Sheep placenta polypeptide in Hu sheep and its use in regulating immune function

Abstract

The present invention discloses a sheep placenta polypeptide in Hu sheep and its use in regulating immune function, and relates to the technical field of active peptides. The present invention uses sheep embryos and sheep placentas as raw materials, removes impurities, performs ultrafine grinding and cell wall breaking treatment, then performs extraction and enzymolysis, macroporous adsorption resin purification, ultrafiltration, and then uses modified dextran gel chromatography to separate. The obtained sheep placenta polypeptide has good sugar removal and decolorization effects; in addition, the present invention also uses the prepared sheep placenta polypeptide in combination with Paecilomyces pyralis powder and tetrahydrofurfuryl propionate, and the obtained composition has good immune function and radiation resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of active peptides, and in particular relates to a sheep placenta polypeptide in Hu sheep and its application in regulating immune function. Background Art

[0002] Since ancient times, the placenta has been valued by famous doctors of all dynasties for its medicinal value and is regarded as a precious Chinese medicine. Compared with other animal placentas, the structure and composition of sheep placenta are most similar to those of human placenta, and it has good nutritional value. Modern medical research has found that sheep placenta contains a large number of biologically active factors such as epidermal growth factor, hyaluronic acid stimulating factor, a variety of enzymes that promote and improve tissue metabolism, 17 amino acids and 14 mineral elements. The active substances extracted and purified from sheep placenta using scientific methods are mainly small molecule polypeptides, which have the effects of improving the body's immunity, anti-aging, anti-cancer, etc., and have a wide range of applications.

[0003] In recent years, two new types of sheep placenta biological products, animal placenta hydrolyzate and placenta extract, have appeared in succession at home and abroad, and have attracted people's attention due to their unique efficacy. Since the chemical method adopts acid-base hydrolysis to prepare active peptides, it will have a destructive effect on amino acids, and the molecular composition of the hydrolyzate is very different and the production cost is high. The production conditions of enzymatic protein hydrolysis are mild, safe and easy to control. Therefore, the present invention combines the hydrolyzate and the extract, which not only retains the natural active substances in the placenta, but also increases its polypeptide and amino acid content, which is a new breakthrough in the development and research of sheep placenta and sheep placenta biological products. Summary of the invention

[0004] The purpose of the present invention is to provide a sheep placenta polypeptide in Hu sheep and its use in regulating immune function. The sheep placenta polypeptide has good sugar removal and decolorization effects.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] A method for preparing sheep placenta polypeptide in Hu sheep, comprising:

[0007] Step 1: Clean and remove impurities from the raw materials;

[0008] Step 2: crushing the raw materials and breaking the cell walls;

[0009] Step 3: extraction and enzymolysis;

[0010] Step 4: purifying with macroporous adsorption resin, ultrafiltration, gel chromatography separation, and drying to obtain sheep placenta polypeptide.

[0011] In the present invention, the gel comprises modified dextran gel; the modified dextran gel is prepared from modified dextran.

[0012] In the present invention, the modified glucan is prepared from 2-pyrimidinethioacetic acid modified glucan.

[0013] Specifically, the method for preparing the above-mentioned sheep placenta polypeptide in vivo comprises the following steps:

[0014] Step 1: Wash and remove impurities from sheep embryos and sheep placentas, then crush them to a particle size of 350-400 μm at -3°C to 3°C, and then place them at -45°C to -50°C for 2-3.5 hours;

[0015] Step 2: Ultrafine grinding at -15°C to -20°C, followed by cell wall breaking treatment until the particle size reaches 1-5 μm;

[0016] Step 3, adding distilled water to the sheep embryo and sheep placenta after cell wall breaking, extracting at 35-40°C for 2-5h, filtering, adding enzyme to the precipitate, enzymolysis at 43-48°C for 2.5-3.5h, filtering with suction, and combining the filtrate after suction filtration with the filtrate after filtration;

[0017] Step 4: Use deionized water to elute the chromatographic column to pass the combined filtrate through a macroporous adsorption resin, and then use an ethanol aqueous solution with a concentration of 30-45wt% as an eluent for elution; enrich the eluate, then concentrate it, and then use an ultrafiltration membrane for ultrafiltration; then separate it through a modified dextran gel, use deionized water as the eluent, the elution rate is 0.3-0.5mL / min, and freeze-dry to obtain sheep placenta polypeptide.

