Key active peptide component of spleen polypeptide as well as fine separation method and application of key active peptide component

Abstract

The invention provides a key active peptide component of spleen polypeptide as well as a fine separation method and application thereof, and belongs to the technical field of protein and active peptides, the fine separation method comprises the following steps: separating out the key active peptide component of the spleen polypeptide from spleen polypeptide trapped fluid with the molecular weight of 1-3kDa by adopting a gel chromatography separation method; the key active peptide component of the spleen polypeptide is used for preparing an immunomodulator, an anti-tumor drug, a tumor postoperative rehabilitation drug or a drug for inhibiting tumor metastasis. It is found that PP-VIII polypeptide with the ultraviolet absorption peak being 258.5 nm is a key active peptide component, the bidirectional immunoregulation activity of the PP-VIII polypeptide is remarkably higher than that of other polypeptides, and meanwhile splenic lymphocyte proliferation and the phagocytic ability of macrophages can be remarkably promoted; the traditional Chinese medicine composition has a very remarkable effect on the recovery of the immunocompetence and the weight of immunosuppressed mice and the index improvement of organs, and has a remarkable inhibition effect on tumors of nude mice bearing melanoma, and the inhibition rate reaches 85% or above.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of proteins and active peptides, and particularly relates to a spleen polypeptide key active peptide component and a fine separation method and application thereof. BACKGROUND

[0002] The spleen is composed of connective tissue and lymphoid tissue, is a complex peripheral lymphoid organ, and is one of the most metabolically active organs in the animal body, has highly organized regions and a complex microcirculation system. The spleen plays a role of filtering blood, storing blood, and storing immune cells in animal tissues. The immune function is the most important function of the spleen. Active substances in the animal spleen, such as polypeptides, ribose, and amino acids, especially small molecule polypeptides such as immune regulatory factors in cells, have become a research hotspot in recent years. The spleen polypeptide, as a main component of the active substance of the spleen, is usually prepared by enzymatic hydrolysis and freeze-thawing.

[0003] In practice, the spleen polypeptide has the effects of bidirectional regulation of immune function of the body, enhancement of cellular immunity, and stimulation of bone marrow cell proliferation, and is used for adjuvant treatment of malignant tumors, respiratory tract and lung infections and the like. However, the research on the spleen polypeptide is mostly limited to the application of the spleen polypeptide mixture, and the research on the basic components, basic pharmacy, and pharmacology is almost blank.

[0004] Therefore, the separation and extraction of the active components in the spleen polypeptide and the evaluation of the activity thereof have important significance for the in-depth development of the spleen polypeptide, and are also key scientific problems urgently to be solved. SUMMARY

[0005] In view of the above problems, the application provides a spleen polypeptide key active peptide component and a fine separation method and application thereof, which not only provides a fine material basis for improving the medicinal value of the hydrolysate of the spleen polypeptide, but also lays a theoretical foundation.

[0006] To achieve the above object, the technical scheme adopted by the application is as follows: A spleen polypeptide key active peptide component, which is a spleen polypeptide key active peptide component with a narrower molecular weight range obtained from a spleen polypeptide retentate with a molecular weight of 1-3 kDa; The spleen polypeptide key active peptide component is a PP-VIII polypeptide and / or a PP-IX polypeptide; The PP-VIII polypeptide has an ultraviolet absorption peak at 258.5 nm; The PP-VIII polypeptide and the PP-IX polypeptide respectively have the effects of improving immune activity and / or inhibiting tumor growth.

[0007] The application discloses a fine separation method of a spleen polypeptide key active peptide component, which adopts gel chromatography separation to separate the spleen polypeptide key active peptide component with a narrower molecular weight range by taking a spleen polypeptide retentate with a molecular weight of 1-3 kDa as raw material. The spleen polypeptide key active peptide component is a PP-VIII polypeptide and / or a PP-IX polypeptide. The PP-VIII polypeptide has an ultraviolet absorption peak at 258.5 nm. The PP-VIII polypeptide and the PP-IX polypeptide have the effects of improving immune activity and / or inhibiting tumor growth respectively.

[0008] Further, the gel chromatography separation adopts agarose gel, dextran gel or polyamide gel.

[0009] Further, the spleen polypeptide retentate with a molecular weight of 1-3 kDa is obtained by adopting membrane combination to separate the spleen polypeptide retentate with a molecular weight of 1-3 kDa from a spleen polypeptide enzyme solution. The membrane combination is a method of adopting multi-stage ultrafiltration membrane combination. The multi-stage ultrafiltration membrane combination is a method of combining a large ultrafiltration membrane system with a small ultrafiltration membrane system to realize the separation of the spleen polypeptide enzyme solution. The large ultrafiltration membrane system and the small ultrafiltration membrane system both adopt polysulfone membrane, polyester membrane or polyamide membrane. The spleen polypeptide enzyme solution is prepared by taking the spleen of an animal (the animal includes but is not limited to pig, cow, sheep, horse and other domesticated poultry) after biochemical treatment.

