Method for separating and purifying protein from Tianshan red deer wrinkled stomach and application

Abstract

The invention discloses a method for separating and purifying protein from Tianshan red deer wrinkled stomach.The method comprises the steps that the Tianshan red deer wrinkled stomachs serve as research objects, adipose tissue and connective tissue of the fresh Tianshan red deer wrinkled stomachs are stripped, and then drying and degreasing treatment is conducted; the method comprises the following steps: extracting, freezing and thawing and centrifuging active proteins by using a freezing and thawing method, dialyzing supernate, freezing and drying to obtain crude protein powder, and purifying by using an ultrafiltration tube, HW-55F size exclusion chromatography and C8 reverse chromatography in combination with high performance liquid chromatography to finally obtain three high-purity Tianshan red deer wrinkled stomach proteins with anti-tumor activity, which are respectively named as S1, S2 and S3. The yields are respectively 1.10%, 1.11% and 0.61%, and the protein contents are 87.14% + / -2.16%, 84.65% + / -3.29% and 82.06% + / -1.25%. The IC50 of the inhibitory activity of the three proteins on HCT-116 colon cancer cells is respectively 55.89 [mu] g / mL + / -2.97, 4.44 [mu] g / mL + / -0.23 and 11.31 [mu] g / mL + / -0.75.

Description

Technical Field

[0001] The invention relates to the field of development and application of natural protein, and in particular to a method for separating and purifying protein from the abomasum of Tianshan red deer and its application. Background Art

[0002] Xinjiang Uygur Autonomous Region is one of the four major pastoral areas in my country, with vast grassland resources and excellent climatic conditions. With the government's support and optimization of animal husbandry policies, Xinjiang's animal husbandry has developed rapidly in recent years. The 2023 Xinjiang Uygur Autonomous Region Government Work Report proposes that Xinjiang will cultivate and expand characteristic and advantageous industries and accelerate the creation of a modern industrial system supported by the "eight major industrial clusters". Tianshan red deer is a subspecies of red deer produced in the Tianshan Mountains of Xinjiang Uygur Autonomous Region. In recent years, Tianshan red deer has also become one of the more popular species in the breeding industry. It is mainly produced in Zhaosu, Tekes, Chabuchaer, Yiwu, Balikun grassland and Mulei in Hami area of ​​Xinjiang, with large numbers and high yields. Tianshan red deer has the characteristics of gentle temperament, tolerance to roughage, strong disease resistance, reproductive ability and adaptability, and high antler production. However, the breeding of Tianshan red deer is mainly used for edible venison and medicinal antlers, while there is waste for some other parts, and sometimes it will cause environmental pollution during the processing process.

[0003] Abomasum refers to the fourth stomach used by ruminants for digestion. It is rich in various digestive enzymes, mucins, nerve regulation-related proteins, and glycoproteins involved in cell communication, which promote the digestion of ruminants. Deer stomach has the effects of strengthening the spleen and digestion, replenishing kidney yang, nourishing yin and nourishing the stomach. It also has therapeutic effects on diseases such as gastritis, stomach pain, and gastric ulcers, and has high medicinal value. Deer stomach is also often used as a delicacy on people's tables, and it is also a very good health food. At present, there have been preliminary studies on the proteins contained in the abomasum of ruminants such as cattle, sheep, and camels. Clinically, there are also lamb stomach extract vitamin B12 capsules with lamb stomach extract as the main raw material, which are mainly used for upper abdominal discomfort, bloating, loss of appetite, etc. caused by chronic gastritis. It also has good effects on infant vomiting and indigestion diarrhea. Compared with sheep, the available area of ​​deer abomasum is larger, and the material basis research of deer abomasum can make better use of it, and it can also be a supplement to lamb abomasum medicine.

[0004] Malignant tumors are the number one killer threatening human health and pose a great threat to human life and health. Although cancer can be alleviated and treated by surgery, chemotherapy, radiotherapy and other means, the surgical cure rate is low, and radiotherapy and chemotherapy methods are prone to serious side effects, most of which will damage normal cells. Therefore, the continuous search for highly effective, low-toxic, and highly specific anti-tumor drugs is one of the hot spots in the medical field. As of 2022, the leading cause of cancer death in my country is lung cancer (733,300 deaths), followed by liver cancer (316,500 deaths), gastric cancer (260,400 deaths), colorectal cancer (240,000 deaths) and esophageal cancer (187,500 deaths), accounting for 67.50% of the total cancer deaths. Because protein drugs have strong targeting and low cytotoxicity, they are very suitable as precursor compounds for finding anti-tumor drugs. There are currently 8 main types of commonly used anti-tumor drugs: cytotoxic, small molecule targeted, antibody, immune checkpoint inhibitor, hormone, antibody conjugate, and other types. The mechanisms by which protein drugs produce anti-tumor activity mainly include inhibiting cancer cell DNA replication, transcription and protein synthesis, affecting tumor cell biochemical metabolism and intracellular signal transduction pathways, affecting the expression of related oncogenes, causing disorders in the mechanisms regulating cell growth and differentiation, and regulating immune function.

[0005] A search using protein extraction and separation and anti-tumor as keywords yielded 313 relevant documents, including 28 patents involving natural products.

[0006] Patent Nos. CN202211153602.1, CN202310521920.7 and CN202410085983.7 extract and separate proteins with anti-tumor activity from lamb abomasum, small intestine and placenta. All three inventions obtain anti-tumor active proteins from ruminant tissues; Patent Nos. CN202310504212.2 and CN202211562019.6 obtain proteins with antioxidant and anti-inflammatory activities from Tianshan red deer abomasum. These five are the previous research results of this research group. The present invention is a further study based on the previous work. Compared with the previous results, the innovation of the present invention lies in the different extraction methods. The extraction methods of the above five patents are room temperature stirring extraction or ultrasound-assisted extraction, and the method used in the present invention is freeze-thaw extraction. Considering the temperature sensitivity of protein, the present invention keeps the temperature below 4°C throughout the experiment. Compared with the other five inventions, the present invention can prevent the degradation of natural protein to a greater extent and retain its intact structure. In addition, the present invention abandons the traditional ion exchange chromatography procedure and also reduces the loss of samples.

[0007] Patents CN201510031233.2, CN202111579470.4 and CN201310467193.7 all use freeze-thaw or low-temperature extraction methods, but only use salting out and gel chromatography to separate samples for separation and purification. Compared with these three inventions, the present invention adds C8 reverse chromatography and further purifies the gel separation site by using the different polarities of different proteins, thereby obtaining proteins with higher purity. Summary of the invention

[0008] The present invention aims to provide a method for separating and purifying protein from the abomasum of the Tianshan red deer and its application. The method takes the abomasum of the Tianshan red deer as the research object. First, the fresh abomasum of the Tianshan red deer is washed, the connective tissue and adipose tissue are peeled off, and then dried and defatted. Freeze-thaw extraction is used, and after centrifugation, the supernatant is dialyzed and freeze-dried to powder to obtain a crude protein extract. Based on the sample content, three anti-tumor proteins with anti-tumor activity are obtained by ultrafiltration tube, HW-55F size exclusion chromatography and C8 reverse chromatography combined with high performance liquid phase purification.

