CN114075544A - Method for preparing de-ubiquitin erythrocyte product based on ultrafiltration centrifugation - Google Patents

Abstract

The invention belongs to the technical field of biological pharmacy, and relates to a method for removing ubiquitin protein in a erythrocyte product, in particular to a method for preparing a de-ubiquitin erythrocyte product based on ultrafiltration and centrifugation. The method of the invention comprises the following steps: adding a substance which recognizes and binds to ubiquitin proteins to the red blood cell preparation; and/or ultrafiltering and centrifuging the erythrocyte product to remove ubiquitin protein. The invention also relates to the effect of the transfusion of the deubiquitinating erythrocyte products on the tumor metastasis and recurrence, which comprises preparing products for adjusting the immune functions of patients, such as the functions of neutrophils, NK cells, DC cells and macrophages, thereby affecting the tumor metastasis and recurrence. The method of the invention has simple operation and lower requirements on equipment and sites, and the blood transfusion product for removing ubiquitin is an innovation of clinical practice, can prevent possible immunoreaction of tumor patients after red blood cell transfusion, and reduces adverse reaction of clinical blood transfusion.

Description

Method for preparing de-ubiquitin erythrocyte product based on ultrafiltration centrifugation

Technical Field

The invention belongs to the technical field of biological pharmacy, and relates to a method for removing ubiquitin protein in a erythrocyte product, in particular to a method for preparing a de-ubiquitin erythrocyte product based on ultrafiltration and centrifugation. The invention also comprises the function of immune regulation of ubiquitin protein in the stored erythrocyte product, in particular to the influence of the ubiquitin protein in the stored erythrocyte product after being removed by using the ultrafiltration centrifugation technology in the process of preparing and preventing tumor metastasis and relapse.

Background

The prior art discloses that perioperative blood transfusion has important significance for clinical treatment of tumor patients, can obviously improve anemia symptoms of the tumor patients, and has certain negative effects. In 1981, Lancet journal reported that perioperative blood transfusion can increase tumor recurrence rate and decrease patient survival rate. Subsequent extensive clinical retrospective studies also demonstrated that perioperative blood transfusion may reduce the long-term survival of tumor patients, such as colon, liver, esophageal, lung, stomach, head and neck, uterine, etc. Several studies have shown that patients may experience immune dysfunction after receiving allogeneic blood transfusion, and that red blood cell transfusion may trigger inflammatory reactions in the body, such as leukocyte activation, neutrophil chemotaxis enhancement, monocyte/macrophage activation, and inflammatory factor release; at the same time, the immune suppression of the recipient, such as the inhibition of NK cell and antigen presenting cell functions, the inhibition of lymphocyte proliferation and macrophage phagocytosis, etc., can be caused. Therefore, metastasis and recurrence of tumors by red blood cell infusion may be associated with transfusion-associated immune modulation reactions.

Research shows that the red blood cells are influenced by various factors in the storage process, and the morphology, the function, the biochemical metabolism and the like of the red blood cells are changed to different degrees, and the change is more obvious along with the prolonging of the storage time.

Research shows that allogeneic blood transfusion can promote tumor metastasis and recurrence and reduce the survival rate of patients; transfusion-related immune modulation may be an important factor in mediating such complications. Among them, storage of red blood cell infusions can promote tumor metastasis. Earlier stage researches prove that the ubiquitin content in the red blood cells is obviously higher than that of other blood cells, and the storage condition and the storage time of the red blood cells can influence the release of the ubiquitin in the red blood cells; along with the prolonging of the storage time of the red blood cells, the ubiquitin concentration is linearly increased, and the ubiquitin content in the whole blood stored for 35 days is more than 40 times of the ubiquitin content in the whole blood stored for 0 day; in vitro experiments prove that ubiquitin can inhibit apoptosis of regulatory T cells, promote chemotactic migration of the regulatory T cells and enhance the immunosuppressive action of the regulatory T cells; animal experiments prove that the extracellular ubiquitin can promote lung metastasis of melanoma cells, reduce the proportion of T cells and NK cells in peripheral blood of mice and reduce the long-term survival rate of tumor-bearing mice; meanwhile, in vitro and in vivo experiments prove that ubiquitin can cause imbalance of Th1/Th2, inhibit Th1 and promote secretion of Th2 cytokines.