[0010] Further, the fine separation method is to take the spleen polypeptide retentate with a molecular weight of 1-3 kDa, first separate the retentate through an ultrafiltration membrane system which can retain components with a molecular weight greater than 2 kDa, collect the retentate with a molecular weight of 2-3 kDa and the dialysate with a molecular weight of 1-2 kDa respectively, then perform gel chromatography separation on the retentate and the dialysate respectively, cut 5 components respectively, and dry to form ten components including the PP-VIII polypeptide and the PP-IX polypeptide. The ultrafiltration membrane system adopts polysulfone membrane, polyester membrane or polyamide membrane; and the drying is freeze drying, vacuum drying or spray drying.

[0011] Further, the fine separation method is to take the spleen polypeptide retentate with a molecular weight of 1-3 kDa, combine ultraviolet spectrum tracking, directly perform gel chromatography separation, and dry to form ten components including the PP-VIII polypeptide and the PP-IX polypeptide. The drying is freeze drying, vacuum drying or spray drying.

[0012] Application of a key active peptide component of spleen polypeptide separated by the above fine separation method, the key active peptide component of spleen polypeptide is used for preparing an immunomodulator; And / or, the key active peptide component of spleen polypeptide is used for preparing an antitumor drug and a postoperative recovery drug of tumor.

[0013] Further, the immunomodulator enhances the immune function in at least one of the following ways: promoting lymphocyte proliferation, increasing immune organ index, enhancing peritoneal macrophage phagocytosis, increasing NK cell (natural killer cell) activity, and regulating serum cytokine levels (increasing IL-2, TNF-alpha, IFN-gamma levels in serum, and reducing IL-10 levels).

[0014] Further, the key active peptide component of spleen polypeptide significantly enhances the biological effect of inhibiting tumor growth by improving immune function, and has potential value as an antitumor drug.

[0015] Further, the key active peptide component of spleen polypeptide is used for preparing an anti-human malignant melanoma drug. Or, the key active peptide component of spleen polypeptide is used for preparing a postoperative recovery drug of human malignant melanoma. Or, the key active peptide component of spleen polypeptide is used for preparing a drug for inhibiting metastasis of human malignant melanoma.

[0016] Further, the key active peptide component of spleen polypeptide has an inhibition rate on tumor growth of more than 85%.

[0017] The key active peptide component of spleen polypeptide, its fine separation method and application have the following beneficial effects: The present application found through activity research that the bidirectional immunomodulatory activity of PP-VIII polypeptide with ultraviolet absorption peak at 258.5 nm is significantly higher than that of other polypeptides, and it can significantly promote the proliferation of spleen lymphocytes and the phagocytosis of macrophages; it has a very significant effect on the recovery of immune capacity and body weight of immunosuppressed mice and the improvement of organ index, and has a significant inhibitory effect on tumors of melanoma-bearing nude mice, with an inhibition rate of more than 85%. The present application separates spleen polypeptides with molecular weight of 1-3 kDa into 9 spleen polypeptides with different ultraviolet absorption peaks by gel chromatography separation technology combined with ultraviolet spectrophotometer monitoring, and determines that the spleen polypeptide PP-VIII with ultraviolet absorption peak at 258.5 nm has excellent immunomodulatory activity and antitumor activity. The present application determines the main component of spleen polypeptide with molecular weight of 1-3 kDa, providing a theoretical basis and practical foundation for further development and research, which is beneficial to improve product quality and efficacy, and has important clinical application value and market prospect. The application provides strong data support for development of spleen polypeptide health products, and more importantly, provides a material and theoretical basis for development of bidirectional immunomodulators and tumor drugs based on the spleen polypeptide, and has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is the influence result of each component polypeptide on the proliferation activity of spleen lymphocytes in the embodiment 7 of the application; Figure 2 is the influence result of each component polypeptide on the phagocytosis capacity of macrophages in the embodiment 7 of the application; Figure 3 is the body weight change result of each group of mice in the embodiment 8 of the application; Figure 4 is the immune organ index result of each group of mice in the embodiment 8 of the application; wherein, the left graph is the thymus index result, and the right graph is the spleen index result; Figure 5 is the spleen lymphocyte induced stimulation transformation index result in the embodiment 8 of the application; Figure 5 The spleen polypeptide control group in the application is the spleen polypeptide stock solution group; Figure 6 is the NK cell killing activity result in the embodiment 8 of the application; Figure 7 is the HE staining result of the spleen tissue of the mice in the embodiment 8 of the application; Figure 8 is the anti-tumor effect in vivo of tumor-bearing mice in the embodiment 9 of the application; wherein, A is a solid tumor under different conditions; B is a body weight change trend graph; C is a tumor volume change graph; and D is a tumor weight comparison result at the end of treatment. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the application will be described clearly and completely below. In the following description, a large number of specific details are set forth in order to facilitate a full understanding of the application, but the application can also be implemented in other ways different from those described herein, and a person skilled in the art can make similar generalizations without departing from the connotation of the application, therefore the application is not limited by the specific embodiments disclosed below.