[0009] The method for preparing high-purity protein from the abomasum of Tianshan red deer of the present invention is carried out according to the following steps:

[0010] a. Wash the fresh abomasum of the Tianshan red deer with clean water, peel off the connective tissue and adipose tissue, cut into 3×3 cm squares, freeze-dry, mix with liquid nitrogen and grind, pass through a 40-mesh sieve to obtain the Tianshan red deer abomasum dry powder;

[0011] b. Soak the dry powder obtained in step a in petroleum ether at 4° C. and maintain it under magnetic stirring for 3 hours, circulate until the petroleum ether becomes colorless to fully remove small polar molecules, defatted and filtered under reduced pressure, and the filter residue is naturally air-dried to obtain defatted Tianshan elk abomasum dry powder;

[0012] c. Soak the powder obtained in step b in a 20 mmol / L phosphate buffer containing 0.3-0.5 mol / L NaCl and pH 3-5 for 3 hours, freeze at -40°C for 10 hours, and then stand at 4°C for 10 hours to thaw, repeat 2-3 times, dialyze the freeze-thaw solution, and freeze-dry to obtain a crude protein extract;

[0013] d. Prepare the crude protein extract in step c into a 3-8 mg / mL protein solution with ultrapure water, fully dissolve it, centrifuge it at 4°C for 10 minutes at a speed of 5000 rpm, and take the supernatant;

[0014] e, the supernatant in step d is loaded into the ultrafiltration tube with a cutoff of 30kDa in batches, centrifuged for 15 minutes, and the speed is set to 2500rcf, and the protein solution on the outer side of the ultrafiltration tube filter membrane is collected after each centrifugation, and the crude protein supernatant is added into the ultrafiltration tube and continued to be centrifuged, after repeating this step 2-3 times, the protein solution on the inner side of the ultrafiltration tube is merged, and the crude protein supernatant is added again, and centrifuged until the crude extract protein solution is all segmented, and then the two parts of the protein solution are freeze-dried respectively to obtain the protein positions of the inner side and the outer side of the ultrafiltration tube filter membrane;

[0015] f. The protein part inside the ultrafiltration tube membrane obtained in step e was prepared into a 5-15 mg / mL protein solution with ultrapure water, and purified by HW-55F size exclusion chromatography, with a sample load of 5 mL and a flow rate of 0.1 mL / min. The sample was collected using an automatic sample collector, and after 1-2 column volumes of elution each time, the sample collection tube was subjected to absorbance detection at a wavelength of 280 nm using an ultraviolet spectrophotometer. The samples in the test tube with the same absorption peak were combined, and the protein part with a molecular weight of 30 kDa-60 kDa was further divided into 4 protein components by HW-55F;

[0016] g. The four protein components obtained in step f were further purified by C8 reverse phase chromatography combined with high performance liquid chromatography, wherein the eluent A phase was ultrapure water containing 0.1% trifluoroacetic acid, and the eluent B phase was chromatographic grade acetonitrile, and the eluent was purified by gradient concentration: 0-5 min, 30% B; 5-8 min, 30%-34% B; 8-10 min, 34%-37% B; 10-20 min, 37%-45% B; 20-25 min, 45% B; 25-28 min, 45%-42% B; 28-30 min, 42%-34% B; 30-32 min, 34%-30% B; 32-40 min, 30% B was used to elute the sample with a flow rate set to 1.5 mL / min. The ultraviolet detector provided by the high performance liquid chromatography was used for real-time monitoring at 280 nm. Symmetrical single peak samples were directly collected, and finally three high-purity Tianshan red deer abomasum proteins were obtained, which were named S1, S2 and S3 respectively.

[0017] The Tianshan red deer abomasum proteins S1, S2 and S3 obtained by the method are used in preparing anti-colon cancer tumor drugs.

[0018] The invention discloses a method for separating and purifying protein from Tianshan red deer abomasum and its application. The method takes Tianshan red deer abomasum as a research object. First, fresh Tianshan red deer abomasum is washed, connective tissue and fat tissue are peeled off, and then dried and defatted. The protein crude extract is obtained by freeze-thaw extraction, and after centrifugation, the supernatant is dialyzed and freeze-dried to a powder state to obtain a protein crude extract, which is stored in a refrigerator at a temperature of -40°C for standby use; three proteins S1, S2 and S3 with anti-tumor activity are obtained by ultrafiltration tube, HW-55F size exclusion chromatography and C8 reverse chromatography combined with high-performance liquid phase purification; the detection and activity evaluation results show that the three proteins S1, S2 and S3 of Tianshan red deer abomasum obtained by the method have molecular weights of 66kDa, 50kDa and 41kDa respectively, and the yields are 1.10%, 1.11% and 0.61% respectively, and the protein contents are 87.14%±2.16, 84.65%±3.29 and 82.06%±1.25. The purity test was 96.24%, 92.58% and 97.37% respectively; amino acid analysis showed that the three proteins S1, S2 and S3 all met the quality protein standards. The IC50 of the three proteins S1, S2 and S3 for inhibiting HCT-116 colon cancer cells were 55.89μg / mL±2.97, 4.44μg / mL±0.23 and 11.31μg / mL±0.75 respectively.

[0019] The method for separating and purifying protein from the abomasum of Tianshan red deer and its application described in the present invention have the advantages of:

[0020] The present invention uses low-temperature freeze-thaw extraction to avoid the extraction of high-temperature proteins. The freeze-thaw extraction method forms ice particles in the cells to increase the salt concentration of the remaining cytosol, causing the cells to swell and break, thereby increasing the extraction rate of intracellular proteins. It is commonly used for animal tissue cell extraction. Low-temperature extraction can prevent the decomposition of proteins during the extraction process; the use of a mixed solution of buffer and sodium chloride can maintain the pH stability of the extract on the one hand, and on the other hand, can also increase the extraction rate of salt-soluble proteins; the present invention uses ultrafiltration tubes, HW-55F size exclusion chromatography and C8 reverse phase chromatography to purify proteins. The use of ultrafiltration tubes avoids the disadvantages of traditional ion exchange chromatography, which has a long separation time and requires dialysis and desalination of the obtained proteins, which may cause protein denaturation. Exclusion chromatography and reverse phase chromatography also purify proteins based on different principles, and can obtain protein samples with higher purity. The preparation method is easy to scale up, has high repeatability, low cost, and is pollution-free.

