Application of alpha2-macroglobulin

Abstract

The invention relates to application of alpha2-macroglobulin. The alpha2-macroglobulin can be used for promoting the interment effect of cells. The invention finds that the alpha2-macroglobulin can be used for promoting the intercellular effect of cells, and further can be possibly used in medicines for treating diseases such as atherosclerosis, inflammatory aging, tumors, chronic obstructive pulmonary disease and systemic lupus erythematosus caused by deficiency of intercellular function.

Description

Technical Field

[0001] The present invention relates to the technical field of macroglobulins, and in particular to an application of α2-macroglobulin. Background Art

[0002] About 200 billion cells die in the human body every day. These dead cell fragments are usually eliminated and digested by tissue-resident phagocytes (such as macrophages) or non-professional phagocytes (such as retinal pigment epithelial cells) through endocytosis, a process also known as "cell burial". The mechanism of cell burial can be divided into three steps: phagocytes recognize the "find me" signal released by apoptotic cells and approach them, phagocyte burial-specific membrane receptors bind to the ligands exposed on the surface of apoptotic cells and initiate cell endocytosis, and phagocytes digest the contents of burial endocytic vesicles. Through cell burial, dead cells in local tissues can be quickly cleared and tissue homeostasis is maintained. When the cell burial function is impaired, a large amount of cell fragments and necrotic nuclei will accumulate in local tissues, and intracellular toxic molecules will be continuously released, resulting in pro-inflammatory signals being stronger than anti-inflammatory signals, which in turn causes an imbalance in the inflammatory response.

[0003] At present, more and more studies have shown that insufficient efferocytosis is an important cause of diseases such as atherosclerosis, inflammatory aging, tumors, chronic obstructive pulmonary disease, and systemic lupus erythematosus. Therefore, drug design targeting efferocytosis has gradually attracted attention, and efferocytosis-promoting molecules are expected to provide new treatments for a variety of refractory inflammatory diseases. Summary of the invention

[0004] In view of the above analysis, an embodiment of the present invention aims to provide an application of α2-macroglobulin to at least solve one of the above problems.

[0005] The invention provides an application of α2-macroglobulin, which can be used to promote the efferocytosis of macrophages.

[0006] Furthermore, the α2-macroglobulin can be used as a drug for treating diseases caused by insufficient macrophage efferocytosis function.

[0007] Furthermore, the α2-macroglobulin is used to prepare a drug for promoting macrophage efferocytosis.

[0008] Furthermore, the purity of the α2-macroglobulin is not less than 60%.

[0009] Furthermore, the α2-macroglobulin is dissolved in a neutral aqueous buffer.

[0010] Furthermore, the concentration of the α2-macroglobulin solution is 200 nM to 3200 nM.

[0011] Furthermore, the raw material for extracting the α2-macroglobulin is plasma or plasma Cohn fraction IV.

[0012] Furthermore, when plasma Cohn method fraction IV is used as the extraction raw material to obtain α2-macroglobulin, the following steps are included:

[0013] S100: Provide extraction raw materials, use ammonium sulfate for salting out,

[0014] Dissolve the plasma Cohn method component IV with physiological saline, take the supernatant after natural precipitation, add ammonium sulfate to the supernatant, stir evenly, centrifuge, take the precipitate and resuspend it with water, filter the resuspended solution with the first filter membrane to obtain the first filtrate, and the volume of the first filtrate is recorded as s;

[0015] S200: Chromatographic pretreatment,

[0016] The first filtrate obtained in step S100 is ultrafiltered and concentrated, and the volume of the concentrate after ultrafiltration is (20% to 50%)*s. The complete removal of sulfate ions is confirmed by titration, and then a first buffer solution with a volume four times that of the concentrate is added, and a second filtrate is obtained by filtering with a second filter membrane;

[0017] S300: Zinc Affinity Chromatography

[0018] The second filtrate obtained in step S200 is subjected to zinc ion affinity chromatography by a protein purifier, which specifically comprises the following steps:

[0019] S301: Fill the chromatography medium into the chromatography column, connect the chromatography column to the protein purification instrument, and use ZnCl2 buffer to chelate Zn 2+ ;

[0020] S302: equilibrate the buffer chromatography medium using a first buffer;

[0021] S303: pumping the second filtrate into the chromatography column to allow the target protein to fully bind to the chromatography medium;

[0022] S304: using a first buffer to elute proteins that are not bound to the chromatography medium to a baseline, and then adding a second buffer to elute non-specifically bound or weakly bound proteins to a baseline;

[0023] S305: using the first eluent to elute the zinc ion binding protein to obtain a chromatography eluent;

[0024] S400: The chromatography eluate is concentrated by ultrafiltration, and then sterilized by filtration using a filter to obtain an α2-macroglobulin stock solution.