[0020] Embodiment 1: A preparation method of a polypeptide cut-off solution with a molecular weight of 1-3 kDa The embodiment is a preparation method of a polypeptide cut-off solution with a molecular weight of 1-3 kDa, including the following specific steps: 1) taking the spleen of an animal (the animal includes but is not limited to pigs, cows, sheep, horses and other farmed poultry) after biochemical treatment, to obtain a spleen polypeptide enzyme hydrolysis stock solution; The spleen polypeptide hydrolysate is subjected to micro solid particles and protein removal by a large ultrafiltration membrane system, and the dialysate is transferred to a small ultrafiltration membrane system for further fractionation. 2) The dialysate obtained in the previous step is subjected to separation by a small ultrafiltration membrane system which can dialyze components with molecular weight below 8 kDa, and the obtained dialysate is used for further fractionation in the next step. 3) The dialysate obtained in the previous step is subjected to separation by a small ultrafiltration membrane system which can dialyze components with molecular weight below 5 kDa, and the obtained dialysate is used for further fractionation in the next step. 4) The dialysate obtained in the previous step is subjected to separation by a small ultrafiltration membrane system which can retain components with molecular weight greater than 3 kDa, and the obtained dialysate is used for further fractionation in the next step. 5) The dialysate obtained in the previous step is subjected to separation by a small ultrafiltration membrane system which can retain components with molecular weight greater than 1 kDa, and a polypeptide retention solution with molecular weight of 1-3 kDa is obtained by retention. The polypeptide retention solution with molecular weight of 1-3 kDa is freeze-dried to obtain spleen polypeptides with molecular weight of 1-3 kDa. The large ultrafiltration membrane system and the small ultrafiltration membrane system both use polysulfone membranes, polyester membranes or polyamide membranes.

[0021] Example 2: A fine separation method for a key active peptide component of spleen polypeptides This embodiment is a fine separation method for a key active peptide component of spleen polypeptides, which includes the following specific steps: S1, the polypeptide retention solution with molecular weight of 1-3 kDa is first subjected to separation by an ultrafiltration membrane system which can retain components with molecular weight greater than 2 kDa, and the retention liquid and the dialysate are collected respectively; wherein the spleen polypeptides in the retention liquid have a molecular weight of 2-3 kDa, and the spleen polypeptides in the dialysate have a molecular weight of 1-2 kDa; wherein the ultrafiltration membrane system uses polysulfone membranes, polyester membranes or polyamide membranes; S2, the spleen polypeptide retention liquid with molecular weight of 2-3 kDa and the spleen polypeptide dialysate with molecular weight of 1-2 kDa obtained in the previous step are subjected to glucose gel chromatography separation respectively, and each takes 5 stages, a total of ten collected components PP-I~PP-X, and ultraviolet spectrum tracking is used; wherein the ultraviolet absorption peak of component PP-VIII is at 258.5 nm; S3, the ten collected components PP-I~PP-X are dehydrated by freeze-drying to obtain PP-I polypeptide~PP-X polypeptide, which is collected and stored.

