WO2022111661A1 - Antibody type biological magnetic microsphere, preparation method therefor and use thereof - Google Patents

Antibody type biological magnetic microsphere, preparation method therefor and use thereof Download PDF

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WO2022111661A1
WO2022111661A1 PCT/CN2021/133735 CN2021133735W WO2022111661A1 WO 2022111661 A1 WO2022111661 A1 WO 2022111661A1 CN 2021133735 W CN2021133735 W CN 2021133735W WO 2022111661 A1 WO2022111661 A1 WO 2022111661A1
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antibody
biotin
magnetic
polymer
biomagnetic
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PCT/CN2021/133735
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French (fr)
Chinese (zh)
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郭敏
徐丽琼
曹平生
唐磊
于雪
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康码（上海）生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification

Definitions

  • the invention belongs to the technical field of biochemistry, and in particular relates to an antibody-type biomagnetic microsphere and a preparation method and application thereof.
  • the separation and purification of protein substances is an important downstream link in the biological drug production process.
  • the effect and efficiency of separation and purification directly affect the quality and production cost of protein drugs.
  • materials such as agarose gel are commonly used as purification columns or purification microsphere carriers at this stage.
  • the three-dimensional porous structure of the gel-like material is beneficial to increase the specific surface area of the material, thereby increasing the sites that can bind to the purification medium and increasing the specific binding amount to the target protein.
  • the porous structure inside the carrier will also increase the retention time of the protein during protein elution, and some discontinuous spaces or dead corners inside the carrier will hinder the Proteins are eluted from within the material, resulting in increased retention. If the protein-binding site is only fixed on the outer surface of the carrier, although the protein product can be prevented from entering the interior of the material, the retention time and retention ratio of the protein during elution can be greatly reduced; but if only the outer surface of the carrier is used, then It will greatly reduce the specific surface area of the carrier, thereby greatly reducing the number of protein binding sites and reducing the purification efficiency.
  • a polymer is a high molecular compound that can be formed by the polymerization of monomer molecules.
  • the polymer product can be rich in a large number of active sites, greatly increasing the number of active sites, and through these active sites, corresponding binding sites can be formed or introduced.
  • the purification medium is mainly fixed by covalent coupling. After the purification column is used for many times, the binding performance of the purification medium will be reduced, and the purification effect will be reduced. Therefore, in order to ensure high purification efficiency and quality, operators need to replace all the fillers in the affinity chromatography column in time. This process not only consumes a large amount of consumables, but also consumes a lot of labor and time, resulting in high purification costs.
  • the present invention provides a biomagnetic microsphere, wherein the biomagnetic microsphere is combined with an anti-target antibody substance, which can be used for the separation and purification of the target substance (including but not limited to protein substances), and can be used for high-throughput Combined with the target, it can effectively reduce the retention ratio of the target during elution, and can also easily replace the purification medium. Purification cost.
  • an anti-target antibody substance which can be used for the separation and purification of the target substance (including but not limited to protein substances), and can be used for high-throughput Combined with the target, it can effectively reduce the retention ratio of the target during elution, and can also easily replace the purification medium. Purification cost.
  • a first aspect of the present invention provides a biomagnetic microsphere, comprising a magnetic microsphere body, and the outer surface of the magnetic microsphere body has at least one polymer with a linear main chain and a branched chain, and the linear main chain One end of the polymer is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the branched end of the polymer of the biomagnetic microsphere is connected with an antibody-type label.
  • the biomagnetic microspheres are called antibody magnetic beads or antibody magnetic microspheres or antibody-type magnetic microspheres.
  • the antibody-type tag is preferably used as a purification medium.
  • the antibody-type tag is any one of an antibody, an antibody fragment, a single chain of an antibody, a fragment of a single chain, an antibody fusion protein, a fusion protein of an antibody fragment, a derivative of any one, or any one of species variant.
  • the antibody-type tag is an anti-protein antibody.
  • the antibody-type tag is an antibody against a fluorescent protein.
  • the antibody-type tag is an antibody against green fluorescent protein or a mutant thereof.
  • the antibody-type tag is a nanobody.
  • the antibody-type tag is an anti-protein nanobody.
  • the antibody-type tag is an anti-protein single-domain antibody.
  • the antibody-type tag is an anti-protein single-domain antibody.
  • the antibody-type tag is an anti-protein VHH antibody.
  • the antibody-type tag is an anti-protein scFV antibody.
  • the antibody-type tag is an anti-fluorescent protein nanobody.
  • the antibody-type tag is a nanobody against green fluorescent protein or a mutant thereof.
  • the antibody-type tag is a Fab fragment.
  • the antibody-type tag is an F(ab')2 fragment.
  • the antibody-type tag is an Fc fragment.
  • the antibody-type tag is linked to the branched end of the polymer through an affinity complex interaction.
  • the affinity complex includes but is not limited to the following situations: biotin and avidin, biotin analogs and avidin, biotin and avidin analogs, biotin analogs and avidin hormone analogs;
  • the avidin is streptavidin, modified streptavidin, streptavidin analog or a combination thereof.
  • the antibody-type tag is linked to the end of the branched chain of the polymer by covalent bonding, supramolecular interaction or a combination thereof.
  • the covalent bond utilizes a dynamic covalent bond; more preferably, the dynamic covalent bond includes an imine bond, an acylhydrazone bond, a disulfide bond or a combination thereof.
  • the supramolecular interaction is selected from the group consisting of: coordination binding, affinity complex interaction, electrostatic adsorption, hydrogen bonding, ⁇ - ⁇ overlapping interaction, hydrophobic interaction and combinations thereof.
  • the interaction of the affinity complex is selected from the group consisting of: biotin-avidin interaction, biotin analog-avidin interaction, biotin-avidin analog interaction, biotin Analog-avidin analog interactions.
  • the branched end of the polymer of the biomagnetic microspheres is connected with biotin or biotin analogs, and the biotin or biotin analogs are used as connecting elements, which are further connected by the binding effect of the affinity complex.
  • Avidin or avidin analogs which still serve as linking elements, further linking the antibody-type tags.
  • the size of the magnetic microsphere body is selected from any one of the following particle size scales or a range between any two particle size scales: 0.1 ⁇ m, 0.15 ⁇ m, 0.2 ⁇ m, 0.25 ⁇ m, 0.3 ⁇ m, 0.35 ⁇ m, 0.4 ⁇ m, 0.45 ⁇ m, 0.5 ⁇ m, 0.55 ⁇ m, 0.6 ⁇ m, 0.65 ⁇ m, 0.7 ⁇ m, 0.75 ⁇ m, 0.8 ⁇ m, 0.85 ⁇ m, 0.9 ⁇ m, 0.95 ⁇ m, 1 ⁇ m, 1.5 ⁇ m, 2 ⁇ m, 2.5 ⁇ m, 3 ⁇ m , 3.5 ⁇ m, 4 ⁇ m, 4.5 ⁇ m, 5 ⁇ m, 5.5 ⁇ m, 6 ⁇ m, 6.5 ⁇ m, 7 ⁇ m, 7.5 ⁇ m, 8 ⁇ m, 8.5 ⁇ m, 9 ⁇ m, 9.5 ⁇ m, 10 ⁇ m, 15 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 35 ⁇ m, 40 ⁇ m, 45 ⁇ m, 50 ⁇ m, 55 ⁇ m, 60 ⁇ m
  • the diameter of the magnetic microsphere body is selected from 0.1-10 ⁇ m.
  • the diameter of the magnetic microsphere body is selected from 0.2 to 6 ⁇ m.
  • the diameter of the magnetic microsphere body is selected from 0.4 to 5 ⁇ m.
  • the diameter of the magnetic microsphere body is selected from 0.5-3 ⁇ m.
  • the diameter of the magnetic microsphere body is selected from 0.2 to 1 ⁇ m.
  • the diameter of the magnetic microsphere body is selected from 0.5-1 ⁇ m.
  • the diameter of the magnetic microsphere body is selected from 1 ⁇ m ⁇ 1 mm.
  • the average diameter of the magnetic microsphere body is 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, and the deviation It is ⁇ 20%, more preferably ⁇ 10%.
  • the main chain of the polymer is a polyolefin main chain, or an acrylic polymer main chain.
  • the acrylic polymers are defined in the "Terms and Terms" section.
  • the polyolefin backbone is also an acrylic polymer backbone (ie, the linear backbone of the polymer is a polyolefin backbone and is provided by the acrylic polymer backbone).
  • the monomer unit of the acrylic polymer is preferably selected from one or a combination of acrylic monomer molecules such as acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, methacrylate, etc. .
  • the acrylic polymer can be obtained by polymerizing one of the above monomers or by copolymerizing a corresponding combination of the above monomers.
  • the branched chain of the polymer covalently binds biotin or biotin analogs through covalent bonds based on functional groups (to obtain biotin magnetic beads), and then passes through the biotin or biotin analogs.
  • the antibody-type tag is attached directly or indirectly.
  • the process of binding the biotin or biotin analogs can be achieved by covalently reacting functional groups contained in the branched chains of the polymer molecules on the outer surface of the biomagnetic microspheres with biotin or biotin analogs.
  • one of the preferred embodiments of the functional group is a specific binding site (for definitions, please refer to the "noun and term" section of the specific embodiment).
  • the functional group-based covalent bond refers to a covalent bond formed by a functional group participating in covalent coupling.
  • the functional group is a carboxyl group, a hydroxyl group, an amino group, a sulfhydryl group, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of the foregoing functional groups.
  • One of the preferred modes of the salt form of the carboxyl group is a sodium salt form such as COONa; the preferred mode of the salt form of the amino group can be an inorganic salt form, or an organic salt form, including but not limited to hydrochloride, hydrofluoride acid salts, etc.
  • the "combination of functional groups” refers to all branches of all polymer molecules on the outer surface of a magnetic microsphere, allowing different functional groups to participate in the formation of covalent bonds; take biotin as an example, that is, a biological All biotin molecules on the outer surface of the prime magnetic microspheres can be covalently linked with different functional groups, but one biotin molecule can only be linked with one functional group.
  • the linear main chain of the polymer is directly covalently coupled to the outer surface of the magnetic microsphere body, or indirectly covalently coupled to the outer surface of the magnetic microsphere body through a linking group .
  • the magnetic microsphere body is a magnetic material wrapped with SiO 2 .
  • SiO2 can be a silane coupling agent with its own active site.
  • the magnetic material is selected from one or a combination of iron oxides, iron compounds, iron alloys, cobalt compounds, cobalt alloys, nickel compounds, nickel alloys, manganese oxides, and manganese alloys.
  • the "fixed to” means that the linear backbone is “fixed to” the outer surface of the magnetic microsphere body in a covalently linked manner.
  • the linear main chain is covalently fixed to the outer surface of the magnetic microsphere body in a direct manner or indirectly via a linker (linking element).
  • the number of the polymer branches is multiple; preferably, at least three.
  • the present invention also discloses a method for preparing the biomagnetic microspheres described in the first aspect, comprising the following steps:
  • the coupling agent is preferably an aminated silane coupling agent.
  • One of the preferred ways is to chemically modify the magnetic microsphere body with a coupling agent.
  • a silane coupling agent can be used to chemically modify the magnetic microsphere body.
  • the silane coupling agent is preferably an aminated silane coupling agent.
  • the polymerization of carbon-carbon double bonds is used to polymerize acrylic monomer molecules (such as sodium acrylate), and the obtained acrylic polymer has a linear main chain and contains functional groups.
  • the polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain to form an acrylic polymer modified magnetic microsphere C.
  • the functional group is a carboxyl group, a hydroxyl group, an amino group, a sulfhydryl group, a formate, an ammonium salt, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of the foregoing functional groups;
  • the " “Combination of functional groups” refers to the functional groups contained in all branches of all polymers on the outer surface of a magnetic microsphere, and the types may be one or more. The meaning of "combination of functional groups" defined in the first aspect is consistent.
  • the functional group is a specific binding site.
  • the biomagnetic microspheres D are biotin-modified.
  • the raw material for providing the antibody-type tag is a covalently linked complex of avidin or an avidin analog and the antibody-type tag.
  • the raw material for providing the antibody-type tag is an avidin-antibody-type tag covalently linked complex.
  • the raw material for providing the antibody-type tag is an avidin-antibody-type tag covalently linked complex.
  • the present invention also discloses the application of the biomagnetic microspheres (antibody magnetic microspheres) described in the first aspect in separation and purification, preferably in the separation and purification of protein substances.
  • One of the cores of the present invention lies in the biomagnetic microsphere structure.
  • polymer molecules with linear main chains are covalently fixed, and these polymer molecules also have a large number of functional branches, so The functionalized branches are linked with a purification medium (antibody-type tag).
  • a purification medium antibody-type tag
  • the type of the purification medium can be selected according to the type of the substance to be purified (target substance).
  • the purification medium When the purification medium is attached to the branched chain of the polymer in the form of an affinity complex with a strong non-covalent interaction; further, the branched backbone between the purification medium and the linear main chain of the polymer may also exist
  • the binding of the affinity complex allows the purification medium to be easily exchanged.
  • the core of the present invention also lies in the construction process (preparation method) of the above-mentioned biomagnetic microsphere structure: by chemical modification of the outer surface, several binding sites are provided on the outer surface of the magnetic bead (the outer surface of the biomagnetic microsphere body), and then the magnetic A single binding site on the outer surface of the bead is covalently linked to a polymer molecule, and the polymer molecule is covalently linked to a single binding site on the outer surface of the magnetic bead through one end of a linear backbone with a large number of side branches distributed along the linear backbone.
  • the side branches carry new binding sites, so as to achieve multiple, dozens, hundreds, hundreds of times, or even thousands of times the amplification of the binding sites, and then according to the specific purification requirements in the polymer branch chain
  • a specific purification medium is attached to the nascent binding site of , in order to achieve the capture of corresponding specific target molecules (especially biochemical molecules).
  • the single binding site of the biomagnetic microspheres can be covalently linked to only one linear polymer backbone or two or more linear backbones, so as not to cause chain stacking and thus the retention ratio Increase is appropriate.
  • only one linear main chain is drawn from one binding site, and at this time, a larger space for the linear main chain can be provided.
  • the surface of magnetic microspheres can be The number of combined purification media is multiplied, ten-fold, dozens of times, hundreds of times, hundreds of times, and even thousands of times, so as to achieve high-throughput binding of the target (one of the preferred ways of the target).
  • the biomagnetic microspheres can efficiently capture the target from the mixed system onto the magnetic microspheres to achieve high-throughput binding, that is, to achieve high-throughput separation.
  • the flexibility of the polymer chain itself can be used, and the polymer chain can flexibly swing in the reaction and purification mixed system, expand the active space of the purification medium, increase the capture rate and binding amount of the protein, and promote the rapid and Fully combined to achieve high efficiency and high throughput.
  • the structural design of the present invention enables the biomagnetic microspheres to achieve efficient elution of the purified target substance during elution, effectively reducing the retention time and retention ratio of the target substance, and achieving high efficiency and high yield.
  • the purification medium (antibody-type tag) can be attached to the end of the branched chain of the polymer.
  • the structure of the polymer can not form a network structure, which does not lead to the accumulation of branched chains, which can avoid discontinuous spaces and dead ends, and avoid the traditional network.
  • the branched chain of the polymer further acts as a spacer, so that the purification medium can be fully distributed in the mixed system, away from the surface of the magnetic microspheres and the internal skeleton of the polymer, It not only increases the efficiency of capturing the target, but also can effectively reduce the retention time and retention ratio of the target in the subsequent elution step, and achieve high-throughput, high-efficiency, and high-proportion separation.
  • the structural design of the present invention can not only utilize the high flexibility of the linear main chain, but also has the advantage of high multiple amplification of the number of branches, so as to better realize the combination of high speed and high flux, high efficiency, high ratio (high yield) separation.
  • the purification medium (antibody-type tag) of the biomagnetic microspheres of the present invention can be connected to the end of the polymer branch on the outer surface of the magnetic bead with a strong non-covalent binding force by means of an affinity complex;
  • the purification medium needs to be renewed or replaced, the purification medium can be easily and quickly eluted from the microspheres and recombined with the new purification medium, and the purification performance of the magnetic microspheres can be quickly restored, so that the biomagnetic microspheres can be regenerated and used for many times. Reduce separation and purification costs.
  • the biomagnetic microspheres of the present invention are convenient to operate and use.
  • the operation is convenient, and only a small magnet can be used to efficiently manipulate the aggregation state and position of the magnetic microspheres, so as to realize the rapid dispersion of the magnetic microspheres in the solution or Rapid precipitation makes the separation and purification of target substances simple and fast, without the need to use large-scale experimental equipment such as high-speed centrifuges, which greatly reduces the cost of separation and purification.
  • the biomagnetic microspheres provided by the present invention are widely used, and the purification medium has selectivity. According to the type of specific purification substrate, the corresponding purification medium can be flexibly carried in the magnetic microsphere system to realize the capture of specific target molecules. Antibody fragments, antibody single chains, nanobodies and other forms of non-whole proteins with smaller molecular size can also be selected as purification media, especially when nanobodies are used as purification media, compared with complete antibodies with whole protein structures, when preparing magnetic microspheres It is easier to obtain a high load on the purification medium; and the purification medium is more likely to fully contact the target with the oscillation of the polymer chain in the mixed system, thereby obtaining a higher binding efficiency.
  • FIG. 1 A schematic diagram of the structure of a biomagnetic microsphere.
  • the antiEGFP nanobody is used as a purification medium, and the antiEGFP nanobody is bound to the end of the branched chain of the brush-like structure by "biotin-avidin-antiEGFP nanobody".
  • the biomagnetic microsphere body is taken as an example of Fe 3 O 4 wrapped by SiO 2 .
  • the number of polymer molecules (4) is only for the sake of simplicity. It does not mean that the number of polymer molecules on the outer surface of the magnetic microsphere is limited to 4, but can be controlled and adjusted according to the content of each raw material in the preparation process. .
  • the number of side chains hanging from the side end of the linear main chain is only for illustrative purposes, and is not intended to limit the number of side chains in the polymer molecule of the present invention.
  • FIG. 1 A flow chart of the preparation method of biomagnetic microspheres, taking nanobody as the purification medium as an example. Among them, the preparation process from amino-modified magnetic microspheres A to biomagnetic microspheres D corresponds to the preparation of biotin magnetic microspheres.
  • FIG. 3 The RFU value test results of biomagnetic microsphere H (a magnetic bead with antiEGFP nanobody) binding to eGFP protein.
  • Biomagnetic microspheres H were incubated with the IVTT supernatant of eGFP protein to bind eGFP protein. Among them, Total corresponds to the IVTT supernatant that has not been treated with biomagnetic microspheres; Flow-through corresponds to the flow-through solution incubated once.
  • FIG. 4 Experimental results of separation and purification of eGFP protein by biomagnetic microspheres H (antiEGFP magnetic beads): biomagnetic microspheres H were incubated with eGFP protein solution and then eluted, eGFP protein was captured and separated from the stock solution, eluted and released to wash In the dehydration, the corresponding SDS-PAGE test results of the eluate containing purified eGFP protein. Among them, M corresponds to the Marker molecular weight marker.
  • SEQ ID No.: 1 the amino acid sequence of Nanobody anti-eGFP, with a length of 117 amino acids.
  • SEQ ID No.: 2 the nucleotide sequence of tamavidin2, 423 bases in length.
  • SEQ ID No.: 3 nucleotide sequence of mScarlet, 693 bases in length.
  • SEQ ID No.:4 the nucleotide sequence of mEGFP, is 714 bases in length.
  • Magnetic beads ferromagnetic or ferromagnetic microspheres with fine particle size, which can also be described as magnetic beads, preferably 0.1 ⁇ m to 1000 ⁇ m in diameter.
  • magnetic beads of the present invention include, but are not limited to: magnetic microspheres A, magnetic microspheres B, magnetic microspheres C, biomagnetic microspheres D (a biotin magnetic bead), biomagnetic microspheres H (a antiEGFP nanobody magnetic beads), biomagnetic microspheres K (antibody magnetic beads).
  • Magnetic microsphere body magnetic beads with modified sites (magnetic microspheres with binding sites).
  • Examples are silica-coated magnetic material particles, more particularly aminated silica-coated magnetic material particles.
  • Magnetic microspheres A Amino-modified magnetic microspheres.
  • Magnetic microspheres B magnetic microspheres containing carbon-carbon double bonds.
  • Magnetic microspheres C acrylic polymer modified magnetic microspheres.
  • Biotin Magnetic Beads Magnetic beads conjugated with biotin or biotin analogs that can specifically bind to substances with avidin-type tags.
  • the advantages include that after the target protein is labeled with avidin or a protein mutant of avidin, it can be expressed in an integrated manner in the form of a fusion protein, and the application method is simple. Also known as biotin magnetic microspheres. Biotin or biotin analogs here can serve as a purification medium and also as a linking element.
  • Biomagnetic Microsphere D A magnetic microsphere bound with biotin or a biotin analog, a biotin magnetic bead. Biotin or biotin analogs here can serve as a purification medium and also as a linking element.
  • Avidin magnetic beads Magnetic beads bound with avidin or avidin analogs, which can specifically bind to substances with biotin-type tags. Also known as avidin magnetic microspheres.
  • Biomagnetic Microspheres K Magnetic beads with antibody-type tags, which can be used to separate and purify targets that can specifically bind to them. Also known as antibody magnetic microspheres or antibody magnetic beads or antibody-type magnetic microspheres.
  • Nanobody magnetic beads Magnetic beads combined with nanobodies can be used to separate and purify targets that can specifically bind to them. Also known as Nanobody Magnetic Microspheres.
  • Biomagnetic Microsphere H a nanobody magnetic bead, a magnetic microsphere (antiEGFP magnetic bead) bound with the nanobody antiEGFP. It can be combined by avidin-antiEGFP covalently linked complex.
  • Polymers including oligomers and polymers in a broad sense in the present invention, have at least three structural units or a molecular weight of at least 500 Da (the molecular weight can be characterized by a suitable method, such as number average molecular weight, weight average molecular weight, viscosity average molecular weight, etc.). molecular weight, etc.).
  • Polyolefin chain refers to a polymer chain without heteroatoms that is only covalently linked by carbon atoms.
  • the main chain of polyolefin in the comb-like structure is mainly involved; for example, the linear main chain of acrylic polymer.
  • Acrylic polymer refers to a homopolymer or copolymer having a -C(COO-)-C- unit structure, and the copolymerization form of the copolymer is not particularly limited, so as to be able to provide a linear main chain and a metered side group COO-
  • heteroatoms are allowed in the linear backbone of the acrylic polymer.
  • other substituents are also allowed on the carbon-carbon double bond, as long as it does not affect the progress of the polymerization reaction, such as methyl substituents (corresponding to -CH 3 C(COO-)-C-).
  • COO- can exist in the form of -COOH, or in the form of a salt (such as a sodium salt), or in the form of a formate (preferably an alkyl formate, such as methyl formate-COOCH 3 , ethyl formate-COOCH 2 CH 3 ; it can also be hydroxyethyl formate-COOCH 2 CH 2 OH) and the like.
  • a salt such as a sodium salt
  • a formate preferably an alkyl formate, such as methyl formate-COOCH 3 , ethyl formate-COOCH 2 CH 3 ; it can also be hydroxyethyl formate-COOCH 2 CH 2 OH
  • Specific structural forms of the -C(COO-)-C-unit structure include but are not limited to -CH(COOH)-CH 2 -, -CH(COONa)-CH 2 -, -MeC(COOH)-CH 2 -, - MeC(COONa)-CH 2 -, -CH(COOCH 3 )-CH 2 -, -CH(COOCH 2 CH 2 OH)-CH 2 -, -MeC(COOCH 3 )-CH 2 -, -MeC(COOCH 2 CH 2 OH)-CH 2 - and the like, or any combination thereof.
  • Me is methyl.
  • On the linear main chain of a polymer molecule there may be only one of the above-mentioned unit structures (corresponding to a homopolymer), or two or more unit structures (corresponding to a copolymer).
  • Branched chain A chain in the present invention that is attached to a branch point and has independent ends.
  • branched chain and side branched chain have the same meaning and can be used interchangeably.
  • branched chain refers to the side chain or side group bonded on the linear main chain of the polymer.
  • the number, length, size, degree of re-branching and other structural characteristics of the branch chain should be avoided to form a network structure as much as possible, so as not to cause the accumulation of branch chains to increase the retention ratio. At this time, the flexible swing of the linear main chain can be smoothly exerted. .
  • Branched-chain skeleton The branched-chain skeleton consists of skeleton atoms connected in sequence by covalent bonds or non-covalent bonds, and is sequentially connected to the main chain of the polymer from the end of the branched chain.
  • the functional groups at the ends of the polymer are attached to the main chain of the polymer through a branched backbone.
  • the intersection of the branched skeleton and the main chain is also the branch point from which the branched chain is drawn.
  • the branched backbone between the antibody-type tag and the linear main chain of the polymer, the antibody-type tag at the end of the polymer branch chain can be sequentially passed through avidin, biotin, propylenediamine residues (-NH-CH 2 CH 2 CH2 -NH-), carbonyl group (residue after the amidation reaction of carboxyl group), and connected to the polyolefin backbone of the polymer.
  • Branched-chain ends including the ends of all branched-chains.
  • the linear main chain in addition to being fixed at one end of the magnetic microsphere body, the other end of the linear main chain must be connected to a branch point, therefore, it is also broadly included in the category of "branch end" of the present invention. Therefore, the polymer attached to the outer surface of the magnetic microsphere body of the present invention has at least one branch point.
  • the functional group of the branch chain of the polymer refers to the reactive group, or the reactive group after activation, which can directly react covalently with the reactive group of other raw materials, or react with the reactive group of other raw materials after activation. A covalent reaction occurs between the groups, resulting in a covalent bond.
  • One of the preferred forms of functional groups of polymer branches is a specific binding site.
  • the direct connection method refers to the connection method in which the interaction occurs directly without the aid of spacer atoms.
  • the form of the interaction includes, but is not limited to: covalent, non-covalent, or a combination thereof.
  • Indirect connection means connection by means of at least one connecting element, in which case at least one spacer atom is involved.
  • the linking elements include, but are not limited to, linking peptides, affinity complex linkages, and the like.
  • Fiberd means of immobilization, immobilization on, immobilization with, immobilization on, etc. refer to the covalent binding method.
  • connection means are not particularly limited, including but not limited to covalent, non-covalent and other means.
  • Covalent method the method of direct bonding by covalent bonds
  • the covalent method includes but not limited to the dynamic covalent method
  • the dynamic covalent method refers to the method of direct bonding by dynamic covalent bonds.
  • Covalent bonds including common covalent bonds such as amide bonds and ester bonds, as well as dynamic covalent bonds with reversible properties.
  • the covalent bonds include dynamic covalent bonds.
  • a dynamic covalent bond is a chemical bond with reversible properties, including but not limited to imine bonds, acylhydrazone bonds, disulfide bonds, or a combination thereof. Those skilled in the chemical arts can understand the meaning.
  • Non-covalent methods including but not limited to supramolecular interaction methods such as coordination binding, affinity complex interaction, electrostatic adsorption, hydrogen bonding, ⁇ - ⁇ overlap, and hydrophobic interaction.
  • Supramolecular interactions including but not limited to coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, ⁇ - ⁇ overlapping interactions, hydrophobic interactions, and combinations thereof.
  • a linking element also referred to as a linking group, refers to an element used to connect two or more non-adjacent groups, including at least one atom.
  • the connection mode between the connecting element and the adjacent group is not particularly limited, including but not limited to covalent mode, non-covalent mode and the like.
  • the internal connection mode of the connecting element is not particularly limited, including but not limited to covalent mode, non-covalent mode and the like.
  • Covalent linking element The spacer atoms from one end of the linking element to the other are covalently linked.
  • the specific binding site refers to a group or structural site with a binding function on the polymer branch chain, and the group or structural site has a specific target.
  • Specific recognition and binding functions, specific binding can be achieved through coordination, complexation, electrostatic force, van der Waals force, hydrogen bond, covalent bond and other binding interactions or other interactions.
  • Covalently linked complex A compound that is directly or indirectly linked by covalent means, also known as a covalent linker.
  • the covalent attachment means include, but are not limited to, covalent bonds, linking peptides, and the like.
  • Avidin-purification medium covalently linked complex a compound that is covalently linked, with avidin at one end and a purification medium at the other end, directly linked by covalent bonds, or indirectly through covalent linking elements connected.
  • Affinity complex a non-covalently linked complex formed by two or more molecules through specific binding, relying on extremely strong affinity, such as: biotin (or biotin analogs) and affinity A complex formed by the interaction of avidin (or avidin).
  • biotin or biotin analogs
  • avidin or avidin
  • Purified substrate also known as target, the substance to be separated from a mixed system.
  • the purification substrate in the present invention is not particularly limited, but the purification substrate is preferably a protein substance (also referred to as a target protein in this case).
  • the purification medium can specifically bind to the purified substrate, thereby capturing the purified substrate, and then separating the purified substrate from the mixed system.
  • the purification medium linked to the branched end of the polymer of the present invention is a functional element having the function of binding the purification substrate. When the purification medium is covalently linked to an adjacent group, it behaves as a functional group with the function of binding the purification substrate.
  • Biotin biotin, can be combined with avidin, and has strong binding force and good specificity.
  • Avidin avidin, which can be combined with biotin, and has strong binding force and good specificity, such as streptavidin (SA), its analogs (such as Tamvavidin2, referred to as Tam2), its modified products, its mutants, etc.
  • SA streptavidin
  • Tam2 Tamvavidin2
  • Biotin analogs refer to non-biotin molecules that can form a specific binding similar to "avidin-biotin" with avidin, preferably one is a polypeptide or protein, such as the one developed by IBA Company Polypeptides containing the WSHPQFEK sequence used in the series (e.g. etc.), and similar polypeptides containing the WNHPQFEK sequence.
  • WN HPQFEK can be regarded as a mutated sequence of WS HPQFEK .
  • Avidin analogs refer to non-avidin molecules that can form a specific binding similar to "avidin-biotin” with biotin, and preferably one is a polypeptide or a protein.
  • the avidin analogs include, but are not limited to, derivatives of avidin, homologs of avidin (homologs), variants of avidin, and the like. Said avidin analogs, such as Tamavidin1, Tamavidin2, etc. (refer to: FEBS Journal, 2009, 276, 1383-1397).
  • Biotin-type tag contains the following units: biotin, avidin analogs that bind to avidin, avidin analogs that bind avidin analogs, and combinations thereof. Biotin-type tags are capable of specifically binding avidin, avidin analogs, or a combination thereof. Therefore, it can be used to separate and purify proteins including but not limited to avidin-type tags.
  • Avidin-type tag contains the following units: avidin, avidin analogs that can bind biotin, avidin analogs that can bind biotin analogs, and combinations thereof. Avidin-type tags are capable of specifically binding biotin, biotin analogs, or a combination thereof. Therefore, it can be used to separate and purify proteins including but not limited to biotin-type tags.
  • the antibody-type tag of the present invention refers to a tag containing an antibody-like substance, which can specifically bind to a corresponding target, such as an antigen.
  • Examples of the antibody-type tag also include antiEGFP antibodies that can specifically bind to eGFP protein.
  • a peptide is a compound in which two or more amino acids are linked by peptide bonds.
  • peptide and peptide segment have the same meaning and can be used interchangeably.
  • Polypeptide a peptide consisting of 10 to 50 amino acids.
  • Protein a peptide composed of more than 50 amino acids.
  • a fusion protein is also a protein.
  • polypeptides, derivatives of proteins any polypeptide or protein involved in the present invention, unless otherwise specified (for example, a specific sequence is specified), should be understood to also include derivatives thereof.
  • the polypeptide derivatives and protein derivatives at least include C-terminal tags, N-terminal tags, and C-terminal and N-terminal tags. Wherein, the C-terminus refers to the COOH end, and the N-terminus refers to the NH2 -terminus, and those skilled in the art will understand its meaning.
  • the tag can be a polypeptide tag or a protein tag.
  • tags include, but are not limited to, histidine tags (generally containing at least 5 histidine residues; such as 6 ⁇ His, HHHHHH; another example, 8 ⁇ His tags), Glu-Glu, c-myc epitopes (EQKLISEEDL ), Tag (DYKDDDDK), Protein C (EDQVDPRLIDGK), Tag-100 (EETARFQPGYRS), V5 Epitope Tag (V5epitope, GKPIPNPLLGLDST), VSV-G (YTDIEMNRLGK), Xpress (DLYDDDDK), Hemagglutinin (hemagglutinin, YPYDVPDYA), ⁇ -Galactosidase ( ⁇ -galactosidase), thioredoxin (thioredoxin), histidine site thioredoxin (His-patch thioredoxin), IgG-binding domain (IgG-binding domain), intein- Intein
  • Protein substances in the present invention, broadly refer to substances containing polypeptides or protein fragments.
  • polypeptide derivatives protein derivatives, glycoproteins, etc. are also included in the category of protein substances.
  • Antibodies and antigens The antibodies and antigens involved in the present invention, unless otherwise specified, should also be understood to include their domains, subunits, fragments, single chains, single chain fragments, and variants.
  • Antibody substances include but are not limited to antibodies, antibody fragments, single chains of antibodies, fragments of single chains, antibody fusion proteins, fusion proteins of antibody fragments, etc. and derivatives and variants thereof, as long as antibodies can be produced - The specific binding of the antigen suffices.
  • Antigen substances include, but are not limited to, antigens known to those skilled in the art and substances capable of exerting antigen functions and specifically binding antibody substances.
  • Anti-protein antibody refers to an antibody that can specifically bind to a protein.
  • Anti-fluorescent protein nanobody refers to a nanobody that can specifically bind to a fluorescent protein.
  • Nanobody also known as single-domain antibody, English single domain antibody (sdAb), or single-chain antibody (single-chain variable fragment), or single-domain antibody, only one heavy chain variable region domain (VHH).
  • scFV It is a small molecule composed of the variable region of the heavy chain of an antibody and the variable region of the light chain under the connection of a peptide chain, and is the smallest functional structural unit with antibody activity.
  • Fab It is the region on the antibody that binds to the antigen. It consists of a constant domain and a variable domain of the heavy chain and the light chain. These domains form a parasite at the amino terminus of the monomer, that is, the antigen-binding site. These two The variable regions bind to epitopes on their specific antigen.
  • F(ab')2 It is the product formed by the antibody under the action of pepsin, which catalyzes the cleavage of the antibody under the hinge region to form F(ab')2 fragment and pFc' fragment. After mild reduction, the F(ab')2 fragment can be split into two Fab' fragments.
  • Homology means having at least 50% homology; preferably at least 60% homology, more preferably at least 70% homology, more preferably at least 75% homology, more preferably at least 80% homology, more preferably at least 85% homology, more preferably at least 90% homology; also such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, At least 97%, at least 98%, at least 99% homology. Examples of description objects are the homologous sequences of the ⁇ sequences mentioned in the present specification. Homology here refers to the similarity in sequence, and may be equivalent to identity in numerical value.
  • Homologues refer to substances with homologous sequences, which can also be called homologues.
  • Variant refers to a substance that has a different structure (including but not limited to minor variations), but still retains or substantially retains the original function or performance.
  • the variants include, but are not limited to, nucleic acid variants, polypeptide variants, and protein variants. Ways to obtain related variants include, but are not limited to, recombination, deletion or deletion, insertion, translocation, substitution and the like of structural units.
  • Such variants include, but are not limited to, modified products, genetically modified products, fusion products, and the like.
  • the methods of genetic modification include but are not limited to gene recombination (corresponding to the gene recombination product), gene deletion or deletion, insertion, frameshift, base substitution and the like.
  • Gene mutation products, also known as gene mutants belong to a type of genetic modification products.
  • One of the preferred ways of the variant is a homologue.
  • Modified products including but not limited to chemically modified products, amino acid modifications, polypeptide modifications, protein modifications, and the like.
  • the chemically modified product refers to a product modified by chemical synthesis methods such as organic chemistry, inorganic chemistry, and polymer chemistry.
  • modification methods include ionization, saltation, desalination, complexation, decomplexation, chelation, dechelation, addition reaction, substitution reaction, elimination reaction, insertion reaction, oxidation reaction, reduction reaction, post-translational modification, etc.
  • Specific examples of the modification method include modification methods such as oxidation, reduction, methylation, demethylation, amination, carboxylation, and sulfuration.
  • mutant refers to a mutant product that can still maintain or substantially maintain the original function or performance, and the number of mutation sites is not particularly limited.
  • the mutants include, but are not limited to, gene mutants, polypeptide mutants, and protein mutants. Mutants are a type of variant. Ways to obtain related mutants include, but are not limited to, recombination, deletion or deletion, insertion, translocation, substitution, and the like of structural units.
  • the structural unit of gene is base, and the structural unit of polypeptide and protein is amino acid.
  • Types of genetic mutations include, but are not limited to, gene deletions or deletions, insertions, frameshifts, base substitutions, and the like.
  • Modified products including but not limited to derivatives, modified products, genetically modified products, fusion products, etc. of the present invention, can maintain their original functions or properties, or can optimize or change their functions or properties.
  • Eluent (take the target protein as an example): Elute the target protein; after elution, the target protein exists in the eluate.
  • Washing solution (take the target protein as an example): Elute impurities such as impurity proteins; after elution, impurity proteins are taken away by the washing solution.
  • Binding capacity binding capacity, such as the binding capacity of magnetic microspheres to a certain protein.
  • IVTT In vitro transcription and translation, in vitro transcription and translation system, a cell-free protein synthesis system.
  • the cell-free protein synthesis system uses exogenous target mRNA or DNA as a protein synthesis template, and can achieve target protein synthesis by artificially controlling the addition of substrates required for protein synthesis and substances related to transcription and translation.
  • the cell-free protein synthesis system of the present invention is not particularly limited, and can be any cell-free protein synthesis system based on yeast cell extract, Escherichia coli cell extract, mammalian cell extract, plant cell extract, and insect cell extract species or any combination.
  • translation-related enzymes refer to the enzyme substances required in the synthesis process from nucleic acid template to protein product, and are not limited to the enzymes required in the translation process.
  • Nucleic acid template also known as genetic template, refers to the nucleic acid sequence as a template for protein synthesis, including DNA template, mRNA template and combinations thereof.
  • RFU Relative Fluorescence Unit
  • eGFP enhanced green fluorescent protein.
  • the eGFP broadly includes wild type and its variants, including but not limited to wild type and its mutants.
  • mEGFP A206K mutant of eGFP.
  • “Optionally” means that there may or may not be, and the selection criterion is based on the technical solution of the present invention.
  • “optional mode” means that as long as it is applicable to the technical solution of the present invention, it can be used to implement the present invention.
  • any combination thereof means “greater than 1" in number, and means a group consisting of the following situations in terms of coverage: “optionally one of them, or a group consisting of at least two of them”.
  • a first aspect of the present invention provides a biomagnetic microsphere, comprising a magnetic microsphere body, and the outer surface of the magnetic microsphere body has at least one polymer with a linear main chain and a branched chain, and the linear main chain One end of the polymer is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the branched end of the polymer of the biomagnetic microsphere is connected with an antibody-type label.
  • the biomagnetic microspheres are called antibody magnetic beads or antibody magnetic microspheres or antibody-type magnetic microspheres.
  • the antibody-type tag can be used either as a purification medium or as a linking element to further connect other types of purification media.
  • the antibody-type tag preferably, serves as a purification medium.
  • FIG. 1 A typical structure of the biomagnetic microspheres is shown in FIG. 1 .
  • a protein material compared with the currently commonly used gel-type porous materials, such as agarose, most of the commercially available microspheres use agarose materials. Porous materials have abundant pore structures, which provide a large specific surface area and provide high binding capacity for purified substrates, but correspondingly, when proteins are adsorbed or eluted, additional protein molecules need to enter or escape inside the porous material. Complex pore channels take more time and are more prone to retention.
  • the binding site for capturing the target protein provided by the present invention only utilizes the outer surface space of the biomagnetic microspheres, and during adsorption and elution, it does not need to go through a complex network channel, and can be directly released to the elution Therefore, the elution time is greatly reduced, the elution efficiency is improved, the retention ratio is reduced, and the purification yield is improved.
  • the volume of the magnetic microsphere body can be any feasible particle size.
  • the diameter of the magnetic microsphere body is any one of the following particle size scales (the deviation can be ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%) or any two particle size scales.
  • the diameter size refers to the average size.
  • the volume of the magnetic microsphere body can be any feasible particle size.
  • the diameter of the magnetic microsphere body is selected from 0.1-10 ⁇ m.
  • the diameter of the magnetic microsphere body is selected from 0.2-6 ⁇ m.
  • the diameter of the magnetic microsphere body is selected from 0.4-5 ⁇ m.
  • the diameter of the magnetic microsphere body is selected from 0.5-3 ⁇ m.
  • the diameter of the magnetic microsphere body is selected from 0.2-1 ⁇ m.
  • the diameter of the magnetic microsphere body is selected from 0.5-1 ⁇ m.
  • the average diameter of the magnetic microsphere body is about 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm,
  • the submultiples may be ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%.
  • the diameter of the magnetic microsphere body is selected from 1 ⁇ m ⁇ 1 mm.
  • the diameter of the magnetic microsphere body is 1 ⁇ m, 10 ⁇ m, 100 ⁇ m, 200 ⁇ m, 500 ⁇ m, 800 ⁇ m, 1000 ⁇ m, and the deviation range can be ⁇ 25%, ⁇ 20%, ⁇ 15%, ⁇ 10%.
  • Different magnetic materials can provide different types of activation sites, which can result in differences in the way they bind to the purification medium, as well as in their ability to disperse and settle with magnets, and can also provide selectivity for the type of purification substrate.
  • the magnetic microsphere body and the magnetic microsphere containing the magnetic microsphere body can be quickly positioned, guided and separated under the action of an external magnetic field, and on the other hand, the surface of the magnetic microsphere can be given more surface by methods such as surface modification or chemical polymerization.
  • Various active functional groups such as hydroxyl, carboxyl, aldehyde group, amino group, etc.
  • magnetic microspheres can also bind antibodies, DNA and other biologically active substances through covalent bonds or non-covalent bonds.
  • the magnetic microsphere body is a magnetic material wrapped with SiO 2 .
  • the SiO 2 coating layer may include a silane coupling agent with its own active site.
  • the magnetic material is selected from the group consisting of: iron compounds (such as iron oxides), iron alloys, cobalt compounds, cobalt alloys, nickel compounds, nickel alloys, manganese oxides, manganese alloys, zinc oxides, gadolinium oxides compounds, chromium oxides, and combinations thereof.