[0025] Furthermore, when plasma is used as the extraction raw material to obtain α2-macroglobulin, the following steps are included:

[0026] Z100: Chromatography Pretreatment

[0027] The plasma is balanced in a water bath, then diluted with a third buffer, and after dilution, filtered with a third filter membrane to obtain a third filtrate;

[0028] Z200: Zinc Affinity Chromatography

[0029] The third filtrate obtained in step Z100 is subjected to zinc ion affinity chromatography by a protein purifier, which specifically comprises the following steps:

[0030] Z201: Fill the chromatography medium into the chromatography column and connect the chromatography column to the protein purifier. Use ZnCl2 buffer to chelate Zn 2+ ;

[0031] Z202: equilibrate the buffer purification medium with a fourth buffer;

[0032] Z203: pumping the third filtrate obtained in step Z100 into the chromatography column to allow the target protein to fully combine with the chromatography medium;

[0033] Z204: Use the fourth buffer to elute the proteins that are not bound to the chromatography medium or the proteins that are weakly bound to the chromatography medium until the baseline is reached;

[0034] Z205: using the second eluent to elute the protein bound to the purification medium to obtain a chromatography eluent;

[0035] Z300: The chromatography eluate is concentrated by ultrafiltration, and then sterilized by filtration using a filter to obtain the α2-macroglobulin stock solution.

[0036] Furthermore, the α2-macroglobulin stock solution is stored by freeze-drying the solution or refrigerating the stock solution at 4°C.

[0037] Compared with the prior art, the present invention can achieve at least one of the beneficial effects: the present invention finds that α2-macroglobulin can be used to promote the efferocytosis of macrophages, and may also be used in drugs for treating diseases such as atherosclerosis, inflammatory aging, tumors, chronic obstructive pulmonary disease, systemic lupus erythematosus, etc. caused by loss of efferocytosis function.

[0038] In the present invention, the above-mentioned technical solutions can also be combined with each other to achieve more preferred combination solutions. Other features and advantages of the present invention will be described in the subsequent description, and some advantages can become obvious from the description, or can be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained through the contents particularly pointed out in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The drawings are only for the purpose of illustrating particular embodiments and are not to be considered limiting of the present invention. Like reference symbols denote like components throughout the drawings.

[0040] Figure 1 It is a graph showing the change in the ability of RAW264.7 cells to phagocytize fluorescently labeled Ecoli cell fragments after the cells were treated with A2M at different concentrations in a specific embodiment (a histogram of fluorescence intensity measured by a microplate reader);

[0041] Figure 2 This is a diagram showing changes in the ability of RAW264.7 cells to phagocytize fluorescently labeled Ecoli cell fragments after the cells were treated with A2M at different concentrations in a specific embodiment (fluorescence imaging image taken with a confocal microscope);

[0042] Figure 3 This is a graph showing the change in the proportion of cells positive for burial after RAW264.7 cells were treated with A2M at different concentrations in a specific embodiment, using 2 micron fluorescently labeled microspheres as indicators;

[0043] Figure 4 This is a graph showing changes in intracellular fluorescence intensity of cell burial-positive cells after RAW264.7 cells were treated with A2M at different concentrations in a specific embodiment, using 2-micron fluorescent-labeled microspheres as indicators;

[0044] Figure 5 This is a graph showing the change in the proportion of cells positive for burial after J774A.1 cells were treated with A2M at different concentrations in a specific embodiment, using 2-micron fluorescent-labeled microspheres as indicators;

