Method for extracting collagen in leather cultural relics for species identification

Abstract

The present application relates to the technical field of collagen detection, and discloses a method for extracting collagen in leather cultural relics for species identification, comprising the following steps: (1) after the leather cultural relics are subjected to impurity removal, degreasing treatment and crushing treatment, ice acetic acid and pepsin are added for extraction to obtain leather cultural relic collagen powder; (2) rawhide collagen powder is extracted; (3) the peptide segments in the rawhide collagen powder are analyzed by using an ultra-high liquid chromatography tandem mass spectrometry method, and the species category is known by comparison; (4) rawhide collagen is used for animal immunization to obtain serum; (5) specific serum is screened and purified by using an ELISA method; and (6) species identification is performed. The acid-enzyme combination method is used to extract collagen, the extraction rate is high, and the extraction effect is good; then, precise positioning of different species characteristic peptide segment sequences is used to realize sensitive immunodetection, and the species is accurately positioned, thereby providing great practical value for immunological micro-trace identification of leather cultural relics.

Description

Technical Field

[0001] This invention belongs to the technical field of collagen detection, and more specifically relates to a method for species identification by extracting collagen from leather artifacts. Background Technology

[0002] The artifacts unearthed from tombs are not only mostly made of silk, but also include leather artifacts of different species from cattle and sheep. Extracting collagen from leather artifacts as a complete antigen, analyzing the characteristic peptide sequences of the proteins, and preparing corresponding specific antibodies to sensitively identify different species of leather artifacts unearthed from tombs is an urgent problem that the current cultural relic protection and micro-trace detection industry needs to solve, and it is of great significance for realizing the micro-trace identification of leather artifacts.

[0003] Given the scarcity and limited quantity of artifacts unearthed from tombs, there is a need to develop a method for extracting collagen from leather artifacts using small quantities and achieving high purity. Based on past experience in collagen extraction, traditional methods mainly include acid extraction, alkaline extraction, enzymatic extraction, high-temperature boiling, and the recently proposed ultrasound-assisted extraction. However, traditional methods have several drawbacks. Acid extraction may dissolve organic matter other than collagen, leading to impurities; furthermore, acid treatment alone usually requires a long time, wasting equipment and resources. Alkaline extraction easily causes denaturation and racemization of collagen, resulting in incomplete protein structures. Enzymatic extraction typically requires large amounts of extraction solution and a long production cycle. High-temperature boiling is time-consuming and difficult to improve purity. Ultrasound-assisted extraction is only used as an auxiliary purification method and does not fundamentally improve the extraction of large protein molecules.

[0004] Proteomics (also translated as proteomics) is the science that studies the composition and changes of proteins in cells, tissues, or organisms, using the proteome as its research object. This technique uses specific chemical reactions to replace any particular protein or peptide, achieving specificity and sensitivity with high selectivity. When processing samples, this technique offers excellent accuracy and sensitivity, enabling the accurate and objective detection of protein expression in high-purity samples, providing precise analytical results. Furthermore, this technique is simple to operate, requiring no complex techniques to obtain satisfactory results, and is time-efficient and cost-effective.

[0005] Proteomics-based immunoantibodies can yield large quantities of highly specific and homogeneous antibodies from relatively impure antigens (cross-linked proteins). Because an "unlimited" supply of homogeneous antibodies can be obtained, they are suitable for immunological analytical methods characterized by labeled antibodies, such as ELISA. Furthermore, the high specificity and singular biological function of monoclonal antibodies enhance the specific recognition function of antigens and antibodies and improve detection sensitivity.

[0006] More than 70% of collagen in mammals is type I collagen. Type I collagen proteins and peptides are almost identical, with differences between species only in a few characteristic sequences on their signature proteins. Traditionally prepared antibodies mostly possess characteristic sequences of the same broad category (both bovine or ovine), and can only specifically recognize antigens of the same type, but cannot accurately identify species. Summary of the Invention

[0007] Therefore, the purpose of this invention is to provide a method for extracting collagen from leather artifacts for species identification, employing an acid-enzyme combined method. This method combines acid and enzymatic methods and has the following advantages: First, under mild enzymatic hydrolysis conditions, collagen extraction is effective without damaging other structures besides collagen, thus preventing impurities. The combination of enzymatic and acid methods requires less extraction solution and has a shorter production cycle. This new method has practical application value in the fields of artifact preservation and identification.

[0008] To accurately identify different species within the same type of sheep—for example, sheep and goats—it is necessary to extract collagen from goat and sheep hides separately, precisely locate the characteristic peptide sequences that distinguish them, and prepare corresponding antibodies. This not only achieves sensitive immunoassay but also accurately identifies the species, providing significant practical value for the immunological micro-trace identification of leather artifacts.

[0009] To achieve the above objectives, the present invention provides a method for extracting collagen from leather artifacts for species identification, characterized by comprising the following steps:

[0010] (1) After removing impurities and defatting the leather artifacts, a pretreated sample was obtained. After crushing the pretreated sample, glacial acetic acid and pepsin were added, and the sample was extracted at 3-5℃ for 24-30h. Then, the sample was centrifuged, dialyzed, and freeze-dried to obtain collagen powder from the leather artifacts.