  • the iron oxide is, for example, magnetite (Fe 3 O 4 ), maghemite ( ⁇ -Fe 2 O 3 ) or a combination of the two oxides, preferably ferric oxide.
  • the magnetic material is selected from: Fe 3 O 4 , ⁇ -Fe 2 O 3 , iron nitride, Mn 3 O 4 , AlNi(Co), FeCrMo, FeAlC, AlNiCo, FeCrCo, ReCo, ReFe, PtCo, MnAlC, CuNiFe, AlMnAg, MnBi, FeNi(Mo), FeSi, FeAl, FeNi(Mo), FeSiAl, BaO ⁇ 6Fe 2 O 3 , SrO ⁇ 6Fe 2 O 3 , PbO ⁇ 6Fe 2 O 3 , GdO and their combination.
  • the Re is a rare earth element, rhenium.
  • the outer surface of the magnetic microsphere body has at least one polymer with a linear main chain and a branched chain, one end of the linear main chain is fixed on the outer surface of the magnetic microsphere body, and the other end of the polymer is free from the magnetic microsphere. body outer surface.
  • the “fixed to” refers to “fixed to” the outer surface of the magnetic microsphere body in a covalently linked manner.
  • the polymer is directly covalently coupled to the outer surface of the magnetic microsphere body, or indirectly covalently coupled to the outer surface of the magnetic microsphere body through a connecting element.
  • the polymer has a linear main chain. At this time, the polymer not only has the high flexibility of the linear main chain, but also has the advantages of high magnification of the number of branches, which can better achieve high-speed, high-throughput binding, and high Efficient, high-ratio (high-yield) separations.
  • the target as a protein substance as an example: for the magnetic microspheres of the present invention, one end of the polymer is covalently coupled to the outer surface of the magnetic microsphere body, and the other ends including all branches and all functional groups are Dissolved in the solution and distributed in the outer space of the magnetic microsphere body, the molecular chain can fully stretch and swing, so that the molecular chain can fully contact with other molecules in the solution, thereby enhancing the capture of the target protein.
  • the target protein When the target protein is eluted from the magnetic microspheres, the target protein can be directly freed from the shackles of the magnetic microspheres and directly enter the eluate.
  • this kind of polymer covalently fixed by one end of the linear main chain (some preferred ways covalently fix a single polymer)
  • Linear main chain in other preferred ways, 2 or 3 linear main chains are covalently drawn from the fixed end of the main chain), which can effectively reduce the stacking of molecular chains, enhance the stretching and swinging of molecular chains in solution, and enhance the capture of target proteins. , reducing the retention ratio and retention time of the target protein during elution.
  • the linear backbone is a polyolefin backbone or an acrylic polymer backbone.
  • the linear main chain of the polymer is an acrylic polymer main chain. It may be a polyolefin main chain (the linear main chain is only carbon atoms), or it may contain hetero atoms (hetero atoms are non-carbon atoms) on the linear main chain.
  • the backbone of the polymer is a polyolefin backbone.
  • the monomer units of the acrylic polymer are acrylic monomer molecules such as acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, methacrylate, or a combination thereof.
  • the acrylic polymer can be obtained by polymerizing one of the above monomers or by copolymerizing a suitable combination of the above monomers.
  • the linear backbone of the polymer is a polyolefin backbone.
  • the polyolefin backbone is the backbone provided by the polymerization product of one of acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, and methacrylate monomers, or a combination thereof
  • the main chain provided by the polymerized product (the main chain provided by the copolymerized product thereof), or the main chain of the copolymerized product formed by the participation of the above-mentioned monomers in the polymerization.
  • the polymerization product of the above-mentioned monomer combination is, for example, acrylic acid-acrylate copolymer, and another example is methyl methacrylate-hydroxyethyl methacrylate copolymer (MMA-HEMA copolymer), acrylic acid-hydroxypropyl acrylate copolymer.
  • MMA-HEMA copolymer methyl methacrylate-hydroxyethyl methacrylate copolymer
  • acrylic acid-hydroxypropyl acrylate copolymer acrylic acid-hydroxypropyl acrylate copolymer.
  • the copolymerization product formed by the above-mentioned monomers participating in the polymerization is, for example, maleic anhydride-acrylic acid copolymer.
  • the linear backbone is a polyolefin backbone and is provided by the backbone of an acrylic polymer.
  • the linear backbone is an acrylic polymer backbone.
  • the main chain of the polymer is an acrylic polymer main chain.
  • a polyolefin main chain (main chain only carbon atoms) may be used, or a hetero atom (hetero atom: non-carbon atom) may be contained in the main chain.
  • the main chain of the polymer is a main chain of a block copolymer containing polyolefin blocks, for example, polyethylene glycol-b-polyacrylic acid copolymer (belonging to the scope of acrylic copolymer). It is preferable to smoothly exert the flexible swing of the linear main chain without causing the accumulation of branched chains and increasing the residence time or/and the ratio.
  • the main chain of the polymer is a polycondensation type main chain.
  • the polycondensation-type main chain refers to a linear main chain that can be formed by a polycondensation reaction between monomer molecules or oligomers; the polycondensation-type main chain can be a homopolymerization type or a copolymerization type.
  • polypeptide chains, polyamino acid chains, etc. Specifically, for example, ⁇ -polylysine chain, ⁇ -polylysine chain, ⁇ -polyglutamic acid, polyaspartic acid chain, etc., aspartic acid/glutamic acid copolymer, and the like.
  • the number of linear backbones to which one binding site on the outer surface of the magnetic microsphere body can be covalently coupled can be one or more.
  • only one linear main chain is drawn from one binding site on the outer surface of the magnetic microsphere body, which can provide a larger space for the linear main chain in this case.
  • only two linear main chains are drawn out from one binding site on the outer surface of the magnetic microsphere body, so as to provide as much space for the linear main chains as possible.
  • the main chain of the polymer one end is covalently coupled to the outer surface of the magnetic bead (the outer surface of the biomagnetic microsphere), and the remaining ends, including all branches and all functional groups, are dissolved in the solution and distributed in the magnetic bead.
  • the molecular chain can fully stretch and swing, so that the molecular chain can fully contact with other molecules in the solution, thereby enhancing the capture of the target protein.
  • the target protein When the target protein is eluted from the magnetic beads, the target protein can be directly freed from the shackles of the magnetic beads and directly enter the eluate;
  • the polymers provided here covalently fixed by one end of the linear backbone (most preferably a single polymer linear backbone covalently fixed, and preferably 2 or 3 linear backbones covalently drawn from the fixed end of the backbone) can effectively reduce the amount of The stacking of molecular chains enhances the stretching and swinging of molecular chains in solution, enhances the capture of target proteins, and reduces the retention ratio and retention time of target proteins during elution.
  • the number of the branched chains is related to the size of the magnetic microsphere body, the skeleton structure type of the polymer, the chain density (especially the branch chain density) of the polymer on the outer surface of the magnetic microsphere body and other factors.
  • the number of polymer branches is plural, at least three.
  • the number of side branches is related to the size of the magnetic microspheres, the length of the polymer backbone, the linear density of side branches along the polymer backbone, and the chain density of the polymer on the outer surface of the magnetic microspheres.
  • the number of polymer branches can be controlled by controlling the feed ratio of the raw materials.
  • the branched polymer has at least 3 branches.
  • Each branch end is independently bound or unbound to the purification medium.
  • each of the branched ends is independently directly bound to the purification medium, or the purification medium is collected indirectly through a linking element.
  • the number of the purification medium may be one or more.
  • one molecule of the branched polymer binds at least 3 purification media.
  • the purification medium is a functional element that specifically captures the target from the mixed system, that is, the purification medium and the target molecule to be separated and purified are capable of specific binding.
  • the captured target molecules can also be eluted and released under suitable conditions, so as to achieve the purpose of separation and purification.
  • the purification medium When the purification medium takes the protein substance as the target substance, it can form a specific binding effect with the target protein itself or the purification tag carried in the target protein.
  • the purification medium in the biomagnetic microspheres is an antibody-type tag.
  • the antibody-type tag is any one of an antibody, a fragment of an antibody, a single chain of an antibody, a fragment of a single chain, an antibody fusion protein, a fusion protein of an antibody fragment, a derivative of any one, or any one of species variant.
  • the antibody-type tag is an anti-protein antibody.
  • the antibody-type tag is an antibody against a fluorescent protein.
  • the antibody-type tag is a Nanobody.
  • the antibody-type tag is an anti-protein nanobody.
  • the antibody-type tag is an anti-protein single domain antibody.
  • the antibody-type tag is an anti-protein single domain antibody.
  • the antibody-type tag is an anti-protein VHH antibody.
  • the antibody-type tag is an anti-protein scFV antibody.
  • the antibody-type tag is an anti-fluorescent protein nanobody.
  • the antibody-type tag is a nanobody against green fluorescent protein or a mutant thereof.
  • the antibody-type tag is a Fab fragment.
  • the antibody-type tag is an F(ab')2 fragment.
  • the antibody-type tag is an Fc fragment.
  • the manner in which the purification medium is linked to the biotin or the biotin analog is not particularly limited.
  • the manner in which the purification medium is linked to the biotin or the biotin analog includes, but is not limited to, covalent bonds, non-covalent bonds (eg, supramolecular interactions), linking elements, or a combination thereof.
  • the covalent bond is a dynamic covalent bond; more preferably, the dynamic covalent bond includes an imine bond, an acylhydrazone bond, a disulfide bond or a combination thereof.
  • the supramolecular interactions are selected from the group consisting of: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, ⁇ - ⁇ overlapping interactions, hydrophobic interactions, and combinations thereof.
  • the purification medium is linked to the branched end of the polymer through a linking element containing an affinity complex.
  • the biotin or biotin analog binds the avidin or avidin analog through an affinity complex interaction, and the purification medium is directly or indirectly linked to the avidin or avidin Vine analogs.
  • the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analog-avidin interaction, biotin-avidin analog interaction, biotin analog Avidin analog interactions.
  • the selection criteria of the affinity complex it has good specificity, strong affinity, and also provides a site for chemical bonding, so that the affinity complex can be covalently linked to the end of the polymer branch, or chemically After modification, it can be covalently linked to the outer surface of the magnetic microsphere body, such as the binding site on the outer surface, the end of the main chain of the linear polymer, and the end of the branched chain of the branched polymer.
  • the combination of the following substances biotin or its analogs and avidin or its analogs, and so on.
  • the avidin is any one or a combination of streptavidin, modified streptavidin, and streptavidin analogs.
  • Tamavidin 1 and Tamavidin2 are proteins with the ability to bind biotin discovered by Yamamoto et al. , 276, 1383-1397), which have a strong affinity for biotin similar to streptavidin.
  • the thermal stability of Tamavidin2 is better than that of streptavidin, and its amino acid sequence can be retrieved from relevant databases, such as UniProt B9A0T7, or the DNA sequence can be optimized by codon conversion and optimization program.
  • biotin analogs such as the WSHPQFEK sequence or its variant sequence, the WRHPQFGG sequence or its variant sequence, and the like.
  • the loading mode includes dynamic covalent bonds and supramolecular interactions (especially the interaction of affinity complexes)
  • a reversible loading mode is formed, and the purification medium can be unloaded from the end of the branch under certain conditions, and then renewed or replaced .
  • the renewal of the purification medium corresponds to the regeneration of the magnetic microspheres, and the types of the purification medium before and after the renewal are the same.
  • the replacement of the purification medium corresponds to the change of the magnetic microspheres, and the types of purification media before and after the replacement are different.
  • biotin, avidin, and a purification medium are sequentially connected to the branched ends of the polymers of the biomagnetic microspheres; wherein, the purification medium is an antibody.
  • the connection mode between the avidin and the purification medium includes, but is not limited to, covalent bonds, non-covalent bonds, connecting elements or combinations thereof.
  • the purification medium is linked to the polymer branched end of the biomagnetic microspheres through the following linking elements: including but not limited to nucleic acid, oligonucleotide, peptide nucleic acid, nucleic acid aptamer, deoxyribonucleic acid, ribonucleic acid , leucine zipper, helix-turn-helix motif, zinc finger motif, biotin, avidin, streptavidin, anti-hapten antibodies, etc., combinations thereof.
  • linking elements including but not limited to nucleic acid, oligonucleotide, peptide nucleic acid, nucleic acid aptamer, deoxyribonucleic acid, ribonucleic acid , leucine zipper, helix-turn-helix motif, zinc finger motif, biotin, avidin, streptavidin, anti-hapten antibodies, etc., combinations thereof.
  • the linking element can also be a double-stranded nucleic acid construct, a duplex, a homohybrid or a heterohybrid (from DNA-DNA, DNA-RNA, DNA-PNA, RNA-RNA, RNA-PNA or PNA-PNA selected homohybrid or heterohybrid), or a combination thereof.
  • the force of the purification medium to capture the target molecule in the reaction and purification mixed system is the specific binding effect between the antibody-type tag and the target.
  • the antibody-type tag is attached to the branched end of the polymer through an affinity complex interaction.
  • the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analog-avidin interaction, biotin-avidin analog interaction, biotin analog Avidin analog interactions.
  • the avidin is streptavidin, modified streptavidin, streptavidin analogs, or a combination thereof.
  • the antibody-type tag is attached to the branched end of the polymer by covalent bonding, supramolecular interaction, or a combination thereof.
  • the covalent bond utilizes a dynamic covalent bond; more preferably, the dynamic covalent bond includes an imine bond, an acylhydrazone bond, a disulfide bond or a combination thereof.
  • the supramolecular interaction is selected from the group consisting of: coordination binding, affinity complex interaction, electrostatic adsorption, hydrogen bonding, ⁇ - ⁇ overlapping interactions, hydrophobic interactions, and combinations thereof.
  • the biomagnetic microsphere includes a magnetic microsphere body, and the outer surface of the magnetic microsphere body has at least one polymer with a linear main chain and a branched chain, and one end of the linear main chain is It is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the branched end of the polymer of the biomagnetic microsphere is connected with biotin or a biotin analog, and the biotin Or biotin analogs are used as connecting elements to further connect avidin or avidin analogs through affinity complex binding, and the avidin or avidin analogs are still used as connecting elements to further connect the antibody type. Label.
  • biotin is connected to the branched end of the polymer of the biomagnetic microspheres, and the biotin is used as a connecting element to further connect avidin or avidin analogs through the binding effect of the affinity complex.
  • the avidin or avidin analog still serves as a linking element to further link the antibody-type tag.
  • Preferred forms of the avidin include any one of streptavidin, modified streptavidin, streptavidin analogs, and combinations thereof.
  • biotin or biotin analog is attached to the branched end of the polymer is not particularly limited.
  • biotin or biotin analog is attached to the branched end of the polymer includes, but is not limited to, covalent bonds, non-covalent bonds (eg, supramolecular interactions), or a combination thereof.
  • the covalent bond is a dynamic covalent bond; more preferably, the dynamic covalent bond includes an imine bond, an acylhydrazone bond, a disulfide bond or a combination thereof.
  • the supramolecular interactions are selected from the group consisting of: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, ⁇ - ⁇ overlapping interactions, hydrophobic interactions, and combinations thereof.
  • the branched chain of the polymer is covalently bound to biotin or biotin analogs through covalent bonds based on functional groups, and biotin or biotin analogs are covalently bound to the branched chains of the polymer. end. It can be obtained by covalently reacting functional groups contained in the branched chains of polymer molecules on the outer surface of biomagnetic microspheres with biotin or biotin analogs.
  • one of the preferred embodiments of the functional group is a specific binding site (for definitions, please refer to the "noun and term" section of the specific embodiment).
  • the functional group-based covalent bond refers to a covalent bond formed by a functional group participating in covalent coupling.
  • the functional group is a carboxyl group, a hydroxyl group, an amino group, a sulfhydryl group, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of the foregoing functional groups.
  • One of the preferred modes of the salt form of the carboxyl group is a sodium salt form such as COONa; the preferred mode of the salt form of the amino group can be an inorganic salt form, or an organic salt form, including but not limited to hydrochloride, hydrofluoride acid salts, etc.
  • the "combination of functional groups” refers to all branches of all polymer molecules on the outer surface of a magnetic microsphere, allowing different functional groups to participate in the formation of covalent bonds; take biotin as an example, that is, a biological All biotin molecules on the outer surface of the prime magnetic microspheres can be covalently linked with different functional groups, but one biotin molecule can only be linked with one functional group.
  • the purification medium When the purification medium is reversibly connected to the end of the polymer branch of the biomagnetic microspheres of the present invention through reversible means such as affinity complexes, the purification medium can be eluted from the end of the polymer branch under appropriate conditions, and then recombined New purification medium.
  • the extremely strong affinity between biotin and streptavidin is a typical binding effect of affinity complexes, which is both stronger than general non-covalent bonds and weaker than covalent bonds, so that both
  • the purification medium antibody-type tag
  • the purification medium can be realized by elution of streptavidin from the specific binding site of biotin when the purification medium needs to be replaced synchronous dissociation, which in turn releases activation sites that can re-associate new avidin-purification media covalently linked complexes (e.g., streptavidin-tagged purification media), enabling rapid bead purification performance recovery, greatly reducing the cost of separation and purification of target compounds.
  • the process of eluting the biomagnetic microspheres modified with the purification medium to remove the covalently linked complex of avidin-purification medium, so as to regain the biotin or biotin analog-modified biomagnetic microspheres we call it Regeneration of biotin magnetic microspheres.
  • the regenerated biotin magnetic microspheres have released biotin active sites, which can re-bond the avidin-purification medium covalently linked complex, and obtain the purification medium-modified biomagnetic microspheres (corresponding to the regeneration of the biomagnetic microspheres). ), which can provide fresh purification media and provide nascent target binding sites. This allows the biotin magnetic microspheres of the present invention to be regenerated, that is, the purification medium can be replaced and then reused.
  • Purified substrates of the present invention refer to the magnetic microspheres of the present invention for capturing separated substances.
  • the purified substrate of the present invention is a substance capable of specifically binding to the antibody-type tag.
  • the purification substrate is a proteinaceous substance.
  • the purification substrate is also called a target protein.
  • the target protein can be a natural protein or a modified product thereof, or an artificial synthetic sequence.
  • the source of the natural protein is not particularly limited, including but not limited to: eukaryotic cells, prokaryotic cells, pathogens; wherein eukaryotic cell sources include but are not limited to: mammalian cells, plant cells, yeast cells, insect cells, nematode cells, and combinations thereof; the mammalian cell sources may include but are not limited to murine sources (including rats, mice, guinea pigs, golden hamsters, hamsters, etc.), rabbit sources, monkey sources, human sources, pig sources, sheep sources, bovine sources source, dog source, horse source, etc.
  • the pathogens include viruses, chlamydia, mycoplasmas, and the like. The viruses include HPV, HBV, TMV, coronavirus, rotavirus, and the like.
  • target protein in the present invention broadly includes polypeptides), fluorescent proteins, enzymes and corresponding zymogens, antibodies, antigens, immunoglobulins, hormones, collagen, polyamino acids , vaccines, etc., partial domains of any of the foregoing proteins, subunits or fragments of any of the foregoing proteins, and variants of any of the foregoing proteins.
  • target protein in the present invention broadly includes polypeptides
  • fluorescent proteins enzymes and corresponding zymogens
  • antibodies antigens, immunoglobulins, hormones, collagen, polyamino acids , vaccines, etc.
  • partial domains of any of the foregoing proteins subunits or fragments of any of the foregoing proteins, and variants of any of the foregoing proteins.
  • subunit or fragment of any of the foregoing proteins includes subunits or fragments of "partial domains of any of the foregoing proteins”.
  • variant of any of the foregoing proteins includes variants of "a partial domain of any of the foregoing proteins, subunits or fragments of any of the foregoing proteins".
  • variant of any of the foregoing proteins includes, but is not limited to, mutants of any of the foregoing proteins. In the present invention, the situation of two or more consecutive "aforesaid" in other positions shall be interpreted similarly.
  • the structure of the target protein can be either a complete structure or selected from corresponding partial domains, subunits, fragments, dimers, multimers, fusion proteins, glycoproteins, and the like.
  • incomplete antibody structures include Nanobodies (heavy chain antibodies lacking light chains, VHH , which retain the full antigen-binding ability of heavy chain antibodies), heavy chain variable regions, complementarity determining regions (CDRs), and the like.
  • the target protein that can be synthesized by the in vitro protein synthesis system of the present invention can be selected from, but not limited to, any of the following proteins, fusion proteins in any combination, and compositions in any combination: luciferase (such as firefly fluorescence peptase), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase (Catalase, for example e.g. murine catalase), actin, antibodies, variable regions of antibodies (e.g.
  • luciferase such as firefly fluorescence peptase
  • GFP green fluorescent protein
  • eGFP enhanced green fluorescent protein
  • YFP yellow fluorescent protein
  • aminoacyl tRNA synthetase aminoacyl tRNA synthetase
  • single chain variable regions of antibodies scFV
  • single chains of antibodies and fragments thereof e.g. heavy chains of antibodies, nanobodies, antibodies light chain
  • alpha-amylase enterobactin A
  • enterobactin A hepatitis C virus E2 glycoprotein
  • insulin and its precursors glucagon-like peptide (GLP-1)
  • interferon including but not limited to interferon Interferon alpha, such as interferon alpha A, interferon beta, interferon gamma, etc.
  • interleukins such as interleukin-1 beta, interleukin 2, interleukin 12, etc.
  • lysozyme serum albumin (including but not limited to human serum Albumin, bovine serum albumin), transthyretin, tyrosinase, xylanase, beta-galactosidase (beta-galactosidase, LacZ, such as E.
  • coli beta-galactosidase coli beta-galactosidase
  • a partial domain of any of the foregoing proteins, a subunit or fragment of any of the foregoing proteins, or a variant of any of the foregoing as defined above, the variants include mutants, such as luciferase mutations mutants of eGFP, which may also be homologs).
  • the aminoacyl-tRNA synthetase for example, human lysine-tRNA synthetase (lysine-tRNA synthetase), human leucine-tRNA synthetase (leucine-tRNA synthetase) and the like.
  • the glyceraldehyde-3-phosphate dehydrogenase is, for example, Arabidopsis glyceraldehyde-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase.
  • the composition in any combination may include any of the aforementioned proteins, and may also include the fusion proteins in any combination.
  • one of GFP, eGFP, mScarlet, or the like, or a target protein with fluorescent properties, such as a similar substance or a mutant thereof, is used to evaluate the protein synthesis ability of the in vitro protein synthesis system.
  • the application fields of the target protein include but are not limited to biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nanoengineering, Cosmetics, food, food additives, nutritional agents, agriculture, feed, daily necessities, washing, environment, chemical dyeing, fluorescent marking and other fields.
  • the magnetic microspheres of the present invention can be used to separate the target protein from its mixed system.
  • the target protein is not limited to one substance, but a combination of multiple substances is allowed, as long as the purpose of purification is to obtain this composition, or the form of this composition can meet the purification requirements.
  • the mixed system containing the target protein is not particularly limited, as long as the purification medium of the magnetic microspheres of the present invention can specifically bind to the target protein; generally, it is also required that the purification medium and other substances other than the target protein in the mixed system are incompatible. There is specific binding or non-specific binding.
  • the mixed system containing the target protein may be a natural source, or may be an artificially constructed or obtained mixed system.
  • proteins can be isolated and purified from commercially available serum.
  • the target protein can be isolated from the post-reaction system of an in vitro protein synthesis system.
  • One of the specific embodiments of the in vitro protein synthesis system also includes but is not limited to, for example, the Escherichia coli-based cell-free protein synthesis system described in WO2016005982A1.
  • Other citations of the present invention, and those described in their direct and indirect citations include, but are not limited to, in vitro cell-free protein synthesis systems based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces marxianus , are also incorporated into the present invention as embodiments of the in vitro protein synthesis system of the present invention.
  • the source cell of the cell extract of the in vitro protein synthesis system is not particularly limited, as long as the target protein can be expressed in vitro.
  • Exogenous proteins disclosed in the prior art that are suitable for prokaryotic cell extracts, eukaryotic cell extracts (preferably yeast cell extracts, and more preferably Kluyveromyces lactis) derived in vitro protein synthesis systems, or suitable for cell
  • the endogenous protein of the prokaryotic cell system and eukaryotic cell system (which can be preferably a yeast cell system, can also be more preferably a Kluyveromyces lactis system) synthesized in the interior can also be synthesized by the in vitro protein synthesis system of the present invention, Or try to synthesize with the in vitro protein synthesis system provided by the present invention.
  • IVTT reaction liquid The liquid after IVTT reaction
  • the IVTT reaction liquid in addition to the expressed target protein, also contains residual reaction raw materials in the IVTT system, especially various factors from cell extracts (such as ribosomes, tRNA, translation-related enzymes, initiation factors, elongation factors, termination factors, etc.).
  • the IVTT reaction solution on the one hand, can provide the target protein for binding to the magnetic beads, and on the other hand, can also provide a mixed system for testing the separation effect of the target protein.
  • the present invention also discloses a method for preparing the biomagnetic microspheres described in the first aspect, comprising the following steps: (i) providing magnetic microspheres (also referred to as biotin magnetic microspheres) combined with biotin or biotin analogs; ball or biotin magnetic beads); (ii) connecting the raw material for providing antibody-type tags with the biotin analog at the end of the polymer branch of the bio-magnetic microspheres to obtain bio-magnetic microspheres bound with antibody-type tags K (antibody magnetic beads).
  • the raw material for providing the antibody-type tag is the antibody-type tag.
  • the raw material for providing the antibody-type tag is a covalently linked complex of avidin or an avidin analog and the antibody-type tag.
  • Step (ii) binds the covalently linked complex of avidin or avidin analog to the antibody-type tag to the end of the polymer branch, the biotin or biotin analog at the end of the polymer branch Binding with the avidin or avidin analogs to form an affinity complex to obtain the biomagnetic microsphere K with the antibody-type label.
  • Preferred forms of the avidin include, but are not limited to, streptavidin, modified streptavidin, streptavidin analogs, and combinations thereof.
  • the raw material for providing the antibody-type tag is an avidin-antibody-type tag covalently linked complex.
  • (iv) replacement of the avidin-purification medium covalently linked complex is included.
  • FIG. 2 A typical preparation method of the biomagnetic microspheres is shown in Fig. 2, taking nanobodies as the purification medium as an example.
  • biotin-conjugated biomagnetic microspheres Take biotin-conjugated biomagnetic microspheres as an example.
  • the biotin magnetic microspheres can be prepared through the following steps: providing SiO 2 -coated magnetic beads (commercially available or homemade), activating modification of SiO 2 , covalently linking the polymer to SiO 2 (the polymer is bound by a linear backbone). One end is covalently attached to SiO2 with a large number of side branches distributed along the polymer backbone), and biotin is covalently attached to the branched ends of the polymer. It should be noted that the above-mentioned links are not required to be completely isolated, and two or three links are allowed to be combined into one link. For example, activated silica-coated magnetic beads (commercially available or homemade) can be directly provided.
  • the biotin magnetic microspheres can be prepared by the following steps: (1) providing or preparing a magnetic microsphere body, the outer surface of the magnetic microsphere body has a reactive group R 1 ; (2) in the reactive group On the basis of the group R 1 , a polymer with a linear main chain and a large number of branched chains is connected, and one end of the linear main chain is covalently connected to the reactive group R 1 ; (3) at the end of the branched chain Link biotin or biotin analogs.
  • the preparation process of the biotin magnetic microspheres can be prepared by the following steps: (1) Provide SiO 2 -coated magnetic microspheres (commercially available or homemade), and carry out Activation and modification of SiO 2 generate reactive group R 1 ; (2) carry out a polymerization reaction on reactive group R 1 (such as using acrylic acid or sodium acrylate as a monomer molecule) to generate a polymer with a linear main chain and a large number of branched chains and a functional group F 1 at the end of the branched chain; (3) connecting biotin or a biotin analog to the functional group F 1 at the end of the branched chain.
  • the polymer covalently connected to the magnetic microsphere body has a linear main chain, one end of the linear main chain is covalently fixed at the reactive group R1, and a large number of side branches are distributed along the main polymer chain.
  • a typical preparation method of the biotin magnetic microspheres comprising the following steps:
  • Step (1) providing a magnetic microsphere body, chemically modifying the magnetic microsphere body, and introducing amino groups into the outer surface of the magnetic microsphere body to form amino-modified magnetic microspheres A.
  • the magnetic microsphere body is chemically modified with a coupling agent.
  • the coupling agent is an aminated silane coupling agent.
  • the magnetic microsphere body is a magnetic material wrapped with SiO 2 , and the magnetic microsphere body is chemically modified by a silane coupling agent; in some preferred modes, the silane coupling agent is aminated silane coupling agent.
  • Step (2) Covalently couple the acrylic acid molecule to the outer surface of the magnetic microsphere A by the covalent reaction between the carboxyl group and the amino group, and introduce a carbon-carbon double bond to form the carbon-carbon double bond-containing magnetic microsphere B.
  • Step (3) polymerize acrylic monomer molecules (such as sodium acrylate) by utilizing the polymerization reaction of carbon-carbon double bonds, and the obtained acrylic polymer is a branched polymer with a linear main chain and functional groups.
  • the polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain to form an acrylic polymer modified magnetic microsphere C. This step can be carried out without adding a crosslinking agent.
  • the functional group F 1 is a carboxyl group, a hydroxyl group, an amino group, a sulfhydryl group, a formate, an ammonium salt, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of the foregoing functional groups ;
  • the "combination of functional groups” refers to the functional groups contained in all the branches of all polymers on the outer surface of a magnetic microsphere, and the types may be one or more. The meaning of "combination of functional groups" defined in the first aspect is consistent.
  • the functional group is a specific binding site.
  • Step (4) Covalently couple biotin or a biotin analog to the end of the polymer branch through the functional group F 1 contained in the branch chain of the polymer to obtain a biotin or biotin analog combined of biomagnetic microspheres (a biotin magnetic microsphere).
  • a biotin magnetic microsphere a biotin magnetic microsphere.
  • an acrylic polymer having a polyacrylic acid backbone.
  • a specific embodiment for preparing the biotin magnetic microspheres is as follows.
  • a silicon dioxide-wrapped triferromagnetic bead is used as the body of the biomagnetic microsphere;
  • APTES aminopropyltriethoxysilane
  • silane coupling agent also a silane coupling agent, more specifically an aminated silane Coupling agent
  • the covalent reaction between amino groups covalently couples the immobilized molecule (acrylic acid molecule, providing a carbon-carbon double bond and a reactive group carboxyl group) to the outer surface of the magnetic bead, thereby introducing the carbon-carbon double bond into the magnetic bead.
  • a carbon-carbon double bond-containing magnetic microsphere B is obtained; then the polymerization of acrylic monomer molecules (such as sodium acrylate) is carried out by the polymerization reaction of carbon-carbon double bonds, and the polymerization product is covalently coupled during the polymerization reaction.
  • acrylic monomer molecules such as sodium acrylate
  • the polymer is connected at SiO2 (covalent connection), and the acrylic polymer modified magnetic microsphere C is obtained; the immobilized molecule here is an acrylic molecule, and one immobilized molecule only leads to one polymer At the same time, only one linear polymer main chain is drawn; taking sodium acrylate as a monomer molecule as an example, the polymerization product is sodium polyacrylate, and its main chain is a linear polyolefin main chain, and there are covalently connected along the main chain.
  • a large number of side chain COONa the functional group contained in the branch is also COONa; the polymerization reaction here does not use cross-linking agents such as N,N'-methylenebisacrylamide (CAS: 110-26-9), To avoid the cross-linking of molecular chains to form a network polymer, the polymer product produces a linear main chain without adding a cross-linking agent. If the molecular chains are cross-linked to form a network polymer, a porous structure will be formed, which affects the elution efficiency of the target protein.
  • cross-linking agents such as N,N'-methylenebisacrylamide (CAS: 110-26-9)
  • the amount of acrylic acid used in the preparation of the magnetic microspheres B is 0.002-20 mol/L.
  • the amount of sodium acrylate used in the preparation of magnetic microspheres C is 0.53-12.76 mol/L.
  • the outer surface of the biomagnetic microspheres can also be modified by activation other than amination.
  • the aminated biomagnetic microspheres (amino-modified magnetic microspheres A) can be further reacted with acid anhydrides or other modified molecules, so as to realize chemical modification of the outer surface of the biomagnetic microspheres by carboxylation or other activation methods.
  • the immobilized molecule is a small molecule that covalently fixes the main chain of the polymer to the outer surface of the magnetic bead.
  • the immobilized small molecule is not particularly limited, as long as one end of the molecule is covalently coupled to the outer surface of the magnetic bead, and the other end can initiate a polymerization reaction, including homopolymerization, copolymerization or polycondensation, or the other end can be copolymerized with a coupled polymer. Linear backbone ends are sufficient.
  • the immobilized molecule allows the extraction of only a single linear polymer chain, and also allows the extraction of two or more linear polymer chains, as long as it does not lead to chain stacking and/or does not lead to an increase in the retention ratio.
  • one immobilized molecule leads only one polymer molecule, and only one linear polymer backbone.
  • the immobilized molecule allows the extraction of only a single polymer linear backbone, and also allows the extraction of two or more polymer linear backbones, as long as it does not lead to chain stacking and/or does not lead to retention ratios Just increase it.
  • one immobilized molecule leads only one polymer molecule, and only one linear polymer backbone.
  • the acrylic monomer molecule as the polymerized monomer unit may also be one of acrylic acid, acrylic acid salt, acrylic acid ester, methacrylic acid, methacrylic acid salt, methacrylic acid ester monomer or its combination.
  • the acrylic polymer can also be replaced by other polymers.
  • the selection criteria are: the formed polymer has a linear main chain, a large number of side branches are distributed along the main chain, and the side branches carry functional groups for subsequent chemical modification; that is, for the outer surface of the magnetic beads One of the binding sites of the polymer, which provides a large number of functional groups through the branches distributed at the side ends of the linear main chain of the polymer.
  • the method of introducing polymer molecules of other alternatives of the above-mentioned polymers to the outer surface of the biomagnetic microsphere according to the chemical structure of the polymer substitute and the type of side branched active groups, a suitable biomagnetic microsphere is selected.
  • the activation and modification method of the outer surface of the sphere, the type of immobilized molecule, the type of monomer molecule, and the appropriate chemical reaction will introduce a large number of active groups located in the branched chain into the outer surface of the biomagnetic microsphere.
  • the functional groups at the end of the branched chains provide activation sites, or connect biotin or biotin analogs.
  • the branched functional group of the polymer molecule can be activated to make it reactive and form an activation site; 1,3-propanediamine can be covalently coupled to the branched polymer Activation site (each monomeric acrylic unit structure can provide one activation site) to form a new functional group (amino group), and then use the amidation covalent reaction between the carboxyl group and the amino group to convert biotin or biotin
  • the analog molecule is covalently coupled to the new functional group at the end of the polymer branch, completing the covalent attachment of biotin or biotin analogs to the branch end of the polymer.
  • biotin or biosimilar modified biomagnetic microspheres D are obtained; one biotin molecule can provide a specific binding site.
  • COONa as the functional group of the branch chain of the polymer as an example
  • sodium acrylate is used as the monomer molecule, and before the covalent reaction with 1,3-propanediamine, the carboxyl group can be activated first, and the existing carboxyl group can be used.
  • Activation method eg: adding EDC 2HCl and NHS.
  • Preparation of magnetic microspheres A To the aqueous solution of silica-coated ferric oxide magnetic microspheres, wash the magnetic microspheres with absolute ethanol, add 3-aminopropyltriethoxysilane (APTES, coupling agent) Ethanol solution, reaction, cleaning, and introducing a large number of amino groups on the outer surface of the magnetic microspheres.
  • APTES 3-aminopropyltriethoxysilane
  • Preparation of magnetic microspheres B To the aqueous solution of acrylic acid, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC ⁇ HCl) and N-hydroxysuccinimide ( NHS) to activate the carboxyl group, and after activation, it was added to the aqueous solution containing magnetic microspheres A.
  • EDC ⁇ HCl 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • Preparation of magnetic microspheres C adding an aqueous solution of acrylic monomer molecules into magnetic microspheres B, adding an initiator, and carrying out a polymerization reaction of carbon-carbon double bonds.
  • the carbon-carbon double bond in the acrylic monomer molecule and the carbon-carbon double bond on the surface of the magnetic microsphere undergo bond opening polymerization, and the acrylic polymer molecule is covalently bonded to the outer surface of the magnetic microsphere, wherein the acrylic polymer contains a carboxyl group
  • the carboxyl-like functional group can exist in the form of carboxyl, formate, formate and the like.
  • One of the preferred ways is to exist in the form of sodium formate, and in this case, sodium acrylate or sodium methacrylate is used as the monomer molecule.
  • Another preferred way is to exist in the form of formate, and at this time, acrylate or methacrylate is used as the monomer molecule. Formate and formate can obtain better reactivity after activation by carboxyl group.
  • Magnetic microsphere C solution add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC ⁇ HCl) and N-hydroxysuccinimide (NHS),
  • EDC ⁇ HCl 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • the carboxyl functional group of the side chain of the polymer molecule on the outer surface of the sphere is activated by the carboxyl group, and then an aqueous solution of propylene diamine is added to carry out a coupling reaction, and propylene diamine is grafted at the position of the side branch carboxyl group of the acrylic polymer molecule,
  • the functional groups of the side chains of the polymer are converted from carboxyl groups to amino groups.
  • Aqueous solution of biotin add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to activate the carboxyl group in the biotin molecule, and then add
  • biotin is covalently bound to the position of the newly-born functional group (amino) of the side chain of the polymer on the outer surface of the magnetic microsphere C, resulting in a large number of side branches in the acrylic polymer Biomagnetic microspheres D connected with biotin molecules, respectively.
  • the method for preparing the above-mentioned biomagnetic microspheres D is as follows:
  • the above solution is added to a pH 7.2-7.5 PBS buffer solution mixed with 0.5-50 mL of magnetic microspheres A, reacted for 1-48 hours, and the magnetic microspheres are washed with distilled water to obtain magnetic microspheres B modified with carbon-carbon double bonds.
  • Magnetic microsphere B Take 0.5-50mL magnetic microsphere B, add 0.5-200mL 5%-30% (w/v) sodium acrylate solution, then add 10 ⁇ L-20mL 2%-20% (w/v) ammonium persulfate solution and 1 ⁇ L-1mL Tetramethylethylenediamine, after 3-60 minutes of reaction, the magnetic microspheres are washed with distilled water to obtain magnetic microspheres C modified with sodium polyacrylate.
  • biotin-modified biomagnetic microspheres D to a fusion protein solution of avidin-antibody-type tag-linked complexes (for example, antiEGFP-mScarlet-Tamvavidin2, an antibody-type fusion protein, and a fusion protein of nanobodies) , mixed and incubated.
  • avidin such as Streptavidin or Tamvavidin2
  • the antibody-type tag is fixed to the end group of the polymer branch on the outer surface of the biomagnetic microsphere D, and the avidin-antibody is obtained.
  • Type-labeled biomagnetic microspheres K Type-labeled biomagnetic microspheres K.
  • the fusion protein of avidin-antibody tag can be obtained by in vitro cell-free protein synthesis by IVTT reaction. At this time, the supernatant obtained after the reaction of the biomagnetic microspheres D and IVTT is mixed, and the antibody-type tag is realized through the specific binding between the biotin on the outer surface of the biomagnetic microspheres D and the avidin fusion protein in the solution. combination.
  • the binding amount of the antibody-type label on the outer surface of the biomagnetic microspheres can be determined by the following method (taking fluorescent protein mScarlet labeling as an example):
  • the antibody-based fusion protein-bound biomagnetic microspheres K are adsorbed and sedimented with a magnet. Then, the liquid phase was separated and collected, which was recorded as the flow-through. At this time, the concentration of the antibody-type fusion protein in the liquid phase decreases.
  • the fluorescence intensity bound on the biomagnetic microspheres was calculated, and the concentration of the antibody-type fusion protein was obtained by conversion.
  • the concentration of the antibody-based fusion protein in the flow-through solution basically does not change compared with the antibody-based fusion protein in the IVTT solution before the incubation of the biomagnetic microspheres, it means that the adsorption of the antibody-based fusion protein by the biomagnetic microspheres tends to increase. At saturation, the corresponding fluorescence values no longer change significantly.
  • the pure product of mScarlet protein can be used to establish a standard curve between the fluorescence value and the concentration of mScarlet protein, so as to quantitatively calculate the antibody-type fusion protein (such as: streptavidin-nanobody, antiEGFP-mScarlet- Tamvavidin2 nanobody fusion protein) content and concentration.
  • the biomagnetic microspheres K with antibody-type tags are used for the separation and purification of the target, and the binding amount of the target can be calculated by the following method: a solution of the biomagnetic microspheres K and the target (take the target protein as an example) (for example, in vitro After the reaction is completed, the target protein is eluted from the magnetic beads using an elution buffer, and the separated target protein exists in the eluate. Use an appropriate method to measure the protein concentration of the target protein in the eluate, and then calculate the yield and yield of the separation and purification.
  • biomagnetic microsphere K biotin-avidin-antibody tag connection method
  • biomagnetic microsphere D "biotin-modified biomagnetic microsphere D”
  • adding denaturation buffer (containing urea) to the biomagnetic microsphere K and sodium dodecyl sulfate) incubate in a metal bath at 95°C, wash off the avidin-antibody tag fusion protein bound to biotin on biomagnetic microspheres K, and obtain regenerated biomagnetic microspheres D (Release the binding site of biotin at the end of the polymer branch), and then add a fresh solution of avidin-antibody-type tag fusion protein (such as antiEGFP-mScarlet-Tamvavidin2) to the regenerated biomagnetic microspheres D The supernatant after the IVTT reaction), so that the biotin-binding site of the released biomagnetic microsphere D re-bonds the new avidin-antibody-type tag, and re-bond
  • the magnetic microspheres of the present invention can be easily settled by a magnet, the liquid phase can be removed, and the adsorbed impurity proteins or/and other impurities can be removed by washing.
  • the magnetic microspheres can be stably suspended in the liquid phase, and can not settle for two days or more. Moreover, it can be stably suspended in the liquid system without continuous stirring.
  • the magnetic microspheres can be controlled to a nanoscale size of several micrometers or even less than 1 micrometer; on the other hand, the graft density of the polymer on the outer surface of the magnetic microspheres can be adjusted, and the hydrophilicity, Structure type, hydrodynamic radius, chain length, number of branches, length of branches, etc., so as to better control the suspension performance of the magnetic microsphere system in the system, and realize the sufficient mixing system of the magnetic microsphere system and the in vitro protein synthesis reaction. touch.
