concentration of 0.4-0.6 mol / L, such as an acetic acid solution with a concentration of 0.4, 0.5, or 0.6 mol / L. The ratio of the precipitate to the resolubilizing solution is 1-5 mg:5-10 ml, and the ratio of the precipitate to the resolubilizing solution can be any one of 1 mg:10 ml, 5 mg:5 ml, 3 mg:8 ml, 2 mg:7 ml or a range value between any two of them. After resolubilization, a collagen resolubilizing solution is obtained.

[0036] In some embodiments, in the dialysis step, the collagen resolubilizing solution and 0.2-0.3 M acetic acid solution are dialyzed at a volume ratio of 1:10-15 for 12-24 hours, and then ultra-pure water is used for dialysis, and the collagen resolubilizing solution and the ultra-pure water are dialyzed at a ratio of 1:10-1:15 for 3 days, and the ultra-pure water is replaced multiple times during the dialysis.

[0037] In some embodiments, the collagen solution is dialyzed against 0.2-0.3 M acetic acid solution at a volume ratio of 1:10, 1:13 or 1:15, and the dialysis time can be any one of 12, 15, 18, 20, 22, 24 h or a range between any two of them.

[0038] In some embodiments, the collagen solution is dialyzed against ultrapure water at a volume ratio of 1:10, 1:13 or 1:15, and the dialysis time is 1, 2 or 3 days.

[0039] In some embodiments, in the pretreatment step, the sample of Tibetan sheep embryo tissue is rinsed to remove residual blood and impurities, and then dehydrated and cut into pieces.

[0040] In some embodiments, in the defatting step,

[0041] The sample and the defatting solution are mixed at a mass-volume ratio of 1:5-1:10, and the sample is soaked in the defatting solution for 2-5 hours, with the defatting solution being replaced every 1-2.5 hours; the defatting solution contains 10 v / v% butanol and 50 v / v% ethanol in water.

[0042] In some embodiments, in the defatting step, the sample and the defatting solution can be mixed at a mass-volume ratio of any one of 1:5, 1:7, 1:9, 1:10 or a range between any two of them. The soaking time can be any one of 2, 3, 4, 5 hours or a range between any two of them.

[0043] In some embodiments, in the impurity removal step, the sample and the sodium hydroxide solution are mixed at a mass-volume ratio of 1:5-1:10, and the sample is soaked in the NaOH solution with a concentration of 0.1-0.2 mol / L for 2-5 hours to remove non-collagen proteins, with the basic solution being replaced every 1-2.5 hours; the residual sodium hydroxide solution is removed by repeated washing with ultrapure water until the pH of the washing water shows neutrality.

[0044] In some embodiments, in the impurity removal step, the sample and the sodium hydroxide solution are mixed at a mass-volume ratio of 1:5, 1:7, 1:9 or 1:10.

[0045] In some embodiments, in the enzyme digestion step, the sample is first homogenized, then mixed with the enzyme solution at a ratio of 1:3-1:5 (w / v) and stirred for extraction for 48-72 hours, and then centrifuged to obtain the supernatant; the enzyme solution contains 1-5% (w / v) pepsin and / or 0.1-0.3% (w / v) trypsin, and the solvent is 0.4-0.6 M acetic acid solution.

[0046] In some embodiments, in the enzymatic digestion step, the sample is mixed with the enzyme solution at a ratio of 1:3, 1:4 or 1:5 (w / v), and the extraction time can be any one of 48, 54, 60, 66 or 72 hours or a range between any two of them. The enzyme solution is a solution containing 1, 2, 3, 4 or 5% (w / v) pepsin, or the enzyme solution is a solution containing 0.1, 0.2 or 0.3% (w / v) trypsin, or the enzyme solution contains 1, 2, 3, 4 or 5% (w / v) pepsin and a solution containing 0.1, 0.2 or 0.3% (w / v) trypsin.

[0047] The present application provides a Tibetan sheep embryo collagen type III prepared by the method.

[0048] The present application provides a Tibetan sheep embryo collagen type III prepared by the method or the use of the Tibetan sheep embryo collagen type III in any one of the following:

[0049] 1) promoting cell proliferation;

[0050] 2) promoting cell adhesion;

[0051] 3) promoting cell migration;

[0052] 4) use in cosmetics;

[0053] 5) use in medical devices;

[0054] 6) use in pharmaceuticals;

[0055] 7) use in tissue engineering and regenerative medicine;

[0056] 8) use in drug delivery carriers;

[0057] 9) use in biomaterials;

[0058] 10) use in food and health products.

[0059] The technical scheme of the present application has the following advantages:

[0060] 1.The preparation method of Tibetan sheep embryo type III collagen provided by the present application, comprising: sequentially performing cation exchange chromatography purification and gel chromatography purification on the extract containing type III collagen extracted from the Tibetan sheep embryo; in the cation exchange chromatography purification step, the column is equilibrated with an acetic acid buffer solution with a pH of 2.5-3.5 and a concentration of 0.4-0.6 M, and then the sample is loaded, and the eluent is a sodium chloride solution, and gradient elution is performed within the concentration range of 0-1 mol / L of the eluent; in the gel chromatography purification step, the eluent is a phosphate buffer solution with a pH of 7.2-7.4 and a concentration of 0.01-0.1 M; the present application finds that after salting-out, the cation exchange chromatography purification is first selected, the column is equilibrated with an acetic acid buffer solution with a pH of 2.5-3.5 and a concentration of 0.4-0.6 M, and then the sample is loaded, and gradient elution is performed with a sodium chloride solution within the concentration range of 0-1 mol / L, which can obtain type III collagen with high purity and high yield on the one hand, and can maintain the natural and non-denatured triple helix structure of type III collagen on the other hand, thereby ensuring the biological activity, avoiding the denaturation and aggregation of type III collagen, and affecting the subsequent gel chromatography purification. Meanwhile, the gel chromatography purification is performed with a phosphate buffer solution with a pH of 7.2-7.4 as the eluent. The eluent matches the pH value of the buffer solution in the cation exchange chromatography purification step and the gradient elution conditions, has appropriate ionic strength and pH value, and can effectively maintain the uniformity of the elution environment, thereby ensuring the stability and efficiency of the entire purification process, and improving the recovery rate of the target protein.

[0061] Further, the Tibetan sheep embryo tissue can be used to prepare type III collagen with complete triple helix structure and high biological activity, which has low extraction cost, good biocompatibility, good thermal stability, cell proliferation, adhesion, migration and other properties, high medical value, and can be applied to the fields of cosmetics, medical devices, drugs, tissue engineering and regenerative medicine, drug delivery carriers, biological materials, food and health products, etc.

[0062] 2.The preparation method of Tibetan sheep embryo type III collagen provided by the present application, in the salting-out step, a mixed solution of sodium chloride and ammonium sulfate is added to the sample for salting-out, wherein the final concentration of ammonium sulfate is 0.5-1 mol / L, and the final concentration of sodium chloride is 0-1 mol / L; the present application finds that in the salting-out step, the conventional sodium chloride salt solution is used to salt out the Tibetan sheep embryo type III collagen, which is easy to cause the denaturation and aggregation of type III collagen, and further affects the subsequent cation exchange chromatography purification, while the present application adds the ammonium sulfate solution with the selected concentration range to the salt solution within the above concentration range, which can salt out a large amount of Tibetan sheep embryo type III collagen precipitate, and can also avoid the denaturation and aggregation of the type III collagen, so that the cation exchange chromatography purification can be smoothly performed after the reconstitution, the exchange column is not easy to be blocked, and the target product with high purity, high yield and activity can be obtained.