[0011] (2) Extract collagen from raw hide using the same method as in step (1) to obtain raw hide collagen powder;

[0012] (3) The peptides in raw skin collagen powder were analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS). The species classification was determined by comparing the characteristic peptide sequences of collagen. The raw skin collagen powder was reconstituted with trichloroacetic acid solution, and the content of peptides in the solution was quantitatively detected using NanoDrop. The reconstituted collagen solution was analyzed using UHPLC-MS / MS. 10X iRT peptides were added, mixed, and injected. Chromatographic separation was performed using the Easy nLC 1200 nano-flow chromatography series. The sample was injected into a trap column and then subjected to gradient separation using a chromatographic analysis column. After peptide separation, DLA mass spectrometry analysis was performed using a Q-Exactive HF-X mass spectrometer. All mass spectrometry data were merged using Spectronaut 16 software and analyzed using a library-free... The DIA (directDIA) method completes database retrieval of DIA mass spectrometry data and quantitative analysis of protein DIA in one step; the database uniprot-collagen-17386-20221026 is compared with uniprot-keratin-7621-20221026.fasta, and the species category is determined by comparing the characteristic peptide sequences of collagen.

[0013] (4) Animals were immunized with a solution containing raw skin collagen to obtain serum: KLH was dissolved in EDTA solution to obtain KLH solution; raw skin collagen powder was completely dissolved in dimethyl sulfoxide, then 1×PBS was added and mixed evenly to obtain collagen solution, which was added dropwise to KLH solution and shaken and placed; dialysis was performed with fresh 1×PBS to obtain protein-KLH; BSA was dissolved in EDTA aqueous solution to obtain BSA solution; protein-KLH was completely dissolved in DMSO, 1×PBS was added and mixed evenly, then BSA solution was added and mixed, gently shaken, placed at room temperature, and then magnetically stirred and dialyzed to obtain protein mixed solution; this protein mixed solution was used as antigen, adjuvant and injection agent, mixed and emulsified, and subcutaneously injected at multiple points in selected mice or rabbits, 0.2 mL at each point. Whole blood was collected after 14 days, serum was separated by high-speed centrifugation and stored at -20℃;

[0014] (5) Screening and purifying specific serum by ELISA to obtain mouse (rabbit) anti-collagen primary antibody: Refer to step (6) to perform gradient detection on the serum obtained in step (4) using ELISA, draw an S-shaped curve, and screen out the serum with the highest positive value and the best titer; wash the affinity chromatography column thoroughly with pure water and 1×PBS flow rate, take the selected serum with the best titer into a centrifuge tube, filter it with a microporous membrane, and repeat once; wash the column with 1×PBS, then connect it to the protein detector, adjust the absorbance reading of the protein detector to 0, turn on the computer HD-A collector, elute with glycine solution and collect the antibody; adjust the pH value of the antibody with sodium bicarbonate in time during the antibody collection process, wash the chromatography column with 1×PBS and pure water, add 20wt% ethanol, seal, and obtain mouse (rabbit) anti-collagen primary antibody, and store it in a low temperature freezer;

[0015] (6) Dilute the collagen powder of the leather artifact and add it to the ELISA plate. Coat it at low temperature overnight, wash it, and then incubate it with bovine serum albumin solution. After washing, incubate it with mouse (rabbit) anti-collagen primary antibody. After washing, incubate it with HRP-labeled goat anti-mouse (rabbit) secondary antibody. After washing, add TMB chromogenic agent A, then add TMB chromogenic agent B, shake well, protect it from light for a period of time, and then add sulfuric acid to stop the reaction. Measure the absorbance with an ELISA reader. When the absorbance is closest to that of the positive group, the species of the leather artifact is the species of raw hide.

[0016] Leather unearthed from tombs that are hundreds or thousands of years old has been tanned and damaged by the environment, so the collagen content is very low. Extraction can be done using methods that are less irritating. However, everyday raw hides contain a lot of fur, and after removing the fur, many other impurities remain.

[0017] The key to successful collagen extraction is the preservation of its triple helix structure. However, as a large molecule, collagen's triple helix structure is easily affected by temperature, denaturing at high temperatures. We employed an acid-enzyme combined extraction method. During enzymatic extraction, pepsin acts on the C-terminus of the disulfide bonds between the heavy chains of the IgG molecule, generating F(ab')2 and pFc fragments. The F(ab')2 fragment retains the biological activity of the corresponding antigen while avoiding potential side effects caused by the antigenicity of the Fc fragment. During acid extraction, glacial acetic acid preserves the triple helix structure of collagen, resulting in high extraction rates and ensuring purity. Therefore, combining these two methods increases purification efficiency while ensuring the triple helix structure of collagen remains intact. The entire process does not utilize expensive reagents. It is low-cost, easy to operate, and highly efficient.

[0018] In step (3), under the influence of nanofluid, TFA can stack with amino acid residues in the protein due to its strong hydrophobic and π-π conjugation interactions, thereby enriching the protein and determining the content of characteristic peptide sequences. The protein sequence quantification using this method is accurate and highly specific.

[0019] In step (4), healthy animals with glossy fur and free movement should be selected for the animal immunization experiment. After selecting the animals, they should be raised for about 2 weeks to eliminate any unqualified animals, so as to ensure the smooth progress of the subsequent animal immunization.

[0020] In step (5), the antibody to be purified can only be used after the titer of the semi-finished product has passed the test. The purified antibody has high purity and can maintain its immunological activity well. The chromatography column can be reused more than 40 times without changing the purification efficiency. Serum, as a half-antibody, has a sequence consistent with the characteristic peptide of the antibody, which can sensitively bind to the antigen and detect the antibody titer.