[0018] For the present invention, in the above step 3, the mass volume ratio of the sheep embryo and sheep placenta after cell wall breaking to distilled water is: 1g:5-10mL; the mass ratio of the precipitate to the enzyme is: 1:0.005-0.015.

[0019] In the present invention, the enzyme used in the above enzymatic hydrolysis process includes at least one of trypsin or neutral protease.

[0020] The invention also discloses the use of the sheep placenta polypeptide prepared by the preparation method in regulating immune function.

[0021] The present invention also discloses a method for preparing a modified dextran gel, comprising:

[0022] Add distilled water to the modified dextran, stir at 90-95°C for 3-5h, keep the ratio of modified dextran to distilled water unchanged during the stirring process, then add potassium chloride, continue stirring at a constant temperature for 25-40min, cool naturally, and obtain a modified dextran gel.

[0023] For the present invention, the mass volume ratio of the modified dextran to distilled water is: 1 g: 3-6 mL; the mass ratio of the modified dextran to potassium chloride is: 1: 1-2.3.

[0024] In the present invention, the modified dextran gel is packed into a column by a wet method and then applied to the separation operation in step four.

[0025] The invention also discloses a method for preparing modified glucan, comprising: subjecting 2-pyrimidinethioacetic acid to chlorination treatment, and then subjecting the 2-pyrimidinethioacetic acid to esterification reaction with glucan to prepare the modified glucan.

[0026] The invention provides a method for preparing modified glucan, wherein 2-pyrimidinethioacetic acid is used as a modifier, the prepared modified glucan is gelled, and then the modified glucan gel is used in the preparation process of sheep placenta polypeptide in vivo, so that the prepared sheep placenta polypeptide in vivo has good sugar removal effect and decolorization effect.

[0027] Specifically, the preparation method of the modified glucan comprises:

[0028] Add 2-pyrimidinethioacetic acid to chloroform (the mass volume ratio of the two is: 1g:100-150mL), then slowly add a chloroform solution of thionyl chloride with a concentration of 0.03-0.04% (v / v), react at 55-65°C for 3.5-5h, and rotary evaporate to obtain 2-pyrimidinethioacetyl chloride; add dextran to dimethyl sulfoxide, then add triethylamine, slowly add 2-pyrimidinethioacetyl chloride under a nitrogen atmosphere, react at 75-85°C for 6-8h, let stand at room temperature for 10-13h, wash with chloroform 3-5 times, wash with ethanol 3-5 times, wash with deionized water 3-5 times, and dry to obtain modified dextran.

[0029] For the present invention, the mass volume ratio of the above-mentioned 2-pyrimidinethioacetic acid to thionyl chloride is: 1g:4-6mL; the mass volume ratio of dextran to dimethyl sulfoxide is: 1g:150-220mL; the mass volume ratio of dextran to triethylamine is: 1g:1.5-1.8mL; the mass volume ratio of dextran to 2-pyrimidinethioacetic acid is: 1:2-3.

[0030] The invention also discloses the use of the modified glucan prepared by the preparation method in preparing the sheep placenta polypeptide in Hu sheep.

[0031] The invention also discloses the use of the modified dextran prepared by the preparation method in a purification process.

[0032] The present invention also provides a composition comprising the sheep placenta polypeptide, Paecilomyces avicularis powder and tetrahydrofurfuryl propionate.

[0033] The invention discloses a composition. Sheep placenta polypeptide, Paecilomyces hymenioides powder and tetrahydrofurfuryl propionate are mixed and compounded for use. The prepared composition has good immune function and anti-radiation performance.

[0034] In the present invention, in the above composition, the mass ratio of sheep placenta polypeptide to Paecilomyces avicularis powder is 1:0.1-0.5.

[0035] In the present invention, in the above composition, the amount of tetrahydrofurfuryl propionate is 15-20 g / ton.

[0036] The invention also discloses the use of the composition in regulating immune function.

