N-formyl peptide MT-ND6 immunoadsorbent as well as preparation method and application thereof

Abstract

The invention discloses an N-formyl peptide MT-ND6 immunoadsorbent as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The invention relates to an N-formyl peptide MT-ND6 immunoadsorbent, the N-formyl peptide MT-ND6 immunoadsorbent comprises a carrier and a ligand, and the carrier and the ligand are covalently connected through an arm; the carrier comprises chloromethylated polystyrene porous microspheres; the ligand comprises an affinity polypeptide; the arm comprises a diamine compound. On the basis of the molecular size and surface structure characteristics of N-formyl peptide MT-ND6, polypeptide ligands with low binding energy and high affinity to the MT-ND6 are screened out, mesoporous polystyrene carrier microspheres are designed and synthesized, then diamine compounds with different molecular weights serve as arms, the affinity polypeptide ligands are covalently linked to the surface of a carrier, and the mesoporous polystyrene carrier microspheres are obtained. And the MT-ND6 immunoadsorbent with high selectivity is prepared.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically to an N-formyl peptide MT-ND6 immunosorbent and a preparation method and application thereof. Background Art

[0002] The study found that the pancreatic tissue of the rat model of severe acute pancreatitis (SAP) was severely lesioned, and the plasma N-formyl peptides MT-ND1, MT-ND3 and MT-ND6 increased significantly (P < 0.05). N-formyl peptide infusion in Wistar rats can induce severe hypotension by activating FPR, releasing nitric oxide and histamine, and can also cause a series of sepsis-like symptoms such as increased body temperature, blood coagulation, increased vascular permeability and organ damage. MT-ND1, MT-ND2, MT-ND3, MT-ND4, MT-ND4L, MT-ND5, MT-ND6, CoxⅠ, CoxⅡ, CoxⅢ, Cytb, ATPase6 and ATPase8 are all amino acid sequences of human mitochondrial N-formyl peptides encoded by the human mitochondrial genome, among which MT-ND6 is the most potent human mitochondrial N-formyl peptide known to date.

[0003] Studies have shown that damage-associated pattern molecules (DAMPs) are produced or released by damaged and dying cells, which can lead to fatal systemic inflammation, activation of the coagulation system, and multiple organ dysfunction syndrome in critical illness. In recent years, hemoperfusion adsorption materials targeting different DAMPs molecules (such as mtDNA, HMGB-1, or histones) have been rapidly developed. Increasing evidence shows that high levels of serum MT-ND6 are significantly associated with disease severity and mortality in critically ill patients.

[0004] Therefore, providing an immunomodulatory strategy for targeted clearance of serum MT-ND6 to reduce inflammation and tissue damage is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides an N-formyl peptide MT-ND6 immunoadsorbent and a preparation method and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] An N-formyl peptide MT-ND6 immunosorbent, comprising a carrier and a ligand, wherein the carrier and the ligand are covalently connected via an arm;

[0008] The carrier includes chloromethylated polystyrene porous microspheres;

[0009] Ligands include affinity peptides;

[0010] The arms include diamine compounds.

[0011] Furthermore, the particle size of the polystyrene porous microspheres is distributed in the range of 300 to 800 μm; the pore size is distributed in the range of 5 to 50 nm, preferably 30 to 50 nm;

[0012] The diamine compound is any one of 1,2-ethylenediamine, 1,3-propylenediamine and 1,8-octanediamine.

[0013] Furthermore, the sequence of the affinity polypeptide ligand is any one of the following:

[0014] Sequence RF: NH 2 -RFVIGF-COOH (RF), as shown in SEQ ID No. 1;

[0015] Sequence VD:NH 2 -VDINLF-COOH (VD), as shown in SEQ ID No. 2;

[0016] Sequence LT: NH 2 -LTARGI-COOH(LT), as shown in SEQ ID No.3.