[0021] In step (6) of the ELISA detection process, it is essential to ensure that the reagents in each well are independent and do not cross-contaminate, so that the final result is a separate and accurate result that can indicate the specific titer of the antibody. This provides a strong guarantee for the specificity of the binding between the antigen and the antibody. HRP secondary antibody, as an enzyme-labeled antibody, is based on the colorimetric principle of horseradish peroxidase (HRP) labeling. Based on the specific binding reaction of HRP-labeled antibody and the multi-oxidation activity of HRP, the HRP-labeled antibody binds to the specific antigen-antibody complex generated by the target antigen. Utilizing the multi-oxidation properties of HRP, an oxygenation reaction is performed, applying the oxidation phenomenon to the in vitro transparent TMB solution system. TMB reacts with iodaldehyde to generate a characteristic blue color, forming a blue precipitate. The size of this blue precipitate represents the amount of antigen and antibody present in the object. Therefore, we can use this method to determine the amount of antigen present, thereby amplifying the antigen signal.

[0022] More preferably, in step (1), the impurity removal and degreasing treatment is as follows: the leather artifact is dried, immersed in a 10-12 wt% sodium chloride solution for 4-6 hours, then removed to remove impurities, washed repeatedly with distilled water, filtered dry, and then immersed in a degreasing agent, which is an aqueous solution containing 6 wt% sodium carbonate and 8 wt% isopropanol, with a bath ratio of 1:1, soaked for 22-28 hours, then removed to remove impurities, proteins and fats, washed repeatedly with distilled water, air-dried naturally, and frozen for later use.

[0023] Sodium chloride soaking can remove non-collagenous components. Sodium chloride solution is easy to prepare, odorless, non-toxic, low in cost, and easy to operate. The main function of degreasing agent is to remove the fatty parts of raw hide. Degreasing agent solution mainly contains 6% sodium carbonate and 8% isopropanol. It is easy to prepare and operate, and does not easily damage the collagen structure in leather artifacts.

[0024] More preferably, in step (2), the ratio of the pretreated raw hide to glacial acetic acid is 22-26 g: 200 mL; and the mass of the pepsin is 0.1-0.2% of the pretreated raw hide.

[0025] Compared to methods for extracting collagen from marine organisms, collagen extraction from cow and sheep hides requires less glacial acetic acid. When the material-to-liquid ratio is too small, extraction is incomplete; conversely, when the ratio is too large, over-extraction occurs, leading to the elution of residual non-collagenous components. Pepsin, naturally present in mammals, provides a gentle extraction, requiring only a small amount for complete extraction. Compared to modern raw hides, poorly preserved leather artifacts exhibit varying degrees of aging, wear, and geological corrosion, all affecting the extent of collagen breakdown. The amount of collagen present is lower, necessitating more powerful extraction methods. However, due to poor preservation conditions, the triple helix structure of the broken-down proteins is not fully intact, necessitating a gentler extraction method. This presents a inherent contradiction. Therefore, selecting the appropriate extraction solution ratio, extraction temperature, and extraction time is crucial for both the extraction yield and integrity of collagen.

[0026] More preferably, in step (2), the centrifugation conditions are 9000-10000 rpm for 10-15 min; and the dialysis time is 24-48 h.

[0027] More preferably, in step (4), the ratio of the amount of KLH and EDTA solution added is 20 mg: 2-3 mL, and the concentration of the EDTA solution is 5 mM; the ratio of the amount of collagen, dimethyl sulfoxide and 1×PBS added is 8-9 mg: 50 μL: 150 μL, and the mass ratio of collagen to KLH is 8-9 mg: 20 mg, and the dialysis time is 2-3 h; the ratio of the amount of protein-KLH, DMSO and 1×PBS added is 5-6 mg: 50 μL: 150 μL, and the stirring dialysis time is 1-2 h; the mixing ratio of the adjuvant and the injection is 1:1.

[0028] More preferably, in step (6), the concentration of the diluted carbonate buffer is 1 μg / mL, and the overnight coating temperature is 3-5℃; the mass fraction of the bovine serum albumin solution is 1%, and the incubation time at 37℃ is 2-3 h; the mouse (rabbit) anti-collagen primary antibody is incubated at 37℃ for 1-2 h, and the HRP-labeled goat anti-mouse (rabbit) secondary antibody is incubated at 37℃ for 1-2 h; the volume ratio of TMB chromogenic agent A to TMB chromogenic agent B is 1:1, and the solution is kept in the dark for 10-15 min, and the absorbance at 450 nm is measured using an ELISA reader.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] (1) The acid-enzyme combined method for extracting collagen has the following advantages: First, under mild enzymatic hydrolysis conditions, the extraction effect of collagen is better, without damaging the structure other than collagen, thus avoiding impurity contamination. The combination of enzymatic and acid methods requires less extraction solution and has a shorter production cycle. This new method has practical application value in the field of cultural relic protection and identification.

[0031] (2) Proteomics (also translated as proteomics) is the science that studies the composition and changes of proteins in cells, tissues, or organisms, using the proteome as the research object. This technology uses specific chemical reactions to replace any specific protein or polypeptide to achieve specificity and sensitivity, and has high selectivity; when processing samples, this technology has excellent accuracy and sensitivity, and can achieve high-purity samples, accurately and objectively detect protein expression, and has precise analytical results; when processing samples, this technology is simple to operate, does not require complex techniques to obtain satisfactory results, and is time-saving and low-cost.

[0032] (3) Proteomics immunoantibodies can be used to obtain a large number of highly specific and homogeneous antibodies from relatively impure antigens (cross-linked proteins). Because an "unlimited" amount of homogeneous antibodies can be obtained, they are suitable for immunological analysis methods characterized by labeled antibodies, such as ELISA. Furthermore, the high specificity and single biological function of monoclonal antibodies improve the specific recognition function of antigens and antibodies and the detection sensitivity.