[0037] The beneficial effects of the present invention include:

[0038] The invention obtains a sheep placenta polypeptide in Hu sheep and its application in immune function regulation. Sheep embryo and sheep placenta are used as raw materials, and after impurities are removed, ultrafine grinding and cell wall breaking treatment are carried out, followed by extraction and enzymolysis, purification by macroporous adsorption resin, ultrafiltration, and then modified dextran gel chromatography separation prepared by modification with 2-pyrimidinethioacetic acid. The obtained sheep placenta polypeptide has good sugar removal effect and decolorization effect. In addition, the invention also compounds the obtained sheep placenta polypeptide with Paecilomyces pyralis powder and tetrahydrofurfuryl propionate for use, and the obtained composition has good immune function and radiation resistance.

[0039] Therefore, the present invention provides a sheep placenta polypeptide in Hu sheep and its use in regulating immune function. The sheep placenta polypeptide has good sugar removal and decolorization effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The infrared spectra test results of the modified dextran and dextran prepared in Example 1;

[0041] Figure 2 The test results of the sugar removal effect in the sheep placenta polypeptide samples prepared in Examples 1 to 4 are as follows;

[0042] Figure 3 The decolorization effect test results of the sheep placenta polypeptide samples prepared in Examples 1 to 4 are as follows;

[0043] Figure 4 The test results of the compositions prepared in Examples 5 to 7 in immune function;

[0044] Figure 5 These are the test results of the radiation resistance of the compositions prepared in Examples 5 to 7. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the present invention more clear, the technical solution of the present invention is further described in detail below in conjunction with specific implementation methods:

[0046] Embodiment 1:

[0047] A method for preparing sheep placenta polypeptide in Hu sheep comprises the following steps:

[0048] Step 1: clean the sheep embryo and sheep placenta, crush them to a particle size of 350 μm at 0°C, and then place them at -45°C for 3 hours;

[0049] Step 2: Ultrafine grinding at -15°C, followed by cell wall breaking treatment until the particle size reaches 3 μm;

[0050] Step 3, adding distilled water to the sheep embryo and sheep placenta after cell wall breaking, extracting at 35°C for 5 hours, filtering, adding trypsin to the precipitate, enzymolysis at 43°C for 3.5 hours, filtering with suction, and combining the filtrate after suction filtration with the filtrate after filtration;

[0051] Step 4: Use deionized water to elute the chromatographic column to pass the combined filtrate through a macroporous adsorption resin, and then use a 30wt% ethanol aqueous solution as an eluent for elution; enrich the eluate, then concentrate it, and then use an ultrafiltration membrane with a molecular weight cutoff of 1KDa for ultrafiltration; then separate it through a modified dextran gel, use deionized water as the eluent, the elution rate is 0.3mL / min, and freeze-dry to obtain sheep placenta polypeptide.

[0052] Among them, in step three, the quantitative ratio of sheep embryos to sheep placentas is: 1:1; the mass volume ratio of sheep embryos and sheep placentas after cell wall breaking treatment to distilled water is: 1g:5mL; the mass ratio of precipitate to enzyme is: 1:0.005.

[0053] A method for preparing a modified dextran gel, comprising:

[0054] Add distilled water to the modified dextran, stir at 90°C for 5h, keep the ratio of modified dextran to distilled water unchanged during stirring, then add potassium chloride, continue stirring at constant temperature for 25min, cool naturally, and obtain modified dextran gel. The modified dextran gel is wet packed into a column and then applied to the separation operation in step 4 above. The mass volume ratio of modified dextran to distilled water is: 1g:3mL; the mass ratio of modified dextran to potassium chloride is: 1:1.

[0055] A method for preparing modified glucan, comprising:

[0056] Add 2-pyrimidinethioacetic acid to chloroform (the mass volume ratio of the two is: 1g:100mL), then slowly add 0.03% (v / v) thionyl chloride chloroform solution, react at 55°C for 5h, and evaporation to obtain 2-pyrimidinethioacetyl chloride; add dextran to dimethyl sulfoxide, then add triethylamine, slowly add 2-pyrimidinethioacetyl chloride under nitrogen atmosphere, then react at 75°C for 8h, place at room temperature for 10h, wash 3 times with chloroform, 3 times with ethanol, 3 times with deionized water, and dry to obtain modified dextran. Among them, the mass volume ratio of 2-pyrimidinethioacetic acid to thionyl chloride is: 1g:4mL; the mass volume ratio of dextran to dimethyl sulfoxide is: 1g:150mL; the mass volume ratio of dextran to triethylamine is: 1g:1.5mL; the mass ratio of dextran to 2-pyrimidinethioacetic acid is: 1:2.