[0017] Further, the following steps are included:

[0018] (1) Preparation of carrier microspheres

[0019] Obtaining chloromethylated polystyrene porous microspheres; wherein the chloromethyl content on the skeleton of the polystyrene porous microspheres is 0.5 to 1 mmol / mL;

[0020] (2) Arm grafting

[0021] Grafting arms onto chloromethylated polystyrene porous microspheres, and aminating the chloromethylated polystyrene porous microspheres with diamine compounds to obtain polystyrene porous microspheres after grafting arms; the amino content of the polystyrene porous microspheres is 0.05 to 0.25 mmol / mL;

[0022] (3) Ligand coupling

[0023] The polystyrene porous microspheres after grafting the arms are covalently linked to the affinity polypeptide ligand to obtain the N-formyl peptide MT-ND6 immunosorbent; the polypeptide ligand content of the N-formyl peptide MT-ND6 immunosorbent after grafting the arms is 5 to 50 μmol / mL.

[0024] Furthermore, step (1) is specifically as follows:

[0025] 27g of monomer styrene, 5g of crosslinking agent divinylbenzene, and 80g of porogen (liquid wax, toluene) were mixed evenly, and 1.2g of initiator benzoyl peroxide was added. After it was completely dissolved, the aqueous phase was added. The aqueous phase was 0.5% polyvinyl alcohol aqueous solution (containing 0.5% NaH 2 PO 4 ·2H 2 O and 0.5%Na 2 HPO 4 12H 2 O); fully stir to form oil droplets of uniform particle size; slowly heat the reaction system to 80°C, maintain at 90°C, and solidify for 6 hours; wash with hot water and dry; alternately extract with petroleum ether and ethanol for 48 hours, and dry to obtain polystyrene microspheres; add 10g (dry weight) of polystyrene microsphere resin to 80mL of chloromethyl ether, stand for swelling for 12 hours, and add 2g of dried ZnCl 2 The mixture was stirred and reacted at 40°C for 12 h, and then washed and dried thoroughly to obtain chloromethylated polystyrene carrier microspheres.

[0026] Furthermore, step (2) is specifically as follows:

[0027] Place 3g of chloromethylated polystyrene microspheres in 15mL of anhydrous N,N-dimethylformamide and shake and soak overnight to fully swell them. Add 15mL of a diamine compound solution with a concentration of 10-12mmoL / mL, heat to 70℃ and react for 12 hours. After thorough washing, obtain the grafted polystyrene porous microspheres for use.

[0028] Furthermore, step (3) is specifically as follows:

[0029] Weigh 1 g of the grafted polystyrene porous microspheres prepared in step (2) and shake them overnight in 5 mL of anhydrous N, N-dimethylformamide to fully swell; dissolve 100-130 mg of affinity polypeptide, 27-36 mg of 1-(3-dimethylaminopropyl-3-ethylcarbodiimide) (EDC), and 20-27 mg of N-hydroxysuccinimide (NHS) in 5 mL of DMF solution, and shake them at 4° C. for 1-2 hours; mix the reaction solution containing the polypeptide, EDC and NHS with the polystyrene carrier, heat it to 37° C., and continue to shake and react for 12-16 hours; after the reaction is completed, vacuum filter and fully wash the product with N, N-dimethylformamide, ethanol and deionized water to obtain the target adsorbent.

[0030] Application of N-formyl peptide MT-ND6 immunosorbent in the preparation of blood purification materials or equipment.

[0031] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects of the present invention are:

[0032] Based on the molecular size and surface structure characteristics of N-formyl peptide MT-ND6, the present invention screens out polypeptide ligands with low binding energy and high affinity for MT-ND6, designs and synthesizes mesoporous polystyrene carrier microspheres, and then uses diamine compounds of different molecular weights as arms to covalently connect the affinity polypeptide ligands to the carrier surface to prepare a highly selective MT-ND6 immunoadsorbent. The immunoadsorbent has a rich mesoporous structure and a high specific surface area, so that MT-ND6 molecules can freely enter the internal pores of the adsorbent, and the synergistic effect of arms of different lengths and MT-ND6 high affinity ligands can effectively improve the adsorption amount and adsorption selectivity of the adsorbent to MT-ND6.