[0033] (4) Traditionally prepared antibodies mostly possess characteristic sequences of the same broad category (both bovine or ovine), and can only specifically recognize antigens of the same type, but cannot accurately identify species. Especially in mammals, type I collagen accounts for the largest proportion. Proteins extracted from different species of animals of the same category contain almost identical amino acid sequences, with subtle differences existing only in one or two amino acid sequences of one or two proteins in different species. However, leather artifacts are preserved for thousands of years, and after the amino acid sequences are decomposed and broken down, not all may remain; only a portion may remain. Therefore, to accurately identify different species within the same type of leather artifact, such as sheep and goats, it is necessary to extract complete collagen from modern goat and sheep skin respectively, and accurately locate the specific peptide sequences of both to prepare corresponding antibodies, thereby further identifying different species of leather artifacts of the same category with relatively complete preservation of characteristic amino acids. In this way, not only is sensitive immunoassay achieved, but species can also be accurately located, providing great practical value for the immunological micro-trace identification of leather artifacts. Attached Figure Description

[0034] Figure 1 This is a TEM image of collagen extracted from leather artifacts in Example 1;

[0035] Figure 2 FTIR image of collagen extracted from leather artifacts in Example 1;

[0036] Figure 3 The ultraviolet-visible absorption spectrum of collagen extracted from leather artifacts in Example 1;

[0037] Figure 4 ELISA specificity graphs for four types of animal immune sera;

[0038] Figure 5 ELISA specificity graph for detecting bovine, sheep, and goat-specific antibodies on leather artifacts (I);

[0039] Figure 6 ELISA specificity graph for detecting bovine, sheep, and goat-specific antibodies on leather artifacts (II).

[0040] Figure 7 ELISA specificity diagram for detecting bovine, sheep and goat-specific antibodies on leather artifacts. Detailed Implementation

[0041] The present invention will be further described in detail below with reference to the embodiments, and the technical content and effects thereof are not limited thereto.

[0042] Example 1

[0043] Step (1): Extraction of collagen from leather artifacts: After drying the leather artifacts, immerse them in a 12wt% sodium chloride solution for 4 hours. Remove them to remove impurities, wash them three times with distilled water, filter them, and then immerse them in a defatting agent (a mixed solution of 6wt% sodium carbonate and 8wt% isopropanol, bath ratio 1:1) for 24 hours. Remove them to remove impurities, proteins, and fats, wash them three times with distilled water, air dry them naturally, and freeze them at -20℃ for later use to obtain a pretreated sample. Weigh 25g of the pretreated sample, crush it with a homogenizer, add 200mL of glacial acetic acid and 0.025g of pepsin, homogenize it with a homogenizer, extract it at 4℃ for 24 hours, centrifuge the extract (9000rpm, 15min), discard the precipitate and take the supernatant, dialyze it under distilled water conditions for 24 hours, freeze-dry it for 48 hours, and store the freeze-dried leather artifact collagen powder at room temperature for later use.

[0044] Step (2): Extraction of raw skin collagen: Extract collagen from raw skin using the same method as in step (1) to obtain raw skin collagen powder.

[0045] Step (3): Selection of characteristic sequences: The raw skin collagen powder was reconstituted with 0.1% TFA solution, and the content of peptides in the solution was quantitatively detected using NanoDrop. The reconstituted collagen solution was analyzed using ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). 1 μL of 10X iRT peptide was added, mixed, and injected. Chromatographic separation was performed using the Easy nLC 1200 nano-flow chromatography series. The sample was injected into the trap column and then subjected to gradient separation through the chromatographic analysis column. After peptide separation, DLA mass spectrometry analysis was performed using a Q-Exactive HF-X mass spectrometer. All mass spectrometry data were merged using Spectronaut 16 software, and the database retrieval and protein DIA quantitative analysis of DIA mass spectrometry data were completed in one step using the Library-free DIA (direct DIA) method. By comparing the database uniprot-collagen-17386-20221026 with uniprot-keratin-7621-20221026.fasta, the species classification of collagen can be determined through the characteristic peptide sequences. For example, the comparison shows that the extracted collagen belongs to goat skin, a major category within sheepskin.

[0046] Step (4): Obtain serum through animal immunization: Since the species of skin was obtained in step (3), antibodies against this species can be prepared. Dissolve 20 mg of condensed amino acid protein (KLH) in 2 mL of 5 mM anticoagulant (EDTA) aqueous solution to obtain KLH solution. Weigh 8 mg of raw skin collagen powder and completely dissolve it in 50 μL of dimethyl sulfoxide (DMSO), then add 150 μL of 1×PBS and mix well to obtain collagen solution. Add the collagen solution dropwise to the KLH solution and shake for 1 h. Replace with fresh 1×PBS and dialyze for 2 h. Place in a 15 mL centrifuge tube and store at 4℃ to obtain protein-KLH. Weigh 20 mg of BSA and dissolve it in 2 mL of 5 mM EDTA aqueous solution to obtain a BSA solution. Separately, dissolve 5 mg of the above-mentioned well-mixed protein-KLH completely in 50 μL LDMSO, add 150 μL of 1×PBS, mix well with 2 mL of BSA solution, gently shake, incubate at room temperature for 1 h, and then dialyze under a magnetic stirrer for 1 h to obtain a protein mixture solution. Use the prepared protein mixture solution as antigen, adjuvant, and injection agent in a 1:1 ratio, mix well, emulsify, and administer 0.2 mL of the mixture subcutaneously at multiple sites to selected immunized rabbits. After 14 days, collect whole blood, centrifuge at high speed to separate serum, and store at -20℃.