[0057] Embodiment 2:

[0058] A method for preparing sheep placenta polypeptide in Hu sheep comprises the following steps:

[0059] Step 1: Wash and remove impurities from sheep embryos and sheep placentas, then crush them to a particle size of 350 μm at -3°C, and then place them at -50°C for 2 hours;

[0060] Step 2: Ultrafine grinding at -20°C, followed by cell wall breaking treatment until the particle size reaches 1 μm;

[0061] Step 3, adding distilled water to the sheep embryo and sheep placenta after cell wall breaking, extracting at 40°C for 2h, filtering, adding trypsin to the precipitate, enzymolysis at 48°C for 2.5h, filtering with suction, and combining the filtrate after suction filtration with the filtrate after filtration;

[0062] Step 4: Use deionized water to elute the chromatographic column to pass the combined filtrate through a macroporous adsorption resin, and then use a 45wt% ethanol aqueous solution as the eluent for elution; enrich the eluate, then concentrate it, and then use an ultrafiltration membrane with a molecular weight cutoff of 1KDa for ultrafiltration; then separate it through a modified dextran gel, use deionized water as the eluent, the elution rate is 0.5mL / min, and freeze-dry to obtain sheep placenta polypeptide.

[0063] Among them, in step three, the mass volume ratio of the sheep embryo and sheep placenta after wall breaking treatment to distilled water is: 1g:10mL; the mass ratio of precipitate to enzyme is: 1:0.015.

[0064] A method for preparing a modified dextran gel, comprising:

[0065] Add distilled water to the modified dextran, stir at 95°C for 3h, keep the ratio of modified dextran to distilled water unchanged during stirring, then add potassium chloride, continue stirring at constant temperature for 40min, cool naturally, and obtain modified dextran gel. The modified dextran gel is wet packed into a column and then applied to the separation operation in step 4 above. The mass volume ratio of modified dextran to distilled water is: 1g:6mL; the mass ratio of modified dextran to potassium chloride is: 1:2.3.

[0066] A method for preparing modified glucan, comprising:

[0067] Add 2-pyrimidinethioacetic acid to chloroform (the mass volume ratio of the two is: 1g:150mL), then slowly add 0.04% (v / v) thionyl chloride chloroform solution, react at 65°C for 3.5h, and evaporation to obtain 2-pyrimidinethioacetyl chloride; add dextran to dimethyl sulfoxide, then add triethylamine, slowly add 2-pyrimidinethioacetyl chloride under nitrogen atmosphere, then react at 85°C for 6h, leave at room temperature for 13h, wash 5 times with chloroform, 5 times with ethanol, 5 times with deionized water, and dry to obtain modified dextran. Among them, the mass volume ratio of 2-pyrimidinethioacetic acid to thionyl chloride is: 1g:6mL; the mass volume ratio of dextran to dimethyl sulfoxide is: 1g:220mL; the mass volume ratio of dextran to triethylamine is: 1g:1.8mL; the mass ratio of dextran to 2-pyrimidinethioacetic acid is: 1:3.

[0068] Embodiment 3:

[0069] A method for preparing sheep placenta polypeptide in Hu sheep comprises the following steps:

[0070] Step 1: Wash and remove impurities from sheep embryos and sheep placentas, then crush them to a particle size of 400 μm at 3°C, and then place them at -48°C for 3 hours;

[0071] Step 2: Ultrafine grinding at -18°C, followed by cell wall breaking treatment until the particle size reaches 5 μm;

[0072] Step 3, adding distilled water to the sheep embryo and sheep placenta after cell wall breaking, extracting at 38°C for 3h, filtering, adding trypsin to the precipitate, enzymolyzing at 45°C for 3h, filtering with suction, and combining the filtrate after suction filtration with the filtrate after filtration;

[0073] Step 4: Use deionized water to elute the chromatographic column to pass the combined filtrate through a macroporous adsorption resin, and then use a 35wt% ethanol aqueous solution as an eluent for elution; enrich the eluate, then concentrate it, and then use an ultrafiltration membrane with a molecular weight cutoff of 1KDa for ultrafiltration; then separate it through a modified dextran gel, use deionized water as the eluent, the elution rate is 0.4mL / min, and freeze-dry to obtain sheep placenta polypeptide.