  • One preferred size of the biomagnetic microspheres of the present invention is about 1 micron.
  • the preparation method of the biomagnetic microspheres K comprises the following steps:
  • the coupling agent is preferably an aminated silane coupling agent.
  • One of the preferred ways is to chemically modify the magnetic microsphere body with a coupling agent.
  • a silane coupling agent can be used to chemically modify the magnetic microsphere body.
  • the silane coupling agent is preferably an aminated silane coupling agent.
  • the polymerization of carbon-carbon double bonds is used to polymerize acrylic monomer molecules (such as sodium acrylate), and the obtained acrylic polymer has a linear main chain and contains functional groups.
  • the polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain to form an acrylic polymer modified magnetic microsphere C.
  • the functional group is a carboxyl group, a hydroxyl group, an amino group, a sulfhydryl group, a formate, an ammonium salt, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of the foregoing functional groups;
  • the " “Combination of functional groups” refers to the functional groups contained in all branches of all polymers on the outer surface of a magnetic microsphere, and the types may be one or more. The meaning of "combination of functional groups" defined in the first aspect is consistent.
  • the functional group is a specific binding site.
  • the raw material for providing the antibody-type tag is avidin or a covalently linked complex of avidin analog and the antibody-type tag; more preferably, the raw material for providing the antibody-type tag is avidin Covalently linked complexes of covalentin-antibody-type tags.
  • the raw material for providing the antibody-type tag is an avidin-antibody-type tag covalently linked complex.
  • the present invention also discloses the application of the antibody magnetic microspheres in separation and purification, preferably in the separation and purification of protein substances (in this case, the antibody-type tag is an anti-protein antibody).
  • silica-coated magnetic microspheres also known as magnetic microsphere bodies, magnetic beads, glass beads
  • Fe 3 O 4 microspheres into a mixed solvent of 310mL ethanol and 125mL water, add 45mL 28% (wt) ammonia water, add 22.5mL ethyl orthosilicate dropwise, stir at room temperature for 24h, and use ethanol after the reaction Wash with water.
  • Ferric oxide microspheres with different particle sizes (about 1 ⁇ m, 10 ⁇ m, and 100 ⁇ m) were used as raw materials to control the particle size of the obtained glass beads.
  • the iron tetroxide microspheres with different particle sizes can be prepared by conventional technical means.
  • the prepared magnetic microspheres are used as basic raw materials for modifying purification media or connecting elements-purification media, so they are also called magnetic microsphere bodies.
  • the prepared magnetic microspheres have a magnetic core, which can control the position through the action of magnetic force, and realize operations such as movement, dispersion, and sedimentation, so it is a generalized magnetic bead.
  • the prepared magnetic microspheres have a coating layer of silica, so they are also called glass beads, which can reduce the adsorption of the following components or components by the magnetic core: polymers, purification media, components of in vitro protein synthesis systems, nucleic acids Templates, protein expression products, etc.
  • the binding efficiency of the target protein can be increased by more than 50% compared with the particle size of 10 ⁇ m when the particle size of the magnetic microspheres is about 1 ⁇ m, and can be increased by more than 80% compared with the particle size of 100 ⁇ m .
  • Silica-coated magnetic microspheres The biotin magnetic beads were prepared by the following procedure.
  • solution X the final concentration is 0.1mol/L 2-morpholineethanesulfonic acid (CAS: 4432-31-9), the aqueous solution of 0.5mol/L NaCl), add 0.04mol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS: 25952-53-8) and 0.04mol N-hydroxysuccinimide (CAS: 6066-82 -6), stirring and mixing at room temperature, stirring and reacting for 15min, using NaHCO 3 solid powder to adjust the pH of the solution to 7.2, adding the above-mentioned pH-adjusted solution to 100 mL of PBS buffer solution with 10 mL of magnetic microspheres A, Mechanical stirring was carried out in a 30°C water bath for 20 hours, the magnetic microspheres were settled with a magnet, the liquid phase was removed, and the magnetic microspheres were washed with 60 m
  • magnetic microspheres B take 1 mL of magnetic microspheres B, add 12 mL of 15% (w/v) sodium acrylate solution, add 450 ⁇ L of 10% ammonium persulfate solution and 45 ⁇ L of tetramethylethylenediamine, react at room temperature for 30 minutes, and use The magnetic microspheres were settled by a magnet, the liquid phase was removed, and the magnetic microspheres were washed with 10 mL of distilled water each time, for a total of 6 times, to obtain magnetic microspheres C (acrylic polymer-modified magnetic microspheres C).
  • the pH of the solution was adjusted to 7.2 with NaHCO 3 solid powder, and added to the above washed solution containing 10 mL of PBS buffer solution.
  • the magnetic microspheres were mechanically stirred in a water bath at 30°C for 20 hours, the magnetic microspheres were sedimented with a magnet, the liquid phase was removed, and each time was washed 10 times with 10 mL of distilled water to obtain biotin-modified biomagnetic microspheres D.
  • antiEGFP is a nanobody with amino acid sequence such as SEQ ID No.: 1.
  • mScarlet is a bright red fluorescent protein, and the corresponding nucleotide sequence is SEQ ID No.:3.
  • Tamvavidin2 an avidin analog, is a protein with the ability to bind biotin.
  • 2009_FEBS_Yamanomo T_Tamavidins--novel avidin-like biotin-binding proteins from the Tamogitake mushroom translation: a novel biotin-binding avidin analog protein from the Tamogitake mushroom), which has Streptavidin has a strong affinity for biotin similar to streptavidin, and in addition, its thermal stability is better than streptavidin.
  • the amino acid sequence of Tamavidin2 can be retrieved from relevant databases, such as UniProt B9A0T7, which contains a total of 141 amino acid residues. After codon conversion and optimization program optimization, the DNA sequence is obtained.
  • the optimized nucleotide sequence is such as SEQ ID NO.:2.
  • antiEGFP-mScarlet-Tamvavidin2 fusion protein also referred to as antiEGFP fusion protein for short, molecular weight 59kDa
  • DNA templates of antiEGFP-mScarlet-Tamvavidin2 fusion protein were constructed by recombinant PCR method. In vitro amplification was performed by RCA method. Then, the fusion protein antiEGFP-mScarlet-Tamvavidin2 was synthesized by the proteinfactory system based on the in vitro cell-free protein synthesis method (D2P technology).
  • the in vitro protein synthesis system (IVTT system) used in the in vitro cell-free protein synthesis method of this example includes the following components (final concentrations): 9.78 mM Tris-HCl, pH 8.0, 80 mM Potassium acetate, 5 mM magnesium acetate, 1.8 mM mixture of nucleoside triphosphates (adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, and uridine triphosphate, each nucleoside triphosphate 1.8mM), 0.7mM mixture of amino acids (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine , cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, each at a concentration of 0.1 mM ), 15
  • the Kluyveromyces lactis extract includes endogenously expressed T7 RNA polymerase.
  • the Kluyveromyces lactis extract is modified in the following manner: using the modified strain based on Kluyveromyces lactis strain ATCC8585; using the method described in CN109423496A, the encoding gene of T7 RNA polymerase is integrated into the genome of Kluyveromyces lactis In the process, a modified strain is obtained so that it can endogenously express T7 RNA polymerase; the cell raw material is cultured with the modified strain, and then the cell extract is prepared.
  • the preparation process of the Kluyveromyces lactis cell extract adopts conventional technical means, and is prepared with reference to the method described in CN109593656A.
  • the preparation steps include: providing an appropriate amount of raw materials for the fermented Kluyveromyces lactis cells, quick-freezing the cells with liquid nitrogen, breaking the cells, and collecting the supernatant by centrifugation to obtain the cell extract.
  • the protein concentration in the obtained Kluyveromyces lactis cell extract was 20-40 mg/mL.
  • IVTT reaction Add 15ng/ ⁇ L DNA template (the encoded protein contains fluorescent label) to the above-mentioned in vitro protein synthesis system, carry out in vitro protein synthesis reaction, mix well and place it in the environment of 25-30 °C for reaction, the reaction time is 6 ⁇ 18h. Synthesize the protein encoded by the DNA template to obtain an IVTT reaction solution containing the protein.
  • the RFU value is measured by the UV absorption method, and the content of the protein can be calculated by combining the standard curve between its concentration and the RFU value.
  • the IVTT reaction solution of the antiEGFP-mScarlet-Tamvavidin2 fusion protein was obtained.
  • the reaction solution of the IVTT was centrifuged at 4000 rpm and 4° C. for 10 min, and the supernatant was retained. Denoted as IVTT supernatant.
  • the incubated magnetic beads are bound with the nanobody anti-eGFP through the connection of the affinity complex (biotin-Tamvavidin2). , denoted as biomagnetic microsphere H, and also denoted as antiEGFP magnetic bead (a nanobody magnetic bead).
  • the nucleotide sequence of the enhanced fluorescent protein eGFP as a purification substrate is shown in SEQ ID NO.: 4, which is the A206K mutant of eGFP, also denoted as mEGFP.
  • the corresponding amount of eGFP protein was obtained by the calculation formula of eGFP, and the load of antiEGFP magnetic beads was calculated to be 17.7 mg/mL (the mass of eGFP protein bound to each milliliter of antiEGFP magnetic beads).
  • the calculated RFU value of eGFP is converted into the calculation formula of protein concentration:
  • X is the protein concentration ( ⁇ g/mL)
  • Y is the RFU fluorescence reading
  • M is the molecular weight of eGFP (26.7 kDa)
  • N is the molecular weight of the fusion protein (59 kDa).
  • Glycine-eluted eGFP was eluted with 100 ⁇ L of 0.1 M glycine pH 2.8, and a tenth volume (10 ⁇ L) of 1 M Tris-HCl pH 8.0 was added to the eluate immediately. The fluorescence value of the eluate was measured, recorded as Elution, and the purity was detected by SDS-PAGE. The results were shown in Figure 4, and the purity was about 95%.

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Abstract

Provided are an antibody type biological magnetic microsphere, a preparation method therefor and the use thereof. In the first aspect, provided is a biological magnetic microsphere. At least one polymer with a linear main chain and branched chain is fixed to the outer surface of the magnetic microsphere body, and a tail end of the branched chain of the polymer fixed on the biological magnetic microsphere is connected to an antibody type tag. Further provided are a method for preparing the biological magnetic microsphere provided on the basis of the first aspect and the use thereof. The biological magnetic microsphere is convenient in terms of operation and use and can be rapidly dispersed and rapidly settled in a solution without the need to use large-scale experimental equipment such as a high-speed centrifuge. The biological magnetic microsphere is an antibody magnetic bead, and the antibody type tag can also be connected to the tail ends of the branched chains of the polymer by means of the interaction of an affinity compound. The biological magnetic microsphere can be extensively used, the antibody type label, as a purification medium, has flexible selectivity, and the biological magnetic microsphere is universally used for the separation and purification of a target object on a large scale.

Description

一种抗体型生物磁性微球及其制备方法和应用Antibody-type biomagnetic microsphere and its preparation method and application 技术领域technical field
本发明属于生物化学技术领域，具体涉及一种抗体型生物磁性微球及其制备方法和应用。The invention belongs to the technical field of biochemistry, and in particular relates to an antibody-type biomagnetic microsphere and a preparation method and application thereof.
背景技术Background technique
蛋白类物质的分离纯化是生物药生产工艺流程中的重要下游环节，分离纯化的效果和效率直接影响蛋白药的质量和生产成本。对于蛋白纯化，现阶段常用琼脂糖凝胶等材料作为纯化柱或纯化微球载体。凝胶类材料的三维多孔结构有利于提高材料的比表面积，从而增加可结合纯化介质的位点，提高对目标蛋白的特异性结合量。载体材料的三维多孔结构虽然可以大幅度地提高蛋白结合位点的数量，但是载体内部的多孔结构亦会在蛋白洗脱时增加蛋白的滞留时间，载体内部的一些不连续空间或死角还会阻碍蛋白从材料内部洗脱出来，导致增加滞留比例。如果将与蛋白结合的位点仅固定在载体的外表面，虽然可以避免蛋白产物进入材料的内部，大幅度减少洗脱时蛋白的滞留时间和滞留比例；但是如果仅仅利用载体的外表面，则会大幅度降低载体的比表面积，从而大幅度降低蛋白的结合位点数量，降低纯化效率。The separation and purification of protein substances is an important downstream link in the biological drug production process. The effect and efficiency of separation and purification directly affect the quality and production cost of protein drugs. For protein purification, materials such as agarose gel are commonly used as purification columns or purification microsphere carriers at this stage. The three-dimensional porous structure of the gel-like material is beneficial to increase the specific surface area of the material, thereby increasing the sites that can bind to the purification medium and increasing the specific binding amount to the target protein. Although the three-dimensional porous structure of the carrier material can greatly increase the number of protein binding sites, the porous structure inside the carrier will also increase the retention time of the protein during protein elution, and some discontinuous spaces or dead corners inside the carrier will hinder the Proteins are eluted from within the material, resulting in increased retention. If the protein-binding site is only fixed on the outer surface of the carrier, although the protein product can be prevented from entering the interior of the material, the retention time and retention ratio of the protein during elution can be greatly reduced; but if only the outer surface of the carrier is used, then It will greatly reduce the specific surface area of the carrier, thereby greatly reducing the number of protein binding sites and reducing the purification efficiency.
聚合物是一种高分子化合物，可以通过单体分子聚合后形成。采用具有活性位点的单体分子进行聚合，聚合产物可以富含大量的活性位点，大幅提高活性位点的数量，可通过这些活性位点，进而形成或者引入相应的结合位点。聚合物的种类和结构多种多样，有分子链相互交联形成网状结构，有单一线性分子链的线型结构，也有拥有众多分支链的支化结构(例如分枝型、树状、梳状、超支化结构等结构)，不同结构类型的聚合物在不同领域各自拥有广泛的应用。A polymer is a high molecular compound that can be formed by the polymerization of monomer molecules. Using monomer molecules with active sites for polymerization, the polymer product can be rich in a large number of active sites, greatly increasing the number of active sites, and through these active sites, corresponding binding sites can be formed or introduced. There are various types and structures of polymers. There are molecular chains cross-linked to form a network structure, a linear structure with a single linear molecular chain, and a branched structure with many branched chains (such as branched, dendritic, comb, etc.). polymers with different structural types have wide applications in different fields.
在现有技术中，用于蛋白分离纯化的纯化柱中，主要采用共价偶联方式固定纯化介质。在纯化柱多次使用后，会导致纯化介质结合性能降低，纯化效果降低。因此，为了保证较高的纯化效率和质量，操作人员需要及时将亲和层析柱中填料全部更换，该过程不仅耗材用量大，而且消耗大量的人工和时间，导致纯化成本高。In the prior art, in the purification column used for protein separation and purification, the purification medium is mainly fixed by covalent coupling. After the purification column is used for many times, the binding performance of the purification medium will be reduced, and the purification effect will be reduced. Therefore, in order to ensure high purification efficiency and quality, operators need to replace all the fillers in the affinity chromatography column in time. This process not only consumes a large amount of consumables, but also consumes a lot of labor and time, resulting in high purification costs.
发明内容SUMMARY OF THE INVENTION
本发明提供了一种生物磁性微球，所述生物磁性微球结合有抗目标物的抗体类物质，可用于目标物(包括但不限于蛋白类物质)的分离纯化，既能高通量地结合目标物，又能有效减少洗脱时目标物的滞留比例，还可以便捷地更换纯化介质，具有快速、高通量、可重复利用、可再生使用的特点，还可大幅度降低目标物的纯化成本。The present invention provides a biomagnetic microsphere, wherein the biomagnetic microsphere is combined with an anti-target antibody substance, which can be used for the separation and purification of the target substance (including but not limited to protein substances), and can be used for high-throughput Combined with the target, it can effectively reduce the retention ratio of the target during elution, and can also easily replace the purification medium. Purification cost.
1.本发明第一方面提供一种生物磁性微球，包括磁性微球本体，所述磁性微球本体外表面具有至少一种带有线性主链和支链的聚合物，所述线性主链的一端固定于磁性微球本体外表面，聚合物的其他端游离于磁性微球本体外表面，所述生物磁性微球的聚合物的支链末端连接有抗体型标签。1. A first aspect of the present invention provides a biomagnetic microsphere, comprising a magnetic microsphere body, and the outer surface of the magnetic microsphere body has at least one polymer with a linear main chain and a branched chain, and the linear main chain One end of the polymer is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the branched end of the polymer of the biomagnetic microsphere is connected with an antibody-type label.
所述生物磁性微球称之为抗体磁珠或抗体磁性微球或抗体型磁性微球。The biomagnetic microspheres are called antibody magnetic beads or antibody magnetic microspheres or antibody-type magnetic microspheres.
所述抗体型标签优选地作为纯化介质。The antibody-type tag is preferably used as a purification medium.
优选方式之一，所述抗体型标签为抗体、抗体的片段、抗体的单链、单链的片段、抗体融合蛋白、抗体片段的融合蛋白中任一种，任一种的衍生物或任一种的变体。In one preferred manner, the antibody-type tag is any one of an antibody, an antibody fragment, a single chain of an antibody, a fragment of a single chain, an antibody fusion protein, a fusion protein of an antibody fragment, a derivative of any one, or any one of species variant.
优选方式之一，所述抗体型标签为抗蛋白的抗体。In one preferred embodiment, the antibody-type tag is an anti-protein antibody.
优选方式之一，所述抗体型标签为抗荧光蛋白的抗体。In one preferred manner, the antibody-type tag is an antibody against a fluorescent protein.
优选方式之一，所述抗体型标签为抗绿色荧光蛋白或其突变体的抗体。In one preferred manner, the antibody-type tag is an antibody against green fluorescent protein or a mutant thereof.
优选方式之一，所述抗体型标签为纳米抗体。In one preferred manner, the antibody-type tag is a nanobody.
优选方式之一，所述抗体型标签为抗蛋白的纳米抗体。In one preferred manner, the antibody-type tag is an anti-protein nanobody.
优选方式之一，所述抗体型标签为抗蛋白的单域抗体。In one preferred manner, the antibody-type tag is an anti-protein single-domain antibody.
优选方式之一，所述抗体型标签为抗蛋白的单结构域抗体。In one preferred embodiment, the antibody-type tag is an anti-protein single-domain antibody.
优选方式之一，所述抗体型标签为抗蛋白的VHH抗体。In one preferred manner, the antibody-type tag is an anti-protein VHH antibody.
优选方式之一，所述抗体型标签为抗蛋白的scFV抗体。In one preferred manner, the antibody-type tag is an anti-protein scFV antibody.
优选方式之一，所述抗体型标签为抗荧光蛋白的纳米抗体。In one preferred manner, the antibody-type tag is an anti-fluorescent protein nanobody.
优选方式之一，所述抗体型标签为抗绿色荧光蛋白或其突变体的纳米抗体。In one preferred manner, the antibody-type tag is a nanobody against green fluorescent protein or a mutant thereof.
优选方式之一，所述抗体型标签为Fab片段。In one preferred manner, the antibody-type tag is a Fab fragment.
优选方式之一，所述抗体型标签为F(ab’)2片段。In one preferred embodiment, the antibody-type tag is an F(ab')2 fragment.
优选方式之一，所述抗体型标签为Fc片段。In one preferred embodiment, the antibody-type tag is an Fc fragment.
优选方式之一，所述抗体型标签通过亲和复合物相互作用连接到所述聚合物的支链末端。In one preferred manner, the antibody-type tag is linked to the branched end of the polymer through an affinity complex interaction.
优选方式之一，所述亲和复合物包括但不限于以下情形：生物素与亲和素、生物素类似物与亲和素、生物素与亲和素类似物、生物素类似物与亲和素类似物；In one preferred manner, the affinity complex includes but is not limited to the following situations: biotin and avidin, biotin analogs and avidin, biotin and avidin analogs, biotin analogs and avidin hormone analogs;
优选方式之一，所述亲和素为链霉亲和素、改性链霉亲和素、链霉亲和素类似物或者其组合。In one preferred manner, the avidin is streptavidin, modified streptavidin, streptavidin analog or a combination thereof.
优选方式之一，所述抗体型标签连接于所述聚合物的支链末端的方式为：共价键合、超分子相互作用或者其组合。In one preferred manner, the antibody-type tag is linked to the end of the branched chain of the polymer by covalent bonding, supramolecular interaction or a combination thereof.
优选方式之一，所述共价键合利用动态共价键；更优选之一，所述动态共价键包括亚胺键、酰腙键、二硫键或者其组合。In one preferred manner, the covalent bond utilizes a dynamic covalent bond; more preferably, the dynamic covalent bond includes an imine bond, an acylhydrazone bond, a disulfide bond or a combination thereof.
优选方式之一，所述超分子相互作用选自：配位结合、亲和复合物相互作用、静电吸附、氢键、π-π重叠作用、疏水相互作用及其组合。In one preferred manner, the supramolecular interaction is selected from the group consisting of: coordination binding, affinity complex interaction, electrostatic adsorption, hydrogen bonding, π-π overlapping interaction, hydrophobic interaction and combinations thereof.
更优选方式之一，所述亲和复合物相互作用选自：生物素-亲和素相互作用、生物素类似物-亲和素相互作用、生物素-亲和素类似物相互作用、生物素类似物-亲和素类似物相互作用。In one of the more preferred manners, the interaction of the affinity complex is selected from the group consisting of: biotin-avidin interaction, biotin analog-avidin interaction, biotin-avidin analog interaction, biotin Analog-avidin analog interactions.
优选方式之一，所述生物磁性微球的聚合物的支链末端连接有生物素或生物素类似物，所述生物素或者生物素类似物作为连接元件，通过亲和复合物结合作用进一步连接亲和素或亲和素类似物，所述亲和素或亲和素类似物仍作为连接元件，进一步连接所述抗体型标签。In one of the preferred ways, the branched end of the polymer of the biomagnetic microspheres is connected with biotin or biotin analogs, and the biotin or biotin analogs are used as connecting elements, which are further connected by the binding effect of the affinity complex. Avidin or avidin analogs, which still serve as linking elements, further linking the antibody-type tags.
优选方式之一，所述磁性微球本体的尺寸选自下述任一种粒径尺度或任两种粒径尺度之间的范围：0.1μm、0.15μm、0.2μm、0.25μm、0.3μm、0.35μm、0.4μm、0.45μm、 0.5μm、0.55μm、0.6μm、0.65μm、0.7μm、0.75μm、0.8μm、0.85μm、0.9μm、0.95μm、1μm、1.5μm、2μm、2.5μm、3μm、3.5μm、4μm、4.5μm、5μm、5.5μm、6μm、6.5μm、7μm、7.5μm、8μm、8.5μm、9μm、9.5μm、10μm、15μm、20μm、25μm、30μm、35μm、40μm、45μm、50μm、55μm、60μm、65μm、70μm、75μm、80μm、85μm、90μm、95μm、100μm、150μm、200μm、250μm、300μm、350μm、400μm、450μm、500μm、550μm、600μm、650μm、700μm、750μm、800μm、850μm、900μm、950μm、1000μm；所述直径尺寸为平均值。In one preferred manner, the size of the magnetic microsphere body is selected from any one of the following particle size scales or a range between any two particle size scales: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35μm, 0.4μm, 0.45μm, 0.5μm, 0.55μm, 0.6μm, 0.65μm, 0.7μm, 0.75μm, 0.8μm, 0.85μm, 0.9μm, 0.95μm, 1μm, 1.5μm, 2μm, 2.5μm, 3μm , 3.5μm, 4μm, 4.5μm, 5μm, 5.5μm, 6μm, 6.5μm, 7μm, 7.5μm, 8μm, 8.5μm, 9μm, 9.5μm, 10μm, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm, 80μm, 85μm, 90μm, 95μm, 100μm, 150μm, 200μm, 250μm, 300μm, 350μm, 400μm, 450μm, 500μm, 550μm, 600μm, 750μm, 700μm 850 μm, 900 μm, 950 μm, 1000 μm; the diameter size is an average value.
优选方式之一，所述磁性微球本体的直径选自0.1～10μm。In one preferred manner, the diameter of the magnetic microsphere body is selected from 0.1-10 μm.
优选方式之一，所述磁性微球本体的直径选自0.2～6μm。In one preferred manner, the diameter of the magnetic microsphere body is selected from 0.2 to 6 μm.
优选方式之一，所述磁性微球本体的直径选自0.4～5μm。In one preferred manner, the diameter of the magnetic microsphere body is selected from 0.4 to 5 μm.
优选方式之一，所述磁性微球本体的直径选自0.5～3μm。In one preferred manner, the diameter of the magnetic microsphere body is selected from 0.5-3 μm.
优选方式之一，所述磁性微球本体的直径选自0.2～1μm。In one preferred manner, the diameter of the magnetic microsphere body is selected from 0.2 to 1 μm.
优选方式之一，所述磁性微球本体的直径选自0.5～1μm。In one preferred manner, the diameter of the magnetic microsphere body is selected from 0.5-1 μm.
优选方式之一，所述磁性微球本体的直径选自1μm～1mm。In one preferred manner, the diameter of the magnetic microsphere body is selected from 1 μm˜1 mm.
优选方式之一，所述磁性微球本体的平均直径为200nm、250nm、300nm、350nm、400nm、450nm、500nm、550nm、600nm、650nm、700nm、750nm、800nm、850nm、900nm、950nm、1000nm，偏差为±20％，更优选±10％。In one preferred manner, the average diameter of the magnetic microsphere body is 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, and the deviation It is ±20%, more preferably ±10%.
优选方式之一，所述聚合物的主链为聚烯烃主链，或者为丙烯酸类聚合物主链。所述丙烯酸类聚合物的定义见“名词和术语”部分。优选方式之一，所述聚烯烃主链同时为丙烯酸类聚合物主链(也即，所述聚合物的线性主链为聚烯烃主链，且由丙烯酸类聚合物的主链提供)。In one preferred manner, the main chain of the polymer is a polyolefin main chain, or an acrylic polymer main chain. The acrylic polymers are defined in the "Terms and Terms" section. In one preferred manner, the polyolefin backbone is also an acrylic polymer backbone (ie, the linear backbone of the polymer is a polyolefin backbone and is provided by the acrylic polymer backbone).
优选之一，所述丙烯酸类聚合物的单体单元优选地选自丙烯酸、丙烯酸盐、丙烯酸酯、甲基丙烯酸、甲基丙烯酸盐、甲基丙烯酸酯等丙烯酸类单体分子之一或其组合。所述丙烯酸类聚合物可以通过上述单体之一聚合得到或者通过上述单体的相应组合共聚得到。Preferably, the monomer unit of the acrylic polymer is preferably selected from one or a combination of acrylic monomer molecules such as acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, methacrylate, etc. . The acrylic polymer can be obtained by polymerizing one of the above monomers or by copolymerizing a corresponding combination of the above monomers.
优选方式之一，所述聚合物的支链通过基于功能基团的共价键共价结合生物素或生物素类似物(得到生物素磁珠)，再通过所述生物素或生物素类似物直接或间接地连接所述抗体型标签。结合所述结合生物素或生物素类似物的过程，可通过生物磁性微球外表面的聚合物分子的支链所含有的功能基团与生物素或生物素类似物进行共价反应而实现。其中，所述功能性基团的优选实施方式之一为特异性结合位点(定义详见具体实施方式的“名词和术语”部分)。In one of the preferred ways, the branched chain of the polymer covalently binds biotin or biotin analogs through covalent bonds based on functional groups (to obtain biotin magnetic beads), and then passes through the biotin or biotin analogs. The antibody-type tag is attached directly or indirectly. The process of binding the biotin or biotin analogs can be achieved by covalently reacting functional groups contained in the branched chains of the polymer molecules on the outer surface of the biomagnetic microspheres with biotin or biotin analogs. Wherein, one of the preferred embodiments of the functional group is a specific binding site (for definitions, please refer to the "noun and term" section of the specific embodiment).
所述基于功能基团的共价键，指由功能基团参与共价偶联形成的共价键。优选地，所述功能基团为羧基、羟基、氨基、巯基、羧基的盐形式、氨基的盐形式、甲酸酯基，或者前述功能基团的组合。所述羧基的盐形式的优选方式之一为钠盐形式如COONa；所述氨基的盐形式的优选方式可以为无机盐形式，也可以为有机盐形式，包括但不限于盐酸盐、氢氟酸盐等形式。所述“功能基团的组合”指一个磁性微球的外表面的所有聚合物分子的所有支链，允许基于不同的功能基团参与形成共价键；以生物素为例，也即一个生物素磁性微球的外表面的所有生物素分子可以分别与不同的功能基团共价连接，不过一个生物素分子仅能与一个功能基团相连接。The functional group-based covalent bond refers to a covalent bond formed by a functional group participating in covalent coupling. Preferably, the functional group is a carboxyl group, a hydroxyl group, an amino group, a sulfhydryl group, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of the foregoing functional groups. One of the preferred modes of the salt form of the carboxyl group is a sodium salt form such as COONa; the preferred mode of the salt form of the amino group can be an inorganic salt form, or an organic salt form, including but not limited to hydrochloride, hydrofluoride acid salts, etc. The "combination of functional groups" refers to all branches of all polymer molecules on the outer surface of a magnetic microsphere, allowing different functional groups to participate in the formation of covalent bonds; take biotin as an example, that is, a biological All biotin molecules on the outer surface of the prime magnetic microspheres can be covalently linked with different functional groups, but one biotin molecule can only be linked with one functional group.
优选方式之一，所述聚合物的线性主链直接共价偶联于所述磁性微球本体的外表面，或者通过连接基团间接地共价偶联于所述磁性微球本体的外表面。In one preferred manner, the linear main chain of the polymer is directly covalently coupled to the outer surface of the magnetic microsphere body, or indirectly covalently coupled to the outer surface of the magnetic microsphere body through a linking group .
优选方式之一，所述磁性微球本体为SiO 2包裹的磁性材料。作为替代，SiO 2可以为自带活性位点的硅烷偶联剂。 In one preferred manner, the magnetic microsphere body is a magnetic material wrapped with SiO 2 . Alternatively, SiO2 can be a silane coupling agent with its own active site.
优选方式之一，所述磁性材料选自铁氧化物、铁化合物、铁合金、钴化合物、钴合金、镍化合物、镍合金、锰氧化物、锰合金之一或者组合。In one preferred manner, the magnetic material is selected from one or a combination of iron oxides, iron compounds, iron alloys, cobalt compounds, cobalt alloys, nickel compounds, nickel alloys, manganese oxides, and manganese alloys.
进一步地，优选为Fe 3O 4、γ-Fe 2O 3、氮化铁、Mn 3O 4、FeCrMo、FeAlC、AlNiCo、FeCrCo、ReCo、ReFe、PtCo、MnAlC、CuNiFe、AlMnAg、MnBi、FeNiMo)、FeSi、FeAl、FeSiAl、MO·6Fe 2O 3、GdO之一或其组合；其中，所述Re为稀土元素；所述M为Ba、Sr、Pb，也即，所述MO·6Fe 2O 3为BaO·6Fe 2O 3、SrO·6Fe 2O 3或PbO·6Fe 2O 3。 Further, preferably Fe 3 O 4 , γ-Fe 2 O 3 , iron nitride, Mn 3 O 4 , FeCrMo, FeAlC, AlNiCo, FeCrCo, ReCo, ReFe, PtCo, MnAlC, CuNiFe, AlMnAg, MnBi, FeNiMo) , FeSi, FeAl, FeSiAl, MO·6Fe 2 O 3 , GdO or a combination thereof; wherein, the Re is a rare earth element; the M is Ba, Sr, Pb, that is, the MO·6Fe 2 O 3 is BaO·6Fe 2 O 3 , SrO·6Fe 2 O 3 or PbO·6Fe 2 O 3 .
所述“固定于”指所述线性主链以共价连接的方式“固定于”磁性微球本体外表面。The "fixed to" means that the linear backbone is "fixed to" the outer surface of the magnetic microsphere body in a covalently linked manner.
优选方式之一，所述线性主链以直接方式共价地固定于或者通过连接基(连接元件)以间接方式共价地固定于所述磁性微球本体外表面。In one preferred manner, the linear main chain is covalently fixed to the outer surface of the magnetic microsphere body in a direct manner or indirectly via a linker (linking element).
优选方式之一，所述聚合物支链的数量为多个；优选地，至少为3个。In one preferred manner, the number of the polymer branches is multiple; preferably, at least three.
2.本发明还公开一种第一方面所述生物磁性微球的制备方法，包括以下步骤：2. The present invention also discloses a method for preparing the biomagnetic microspheres described in the first aspect, comprising the following steps:
(1)对磁性微球本体进行化学修饰，将氨基引入到磁性微球本体的外表面，形成氨基修饰磁性微球A；当所述磁性微球本体为SiO 2包裹的磁性材料时，所述偶联剂优选为氨基化硅烷偶联剂。 (1) chemically modifying the magnetic microsphere body, and introducing amino groups to the outer surface of the magnetic microsphere body to form amino-modified magnetic microspheres A; when the magnetic microsphere body is a magnetic material wrapped by SiO 2 , the The coupling agent is preferably an aminated silane coupling agent.
优选方式之一，利用偶联剂对磁性微球本体进行化学修饰。One of the preferred ways is to chemically modify the magnetic microsphere body with a coupling agent.
当所述磁性微球本体为SiO 2包裹的磁性材料时，可利用硅烷偶联剂对磁性微球本体进行化学修饰。所述硅烷偶联剂优选为氨基化硅烷偶联剂。 When the magnetic microsphere body is a magnetic material wrapped with SiO 2 , a silane coupling agent can be used to chemically modify the magnetic microsphere body. The silane coupling agent is preferably an aminated silane coupling agent.
(2)利用羧基与氨基之间的共价反应将丙烯酸分子共价偶联到所述磁性微球A的外表面，引入碳碳双键，形成含碳碳双键磁性微球B。(2) Covalently coupling acrylic acid molecules to the outer surface of the magnetic microspheres A by the covalent reaction between carboxyl groups and amino groups, and introducing carbon-carbon double bonds to form magnetic microspheres B containing carbon-carbon double bonds.
(3)在不加交联剂的条件下，利用碳碳双键的聚合反应，将丙烯酸类单体分子(如丙烯酸钠)进行聚合，获得的丙烯酸类聚合物具有线性主链和含有功能基团的支链，聚合物通过线性主链的一端共价偶联于磁性微球B的外表面，形成丙烯酸类聚合物修饰磁性微球C。(3) Under the condition of not adding a crosslinking agent, the polymerization of carbon-carbon double bonds is used to polymerize acrylic monomer molecules (such as sodium acrylate), and the obtained acrylic polymer has a linear main chain and contains functional groups. The polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain to form an acrylic polymer modified magnetic microsphere C.
所述丙烯酸类单体分子、聚合物支链的功能基团的定义见“名词和术语”部分。The definitions of the acrylic monomer molecules and the functional groups of the polymer branches are shown in the section "Terms and Terms".
优选地，所述功能基团为羧基、羟基、氨基、巯基、甲酸盐、铵盐、羧基的盐形式、氨基的盐形式、甲酸酯基，或前述功能基团的组合；所述“功能基团的组合”指一个磁性微球的外表面的所有聚合物的所有支链所含有的功能基团，其种类可以为一种或一种以上。与第一方面定义的“功能基团的组合”的含义是一致的。Preferably, the functional group is a carboxyl group, a hydroxyl group, an amino group, a sulfhydryl group, a formate, an ammonium salt, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of the foregoing functional groups; the " "Combination of functional groups" refers to the functional groups contained in all branches of all polymers on the outer surface of a magnetic microsphere, and the types may be one or more. The meaning of "combination of functional groups" defined in the first aspect is consistent.
另优选地，所述功能基团为特异性结合位点。Also preferably, the functional group is a specific binding site.
(4)通过所述聚合物的支链含有的功能基团，将生物素或生物素类似物共价偶联到聚合物支链末端，得到结合有生物素或生物素类似物的生物磁性微球D。(4) Covalently couple biotin or biotin analog to the end of the polymer branch through the functional group contained in the branched chain of the polymer, to obtain a biomagnetic microparticle combined with biotin or biotin analog ball D.
(5)将提供抗体型标签的原料与所述生物磁性微球D中所述聚合物支链末端的生物素或生物素类似物相连接，得到所述生物磁性微球(一种抗体磁性微球)。(5) linking the raw material that provides the antibody-type label with the biotin or biotin analog at the end of the polymer branch in the biomagnetic microsphere D to obtain the biomagnetic microsphere (an antibody magnetic microsphere). ball).
独立地可选地，包括(6)磁铁沉降生物磁性微球，去除液相，清洗。Independently and optionally, including (6) magnet sedimentation of biomagnetic microspheres, removal of liquid phase, and cleaning.
优选方式之一，所述生物磁性微球D为生物素修饰类型。In one preferred manner, the biomagnetic microspheres D are biotin-modified.
优选方式之一，所述提供抗体型标签的原料为亲和素或亲和素类似物与所述抗体型标签的共价连接复合物。In one preferred manner, the raw material for providing the antibody-type tag is a covalently linked complex of avidin or an avidin analog and the antibody-type tag.
更优选地，所述提供抗体型标签的原料为亲和素-抗体型标签共价连接复合物。此时，独立地可选地，包括所述亲和素-抗体型标签共价连接复合物的更换。More preferably, the raw material for providing the antibody-type tag is an avidin-antibody-type tag covalently linked complex. At this point, independently and optionally, replacement of the covalently linked complex of the avidin-antibody-type tag is included.
3.本发明还公开第一方面所述生物磁性微球(抗体磁性微球)在分离纯化中的应用，优选在蛋白类物质的分离纯化中的应用。3. The present invention also discloses the application of the biomagnetic microspheres (antibody magnetic microspheres) described in the first aspect in separation and purification, preferably in the separation and purification of protein substances.
本发明的主要优点和积极效果包括：The main advantages and positive effects of the present invention include:
本发明的核心之一在于生物磁性微球结构，在磁性微球本体的外表面，共价地固定具有线性主链的聚合物分子，这些聚合物分子还带有大量功能化的支链，所述功能化的支链连接有纯化介质(抗体型标签)。通过上述结构，在生物磁性微球的外表面提供大量的悬挂于聚合物线性主链侧端的纯化介质，既避免因传统网状结构导致的高滞留比例，还克服比表面积的局限能够提供大量的目标物结合位点。其中，纯化介质的种类可根据待纯化物质(目标物)的种类进行选择。当纯化介质以亲和复合物的形式，以一种非共价的强相互作用连接于聚合物的支链时；进一步地，纯化介质与聚合物线性主链之间的支链骨架还可以存在亲和复合物的结合作用，使得纯化介质可以被便捷地更换。结合磁性微球的制备及原理部分的阐述进行理解。One of the cores of the present invention lies in the biomagnetic microsphere structure. On the outer surface of the magnetic microsphere body, polymer molecules with linear main chains are covalently fixed, and these polymer molecules also have a large number of functional branches, so The functionalized branches are linked with a purification medium (antibody-type tag). Through the above structure, a large amount of purification medium suspended on the side end of the linear main chain of the polymer is provided on the outer surface of the biomagnetic microsphere, which not only avoids the high retention ratio caused by the traditional network structure, but also overcomes the limitation of the specific surface area. target binding site. Among them, the type of the purification medium can be selected according to the type of the substance to be purified (target substance). When the purification medium is attached to the branched chain of the polymer in the form of an affinity complex with a strong non-covalent interaction; further, the branched backbone between the purification medium and the linear main chain of the polymer may also exist The binding of the affinity complex allows the purification medium to be easily exchanged. Combined with the preparation of magnetic microspheres and the explanation of the principle part to understand.
本发明的核心还在于上述生物磁性微球结构的构建过程(制备方法)：通过外表面化学修饰，在磁珠外表面(生物磁性微球本体外表面)提供若干的结合位点，然后在磁珠外表面的单个结合位点共价连接聚合物分子，聚合物分子通过线性主链的一端与磁珠外表面的单个结合位点共价连接，沿着线性主链分布有大量的侧支链，侧支链中携带有新生的结合位点，从而实现结合位点的多倍、数十倍、上百倍、数百倍、甚至上千倍地放大，再根据具体纯化需求在聚合物支链的新生结合位点处连接特定的纯化介质，以实现对应特定目标分子(特别是生物化学分子)的捕获。此外，生物磁性微球的单个结合位点，既可以仅共价连接一条线性聚合物主链，也可以共价连接两条或更多条线性主链，以不会导致链堆积以致于滞留比例增大为宜。The core of the present invention also lies in the construction process (preparation method) of the above-mentioned biomagnetic microsphere structure: by chemical modification of the outer surface, several binding sites are provided on the outer surface of the magnetic bead (the outer surface of the biomagnetic microsphere body), and then the magnetic A single binding site on the outer surface of the bead is covalently linked to a polymer molecule, and the polymer molecule is covalently linked to a single binding site on the outer surface of the magnetic bead through one end of a linear backbone with a large number of side branches distributed along the linear backbone. , the side branches carry new binding sites, so as to achieve multiple, dozens, hundreds, hundreds of times, or even thousands of times the amplification of the binding sites, and then according to the specific purification requirements in the polymer branch chain A specific purification medium is attached to the nascent binding site of , in order to achieve the capture of corresponding specific target molecules (especially biochemical molecules). In addition, the single binding site of the biomagnetic microspheres can be covalently linked to only one linear polymer backbone or two or more linear backbones, so as not to cause chain stacking and thus the retention ratio Increase is appropriate.
优选地，一个结合位点仅引出一条线性主链，此时能够为线性主链提供较大的活动空间。Preferably, only one linear main chain is drawn from one binding site, and at this time, a larger space for the linear main chain can be provided.
另优选地，一个结合位点仅引出两条线性主链，尽可能地为线性主链提供较大的活动空间。Also preferably, only two linear backbones are drawn out from one binding site, so as to provide as much space for the linear backbones as possible.