[0045] Figure 6 This is a graph showing changes in intracellular fluorescence intensity of cell burial-positive cells after J774A.1 cells were treated with A2M at different concentrations in a specific embodiment, using 2-micron fluorescently labeled microspheres as indicators;

[0046] Figure 7 This is a flow cytometric detection diagram of RAW264.7 cells phagocytosing 2-micron fluorescent-labeled microspheres after the cells were treated with A2M at different concentrations in a specific embodiment;

[0047] Figure 8 This is a flow cytometric detection diagram of J774A.1 cells phagocytosing 2-micron fluorescent-labeled microspheres after the cells were treated with different concentrations of A2M in a specific embodiment. DETAILED DESCRIPTION

[0048] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, wherein the accompanying drawings constitute a part of the present invention and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not used to limit the scope of the present invention.

[0049] In the description of the embodiments of the present invention, it should be noted that, unless otherwise clearly specified and limited, the term "connected" should be understood in a broad sense, for example, it can be a fixed connection, or a detachable connection, or an integral connection, which can be a mechanical connection, or an electrical connection, which can be a direct connection, or can be indirectly connected through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0050] The terms "top", "bottom", "above", "lower", and "on" used throughout the description are relative to the relative positions of components of a device, such as the relative positions of the top and bottom substrates within a device. It is understood that devices are multifunctional regardless of their orientation in space.

[0051] The common working surface of the present invention can be a plane or a curved surface, can be inclined, or can be horizontal. For the convenience of description, the embodiment of the present invention is placed on a horizontal plane and used on the horizontal plane, and "high and low" and "up and down" are defined in this way.

[0052] A specific embodiment of the present invention discloses an application of α2-macroglobulin, which can be used to promote the efferocytosis of macrophages, that is, α2-macroglobulin can be used in tissues, organs, and systems with insufficient efferocytosis, that is, α2-macroglobulin can be used in drugs that promote the efferocytosis of macrophages, and can act on the treatment of tissues, organs, and systems with insufficient efferocytosis. Furthermore, the α2-macroglobulin may also be used to treat diseases caused by insufficient efferocytosis, such as atherosclerosis, inflammatory aging, tumors, chronic obstructive pulmonary disease, systemic lupus erythematosus, and other diseases.

[0053] α2-macroglobulin (A2M) is the protein with the largest molecular weight in plasma. It is synthesized by hepatocytes and monocyte-macrophage system. Its number in Uniprot database is P01023.

[0054] The invention provides an application of α2-macroglobulin, which includes using α2-macroglobulin to prepare a drug for promoting cell efferocytosis.

[0055] When the α2-macroglobulin is used to prepare a drug for promoting cell efferocytosis, the purity of the α2-macroglobulin is not less than 60%.

[0056] When the α2-macroglobulin is used to prepare a drug for promoting cell efferocytosis, the α2-macroglobulin is dissolved in a neutral aqueous buffer.

[0057] Preferably, when the α2-macroglobulin is used to prepare a drug for promoting cell efferocytosis, its concentration is 200 nM to 3200 nM, where nM refers to nmol / L.

[0058] The extraction material of α2-macroglobulin is plasma or plasma Cohn fraction IV. Preferably, the extraction material of α2-macroglobulin is plasma Cohn fraction IV precipitate or fresh frozen plasma. Exemplarily, the extraction material is human plasma Cohn fraction IV precipitate or human fresh frozen plasma.

[0059] When plasma fraction Cohn method fraction IV (such as human plasma fraction IV precipitation) is used as the extraction raw material to obtain α2-macroglobulin, the method comprises the following steps:

[0060] S100: Provide extraction raw materials, use ammonium sulfate for salting out,

[0061] Dissolve the plasma Cohn method component IV with physiological saline, take the supernatant after natural precipitation, add ammonium sulfate to the supernatant, stir evenly, centrifuge, take the precipitate and resuspend it with water, filter the resuspended solution with the first filter membrane to obtain the first filtrate, and the volume of the first filtrate is recorded as s;

[0062] S200: Chromatographic pretreatment,

[0063] The first filtrate obtained in step S100 is ultrafiltered and concentrated. The volume of the concentrate after ultrafiltration is (20% to 50%)*s. The complete removal of sulfate ions is confirmed by titration, and then a first buffer solution with a volume four times that of the concentrate is added, and the second filtrate is obtained by filtering with a second filter membrane; S300: zinc ion affinity chromatography