[0074] Among them, in step three, the mass volume ratio of the sheep embryo and sheep placenta after wall breaking treatment to distilled water is: 1g:7mL; the mass ratio of precipitate to enzyme is: 1:0.01.

[0075] A method for preparing a modified dextran gel, comprising:

[0076] Add distilled water to the modified dextran, stir at 92°C for 4 hours, keep the ratio of modified dextran to distilled water unchanged during the stirring process, then add potassium chloride, continue stirring at a constant temperature for 30 minutes, and cool naturally to obtain a modified dextran gel. The modified dextran gel is wet packed into a column and then applied to the separation operation in the above step 4. The mass volume ratio of modified dextran to distilled water is: 1g:4mL; the mass ratio of modified dextran to potassium chloride is: 1:2.

[0077] A method for preparing modified glucan, comprising:

[0078] Add 2-pyrimidinethioacetic acid to chloroform (the mass volume ratio of the two is: 1g:130mL), then slowly add 0.035% (v / v) thionyl chloride chloroform solution, react at 60°C for 4h, and evaporation to obtain 2-pyrimidinethioacetyl chloride; add dextran to dimethyl sulfoxide, then add triethylamine, slowly add 2-pyrimidinethioacetyl chloride under nitrogen atmosphere, then react at 80°C for 7h, leave at room temperature for 12h, wash 4 times with chloroform, 4 times with ethanol, 4 times with deionized water, and dry to obtain modified dextran. Among them, the mass volume ratio of 2-pyrimidinethioacetic acid to thionyl chloride is: 1g:5mL; the mass volume ratio of dextran to dimethyl sulfoxide is: 1g:200mL; the mass volume ratio of dextran to triethylamine is: 1g:1.6mL; the mass ratio of dextran to 2-pyrimidinethioacetic acid is: 1:2.5.

[0079] Embodiment 4:

[0080] The difference between the method for preparing sheep placenta polypeptide in Hu sheep and Example 1 is that dextran gel is used instead of modified dextran gel.

[0081] Embodiment 5:

[0082] A composition comprises sheep placenta polypeptide, Paecilomyces avicularis powder and tetrahydrofurfuryl propionate; wherein the mass ratio of the sheep placenta polypeptide to the Paecilomyces avicularis powder is 1:0.2; and in the composition, the dosage of tetrahydrofurfuryl propionate is 20 g / ton.

[0083] The sheep placenta polypeptide is the same as the sheep placenta polypeptide prepared in Example 1.

[0084] Embodiment 6:

[0085] The difference between a composition and Example 5 is that tetrahydrofurfuryl propionate is not added.

[0086] Embodiment 7:

[0087] The difference between a composition and Example 5 is that tetrahydrofurfuryl alcohol is used instead of tetrahydrofurfuryl propionate.

[0088] Test example:

[0089] 1. Infrared spectrum test

[0090] The samples were analyzed and tested using Fourier transform infrared spectrometer and potassium bromide tablet method.

[0091] The modified dextran and dextran prepared in Example 1 were subjected to the above test, and the results were as follows: Figure 1 As shown by Figure 1 It can be seen that compared with the infrared spectrum of dextran, the infrared spectrum of modified dextran is at 1576cm -1 The presence of the infrared characteristic absorption peak of the pyrimidine group indicates that 2-pyrimidinethioacetic acid is involved in the formation reaction of modified dextran.

[0092] 2. Sugar removal effect test

[0093] The polysaccharide content in the sheep placenta polypeptide samples was tested by high performance liquid chromatography.