[0047] Step (5): ELISA screening of specific serum and antibody purification: The serum from step (4) was subjected to gradient detection using the ELISA method (see step 6 for specific operation), and an S-shaped curve was plotted to screen out the serum with the highest positive value and best titer. The affinity chromatography column was thoroughly washed with 20 mL of pure water and 1×PBS at a flow rate of 70 mL / h. 10 mL of the selected serum with the best titer was placed in a 50 mL centrifuge tube and filtered through a 0.45 μm pore size and 25 mm diameter microporous membrane, and the process was repeated once. The column was washed with 20 mL of 1×PBS at a flow rate of 70 mL / h. After 10 min, the column was connected to a protein analyzer, the absorbance reading of the protein analyzer was adjusted to 0, the HD-A collector was turned on, and the antibody was eluted and collected with glycine solution (pH 2.7, 0.2 M). During antibody collection, the pH of the antibody was adjusted to around 7 with 1M sodium bicarbonate. The chromatography column was washed with 20 mL of 1×PBS and pure water at a rate of 70 mL / h. 20% ethanol was added, the column was sealed, and stored at -20℃ to obtain rabbit anti-collagen primary antibody.

[0048] Step (6): Species identification of leather artifacts: Dilute the collagen powder from the leather artifacts to 1 μg / mL with carbonate buffer (CB9.6), add 100 μL to each well of an ELISA plate, and incubate overnight at 4°C. Wash with 200 μL of phosphate buffer (PBS7.4) for 3 minutes each time, for a total of 3 times. Add 200 μL of 1 wt% bovine serum albumin solution (BSA) to each well and incubate at 37°C for 2 hours. Wash with 200 μL of PBS7.4 for 3 minutes each time, for a total of 3 times. Add 100 μL of rabbit anti-collagen primary antibody (prepared in step 5) and incubate at 37°C for 1 hour. Wash with 200 μL of PBS7.4 for 3 minutes each time, for a total of 3 times. Add 100 μL of HRP-labeled goat anti-rabbit secondary antibody and incubate at 37°C for 1 hour. Wash with 200 μL of PBS 7.4 for 3 minutes each time, for a total of 5 times. Then add 50 μL of TMB chromogenic reagent A to each well, followed by 50 μL of TMB chromogenic reagent B. Shake well and incubate in the dark for 10 minutes. Finally, add 100 μL of sulfuric acid to stop the reaction. Measure the absorbance at 450 nm using a microplate reader.

[0049] Example 2

[0050] The species identification of leather artifacts was performed according to the method in Example 1. In step (5), the prepared animal serum was tested by ELISA (first step). The specific steps are as follows:

[0051] Step (5): ELISA screening of specific serum and antibody purification: The serum from step (4) was subjected to gradient detection using the ELISA method (specific operation refers to step 6), and an S-shaped curve was plotted to screen out the serum with the highest positive value and best titer. Collagen was extracted from leather artifacts using the method in step 2), diluted to 1 μg / mL with carbonate buffer (CB9.6), and added to an ELISA plate, 100 μL per well, and incubated overnight at 4°C. Washed with 200 μL of phosphate buffer (PBS7.4) for 3 minutes each time, for a total of 3 times, and 200 μL of 1 wt% bovine serum albumin solution (BSA) was added to each well, and incubated at 37°C for 2 h. Washed with 200 μL of PBS7.4 for 3 minutes each time, for a total of 3 times, and 100 μL of rabbit anti-collagen primary antibody (prepared in step 5, 4 control groups were prepared respectively) was added, and incubated at 37°C for 1 h. Wash with 200 μL of PBS 7.4 for 3 minutes each time, for a total of 3 times. Add 100 μL of HRP-labeled goat anti-mouse (rabbit) secondary antibody and incubate at 37°C for 1 hour. Wash with 200 μL of PBS 7.4 for 3 minutes each time, for a total of 5 times. Then add 50 μL of TMB chromogenic reagent A to each well, followed by 50 μL of TMB chromogenic reagent B. Shake well and incubate in the dark for 10 minutes. Finally, add 100 μL of sulfuric acid to stop the incubation. Measure the absorbance at 450 nm using a microplate reader. Figure 4 As shown, the serum with the test result closest to the positive value is the best serum.

[0052] Example 3

[0053] The species identification of leather artifacts was carried out according to the method in Example 1. For the identification of leather artifact I, raw hides of cattle, sheep, and goats were used to prepare raw hide collagen powder and specific antibodies for each. The steps for species identification are as follows:

[0054] Step (6): Dilute the collagen powder from the leather artifacts to 1 μg / mL with carbonate buffer (CB9.6), add 100 μL to each well of an ELISA plate, and incubate overnight at 4°C. Wash with 200 μL of phosphate buffer (PBS7.4) for 3 minutes each time, for a total of 3 times. Add 200 μL of 1 wt% bovine serum albumin solution (BSA) to each well and incubate at 37°C for 2 hours. Wash with 200 μL of PBS7.4 for 3 minutes each time, for a total of 3 times. Add 100 μL of bovine, sheep, or goat rabbit anti-collagen primary antibodies (prepared according to step 5) to each well and incubate at 37°C for 1 hour. Wash with 200 μL of PBS7.4 for 3 minutes each time, for a total of 3 times. Add 100 μL of HRP-labeled goat anti-mouse (rabbit) secondary antibody and incubate at 37°C for 1 hour. Wash with 200 μL of PBS 7.4 for 3 minutes each time, for a total of 5 times. Then add 50 μL of TMB chromogenic reagent A to each well, followed by 50 μL of TMB chromogenic reagent B. Shake well and incubate in the dark for 10 minutes. Finally, add 100 μL of sulfuric acid to stop the reaction. Measure the absorbance at 450 nm using a microplate reader. Figure 5 As shown, the results indicate that the absorbance value of the goatskin is close to the positive value (the positive value is the data obtained by ELISA detection of goat collagen polypeptide solution and corresponding antibody provided by Cambridge University), and this leather artifact I is goatskin.