本发明的主要优点和积极效果还包括：The main advantages and positive effects of the present invention also include:
(1)本发明的结构设计，通过携带大量支链特殊结构的聚合物包裹磁性微球表面，克服比表面积的局限提供大量的纯化介质(抗体型标签)结合位点，将磁性微球表面可结合的纯化介质的数量多倍、十多倍、数十倍、上百倍、数百倍、甚至上千倍地放大，进而实现高通量的结合目标物(所述目标物的优选方式之一为目标蛋白)；使得生物磁性微球能高效地将目标物从混合体系中捕获到磁性微球上，实现高通量地结合，也即实现高通量 的分离。(1) In the structural design of the present invention, by wrapping the surface of magnetic microspheres with polymers with a large number of branched special structures, overcoming the limitation of specific surface area and providing a large number of binding sites for purification media (antibody tags), the surface of magnetic microspheres can be The number of combined purification media is multiplied, ten-fold, dozens of times, hundreds of times, hundreds of times, and even thousands of times, so as to achieve high-throughput binding of the target (one of the preferred ways of the target). As the target protein); the biomagnetic microspheres can efficiently capture the target from the mixed system onto the magnetic microspheres to achieve high-throughput binding, that is, to achieve high-throughput separation.
(2)可以利用聚合物链本身的柔顺性，聚合物链能够在反应纯化混合体系中柔性摆动，扩大纯化介质的活动空间，增大对蛋白的捕获速率和结合量，促进目标物的快速、充分结合，实现高效率、高通量。(2) The flexibility of the polymer chain itself can be used, and the polymer chain can flexibly swing in the reaction and purification mixed system, expand the active space of the purification medium, increase the capture rate and binding amount of the protein, and promote the rapid and Fully combined to achieve high efficiency and high throughput.
(3)本发明的结构设计，使得生物磁性微球能在洗脱时实现被纯化的目标物的高效洗脱，有效减少目标物的滞留时间和滞留比例，实现高效率和高产率。纯化介质(抗体型标签)可以连接在聚合物的支链末端，一方面，聚合物的结构可以不形成网状结构，不导致支链堆积，可避免出现不连续空间和死角，避免传统网状结构导致的高滞留时间和高滞留比例；另一方面，聚合物的支链进一步起到空间间隔作用，使纯化介质能够充分分布在混合体系中，远离磁性微球表面以及聚合物的内部骨架，既增大捕获目标物的效率，还可以在后续洗脱步骤时，有效减少目标物的滞留时间和滞留比例，实现高通量、高效率、高比例的分离。本发明的结构设计既能利用线性主链的高柔顺性，又兼具支链数量的高倍数放大优势，更好地实现高速率、高通量的结合，高效率、高比例(高产率)的分离。(3) The structural design of the present invention enables the biomagnetic microspheres to achieve efficient elution of the purified target substance during elution, effectively reducing the retention time and retention ratio of the target substance, and achieving high efficiency and high yield. The purification medium (antibody-type tag) can be attached to the end of the branched chain of the polymer. On the one hand, the structure of the polymer can not form a network structure, which does not lead to the accumulation of branched chains, which can avoid discontinuous spaces and dead ends, and avoid the traditional network. High retention time and high retention ratio caused by the structure; on the other hand, the branched chain of the polymer further acts as a spacer, so that the purification medium can be fully distributed in the mixed system, away from the surface of the magnetic microspheres and the internal skeleton of the polymer, It not only increases the efficiency of capturing the target, but also can effectively reduce the retention time and retention ratio of the target in the subsequent elution step, and achieve high-throughput, high-efficiency, and high-proportion separation. The structural design of the present invention can not only utilize the high flexibility of the linear main chain, but also has the advantage of high multiple amplification of the number of branches, so as to better realize the combination of high speed and high flux, high efficiency, high ratio (high yield) separation.
(4)本发明生物磁性微球的纯化介质(抗体型标签)可以通过亲和复合物的方式，以一种非共价的强结合力连接于磁珠外表面的聚合物支链末端；在需要更新、更换纯化介质时，能够方便、快捷地将纯化介质从微球上洗脱并重新结合新的纯化介质，快速恢复磁性微球纯化性能，使得生物磁性微球可以多次再生使用，从而降低分离纯化成本。(4) The purification medium (antibody-type tag) of the biomagnetic microspheres of the present invention can be connected to the end of the polymer branch on the outer surface of the magnetic bead with a strong non-covalent binding force by means of an affinity complex; When the purification medium needs to be renewed or replaced, the purification medium can be easily and quickly eluted from the microspheres and recombined with the new purification medium, and the purification performance of the magnetic microspheres can be quickly restored, so that the biomagnetic microspheres can be regenerated and used for many times. Reduce separation and purification costs.
(5)本发明生物磁性微球操作使用便捷。将结合了目标物的磁性微球从体系中分离时，操作便捷，仅需利用一小块磁铁便能高效地操纵磁性微球的聚集状态和位置，实现磁性微球在溶液中的快速分散或快速沉聚，使目标物的分离纯化变得简单、快捷，无需使用高速离心机等大型实验设备，大大地降低分离纯化成本。(5) The biomagnetic microspheres of the present invention are convenient to operate and use. When the magnetic microspheres combined with the target are separated from the system, the operation is convenient, and only a small magnet can be used to efficiently manipulate the aggregation state and position of the magnetic microspheres, so as to realize the rapid dispersion of the magnetic microspheres in the solution or Rapid precipitation makes the separation and purification of target substances simple and fast, without the need to use large-scale experimental equipment such as high-speed centrifuges, which greatly reduces the cost of separation and purification.
(6)本发明提供的生物磁性微球用途广泛，纯化介质具有可选择性。可根据具体的纯化底物的种类，在磁性微球系统中灵活地搭载相应的纯化介质，实现对特定目标分子的捕获。还可选择分子尺寸较小的抗体片段、抗体单链、纳米抗体等非全蛋白的形式作为纯化介质，特别是纳米抗体作为纯化介质时，相对于全蛋白结构的完整抗体，制备磁性微球时更容易获得对纯化介质的高载量；且所述纯化介质更容易在混合体系中随聚合物链的摆动而充分接触目标物，从而获得更高的结合效率。(6) The biomagnetic microspheres provided by the present invention are widely used, and the purification medium has selectivity. According to the type of specific purification substrate, the corresponding purification medium can be flexibly carried in the magnetic microsphere system to realize the capture of specific target molecules. Antibody fragments, antibody single chains, nanobodies and other forms of non-whole proteins with smaller molecular size can also be selected as purification media, especially when nanobodies are used as purification media, compared with complete antibodies with whole protein structures, when preparing magnetic microspheres It is easier to obtain a high load on the purification medium; and the purification medium is more likely to fully contact the target with the oscillation of the polymer chain in the mixed system, thereby obtaining a higher binding efficiency.
附图说明Description of drawings
图1.一种生物磁性微球的结构示意图。以antiEGFP纳米抗体作为纯化介质示意，antiEGFP纳米抗体通过“生物素-亲和素-antiEGFP纳米抗体的方式”结合在刷状结构的支链末端。其中，生物磁性微球本体以SiO 2包裹的Fe 3O 4为例。图中，聚合物分子数(4个)仅为简明示意起见，并非意味着磁性微球外表面的聚合物分子数量局限于4个，而是可以根据制备过程中各原料的含量进行控制和调节。同理，悬挂于线性主链侧端的支链的数量也仅起示意作用，并非对本发明聚合物分子侧支链数量的限定。 Figure 1. A schematic diagram of the structure of a biomagnetic microsphere. The antiEGFP nanobody is used as a purification medium, and the antiEGFP nanobody is bound to the end of the branched chain of the brush-like structure by "biotin-avidin-antiEGFP nanobody". Among them, the biomagnetic microsphere body is taken as an example of Fe 3 O 4 wrapped by SiO 2 . In the figure, the number of polymer molecules (4) is only for the sake of simplicity. It does not mean that the number of polymer molecules on the outer surface of the magnetic microsphere is limited to 4, but can be controlled and adjusted according to the content of each raw material in the preparation process. . Similarly, the number of side chains hanging from the side end of the linear main chain is only for illustrative purposes, and is not intended to limit the number of side chains in the polymer molecule of the present invention.
图2.一种生物磁性微球的制备方法流程图，以纳米抗体作为纯化介质为例。其中，从氨基修饰磁性微球A至生物磁性微球D的制备过程对应生物素磁性微球的制备。Figure 2. A flow chart of the preparation method of biomagnetic microspheres, taking nanobody as the purification medium as an example. Among them, the preparation process from amino-modified magnetic microspheres A to biomagnetic microspheres D corresponds to the preparation of biotin magnetic microspheres.
图3.生物磁性微球H(一种带有antiEGFP纳米抗体的磁珠)结合eGFP蛋白的RFU 值测试结果。将生物磁性微球H与eGFP蛋白的IVTT上清液孵育，结合eGFP蛋白。其中，Total对应未经生物磁性微球处理的IVTT上清液；Flow-through对应孵育一次的流穿液。Figure 3. The RFU value test results of biomagnetic microsphere H (a magnetic bead with antiEGFP nanobody) binding to eGFP protein. Biomagnetic microspheres H were incubated with the IVTT supernatant of eGFP protein to bind eGFP protein. Among them, Total corresponds to the IVTT supernatant that has not been treated with biomagnetic microspheres; Flow-through corresponds to the flow-through solution incubated once.
图4.生物磁性微球H(antiEGFP磁珠)分离纯化eGFP蛋白的实验结果：生物磁性微球H与eGFP蛋白溶液孵育后进行洗脱，将eGFP蛋白从原液中捕获分离、洗脱释放到洗脱液中，相应的含纯化eGFP蛋白的洗脱液的SDS-PAGE测试结果。其中，M对应Marker分子量标记。Figure 4. Experimental results of separation and purification of eGFP protein by biomagnetic microspheres H (antiEGFP magnetic beads): biomagnetic microspheres H were incubated with eGFP protein solution and then eluted, eGFP protein was captured and separated from the stock solution, eluted and released to wash In the dehydration, the corresponding SDS-PAGE test results of the eluate containing purified eGFP protein. Among them, M corresponds to the Marker molecular weight marker.
核苷酸和/或氨基酸序列表Nucleotide and/or Amino Acid Sequence Listing
SEQ ID No.:1，纳米抗体anti-eGFP的氨基酸序列，长度为117个氨基酸。SEQ ID No.: 1, the amino acid sequence of Nanobody anti-eGFP, with a length of 117 amino acids.
SEQ ID No.:2，tamavidin2的核苷酸序列，长度423个碱基。SEQ ID No.: 2, the nucleotide sequence of tamavidin2, 423 bases in length.
SEQ ID No.:3，mScarlet的核苷酸序列，长度为693个碱基。SEQ ID No.: 3, nucleotide sequence of mScarlet, 693 bases in length.
SEQ ID No.:4，mEGFP的核苷酸序列，长度为714个碱基。SEQ ID No.:4, the nucleotide sequence of mEGFP, is 714 bases in length.
具体实施方式Detailed ways
下面结合具体实施方式和实施例，进一步阐述本发明，请一并参阅附图。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，优先按照、参考上文所述的具体实施方式指引的条件，然后可按照常规条件，例如“Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)”、《无细胞蛋白合成实验手册》“Edited by Alexander S.Spirin and James R.Swartz.Cell-free protein synthesis:methods and protocols[M].2008”等文献中所述的实验条件，或按照制造厂商所建议的条件。The present invention will be further described below with reference to the specific embodiments and examples, and please refer to the accompanying drawings. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, preferentially follow, refer to the conditions of the specific embodiment guidelines described above, and then can follow normal conditions, such as "Sambrook et al., Molecular Cloning: Laboratory Manual (New York). : Cold Spring Harbor Laboratory Press, 1989)", "Cell-free Protein Synthesis Laboratory Manual" "Edited by Alexander S.Spirin and James R.Swartz.Cell-free protein synthesis:methods and protocols[M].2008" and other documents The experimental conditions described, or as suggested by the manufacturer.
除非另外说明，否则本发明中提及的百分比和份数是重量百分比和重量份数。Unless otherwise stated, the percentages and parts mentioned in the present invention are weight percentages and parts by weight.
如无特别说明，则本发明实施例中所用的材料和试剂均为市售产品。Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products.
本申请中的温度单位如无特殊说明，均为摄氏度(℃)。The temperature units in this application are all degrees Celsius (°C) unless otherwise specified.
名词和术语nouns and terms
以下是针对本发明所采用的部分相关“名词”、“术语”的含义进行的解释或说明，以便于更好地理解本发明。相应的解释或说明适用于本发明的全文，既适用于下文，也适用于上文。本发明中涉及引用文献时，相关术语、名词、短语在引用文献中的定义也一并被引用，但是，与本发明中的定义相冲突时，以本发明中的定义为准。在引用文献中的定义与本发明中的定义发生冲突时，并不影响所引用的成分、物质、组合物、材料、体系、配方、种类、方法、设备等以引用文献中确定的内容为准。The following is an explanation or description of the meanings of some related "nouns" and "terms" used in the present invention, so as to facilitate a better understanding of the present invention. Corresponding explanations or illustrations apply to the entirety of the invention, both below and above. When references are involved in the present invention, the definitions of related terms, nouns, and phrases in the cited documents are also cited together, but in the event of a conflict with the definitions in the present invention, the definitions in the present invention shall prevail. In the event of a conflict between the definitions in the cited documents and the definitions in the present invention, it does not affect the cited ingredients, substances, compositions, materials, systems, formulations, types, methods, equipment, etc., and the content determined in the cited documents shall prevail .
磁珠：具有细小粒径的强磁性或可被强磁性化的微球，也可描述为磁珠，直径尺寸优选0.1μm～1000μm。本发明的磁珠实例包括但不限于：磁性微球A、磁性微球B、磁性微球C、生物磁性微球D(一种生物素磁珠)、生物磁性微球H(一种带有antiEGFP纳米抗体的磁珠)、生物磁性微球K(抗体磁珠)。Magnetic beads: ferromagnetic or ferromagnetic microspheres with fine particle size, which can also be described as magnetic beads, preferably 0.1 μm to 1000 μm in diameter. Examples of magnetic beads of the present invention include, but are not limited to: magnetic microspheres A, magnetic microspheres B, magnetic microspheres C, biomagnetic microspheres D (a biotin magnetic bead), biomagnetic microspheres H (a antiEGFP nanobody magnetic beads), biomagnetic microspheres K (antibody magnetic beads).
磁性微球本体：具有被修饰位点的磁珠(具有可结合位点的磁性微球)。例如二氧化 硅包裹的磁性材料颗粒，更具体地如氨基化的二氧化硅包裹的磁性材料颗粒。Magnetic microsphere body: magnetic beads with modified sites (magnetic microspheres with binding sites). Examples are silica-coated magnetic material particles, more particularly aminated silica-coated magnetic material particles.
磁性微球A：氨基修饰磁性微球。Magnetic microspheres A: Amino-modified magnetic microspheres.
磁性微球B：含碳碳双键的磁性微球。Magnetic microspheres B: magnetic microspheres containing carbon-carbon double bonds.
磁性微球C：丙烯酸类聚合物修饰磁性微球。Magnetic microspheres C: acrylic polymer modified magnetic microspheres.
生物素磁珠：结合有生物素或生物素类似物的磁珠，能够特异性结合带有亲和素型标签的物质。其优点包括，目标蛋白用亲和素或亲和素的蛋白突变体进行标记后，能够以融合蛋白的方式一体化表达，应用方式简便。也称为生物素磁性微球。这里的生物素或生物素类似物可以作为一种纯化介质，也可以作为连接元件。Biotin Magnetic Beads: Magnetic beads conjugated with biotin or biotin analogs that can specifically bind to substances with avidin-type tags. The advantages include that after the target protein is labeled with avidin or a protein mutant of avidin, it can be expressed in an integrated manner in the form of a fusion protein, and the application method is simple. Also known as biotin magnetic microspheres. Biotin or biotin analogs here can serve as a purification medium and also as a linking element.
生物磁性微球D：一种结合有生物素或生物素类似物的磁性微球，一种生物素磁珠。这里的生物素或生物素类似物可以作为一种纯化介质，也可以作为连接元件。Biomagnetic Microsphere D: A magnetic microsphere bound with biotin or a biotin analog, a biotin magnetic bead. Biotin or biotin analogs here can serve as a purification medium and also as a linking element.
亲和素磁珠：结合有亲和素或亲和素类似物的磁珠，能够特异性结合带有生物素型标签的物质。也称为亲和素磁性微球。Avidin magnetic beads: Magnetic beads bound with avidin or avidin analogs, which can specifically bind to substances with biotin-type tags. Also known as avidin magnetic microspheres.
生物磁性微球K：结合抗体型标签的磁珠，可以用于分离纯化能够与之特异性结合的目标物。也称为抗体磁性微球或抗体磁珠或抗体型磁性微球。Biomagnetic Microspheres K: Magnetic beads with antibody-type tags, which can be used to separate and purify targets that can specifically bind to them. Also known as antibody magnetic microspheres or antibody magnetic beads or antibody-type magnetic microspheres.
纳米抗体磁珠：结合由纳米抗体的磁珠，可以用于分离纯化能够与之特异性结合的目标物。也称为纳米抗体磁性微球。Nanobody magnetic beads: Magnetic beads combined with nanobodies can be used to separate and purify targets that can specifically bind to them. Also known as Nanobody Magnetic Microspheres.
生物磁性微球H：一种纳米抗体磁珠，一种结合有纳米抗体antiEGFP的磁性微球(antiEGFP磁珠)。可以由亲和素-antiEGFP共价连接复合物结合而成。Biomagnetic Microsphere H: a nanobody magnetic bead, a magnetic microsphere (antiEGFP magnetic bead) bound with the nanobody antiEGFP. It can be combined by avidin-antiEGFP covalently linked complex.
聚合物，本发明中广义地包括寡聚物和高聚物，至少具有三个结构单元或者分子量至少为500Da(所述分子量可以采用适合的表征方式，比如数均分子量、重均分子量、粘均分子量等)。Polymers, including oligomers and polymers in a broad sense in the present invention, have at least three structural units or a molecular weight of at least 500 Da (the molecular weight can be characterized by a suitable method, such as number average molecular weight, weight average molecular weight, viscosity average molecular weight, etc.). molecular weight, etc.).
聚烯烃链：指仅由碳原子共价连接而成的不含杂原子的聚合物链。本发明中，主要涉及梳状结构中的聚烯烃主链；比如丙烯酸类聚合物的线性主链。Polyolefin chain: refers to a polymer chain without heteroatoms that is only covalently linked by carbon atoms. In the present invention, the main chain of polyolefin in the comb-like structure is mainly involved; for example, the linear main chain of acrylic polymer.
丙烯酸类聚合物：指具有-C(COO-)-C-单元结构的均聚物或共聚物，所述共聚物的共聚形式没有特别限制，以能够提供线性主链和计量的侧基COO-为宜；所述丙烯酸类聚合物的线性主链中允许含有杂原子。其中，碳碳双键上还允许存在其他的取代基，只要不影响聚合反应的进行即可，例如甲基取代基(对应-CH 3C(COO-)-C-)。其中，COO-的存在形式可以为-COOH，也可以为盐形式(如钠盐)，还可以为甲酸酯形式(优选甲酸烷基酯，例如甲酸甲酯-COOCH 3，甲酸乙酯-COOCH 2CH 3；还可以为甲酸羟乙酯-COOCH 2CH 2OH)等。-C(COO-)-C-单元结构的具体结构形式包括但不限于-CH(COOH)-CH 2-、-CH(COONa)-CH 2-、-MeC(COOH)-CH 2-、-MeC(COONa)-CH 2-、-CH(COOCH 3)-CH 2-、-CH(COOCH 2CH 2OH)-CH 2-、-MeC(COOCH 3)-CH 2-、-MeC(COOCH 2CH 2OH)-CH 2-等中任一种或其任意组合。其中，Me为甲基。一个聚合物分子的线性主链上，可以仅有一种上述单元结构(对应均聚物)，也可以含有两种或两种以上的单元结构(对应共聚物)。 Acrylic polymer: refers to a homopolymer or copolymer having a -C(COO-)-C- unit structure, and the copolymerization form of the copolymer is not particularly limited, so as to be able to provide a linear main chain and a metered side group COO- Preferably; heteroatoms are allowed in the linear backbone of the acrylic polymer. Among them, other substituents are also allowed on the carbon-carbon double bond, as long as it does not affect the progress of the polymerization reaction, such as methyl substituents (corresponding to -CH 3 C(COO-)-C-). Wherein, COO- can exist in the form of -COOH, or in the form of a salt (such as a sodium salt), or in the form of a formate (preferably an alkyl formate, such as methyl formate-COOCH 3 , ethyl formate-COOCH 2 CH 3 ; it can also be hydroxyethyl formate-COOCH 2 CH 2 OH) and the like. Specific structural forms of the -C(COO-)-C-unit structure include but are not limited to -CH(COOH)-CH 2 -, -CH(COONa)-CH 2 -, -MeC(COOH)-CH 2 -, - MeC(COONa)-CH 2 -, -CH(COOCH 3 )-CH 2 -, -CH(COOCH 2 CH 2 OH)-CH 2 -, -MeC(COOCH 3 )-CH 2 -, -MeC(COOCH 2 CH 2 OH)-CH 2 - and the like, or any combination thereof. Wherein, Me is methyl. On the linear main chain of a polymer molecule, there may be only one of the above-mentioned unit structures (corresponding to a homopolymer), or two or more unit structures (corresponding to a copolymer).
丙烯酸类单体分子：可用于合成上述丙烯酸类聚合物的单体分子，具有C(COO-)＝C的基本结构，举例如，CH(COOH)＝CH 2、CH(COONa)＝CH 2、CH 3C(COOH)＝CH 2、CH 3C(COONa)＝CH 2、CH(COOCH 3)＝CH 2、CH(COOCH 2CH 2OH)＝CH 2、CH 3C(COOCH 3)＝CH 2、CH 3C(COOCH 2CH 2OH)＝CH 2等。 Acrylic monomer molecule: a monomer molecule that can be used to synthesize the above-mentioned acrylic polymer, with the basic structure of C(COO-)=C, for example, CH(COOH)=CH 2 , CH(COONa)=CH 2 , CH 3 C(COOH)=CH 2 , CH 3 C(COONa)=CH 2 , CH(COOCH 3 )=CH 2 , CH(COOCH 2 CH 2 OH)=CH 2 , CH 3 C(COOCH 3 )=CH 2. CH 3 C(COOCH 2 CH 2 OH)=CH 2 and the like.
支链：本发明中连接于支化点且具有独立末端的链。本发明中支链、侧支链具有相同含义，可以互换使用。本发明中，支链是指聚合物线性主链上键合的侧链或侧基，对于支链的长度、大小无特殊要求，可以是羧基、羟基、氨基等短支链，也可以是包含原子数较多的长支链。支链的结构无特殊要求，可以是线型的，也可以是具有分支结构的支链。支链也可以包含另外的侧链或侧基。支链的数量、长度、大小、再分支的程度等结构特征，以尽量不形成网状结构，不导致支链堆积以致于滞留比例增大为宜，此时可以顺利发挥线性主链的柔性摆动。Branched chain: A chain in the present invention that is attached to a branch point and has independent ends. In the present invention, branched chain and side branched chain have the same meaning and can be used interchangeably. In the present invention, branched chain refers to the side chain or side group bonded on the linear main chain of the polymer. There is no special requirement for the length and size of the branched chain. Long chains with more atoms. There is no special requirement for the structure of the branched chain, and it can be linear or branched with a branched structure. Branches may also contain additional side chains or side groups. The number, length, size, degree of re-branching and other structural characteristics of the branch chain should be avoided to form a network structure as much as possible, so as not to cause the accumulation of branch chains to increase the retention ratio. At this time, the flexible swing of the linear main chain can be smoothly exerted. .
支链骨架：支链骨架由骨架原子以共价键或者非共价结合方式依次相连而成，从支链末端依次连接到聚合物的主链。通过支链骨架，聚合物末端的功能性基团，被连接到聚合物的主链。支链骨架与主链的交叉点也即引出支链的支化点。比如，抗体型标签与聚合物线性主链之间的支链骨架，聚合物支链末端的抗体型标签可以依次通过亲和素、生物素、丙二胺残基(-NH-CH 2CH 2CH 2-NH-)、羰基(羧基进行酰胺化反应后的残基)而连接到聚合物的聚烯烃主链。 Branched-chain skeleton: The branched-chain skeleton consists of skeleton atoms connected in sequence by covalent bonds or non-covalent bonds, and is sequentially connected to the main chain of the polymer from the end of the branched chain. The functional groups at the ends of the polymer are attached to the main chain of the polymer through a branched backbone. The intersection of the branched skeleton and the main chain is also the branch point from which the branched chain is drawn. For example, the branched backbone between the antibody-type tag and the linear main chain of the polymer, the antibody-type tag at the end of the polymer branch chain can be sequentially passed through avidin, biotin, propylenediamine residues (-NH-CH 2 CH 2 CH2 -NH-), carbonyl group (residue after the amidation reaction of carboxyl group), and connected to the polyolefin backbone of the polymer.
支链末端，包括所有支链的末端。对于线性主链，除了固定在磁性微球本体的一端外，线性主链的另外一端必然连接于一个支化点，因此，也被广义地包括在本发明的“支链末端”范畴之内。因此，本发明的连接于磁性微球本体外表面的聚合物至少具有1个支化点。Branched-chain ends, including the ends of all branched-chains. For the linear main chain, in addition to being fixed at one end of the magnetic microsphere body, the other end of the linear main chain must be connected to a branch point, therefore, it is also broadly included in the category of "branch end" of the present invention. Therefore, the polymer attached to the outer surface of the magnetic microsphere body of the present invention has at least one branch point.
聚合物支链的功能基团：是指具有反应活性，或者经活化后具有反应活性，能够直接地与其他原料的反应性基团发生共价反应，或者经活化后与其他原料的反应性基团发生共价反应，进而生成共价键连接。聚合物支链的功能基团，其优选方式之一为特异性结合位点。The functional group of the branch chain of the polymer: refers to the reactive group, or the reactive group after activation, which can directly react covalently with the reactive group of other raw materials, or react with the reactive group of other raw materials after activation. A covalent reaction occurs between the groups, resulting in a covalent bond. One of the preferred forms of functional groups of polymer branches is a specific binding site.
直接连接方式，指不借助于间隔原子，直接发生相互作用的连接方式。所述相互作用的形式包括但不限于：共价方式、非共价方式或者其组合方式。The direct connection method refers to the connection method in which the interaction occurs directly without the aid of spacer atoms. The form of the interaction includes, but is not limited to: covalent, non-covalent, or a combination thereof.
间接连接方式，指借助于至少一个连接元件而形成的连接方式，此时涉及至少一个间隔原子。所述连接元件包括但不限于：连接肽、亲和复合物连接等。Indirect connection means connection by means of at least one connecting element, in which case at least one spacer atom is involved. The linking elements include, but are not limited to, linking peptides, affinity complex linkages, and the like.
固定、固定于、固定有、固定在等“固定”方式，指共价的结合方式。"Fixed" means of immobilization, immobilization on, immobilization with, immobilization on, etc. refer to the covalent binding method.
带有、连接有、连接于、连接在、结合、捕捉、捕获到等“连接”/“结合”方式，没有特别限定，包括但不限于共价方式、非共价方式等方式。With, connected with, connected to, connected to, combined, captured, captured, etc. "connected"/"connected" means are not particularly limited, including but not limited to covalent, non-covalent and other means.
共价方式：以共价键直接键合的方式，所述共价方式中包括但不限于动态共价方式，所述动态共价方式指以动态共价键直接键合的方式。Covalent method: the method of direct bonding by covalent bonds, the covalent method includes but not limited to the dynamic covalent method, and the dynamic covalent method refers to the method of direct bonding by dynamic covalent bonds.
共价键：包括酰胺键、酯键等常见的共价键，还包括具有可逆性质的动态共价键。所述共价键中包括动态共价键。动态共价键是一种具有可逆性质的化学键，包括但不限于亚胺键、酰腙键、二硫键或者其组合。化学领域的技术人员可以理解其含义。Covalent bonds: including common covalent bonds such as amide bonds and ester bonds, as well as dynamic covalent bonds with reversible properties. The covalent bonds include dynamic covalent bonds. A dynamic covalent bond is a chemical bond with reversible properties, including but not limited to imine bonds, acylhydrazone bonds, disulfide bonds, or a combination thereof. Those skilled in the chemical arts can understand the meaning.
非共价方式：包括但不限于配位结合、亲和复合物相互作用、静电吸附、氢键、π-π重叠作用、疏水相互作用等超分子相互作用方式。Non-covalent methods: including but not limited to supramolecular interaction methods such as coordination binding, affinity complex interaction, electrostatic adsorption, hydrogen bonding, π-π overlap, and hydrophobic interaction.
超分子相互作用：包括但不限于配位结合、亲和复合物相互作用、静电吸附、氢键、π-π重叠作用、疏水相互作用、及其组合。Supramolecular interactions: including but not limited to coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, π-π overlapping interactions, hydrophobic interactions, and combinations thereof.
连接元件，也称为连接基团，指用于连接两个或两个以上不相邻基团的元件，至少包括一个原子。所述连接元件与相邻基团之间的连接方式，没有特别限定，包括但不限于共 价方式、非共价方式等方式。所述连接元件的内部连接方式，没有特别限定，包括但不限于共价方式、非共价方式等方式。A linking element, also referred to as a linking group, refers to an element used to connect two or more non-adjacent groups, including at least one atom. The connection mode between the connecting element and the adjacent group is not particularly limited, including but not limited to covalent mode, non-covalent mode and the like. The internal connection mode of the connecting element is not particularly limited, including but not limited to covalent mode, non-covalent mode and the like.
共价连接元件：从连接元件一端到另一端的间隔原子之间均以共价方式相连接。Covalent linking element: The spacer atoms from one end of the linking element to the other are covalently linked.
特异性结合位点：本发明中，所述特异性结合位点，是指聚合物支链上的具备结合功能的基团或结构部位，该基团或结构部位具备对于某种特定目标物的特异性识别、结合功能，特异性结合可以通过配位、络合、静电力、范德华力、氢键、共价键等结合作用或者其他相互作用实现。Specific binding site: In the present invention, the specific binding site refers to a group or structural site with a binding function on the polymer branch chain, and the group or structural site has a specific target. Specific recognition and binding functions, specific binding can be achieved through coordination, complexation, electrostatic force, van der Waals force, hydrogen bond, covalent bond and other binding interactions or other interactions.
共价连接复合物：通过共价方式直接或间接地连接而成的化合物，也称为共价连接物。所述共价连接方式包括但不限于共价键、连接肽，等。Covalently linked complex: A compound that is directly or indirectly linked by covalent means, also known as a covalent linker. The covalent attachment means include, but are not limited to, covalent bonds, linking peptides, and the like.
亲和素-纯化介质共价连接复合物：以共价方式连接而成的化合物，一端为亲和素，另一端为纯化介质，两者通过共价键直接连接，或者通过共价连接元件间接相连接。Avidin-purification medium covalently linked complex: a compound that is covalently linked, with avidin at one end and a purification medium at the other end, directly linked by covalent bonds, or indirectly through covalent linking elements connected.
亲和复合物：由两个或两个以上的分子通过特异性结合作用，依靠极强的亲和力而形成的非共价连接的复合物，比如：生物素(或生物素类似物)与亲和素(或亲和素类物)的相互作用所形成的复合物。生物素与亲和素的亲和复合物的结合方式为本领域技术人员所熟知。Affinity complex: a non-covalently linked complex formed by two or more molecules through specific binding, relying on extremely strong affinity, such as: biotin (or biotin analogs) and affinity A complex formed by the interaction of avidin (or avidin). The binding mode of biotin and avidin affinity complexes is well known to those skilled in the art.
纯化底物，也称为目标物，待从混合体系中分离出来的物质。本发明中的纯化底物没有特别限制，但优选纯化底物为蛋白类物质(此时也称为目标蛋白)。Purified substrate, also known as target, the substance to be separated from a mixed system. The purification substrate in the present invention is not particularly limited, but the purification substrate is preferably a protein substance (also referred to as a target protein in this case).
纯化介质，能够和纯化底物进行特异性结合，从而捕捉纯化底物，进而能够将纯化底物从混合体系中分离出来。连接于本发明聚合物的支链末端的纯化介质，为一种具有结合纯化底物功能的功能元件。当纯化介质以共价方式与相邻基团连接时，则表现为一种具有结合纯化底物功能的功能基。The purification medium can specifically bind to the purified substrate, thereby capturing the purified substrate, and then separating the purified substrate from the mixed system. The purification medium linked to the branched end of the polymer of the present invention is a functional element having the function of binding the purification substrate. When the purification medium is covalently linked to an adjacent group, it behaves as a functional group with the function of binding the purification substrate.
生物素：biotin，可与亲和素结合，且结合力强、特异性好。Biotin: biotin, can be combined with avidin, and has strong binding force and good specificity.
亲和素：avidin，可与生物素结合，且结合力强、特异性好，如链霉亲和素(Streptavidin，简称SA)、其类似物(如Tamvavidin2，简称Tam2)、其改性产物、其突变体等。Avidin: avidin, which can be combined with biotin, and has strong binding force and good specificity, such as streptavidin (SA), its analogs (such as Tamvavidin2, referred to as Tam2), its modified products, its mutants, etc.
生物素类似物，指能够与亲和素形成类似于“亲和素-生物素”特异性结合的非生物素分子，优选之一为多肽或蛋白，比如IBA公司开发的
Figure PCTCN2021133735-appb-000001
系列中使用的含有WSHPQFEK序列的多肽(如
Figure PCTCN2021133735-appb-000002
等)，以及类似的含有WNHPQFEK序列的多肽。W NHPQFEK可视为W SHPQFEK的突变序列。 Biotin analogs refer to non-biotin molecules that can form a specific binding similar to "avidin-biotin" with avidin, preferably one is a polypeptide or protein, such as the one developed by IBA Company
Figure PCTCN2021133735-appb-000001
Polypeptides containing the WSHPQFEK sequence used in the series (e.g.
Figure PCTCN2021133735-appb-000002
etc.), and similar polypeptides containing the WNHPQFEK sequence. WN HPQFEK can be regarded as a mutated sequence of WS HPQFEK .
亲和素类似物，指能够与生物素形成类似于“亲和素-生物素”特异性结合的非亲和素分子，优选之一为多肽或蛋白。所述亲和素类似物包括但不限于亲和素的衍生物、亲和素的同源性物质(同源体)、亲和素的变体等。所述亲和素类似物，例如Tamavidin1、Tamavidin2，等(可参考文献：FEBS Journal,2009,276,1383-1397)。Avidin analogs refer to non-avidin molecules that can form a specific binding similar to "avidin-biotin" with biotin, and preferably one is a polypeptide or a protein. The avidin analogs include, but are not limited to, derivatives of avidin, homologs of avidin (homologs), variants of avidin, and the like. Said avidin analogs, such as Tamavidin1, Tamavidin2, etc. (refer to: FEBS Journal, 2009, 276, 1383-1397).
生物素型标签：所述生物素型标签中含有以下单元：生物素、可结合亲和素的亲和素类似物、可结合亲和素类似物的亲和素类似物，及其组合。生物素型标签能够特异性结合亲和素、亲和素类似物或者其组合。因此可以用来分离纯化包括但不限于被亲和素型标签标记的蛋白类物质。Biotin-type tag: The biotin-type tag contains the following units: biotin, avidin analogs that bind to avidin, avidin analogs that bind avidin analogs, and combinations thereof. Biotin-type tags are capable of specifically binding avidin, avidin analogs, or a combination thereof. Therefore, it can be used to separate and purify proteins including but not limited to avidin-type tags.
亲和素型标签：所述亲和素型标签中含有以下单元：亲和素、可结合生物素的亲和素类似物、可结合生物素类似物的亲和素类似物，及其组合。亲和素型标签能够特异性结合 生物素、生物素类似物或者其组合。因此可以用来分离纯化包括但不限于被生物素型标签标记的蛋白类物质。Avidin-type tag: The avidin-type tag contains the following units: avidin, avidin analogs that can bind biotin, avidin analogs that can bind biotin analogs, and combinations thereof. Avidin-type tags are capable of specifically binding biotin, biotin analogs, or a combination thereof. Therefore, it can be used to separate and purify proteins including but not limited to biotin-type tags.
抗体型标签：本发明的抗体型标签，指含有抗体类物质的标签，其能够特异性结合相应的目标物，所述目标物如抗原。所述抗体型标签的举例还包括可以特异性结合eGFP蛋白的antiEGFP抗体。Antibody-type tag: The antibody-type tag of the present invention refers to a tag containing an antibody-like substance, which can specifically bind to a corresponding target, such as an antigen. Examples of the antibody-type tag also include antiEGFP antibodies that can specifically bind to eGFP protein.
肽，是两个或两个以上氨基酸以肽键相连的化合物。本发明中，肽与肽段具有同等含义，可互换使用。A peptide is a compound in which two or more amino acids are linked by peptide bonds. In the present invention, peptide and peptide segment have the same meaning and can be used interchangeably.
多肽，10～50个氨基酸组成的肽。Polypeptide, a peptide consisting of 10 to 50 amino acids.
蛋白，50个以上的氨基酸组成的肽。融合蛋白也是一种蛋白。Protein, a peptide composed of more than 50 amino acids. A fusion protein is also a protein.
多肽的衍生物、蛋白的衍生物：本发明涉及的任一种多肽或蛋白，如无特别说明(例如指定具体序列)，应理解还包括其衍生物。所述多肽的衍生物、蛋白的衍生物，至少包括含有C端标签、含有N端标签、含有C端及N端标签。其中，C端指COOH端，N端指NH 2端，本领域技术人员理解其含义。所述标签可以为多肽标签，也可以为蛋白标签。一些标签举例包括但不限于，组氨酸标签(一般含有至少5个组氨酸残基；比如6×His,HHHHHH；又比如8×His标签)、Glu-Glu、c-myc表位(EQKLISEEDL)、
Figure PCTCN2021133735-appb-000003
标签(DYKDDDDK)、蛋白C(EDQVDPRLIDGK)、Tag-100(EETARFQPGYRS)、V5表位标记(V5epitope,GKPIPNPLLGLDST)、VSV-G(YTDIEMNRLGK)、Xpress(DLYDDDDK)、血凝素(hemagglutinin,YPYDVPDYA)、β-半乳糖苷酶(β-galactosidase)、硫氧还原蛋白(thioredoxin)、组氨酸位点硫氧还原蛋白(His-patch thioredoxin)、IgG结合域(IgG-binding domain)、内含肽-几丁质结合域(intein-chitin binding domain)、T7基因10(T7gene 10)、谷胱甘肽S-转移酶(glutathione-S-transferase,GST)、绿色荧光蛋白(GFP)、麦芽糖结合蛋白(maltose binding protein,MBP)等。 Derivatives of polypeptides, derivatives of proteins: any polypeptide or protein involved in the present invention, unless otherwise specified (for example, a specific sequence is specified), should be understood to also include derivatives thereof. The polypeptide derivatives and protein derivatives at least include C-terminal tags, N-terminal tags, and C-terminal and N-terminal tags. Wherein, the C-terminus refers to the COOH end, and the N-terminus refers to the NH2 -terminus, and those skilled in the art will understand its meaning. The tag can be a polypeptide tag or a protein tag. Some examples of tags include, but are not limited to, histidine tags (generally containing at least 5 histidine residues; such as 6×His, HHHHHH; another example, 8×His tags), Glu-Glu, c-myc epitopes (EQKLISEEDL ),
Figure PCTCN2021133735-appb-000003
Tag (DYKDDDDK), Protein C (EDQVDPRLIDGK), Tag-100 (EETARFQPGYRS), V5 Epitope Tag (V5epitope, GKPIPNPLLGLDST), VSV-G (YTDIEMNRLGK), Xpress (DLYDDDDK), Hemagglutinin (hemagglutinin, YPYDVPDYA), β -Galactosidase (β-galactosidase), thioredoxin (thioredoxin), histidine site thioredoxin (His-patch thioredoxin), IgG-binding domain (IgG-binding domain), intein- Intein-chitin binding domain (intein-chitin binding domain), T7 gene 10 (T7gene 10), glutathione S-transferase (glutathione-S-transferase, GST), green fluorescent protein (GFP), maltose binding protein (maltose) binding protein, MBP) and so on.
蛋白类物质，本发明中，广义地指含有多肽或蛋白片段的物质。比如，多肽衍生物、蛋白衍生物、糖蛋白等也包含在蛋白类物质的范畴之内。Protein substances, in the present invention, broadly refer to substances containing polypeptides or protein fragments. For example, polypeptide derivatives, protein derivatives, glycoproteins, etc. are also included in the category of protein substances.
抗体、抗原：本发明涉及的抗体、抗原，如无特别说明，应理解还包括其结构域、亚基、片段、单链、单链片段、变体。比如，涉及“抗体”，如无特别说明，还包括其片段、重链、缺失轻链的重链(如纳米抗体)、互补决定区(CDR)等。比如，涉及“抗原”，如无特别说明，还包括抗原决定基(epitope)、表位肽。Antibodies and antigens: The antibodies and antigens involved in the present invention, unless otherwise specified, should also be understood to include their domains, subunits, fragments, single chains, single chain fragments, and variants. For example, reference to "antibody", unless otherwise specified, also includes fragments thereof, heavy chains, heavy chains lacking light chains (such as nanobodies), complementarity determining regions (CDRs), and the like. For example, reference to "antigen", unless otherwise specified, also includes epitope and epitope peptide.
抗体类物质，本发明中，包括但不限于抗体、抗体的片段、抗体的单链、单链的片段、抗体融合蛋白、抗体片段的融合蛋白等及其衍生物与变体，只要能够产生抗体-抗原的特异性结合作用即可。Antibody substances, in the present invention, include but are not limited to antibodies, antibody fragments, single chains of antibodies, fragments of single chains, antibody fusion proteins, fusion proteins of antibody fragments, etc. and derivatives and variants thereof, as long as antibodies can be produced - The specific binding of the antigen suffices.
抗原类物质，本发明中，包括但不限于，本领域技术人员所知的抗原以及能够发挥抗原功能、特异性结合抗体类物质的物质。Antigen substances, in the present invention, include, but are not limited to, antigens known to those skilled in the art and substances capable of exerting antigen functions and specifically binding antibody substances.
抗蛋白的抗体：指能够与某一蛋白进行特异性结合的抗体。Anti-protein antibody: refers to an antibody that can specifically bind to a protein.
抗荧光蛋白的纳米抗体：指能够与某一荧光蛋白进行特异性结合的纳米抗体。Anti-fluorescent protein nanobody: refers to a nanobody that can specifically bind to a fluorescent protein.