The existing common protein removal methods have the advantages and disadvantages, and the affinity chromatography technology has higher specificity for removing protein but needs to prepare a specific ligand of a target protein; the use of a precipitant in the precipitation process may alter the original properties of the solution; isoelectric trapping, preparative electrophoresis and liquid chromatography are relatively complex in operation, the possibility of changing the original properties of the solution exists, and the limitation of instruments and fields is large. Based on the current state of the art, the inventors of the present application intend to provide a method for removing ubiquitin proteins from a red blood cell preparation, and particularly, a method for preparing a de-ubiquitin red blood cell preparation based on ultrafiltration and centrifugation. The method with operability and efficiency of the invention is very important for removing protein in blood products and improving the using effect of the blood products.

Disclosure of Invention

The invention aims to provide a method for removing ubiquitin protein in a red blood cell preparation based on the current situation of the prior art, in particular to a method for preparing a de-ubiquitin red blood cell preparation based on ultrafiltration and centrifugation.

The early stage research of the invention shows that animal experiments prove that the storage time of the blood preparation is one of the important factors for mediating the adverse effects of blood transfusion, and the aged red blood cell transfusion can promote tumor metastasis. Therefore, we speculate that various biological mediators in the red blood cell preparation may participate in the regulation of the immune response of the organism, and the storage time of the red blood cells influences the content of the biological mediators, thereby influencing the immune function of the organism in pathological states. The research shows that the damage of the red blood cells is increased and the ubiquitin protein content in the suspension red blood cell preserving fluid is obviously increased along with the prolonging of the storage time. Ubiquitin (Ub) is ubiquitous in all eukaryotic cells as a highly conserved small molecule protein (8.5Kd) containing 76 amino acids. The research shows that the extracellular ubiquitin has the functions of resisting inflammation, inhibiting the immune response of the organism, simultaneously influencing the apoptosis, the proliferation and the differentiation and the like. The 50-59 peptide segment of ubiquitin has the immunosuppressive activity equivalent to that of cyclosporine, and can inhibit cellular immunity and humoral immunity; ubiquitin infusion can prolong survival of mouse allogenic skin grafts. In conclusion, we speculate that the remarkably increased ubiquitin in the stored erythrocyte preparation may affect the immune function of the tumor patients, and mediate adverse effects such as tumor recurrence and metastasis. Therefore, the invention intends that the prepared erythrocyte product can be used for preventing the recurrence and the metastasis of the tumor by removing the ubiquitin protein in the stored erythrocyte product.

After comprehensively analyzing and testing the advantages and disadvantages and operation possibility of the existing methods, the invention primarily realizes the purpose of removing ubiquitin protein in the stored erythrocyte products by adopting the ultrafiltration centrifugation technology. The ultrafiltration centrifugation technology is a technology for selectively separating proteins according to the molecular weight by a microporous structure on the surface of a membrane under the action of centrifugal force, and is widely applied to concentration of microorganism samples containing antigens, antibodies, enzymes, nucleic acids, phages and the like; purifying macromolecular components in the tissue culture extracting solution or the cell lysate and removing primers, joints or molecular markers in the reaction solution. Meanwhile, the technology is already applied to extract exosomes, microparticles and other components in blood. Most of proteins in plasma are removed in the process of preparing the stored red blood cells, and the molecular weights of residual hemoglobin and lipoprotein in the preservation solution are both obviously greater than those of ubiquitin protein, so that the aim of removing ubiquitin protein can be achieved by using ultrafiltration centrifugal tubes with different molecular weight cut-off. Finally, the ultrafiltration and centrifugation technology is simple and convenient to operate, and has lower requirements on equipment and places, so that the feasibility of removing ubiquitin protein in the stored erythrocyte product by using the method is higher. The present invention has been completed based on the above-mentioned studies.