[0064] The second filtrate obtained in step S200 is subjected to zinc ion affinity chromatography by a protein purifier, which specifically comprises the following steps:

[0065] S301: Fill the chromatography medium into the chromatography column, connect the chromatography column to the protein purification instrument, and use ZnCl2 buffer to chelate Zn 2+ ;

[0066] S302: equilibrate the buffer chromatography medium using a first buffer;

[0067] S303: pumping the second filtrate into the chromatography column to allow the target protein to fully bind to the chromatography medium;

[0068] S304: using the first buffer to elute the protein not bound to the chromatography medium, eluting to the baseline, and then adding the second buffer to elute the non-specifically bound or weakly bound protein, eluting to the baseline; S305: using the first eluent to elute the zinc ion-bound protein to obtain a chromatography eluent;

[0069] S400: The chromatographic eluate is ultrafiltered and concentrated using a hollow fiber column, and then sterilized by filtration using a filter to obtain an α2-macroglobulin stock solution.

[0070] In step S100, the plasma Cohn fraction IV is the IV fraction obtained by the Cohn separation method, and may also be referred to as Cohn fraction IV precipitate, Cohn fraction IV, Cohn fraction IV precipitate, or Cohn fraction IV.

[0071] The physiological saline is a 0.9% sodium chloride solution. The mass volume ratio (g:mL) of plasma Cohn method component IV to physiological saline is 1:5, that is, 1g of Cohn component IV precipitate is dissolved in 5mL of physiological saline, and the dissolution process is stirred by a reactor at a stirring speed of 150-200r / min. After dissolution and natural precipitation, the supernatant is taken, and the volume of the supernatant is recorded as a.

[0072] Add ammonium sulfate to the supernatant at a ratio of 1:0.313 (mL:g) and stir thoroughly for 2 hours. Determine whether the sulfate radical has been completely removed by barium chloride titration, that is, take a small amount of ultrafiltration solution and add barium chloride solution dropwise. If no white precipitate is produced, the sulfate radical has been completely removed.

[0073] Use a high-speed centrifuge to centrifuge at a speed of 22500r / min for 10min, obtain the centrifugal precipitate and then resuspend it in 50%*a distilled water.

[0074] The pore size of the first filter membrane is less than or equal to 0.8 μm.

[0075] Specifically, the plasma component IV precipitate was dissolved with physiological saline at a mass volume ratio (g:mL) of 1:5, and the supernatant was taken after natural precipitation (the volume of the supernatant was recorded as a), and ammonium sulfate was added to the supernatant at a ratio of 1:0.313 (mL:g), and the mixture was fully stirred for 2 hours. The mixture was centrifuged in a high-speed centrifuge at a speed of 22500 r / min for 10 minutes to obtain the centrifugal precipitate, which was then resuspended in 50%*a distilled water and filtered through a first filter membrane with a pore size of less than or equal to 0.8 μm to obtain the first filtrate, and the volume of the first filtrate was recorded as s.

[0076] Step S200: The first buffer solution is a NaH2PO4 buffer solution. Specifically, the first buffer solution includes NaH2PO4 and NaCl. The concentration of NaH2PO4 is 0.02 mol / L, the concentration of NaCl is 0.5 mol / L, the solvent is deionized water, and the pH of the buffer solution is 6.0.

[0077] The pore size of the second filter membrane is less than or equal to 0.45 μm.

[0078] The material of the first filter membrane and the second filter membrane is one of polypropylene, polyethersulfone, mixed cellulose ester, polyvinylidene fluoride membrane, nylon, etc. The pore size of the second filter membrane is smaller than or equal to the pore size of the first filter membrane.

[0079] Specifically, the first filtrate obtained in step S100 is ultrafiltrated and concentrated, and the volume of the concentrate after ultrafiltration is (20% to 50%)*s. The titration method is used to confirm the complete removal of sulfate ions, and then a first buffer solution with a volume four times that of the concentrate is added, and a second filter membrane with a pore size less than or equal to 0.45 μm is used to obtain a second filtrate; wherein the first buffer solution is an aqueous buffer solution including 0.02 M NaH2PO4 and 0.5 M NaCl, and the pH is 6.0.