[0063] 3. The application provides a preparation method of Tibetan sheep embryo type III collagen, and the defatting solution contains 10% butanol and 50% ethanol aqueous solution; the application finds that the conventional 5-30% butanol solution is used for defatting the Tibetan sheep embryo tissue, which easily affects the complete triple helix structure of the Tibetan sheep embryo type III collagen and affects the activity, and the application adds the selected concentration of the ethanol solution on the basis of the selected concentration of the butanol solution for compounding, so that the complete triple helix structure of the Tibetan sheep embryo type III collagen is maintained in the defatting process, and the biological activity is ensured. BRIEF DESCRIPTION OF DRAWINGS

[0064] In order to more clearly illustrate the specific embodiments of the application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the specific embodiments or the prior art description. Obviously, the drawings described below are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0065] Figure 1 Figure is the observation diagram of the Tibetan sheep embryo type III collagen solution prepared in the embodiment 1 of the application; a is the type III collagen solution prepared by crude extraction, and b is the type III collagen solution prepared by purification;

[0066] Figure 2 Figure is the freeze-dried sample of the Tibetan sheep embryo type III collagen prepared in the embodiment 1 of the application; a is the freeze-dried sample of the type III collagen solution prepared by crude extraction, and b is the freeze-dried sample of the type III collagen solution prepared by purification;

[0067] Figure 3 Figure is the electrophoresis diagram of the Tibetan sheep embryo type III collagen prepared in the embodiment 1 of the application; lanes 1-2 are the bands corresponding to the collagen crude extraction mixture, and lanes 3-4 are the bands corresponding to the collagen mixture after salting-out;

[0068] Figure 4 Figure is the electrophoresis diagram of the Tibetan sheep embryo type III collagen prepared in the embodiment 1 of the application; lanes 1-5 are all the bands of the collagen solution finally obtained in the step (8);

[0069] Figure 5 Figure is the liquid chromatography-mass spectrometry diagram of the unique peptide segment amino acid sequence GSPGPQGLKGENGKPGPSGQNGER of the type III collagen obtained by the liquid chromatography-mass spectrometry technology of the Tibetan sheep embryo type III collagen prepared in the embodiment 1 of the application;

[0070] Figure 6The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment amino acid sequence NGDRGETGPAGPSGAPGPAGSR of the collagen type III protein prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by liquid chromatography-mass spectrometry technology;

[0071] Figure 7 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment amino acid sequence GSPGPQGIKGENGKPGPSGQNGER of the collagen type III protein prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by liquid chromatography-mass spectrometry technology;

[0072] Figure 8 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment amino acid sequence GAPGPQGPRGDKGETGER of the collagen type III protein prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by liquid chromatography-mass spectrometry technology;

[0073] Figure 9 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment amino acid sequence GDRGETGPAGPPGAPGAPGAPGPVGPAGK of the collagen type III protein prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by liquid chromatography-mass spectrometry technology;

[0074] Figure 10 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment amino acid sequence GERGFPGERGVQGPPGPAGPR of the collagen type III protein prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by liquid chromatography-mass spectrometry technology;

[0075] Figure 11 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment amino acid sequence GEPGPTGIQGPPGPAGEEGKR of the collagen type III protein prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by liquid chromatography-mass spectrometry technology;

[0076] Figure 12 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment amino acid sequence GERGPPGESGAAGPTGPIGSR of the collagen type III protein prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by liquid chromatography-mass spectrometry technology;

[0077] Figure 13 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment amino acid sequence GPTGDPGKAGEKGHAGLAGPR of the collagen type III protein prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by liquid chromatography-mass spectrometry technology;

[0078] Figure 14The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment amino acid sequence HGSRGEPGPVGAVGPAGAVGPR of the collagen type III prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by liquid chromatography-mass spectrometry technology;

[0079] Figure 15 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment amino acid sequence GPVGPSGPPGK of the collagen type III prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by liquid chromatography-mass spectrometry technology;

[0080] Figure 16 The ultraviolet absorption spectrum diagram of the collagen type III prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by ultraviolet absorption spectrum technology;

[0081] Figure 17 The Fourier infrared spectrum diagram of the collagen type III prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by Fourier infrared spectrum technology;

[0082] Figure 18 The amide I region secondary structure absorption peak of the collagen type III prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by infrared spectrum technology;

[0083] Figure 19 The relative content of the secondary structure of the collagen type III prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by infrared spectrum technology;

[0084] Figure 20 The scanning electron microscope diagram of the collagen type III prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by scanning electron microscope technology;

[0085] Figure 21 The Raman spectrum diagram of the collagen type III prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by Raman spectrum technology;

[0086] Figure 22 The circular dichroism spectrum diagram of the collagen type III prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by circular dichroism spectrum technology;

[0087] Figure 23 The circular secondary structure content of the collagen type III prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by circular dichroism spectrum technology;

[0088] Figure 24 The differential scanning calorimetry result of the collagen type III prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by differential scanning calorimetry technology;

[0089] Figure 25 The X-ray diffraction spectrum of the collagen type III prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by X-ray diffraction spectrum technology;

[0090] Figure 26 The live / dead cell staining result under the treatment of the collagen type III prepared from the Tibetan sheep embryo in the embodiment 1 of the present application is obtained by live / dead cell staining technology.

[0091] Figure 27 The number of living cells treated by the collagen type III prepared from Tibetan sheep embryos in Example 1 of the present application is counted;

[0092] Figure 28 The cell proliferation promotion experiment results of the collagen type III prepared from Tibetan sheep embryos in Example 1 of the present application are shown;

[0093] Figure 29 The cell viability improvement experiment results of the collagen type III prepared from Tibetan sheep embryos in Example 1 of the present application are shown;

[0094] Figure 30 The results of quantitatively counting the cell migration rate in the scratch test of the collagen type III prepared from Tibetan sheep embryos in Example 1 of the present application are shown;

[0095] Figure 31 The scratch test results of the collagen type III prepared from Tibetan sheep embryos in Example 1 of the present application at 0.1 mg / ml and the control group are shown;

[0096] Figure 32 The scratch test results of the collagen type III prepared from Tibetan sheep embryos in Example 1 of the present application at 0.5 mg / ml and 1 mg / ml are shown;

[0097] In the above figures, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001. The data processing method is as follows: data analysis and drawing use GraphPad Prism 8 software, component difference analysis uses the built-in Two-way ANOVA function of the software, all data are expressed as mean ± SD, and the difference between groups is considered statistically significant when p<0.05. DETAILED DESCRIPTION

[0098] The following examples are provided to better further understand the present application and are not limited to the best mode, and do not constitute a limitation on the content and scope of protection of the present application. Any person under the inspiration of the present application or the combination of the present application with other prior art features will fall within the scope of protection of the present application.

[0099] If the specific experimental steps or conditions are not specified in the examples, the operation or conditions can be carried out according to the conventional experimental steps described in the literature in the art. If the reagents or instruments used are not specified by the manufacturer, they are conventional reagent products that can be obtained by purchase.

[0100] The Tibetan sheep embryo tissue involved in the following examples is purchased from Lanzhou Mingde Pharmaceutical Co., Ltd.

[0101] Preparation of Tibetan sheep embryo triple helix type III collagen

[0102] The present embodiment provides a preparation method of Tibetan sheep embryo triple helix type III collagen, comprising the following steps:

[0103] (1) Pretreatment: Place the Tibetan sheep embryo tissue at 4°C for thawing, rinse the animal tissue with distilled water to remove residual blood and impurities, wash multiple times and remove excess water, then weigh and label the tissue sample and cut it into about 3x3 cm. The pretreatment process is carried out at 4°C.

[0104] (2) Degreasing: Mix the sample and the degreasing solution at a ratio of 1:10 (w / v, g / mL), soak the pretreated sample in step (1) with the degreasing solution for 5 hours, and replace the degreasing solution every 2.5 hours. After degreasing, rinse the animal tissue multiple times with distilled water. The degreasing solution is an aqueous solution containing 10 v / v% butanol and 50 v / v% ethanol. The degreasing process is carried out at 4°C.

[0105] (3) Impurity removal: Mix the sample in step (2) with a sodium hydroxide solution at a ratio of 1:10 (w / v, g / mL), soak the sample in a 0.1 mol / L NaOH solution for 5 hours to remove non-collagen proteins, and replace the alkaline solution every 2.5 hours; repeatedly rinse with ultrapure water to remove residual sodium hydroxide solution until the pH of the rinse water shows neutrality. The impurity removal process is carried out at 4°C.