The invention provides a method for removing ubiquitin protein in a red blood cell product, which comprises the following steps:

adding a substance which recognizes and binds to ubiquitin proteins to the red blood cell preparation; and/or

And (4) carrying out ultrafiltration and centrifugation on the erythrocyte product to remove ubiquitin protein.

The substance capable of recognizing ubiquitin protein and binding ubiquitin protein can be antibody of ubiquitin protein, active fragment of antibody of ubiquitin protein or conjugate containing active fragment of antibody of ubiquitin protein.

The ultrafiltration and centrifugation of the erythrocyte preparation comprises the following steps:

centrifuging the red blood cell preparation at low speed; and/or

Removing supernatant of erythrocyte product, ultrafiltering and centrifuging to obtain concentrated solution I and filtrate I, and keeping the concentrated solution I, wherein the speed of ultrafiltration and centrifugation is not less than 10000 g;

the red blood cell product without ubiquitin protein comprises a red blood cell product after low-speed centrifugation and/or a concentrated solution I of the red blood cell product after ultrafiltration and centrifugation.

Centrifuging the filtrate I again to obtain a concentrated solution II and a filtrate II, and mixing the concentrated solution I and the filtrate II to obtain a supernatant of the de-ubiquitin red blood cells;

the centrifugation speed of the second centrifugation is usually not less than 1000 g;

the red blood cell preparation for removing ubiquitin protein comprises one or more of the following components:

the red blood cell product after low-speed centrifugation,

concentrated solution I obtained after the erythrocyte product is ultrafiltered and centrifuged,

and (3) filtrate II of the erythrocyte product after ultrafiltration and re-centrifugation.

Preferably, the ultrafiltration tube is inverted during centrifugation again.

The ultrafiltration centrifugation speed can be 10000g-50000g, and can correspond to 5000rpm-100000rpm according to different centrifuge structures and models. For example, it may be 12000g, 14000g, 15000g, 16000g, 18000g, or 20000rpm, 30000rpm, 40000rpm, 50000rpm, etc.

The speed of the re-centrifugation can be 1000g-3000g, and can correspond to 800rpm-5000rpm according to different structures and models of centrifuges. For example, it may be 1200g, 1400g, 1500g, 1600g, 1800g, 2000g, 2500g, or 1000rpm, 1500rpm, 2000rpm, 2500rpm, 3000rpm, 4000rpm, etc.

Experiments show that the method can remove ubiquitin in blood products. For example, in a preferred embodiment of the present invention, the method of the present invention is capable of removing almost all ubiquitin proteins in red blood cell preparations as measured by the Elisa method.

The invention also comprises a red blood cell preparation which does not contain the active fragment of the ubiquitin protein.

Preferably, the erythrocyte product is obtained by the ultrafiltration and centrifugation method.

The method of the invention can be used for removing ubiquitin protein, active fragments of ubiquitin protein or substances combined with the active fragments of ubiquitin protein in red blood cell preparations.

The invention also comprises the application of the obtained red blood cell product of the deubiquitinating protein in preparing anti-tumor drugs.

Preferably, the anti-tumor drug comprises a drug for inhibiting tumor metastasis and a drug for preventing or relieving tumor recurrence.

The red blood cell product of the deubiquitinating protein obtained by the method can be used for assisting and promoting the activity of immune cells.

For example, methods for altering the effect of deubiquitinating red blood cell infusion on immune cell function in tumor samples include:

the effect of deubiquitinating erythrocyte infusion on neutrophil function in tumor patients;

the effect of deubiquitinating erythrocyte infusion on NK cell function in tumor patients;

the effect of deubiquitinating erythrocyte infusion on DC cell function in tumor patients;

the effect of deubiquitinating erythrocyte infusion on macrophage function in tumor patients;

also included are the effects of deubiquitinating red blood cell infusion on tumor metastasis and recurrence.

The deubiquitinating erythrocyte transfusion can enhance the activity of immune cells and improve the function of the immune cells. Animal experiments show that the deubiquitinating erythrocyte infusion can inhibit tumor metastasis and relapse.