[0055] Example 5

[0056] The species identification of leather artifacts was carried out according to the method in Example 1. For the identification of leather artifacts (Type II), raw hides of cattle, sheep, and goats were used to prepare raw hide collagen powder and specific antibodies for each. The steps for species identification are as follows:

[0057] Step (6): Extract collagen from the leather artifacts using the method in Step 2), dilute to 1 μg / mL with carbonate buffer (CB9.6), add 100 μL to each well of an ELISA plate, and incubate overnight at 4°C. Wash with 200 μL of phosphate buffer (PBS7.4) for 3 minutes each time, for a total of 3 times. Add 200 μL of 1 wt% bovine serum albumin solution (BSA) to each well and incubate at 37°C for 2 hours. Wash with 200 μL of PBS7.4 for 3 minutes each time, for a total of 3 times. Add 100 μL of bovine, sheep, and goat / rabbit anti-collagen primary antibodies (prepared in Step 5) to each well of the ELISA plate and incubate at 37°C for 1 hour. Wash with 200 μL of PBS7.4 for 3 minutes each time, for a total of 3 times. Add 100 μL of HRP-labeled goat anti-mouse (rabbit) secondary antibody and incubate at 37°C for 1 hour. Wash with 200 μL of PBS 7.4 for 3 minutes each time, for a total of 5 times. Then add 50 μL of TMB chromogenic reagent A to each well, followed by 50 μL of TMB chromogenic reagent B. Shake well and incubate in the dark for 10 minutes. Finally, add 100 μL of sulfuric acid to stop the reaction. Measure the absorbance at 450 nm using a microplate reader. Figure 6 As shown, the results indicate that the detection value of sheepskin is close to the positive value (the positive value is the data obtained by ELISA detection of sheep collagen polypeptide solution and corresponding antibody provided by Cambridge University), and this leather artifact II is sheepskin.

[0058] Example 6

[0059] The species identification of leather artifacts was carried out according to the method in Example 1. For the identification of leather artifacts (Type III), raw hides of cattle, sheep, and goats were used to prepare raw hide collagen powder and specific antibodies for each. The steps for species identification are as follows:

[0060] Step (6): Extract collagen from the leather artifacts using the method in Step 2), dilute to 1 μg / mL with carbonate buffer (CB9.6), add 100 μL to each well of an ELISA plate, and incubate overnight at 4°C. Wash with 200 μL of phosphate buffer (PBS7.4) for 3 minutes each time, for a total of 3 times. Add 200 μL of 1 wt% bovine serum albumin solution (BSA) to each well and incubate at 37°C for 2 hours. Wash with 200 μL of PBS7.4 for 3 minutes each time, for a total of 3 times. Add 100 μL of bovine, sheep, and goat / rabbit anti-collagen primary antibodies (prepared in Step 5) to each well of the ELISA plate and incubate at 37°C for 1 hour. Wash with 200 μL of PBS7.4 for 3 minutes each time, for a total of 3 times. Add 100 μL of HRP-labeled goat anti-mouse (rabbit) secondary antibody and incubate at 37°C for 1 hour. Wash with 200 μL of PBS 7.4 for 3 minutes each time, for a total of 5 times. Then add 50 μL of TMB chromogenic reagent A to each well, followed by 50 μL of TMB chromogenic reagent B. Shake well and incubate in the dark for 10 minutes. Finally, add 100 μL of sulfuric acid to stop the reaction. Measure the absorbance at 450 nm using a microplate reader. Figure 7 As shown, the results indicate that the detection value of the cowhide is close to the positive value (the positive value is the data obtained by ELISA detection of bovine collagen polypeptide solution and corresponding antibody provided by Cambridge University), and this leather artifact III is cowhide.

[0061] Comparative Example 1

[0062] The leather species identification was performed according to the method in Example 1, but only glacial acetic acid was added during collagen extraction. The specific steps are as follows:

[0063] Step (2): Collagen extraction: Weigh 25g of pretreated raw hide, crush it with a crusher, add 200mL of glacial acetic acid, homogenize it with a homogenizer, extract at 4℃ for 24h, centrifuge the extract (9000rpm, 15min), discard the precipitate and take the supernatant, dialyze under distilled water for 24h, freeze dry for 48h, and store the freeze-dried collagen powder at room temperature for later use.

[0064] Comparative Example 2

[0065] The leather species identification was performed according to the method in Example 1, but only pepsin was added during collagen extraction. The specific steps are as follows:

[0066] Step (2): Collagen extraction: Weigh 25g of pretreated raw hide, crush it with a crusher, add 0.025g of pepsin, homogenize it with a homogenizer, extract at 4℃ for 24h, centrifuge the extract (9000rpm, 15min), discard the precipitate and take the supernatant, dialyze under distilled water for 24h, freeze dry for 48h, store the freeze-dried collagen powder at room temperature for later use.