[0080] In step S300, the protein purifier used is AKTA purifier explorer 100 produced by GE.

[0081] The chromatography medium is also called a purification medium. Exemplarily, the chromatography medium is an IDA metal chelate affinity chromatography medium, an NTA affinity chromatography medium, a chitosan affinity chromatography medium, or the like.

[0082] The concentration of ZnCl2 buffer is 0.2 mol / L.

[0083] The first buffer solution is a NaH2PO4 buffer solution. Specifically, the first buffer solution includes NaH2PO4 and NaCl. The concentration of NaH2PO4 is 0.02 mol / L, the concentration of NaCl is 0.5 mol / L, the solvent is deionized water, and the pH value of the buffer solution is 6.0.

[0084] The second buffer solution includes NaH2PO4 and NaCl, the concentration of NaH2PO4 is 0.02 mol / L, the concentration of NaCl is 0.5 mol / L, the solvent is deionized water, and the pH value of the buffer solution is 5.3.

[0085] The pH gradient elution of the present invention utilizes the charge characteristics of proteins at different pH values ​​for separation. When the pH value changes, the surface charge of the proteins will also change accordingly, which changes their binding ability on the ion exchange chromatography column. By gradually changing the pH of the elution buffer, the elution of the target protein can be achieved.

[0086] The first eluent is a 0.1M EDTA-2Na aqueous solution with a pH of 6.7. That is, the first eluent includes EDTA-2Na, the concentration of EDTA-2Na is 0.1 mol / L, the solvent is deionized water, and the pH of the eluent is 6.7.

[0087] The EDTA in the first eluent is a strong metal ion chelator that can form stable complexes with a variety of metal ions. During the elution process, EDTA competitively replaces the coordination bond between the protein and the zinc ion by forming a more stable complex with the zinc ion, thereby eluting the target protein from the affinity chromatography medium. Since the affinity of EDTA to metal ions is usually stronger than that of the amino acid residues in the protein, the target protein bound to the medium can be effectively eluted.

[0088] Specifically, the second filtrate obtained in step S200 is subjected to zinc ion affinity chromatography by a protein purifier, which specifically includes the following steps:

[0089] S301: Fill the IDA metal chelate affinity chromatography medium into the chromatography column, connect the chromatography column to the protein purification instrument, and use 0.2 M ZnCl2 buffer to chelate Zn 2+ ;

[0090] S302: using a first buffer (i.e., an aqueous buffer of 0.02 M NaH2PO4 and 0.5 M NaCl, pH 6.0) to balance the buffer purification medium;

[0091] S303: pumping the second filtrate into the chromatography column to allow the target protein to fully bind to the chromatography medium, i.e., the A2M molecule contains a zinc ion binding domain, and after chelating zinc ions on the chromatography medium, A2M can be adsorbed and bound to the chromatography medium;

[0092] S304: using a first buffer to elute proteins not bound to the chromatography medium to a baseline, and then adding a second buffer to elute non-specifically bound or weakly bound proteins to a baseline; wherein the second buffer is an aqueous buffer comprising 0.02 M NaH2PO4 and 0.5 M NaCl, with a pH of 5.3;

[0093] S305: using a first eluent to elute the zinc ion binding protein to obtain a chromatography eluent; the first eluent is a 0.1 M EDTA-2Na aqueous solution with a pH of 6.7;

[0094] In the step S400, the chromatography eluate is ultrafiltered and concentrated using a hollow fiber column (UFP-300-C-3A, Cytiva), and then sterilized by filtration using a filter (Merck Millipore, SLGP033RB).