The invention removes ubiquitin protein in erythrocyte products through ultrafiltration centrifugal tubes with different molecular weight cut-off, and experiment observation shows that the unhubulited erythrocytes have influence on tumor progression and immunoregulation of liver cancer rats; in vitro experiments investigated the effect of de-ubiquitin stored erythrocyte supernatants on immune cell function. The achievement of the invention is helpful to elucidate the possible mechanism of regulating the immune function of the liver cancer patient by storing the red blood cells to promote the tumor metastasis and recurrence, lays experimental and theoretical foundation for the application of the deubiquitinating blood in clinical blood transfusion practice, and the deubiquitinating red blood cell transfusion is an innovation in human blood transfusion history and certainly brings far-reaching influence on the reduction of adverse reaction of clinical blood transfusion.

For the purpose of facilitating understanding, the invention will hereinafter be described in detail by way of embodiments with reference to the accompanying drawings. It is to be expressly understood that the description is illustrative only and is not intended as a definition of the limits of the invention. Many variations and modifications of the present invention will be apparent to those skilled in the art in light of the teachings of this specification. In addition, the present invention incorporates publications which are intended to more clearly describe the invention, and which are incorporated herein by reference in their entirety as if reproduced in their entirety.

Drawings

FIG. 1. ubiquitin concentration increased significantly with storage time during whole blood storage.

FIG. 2 infusion of stored blood promotes pulmonary metastasis of melanoma tumor-bearing mice tumors.

(A) Fresh lung tissue from tumor-bearing mice of control, D0 and D15 groups on day 21 post-transfusion. (B) HE staining of lung pathological sections of tumor-bearing mice. (C) Statistics of the relative number of tumor metastasis foci inside lung tissue. (D) And (5) counting the number of tumor lesions on the surface of the lung tissue. (E) Statistics of relative weights of lung tissues in tumor-bearing mice.

FIG. 3 ubiquitin promotes tumor progression in tumor bearing mice, reducing long-term survival in tumor bearing mice.

(A) And (3) analyzing the tumor metastasis condition of the tumor-bearing mice by using a living body fluorescence imaging system. (B) Statistical analysis of luciferase intensity in tumor-bearing mice live imaging. (C) Analysis of the rate of body weight gain of each group of tumor-bearing mice. (D) Long-term survival analysis of each group of tumor-bearing mice.

FIG. 4. Effect of ubiquitin in stored blood on the secretion of TH1/TH2 cytokines from peripheral blood of tumor-bearing mice.

(A) Effect of TH1/TH2 cytokine expression in peripheral blood of tumor-bearing mice after transfusion. (B) The effect of ubiquitin in the storage blood on the expression of TH1/TH2 cytokines in the peripheral blood of tumor-bearing mice.

FIG. 5. Effect of ubiquitin in stored blood on the expression of T, B peripheral blood and subsets of NK and T cells in tumor-bearing mice.

(A) Effect of ubiquitin in storage blood on peripheral blood T, B and NK cell expression in tumor-bearing mice. (B) Analysis of T cell subsets (CD4+ T and CD8+ T) expression in peripheral blood of tumor-bearing mice.

FIG. 6. step of preparing deubiquitinating erythrocytes by ultrafiltration and centrifugation.

FIG. 7. Ultrafiltration centrifuge tube application procedure.

Detailed Description

Example 1 removal of ubiquitin proteins from stored erythrocytes by ultrafiltration centrifugation

Removing ubiquitin by an ultrafiltration centrifugal tube: centrifuging human suspended red blood cells stored for 35 days at a low speed, separating the obtained supernatant by an ultrafiltration centrifugal tube (30K) to obtain a concentrated solution 1 and a filtrate 1, centrifuging the filtrate 1 by an ultrafiltration centrifugal tube (2K) again to obtain a concentrated solution 2 and a filtrate 2, and mixing the concentrated solution 1 and the filtrate 2 to obtain a supernatant of the de-ubiquitin red blood cells for later use;

and detecting the ubiquitin concentration in the supernatant by using an ELISA ubiquitin kit and optimizing the experimental conditions.