[0106] (4) Enzymatic digestion: Homogenize the sample in step (3) with a tissue homogenizer, mix the homogenized sample with an enzyme solution at a ratio of 1:5 (w / v, g / mL) and stir for 72 hours, then centrifuge and take the supernatant; the enzyme solution contains 1% (w / v) pepsin and 0.1% (w / v) trypsin, and the solvent is 0.5 M acetic acid solution. The enzymatic digestion process is carried out at 4°C.

[0107] (5) Salting-out: Add a mixed solution of sodium chloride and ammonium sulfate to the supernatant obtained in step (4), and the final concentration of the mixed solution is 0.8 mol / L and the final concentration of ammonium sulfate is 0.8 mol / L, and the collagen is precipitated after salting-out for 24 hours. Centrifuge and take the precipitate, mix the precipitate with 0.5 mol / L acetic acid solution at a ratio of 1 mg:5 ml, and resuspend the precipitate in 0.5 mol / L acetic acid solution. Pour the resuspended solution into a cellulose dialysis membrane for dialysis, first dialyze the resuspended solution against 15 volumes of 0.2 mol / L acetic acid solution for 24 hours, then dialyze against ultrapure water for 3 days, and dialyze against 15 volumes of ultrapure water, and replace the ultrapure water multiple times during dialysis to obtain a collagen solution. The salting-out and dialysis processes are both carried out at 4°C.

[0108] (6) SP Sepharose HP ion exchange chromatography purification: SP Sepharose HP cation exchange column (specification: particle size 24-44 μm, 26 mm x 20 cm) was packed, and the ion exchange column was equilibrated with acetic acid buffer (concentration 0.4 M) at pH = 3.0, then the collagen solution in step (5) was directly loaded, the loading amount was 110 mg / ml column volume, then gradient elution was performed with 0, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.5 M, 1 M NaCl aqueous solution, the flow rate was 1.0 ml / min, the pressure was not higher than 0.15 Mpa, and the sample in the 0.1-0.3 M NaCl solution interval was collected.

[0109] (7) Gel chromatography: Sephadex G-200 gel chromatography (specification: particle size 40-120 μm, 16 mm x 100 cm) was used as the filler, and the sample collected in step (6), i.e. the sample eluted and collected in different concentrations of eluent in the 0.1-0.3 M interval, was purified by gel chromatography, the loading amount was 3% of the column volume, the eluent was phosphate buffer solution (concentration 0.05 M) at pH = 7.3, the flow rate was 0.4 ml / min, the pressure was not higher than 0.15 Mpa, 1 tube was collected every 10 min, detection was performed at 230 nm, and the absorbance curve was drawn, the eluent of the 2nd-3rd tubes was collected from the 1st tube at 0 min.

[0110] (8) Desalination and concentration: the mixed collagen solution extracted in step (7) was first concentrated by a 30K ultrafiltration tube, and then the separated collagen solution was dialyzed again by a 100 kDa dialysis bag to remove salt, and the obtained high-purity Tibetan sheep embryo type III collagen solution was subjected to desalination and concentration treatment.

[0111] Sample treatment and analysis:

[0112] (1) The high-purity Tibetan sheep embryo type III collagen solution obtained above was inverted in a uniform culture dish for observation, and the results are shown in Figure 1 , wherein Figure 1 a is the type III collagen solution prepared by crude extraction (the collagen solution obtained after step (5) ends), which is translucent, Figure 1 b is the type III collagen solution prepared by purification (the collagen solution obtained after step (7) ends, concentrated by a 30K ultrafiltration tube), which is milky white, indicating that the type III collagen solution has high concentration and content. The purified type III collagen solution was pre-frozen at -20℃ for 24 h, and then placed in a freeze-drying machine for 24 h to obtain a collagen freeze-dried sample for storage, and the freeze-dried sample is as shown inFigure 2 As shown in Figure 2 As can be seen from a, b in FIG. 6, the low-temperature freeze-dried collagen type III solution forms a sponge-like or fibrous structure, which is likely to be formed due to the crystallization or aggregation of collagen during freeze-drying. The freeze-dried sample is usually white, indicating that the sample has high purity.

[0113] (2) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE):

[0114] Tris-glycine electrophoresis buffer, 8% acrylamide separation gel, 5% acrylamide concentration gel were used. The sample was dissolved in 0.5M acetic acid at a concentration of 1mg / mL, and after adding the loading dye (bromophenol blue stain), the sample was denatured at 100°C for 15min. Under reducing conditions, 30mA constant current electrophoresis was performed for 3.5h, and after fixation, staining and decolorization, the gel imaging system was used for analysis, and then semi-quantitative analysis was performed using Image J software.

[0115] The results are shown in Figure 3 As can be seen from FIG. 6, collagen type III is composed of three α1(III) chains. As can be seen from the figure, the bands in lanes 1 and 2 are the crude collagen mixture (after step (4) and before step (5) salting-out), and the bands in lanes 3 and 4 are the collagen mixture after salting-out. The protein bands in the first four lanes are more and the purity is lower. Figure 4 As can be seen from FIG. 6, lanes 1-5 are the final collagen solution obtained in step (8), with a molecular weight of about 130kDa, which is the α1(III) chain of collagen type III, accounting for about 80% of the total collagen type III (obtained by gray scale analysis of collagen SDS-PAGE gel images using Image software); the β chain of the α chain with a molecular weight of about 250kDa accounts for about 20% of the total collagen; there are basically no other bands below 130kDa, indicating that the structure of the collagen is well preserved and has not been decomposed into polypeptides or small molecule hydrolyzed collagen, and the purity is high.

[0116] (3) Liquid chromatography-mass spectrometry (LC-MS):

[0117] The target protein band near 130 kDa was cut, the protein band was washed with ddH2O for 3 times, 300 μL decolorizing solution was added for decolorization, washed with water for 4 times, then the gel strip was washed with 300 ul 25 mM ammonium bicarbonate, 50% acetonitrile and 100% acetonitrile for 2 times, and finally the gel was dehydrated until the gel block became white. 50 μl of 10 mM DTT solution (dithiothreitol) was added for reduction at 37°C for 2 hours, and when the temperature decreased to room temperature, an equal volume of 50 mM IAA solution (commercially available) was added for alkylation for 30 min in the dark, then the gel strip was washed with 300 ul 25 mM ammonium bicarbonate, 50% acetonitrile and 100% acetonitrile for 2 times, and finally the gel was dehydrated until the gel block became white. 0.01 ug / ul proteomics grade trypsin 15-40 ul was added, and after the gel was fully absorbed and became transparent on ice, 50 mM NH4HCO3 (containing 10% ACN solution) 30-40 ul was added for covering; digestion at 37°C overnight. After enzymatic hydrolysis, the supernatant was transferred to another new EP tube, 100 μL extraction solution (67% acetonitrile containing 2% formic acid) was added to the remaining gel block, and 37°C was kept for 30 min, then ultrasonic was performed for 15 min, centrifuged, the supernatant was combined and concentrated by centrifugation and dried, and then mass spectrometry analysis was performed. After the enzymatic polypeptide sample was dried by centrifugation, it was re-dissolved in mobile phase A (0.1% formic acid / water) and bottled for loading, and online mass spectrometry analysis was performed. After the dissolved sample was loaded onto a C18 pre-column with a suitable volume, it was then desalted with 20 ul volume. After desalination on the pre-column, the sample was separated by an analytical column, and the analytical column was a C18 reversed-phase chromatographic column. The gradient used in the experiment was that the mobile phase B (80% acetonitrile, 0.1% formic acid) was increased from 5% to 40% within 15 min.

[0118] The results are as follows:

[0119] Figure 5 The liquid chromatography-mass spectrometry diagram of the unique peptide segment of type III collagen protein obtained by liquid chromatography-mass spectrometry technology for type III collagen protein is GSPGPQGLKGENGKPGPSGQNGER.

[0120] Figure 6 The liquid chromatography-mass spectrometry diagram of the unique peptide segment of type III collagen protein obtained by liquid chromatography-mass spectrometry technology for type III collagen protein is NGDRGETGPAGPSGAPGPAGSR.

[0121] Figure 7 The liquid chromatography-mass spectrometry diagram of the unique peptide segment of type III collagen protein obtained by liquid chromatography-mass spectrometry technology for type III collagen protein is GSPGPQGIKGENGKPGPSGQNGER.