[0021] The innovation of the present invention is that, in combination with the characteristics of proteins, the extraction and purification methods are improved on the basis of the original extraction method, thereby reducing the steps in the experimental process that may affect the structure and activity of the compound, while reducing the experimental cost as much as possible and obtaining active protein compounds to the greatest extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention studies the flow chart;

[0023] Figure 2 The present invention uses HW-55F gel separation chromatogram;

[0024] Figure 3 This is the chromatogram of S1 purified by C8 reverse phase chromatography combined with high performance liquid chromatography in the present invention;

[0025] Figure 4 This is the chromatogram of S2 purified by C8 reverse phase chromatography combined with high performance liquid chromatography in the present invention;

[0026] Figure 5 This is the chromatogram of S3 purified by C8 reverse phase chromatography combined with high performance liquid chromatography in the present invention;

[0027] Figure 6 The 15% SDS-polyacrylamide gel electrophoresis diagram of the three anti-tumor active proteins in the invention, wherein the first lane is the protein standard, the second lane is protein S1, the third lane is protein S2, and the fourth lane is protein S3;

[0028] Figure 7 is the Fourier infrared detection spectrum of S1 in the present invention;

[0029] Figure 8 is the Fourier infrared detection spectrum of S2 in the present invention;

[0030] Fig. 9 is the Fourier infrared detection spectrum of S3 in the present invention;

[0031] Fig.10 The inhibition rates of proteins S1, S2 and S3 of the present invention on HCT-116 colon cancer cells at different concentrations. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with examples and drawings, but the uses and purposes of these exemplary embodiments are only used to list the present invention and do not constitute any form of limitation on the actual protection scope of the present invention, nor do they limit the protection scope of the present invention to them.

[0033] Example 1

[0034] a. Wash the fresh abomasum of the Tianshan red deer with clean water, peel off the connective tissue and adipose tissue, cut into 3×3 cm squares, freeze-dry, mix with liquid nitrogen and grind, pass through a 40-mesh sieve to obtain the Tianshan red deer abomasum dry powder;

[0035] b. Soak the dry powder obtained in step a in petroleum ether at 4° C. and stir it magnetically for 3 hours, circulate it until the petroleum ether becomes colorless to fully remove small polar molecules, defatted and filtered under reduced pressure, and the filter residue was naturally air-dried to obtain defatted Tianshan elk abomasum dry powder;

[0036] c. Soak the powder obtained in step b in a 20 mmol / L phosphate buffer containing 0.3 mol / L NaCl, pH 4 for 3 hours, freeze at -40°C for 10 hours, and then stand at 4°C for 10 hours to thaw, repeat twice, dialyze the freeze-thaw solution, and freeze-dry to obtain a crude protein extract;

[0037] d. Prepare the crude protein extract in step c into a 3 mg / mL protein solution with ultrapure water, fully dissolve it, centrifuge it at 4°C for 10 minutes at a speed of 5000 rpm, and take the supernatant;

[0038] e, the supernatant in step d is loaded into the ultrafiltration tube with a cutoff of 30kDa in batches, centrifuged for 15 minutes, and the speed is set to 2500rcf, and the protein solution on the outer side of the ultrafiltration tube filter membrane is collected after each centrifugation, and the crude protein supernatant is added into the ultrafiltration tube and continued to be centrifuged, after repeating this step 2-3 times, the protein solution on the inner side of the ultrafiltration tube is merged, and the crude protein supernatant is added again, and centrifuged until the crude extract protein solution is all segmented, and then the two parts of the protein solution are freeze-dried respectively to obtain the protein positions of the inner side and the outer side of the ultrafiltration tube filter membrane;

[0039] f. The protein part inside the ultrafiltration tube membrane obtained in step e was prepared into a 5 mg / mL protein solution with ultrapure water, and purified by HW-55F size exclusion chromatography, with a sample load of 5 mL and a flow rate of 0.1 mL / min. The sample was collected using an automatic sample collector, and after 1-2 column volumes of elution each time, the sample collection tube was subjected to absorbance detection at a wavelength of 280 nm using an ultraviolet spectrophotometer. The samples in the test tube with the same absorption peak were combined, and the protein part with a molecular weight of 30 kDa-60 kDa was further divided into 4 protein components by HW-55F;

[0040] g. The four protein components obtained in step f were further purified by C8 reverse phase chromatography combined with high performance liquid chromatography, wherein the eluent A phase was ultrapure water containing 0.1% trifluoroacetic acid (TFA), and the eluent B phase was pure acetonitrile (chromatographic grade), and the gradient concentrations were 0-5 min, 30% B; 5-8 min, 30%-34% B; 8-10 min, 34%-37% B; 10-20 min, 37%-45% B; 20-25 min, 45% B; 25-28 min, 45%-42% B; 28-30 min, 42%-34% B; 30-32 min, 34%-30% B; 32-40 min, 30% B eluted the sample with a flow rate of 1.5 mL / min, and used the UV detector of the HPLC to monitor in real time at 280 nm, directly collecting symmetrical single peak samples, and finally obtained three high-purity proteins, named S1, S2, and S3;

[0041] The medium-to-high purity proteins S1, S2 and S3 obtained in step g were subjected to high performance liquid chromatography purity analysis, chemical property detection, amino acid composition analysis, infrared characteristic functional group detection, liquid chromatography-mass spectrometry homologous protein detection and anti-tumor activity screening.

[0042] Example 2

[0043] a. Wash the fresh abomasum of the Tianshan red deer with clean water, peel off the connective tissue and adipose tissue, cut into 3×3 cm squares, freeze-dry, mix with liquid nitrogen and grind, pass through a 40-mesh sieve to obtain the Tianshan red deer abomasum dry powder;

[0044] b. Soak the dry powder obtained in step a in petroleum ether at 4° C. and maintain it under magnetic stirring for 3 hours, circulate until the petroleum ether becomes colorless to fully remove small polar molecules, defatted and filtered under reduced pressure, and the filter residue is naturally air-dried to obtain defatted Tianshan elk abomasum dry powder;

[0045] c. Soak the powder obtained in step b in a 20 mmol / L phosphate buffer containing 0.45 mol / L NaCl and pH 4 for 3 hours, freeze at -40°C for 10 hours, and then stand at 4°C for 10 hours to thaw, repeat twice, dialyze the freeze-thaw solution, and freeze-dry to obtain a crude protein extract;

[0046] d. Prepare the crude protein extract in step c into 8 mg / mL protein solution with ultrapure water, fully dissolve it, centrifuge it at 4°C for 10 minutes at a speed of 5000 rpm, and take the supernatant;