Example 2 Effect of Deubiquitinated stock erythrocyte supernatant on immune cell function

Removing ubiquitin protein in a stored erythrocyte product by using an ultrafiltration centrifugation technology;

inducing leukemia cell line NB4 to differentiate into granulocytes by using all-trans retinoic acid (ATRA), incubating the stored erythrocyte supernatant without ubiquitin with granulocytes, and detecting the immune function change:

phagocytic function: flow Cytometry (FCM) detects the phagocytic capacity of neutrophils on Candida albicans,

chemotactic function: the cell of the Transwell is arranged in the chamber,

ROS generation: FCM measures the fluorescence intensity of the molecular probe CM-H2 DCFDA.

And thirdly, after the supernatant of the stored red blood cells with the ubiquitin removed and the human NK cell line NK-92 are incubated together, detecting the change of the killing activity:

detecting the killing activity of the NK cells to K562 cells by a Lactate Dehydrogenase (LDH) release method,

FCM detects NK cell perforin, granzyme B and CD107a expression.

And fourthly, after the supernatant of the stored red blood cells with the ubiquitin removed and the dendritic cell line DC2.4 of the mouse are incubated together, detecting the change of the antigen presenting function:

FCM measures DC2.4 cell surface (MHC class II/CD 86/CD80) expression,

allogeneic Mixed Lymphocyte Reaction (MLR) to examine the ability of DC2.4 cells to stimulate proliferation of allogeneic T lymphocytes,

ELISA was used to detect IFN-. gamma.content in cell supernatants.

Inducing human monocyte THP-1 to differentiate into macrophage by utilizing phorbol lipid (PMA), and detecting the immune function change of the macrophage after co-incubating the supernatant of the stored red blood cell with the ubiquitin removed and the macrophage:

phagocytic ability: the neutral red test is carried out in the presence of a catalyst,

polarization state: FCM measures macrophage feature molecule expression (CD68/CD163/CD86),

ELISA detects the content of cell factors (TGF-beta/IL-10/TNF-alpha/IL-12) in cell supernatant,

macrophage marker (CCL3/CCL18/CCL22/iNOS) expression was detected by PCR.

Example 3 Effect of Deubiquitinated stored Red blood cells on liver cancer progression and immune modulation

Firstly, a Wistar rat liver cancer model is established by intragastric gavage of 0.2% Diethylnitrosamine (DEN) solution;

preparing and storing red blood cells: collecting blood from heart of Wistar rat, removing leukocyte and plasma, adding 14% CPDA protective solution to obtain suspension erythrocyte, and storing at 4 deg.C for 14 days;

thirdly, removing ubiquitin protein in the protective solution for storing the red blood cells by ultrafiltration and centrifugation;

fourthly, the stored red blood cells with the ubiquitin removed are returned to Wistar liver cancer rats through tail veins, and HE staining is carried out to dynamically observe liver cancer metastasis;

collecting peripheral blood of a Wistar liver cancer rat, and detecting the proportion and the type of neutrophils, NK cells, DC cells and macrophages by FCM;

sixthly, collecting peripheral blood of the Wistar liver cancer rat, separating neutral granulocytes, NK cells, DC cells and macrophages by magnetic beads, and detecting the change of the cellular immune function by the method in 2.2;

collecting peripheral blood of Wistar liver cancer rat, and detecting the content of cell factors (TGF-beta/IL-10/TNF-alpha and the like) in blood serum by ELISA.

And (3) detecting the phagocytic capacity of the neutrophil granulocytes: counting and re-suspending Candida albicans to concentration of 300 × 106/ml; adding candida albicans into a culture medium containing 1.5 mu g/ml 5(6) -FAM-SE fluorescence, incubating at 4 ℃ in a dark place for 30min under continuous stirring, and adding an equal volume of 50mM glycine buffer solution for termination; ③ treatment with neutrophils: (6) -FAM-SE-labeled c. albicans ═ 1: 3, mixing, and incubating at 4 ℃ for 30min in dark. Transferring the mixture into a flow tube, adding an isometric TB solution, placing on ice for 1min, and performing up-flow detection.