纳米抗体(nanobody)：又称单域抗体，英文single domain antibody(sdAb)，或称为单链抗体(single-chain variable fragment)，或单结构域抗体，仅有一个重链可变区结构域(VHH)。Nanobody (nanobody): also known as single-domain antibody, English single domain antibody (sdAb), or single-chain antibody (single-chain variable fragment), or single-domain antibody, only one heavy chain variable region domain (VHH).
scFV：是由抗体重链的可变区与轻链的可变区在一段肽链的连接下构成的小分子，是具有抗体活性的最小功能结构单位。scFV: It is a small molecule composed of the variable region of the heavy chain of an antibody and the variable region of the light chain under the connection of a peptide chain, and is the smallest functional structural unit with antibody activity.
Fab：是抗体上与抗原结合的区域，它由重链和轻链各一个常域和一个变域组成，这些结构域在单体的氨基末端形成副位，即抗原结合位点，这两个可变区域结合在它们的特定抗原上的表位上。Fab: It is the region on the antibody that binds to the antigen. It consists of a constant domain and a variable domain of the heavy chain and the light chain. These domains form a parasite at the amino terminus of the monomer, that is, the antigen-binding site. These two The variable regions bind to epitopes on their specific antigen.
F(ab’)2：是抗体在胃蛋白酶作用下形成的产物，胃蛋白酶催化抗体在铰链区域下方裂解，形成F(ab’)2片段和pFc’片段。轻度还原后，F(ab’)2片段可分裂为两个Fab’片段。F(ab')2: It is the product formed by the antibody under the action of pepsin, which catalyzes the cleavage of the antibody under the hinge region to form F(ab')2 fragment and pFc' fragment. After mild reduction, the F(ab')2 fragment can be split into two Fab' fragments.
同源性(homology)，如没有特别说明，指具有至少50％同源性；优选至少60％同源性，更优选至少70％同源性，更优选至少75％同源性，更优选至少80％同源性，更优选至少85％同源性，更优选至少90％同源性；还比如至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％的同源性。描述对象举例如本发明书提及的Ω序列的同源序列。这里的同源性指序列上的相似性，数值上可以等同相似性(identity)。Homology, unless otherwise specified, means having at least 50% homology; preferably at least 60% homology, more preferably at least 70% homology, more preferably at least 75% homology, more preferably at least 80% homology, more preferably at least 85% homology, more preferably at least 90% homology; also such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, At least 97%, at least 98%, at least 99% homology. Examples of description objects are the homologous sequences of the Ω sequences mentioned in the present specification. Homology here refers to the similarity in sequence, and may be equivalent to identity in numerical value.
同源物，指具有同源性序列的物质，也可称为同源体。Homologues refer to substances with homologous sequences, which can also be called homologues.
“变体”，variant，指具有不同结构(包括但不限于进行微小变异)，但仍能保持或基本保持原有功能或性能的物质。所述变体包括但不限于核酸变体、多肽变体、蛋白变体。获得相关变体的方式包括但不限于结构单元的重组、删除或缺失、插入、移位、置换等。所述变体包括但不限于经修饰的产物、基因改造产物、融合产物等。为获得基因改造产物，进行基因改造的方式包括但不限于基因重组(对应基因重组产物)、基因删除或缺失、插入、移码、碱基置换等。基因突变产物，也称为基因突变体，属于基因改造产物的一种类型。所述变体的优选方式之一是同源体。"Variant" refers to a substance that has a different structure (including but not limited to minor variations), but still retains or substantially retains the original function or performance. The variants include, but are not limited to, nucleic acid variants, polypeptide variants, and protein variants. Ways to obtain related variants include, but are not limited to, recombination, deletion or deletion, insertion, translocation, substitution and the like of structural units. Such variants include, but are not limited to, modified products, genetically modified products, fusion products, and the like. In order to obtain the genetically modified product, the methods of genetic modification include but are not limited to gene recombination (corresponding to the gene recombination product), gene deletion or deletion, insertion, frameshift, base substitution and the like. Gene mutation products, also known as gene mutants, belong to a type of genetic modification products. One of the preferred ways of the variant is a homologue.
经修饰的产物：包括但不限于化学修饰产物、氨基酸修饰物、多肽修饰物、蛋白修饰物等。所述化学修饰产物指采用有机化学、无机化学、高分子化学等化学合成方法进行改造的产物。修饰方法举例如离子化、盐化、脱盐化、络合、解络合、螯合、解螯合、加成反应、取代反应、消除反应、插入反应、氧化反应、还原反应、翻译后修饰等修饰方法，具体举例如氧化、还原化、甲基化、去甲基化、氨基化、羧基化、硫化等修饰方法。Modified products: including but not limited to chemically modified products, amino acid modifications, polypeptide modifications, protein modifications, and the like. The chemically modified product refers to a product modified by chemical synthesis methods such as organic chemistry, inorganic chemistry, and polymer chemistry. Examples of modification methods include ionization, saltation, desalination, complexation, decomplexation, chelation, dechelation, addition reaction, substitution reaction, elimination reaction, insertion reaction, oxidation reaction, reduction reaction, post-translational modification, etc. Specific examples of the modification method include modification methods such as oxidation, reduction, methylation, demethylation, amination, carboxylation, and sulfuration.
“突变体”，mutant，本发明中如无特别说明，指仍能保持或基本保持原有功能或性能的突变产物，对突变位点的数量没有特别限制。所述突变体包括但不限于基因突变体、多肽的突变体、蛋白的突变体。突变体是变体的一种类型。获得相关突变体的方式包括但不限于结构单元的重组、删除或缺失、插入、移位、置换等。基因的结构单位为碱基，多肽和蛋白的结构单元为氨基酸。基因突变的类型包括但不限于基因删除或缺失、插入、移码、碱基置换等。"Mutant", mutant, unless otherwise specified in the present invention, refers to a mutant product that can still maintain or substantially maintain the original function or performance, and the number of mutation sites is not particularly limited. The mutants include, but are not limited to, gene mutants, polypeptide mutants, and protein mutants. Mutants are a type of variant. Ways to obtain related mutants include, but are not limited to, recombination, deletion or deletion, insertion, translocation, substitution, and the like of structural units. The structural unit of gene is base, and the structural unit of polypeptide and protein is amino acid. Types of genetic mutations include, but are not limited to, gene deletions or deletions, insertions, frameshifts, base substitutions, and the like.
“改性”产物，包括但不限于本发明的衍生物、经修饰的产物、基因改造产物、融合产物等，可以保持原有的功能或性能，也可以优化、改变其功能或性能。"Modified" products, including but not limited to derivatives, modified products, genetically modified products, fusion products, etc. of the present invention, can maintain their original functions or properties, or can optimize or change their functions or properties.
洗脱液(以目标蛋白为例)：洗脱目标蛋白；经洗脱后，目标蛋白存在于洗脱液中。Eluent (take the target protein as an example): Elute the target protein; after elution, the target protein exists in the eluate.
洗涤液(以目标蛋白为例)：洗脱杂蛋白等杂质；经洗脱后，杂蛋白被洗涤液带走。Washing solution (take the target protein as an example): Elute impurities such as impurity proteins; after elution, impurity proteins are taken away by the washing solution.
结合力：结合能力，如磁性微球与某一蛋白的结合能力。Binding capacity: binding capacity, such as the binding capacity of magnetic microspheres to a certain protein.
IVTT：In vitro transcription and translation，体外转录与翻译系统，一种无细胞蛋白合 成体系。无细胞蛋白合成体系是以外源目的mRNA或DNA为蛋白质合成模板，通过人工控制补加蛋白质合成所需的底物和转录、翻译相关蛋白因子等物质，能实现目的蛋白质的合成。本发明的无细胞蛋白合成体系无特别限制，可以是基于酵母细胞提取物、大肠杆菌细胞提取物、哺乳动物细胞提取物、植物细胞提取物、昆虫细胞提取物的无细胞蛋白合成体系的任一种或任意组合。IVTT: In vitro transcription and translation, in vitro transcription and translation system, a cell-free protein synthesis system. The cell-free protein synthesis system uses exogenous target mRNA or DNA as a protein synthesis template, and can achieve target protein synthesis by artificially controlling the addition of substrates required for protein synthesis and substances related to transcription and translation. The cell-free protein synthesis system of the present invention is not particularly limited, and can be any cell-free protein synthesis system based on yeast cell extract, Escherichia coli cell extract, mammalian cell extract, plant cell extract, and insect cell extract species or any combination.
本发明中，“翻译相关酶”，translation-related enzymes(TRENs)，指从核酸模板到蛋白质产物合成过程中所需的酶物质，不局限于翻译过程需要的酶。核酸模板：也称为遗传模板，指作为蛋白合成模板的核酸序列，包括DNA模板、mRNA模板及其组合。In the present invention, "translation-related enzymes", translation-related enzymes (TRENs), refer to the enzyme substances required in the synthesis process from nucleic acid template to protein product, and are not limited to the enzymes required in the translation process. Nucleic acid template: also known as genetic template, refers to the nucleic acid sequence as a template for protein synthesis, including DNA template, mRNA template and combinations thereof.
流穿液：磁珠与含目标蛋白的体系进行孵育后所收集的清液，其中含有未被磁珠捕获的残余的目标蛋白。Flow-through: The supernatant collected after the magnetic beads were incubated with the target protein-containing system, which contained the residual target protein that was not captured by the magnetic beads.
RFU，相对荧光单位值(Relative Fluorescence Unit)。RFU, Relative Fluorescence Unit.
eGFP：增强型绿色荧光蛋白(enhanced green fluorescence protein)。本发明中，所述eGFP广义地包括野生型及其变体，包括但不限于野生型及其突变体。eGFP: enhanced green fluorescent protein. In the present invention, the eGFP broadly includes wild type and its variants, including but not limited to wild type and its mutants.
mEGFP：eGFP的A206K突变体。mEGFP: A206K mutant of eGFP.
“可选地”，表示可以有，也可以无，以能够实现本发明的技术方案的为选择标准。本发明中，“可选方式”，表示只要适用于本发明的技术方案，就可以用来实施本发明。"Optionally" means that there may or may not be, and the selection criterion is based on the technical solution of the present invention. In the present invention, "optional mode" means that as long as it is applicable to the technical solution of the present invention, it can be used to implement the present invention.
本发明中，“优选(比如，prefer,preferable,preferably,preferred等)”、“较佳”、“更优选”、“更佳”、“最优选”等优选实施方式，不构成对发明的涵盖范围及保护范围的任何意义上的限制，并非用于限定本发明的范围和实施方式，仅用于提供一些实施方式作为举例。In the present invention, "preferred (for example, preferred, preferred, preferred, preferred, etc.)", "preferred", "more preferred", "better", "most preferred" and other preferred embodiments do not constitute the coverage of the invention Any limitation in the scope and protection scope is not used to limit the scope and embodiments of the present invention, but is only used to provide some embodiments as examples.
本发明的描述中，对于“优选之一”、“优选方式之一”、“优选实施方式之一”、“优选例之一”、“优选例”、“在一优选的实施方式中”、“一些优选例中”、“一些优选方式中”、“优选为”、“优选”、“优选地”、“更优选”、“更优地”、“进一步优选”、“最优选”等优选方式，以及“实施方式之一”、“方式之一”、“示例”、“具体示例”、“举例如”、“作为举例”、“例如”、“比如”、“如”等示意的列举方式，同样不构成对发明的涵盖范围及保护范围的任何意义上的限制，且各方式所描述的具体特征包含于本发明的至少一个具体实施方式中。本发明中，各方式所描述的具体特征可以在任何的一个或者多个具体实施方式中以合适的方式结合。本发明中，各优选方式对应的技术特征或技术方案也可以通过任意合适的方式结合。In the description of the present invention, for "one of the preferred", "one of the preferred modes", "one of the preferred embodiments", "one of the preferred examples", "preferred example", "in a preferred embodiment", "In some preferred cases", "in some preferred modes", "preferably", "preferably", "preferably", "more preferred", "more preferably", "further preferred", "most preferred", etc. are preferred Ways, and "one of the embodiments", "one of the ways", "example", "specific example", "for example", "as an example", "for example", "such as", "as", etc. manners also do not limit the scope of coverage and protection of the invention in any sense, and the specific features described in each manner are included in at least one specific embodiment of the present invention. In the present invention, the specific features described in the various embodiments may be combined in any suitable manner in any one or more of the specific embodiments. In the present invention, the technical features or technical solutions corresponding to each preferred mode may also be combined in any suitable manner.
本发明中，“其任意组合”，在数量上表示“大于1”，在涵盖范围上表示以下情形构成的组：“任选其中一个，或者任选其中至少两个构成的组”。In the present invention, "any combination thereof" means "greater than 1" in number, and means a group consisting of the following situations in terms of coverage: "optionally one of them, or a group consisting of at least two of them".
本发明中，“一个或多个”、“一种或多种”等“一或多”的描述，与“至少一个”、“至少一种”、“其组合”、“或其组合”、“及其组合”、“或其任意组合”、“及其任意组合”等具有相同含义，可以互换使用，表示数量上等于“1”或“大于1”。In the present invention, the description of "one or more" such as "one or more", "one or more", etc., is different from "at least one", "at least one", "the combination thereof", "or the combination thereof", "And any combination thereof", "or any combination thereof", "and any combination thereof" and the like have the same meaning and can be used interchangeably, indicating that the number is equal to "1" or "greater than 1".
本发明中，采用“或/和”、“和/或”表示“任选其一或者任选其组合”，也表示至少其一。In the present invention, the use of "or/and" and "and/or" means "optionally one or a combination thereof", and also means at least one of them.
本发明所述的“通常”、“常规”、“一般”、“经常”、“往往”等方式描述的现 有技术手段，也都被引用作为本发明内容的参考，如无特别说明，可视为本发明的部分技术特征的优选方式之一，且需要注意的是，不构成对发明的涵盖范围及保护范围的任何意义上的限制。The prior art means described in the present invention in the manner of "usually", "conventional", "general", "often", "often", etc. are also cited as references for the content of the present invention. It is regarded as one of the preferred modes of some technical features of the present invention, and it should be noted that it does not constitute any limitation on the scope of coverage and protection of the invention.
在本发明提及的所有文献及这些文献直接引用或者间接引用的文献，都在本申请中被引用作为参考，就如同每一篇文献被单独引用作为参考那样。All documents mentioned in this application, and documents cited directly or indirectly by these documents, are hereby incorporated by reference in this application as if each document were individually incorporated by reference.
应理解，在本发明范围内，本发明的上述各技术特征和在下文(包括但不限于实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案，只要能够用于实施本发明即可。限于篇幅，不再一一累述。It should be understood that, within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (including but not limited to the embodiments) can be combined with each other to form new or preferred technical solutions , as long as it can be used to implement the present invention. Due to space limitations, we will not repeat them one by one.
1.本发明第一方面提供一种生物磁性微球，包括磁性微球本体，所述磁性微球本体外表面具有至少一种带有线性主链和支链的聚合物，所述线性主链的一端固定于磁性微球本体外表面，聚合物的其他端游离于磁性微球本体外表面，所述生物磁性微球的聚合物的支链末端连接有抗体型标签。1. A first aspect of the present invention provides a biomagnetic microsphere, comprising a magnetic microsphere body, and the outer surface of the magnetic microsphere body has at least one polymer with a linear main chain and a branched chain, and the linear main chain One end of the polymer is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the branched end of the polymer of the biomagnetic microsphere is connected with an antibody-type label.
所述生物磁性微球称之为抗体磁珠或抗体磁性微球或抗体型磁性微球。The biomagnetic microspheres are called antibody magnetic beads or antibody magnetic microspheres or antibody-type magnetic microspheres.
所述抗体型标签，既可以作为纯化介质，也可以作为连接元件进一步连接其它类型的纯化介质。The antibody-type tag can be used either as a purification medium or as a linking element to further connect other types of purification media.
所述抗体型标签，优选地，作为纯化介质。The antibody-type tag, preferably, serves as a purification medium.
所述生物磁性微球的一种典型结构如图1所示。A typical structure of the biomagnetic microspheres is shown in FIG. 1 .
以目标物为蛋白类物质为例：与目前常用的凝胶类多孔材料相比，比如琼脂糖类，大部分市售微球采用琼脂糖类材料。多孔材料拥有丰富的孔隙结构，从而提供大的比表面积，为纯化底物提供高的结合量，但是相应地，对蛋白质进行吸附或洗脱时，需要蛋白分子额外地进入或逸出多孔材料内部复杂的孔隙通道，需要花费更多的时间，也更容易滞留。相比而言，本发明提供的捕获目标蛋白的结合位点仅利用生物磁性微球的外表面空间，且进行吸附和洗脱时，不需要经过复杂的网状通道，可直接释放到洗脱液中，从而大大减少了洗脱时间，提高了洗脱效率，降低了滞留比例，提高了纯化产率。Taking the target as a protein material as an example: compared with the currently commonly used gel-type porous materials, such as agarose, most of the commercially available microspheres use agarose materials. Porous materials have abundant pore structures, which provide a large specific surface area and provide high binding capacity for purified substrates, but correspondingly, when proteins are adsorbed or eluted, additional protein molecules need to enter or escape inside the porous material. Complex pore channels take more time and are more prone to retention. In contrast, the binding site for capturing the target protein provided by the present invention only utilizes the outer surface space of the biomagnetic microspheres, and during adsorption and elution, it does not need to go through a complex network channel, and can be directly released to the elution Therefore, the elution time is greatly reduced, the elution efficiency is improved, the retention ratio is reduced, and the purification yield is improved.
1.1.磁性微球本体1.1. Magnetic microsphere body
本发明中，所述磁性微球本体的体积可以为任意可行的粒径尺寸。In the present invention, the volume of the magnetic microsphere body can be any feasible particle size.
较小的颗粒尺寸，有助于实现磁性微球在混合体系中悬浮，更充分地与蛋白产物相接触，提高对蛋白产物的捕获效率和结合率。一些优选方式中，所述磁性微球本体的直径尺寸为下述任一种粒径尺度(偏差可以为±25％、±20％、±15％、±10％)或任两种粒径尺度之间的范围：0.1μm、0.15μm、0.2μm、0.25μm、0.3μm、0.35μm、0.4μm、0.45μm、0.5μm、0.55μm、0.6μm、0.65μm、0.7μm、0.75μm、0.8μm、0.85μm、0.9μm、0.95μm、1μm、1.5μm、2μm、2.5μm、3μm、3.5μm、4μm、4.5μm、5μm、5.5μm、6μm、6.5μm、7μm、7.5μm、8μm、8.5μm、9μm、9.5μm、10μm、15μm、20μm、25μm、30μm、35μm、40μm、45μm、50μm、55μm、60μm、65μm、70μm、75μm、80μm、85μm、90μm、95μm、100μm、150μm、200μm、250μm、300μm、350μm、400μm、450μm、500μm、550μm、600μm、650μm、700μm、750μm、800μm、850μm、900μm、950μm、1000μm。 如无特别说明，所述直径尺寸指的是平均尺寸。The smaller particle size is helpful to realize the suspension of the magnetic microspheres in the mixed system, more fully contact with the protein product, and improve the capture efficiency and binding rate of the protein product. In some preferred modes, the diameter of the magnetic microsphere body is any one of the following particle size scales (the deviation can be ±25%, ±20%, ±15%, ±10%) or any two particle size scales. Range between: 0.1μm, 0.15μm, 0.2μm, 0.25μm, 0.3μm, 0.35μm, 0.4μm, 0.45μm, 0.5μm, 0.55μm, 0.6μm, 0.65μm, 0.7μm, 0.75μm, 0.8μm, 0.85μm, 0.9μm, 0.95μm, 1μm, 1.5μm, 2μm, 2.5μm, 3μm, 3.5μm, 4μm, 4.5μm, 5μm, 5.5μm, 6μm, 6.5μm, 7μm, 7.5μm, 8μm, 8.5μm, 9μm a 350μm, 400μm, 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, 1000μm. Unless otherwise specified, the diameter size refers to the average size.
所述磁性微球本体的体积可以为任意可行的粒径尺寸。The volume of the magnetic microsphere body can be any feasible particle size.
一些优选方式中，所述磁性微球本体的直径选自0.1～10μm。In some preferred embodiments, the diameter of the magnetic microsphere body is selected from 0.1-10 μm.
一些优选方式中，所述磁性微球本体的直径选自0.2～6μm。In some preferred embodiments, the diameter of the magnetic microsphere body is selected from 0.2-6 μm.
一些优选方式中，所述磁性微球本体的直径选自0.4～5μm。In some preferred embodiments, the diameter of the magnetic microsphere body is selected from 0.4-5 μm.
一些优选方式中，所述磁性微球本体的直径选自0.5～3μm。In some preferred embodiments, the diameter of the magnetic microsphere body is selected from 0.5-3 μm.
一些优选方式中，所述磁性微球本体的直径选自0.2～1μm。In some preferred embodiments, the diameter of the magnetic microsphere body is selected from 0.2-1 μm.
一些优选方式中，所述磁性微球本体的直径选自0.5～1μm。In some preferred embodiments, the diameter of the magnetic microsphere body is selected from 0.5-1 μm.
一些优选方式中，所述磁性微球本体的平均直径约为200nm、250nm、300nm、350nm、400nm、450nm、500nm、550nm、600nm、650nm、700nm、750nm、800nm、850nm、900nm、950nm、1000nm，所述约数可以为±25％、±20％、±15％、±10％。In some preferred manners, the average diameter of the magnetic microsphere body is about 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, The submultiples may be ±25%, ±20%, ±15%, ±10%.
一些优选方式中，所述磁性微球本体的直径选自1μm～1mm。In some preferred embodiments, the diameter of the magnetic microsphere body is selected from 1 μm˜1 mm.
一些优选方式中，所述磁性微球本体的直径如1μm、10μm、100μm、200μm、500μm、800μm、1000μm，偏差范围可以为±25％、±20％、±15％、±10％。In some preferred embodiments, the diameter of the magnetic microsphere body is 1 μm, 10 μm, 100 μm, 200 μm, 500 μm, 800 μm, 1000 μm, and the deviation range can be ±25%, ±20%, ±15%, ±10%.
不同的磁性材料能够提供不同类型的活化位点，可产生结合纯化介质方式的差异，并且用磁铁进行分散和沉降的能力也有所不同，还可以对纯化底物类型产生选择性。Different magnetic materials can provide different types of activation sites, which can result in differences in the way they bind to the purification medium, as well as in their ability to disperse and settle with magnets, and can also provide selectivity for the type of purification substrate.
磁性微球本体及包含磁性微球本体的磁性微球，一方面可在外加磁场的作用下快速定位、导向和分离，另一方面可通过表面改性或化学聚合等方法赋予磁性微球表面多种活性功能基团，如羟基、羧基、醛基、氨基等，此外，磁性微球还可以通过共价键或非共价键方式结合抗体、DNA等生物活性物质。The magnetic microsphere body and the magnetic microsphere containing the magnetic microsphere body can be quickly positioned, guided and separated under the action of an external magnetic field, and on the other hand, the surface of the magnetic microsphere can be given more surface by methods such as surface modification or chemical polymerization. Various active functional groups, such as hydroxyl, carboxyl, aldehyde group, amino group, etc., in addition, magnetic microspheres can also bind antibodies, DNA and other biologically active substances through covalent bonds or non-covalent bonds.
一些优选方式中，所述磁性微球本体为SiO 2包裹的磁性材料。其中，SiO 2包裹层可以包括自带活性位点的硅烷偶联剂。 In some preferred manners, the magnetic microsphere body is a magnetic material wrapped with SiO 2 . Wherein, the SiO 2 coating layer may include a silane coupling agent with its own active site.
一些优选方式中，所述磁性材料选自：铁化合物(如铁氧化物)、铁合金、钴化合物、钴合金、镍化合物、镍合金、锰氧化物、锰合金、锌的氧化物、钆的氧化物、铬的氧化物及其组合。In some preferred modes, the magnetic material is selected from the group consisting of: iron compounds (such as iron oxides), iron alloys, cobalt compounds, cobalt alloys, nickel compounds, nickel alloys, manganese oxides, manganese alloys, zinc oxides, gadolinium oxides compounds, chromium oxides, and combinations thereof.
一些优选方式中，所述氧化铁为如磁铁矿(Fe 3O 4)、磁赤铁矿(γ-Fe 2O 3)或所述两种氧化物的组合，优选为四氧化三铁。 In some preferred modes, the iron oxide is, for example, magnetite (Fe 3 O 4 ), maghemite (γ-Fe 2 O 3 ) or a combination of the two oxides, preferably ferric oxide.
一些优选方式中，所述磁性材料选自：Fe 3O 4、γ-Fe 2O 3、氮化铁、Mn 3O 4、AlNi(Co)、FeCrMo、FeAlC、AlNiCo、FeCrCo、ReCo、ReFe、PtCo、MnAlC、CuNiFe、AlMnAg、MnBi、FeNi(Mo)、FeSi、FeAl、FeNi(Mo)、FeSiAl、BaO·6Fe 2O 3、SrO·6Fe 2O 3、PbO·6Fe 2O 3、GdO及其组合。其中，所述Re为一种稀土元素，铼。 In some preferred manners, the magnetic material is selected from: Fe 3 O 4 , γ-Fe 2 O 3 , iron nitride, Mn 3 O 4 , AlNi(Co), FeCrMo, FeAlC, AlNiCo, FeCrCo, ReCo, ReFe, PtCo, MnAlC, CuNiFe, AlMnAg, MnBi, FeNi(Mo), FeSi, FeAl, FeNi(Mo), FeSiAl, BaO·6Fe 2 O 3 , SrO·6Fe 2 O 3 , PbO·6Fe 2 O 3 , GdO and their combination. Wherein, the Re is a rare earth element, rhenium.
1.2.提供大量支链末端的聚合物结构1.2. Polymer structures that provide a large number of branched ends
所述磁性微球本体外表面具有至少一种带有线性主链和支链的聚合物，所述线性主链的一端固定于磁性微球本体外表面，聚合物的其他端游离于磁性微球本体外表面。The outer surface of the magnetic microsphere body has at least one polymer with a linear main chain and a branched chain, one end of the linear main chain is fixed on the outer surface of the magnetic microsphere body, and the other end of the polymer is free from the magnetic microsphere. body outer surface.
所述“固定于”指以共价连接的方式“固定于”磁性微球本体的外表面。The "fixed to" refers to "fixed to" the outer surface of the magnetic microsphere body in a covalently linked manner.
一些优选方式中，所述聚合物直接地共价偶联于所述磁性微球本体的外表面，或者通过连接元件间接地共价偶联于所述磁性微球本体的外表面。In some preferred modes, the polymer is directly covalently coupled to the outer surface of the magnetic microsphere body, or indirectly covalently coupled to the outer surface of the magnetic microsphere body through a connecting element.
所述聚合物具有线性主链，此时，聚合物既具有线性主链的高柔顺性，又具有支链数 量的高倍数放大优点，能够更好地实现高速率、高通量的结合，高效率、高比例(高产率)的分离。The polymer has a linear main chain. At this time, the polymer not only has the high flexibility of the linear main chain, but also has the advantages of high magnification of the number of branches, which can better achieve high-speed, high-throughput binding, and high Efficient, high-ratio (high-yield) separations.
以目标物为蛋白类物质为例：对于本发明的磁性微球，聚合物的一端共价偶联于磁性微球本体的外表面，包括所有支链和所有功能基团在内的其余端均溶于溶液中，分布在磁性微球本体的外部空间，分子链可充分伸展和摆动，使得分子链可与溶液中的其它分子充分接触，进而能够加强对目标蛋白的捕获。将目标蛋白从磁性微球上洗脱时，则能让目标蛋白直接摆脱磁性微球的束缚，直接进入到洗脱液中。相比于物理缠绕在磁性微球本体外表面或者与磁性微球本体一体形成的聚合物，这种通过线性主链一端共价固定的聚合物(一些优选方式中共价地固定单一的一条聚合物线性主链，另一些优选方式中主链固定端共价地引出2或3条线性主链)可有效减少分子链的堆叠，加强分子链在溶液中的伸展与摆动，增强对目标蛋白的捕获，减少洗脱时目标蛋白的滞留比例和滞留时间。Taking the target as a protein substance as an example: for the magnetic microspheres of the present invention, one end of the polymer is covalently coupled to the outer surface of the magnetic microsphere body, and the other ends including all branches and all functional groups are Dissolved in the solution and distributed in the outer space of the magnetic microsphere body, the molecular chain can fully stretch and swing, so that the molecular chain can fully contact with other molecules in the solution, thereby enhancing the capture of the target protein. When the target protein is eluted from the magnetic microspheres, the target protein can be directly freed from the shackles of the magnetic microspheres and directly enter the eluate. Compared with the polymer physically wound on the outer surface of the magnetic microsphere body or formed integrally with the magnetic microsphere body, this kind of polymer covalently fixed by one end of the linear main chain (some preferred ways covalently fix a single polymer) Linear main chain, in other preferred ways, 2 or 3 linear main chains are covalently drawn from the fixed end of the main chain), which can effectively reduce the stacking of molecular chains, enhance the stretching and swinging of molecular chains in solution, and enhance the capture of target proteins. , reducing the retention ratio and retention time of the target protein during elution.
1.2.1.本发明提供的生物磁性微球的聚合物的主链1.2.1. The main chain of the polymer of the biomagnetic microspheres provided by the present invention
一些优选方式中，所述线性主链为聚烯烃主链或者丙烯酸类聚合物主链。In some preferred embodiments, the linear backbone is a polyolefin backbone or an acrylic polymer backbone.
另一些优选方式中，所述聚合物的线性主链为丙烯酸类聚合物主链。可以为聚烯烃主链(线性主链仅为碳原子)，也可以在线性主链上含有杂原子(杂原子为非碳原子)。In other preferred embodiments, the linear main chain of the polymer is an acrylic polymer main chain. It may be a polyolefin main chain (the linear main chain is only carbon atoms), or it may contain hetero atoms (hetero atoms are non-carbon atoms) on the linear main chain.
一些优选方式中，所述聚合物的主链为聚烯烃主链。所述丙烯酸类聚合物的单体单元为丙烯酸、丙烯酸盐、丙烯酸酯、甲基丙烯酸、甲基丙烯酸盐、甲基丙烯酸酯等丙烯酸类单体分子或其组合。所述丙烯酸类聚合物可以通过上述单体之一聚合得到或者通过上述单体的适当组合共聚得到。In some preferred embodiments, the backbone of the polymer is a polyolefin backbone. The monomer units of the acrylic polymer are acrylic monomer molecules such as acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, methacrylate, or a combination thereof. The acrylic polymer can be obtained by polymerizing one of the above monomers or by copolymerizing a suitable combination of the above monomers.
一些优选方式中，所述聚合物的线性主链为聚烯烃主链。具体地，例如，所述聚烯烃主链为丙烯酸、丙烯酸盐、丙烯酸酯、甲基丙烯酸、甲基丙烯酸盐、甲基丙烯酸酯类单体之一的聚合产物提供的主链，或其组合的聚合产物提供的主链(其共聚产物提供的主链)，或者上述单体参与聚合形成的共聚产物的主链。上述单体组合的聚合产物，举例如丙烯酸-丙烯酸酯共聚物，又如甲基丙烯酸甲酯-甲基丙烯酸羟乙酯共聚物(MMA-HEMA共聚物)、丙烯酸-丙烯酸羟丙酯共聚物。上述单体参与聚合形成的共聚产物，举例如马来酸酐-丙烯酸共聚物。In some preferred embodiments, the linear backbone of the polymer is a polyolefin backbone. Specifically, for example, the polyolefin backbone is the backbone provided by the polymerization product of one of acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, and methacrylate monomers, or a combination thereof The main chain provided by the polymerized product (the main chain provided by the copolymerized product thereof), or the main chain of the copolymerized product formed by the participation of the above-mentioned monomers in the polymerization. The polymerization product of the above-mentioned monomer combination is, for example, acrylic acid-acrylate copolymer, and another example is methyl methacrylate-hydroxyethyl methacrylate copolymer (MMA-HEMA copolymer), acrylic acid-hydroxypropyl acrylate copolymer. The copolymerization product formed by the above-mentioned monomers participating in the polymerization is, for example, maleic anhydride-acrylic acid copolymer.
一些优选方式中，所述线性主链为聚烯烃主链，且由丙烯酸类聚合物的主链提供。In some preferred embodiments, the linear backbone is a polyolefin backbone and is provided by the backbone of an acrylic polymer.
一些优选方式中，所述线性主链为丙烯酸类聚合物主链。In some preferred embodiments, the linear backbone is an acrylic polymer backbone.
另一些优选方式中，所述聚合物的主链为丙烯酸类聚合物主链。可以为聚烯烃主链(主链仅为碳原子)，也可以在主链上含有杂原子(杂原子：非碳原子)。In other preferred embodiments, the main chain of the polymer is an acrylic polymer main chain. A polyolefin main chain (main chain only carbon atoms) may be used, or a hetero atom (hetero atom: non-carbon atom) may be contained in the main chain.
另一些优选方式中，所述聚合物的主链为含有聚烯烃嵌段的嵌段共聚物主链，例如，聚乙二醇-b-聚丙烯酸共聚物(属于丙烯酸类共聚物的范围)。以顺利发挥线性主链的柔性摆动，不会导致支链堆积，不会致使滞留时间或/和比例增大为宜。In other preferred embodiments, the main chain of the polymer is a main chain of a block copolymer containing polyolefin blocks, for example, polyethylene glycol-b-polyacrylic acid copolymer (belonging to the scope of acrylic copolymer). It is preferable to smoothly exert the flexible swing of the linear main chain without causing the accumulation of branched chains and increasing the residence time or/and the ratio.
另一些优选方式中，所述聚合物的主链为缩聚型主链。所述缩聚型主链指可以通过单体分子或者低聚物之间的缩聚反应形成的线性主链；所述缩聚型主链可以是均聚型，也可以是共聚型。例如多肽链、聚氨基酸链等。具体地，例如ε-聚赖氨酸链、α-聚赖氨酸链、γ-聚谷氨酸、聚天门冬氨酸链等、天冬氨酸/谷氨酸共聚物等。In other preferred embodiments, the main chain of the polymer is a polycondensation type main chain. The polycondensation-type main chain refers to a linear main chain that can be formed by a polycondensation reaction between monomer molecules or oligomers; the polycondensation-type main chain can be a homopolymerization type or a copolymerization type. For example, polypeptide chains, polyamino acid chains, etc. Specifically, for example, ε-polylysine chain, α-polylysine chain, γ-polyglutamic acid, polyaspartic acid chain, etc., aspartic acid/glutamic acid copolymer, and the like.
磁性微球本体外表面的一个结合位点可以共价偶联的线性主链的数量可以为1个或更多个。The number of linear backbones to which one binding site on the outer surface of the magnetic microsphere body can be covalently coupled can be one or more.
一些优选方式中，磁性微球本体外表面的一个结合位点仅引出一条线性主链，此时能够为线性主链提供较大的活动空间。In some preferred manners, only one linear main chain is drawn from one binding site on the outer surface of the magnetic microsphere body, which can provide a larger space for the linear main chain in this case.
另一些优选方式中，磁性微球本体外表面的一个结合位点仅引出两条线性主链，尽可能地为线性主链提供较大的活动空间。In other preferred embodiments, only two linear main chains are drawn out from one binding site on the outer surface of the magnetic microsphere body, so as to provide as much space for the linear main chains as possible.
聚合物的主链，一端共价偶联于磁珠外表面(生物磁性微球外表面)，包括所有支链和所有功能基团在内的其余端均溶解于溶液中，分布在磁珠的外部空间，分子链可充分伸展和摆动，使得分子链可与溶液中的其他分子充分接触，进而能够加强对目标蛋白的捕获。将目标蛋白从磁珠上洗脱时，则能让目标蛋白直接摆脱磁珠的束缚，直接进入洗脱液中；相比于物理缠绕在磁珠外表面或者与磁珠一体形成的聚合物，此处提供的通过线性主链一端共价固定的聚合物(最优选共价固定单一的一条聚合物线性主链，另优选主链固定端共价引出2或3条线性主链)可有效减少分子链的堆叠，加强分子链在溶液中的伸展与摆动，增强对目标蛋白的捕获，减少洗脱时目标蛋白的滞留比例和滞留时间。The main chain of the polymer, one end is covalently coupled to the outer surface of the magnetic bead (the outer surface of the biomagnetic microsphere), and the remaining ends, including all branches and all functional groups, are dissolved in the solution and distributed in the magnetic bead. In the external space, the molecular chain can fully stretch and swing, so that the molecular chain can fully contact with other molecules in the solution, thereby enhancing the capture of the target protein. When the target protein is eluted from the magnetic beads, the target protein can be directly freed from the shackles of the magnetic beads and directly enter the eluate; The polymers provided here covalently fixed by one end of the linear backbone (most preferably a single polymer linear backbone covalently fixed, and preferably 2 or 3 linear backbones covalently drawn from the fixed end of the backbone) can effectively reduce the amount of The stacking of molecular chains enhances the stretching and swinging of molecular chains in solution, enhances the capture of target proteins, and reduces the retention ratio and retention time of target proteins during elution.
1.2.2.本发明提供的生物磁性微球的聚合物支链1.2.2. The polymer branch of the biomagnetic microspheres provided by the present invention
所述支链的数量与磁性微球本体的大小、聚合物的骨架结构类型、聚合物在磁性微球本体外表面的链密度(特别是支链密度)等因素相关。The number of the branched chains is related to the size of the magnetic microsphere body, the skeleton structure type of the polymer, the chain density (especially the branch chain density) of the polymer on the outer surface of the magnetic microsphere body and other factors.
聚合物支链的数量为多个，至少为3个。侧支链的数量与磁性微球的大小、聚合物主链的长度、沿聚合物主链的侧支链的线密度相关、聚合物在磁性微球外表面的链密度等因素相关。聚合物支链的数量可以通过控制原料的投料比进行控制。The number of polymer branches is plural, at least three. The number of side branches is related to the size of the magnetic microspheres, the length of the polymer backbone, the linear density of side branches along the polymer backbone, and the chain density of the polymer on the outer surface of the magnetic microspheres. The number of polymer branches can be controlled by controlling the feed ratio of the raw materials.
所述支链型聚合物具有至少3个支链。The branched polymer has at least 3 branches.
各个支链末端各自独立地结合或者未结合纯化介质。Each branch end is independently bound or unbound to the purification medium.
当支链末端结合有纯化介质时，各个支链末端各自独立地直接结合纯化介质，或者通过连接元件间接地集合纯化介质。When the branched ends are bound to the purification medium, each of the branched ends is independently directly bound to the purification medium, or the purification medium is collected indirectly through a linking element.
当支链末端结合有纯化介质时，纯化介质的数量可以为1个或更多个。When a purification medium is bound to the end of the branched chain, the number of the purification medium may be one or more.
一些优选方式中，一分子所述支链型聚合物至少结合3个纯化介质。In some preferred embodiments, one molecule of the branched polymer binds at least 3 purification media.
1.3.纯化介质(本发明为抗体型标签)1.3. Purification medium (antibody tag in the present invention)
1.3.1.纯化介质，是从混合体系中特异性地捕获目标物的功能性元件，也即所述纯化介质和待被分离纯化的目标物分子之间能够进行特异性结合。被捕获的目标物分子还能在合适条件下被洗脱释放，从而实现分离纯化的目的。1.3.1. The purification medium is a functional element that specifically captures the target from the mixed system, that is, the purification medium and the target molecule to be separated and purified are capable of specific binding. The captured target molecules can also be eluted and released under suitable conditions, so as to achieve the purpose of separation and purification.
所述纯化介质以蛋白类物质为目标物时，与目标蛋白本身或者目标蛋白中携带的纯化标签，可以相互形成特异性结合作用。When the purification medium takes the protein substance as the target substance, it can form a specific binding effect with the target protein itself or the purification tag carried in the target protein.
所述生物磁性微球中的纯化介质为抗体型标签。The purification medium in the biomagnetic microspheres is an antibody-type tag.
一些优选方式中，所述抗体型标签为抗体、抗体的片段、抗体的单链、单链的片段、抗体融合蛋白、抗体片段的融合蛋白中任一种，任一种的衍生物或任一种的变体。In some preferred embodiments, the antibody-type tag is any one of an antibody, a fragment of an antibody, a single chain of an antibody, a fragment of a single chain, an antibody fusion protein, a fusion protein of an antibody fragment, a derivative of any one, or any one of species variant.
一些优选方式中，所述抗体型标签为抗蛋白的抗体。In some preferred embodiments, the antibody-type tag is an anti-protein antibody.
一些优选方式中，所述抗体型标签为抗荧光蛋白的抗体。In some preferred embodiments, the antibody-type tag is an antibody against a fluorescent protein.
一些优选方式中，所述抗体型标签为纳米抗体。In some preferred embodiments, the antibody-type tag is a Nanobody.
一些优选方式中，所述抗体型标签为抗蛋白的纳米抗体。In some preferred embodiments, the antibody-type tag is an anti-protein nanobody.
一些优选方式中，所述抗体型标签为抗蛋白的单域抗体。In some preferred embodiments, the antibody-type tag is an anti-protein single domain antibody.
一些优选方式中，所述抗体型标签为抗蛋白的单结构域抗体。In some preferred embodiments, the antibody-type tag is an anti-protein single domain antibody.
一些优选方式中，所述抗体型标签为抗蛋白的VHH抗体。In some preferred embodiments, the antibody-type tag is an anti-protein VHH antibody.
一些优选方式中，所述抗体型标签为抗蛋白的scFV抗体。In some preferred embodiments, the antibody-type tag is an anti-protein scFV antibody.
一些优选方式中，所述抗体型标签为抗荧光蛋白的纳米抗体。In some preferred embodiments, the antibody-type tag is an anti-fluorescent protein nanobody.
一些优选方式中，所述抗体型标签为抗绿色荧光蛋白或其突变体的纳米抗体。In some preferred embodiments, the antibody-type tag is a nanobody against green fluorescent protein or a mutant thereof.
一些优选方式中，所述抗体型标签为Fab片段。In some preferred embodiments, the antibody-type tag is a Fab fragment.
一些优选方式中，所述抗体型标签为F(ab’)2片段。In some preferred embodiments, the antibody-type tag is an F(ab')2 fragment.
一些优选方式中，所述抗体型标签为Fc片段。In some preferred embodiments, the antibody-type tag is an Fc fragment.
1.3.2.纯化介质的负载方式1.3.2. Loading method of purification medium
所述纯化介质连接到所述生物素或所述生物素类似物的方式没有特别限制。The manner in which the purification medium is linked to the biotin or the biotin analog is not particularly limited.