[0033] Adsorption experiments show that the immunosorbent provided by the present invention has a high adsorption capacity for N-formyl peptide MT-ND6 in serum, and the MT-ND6 immunosorbent coupled with a polypeptide ligand has no obvious effect on the adsorption performance of TNF-α and IL-6, and can therefore be used as an effective MT-ND6 selective hemoperfusion adsorbent.

[0034] The adsorbent and preparation method of the present invention are simple, low in cost, and the obtained material has good blood compatibility and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0036] Figure 1 The molecular size (left) and structural characteristics (right) of MT-ND6;

[0037] Figure 2 APTT and PT values ​​of adsorbents;

[0038] Figure 3 Overall morphology (left) and surface structure (right) of adsorbent 1; DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The carrier, ligand and arm of the adsorbent are three key factors that affect the adsorption effect. The pore size of the carrier determines whether the target molecule can freely enter the gap, the specific surface area determines the effective adsorption area of ​​the adsorbent for the target molecule, the ligand determines the adsorption selectivity or adsorption capacity of the target molecule, and the arm determines the steric hindrance when the adsorbent interacts with the target molecule.

[0041] Based on the current situation that there is no highly selective adsorption material for N-formyl peptide MT-ND6 on the market, the present invention proposes an N-formyl peptide MT-ND6 immunoadsorbent, including a carrier and a ligand, wherein the carrier and the ligand are covalently connected through an arm; the carrier includes chloromethylated polystyrene porous microspheres; the ligand includes an affinity polypeptide ligand; and the arm includes a diamine compound.

[0042] [Carrier]

[0043] Chloromethylated polystyrene porous microspheres are copolymers formed by using styrene and divinylbenzene as monomers and are spherical in shape. Chloromethylated polystyrene porous microspheres can be prepared by existing methods.

[0044] A typical example is polystyrene-divinylbenzene copolymer.

[0045] In one embodiment, the particle size distribution of the polystyrene porous microspheres is between 300 and 800 μm; the carrier has a rich pore structure, and the pore size distribution is between 5 and 50 nm, preferably between 30 and 50 nm; the chloromethyl content on the polystyrene carrier is 0.5 to 1 mmol / mL.

[0046] [Ligand]

[0047] Affinity polypeptide ligand, the sequence is:

[0048] Sequence RF: NH 2 -RFVIGF-COOH (RF), as shown in SEQ ID No. 1;

[0049] Sequence VD:NH 2 -VDINLF-COOH (VD), as shown in SEQ ID No. 2;

[0050] Sequence LT: NH 2 -LTARGI-COOH (LT), as shown in SEQ ID No. 3;

[0051] The purity of the peptide is 95-99%, obtained by solid phase synthesis. The C-terminus of the peptide contains a carboxyl active group, which can be covalently linked to the carrier through the arm.

[0052] Screening of affinity peptide ligands: MT-ND6 sequence was extracted from the crystal structure of MT-ND6 complex protein.

[0053] The amino acid sequence of MT-ND6 (174 residues) is shown in SEQ ID No. 4:

[0054] MMYALFLLSVGLVMGFVGFSSKPPSPIYGGLVLIVSGVVGCVIILNFGGGYMGLMVFLIYLGGMMVVFGYTTAMAIEEYPEAWGSGVEVLVSVLVGLAMEVGLVLWVKEYDGVVVVVNFNSVGSWMIYEGEGSGLIREDPIGAGALYDYGRWLVVVTGWTLFVGVYIVIEIARGN;

[0055] Obtain its 3D structure diagram, molecular weight, molecular size, surface hydrophilicity and other parameters (see Appendix Figure 1 ). According to its molecular structure characteristics and receptor structure characteristics, short peptide fragments were designed to dock with MT-ND6 molecules, and three peptides RF, VD and LT with low binding energy and high affinity were screened, and their binding energies were -7.6kcal / mol, -7.5kcal / mol and -7.2kcal / mol respectively.

[0056] [arm]

[0057] The arms are typically, but not limited to, diamine compounds (eg, 1,2-ethylenediamine, 1,3-propylenediamine, 1,8-octanediamine).