[0022] Example 3: A fine separation method for a key active peptide component of spleen polypeptides This embodiment is a fine separation method for a key active peptide component of spleen polypeptides, which includes the following specific steps: S1, the spleen polypeptide enzyme solution is first separated by an ultrafiltration membrane system that can intercept components with a molecular weight greater than 5 kDa, and the intercepting liquid and the dialysis liquid are collected respectively; wherein the molecular weight of the spleen polypeptide in the intercepting liquid is greater than 5 kDa, and the molecular weight of the spleen polypeptide in the dialysis liquid is less than or equal to 5 kDa; S2, the dialysis liquid with a molecular weight less than or equal to 5 kDa obtained in the previous step is separated by an ultrafiltration membrane system that can intercept components with a molecular weight greater than 3 kDa, and the intercepting liquid and the dialysis liquid are collected respectively; wherein the molecular weight of the spleen polypeptide in the intercepting liquid is 3-5 kDa, and the molecular weight of the spleen polypeptide in the dialysis liquid is less than or equal to 3 kDa; S3, the dialysis liquid with a molecular weight less than or equal to 3 kDa obtained in the previous step is separated by an ultrafiltration membrane system that can intercept components with a molecular weight greater than 2 kDa, and the intercepting liquid and the dialysis liquid are collected respectively; wherein the molecular weight of the spleen polypeptide in the intercepting liquid is 2-3 kDa, and the molecular weight of the spleen polypeptide in the dialysis liquid is less than or equal to 2 kDa; S4, the dialysis liquid with a molecular weight less than or equal to 2 kDa obtained in the previous step is separated by an ultrafiltration membrane system that can intercept components with a molecular weight greater than 1 kDa, and the intercepting liquid and the dialysis liquid are collected respectively; wherein the molecular weight of the spleen polypeptide in the intercepting liquid is 1-2 kDa, and the molecular weight of the spleen polypeptide in the dialysis liquid is less than or equal to 1 kDa; In steps S1-S4, the ultrafiltration membrane system uses a polysulfone membrane, a polyester membrane or a polyamide membrane; S5, the intercepting liquid with a molecular weight of 1-2 KDa obtained in step S4 and the intercepting liquid with a molecular weight of 2-3 KDa obtained in step S3 are subjected to glucose gel chromatography separation respectively, and 5 fractions are collected respectively, a total of ten collected components PP-I-PP-X, and ultraviolet spectrum tracking is performed; wherein the ultraviolet absorption peak of component PP-VIII is at 258.5 nm; S6, the ten collected components PP-I-PP-X are dehydrated by a spray drying method to obtain PP-I polypeptide-PP-X polypeptide, which is collected and stored.

[0023] Example 4: A fine separation method of a key active peptide component of spleen polypeptide The present embodiment is a fine separation method of a key active peptide component of spleen polypeptide, which comprises the following specific steps: S1, the spleen polypeptide enzyme solution is first separated by an ultrafiltration membrane system that can intercept components with a molecular weight greater than 5 kDa, and the intercepting liquid and the dialysis liquid are collected respectively; wherein the molecular weight of the spleen polypeptide in the intercepting liquid is greater than 5 kDa, and the molecular weight of the spleen polypeptide in the dialysis liquid is less than or equal to 5 kDa; S2, the molecular weight of less than or equal to 5kDa spleen polypeptide dialysis fluid obtained in the previous step, through the ultrafiltration membrane system can intercept the molecular weight of more than 3kDa components are separated, respectively, the intercept and dialysate; wherein the intercept liquid molecular weight of 3~5kDa spleen polypeptide, dialysate molecular weight of less than or equal to 3kDa spleen polypeptide; S3, the molecular weight of less than or equal to 3kDa spleen polypeptide dialysis fluid obtained in the previous step, through the ultrafiltration membrane system can intercept the molecular weight of more than 1kDa components are separated, respectively, the intercept and dialysate; wherein the intercept liquid molecular weight of 1~3kDa spleen polypeptide, dialysate molecular weight of less than or equal to 1kDa spleen polypeptide; S4, the molecular weight of 1~3kDa spleen polypeptide intercept liquid obtained in the previous step, with ultraviolet spectrum tracking, glucose gel chromatography separation, a total of ten collection components PP-I~PP-X; wherein, the ultraviolet absorption peak of component PP-VIII is at 258.5nm; S5, the ten collection components PP-I~PP-X obtained are dehydrated by spray drying, respectively, to obtain PP-I polypeptide~PP-X polypeptide, which are collected and stored.

[0024] Example 5 A fine separation method of a key active peptide component of spleen polypeptide The preparation method of the five components of spleen polypeptide in this example is basically the same as that in example 4, except that in step S4 of this example, the 1~3kDa spleen polypeptide intercept liquid is subjected to agarose gel chromatography separation with ultraviolet spectrum tracking, and a total of ten collection components PP-I~PP-X are obtained; in step S5, the ten collection components PP-I~PP-X obtained are dehydrated by vacuum drying, respectively, to obtain PP-I polypeptide~PP-X polypeptide, which are collected and stored. The ultraviolet absorption peak of PP-VIII polypeptide is at 258.5nm.

[0025] Example 6 A fine separation method of a key active peptide component of spleen polypeptide The preparation method of the five components of spleen polypeptide in this example is basically the same as that in example 3, except that in step S5 of this example, the 1~2KDa spleen polypeptide intercept liquid and the 2~3KDa spleen polypeptide intercept liquid are subjected to polyacrylamide gel chromatography separation, respectively, and each intercepts 5 segments, a total of ten collection components PP-I~PP-X are obtained, which are tracked by ultraviolet spectrum; in step S6, the ten collection components PP-I~PP-X obtained are dehydrated by spray drying, respectively, to obtain PP-I polypeptide~PP-X polypeptide, which are collected and stored. The ultraviolet absorption peak of PP-VIII polypeptide is at 258.5nm.