[0047] e, the supernatant in step d is loaded into the ultrafiltration tube with a cutoff of 30kDa in batches, centrifuged for 15 minutes, and the speed is set to 2500rcf, and the protein solution on the outer side of the ultrafiltration tube filter membrane is collected after each centrifugation, and the crude protein supernatant is added into the ultrafiltration tube and continued to be centrifuged, after repeating this step 2-3 times, the protein solution on the inner side of the ultrafiltration tube is merged, and the crude protein supernatant is added again, and centrifuged until the crude extract protein solution is all segmented, and then the two parts of the protein solution are freeze-dried respectively to obtain the protein positions of the inner side and the outer side of the ultrafiltration tube filter membrane;

[0048] f. The protein part inside the ultrafiltration tube membrane obtained in step e was prepared into a 15 mg / mL protein solution with ultrapure water, and purified by HW-55F size exclusion chromatography, with a sample load of 5 mL and a flow rate of 0.1 mL / min. The sample was collected using an automatic sample collector, and after 1-2 column volumes of elution each time, the sample collection tube was subjected to absorbance detection at a wavelength of 280 nm using an ultraviolet spectrophotometer. The samples in the test tube with the same absorption peak were combined, and the protein part with a molecular weight of 30-60 kDa was further divided into 4 protein components by HW-55F;

[0049] g. The four protein components obtained in step f were further purified by C8 reverse phase chromatography combined with high performance liquid chromatography, wherein the eluent A phase was ultrapure water containing 0.1% trifluoroacetic acid (TFA), and the eluent B phase was pure acetonitrile (chromatographic grade), and the gradient concentrations were 0-5 min, 30% B; 5-8 min, 30%-34% B; 8-10 min, 34%-37% B; 10-20 min, 37%-45% B; 20-25 min, 45% B; 25-28 min, 45%-42% B; 28-30 min, 42%-34% B; 30-32 min, 34%-30% B; 32-40 min, 30% B eluted the sample with a flow rate of 1.5 mL / min, and used the UV detector of the HPLC to monitor in real time at 280 nm, directly collecting symmetrical single peak samples, and finally obtained three high-purity proteins, named S1, S2, and S3;

[0050] The high-purity proteins S1, S2, and S3 in step g are subjected to high-performance liquid phase purity analysis, chemical property detection, amino acid composition analysis, infrared characteristic functional group detection, liquid chromatography-mass spectrometry homologous protein detection, and anti-tumor activity screening.

[0051] Example 3

[0052] a. Wash the fresh abomasum of the Tianshan red deer with clean water, peel off the connective tissue and adipose tissue, cut into 3×3 cm squares, freeze-dry, mix with liquid nitrogen and grind, pass through a 40-mesh sieve to obtain the Tianshan red deer abomasum dry powder;

[0053] b. Soak the dry powder obtained in step a in petroleum ether at 4° C. and maintain it under magnetic stirring for 3 hours, circulate until the petroleum ether becomes colorless to fully remove small polar molecules, defatted and filtered under reduced pressure, and the filter residue is naturally air-dried to obtain defatted Tianshan elk abomasum dry powder;

[0054] c. Soak the powder obtained in step b in a 20 mmol / L phosphate buffer containing 0.5 mol / L NaCl, pH 4 for 3 hours, freeze at -40°C for 10 hours, and then stand at 4°C for 10 hours to thaw, repeat 3 times, dialyze the freeze-thaw solution, and freeze-dry to obtain a crude protein extract;

[0055] d. Prepare the crude protein extract in step c into a 5 mg / mL protein solution with ultrapure water, fully dissolve it, centrifuge it at 4°C for 10 minutes at a speed of 5000 rpm, and take the supernatant;

[0056] e, the supernatant in step d is loaded into the ultrafiltration tube with a cutoff of 30kDa in batches, centrifuged for 15 minutes, and the speed is set to 2500rcf, and the protein solution on the outer side of the ultrafiltration tube filter membrane is collected after each centrifugation, and the crude protein supernatant is added into the ultrafiltration tube and continued to be centrifuged, after repeating this step 2-3 times, the protein solution on the inner side of the ultrafiltration tube is merged, and the crude protein supernatant is added again, and centrifuged until the crude extract protein solution is all segmented, and then the two parts of the protein solution are freeze-dried respectively to obtain the protein positions of the inner side and the outer side of the ultrafiltration tube filter membrane;

[0057] f. The protein part inside the ultrafiltration tube membrane obtained in step e was prepared into a 10 mg / mL protein solution with ultrapure water, and purified by HW-55F size exclusion chromatography, with a sample load of 5 mL and a flow rate of 0.1 mL / min. The sample was collected using an automatic sample collector, and after 1-2 column volumes of elution each time, the sample collection tube was subjected to absorbance detection at a wavelength of 280 nm using an ultraviolet spectrophotometer. The samples in the test tube with the same absorption peak were combined, and the protein part with a molecular weight of 30-60 kDa was further divided into 4 protein components by HW-55F;

[0058] g. The four protein components obtained in step f were further purified by C8 reverse phase chromatography combined with high performance liquid chromatography, wherein the eluent A phase was ultrapure water containing 0.1% trifluoroacetic acid (TFA), and the eluent B phase was pure acetonitrile (chromatographic grade), and the gradient concentrations were 0-5 min, 30% B; 5-8 min, 30%-34% B; 8-10 min, 34%-37% B; 10-20 min, 37%-45% B; 20-25 min, 45% B; 25-28 min, 45%-42% B; 28-30 min, 42%-34% B; 30-32 min, 34%-30% B; 32-40 min, 30% B eluted the sample with a flow rate of 1.5 mL / min, and used the UV detector of the HPLC to monitor in real time at 280 nm, directly collecting symmetrical single peak samples, and finally obtained three high-purity proteins, named S1, S2, and S3;

[0059] The high-purity proteins S1, S2, and S3 in step g are subjected to high-performance liquid phase purity analysis, chemical property detection, amino acid composition analysis, infrared characteristic functional group detection, liquid chromatography-mass spectrometry homologous protein detection, and anti-tumor activity screening.

