CN113564213A - Antibody type biomagnetic microsphere and preparation method and application thereof - Google Patents

Abstract

The invention discloses an antibody type biomagnetic microsphere and a preparation method and application thereof. The invention provides a biomagnetic microsphere, wherein at least one polymer with a linear main chain and a branched chain is fixed on the outer surface of a magnetic microsphere body, and an antibody type label is connected to the tail end of the branched chain of the polymer of the biomagnetic microsphere. The invention also provides a preparation method and application of the biomagnetic microspheres based on the first aspect. The biomagnetic microspheres are convenient to operate and use, can be rapidly dispersed and rapidly precipitated in a solution, and do not need large experimental equipment such as a high-speed centrifuge; the biomagnetic microsphere is an antibody magnetic bead, and the antibody type label can also be connected to the branched chain end of the polymer through affinity complex interaction; the antibody type marker serving as a purification medium has flexible selectivity, and is widely and massively applied to separation and purification of a target substance.

Description

Antibody type biomagnetic microsphere and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biochemistry, and particularly relates to an antibody type biomagnetic microsphere and a preparation method and application thereof.

Background

The separation and purification of protein substances is an important downstream link in the production process flow of biological medicines, and the separation and purification effect and efficiency directly influence the quality and production cost of the protein medicines. For protein purification, agarose gel and other materials are commonly used as purification columns or purification microsphere carriers at present. The three-dimensional porous structure of the gel material is beneficial to improving the specific surface area of the material, thereby increasing the sites capable of being combined with a purification medium and improving the specific binding capacity 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 can also increase the retention time of the protein during protein elution, and discontinuous spaces or dead spaces inside the carrier can also prevent the protein from being eluted from the material, thereby increasing the retention ratio. If the binding sites with the protein are 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 the retention ratio of the protein during elution are greatly reduced; however, if only the outer surface of the carrier is used, the specific surface area of the carrier is greatly reduced, and the number of binding sites of the protein is greatly reduced, thereby reducing the purification efficiency.

The polymer is a high molecular compound and can be formed by polymerizing monomer molecules. The monomer molecules with active sites are adopted for polymerization, the polymerization product can be rich in a large number of active sites, the number of the active sites is greatly increased, and corresponding binding sites can be formed or introduced through the active sites. The polymer has various types and structures, molecular chains are mutually crosslinked to form a net structure, the linear structure of a single linear molecular chain is adopted, the branched structure with a plurality of branched chains (such as structures of branched structures, dendritic structures, comb-shaped structures, hyperbranched structures and the like) is also adopted, and the polymers with different structural types have wide application in different fields.

In the prior art, purification columns for protein separation and purification mainly use covalent coupling to fix the purification medium. After the purification column is used for a plurality of times, the binding performance of the purification medium is reduced, and the purification effect is reduced. Therefore, in order to guarantee higher purification efficiency and quality, operating personnel need in time with the whole changes of filler in the affinity chromatography column, and this process not only consumptive material quantity is big, consumes a large amount of manual works and time moreover, leads to the purification with high costs.

Disclosure of Invention

The invention provides a biomagnetic microsphere, which is combined with an antibody substance for resisting a target substance, can be used for separating and purifying the target substance (including but not limited to protein substances), can be combined with the target substance in a high-flux manner, can effectively reduce the retention ratio of the target substance during elution, can conveniently replace a purification medium, has the characteristics of rapidness, high flux, reusability and reusability, and can greatly reduce the purification cost of the target substance.

1. The invention provides a biomagnetic microsphere, which comprises a magnetic microsphere body, wherein the outer surface of the magnetic microsphere body is provided with 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, the other end of the polymer is dissociated on the outer surface of the magnetic microsphere body, and the tail end of the branched chain of the polymer of the biomagnetic microsphere is connected with an antibody type label.

The biomagnetic microspheres are called antibody magnetic beads, antibody magnetic microspheres or antibody type magnetic microspheres.

The antibody type tag preferably serves as a purification medium.

In a preferred embodiment, the antibody-type tag is any one of an antibody, a fragment of an antibody, a single chain fragment, an antibody fusion protein, a fusion protein of an antibody fragment, a derivative of any one, or a variant of any one.

In a preferred embodiment, the antibody type tag is an anti-protein antibody.

In a preferred embodiment, the antibody-type tag is an antibody against a fluorescent protein.

In a preferred embodiment, the antibody type tag is an antibody against green fluorescent protein or a mutant thereof.

In a preferred embodiment, the antibody-type tag is a nanobody.

In one preferred embodiment, the antibody-type tag is a nanobody against a protein.

In a preferred embodiment, the antibody type tag is a single domain antibody against a protein.

In a preferred embodiment, the antibody-type tag is a single domain antibody against a protein.

In a preferred embodiment, the antibody type tag is a VHH antibody against a protein.

In a preferred embodiment, the antibody type tag is an anti-protein scFV antibody.

In a preferred embodiment, the antibody-type tag is a nanobody against a fluorescent protein.

In one preferred embodiment, the antibody type tag is a nanobody against green fluorescent protein or a mutant thereof.

In a preferred embodiment, the antibody type tag is a Fab fragment.

In a preferred embodiment, the antibody-type tag is a F (ab') 2 fragment.

In a preferred embodiment, the antibody-type tag is an Fc fragment.

In one preferred form, the antibody-type tag is attached to the branched end of the polymer by affinity complex interaction.

In one of the preferred modes, the affinity complex includes but is not limited to the following cases: biotin and avidin, biotin analogue and avidin, biotin and avidin analogue, biotin analogue and avidin analogue;

in one preferred embodiment, the avidin is streptavidin, modified streptavidin, a streptavidin analog, or a combination thereof.

In a preferred embodiment, the antibody-type tag is attached to the end of a branch of the polymer in such a manner that: covalent bonding, supramolecular interactions, or combinations thereof.

In one preferred embodiment, the covalent bonding utilizes a dynamic covalent bond; more preferably, the dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In a preferred form, the supramolecular interaction is selected from the group consisting of: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions, and combinations thereof.

In one of the more preferred modes, the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction.

In one preferred embodiment, the polymer of the biomagnetic microspheres is linked with biotin or a biotin analogue at the end of a branch chain, the biotin or the biotin analogue is used as a linking element to further link avidin or an avidin analogue through affinity complex binding, and the avidin or the avidin analogue is still used as a linking element to further link the antibody type tag.

In one preferred embodiment, the size of the magnetic microsphere body is selected from any one of the following particle size scales or a range between any two of the following particle size scales: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 65 μm, 40 μm, 45 μm, 50 μm, 25 μm, 1 μm, 5 μm, 1 μm, 5 μm, and a, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm; the diameter dimensions are averages.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.1 to 10 μm.

In one preferred mode, the diameter of the magnetic microsphere body is selected from 0.2 to 6 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.4 to 5 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.5 to 3 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.2 to 1 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 0.5 to 1 μm.

In one preferred embodiment, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm.

In a preferred embodiment, the magnetic microsphere body has an average diameter of 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, or 1000nm, with a deviation of ± 20%, more preferably ± 10%.

In one preferred embodiment, the polymer main chain is a polyolefin main chain or an acrylic polymer main chain. The acrylic polymer is defined in the noun and term section. In one preferred form, the polyolefin backbone is simultaneously an acrylic polymer backbone (i.e., the linear backbone of the polymer is the polyolefin backbone and is provided by the backbone of the acrylic polymer).

Preferably, the monomer unit of the acrylic polymer is selected from one or a combination of acrylic monomer molecules such as acrylic acid, acrylate, methacrylic acid, methacrylate and the like. The acrylic polymer may be obtained by polymerization of one of the above monomers or by copolymerization of a corresponding combination of the above monomers.

In one preferred embodiment, the side chain of the polymer is covalently bonded to biotin or a biotin analogue through a covalent bond based on a functional group (to obtain biotin magnetic beads), and then the antibody-type tag is directly or indirectly connected to the biotin or a biotin analogue through the side chain. The process of binding biotin or biotin analogue can be realized by covalent reaction of functional groups contained in the branched chains of the polymer molecules on the outer surface of the biomagnetic microsphere and biotin or biotin analogue. Among the preferred embodiments of the functional group is a specific binding site (defined in detail in the "noun and term" section of the detailed description).

The covalent bond based on the functional group refers to a covalent bond formed by the functional group participating in covalent coupling. Preferably, the functional group is carboxyl, hydroxyl, amino, mercapto, a salt form of carboxyl, a salt form of amino, a formate group, or a combination of the foregoing functional groups. One of the preferred forms of the salt of the carboxyl group is the sodium salt form such as COONa; the salt form of the amino group may be preferably an inorganic salt form or an organic salt form, including, but not limited to, hydrochloride, hydrofluoride, and the like. The combination of the functional groups refers to all branched chains of all polymer molecules on the outer surface of one magnetic microsphere, and allows the participation in the formation of covalent bonds based on different functional groups; in the case of biotin, all biotin molecules on the outer surface of one biotin magnetic microsphere may be covalently linked to different functional groups, but one biotin molecule can be linked to only one functional group.

In one preferred embodiment, the linear backbone of the polymer is covalently coupled to the outer surface of the magnetic microsphere body directly or indirectly via a linking group.

In one preferred embodiment, the magnetic microsphere body is SiO₂A wrapped magnetic material. Alternatively, SiO₂Can be a silane coupling agent with an active site.

In a preferred embodiment, the magnetic material is selected from one or a combination of iron oxide, iron compound, iron alloy, cobalt compound, cobalt alloy, nickel compound, nickel alloy, manganese oxide, and manganese alloy.

Further, Fe is preferable₃O₄、γ-Fe₂O₃Iron nitride, Mn₃O₄、FeCrMo、FeAlC、AlNiCo、FeCrCo、ReCo、ReFe、PtCo、MnAlC、CuNiFe、AlMnAg、MnBi、FeNiMo)、FeSi、FeAl、FeSiAl、MO·6Fe₂O₃One or a combination of GdO; wherein Re is a rare earth element; m is Ba, Sr, Pb, i.e., MO.6Fe₂O₃Is BaO.6Fe₂O₃、SrO·6Fe₂O₃Or PbO.6Fe₂O₃。

The term "immobilized" refers to the linear backbone being "immobilized" on the outer surface of the magnetic microsphere body by covalent bonding.

In one preferred form, the linear backbone is covalently attached to the outer surface of the magnetic microsphere body either directly or indirectly through a linker (linking element).

In one preferred embodiment, the number of the polymer branches is two or more; preferably at least 3.

2. The invention also discloses a preparation method of the biomagnetic microspheres in the first aspect, which comprises the following steps:

(1) chemically modifying the magnetic microsphere body, and introducing amino to the outer surface of the magnetic microsphere body to form an amino modified magnetic microsphere A; when the magnetic microsphere body is SiO₂In the case of the coated magnetic material, the coupling agent is preferably an aminosilicone coupling agent.

In one preferred embodiment, the magnetic microsphere body is chemically modified by a coupling agent.

When the magnetic microsphere body is SiO₂When the magnetic material is wrapped, the magnetic microsphere body can be chemically modified by using a silane coupling agent. The silane coupling agent is preferably an amino silane coupling agent.

(2) Covalently coupling acrylic acid molecules to the outer surface of the magnetic microsphere A by utilizing covalent reaction between carboxyl and amino, and introducing carbon-carbon double bonds to form a carbon-carbon double bond-containing magnetic microsphere B.

(3) Under the condition of not adding a cross-linking agent, polymerizing acrylic monomer molecules (such as sodium acrylate) by utilizing the polymerization reaction of carbon-carbon double bonds to obtain an acrylic polymer, wherein the obtained acrylic polymer has a linear main chain and a branched chain containing functional groups, and the polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain to form the acrylic polymer modified magnetic microsphere C.

The definition of the functional groups of the acrylic monomer molecules and the polymer branches is shown in the noun and term part.

Preferably, the functional group is carboxyl, hydroxyl, amino, mercapto, formate, ammonium salt, salt form of carboxyl, salt form of amino, formate group, or a combination of the foregoing functional groups; the "combination of functional groups" refers to the functional groups contained in all the branched chains of all the polymers on the outer surface of one magnetic microsphere, and the types of the functional groups can be one or more. The meaning of "combination of functional groups" as defined in the first aspect is identical.

Further preferably, the functional group is a specific binding site.

(4) And covalently coupling biotin or biotin analogues to the tail ends of the branched chains of the polymer through functional groups contained in the branched chains of the polymer to obtain the biomagnetic microspheres D combined with the biotin or biotin analogues.

(5) And (2) connecting a raw material for providing an antibody type label with biotin or biotin analogues at the tail end of the polymer branched chain in the biomagnetic microsphere D to obtain the biomagnetic microsphere (an antibody magnetic microsphere).

Independently and optionally, the method comprises (6) settling the biomagnetic microspheres by using a magnet, removing the liquid phase and washing.

In a preferred mode, the biomagnetic microspheres D are of a biotin modification type.

In a preferred embodiment, the source material for providing the antibody-type tag is a covalently linked complex of avidin or an avidin analog and the antibody-type tag.

More preferably, the source material for providing the antibody-type tag is an avidin-antibody-type tag covalent linkage complex. At this point, independently optionally, a replacement of the avidin-antibody type tag covalent linkage complex is included.

3. The invention also discloses application of the biomagnetic microspheres (antibody magnetic microspheres) in the first aspect in separation and purification, preferably application in separation and purification of protein substances.

The main advantages and positive effects of the invention include:

one of the cores of the present invention is the biomagnetic microsphere structure, on the outer surface of the magnetic microsphere body, polymer molecules with linear main chains are covalently fixed, and the polymer molecules also have a large number of functionalized branched chains, and the functionalized branched chains are connected with a purification medium (antibody type label). Through the structure, a large amount of purification media suspended at the side end of the linear main chain of the polymer are provided on the outer surface of the biomagnetic microsphere, so that high retention ratio caused by the traditional net structure is avoided, and a large amount of target binding sites can be provided by overcoming the limitation of specific surface area. The kind of the purification medium can be selected according to the kind of the substance to be purified (target substance). When the purification medium is attached to the polymer arms in the form of an affinity complex, with a strong non-covalent interaction; further, the branched backbone between the purification media and the linear backbone of the polymer may also present a binding effect of an affinity complex, such that the purification media can be easily replaced. The preparation of the magnetic microspheres and the explanation of the principle part are combined for understanding.

The core of the invention is also the construction process (preparation method) of the biomagnetic microsphere structure: through chemical modification of the outer surface, a plurality of binding sites are provided on the outer surface of a magnetic bead (the outer surface of a biomagnetic microsphere body), then polymer molecules are covalently connected to the single binding sites on the outer surface of the magnetic bead, the polymer molecules are covalently connected to the single binding sites on the outer surface of the magnetic bead through one end of a linear main chain, a large number of side branched chains are distributed along the linear main chain, and the side branched chains carry nascent binding sites, so that the amplification of the binding sites is multiple times, dozens of times, hundreds of times and even thousands of times, and then a specific purification medium is connected to the nascent binding sites of the polymer branched chains according to specific purification requirements, so as to realize the capture of corresponding specific target molecules (particularly biochemical molecules). In addition, the single binding site of the biomagnetic microsphere can be covalently linked to only one linear polymer backbone, or can be covalently linked to two or more linear backbones, so that the chain accumulation is not caused, and the retention ratio is preferably increased.