[0026] Example 7 In vitro immune activity of ten spleen polypeptide separation components 1) Mouse spleen lymphocyte proliferation experiment A Kunming mouse weighing between 18 to 20 grams was euthanized and the spleen was removed to prepare a spleen lymphocyte suspension. The cell concentration was adjusted to 1 x 10 6 The spleen lymphocytes were added to the 96-well plate at 100 μL / well. The PP-I and PP-X polypeptides obtained in Example 1 were each assigned a concentration of 0.6 mg / mL and added to the 96-well plate at a volume of 100 μL / well. The positive control group used ConA (concanavalin A) at a concentration of 0.6 mg / mL and the blank group used RPMI-1640 base medium. The plate was incubated in a 37°C incubator with 5% CO2for 68 h. After adding cck-8 solution and mixing, the plate was incubated for an additional 4 h. Finally, the optical density (OD) value at 450 nm was measured using a microplate reader. The proliferation rate of the spleen lymphocytes was calculated using the following formula I: Proliferation rate (%) = [(OD 处理 - OD 空白 ) / OD 空白 ] x 100% Formula I The results of the spleen lymphocyte proliferation rate are shown in Figure 1 and Table 1.

[0027] Table 1. Proliferation rate of spleen lymphocytes after polypeptide treatment

[0028] The results show that the ten components of PP-I and PP-X polypeptides can improve the proliferation ability of spleen lymphocytes, and the proliferation activity of PP-VIII and PP-IX polypeptides is higher.

[0029] 2) Mouse peritoneal macrophage phagocytosis of neutral red experiment A Kunming mouse weighing between 18 to 20 grams was euthanized and the spleen was removed to prepare a spleen lymphocyte suspension. The cell concentration was adjusted to 1 x 10 6The macrophage cells 100 μL / well were added to the 96-well plate, and the ten components of the PP-I polypeptide and PP-X polypeptide obtained in Example 1 were dispensed at a concentration of 0.6 mg / mL, and added to the 96-well plate at a volume of 100 μL / well for culture, as the PP-I group and the PP-X group. The blank control group and the positive control group were added with RPMI-1640 basic culture medium and LPS (lipopolysaccharide), respectively, and incubated at 37°C in a CO2volume fraction of 5% for 48 h. After the incubation was completed, 100 μL of 0.1% neutral red saline was added to each well, and after 2-4 h, the supernatant was removed, 200 μL of PBS buffer was added, and after 3 min of standing, the supernatant was removed, and the operation was repeated twice, and finally 200 μL of cell lysis solution was added, mixed, and the absorbance value at 540 nm was measured to evaluate the phagocytosis efficiency of the macrophage cells on the neutral red, and the results are shown in Table 2. Figure 2 and Table 2.

[0030] Table 2 Phagocytosis rate of macrophage cells after polypeptide of each component

[0031] The results prove that the ten components of the PP-I polypeptide and the PP-X polypeptide can improve the phagocytosis ability of the macrophage cells, and the phagocytosis ability of the PP-VIII polypeptide and the PP-IX polypeptide is stronger.

[0032] Example 8 Effect of PP-VIII polypeptide on immune function of immunosuppressed mice 1) Experimental animal grouping and treatment Thirty Kunming mice with a body weight of 18-20 g were evenly divided into five groups, each group of 6, which were normal group, immunosuppressed group, positive control group, spleen polypeptide stock solution group, and PP-VIII group. Except for the normal group, the mice in the other groups were pre-injected with cyclophosphamide CTX intraperitoneally, with a dose of 80 mg / kg, and continuous administration for five days. During the administration period, the spleen polypeptide stock solution group and the PP-VIII group were respectively given 50 mg / (kg·d) of spleen polypeptide enzymatic stock solution and PP-VIII polypeptide by gavage every day, and the positive control group was given 40 mg / (kg·d) of ConA (concanavalin A) by gavage every day. The body weight of the mice was measured every two days, and the body weight change graph was drawn, and the results are shown in Table 1. Figure 3

[0033] The experimental results show that after the CTX (cyclophosphamide) modeling for 5 days, the body weight of the mice in the four groups except the normal group decreased. After the administration began, the body weight of the mice began to rise, and the use of PP-VIII polypeptide was the most significant in promoting the body weight of the mice to rise.