[0067] Comparative Example 3

[0068] The leather species identification was performed according to the method in Example 1, but the extraction time for collagen extraction was 8 hours. The specific steps are as follows:

[0069] Step (2): Collagen extraction: Weigh 25g of pretreated raw hide, crush it with a crusher, add 200mL of glacial acetic acid and 0.025g of pepsin, homogenize with a homogenizer, extract at 4℃ for 8h, centrifuge the extract (9000rpm, 15min), discard the precipitate and take the supernatant, dialyze under distilled water for 24h, freeze dry for 48h, and store the freeze-dried collagen powder at room temperature for later use.

[0070] Comparative Example 4

[0071] The leather species identification was performed according to the method in Example 1, but the extraction time for collagen extraction was 16 hours. The specific steps are as follows:

[0072] Step (2): Collagen extraction: Weigh 25g of pretreated raw hide, crush it with a crusher, add 200mL of glacial acetic acid and 0.025g of pepsin, homogenize with a homogenizer, extract at 4℃ for 16h, centrifuge the extract (9000rpm, 15min), discard the precipitate and take the supernatant, dialyze under distilled water for 24h, freeze dry for 48h, and store the freeze-dried collagen powder at room temperature for later use.

[0073] Comparative Example 5

[0074] The species identification of leather artifacts was performed according to the method in Example 1, but the extraction time for collagen extraction was 32 hours. The specific steps are as follows:

[0075] Step (2): Collagen extraction: Weigh 25g of pretreated raw hide, crush it with a crusher, add 200mL of glacial acetic acid and 0.025g of pepsin, homogenize with a homogenizer, extract at 4℃ for 32h, centrifuge the extract (9000rpm, 15min), discard the precipitate and take the supernatant, dialyze under distilled water for 24h, freeze dry for 48h, and store the freeze-dried collagen powder at room temperature for later use.

[0076] Table 1

[0077] Example 1 25g 12.525g 50.1％ Comparative Example 1 25g 10.575g 42.3％ Comparative Example 2 25g 8.7525g 35.01％ Comparative Example 3 25g 8.7475g 34.9％ Comparative Example 4 25g 11.3g 45.2％ Comparative Example 5 25g 10.075g 40.3％

[0078] As shown in Table 1, when acid and enzyme are present simultaneously, the extraction rate gradually increases with increasing extraction time. The antibody with the highest extraction rate was obtained, and proteomics analysis was used to obtain characteristic peptide sequences of different species, leading to the preparation of corresponding specific antibodies.

[0079] like Figure 1 As shown in the TEM image, the collagen microstructure has distinct pores, indicating that collagen has a good fibrous structure.

[0080] like Figure 2 As shown, the infrared spectrum indicates that at 3323 cm⁻¹ -1 At 2979 cm⁻¹, collagen exhibits a characteristic absorption peak, the amide A band. This is due to the stretching vibrations of the NH group in the amino group and the OH group in the carboxyl group, which are characteristic peaks of collagen. -1 At 1652 cm⁻¹, collagen exhibits a characteristic absorption peak, the amide B band, due to the CH stretching vibration; at 1652 cm⁻¹... -1 At 1550 cm⁻¹, a characteristic absorption peak, the amide I band, is observed in collagen. This is due to the stretching vibration of C=O, which is related to the secondary structure of collagen, indicating that the collagen structure has not been disrupted. -1 At 1236 cm⁻¹, collagen exhibits a characteristic absorption peak, the amide II band, which is due to the NH bending and CH bending vibrations in the collagen structure; at 1236 cm⁻¹... -1 At this point, collagen exhibits a characteristic absorption peak, the amide III band, due to the CN stretching vibration at 1452 cm⁻¹. -1 ~1236cm -1 The nearby absorption peaks indicate that the triple helix structure of the extracted collagen is intact. Overall, the infrared spectrum confirms that the extracted collagen is type I collagen.

[0081] like Figure 3 As shown, the UV-Vis absorption spectrum indicates that the maximum absorption peak is around 238 nm. The collagen macromolecular chain structure contains chromogenic groups such as -C=O, -COOH, and -NH2. The n→π* transitions of these groups form an absorption peak at 230 nm. Observing the area around 280 nm, no obvious absorption peaks are observed for any of these three groups, indicating a low content of aromatic amino acids and high purity of the extracted collagen. In summary, the presence of a maximum absorption peak around 238 nm is consistent with the characteristics of collagen.

[0082] like Figure 4The results shown are the ELISA screening results of serum extracted from animals immunized with collagen extracted under optimal conditions. Among them, the OD value of serum 4 is closest to that of the positive control, indicating that its specificity and titer are superior. It is regarded as the best serum, and the purified antibody is the best antibody.