[0122] Figure 8The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment with the amino acid sequence of GDRGETGPAGPPGAPGAPGAPGPVGPAGK obtained by liquid chromatography-mass spectrometry for collagen type III.

[0123] Figure 9 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment with the amino acid sequence of GDRGETGPAGPPGAPGAPGAPGPVGPAGK obtained by liquid chromatography-mass spectrometry for collagen type III.

[0124] Figure 10 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment with the amino acid sequence of GERGFPGERGVQGPPGPAGPR obtained by liquid chromatography-mass spectrometry for collagen type III.

[0125] Figure 11 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment with the amino acid sequence of GEPGPTGIQGPPGPAGEEGKR obtained by liquid chromatography-mass spectrometry for collagen type III.

[0126] Figure 12 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment with the amino acid sequence of GERGPPGESGAAGPTGPIGSR obtained by liquid chromatography-mass spectrometry for collagen type III.

[0127] Figure 13 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment with the amino acid sequence of GPTGDPGKAGEKGHAGLAGPR obtained by liquid chromatography-mass spectrometry for collagen type III.

[0128] Figure 14 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment with the amino acid sequence of HGSRGEPGPVGAVGPAGAVGPR obtained by liquid chromatography-mass spectrometry for collagen type III.

[0129] Figure 15 The liquid chromatography-mass spectrometry diagram of the unique collagen type III peptide segment with the amino acid sequence of GPVGPSGPPGK obtained by liquid chromatography-mass spectrometry for collagen type III.

[0130] As can be seen from the above, these peptide segment amino acid sequences conform to the general composition pattern (Gly-X-Y) of collagen type III reported in the literature.

[0131] (4), amino acid composition analysis:

[0132] The purified collagen sample (100 mg) was hydrolyzed in 5 mL of 6M HCl in an oven at 105°C for 24 h. The hydrolyzate was neutralized to neutral with 5 mL of 6M NaOH solution, centrifuged at 4000 r / min for 10 min, and 0.5 mL of supernatant was derivatized and added with 0.5 mL of pH=9.0 sodium bicarbonate buffer solution and DNFB (2,4-dinitrofluorobenzene) respectively, and placed in a 60±0.5°C water bath, avoiding light for 60 min. It was immediately cooled to room temperature with cold water, 3.5 mL of pH=7.0 phosphate buffer solution was added, mixed thoroughly, and reacted at room temperature for 15 min in the dark. The supernatant was filtered through a 0.22 μm filter membrane, and liquid chromatography (Agilent HPLC1200, C18 chromatographic column: 250×4.6 mm×5 μm, column temperature: 38°C, wavelength: 360 nm, flow rate: 1 mL / min, injection volume: 20 μL) was used for determination. The mobile phase included A: acetonitrile:methanol=90:10 B: 0.02M sodium dihydrogen phosphate + disodium hydrogen phosphate. The amino acid composition was the proportion of single amino acid in total amino acid.

[0133] The amino acid composition analysis results of the prepared collagen type III are shown in Table 1. From the identified amino acid composition, it is analyzed that different amino acids present in the extracted collagen type III sample include glycine (Gly), proline (Pro), and hydroxyproline (Hyp), etc. The contents of tryptophan (Ser), histidine (His), threonine (Thr), valine (Val), and tyrosine (Tyr) are low. These amino acid compositions are similar to the collagen type III extracted from various sources reported in the literature. Cysteine (Cys), hydroxylysine (Hyl), phenylalanine (Phe), methionine (Met), isoleucine (lle), and phenylalanine (Phe) were not found in this study, which may be destroyed in the acid hydrolysis process.

[0134] Table 1, Amino acid composition analysis of collagen type III

[0135]

[0137]

[0138]

[0139] (5) Ultraviolet absorption spectrum analysis (UV): Figure 16 In order to identify the presence of collagen, the purified collagen sample was dissolved into 1 mg / mL with 0.5M acetic acid, and the supernatant was obtained by centrifugation at 12000 rpm for 5 min at 4°C, and then analyzed by ultraviolet-visible spectrophotometer, with a measurement range of 190-400 nm and a scanning rate of 2 nm / min, using 0.5M acetic acid to obtain the baseline, and using Origin software to complete the Gaussian curve fitting.

[0140]

[0139] The results are as follows Figure 16 As shown, due to the presence of -CO-NH-, most proteins exhibit strong absorption peaks below 230 nm and at 280 nm. The figure shows no obvious absorption peak near 280 nm, but a strong absorption peak at 230 nm. This is consistent with the reported absorption peak of collagen (around 225 nm), a characteristic UV absorption peak of collagen, indicating that the extracted protein is collagen. Furthermore, the low content of aromatic amino acids (tryptophan, tyrosine, and phenylalanine) (no strong aromatic absorption peak at 280 nm) indicates minimal non-collagen contamination, suggesting high purity of the extracted collagen in this example.

[0140] (6) Fourier transform infrared spectroscopy analysis (FTIR):

[0141] To identify the intact triple helix structure of collagen, completely dried collagen samples were ground evenly with potassium bromide in a mortar, using potassium bromide as a blank control. The mixture was heated at 500 cm⁻¹. -1 and 4000cm -1 Spectral scanning was performed on the spectral region between [specific parameters]. A resolution of 2.0 cm⁻¹ was used. -1 The average of the three scans was taken. Gaussian curve fitting was performed using Origin software.

[0142] The results are as follows Figure 17 As shown, at 3306cm -1 There is an absorption peak at 3400-3440 cm⁻¹, which is the absorption peak of the amide A band and is related to the stretching vibration of NH₄⁺. A free NH₄⁺ molecule has an absorption peak at 3400-3440 cm⁻¹. -1 Within a certain range, tensile vibrations occur, and when these vibrations interact with hydrogen bonds to form -NH2, the resulting structure shifts to 3300 cm⁻¹. -1 The absorption peak of amide B appears at 2928 cm⁻¹. -1 This is due to the asymmetric stretching of -CH2. The absorption peak of the amide I band of sheep placental collagen is at 1658 cm⁻¹. -1 The stretching vibration of C=O is related to the characteristic frequency of the amide I band. The amide II band of sheep placental collagen is located at 1548 cm⁻¹. -1 The amide III band is located at 1238 cm⁻¹ -1 At this point, the NH bending vibration couples with the CN and CH stretching vibrations, respectively, producing characteristic peaks for the amide II and amide III bands. The absorption peak of the amide I band in sheep placental collagen belongs to the α-helical structure absorption peak, and the β-sheet structure absorption peak also coincides with the absorption peak of the amide III band. The intensity ratio of the amide III band (1238 cm⁻¹) is... -1 and 1548cm -1This indicates the integrity of the triple helix structure. A ratio of approximately 1 generally indicates the integrity of the triple helix structure. The type III collagen shows a ratio of approximately 1, proving that type III collagen has a complete triple helix structure.

[0143] like Figure 18 As shown, the absorption wavelength of type III collagen amide I region is 1600-1700 cm⁻¹. -1 This is attributed to the stretching vibrations of the C=O and NH bonds, which are related to the secondary structure of type III collagen. The figure shows four small peaks in the amide I region, corresponding to different absorption wavelengths and representing different secondary structures, as determined by fitting analysis.

[0144] like Figure 19 As shown in the figure, the α-helix has the highest relative content in the secondary structure of type III collagen, followed by the β-sheet structure, proving that type III collagen has a complete helical structure.

[0145] (7) Scanning electron microscopy (SEM) analysis:

[0146] To observe the structure of the freeze-dried collagen samples, the freeze-dried samples were coated with gold before observation, and the microstructure of the samples was evaluated at a magnification of 15KV electron acceleration voltage.

[0147] The results are as follows Figure 20 As shown, the structure of the freeze-dried collagen sample was observed by scanning electron microscopy (SEM). The extracted type III collagen has a fine fibrous network structure characteristic of natural collagen.

[0148] (8) Raman spectroscopy analysis:

[0149] To analyze the secondary structure of type III collagen, the lyophilized sample was measured using a Raman spectrometer (Nicoletraman 960) at a wavenumber of 4000 cm⁻¹. -1 -400cm -1 The resolution is 4cm. -1 The number of scans was 20.