Preferably, one binding site leads out only one linear backbone, which in this case provides a larger space for the linear backbone to move.

Preferably, one binding site leads out only two linear backbones, providing as much space for movement of the linear backbones as possible.

The main advantages and positive effects of the invention also include:

(1) according to the structural design, the polymer carrying a large number of branched chains with special structures wraps the surface of the magnetic microsphere, so that the limitation of specific surface area is overcome, a large number of purified medium (antibody type label) binding sites are provided, the number of the purified medium which can be bound on the surface of the magnetic microsphere is multiplied by multiple times, more than ten times, more than one hundred times and even more than one thousand times, and then a high-flux binding target object is realized (one of the optimal modes of the target object is target protein); so that the biomagnetic microspheres can capture target substances from a mixed system onto the magnetic microspheres efficiently, and high-flux combination, namely high-flux separation, is realized.

(2) The flexibility of the polymer chain can be utilized, the polymer chain can flexibly swing in a reaction and purification mixed system, the activity space of a purification medium is enlarged, the capture rate and the combination amount of protein are increased, the rapid and sufficient combination of a target object is promoted, and the high efficiency and the high flux are realized.

(3) The structural design of the invention ensures that the biomagnetic microspheres can realize the high-efficiency elution of the purified target during the elution, effectively reduces the detention time and the detention proportion of the target and realizes high efficiency and high yield. The purification medium (antibody type label) can be connected to the tail end of the branched chain of the polymer, on one hand, the structure of the polymer can not form a net structure, so that the branched chain is not accumulated, discontinuous space and dead angle can be avoided, and high detention time and high detention ratio caused by the traditional net structure are avoided; on the other hand, the branched chains of the polymer further play a space separation role, so that the purification medium can be fully distributed in the mixed system and is far away from the surfaces of the magnetic microspheres and the internal skeleton of the polymer, the efficiency of capturing the target object is increased, the retention time and the retention proportion of the target object can be effectively reduced in the subsequent elution step, and the separation with high flux, high efficiency and high proportion is realized. The structural design of the invention can utilize the high flexibility of the linear main chain and has the advantage of high magnification of the number of the branched chains, thereby better realizing the combination of high speed and high flux and the separation of high efficiency and high proportion (high yield).

(4) The purification medium (antibody type label) of the biomagnetic microsphere can be connected to the tail end of a polymer branched chain on the outer surface of a magnetic bead in a non-covalent strong binding force manner in an affinity compound manner; when the purification medium needs to be updated and replaced, the purification medium can be conveniently and quickly eluted from the microspheres and recombined with the new purification medium, and the purification performance of the magnetic microspheres can be quickly recovered, so that the biological magnetic microspheres can be repeatedly regenerated and used, and the separation and purification cost is reduced.

(5) The biological magnetic microsphere is convenient to operate and use. When the magnetic microspheres combined with the target object are separated from the system, the operation is convenient, the aggregation state and the position of the magnetic microspheres can be efficiently controlled by only using a small magnet, the rapid dispersion or rapid sedimentation of the magnetic microspheres in the solution is realized, the separation and purification of the target object are simple and rapid, large-scale experimental equipment such as a high-speed centrifuge and the like is not needed, and the separation and purification cost is greatly reduced.

(6) The biological magnetic microsphere provided by the invention has wide application, and the purification medium is selective. According to the specific type of the purified substrate, a corresponding purification medium can be flexibly carried in the magnetic microsphere system, so that the capture of specific target molecules is realized. The non-holoprotein forms such as antibody fragments with smaller molecular size, antibody single chains, nano antibodies and the like can also be selected as the purification medium, and particularly when the nano antibody is used as the purification medium, the high-load capacity of the purification medium can be more easily obtained when the magnetic microspheres are prepared compared with the holoprotein structural complete antibody; and the purification medium is easier to contact with the target substance sufficiently with the oscillation of the polymer chain in the mixed system, thereby obtaining higher binding efficiency.

Drawings

FIG. 1 is a schematic structural diagram of a biomagnetic microsphere. The anti EGFP nano antibody is taken as a purification medium, and is combined at the tail end of a branched chain of a brush-shaped structure in a biotin-avidin-anti EGFP nano antibody mode. Wherein the biomagnetic microsphere body is made of SiO₂Encapsulated Fe₃O₄For example. In the figure, the number of polymer molecules (4) is only used for the sake of simplicity and illustration, and 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 branches pendant from the side ends of the linear backbone is also illustrative and is not intended to limit the number of side branches of the polymer molecules of the present invention.

Fig. 2 is a flow chart of a preparation method of a biomagnetic microsphere, taking a nano antibody as an example of a purification medium. Wherein, the preparation process from the amino modified magnetic microsphere A to the biological magnetic microsphere D corresponds to the preparation of the biotin magnetic microsphere.

FIG. 3 shows the RFU value test result of binding of biomagnetic microsphere H (a magnetic bead with anti EGFP nano-antibody) to eGFP protein. And (3) incubating the biomagnetic microspheres H with IVTT supernatant of the eGFP protein, and combining the eGFP protein. Wherein, Total corresponds to IVTT supernatant which is not processed by the biomagnetic microspheres; flow-through corresponds to Flow-through incubated once.

FIG. 4 shows the result of the experiment for separating and purifying eGFP protein by using biomagnetic microsphere H (anti EGFP magnetic bead): and (3) carrying out elution after the biological magnetic microspheres H and the eGFP solution are incubated, capturing, separating and eluting the eGFP from the stock solution, releasing the eGFP into the eluent, and correspondingly carrying out SDS-PAGE test on the eluent containing the purified eGFP. Wherein M corresponds to Marker molecular weight Marker.

Nucleotide and/or amino acid sequence listing

SEQ ID No. 1, the amino acid sequence of the nano antibody anti-eGFP, the length is 117 amino acids.

SEQ ID No. 2, nucleotide sequence of tamavidin2, 423 bases in length.

Nucleotide sequence of mScarlet, 693 bases in length, SEQ ID No. 3.

SEQ ID No. 4, the nucleotide sequence of mEGFP, 714 bases in length.

Detailed Description

The invention will be further elucidated with reference to the embodiments and examples, to which reference is made. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, without specific conditions being noted in the following examples, are preferably carried out according to, with reference to, the conditions as indicated in the specific embodiments described above, and may then be carried out according to conventional conditions, for example "Sambrook et al, molecular cloning: a Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), "A Laboratory Manual for cell-free protein Synthesis" Experimental Manual for expressed by Alexander S.Spirin and James R.Swartz.cell-free protein synthesis: methods and protocols [ M ] 2008 ", etc., or according to the conditions recommended by the manufacturer.

Unless otherwise indicated, percentages and parts referred to in this invention are percentages and parts by weight.

Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

The temperature units in this application are, unless otherwise specified, degrees Celsius (. degree. C.).

Nouns and terms

The following is an explanation or description of the meanings of the partially related terms or terms used in the present invention in order to facilitate a better understanding of the present invention. The corresponding explanations or illustrations apply to the present invention in its entirety, both as follows and as described above. In the present invention, when a cited document is referred to, the definitions of the related terms, nouns and phrases in the cited document are also incorporated, but in case of conflict with the definitions in the present invention, the definitions in the present invention shall control. In the event that a definition in a cited reference conflicts with a definition in the present disclosure, the cited components, materials, compositions, materials, systems, formulations, species, methods, devices, etc. are not to be construed as limiting.

Magnetic beads: the ferromagnetic or magnetizable microspheres, which can also be described as magnetic beads, have a fine particle size, preferably in the range from 0.1 μm to 1000. mu.m in diameter. Examples of magnetic beads of the present invention include, but are not limited to: magnetic microsphere A, magnetic microsphere B, magnetic microsphere C, biomagnetic microsphere D (a biotin magnetic bead), biomagnetic microsphere H (a magnetic bead with anti EGFP nano antibody), and biomagnetic microsphere K (an antibody magnetic bead).

A magnetic microsphere body: magnetic beads with modified sites (magnetic microspheres with bindable sites). For example silica coated magnetic material particles, more specifically as 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: the acrylic polymer modifies the magnetic microsphere.

Biotin magnetic beads: the magnetic beads to which biotin or a biotin analogue is bound are capable of specifically binding to a substance having an avidin-type label. The advantages include that the target protein can be expressed integrally in a fusion protein mode after being marked by the avidin or the avidin protein mutant, and the application mode is simple and convenient. Also known as biotin magnetic microspheres. The biotin or biotin analogue can be used as a purification medium and also as a linker element.

And (3) biological magnetic microspheres D: a magnetic microsphere combined with biotin or biotin analogues, a biotin magnetic bead. The biotin or biotin analogue can be used as a purification medium and also as a linker element.

Avidin magnetic beads: the magnetic beads to which avidin or an avidin analog is bound are capable of specifically binding a substance having a biotin-type label. Also known as avidin magnetic microspheres.

The biomagnetic microsphere K: the antibody-type tag-bound magnetic beads can be used for separation and purification of a target capable of specifically binding thereto. Also called antibody magnetic microspheres or antibody magnetic beads or antibody type magnetic microspheres.

Nano antibody magnetic beads: the magnetic beads combined with the nano-antibodies can be used for separating and purifying target substances capable of being specifically combined with the nano-antibodies. Also known as nanobody magnetic microspheres.

And (3) biological magnetic microspheres H: a nano antibody magnetic bead, a magnetic microsphere (anti EGFP magnetic bead) combined with a nano antibody anti EGFP. Can be combined by an avidin-anti EGFP covalent connecting complex.

Polymers, broadly included in the present invention are oligomers and polymers having at least three structural units or a molecular weight of at least 500Da (which molecular weight may be characterized in a suitable manner, such as number average molecular weight, weight average molecular weight, viscosity average molecular weight, etc.).

Polyolefin chain: refers to a polymer chain free of heteroatoms covalently linked only by carbon atoms. The invention mainly relates to a polyolefin main chain in a comb structure; such as the linear backbone of an acrylic polymer.

Acrylic polymer: refers to a homopolymer or copolymer having a structure of-C (COO-) -C-unit, the copolymerization form of said copolymer is not particularly limited, preferably capable of providing a linear main chain and a metered amount of pendant group COO-; the acrylic polymer is allowed to contain a hetero atom in the linear main chain. Wherein the carbon-carbon double bond may also allow the presence of other substituents, as long as the progress of the polymerization reaction is not impaired, e.g. methyl substituents (corresponding to-CH)₃C (COO-) -C-). Wherein COO-can be present in the form of-COOH, in the form of a salt (e.g., sodium salt), or in the form of a formate (preferably an alkyl formate, such as methyl formate-COOCH)₃Ethyl formate-COOCH₂CH₃(ii) a May also be hydroxyethyl formate-COOCH₂CH₂OH), and the like. Specific structural forms of the-C (COO-) -C-unit structure include, but are not limited to, -CH (COOH) -CH₂-、-CH(COONa)-CH₂-、-MeC(COOH)-CH₂-、-MeC(COONa)-CH₂-、-CH(COOCH₃)-CH₂-、-CH(COOCH₂CH₂OH)-CH₂-、-MeC(COOCH₃)-CH₂-、-MeC(COOCH₂CH₂OH)-CH₂-any one of the like or any combination thereof. Wherein Me is methyl. The linear main chain of one polymer molecule may have only one kind of the above-mentioned unit structure (corresponding to a homopolymer), or may have two or more kinds of unit structures (corresponding to a copolymer).

Acrylic monomer molecule: the monomer molecule which can be used for synthesizing the acrylic polymer has a basic structure of C (COO ═ C, and examples thereof include CH (cooh) ═ CH₂、CH(COONa)＝CH₂、CH₃C(COOH)＝CH₂、CH₃C(COONa)＝CH₂、CH(COOCH₃)＝CH₂、CH(COOCH₂CH₂OH)＝CH₂、CH₃C(COOCH₃)＝CH₂、CH₃C(COOCH₂CH₂OH)＝CH₂And the like.

Branched chain: chains of the present invention are attached to the branch point and have independent ends. In the present invention, the branched chain and the side chain have the same meaning and may be used interchangeably. In the present invention, the branched chain means a side chain or a side group bonded to the linear main chain of the polymer, and may be a short branched chain such as a carboxyl group, a hydroxyl group, an amino group, or a long branched chain containing a large number of atoms, without any particular requirement for the length or size of the branched chain. The structure of the branched chain is not particularly limited, and the branched chain may be linear or branched with a branched structure. The branches may also contain additional side chains or side groups. The number, length, size, degree of re-branching, and other structural features of the branched chains are preferably such that the net structure is not formed as much as possible, and the branched chains are not accumulated to increase the retention ratio, and in this case, the flexible swing of the linear backbone can be smoothly exerted.

Branched chain skeleton: the branched chain skeleton is formed by connecting skeleton atoms in sequence in a covalent bond or non-covalent bond mode, and is connected to the main chain of the polymer in sequence from the tail end of the branched chain. The functional group at the end of the polymer is linked to the main chain of the polymer via a branched backbone. The cross point of the branched skeleton and the main chain is the branching point of the leading-out branched chain. For example,the antibody type tag at the tail end of the polymer branch chain can sequentially pass through avidin, biotin and propane diamine residue (-NH-CH)₂CH₂CH₂-NH-), carbonyl (residue after amidation of the carboxyl group) to the polyolefin backbone of the polymer.

The end of the branch includes the end of all branches. In the case of a linear main chain, in addition to being fixed to one end of the magnetic microsphere body, the other end of the linear main chain must be connected to a branch point, and thus, is also broadly included in the scope of the "branched 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 1 branch point.

Functional groups of polymer branches: the functional group has reactivity, or has reactivity after being activated, and can directly carry out covalent reaction with reactive groups of other raw materials, or carry out covalent reaction with reactive groups of other raw materials after being activated, so as to generate covalent bond connection. One of the preferred ways to function as a functional group for the polymer branches is a specific binding site.

Direct linkage refers to linkage in which interaction occurs directly without the aid of spacer atoms. Forms of such interactions include, but are not limited to: covalent means, non-covalent means, or a combination thereof.

Indirect attachment means by means of at least one attachment element, in which case at least one spacer atom is involved. The connecting elements include, but are not limited to: linker peptides, affinity complex linkages, and the like.

Immobilization, immobilized, immobilization, and the like "immobilization" means a covalent bonding means.

The "linkage"/"binding" means such as carrying, linking, binding, capturing, etc. is not particularly limited and includes, but is not limited to, covalent means, non-covalent means, etc.

Covalent mode: the covalent bond is directly bonded, and the covalent bond includes, but is not limited to, dynamic covalent bond, which means direct bonding by dynamic covalent bond.

Covalent bond: the method comprises common covalent bonds such as amide bonds and ester bonds, and also comprises dynamic covalent bonds with reversible properties. The covalent bond comprises a dynamic covalent bond. A dynamic covalent bond is a chemical bond with reversible properties including, but not limited to, imine bonds, acylhydrazone bonds, disulfide bonds, or combinations thereof. The meaning of which is understood by those skilled in the chemical arts.

Non-covalent means: including but not limited to, coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions and other supramolecular interaction modes.

Supramolecular interaction: including but not limited to coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions, and combinations thereof.