[0095] When plasma is used as the extraction raw material to obtain α2-macroglobulin, the method comprises the following steps:

[0096] Z100: Chromatography Pretreatment

[0097] The plasma is balanced in a water bath, then diluted with a third buffer, and after dilution, filtered with a third filter membrane to obtain a third filtrate;

[0098] Z200: Zinc Affinity Chromatography

[0099] The third filtrate obtained in step Z100 is subjected to zinc ion affinity chromatography by a protein purifier, which specifically comprises the following steps:

[0100] Z201: Fill the chromatography medium into the chromatography column and connect the chromatography column to the protein purifier. Use ZnCl2 buffer to chelate Zn 2+ ;

[0101] Z202: equilibrate the buffer purification medium with a fourth buffer;

[0102] Z203: pumping the third filtrate obtained in step Z100 into the chromatography column to allow the target protein to fully combine with the chromatography medium;

[0103] Z204: Use the fourth buffer to elute the proteins that are not bound to the chromatography medium or the proteins that are weakly bound to the chromatography medium until the baseline is reached;

[0104] Z205: using the second eluent to elute the protein bound to the purification medium to obtain a chromatography eluent;

[0105] Z300: The chromatography eluate is concentrated by ultrafiltration, and then sterilized by filtration using a filter to obtain the α2-macroglobulin stock solution.

[0106] Preferably, fresh frozen plasma, such as human fresh frozen plasma, is used.

[0107] In step Z100, the temperature of the water bath is 30-37°C (preferably 37°C).

[0108] The third buffer comprises Tris-HCl and NaCl, the concentration of Tris-HCl is 0.02 mol / L, the concentration of NaCl is 0.5 mol / L, the solvent is deionized water, and the pH of the buffer is 6.0. That is, the third buffer comprises 0.02 M Tris-HCl and 0.5 M NaCl aqueous buffer, and the pH is 6.0.

[0109] After the plasma is equilibrated in a water bath, it is diluted with the third buffer at a volume ratio of 1:2 to 1:9 (preferably 1:2).

[0110] The pore size of the third filter membrane is less than or equal to 0.45 μm.

[0111] Specifically, the plasma is equilibrated in a water bath at 30-37°C (preferably 37°C), and then diluted with a third buffer solution at a volume ratio of 1:2-1:9 (preferably 1:2). After dilution, the third filtrate is filtered through a third filter membrane with a pore size less than or equal to 0.45 μm to obtain a third filtrate, wherein the third buffer solution is an aqueous buffer solution including 0.02 M Tris-HCl and 0.5 M NaCl, and has a pH of 6.0.

[0112] In step Z200, the protein purifier used was AKTA pure 150L from Cytiva.

[0113] The chromatography medium is also called a purification medium. Exemplarily, the chromatography medium is an IDA metal chelate affinity chromatography medium, an NTA affinity chromatography medium, a chitosan affinity chromatography medium, or the like.

[0114] The concentration of ZnCl2 buffer is 0.2 mol / L.

[0115] The fourth buffer comprises imidazole, NaCl and Tris-HCl, the concentration of imidazole is 20 mmol / L, the concentration of NaCl is 0.5 mol / L, the concentration of Tris-HCl is 0.02 mol / L, the solvent is deionized water, and the pH of the buffer is 6.0. That is, the fourth buffer comprises 20 mM imidazole, 0.5 M NaCl and 0.02 M Tris-HCl aqueous buffer, and the pH is 6.0.

[0116] The second eluent includes imidazole, NaCl and Tris-HCl, the concentration of imidazole is 100mmol / L, the concentration of NaCl is 0.5mol / L, the concentration of Tris-HCl is 0.02mol / L, the solvent is deionized water, and the pH of the buffer is 6.0. That is, the second eluent is an aqueous buffer including 100mM imidazole, 0.5M NaCl, and 0.02M Tris-HCl, and the pH is 6.0.

[0117] The chromatographic eluate was concentrated by ultrafiltration using a hollow fiber column (UFP-300-E-3MA, Cytiva), and then sterilized by filtration using a filter (Merck Millipore, SLGP033RB) to obtain a stock solution of α2-macroglobulin.