[0060] Example 4

[0061] a. Wash the fresh abomasum of the Tianshan red deer with clean water, peel off the connective tissue and adipose tissue, cut into 3×3 cm squares, freeze-dry, mix with liquid nitrogen and grind, pass through a 40-mesh sieve to obtain the Tianshan red deer abomasum dry powder;

[0062] b. Soak the dry powder obtained in step a in petroleum ether at 4° C. and maintain it under magnetic stirring for 3 hours, circulate until the petroleum ether becomes colorless to fully remove small polar molecules, defatted and filtered under reduced pressure, and the filter residue is naturally air-dried to obtain defatted Tianshan elk abomasum dry powder;

[0063] c. Soak the powder obtained in step b in a 20 mmol / L phosphate buffer containing 0.3 mol / L NaCl, pH 4 for 3 hours, freeze at -40°C for 10 hours, and then stand at 4°C for 10 hours to thaw, repeat 3 times, dialyze the freeze-thaw solution, and freeze-dry to obtain a crude protein extract;

[0064] d. Prepare the crude protein extract in step c into a 5 mg / mL protein solution with ultrapure water, fully dissolve it, centrifuge it at 4°C for 10 minutes at a speed of 5000 rpm, and take the supernatant;

[0065] e, the supernatant in step d is loaded into the ultrafiltration tube with a cutoff of 30kDa in batches, centrifuged for 15 minutes, and the speed is set to 2500rcf, and the protein solution on the outer side of the ultrafiltration tube filter membrane is collected after each centrifugation, and the crude protein supernatant is added into the ultrafiltration tube and continued to be centrifuged, after repeating this step 2-3 times, the protein solution on the inner side of the ultrafiltration tube is merged, and the crude protein supernatant is added again, and centrifuged until the crude extract protein solution is all segmented, and then the two parts of the protein solution are freeze-dried respectively to obtain the protein positions of the inner side and the outer side of the ultrafiltration tube filter membrane;

[0066] f. The protein part inside the ultrafiltration tube membrane obtained in step e was prepared into a 15 mg / mL protein solution with ultrapure water, and purified by HW-55F size exclusion chromatography, with a sample load of 5 mL and a flow rate of 0.1 mL / min. The sample was collected using an automatic sample collector, and after 1-2 column volumes of elution each time, the sample collection tube was subjected to absorbance detection at a wavelength of 280 nm using an ultraviolet spectrophotometer. The samples in the test tube with the same absorption peak were combined, and the protein part with a molecular weight of 30 kDa-60 kDa was further divided into 4 protein components by HW-55F;

[0067] g. The four protein components obtained in step f were further purified by C8 reverse phase chromatography combined with high performance liquid chromatography, wherein the eluent phase A was ultrapure water containing 0.1% trifluoroacetic acid (TFA), and the phase B was pure acetonitrile (chromatographic grade). The sample was eluted by gradient concentration: 0-5min, 30% B; 5-8min, 30%-34% B; 8-10min, 34%-37% B; 10-20min, 37%-45% B; 20-25min, 45% B; 25-28min, 45%-42% B; 28-30min, 42%-34% B; 30-32min, 34%-30% B; 32-40min, 30% B, the flow rate was set to 1.5mL / min, and the high performance liquid phase was monitored in real time at 280nm using the UV detector provided, and symmetrical single symmetrical peak samples were directly collected, and finally three high-purity proteins were obtained, which were named S1, S2, and S3 respectively;

[0068] The high-purity proteins S1, S2, and S3 in step g are subjected to high-performance liquid phase purity analysis, chemical property detection, amino acid composition analysis, infrared characteristic functional group detection, liquid chromatography-mass spectrometry homologous protein detection, and anti-tumor activity screening.

[0069] Example 5

[0070] a. Wash the fresh abomasum of the Tianshan red deer with clean water, peel off the connective tissue and adipose tissue, cut into 3×3 cm squares, freeze-dry, mix with liquid nitrogen and grind, pass through a 40-mesh sieve to obtain the Tianshan red deer abomasum dry powder;

[0071] b. Soak the dry powder obtained in step a in petroleum ether at 4° C. and maintain it under magnetic stirring for 3 hours, circulate until the petroleum ether becomes colorless to fully remove small polar molecules, defatted and filtered under reduced pressure, and the filter residue is naturally air-dried to obtain defatted Tianshan elk abomasum dry powder;

[0072] c. Soak the powder obtained in step b in a 20 mmol / L phosphate buffer containing 0.45 mol / L NaCl and pH 4 for 3 hours, freeze at -40°C for 10 hours, and then stand at 4°C for 10 hours to thaw, repeat 3 times, dialyze the freeze-thaw solution, and freeze-dry to obtain a crude protein extract;

[0073] d. Prepare the crude protein extract in step c into 8 mg / mL protein solution with ultrapure water, fully dissolve it, centrifuge it at 4°C for 10 minutes at a speed of 5000 rpm, and take the supernatant;

[0074] e, the supernatant in step d is loaded into the ultrafiltration tube with a cutoff of 30kDa in batches, centrifuged for 15 minutes, and the speed is set to 2500rcf, and the protein solution on the outer side of the ultrafiltration tube filter membrane is collected after each centrifugation, and the crude protein supernatant is added into the ultrafiltration tube and continued to be centrifuged, after repeating this step 2-3 times, the protein solution on the inner side of the ultrafiltration tube is merged, and the crude protein supernatant is added again, and centrifuged until the crude extract protein solution is all segmented, and then the two parts of the protein solution are freeze-dried respectively to obtain the protein positions of the inner side and the outer side of the ultrafiltration tube filter membrane;

[0075] f. The protein part inside the ultrafiltration tube membrane obtained in step e was prepared into a 15 mg / mL protein solution with ultrapure water, and purified by HW-55F size exclusion chromatography, with a sample load of 5 mL and a flow rate of 0.1 mL / min. The sample was collected using an automatic sample collector, and after 1-2 column volumes of elution each time, the sample collection tube was subjected to absorbance detection at a wavelength of 280 nm using an ultraviolet spectrophotometer. The samples in the test tube with the same absorption peak were combined, and the protein part with a molecular weight of 30 kDa-60 kDa was further divided into 4 protein components by HW-55F;

[0076] g. The four protein components obtained in step f were further purified by C8 reverse phase chromatography combined with high performance liquid chromatography, wherein the eluent A phase was ultrapure water containing 0.1% trifluoroacetic acid (TFA), and the B phase was pure acetonitrile (chromatographic grade). The samples were eluted by gradient concentrations of 0-5 min, 30% B; 5-8 min, 30%-34% B; 8-10 min, 34%-37% B; 10-20 min, 37%-45% B; 20-25 min, 45% B; 25-28 min, 45%-42% B; 28-30 min, 42%-34% B; 30-32 min, 34%-30% B; 32-40 min, 30% B, the flow rate was set to 1.5 mL / min, and the high performance liquid chromatography was used for real-time monitoring at 280 nm to directly collect symmetrical single symmetrical peak samples. Finally, three high-purity proteins were obtained, named S1, S2, and S3;

[0077] The high-purity proteins S1, S2 and S3 in step g are subjected to high-performance liquid phase purity analysis, chemical property detection, amino acid composition analysis, infrared characteristic functional group detection, liquid chromatography-mass spectrometry homologous protein detection and anti-tumor activity screening.