所述纯化介质连接到所述生物素或所述生物素类似物的连接方式包括但不限于：共价键、非共价键(如超分子相互作用)、连接元件，或者其组合。The manner in which the purification medium is linked to the biotin or the biotin analog includes, but is not limited to, covalent bonds, non-covalent bonds (eg, supramolecular interactions), linking elements, or a combination thereof.
一些优选方式中，所述共价键为动态共价键；更优选之一，所述动态共价键包括亚胺键、酰腙键、二硫键或者其组合。In some preferred modes, the covalent bond is a dynamic covalent bond; more preferably, the dynamic covalent bond includes an imine bond, an acylhydrazone bond, a disulfide bond or a combination thereof.
一些优选方式中，所述超分子相互作用选自：配位结合、亲和复合物相互作用、静电吸附、氢键、π-π重叠作用、疏水相互作用、及其组合。In some preferred modes, the supramolecular interactions are selected from the group consisting of: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, π-π overlapping interactions, hydrophobic interactions, and combinations thereof.
所述生物磁性微球的一些优选方式中，所述纯化介质通过含有亲和复合物的连接元件连接到所述聚合物的支链末端。In some preferred embodiments of the biomagnetic microspheres, the purification medium is linked to the branched end of the polymer through a linking element containing an affinity complex.
一些优选方式中，所述生物素或生物素类似物通过亲和复合物相互作用结合亲和素或亲和素类似物，所述纯化介质直接或间接地连接于所述亲和素或亲和素类似物。In some preferred modes, the biotin or biotin analog binds the avidin or avidin analog through an affinity complex interaction, and the purification medium is directly or indirectly linked to the avidin or avidin Vine analogs.
一些优选方式中，所述亲和复合物相互作用选自：生物素-亲和素相互作用、生物素类似物-亲和素相互作用、生物素-亲和素类似物相互作用、生物素类似物-亲和素类似物相互作用。In some preferred modes, the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analog-avidin interaction, biotin-avidin analog interaction, biotin analog Avidin analog interactions.
一些优选方式中，亲和复合物选取标准：具有特异性好、亲和力强，还提供一个可供化学键连的位点，使得亲和复合物能够共价连接到聚合物支链末端，或者经化学修饰后能够共价连接到磁性微球本体的外表面，比如外表面的结合位点、线性聚合物的主链末端、支链型聚合物的支链末端。比如以下物质的组合：生物素或其类似物与亲和素或其类似物、等等。In some preferred manners, the selection criteria of the affinity complex: it has good specificity, strong affinity, and also provides a site for chemical bonding, so that the affinity complex can be covalently linked to the end of the polymer branch, or chemically After modification, it can be covalently linked to the outer surface of the magnetic microsphere body, such as the binding site on the outer surface, the end of the main chain of the linear polymer, and the end of the branched chain of the branched polymer. For example, the combination of the following substances: biotin or its analogs and avidin or its analogs, and so on.
一些优选方式中，所述亲和素为链霉亲和素、改性的链霉亲和素、链霉亲和素类似物中任一种或其组合。In some preferred embodiments, the avidin is any one or a combination of streptavidin, modified streptavidin, and streptavidin analogs.
所述亲和素类似物，比如tamavidin 1、tamavidin 2等。Tamavidin 1和Tamavidin2是Yamamoto等人于2009年发现的一种具有结合生物素能力的蛋白(Takakura Y et al.Tamavidins:Novel avidin-like biotin-binding proteins from the Tamogitake mushroom[J].FEBS Journal,2009,276,1383-1397)，它们具有与链霉亲和素相似的很强的生物素亲和力。Tamavidin2的热稳定性优于链霉亲和素，其氨基酸序列可以从相关数据库检索到，如UniProt B9A0T7，也可以经密码子转换，优化程序优化得到DNA序列。The avidin analogs, such as tamavidin 1, tamavidin 2 and the like. Tamavidin 1 and Tamavidin2 are proteins with the ability to bind biotin discovered by Yamamoto et al. , 276, 1383-1397), which have a strong affinity for biotin similar to streptavidin. The thermal stability of Tamavidin2 is better than that of streptavidin, and its amino acid sequence can be retrieved from relevant databases, such as UniProt B9A0T7, or the DNA sequence can be optimized by codon conversion and optimization program.
所述生物素类似物，比如WSHPQFEK序列或其变体序列，WRHPQFGG序列或其变体序列，等。The biotin analogs, such as the WSHPQFEK sequence or its variant sequence, the WRHPQFGG sequence or its variant sequence, and the like.
当加载方式中包括动态共价键、超分子相互作用(特别是亲和复合物相互作用)时，形成可逆的加载方式，纯化介质可以在一定条件下从支链末端卸载，进而进行更新或更换。When the loading mode includes dynamic covalent bonds and supramolecular interactions (especially the interaction of affinity complexes), a reversible loading mode is formed, and the purification medium can be unloaded from the end of the branch under certain conditions, and then renewed or replaced .
纯化介质的更新，对应磁性微球的再生，更新前后纯化介质的种类相同。The renewal of the purification medium corresponds to the regeneration of the magnetic microspheres, and the types of the purification medium before and after the renewal are the same.
纯化介质的更换，对应磁性微球的改变，更换前后纯化介质的种类不同。The replacement of the purification medium corresponds to the change of the magnetic microspheres, and the types of purification media before and after the replacement are different.
一些优选方式中，所述生物磁性微球的聚合物的支链末端依次连接有生物素、亲和素、纯化介质；其中，所述纯化介质为抗体。所述亲和素与纯化介质之间的连接方式包括但不限于：共价键、非共价键、连接元件或者其组合。In some preferred modes, biotin, avidin, and a purification medium are sequentially connected to the branched ends of the polymers of the biomagnetic microspheres; wherein, the purification medium is an antibody. The connection mode between the avidin and the purification medium includes, but is not limited to, covalent bonds, non-covalent bonds, connecting elements or combinations thereof.
一些优选方式中，所述纯化介质通过以下连接元件连接到生物磁性微球的聚合物支链末端：包括但不限于核酸、寡核苷酸、肽核酸、核酸适体、脱氧核糖核酸、核糖核酸、亮氨酸拉链、螺旋-转角-螺旋基序、锌指基序、生物素、抗生物素蛋白、抗生蛋白链菌素、抗半抗原抗体、等、其组合。当然，所述连接元件也可以是双链核酸构建体、双螺旋、同型杂交物或异型杂交物(从DNA-DNA、DNA-RNA、DNA-PNA、RNA-RNA、RNA-PNA或PNA-PNA中选择的同型杂交物或异型杂交物)、或者其组合。In some preferred modes, the purification medium is linked to the polymer branched end of the biomagnetic microspheres through the following linking elements: including but not limited to nucleic acid, oligonucleotide, peptide nucleic acid, nucleic acid aptamer, deoxyribonucleic acid, ribonucleic acid , leucine zipper, helix-turn-helix motif, zinc finger motif, biotin, avidin, streptavidin, anti-hapten antibodies, etc., combinations thereof. Of course, the linking element can also be a double-stranded nucleic acid construct, a duplex, a homohybrid or a heterohybrid (from DNA-DNA, DNA-RNA, DNA-PNA, RNA-RNA, RNA-PNA or PNA-PNA selected homohybrid or heterohybrid), or a combination thereof.
1.3.3.纯化介质的作用机理1.3.3. The mechanism of action of the purification medium
纯化介质捕获所述反应纯化混合体系中的目标物分子的作用力为所述抗体型标签与目标物之间的特异性结合作用。The force of the purification medium to capture the target molecule in the reaction and purification mixed system is the specific binding effect between the antibody-type tag and the target.
1.4.优选的实施方式1.4. Preferred Embodiments
一些优选方式中，所述抗体型标签通过亲和复合物相互作用连接到所述聚合物的支链末端。In some preferred embodiments, the antibody-type tag is attached to the branched end of the polymer through an affinity complex interaction.
一些优选方式中，所述亲和复合物相互作用选自：生物素-亲和素相互作用、生物素类似物-亲和素相互作用、生物素-亲和素类似物相互作用、生物素类似物-亲和素类似物相互作用。In some preferred modes, the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analog-avidin interaction, biotin-avidin analog interaction, biotin analog Avidin analog interactions.
一些优选方式中，所述亲和素为链霉亲和素、改性链霉亲和素、链霉亲和素类似物或者其组合。In some preferred embodiments, the avidin is streptavidin, modified streptavidin, streptavidin analogs, or a combination thereof.
一些优选方式中，所述抗体型标签连接于所述聚合物的支链末端的方式为：共价键合、超分子相互作用或者其组合。In some preferred modes, the antibody-type tag is attached to the branched end of the polymer by covalent bonding, supramolecular interaction, or a combination thereof.
一些优选方式中，所述共价键合利用动态共价键；更优选之一，所述动态共价键包括亚胺键、酰腙键、二硫键或者其组合。In some preferred modes, the covalent bond utilizes a dynamic covalent bond; more preferably, the dynamic covalent bond includes an imine bond, an acylhydrazone bond, a disulfide bond or a combination thereof.
一些优选方式中，所述超分子相互作用选自：配位结合、亲和复合物相互作用、静电吸附、氢键、π-π重叠作用、疏水相互作用及其组合。In some preferred modes, the supramolecular interaction is selected from the group consisting of: coordination binding, affinity complex interaction, electrostatic adsorption, hydrogen bonding, π-π overlapping interactions, hydrophobic interactions, and combinations thereof.
1.5.具体的优选实施方式1.5. Specific preferred embodiments
一种优选实施方式为，所述生物磁性微球包括磁性微球本体，所述磁性微球本体外表面具有至少一种带有线性主链和支链的聚合物，所述线性主链的一端固定于磁性微球本体外表面，聚合物的其他端游离于磁性微球本体外表面，所述生物磁性微球的聚合物的支链末端连接有生物素或生物素类似物，所述生物素或者生物素类似物作为连接元件，通过亲和复合物结合作用进一步连接亲和素或亲和素类似物，所述亲和素或亲和素类似物仍作为 连接元件，进一步连接所述抗体型标签。A preferred embodiment is that the biomagnetic microsphere includes a magnetic microsphere body, and the outer surface of the magnetic microsphere body has at least one polymer with a linear main chain and a branched chain, and one end of the linear main chain is It is fixed on the outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the branched end of the polymer of the biomagnetic microsphere is connected with biotin or a biotin analog, and the biotin Or biotin analogs are used as connecting elements to further connect avidin or avidin analogs through affinity complex binding, and the avidin or avidin analogs are still used as connecting elements to further connect the antibody type. Label.
更优选地，所述生物磁性微球的聚合物的支链末端连接有生物素，所述生物素作为连接元件，通过亲和复合物结合作用进一步连接亲和素或亲和素类似物，所述亲和素或亲和素类似物仍作为连接元件，进一步连接所述抗体型标签。More preferably, biotin is connected to the branched end of the polymer of the biomagnetic microspheres, and the biotin is used as a connecting element to further connect avidin or avidin analogs through the binding effect of the affinity complex. The avidin or avidin analog still serves as a linking element to further link the antibody-type tag.
所述亲和素的的优选方包括链霉亲和素、改性链霉亲和素、链霉亲和素类似物中任一项及其组合。Preferred forms of the avidin include any one of streptavidin, modified streptavidin, streptavidin analogs, and combinations thereof.
生物素或生物素类似物的结合方式Conjugation of biotin or biotin analogs
所述生物素或生物素类似物连接到所述聚合物的支链末端的方式没有特别限制。The manner in which the biotin or biotin analog is attached to the branched end of the polymer is not particularly limited.
所述生物素或生物素类似物连接于所述聚合物的支链末端的方式包括但不限于：共价键、非共价键(如超分子相互作用)或者其组合。The manner in which the biotin or biotin analog is attached to the branched end of the polymer includes, but is not limited to, covalent bonds, non-covalent bonds (eg, supramolecular interactions), or a combination thereof.
一些优选方式中，所述共价键为动态共价键；更优选之一，所述动态共价键包括亚胺键、酰腙键、二硫键或者其组合。In some preferred modes, the covalent bond is a dynamic covalent bond; more preferably, the dynamic covalent bond includes an imine bond, an acylhydrazone bond, a disulfide bond or a combination thereof.
一些优选方式中，所述超分子相互作用选自：配位结合、亲和复合物相互作用、静电吸附、氢键、π-π重叠作用、疏水相互作用、及其组合。In some preferred modes, the supramolecular interactions are selected from the group consisting of: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, π-π overlapping interactions, hydrophobic interactions, and combinations thereof.
一些优选方式中，所述聚合物的支链，通过基于功能基团的共价键共价结合生物素或生物素类似物，将生物素或生物素类似物共价键合在聚合物支链末端。可通过生物磁性微球外表面的聚合物分子的支链所含有的功能基团与生物素或生物素类似物进行共价反应而获得。其中，所述功能性基团的优选实施方式之一为特异性结合位点(定义详见具体实施方式的“名词和术语”部分)。In some preferred modes, the branched chain of the polymer is covalently bound to biotin or biotin analogs through covalent bonds based on functional groups, and biotin or biotin analogs are covalently bound to the branched chains of the polymer. end. It can be obtained by covalently reacting functional groups contained in the branched chains of polymer molecules on the outer surface of biomagnetic microspheres with biotin or biotin analogs. Wherein, one of the preferred embodiments of the functional group is a specific binding site (for definitions, please refer to the "noun and term" section of the specific embodiment).
所述基于功能基团的共价键，指由功能基团参与共价偶联形成的共价键。优选地，所述功能基团为羧基、羟基、氨基、巯基、羧基的盐形式、氨基的盐形式、甲酸酯基，或者前述功能基团的组合。所述羧基的盐形式的优选方式之一为钠盐形式如COONa；所述氨基的盐形式的优选方式可以为无机盐形式，也可以为有机盐形式，包括但不限于盐酸盐、氢氟酸盐等形式。所述“功能基团的组合”指一个磁性微球的外表面的所有聚合物分子的所有支链，允许基于不同的功能基团参与形成共价键；以生物素为例，也即一个生物素磁性微球的外表面的所有生物素分子可以分别与不同的功能基团共价连接，不过一个生物素分子仅能与一个功能基团相连接。The functional group-based covalent bond refers to a covalent bond formed by a functional group participating in covalent coupling. Preferably, the functional group is a carboxyl group, a hydroxyl group, an amino group, a sulfhydryl group, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of the foregoing functional groups. One of the preferred modes of the salt form of the carboxyl group is a sodium salt form such as COONa; the preferred mode of the salt form of the amino group can be an inorganic salt form, or an organic salt form, including but not limited to hydrochloride, hydrofluoride acid salts, etc. The "combination of functional groups" refers to all branches of all polymer molecules on the outer surface of a magnetic microsphere, allowing different functional groups to participate in the formation of covalent bonds; take biotin as an example, that is, a biological All biotin molecules on the outer surface of the prime magnetic microspheres can be covalently linked with different functional groups, but one biotin molecule can only be linked with one functional group.
1.6.纯化介质的再生与再利用1.6. Regeneration and reuse of purification media
当纯化介质通过亲和复合物等可逆方式连接在本发明生物磁性微球的聚合物支链末端时，可以在适当的条件下，将纯化介质从聚合物支链末端洗脱下来，进而重新结合新的纯化介质。When the purification medium is reversibly connected to the end of the polymer branch of the biomagnetic microspheres of the present invention through reversible means such as affinity complexes, the purification medium can be eluted from the end of the polymer branch under appropriate conditions, and then recombined New purification medium.
以亲和复合物相互作用为生物素和链霉亲和素之间的亲和复合物作用力为例。Take the affinity complex interaction as the affinity complex interaction between biotin and streptavidin as an example.
生物素和链霉亲和素之间极强的亲和力是一种典型的亲和复合物的结合作用，既强于一般非共价键作用，同时又弱于共价键作用，从而既能使纯化介质(抗体型标签)牢固的结合在磁珠外表面的聚合物支链末端，又能在需要更换纯化介质时通过将链霉亲和素从生物素的特异性结合位置洗脱实现纯化介质的同步脱离，进而释放出可重新结合新的亲和素-纯化介质共价连接复合物(例如带有链霉亲和素标签的纯化介质)的活化位点，从而实现磁珠纯化性能的快速恢复，大幅降低目标物分离纯化成本。对修饰有纯化介质的生物磁性 微球进行洗脱，去除亲和素-纯化介质共价连接复合物，从而重新获得生物素或生物素类似物修饰的生物磁性微球的过程，我们称之为生物素磁性微球的再生。再生的生物素磁性微球具有被释放的生物素活性位点，能够重新结合亲和素-纯化介质共价连接复合物，再次得到纯化介质修饰的生物磁性微球(对应生物磁性微球的再生)，可提供新鲜的纯化介质，提供新生的目标物结合位点。这就使得本发明的生物素磁性微球可以再生使用，也即可以更换纯化介质后再利用。The extremely strong affinity between biotin and streptavidin is a typical binding effect of affinity complexes, which is both stronger than general non-covalent bonds and weaker than covalent bonds, so that both The purification medium (antibody-type tag) is firmly bound to the end of the polymer branch on the outer surface of the magnetic bead, and the purification medium can be realized by elution of streptavidin from the specific binding site of biotin when the purification medium needs to be replaced synchronous dissociation, which in turn releases activation sites that can re-associate new avidin-purification media covalently linked complexes (e.g., streptavidin-tagged purification media), enabling rapid bead purification performance recovery, greatly reducing the cost of separation and purification of target compounds. The process of eluting the biomagnetic microspheres modified with the purification medium to remove the covalently linked complex of avidin-purification medium, so as to regain the biotin or biotin analog-modified biomagnetic microspheres, we call it Regeneration of biotin magnetic microspheres. The regenerated biotin magnetic microspheres have released biotin active sites, which can re-bond the avidin-purification medium covalently linked complex, and obtain the purification medium-modified biomagnetic microspheres (corresponding to the regeneration of the biomagnetic microspheres). ), which can provide fresh purification media and provide nascent target binding sites. This allows the biotin magnetic microspheres of the present invention to be regenerated, that is, the purification medium can be replaced and then reused.
1.7.纯化底物(目标物)1.7. Purification of substrates (targets)
本发明的纯化底物指本发明的磁性微球用来捕捉分离的物质。Purified substrates of the present invention refer to the magnetic microspheres of the present invention for capturing separated substances.
本发明的纯化底物为能够与所述抗体型标签产生特异性结合作用的物质。The purified substrate of the present invention is a substance capable of specifically binding to the antibody-type tag.
一些优选方式中，所述纯化底物为蛋白类物质。所述纯化底物为蛋白类物质时，纯化底物也称为目标蛋白。In some preferred embodiments, the purification substrate is a proteinaceous substance. When the purification substrate is a protein substance, the purification substrate is also called a target protein.
1.7.1.目标蛋白的类型1.7.1. Types of target proteins
所述目标蛋白可以为天然蛋白或其改造产物，也可以为人工合成序列。所述天然蛋白的来源没有特别限制，包括但不限于：真核细胞、原核细胞、病原体；其中真核细胞来源包括但不限于：哺乳动物细胞、植物细胞、酵母细胞、昆虫细胞、线虫细胞、及其组合；所述哺乳动物细胞来源可以包括但不限于鼠源(包括大鼠、小鼠、豚鼠、金地鼠、仓鼠等)、兔源、猴源、人源、猪源、羊源、牛源、狗源、马源等。所述病原体包括病毒、衣原体、支原体等。所述病毒包括HPV、HBV、TMV、冠状病毒、轮状病毒，等。The target protein can be a natural protein or a modified product thereof, or an artificial synthetic sequence. The source of the natural protein is not particularly limited, including but not limited to: eukaryotic cells, prokaryotic cells, pathogens; wherein eukaryotic cell sources include but are not limited to: mammalian cells, plant cells, yeast cells, insect cells, nematode cells, and combinations thereof; the mammalian cell sources may include but are not limited to murine sources (including rats, mice, guinea pigs, golden hamsters, hamsters, etc.), rabbit sources, monkey sources, human sources, pig sources, sheep sources, bovine sources source, dog source, horse source, etc. The pathogens include viruses, chlamydia, mycoplasmas, and the like. The viruses include HPV, HBV, TMV, coronavirus, rotavirus, and the like.
所述目标蛋白的类型包括但不限于多肽(本发明中“目标蛋白”广义地包括多肽)、荧光类蛋白、酶及相应的酶原、抗体、抗原、免疫球蛋白、激素、胶原、聚氨基酸、疫苗等，前述任一种蛋白的部分结构域，前述任一种蛋白的亚基或片段，以及前述任一种蛋白的变体。所述“前述任一种蛋白的亚基或片段”包括“前述任一种蛋白的部分结构域”的亚基或片段。所述“前述任一种蛋白的变体”包括“前述任一种蛋白的部分结构域、前述任一种蛋白的亚基或片段”的变体。所述“前述任一种蛋白的变体”包括但不限于前述任一种蛋白的突变体。本发明中，其它位置的连续两个或两个以上“前述”的情形，含义做类似解释。The types of the target protein include but are not limited to polypeptides ("target protein" in the present invention broadly includes polypeptides), fluorescent proteins, enzymes and corresponding zymogens, antibodies, antigens, immunoglobulins, hormones, collagen, polyamino acids , vaccines, etc., partial domains of any of the foregoing proteins, subunits or fragments of any of the foregoing proteins, and variants of any of the foregoing proteins. The "subunit or fragment of any of the foregoing proteins" includes subunits or fragments of "partial domains of any of the foregoing proteins". The "variant of any of the foregoing proteins" includes variants of "a partial domain of any of the foregoing proteins, subunits or fragments of any of the foregoing proteins". The "variant of any of the foregoing proteins" includes, but is not limited to, mutants of any of the foregoing proteins. In the present invention, the situation of two or more consecutive "aforesaid" in other positions shall be interpreted similarly.
所述目标蛋白的结构，既可以为完整结构，也可以选自相应的部分结构域、亚基、片段、二聚体、多聚体、融合蛋白、糖蛋白等。不完整的抗体结构的举例如，纳米抗体(缺失轻链的重链抗体，V HH，保留了重链抗体完整的抗原结合能力)、重链可变区、互补决定区(CDR)等。 The structure of the target protein can be either a complete structure or selected from corresponding partial domains, subunits, fragments, dimers, multimers, fusion proteins, glycoproteins, and the like. Examples of incomplete antibody structures include Nanobodies (heavy chain antibodies lacking light chains, VHH , which retain the full antigen-binding ability of heavy chain antibodies), heavy chain variable regions, complementarity determining regions (CDRs), and the like.
例如，本发明所述体外蛋白合成体系可合成的目标蛋白，可以选自包括但不限于以下任一种蛋白、任意组合方式的融合蛋白、任意组合方式的组合物：荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白(GFP)、增强绿色荧光蛋白(eGFP)、黄色荧光蛋白(YFP)、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶(Catalase，举例如鼠过氧化氢酶)、肌动蛋白、抗体、抗体的可变区域(如抗体的单链可变区域，scFV)、抗体的单链及其片段(如抗体的重链、纳米抗体、抗体的轻链)、α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素及其前体、胰高血糖素样肽(GLP-1)、干扰素(包括但不限于干扰素α，如干扰素αA、干扰素β、干扰素γ等)、白介素(如白细胞介素-1β、白介素2、白介素 12，等)、溶菌酶素、血清白蛋白(包括但不限于人血清白蛋白、牛血清白蛋白)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶、β-半乳糖苷酶(β-galactosidase,LacZ，举例如大肠杆菌β-半乳糖苷酶)，等，前述任一种蛋白的部分结构域，前述任一种蛋白的亚基或片段，或前述任一种的变体(如前述定义，所述变体包括突变体，举例如萤光素酶突变体、eGFP的突变体，所述变体还可以是同源体)。所述氨酰tRNA合成酶，举例如人赖氨酸-tRNA合成酶(lysine-tRNA synthetase)、人亮氨酸-tRNA合成酶(leucine-tRNA synthetase)等。所述甘油醛-3-磷酸脱氢酶，举例如拟南芥甘油醛3-磷酸脱氢酶，glyceraldehyde-3-phosphate dehydrogenase。还可参考专利文献CN109423496A。所述任意组合方式的组合物，可以包括前述任一种蛋白，也可以包括前述任意组合方式的融合蛋白。For example, the target protein that can be synthesized by the in vitro protein synthesis system of the present invention can be selected from, but not limited to, any of the following proteins, fusion proteins in any combination, and compositions in any combination: luciferase (such as firefly fluorescence peptase), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase (Catalase, for example e.g. murine catalase), actin, antibodies, variable regions of antibodies (e.g. single chain variable regions of antibodies, scFV), single chains of antibodies and fragments thereof (e.g. heavy chains of antibodies, nanobodies, antibodies light chain), alpha-amylase, enterobactin A, hepatitis C virus E2 glycoprotein, insulin and its precursors, glucagon-like peptide (GLP-1), interferon (including but not limited to interferon Interferon alpha, such as interferon alpha A, interferon beta, interferon gamma, etc.), interleukins (such as interleukin-1 beta, interleukin 2, interleukin 12, etc.), lysozyme, serum albumin (including but not limited to human serum Albumin, bovine serum albumin), transthyretin, tyrosinase, xylanase, beta-galactosidase (beta-galactosidase, LacZ, such as E. coli beta-galactosidase), etc. , a partial domain of any of the foregoing proteins, a subunit or fragment of any of the foregoing proteins, or a variant of any of the foregoing (as defined above, the variants include mutants, such as luciferase mutations mutants of eGFP, which may also be homologs). The aminoacyl-tRNA synthetase, for example, human lysine-tRNA synthetase (lysine-tRNA synthetase), human leucine-tRNA synthetase (leucine-tRNA synthetase) and the like. The glyceraldehyde-3-phosphate dehydrogenase is, for example, Arabidopsis glyceraldehyde-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase. Reference can also be made to patent document CN109423496A. The composition in any combination may include any of the aforementioned proteins, and may also include the fusion proteins in any combination.
一些优选方式中，采用GFP、eGFP、mScarlet等之一，或其类似物质、或其突变体等具有荧光性质的目标蛋白对所述体外蛋白合成体系的蛋白合成能力进行评估。In some preferred manners, one of GFP, eGFP, mScarlet, or the like, or a target protein with fluorescent properties, such as a similar substance or a mutant thereof, is used to evaluate the protein synthesis ability of the in vitro protein synthesis system.
所述目标蛋白的应用领域包括但不限于生物医药、分子生物、医学、体外检测、医疗诊断、再生医学、生物工程、组织工程、干细胞工程、基因工程、聚合物工程、表面工程、纳米工程、化妆品、食品、食品添加剂、营养剂、农业、饲料、生活用品、洗涤、环境、化学染色、荧光标记等领域。The application fields of the target protein include but are not limited to biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nanoengineering, Cosmetics, food, food additives, nutritional agents, agriculture, feed, daily necessities, washing, environment, chemical dyeing, fluorescent marking and other fields.
1.7.2.含有目标蛋白的混合体系1.7.2. Mixed system containing target protein
本发明的磁性微球可用于将目标蛋白从其混合体系中分离出来。所述目标蛋白不限于一种物质，允许是多种物质的组合，只要纯化的目的是为了获得这种组合物，或者这种组合物的形式可以满足纯化需求。The magnetic microspheres of the present invention can be used to separate the target protein from its mixed system. The target protein is not limited to one substance, but a combination of multiple substances is allowed, as long as the purpose of purification is to obtain this composition, or the form of this composition can meet the purification requirements.
含有目标蛋白的混合体系没有特别限制，只要本发明的磁性微球的纯化介质能够与目标蛋白特异性结合即可；通常地，还需要所述纯化介质与混合体系中目标蛋白以外的其他物质不存在特异性结合或非特异性结合作用。The mixed system containing the target protein is not particularly limited, as long as the purification medium of the magnetic microspheres of the present invention can specifically bind to the target protein; generally, it is also required that the purification medium and other substances other than the target protein in the mixed system are incompatible. There is specific binding or non-specific binding.
本发明的实施例中，所述含有目标蛋白的混合体系可以为天然来源，也可以为人工构建或得到的混合体系。In the embodiment of the present invention, the mixed system containing the target protein may be a natural source, or may be an artificially constructed or obtained mixed system.
比如，可以从市售血清中分离纯化特定蛋白。For example, specific proteins can be isolated and purified from commercially available serum.
比如，可以从体外蛋白合成体系的反应后的体系中分离目标蛋白。For example, the target protein can be isolated from the post-reaction system of an in vitro protein synthesis system.
可采用已公开的体外无细胞蛋白合成方法，参照专利文献CN 201610868691.6、WO2018161374A1、KR20190108180A、CN108535489B等。概括地，由细胞提取物(含基因组整合表达的RNA聚合酶)、DNA模板、能量体系(如：磷酸肌酸-磷酸肌酸激酶体系)、镁离子、钠离子、聚乙二醇等组分构建成IVTT体系，28～30℃条件下进行IVTT反应，反应时间8～12小时。反应结束后，所得到的IVTT反应液中含有所述DNA模板所编码的融合蛋白。Published in vitro cell-free protein synthesis methods can be used, refer to patent documents CN 201610868691.6, WO2018161374A1, KR20190108180A, CN108535489B, etc. In general, it is composed of cell extracts (including RNA polymerase integrated and expressed in the genome), DNA templates, energy systems (such as: phosphocreatine-phosphocreatine kinase system), magnesium ions, sodium ions, polyethylene glycol and other components The IVTT system was constructed, and the IVTT reaction was carried out under the condition of 28-30°C, and the reaction time was 8-12 hours. After the reaction, the obtained IVTT reaction solution contains the fusion protein encoded by the DNA template.
所述体外蛋白合成体系的具体实施方式之一，还包括但不限于，例如WO2016005982A1所记载的基于大肠杆菌的无细胞蛋白合成体系。本发明的其它引用文献、其直接及间接引用文献中所记载的包括但不限于基于麦胚细胞、兔网织红细胞、酿酒酵母、毕赤酵母、马克斯克鲁维酵母的体外无细胞蛋白合成体系，也均作为本发明的体外蛋白合成体系的实施方式纳入本发明。举例如，文献“Lu,Y.Advances in Cell-Free Biosynthetic Technology.Current Developments in Biotechnology and Bioengineering,2019, Chapter 2,23-45”中包括但不限于“2.1Systems and Advantages”部分第27-28页所引用文献中记载的体外无细胞蛋白合成体系，均可作为实施本发明的体外蛋白合成体系。举例如(除非和本发明相冲突，否则，下述文献及其引用文献以全部内容、全部目的被引用)，文献CN106978349A、CN108535489A、CN108690139A、CN108949801A、CN108642076A、CN109022478A、CN109423496A、CN109423497A、CN109423509A、CN109837293A、CN109971783A、CN109988801A、CN109971775A、CN110093284A、CN110408635A、CN110408636A、CN110551745A、CN110551700A、CN110551785A、CN110819647A、CN110845622、CN110938649A、CN110964736A、CN111378706A、CN111378707A、CN111378708A、CN111718419A、CN111748569A、CN2019107298813、CN2019112066163、CN2018112862093(CN111118065A)、CN2019114181518、CN2020100693833、CN2020101796894、CN202010269333X、CN2020102693382及其引用文献中记载的体外无细胞蛋白合成体系、DNA模板的构建和扩增方法，均可作为实施本发明的体外蛋白合成体系、本发明的DNA模板的构建和扩增方法。One of the specific embodiments of the in vitro protein synthesis system also includes but is not limited to, for example, the Escherichia coli-based cell-free protein synthesis system described in WO2016005982A1. Other citations of the present invention, and those described in their direct and indirect citations include, but are not limited to, in vitro cell-free protein synthesis systems based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces marxianus , are also incorporated into the present invention as embodiments of the in vitro protein synthesis system of the present invention. For example, the document "Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45" includes but is not limited to pages 27-28 of "2.1 Systems and Advantages" The in vitro cell-free protein synthesis systems described in the cited documents can all be used as the in vitro protein synthesis systems for implementing the present invention.举例如(除非和本发明相冲突，否则，下述文献及其引用文献以全部内容、全部目的被引用)，文献CN106978349A、CN108535489A、CN108690139A、CN108949801A、CN108642076A、CN109022478A、CN109423496A、CN109423497A、CN109423509A、CN109837293A、 CN109971783A、CN109988801A、CN109971775A、CN110093284A、CN110408635A、CN110408636A、CN110551745A、CN110551700A、CN110551785A、CN110819647A、CN110845622、CN110938649A、CN110964736A、CN111378706A、CN111378707A、CN111378708A、CN111718419A、CN111748569A、CN2019107298813、CN2019112066163、CN2018112862093(CN111118065A)、CN2019114181518、CN2020100693833、CN2020101796894 , CN202010269333X, CN2020102693382 and the in vitro cell-free protein synthesis system, the construction and amplification method of DNA template recorded in the cited literature, all can be used as the in vitro protein synthesis system of the present invention, the construction and amplification method of DNA template of the present invention .
所述体外蛋白合成体系的细胞提取物的来源细胞没有特别限制，只要能够体外表达所述目标蛋白即可。现有技术已公开的适用原核细胞提取物、真核细胞提取物(可以优选酵母细胞提取物，还可以更优先乳酸克鲁维酵母)来源的体外蛋白合成体系的外源蛋白，或者适用于细胞内合成的原核细胞体系、真核细胞体系(可以优选为酵母细胞体系，还可以更优选为乳酸克鲁维酵母体系)的内源蛋白，也均可以采用本发明的体外蛋白合成体系进行合成，或者尝试用本发明提供的体外蛋白合成体系进行合成。The source cell of the cell extract of the in vitro protein synthesis system is not particularly limited, as long as the target protein can be expressed in vitro. Exogenous proteins disclosed in the prior art that are suitable for prokaryotic cell extracts, eukaryotic cell extracts (preferably yeast cell extracts, and more preferably Kluyveromyces lactis) derived in vitro protein synthesis systems, or suitable for cell The endogenous protein of the prokaryotic cell system and eukaryotic cell system (which can be preferably a yeast cell system, can also be more preferably a Kluyveromyces lactis system) synthesized in the interior can also be synthesized by the in vitro protein synthesis system of the present invention, Or try to synthesize with the in vitro protein synthesis system provided by the present invention.
所述体外蛋白合成体系的优选方式之一为IVTT体系。进行IVTT反应后的液体(记为IVTT反应液)，除了含有所表达的目标的蛋白外，还含有IVTT体系中残余反应原料，特别含有来自细胞提取物的各种因子(比如核糖体、tRNA、翻译相关酶、起始因子、延伸因子、终止因子等)。所述IVTT反应液，一方面能够提供用于与磁珠结合的目标蛋白，另一方面还可以提供用于检验目标蛋白分离效果的混合体系。One of the preferred modes of the in vitro protein synthesis system is the IVTT system. The liquid after IVTT reaction (referred to as IVTT reaction liquid), in addition to the expressed target protein, also contains residual reaction raw materials in the IVTT system, especially various factors from cell extracts (such as ribosomes, tRNA, translation-related enzymes, initiation factors, elongation factors, termination factors, etc.). The IVTT reaction solution, on the one hand, can provide the target protein for binding to the magnetic beads, and on the other hand, can also provide a mixed system for testing the separation effect of the target protein.
2.本发明还公开一种第一方面所述生物磁性微球的制备方法，包括以下步骤：(i)提供结合有生物素或生物素类似物的磁性微球(也称为生物素磁性微球或生物素磁珠)；(ii)将提供抗体型标签的原料与所述生物磁性微球的聚合物支链末端的生物素类似物相连接，得到结合有抗体型标签的生物磁性微球K(抗体磁珠)。2. The present invention also discloses a method for preparing the biomagnetic microspheres described in the first aspect, comprising the following steps: (i) providing magnetic microspheres (also referred to as biotin magnetic microspheres) combined with biotin or biotin analogs; ball or biotin magnetic beads); (ii) connecting the raw material for providing antibody-type tags with the biotin analog at the end of the polymer branch of the bio-magnetic microspheres to obtain bio-magnetic microspheres bound with antibody-type tags K (antibody magnetic beads).
一些优选方式中，所述提供抗体型标签的原料为所述抗体型标签。In some preferred embodiments, the raw material for providing the antibody-type tag is the antibody-type tag.
一些优选方式中，所述提供抗体型标签的原料为亲和素或亲和素类似物与所述抗体型标签的共价连接复合物。第(ii)步将亲和素或亲和素类似物与所述抗体型标签的共价连接复合物结合到聚合物支链末端，所述聚合物支链末端的生物素或生物素类似物与所述亲和素或亲和素类似物之间形成亲和复合物的结合作用，获得带有所述抗体型标签的生物磁性微球K。所述亲和素的优选方式包括但不限于链霉亲和素、改性链霉亲和素、链霉亲和素类似物及其组合。In some preferred embodiments, the raw material for providing the antibody-type tag is a covalently linked complex of avidin or an avidin analog and the antibody-type tag. Step (ii) binds the covalently linked complex of avidin or avidin analog to the antibody-type tag to the end of the polymer branch, the biotin or biotin analog at the end of the polymer branch Binding with the avidin or avidin analogs to form an affinity complex to obtain the biomagnetic microsphere K with the antibody-type label. Preferred forms of the avidin include, but are not limited to, streptavidin, modified streptavidin, streptavidin analogs, and combinations thereof.
独立地可选地，包括(iii)磁铁沉降生物磁性微球K，去除液相，清洗；Independently and optionally, including (iii) magnet sedimentation of biomagnetic microspheres K, removal of liquid phase, and cleaning;
更优选地，所述提供抗体型标签的原料为亲和素-抗体型标签共价连接复合物。此时， 独立地可选地，包括(iv)所述亲和素-纯化介质共价连接复合物的更换。More preferably, the raw material for providing the antibody-type tag is an avidin-antibody-type tag covalently linked complex. At this point, independently and optionally, (iv) replacement of the avidin-purification medium covalently linked complex is included.
一种典型的所述生物磁性微球的制备方法参考图2，以纳米抗体作为纯化介质为例。A typical preparation method of the biomagnetic microspheres is shown in Fig. 2, taking nanobodies as the purification medium as an example.
2.1.制备生物素磁性微球2.1. Preparation of biotin magnetic microspheres
以结合有生物素的生物磁性微球为例。Take biotin-conjugated biomagnetic microspheres as an example.
所述生物素磁性微球可通过以下环节制备得到：提供SiO 2包裹的磁珠(市售或者自制)、SiO 2的活化修饰、聚合物共价连接到SiO 2(聚合物通过线性主链的一端共价连接到SiO 2，且沿聚合物主链分布大量侧支链)、生物素共价连接到聚合物的支链末端。需要说明的是，上述环节并不要求完全孤立，允许两个或三个环节合并为一个环节，例如，可以直接提供已活化的二氧化硅包裹的磁珠(市售或者自制)。 The biotin magnetic microspheres can be prepared through the following steps: providing SiO 2 -coated magnetic beads (commercially available or homemade), activating modification of SiO 2 , covalently linking the polymer to SiO 2 (the polymer is bound by a linear backbone). One end is covalently attached to SiO2 with a large number of side branches distributed along the polymer backbone), and biotin is covalently attached to the branched ends of the polymer. It should be noted that the above-mentioned links are not required to be completely isolated, and two or three links are allowed to be combined into one link. For example, activated silica-coated magnetic beads (commercially available or homemade) can be directly provided.
所述生物素磁性微球可通过以下步骤制备：(1)提供或者制备磁性微球本体，所述磁性微球本体的外表面具有反应性基团R 1；(2)在所述反应性基团R 1的基础上连接上具有线性主链和大量支链的聚合物，所述线性主链的一端与所述反应性基团R 1共价连接；(3)在所述支链的末端连接生物素或生物素类似物。 The biotin magnetic microspheres can be prepared by the following steps: (1) providing or preparing a magnetic microsphere body, the outer surface of the magnetic microsphere body has a reactive group R 1 ; (2) in the reactive group On the basis of the group R 1 , a polymer with a linear main chain and a large number of branched chains is connected, and one end of the linear main chain is covalently connected to the reactive group R 1 ; (3) at the end of the branched chain Link biotin or biotin analogs.
以SiO 2包裹的磁性材料作为磁性微球本体为例，所述生物素磁性微球的制备过程可通过以下步骤制备：(1)提供SiO 2包裹的磁性微球(市售或者自制)，进行SiO 2的活化修饰生成反应性基团R 1；(2)在反应性基团R 1进行聚合反应(比如以丙烯酸或丙烯酸钠作为单体分子)，生成具有线性主链和大量支链的聚合物，并在所述支链的末端带有功能基团F 1；(3)将生物素或生物素类似物连接到支链末端的功能基团F 1处。此时，在磁性微球本体共价连接的聚合物具有线性主链，线性主链的一端共价固定在反应性基团R 1处，并沿聚合物主链分布大量侧支链。 Taking the SiO 2 -coated magnetic material as the magnetic microsphere body as an example, the preparation process of the biotin magnetic microspheres can be prepared by the following steps: (1) Provide SiO 2 -coated magnetic microspheres (commercially available or homemade), and carry out Activation and modification of SiO 2 generate reactive group R 1 ; (2) carry out a polymerization reaction on reactive group R 1 (such as using acrylic acid or sodium acrylate as a monomer molecule) to generate a polymer with a linear main chain and a large number of branched chains and a functional group F 1 at the end of the branched chain; (3) connecting biotin or a biotin analog to the functional group F 1 at the end of the branched chain. At this time, the polymer covalently connected to the magnetic microsphere body has a linear main chain, one end of the linear main chain is covalently fixed at the reactive group R1, and a large number of side branches are distributed along the main polymer chain.
2.1.2.典型举例2.1.2. Typical example
一种典型的所述生物素磁性微球的制备方法，包括以下步骤：A typical preparation method of the biotin magnetic microspheres, comprising the following steps:
步骤(1)：提供磁性微球本体，对磁性微球本体进行化学修饰，将氨基引入到磁性微球本体的外表面，形成氨基修饰磁性微球A。Step (1): providing a magnetic microsphere body, chemically modifying the magnetic microsphere body, and introducing amino groups into the outer surface of the magnetic microsphere body to form amino-modified magnetic microspheres A.
一些优选方式中，利用偶联剂对磁性微球本体进行化学修饰。In some preferred manners, the magnetic microsphere body is chemically modified with a coupling agent.
一些优选方式中，所述偶联剂为氨基化硅烷偶联剂。In some preferred embodiments, the coupling agent is an aminated silane coupling agent.