[0118] Specifically, the third filtrate obtained in step Z100 is subjected to zinc ion affinity chromatography by a protein purifier, which specifically includes the following steps:

[0119] Z201: IDA metal chelate affinity chromatography medium (i.e., purification medium) is loaded into a chromatography column, and the chromatography column is connected to a protein purifier. Zn is chelated using 0.2 M ZnCl2 buffer. 2+ ;

[0120] Z202: using a fourth buffer to balance the buffer purification medium, wherein the fourth buffer is an aqueous buffer comprising 20 mM imidazole, 0.5 M NaCl and 0.02 M Tris-HCl, with a pH of 6.0;

[0121] Z203: pumping the third filtrate obtained in step Z100 into the chromatography column to allow the target protein to fully combine with the chromatography medium;

[0122] Z204: Use the fourth buffer to elute the proteins that are not bound to the chromatography medium or the proteins that are weakly bound to the chromatography medium until the baseline is reached;

[0123] Z205: Use a second eluent to elute the protein bound to the purification medium to obtain a chromatography eluent, wherein the second eluent is an aqueous buffer solution including 100 mM imidazole, 0.5 M NaCl, and 0.02 M Tris-HCl, with a pH of 6.0.

[0124] In step Z300, the chromatography eluate is ultrafiltered and concentrated using a hollow fiber column (UFP-300-E-3MA, Cytiva), and then filtered and sterilized using a filter (Merck Millipore, SLGP033RB) to obtain an α2-macroglobulin stock solution.

[0125] The A2M stock solutions extracted by the above two methods can be stored by freeze-drying the solutions or refrigerating the stock solutions at 4°C.

[0126] The promoting effect of A2M on macrophage efferocytosis was verified by cell phagocytosis of fluorescently labeled E. coli cell fragments and 2.0 μm fluorescent microspheres. This is a classic experimental design for evaluating the level of cell efferocytosis. The two fluorescent particles are commonly used efferocytosis indicators.

[0127]

Experiment 1

[0128] RAW264.7 cells were cultured at 1×10 6The cells were plated at a density of 100 μL / mL in a 96-well plate, with 100 μL of cell suspension in each well, and cultured for 24 hours. After 24 hours of cell adhesion, the culture medium in the wells was discarded, and 100 μL of A2M solution of different concentrations was added to each well. A control group (no drug treatment, i.e., control group) and experimental groups (200nM, 800nM group, and 3200nM group) were set up, with four replicate wells in each group; after 24 hours of A2M treatment, the solution in the wells was discarded, and 100 μL of 0.5 mg / ml fluorescently labeled E. coli cell fragment solution was added to each well and co-cultured with the cells for 2 hours, then discarded and trypan blue was added to quench the fluorescent particles adhered to the cell membrane. Wash twice with PBS, and then measure its fluorescence intensity using a multifunctional microplate reader (excitation wavelength 480nm, detection wavelength 520nm). The results are as follows: Figure 1 The confocal fluorescence microscope was used to photograph the fluorescent biological particles of E. coli in the cells. The results are shown in Figure 2 shown.

[0129] from Figure 1 and Figure 2 It can be seen that A2M solution in the range of 800nmol / L to 3200nmol / L can promote RAW264.7 macrophages to phagocytize E.coli fluorescent bioparticles, and its promotion ability is 11.6-18.5 times that of the Control group.

[0130]

Experiment 2

[0131] RAW264.7 and J774A.1 cells were cultured at 6 × 10 5 / mL were plated in 24-well plates, with 1ml of cell suspension in each well, and cultured for 24 hours. After 24 hours of cell adhesion, the culture medium in the wells was discarded, and 1ml of A2M solution of different concentrations was added to each well. A control group (no drug treatment, i.e., control group) and experimental groups (200nM, 400nM, 800nM, 1600nM and 3200nM groups) were set up, with three replicate wells in each group. After 24 hours of A2M treatment, the solution in the wells was discarded, and 2.0μm fluorescent microspheres were used as cell burial indicators. The cells were co-cultured with 2.0μm fluorescent microspheres at a ratio of 1:5 for 4 hours. After the culture was completed, the excess microspheres that were not engulfed were washed several times with PBS. The proportion of positive cells that engulfed the microspheres (i.e., the proportion of cell burial positive cells) and the intracellular fluorescence intensity were detected by flow cytometry. The results are as follows Figures 3 to 8 shown.

[0132] from Figure 3 to Figure 8It can be seen that A2M solution in the range of 200nM-3200nM can increase the proportion of positive cells that play an efferocytic role in the cell populations of RWA264.7 macrophages and J774A.1 macrophages, and promote the specific efferocytic level of efferocytic positive cells (i.e., the ability to endocytose substrates).