[0034] 2) Determination of thymus and spleen indices ​After the end of administration of mice, euthanize each group of mice. Place the mice in a supine position, dissect and collect the spleen and thymus of each group of mice, and weigh them to calculate the organ index, as follows: Organ index (%) = [organ weight (spleen, thymus) (mg) / body weight (g)] x 100%; The organ index results are shown in Table 1. Figure 4 As shown in Table 1, the results show that the spleen polypeptide (spleen polypeptide stock solution group and PP-VIII group) has a significant effect on improving the immune organ (spleen and thymus) index of immunosuppressed mice induced by cyclophosphamide (CTX). The immune organ index of the immunosuppression group is significantly lower than that of the normal group. The immune organ index of the spleen polypeptide stock solution group, the PP-VIII group and the positive control group is significantly increased. The spleen index of the PP-VIII group is significantly higher than that of the spleen polypeptide stock solution group, indicating that the PP-VIII polypeptide has a significant repair effect on the immune organ damage caused by CTX, and the repair effect is significantly higher than that of the existing spleen polypeptide stock solution.

[0035] 3) Measurement of spleen lymphocyte-induced transformation index Each group of experimental mice was euthanized and the spleen was removed and immediately soaked in physiological saline. The spleen was transferred to a 200-mesh nylon net, ground and washed with PBS to obtain a cell suspension. The supernatant was discarded by centrifugation, 3 mL of red blood cell lysate was added to the precipitated cells, and after standing for 5 minutes, centrifugation was performed again, and the supernatant was discarded. The cells were resuspended in RPMI-1640 medium containing 10% fetal bovine serum and placed in a cell culture bottle. The cells were further incubated at 37°C, 5% CO2 for 3 hours to stabilize the cell state.

[0036] The cell precipitate after centrifugation was resuspended to prepare a lymphocyte suspension. The cell suspension was counted using a cell counting plate and the concentration was adjusted to 4 x 10 6 / mL. Take 200 μL of lymphocyte suspension and add 12 μL of ConA and mix well. Take the uniformly mixed cell suspension, add 100 μL per well to a 96-well plate, and 6 parallel wells per group. The cells were further incubated at 37°C, 5% CO2 for 48 hours. After incubation, 10 μL of CCK-8 reagent was added to each well, and after 2 hours of incubation, the optical density (OD) value was measured using a microplate reader to evaluate cell proliferation. The results are shown in Table 2. Figure 5

[0037] ​In order to study the effect of each component of spleen polypeptide on cellular immunity, the stimulation transformation index of spleen lymphocytes of each group of mice under the induction of ConA was determined. After induction by ConA, the stimulation index of the spleen polypeptide stock solution group and the PP-VIII group increased significantly, and the stimulation index of the PP-VIII group was significantly higher than that of the spleen polypeptide stock solution group. It is confirmed that the proliferation ability of spleen lymphocytes in the PP-VIII group is improved, which is especially obvious for mice with weak immune function, indicating that the effect of PP-VIII polypeptide on cellular immunity is significantly higher than that of the spleen polypeptide enzyme solution.

[0038] 4) NK cell killing activity Preparation of YAC-1 cell suspension: Before the formal experiment, YAC-1 cells need to be cultured to the exponential growth phase, and the YAC-1 cells in the exponential growth phase are taken to adjust the cell concentration to 1x10 / mL for standby.

[0039] Determination of NK cell killing activity: In a 96-well plate, sample addition and mixing were performed according to the following scheme, with 3 parallel sets in each well, and incubation was carried out at 37°C, saturated water vapor, 5% carbon dioxide for 4h.

[0040] Among them, 100μL of YAC-1 cell suspension and 100μL of RPMI 1640 culture solution were added to the natural release hole; 100μL of YAC-1 cell suspension and +100μL of 2.5% Triton X-100 solution were added to the maximum release hole; 100μL of YAC-1 cell suspension and 100μL of normal group mouse spleen cell suspension were added to the normal group release hole; 100μL of YAC-1 cell suspension and 100μL of PP-VIII group mouse spleen cell suspension were added to the PP-VIII group experimental hole; 100μL of YAC-1 cell suspension and 100μL of positive control group mouse spleen cell suspension were added to the positive control hole; 100μL of YAC-1 cell suspension and 100μL of spleen polypeptide stock solution group mouse spleen cell suspension were added to the spleen polypeptide stock solution group control hole; 100μL of YAC-1 cell suspension and 100μL of immune suppression group mouse spleen cell suspension were added to the immune suppression group control hole; After completion of the culture, 100μL of supernatant was collected from each hole, lactate dehydrogenase release was determined using a lactate dehydrogenase activity assay kit, and mouse NK cell killing activity was calculated, as shown in Figure 6 .