[0094] The above test was performed on the sheep placenta polypeptide samples prepared in Examples 1 to 4. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that compared with Example 4, the polysaccharide content in Example 1 is significantly reduced, indicating that after using 2-pyrimidinethioacetic acid to modify dextran, the modified dextran is used in the separation operation of the sheep placenta polypeptide in the body of Hu sheep, so that the polysaccharide content in the prepared sheep placenta polypeptide is significantly reduced, further illustrating that the modified dextran has a good purification effect on the sheep placenta polypeptide in the body of Hu sheep.

[0095] 3. Decolorization effect test

[0096] The liquid before and after gel separation was tested using a spectrophotometer.

[0097] S / %=[(T0-T1) / T0]×100%

[0098] Wherein, S is the decolorization rate; T0 is the absorbance of the liquid before gel separation; T1 is the absorbance of the liquid after gel separation.

[0099] The above test was performed on the sheep placenta polypeptide samples prepared in Examples 1 to 4. The results are as follows: Figure 3 As shown. Figure 3 It can be seen that compared with Example 4, the decolorization rate of Example 1 is significantly improved, indicating that after using 2-pyrimidinethioacetic acid to modify dextran, the modified dextran is used in the separation operation of sheep placenta polypeptide in the body of Hu sheep, which has a good decolorization effect on the sheep placenta polypeptide in the body of Hu sheep.

[0100] 4. Immune function test

[0101] Healthy Kunming mice were selected, with 20 mice in each group, half female and half male, weighing 18-22g. Distilled water was added to the composition sample to prepare a suspension with a concentration of 0.5g / kg. The mice were gavaged with 0.2mL / 10g for 30 consecutive days, once a day. The control group was gavaged with distilled water. One hour after the last gavage, Indian ink diluted 4 times with physiological saline was injected through the tail vein at a volume of 0.1mL / 10g for 2min (denoted as t2) and 10min (denoted as t 10 ) were taken from the medial canthal venous plexus, and then added to 2 mL of 0.1 wt% sodium carbonate solution. After thorough mixing, the optical density at 600 nm was measured by ultraviolet spectrophotometer. The 0.1 wt% sodium carbonate solution was used as a blank control for zero calibration. Then, the mice were dissected and the phagocytic index was calculated as follows:

[0102] R = [(lgOD2)-(lgOD 10 )] / (t 10 -t2)

[0103] Where R is the carbon clearance index; lgOD2 is the optical density of the blood sample taken at 2 minutes; lgOD 10 is the optical density of the blood sample taken at 10 min.

[0104] N=[M0 / (M1+M2)]×

[0105] Wherein, N is the phagocytic index; M0 is the weight of the mouse; M1 is the weight of the liver of the dissected mouse; M2 is the weight of the spleen of the dissected mouse.

[0106] The compositions obtained in Examples 5 to 7 were tested as above. Figure 4 As shown. Figure 4It can be seen that compared with Example 6, the phagocytic index of Example 5 is significantly increased, indicating that adding tetrahydrofurfuryl propionate to the composition is helpful to improve the immune performance of the composition; compared with Example 7, the phagocytic index of Example 5 is also increased, indicating that the immune effect of adding tetrahydrofurfuryl propionate to the composition is better than the effect of adding tetrahydrofurfuryl alcohol to the composition.

[0107] 5. Radiation resistance test

[0108] Healthy Kunming mice were selected, with 20 mice in each group, half of which were female and half were male, weighing 18-22 g. Distilled water was added to the composition sample to prepare a suspension with a concentration of 0.5 g / kg. The mice were gavaged with 0.2 mL / 10 g for 5 consecutive days, once a day. The control group was gavaged with distilled water, and then irradiated. The irradiation conditions were 60 The Co-γ ray source had an irradiation dose of 8 Gy. During the irradiation period, mice were gavaged 1 hour after irradiation with a volume of 0.2 mL / 10 g, once a day. The survival of the mice was observed within 30 days.

[0109] The compositions obtained in Examples 5 to 7 were tested as above. Figure 5 As shown. Figure 5 It can be seen that compared with Example 6, Example 5 has a significantly increased survival time, indicating that adding tetrahydrofurfuryl propionate to the composition helps to improve the radiation resistance of the composition; compared with Example 7, Example 5 has a slightly increased survival time, indicating that the radiation resistance of adding tetrahydrofurfuryl propionate to the composition is better than the effect of adding tetrahydrofurfuryl alcohol to the composition.