[0150] The results are as follows Figure 21 As shown, the frequencies of amide I and amide III bands can detect differences in secondary structure. This region, along with the hydrocarbon stretching vibration region (i.e., the lipid region), the proline and hydroxyproline regions, are the three main regions of conformational changes in collagen structure. A band at 1660 cm⁻¹ can be observed. -1 The amide I band represents peptide chain vibrations, primarily derived from C=O stretching vibrations, CN stretching vibrations, and NH bending vibrations, indicating the presence of normal protein secondary structure; the amide III band (1241 cm⁻¹) represents peptide chain vibrations. -1) mainly associated with C-N stretching and N-H bending, C-C stretching and C-H bending.

[0151] (9) Circular dichroism (CD):

[0152] The samples of collagen with a final concentration of 1 mg / ml were prepared using 0.5 M acetic acid, and then transferred to the sample cell of the CD spectrometer with a 1 mm light path, which can be scanned in the range of 190-260 nm. The scanning speed is 50 nm / min, the scanning interval is 1 nm, and the temperature is 25°C.

[0153] The results are shown in Figure 22 CD is a common method for characterizing protein secondary structure and determining the triple helix structure of collagen. The CD results of the prepared type III collagen are shown in the figure. Type III collagen has a negative peak and a positive peak at 198 nm and 222 nm, respectively, indicating that the collagen retains its natural, non-denatured triple helix structure.

[0154] Figure 23 To estimate the relative content of type III collagen secondary structure according to the CD spectrum, it can be seen from the results that the relative content of alpha helix is the highest, followed by the beta sheet structure. The results are consistent with the estimation of the relative content of the secondary structure by infrared spectroscopy.

[0155] (10) Differential scanning calorimetry (DSC):

[0156] Weigh about 3-5 mg of a single sample and transfer it to an aluminum DSC crucible, then seal it, then use a suitable DSC instrument to quantify the thermal transition (Tmax), and use an empty crucible as a reference control. Heat the sample at a rate of 1°C / min from 0-75°C.

[0157] The results are shown in Figure 24 Thermal stability is a method for characterizing the stability and characteristic triple helix structure of collagen. The thermal denaturation curve of the prepared type III collagen is shown in the figure. As can be seen from the figure, there is a thermal absorption peak, the denaturation temperature (Td) is 40.26°C, and the melting temperature (Tm) is 83.13°C, which is consistent with the characteristics of animal collagen reported in the literature. The denaturation temperature is higher than that of collagen extracted from marine animals, and is more suitable for use in the field of medical materials, etc.

[0158] (11) X-ray diffraction: Measurement is carried out using XRD instrument in powder mode under Cu-Kα radiation. The scanning range (2θ) is set between 5 and 50°, and the scanning rate is 2° / min.

[0159] The results are shown in Figure 25As shown, the XRD of collagen shows diffraction peaks at 7.7°, 21°, 31°. The peak value around 7° shows the distance between the molecular chains of collagen fibers. According to the Bragg formula, the distance between the molecular chains of collagen fibers is calculated to be 1.11 nm. The second diffraction peak is found to be a wide peak around 20°, which reflects the diffuse scattering caused by many structural layers of collagen fibers, and indicates the characteristic interchain spacing of collagen triple helix, indicating the integrity of the triple helix structure of collagen.

[0160] In summary, the Tibetan sheep embryo type III collagen prepared in this embodiment has a complete triple helix structure, the molecular weight of the a1(III) chain is about 130 kDa, the molecular weight of the β chain formed by two a chain dimer peptide chains is about 250 kDa, accounting for about 20% of the total type III collagen content; it contains the characteristic amino acid sequence of collagen, showing a "Gly-X-Y" repeating pattern, and glycine accounts for about 1 / 3 of the total number of collagen amino acids.

[0161] Further identification shows that the type III collagen has a characteristic fine fibrous structure of natural collagen.

[0162] Further identification shows that the type III collagen has high purity and good thermal stability, with a denaturation temperature (Td) of 40.26°C and a melting temperature (Tm) of 83.13°C, which is consistent with the characteristics of animal collagen reported in the literature, and the denaturation temperature is higher than that of collagen extracted from marine animals, and is more suitable for use in the field of medical materials.

[0163] Example 2

[0164] The present embodiment provides a preparation method of Tibetan sheep embryo triple helix type III collagen, comprising the following steps:

[0165] (1) Pretreatment: Place the Tibetan sheep embryo tissue at 4°C for thawing, wash the animal tissue with distilled water to remove residual blood and impurities, wash multiple times and remove excess water, then weigh and label the tissue sample and cut it into about 3x3 cm. The pretreatment process is carried out at 4°C.

[0166] (2) Defatting: Mix the sample and the defatting solution at a ratio of 1:5 (w / v, g / ml), and soak the pretreated sample in step (1) in the defatting solution for 2 hours, replacing the defatting solution every 1 hour. After defatting, wash the animal tissue multiple times with distilled water. The defatting solution is an aqueous solution containing 10 v / v% butanol and 50 v / v% ethanol. The defatting process is carried out at 4°C.

[0167] (3) Impurity removal: the sample in step (2) and sodium hydroxide solution are mixed according to the ratio of 1:5 (w / v, g / ml), the sample is soaked in 0.2 mol / L NaOH solution for 2 hours to remove non-collagen protein, and the alkaline solution is replaced every 1 hour; the residual sodium hydroxide solution is removed by repeatedly washing with ultrapure water until the pH value of the washing water shows neutral. The impurity removal process is carried out at 4°C.

[0168] (4) Enzymatic digestion: the sample in step (3) is homogenized with a tissue homogenizer, and the homogenized sample is mixed with an enzyme solution according to the ratio of 1:3 (w / v, g / ml) and stirred for extraction for 48 hours, and then the supernatant is obtained by centrifugation; the enzyme solution contains 5% (w / v) pepsin and 0.3% (w / v) trypsin, and the solvent is 0.4 M acetic acid solution. The enzyme digestion process is carried out at 4°C.

[0169] (5) Salting-out: a mixed solution of sodium chloride and ammonium sulfate is added to the supernatant obtained in step (4), and the final concentration of the mixed solution is 0.5 mol / L and the final concentration of ammonium sulfate is 1 mol / L, and the collagen is precipitated by salting-out for 12 hours, and the precipitate is obtained by centrifugation, and the precipitate is mixed with 0.4 mol / L acetic acid solution according to the ratio of 5 mg:10 ml, and the precipitate is dissolved in 0.4 mol / L acetic acid solution. The redissolved solution is inverted in a cellulose dialysis membrane for dialysis, first dialyzed with 10 times the volume of 0.3 mol / L acetic acid solution for 12 h, and then dialyzed with ultrapure water for 3 d, and dialyzed with 10 times the volume of ultrapure water, and the collagen solution is obtained. The salting-out and dialysis processes are carried out at 4°C.

[0170] (6) SP Sepharose HP ion exchange chromatography purification: SP Sepharose HP cation exchange column filler is used, the ion exchange column is equilibrated with acetic acid buffer solution with pH=2.5, the collagen solution obtained in step (5) is directly loaded, the loading amount is 75 mg / ml column volume, gradient elution is carried out with 0, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.5 M, 1 M sodium chloride solution, the flow rate is 2.0 ml / min, the pressure is not higher than 0.15 Mpa, and the samples in the range of 0.1-0.3 M sodium chloride solution are collected.

[0171] (7) The filler used in gel chromatography is Sephadex G-200 gel chromatography (specification: particle size 40-120 μm, 16 mm x 100 cm) purification, the sample collected in step (6) is eluted and collected, i.e. different concentrations of eluent in 0.1-0.3 M, and the sample is subjected to gel chromatography purification, the loading amount is 1% of the column volume, the eluent is phosphate buffer solution (concentration 0.01 M) with pH = 7.2, the flow rate is 0.25 ml / min, the pressure is not higher than 0.15 Mpa, one tube is collected every 10 min, and the absorbance curve is detected at 230 nm and drawn. The eluent of the 2nd and 3rd tubes is collected from the 1st tube. The 2nd and 3rd tubes collected are subjected to SDS-PAGE and LC-MS / MS detection to determine whether they are target type III collagen, and the eluent of the target type III collagen detected is mixed with the eluent of the sample collected after elution.