[0078] Example 6

[0079] a. Wash the fresh abomasum of the Tianshan red deer with clean water, peel off the connective tissue and adipose tissue, cut into 3×3 cm squares, freeze-dry, mix with liquid nitrogen and grind, pass through a 40-mesh sieve to obtain the Tianshan red deer abomasum dry powder;

[0080] b. Soak the dry powder obtained in step a in petroleum ether at 4° C. and maintain it under magnetic stirring for 3 hours, circulate until the petroleum ether becomes colorless to fully remove small polar molecules, defatted and filtered under reduced pressure, and the filter residue is naturally air-dried to obtain defatted Tianshan elk abomasum dry powder;

[0081] c. Soak the powder obtained in step b in a 20 mmol / L phosphate buffer containing 0.5 mol / L NaCl, pH 4 for 3 hours, freeze at -40°C for 10 hours, and then stand at 4°C for 10 hours to thaw, repeat 3 times, dialyze the freeze-thaw solution, and freeze-dry to obtain a crude protein extract;

[0082] d. Prepare the crude protein extract in step c into 8 mg / mL protein solution with ultrapure water, fully dissolve it, centrifuge it at 4°C for 10 minutes at a speed of 5000 rpm, and take the supernatant;

[0083] e, the supernatant in step d is loaded into the ultrafiltration tube with a cutoff of 30kDa in batches, centrifuged for 15 minutes, and the speed is set to 2500rcf, and the protein solution on the outer side of the ultrafiltration tube filter membrane is collected after each centrifugation, and the crude protein supernatant is added into the ultrafiltration tube and continued to be centrifuged, after repeating this step 2-3 times, the protein solution on the inner side of the ultrafiltration tube is merged, and the crude protein supernatant is added again, and centrifuged until the crude extract protein solution is all segmented, and then the two parts of the protein solution are freeze-dried respectively to obtain the protein positions of the inner side and the outer side of the ultrafiltration tube filter membrane;

[0084] f. The protein part inside the ultrafiltration tube membrane obtained in step e was prepared into a 12 mg / mL protein solution with ultrapure water, and purified by HW-55F size exclusion chromatography, with a sample load of 5 mL and a flow rate of 0.1 mL / min. The sample was collected using an automatic sample collector, and after 1-2 column volumes of elution each time, the sample collection tube was subjected to absorbance detection at a wavelength of 280 nm using an ultraviolet spectrophotometer. The samples in the test tube with the same absorption peak were combined, and the protein part with a molecular weight of 30-60 kDa was further divided into 4 protein components by HW-55F;

[0085] g. The four protein components obtained in step f were further purified by C8 reverse phase chromatography combined with high performance liquid chromatography, wherein the eluent A phase was ultrapure water containing 0.1% trifluoroacetic acid (TFA), and the eluent B phase was pure acetonitrile (chromatographic grade), and the gradient concentrations were 0-5 min, 30% B; 5-8 min, 30%-34% B; 8-10 min, 34%-37% B; 10-20 min, 37%-45% B; 20-25 min, 45% B; 25-28 min, 45%-42% B; 28-30 min, 42%-34% B; 30-32 min, 34%-30% B; 32-40 min, 30% B eluted the sample with a flow rate of 1.5 mL / min, and used the UV detector of the HPLC to monitor in real time at 280 nm, directly collecting symmetrical single peak samples, and finally obtained three high-purity proteins, named S1, S2, and S3;

[0086] The high-purity proteins S1, S2 and S3 obtained in step g are subjected to high-performance liquid phase purity analysis, chemical property detection, amino acid composition analysis, infrared characteristic functional group detection, liquid chromatography-mass spectrometry homologous protein detection and anti-tumor activity screening.

[0087] Example 7

[0088] The crude extract obtained in step b was extracted by conventional stirring method, the extraction solution was 20 mmol / L phosphate buffer containing 0.45 mol / L NaCl, pH 4, and the extraction was performed 3 times at room temperature. The extract was dialyzed and freeze-dried to obtain a stirred protein crude extract; the protein content and anti-tumor activity of the stirred crude extract and the crude extracts obtained in Examples 1-5 were measured as shown in Table 1, and the high-purity protein and protein crude extract obtained in Example 5 were selected for protein content measurement as shown in Table 1:

[0089] Experimental methods:

[0090] The protein content was detected using a BCA kit, with standard bovine serum albumin as the standard curve. The standard was prepared into protein solutions of 1.2 mg / mL, 1 mg / mL, 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL, 0.2 mg / mL and 0 mg / mL. To ensure that the sample protein concentration fell as far as possible at the middle point of the standard curve, the sample concentration was set to 2 mg / mL. According to the required amount of working solution, working solution A and working solution B were mixed in a ratio of 50:1, shaken and stored in the dark (prepared and used immediately). 100 μL of protein solution (standard solution and test protein solution) and 800 μL of working mixture were added to a .5mL centrifuge tube. Each experiment was repeated 3 times in parallel. After reacting at 37°C in a desktop drying oven for 30 minutes, 250 μL of the mixed reaction solution was transferred to a 96-well plate. The absorbance was detected at a wavelength of 562 nm using an ELISA reader. A standard curve was made with the standard protein concentration and absorbance as the independent variable and the dependent variable. The sample absorbance was substituted into the standard curve to obtain the sample protein concentration (mg / mL). The sample protein content was obtained by formula 1.

[0091]

[0092] Note: C1 is the sample protein concentration calculated by inserting the test sample into the standard curve, and C0 is the sample concentration of the test sample;

[0093] The antitumor activity of the two crude extracts was screened by thiazolyl blue colorimetry. The samples were prepared into 1 mg / mL protein solution with cell culture medium. The cells in the logarithmic growth phase were digested and resuspended, and then seeded in 96-well plates at the corresponding cell density. 100 μL of cell solution was added to each well. After culturing in a cell culture incubator at 37°C, 5% CO2 and 95% humidity for 24 hours, 20 μL of the test compound was added, the supernatant was discarded, and 100 μL of protein solution of different concentrations was added; 3-6 replicate wells were set up and a cell-free background group, a solvent control group and a positive control group were set up separately; after continuing to culture at 37°C and 5% CO2 for 48 hours, the supernatant was discarded, 100 μL / well thiazolyl blue solution was added, and the culture was continued for 2-4 hours; the supernatant was discarded, and 150 μL of each well was added DMSO was added and the mixture was shaken at a low speed for 10 min to dissolve the crystals. After the formazan was dissolved, the absorbance (OD value) at 570 nm was detected by an ELISA reader. The inhibition rate of the compound on tumor cell growth was calculated using formula 3.

[0094]

[0095] Table 1 Protein contents of three high-purity proteins and freeze-thaw crude extracts from Tianshan red deer abomasum

[0096]

[0097] Note: In the tumor cell inhibition ability, the more * the stronger the inhibition ability;

[0098] It can be seen from Table 1 that among the crude protein extracts obtained by the five different methods in Examples 1 to 5, the protein extraction rate of Example 5 was the highest, so the three high-purity proteins S1, S2 and S3 obtained by purifying the crude extract obtained in Example 5 were further analyzed; in addition, the crude protein extract obtained by the conventional stirring method in Example 7 was weaker than the crude extract obtained by freeze-thaw in terms of protein extraction rate and activity, which proves that the freeze-thaw method is one of the effective methods for preparing active abomasum protein.