[0058] The present invention uses chloromethylated polystyrene porous microspheres as carriers, affinity polypeptides as ligands, and uses diamine compound arms to covalently couple affinity polypeptide ligands to porous polystyrene carrier microspheres, so that affinity polypeptide ligands are exposed outside the pores, thereby obtaining a highly selective N-formyl peptide MT-ND6 immunosorbent. Compared with natural carrier materials, this adsorbent has a higher specific surface area and a developed pore structure, which is more conducive to the target molecule MT-ND6 freely entering the pores; the introduction of arms effectively increases the action sites of ligands and target molecules, which can further improve the adsorption capacity and adsorption selectivity; compared with traditional ligands such as monoclonal antibodies / polyclonal antibodies, the affinity polypeptide ligands screened by molecular docking have higher chemical stability and thermal stability, are easy to store and have a lower price. Therefore, the MT-ND6 immunosorbent prepared in this way can be used as an ideal highly selective hemoperfusion adsorption material.

[0059] According to a second aspect of the present invention, there is provided a method for preparing the above-mentioned N-formyl peptide MT-ND6 immunosorbent, comprising the following steps:

[0060] (a) obtaining chloromethylated polystyrene porous microspheres;

[0061] (b) Grafting arms onto chloromethylated polystyrene porous microspheres;

[0062] (c) covalently linking the polystyrene porous microspheres after grafting the arms and the affinity polypeptide ligand to obtain the N-formyl peptide MT-ND6 immunoadsorbent.

[0063] Specifically, the method for preparing an adsorbent for removing MT-ND6 by whole blood perfusion comprises the following steps:

[0064] Microsphere carrier (polystyrene-divinylbenzene copolymer) synthesis:

[0065] The carrier microspheres were synthesized by suspension polymerization:

[0066] Specifically include: evenly mix the monomer styrene, crosslinking agent divinylbenzene, porogen liquid wax and toluene, add the initiator benzoyl peroxide, transfer to a three-necked round-bottom flask after complete dissolution. Add a certain amount of water phase to the system and stir thoroughly to form oil droplets of uniform particle size. Slowly heat the reaction system to 80°C and maintain for 3h. Rapidly heat to 90°C and solidify for 3-6h. Wash with hot water and dry. Alternately extract with petroleum ether and ethanol in a Soxhlet extractor for 48h, and dry to obtain polystyrene microspheres.

[0067] The mass ratio of monomer, porogen, crosslinker and initiator is 27:80:50:1.2; the aqueous phase is 0.5% polyvinyl alcohol aqueous solution (containing 0.5% NaH 2 PO 4 ·2H 2 O and 0.5% Na 2 HPO 4 12H 2 O). 10 g (dry weight) of polystyrene microsphere resin was added to 80 mL of chloromethyl ether and allowed to swell for 12 h. 2 g of dried ZnCl was added to the reaction system. 2 The reaction was stirred at 40°C for 12 hours, and the chloromethylated polystyrene carrier microspheres were obtained after washing and drying.

[0068] (2) Arm grafting

[0069] The carrier is aminated with a diamine compound (eg, 1,2-ethylenediamine, 1,3-propylenediamine, 1,8-octanediamine) to facilitate the subsequent coupling of an affinity polypeptide ligand.

[0070] Specifically, the method comprises: placing the chloromethylated polystyrene microspheres in anhydrous N,N-dimethylformamide and shaking and soaking them overnight to fully swell them, adding a diamine compound solution, and then heating the solution to 70° C. to react for 12 hours, and then fully washing the microspheres for later use.

[0071] (3) Chemical coupling of high-affinity peptide ligands

[0072] The microsphere carrier after the arm grafting in step (2) is coupled with the affinity polypeptide ligand, and the coupling agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS).

[0073] Specifically, the method includes: placing the carrier in DMF and shaking overnight to fully swell it; dissolving the affinity polypeptide and coupling agent in DMF, shaking at 4°C for 0.5 to 2 hours, and then adding different volumes of polypeptide DMF solution to the carrier, heating to 37°C, and continuing to shake for 8 to 24 hours. After the reaction is completed, vacuum filtration is performed, and the product is fully washed with ethanol and deionized water to obtain the MT-ND6 immunosorbent.