[0172] (8) Desalination and concentration: the mixed collagen protein solution extracted in step (7) is first concentrated by using a 30K ultrafiltration tube, and finally the collagen protein solution separated is dialyzed by using a 100 kDa dialysis belt to desalt again, and the high-purity Tibetan sheep embryo type III collagen protein solution obtained is subjected to desalination and concentration treatment.

[0173] Example 3

[0174] The present embodiment provides a preparation method of Tibetan sheep embryo triple helix type III collagen, comprising the following steps:

[0175] (1) Pretreatment: the Tibetan sheep embryo tissue is thawed at 4°C, the animal tissue is washed with distilled water to remove residual blood and impurities, the tissue sample is weighed, labeled and cut into about 3 x 3 cm after multiple washing and removal of excess water. The pretreatment process is carried out at 4°C.

[0176] (2) Degreasing: the sample and the degreasing solution are mixed at a ratio of 1:8 (w / v, g / ml), the pretreated sample in step (1) is soaked in the degreasing solution for 3 hours, and the degreasing solution is replaced every 2 hours. After degreasing, the animal tissue is washed multiple times with distilled water. The degreasing solution is a water solution containing 10 v / v% butanol and 50 v / v% ethanol. The degreasing process is carried out at 4°C.

[0177] (3) Impurity removal: the sample in step (2) and the sodium hydroxide solution are mixed at a ratio of 1:8 (w / v, g / ml), the sample is soaked in 0.2 mol / L NaOH solution for 2 hours to remove non-collagen proteins, and the alkaline solution is replaced every 1 hour; the residual sodium hydroxide solution is repeatedly washed with ultrapure water until the pH value of the washing water shows neutral. The impurity removal process is carried out at 4°C.

[0178] (4) Enzymatic digestion: homogenize the sample in step (3) with a tissue homogenizer, mix the homogenized sample with an enzyme solution according to 1:4 (w / v, g / ml) and stir for 60 hours, then centrifuge to obtain the supernatant; the enzyme solution contains 3% (w / v) pepsin and 0.2% (w / v) trypsin, and the solvent is 0.6 M acetic acid solution. The enzymatic digestion process is carried out at 4°C.

[0179] (5) Salting-out: add an ammonium sulfate solution to the supernatant obtained in step (4), and the final concentration of ammonium sulfate in the mixed solution is 0.5 moL / L, and the salting-out is carried out for 18 hours to precipitate the collagen, and the precipitate is obtained by centrifugation, and the precipitate is mixed with 0.6 moL / L acetic acid solution according to the ratio of 3 mg:7 ml, and the precipitate is dissolved in 0.6 moL / L acetic acid solution. The redissolved solution is inverted in a cellulose dialysis membrane for dialysis, first dialyzed against 13 volumes of 0.3 moL / L acetic acid for 18 hours, then dialyzed against ultrapure water for 3 days, and dialyzed against 13 volumes of ultrapure water, and the collagen solution is obtained. The salting-out and dialysis processes are carried out at 4°C.

[0180] (6) SP Sepharose HP ion exchange chromatography purification: use SP Sepharose HP cation exchange column filler, sample in acetic acid buffer solution at pH=3.5, sample amount is 150 mg / ml column volume, use 0, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.5 M, 1 M NaCl solution gradient elution, flow rate is 1.5 ml / min, pressure is not higher than 0.15 Mpa, collect samples in 0.1-0.3 M NaCl solution interval.

[0181] (7) Gel chromatography: the filler used is Sephadex G-200 gel chromatography (specification: particle size 40-120 μm, 16 mm x 100 cm) purification, the sample collected in step (6), i.e. the eluate collected in different concentrations of 0.1-0.3 M, is subjected to gel chromatography purification, the sample amount is 5% of the column volume, the eluent is pH=7.4 phosphate buffer solution (concentration is 0.1 M), the flow rate is 0.5 ml / min, the pressure is not higher than 0.15 Mpa, every 10 min collects 1 tube, detects at 230 nm and draws the absorbance curve, and collects the eluate of the 2nd-3rd tubes from the start of 0 min.

[0182] (8) Desalting and concentration: the mixed collagen solution extracted in step (7) is concentrated by using a 30K ultrafiltration tube, and the separated collagen solution is dialyzed again by using a 100kDa dialysis bag to remove salt, and the obtained high-purity Tibetan sheep embryo type III collagen solution is desalted and concentrated.

[0183] Comparative Example 1

[0184] The difference between the present comparative example and Example 1 is that in the cation exchange chromatography purification step, the eluent is replaced by potassium chloride solution.

[0185] Comparative Example 2

[0186] The difference between the present comparative example and Example 1 is that in the cation exchange chromatography purification step, the sample is loaded in a phosphate buffer with a pH of 5.

[0187] Comparative Example 3

[0188] The difference between the present comparative example and Example 1 is that in the cation exchange chromatography purification step, the gradient elution condition is to use 0mol / L, 0.1mol / L, 0.2mol / L, 0.3mol / L, 0.4mol / L, 0.5mol / L, 0.6mol / L, 0.7mol / L, 0.8mol / L, 0.9mol / L, 1mol / L, 1.1mol / L, 1.2mol / L, 1.3mol / L sodium chloride solution for elution in turn.

[0189] Comparative Example 4

[0190] The difference between the present comparative example and Example 1 is that in the cation exchange chromatography purification step, the gradient elution condition is to use 0mol / L, 0.05mol / L, 0.08mol / L, 0.11mol / L, 0.14mol / L, 0.17mol / L, 0.20mol / L, 0.23mol / L, 0.28mol / L, 0.31mol / L, 0.34mol / L, 0.37mol / L, 0.40mol / L, 0.43mol / L, 0.46mol / L, 0.46mol / L, 0.49mol / L, 0.52mol / L, 0.55mol / L, 0.7mol / L, 0.85mol / L, 1mol / L sodium chloride solution for elution in turn.

[0191] Comparative Example 5

[0192] The difference between the present comparative example and Example 1 is that in the gel chromatography purification step, the eluent is a phosphate buffer with a pH of 7.0 and a concentration of 10mM.

[0193] Comparative Example 6

[0194] The difference between the present comparative example and Example 1 is that in the salting-out step, a sodium chloride solution with a final concentration of 1 mol / L is used for salting-out.

[0195] Comparative Example 7

[0196] The difference between the present comparative example and Example 1 is that in the salting-out step, an ammonium sulfate solution with a final concentration of 1.5 mol / L is used for salting-out.

[0197] Comparative Example 8

[0198] The difference between the present comparative example and Example 1 is that in the salting-out step, a mixed solution of ammonium sulfate with a final concentration of 1.5 mol / L and sodium chloride with a final concentration of 1 mol / L is used for salting-out.

[0199] Comparative Example 9

[0200] The difference between the present comparative example and Example 1 is that in the defatting step, the defatting solution is a 10 v / v% butanol aqueous solution.

[0201] Experimental Example 1

[0202] This experimental example investigates the purity, yield and activity of the triple helix type III collagen protein prepared in Example 1 and Comparative Examples 1-10.

[0203] Purity detection method: preliminary purity detection and identification is performed according to the gray scale ratio of the collagen protein SDS-PAGE band. Then, size exclusion chromatography (SEC) column is used to detect the purity of the collagen protein: chromatographic conditions: Welch Xtimate SEC120 7.8x300mm, 5μm; mobile phase: triethylamine 1.72g, anhydrous sodium acetate 11.48g, EDTA 1mg dissolved in 1L water, and the pH is adjusted to 4.95 with phosphoric acid; flow rate: 0.8ml / min; detection wavelength: 230nm; injection volume 10μl.

[0204] Yield: the calculation formula of the collagen protein yield (%) is (the content of hydroxyproline in the extracted collagen protein / the content of hydroxyproline in the Tibetan sheep embryo tissue)x100%. The content of hydroxyproline is detected and calculated using a commercially available hydroxyproline detection kit.