[0133] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. An application of α2-macroglobulin, characterized in that: Can be used to promote macrophage efferocytosis.

2. The use according to claim 1, characterized in that: The invention can be used as a drug for treating diseases caused by insufficient macrophage efferocytosis function.

3. The use according to claim 1, characterized in that: The invention comprises α2-macroglobulin for preparing a drug for promoting macrophage efferocytosis.

4. The use according to claim 3, characterized in that: The purity of the α2-macroglobulin is not less than 60%.

5. The use according to claim 3, characterized in that: The α2-macroglobulin is dissolved in a neutral aqueous buffer.

6. The use according to claim 5, characterized in that: The concentration of the α2-macroglobulin solution is 200 nM to 3200 nM.

7. The use according to any one of claims 1 to 6, characterized in that: The raw material for extracting α2-macroglobulin is plasma or plasma Cohn fraction IV.

8. The use according to claim 7, characterized in that: When plasma Cohn method fraction IV is used as the extraction raw material to obtain α2-macroglobulin, the method comprises the following steps: S100: Provide extraction raw materials, use ammonium sulfate for salting out, Dissolve the plasma Cohn method component IV with physiological saline, take the supernatant after natural precipitation, add ammonium sulfate to the supernatant, stir evenly, centrifuge, take the precipitate and resuspend it with water, filter the resuspended solution with the first filter membrane to obtain the first filtrate, and the volume of the first filtrate is recorded as s; S200: Chromatographic pretreatment, The first filtrate obtained in step S100 is ultrafiltered and concentrated, and the volume of the concentrate after ultrafiltration is (20% to 50%)*s. The complete removal of sulfate ions is confirmed by titration, and then a first buffer solution with a volume four times that of the concentrate is added, and a second filtrate is obtained by filtering with a second filter membrane; S300: Zinc Affinity Chromatography The second filtrate obtained in step S200 is subjected to zinc ion affinity chromatography by a protein purifier, which specifically comprises the following steps: S301: Fill the chromatography medium into the chromatography column, connect the chromatography column to the protein purification instrument, and use ZnCl2 buffer to chelate Zn 2+ ; S302: equilibrate the buffer chromatography medium using a first buffer; S303: pumping the second filtrate into the chromatography column to allow the target protein to fully bind to the chromatography medium; S304: using a first buffer to elute proteins that are not bound to the chromatography medium to a baseline, and then adding a second buffer to elute non-specifically bound or weakly bound proteins to a baseline; S305: using the first eluent to elute the zinc ion binding protein to obtain a chromatography eluent; S400: The chromatography eluate is concentrated by ultrafiltration, and then sterilized by filtration using a filter to obtain an α2-macroglobulin stock solution.

9. The use according to claim 7, characterized in that: When plasma is used as the extraction raw material to obtain α2-macroglobulin, the method comprises the following steps: Z100: Chromatography Pretreatment The plasma is balanced in a water bath, then diluted with a third buffer, and after dilution, filtered with a third filter membrane to obtain a third filtrate; Z200: Zinc Affinity Chromatography The third filtrate obtained in step Z100 is subjected to zinc ion affinity chromatography by a protein purifier, which specifically comprises the following steps: Z201: Fill the chromatography medium into the chromatography column and connect the chromatography column to the protein purifier. Use ZnCl2 buffer to chelate Zn 2+ ; Z202: equilibrate the buffer purification medium with a fourth buffer; Z203: pumping the third filtrate obtained in step Z100 into the chromatography column to allow the target protein to fully combine with the chromatography medium; Z204: Use the fourth buffer to elute the proteins that are not bound to the chromatography medium or the proteins that are weakly bound to the chromatography medium until the baseline is reached; Z205: using the second eluent to elute the protein bound to the purification medium to obtain a chromatography eluent; Z300: The chromatography eluate is concentrated by ultrafiltration, and then sterilized by filtration using a filter to obtain the α2-macroglobulin stock solution.

10. The use according to claim 8 or 9, characterized in that: The α2-macroglobulin stock solution was stored by lyophilizing the solution or refrigerating the stock solution at 4°C.

Images