[0074] The volume ratio of the microsphere carrier to DMF in the reaction system is 1:8-15; the concentration of the affinity polypeptide in the reaction system is 5-50 mg / mL; and the molar ratio of the affinity polypeptide, EDC and NHS is 1:1.5-2:0.75-2.

[0075] According to the third aspect of the present invention, there is provided a use of the above-mentioned N-formyl peptide MT-ND6 immunoadsorbent or the N-formyl peptide MT-ND6 immunoadsorbent prepared by the above-mentioned preparation method in a blood purification material or device.

[0076] The adsorbent provided by the present invention can be used for plasma or whole blood perfusion to remove MT-ND6 in patients, and is used to treat severe acute pancreatitis, sepsis and other diseases. The device is, for example, a blood perfusion device.

[0077] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0078] Implementation 1

[0079] Preparation of carrier microspheres

[0080] 27g of monomer styrene, 50g of crosslinking agent divinylbenzene and 80g of porogen (30g of liquid wax and 50g of toluene) were mixed evenly, and 1.2g of initiator benzoyl peroxide was added. After it was completely dissolved, the aqueous phase, i.e., 0.5% polyvinyl alcohol aqueous solution (containing 0.5% NaH 2 PO 4 ·2H 2 O and 0.5% Na 2 HPO 4 12H 2O). Stir thoroughly to form oil droplets of uniform size. Slowly heat the reaction system to 80°C and maintain for 3 hours. Rapidly heat to 90°C and solidify for 6 hours. Wash with hot water and dry. Alternately dry with petroleum ether and ethanol in a Soxhlet extractor to obtain polystyrene microspheres. Add 10g (dry weight) of polystyrene microsphere resin to 80mL of chloromethyl ether, let it stand for 12 hours to swell, and add 2g of dried ZnCl 2 The mixture was stirred and reacted at 40°C for 12 h, and then washed and dried thoroughly to obtain chloromethylated polystyrene carrier microspheres.

[0081] (2) Arm grafting

[0082] 3g of chloromethylated polystyrene microspheres were placed in 15mL of anhydrous N,N-dimethylformamide and shaken and soaked overnight to fully swell, and 15mL of 10mmoL / mL 1,2-ethylenediamine solution was added, and then the temperature was raised to 70℃ for reaction for 12 hours, and the microspheres were fully washed and used for later use. The terminal amino group content was 0.23mmol / mL.

[0083] (3) Ligand coupling

[0084] Weigh 1g of the carrier prepared in step (2) and shake it in 5mL of anhydrous N,N-dimethylformamide overnight to allow it to fully swell. Dissolve 100mg of affinity polypeptide RF, 35.95mg of EDC, and 26.63mg of NHS in 5mL of DMF solution and shake at 4°C for 1 hour. Mix the reaction solution containing the polypeptide, EDC and NHS with the polystyrene carrier, heat it to 37°C, and continue to shake for 12 hours. After the reaction is completed, vacuum filter and wash the product thoroughly with N,N-dimethylformamide, ethanol and deionized water to obtain the target adsorbent (adsorbent 1). Figure 3 shown.

[0085] Implementation 2

[0086] Preparation of carrier microspheres

[0087] 27g of monomer styrene, 50g of crosslinking agent divinylbenzene and 80g of porogen (30g of liquid wax and 50g of toluene) were mixed evenly, and 1.2g of initiator benzoyl peroxide was added. After it was completely dissolved, the aqueous phase, i.e., 0.5% polyvinyl alcohol aqueous solution (containing 0.5% NaH 2 PO 4 ·2H 2 O and 0.5% Na 2 HPO 4 12H 2O). Stir thoroughly to form oil droplets of uniform size. Slowly heat the reaction system to 80°C and maintain for 3 hours. Rapidly heat to 90°C and solidify for 6 hours. Wash with hot water and dry. Alternately extract with petroleum ether and ethanol in a Soxhlet extractor for 48 hours, and dry to obtain polystyrene microspheres. Add 10g (dry weight) of polystyrene microsphere resin to 80mL of chloromethyl ether, let it stand for 12 hours to swell, and add 2g of dried ZnCl 2 The mixture was stirred and reacted at 40°C for 12 h, and then washed and dried thoroughly to obtain chloromethylated polystyrene carrier microspheres.