[0041] The experimental results show that: spleen polypeptide (spleen polypeptide stock solution group and PP-VIII group) can significantly improve the activity of natural killer cells in mice. Compared with the normal group, the activity of NK cells in the immunosuppressed group of mice decreased significantly. The activity of NK cells in the spleen polypeptide stock solution group and the PP-VIII group was significantly enhanced compared with the immunosuppressed group. At the same time, the activity of natural killer cells in the PP-VIII group was significantly higher than that in the spleen polypeptide stock solution group, indicating that the PP-VIII polypeptide can greatly enhance the body's immunity, and its ability to enhance the body's immunity is significantly higher than that of the spleen polypeptide stock solution.

[0042] 5) Determination of serum cytokine content The whole blood of each group of mice was taken, centrifuged at 3000 rpm / min for 15 min, and the supernatant was stored at -20℃. The concentration of interleukin-2 (IL-2), interleukin-10 (IL-10), interferon-γ (IFN-γ) and tumor necrosis factor alpha (TNF-α) was quantitatively analyzed according to the ELISA kit instructions, and the results are shown in Table 3.

[0043] Table 3 PP-VIII on the average level of serum cytokines in immunosuppressed mice (unit pg / mL)

[0044] As can be seen from Table 3, the spleen polypeptide stock solution group and the PP-VIII group have a significant effect on the average level of serum cytokines in the immunosuppressed group of mice. At the same time, the PP-VIII polypeptide also has a significant bidirectional regulation effect, such as IL-2, TNF-α, IFN-γ, and the level of IL-10 is significantly reduced, and its effect is obviously better than that of the spleen polypeptide stock solution.

[0045] 6) Pathological observation of spleen tissue The weighed spleen samples of each group were immersed in 4% paraformaldehyde solution for 24 hours. The spleen tissue was paraffin-embedded and sectioned; after removing the paraffin by xylene treatment, the section was dehydrated in a graded ethanol solution; the section was stained with hematoxylin for 5 minutes. The same duration was 5 minutes. In the washing step between the staining, double distilled water was used for cleaning. The final step is to encapsulate the section with neutral gum, and use a microscope to observe and record the image, as shown in Figure 7 .

[0046] It can be seen that in normal group of mice, the tissue structure of the spleen remains clear and complete. In contrast, the spleen tissue cell distribution of the immunosuppression group of mice becomes sparse, the white pulp area shrinks, and the white and red pulp boundary becomes blurred. Compared with the immunosuppression group, the spleen tissue structure of the PP-VIII group of mice is restored, the cell arrangement begins to become orderly, the number of lymphoid follicles is gradually restored, the number of lymphocytes increases, and the boundary between white and red pulp begins to become clear. And the recovery effect of the PP-VIII group is better than that of the spleen polypeptide stock solution group.

[0047] Example 9 Inhibition of PP-VIII polypeptide on melanoma tumor-bearing nude mice I. Experimental method 1) Establishment of A375 tumor-bearing nude mice melanoma model and grouping and treatment The human malignant melanoma cell line (A375) was cultured to the logarithmic growth phase, and the A375 cells in good growth state were taken and diluted with sterile PBS solution, and the dilution concentration was 1×10 7 Each mouse was injected with 0.15 mL. After 5 to 7 days of culture, when the average diameter of the tumor in the inoculation area reached 5 mm, the model was considered to be successfully constructed, and the success rate was close to 100%.

[0048] After the tumor model was successfully constructed, the tumor-bearing nude mice were randomly divided into normal group, model group, positive control group, 1-3KD ultrafiltration group, PP-VIII group and spleen polypeptide stock solution group, each group consisting of 3 mice. Every two days at a fixed time, different groups were given corresponding drugs by intraperitoneal injection. The normal group received 0.15 mL of normal saline injection, and the spleen polypeptide stock solution group, 1-3kDa ultrafiltration group and PP-VIII group were respectively given 50 mg / kg of spleen polypeptide enzyme solution, 1-3kDa spleen polypeptide and PP-VIII polypeptide per kilogram of body weight by gavage, and the positive control group was injected with 10 mg of vemurafenib per kilogram of body weight per day, for 14 days.

[0049] 2) Measurement of mouse signs and tumor During the 14-day drug treatment period, the body weight and tumor size of each group of mice were measured every two days, and the body weight and tumor volume trend graphs were drawn according to these data, as shown in Figure 8 .