[0099] Example 8

[0100] The protein content of the high-purity protein obtained in Example 5 was determined using the method described in Example 7 (Table 2):

[0101] Table 2 Contents of three kinds of high-purity proteins in Tianshan red deer stomach

[0102]

[0103] It can be seen from Table 2 that the protein contents of the three high-purity proteins S1, S2 and S3 are all above 80%, and the protein purity gradually increases during the purification process.

[0104] Example 9

[0105] The high-purity protein obtained in Example 5 was selected for molecular weight estimation and purity identification:

[0106] Experimental methods:

[0107] The molecular weights of the three proteins S1, S2 and S3 were estimated by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE); 4% stacking gel and 15% separation gel were used, and 20 μL of protein sample and 3.5 μL of molecular weight marker were added to different lanes of the gel under non-reducing conditions for analysis, the stacking gel was run at 75V, and the separation gel was run at 120V until the sample reached the bottom of the separation gel and was stained with Coomassie blue; the molecular weights of the three proteins S1, S2 and S3 were estimated by comparison with the molecular weight marker bands; the purity of the samples was detected by high performance liquid chromatography using a TSKgel G3000PWXl analytical column (TOSOH, Shanghai), the eluent was ultrapure water, and the flow rate was 0.8 mL / min;

[0108] The results showed that the molecular weights of the three proteins S1, S2 and S3 were 66 kDa, 50 kDa and 41 kDa, and the purity of the three proteins was 96.24%, 92.58% and 97.37% according to the liquid phase results.

[0109] Example 10

[0110] The high-purity protein obtained in Example 5 was selected for infrared characteristic peak analysis:

[0111] Experimental methods:

[0112] Take a small amount of sample and mix with dry potassium bromide powder, grind, press at 10-20MPa for 2 minutes to make a translucent sheet, and use the same method to treat KBr powder alone as a blank background; -1 The infrared spectra of the three high-purity proteins were obtained by scanning in the frequency range, and the relevant functional group information was derived. In addition, PeakFit v4.12 was used to analyze the amide I band of the infrared spectrum (1700-1600cm -1 ) region to perform baseline correction, decurling and second-order derivative fitting to determine the proportion of various secondary structures of the protein;

[0113] The three proteins S1, S2 and S3 all showed characteristic peaks of proteins: at 3400-3200cm -1 The broad and strong absorption peaks are caused by the stretching vibration of NH and OH; at 1690-1630cm -1 The absorption peaks from C=O stretching vibration and 1400-1060cm -1The absorption peak is caused by CN stretching vibration; the absorption peak is caused by NH bending vibration at around 1500cm-1; 3400cm -1 Up to 3200cm -1 The broad and strong absorption peaks between 1650cm and 1650cm may be caused by the stretching vibration of NH. -1 The absorption peaks on the left and right are caused by the asymmetric vibration of C=O acetylamino; at about 1550cm -1 It is the absorption peak produced by NH angle vibration. Secondary structure analysis shows that all three proteins have four structures: α-helix, β-sheet, β-turn and random coil (Table 3);

[0114] Table 3 Secondary structure analysis of three high-purity proteins

[0115]

[0116] Among them, S2 is mainly α-helix and S3 is mainly β-fold.

[0117] Embodiment 11

[0118] The high-purity protein obtained in Example 5 was selected for amino acid composition analysis (Table 4):

[0119] Experimental methods:

[0120] Sample acid hydrolysis: 8 mg of sample was placed in a 20 mL screw cap (with built-in septum) headspace bottle, 10 mL of 0.1% phenol and 6 mol / L hydrochloric acid aqueous solution were added, the lid was tightened, the mixture was shaken and mixed, and the reaction was carried out at 110°C for 24 hours. After the reaction was completed, the reaction solution was transferred to a 100 mL distillation bottle, and the reaction solution was evaporated to near dryness under reduced pressure at 75°C to remove the residual hydrochloric acid in the reaction solution. Then, 12 mL of 0.2 mol / L hydrochloric acid aqueous solution was used to dissolve the residue three times, and the residue was transferred to a 20 mL glass stoppered graduated test tube, and then the volume was made up to 20 mL with pure water for derivatization;

[0121] Phenyl isothiocyanate derivatization: Take 20 μL of the sample solution after acid hydrolysis, place it in a 1.5 mL centrifuge tube, add 100 μL of 1 mol / L triethylamine acetonitrile solution and 100 μL of 0.2 mol / L phenyl isothiocyanate acetonitrile solution, mix well, react at room temperature for 1 hour, then add 400 μL of n-hexane, tighten the lid and shake vigorously for 5-10 seconds, let it stand to separate, take 200 μL of the lower layer solution and mix it with 800 μL of water, filter it with a 0.22 μm syringe filter, and wait for analysis;

[0122] HPLC amino acid composition analysis: According to the instruction manual of Diamonsil AAA amino acid analyzer, the derived amino acid mixed standard and sample were analyzed by HPLC, and the types of amino acids contained in the sample were determined by comparing the peak elution time of the sample spectrum with the peak elution time of different amino acids in the mixed standard; the relative molar ratio of amino acids contained in the sample was determined by comparing the peak area ratio of each peak in the sample spectrum;

[0123] The occupancy rates of the four essential amino acids in the protein are 32.4%, 43.26%, 47.63% and 58.76% respectively, which meet the standards of high-quality protein and can be absorbed and utilized as a nutritional supplement;

[0124] Table 4 Relative molar ratio of amino acids in three high-purity proteins of Tianshan red deer abomasal mucosa

[0125]

[0126] Note: “*” indicates essential amino acids, and “#” indicates hydrophobic amino acids.