[0088] (2) Arm grafting

[0089] 3g of chloromethylated polystyrene microspheres were placed in 15mL of anhydrous N,N-dimethylformamide and shaken and soaked overnight to fully swell, and 15mL of 1,3-propylenediamine solution with a concentration of 12mmol / mL was added, and then the temperature was raised to 70℃ for reaction for 12 hours, and the microspheres were fully washed and used for later use. The terminal amino group content was 0.19mmol / mL.

[0090] (3) Ligand coupling

[0091] Weigh 1g of the carrier prepared in step (2) and shake it overnight in 5mL of anhydrous N,N-dimethylformamide to fully swell it. Dissolve 105mg of affinity polypeptide VD, 27.82mg of EDC, and 20.61mg of NHS in 5mL of DMF solution and shake for 1.5 hours at 4°C. Mix the reaction solution containing the polypeptide, EDC, and NHS with the polystyrene carrier, heat it to 37°C, and continue to shake for 14 hours. After the reaction is completed, vacuum filter and wash the product thoroughly with N,N-dimethylformamide, ethanol, and deionized water to obtain the target adsorbent (adsorbent 2).

[0092] Example 3

[0093] Preparation of carrier microspheres

[0094] 27g of monomer styrene, 50g of crosslinking agent divinylbenzene and 80g of porogen (30g of liquid wax and 50g of toluene) were mixed evenly, and 1.2g of initiator benzoyl peroxide was added. After it was completely dissolved, the aqueous phase, i.e., 0.5% polyvinyl alcohol aqueous solution (containing 0.5% NaH 2 PO 4 ·2H 2 O and 0.5% Na 2 HPO 4 12H 2O). Stir thoroughly to form oil droplets of uniform size. Slowly heat the reaction system to 80°C and maintain for 3 hours. Rapidly heat to 90°C and solidify for 6 hours. Wash with hot water and dry. Alternately extract with petroleum ether and ethanol in a Soxhlet extractor for 48 hours, and dry to obtain polystyrene microspheres. Add 10g (dry weight) of polystyrene microsphere resin to 80mL of chloromethyl ether, let it stand for 12 hours to swell, and add 2g of dried ZnCl 2 The mixture was stirred and reacted at 40°C for 12 h, and then washed and dried thoroughly to obtain chloromethylated polystyrene carrier microspheres.

[0095] (2) Arm grafting

[0096] 3g of chloromethylated polystyrene microspheres were placed in 15mL of anhydrous N,N-dimethylformamide and shaken and soaked overnight to fully swell, and 15mL of 1,8-octanediamine solution with a concentration of 12mmoL / mL was added, and then the temperature was raised to 70℃ for reaction for 12 hours, and the microspheres were fully washed and used for later use. The terminal amino group content was 0.12mmol / mL.

[0097] (3) Ligand coupling

[0098] Weigh 1g of the carrier prepared in step (2) and shake it overnight in 5mL of anhydrous N,N-dimethylformamide to fully swell it. Dissolve 129.66mg of affinity polypeptide LT, 32.35mg of EDC, and 23.93mg of NHS in 5mL of DMF solution and shake for 2 hours at 4°C. Mix the reaction solution containing polypeptide, EDC and NHS with the polystyrene carrier, heat it to 37°C, and continue to shake for 16 hours. After the reaction is completed, vacuum filter and wash the product thoroughly with N,N-dimethylformamide, ethanol and deionized water to obtain the target adsorbent (adsorbent 3).