Neutrophil chemotactic capacity assay: adding 600ul of chemotactic factor and serum-free culture solution into a lower chamber of a transwell chamber; adding 100ul of cells and serum-free culture solution into the upper chamber and incubating for 2 hours; taking out the upper chamber, wiping off the cells on the membrane, centrifuging to centrifuge the cells adhered to the membrane to the lower chamber, and counting.

And (3) ROS detection: resuspending neutrophils by using 1 XHBSS, and adjusting the cell density to 5X 106/ml; adding 100 mul of cell suspension into a 96-pore plate, standing at 37 ℃ for 10min, adding a molecular probe CM-H2DCFDE with the final concentration of 2.5 mu mol/L into each pore, and incubating for 10min at 37 ℃ in a dark place; thirdly, rinsing the culture solution, resuspending the cells, standing at 37 ℃ for 20min, and adding PMA with the final concentration of 300nmol/L for stimulation; adding precooled PBS to stop the reaction, rinsing the reaction by PBS, resuspending the cells, and detecting the cells in an up-flow mode.

And (3) detecting the killing activity of NK cells: firstly, using RPMI1640 containing 10% calf serum to resuspend NK cells to be 2 x 109/L, and resuspending K562 cells in logarithmic growth phase to be 2 x 108/L; mixing equal amounts of NK and K562 cells, and incubating in a 5% CO2 incubator at 37 ℃ for 6 h; ③ centrifuging at 1500r/min for 10min, collecting the supernatant, and measuring the LDH activity by a full-automatic biochemical analyzer.

NK cell perforin, granzyme B and CD107a expression assay: PBS resuspending NK cells to be 1 × 1010/L, adding 20ul of FITC labeled CD56 antibody into each tube, and incubating for 30min in a dark place; adding 100ul of fixing solution, incubating for 15min in the dark at room temperature, and washing for 1 time by PBS; ③ after 100ul of membrane-breaking liquid is added into each tube, 100ul of PE-labeled anti-Perforin, anti-granzyme B antibody and APC-labeled CD107a antibody are respectively added, and incubation is carried out for 15min at room temperature in a dark place; and fourthly, rinsing by PBS and carrying out up-flow detection after cell resuspension.

Allogenic mixed lymphocyte reaction: separating T cell, inoculating 1 × 105/hole in 96 hole plate; ② after mitomycin (25. mu.g/ml) stimulates DC2.4 cells for 30min, RPMI1640 was washed 3 times. ③ mixing DC2.4 cells and T cells in a ratio of 1: 10 to obtain a final volume of 200. mu.l; fourthly, co-culturing the DC2.4 cells and the T cells for 72 hours, and adding 3H-TdR in the last 12 hours of culture; collecting cells on glass fiber paper, drying, measuring pulse number per minute by beta-liquid scintillation analyzer, and detecting T lymphocyte proliferation reaction.

And (3) detecting the phagocytic capacity of macrophages: PMA induces THP-1 cells to be macrophages, then 0.1% neutral red physiological saline solution is added, and the culture is continued for 20 min; ② removing supernatant, washing with warm PBS for 3 times, adding 0.2ml of cell lysis solution into each hole, and standing for 2-3h at room temperature; thirdly, after the cells are dissolved, the enzyme-labeling instrument detects the absorbance at the wavelength of 540 nm.

FC detection macrophage proportion and typing: digesting and collecting cells, and resuspending and washing the cells by PBS; ② adding 5ul CD86 antibody, incubating for 30min at 4 ℃ in the dark; ③ resuspending the cells by PBS, washing the cells, adding 100 mul Reagent A fixing solution to resuspend the cells, and incubating for 15min at room temperature in a dark place; adding 100 mul Reagent B fixing liquid to resuspend the cells after PBS is resuspended and the cells are washed, adding 5 mul CD68 and 20 mul CD163 antibody, and incubating for 30min at 4 ℃ in a dark place; fifthly, resuspending PBS and washing the cells, adding 500 mul PBS to resuspend the cells, and then detecting on a machine.