Linking elements, also referred to as linking groups, refer to elements used to link two or more non-adjacent groups, including at least one atom. The linking means between the linking member and the adjacent group is not particularly limited, and includes, but is not limited to, covalent means, non-covalent means, and the like. The internal connection of the linking member is not particularly limited, and includes, but is not limited to, covalent and non-covalent.

A covalent linking element: the spacer atoms from one end of the linker to the other are all covalently linked.

Specific binding site: in the present invention, the specific binding site refers to a group or a structural site having a binding function on a polymer branch chain, the group or the structural site having a specific recognition and binding function for a specific target, and the specific binding can be achieved by a binding action such as coordination, complexation, electrostatic force, van der waals force, hydrogen bond, covalent bond, or other interaction.

Covalent attachment of the complex: compounds that are linked directly or indirectly by covalent means are also referred to as covalent linkers. Such covalent attachment means include, but are not limited to, covalent bonds, linking peptides, and the like.

Avidin-purification medium covalent linking complex: the compound formed by covalent connection has one end of avidin and the other end of purification medium, and the two are directly connected through covalent bonds or indirectly connected through covalent connecting elements.

Affinity complex: non-covalently linked complexes formed by two or more molecules through specific binding interactions, relying on extremely strong affinity, such as: the complex formed by the interaction of biotin (or a biotin analogue) with avidin (or an avidin-like substance). The manner of binding of biotin to an affinity complex of avidin is well known to those skilled in the art.

Purifying the substrate, also called target, material to be separated from the mixed system. The purification substrate in the present invention is not particularly limited, but preferably the purification substrate is a protein-based substance (in this case, also referred to as a target protein).

A purification medium capable of specifically binding to the purification substrate to capture the purification substrate, thereby separating the purification substrate from the mixed system. The purification medium attached to the end of the branch of the polymer of the invention is a functional element having the function of binding a 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: the biotin can be combined with avidin, and has strong binding force and good specificity.

Avidin: avidin, which can bind biotin with strong binding force and good specificity, is used as Streptavidin (Streptavidin, abbreviated as SA), analogues thereof (Tamvavidin 2, abbreviated as Tam2), modified products thereof, mutants thereof, and the like.

Biotin analogues, meaning non-biotin molecules capable of forming a specific binding with avidin similar to "avidin-biotin", preferably one of them being a polypeptide or protein, such as those developed by IBA

Polypeptides comprising the WSHPQFEK sequence used in the series (e.g.

II、

Etc.), and similar polypeptides containing the WNHPQFEK sequence. WNHPQFEK can be regarded as WSMutated sequences of HPQFEK.

Avidin analogs, which refer to non-avidin molecules capable of forming specific binding with biotin similar to "avidin-biotin", preferably one of which is a polypeptide or protein. The avidin analogs include, but are not limited to, derivatives of avidin, homologous species of avidin (homologues), variants of avidin, and the like. Such avidin analogs are, for example, Tamavidin1, Tamavidin2, etc. (ref.: FEBS Journal,2009,276, 1383-.

Biotin-type label: the biotin type label comprises the following units: biotin, an avidin analog capable of binding avidin analogs, and combinations thereof. The biotin-type tag is capable of specifically binding avidin, an avidin analog, or a combination thereof. Therefore, the method can be used for separating and purifying protein substances including but not limited to protein substances marked by avidin type labels.

Avidin-type tag: the avidin type tag comprises the following units: avidin, avidin analogs that bind biotin analogs, and combinations thereof. The avidin-type tag is capable of specifically binding biotin, a biotin analog, or a combination thereof. Therefore, the method can be used for separating and purifying protein substances including but not limited to protein substances labeled by biotin type labels.

Antibody type tag: the antibody-type tag of the present invention refers to a tag containing an antibody-type substance, which is capable of specifically binding to a corresponding target, such as an antigen. Examples of such antibody-type tags also include anti eGFP antibodies that can specifically bind eGFP protein.

A peptide is a compound in which two or more amino acids are linked by peptide bonds. In the present invention, the peptide and the peptide fragment have the same meaning and may be used interchangeably.

Polypeptide, peptide composed of 10-50 amino acids.

Protein, peptide composed of more than 50 amino acids. The fusion protein is also a protein.

Derivatives of polypeptides, derivatives of proteins: any polypeptide or protein to which the present invention relates, unless otherwise specified (e.g., specifying a particular sequence), is understood to also include derivatives thereof. The derivatives of the polypeptide and the derivatives of the protein at least comprise C-terminal tags, N-terminal tags, C-terminal tags and N-terminal tags. Wherein, C terminal refers to COOH terminal, N terminal refers to NH₂The meaning of which is understood by those skilled in the art. The label can be a polypeptide label or a protein label. Some examples of tags include, but are not limited to, histidine tags (typically containing at least 5 histidine residues; such as the 6 XHis, HHHHHHHHHH; such as the 8 XHis tag), Glu-Glu, c-myc epitopes (EQKLISEEDL),

A Tag (DYKDDDDK), a protein C (EDQVDPRLIDGK), Tag-100(EETARFQPGYRS), a V5 epitope Tag (V5 epitope, GKPIPNPLLGLDST), VSV-G (YTDIEMNRLGK), Xpress (DLYDDDDK), hemagglutinin (YPYDVPDYA), beta-galactosidase (beta-galactosidase), thioredoxin (thioredoxin), histidine-site thioredoxin (His-notch thioredoxin), IgG-binding domain (IgG-binding domain), intein-chitin binding domain (intein-chitin binding domain), T7 gene 10(T7 gene 10), glutathione S-transferase (glutathione-S-transferase, GST), green fluorescent protein (GST), maltose binding protein (maltose binding protein, MBP), and the like.

Protein-based substances, in the present invention, broadly refer to substances containing polypeptides or protein fragments. For example, polypeptide derivatives, protein derivatives, glycoproteins, and the like are also included in the category of protein substances.

Antibody, antigen: the present invention relates to antibodies, antigens, and, unless otherwise specified, domains, subunits, fragments, single chains, single chain fragments, variants thereof are also understood to be encompassed. For example, reference to an "antibody" includes, unless otherwise specified, fragments thereof, heavy chains lacking light chains (e.g., nanobodies), Complementarity Determining Regions (CDRs), and the like. For example, reference to "antigen" includes, unless otherwise specified, epitopes (epitopes), epitope peptides.

The antibody substance, including but not limited to antibodies, fragments of antibodies, single chains of antibodies, single chain fragments, antibody fusion proteins, fusion proteins of antibody fragments, and the like, and derivatives and variants thereof, of the present invention may be any substance that can produce antibody-antigen specific binding.

The antigenic substances, as used herein, include, but are not limited to, antigens known to those skilled in the art and substances capable of performing an antigenic function and specifically binding to the antibody substances.

Anti-protein antibodies: refers to an antibody that specifically binds to a protein.

Nanobody against fluorescent protein: refers to a nanobody capable of specific binding to a fluorescent protein.

Nanobody (nanobody): also known as single domain antibodies (sdabs), or single-chain antibodies (single-chain variable fragments), or single domain antibodies, have only one heavy chain variable domain (VHH).

scFV: is a small molecule formed by connecting the variable region of the heavy chain and the variable region of the light chain of an antibody under a section of peptide chain, and is the smallest functional structural unit with the activity of the antibody.

Fab: is the antigen-binding region of an antibody, which consists of a constant and a variable domain of each of the heavy and light chains, which domains form a paratope at the amino terminus of the monomer, the antigen-binding site, and which variable domains bind to epitopes on their particular antigen.

F (ab') 2: is the product of antibody formation by pepsin which catalyzes antibody cleavage below the hinge region to form an F (ab ') 2 fragment and a pf' fragment. After mild reduction, the F (ab ') 2 fragment can be split into two Fab' fragments.

Homology (homology), unless otherwise specified, means 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. The description object is exemplified by homologous sequences such as the omega sequences mentioned in the present description. Homology here refers to similarity in sequence, and may be equal in numerical similarity (identity).

Homologs, which refer to substances having homologous sequences, may also be referred to as homologues.

"variant," or "variant," refers to a substance that has a different structure (including, but not limited to, minor variations) but retains or substantially retains its original function or property. Such variants include, but are not limited to, nucleic acid variants, polypeptide variants, protein variants. Means for obtaining related variants include, but are not limited to, recombination, deletion or deletion, insertion, displacement, substitution, etc. of the building blocks. Such variants include, but are not limited to, modified products, genetically engineered products, fusion products, and the like. To obtain the gene modification product, the gene modification can be performed by, but not limited to, gene recombination (corresponding to the gene recombination product), gene deletion or deletion, insertion, frame shift, base substitution, and the like. Gene mutation products, also called gene mutants, belong to one type of gene modification products. One of the preferred modes of such variants is a homologue.

Modified product: including but not limited to chemically modified products, amino acid modifications, polypeptide modifications, protein modifications, and the like. The chemical modification product refers to a product modified by chemical synthesis methods such as organic chemistry, inorganic chemistry, polymer chemistry and the like. Examples of the modification method include ionization, salinization, desalinization, complexation, decomplexing, chelation, decomplexing, addition reaction, substitution reaction, elimination reaction, insertion reaction, oxidation reaction, reduction reaction, and post-translational modification, and specific examples thereof include oxidation, reduction, methylation, demethylation, amination, carboxylation, and vulcanization.

"mutant", mutant, as used herein, unless otherwise specified, refers to a mutant product that retains or substantially retains its original function or property, and the number of mutation sites is not particularly limited. Such mutants include, but are not limited to, gene mutants, polypeptide mutants, and protein mutants. Mutants are one type of variant. Means for obtaining relevant mutants include, but are not limited to, recombination, deletion or deletion of structural units, insertion, displacement, substitution, and the like. The structural unit of the gene is basic group, and the structural units of the polypeptide and the protein are amino acid. Types of gene 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 engineered products, fusion products, etc., of the present invention, can retain their original function or property, and can optimize, alter their function or property.

Eluent (target protein for example): eluting the target protein; after elution, the target protein is present in the eluent.

Washing solution (taking target protein as an example): eluting impurities such as impure protein and the like; after elution, the impure protein is carried away by the washing liquid.

The binding force is as follows: binding capacity, e.g., binding capacity of magnetic microspheres to a protein.

IVTT: in vitro transcription and translation, an In vitro transcription and translation system, a cell-free protein synthesis system. The cell-free protein synthesis system takes exogenous target mRNA or DNA as a protein synthesis template, and can realize the synthesis of target protein by artificially controlling and supplementing substrates required by protein synthesis, substances such as transcription and translation related protein factors and the like. The cell-free protein synthesis system of the present invention is not particularly limited, and may be any one or any combination of cell-free protein synthesis systems based on yeast cell extracts, escherichia coli cell extracts, mammalian cell extracts, plant cell extracts, insect cell extracts.

In the present invention, the term "translation-related enzymes" (TRENs) refers to an enzyme substance required in the synthesis process from a nucleic acid template to a protein product, and is not limited to an enzyme required in the translation process. Nucleic acid template: also referred to as genetic template, refers to a nucleic acid sequence that serves as a template for protein synthesis, including DNA templates, mRNA templates, and combinations thereof.

Flow-through liquid: and (3) the clear liquid collected after the magnetic beads are incubated with the system containing the target protein, wherein the clear liquid contains residual target protein which is not captured by the magnetic beads.

RFU, Relative Fluorescence Unit value (Relative Fluorescence Unit).

eGFP: enhanced green fluorescence protein (enhanced green fluorescence protein). In the present invention, the eGFP broadly includes wild-type and variants thereof, including but not limited to wild-type and mutants thereof.

mEGFP: a206K mutant of eGFP.

"optionally" means that there may or may not be any selection criterion that can implement the technical solution of the present invention. In the present invention, the term "optional" means that the present invention can be implemented as long as it is applied to the technical means of the present invention.

In the present invention, preferred embodiments such as "preferred" (e.g., preferred, preferable, preferably, preferred, etc.), "preferred", "more preferred", "better", "most preferred", etc. do not limit the scope and protection of the invention in any sense, do not limit the scope and embodiments of the invention, and are provided as examples only.

In the description of the invention, references to "one of the preferred", "in a preferred embodiment", "some preferred", "preferably", "preferred", "more preferred", "further preferred", "most preferred", etc. preferred, and references to "one of the embodiments", "one of the modes", "an example", "a specific example", "an example", "by way of example", "for example", "such as", "such", etc. do not constitute any limitation in any sense to the scope of coverage and protection of the invention, and the particular features described in each mode are included in at least one embodiment of the invention. The particular features described in connection with the various modes can be combined in any suitable manner in any one or more of the particular embodiments of the invention. In the present invention, the technical features or technical aspects corresponding to the respective preferred embodiments may be combined in any suitable manner.

In the present invention, "any combination thereof" means "more than 1" in number, and means a group consisting of the following cases in an inclusive range: "optionally one of them, or optionally a group of at least two of them".

In the present invention, the description of "one or more", etc. "has the same meaning as" at least one "," a combination thereof "," or a combination thereof "," and a combination thereof "," or any combination thereof "," any combination thereof ", etc., and may be used interchangeably to mean" 1 "or" greater than 1 "in number.

In the present invention, "and/or" means "either one of them or any combination thereof, and also means at least one of them.

The prior art means described in the modes of "usually", "conventionally", "generally", "often", etc. are also referred to as the content of the present invention, and if not specifically stated, they may be regarded as one of the preferred modes of the partial technical features of the present invention, and it should be noted that they do not constitute any limitation to the scope of the invention and the protection scope.

All documents cited herein, and documents cited directly or indirectly by such documents, are hereby incorporated by reference into this application as if each were individually incorporated by reference.

It is to be understood that within the scope of the present invention, each of the above-described technical features of the present invention and each of the technical features described in detail below (including but not limited to the examples) may be combined with each other to constitute a new or preferred technical solution as long as it can be used for implementing the present invention. Not described in detail.

1. The invention provides a biomagnetic microsphere, which comprises a magnetic microsphere body, wherein the outer surface of the magnetic microsphere body is provided with 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, the other end of the polymer is dissociated on the outer surface of the magnetic microsphere body, and the tail end of the branched chain of the polymer of the biomagnetic microsphere is connected with an antibody type label.

The biomagnetic microspheres are called antibody magnetic beads, antibody magnetic microspheres or antibody type magnetic microspheres.

The antibody type label can be used as a purification medium, and can also be used as a connecting element for further connecting other types of purification media.

The antibody-type tag preferably acts as a purification medium.

A typical structure of the biomagnetic microspheres is shown in FIG. 1.

Taking the target as a protein substance as an example: compared with the gel porous materials commonly used at present, such as agaroses, most of the commercially available microspheres adopt the agaroses. The porous material possesses a rich pore structure, thereby providing a large specific surface area and a high binding capacity for a purified substrate, but accordingly, when proteins are adsorbed or eluted, protein molecules are required to additionally enter or escape from complex pore channels inside the porous material, which takes more time and is easier to retain. In contrast, the binding site for capturing the target protein provided by the invention only utilizes the outer surface space of the biomagnetic microspheres, and can be directly released into eluent without passing through a complex reticular channel during adsorption and elution, so that the elution time is greatly reduced, the elution efficiency is improved, the retention ratio is reduced, and the purification yield is improved.

1.1. Magnetic microsphere body

In the present invention, the volume of the magnetic microsphere body can be any feasible particle size.