[0127] Example 12

[0128] The high-purity protein obtained in Example 5 was selected for homologous protein analysis:

[0129] Experimental methods:

[0130] The total protein of the two crude extracts was identified by LC-MS / MS; the protein band was cut out from the SDS-PAGE gel and placed in a centrifuge tube with a cap, the gel band was fully decolorized and crushed, then trypsin buffer was added, and the reaction was carried out at 37°C for 16-18 hours before capillary high performance liquid chromatography separation (Zorbax 300SB-C18 column); phase A: 0.1% formic acid aqueous solution, phase B: 0.1% formic acid acetonitrile aqueous solution (acetonitrile volume fraction is 84%), using a gradient elution program: 0-50 minutes, 4%-50% B; 50-54 minutes, 50%-100% B; 54-60 minutes, 100% B; the separated enzymatic hydrolysate was analyzed by mass spectrometry using a QExactive HF-X mass spectrometer, the analysis time: 60 minutes, the detection method: positive ion, the mass-to-charge ratio of the polypeptide and the polypeptide fragments was obtained by full scanning and collecting 10 fragment spectra; using MaxQuant 1.5.5.1 The software visualizes the spectrum results and uses Cervus elaphus as the species limit in the UniPort database to search for proteins with the same characteristic peptides as the peptides detected by mass spectrometry to obtain the final results of protein identification and quantitative analysis;

[0131] The results showed that S1 had high homology with albumin (A0A212D5P0) and actin (A0A212CUK9), with similarities of 39% and 19.4%, respectively; S2 had high homology with acetaldehyde dehydrogenase (A0A212C5A0) and protein disulfide isomerase (A0A212DB37), with homology of 27.4% and 24.8%, respectively; S3 had high homology with thiol oxidase (A0A212CGS9) and Rab GDP dissociation inhibitor (A0A212CAH5), with similarities of 45.28% and 29.03%, respectively. At the same time, the three proteins also had similarities with homologous proteins in secondary structure.

[0132] Example 13

[0133] The high-purity protein obtained in Example 5 was screened for anti-tumor activity (HCT-116 cell inhibitory activity) using the method described in Example 7:

[0134] The results showed that the IC values ​​of the three proteins S1, S2 and S3 for the inhibitory activity against HCT-116 50 The IC values ​​of the frozen-thawed protein extract on tumor cells in Example 5 were 55.89 μg / mL±2.97, 4.44 μg / mL±0.23 and 11.31 μg / mL±0.75 respectively. 50 It was 12.91±0.65μg / mL. The anti-tumor activity gradually increased with the increase of sample purity. Several anti-tumor proteins had additive or synergistic effects when producing active effects.

[0135] The present invention discloses a method for separating and purifying protein from the abomasum of the Tianshan red deer and its application. The method firstly uses the freeze-thaw method to extract the washed, defatted and dehydrated abomasum of the Tianshan red deer to obtain a crude protein extract; the crude extract is dissolved in ultrapure water, and the sample is divided into a large molecular weight portion and a small molecular weight portion using an ultrafiltration tube; the large molecular weight portion with a higher content of the sample is purified using HW-55F gel chromatography, and further purified using C8 reverse phase chromatography combined with high performance liquid phase to obtain three high-purity anti-tumor protein substances with yields of 1.10%, 1.11% and 0.61%, respectively, and molecular weights of 66kDa, 50kDa and 41kDa. The protein contents of the three proteins were 87.14%±2.16, 84.65%±3.29 and 82.06%±1.25 through component detection, and the characteristic peaks of amide bonds detected by infrared further illustrate their protein structures and secondary structure compositions. The amino acid composition shows that the three proteins meet the standards of high-quality proteins. The anti-tumor activity was evaluated, and the IC values ​​of the three proteins for inhibiting HCT-116 colon cancer cells were 0.1%, ... 50The separation and preparation methods of the present invention are more scientific, and a high-purity protein with strong anti-tumor activity is obtained. The production process is simpler and more reasonable, and provides a certain raw material basis for the development of anti-tumor drugs, especially anti-colon cancer drugs.

Claims

1. A method for preparing high-purity protein from the abomasum of Tianshan red deer, characterized in that The method proceeds in the following steps: a. Wash the fresh abomasum of the Tianshan red deer with clean water, peel off the connective tissue and adipose tissue, cut into 3×3 cm squares, freeze-dry, mix with liquid nitrogen and grind, pass through a 40-mesh sieve to obtain the Tianshan red deer abomasum dry powder; b. Soak the dry powder obtained in step a in petroleum ether at 4° C. and maintain it under magnetic stirring for 3 hours, circulate until the petroleum ether becomes colorless to fully remove small polar molecules, defatted and filtered under reduced pressure, and the filter residue is naturally air-dried to obtain defatted Tianshan elk abomasum dry powder; c. Soak the powder obtained in step b in a 20 mmol / L phosphate buffer containing 0.3-0.5 mol / L NaCl and pH 3-5 for 3 hours, freeze at -40°C for 10 hours, and then stand at 4°C for 10 hours to thaw, repeat 2-3 times, dialyze the freeze-thaw solution, and freeze-dry to obtain a crude protein extract; d. Prepare the crude protein extract in step c into a 3-8 mg / mL protein solution with ultrapure water, fully dissolve it, centrifuge it at 4°C for 10 minutes at a speed of 5000 rpm, and take the supernatant; e. The supernatant in step d was loaded into an ultrafiltration tube with a cutoff of 30 kDa in batches, and centrifuged for 15 minutes at a speed of 2500 rcf. After each centrifugation, the protein solution on the outside of the ultrafiltration tube filter membrane was collected, and the crude protein supernatant was added into the ultrafiltration tube for continued centrifugation. After repeating this step 2-3 times, the protein solution on the inside of the ultrafiltration tube was merged, and the crude protein supernatant was added again, and centrifuged until the crude extract protein solution was all segmented, and then the two parts of the protein solution were freeze-dried respectively to obtain two kinds of dried protein parts on the inside and outside of the ultrafiltration tube filter membrane; f. The protein part inside the ultrafiltration tube membrane obtained in step e was prepared into a 5-15 mg / mL protein solution with ultrapure water, and purified by HW-55F size exclusion chromatography, with a sample volume of 5 mL and a flow rate of 0.1 mL / min. The sample was collected using an automatic sample collector. After 1-2 column volumes of elution each time, the sample collection tube was subjected to absorbance detection at a wavelength of 280 nm using an ultraviolet spectrophotometer. The samples in the test tube with the same absorption peak were combined, and the protein part with a molecular weight of 30 kDa-60 kDa was further divided into 4 protein components by HW-55F; g. The four protein components obtained in step f were further purified by C8 reverse phase chromatography combined with high performance liquid chromatography, wherein the eluent A phase was ultrapure water containing 0.1% trifluoroacetic acid, and the eluent B phase was chromatographic grade acetonitrile. The sample was eluted by gradient concentrations of 0-5 min, 30% B; 5-8 min, 30%-34% B; 8-10 min, 34%-37% B; 10-20 min, 37%-45% B; 20-25 min, 45% B; 25-28 min, 45%-42% B; 28-30 min, 42%-34% B; 30-32 min, 34%-30% B; 32-40 min, 30% B, and the flow rate was set to 1.5 mL / min. The sample was eluted at 280 °C using the UV detector provided by the high performance liquid chromatography. Real-time monitoring was performed at 400 nm and symmetrical single peak samples were directly collected. Finally, three high-purity Tianshan red deer abomasum proteins were obtained and named S1, S2 and S3 respectively.

2. Use of the Tianshan red deer abomasum proteins S1, S2 and S3 obtained according to the method of claim 1 in the preparation of anti-colon cancer drugs.

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