一些优选方式中，所述磁性微球本体为SiO 2包裹的磁性材料，利用硅烷偶联剂对磁性微球本体进行化学修饰；所述硅烷偶联剂在一些优选方式中为氨基化硅烷偶联剂。 In some preferred modes, the magnetic microsphere body is a magnetic material wrapped with SiO 2 , and the magnetic microsphere body is chemically modified by a silane coupling agent; in some preferred modes, the silane coupling agent is aminated silane coupling agent.
步骤(2)：利用羧基与氨基之间的共价反应将丙烯酸分子共价偶联到所述磁性微球A的外表面，引入碳碳双键，形成含碳碳双键磁性微球B。Step (2): Covalently couple the acrylic acid molecule to the outer surface of the magnetic microsphere A by the covalent reaction between the carboxyl group and the amino group, and introduce a carbon-carbon double bond to form the carbon-carbon double bond-containing magnetic microsphere B.
步骤(3)：利用碳碳双键的聚合反应，将丙烯酸类单体分子(如丙烯酸钠)进行聚合，获得的丙烯酸类聚合物为支链型聚合物，具有线性主链和含有功能基团F 1的支链，聚合物通过线性主链的一端共价偶联于磁性微球B的外表面，形成丙烯酸类聚合物修饰磁性微球C。该步骤可以在不加交联剂的条件下进行。 Step (3): polymerize acrylic monomer molecules (such as sodium acrylate) by utilizing the polymerization reaction of carbon-carbon double bonds, and the obtained acrylic polymer is a branched polymer with a linear main chain and functional groups. For the branched chain of F 1 , the polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain to form an acrylic polymer modified magnetic microsphere C. This step can be carried out without adding a crosslinking agent.
所述丙烯酸类单体分子、聚合物支链的功能基团的定义见“名词和术语”部分。The definitions of the acrylic monomer molecules and the functional groups of the polymer branches are shown in the section "Terms and Terms".
一些优选方式中，所述功能基团F 1为羧基、羟基、氨基、巯基、甲酸盐、铵盐、羧基的盐形式、氨基的盐形式、甲酸酯基，或前述功能基团的组合；所述“功能基团的组合”指一个磁性微球的外表面的所有聚合物的所有支链所含有的功能基团，其种类可以为一种 或一种以上。与第一方面定义的“功能基团的组合”的含义是一致的。 In some preferred modes, the functional group F 1 is a carboxyl group, a hydroxyl group, an amino group, a sulfhydryl group, a formate, an ammonium salt, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of the foregoing functional groups ; The "combination of functional groups" refers to the functional groups contained in all the branches of all polymers on the outer surface of a magnetic microsphere, and the types may be one or more. The meaning of "combination of functional groups" defined in the first aspect is consistent.
另一些优选方式中，所述功能基团为特异性结合位点。In other preferred embodiments, the functional group is a specific binding site.
步骤(4)：通过所述聚合物的支链含有的功能基团F 1，将生物素或生物素类似物共价偶联到聚合物支链末端，得到结合有生物素或生物素类似物的生物磁性微球(一种生物素磁性微球)。制备得到的生物磁性微球中，通过丙烯酸类聚合物(具有聚丙烯酸骨架)提供可结合生物素的大量位点。 Step (4): Covalently couple biotin or a biotin analog to the end of the polymer branch through the functional group F 1 contained in the branch chain of the polymer to obtain a biotin or biotin analog combined of biomagnetic microspheres (a biotin magnetic microsphere). In the prepared biomagnetic microspheres, a large number of biotin-binding sites are provided by an acrylic polymer (having a polyacrylic acid backbone).
2.1.3.具体实施方式2.1.3. Specific implementation
制备所述生物素磁性微球的一种具体实施方式如下。A specific embodiment for preparing the biotin magnetic microspheres is as follows.
具体地，以丙烯酸类聚合物提供线性主链和大量支链为例，本发明提供如下一种具体实施方式：以二氧化硅包裹的四氧化三铁磁珠作为生物磁性微球的本体；先利用偶联剂3-氨基丙基三乙氧基硅烷(APTES，CAS：919-30-2，一种氨基化偶联剂，也是一种硅烷偶联剂，更具体的为一种氨基化硅烷偶联剂)对二氧化硅包裹的四氧化三铁磁珠进行化学修饰，将氨基引入到磁珠的外表面，完成对SiO 2的活化修饰，得到氨基修饰磁性微球A；然后利用羧基与氨基之间的共价反应将固定化分子(丙烯酸分子，提供一个碳碳双键和一个反应性基团羧基)共价偶联到磁珠外表面，从而将碳碳双键引入到磁珠的外表面，得到含碳碳双键磁性微球B；然后再利用碳碳双键的聚合反应进行丙烯酸类单体分子(如丙烯酸钠)的聚合，进行聚合反应的同时把聚合产物共价偶联到磁珠外表面，完成在SiO 2处连接聚合物(共价连接方式)，得到丙烯酸类聚合物修饰磁性微球C；这里的固定化分子为丙烯酸分子，一个固定化分子仅引出一个聚合物分子，同时也仅引出一条聚合物线性主链；以丙烯酸钠作为单体分子为例，聚合产物为聚丙烯酸钠，其主链为线性的聚烯烃主链，并且沿着主链共价连接有大量侧支链COONa，支链含有的功能基团亦为COONa；此处的聚合反应并不使用N,N'-亚甲基双丙烯酰胺(CAS：110-26-9)等交联剂，避免分子链相互交联成网状聚合物，而是在不加交联剂的条件下使聚合产物产生线状主链。如果分子链相互交联成网状聚合物，则会形成多孔结构，影响目标蛋白的洗脱效率。 Specifically, taking the acrylic polymer providing a linear main chain and a large number of branched chains as an example, the present invention provides the following specific embodiment: a silicon dioxide-wrapped triferromagnetic bead is used as the body of the biomagnetic microsphere; Using coupling agent 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2, an aminated coupling agent, also a silane coupling agent, more specifically an aminated silane Coupling agent) chemically modified the silica-wrapped ferrite magnetic beads, and introduced amino groups to the outer surface of the magnetic beads to complete the activation modification of SiO 2 to obtain amino-modified magnetic microspheres A; The covalent reaction between amino groups covalently couples the immobilized molecule (acrylic acid molecule, providing a carbon-carbon double bond and a reactive group carboxyl group) to the outer surface of the magnetic bead, thereby introducing the carbon-carbon double bond into the magnetic bead. On the outer surface, a carbon-carbon double bond-containing magnetic microsphere B is obtained; then the polymerization of acrylic monomer molecules (such as sodium acrylate) is carried out by the polymerization reaction of carbon-carbon double bonds, and the polymerization product is covalently coupled during the polymerization reaction. To the outer surface of the magnetic bead, the polymer is connected at SiO2 (covalent connection), and the acrylic polymer modified magnetic microsphere C is obtained; the immobilized molecule here is an acrylic molecule, and one immobilized molecule only leads to one polymer At the same time, only one linear polymer main chain is drawn; taking sodium acrylate as a monomer molecule as an example, the polymerization product is sodium polyacrylate, and its main chain is a linear polyolefin main chain, and there are covalently connected along the main chain. A large number of side chain COONa, the functional group contained in the branch is also COONa; the polymerization reaction here does not use cross-linking agents such as N,N'-methylenebisacrylamide (CAS: 110-26-9), To avoid the cross-linking of molecular chains to form a network polymer, the polymer product produces a linear main chain without adding a cross-linking agent. If the molecular chains are cross-linked to form a network polymer, a porous structure will be formed, which affects the elution efficiency of the target protein.
一些优选方式中，制备磁性微球B时所用丙烯酸的用量为0.002～20mol/L。In some preferred modes, the amount of acrylic acid used in the preparation of the magnetic microspheres B is 0.002-20 mol/L.
一些优选方式中，制备磁性微球C时所用丙烯酸钠的用量为0.53～12.76mol/L。In some preferred modes, the amount of sodium acrylate used in the preparation of magnetic microspheres C is 0.53-12.76 mol/L.
所述生物磁性微球的外表面还可以采用氨基化以外的其他活化修饰方式。比如，上述氨基化的生物磁性微球(氨基修饰磁性微球A)，还可进一步与酸酐或其他修饰分子反应，从而实现对生物磁性微球外表面羧基化或其他活化方式的化学修饰。The outer surface of the biomagnetic microspheres can also be modified by activation other than amination. For example, the aminated biomagnetic microspheres (amino-modified magnetic microspheres A) can be further reacted with acid anhydrides or other modified molecules, so as to realize chemical modification of the outer surface of the biomagnetic microspheres by carboxylation or other activation methods.
所述固定化分子，是将聚合物的主链共价固定到磁珠外表面的小分子。固定化小分子没有特别限制，只要满足其一端共价偶联到磁珠外表面，另一端能够引发聚合反应，包括均聚反应或者共聚反应或者缩聚反应，或者另一端能够共聚偶联聚合物的线性主链末端即可。The immobilized molecule is a small molecule that covalently fixes the main chain of the polymer to the outer surface of the magnetic bead. The immobilized small molecule is not particularly limited, as long as one end of the molecule is covalently coupled to the outer surface of the magnetic bead, and the other end can initiate a polymerization reaction, including homopolymerization, copolymerization or polycondensation, or the other end can be copolymerized with a coupled polymer. Linear backbone ends are sufficient.
所述固定化分子，允许仅引出单一的一条聚合物线性主链，也允许引出两条或更多条聚合物线性主链，只要不导致链堆积和/或不导致滞留比例增大即可。优选地，一个固定化分子仅引出一个聚合物分子，并仅引出一条聚合物线性主链。The immobilized molecule allows the extraction of only a single linear polymer chain, and also allows the extraction of two or more linear polymer chains, as long as it does not lead to chain stacking and/or does not lead to an increase in the retention ratio. Preferably, one immobilized molecule leads only one polymer molecule, and only one linear polymer backbone.
一些优选方式中，所述固定化分子，允许仅引出单一的一条聚合物线性主链，也允许引出两条或更多条聚合物线性主链，只要不导致链堆积和/或不导致滞留比例增大即 可。优选地，一个固定化分子仅引出一个聚合物分子，并仅引出一条聚合物线性主链。In some preferred modes, the immobilized molecule allows the extraction of only a single polymer linear backbone, and also allows the extraction of two or more polymer linear backbones, as long as it does not lead to chain stacking and/or does not lead to retention ratios Just increase it. Preferably, one immobilized molecule leads only one polymer molecule, and only one linear polymer backbone.
另一些优选方式中，作为聚合的单体单元的所述丙烯酸类单体分子还可以为丙烯酸、丙烯酸盐、丙烯酸酯、甲基丙烯酸、甲基丙烯酸盐、甲基丙烯酸酯类单体之一或其组合。In other preferred modes, the acrylic monomer molecule as the polymerized monomer unit may also be one of acrylic acid, acrylic acid salt, acrylic acid ester, methacrylic acid, methacrylic acid salt, methacrylic acid ester monomer or its combination.
作为本发明的其他实施方式，所述丙烯酸类聚合物还可以用其他聚合物代替。选取的标准是：所形成的聚合物具有一条线性主链，沿着主链分布大量侧支链，并且在侧支链上携带有功能基团可供后续化学修饰；也即针对磁珠外表面的一个结合位点，通过分布于聚合物线性主链侧端的支链提供大量的功能基团。例如ε-聚赖氨酸链、α-聚赖氨酸链、γ-聚谷氨酸、聚天门冬氨酸链、天冬氨酸/谷氨酸共聚物等结构。As other embodiments of the present invention, the acrylic polymer can also be replaced by other polymers. The selection criteria are: the formed polymer has a linear main chain, a large number of side branches are distributed along the main chain, and the side branches carry functional groups for subsequent chemical modification; that is, for the outer surface of the magnetic beads One of the binding sites of the polymer, which provides a large number of functional groups through the branches distributed at the side ends of the linear main chain of the polymer. For example, ε-polylysine chain, α-polylysine chain, γ-polyglutamic acid, polyaspartic acid chain, aspartic acid/glutamic acid copolymer and other structures.
将上述聚合物的其他替代方式的聚合物分子引入到所述生物磁性微球外表面的方法：根据聚合物替代物的化学结构及其侧支链活性基团的类型，选择适合的生物磁性微球外表面的活化修饰方式、固定化分子的种类、单体分子类型，进行合适的化学反应将大量的位于支链的活性基团引入到生物磁性微球的外表面。The method of introducing polymer molecules of other alternatives of the above-mentioned polymers to the outer surface of the biomagnetic microsphere: according to the chemical structure of the polymer substitute and the type of side branched active groups, a suitable biomagnetic microsphere is selected. The activation and modification method of the outer surface of the sphere, the type of immobilized molecule, the type of monomer molecule, and the appropriate chemical reaction will introduce a large number of active groups located in the branched chain into the outer surface of the biomagnetic microsphere.
将丙烯酸类聚合物分子(如：聚丙烯酸钠线性分子链)共价偶联到磁珠外表面后，以支链末端的功能基团提供活化位点，或者在连接生物素或生物素类似物分子之前，根据反应需要，可以对聚合物分子的支链功能基团进行活化，使其具有反应活性，形成活化位点；将1,3-丙二胺共价偶联到聚合物支链的活化位点(每个单体丙烯酸类单元结构可提供一个活化位点)，形成新的功能基团(氨基)，然后再利用羧基与氨基之间的酰胺化共价反应将生物素或生物素类似物分子共价偶联到聚合物支链末端的新功能基团，完成生物素或生物素类似物共价连接到聚合物的支链末端。以生物素作为纯化介质为例，获得生物素或生物类似物修饰的生物磁性微球D；一个生物素分子能够提供一个特异性结合位点。以聚合物支链的功能基团为COONa为例，此时采用丙烯酸钠作为单体分子，与1,3-丙二胺进行共价反应前，可以先进行羧基活化，可采用现有的羧基活化方法，例如：加入EDC 2HCl和NHS。After covalently coupling acrylic polymer molecules (such as: sodium polyacrylate linear molecular chains) to the outer surface of the magnetic beads, the functional groups at the end of the branched chains provide activation sites, or connect biotin or biotin analogs. Before the molecule, according to the needs of the reaction, the branched functional group of the polymer molecule can be activated to make it reactive and form an activation site; 1,3-propanediamine can be covalently coupled to the branched polymer Activation site (each monomeric acrylic unit structure can provide one activation site) to form a new functional group (amino group), and then use the amidation covalent reaction between the carboxyl group and the amino group to convert biotin or biotin The analog molecule is covalently coupled to the new functional group at the end of the polymer branch, completing the covalent attachment of biotin or biotin analogs to the branch end of the polymer. Taking biotin as a purification medium as an example, biotin or biosimilar modified biomagnetic microspheres D are obtained; one biotin molecule can provide a specific binding site. Taking COONa as the functional group of the branch chain of the polymer as an example, at this time, sodium acrylate is used as the monomer molecule, and before the covalent reaction with 1,3-propanediamine, the carboxyl group can be activated first, and the existing carboxyl group can be used. Activation method, eg: adding EDC 2HCl and NHS.
2.1.3.1.制备丙烯酸类聚合物修饰磁性微球2.1.3.1. Preparation of acrylic polymer-modified magnetic microspheres
制备磁性微球A：对二氧化硅包裹的四氧化三铁磁性微球的水溶液，用无水乙醇清洗磁性微球，加入3-氨基丙基三乙氧基硅烷(APTES，偶联剂)的乙醇溶液，反应，清洗，在磁性微球外表面引入大量氨基。Preparation of magnetic microspheres A: To the aqueous solution of silica-coated ferric oxide magnetic microspheres, wash the magnetic microspheres with absolute ethanol, add 3-aminopropyltriethoxysilane (APTES, coupling agent) Ethanol solution, reaction, cleaning, and introducing a large number of amino groups on the outer surface of the magnetic microspheres.
制备磁性微球B：向丙烯酸的水溶液中，加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC·HCl)和N-羟基琥珀酰亚胺(NHS)进行羧基活化，活化后加入至含磁性微球A的水溶液中。丙烯酸上活化的羧基和磁性微球外表面的氨基形成共价键连(酰胺键)，在磁性微球外表面引入大量碳碳双键。Preparation of magnetic microspheres B: To the aqueous solution of acrylic acid, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide ( NHS) to activate the carboxyl group, and after activation, it was added to the aqueous solution containing magnetic microspheres A. The activated carboxyl group on the acrylic acid forms a covalent bond (amide bond) with the amino group on the outer surface of the magnetic microsphere, and a large number of carbon-carbon double bonds are introduced into the outer surface of the magnetic microsphere.
制备磁性微球C：将丙烯酸类单体分子的水溶液加入到磁性微球B中，添加引发剂，进行碳碳双键的聚合反应。丙烯酸类单体分子中的碳碳双键和磁性微球表面的碳碳双键进行开键聚合，丙烯酸类聚合物分子共价键连至磁性微球外表面，其中，丙烯酸类聚合物含有羧基类功能基团；所述羧基类功能基团可以以羧基、甲酸盐、甲酸酯等形式存在。优选方式之一，以甲酸钠方式存在，此时采用如丙烯酸钠或甲基丙烯酸钠作为单体分子。另优选方式之一，以甲酸酯方式存在，此时采用如丙烯酸酯或甲基丙烯酸酯作为单体分子。甲酸盐和甲酸酯可以在经羧基活化后获得较好的反应活性。Preparation of magnetic microspheres C: adding an aqueous solution of acrylic monomer molecules into magnetic microspheres B, adding an initiator, and carrying out a polymerization reaction of carbon-carbon double bonds. The carbon-carbon double bond in the acrylic monomer molecule and the carbon-carbon double bond on the surface of the magnetic microsphere undergo bond opening polymerization, and the acrylic polymer molecule is covalently bonded to the outer surface of the magnetic microsphere, wherein the acrylic polymer contains a carboxyl group The carboxyl-like functional group can exist in the form of carboxyl, formate, formate and the like. One of the preferred ways is to exist in the form of sodium formate, and in this case, sodium acrylate or sodium methacrylate is used as the monomer molecule. Another preferred way is to exist in the form of formate, and at this time, acrylate or methacrylate is used as the monomer molecule. Formate and formate can obtain better reactivity after activation by carboxyl group.
2.1.3.2.制备生物磁性微球D(生物素修饰)2.1.3.2. Preparation of biomagnetic microspheres D (biotin-modified)
磁性微球C的溶液：加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC·HCl)和N-羟基琥珀酰亚胺(NHS)，对微球外表面的聚合物分子侧支链的羧基类功能基团进行羧基活化，然后加入丙二胺的水溶液，进行偶合反应，在丙烯酸类聚合物分子的侧支链羧基位置接枝丙二胺，将聚合物侧支链的功能基团由羧基转化为氨基。Magnetic microsphere C solution: add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS), The carboxyl functional group of the side chain of the polymer molecule on the outer surface of the sphere is activated by the carboxyl group, and then an aqueous solution of propylene diamine is added to carry out a coupling reaction, and propylene diamine is grafted at the position of the side branch carboxyl group of the acrylic polymer molecule, The functional groups of the side chains of the polymer are converted from carboxyl groups to amino groups.
生物素的水溶液：加入1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺，对生物素分子中的羧基进行活化，然后加入到含有磁性微球C的水溶液中，在磁性微球C外表面的聚合物的侧支链的新生功能基团(氨基)位置共价结合生物素，得到在丙烯酸类聚合物的大量侧支链分别连接有生物素分子的生物磁性微球D。Aqueous solution of biotin: add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to activate the carboxyl group in the biotin molecule, and then add In the aqueous solution containing magnetic microspheres C, biotin is covalently bound to the position of the newly-born functional group (amino) of the side chain of the polymer on the outer surface of the magnetic microsphere C, resulting in a large number of side branches in the acrylic polymer Biomagnetic microspheres D connected with biotin molecules, respectively.
2.1.3.3.优选例2.1.3.3. Preferred example
一些优选方式中，制备上述生物磁性微球D的方法如下：In some preferred modes, the method for preparing the above-mentioned biomagnetic microspheres D is as follows:
首先，量取0.5～1000mL(20％，v/v)二氧化硅包裹的四氧化三铁磁性微球的水溶液，用无水乙醇清洗磁性微球，将10～300mL 3-氨基丙基三乙氧基硅烷(APTES，CAS：919-30-2)的乙醇溶液(5％～50％，v/v)加入到上述清洗后的磁性微球中，反应2～72小时，用无水乙醇和蒸馏水清洗磁性微球，获得氨基修饰磁性微球A。First, measure 0.5-1000mL (20%, v/v) aqueous solution of ferric oxide magnetic microspheres coated with silica, wash the magnetic microspheres with absolute ethanol, and add 10-300mL 3-aminopropyltriethyl Ethanol solution (5%-50%, v/v) of oxysilane (APTES, CAS: 919-30-2) was added to the above-mentioned cleaned magnetic microspheres, and reacted for 2-72 hours. The magnetic microspheres were washed with distilled water to obtain amino-modified magnetic microspheres A.
移取1.0×10 -4～1mol丙烯酸，加入到pH4～6溶液X中(溶液X：终浓度为0.01～1mol/L 2-吗啉乙磺酸(CAS：4432-31-9)、0.1～2mol/L NaCl的水溶液)，加入0.001～0.5mol 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC·HCl，CAS：25952-53-8)和0.001～0.5mol N-羟基琥珀酰亚胺(NHS,CAS：6066-82-6)，反应3～60min。将上述溶液加入到混有0.5～50mL磁性微球A的pH7.2～7.5PBS缓冲溶液中，反应1～48小时，用蒸馏水清洗磁性微球，获得碳碳双键修饰的磁性微球B。 Pipette 1.0×10 -4 ～1 mol of acrylic acid and add it to pH4～6 solution X (solution X: the final concentration is 0.01～1mol/L 2-morpholineethanesulfonic acid (CAS: 4432-31-9), 0.1～ 2mol/L NaCl aqueous solution), add 0.001～0.5mol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, CAS: 25952-53-8) and 0.001～0.5mol N-hydroxysuccinimide (NHS, CAS: 6066-82-6), react for 3～60min. The above solution is added to a pH 7.2-7.5 PBS buffer solution mixed with 0.5-50 mL of magnetic microspheres A, reacted for 1-48 hours, and the magnetic microspheres are washed with distilled water to obtain magnetic microspheres B modified with carbon-carbon double bonds.
取0.5～50mL磁性微球B，加入0.5～200mL 5％～30％(w/v)丙烯酸钠溶液，再加入10μL～20mL 2％～20％(w/v)过硫酸铵溶液和1μL～1mL四甲基乙二胺，反应3～60分钟后用蒸馏水清洗磁性微球，获得聚丙烯酸钠修饰的磁性微球C。Take 0.5-50mL magnetic microsphere B, add 0.5-200mL 5%-30% (w/v) sodium acrylate solution, then add 10μL-20mL 2%-20% (w/v) ammonium persulfate solution and 1μL-1mL Tetramethylethylenediamine, after 3-60 minutes of reaction, the magnetic microspheres are washed with distilled water to obtain magnetic microspheres C modified with sodium polyacrylate.
取0.5～50mL磁性微球C转移至pH4～6溶液X中，加入0.001～0.5mol 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC·HCl)和0.001～0.5mol N-羟基琥珀酰亚胺(NHS)，反应3～60min。然后加入溶有0.0001～1mol 1,3-丙二胺的pH7.2～7.5PBS缓冲溶液，反应1～48小时。用蒸馏水清洗后加入PBS缓冲溶液，将磁性微球C中聚合物侧支链的COONa转化为氨基功能基团；称取1.0×10 -6～3.0310 -4mol生物素至溶液X中，加入2.0×10 -6～1.5×10 -3mol 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和2.0×10 -6～1.5×10 -3mol N-羟基琥珀酰亚胺，反应3～60min。然后加入至上述清洗后的磁性微球溶液中，反应1～48小时，蒸馏水清洗后得到生物素修饰的磁性微球D。 Transfer 0.5-50 mL of magnetic microspheres C to pH 4-6 solution X, add 0.001-0.5 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and 0.001～0.5mol N-hydroxysuccinimide (NHS), and react for 3～60min. Then, a pH 7.2-7.5 PBS buffer solution in which 0.0001-1 mol of 1,3-propanediamine was dissolved was added, and the reaction was carried out for 1-48 hours. After washing with distilled water, add PBS buffer solution to convert COONa of polymer side chain in magnetic microsphere C into amino functional group; weigh 1.0×10 -6 ~ 3.0310 -4 mol biotin into solution X, add 2.0 ×10 -6 ～1.5×10 -3 mol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 2.0×10 -6 ～1.5×10 -3 mol N-hydroxyl Succinimide, the reaction is 3～60min. Then, it is added to the above-mentioned cleaned magnetic microsphere solution, reacted for 1 to 48 hours, and washed with distilled water to obtain biotin-modified magnetic microsphere D.
2.2.基于生物磁性微球D制备所述抗体磁性微球的优选实施方式2.2. The preferred embodiment of preparing the antibody magnetic microspheres based on the biomagnetic microspheres D
将生物素修饰的生物磁性微球D加入到亲和素-抗体型标签连接复合物(例如，antiEGFP-mScarlet-Tamvavidin2，一种抗体型融合蛋白，且为纳米抗体的融合蛋白)的融合蛋白溶液中，混合孵育。通过亲和素(如Streptavidin或Tamvavidin2)与生物素特异性结合，将所述抗体型标签固定到生物磁性微球D的外表面的聚合物支链的端基，得到结合有亲和素-抗体型标签的生物磁性微球K。其中，亲和素-抗体型标签的融合蛋白，可通过 IVTT反应进行体外无细胞蛋白合成获得。此时，生物磁性微球D与IVTT反应后所得上清液混合，通过生物磁性微球D外表面的生物素与溶液中的亲和素融合蛋白之间的特异性结合作用，实现抗体型标签的结合。Add biotin-modified biomagnetic microspheres D to a fusion protein solution of avidin-antibody-type tag-linked complexes (for example, antiEGFP-mScarlet-Tamvavidin2, an antibody-type fusion protein, and a fusion protein of nanobodies) , mixed and incubated. Through the specific binding of avidin (such as Streptavidin or Tamvavidin2) to biotin, the antibody-type tag is fixed to the end group of the polymer branch on the outer surface of the biomagnetic microsphere D, and the avidin-antibody is obtained. Type-labeled biomagnetic microspheres K. Among them, the fusion protein of avidin-antibody tag can be obtained by in vitro cell-free protein synthesis by IVTT reaction. At this time, the supernatant obtained after the reaction of the biomagnetic microspheres D and IVTT is mixed, and the antibody-type tag is realized through the specific binding between the biotin on the outer surface of the biomagnetic microspheres D and the avidin fusion protein in the solution. combination.
2.3.生物磁性微球外表面的抗体型标签结合量可以通过以下方法确定(以荧光蛋白mScarlet标记为例)：2.3. The binding amount of the antibody-type label on the outer surface of the biomagnetic microspheres can be determined by the following method (taking fluorescent protein mScarlet labeling as an example):
首先，在抗体型融合蛋白的溶液与生物素磁珠(以生物素磁性微球为例)的结合反应结束后，用磁铁将结合了抗体型融合蛋白的生物磁性微球K吸附、沉降。然后，分离收集液相，记为流穿液。此时，液相中的抗体型融合蛋白浓度减少。通过测定结合生物磁性微球前后IVTT反应所得上清液中荧光值的变化值，计算得到结合在生物磁性微球上的荧光强度，换算得出抗体型融合蛋白浓度。当流穿液中的抗体型融合蛋白浓度，与生物磁性微球孵育前的IVTT溶液中的抗体型融合蛋白相比，基本不再变化时，意味着生物磁性微球对抗体型融合蛋白的吸附趋于饱和，相应的的荧光值不再有明显改变。可以采用mScarlet蛋白的纯品，建立荧光值与mScarlet蛋白浓度的标准曲线，从而定量计算结合在生物磁性微球上的抗体型融合蛋白(如：链霉亲和素-纳米抗体，antiEGFP-mScarlet-Tamvavidin2纳米抗体融合蛋白)的含量、浓度。First, after the binding reaction between the antibody-based fusion protein solution and biotin magnetic beads (take biotin magnetic microspheres as an example), the antibody-based fusion protein-bound biomagnetic microspheres K are adsorbed and sedimented with a magnet. Then, the liquid phase was separated and collected, which was recorded as the flow-through. At this time, the concentration of the antibody-type fusion protein in the liquid phase decreases. By measuring the change value of the fluorescence value in the supernatant obtained from the IVTT reaction before and after binding the biomagnetic microspheres, the fluorescence intensity bound on the biomagnetic microspheres was calculated, and the concentration of the antibody-type fusion protein was obtained by conversion. When the concentration of the antibody-based fusion protein in the flow-through solution basically does not change compared with the antibody-based fusion protein in the IVTT solution before the incubation of the biomagnetic microspheres, it means that the adsorption of the antibody-based fusion protein by the biomagnetic microspheres tends to increase. At saturation, the corresponding fluorescence values no longer change significantly. The pure product of mScarlet protein can be used to establish a standard curve between the fluorescence value and the concentration of mScarlet protein, so as to quantitatively calculate the antibody-type fusion protein (such as: streptavidin-nanobody, antiEGFP-mScarlet- Tamvavidin2 nanobody fusion protein) content and concentration.
带有抗体型标签的生物磁性微球K进行目标物的分离纯化，可通过以下方法计算目标物结合量：将生物磁性微球K与目标物(以目标蛋白为例)的溶液(比如采用体外蛋白合成体系表达目标蛋白获得)进行孵育，反应结束后，使用洗脱缓冲液将目标蛋白从磁珠上洗脱下来，分离得到的目标蛋白存在于洗脱液中。采用适当的方法测量洗脱液中目标蛋白的蛋白浓度，进而计算得到分离纯化的产量、产率。The biomagnetic microspheres K with antibody-type tags are used for the separation and purification of the target, and the binding amount of the target can be calculated by the following method: a solution of the biomagnetic microspheres K and the target (take the target protein as an example) (for example, in vitro After the reaction is completed, the target protein is eluted from the magnetic beads using an elution buffer, and the separated target protein exists in the eluate. Use an appropriate method to measure the protein concentration of the target protein in the eluate, and then calculate the yield and yield of the separation and purification.
2.4.纯化介质的更换2.4. Replacement of purification medium
洗脱更换抗体型标签：洗脱亲和素的同时实现所述抗体型标签的同步脱离，因此，也是亲和素-抗体型标签的更换。Elution and replacement of antibody-type tags: synchronous detachment of the antibody-type tags is achieved while avidin is eluted, so it is also the replacement of avidin-antibody-type tags.
以基于“生物素修饰的生物磁性微球D”的生物磁性微球K(生物素-亲和素-抗体型标签连接方式)为例，向生物磁性微球K中加入变性缓冲液(含有尿素和十二烷基硫酸钠)，95℃金属浴孵育，洗脱掉与生物磁性微球K上的生物素相结合的亲和素-抗体型标签的融合蛋白，得到再生的生物磁性微球D(释放出聚合物支链末端的生物素的结合位点)，然后向再生的生物磁性微球D中加入新鲜的含有亲和素-抗体型标签的融合蛋白的溶液(如antiEGFP-mScarlet-Tamvavidin2的IVTT反应后的上清液)，使被释放的生物磁性微球D的生物素结合位点重新结合新的亲和素-抗体型标签，重新在生物素与亲和素(如Tamvavidin2)之间形成非共价的特异性结合作用，从而实现抗体型标签的更换，得到再生的生物磁性微球K。Taking the biomagnetic microsphere K (biotin-avidin-antibody tag connection method) based on "biotin-modified biomagnetic microsphere D" as an example, adding denaturation buffer (containing urea) to the biomagnetic microsphere K and sodium dodecyl sulfate), incubate in a metal bath at 95°C, wash off the avidin-antibody tag fusion protein bound to biotin on biomagnetic microspheres K, and obtain regenerated biomagnetic microspheres D (Release the binding site of biotin at the end of the polymer branch), and then add a fresh solution of avidin-antibody-type tag fusion protein (such as antiEGFP-mScarlet-Tamvavidin2) to the regenerated biomagnetic microspheres D The supernatant after the IVTT reaction), so that the biotin-binding site of the released biomagnetic microsphere D re-bonds the new avidin-antibody-type tag, and re-bonds the biotin and avidin (such as Tamvavidin2) A non-covalent specific binding effect is formed between the two, thereby realizing the replacement of the antibody-type label, and obtaining the regenerated biomagnetic microsphere K.
2.5.磁性微球的位置控制2.5. Position control of magnetic microspheres
在制得本发明所述生物磁性微球后，可以简便地利用磁铁沉降磁性微球，去除液相，清洗去除吸附的杂蛋白或/和其它杂质。After the biomagnetic microspheres of the present invention are prepared, the magnetic microspheres can be easily settled by a magnet, the liquid phase can be removed, and the adsorbed impurity proteins or/and other impurities can be removed by washing.
通过控制磁性微球的尺寸和聚合物的化学参数与结构参数，所述磁性微球可以稳定地悬浮在液相中，能够在两天甚至更久的时间内不沉降。而且可以实现在不进行持续搅拌的条件下，稳定地悬浮在液态体系中。一方面，磁性微球可以控制在几微米甚至1微米以下 的纳米级尺寸，另一方面，可以调节磁性微球外表面的聚合物的接枝密度，还可以调节聚合物本身的亲水性、结构类型、流体力学半径、链长度、支链数量、支链长度等特征，从而更好地控制磁性微球系统在体系中的悬浮性能，实现磁性微球系统与体外蛋白合成反应混合体系的充分接触。本发明的生物磁性微球的一种优选的尺寸为约1微米。By controlling the size of the magnetic microspheres and the chemical and structural parameters of the polymer, the magnetic microspheres can be stably suspended in the liquid phase, and can not settle for two days or more. Moreover, it can be stably suspended in the liquid system without continuous stirring. On the one hand, the magnetic microspheres can be controlled to a nanoscale size of several micrometers or even less than 1 micrometer; on the other hand, the graft density of the polymer on the outer surface of the magnetic microspheres can be adjusted, and the hydrophilicity, Structure type, hydrodynamic radius, chain length, number of branches, length of branches, etc., so as to better control the suspension performance of the magnetic microsphere system in the system, and realize the sufficient mixing system of the magnetic microsphere system and the in vitro protein synthesis reaction. touch. One preferred size of the biomagnetic microspheres of the present invention is about 1 micron.
2.6.制备抗体磁珠的具体优选例2.6. Specific preferred examples for preparing antibody magnetic beads
一些优选例中，所述生物磁性微球K的制备方法，包括以下步骤：In some preferred examples, the preparation method of the biomagnetic microspheres K comprises the following steps:
(1)对磁性微球本体进行化学修饰，将氨基引入到磁性微球本体的外表面，形成氨基修饰磁性微球A；当所述磁性微球本体为SiO 2包裹的磁性材料时，所述偶联剂优选为氨基化硅烷偶联剂。 (1) chemically modifying the magnetic microsphere body, and introducing amino groups to the outer surface of the magnetic microsphere body to form amino-modified magnetic microspheres A; when the magnetic microsphere body is a magnetic material wrapped by SiO 2 , the The coupling agent is preferably an aminated silane coupling agent.
优选方式之一，利用偶联剂对磁性微球本体进行化学修饰。One of the preferred ways is to chemically modify the magnetic microsphere body with a coupling agent.
当所述磁性微球本体为SiO 2包裹的磁性材料时，可利用硅烷偶联剂对磁性微球本体进行化学修饰。所述硅烷偶联剂优选为氨基化硅烷偶联剂。 When the magnetic microsphere body is a magnetic material wrapped with SiO 2 , a silane coupling agent can be used to chemically modify the magnetic microsphere body. The silane coupling agent is preferably an aminated silane coupling agent.
(2)利用羧基与氨基之间的共价反应将丙烯酸分子共价偶联到所述磁性微球A的外表面，引入碳碳双键，形成含碳碳双键磁性微球B。(2) Covalently coupling acrylic acid molecules to the outer surface of the magnetic microspheres A by the covalent reaction between carboxyl groups and amino groups, and introducing carbon-carbon double bonds to form magnetic microspheres B containing carbon-carbon double bonds.
(3)在不加交联剂的条件下，利用碳碳双键的聚合反应，将丙烯酸类单体分子(如丙烯酸钠)进行聚合，获得的丙烯酸类聚合物具有线性主链和含有功能基团的支链，聚合物通过线性主链的一端共价偶联于磁性微球B的外表面，形成丙烯酸类聚合物修饰磁性微球C。(3) Under the condition of not adding a crosslinking agent, the polymerization of carbon-carbon double bonds is used to polymerize acrylic monomer molecules (such as sodium acrylate), and the obtained acrylic polymer has a linear main chain and contains functional groups. The polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain to form an acrylic polymer modified magnetic microsphere C.
所述丙烯酸类单体分子、聚合物支链的功能基团的定义见“名词和术语”部分。The definitions of the acrylic monomer molecules and the functional groups of the polymer branches are shown in the section "Terms and Terms".
优选地，所述功能基团为羧基、羟基、氨基、巯基、甲酸盐、铵盐、羧基的盐形式、氨基的盐形式、甲酸酯基，或前述功能基团的组合；所述“功能基团的组合”指一个磁性微球的外表面的所有聚合物的所有支链所含有的功能基团，其种类可以为一种或一种以上。与第一方面定义的“功能基团的组合”的含义是一致的。Preferably, the functional group is a carboxyl group, a hydroxyl group, an amino group, a sulfhydryl group, a formate, an ammonium salt, a salt form of a carboxyl group, a salt form of an amino group, a formate group, or a combination of the foregoing functional groups; the " "Combination of functional groups" refers to the functional groups contained in all branches of all polymers on the outer surface of a magnetic microsphere, and the types may be one or more. The meaning of "combination of functional groups" defined in the first aspect is consistent.
另优选地，所述功能基团为特异性结合位点。Also preferably, the functional group is a specific binding site.
(4)通过所述聚合物的支链含有的功能基团，将生物素或生物素类似物共价偶联到聚合物支链末端，得到结合有生物素或生物素类似物的生物磁性微球D(生物素或其类似物修饰的生物磁性微球D)。(4) Covalently couple biotin or biotin analog to the end of the polymer branch through the functional group contained in the branched chain of the polymer, to obtain a biomagnetic microparticle combined with biotin or biotin analog Sphere D (biomagnetic microsphere D modified with biotin or its analogs).
(5)将提供抗体型标签的原料与所述生物磁性微球D中所述聚合物支链末端的生物素或生物素类似物相连接，得到所述讼生物磁性微球K(抗体磁性微球)。(5) linking the raw material that provides the antibody-type label with the biotin or biotin analog at the end of the polymer branch in the biomagnetic microsphere D to obtain the biomagnetic microsphere K (antibody magnetic microsphere K). ball).
独立地可选地，包括(6)磁铁沉降生物磁性微球，去除液相，清洗。Independently and optionally, including (6) magnet sedimentation of biomagnetic microspheres, removal of liquid phase, and cleaning.
优选方式之一，所述提供抗体型标签的原料为亲和素或亲和素类似物与所述抗体型标签的共价连接复合物；更优选地，所述提供抗体型标签的原料为亲和素-抗体型标签共价连接复合物。In one preferred manner, the raw material for providing the antibody-type tag is avidin or a covalently linked complex of avidin analog and the antibody-type tag; more preferably, the raw material for providing the antibody-type tag is avidin Covalently linked complexes of covalentin-antibody-type tags.
独立地可选地，包括所述亲和素-抗体型标签共价连接复合物的更换。Independently and optionally, replacement of the covalently linked complex of the avidin-antibody-type tag is included.
优选地，所述提供抗体型标签的原料为亲和素-抗体型标签共价连接复合物。Preferably, the raw material for providing the antibody-type tag is an avidin-antibody-type tag covalently linked complex.
3.本发明还公开所述抗体磁性微球在分离纯化中的应用，优选在蛋白类物质的分离纯化中的应用(此时抗体型标签为抗蛋白的抗体)。3. The present invention also discloses the application of the antibody magnetic microspheres in separation and purification, preferably in the separation and purification of protein substances (in this case, the antibody-type tag is an anti-protein antibody).
实施例1生物磁性微球D的制备(结合生物素)Example 1 Preparation of biomagnetic microspheres D (combined with biotin)
制备二氧化硅包裹的磁性微球(也称为磁性微球本体、磁珠、玻璃珠)Preparation of silica-coated magnetic microspheres (also known as magnetic microsphere bodies, magnetic beads, glass beads)
将20g Fe 3O 4微球置入310mL乙醇和125mL水的混合溶剂中，加入45mL 28％(wt)氨水，逐滴加入22.5mL正硅酸乙酯，室温下搅拌反应24h，反应后用乙醇和水进行清洗。使用不同粒径(约1μm、10μm、100μm)的四氧化三铁微球作为原料，控制所得玻璃珠的粒径大小。所述不同粒径的四氧化三铁微球可通过常规技术手段制备。 Put 20g Fe 3 O 4 microspheres into a mixed solvent of 310mL ethanol and 125mL water, add 45mL 28% (wt) ammonia water, add 22.5mL ethyl orthosilicate dropwise, stir at room temperature for 24h, and use ethanol after the reaction Wash with water. Ferric oxide microspheres with different particle sizes (about 1 μm, 10 μm, and 100 μm) were used as raw materials to control the particle size of the obtained glass beads. The iron tetroxide microspheres with different particle sizes can be prepared by conventional technical means.
所制得的磁性微球用作修饰纯化介质或连接元件-纯化介质的基础原料，因此也称为磁性微球本体。The prepared magnetic microspheres are used as basic raw materials for modifying purification media or connecting elements-purification media, so they are also called magnetic microsphere bodies.
所制得的磁性微球具有磁性核心，能够通过磁力作用进行位置控制，实现移动、分散、沉降等操作，因此是一种广义的磁珠。The prepared magnetic microspheres have a magnetic core, which can control the position through the action of magnetic force, and realize operations such as movement, dispersion, and sedimentation, so it is a generalized magnetic bead.
所制得的磁性微球具有二氧化硅的包裹层，因此也称为玻璃珠，可以降低磁性核心对以下成分或组分的吸附：聚合物、纯化介质、体外蛋白合成体系各组分、核酸模板、蛋白表达产物等。The prepared magnetic microspheres have a coating layer of silica, so they are also called glass beads, which can reduce the adsorption of the following components or components by the magnetic core: polymers, purification media, components of in vitro protein synthesis systems, nucleic acids Templates, protein expression products, etc.