The smaller particle size is beneficial to realizing that the magnetic microspheres are suspended in a mixing system and are more fully contacted with protein products, and the capture efficiency and the binding rate of the protein products are improved. In some preferred modes, the diameter size of the magnetic microsphere body is any one of the following particle size scales (deviation may be ± 25%, ± 20%, ± 15%, ± 10%) or a range between any two particle size scales: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 65 μm, 40 μm, 45 μm, 50 μm, 25 μm, 1 μm, 5 μm, 1 μm, 5 μm, and a, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm. Unless otherwise specified, the diameter size refers to an average size.

The volume of the magnetic microsphere body can be any feasible particle size.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.1-10 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.2-6 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.4-5 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.5-3 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.2-1 μm.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 0.5-1 μm.

In some preferred embodiments, the average diameter of the magnetic microsphere body is about 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, 1000nm, and the submultiples may be ± 25%, ± 20%, ± 15%, and ± 10%.

In some preferred modes, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm.

In some preferred modes, 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%.

Different magnetic materials can provide different types of activation sites, can create differences in the manner in which the purification media are bound, and can also differ in the ability to disperse and settle with a magnet, and can also create selectivity for the type of substrate being purified.

The magnetic microsphere body and the magnetic microsphere comprising the magnetic microsphere body can be quickly positioned, guided and separated under the action of an external magnetic field, and can be endowed with various active functional groups such as hydroxyl, carboxyl, aldehyde group, amino and the like on the surface of the magnetic microsphere by surface modification or chemical polymerization and other methods.

In some preferred modes, the magnetic microsphere body is SiO₂A wrapped magnetic material. Wherein, SiO₂The wrapping layer may include a silane coupling agent with its own active site.

In some preferred forms, the magnetic material is selected from: iron compounds (e.g., iron oxides), iron alloys, cobalt compounds, cobalt alloys, nickel compounds, nickel alloys, manganese oxides, manganese alloys, zinc oxides, gadolinium oxides, chromium oxides, and combinations thereof.

In some preferred embodiments, the iron oxide is, for example, magnetite (Fe)₃O₄) Maghemite (gamma-Fe)₂O₃) Or a combination of the two oxides, preferably ferroferric oxide.

In some preferred forms, the magnetic material is selected from: fe₃O₄、γ-Fe₂O₃Iron nitride, Mn₃O₄、AlNi(Co)、FeCrMo、FeAlC、AlNiCo、FeCrCo、ReCo、ReFe、PtCo、MnAlC、CuNiFe、AlMnAg、MnBi、FeNi(Mo)、FeSi、FeAl、FeNi(Mo)、FeSiAl、BaO·6Fe₂O₃、SrO·6Fe₂O₃、PbO·6Fe₂O₃GdO, and combinations thereof. Wherein, the Re is a rare earth element, rhenium.

1.2. Polymer structures providing a high number of branch ends

The outer surface of the magnetic microsphere body is provided with 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 outer surface of the magnetic microsphere body.

The term "immobilized" refers to being "immobilized" on the outer surface of the magnetic microsphere body by covalent bonding.

In some preferred embodiments, the polymer is covalently coupled to the outer surface of the magnetic microsphere body directly or indirectly through a linking element.

The polymer has a linear main chain, and at the moment, the polymer has the advantages of high flexibility of the linear main chain and high magnification of the number of branched chains, and can better realize the combination of high speed and high flux, and the separation of high efficiency and high proportion (high yield).

Taking the target as a protein substance as an example: for the magnetic microsphere, one end of the polymer is covalently coupled to the outer surface of the magnetic microsphere body, the rest ends including all branched chains and all functional groups are dissolved in the solution and distributed in the outer space of the magnetic microsphere body, and the molecular chain can be fully stretched and swung, so that the molecular chain can be fully contacted with other molecules in the solution, and the capture of the target protein can be further enhanced. When the target protein is eluted from the magnetic microspheres, the target protein can directly get rid of the constraint of the magnetic microspheres and directly enter the eluent. Compared with the polymer physically wound on the outer surface of the magnetic microsphere body or integrally formed with the magnetic microsphere body, the polymer covalently fixed through one end of the linear main chain (in some preferred modes, a single linear main chain of the polymer is covalently fixed, and in other preferred modes, 2 or 3 linear main chains are covalently led out from the fixed end of the main chain) can effectively reduce the stacking of the molecular chain, strengthen the stretching and swinging of the molecular chain in the solution, strengthen the capture of the target protein, and reduce the retention ratio and the retention time of the target protein during elution.

1.2.1. The polymer main chain of the biomagnetic microsphere provided by the invention

In some preferred embodiments, the linear backbone is a polyolefin backbone or an acrylic polymer backbone.

In other preferred embodiments, the linear backbone of the polymer is an acrylic polymer backbone. The polyolefin main chain may be a linear main chain containing only carbon atoms, or may contain hetero atoms (hetero atoms are non-carbon atoms) in the linear main chain.

In some preferred embodiments, the backbone of the polymer is a polyolefin backbone. The monomer unit of the acrylic polymer is acrylic acid, acrylate, methacrylic acid, methacrylate and other acrylic monomer molecules or a combination thereof. The acrylic polymer may be obtained by polymerization of one of the above monomers or by copolymerization of an appropriate combination of the above monomers.

In some preferred embodiments, the linear backbone of the polymer is a polyolefin backbone. Specifically, for example, the polyolefin backbone is a backbone provided by a polymerization product of one of acrylic acid, acrylate, methacrylic acid, methacrylate, or a combination thereof (a backbone provided by a copolymerization product thereof), or a backbone of a copolymerization product formed by polymerization of the above monomers. The polymerization product of the above monomer combination is exemplified by acrylic acid-acrylic ester copolymer, and also methyl methacrylate-hydroxyethyl methacrylate copolymer (MMA-HEMA copolymer), acrylic acid-hydroxypropyl acrylate copolymer. The above-mentioned monomers participate in the polymerization to form a copolymerization product, such as maleic anhydride-acrylic acid copolymer, for example.

In some preferred embodiments, the linear backbone is a polyolefin backbone and is provided by the backbone of an acrylic polymer.

In some preferred embodiments, the linear backbone is an acrylic polymer backbone.

In other preferred embodiments, the backbone of the polymer is an acrylic polymer backbone. May be a main chain of the polyolefin (main chain is only carbon atoms) or may contain a hetero atom (hetero atom: non-carbon atom) in the main chain.

In other preferred embodiments, the polymer backbone is a block copolymer backbone comprising polyolefin blocks, for example, a polyethylene glycol-b-polyacrylic acid copolymer (within the scope of acrylic copolymers). It is preferable that the flexible swing of the linear main chain is smoothly exerted, that the accumulation of the branched chain is not caused, and that the residence time or/and the ratio is not increased.

In other preferred embodiments, the backbone of the polymer is a condensation-polymerized backbone. The condensation polymerization type main chain refers to a linear main chain which can be formed by condensation polymerization between monomer molecules or oligomers; the polycondensation main chain may be of a homo-type or a co-type. Such as polypeptide chains, polyamino acid chains, and the like. Specifically, for example, an epsilon-polylysine chain, an alpha-polylysine chain, gamma-polyglutamic acid, polyaspartic acid chain, etc., an aspartic acid/glutamic acid copolymer, etc.

The number of linear backbones to which one binding site of the outer surface of the magnetic microsphere body may be covalently coupled may be 1 or more.

In some preferred modes, only one linear main chain is led out from one binding site on the outer surface of the magnetic microsphere body, so that a larger movement space can be provided for the linear main chain.

In other preferred modes, only two linear main chains are led out from one binding site on the outer surface of the magnetic microsphere body, and a larger movement space is provided for the linear main chains as much as possible.

One end of a main chain of the polymer is covalently coupled to the outer surface of the magnetic bead (the outer surface of the biomagnetic microsphere), the rest ends including all branched chains and all functional groups are dissolved in the solution and distributed in the outer space of the magnetic bead, and the molecular chain can be fully stretched and swung, so that the molecular chain can be fully contacted with other molecules in the solution, and the capture of the target protein can be further enhanced. When the target protein is eluted from the magnetic beads, the target protein can directly get rid of the constraint of the magnetic beads and directly enter the eluent; compared with the polymer physically wound on the outer surface of the magnetic bead or integrally formed with the magnetic bead, the polymer covalently fixed through one end of the linear main chain (most preferably, a single linear main chain of the polymer is covalently fixed, and in addition, the fixed end of the main chain is covalently led out 2 or 3 linear main chains) provided by the method can effectively reduce the stacking of the molecular chain, strengthen the stretching and swinging of the molecular chain in the solution, enhance the capture of the target protein, and reduce the retention ratio and the retention time of the target protein during elution.

1.2.2. The polymer branched chain of the biomagnetic microsphere provided by the invention

The number of the branched chains is related to factors such as the size of the magnetic microsphere body, the type of the skeleton structure of the polymer, the chain density (particularly the branched chain density) of the polymer on the outer surface of the magnetic microsphere body and the like.

The number of polymer branches is plural, at least 3. The number of side branches is related to the size of the magnetic microsphere, the length of the polymer main chain, the linear density of the side branches along the polymer main chain, the chain density of the polymer on the outer surface of the magnetic microsphere and other factors. The amount 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.

When the branch termini are bound to the purification medium, each branch terminus is independently bound directly to the purification medium or is indirectly joined to the purification medium through a linking element.

When the purification medium is bound to the end of the branched chain, the number of the purification medium may be 1 or more.

In some preferred embodiments, at least 3 purification media are bound to one molecule of the branched polymer.

1.3. Purification media (antibody type label according to the invention)

1.3.1. The purification medium is a functional element for specifically capturing the target from the mixed system, i.e. the purification medium and the target molecule to be separated and purified can be specifically combined. The captured target molecules can be eluted and released under proper conditions, so that the purposes of separation and purification are achieved.

When the protein substance is taken as a target object, the purification medium and the target protein or a purification label carried in the target protein can mutually form specific binding action.

The purification medium in the biomagnetic microspheres is an antibody type label.

In some preferred embodiments, the antibody-type tag is any one of an antibody, a fragment of an antibody, a single chain fragment, an antibody fusion protein, a fusion protein of an antibody fragment, a derivative of any one, or a variant of any one.

In some preferred embodiments, the antibody-type tag is an anti-protein antibody.

In some preferred embodiments, the antibody-type tag is an anti-fluorescent protein antibody.

In some preferred embodiments, the antibody-type tag is a nanobody.

In some preferred embodiments, the antibody-type tag is a nanobody against a protein.

In some preferred embodiments, the antibody type tag is a single domain antibody against a protein.

In some preferred embodiments, the antibody-type tag is a single domain antibody against a protein.

In some preferred forms, the antibody type tag is a VHH antibody directed against a protein.

In some preferred embodiments, the antibody type tag is an anti-protein scFV antibody.

In some preferred embodiments, the antibody-type tag is a nanobody against a fluorescent protein.

In some preferred modes, the antibody type tag is a nanobody against green fluorescent protein or a mutant thereof.

In some preferred embodiments, the antibody type tag is a Fab fragment.

In some preferred embodiments, the antibody-type tag is a F (ab') 2 fragment.

In some preferred embodiments, the antibody-type tag is an Fc fragment.

1.3.2. Loading mode of purification medium

The manner in which the purification medium is attached to the biotin or the biotin analogue is not particularly limited.

The attachment means of the purification medium to the biotin or the biotin analogue include, but are not limited to: covalent bonds, non-covalent bonds (e.g., supramolecular interactions), linking elements, or combinations thereof.

In some preferred forms, the covalent bond is a dynamic covalent bond; more preferably, the dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In some preferred forms, the supramolecular interaction is selected from the group consisting of: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions, and combinations thereof.

In some preferred forms of the biomagnetic microspheres, the purification medium is attached to the branched ends of the polymer via a linking element comprising an affinity complex.

In some preferred forms, the biotin or biotin analogue binds to avidin or avidin analogue via an affinity complex interaction, and the purification medium is directly or indirectly attached to the avidin or avidin analogue.

In some preferred forms, the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction.

In some preferred embodiments, the affinity complex selection criteria are: the magnetic microsphere has good specificity and strong affinity, and also provides a site for chemical bonding, so that the affinity compound can be covalently connected to the tail end of a branched chain of a polymer, or can be covalently connected to the outer surface of the magnetic microsphere body after chemical modification, such as a binding site of the outer surface, the tail end of a main chain of a linear polymer and the tail end of a branched chain type polymer. Such as a combination of: biotin or an analog thereof and avidin or an analog thereof, and the like.

In some preferred embodiments, the avidin is any one of streptavidin, modified streptavidin, streptavidin analogs, or a combination thereof.

Such avidin analogs, e.g., tamavidin1, tamavidin2, and the like. Tamavidin1 and Tamavidin2 are proteins found by Yamamoto et al in 2009 to have the ability to bind biotin (Takakura Y et al Tamavidins: Novel avidin-like biotin-binding proteins from the tamogitateke mushroom [ J ]. FEBS Journal,2009,276,1383-1397), which have a strong affinity for biotin similar to streptavidin. The thermal stability of Tamavidin2 is superior to that of streptavidin, and its amino acid sequence may be retrieved from relevant database, such as UniProt B9A0T7, or optimized with codon conversion and optimizing program to obtain DNA sequence.

Such as a WSHPQFEK sequence or a variant sequence thereof, a WRHPQFGG sequence or a variant sequence thereof, and the like.

When the loading mode comprises dynamic covalent bonds and supermolecular interactions (especially affinity complex interactions), a reversible loading mode is formed, and the purification medium can be unloaded from the tail end of the branched chain under certain conditions, so as to be updated or replaced.

And (4) updating the purified medium, wherein the purified medium is the same in type before and after updating corresponding to the regeneration of the magnetic microspheres.

The purified medium is replaced, and the types of the purified medium before and after replacement are different corresponding to the change of the magnetic microspheres.

In some preferred modes, the tail end of a branched chain of the polymer of the biomagnetic microsphere is sequentially connected with biotin, avidin and a purification medium; wherein the purification medium is an antibody. The means of linkage between the avidin and the purification medium include, but are not limited to: covalent bonds, non-covalent bonds, linking elements, or combinations thereof.

In some preferred embodiments, the purification medium is attached to the biomagnetic microspheres at the end of a polymer branch via the following attachment elements: including, but not limited to, nucleic acids, oligonucleotides, peptide nucleic acids, aptamers, deoxyribonucleic acids, ribonucleic acids, leucine zippers, helix-turn-helix motifs, zinc finger motifs, biotin, avidin, streptavidin, anti-hapten antibodies, and the like, combinations thereof. Of course, the linking element may also be a double stranded nucleic acid construct, a duplex, a homo-or hetero-hybrid (a homo-or hetero-hybrid selected from DNA-DNA, DNA-RNA, DNA-PNA, RNA-RNA, RNA-PNA or PNA-PNA), or a combination thereof.

1.3.3. Mechanism of action of the purification Medium

The action force of the purification medium for capturing the target substance molecules in the reaction and purification mixed system is the specific binding action between the antibody type label and the target substance.

1.4. Preferred embodiments of the invention

In some preferred embodiments, the antibody-type tag is attached to the branched end of the polymer via affinity complex interaction.