[0099] Adsorption effect test

[0100] The polystyrene carrier without ligand coupling was used as the control (adsorbent 4), and the above three adsorbents were taken respectively, and the serum of rats with severe acute pancreatitis SAP was added at an adsorption ratio (volume ratio of adsorbent to serum) of 1:6. After the adsorption system was sealed, it was shaken at 60 rpm in a 37°C shaker for 2 hours, and then the remaining MT-ND6 in the serum was detected and the adsorption rate was calculated; the adsorption rates of TNF-α and IL-6 were also calculated. The adsorption effects of each adsorbent are shown in Table 1.

[0101] Table 1 Adsorption performance of adsorbents for MT-ND6, TNF-α and IL-6 in SAP rat serum

[0102]

[0103] The results in Table 1 show that after the immobilization of high-affinity peptide ligands, the adsorption rate of MT-ND6 in SAP serum by the immunosorbent increased by about 1.5 to 3 times, but had no significant effect on the adsorption rate of TNF-α and IL-6.

[0104] Hemolysis test

[0105] Hemolysis experiment: Take fresh blood from big-eared white rabbits and mix it in heparin anticoagulation blood collection tubes, and dilute it with normal saline to make a red blood cell suspension. The experiment is divided into three groups: ① Negative control group: add 5mL normal saline to the centrifuge tube; ② Positive control group: add 5mL deionized water to the centrifuge tube; ③ Experimental group: add 5mL normal saline and 2g adsorbent to the centrifuge tube. Then add 100μL of the above-prepared diluted blood to each group of centrifuge tubes, incubate in a 37℃ water bath for 1h (gently mix once every 15min). Centrifuge the mixture at 2500r / min for 5min, take the supernatant and use a multifunctional microplate reader to measure the absorbance value at 545nm, and calculate the hemolysis rate.

[0106] Table 2 Hemolytic properties of adsorbents

[0107]

[0108]

[0109] The results in Table 2 show that the hemolysis rates of adsorbent 1, adsorbent 2, adsorbent 3 and adsorbent 4 were 0.26±0.08%, 0, 0.40±0.09% and 0.13±0.03%, respectively, which were all lower than the standard value of 5% for clinical application. This shows that chemical immobilization of peptide ligands does not increase the hemolysis risk of the adsorbent, and PS immunosorbents loaded with different peptides meet the clinical application standards of biomedical materials and can be used for whole blood perfusion.

[0110] Coagulation test

[0111] Fresh plasma comes from healthy Japanese white rabbits. After blood is collected from the heart, the blood and sodium citrate anticoagulant are gently mixed at a ratio of 1:9 (v:v), centrifuged at 3000r / min for 10min, and the supernatant is aspirated to be fresh plasma.

[0112] Weigh 100 mg of each wet adsorbent and place it in a 5 mL centrifuge tube. Add 2 mL of fresh plasma and shake at 60 rpm for 15 min at 37 °C. Take out the supernatant and use an automatic coagulation analyzer to detect the APTT and PT values ​​of the plasma supernatant (see attached). Figure 2 ).

[0113] Figure 2The results showed that after direct contact with the adsorbent, the changes in the APTT values ​​of rabbit plasma in each adsorbent sample group were all within 3 seconds compared with the blank control group; while the PT values ​​of each sample group showed no significant changes compared with the blank control, which means that the adsorbent material we prepared had no significant effect on the coagulation indicators APTT and PT values ​​of fresh rabbit plasma, indicating that the immunosorbent loaded with different polypeptides through diamine arms has good anti-coagulation properties.

[0114] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An N-formyl peptide MT-ND6 immunoadsorbent, characterized in that: The N-formyl peptide MT-ND6 immunosorbent comprises a carrier and a ligand, and the carrier and the ligand are covalently connected via an arm; The carrier includes chloromethylated polystyrene porous microspheres; Ligands include affinity peptides; The arms include diamine compounds.

2. The adsorbent according to claim 1, characterized in that The particle size of the polystyrene porous microspheres is distributed in the range of 300 to 800 μm; the pore size is distributed in the range of 5 to 50 nm, preferably 30 to 50 nm; The diamine compound is any one of 1,2-ethylenediamine, 1,3-propylenediamine and 1,8-octanediamine.