[0083] The above embodiments of the present invention are merely illustrative examples and are not intended to limit the implementation of the invention. Those skilled in the art can make other variations and modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for species identification by extracting collagen from leather artifacts, characterized in that, Includes the following steps: (1) After removing impurities and defatting the leather artifacts, a pretreated sample was obtained; after crushing the pretreated sample, glacial acetic acid and pepsin were added. The ratio of pretreated sample to glacial acetic acid was 22~26g:200mL, and the mass of pepsin was 0.1~0.2% of the pretreated sample. Extraction was carried out at 3~5℃ for 24~30h, followed by centrifugation, dialysis, and freeze-drying to obtain collagen powder from the leather artifacts. In step (1), the impurity removal and degreasing treatment is as follows: the leather artifact is dried, immersed in a 10-12 wt% sodium chloride solution for 4-6 hours, then removed to remove impurities, washed repeatedly with distilled water, filtered dry, and then immersed in a degreasing agent, which is an aqueous solution containing 6 wt% sodium carbonate and 8 wt% isopropanol, with a bath ratio of 1:1, soaked for 22-28 hours, then removed to remove impurities, proteins and fats, washed repeatedly with distilled water, air-dried naturally, and frozen for later use; (2) Extract collagen from raw hide using the same method as in step (1) to obtain raw hide collagen powder; (3) The raw skin collagen powder was reconstituted with trichloroacetic acid solution, and the content of peptides in the solution was quantitatively detected using NanoDrop. The reconstituted collagen solution was analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. 10X iRT peptides were added, mixed and injected, and chromatographic separation was performed using the Easy nLC 1200 nano-flow chromatography series. The sample was injected into the Trap Column and then subjected to gradient separation by the chromatographic analysis column. After peptide separation, DLA mass spectrometry analysis was performed using a Q-Exactive HF-X mass spectrometer. All mass spectrometry data were merged using the software Spectronaut 16, and the database retrieval of DIA mass spectrometry data and quantitative analysis of protein DIA were completed in one step using the Library-free DIA method. The search database uniprot-collagen-17386-20221026 was compared with uniprot-keratin-7621-20221026.fasta, and the species category was determined by comparing the characteristic peptide sequences of collagen. (4) Dissolve the aggregated amino acid protein KLH in EDTA solution to obtain KLH solution; select three types of raw hides, namely bovine, sheep and goat, to prepare raw hide collagen powder and specific antibodies respectively. Dissolve the raw hide collagen powder completely in dimethyl sulfoxide, then add 1×PBS, mix well to obtain collagen solution, add it dropwise to KLH solution, shake and let stand; replace with new 1×PBS and dialyze to obtain protein-KLH; dissolve BSA in EDTA aqueous solution to obtain BSA solution; dissolve protein-KLH completely in DMSO, add 1×PBS and mix well, then add BSA solution and mix, shake gently, let stand at room temperature, then magnetically stir and dialyze to obtain protein mixed solution; use this protein mixed solution as antigen, adjuvant and injection agent, mix and extract, emulsify, and inject 0.2 mL at multiple points into selected mice or rabbits. Collect whole blood after 14 days, centrifuge at high speed to separate serum, and store at -20℃; In step (4), the ratio of KLH to EDTA solution is 20 mg: 2-3 mL, and the concentration of EDTA solution is 5 mM; the ratio of collagen, dimethyl sulfoxide, and 1×PBS is 8-9 mg: 50 μL: 150 μL, and the mass ratio of collagen to KLH is 8-9 mg: 20 mg, and the dialysis time is 2-3 h; the ratio of protein-KLH, DMSO, and 1×PBS is 5-6 mg: 50 μL: 150 μL, and the dialysis time is 1-2 h; the ratio of adjuvant to injection is 1:

1. (5) Specific serum was screened by ELISA and purified to obtain mouse or rabbit anti-collagen primary antibodies; (6) Dilute the collagen powder of the leather artifact and add it to the ELISA plate. Coat it at low temperature overnight, wash it, and then incubate it with bovine serum albumin solution. After washing, incubate it with mouse or rabbit anti-collagen primary antibody. After washing, incubate it with HRP-labeled goat anti-mouse or rabbit secondary antibody. After washing, add TMB chromogenic agent A, then add TMB chromogenic agent B, shake well, protect it from light for a period of time, and then add sulfuric acid to stop the reaction. Measure the absorbance with an ELISA reader. When the absorbance is closest to that of the positive group, the species of the leather artifact is the species of the raw hide.

2. The method for extracting collagen from leather artifacts for species identification as described in claim 1, characterized in that, In step (1), the centrifugation conditions are 9000~10000 rpm for 10~15 min; the dialysis time is 24~48 h.

3. The method for extracting collagen from leather artifacts for species identification as described in claim 1, characterized in that, The specific steps of step (5) are as follows: Refer to step (6) to perform gradient detection on the serum obtained in step (4) using the ELISA method, draw an S-shaped curve, and screen out the serum with the highest positive value and the best titer; wash the affinity chromatography column thoroughly with pure water and 1×PBS flow rate in sequence, take the serum with the best titer selected into a centrifuge tube, filter it with a microporous membrane, and repeat once; wash the column with 1×PBS, then connect it to the protein detector, adjust the absorbance reading of the protein detector to 0, turn on the computer HD-A collector, elute with glycine solution and collect the antibody; during the antibody collection process, adjust the pH value of the antibody with sodium bicarbonate in time, wash the chromatography column with 1×PBS and pure water, add 20wt% ethanol, seal, and obtain mouse or rabbit anti-collagen primary antibody, which is then stored in the refrigerator.

4. The method for extracting collagen from leather artifacts for species identification as described in claim 1, characterized in that, In step (6), the collagen powder from the leather artifacts is diluted to 1 μg / mL with carbonate buffer and coated overnight at 3-5°C; the bovine serum albumin solution has a mass fraction of 1% and is incubated at 37°C for 2-3 hours; the mouse or rabbit anti-collagen primary antibody is incubated at 37°C for 1-2 hours, and the HRP-labeled goat anti-mouse or rabbit secondary antibody is incubated at 37°C for 1-2 hours; the volume ratio of TMB chromogenic agent A to TMB chromogenic agent B is 1:1, and the mixture is kept in the dark for 10-15 minutes, and the absorbance at 450 nm is measured using an ELISA reader.

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