[0110] The conventional techniques in the above embodiments are prior arts known to those skilled in the art, and thus will not be described in detail here.

[0111] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing sheep placenta polypeptide in Hu sheep, comprising: Step 1: Clean and remove the sheep embryo and sheep placenta; Step 2: crushing at -3°C to 3°C to a particle size of 350-400 μm; cell wall disruption treatment to a particle size of 1-5 μm; Step 3, adding distilled water to the sheep embryo and sheep placenta after cell wall breaking, extracting at 35-40°C for 2-5h, filtering, adding trypsin to the precipitate, enzymolyzing at 43-48°C for 2.5-3.5h, filtering with suction, and combining the filtrate after suction filtration with the filtrate after filtration; Step 4: Purification with macroporous adsorption resin, using ethanol aqueous solution as eluent for elution treatment, enriching the eluate, then concentrating it, and then using an ultrafiltration membrane with a molecular weight cutoff of 1KDa for ultrafiltration treatment; using deionized water as eluent for gel chromatography separation, the elution rate is 0.3-0.5mL / min; drying, and obtaining sheep placenta polypeptide; The gel comprises a modified dextran gel; In the preparation of the modified dextran gel, distilled water is added to the modified dextran, and the mixture is stirred at 90-95° C. for 3-5 hours. During the stirring process, the ratio of the modified dextran to the distilled water is kept constant, and then potassium chloride is added, and the mixture is stirred at a constant temperature for 25-40 minutes, and cooled naturally to obtain the modified dextran gel; the mass volume ratio of the modified dextran to the distilled water is: 1 g: 3-6 mL; the mass ratio of the modified dextran to the potassium chloride is: 1: 1-2.3; In the preparation of modified dextran, 2-pyrimidinethioacetic acid is added to chloroform, and the mass volume ratio of the two is: 1g:100-150mL, and then a chloroform solution of thionyl chloride with a concentration of 0.03-0.04% (v / v) is slowly added, and the reaction is carried out at 55-65°C for 3.5-5h, and 2-pyrimidinethioacetyl chloride is obtained by rotary evaporation; dextran is added to dimethyl sulfoxide, and then triethylamine is added. Under a nitrogen atmosphere, 2-pyrimidinethioacetyl chloride is slowly added, and the reaction is carried out at 75-85°C. 6-8h, placed at room temperature for 10-13h, washed with chloroform 3-5 times, washed with ethanol 3-5 times, washed with deionized water 3-5 times, and dried to obtain modified dextran; the mass volume ratio of 2-pyrimidinethioacetic acid to thionyl chloride is: 1g:4-6mL; the mass volume ratio of dextran to dimethyl sulfoxide is: 1g:150-220mL; the mass volume ratio of dextran to triethylamine is: 1g:1.5-1.8mL; the mass ratio of dextran to 2-pyrimidinethioacetic acid is: 1:2-3.

2. A method for preparing a modified glucan, comprising: Add 2-pyrimidinethioacetic acid to chloroform, the mass volume ratio of the two is: 1g:100-150mL, then slowly add 0.03-0.04% (v / v) thionyl chloride chloroform solution, react at 55-65℃ for 3.5-5h, and rotary evaporate to obtain 2-pyrimidinethioacetyl chloride; add dextran to dimethyl sulfoxide, then add triethylamine, and slowly add 2-pyrimidinethioacetyl chloride under nitrogen atmosphere, and react at 75-85℃ for 6-8h. The mixture was placed at room temperature for 10-13 hours, washed with chloroform for 3-5 times, washed with ethanol for 3-5 times, washed with deionized water for 3-5 times, and dried to obtain modified dextran; the mass volume ratio of 2-pyrimidinethioacetic acid to thionyl chloride was 1 g: 4-6 mL; the mass volume ratio of dextran to dimethyl sulfoxide was 1 g: 150-220 mL; the mass volume ratio of dextran to triethylamine was 1 g: 1.5-1.8 mL; the mass ratio of dextran to 2-pyrimidinethioacetic acid was 1: 2-3.

3. Use of the modified glucan prepared by the preparation method of claim 2 in the preparation of sheep placenta polypeptide in Hu sheep.

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