经过多次实验表明，磁性微球粒径约1μm时，易悬浮性，悬浮的持久性，以及对蛋白的结合效率都最好。用IVTT反应液提供目标蛋白的混合体系，对于目标蛋白的结合效率，磁性微球粒径约1μm时，相较于粒径10μm可提高50％以上，相较于粒径100μm可提高80％以上。After many experiments, it has been shown that when the particle size of the magnetic microspheres is about 1 μm, the ease of suspension, the persistence of suspension, and the binding efficiency to proteins are the best. Using the IVTT reaction solution to provide the mixed system of the target protein, the binding efficiency of the target protein can be increased by more than 50% compared with the particle size of 10 μm when the particle size of the magnetic microspheres is about 1 μm, and can be increased by more than 80% compared with the particle size of 100 μm .
以二氧化硅包裹的磁性微球通过以下步骤制备生物素磁珠。Silica-coated magnetic microspheres The biotin magnetic beads were prepared by the following procedure.
首先，量取50mL二氧化硅包裹的四氧化三铁磁性微球(磁性微球的粒径约为1μm)的水溶液，其固含量20％(v/v)，用磁铁沉降磁性微球，去除液相，每次用60mL无水乙醇清洗磁性微球，总共清洗5次。将100mL过量的3-氨基丙基三乙氧基硅烷(APTES，CAS：919-30-2)的乙醇溶液(25％，v/v)加入到上述清洗后的磁性微球中，在50℃水浴下机械搅拌48小时，之后在70℃水浴下机械搅拌2小时，用磁铁沉降磁性微球，去除液相，每次用60mL无水乙醇清洗磁性微球，总共清洗2次，然后每次用60mL蒸馏水清洗磁性微球，重复清洗3次，得磁性微球A。First, measure 50 mL of an aqueous solution of ferric oxide magnetic microspheres (the particle size of the magnetic microspheres is about 1 μm) wrapped in silica, the solid content of which is 20% (v/v), and settle the magnetic microspheres with a magnet to remove In the liquid phase, the magnetic microspheres were washed with 60 mL of absolute ethanol each time, for a total of 5 times. 100 mL of excess 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) in ethanol solution (25%, v/v) was added to the above cleaned magnetic microspheres at 50 °C Mechanical stirring in a water bath for 48 hours, followed by mechanical stirring in a water bath at 70°C for 2 hours, the magnetic microspheres were settled with a magnet, the liquid phase was removed, and the magnetic microspheres were washed with 60 mL of absolute ethanol each time, for a total of 2 times, and then each time with The magnetic microspheres were washed with 60 mL of distilled water, and the cleaning was repeated 3 times to obtain magnetic microspheres A.
其次，移取0.01mol丙烯酸加入100mL溶液X中(溶液X：终浓度为0.1mol/L 2-吗啉乙磺酸(CAS：4432-31-9)、0.5mol/L NaCl的水溶液)，加入0.04mol 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(CAS：25952-53-8)和0.04mol N-羟基琥珀酰亚胺(CAS：6066-82-6)，室温下搅拌混匀，搅拌反应15min，用NaHCO 3固体粉末将溶液的pH调至7.2，将调好pH的上述溶液加入到加有10mL磁性微球A的100mL PBS缓冲溶液中，在30℃水浴下机械搅拌20小时，用磁铁沉降磁性微球，去除液相，每次用60mL蒸馏水清洗磁性微球，重复清洗6次，得磁性微球B。 Next, pipette 0.01mol of acrylic acid into 100mL of solution X (solution X: the final concentration is 0.1mol/L 2-morpholineethanesulfonic acid (CAS: 4432-31-9), the aqueous solution of 0.5mol/L NaCl), add 0.04mol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (CAS: 25952-53-8) and 0.04mol N-hydroxysuccinimide (CAS: 6066-82 -6), stirring and mixing at room temperature, stirring and reacting for 15min, using NaHCO 3 solid powder to adjust the pH of the solution to 7.2, adding the above-mentioned pH-adjusted solution to 100 mL of PBS buffer solution with 10 mL of magnetic microspheres A, Mechanical stirring was carried out in a 30°C water bath for 20 hours, the magnetic microspheres were settled with a magnet, the liquid phase was removed, and the magnetic microspheres were washed with 60 mL of distilled water each time, and the cleaning was repeated 6 times to obtain magnetic microspheres B.
第三，取1mL磁性微球B，加入12mL 15％(w/v)的丙烯酸钠溶液，加入450μL 10％的过硫酸铵溶液和45μL的四甲基乙二胺，室温下反应30分钟，用磁铁沉降磁性微球，去除液相，每次用10mL蒸馏水清洗磁性微球，总共清洗6次，得磁性微球C(丙烯酸类聚合物修饰的磁性微球C)。Third, take 1 mL of magnetic microspheres B, add 12 mL of 15% (w/v) sodium acrylate solution, add 450 μL of 10% ammonium persulfate solution and 45 μL of tetramethylethylenediamine, react at room temperature for 30 minutes, and use The magnetic microspheres were settled by a magnet, the liquid phase was removed, and the magnetic microspheres were washed with 10 mL of distilled water each time, for a total of 6 times, to obtain magnetic microspheres C (acrylic polymer-modified magnetic microspheres C).
第四，将所合成的磁性微球C转移至10mL的溶液X中，加入0.004mol 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和0.004mol N-羟基琥珀酰亚胺，室温下搅拌混匀，搅拌反应15min，用磁铁沉降磁性微球，去除液相，每次用10mL蒸馏水清洗3次；移取4.0×10 -4 mol 1,3-丙二胺溶于10mL的PBS缓冲溶液中，加入至清洗后的磁性微球中，在30℃水浴下机械搅拌20小时，用磁铁沉降磁性微球，去除液相，每次用10mL蒸馏水清洗6次，加入10mL PBS缓冲溶液；称取2.5×10 -4mol生物素，加入10mL溶液X，再加入1.0×10 -3mol 1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和0.001mol N-羟基琥珀酰亚胺，室温下搅拌混匀，搅拌反应15min，用NaHCO 3固体粉末将溶液的调至pH7.2，加入至上述清洗后的含有10mL PBS缓冲溶液的磁性微球中，在30℃水浴下机械搅拌20小时，用磁铁沉降磁性微球，去除液相，每次用10mL蒸馏水清洗10次，得到生物素修饰的生物磁性微球D。 Fourth, transfer the synthesized magnetic microspheres C to 10 mL of solution X, add 0.004mol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.004mol N- Hydroxysuccinimide, stir and mix at room temperature, stir and react for 15 min, settle the magnetic microspheres with a magnet, remove the liquid phase, and wash 3 times with 10 mL of distilled water each time; pipette 4.0×10 -4 mol of 1,3-propanedi The amine was dissolved in 10 mL of PBS buffer solution, added to the cleaned magnetic microspheres, mechanically stirred in a 30°C water bath for 20 hours, the magnetic microspheres were sedimented with a magnet, the liquid phase was removed, and washed 6 times with 10 mL of distilled water each time, Add 10mL PBS buffer solution; weigh 2.5×10 -4 mol biotin, add 10mL solution X, and then add 1.0×10 -3 mol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide Hydrochloride and 0.001mol N-hydroxysuccinimide were stirred and mixed at room temperature, and the reaction was stirred for 15min. The pH of the solution was adjusted to 7.2 with NaHCO 3 solid powder, and added to the above washed solution containing 10 mL of PBS buffer solution. The magnetic microspheres were mechanically stirred in a water bath at 30°C for 20 hours, the magnetic microspheres were sedimented with a magnet, the liquid phase was removed, and each time was washed 10 times with 10 mL of distilled water to obtain biotin-modified biomagnetic microspheres D.
实施例2结合纳米抗体anti-eGFP的生物磁性微球H的制备(以纳米抗体anti-eGFP为纯化介质，以生物素-亲和素的亲和复合物作为连接元件)Example 2 Preparation of biomagnetic microspheres H combined with nanobody anti-eGFP (with nanobody anti-eGFP as purification medium and biotin-avidin affinity complex as connecting element)
2.1.合成antiEGFP-mScarlet-亲和素融合蛋白(antiEGFP-mScarlet-Tamvavidin2融合蛋白，一种纳米抗体的融合蛋白)2.1. Synthesis of antiEGFP-mScarlet-avidin fusion protein (antiEGFP-mScarlet-Tamvavidin2 fusion protein, a nanobody fusion protein)
首先构建包括三段基因序列的antiEGFP-mScarlet-Tamvavidin2融合蛋白的DNA模板。Firstly, the DNA template of antiEGFP-mScarlet-Tamvavidin2 fusion protein including three gene sequences was constructed.
其中，antiEGFP为氨基酸序列如SEQ ID No.:1的纳米抗体。Wherein, antiEGFP is a nanobody with amino acid sequence such as SEQ ID No.: 1.
其中，mScarlet是一种明亮的红色荧光蛋白，相应核苷酸序列为SEQ ID No.:3.Among them, mScarlet is a bright red fluorescent protein, and the corresponding nucleotide sequence is SEQ ID No.:3.
Tamvavidin2，一种亲和素类似物，是一种具有结合生物素能力的蛋白。Yamamoto等人于2009年发现(2009_FEBS_Yamanomo T_Tamavidins--novel avidin-like biotin-binding proteins from the Tamogitake mushroom，译文：一种来自Tamogitake菌菇的结合生物素的新型亲和素类似物蛋白)，它具有与链霉亲和素相似的很强的生物素亲和力，另外，其热稳定性优于链霉亲和素。Tamvavidin2, an avidin analog, is a protein with the ability to bind biotin. Discovered in 2009 by Yamamoto et al. (2009_FEBS_Yamanomo T_Tamavidins--novel avidin-like biotin-binding proteins from the Tamogitake mushroom, translation: a novel biotin-binding avidin analog protein from the Tamogitake mushroom), which has Streptavidin has a strong affinity for biotin similar to streptavidin, and in addition, its thermal stability is better than streptavidin.
Tamavidin2的氨基酸序列可从相关数据库检索到，如UniProt B9A0T7，共含有141个氨基酸残基，经密码子转换，优化程序优化得到DNA序列，优化后的核苷酸序列如SEQ ID NO.:2。The amino acid sequence of Tamavidin2 can be retrieved from relevant databases, such as UniProt B9A0T7, which contains a total of 141 amino acid residues. After codon conversion and optimization program optimization, the DNA sequence is obtained. The optimized nucleotide sequence is such as SEQ ID NO.:2.
采用重组PCR方法分别构建antiEGFP-mScarlet-Tamvavidin2融合蛋白(也简记为antiEGFP融合蛋白，分子量59kDa)的DNA模板。采用RCA法进行体外扩增。然后采用基于体外无细胞蛋白合成方法(D2P技术)的proteinfactory系统合成融合蛋白antiEGFP-mScarlet-Tamvavidin2。The DNA templates of antiEGFP-mScarlet-Tamvavidin2 fusion protein (also referred to as antiEGFP fusion protein for short, molecular weight 59kDa) were constructed by recombinant PCR method. In vitro amplification was performed by RCA method. Then, the fusion protein antiEGFP-mScarlet-Tamvavidin2 was synthesized by the proteinfactory system based on the in vitro cell-free protein synthesis method (D2P technology).
本实施例的体外无细胞蛋白合成方法中使用的体外蛋白合成体系(IVTT体系)包括以下组分(终浓度)：9.78mM的pH8.0三羟甲基氨基甲烷盐酸(Tris-HCl)，80mM醋酸钾，5mM醋酸镁，1.8mM核苷三磷酸混合物(腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸，每种核苷三磷酸的浓度均为1.8mM)，0.7mM的氨基酸混合物(甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸，每种氨基酸各自浓度均为0.1mM)，15mM葡萄糖，320mM麦芽糊精(以葡萄糖单元计量摩尔浓度，对应约52mg/mL)，24mM磷酸三钾，2％(w/v)聚乙二醇8000，最后加入50％体积的细胞提取物(具体为酵母细胞提取物，更具体地为乳酸克鲁维酵母细胞提取物)。The in vitro protein synthesis system (IVTT system) used in the in vitro cell-free protein synthesis method of this example includes the following components (final concentrations): 9.78 mM Tris-HCl, pH 8.0, 80 mM Potassium acetate, 5 mM magnesium acetate, 1.8 mM mixture of nucleoside triphosphates (adenosine triphosphate, guanosine triphosphate, cytosine triphosphate, and uridine triphosphate, each nucleoside triphosphate 1.8mM), 0.7mM mixture of amino acids (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine , cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, each at a concentration of 0.1 mM ), 15 mM glucose, 320 mM maltodextrin (molarity in glucose units, corresponding to about 52 mg/mL), 24 mM tripotassium phosphate, 2% (w/v) polyethylene glycol 8000, and finally 50% volume of cell extraction (specifically, yeast cell extract, more specifically, Kluyveromyces lactis cell extract).
其中，所述乳酸克鲁维酵母提取物中包括内源性表达的T7RNA聚合酶。所述乳酸克 鲁维酵母提取物经过以下方式改造：采用基于乳酸克鲁维酵母菌株ATCC8585的改造菌株；采用CN109423496A所记载的方法，将T7RNA聚合酶的编码基因整合到乳酸克鲁维酵母的基因组中，获得改造菌株，使其可以内源性表达T7RNA聚合酶；以改造菌株培养出细胞原料，然后制备细胞提取物。乳酸克鲁维酵母细胞提取物的制备过程采用常规技术手段，参考CN109593656A记载的方法制备。制备步骤概括而言，包括：提供经发酵培养的乳酸克鲁维酵母细胞的适量原料，用液氮将细胞速冻，将细胞打碎，离心收集上清液，即可得到细胞提取物。所得乳酸克鲁维酵母细胞提取物中的蛋白浓度为20～40mg/mL。Wherein, the Kluyveromyces lactis extract includes endogenously expressed T7 RNA polymerase. The Kluyveromyces lactis extract is modified in the following manner: using the modified strain based on Kluyveromyces lactis strain ATCC8585; using the method described in CN109423496A, the encoding gene of T7 RNA polymerase is integrated into the genome of Kluyveromyces lactis In the process, a modified strain is obtained so that it can endogenously express T7 RNA polymerase; the cell raw material is cultured with the modified strain, and then the cell extract is prepared. The preparation process of the Kluyveromyces lactis cell extract adopts conventional technical means, and is prepared with reference to the method described in CN109593656A. In general, the preparation steps include: providing an appropriate amount of raw materials for the fermented Kluyveromyces lactis cells, quick-freezing the cells with liquid nitrogen, breaking the cells, and collecting the supernatant by centrifugation to obtain the cell extract. The protein concentration in the obtained Kluyveromyces lactis cell extract was 20-40 mg/mL.
IVTT反应：向上述体外蛋白合成体系中加入15ng/μL DNA模板(所编码的蛋白中含有荧光标记)，进行体外蛋白质合成反应，混匀后放置在25～30℃环境中反应，反应时间为6～18h。合成所述DNA模板编码的蛋白，得到含有所述蛋白的IVTT反应液。采用紫外吸收法测量RFU值，结合其浓度与RFU值的标准曲线，可计算所述蛋白的含量。IVTT reaction: Add 15ng/μL DNA template (the encoded protein contains fluorescent label) to the above-mentioned in vitro protein synthesis system, carry out in vitro protein synthesis reaction, mix well and place it in the environment of 25-30 °C for reaction, the reaction time is 6 ~18h. Synthesize the protein encoded by the DNA template to obtain an IVTT reaction solution containing the protein. The RFU value is measured by the UV absorption method, and the content of the protein can be calculated by combining the standard curve between its concentration and the RFU value.
IVTT反应结束后，得到antiEGFP-mScarlet-Tamvavidin2融合蛋白的IVTT反应液。将该IVTT的反应液分别经过4000rpm，4℃，离心10min，保留上清液。记为IVTT上清液。After the IVTT reaction, the IVTT reaction solution of the antiEGFP-mScarlet-Tamvavidin2 fusion protein was obtained. The reaction solution of the IVTT was centrifuged at 4000 rpm and 4° C. for 10 min, and the supernatant was retained. Denoted as IVTT supernatant.
2.2.制备结合纳米抗体anti-eGFP的生物磁性微球H2.2. Preparation of biomagnetic microspheres H with nanobody anti-eGFP
吸取30μL 10％(w/v)实施例1制备的生物素修饰的生物磁性微球D，用结合/洗涤缓冲液(10mM Na 2HPO 4pH 7.4，2mM KH 2PO 4，140mM NaCl，2.6mM KCl)洗涤3次后备用。 Pipette 30 μL of 10% (w/v) biotin-modified biomagnetic microspheres D prepared in Example 1 with binding/washing buffer (10 mM Na 2 HPO 4 pH 7.4, 2 mM KH 2 PO 4 , 140 mM NaCl, 2.6 mM KCl) was washed 3 times and used for later use.
取2mL含有antiEGFP-mScarlet-Tamvavidin2融合蛋白的IVTT上清液(RFU值为2400)与上述生物磁性微球D在4℃条件下旋转孵育1小时，收集清液，即为流穿液(RFU值为1700)。流穿液中含有未被微球结合的剩余融合蛋白。RFU值的测试条件为：激发波长(Ex)为569nm，发射波长(Em)为593nm。Take 2 mL of the IVTT supernatant containing antiEGFP-mScarlet-Tamvavidin2 fusion protein (RFU value 2400) and the above-mentioned biomagnetic microspheres D for 1 hour rotation and incubation at 4 °C, and collect the supernatant, which is the flow-through liquid (RFU value). 1700). The flow-through contains the remaining fusion protein that is not bound by the microspheres. The test conditions of RFU value are: excitation wavelength (Ex) is 569 nm, emission wavelength (Em) is 593 nm.
通过上述将生物磁性微球D与含有所述antiEGFP融合蛋白的IVTT上清液进行孵育的过程，被孵育的磁珠通过亲和复合物(生物素-Tamvavidin2)连接方式结合有纳米抗体anti-eGFP，记为生物磁性微球H，也记为antiEGFP磁珠(一种纳米抗体磁珠)。Through the above process of incubating the biomagnetic microspheres D with the IVTT supernatant containing the antiEGFP fusion protein, the incubated magnetic beads are bound with the nanobody anti-eGFP through the connection of the affinity complex (biotin-Tamvavidin2). , denoted as biomagnetic microsphere H, and also denoted as antiEGFP magnetic bead (a nanobody magnetic bead).
2.3.antiEGFP磁珠结合eGFP蛋白的载量的测试(以eGFP作为目标蛋白，eGFP过量)2.3. Test of the capacity of antiEGFP magnetic beads bound to eGFP protein (eGFP is used as the target protein, eGFP is excessive)
作为纯化底物(目标物、目标蛋白)的增强型荧光蛋白eGFP的核苷酸序列如SEQ ID NO.:4所示，是eGFP的A206K突变体，也记为mEGFP。The nucleotide sequence of the enhanced fluorescent protein eGFP as a purification substrate (target, target protein) is shown in SEQ ID NO.: 4, which is the A206K mutant of eGFP, also denoted as mEGFP.
吸取30μL 10％(w/v)本实施例2.2.制备的antiEGFP磁珠，用结合/洗涤缓冲液洗涤3次，备用。Aspirate 30 μL of 10% (w/v) antiEGFP magnetic beads prepared in Example 2.2., wash three times with binding/washing buffer, and set aside.
取2mL eGFP蛋白的IVTT反应液(DNA模板中编码eGFP的核苷酸序列如SEQ ID No.:4)，测量其荧光值，记为Total的荧光值。将其与前述洗涤过的3μL antiEGFP磁珠混合，旋转孵育1小时，未与磁珠结合的上清液记为Flow-through，测量其荧光值。根据表1的荧光值测试结果，利用eGFP的计算公式得到相应的eGFP蛋白的量，计算得到antiEGFP磁珠的载量为17.7mg/mL(每毫升antiEGFP磁珠结合的eGFP蛋白的质量)。Take 2 mL of the IVTT reaction solution of eGFP protein (the nucleotide sequence encoding eGFP in the DNA template is such as SEQ ID No.: 4), measure its fluorescence value, and record it as the fluorescence value of Total. It was mixed with the previously washed 3 μL antiEGFP magnetic beads, and incubated with rotation for 1 hour. The supernatant that was not bound to the magnetic beads was recorded as Flow-through, and its fluorescence value was measured. According to the test results of the fluorescence value in Table 1, the corresponding amount of eGFP protein was obtained by the calculation formula of eGFP, and the load of antiEGFP magnetic beads was calculated to be 17.7 mg/mL (the mass of eGFP protein bound to each milliliter of antiEGFP magnetic beads).
根据纯化的eGFP拟定标准曲线，推算出的eGFP的RFU值换算成蛋白质量浓度的计算式为：According to the standard curve of purified eGFP, the calculated RFU value of eGFP is converted into the calculation formula of protein concentration:
Figure PCTCN2021133735-appb-000004
Figure PCTCN2021133735-appb-000004
其中，X为蛋白质量浓度(μg/mL)，Y为RFU荧光读数，M为eGFP的分子量(26.7kDa)，N为融合蛋白的分子量(59kDa)。Wherein, X is the protein concentration (μg/mL), Y is the RFU fluorescence reading, M is the molecular weight of eGFP (26.7 kDa), and N is the molecular weight of the fusion protein (59 kDa).
表1 antiEGFP磁珠结合eGFP蛋白的载量测试结果Table 1 Loading test results of antiEGFP magnetic beads bound to eGFP protein
Figure PCTCN2021133735-appb-000005
Figure PCTCN2021133735-appb-000005
2.4.antiEGFP磁珠纯化eGFP蛋白的结合效率(以eGFP作为目标蛋白，磁珠过量)2.4. Binding efficiency of eGFP protein purified by antiEGFP magnetic beads (with eGFP as the target protein, the magnetic beads are in excess)
取本实施例2.2制备的antiEGFP磁珠，用结合/洗涤缓冲液洗涤3次，备用。Take the antiEGFP magnetic beads prepared in Example 2.2, wash three times with binding/washing buffer, and set aside.
取1mL eGFP蛋白的IVTT反应液(DNA模板中编码eGFP的核苷酸序列如SEQ ID No.:4)，测量其荧光值，记为Total。将其与过量的antiEGFP磁珠混合，旋转孵育1小时，收集清夜，记为Flow-through，测量其荧光值。磁珠用结合/洗涤缓冲液1mL洗涤2次，每次洗涤4℃孵育旋转10分钟，所得洗涤液分别记为Washing1、Washing2，并分别测量其荧光值。经孵育的磁珠结合了目标蛋白eGFP。上述各荧光值的测量结果如图3和表2所示。结果表明，用本方案制备的抗eGFP的磁珠可以有效结合并洗脱得到eGFP蛋白。根据IVTT上清液及流穿液的荧光值，计算得到上述孵育1小时后，antiEGFP磁珠对目标蛋白eGFP的结合效率为98.2％。Take 1 mL of the IVTT reaction solution of eGFP protein (the nucleotide sequence encoding eGFP in the DNA template is such as SEQ ID No.: 4), measure its fluorescence value, and denote it as Total. It was mixed with excess antiEGFP magnetic beads, incubated with rotation for 1 hour, collected the clear night, recorded as Flow-through, and measured its fluorescence value. The magnetic beads were washed twice with 1 mL of binding/washing buffer, and each wash was incubated and rotated at 4°C for 10 minutes. The obtained washing solutions were recorded as Washing1 and Washing2, respectively, and their fluorescence values were measured. The incubated magnetic beads bind the target protein eGFP. The measurement results of the above-mentioned fluorescence values are shown in FIG. 3 and Table 2. The results show that the anti-eGFP magnetic beads prepared by this protocol can effectively bind and elute eGFP protein. According to the fluorescence values of the IVTT supernatant and the flow-through liquid, it was calculated that the binding efficiency of the antiEGFP magnetic beads to the target protein eGFP after 1 hour of incubation was 98.2%.
表2 antiEGFP磁珠纯化eGFP蛋白的结合效率测试Table 2 Binding efficiency test of eGFP protein purified by antiEGFP magnetic beads
Figure PCTCN2021133735-appb-000006
Figure PCTCN2021133735-appb-000006
用100μL 0.1M pH2.8的甘氨酸洗脱甘氨酸洗脱eGFP，并立即向洗脱液中加入十分之一体积(10μL)的1M pH 8.0的Tris-HCl。测量洗脱液的荧光值，记为Elution，并用SDS-PAGE检测纯度，结果如图4所示，纯度约为95％。Glycine-eluted eGFP was eluted with 100 μL of 0.1 M glycine pH 2.8, and a tenth volume (10 μL) of 1 M Tris-HCl pH 8.0 was added to the eluate immediately. The fluorescence value of the eluate was measured, recorded as Elution, and the purity was detected by SDS-PAGE. The results were shown in Figure 4, and the purity was about 95%.
应当理解，上述仅为本发明的部分优选实施例，本发明并不仅限于上述实施例的内容。对于本领域中的技术人员来说，在本发明技术方案的构思范围内或指导、启示下，可以有各种变化和更改，所作的任何具有等同技术效果的变化和更改，均在本发明保护范围之内。It should be understood that the above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the concept of the technical solution of the present invention or under the guidance and inspiration, and any changes and modifications made with equivalent technical effects are protected by the present invention. within the range.

Claims (13)

  1. 一种生物磁性微球，包括磁性微球本体，其特征在于：所述磁性微球本体外表面具有至少一种带有线性主链和支链的聚合物，所述线性主链的一端固定于磁性微球本体外表面，聚合物的其他端游离于磁性微球本体外表面，所述生物磁性微球的聚合物的支链末端连接有抗体型标签。A biological magnetic microsphere, comprising a magnetic microsphere body, characterized in that: the outer surface of the magnetic microsphere body has at least one polymer with a linear main chain and a branched chain, and one end of the linear main chain is fixed on the The outer surface of the magnetic microsphere body, the other end of the polymer is free from the outer surface of the magnetic microsphere body, and the branched end of the polymer of the biomagnetic microsphere is connected with an antibody-type label.
  2. 根据权利要求1所述生物磁性微球，其特征在于：所述抗体型标签为抗体、抗体的片段、抗体的单链、单链的片段、抗体融合蛋白、抗体片段的融合蛋白中任一种，任一种的衍生物或任一种的变体；The biomagnetic microsphere according to claim 1, wherein the antibody-type label is any one of an antibody, a fragment of an antibody, a single chain of an antibody, a fragment of a single chain, an antibody fusion protein, and a fusion protein of an antibody fragment , a derivative of any or a variant of any;
    优选之一，所述抗体型标签为抗蛋白的抗体；Preferably, the antibody-type tag is an anti-protein antibody;
    优选之一，所述抗体型标签为抗荧光蛋白的抗体；Preferably, the antibody-type tag is an antibody against a fluorescent protein;
    优选之一，所述抗体型标签为抗绿色荧光蛋白或其突变体的抗体；Preferably, the antibody-type tag is an antibody against green fluorescent protein or a mutant thereof;
    优选之一，所述抗体型标签为纳米抗体；Preferably, the antibody-type label is a nanobody;
    优选之一，所述抗体型标签为抗蛋白的纳米抗体；Preferably, the antibody-type tag is an anti-protein nanobody;
    优选之一，所述抗体型标签为抗蛋白的单域抗体；Preferably, the antibody-type tag is an anti-protein single-domain antibody;
    优选之一，所述抗体型标签为抗蛋白的单结构域抗体；Preferably, the antibody-type tag is an anti-protein single-domain antibody;
    优选之一，所述抗体型标签为抗蛋白的VHH抗体；Preferably, the antibody-type tag is an anti-protein VHH antibody;
    优选之一，所述抗体型标签为抗蛋白的scFV抗体；Preferably, the antibody-type tag is an anti-protein scFV antibody;
    优选之一，所述抗体型标签为抗荧光蛋白的纳米抗体；Preferably, the antibody-type label is an anti-fluorescent protein nanobody;
    优选之一，所述抗体型标签为抗绿色荧光蛋白或其突变体的纳米抗体；Preferably, the antibody-type label is a nanobody against green fluorescent protein or a mutant thereof;
    优选之一，所述抗体型标签为Fab片段；Preferably, the antibody-type tag is a Fab fragment;
    优选之一，所述抗体型标签为F(ab’)2片段；Preferably, the antibody-type tag is an F(ab')2 fragment;
    优选之一，所述抗体型标签为Fc片段。Preferably, the antibody-type tag is an Fc fragment.
  3. 根据权利要求1-2中任一项所述生物磁性微球，其特征在于：所述抗体型标签通过亲和复合物相互作用连接到所述聚合物的支链末端。The biomagnetic microsphere according to any one of claims 1-2, wherein the antibody-type tag is linked to the branched end of the polymer through an affinity complex interaction.
  4. 根据权利要求3所述生物磁性微球，其特征在于：所述亲和复合物相互作用选自：生物素-亲和素相互作用、生物素类似物-亲和素相互作用、生物素-亲和素类似物相互作用、生物素类似物-亲和素类似物相互作用；The biomagnetic microsphere according to claim 3, wherein the interaction of the affinity complex is selected from the group consisting of: biotin-avidin interaction, biotin analog-avidin interaction, biotin-avidin interaction Interactions with andtin analogs, biotin analogs - avidin analogs interactions;
    优选之一，所述亲和素为链霉亲和素、改性链霉亲和素、链霉亲和素类似物或者其组合；Preferably, the avidin is streptavidin, modified streptavidin, streptavidin analogs or a combination thereof;
    优选之一，所述生物磁性微球的聚合物的支链末端连接有生物素或生物素类似物，所述生物素或者生物素类似物作为连接元件，通过亲和复合物结合作用进一步连接亲和素或亲和素类似物，所述亲和素或亲和素类似物仍作为连接元件，进一步连接所述抗体型标签。Preferably, the branched end of the polymer of the biomagnetic microsphere is connected with biotin or a biotin analog, and the biotin or biotin analog is used as a connecting element to further connect the affinity through the binding effect of the affinity complex. Avidin or avidin analogs, which still serve as linking elements, further link the antibody-type tags.
  5. 根据权利要求1-4中任一项所述生物磁性微球，其特征在于：所述抗体型标签连接于所述聚合物的支链末端的方式为：共价键合、超分子相互作用或者其组合；The biomagnetic microsphere according to any one of claims 1-4, wherein the antibody-type tag is connected to the branched chain end of the polymer in a manner of covalent bonding, supramolecular interaction or its combination;
    优选之一，所述共价键合利用动态共价键；更优选之一，所述动态共价键包括亚胺键、酰腙键、二硫键或者其组合；Preferably one, the covalent bonding utilizes dynamic covalent bonds; more preferably one, the dynamic covalent bonds include imine bonds, acylhydrazone bonds, disulfide bonds or combinations thereof;
    优选之一，所述超分子相互作用选自：配位结合、亲和复合物相互作用、静电吸附、 氢键、π-π重叠作用、疏水相互作用及其组合；Preferably, the supramolecular interaction is selected from the group consisting of: coordination binding, affinity complex interaction, electrostatic adsorption, hydrogen bonding, π-π overlapping interaction, hydrophobic interaction and combinations thereof;
    更优选之一，所述亲和复合物相互作用选自：生物素-亲和素相互作用、生物素类似物-亲和素相互作用、生物素-亲和素类似物相互作用、生物素类似物-亲和素类似物相互作用。More preferably, the interaction of the affinity complex is selected from the group consisting of: biotin-avidin interaction, biotin analog-avidin interaction, biotin-avidin analog interaction, biotin analog Avidin analog interactions.
  6. 根据权利要求1-5中任一项所述生物磁性微球，其特征在于：所述磁性微球本体的尺寸选自下述任一种粒径尺度或任两种粒径尺度之间的范围：0.1μm、0.15μm、0.2μm、0.25μm、0.3μm、0.35μm、0.4μm、0.45μm、0.5μm、0.55μm、0.6μm、0.65μm、0.7μm、0.75μm、0.8μm、0.85μm、0.9μm、0.95μm、1μm、1.5μm、2μm、2.5μm、3μm、3.5μm、4μm、4.5μm、5μm、5.5μm、6μm、6.5μm、7μm、7.5μm、8μm、8.5μm、9μm、9.5μm、10μm、15μm、20μm、25μm、30μm、35μm、40μm、45μm、50μm、55μm、60μm、65μm、70μm、75μm、80μm、85μm、90μm、95μm、100μm、150μm、200μm、250μm、300μm、350μm、400μm、450μm、500μm、550μm、600μm、650μm、700μm、750μm、800μm、850μm、900μm、950μm、1000μm；所述直径尺寸为平均值；The biomagnetic microsphere according to any one of claims 1 to 5, wherein the size of the magnetic microsphere body is selected from any one of the following particle size scales or a range between any two particle size scales : 0.1μm, 0.15μm, 0.2μm, 0.25μm, 0.3μm, 0.35μm, 0.4μm, 0.45μm, 0.5μm, 0.55μm, 0.6μm, 0.65μm, 0.7μm, 0.75μm, 0.8μm, 0.85μm, 0.9 μm, 0.95μm, 1μm, 1.5μm, 2μm, 2.5μm, 3μm, 3.5μm, 4μm, 4.5μm, 5μm, 5.5μm, 6μm, 6.5μm, 7μm, 7.5μm, 8μm, 8.5μm, 9μm, 9.5μm, 10μm, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm, 50μm, 55μm, 60μm, 65μm, 70μm, 75μm, 80μm, 85μm, 90μm, 95μm, 100μm, 150μm, 200μm, 250μm, 300μm, 350μm 450μm, 500μm, 550μm, 600μm, 650μm, 700μm, 750μm, 800μm, 850μm, 900μm, 950μm, 1000μm; the diameter size is an average value;
    优选方式之一，所述磁性微球本体的直径选自0.1～10μm；In one preferred manner, the diameter of the magnetic microsphere body is selected from 0.1 to 10 μm;
    优选方式之一，所述磁性微球本体的直径选自0.2～6μm；In one preferred manner, the diameter of the magnetic microsphere body is selected from 0.2 to 6 μm;
    优选方式之一，所述磁性微球本体的直径选自0.4～5μm；In one preferred manner, the diameter of the magnetic microsphere body is selected from 0.4 to 5 μm;
    优选方式之一，所述磁性微球本体的直径选自0.5～3μm；In one preferred manner, the diameter of the magnetic microsphere body is selected from 0.5 to 3 μm;
    优选方式之一，所述磁性微球本体的直径选自0.2～1μm；In one preferred manner, the diameter of the magnetic microsphere body is selected from 0.2 to 1 μm;
    优选方式之一，所述磁性微球本体的直径选自0.5～1μm；In one preferred manner, the diameter of the magnetic microsphere body is selected from 0.5 to 1 μm;
    优选方式之一，所述磁性微球本体的直径选自1μm～1mm；In one preferred manner, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm;
    优选方式之一，所述磁性微球本体的平均直径为200nm、250nm、300nm、350nm、400nm、450nm、500nm、550nm、600nm、650nm、700nm、750nm、800nm、850nm、900nm、950nm、1000nm，偏差为±20％，更优选±10％。In one preferred manner, the average diameter of the magnetic microsphere body is 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, and the deviation It is ±20%, more preferably ±10%.
  7. 根据权利要求1-6中任一项所述生物磁性微球，其特征在于：所述聚合物的线性主链为聚烯烃主链或者丙烯酸类聚合物主链；The biomagnetic microsphere according to any one of claims 1-6, wherein the linear main chain of the polymer is a polyolefin main chain or an acrylic polymer main chain;
    优选之一，所述聚合物的线性主链为聚烯烃主链，且由丙烯酸类聚合物的主链提供；Preferably, the linear backbone of the polymer is a polyolefin backbone, and is provided by the backbone of an acrylic polymer;
    更优选之一，所述丙烯酸类聚合物的单体单元为丙烯酸、丙烯酸盐、丙烯酸酯、甲基丙烯酸、甲基丙烯酸盐、甲基丙烯酸酯之一或者其组合。More preferably, the monomer unit of the acrylic polymer is one of acrylic acid, acrylate, acrylate, methacrylic acid, methacrylate, methacrylate, or a combination thereof.
  8. 根据权利要求1所述生物磁性微球，其特征在于：所述聚合物的支链通过基于功能基团的共价键共价结合所述生物素或生物素类似物，再通过所述生物素或生物素类似物直接或间接地连接所述抗体型标签；The biomagnetic microsphere according to claim 1, wherein the branched chain of the polymer covalently binds to the biotin or biotin analog through a functional group-based covalent bond, and then passes through the biotin or a biotin analog directly or indirectly linked to the antibody-type tag;
    优选地，所述基于功能基团的共价键，指由功能基团参与共价偶联形成的共价键，其中，所述功能基团为羧基、羟基、氨基、巯基、羧基的盐形式、氨基的盐形式、甲酸酯基，或前述功能基团的组合。Preferably, the functional group-based covalent bond refers to a covalent bond formed by a functional group participating in covalent coupling, wherein the functional group is in the form of a salt of a carboxyl group, a hydroxyl group, an amino group, a sulfhydryl group, or a carboxyl group. , a salt form of an amino group, a formate group, or a combination of the foregoing functional groups.
  9. 根据权利要求1-8中任一项所述生物磁性微球，其特征在于：所述聚合物的线性主链直接地共价偶联于所述磁性微球本体的外表面，或者通过连接基团间接地共价偶联于所述磁性微球本体的外表面。The biomagnetic microsphere according to any one of claims 1-8, characterized in that: the linear main chain of the polymer is directly covalently coupled to the outer surface of the magnetic microsphere body, or through a linking group The group is indirectly covalently coupled to the outer surface of the magnetic microsphere body.
  10. 根据权利要求1-9中任一项所述生物磁性微球，其特征在于：所述磁性微球本体为SiO 2包裹的磁性材料； The biomagnetic microsphere according to any one of claims 1-9, characterized in that: the magnetic microsphere body is a magnetic material wrapped by SiO 2 ;
    优选地，所述磁性材料为铁氧化物、铁化合物、铁合金、钴化合物、钴合金、镍化合物、镍合金、锰氧化物、锰合金或者其组合；Preferably, the magnetic material is iron oxide, iron compound, iron alloy, cobalt compound, cobalt alloy, nickel compound, nickel alloy, manganese oxide, manganese alloy or a combination thereof;
    更优选地，所述磁性材料为Fe 3O 4、γ-Fe 2O 3、氮化铁、Mn 3O 4、FeCrMo、FeAlC、AlNiCo、FeCrCo、ReCo、ReFe、PtCo、MnAlC、CuNiFe、AlMnAg、MnBi、FeNiMo、FeSi、FeAl、FeSiAl、BaO·6Fe 2O 3、SrO·6Fe 2O 3或PbO·6Fe 2O 3、GdO或者其组合。 More preferably, the magnetic material is Fe 3 O 4 , γ-Fe 2 O 3 , iron nitride, Mn 3 O 4 , FeCrMo, FeAlC, AlNiCo, FeCrCo, ReCo, ReFe, PtCo, MnAlC, CuNiFe, AlMnAg, MnBi, FeNiMo , FeSi, FeAl, FeSiAl , BaO.6Fe2O3 , SrO.6Fe2O3 , or PbO.6Fe2O3 , GdO , or a combination thereof.
  11. 如权利要求3所述生物磁性微球的制备方法，其特征在于：包括以下步骤：The preparation method of biological magnetic microspheres as claimed in claim 3, is characterized in that: comprises the following steps:
    (1)利用氨基化硅烷偶联剂对磁性微球本体进行化学修饰，将氨基引入到磁性微球本体的外表面，形成氨基修饰磁性微球A；(1) The magnetic microsphere body is chemically modified with an aminoated silane coupling agent, and amino groups are introduced into the outer surface of the magnetic microsphere body to form amino-modified magnetic microspheres A;
    所述磁性微球本体为SiO 2包裹的磁性材料； The magnetic microsphere body is a magnetic material wrapped by SiO 2 ;
    (2)利用羧基与氨基之间的共价反应将丙烯酸分子共价偶联到所述磁性微球A的外表面，引入碳碳双键，形成含碳碳双键磁性微球B；(2) utilizing the covalent reaction between the carboxyl group and the amino group to covalently couple the acrylic acid molecule to the outer surface of the magnetic microsphere A, and introduce a carbon-carbon double bond to form the carbon-carbon double bond-containing magnetic microsphere B;
    (3)在不加交联剂的条件下，利用碳碳双键的聚合反应，将丙烯酸类单体分子进行聚合，获得的丙烯酸类聚合物具有线性主链和含有功能基团的支链，聚合物通过线性主链的一端共价偶联于磁性微球B的外表面；形成丙烯酸类聚合物修饰磁性微球C；(3) Under the condition of not adding a crosslinking agent, using the polymerization reaction of carbon-carbon double bonds, the acrylic monomer molecules are polymerized, and the obtained acrylic polymer has a linear main chain and a branched chain containing functional groups, The polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain; the acrylic polymer modified magnetic microsphere C is formed;
    (4)通过所述聚合物支链含有的功能基团，将生物素或生物素类似物共价偶联到聚合物支链末端，得到生物素或生物素类似物修饰的生物磁性微球D；(4) Covalently couple biotin or a biotin analog to the end of the polymer branch through the functional group contained in the polymer branch to obtain biotin or biotin analog modified biomagnetic microspheres D ;
    (5)将提供抗体型标签的原料与所述生物磁性微球D中所述聚合物支链末端的生物素或生物素类似物相连接，得到所述生物磁性微球；(5) linking the raw material that provides the antibody-type label with the biotin or biotin analog at the end of the polymer branch in the biomagnetic microsphere D to obtain the biomagnetic microsphere;
    独立地可选地，包括(6)磁铁沉降生物磁性微球，去除液相，清洗；Independently and optionally, including (6) magnet sedimentation of biomagnetic microspheres, removal of liquid phase, and cleaning;
    优选方式之一，所述提供抗体型标签的原料为亲和素或亲和素类似物与所述抗体型标签的共价连接复合物；In one preferred manner, the raw material for providing the antibody-type tag is a covalently linked complex of avidin or an avidin analog and the antibody-type tag;
    更优选地，所述提供抗体型标签的原料为亲和素-抗体型标签共价连接复合物。More preferably, the raw material for providing the antibody-type tag is an avidin-antibody-type tag covalently linked complex.
  12. 如权利要求11所述生物磁性微球的制备方法，其特征在于：包括所述亲和素-抗体型标签共价连接复合物的更换。The method for preparing biomagnetic microspheres according to claim 11, characterized in that it comprises the replacement of the avidin-antibody tag covalently linked complex.
  13. 根据权利要求1-10中任一项所述生物磁性微球在分离纯化中的应用，优选在蛋白类物质的分离纯化中的应用。Application of the biomagnetic microspheres in separation and purification according to any one of claims 1-10, preferably in the separation and purification of protein substances.
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