In some preferred forms, the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction.

In some preferred embodiments, the avidin is streptavidin, modified streptavidin, a streptavidin analog, or a combination thereof.

In some preferred embodiments, the antibody-type tag is attached to the end of a branch of the polymer by: covalent bonding, supramolecular interactions, or combinations thereof.

In some preferred forms, the covalent bonding utilizes a dynamic covalent bond; more preferably, the dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In some preferred forms, the supramolecular interaction is selected from the group consisting of: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions, and combinations thereof.

1.5. Detailed description of the preferred embodiments

In a preferred embodiment, the biomagnetic microspheres comprise a magnetic microsphere body, the outer surface of the magnetic microsphere body is provided with 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, the other end of the polymer is free from the outer surface of the magnetic microsphere body, the end of the branched chain of the polymer of the biomagnetic microspheres is connected with biotin or a biotin analogue, the biotin or the biotin analogue is used as a connecting element and is further connected with avidin or an avidin analogue through affinity complex binding, and the avidin or the avidin analogue is still used as the connecting element and is further connected with the antibody type labels.

More preferably, the polymer of the biomagnetic microspheres is connected with biotin at the end of a branch chain, the biotin serves as a connecting element and is further connected with avidin or an avidin analogue through affinity complex binding, and the avidin or the avidin analogue still serves as a connecting element and is further connected with the antibody type tag.

Preferred formulations of the avidin include any of streptavidin, modified streptavidin, streptavidin analogs, and combinations thereof.

Binding mode of biotin or biotin analogue

The manner in which the biotin or biotin analogue is attached to the branched ends of the polymer is not particularly limited.

The means by which the biotin or biotin analogue is attached to the end of the branch of the polymer include, but are not limited to: covalent bonds, non-covalent bonds (e.g., supramolecular interactions), or combinations thereof.

In some preferred forms, the covalent bond is a dynamic covalent bond; more preferably, the dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof.

In some preferred forms, the supramolecular interaction is selected from the group consisting of: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions, and combinations thereof.

In some preferred embodiments, the branches of the polymer are covalently bound to biotin or a biotin analogue via a functional group-based covalent bond, whereby the biotin or biotin analogue is covalently bound to the ends of the polymer branches. Can be obtained by covalent reaction of functional groups contained in branched chains of polymer molecules on the outer surface of the biomagnetic microsphere and biotin or biotin analogues. Among the preferred embodiments of the functional group is a specific binding site (defined in detail in the "noun and term" section of the detailed description).

The covalent bond based on the functional group refers to a covalent bond formed by the functional group participating in covalent coupling. Preferably, the functional group is carboxyl, hydroxyl, amino, mercapto, a salt form of carboxyl, a salt form of amino, a formate group, or a combination of the foregoing functional groups. One of the preferred forms of the salt of the carboxyl group is the sodium salt form such as COONa; the salt form of the amino group may be preferably an inorganic salt form or an organic salt form, including, but not limited to, hydrochloride, hydrofluoride, and the like. The combination of the functional groups refers to all branched chains of all polymer molecules on the outer surface of one magnetic microsphere, and allows the participation in the formation of covalent bonds based on different functional groups; in the case of biotin, all biotin molecules on the outer surface of one biotin magnetic microsphere may be covalently linked to different functional groups, but one biotin molecule can be linked to only one functional group.

1.6. Regeneration and reuse of purification media

When the purification medium is connected to the end of the polymer branch chain of the biomagnetic microsphere of the invention in a reversible mode such as affinity complex, the purification medium can be eluted from the end of the polymer branch chain under proper conditions, and then new purification medium is recombined.

Take the example of affinity complex interaction as the affinity complex interaction force between biotin and streptavidin.

The strong affinity between biotin and streptavidin is the binding effect of a typical affinity complex, which is stronger than the general non-covalent bond effect and weaker than the covalent bond effect, so that a purification medium (antibody type tag) can be firmly bound at the tail end of a polymer branched chain on the outer surface of a magnetic bead, and the streptavidin can be eluted from the specific binding position of the biotin to realize synchronous separation of the purification medium when the purification medium needs to be replaced, so that an activation site which can be recombined with a new avidin-purification medium covalent bond complex (such as a purification medium with a streptavidin tag) is released, thereby realizing the rapid recovery of the purification performance of the magnetic bead, and greatly reducing the separation and purification cost of a target. The process of eluting the biomagnetic microspheres modified with the purification medium and removing the avidin-purification medium covalent linkage compound to recover the biomagnetic microspheres modified with biotin or biotin analogues is called as the regeneration of the biotin magnetic microspheres. The regenerated biotin magnetic microspheres have released biotin active sites and can be recombined with avidin-purification medium covalent linkage complexes to obtain purification medium modified biomagnetic microspheres (corresponding to the regeneration of the biomagnetic microspheres), so that fresh purification medium can be provided, and new target binding sites can be provided. Therefore, the biotin magnetic microspheres of the invention can be recycled, namely, the purified medium can be replaced for reuse.

1.7. Purification of the substrate (target)

The purification substrate of the present invention refers to the magnetic microspheres of the present invention used to capture the isolated substance.

The purification substrate of the present invention is a substance capable of producing a specific binding effect with the antibody-type tag.

In some preferred embodiments, the purification substrate is a proteinaceous substance. When the purification substrate is a protein substance, the purification substrate is also referred to as a target protein.

1.7.1. Type of protein of interest

The target protein can be a natural protein or an altered product thereof, and can also be an artificially synthesized sequence. The source of the native 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 source can include, but is not limited to, murine (including rat, mouse, guinea pig, hamster, etc.), rabbit, monkey, human, pig, sheep, cow, dog, horse, etc. The pathogens include viruses, chlamydia, mycoplasma, etc. The viruses include HPV, HBV, TMV, coronavirus, rotavirus, etc.

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, collagens, polyamino acids, vaccines, etc., partial domains of any of the foregoing, subunits or fragments of any of the foregoing, and variants of any of the foregoing. The "subunit or fragment of any one of the aforementioned proteins" includes a subunit or fragment of "a partial domain of any one of the aforementioned proteins". The "variant of any one of the aforementioned proteins" includes a variant of "a partial domain of any one of the aforementioned proteins, a subunit or fragment of any one of the aforementioned proteins". Such "variants of any of the foregoing proteins" include, but are not limited to, mutants of any of the foregoing proteins. In the present invention, the meanings of two or more "preceding" cases in succession in other positions are similarly explained.

The structure of the target protein can be a complete structure, and can also be selected from corresponding partial domains, subunits, fragments, dimers, multimers, fusion proteins, glycoproteins and the like. Examples of incomplete antibody structures are nanobodies (heavy chain antibody lacking light chain, V)_HH, retains the full antigen binding ability of the heavy chain antibody), the heavy chain variable region, the Complementarity Determining Region (CDR), and the like.

For example, the target protein synthesized by the in vitro protein synthesis system of the present invention can be selected from the group consisting of, but not limited to, any one of the following proteins, fusion proteins in any combination, and compositions in any combination: luciferase (e.g., firefly luciferase), Green Fluorescent Protein (GFP), enhanced green fluorescent protein (eGFP), Yellow Fluorescent Protein (YFP), aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, Catalase (Catalase, e.g., murine Catalase), actin, antibody, variable region of antibody (e.g., single chain variable region of antibody, scFV), single chain of antibody and fragment thereof (e.g., heavy chain of antibody, nanobody, light chain of antibody), alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin and precursors thereof, glucagon-like peptide (GLP-1), interferon (including but not limited to interferon alpha, e.g., interferon alpha A, interferon beta, interferon gamma, etc.), interleukin (e.g., interleukin-1 beta, interleukin 2, interleukin 12, etc.),(s), Lysozyme, serum albumin (including but not limited to human serum albumin, bovine serum albumin), transthyretin, tyrosinase, xylanase, beta-galactosidase (β -galactosidase, LacZ, such as e.g. e.coli β -galactosidase), etc., a partial domain of any of the foregoing, a subunit or fragment of any of the foregoing, or a variant of any of the foregoing (as defined above, including mutants, such as, for example, luciferase mutants, eGFP mutants, which may also be homologous). Examples of the aminoacyl tRNA synthetase include human lysine-tRNA synthetase (lysine-tRNA synthetase), human leucine-tRNA synthetase (leucine-tRNA synthetase), and the like. Examples of the glyceraldehyde-3-phosphate dehydrogenase include Arabidopsis glyceraldehyde-3-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Reference may also be made to patent document CN 109423496A. The composition in any combination may comprise any one of the proteins described above, and may also comprise a fusion protein in any combination of the proteins described above.

In some preferred embodiments, the protein synthesis capacity of the in vitro protein synthesis system is evaluated by using a target protein having fluorescent properties, such as one of GFP, eGFP, mSCarlet, etc., or an analogous substance thereof, or a mutant thereof.

The application fields of the target protein include but are not limited to the fields of biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nano engineering, cosmetics, food additives, nutritional agents, agriculture, feed, living goods, washing, environment, chemical dyeing, fluorescent labeling and the like.

1.7.2. Mixed system containing target protein

The magnetic microspheres of the invention can be used for separating target protein from a mixed system thereof. The target protein is not limited to one substance, and may be a combination of substances as long as the purpose of purification is to obtain such a composition, or the form of such a composition may satisfy 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; it is also generally desirable that the purification medium does not have specific or non-specific binding to other substances than the target protein in the mixed system.

In the embodiment of the invention, the mixed system containing the target protein can be a natural source, and can also be an artificially constructed or obtained mixed system.

For example, a specific protein can be isolated and purified from commercially available serum.

For example, the target protein can be isolated from the reacted system of the in vitro protein synthesis system.

The published in vitro cell-free protein synthesis methods can be used, for example, see patent documents CN 201610868691.6, WO2018161374a1, KR20190108180A, and CN 108535489B. In general terms, an IVTT system is constructed by components such as a cell extract (containing RNA polymerase expressed by genome integration), a DNA template, an energy system (such as a phosphocreatine-phosphocreatine kinase system), magnesium ions, sodium ions, polyethylene glycol and the like, and IVTT reaction is carried out at the temperature of 28-30 ℃ for 8-12 hours. After the reaction is finished, the obtained IVTT reaction liquid contains the fusion protein coded by the DNA template.

One embodiment of the in vitro protein synthesis system further includes, but is not limited to, for example, the cell-free E.coli-based protein synthesis system described in WO2016005982A 1. Other citations of the present invention, including but not limited to in vitro cell-free protein synthesis systems based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, as described in direct and indirect citations thereof, are also incorporated herein as embodiments of the in vitro protein synthesis system of the present invention. For example, the in vitro Cell-Free protein synthesis system described in the "Lu, Y.Advances in Cell-Free biosynthestic technology. Current Developments in Biotechnology and Bioengineering,2019, Chapter 2, 23-45" section, including but not limited to the "2.1 Systems and Advantages" section, pages 27-28, can be used as an in vitro protein synthesis system for carrying out the present invention. For example, documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109423509A, CN109837293A, CN109971783A, CN109988801A, CN109971775A, CN 36568472, CN 110568422, CN109971775A, CN 0720723672, CN 2019198813, CN2019112066163, CN2018112862093(CN 109971775A), CN 20191819181518, CN2020100693833, CN2020101796894, CN109971775A, CN2020102693382 and cited documents, and the in vitro cell-free protein amplification system, DNA template, and the method of the present invention can be used as the in vitro protein amplification system and the method 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. The exogenous proteins disclosed in the prior art and suitable for in vitro protein synthesis systems derived from prokaryotic cell extracts and eukaryotic cell extracts (yeast cell extracts can be preferred, and kluyveromyces lactis can be more preferred), or the endogenous proteins suitable for prokaryotic cell systems and eukaryotic cell systems (yeast cell systems can be preferred, and kluyveromyces lactis can be more preferred) synthesized in cells can be synthesized by using the in vitro protein synthesis system disclosed by the invention, or synthesized by using the in vitro protein synthesis system provided by the invention.

One of the preferred modes of the in vitro protein synthesis system is the IVTT system. The liquid after the IVTT reaction (referred to as IVTT reaction liquid) contains not only the expressed target protein but also residual reaction materials in the IVTT system, and in particular, various factors derived from cell extracts (such as ribosomes, tRNA, translation-related enzymes, initiation factors, elongation factors, termination factors, and the like). The IVTT reaction liquid can provide a target protein for being combined with magnetic beads on one hand, and can also provide a mixed system for testing the separation effect of the target protein on the other hand.

2. The invention also discloses a preparation method of the biomagnetic microspheres in the first aspect, which comprises the following steps: (i) providing magnetic microspheres (also referred to as biotin magnetic microspheres or biotin magnetic beads) to which biotin or a biotin analogue is bound; (ii) and connecting the raw material for providing the antibody type label with the biotin analogue at the tail end of the polymer branched chain of the biomagnetic microsphere to obtain the biomagnetic microsphere K (antibody magnetic bead) combined with the antibody type label.

In some preferred forms, the antibody type label is provided from a raw material.

In some preferred embodiments, the source material for providing the antibody-type tag is a covalently linked complex of avidin or an avidin analog and the antibody-type tag. And (ii) binding a covalent connection complex of avidin or avidin analogue and the antibody type label to the tail end of a polymer branch chain, and forming the binding action of an affinity complex between biotin or biotin analogue at the tail end of the polymer branch chain and the avidin or avidin analogue to obtain the biomagnetic microspheres K with the antibody type label. Preferred modes of such avidin include, but are not limited to, streptavidin, modified streptavidin, streptavidin analogs, and combinations thereof.

(ii) independently optionally, (iii) magnet sedimentation of the biomagnetic microspheres K, removal of the liquid phase, washing;

more preferably, the source material for providing the antibody-type tag is an avidin-antibody-type tag covalent linkage complex. At this point, independently optionally, (iv) replacement of the avidin-purification medium covalent linkage complex.

A typical preparation method of the biomagnetic microspheres refers to fig. 2, and a nanobody is taken as an example of a purification medium.

2.1. Preparation of biotin magnetic microspheres

Take the biological magnetic microsphere combined with biotin as an example.

The biotin magnetic microsphere can be prepared by the following steps: providing SiO₂Coated magnetic beads (commercially available or self-made), SiO₂Activated modification of (3), covalent attachment of Polymer to SiO₂(the polymer is covalently attached to the SiO through one end of the linear backbone₂And a plurality of side branches distributed along the polymer backbone), biotin is covalently attached to the ends of the branches 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 home-made) may be directly provided.

The biotin magnetic microsphere can be prepared by the following steps: (1) providing or preparing a magnetic microsphere body, wherein the outer surface of the magnetic microsphere body is provided with a reactive group R₁(ii) a (2) At the reactive group R₁Is linked to a polymer having a linear main chain and a plurality of branches, one end of the linear main chain being linked to the reactive group R₁Covalent attachment; (3) biotin or a biotin analogue is attached to the end of the branch.