3. The N-formyl peptide MT-ND6 immunoadsorbent according to claim 1, characterized in that Said The sequence of the affinity peptide ligand is any of the following: Sequence RF: NH2-RFVIGF-COOH, as shown in SEQ ID No. 1; Sequence VD: NH2-VDINLF-COOH, as shown in SEQ ID No. 2; Sequence LT: NH2-LTARGI-COOH, as shown in SEQ ID No.

3.

4. The method for preparing the N-formyl peptide MT-ND6 immunosorbent according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Preparation of carrier microspheres Obtaining chloromethylated polystyrene porous microspheres; wherein the chloromethyl content on the skeleton of the polystyrene porous microspheres is 0.5 to 1 mmol / mL; (2) Arm grafting Grafting arms onto chloromethylated polystyrene porous microspheres, and aminating the chloromethylated polystyrene porous microspheres with diamine compounds to obtain polystyrene porous microspheres after grafting arms; the amino content of the polystyrene porous microspheres is 0.05 to 0.25 mmol / mL; (3) Ligand coupling The polystyrene porous microspheres after grafting the arms are covalently linked to the affinity polypeptide ligand to obtain the N-formyl peptide MT-ND6 immunosorbent; the polypeptide ligand content of the N-formyl peptide MT-ND6 immunosorbent after grafting the arms is 5 to 50 μmol / mL.

5. The method for preparing the N-formyl peptide MT-ND6 immunosorbent according to claim 4, characterized in that: Step (1) is specifically as follows: 27g of monomer styrene, 50g of cross-linking agent divinylbenzene, and 80g of porogen are mixed evenly, and 1.2g of initiator benzoyl peroxide is added. After it is completely dissolved, an aqueous phase is added, wherein the aqueous phase is a 0.5% polyvinyl alcohol aqueous solution (containing 0.5% NaH2PO4·2H2O and 0.5% Na2HPO4·12H2O); the mixture is stirred thoroughly to form oil droplets of uniform particle size; the reaction system is slowly heated to 80°C and maintained for 3h; the temperature is quickly raised to 90°C and cured for 6h; hot water washing and drying are performed; petroleum ether and ethanol are alternately extracted for 48h, and polystyrene microspheres are obtained by drying; 10g (dry weight) of polystyrene microsphere resin is added to 80mL of chloromethyl ether, and the mixture is allowed to stand for swelling for 12h, 2g of dried ZnCl2 is added to the reaction system, the reaction is stirred at 40°C for 12h, and the chloromethylated polystyrene carrier microspheres are obtained by thorough washing and drying.

6. The method for preparing the N-formyl peptide MT-ND6 immunosorbent according to claim 4, characterized in that: Step (2) is specifically as follows: Place 3g of chloromethylated polystyrene microspheres in 15mL of anhydrous N,N-dimethylformamide and shake and soak overnight to fully swell them. Add 15mL of a diamine compound solution with a concentration of 10-12mmoL / mL, heat to 70℃ and react for 12 hours. After thorough washing, obtain the grafted polystyrene porous microspheres for use.

7. The method for preparing the N-formyl peptide MT-ND6 immunosorbent according to claim 4, characterized in that: Step (3) is specifically as follows: Weigh 1 g of the grafted polystyrene porous microspheres prepared in step (2) and shake them in 5 mL of anhydrous N, N-dimethylformamide overnight to fully swell them; dissolve 100-130 mg of affinity polypeptide, 27-36 mg of EDC, and 20-27 mg of NHS in 5 mL of DMF solution, and shake them at 4°C for 1-2 hours; mix the reaction solution containing polypeptide, EDC and NHS with the polystyrene carrier, heat it to 37°C, and continue to shake and react for 12-16 hours; after the reaction is completed, vacuum filter and fully wash the product with N, N-dimethylformamide, ethanol and deionized water to obtain the target adsorbent.

8. Use of the N-formyl peptide MT-ND6 immunoadsorbent according to any one of claims 1 to 3 in the preparation of blood purification materials or equipment.

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