ELISA detection of cytokine content: adding 100 mul of cell supernatant or serum samples into each hole, sealing the reaction holes, incubating in an incubator at 37 ℃ for 2 hours, and washing the plate for 4 times; adding 100 mul of biotinylation antibody working solution into each hole, sealing the reaction hole, incubating in an incubator at 37 ℃ for 60min, and washing the plate for 5 times; ③ adding 100 mul of the working solution of the enzyme conjugate into each hole, sealing the reaction holes, incubating for 30min at 37 ℃ in the dark, and washing the plate for 5 times; adding 100 mul of chromogenic substrate into each hole, and incubating for 15min at 37 ℃ in a dark place; fifthly, adding 100ul of stop solution into each hole, and detecting the OD450 value by an enzyme-labeling instrument after uniformly mixing.

qPCR detection of target gene mRNA expression: firstly, primer sequences are designed and synthesized by Shanghai worker; ② extracting total RNA; reverse transcription of RNA into cDNA with the reverse transcription kit; and fourthly, mixing the primer, the cDNA and the PCR mix and then detecting on a machine.

Establishing a rat liver cancer model: wistar rats were given a 0.2% DEN lavage and dosed at 10mg/kg body weight, 1.5-2.0ml/d per dose, 5 times/week, and discontinued by 14 weeks.

The above description is only for the specific embodiments of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present disclosure should be covered within the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims (9)

1. A method for preparing a deubiquitinating erythrocyte product based on ultrafiltration and centrifugation is characterized by comprising the following steps:

adding a substance which recognizes and binds to ubiquitin proteins to the red blood cell preparation; and/or

And (4) carrying out ultrafiltration and centrifugation on the erythrocyte product to remove ubiquitin protein.

2. The method according to claim 1, wherein the substance that recognizes and binds to ubiquitin protein is an antibody against ubiquitin protein, an active fragment of an antibody against ubiquitin protein, or a conjugate containing an active fragment of an antibody against ubiquitin protein.

3. The method of claim 1, wherein said ultra-filtration centrifugation of said red blood cell preparation comprises the steps of:

centrifuging the red blood cell preparation at low speed; and/or

Taking supernatant of a erythrocyte product, performing ultrafiltration and centrifugation to obtain a concentrated solution I and a filtrate I, and reserving the concentrated solution I; wherein the speed of ultrafiltration centrifugation is not less than 10000 g;

the red blood cell product without ubiquitin protein comprises a red blood cell product after low-speed centrifugation and/or a concentrated solution I of the red blood cell product after ultrafiltration and centrifugation.

4. The method of claim 3, wherein the filtrate I is centrifuged again to obtain a concentrate II and a filtrate II, and the concentrate I and the filtrate II are mixed to obtain a supernatant of de-ubiquitinated erythrocytes;

the centrifugation speed of the second centrifugation is 1000g-3000 g;

the red blood cell preparation for removing ubiquitin protein comprises the red blood cell preparation after low-speed centrifugation and/or one or two of the following components:

concentrated solution I obtained after the erythrocyte product is ultrafiltered and centrifuged,

and (3) obtaining filtrate II after the filtrate of the erythrocyte product after ultrafiltration and centrifugation is centrifuged again.

5. The method of claim 4, wherein the ultrafiltration centrifugation rate is 14000g and the recentrifugation rate is 2000 g.

6. A red blood cell preparation, wherein said red blood cell preparation does not contain an active fragment of ubiquitin protein.

7. Use of the method according to any one of claims 1 to 5 for the preparation of a substance for the removal of ubiquitin proteins, active fragments of ubiquitin proteins or active fragments of conjugated ubiquitin proteins from a preparation of red blood cells.

8. The use according to claim 7, characterized in that the red blood cell preparation of deubiquitinating protein obtained by the method is used for preparing anti-tumor drugs; the anti-tumor medicament comprises a medicament for inhibiting tumor metastasis or recurrence.

9. The use according to claim 7, characterized in that said red blood cell preparation of deubiquitinating protein obtained by said method is used for preparing preparations for assisting the promotion of the activity of immune cells.

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