With SiO₂The wrapped magnetic material is taken as an example of a magnetic microsphere body, and the preparation process of the biotin magnetic microsphere can be prepared through the following steps: (1) providing SiO₂Coated magnetic microspheres (commercially available or self-made) were subjected to SiO₂Activated modification of (2) to form a reactive group R₁(ii) a (2) At the reactive group R₁Carrying out a polymerization reaction (for example, using acrylic acid or sodium acrylate as monomer molecules) to form a polymer having a linear main chain and a plurality of branches, and having a functional group F at the end of the branch₁；(3) Functional group F for linking biotin or biotin analogue to the end of the branch₁To (3). In this case, the polymer covalently bonded to the magnetic microsphere body has a linear main chain, one end of which is covalently fixed to the reactive group R₁And a plurality of pendant side chains distributed along the polymer backbone.

2.1.2. Typical examples

A typical preparation method of the biotin magnetic microspheres comprises the following steps:

step (1): providing a magnetic microsphere body, carrying out chemical modification on the magnetic microsphere body, and introducing amino to the outer surface of the magnetic microsphere body to form the amino modified magnetic microsphere A.

In some preferred modes, the magnetic microsphere body is chemically modified by using a coupling agent.

In some preferred embodiments, the coupling agent is an aminosilicone coupling agent.

In some preferred modes, the magnetic microsphere body is SiO₂The wrapped magnetic material is prepared by chemically modifying a magnetic microsphere body by using a silane coupling agent; the silane coupling agent is in some preferred forms an amino silane coupling agent.

Step (2): covalently coupling acrylic acid molecules to the outer surface of the magnetic microsphere A by utilizing covalent reaction between carboxyl and amino, and introducing carbon-carbon double bonds to form a carbon-carbon double bond-containing magnetic microsphere B.

And (3): polymerizing acrylic monomer molecules (such as sodium acrylate) by utilizing the polymerization reaction of carbon-carbon double bonds to obtain a branched-chain acrylic polymer which has a linear main chain and contains a functional group F₁The polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain to form the acrylic polymer modified magnetic microsphere C. This step can be carried out without addition of a crosslinking agent.

The definition of the functional groups of the acrylic monomer molecules and the polymer branches is shown in the noun and term part.

In some preferred modes, the functional group F₁Is a carboxyl group, a hydroxyl group,Amino, mercapto, formate, ammonium salt, salt form of carboxyl, salt form of amino, formate group, or a combination of the foregoing functional groups; the "combination of functional groups" refers to the functional groups contained in all the branched chains of all the polymers on the outer surface of one magnetic microsphere, and the types of the functional groups can be one or more. The meaning of "combination of functional groups" as defined in the first aspect is identical.

In other preferred embodiments, the functional group is a specific binding site.

And (4): functional group F contained by a branch of the polymer₁Biotin or a biotin analogue is covalently coupled to the end of the polymer branch chain to obtain a biomagnetic microsphere (a biotin magnetic microsphere) to which biotin or a biotin analogue is bound. In the prepared biological magnetic microsphere, a large number of sites capable of being combined with biotin are provided by acrylic polymers (with polyacrylic acid skeletons).

2.1.3. Detailed description of the preferred embodiments

One specific embodiment of the preparation of the biotin magnetic microspheres is as follows.

Specifically, taking an example in which an acrylic polymer provides a linear main chain and a large number of branches, the present invention provides one embodiment as follows: the ferroferric oxide magnetic beads coated by silicon dioxide are used as a body of the biological magnetic microsphere; firstly, chemically modifying a silicon dioxide-coated ferroferric oxide magnetic bead by using a 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), introducing amino to the outer surface of the magnetic bead to finish the SiO reaction₂Activating and modifying to obtain amino modified magnetic microspheres A; then, covalently coupling immobilized molecules (acrylic acid molecules, providing a carbon-carbon double bond and a reactive group carboxyl) to the outer surface of the magnetic bead by utilizing a covalent reaction between carboxyl and amino, so that the carbon-carbon double bond is introduced to the outer surface of the magnetic bead to obtain a carbon-carbon double bond-containing magnetic microsphere B; then polymerizing acrylic monomer molecules (such as sodium acrylate) by using polymerization reaction of carbon-carbon double bondAt the same time, covalently coupling the polymerization product to the outer surface of the magnetic bead to complete the reaction in SiO₂Connecting a polymer (covalent connection mode) to obtain the acrylic polymer modified magnetic microsphere C; the immobilized molecules are acrylic acid molecules, one immobilized molecule only leads out one polymer molecule, and simultaneously only leads out one polymer linear main chain; taking sodium acrylate as an example of a monomer molecule, the polymerization product is sodium polyacrylate, the main chain of the sodium polyacrylate is a linear polyolefin main chain, and a large number of side chain COONa are covalently connected along the main chain, and the functional group contained in the side chain is also COONa; in the polymerization reaction, a cross-linking agent such as N, N' -methylenebisacrylamide (CAS: 110-26-9) is not used, and molecular chains are prevented from being cross-linked with each other to form a network polymer, but a linear main chain is generated in the polymerization product under the condition of not adding the cross-linking agent. If the molecular chains are crosslinked into a network polymer, a porous structure is formed, and the elution efficiency of the target protein is influenced.

In some preferred modes, the amount of the acrylic acid used for preparing the magnetic microspheres B is 0.002-20 mol/L.

In some preferred modes, the amount of the sodium acrylate used for preparing the magnetic microspheres C is 0.53-12.76 mol/L.

The external surface of the biomagnetic microsphere can also adopt other activation modification modes besides amination. For example, the aminated biomagnetic microspheres (amino modified magnetic microspheres a) can further react with acid anhydride or other modified molecules, so as to implement chemical modification of the external surface carboxylation or other activation modes of the biomagnetic microspheres.

The immobilized molecules are small molecules which are used for covalently fixing the main chain of the polymer to the outer surface of the magnetic beads. The immobilized small molecule is not particularly limited as long as one end of the immobilized small molecule is covalently coupled to the outer surface of the magnetic bead, the other end of the immobilized small molecule can initiate polymerization reaction, including homopolymerization reaction, copolymerization reaction or polycondensation reaction, or the other end of the immobilized small molecule can be copolymerized with the end of the linear main chain of the coupled polymer.

The immobilized molecules allow for the extraction of only a single polymeric linear backbone, as well as two or more polymeric linear backbones, as long as they do not result in chain stacking and/or do not result in an increase in the retention ratio. Preferably, one immobilized molecule leads out only one polymer molecule and only one polymer linear backbone.

In some preferred embodiments, the immobilized molecule allows for the extraction of only a single polymeric linear backbone, as well as two or more polymeric linear backbones, as long as it does not result in chain stacking and/or does not result in an increase in the retention ratio. Preferably, one immobilized molecule leads out only one polymer molecule and only one polymer linear backbone.

In other preferred embodiments, the acrylic monomer molecule as a polymerized monomer unit may also be one of acrylic acid, acrylate, methacrylic acid, methacrylate type monomers or a combination thereof.

In another embodiment of the present invention, the acrylic polymer may be replaced with another polymer. The criteria chosen were: the formed polymer has a linear main chain, a large number of side branched chains are distributed along the main chain, and functional groups are carried on the side branched chains for subsequent chemical modification; namely, aiming at a binding site on the outer surface of the magnetic bead, a large number of functional groups are provided through branched chains distributed at the side end of a linear main chain of the polymer. Such as epsilon-polylysine chains, alpha-polylysine chains, gamma-polyglutamic acid, polyaspartic acid chains, aspartic acid/glutamic acid copolymers, and the like.

A method for introducing polymer molecules of other alternative ways of the above-mentioned polymers to the outer surface of the biomagnetic microspheres: according to the chemical structure of the polymer substitute and the type of the side chain active group thereof, selecting a proper activation modification mode of the external surface of the biomagnetic microsphere, the type of immobilized molecules and the type of monomer molecules, and carrying out proper chemical reaction to introduce a large amount of active groups positioned on the side chain into the external surface of the biomagnetic microsphere.

Covalently coupling acrylic polymer molecules (such as sodium polyacrylate linear molecular chains) to the outer surface of the magnetic beads, and then providing an activation site with a functional group at the tail end of a branched chain, or activating the branched chain functional group of the polymer molecules according to reaction requirements before connecting biotin or biotin analogue molecules to ensure that the polymer molecules have reaction activity and form the activation site; covalently coupling 1, 3-propanediamine to the activated sites of the polymer arms (each monomeric acrylic unit structure may provide one activated site) to form a new functional group (amino group), and then covalently coupling biotin or biotin analogue molecules to the new functional group at the end of the polymer arm by amidation covalent reaction between carboxyl and amino groups to complete covalent attachment of biotin or biotin analogue to the end of the polymer arm. Taking biotin as a purification medium as an example, obtaining biotin or biological analogue modified biological magnetic microspheres D; a biotin molecule can provide a specific binding site. Taking the functional group of the polymer branch chain as COONa as an example, in this case, sodium acrylate is used as a monomer molecule, and before the covalent reaction with 1, 3-propane diamine, carboxyl activation can be performed first, and the existing carboxyl activation method can be used, for example: EDC. HCl and NHS were added.

2.1.3.1. Preparation of acrylic polymer modified magnetic microspheres

Preparing magnetic microspheres A: washing the magnetic microspheres with water solution of ferroferric oxide magnetic microspheres wrapped by silicon dioxide by using absolute ethyl alcohol, adding an ethanol solution of 3-aminopropyltriethoxysilane (APTES, coupling agent), reacting, washing, and introducing a large amount of amino groups on the outer surfaces of the magnetic microspheres.

Preparing magnetic microspheres B: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and N-hydroxysuccinimide (NHS) were added to an aqueous solution of acrylic acid to activate the carboxyl group, and then added to an aqueous solution containing magnetic microspheres A after activation. The activated carboxyl on the acrylic acid and the amino on the outer surface of the magnetic microsphere form covalent bond connection (amido bond), and a large amount of carbon-carbon double bonds are introduced into the outer surface of the magnetic microsphere.

Preparing magnetic microspheres C: and adding the aqueous solution of acrylic monomer molecules into the magnetic microspheres B, and adding an initiator to perform polymerization reaction of carbon-carbon double bonds. C-C double bonds in acrylic monomer molecules and C-C double bonds on the surfaces of the magnetic microspheres are subjected to open bond polymerization, and acrylic polymer molecules are covalently bonded to the outer surfaces of the magnetic microspheres, wherein the acrylic polymer contains carboxyl functional groups; the carboxyl functional group can exist in the form of carboxyl, formate, etc. In one of the preferred forms, sodium formate is present, in which case, for example, sodium acrylate or sodium methacrylate is used as monomer molecule. In another preferred embodiment, the monomer is present as a formate ester, in which case, for example, an acrylate or methacrylate ester is used as the monomer molecule. Formate and formate can obtain better reactivity after being activated by carboxyl.

2.1.3.2. Preparation of biomagnetic microspheres D (Biotin modification)

Solution of magnetic microspheres C: adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC & HCl) and N-hydroxysuccinimide (NHS), activating carboxyl functional groups of side branches of polymer molecules on the outer surface of the microspheres, adding an aqueous solution of propylene diamine, performing a coupling reaction, grafting the propylene diamine at the positions of the side branches of the acrylic polymer molecules, and converting the functional groups of the side branches of the polymer into amino groups from the carboxyl groups.

Aqueous solution of biotin: adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to activate carboxyl in biotin molecules, then adding the activated carboxyl into an aqueous solution containing magnetic microspheres C, and covalently bonding biotin at the position of a nascent functional group (amino) of a side chain of a polymer on the outer surface of the magnetic microspheres C to obtain biomagnetic microspheres D with a large number of side chains of an acrylic polymer respectively connected with the biotin molecules.

2.1.3.3. Preferred embodiment(s) of the invention

In some preferred embodiments, the method for preparing the biomagnetic microspheres D comprises the following steps:

firstly, 0.5-1000 mL (20%, v/v) of aqueous solution of silicon dioxide-coated ferroferric oxide magnetic microspheres is measured, the magnetic microspheres are washed by absolute ethyl alcohol, 10-300 mL of ethanol solution (5% -50%, v/v) of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) is added into the washed magnetic microspheres, reaction is carried out for 2-72 hours, and the magnetic microspheres are washed by absolute ethyl alcohol and distilled water, so that amino modified magnetic microspheres A are obtained.

Removing 1.0X 10^-4About 1mol of acrylic acid is added into a solution X with the pH value of 4-6 (the solution X is an aqueous solution with the final concentration of 0.01-1 mol/L2-morpholine ethanesulfonic acid (CAS: 4432-31-9) and 0.1-2 mol/L NaCl), 0.001-0.5 mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl, CAS: 25952-53-8) and 0.001-0.5 mol of N-hydroxysuccinimide (NHS, CAS: 6066-82-6) are added, and the reaction is carried out for 3-60 min. And adding the solution into PBS buffer solution with the pH value of 7.2-7.5 mixed with 0.5-50 mL of magnetic microspheres A, reacting for 1-48 hours, and washing the magnetic microspheres with distilled water to obtain the carbon-carbon double bond modified magnetic microspheres B.

And (3) taking 0.5-50 mL of magnetic microsphere B, adding 0.5-200 mL of 5-30% (w/v) sodium acrylate solution, then adding 10-20 mL of 2-20% (w/v) ammonium persulfate solution and 1-1 mL of tetramethylethylenediamine, reacting for 3-60 minutes, and then washing the magnetic microsphere with distilled water to obtain the sodium polyacrylate modified magnetic microsphere C.

Transferring 0.5-50 mL of magnetic microsphere C into a solution X with the pH value of 4-6, adding 0.001-0.5 mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 0.001-0.5 mol of N-hydroxysuccinimide (NHS), and reacting for 3-60 min. Then adding PBS buffer solution with 0.0001-1 mol of 1, 3-propane diamine and pH7.2-7.5, and reacting for 1-48 hours. Washing with distilled water, adding PBS buffer solution, and converting COONa of a side branch chain of a polymer in the magnetic microsphere C into an amino functional group; weighing 1.0 × 10^-6～3.0×10^-4Adding biotin into the solution X in mol, adding 2.0X 10^-6～1.5×10^{- 3}mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 2.0X 10^-6～1.5×10^-3And (3) mol of N-hydroxysuccinimide, and reacting for 3-60 min. And then adding the mixture into the cleaned magnetic microsphere solution, reacting for 1-48 hours, and cleaning with distilled water to obtain the biotin-modified magnetic microsphere D.

2.2. Preferred embodiment of preparing the antibody magnetic microsphere based on the biomagnetic microsphere D

The biotin-modified biomagnetic microspheres D are added into a fusion protein solution of an avidin-antibody type tag linkage complex (e.g., anti EGFP-mScelet-Tamvavidin 2, an antibody type fusion protein, and a fusion protein of a nanobody), and mixed and incubated. The antibody type label is fixed on the terminal group of the polymer branch chain of the outer surface of the biomagnetic microsphere D by the specific combination of avidin (such as Streptavidin or Tamvavidin2) and biotin, so as to obtain the biomagnetic microsphere K combined with the avidin-antibody type label. Wherein, the fusion protein of the avidin-antibody type label can be obtained by in vitro cell-free protein synthesis through IVTT reaction. At the moment, supernatant obtained after the reaction of the biomagnetic microspheres D and the IVTT is mixed, and the antibody type label is combined through the specific binding action between the biotin on the outer surfaces of the biomagnetic microspheres D and the avidin fusion protein in the solution.