[0050] II. Anti-tumor experimental results From Figure 8It can be seen that, by the effect of anti-melanoma A375 in vivo, the PP-VIII group, the 1-3 kDa ultrafiltration group, and the spleen polypeptide stock solution group all showed significant anti-melanoma activity, and the tumor growth inhibition rates were 85.2%, 62.5%, and 51.3%, respectively, and the PP-VIII group was more significant than the 1-3 kDa group and the spleen polypeptide stock solution group. During the treatment, the body weight of the mice remained stable, which indicated that the spleen polypeptide had good safety in vivo. These results further illustrated the superior effect of the PP-VIII polypeptide in treating tumors in vivo, and it was significantly better than the spleen polypeptide enzyme solution and the spleen polypeptide with a molecular weight of 1-3 kDa.

[0051] Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

Claims

1. A key active peptide component of a spleen polypeptide, characterized in that, The key active peptide component of the spleen polypeptide is a key active peptide component of the spleen polypeptide with a narrower molecular weight range obtained from spleen polypeptide retrieval solution with a molecular weight of 1~3kDa. The key active peptide components of the spleen polypeptide are PP-VIII polypeptide and / or PP-IX polypeptide; The ultraviolet absorption peak of the PP-VIII polypeptide is at 258.5 nm; The PP-VIII polypeptide and PP-IX polypeptide respectively have the effects of enhancing immune activity and / or inhibiting tumor growth.

2. A method for fine separation of key active peptide components of spleen polypeptide according to claim 1, characterized in that, The fine separation method uses spleen polypeptide retrieval solution with a molecular weight of 1~3kDa as raw material and employs gel chromatography to separate the key active peptide components of spleen polypeptide with a narrower molecular weight range. The key active peptide components of the spleen polypeptide are PP-VIII polypeptide and / or PP-IX polypeptide; The ultraviolet absorption peak of the PP-VIII polypeptide is at 258.5 nm; The PP-VIII polypeptide and PP-IX polypeptide respectively have the effects of enhancing immune activity and / or inhibiting tumor growth.

3. The method for fine separation of key active peptide components of spleen polypeptide according to claim 2, characterized in that, Gel chromatography separation uses agarose gel, dextran gel or polyamide gel.

4. The method for fine separation of key active peptide components of spleen polypeptide according to claim 2 or 3, characterized in that, The spleen polypeptide retentate with a molecular weight of 1~3kDa was obtained by fractionating the spleen polypeptide retentate with a molecular weight of 1~3kDa using the spleen polypeptide enzymatic hydrolysis stock solution as raw material and by membrane assembly method.

5. The method for fine separation of key active peptide components of spleen polypeptide according to claim 2 or 3, characterized in that, The fine separation method involves taking a spleen polypeptide retentate with a molecular weight of 1-3 kDa, first separating it through an ultrafiltration membrane system capable of retaining components with a molecular weight greater than 2 kDa, collecting the retentate containing spleen polypeptide with a molecular weight of 2-3 kDa and the dialysate containing spleen polypeptide with a molecular weight of 1-2 kDa respectively, then separating the retentate and dialysate by tandem gel chromatography, extracting 5 fractions from each, drying them to form ten fractions including PP-VIII polypeptide and PP-IX polypeptide.

6. The method for fine separation of key active peptide components of spleen polypeptide according to claim 2 or 3, characterized in that, The fine separation method involves taking a spleen polypeptide retentate with a molecular weight of 1-3 kDa, combining it with ultraviolet spectroscopy tracking, and directly performing gel chromatography separation, followed by drying to form ten components including PP-VIII polypeptide and PP-IX polypeptide.

7. The application of the key active peptide component of the spleen polypeptide according to claim 1, characterized in that, The key active peptide component of the spleen polypeptide is used to prepare immunomodulators; And / or, the key active peptide component of the spleen polypeptide is used to prepare anti-tumor drugs and post-tumor rehabilitation drugs.

8. The application of the key active peptide component of the spleen polypeptide according to claim 7, characterized in that, The immunomodulator enhances immune function by at least one of the following methods: promoting lymphocyte proliferation, increasing immune organ indices, enhancing the phagocytic capacity of peritoneal macrophages, increasing NK cell activity, and regulating serum cytokine levels.

9. The application of the key active peptide component of the spleen polypeptide according to claim 7 or 8, characterized in that, The key active peptide component of the spleen polypeptide inhibits tumor growth by improving immune function.

10. The application of the key active peptide component of the spleen polypeptide according to claim 7 or 8, characterized in that, The key active peptide component of the spleen polypeptide is used to prepare an anti-human malignant melanoma drug. Alternatively, the key active peptide component of the spleen polypeptide may be used to prepare a postoperative rehabilitation drug for human malignant melanoma. Alternatively, the key active peptide component of the spleen polypeptide may be used to prepare a drug that inhibits the metastasis of human malignant melanoma; The key active peptide component of the spleen polypeptide has an inhibition rate of more than 85% on tumor growth.

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