[0205] Collagen protein activity detection method: MTT method and scratch test migration rate (quantified by Image software). The migration rate calculation formula is cell migration rate (100%) = [(A0-A 24 ) / A0]x 100%, A0: initial scratch area (0 hour, t=0), A 24 : scratch remaining area after 24 hours.

[0206] Results are as follows in Table 2:

[0207] Table 2, detection effect

[0208]

[0209]

[0210] From the above table, compared with Comparative Example 1, in the cation exchange chromatography purification, the purity, yield and protein activity of Example 1 are significantly improved by replacing potassium chloride with sodium chloride. The reason may be that the elution ability depends not only on the salt concentration, but also on the hydration radius and charge density of the ion. The hydration radius of Na + is smaller than that of K + , and it is easier to compete with collagen to bind to the cation exchange medium, so under the same concentration, the elution ability of NaCl is stronger, and efficient elution can be achieved under milder conditions, reducing the risk of protein denaturation and aggregation. Collagen is sensitive to the ionic environment, and the ionic strength provided by NaCl is milder, which helps to maintain its triple helix structure and biological activity. In contrast, KCl may cause changes in protein conformation or slight precipitation under certain conditions, affecting the recovery of activity.

[0211] Compared with Comparative Example 2, in the cation exchange chromatography purification, the purity, yield and protein activity of Example 1 are significantly improved by adjusting the sample to pH 3 acetic acid buffer after equilibration in pH 5 phosphate buffer. The reason may be that the pI of type III collagen is 6.0-7.0, and its net positive charge is much higher at pH 3 than at pH 5; higher positive charge makes collagen bind more firmly to the sulfonic acid group (-SO3 - ) of the CEX medium, while most host proteins (pI 4-6) still have a net negative charge at pH 3, and the flow-through impurities are greatly reduced; at pH 3, acetic acid is mainly in the form of CH3COOH molecules, and its carboxyl group can act as a hydrogen bond donor, competing with the hydroxyl group on the medium skeleton for hydrogen bonds, blocking the irreversible adsorption of the hydrophobic "patch" of collagen to the medium. Phosphate (H2PO4 - ) is negatively charged and easily forms "salt bridge-aggregation" complexes with the positive charge of the protein, resulting in tailing and reduced yield.

[0212] Compared with Comparative Examples 3 and 4, in the cation exchange chromatography purification, the purity, yield and protein activity of Example 1 can be significantly improved by eluting with a gradient concentration of sodium chloride solution. The reason may be that elution with a gradient of too high concentration of sodium chloride solution or a gradient of too low concentration of sodium chloride solution is difficult to maintain the natural, non-denatured triple helix structure of the target collagen, reducing the biological activity, thus leading to easy denaturation and aggregation, ultimately reducing the purity, yield and protein activity.

[0213] Compared to Comparative Example 5, in the gel chromatography purification, the elution buffer used in Example 1, which was pH 7.0 phosphate buffer at a concentration of 10 mM, was replaced with pH 7.2 phosphate buffer at a concentration of 10 mM. The purity, yield, and protein activity were significantly improved. This may be because pH 7.2 allows the net charge, hydrogen bonds, and metal bridging of type III collagen to fall within the "minimum aggregation, maximum solubility" window. Therefore, a slight adjustment of pH can simultaneously improve purity, yield, and activity without changing the ionic strength or buffer system.

[0214] Compared with Comparative Examples 6-8, in the salting-out step, it was found that using ammonium sulfate and sodium chloride in combination with specific concentration ranges for salting-out was more effective than using ammonium sulfate or sodium chloride alone, or excessively high concentrations of ammonium sulfate and sodium chloride. This method was able to salt out the target collagen while maintaining its activity. However, using ammonium sulfate or sodium chloride alone, or excessively high concentrations of ammonium sulfate and sodium chloride, could not guarantee the activity of the target collagen. As a result, in subsequent purification steps, the target collagen was prone to denaturation and aggregation, leading to blockage and affecting the purity and yield of the purification.

[0215] Compared with Comparative Example 9, Example 1 uses an aqueous solution containing 10% butanol and 50% ethanol in the defatting step, which can ensure the integrity of the triple helix structure of the target collagen and maintain its activity. However, in Comparative Example 9, the use of 50% ethanol alone affects the integrity of the triple helix structure of the target collagen, damages its activity, and ultimately leads to a decrease in product purity, yield and activity.

[0216] Experiment Example 2 Biocompatibility

[0217] This experiment investigated the biocompatibility of the Tibetan sheep embryo triple-helix type III collagen prepared in Example 1.

[0218] Frozen cells (NIH-3T3) were rapidly thawed in a 37°C water bath. After thawing, the cell suspension was thoroughly mixed by pipetting and transferred to EP tubes containing 3 mL of complete culture medium (DMEM medium containing 10% fetal bovine serum and 100 U / mL penicillin / streptomycin). The tubes were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in 1 mL of complete culture medium and seeded into new culture flasks, with the medium replenished to the required level. The cells were cultured at 37°C and 5% CO2, with the medium changed every 2 days. Four groups were established: commercially available collagen (purchased from Nanjing Sinobiotech Co., Ltd.) - control (0.1 mg / mL), purified type III collagen prepared in Example 1 (0.1 mg / mL, 0.5 mg / mL, 1 mg / mL), and 100 μL of each collagen were placed in 24-well plates, with 4 × 10⁶ cells seeded per well. 4Cells were cultured in a controlled group containing only culture medium and cultured cells. The experimental group contained the sample from the previous example. After 4 days of culture, the cells were added to a live / dead cell staining solution, and the green (492 nm) and red (545 nm) fluorescence of the cells were observed using a confocal microscope. Cell proliferation rate (%) = (OD value of control group - OD value of control group) / (OD value of experimental group - OD value of control group) × 100%.

[0219] The results are as follows Figure 26 and Figure 27 As shown in the figure, it can be observed that compared with the control (0.1 mg / ml) and the prepared type III collagen (0.1 mg / ml), the type III collagen prepared in this invention has more live cells. Furthermore, as the collagen concentration increases, the number of live cells gradually increases, and dead cells are almost not observed, indicating that the type III collagen prepared in this invention has better biocompatibility.

[0220] Application of Experiment Example 3

[0221] This experimental example investigated the cell proliferation-promoting effect of the Tibetan sheep embryo triple-helix type III collagen prepared in Example 1.

[0222] Cell proliferation was measured using the CCK-8 assay. Commercially available collagen (purchased from Nanjing Sinobiotech Co., Ltd.) standard (0.1 mg / ml) and collagen solutions prepared in Example 1 (0.1 mg / ml, 0.5 mg / ml, 1 mg / ml) were placed in each well of a 24-well plate, with 100 μL per well and 4 × 10⁶ cells / well seeded per well. 4 Cells (NIH-3T3). At specified time points, the culture medium was replaced with 1 mL of complete culture medium (DMEM) containing 10% CCK-8 reagent. After incubation for 1 day, 4 days, and 7 days, the absorbance of 100 μL of supernatant at 450 nm was measured using a microplate reader. Each group was divided into three replicates, and the average value was taken.

[0223] The results are as follows Figure 28 As shown, the proliferation of cells under different concentrations of type III collagen was quantitatively evaluated using the CCK-8 assay. As the culture time increased from day 1 to day 7, cell viability increased at all concentrations, indicating that different concentrations of type III collagen had no significant cytotoxicity. From day 4 onwards, the OD 450 value of 1 mg / ml type III collagen was significantly higher than that of other groups, demonstrating its ability to promote cell proliferation, indicating that the type III collagen prepared in this invention significantly promotes cell proliferation.

[0224] Application of Experiment Example 4

[0225] This experimental example investigated the effect of Tibetan sheep embryo triple-helix type III collagen prepared in Example 1 on improving cell viability.

[0226] Using CellTiter-Lumi TM Cell viability was assessed using the Steady luminescence cell viability assay kit. Commercially available collagen (purchased from Nanjing Sinobiotech Co., Ltd.) standard (0.1 mg / ml) and collagen solutions prepared in Example 1 (0.1 mg / ml, 0.5 mg / ml, 1 mg / ml) were placed in each well of a 24-well plate, with 100 μL per well and 4 × 10⁶ cells / well seeded per well. 4 Cells (NIH-3T3). Relative cell viability was recorded at specified time points (1, 4, 7 days) based on chemiluminescence readings using the reagent manufacturer's method. Three replicates were performed for each group, and the average value was taken.