2.3. The amount of antibody-type tag bound to the outer surface of the biomagnetic microspheres can be determined by the following method (using the fluorescent protein mScarlet as an example):

first, after the binding reaction between a solution of the antibody-type fusion protein and biotin magnetic beads (biotin magnetic microspheres, for example) is completed, the antibody-type fusion protein-bound biomagnetic microspheres K are adsorbed and settled by a magnet. The liquid phase was then collected separately and noted as flow-through. At this time, the concentration of the antibody-type fusion protein in the liquid phase was decreased. And (3) calculating the change value of the fluorescence value in the supernatant obtained by IVTT reaction before and after combination with the biomagnetic microspheres to obtain the fluorescence intensity combined on the biomagnetic microspheres, and converting to obtain the concentration of the antibody type fusion protein. When the concentration of the antibody type fusion protein in the flow-through liquid is basically unchanged compared with that of the antibody type fusion protein in the IVTT solution before the biological magnetic microsphere is incubated, the fact that the adsorption of the biological magnetic microsphere on the antibody type fusion protein is saturated means that the corresponding fluorescence value is not obviously changed any more. A standard curve of fluorescence value and concentration of the mScarlet protein can be established by adopting a pure mScarlet protein, so that the content and concentration of the antibody type fusion protein (such as streptavidin-nanobody, anti EGFP-mScarlet-Tamvavidin2 nanobody fusion protein) bound on the biomagnetic microspheres can be quantitatively calculated.

The biomagnetic microspheres K with the antibody type labels are used for separating and purifying the target substance, and the binding capacity of the target substance can be calculated by the following method: and (2) incubating the biomagnetic microspheres K with a solution (for example, obtained by expressing the target protein by using an in-vitro protein synthesis system) of a target object (taking the target protein as an example), eluting the target protein from the magnetic beads by using an elution buffer solution after the reaction is finished, and allowing the separated target protein to exist in the eluent. And measuring the protein concentration of the target protein in the eluent by adopting a proper method, and further calculating to obtain the yield and the yield of separation and purification.

2.4. Replacement of purification media

Antibody type label was changed by elution: the simultaneous elution of avidin and the simultaneous detachment of the antibody-type tag is thus also a replacement of the avidin-antibody-type tag.

Taking the biomagnetic microsphere K (biotin-avidin-antibody type label connection mode) based on the biotin-modified biomagnetic microsphere D as an example, adding a denaturation buffer solution (containing urea and sodium dodecyl sulfate) into the biomagnetic microsphere K, incubating the biomagnetic microsphere K in a metal bath at 95 ℃, eluting the avidin-antibody type label fusion protein combined with the biotin on the biomagnetic microsphere K to obtain a regenerated biomagnetic microsphere D (releasing the biotin binding site at the tail end of a polymer branch chain), adding a fresh avidin-antibody type label fusion protein-containing solution (such as supernatant obtained after IVTT reaction of anti-EGFP-mSacrlet-Tamvavidin 2) into the regenerated biomagnetic microsphere D, and enabling the biotin binding site of the released biomagnetic microsphere D to be recombined with a new avidin-antibody type label, and a noncovalent specific binding effect is formed between biotin and avidin (such as Tamvavidin2) again, so that the antibody type label is replaced, and the regenerated biomagnetic microsphere K is obtained.

2.5. Position control of magnetic microspheres

After the biological magnetic microsphere is prepared, the magnetic microsphere can be simply and conveniently settled by using a magnet, the liquid phase is removed, and the adsorbed foreign protein or/and other impurities are removed by cleaning.

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 a liquid phase and can not settle within two days or even longer. And can be stably suspended in a liquid system without continuous stirring. On one hand, the magnetic microsphere can be controlled to be in a nanometer size of several micrometers or even less than 1 micrometer, on the other hand, the grafting density of the polymer on the outer surface of the magnetic microsphere can be adjusted, and the characteristics of the hydrophilicity, the structure type, the hydrodynamic radius, the chain length, the number of branched chains, the length of the branched chains and the like of the polymer can be adjusted, so that the suspension performance of the magnetic microsphere system in the system can be better controlled, and the full contact between the magnetic microsphere system and an in vitro protein synthesis reaction mixed system can be realized. One preferred size of the biomagnetic microspheres of the present invention is about 1 micron.

2.6. Specific preferred example of producing antibody magnetic beads

In some preferred embodiments, the preparation method of the biomagnetic microspheres K comprises the following steps:

(1) chemically modifying the magnetic microsphere body, and introducing amino to the outer surface of the magnetic microsphere body to form an amino modified magnetic microsphere A; when the magnetic microsphere body is SiO₂In the case of the coated magnetic material, the coupling agent is preferably an aminosilicone coupling agent.

In one preferred embodiment, the magnetic microsphere body is chemically modified by a coupling agent.

When the magnetic microsphere body is SiO₂When the magnetic material is wrapped, the magnetic microsphere body can be chemically modified by using a silane coupling agent. The silane coupling agent is preferably an amino silane coupling agent.

(2) Covalently coupling acrylic acid molecules to the outer surface of the magnetic microsphere A by utilizing covalent reaction between carboxyl and amino, and introducing carbon-carbon double bonds to form a carbon-carbon double bond-containing magnetic microsphere B.

(3) Under the condition of not adding a cross-linking agent, polymerizing acrylic monomer molecules (such as sodium acrylate) by utilizing the polymerization reaction of carbon-carbon double bonds to obtain an acrylic polymer, wherein the obtained acrylic polymer has a linear main chain and a branched chain containing functional groups, and the polymer is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain to form the acrylic polymer modified magnetic microsphere C.

The definition of the functional groups of the acrylic monomer molecules and the polymer branches is shown in the noun and term part.

Preferably, the functional group is carboxyl, hydroxyl, amino, mercapto, formate, ammonium salt, salt form of carboxyl, salt form of amino, formate group, or a combination of the foregoing functional groups; the "combination of functional groups" refers to the functional groups contained in all the branched chains of all the polymers on the outer surface of one magnetic microsphere, and the types of the functional groups can be one or more. The meaning of "combination of functional groups" as defined in the first aspect is identical.

Further preferably, the functional group is a specific binding site.

(4) Biotin or biotin analogue is covalently coupled to the tail end of the branched chain of the polymer through a functional group contained in the branched chain of the polymer, so that the biomagnetic microsphere D (the biomagnetic microsphere D modified by biotin or the analogue thereof) combined with the biotin or the biotin analogue is obtained.

(5) And (3) connecting a raw material for providing an antibody type label with biotin or biotin analogues at the tail ends of the polymer branched chains in the biomagnetic microspheres D to obtain the litigation biomagnetic microspheres K (antibody magnetic microspheres).

Independently and optionally, the method comprises (6) settling the biomagnetic microspheres by using a magnet, removing the liquid phase and washing.

In a preferred embodiment, the raw material for providing the antibody-type tag is a covalent linkage complex of avidin or an avidin analog and the antibody-type tag; more preferably, the source material for providing the antibody-type tag is an avidin-antibody-type tag covalent linkage complex.

Independently optionally, comprising replacement of the avidin-antibody type tag covalent linkage complex.

Preferably, the source material for providing the antibody-type tag is an avidin-antibody-type tag covalent linkage complex.

3. The invention also discloses application of the antibody magnetic microsphere in separation and purification, preferably application in separation and purification of protein substances (in the case that the antibody type label is an anti-protein antibody).

EXAMPLE 1 preparation of biomagnetic microspheres D (conjugated Biotin)

Preparation of silica-coated magnetic microspheres (also known as magnetic microsphere bodies, magnetic beads, glass beads)

20g of Fe₃O₄The microspheres are put into a mixed solvent of 310mL of ethanol and 125mL of water, 45mL of 28 percent (wt) ammonia water is added, 22.5mL of tetraethoxysilane is added dropwise, the mixture is stirred and reacted for 24 hours at room temperature, and the mixture is washed by ethanol and water after the reaction. Ferroferric oxide microspheres with different particle sizes (about 1 micron, 10 microns and 100 microns) are used as raw materials, and the particle size of the obtained glass beads is controlled. The ferroferric oxide microspheres with different particle sizes can be prepared by a conventional technical means.

The magnetic microspheres produced are used as a base material for modifying purification media or connecting elements-purification media and are therefore also referred to as magnetic microsphere bodies.

The prepared magnetic microsphere has a magnetic core, can be subjected to position control under the action of magnetic force, and realizes operations such as movement, dispersion, sedimentation and the like, so that the magnetic microsphere is a generalized magnetic bead.

The prepared magnetic microsphere has a coating layer of silicon dioxide, so the magnetic microsphere is also called as glass bead, and can reduce the adsorption of the magnetic core on the following components or components: polymer, purification medium, components of in vitro protein synthesis system, nucleic acid template, protein expression product, etc.

Multiple experiments show that the magnetic microsphere has the best suspension property, suspension durability and protein combination efficiency when the particle size is about 1 mu m. The IVTT reaction liquid is used for providing a mixed system of target protein, and for the combination efficiency of the target protein, when the grain size of the magnetic microsphere is about 1 mu m, the grain size can be improved by more than 50% compared with 10 mu m, and can be improved by more than 80% compared with 100 mu m.

The magnetic microsphere coated with silicon dioxide is used for preparing biotin magnetic beads through the following steps.

Firstly, 50mL of aqueous solution of silicon dioxide-coated ferroferric oxide magnetic microspheres (the particle size of the magnetic microspheres is about 1 μm) with solid content of 20% (v/v) is measured, the magnetic microspheres are settled by a magnet, liquid phase is removed, 60mL of absolute ethyl alcohol is used for cleaning the magnetic microspheres each time, and the total cleaning is carried out for 5 times. 100mL of an excessive ethanol solution (25%, v/v) of 3-aminopropyltriethoxysilane (APTES, CAS: 919-30-2) was added to the washed magnetic microspheres, and the mixture was mechanically stirred in a water bath at 50 ℃ for 48 hours, then in a water bath at 70 ℃ for 2 hours, the magnetic microspheres were settled with a magnet, the liquid phase was removed, the magnetic microspheres were washed with 60mL of absolute ethanol each time, 2 times in total, then with 60mL of distilled water each time, and the washing was repeated 3 times to obtain magnetic microspheres A.

Secondly, 0.01mol of acrylic acid is transferred and added into 100mL of solution X (solution X: the aqueous solution of 2-morpholine ethanesulfonic acid (CAS: 4432-31-9) with the final concentration of 0.1mol/L and NaCl 0.5 mol/L), 0.04mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (CAS: 25952-53-8) and 0.04mol of N-hydroxysuccinimide (CAS: 6066-82-6) are added, stirred and mixed evenly at room temperature, stirred and reacted for 15min, NaHCO is used for reaction₃Adjusting the pH of the solution to 7.2 by using solid powder, adding the solution with the adjusted pH into 100mL of PBS buffer solution added with 10mL of magnetic microspheres A, mechanically stirring for 20 hours in a water bath at 30 ℃, settling the magnetic microspheres by using a magnet, removing a liquid phase, washing the magnetic microspheres by using 60mL of distilled water each time, and repeatedly washing for 6 times to obtain magnetic microspheres B.

Thirdly, taking 1mL of the magnetic microsphere B, adding 12mL of 15% (w/v) sodium acrylate solution, adding 450 muL of 10% ammonium persulfate solution and 45 muL of tetramethylethylenediamine, reacting for 30 minutes at room temperature, settling the magnetic microsphere by using a magnet, removing a liquid phase, washing the magnetic microsphere by using 10mL of distilled water each time, and washing for 6 times in total to obtain the magnetic microsphere C (the magnetic microsphere C modified by the acrylic polymer).

Fourthly, transferring the synthesized magnetic microspheres C into 10mL of solution X, adding 0.004mol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.004mol of N-hydroxysuccinimide, stirring and uniformly mixing at room temperature, stirring for reacting for 15min, settling the magnetic microspheres by using a magnet, removing a liquid phase, and washing 3 times by using 10mL of distilled water each time; removing 4.0X 10^-4mol 1, 3-propanediamine is dissolved in 10mL PBS buffer solution, added to the washed solutionMechanically stirring the magnetic microspheres in a water bath at 30 ℃ for 20 hours, settling the magnetic microspheres by using a magnet, removing a liquid phase, washing the magnetic microspheres for 6 times by using 10mL of distilled water each time, and adding 10mL of PBS (phosphate buffer solution); weighing 2.5X 10^-4mol biotin, 10mL of solution X, 1.0X 10^-3mixing mol 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.001mol N-hydroxysuccinimide at room temperature, stirring for reaction for 15min, and reacting with NaHCO₃Adjusting the pH value of the solution to 7.2, adding the solution into the washed magnetic microspheres containing 10mL of PBS buffer solution, mechanically stirring the solution in a water bath at 30 ℃ for 20 hours, settling the magnetic microspheres by using a magnet, removing a liquid phase, and washing the magnetic microspheres 10 times by using 10mL of distilled water each time to obtain the biotin-modified biomagnetic microspheres D.

Example 2 preparation of biomagnetic microspheres H binding to Nanobody anti-eGFP (Nanobody anti-eGFP as purification media, biotin-avidin affinity complexes as connecting elements)

2.1. Synthesis of anti EGFP-mScelet-avidin fusion protein (anti EGFP-mScelet-Tamvavidin 2 fusion protein, a fusion protein of Nano antibody)

Firstly, a DNA template of an anti EGFP-mScarlet-Tamvavidin2 fusion protein comprising three sections of gene sequences is constructed.

Wherein, the anti EGFP is a nano antibody with an amino acid sequence shown as SEQ ID No. 1.

Wherein the 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. Yamamoto et al found in 2009 (2009_ FEBS _ Yamanoo T _ Tamavidins- -novel avidin-like biotin-binding proteins from the Tamogitake mushroom, a translation: a novel biotin-binding avidin-analog protein from Tamogitake mushroom) that it has a strong biotin affinity similar to streptavidin, and in addition, its thermal stability is superior to streptavidin.

The amino acid sequence of Tamavidin2 can be retrieved from a relational database, such as UniProt B9A0T7, which contains 141 amino acid residues in total, and is optimized by a codon conversion and optimization program to obtain a DNA sequence, wherein the optimized nucleotide sequence is shown as SEQ ID No. 2.

DNA templates of anti EGFP-mScarlet-Tamvavidin2 fusion protein (also abbreviated as anti EGFP fusion protein with molecular weight of 59kDa) are respectively constructed by adopting a recombinant PCR method. RCA method is adopted for in vitro amplification. Then, a protein factor system based on an in vitro cell-free protein synthesis method (D2P technology) is adopted to synthesize the fusion protein anti EGFP-mScarlet-Tamvavidin 2.

The in vitro protein synthesis system (IVTT system) used in the in vitro cell-free protein synthesis method of this example comprises the following components (final concentrations): 9.78mM Tris-HCl pH8.0, 80mM potassium acetate, 5mM magnesium acetate, 1.8mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a concentration of 1.8mM), 0.7mM amino acid mixture (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 0.1mM), 15mM glucose, 320mM maltodextrin (molar concentration in glucose units, corresponding to about 52mg/mL), 24mM tripotassium phosphate, 2% (w/v) polyethylene glycol 8000, finally, 50% by volume of cell extract (in particular yeast cell extract, more in particular kluyveromyces lactis cell extract) is added.