[0227] The results are as follows Figure 29 As shown, chemiluminescence studies revealed that from day 1, the ATP luminescence value of 1 mg / ml type III collagen was significantly higher than that of other groups, indicating that it had the strongest cell viability and promoted cell growth and diffusion, which is consistent with the results of the live-dead cell experiment.

[0228] Application of Experiment Example 5

[0229] This experimental example investigated the cell migration-promoting effect of the Tibetan sheep embryo triple-helix type III collagen prepared in Example 1.

[0230] Cell scratching: Using a marker pen, draw evenly spaced horizontal lines across the back of a 24-well plate, approximately every 0.5–1.0 cm, passing through each well. At least three lines should pass through each well. Place 100 μL of commercially available collagen (purchased from Nanjing Sinobiotech Co., Ltd.) standard (0.1 mg / ml) and collagen solutions prepared in Example 1 (0.1 mg / ml, 0.5 mg / ml, 1 mg / ml) into each well of the 24-well plate. Add approximately 4 × 10⁻⁶ cells / well to each well. 4 Cells (NIH-3T3) were selected, with the standard being that they could congregate overnight. The next day, using a 100 μl pipette tip, a streak was made perpendicular to the horizontal line on the reverse side of the cell line in the confluent monolayer. Cells were washed three times with PBS to remove the streaked cells, and serum-free medium was added. Three replicates were made per group, and the cells were incubated at 37°C with 5% CO2. Samples were taken and photographed at 0 and 24 days for analysis. Cell migration rate (100%) = [(A0-A...] 24 [) / A0]x 100%, A0: initial scratch area (0 hours, t=0), A 24 The remaining area of ​​the scratch after 24 hours.

[0231] The results are as follows Figures 30-32As shown in the figures, it can be observed that the scratch recovery degree of the cells cultured with the prepared collagen type III solution of different concentrations for 24 h is significantly faster than that of the control group, and the scratch recovery is faster with the increase of the concentration. The quantitative statistical results show that the collagen concentration of 1 mg / ml is the best, indicating that the triple helix collagen type III has a migration promoting activity on NIH / 3T3 cells.

[0232] Obviously, the above examples are only examples for clearly illustrating but not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments need not and cannot be exhausted. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A method for preparing type III collagen from Tibetan sheep embryos, characterized in that, include: The extract containing type III collagen from Tibetan sheep embryos was purified sequentially by cation exchange chromatography and gel chromatography. In the cation exchange chromatography purification step, an acetic acid buffer solution with a pH of 2.5–3.5 and a concentration of 0.4–0.6 M is used for equilibration before loading the sample. The eluent is a sodium chloride solution, and gradient elution is performed within the concentration range of 0–1 mol / L. In the gel chromatography purification step, the eluent is a phosphate buffer solution with a pH of 7.2-7.4 and a concentration of 0.01-0.1M.

2. The method for preparing type III collagen from Tibetan sheep embryos according to claim 1, characterized in that, The cation exchange chromatography purification step includes at least one of the following: (1) The gradient elution conditions are as follows: sodium chloride solutions with concentrations of 0 mol / L, 0.1 mol / L, 0.15 mol / L, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.5 mol / L and 1 mol / L are used for gradient elution. (2) Collect the eluent with a concentration of 0.1 mol / L to 0.3 mol / L; (3) The elution rate of the different concentrations of eluent is 1.0 to 2.0 mL / min; (4) Cation exchange chromatography was performed using SP Sepharose HP ion exchange chromatography. (5) The pressure shall not exceed 0.3 MPa; (6) The loading volume is 75-150 mg / ml column volume.

3. The method for preparing type III collagen from Tibetan sheep embryos according to claim 1 or 2, characterized in that, The gel chromatography purification step includes at least one of the following: (1) Collect the effluent from 10-20 minutes; (2) The flow rate of the eluent during the elution process is 0.25-0.5 ml / min; (3) The gel chromatography packing material used was Sephadex G-200 gel chromatography; (4) The pressure shall not exceed 0.15 MPa; (5) The sample loading amount is 1%-5% of the column volume.

4. The method for preparing Tibetan sheep embryo type III collagen according to any one of claims 1-3, characterized in that, The preparation steps of the extract containing type III collagen extracted from Tibetan sheep embryos include: pretreatment, defatting, impurity removal, enzymatic digestion or salting out; And / or, the gel chromatography purification process further includes a desalting and concentration step.

5. The method for preparing type III collagen from Tibetan sheep embryos according to claim 4, characterized in that, In the salting-out step, a mixed solution of sodium chloride and ammonium sulfate is added to the sample for salting-out, wherein the final concentration of ammonium sulfate is 0.5-1 mol / L and the final concentration of sodium chloride is 0-1 mol / L. And / or, in the salting-out step, the salting-out time is 12-24 hours; And / or, in the salting-out step, after the salting-out is completed, the step of redissolving and dialysis of the precipitate obtained by salting-out is further included; the redissolving solution is a 0.4-0.6 mol / L acetic acid solution, and the ratio of precipitate to redissolving solution is 1-5 mg: 5-10 ml, to obtain a collagen redissolving solution; in the dialysis step, the collagen redissolving solution and 0.2-0.3 M acetic acid solution are dialyzed at a volume ratio of 1:10-15 for 12-24 h, and then dialyzed with ultrapure water at a volume ratio of 1:10-1:15 for 1-3 days, with the ultrapure water being changed multiple times during the dialysis period.

6. The method for preparing Tibetan sheep embryo type III collagen according to claim 4 or 5, characterized in that, In the pretreatment step, the sample of Tibetan sheep embryo tissue is rinsed to remove residual blood and impurities, then the water is removed and it is cut into pieces.

7. The method for preparing Tibetan sheep embryo type III collagen according to any one of claims 4-6, characterized in that, In the degreasing step The sample and degreasing solution are mixed at a mass-to-volume ratio of 1:5 to 1:

10. The sample is then immersed in the degreasing solution for 2 to 5 hours, with the degreasing solution being replaced every 1 to 2.5 hours. The degreasing solution contains an aqueous solution of 10 v / v% butanol and 50 v / v% ethanol. The mass-to-volume ratio is g / mL or kg / L. And / or, in the impurity removal step, the sample and sodium hydroxide solution are mixed at a mass-to-volume ratio of 1:5 to 1:10, and the sample is soaked in a NaOH solution with a concentration of 0.1 to 0.2 mol / L for 2 to 5 hours to remove non-collagenous proteins. The alkaline solution is replaced every 1 to 2.5 hours. The sample is repeatedly rinsed with ultrapure water to remove residual sodium hydroxide solution until the pH of the rinsing water is neutral. The mass-to-volume ratio is g / mL or kg / L.

8. The method for preparing Tibetan sheep embryo type III collagen according to any one of claims 4-7, characterized in that, In the enzymatic digestion step, the sample is first homogenized, then mixed with an enzyme solution at a mass-to-volume ratio of 1:3 to 1:5 and stirred for 48 to 72 hours. Subsequently, the sample is centrifuged and the supernatant is collected. The enzyme solution contains 1 to 5% (w / v) pepsin and / or 0.1 to 0.3% (w / v) trypsin, and the solvent is 0.4 to 0.6 M acetic acid solution. The mass-to-volume ratio is g / mL or kg / L.

9. A Tibetan sheep embryo type III collagen prepared by the method for preparing Tibetan sheep embryo type III collagen according to any one of claims 1-8.

10. The use of Tibetan sheep embryo type III collagen prepared by the method of any one of claims 1-8, or the Tibetan sheep embryo type III collagen of claim 9, in any one of the following: 1) Promotes cell proliferation; 2) Promotes cell adhesion; 3) Promotes cell migration; 4) Uses in cosmetics; 5) Uses in medical devices; 6) Uses in pharmaceuticals; 7) Applications in tissue engineering and regenerative medicine; 8) Uses in drug delivery vehicles; 9) Applications in biomaterials; 10) Uses in food and health products.

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