Wherein the Kluyveromyces lactis extract comprises endogenously expressed T7 RNA polymerase. The Kluyveromyces lactis extract is modified in the following way: adopting a modified strain based on a Kluyveromyces lactis strain ATCC 8585; integrating a coding gene of T7 RNA polymerase into a genome of Kluyveromyces lactis by adopting the method described in CN109423496A to obtain a modified strain, so that the modified strain can endogenously express T7 RNA polymerase; culturing cell material with the modified strain, and preparing cell extract. The preparation process of the kluyveromyces lactis cell extract adopts conventional technical means, and refers to the method recorded in CN 109593656A. The preparation steps, in summary, include: providing appropriate amount of raw materials of Kluyveromyces lactis cells cultured by fermentation, quickly freezing the cells with liquid nitrogen, crushing the cells, centrifuging, and collecting supernatant to obtain cell extract. The protein concentration of the obtained kluyveromyces lactis cell extract is 20-40 mg/mL.

IVTT reaction: and adding a 15 ng/mu L DNA template (the coded protein contains a fluorescent label) into the in-vitro protein synthesis system to perform in-vitro protein synthesis reaction, uniformly mixing, and placing in an environment with the temperature of 25-30 ℃ for reaction for 6-18 h. Synthesizing the protein coded by the DNA template to obtain IVTT reaction liquid containing the protein. The RFU value is measured by adopting an ultraviolet absorption method, and the content of the protein can be calculated by combining a standard curve of the concentration and the RFU value.

After the IVTT reaction is finished, obtaining IVTT reaction liquid of the anti EGFP-mScarlet-Tamvavidin2 fusion protein. The reaction solution of IVTT is centrifuged for 10min at 4000rpm and 4 ℃ respectively, and the supernatant is reserved. Record as IVTT supernatant.

2.2. Preparation of biomagnetic microsphere H combined with nano antibody anti-eGFP

Aspirate 30. mu.L of 10% (w/v) biotin-modified biomagnetic microspheres D prepared in example 1 with binding/washing buffer (10mM Na)₂HPO₄ pH 7.4，2mM KH₂PO₄140mM NaCl, 2.6mM KCl) for use after 3 washes.

2mL of IVTT supernatant (RFU value of 2400) containing the anti EGFP-mScarlet-Tamvavidin2 fusion protein and the biomagnetic microspheres D are subjected to rotary incubation for 1 hour at 4 ℃, and the supernatant is collected, namely the flow-through solution (RFU value of 1700). The flow-through contains the remaining fusion protein unbound by the microspheres. The test conditions for RFU values were: the excitation wavelength (Ex) was 569nm and the emission wavelength (Em) was 593 nm.

Through the process of incubating the biomagnetic microsphere D and the IVTT supernatant containing the anti EGFP fusion protein, the incubated magnetic beads are combined with a nano antibody anti-eGFP in an affinity complex (biotin-Tamvavidin 2) connection mode and are marked as biomagnetic microspheres H and anti EGFP magnetic beads (nano antibody magnetic beads).

Testing of the Loading of anti EGFP magnetic beads bound to eGFP protein (eGFP as target protein, eGFP excess)

The nucleotide sequence of the enhanced fluorescent protein eGFP used as a purification substrate (target object, target protein) is shown in SEQ ID NO. 4, is an A206K mutant of eGFP and is also marked as mEGFP.

mu.L of 10% (w/v) anti EGFP magnetic beads prepared in this example 2.2. were pipetted and washed 3 times with binding/washing buffer for use.

The fluorescence value of 2mL of IVTT reaction solution of eGFP protein (the nucleotide sequence of the DNA template coding eGFP is shown in SEQ ID No.:4) was measured and recorded as the fluorescence value of Total. This was mixed with the previously washed 3. mu.L of anti EGFP magnetic beads, and the mixture was incubated for 1 hour by rotation, and the supernatant which was not bound to the magnetic beads was designated as Flow-through, and the fluorescence value was measured. According to the fluorescence value test results in table 1, the amount of eGFP protein is calculated by using the eGFP calculation formula, and the loading capacity of the anti eGFP magnetic beads is calculated to be 17.7mg/mL (mass of eGFP protein bound to each mL of anti eGFP magnetic beads).

A standard curve is drawn according to the purified eGFP, and the calculated RFU value of the eGFP is converted into the protein mass concentration by the calculation formula:

wherein X is the protein mass concentration (. mu.g/mL), Y is the RFU fluorescence reading, M is the molecular weight of eGFP (26.7kDa), and N is the molecular weight of the fusion protein (59 kDa).

TABLE 1 results of the loading test of anti EGFP magnetic beads combined with eGFP protein

Purification of eGFP binding efficiency with anti EGFP magnetic beads (eGFP as target protein, magnetic bead excess)

The anti EGFP magnetic beads prepared in example 2.2 were washed 3 times with binding/washing buffer for use.

1mL of IVTT reaction solution of eGFP protein (the nucleotide sequence of the DNA template coding eGFP is shown as SEQ ID No.:4) was taken, and the fluorescence value was measured and recorded as Total. This was mixed with an excess of anti EGFP magnetic beads, incubated for 1 hour with rotation, and the clear solution was collected and recorded as Flow-through and the fluorescence was measured. The magnetic beads were washed 2 times with 1mL of binding/Washing buffer, and the beads were incubated and rotated at 4 ℃ for 10 minutes for each Washing, and the Washing solutions were recorded as Washing1 and Washing2, and the fluorescence values thereof were measured. The incubated magnetic beads bound the target protein eGFP. The measurement results of the fluorescence values are shown in fig. 3 and table 2. The result shows that the magnetic beads for resisting the eGFP prepared by the scheme can be effectively combined and eluted to obtain the eGFP. And calculating the binding efficiency of the anti EGFP magnetic beads to the target protein eGFP after the incubation for 1 hour according to the fluorescence values of the IVTT supernatant and the flow-through liquid to be 98.2%.

Table 2 testing of binding efficiency of anti eGFP magnetic bead purified eGFP protein

eGFP was eluted with 100. mu.L of 0.1M glycine, pH2.8, and one-tenth volume (10. mu.L) of 1M Tris-HCl pH8.0 was immediately added to the eluate. The fluorescence value of the eluate was measured and recorded as "Elution" and the purity was checked by SDS-PAGE, and the purity was about 95% as shown in FIG. 4.

It should be understood that the above description is only a partial description of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made which will achieve the same technical effects within the spirit or scope of the invention and the scope of the invention is to be determined by the appended claims.

Sequence listing

<110> Kangma (Shanghai) Biotech Co., Ltd

<120> antibody type biomagnetic microspheres and preparation method and application thereof

<130> 2020

<141> 2020-11-27

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ttgtcaccac aattcatgta tggttctaga gctttcatta agcatccagc tgatattcca 240

gattactata agcaatcatt cccagaaggt ttcaagtggg aaagagttat gaattttgaa 300

gatggtggtg ctgttactgt tactcaagat acttcattgg aagatggtac tttgatctat 360

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Claims (13)

1. A biological magnetic microsphere comprises a magnetic microsphere body, and is characterized in that: the external surface of the magnetic microsphere body is provided with at least one polymer with a linear main chain and a branched chain, one end of the linear main chain is fixed on the external surface of the magnetic microsphere body, the other end of the polymer is free from the external surface of the magnetic microsphere body, and the tail end of the branched chain of the polymer of the biomagnetic microsphere is connected with an antibody type label.

2. The biomagnetic microsphere of claim 1, wherein: the antibody type tag is any one of an antibody, a fragment of the antibody, a single chain fragment, an antibody fusion protein and a fusion protein of the antibody fragment, a derivative of any one or a variant of any one;

preferably, the antibody type tag is an anti-protein antibody;

preferably, the antibody type tag is an anti-fluorescent protein antibody;

preferably, the antibody type tag is an antibody against green fluorescent protein or a mutant thereof;

preferably, the antibody type tag is a nanobody;

preferably, the antibody type tag is a nano antibody against protein;

preferably, the antibody type tag is a single domain antibody against a protein;

preferably, the antibody type tag is a single domain antibody against a protein;

preferably, the antibody type tag is an anti-protein VHH antibody;

preferably, the antibody type tag is an anti-protein scFV antibody;

preferably, the antibody type label is a nano antibody of anti-fluorescent protein;

preferably, the antibody type tag is a nano antibody against green fluorescent protein or a mutant thereof;

preferably, the antibody type tag is a Fab fragment;

preferably, the antibody type tag is a F (ab') 2 fragment;

preferably, the antibody type tag is an Fc fragment.

3. The biomagnetic microsphere according to any one of claims 1-2, wherein: the antibody-type tag is attached to the branched end of the polymer via affinity complex interactions.

4. The biomagnetic microsphere of claim 3, wherein: the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction;

preferably, the avidin is streptavidin, modified streptavidin, a streptavidin analog or a combination thereof;

preferably, the polymer of the biomagnetic microspheres is connected with biotin or biotin analogue at the end of a branched chain, the biotin or biotin analogue is used as a connecting element to further connect avidin or avidin analogue through affinity complex binding, and the avidin or avidin analogue is still used as a connecting element to further connect the antibody type tag.

5. The biomagnetic microspheres according to any one of claims 1-4, wherein: the antibody type tag is attached to the end of the branch of the polymer in a manner that: covalent bonding, supramolecular interactions, or combinations thereof;

preferably, the covalent bonding utilizes a dynamic covalent bond; more preferably, the dynamic covalent bond comprises an imine bond, an acylhydrazone bond, a disulfide bond, or a combination thereof;

preferably one, said supramolecular interaction is selected from: coordination binding, affinity complex interactions, electrostatic adsorption, hydrogen bonding, pi-pi overlap, hydrophobic interactions, and combinations thereof;

more preferably, the affinity complex interaction is selected from the group consisting of: biotin-avidin interaction, biotin analogue-avidin interaction, biotin-avidin analogue interaction, biotin analogue-avidin analogue interaction.

6. The biomagnetic microspheres according to any one of claims 1-5, 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 of the following particle size scales: 0.1 μm, 0.15 μm, 0.2 μm, 0.25 μm, 0.3 μm, 0.35 μm, 0.4 μm, 0.45 μm, 0.5 μm, 0.55 μm, 0.6 μm, 0.65 μm, 0.7 μm, 0.75 μm, 0.8 μm, 0.85 μm, 0.9 μm, 0.95 μm, 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm, 5 μm, 5.5 μm, 6 μm, 6.5 μm, 7 μm, 7.5 μm, 8 μm, 8.5 μm, 9 μm, 9.5 μm, 10 μm, 15 μm, 20 μm, 25 μm, 30 μm, 65 μm, 40 μm, 45 μm, 50 μm, 25 μm, 1 μm, 5 μm, 1 μm, 5 μm, and a, 650 μm, 700 μm, 750 μm, 800 μm, 850 μm, 900 μm, 950 μm, 1000 μm; the diameter sizes are averages;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.1-10 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.2-6 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.4-5 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.5-3 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.2-1 μm;

in one preferable mode, the diameter of the magnetic microsphere body is selected from 0.5-1 μm;

in one preferred mode, the diameter of the magnetic microsphere body is selected from 1 μm to 1 mm;

in a preferred embodiment, the magnetic microsphere body has an average diameter of 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm, 550nm, 600nm, 650nm, 700nm, 750nm, 800nm, 850nm, 900nm, 950nm, or 1000nm, with a deviation of ± 20%, more preferably ± 10%.

7. The biomagnetic microsphere according to any one of claims 1 to 6, wherein: the linear backbone of the polymer is a polyolefin backbone or an acrylic polymer backbone;

preferably, the linear backbone of the polymer is a polyolefin backbone and is provided by the backbone of an acrylic polymer;

more preferably, the monomer unit of the acrylic polymer is one of acrylic acid, acrylate, methacrylic acid, methacrylate or a combination thereof.

8. The biomagnetic microsphere of claim 1, wherein: the branched chain of the polymer is covalently bonded with the biotin or biotin analogue through a covalent bond based on a functional group, and then the antibody type tag is directly or indirectly connected with the biotin or biotin analogue;

preferably, the covalent bond based on a functional group refers to a covalent bond formed by the functional group participating in covalent coupling, wherein the functional group is carboxyl, hydroxyl, amino, sulfhydryl, salt form of carboxyl, salt form of amino, formate, or a combination of the foregoing functional groups.

9. The biomagnetic microsphere according to any one of claims 1 to 8, wherein: the linear backbone of the polymer is covalently coupled to the outer surface of the magnetic microsphere body either directly or indirectly through a linking group.

10. The biomagnetic microsphere according to any one of claims 1 to 9, wherein: the magnetic microsphere body is SiO₂A wrapped magnetic material;

preferably, the magnetic material is an iron oxide, an iron compound, an iron alloy, a cobalt compound, a cobalt alloy, a nickel compound, a nickel alloy, a manganese oxide, a manganese alloy, or a combination thereof;

more preferably, the magnetic material is Fe₃O₄、γ-Fe₂O₃Iron nitride, Mn₃O₄、FeCrMo、FeAlC、AlNiCo、FeCrCo、ReCo、ReFe、PtCo、MnAlC、CuNiFe、AlMnAg、MnBi、FeNiMo、FeSi、FeAl、FeSiAl、BaO·6Fe₂O₃、SrO·6Fe₂O₃Or PbO.6Fe₂O₃GdO, or a combination thereof.

11. The method for preparing the biomagnetic microspheres according to claim 3, wherein the biomagnetic microspheres comprise: the method comprises the following steps:

(1) chemically modifying the magnetic microsphere body by using an amino silane coupling agent, and introducing amino to the outer surface of the magnetic microsphere body to form an amino modified magnetic microsphere A;

the magnetic microsphere body is SiO₂A wrapped magnetic material;

(2) covalently coupling acrylic acid molecules to the outer surface of the magnetic microsphere A by utilizing covalent reaction between carboxyl and amino, and introducing carbon-carbon double bonds to form a carbon-carbon double bond-containing magnetic microsphere B;

(3) under the condition of not adding a cross-linking agent, polymerizing acrylic monomer molecules by utilizing the polymerization reaction of carbon-carbon double bonds to obtain an acrylic polymer, wherein the acrylic polymer has a linear main chain and a branched chain containing functional groups, and is covalently coupled to the outer surface of the magnetic microsphere B through one end of the linear main chain; forming acrylic polymer modified magnetic microspheres C;

(4) covalently coupling biotin or biotin analogue to the tail end of the polymer branched chain through a functional group contained in the polymer branched chain to obtain biotin or biotin analogue modified biomagnetic microspheres D;

(5) connecting a raw material for providing an antibody type label with biotin or biotin analogues at the tail end of the polymer branched chain in the biomagnetic microsphere D to obtain the biomagnetic microsphere;

independently and optionally, comprises (6) magnet sedimentation of the biomagnetic microspheres, liquid phase removal and washing;

in a preferred embodiment, the raw material for providing the antibody-type tag is a covalent linkage complex of avidin or an avidin analog and the antibody-type tag;

more preferably, the source material for providing the antibody-type tag is an avidin-antibody-type tag covalent linkage complex.

12. The method for preparing biomagnetic microspheres according to claim 11, wherein the biomagnetic microspheres comprise: including replacement of the avidin-antibody type tag covalent linkage complex.

13. Use of the biomagnetic microspheres according to any one of claims 1-10 for separation and purification, preferably for separation and purification of proteinaceous substances.

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